Series Editor: Olga GolubnitschajaAdvances in Predictive, Preventive and Personalised Medicine

Meral Özgüç Editor

Rare DiseasesIntegrative PPPM Approach as the Medicine of the Future

Rare Diseases

Advances in Predictive, Preventive and Personalised Medicine

Volume 6

Series Editor:

Olga Golubnitschaja

Managing Editor:

Kristina Yeghiazaryan

For further volumes:http://www.springer.com/series/10051

Meral Özgüç Editor

Rare Diseases

Integrative PPPM Approach as the Medicine of the Future

ISSN 2211-3495 ISSN 2211-3509 (electronic) ISBN 978-94-017-9213-4 ISBN 978-94-017-9214-1 (eBook) DOI 10.1007/978-94-017-9214-1 Springer Dordrecht Heidelberg New York London

Library of Congress Control Number: 2014948033

© Springer Science+Business Media Dordrecht 2015 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifi cally the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfi lms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. Exempted from this legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifi cally for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the Publisher’s location, in its current version, and permission for use must always be obtained from Springer. Permissions for use may be obtained through RightsLink at the Copyright Clearance Center. Violations are liable to prosecution under the respective Copyright Law. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specifi c statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. While the advice and information in this book are believed to be true and accurate at the date of publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein.

Printed on acid-free paper

Springer is part of Springer Science+Business Media (www.springer.com)

Editor Meral Özgüç Faculty of Medicine, Medical Biology Hacettepe University Ankara , Turkey

v

What This Book Series Is About…

Current Healthcare: What Is Behind the Issue?

For many acute and chronic disorders, the current healthcare outcomes are considered as being inadequate: global fi gures cry for preventive measures and personalised treatments. In fact, severe chronic pathologies such as cardiovascu-lar disorders, diabetes and cancer are treated after onset of the disease, frequently at near end-stages. Pessimistic prognosis considers pandemic scenario for type 2 diabetes mellitus, neurodegenerative disorders and some types of cancer over the next 10–20 years followed by the economic disaster of healthcare systems in a global scale.

Advanced Healthcare Tailored to the Person: What Is Beyond the Issue?

Advanced healthcare promotes the paradigm change from delayed interventional to predictive medicine tailored to the person, from reactive to preventive medicine and from disease to wellness. The innovative predictive, preventive and personalised medicine (PPPM) is emerging as the focal point of efforts in healthcare aimed at curbing the prevalence of both communicable and non-communicable diseases such as diabetes, cardiovascular diseases, chronic respiratory diseases, cancer and dental pathologies. The cost-effective management of diseases and the critical role of PPPM in modernisation of healthcare have been acknowledged as priorities by global and regional organisations and health-related institutions such as the Organisation of United Nations, the European Union and the National Institutes of Health.

vi

Why Integrative Medical Approach by PPPM as the Medicine of the Future?

PPPM is the new integrative concept in healthcare sector that enables to predict individual predisposition before onset of the disease, to provide targeted preventive measures and create personalised treatment algorithms tailored to the person. The expected outcomes are conducive to more effective population screening, prevention early in childhood, identifi cation of persons at risk, stratifi cation of patients for the optimal therapy planning, and prediction and reduction of adverse drug-drug or drug-disease interactions relying on emerging technologies, such as pharmacogenetics, pathology-specifi c molecular patterns, sub-cellular imaging, disease modelling, individual patient profi les, etc. Integrative approach by PPPM is considered as the medicine of the future. Being at the forefront of the global efforts, the European Association for Predictive, Preventive and Personalised Medicine (EPMA, http://www.epmanet.eu/ ) promotes the integrative concept of PPPM among healthcare stakeholders, governmental institutions, educa-tors, funding bodies, patient organisations and in the public domain.

Current Book Series , published by Springer in collaboration with EPMA, over-view multidisciplinary aspects of advanced bio-medical approaches and innovative technologies. Integration of individual professional groups into the overall concept of PPPM is a particular advantage of this book series. Expert recommendations focus on the cost-effective management tailored to the person in health and disease. Innovative strategies are considered for standardisation of healthcare services. New guidelines are proposed for medical ethics, treatment of rare diseases, innovative approaches to early and predictive diagnostics, patient stratifi cation and targeted prevention in healthy individuals, persons at risk, individual patient groups, sub- populations, institutions, healthcare economy and marketing.

Prof. Dr. Olga Golubnitschaja

Book Series Editor

Dr. Golubnitschaja , Department of Radiology, Medical Faculty of the University in Bonn, Germany, has studied journalism, biotechnology and medicine and has

What This Book Series Is About…

vii

been awarded fellowships for biomedical research in Paediatrics and Neurosciences (Medical Centres in Austria, Russia, UK, Germany, the Netherlands, and Switzerland). She is well-cited in the research fi elds of “gene hunting” and “sub-tractive hybridisation” applied to predictive prenatal and postnatal diagnostics pub-lished as O. Labudova in years 1990–2000. Dr. Golubnitschaja is an expert in molecular diagnostics actively publishing in the fi elds of ophthalmic diseases, neu-rodegenerative pathologies, cancer, cardiovascular disease, Diabetes mellitus, hyperhomocysteinemia, etc. She is the cofounder of the theory of individual patient profi les, author of fundamental works in systems medicine (holistic approach con-sidering molecular patterns at epi/genomic, transcriptional and post/translational levels). Dr. Golubnitschaja holds appointments, at the rank of Professor, at several European Universities and in International Programmes for Personalised Medicine, and is author of more than 300 international publications in the fi eld. Her awards include: National and International Fellowship of the Alexander von Humboldt-Foundation, Highest Prize in Medicine and Eiselsberg- Prize in Austria. She is Secretary-General of the “European Association for Predictive, Preventive and Personalised Medicine” (EPMA in Brussels, www.epmanet.eu ), Editor-in-Chief of The EPMA-Journal (BioMed Central, London); Book Editor of Predictive Diagnostics and Personalized Treatment: Dream or Reality , Nova Science Publishers, New York 2009; Book Co-editor Personalisierte Medizin , Health Academy, Dresden 2010; Book Series Editor of Advances in Predictive, Preventive and Personalised Medicine , Springer 2012; European Representative in the EDR-Network at the NIH/NCI, http://edrn.nci.nih.gov/ ; Advisor and Evaluator of proj-ects dedicated to personalised medicine at the EU-Commission in Brussels, NIH/NCI, Washington, DC, USA, and Foundations and National Ministries of Health in several countries worldwide.

What This Book Series Is About…

ix

Pref ace

Rare diseases (RDs) or orphan diseases are a group of diseases with a low prevalance. In EU, a disease is classifi ed as rare if it affects less than 5 persons per 10,000 individuals. Globally, there are more than 300 million individuals affected by rare diseases. From about 7,000 RDs, still more than half have no identifi ed causative gene or a diagnosis.

Almost 80 % of RDs have a genetic origin with symptoms appearing in prenatal and early postnatal periods. Amongst RDs there are rare cancer types, congenital malformations, and consequently developed infectious diseases. These are usually severe, chronic and life threatening pathologies, which from case-to case vary dramatically in the corresponding grade of clinical severity and by the individual outcome. Due to the wide spectrum of RDs and a lack of suffi cient knowledge about individual RDs, the correct diagnosis is diffi cult to make. Furthermore, currently there are no appropriate treatment approaches for most of the RDs. The only reason-able approach seems to be a development of methods for early diagnosis of RDs that might lead to the creation of the optimal care management saving lives and improv-ing life quality within the patient cohort.

Due to unfavoured economical aspects, such as a limited number of responding patients, problematic conduction of corresponding clinical trials and consequently high costs of potential treatment, the drug development for RD is currently stagnat-ing, and manufacturers are not really motivated to bring new products to the market. Consequently, there is a real need for R&D in fi nding new drugs well regulated by healthcare responsible institutions guided by new guidelines for effective treatments tailored to the person diagnosed with RD.

The improvement in RDs healthcare is initiated by legislations in EU and the USA to create an integrative medical approach for RDs.

How is the emerging paradigm of PPPM related to the healthcare of RDs? Due to the molecular background of most RD pathologies, it is expected that the multimodal approach (*omics, pharmacogenetics, medical imaging, etc.) with multidisciplinary professionals should be instrumental for the “personalisation” to diagnose individual RDs, to create effective preventive measures and to develop targeted therapies – the integrative medical approach by predictive, preventive and

x

personalised medicine (PPPM). Recent achievements in bio/medical sciences let us trust in a prompt translation of innovative technologies into daily clinical practice.

Besides its general add-value for the public health promotion, the advanced RD healthcare provides an excellent “proof-of-principles” for the personalisation of healthcare systems in a global scale. RDs is an important source for related scien-tifi c, methodological and technological progress: all medical branches may benefi t from comprehensive efforts if made in the promotion of the scientifi c and techno-logical fi eld of RDs including ethical considerations, creation of the robust platform for the professional communication, synergies with patient organisations, “doctor- patient” collaboration and new philosophy of integrative medicine by PPPM to advance current healthcare.

The European Association for Predictive, Preventive and Personalised Medicine (EPMA) actively promotes the scientifi c and technological efforts, expert recom-mendations and creation of new guidelines in the fi eld of RD healthcare. This initia-tive has been triggered through The EPMA Journal launched in 2010 as the professional forum in PPPM. The book series Advances in PPPM overview multi-disciplinary aspects of advanced bio/medical approaches and innovative technolo-gies aiming at remarkable improvements in healthcare performance. Integration of individual professional groups into the overall concept of PPPM is a particular advantage of this book series.

The current book is dedicated to all aspect related to the prediction, prevention and personalised treatments of RDs . This volume is intended to serve as a reference source for scientifi c and medical centres involved in the fi eld with a special empha-sis on healthcare promotion and innovations intended to combat RDs, save the affected lives and enhance the life quality of this patient cohort.

I wish to thank the book contributors, book series editor and Springer for the excellent performance and highest professional level in the book preparation. I express this cordial thank on behalf of all the patients with rare diseases to whom I would like to dedicate this book “Rare Diseases: Integrative PPPM Approach as the Medicine of the Future ”.

Ankara, Turkey Meral ÖzgüçEditor

Preface

xi

Contents

National Plans on Rare Diseases .................................................................... 1 Domenica Taruscio , Amalia Egle Gentile , Marta De Santis , Rita Ferrelli , Rosa Giuseppa Frazzica , Georgi Iskrov , and Rumen Stefanov

Biobanking for Rare Diseases – Impact on Personalised Medicine ............................................................................... 23 Jeanne-Hélène di Donato

Emerging Technologies for Gene Identifi cation in Rare Diseases .............. 33 Filippo Beleggia and Bernd Wollnik

Personalized Medicine for Hereditary Deafness .......................................... 47 Jessica Ordóñez , Oscar Diaz-Horta , and Mustafa Tekin

Mitochondrial Diseases ................................................................................... 61 Maria Judit Molnar and Klara Pentelenyi

Complexity of Genotype-Phenotype Correlations in Mendelian Disorders: Lessons from Gaucher Disease ............................ 69 Nima Moaven , Nahid Tayebi , Ehud Goldin , and Ellen Sidransky

Enzyme Replacement Therapy in Lysosomal Storage Diseases ................. 91 Vassili Valayannopoulos

Rare Cancers ................................................................................................... 109 Nikolajs Zeps and Chris Hemmings

Adeno-Associated Virus Gene Therapy and Its Application to the Prevention and Personalised Treatment of Rare Diseases ................ 131 Konstantina Grosios , Harald Petry , and Jacek Lubelski

xii

Induced Pluripotency for the Study of Disease Mechanisms and Cell Therapy .................................................... 159 Toivo Maimets

Author Index.................................................................................................... 175

Subject Index ................................................................................................... 205

Contents

xiii

Filippo Beleggia Institute of Human Genetics, University Medical Faculty, University of Cologne, Cologne, Germany

Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany

Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany

Marta De Santis National Centre for Rare Diseases, Istituto Superiore di Sanità, Rome, Italy

Jeanne-Hélène di Donato 3C-R, Castelginest, France

Oscar Diaz-Horta Dr. John T. Macdonald Foundation Department of Human Genetics and John P. Hussman Institute for Human Genomics, University of Miami Miller School of Medicine, Miami, FL, USA

Rita Ferrelli National Centre for Rare Diseases, Istituto Superiore di Sanità, Rome, Italy

Rosa Giuseppa Frazzica National Centre for Rare Diseases, Istituto Superiore di Sanità, Rome, Italy

Amalia Egle Gentile National Centre for Rare Diseases, Istituto Superiore di Sanità, Rome, Italy

Ehud Goldin Section on Molecular Neurogenetics, Medical Genetics Branch, National Human Genome Research Institute (NHGRI), National Institutes of Health (NIH), Bethesda, MD, USA

Konstantina Grosios uniQure B.V, Amsterdam, The Netherlands

Chris Hemmings School of Surgery, University of Western Australia, Crawley, Australia

Contributors

xiv

Department of Anatomic Pathology, St John of God Pathology , Subiaco , Australia

Georgi Iskrov Clinical and Information Centre for Rare Diseases, Plovdiv, Bulgaria

Jacek Lubelski uniQure B.V, Amsterdam, The Netherlands

Toivo Maimets Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia

Nima Moaven Section on Molecular Neurogenetics, Medical Genetics Branch, National Human Genome Research Institute (NHGRI), National Institutes of Health (NIH), Bethesda, MD, USA

Maria Judit Molnar Institute of Genomic Medicine and Rare Disorders, Semmelweis University, Budapest, Hungary

Jessica Ordóñez Dr. John T. Macdonald Foundation Department of Human Genetics and John P. Hussman Institute for Human Genomics, University of Miami Miller School of Medicine, Miami, FL, USA

Klara Pentelenyi Institute of Genomic Medicine and Rare Disorders, Semmelweis University, Budapest, Hungary

Harald Petry uniQure B.V, Amsterdam, The Netherlands

Ellen Sidransky Section on Molecular Neurogenetics, Medical Genetics Branch, National Human Genome Research Institute (NHGRI), National Institutes of Health (NIH), Bethesda, MD, USA

Rumen Stefanov Clinical and Information Centre for Rare Diseases, Plovdiv, Bulgaria

Domenica Taruscio National Centre for Rare Diseases, Istituto Superiore di Sanità, Rome, Italy

Nahid Tayebi Section on Molecular Neurogenetics, Medical Genetics Branch, National Human Genome Research Institute (NHGRI), National Institutes of Health (NIH), Bethesda, MD, USA

Mustafa Tekin Dr. John T. Macdonald Foundation Department of Human Genetics and John P. Hussman Institute for Human Genomics, University of Miami Miller School of Medicine, Miami, FL, USA

Vassili Valayannopoulos Reference Center for Inherited Metabolic Disease in Children and Adults (MaMEA) and IMAGINE Institute, Necker-Enfants Malades Hospital and Paris Descartes University, Paris Cedex 15, France

Bernd Wollnik Institute of Human Genetics, University Medical Faculty, University of Cologne, Cologne, Germany,

Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany

Contributors

xv

Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany

Nikolajs Zeps Bendat Family Comprehensive Cancer Centre, St John of God HealthCare, Subiaco, WA, Australia

School of Surgery, University of Western Australia, Crawley, Australia

Contributors

xvii

About the Editor

Dr. Özgüç is currently Professor of Medical Biology at the Faculty of Medicine, Hacettepe University, Ankara, Turkey, where she received her Ph.D. in Medical Biology in 1985. She completed her undergraduate studies at Fairleigh Dickinson University in chemistry and received masters degree in biochemistry from Columbia University in the USA. Immediately afterwards, she worked for 3 years as Research Associate in the Department of Research Hematology, Childrens Hospital of Philadelphia, before joining the Hacettepe faculty in 1980. She served as Assistant Dean of the Faculty of Medicine (2006–2009), and currently she is the Director of the Hacettepe DNA/Cell Bank for Rare Diseases-Center for Genomics.

Her work, which is supported by State Planning Agency, Scientifi c and Technical Research Council of Turkey (TÜBİTAK) and through international grants, is focused on a genomic medicine approach for the study of rare diseases. She is actively involved in the formulation of national policies to create an awareness of and to promote genomics in public health. Her scientifi c publications concentrate in

Prof. Dr. Meral Özgüç

xviii

the area of identifi cation of new disease genes and genomic variants and development of diagnostic tests. She is also involved in networking activities, governance and bioethical aspects of sample acquisition and data management of biobanks. She is member of European, Middle Eastern and African Society for Biopreservation and Biobanking.

She has worked as a member of various international committees involved in genomics and health such as OECD – Working Party on Biotechnology and Human Health Related Biotechnologies, ESF – Integrated Approaches to Functional Genomics, EC-FP6 Genomics and Biotechnology for Health (National Contact Point), and UNESCO-International Bioethics Committee. Currently she is the chair of the Bioethics Committee of the Turkish National Commission for UNESCO and serves as a member of National EPMA Board in the section for Neonatal Diagnostics and Population Screening.

About the Editor

1M. Özgüç (ed.), Rare Diseases: Integrative PPPM Approach as the Medicine of the Future, Advances in Predictive, Preventive and Personalised Medicine 6,DOI 10.1007/978-94-017-9214-1_1, © Springer Science+Business Media Dordrecht 2015

Abstract This paper aims to describe the state-of-the-art of national plans for rare diseases (RD) in EU Member States, pointing out common as well as country- specifi c features and approaches. It critically assesses the national achievements in this fi eld, drawing conclusions to further strengthen the process of planning, implementing and evaluating RD national policies.

A national plan for RD can be defi ned as an offi cial strategic public health document, issued by the government, containing specifi c priorities, objectives, strategies, actions, a timetable for implementation and a dedicated budget. For the last decade, RD have steadily emerged as top public health priority of the EU health policy. RD national plans are consistently being identifi ed as the main strategic instrument to address the complex RD issues. This political tool is advanced at both EU and Member State level, because it provides a uniform approach for the implementation of the common EU objective: ensuring equal access and availability of prevention, diagnosis, treatment and rehabilitation to people with RD. However, at the same time it is fl exible enough to give opportunities to the national authorities to adopt country-specifi c measures as well.

Since the fi rst national plan for RD in France, dating back to 2004, such strategic public health documents are now being elaborated in a growing number of countries and, virtually, all EU Member States are working on drafting and adopting RD national plans in accordance with EU recommendations. Meanwhile, the European Commission has supported and guided national authorities, namely through the European Project for Rare Diseases National Plans Development (EUROPLAN). EUROPLAN has effectively stimulated expertise sharing and consensus building, ensuring that all Member States RD activities are coherent and consistent with the

National Plans on Rare Diseases

Domenica Taruscio , Amalia Egle Gentile , Marta De Santis , Rita Ferrelli , Rosa Giuseppa Frazzica , Georgi Iskrov , and Rumen Stefanov

D. Taruscio (*) • A. E. Gentile • M. De Santis • R. Ferrelli • R. G. Frazzica National Centre for Rare Diseases , Istituto Superiore di Sanità , Rome , Italy e-mail: domenica.taruscio@iss.it

G. Iskrov • R. Stefanov Clinical and Information Centre for Rare Diseases , Plovdiv , Bulgaria

2

EU policy and with the current RD best practices. Furthermore, EUROPLAN has provided relevant support regarding two key aspects of RD national plans: the processes of planning and that of evaluating RD plans. As a policy strategy, RD national plan’s overall success heavily depends on the initial planning’s effectiveness and on the fi nal evaluation’s results. As well, the availability of dedicated funds plays a crucial role in the implementation of NP for RD.

Keywords Rare diseases • EU policy • National plans • EU collaboration • Health policy • Council’s Recommendation • EUROPLAN

1 Introduction

In the European Union (EU), rare diseases (RD) are offi cially defi ned as disorders affecting no more than 5 per 10,000 persons [ 1 ]. These are life-threatening or chron-ically debilitating diseases with a low prevalence and a high level of complexity. Despite their rarity, there are between 5,000 and 8,000 RD that affect millions of people around the world [ 1 ]. While single RD are characterised by a low preva-lence, at the same time, knowledge and expertise on these topics are also scarce. Likewise, RD receive limited attention by public health systems and by the society in general. Because of these combined specifi cities, RD require a global strategic approach, based on cooperation and collaboration, in order to prevent signifi cant morbidity or avoidable premature mortality, and to improve the quality of life and the socioeconomic potential of affected persons and that of their families.

For the last decade, RD have steadily emerged as a top public health priority of the EU health policy making. Two legal documents are key to for RD at European level: the Commission Communication 679 of 11 November 2008 [ 2 ] and the Council Recommendation on an action in the fi eld of rare diseases, of 9 June 2009 [ 1 ]. Both documents have recommended that Member States develop a National Plan or a Strategy for RD by the end of 2013. These documents put the basis of a common EU strategy, which aims to answer the legitimate claims of the RD com-munity, most notably, improved access to information, appropriate and timely diag-nosis, and effective care.

RD National Plans are therefore the common denominator of current public health policy and concerns for RD across Europe. While National Plans common objective is to ensure equal access and availability of services for the prevention, diagnosis, treatment and rehabilitation to people with RD, National Authorities can choose the specifi c measures to be dealt with in their Plan. National Plans are stra-tegic documents that attempt to optimise the limited resources for RD (both human, fi nancial and material), using them in the most effective and effi cient manner to achieve the planned objectives. This is a key factor for national health systems, which often struggle to meet the criteria of effi ciency and cost-effectiveness of public funds utilisation. Thus, RD National Plans create added value not only for the RD stakeholders, but for the entire society as well.

D. Taruscio et al.

3

The policy documents issued by the European Commission have given a major impulse to Member States concerning RD. Some Countries have already adopted National Plans or Strategies while France is implementing its second Plan. Meanwhile, virtually all other Member States are at some point of drafting and/or approving such policy document. This paper aims to describe the state-of-the-art of National Plans in the EU Member States, underlining common as well as country-specifi c features and approaches used throughout the process. Furthermore, it critically assesses the national achievements in the fi eld of RD drawing conclusions that may be useful to further strengthen the process of planning, implementing and evaluating RD national policies across Europe.

2 EU Countries with National Plans at a Glance

The rarity of a disease presents a number of challenges for research and for patient care, which the health systems only partially meet. RD are not limited by geographical or historical boundaries and global partnerships are rapidly expanding across the RD community. Accordingly, as a transnational community of different countries, the EU is working to develop a common framework, encouraging initiatives at European, as well as at Member States level, promoting the development of National Plans and strategies to tackle the complexity of RD and obtain lasting improvements.

A National Plan for RD can be defi ned as an offi cial strategic public health document, developed by the government, containing specifi c priorities, objectives, actions, a timetable for implementation, and a dedicated budget.

The Council of the EU Health Ministers, acknowledging the need to act in the area of RD [ 1 ], issued a number of recommendations for actions to be considered by the Member States in the planning process. These include:

• Integration of relevant national actions in the fi eld of RD into comprehensive plans or strategies, to be issued no later than 2013, in order to improve the coordination and coherence of national, regional and local initiatives addressing RD and to strengthen the cooperation between clinical and research professionals;

• Use of an appropriate tool for classifi cation and coding in order to improve the visibility of RD and their recognition in the national health systems;

• Selection of qualifi ed Centres of Expertise for diagnosis and care of RD and promotion of their participation in European Reference Networks, in order to facilitate cooperation among Member States;

• Identifi cation of current research activities and resources dedicated to RD research: defi nition of the needs, the priorities and the fi nancing schemes to support research and facilitate its coordination at national, Community and regional levels;

• Gathering expertise at Community level in order to facilitate sharing of best practices for diagnosis and care, adequate education and training for health professionals, guidelines on diagnostic tests and population screening. As well, sharing national assessment reports on orphan drug is an added value.

National Plans on Rare Diseases

4

The EU Recommendations refl ect the achievements of the ongoing action and commitment of the European Commission. In fact, in order to raise the attention and improve the information on RD, the European Commission has, since 1997, given a high priority to projects that can support the development of a common EU framework. The foreseen actions include: collecting information on Centres of Expertise, setting European registries and networks of experts on RD, and developing consensus guide-lines for newborn screening. The Council Document also recommends the involvement of patient representatives in the development of policies and in other activities aiming at patient empowerment, such as awareness-raising, capacity-building and training, exchange of information and best practices and support of isolated patients [ 1 ].

Meanwhile, the European Commission has co-funded the European Project for Rare Diseases National Plans Development (EUROPLAN, www.europlanproject.eu ) coordinated by the National Centre for Rare Diseases – Italian National Institute of Health since 2008. The project’s main objectives are: to promote and implement National Plans or Strategies for RD and to share relevant experiences within Countries, linking national efforts with a common strategy at European level (see Table 1 ) [ 3 ].

EUROPLAN is a process, more than a project. It involves all stakeholders (institutions, patients, health personnel, industries), stimulating a discussion, reaching a consensus and generating a momentum for National Plans. These should defi ne the most relevant actions in the fi eld of RD to be undertaken in each country. The added value of EUROPLAN consists in its “double-level” approach [ 3 ]: on one hand the Plan is generated with a top-down approach while inputs for its develop-ment use a bottom-up fl ow.

In EUROPLAN, the patients’ advocates play an important role: they voice the patients’ needs and expectations and ensure that patients participate in the process as equal partners.

Today, the active role of patients’ representatives is recognised as a major contribution to innovation and as a catalyser for cooperation and sustainable development. As a matter of fact, EUROPLAN could be defi ned as “litmus”. It is a proof of how the collaboration between Institutions and patient organisations can accelerate the development and implementation of RD National Plans. This partnership is possible thanks to the empowerment of patients, which is the process of increasing the capacity of individuals or groups to make informed choices and to transform those choices into desired actions and outcomes [ 4 ]. The “empowerment of patients” is a prerequisite for health. As well, a proactive partnership and a patient self-care strategy can improve health outcomes and the quality of life among the chronically ill [ 5 ]. Empowerment relates to the individual as “self-realisation” and as “identity formation”. The main features of empowerment are:

• Creating a positive self-image; • Availability of a range of options; • Own decision making power; • Assertiveness; • Ability to make change; • Ability to discern and listen;

D. Taruscio et al.

5

Table 1 EUROPLAN. The European Project for Rare Diseases National Plan Development [ 3 ]

National Plans on Rare Diseases

6

The goal of the empowerment of patients is to break the silence and reduce the discrimination or the stigmatisation that often surround RD and those affected by these diseases. Empowerment becomes possible when:

– an informed patient becomes an “expert” patient; – a patient gains control, e.g. through the self-management of treatment; – there exist effective equal opportunities.

Empowerment it is also about quality of life. RD patients and their support organisations are among the most empowered groups in the health sector, mainly as a result of their own fi ght for recognition and for better care [ 6 ]. The empowerment of patient organisations is included in all existing plans/strategies for RD in EU Countries.

As previously mentioned, some European Member States are in the process of adopting National Plans for RD, integrated within a coherent European policy framework. Among the 27 EU Member States, only 8 Countries have already adopted National Plans or strategies of different complexity and with different aims: France, Bulgaria, Czech Republic, Greece, Netherlands, Portugal, Slovenia, and Spain. Among them, France is the only EU Member State that is implementing the second national RD plan.

All other EU Member States are currently preparing their plans or strategies [ 3 , 7 ] while some Countries are awaiting for a formal approval of their Plan. Documents and activities undertaken in the EU Member States regarding RD are available in the EUROPLAN website ( www.europlanproject.eu ), and in the EU Committee of Experts on Rare Diseases (EUCERD) website ( www.eucerd.eu ).

A brief overview of the existing National Plans/Strategies in Europe is provided below. For each plan, the similarities and differences in their complexity are highlighted, as well as their aims and the main programmed actions.

2.1 Bulgaria

The goal of the Bulgarian National Plan for Rare Diseases 2009–2013 (Genetic, congenital malformations and non-hereditary diseases) [ 8 ] is to create an adequate institutional framework and mechanisms for the provision of timely prevention, diagnostics, optimal treatment and rehabilitation of patients with RD.

The Plan’s identifi ed priorities are:

1. Collection of epidemiological data for RD in Bulgaria through the establishment of a National Register;

2. Improvement of the prevention of RD of genetic origin by extending the actual screening programs;

3. Improvement of the prevention and diagnosis of RD by the introduction of new genetic tests, decentralisation of laboratory activities, and facilitated access to genetic counselling;

4. Integrated approach to the implementation of prevention, diagnostics, treatment, and social integration of patients with RD and their families;

D. Taruscio et al.

7

5. Improvement of professional qualifi cation of medical specialists in the fi eld of early diagnosis and prevention of RD;

6. Feasibility study on the necessity, opportunity, and criteria for the establishment in the Country of a reference centre for RD of functional type;

7. Organisation of a national public awareness campaign on RD and on their prevention;

8. Support and collaboration with non-governmental organisations and with the RD patient organisations;

9. Close collaboration with other EU Member States working to achieve the purpose of the plan and with the Rare Disease Task Force at DG SANCO, EC.

2.2 Czech Republic

The National Strategy for Rare Diseases of the Czech Republic (2010–2020) [ 9 ] intends to ensure the effective diagnosis and treatment of RD, to guarantee the accessibility of high-quality health care to all patients with RD and to promote their social integration on the basis of equal treatment and solidarity. The purpose of the national strategy is also to make use of expert cooperation with other countries, to enable Czech patients to take part in international clinical studies of new medicines, including treatment abroad in strictly identifi ed cases, when it is not possible to obtain suitable specialised care in the Country.

The proposed national strategy sums up the issue of RD from the EU’s and from the Czech Republic’s point of view and proposes major targets and measures for improving the situation in the Country. These targets and measures are subsequently specifi ed in more details in Czech Republic’s National Plan for Rare Diseases (2012–2014) [ 10 ], which establishes sub-tasks, instruments, responsibilities, dates and indicators for fulfi lling the individual tasks.

The measures proposed regard:

• Improved information on RD; • Education in the fi eld of RD; • Improved diagnosis and screening of RD; • Improvement of the quality of treatment and care; • Improvement of quality of life and social integration of people with RD; • Support for science and research in the fi eld of RD; • Harmonisation and development of data collection and biological sampling

in connection with RD; • Development of international RD patient organisations; • Cooperation with the World Health Organisation; • Supporting and strengthening the role of RD patient organisations; • Supporting patients with RD to participate in clinical tests of new medicinal

products at European level; • Cooperation with the European Commission’s EUROPLAN project; • Sustainable activities in the fi eld of RD; • Setting up an inter-ministerial working group on RD.

National Plans on Rare Diseases

8

2.3 France

The Second National Plan on Rare Diseases of France (2011–2014) [ 11 ] is built on three main objectives and relative operational measures.

Objective 1. Improving the quality of care the patient :

• Improving access to diagnosis and care of patients suffering from RD; • Optimising assessment procedures and funding of centres of reference for RD; • Accelerating the draft of national protocols for diagnosis and treatment; • Ensuring quality of pharmaceutical treatment for every RD patient; • Developing linkages between actors in the management and coaching; • Improving the practice of health professionals; • Make information accessible through dissemination; • Use Orphanet as a tool for information and research.

Objective 2. Developing research on RD :

• Creating a national research interface with public and private actors; • Promoting tools to increase the knowledge on RD and to allocate in Agence

Nationale de la Recherche (ANR) programmes a minimum amount dedicated to RD research;

• Promoting the development of therapeutic trials; • Promoting translational clinical research and therapy.

Objective 3. Amplifying European and international cooperation :

• Promoting expertise sharing internationally, through European Reference Networks;

• Improving the ability to conduct multinational clinical trials, access to diag-nostic tests available at European level and to quality control testing;

• Improving access to diagnosis, care and support, research and information on RD in structuring the European and international cooperation.

2.4 Greece

The National Plan of Action for Rare Diseases (2008–2012) [ 12 ] of Greece was published in Athens in 2008. It is based on PESPA (the Greek Alliance for RD) recommendations, it derives from the French National Plan, and was modifi ed by the Greek Ministry of Health (2008). The Plan, has not yet been implemented, is based on the following strategic priorities:

• Recognition of the specifi cities of RD; • Systematic monitoring of RD epidemiology and institution of a registry of

RD patients; • Developing information for patients, health professionals and the general public

concerning RD;

D. Taruscio et al.

9

• Improving the quality of all services involved for timely diagnosis, early intervention, treatment and rehabilitation of RD patients;

• Increasing access to screening; • Promoting research and innovation on RD, regarding treatment in particular; • Responding to the needs of people suffering from RD, especially for new and

more effective treatments; Developing a common platform of action at national level in the sector of RD and participating in equivalent European networks.

2.5 Portugal

Portugal’s National Programme for Rare Diseases (2008–2015) [ 13 ] pursues the following main objectives:

• Improving national responses to the unmet health needs of RD patients and their families;

• Improving the quality and the equity of health care provided to RD patients.

The main strategies of the Portuguese Plan are grouped into three main areas:

1. Intervention The intervention strategies are considered more relevant to the development and the implementation of the NP:

• Creating a national network of reference centres for RD; • Improving the access to appropriate care for people with RD; • Improving the mechanisms for integrated management of RD; • Improving the answers to the needs of patients and families; • Increasing the awareness and strengthening the knowledge of RD by pro-

moting initiatives in RD research; • Promoting innovation and accessibility to treatment for RD patients; • Ensuring transnational cooperation within the EU and the Community of

Portuguese Speaking Countries (CPLP).

2. Training The training strategies include targeted training of health professionals, the academic community, RD patients and their families, and the general population-in order to enable them to manage effectively and control RD. The social partners can make important contributions in various strategies.

3. Collection and analysis of data The collection and analysis of data are actions intended to improve knowledge about RD throughout the life cycle. In order to achieve this, different agencies should be mobilised and public and private funding of R & D in health sciences should be raised.

National Plans on Rare Diseases

10

2.6 Slovenia

The Work Plan for Rare Diseases [ 14 ] of Slovenia (2011) is considered an opportunity for better coordination of efforts of all partners involved to achieve, comprehensive, accessible, timely and patient centred health care. A Coordination Group annually prepares a report on the implementation of the plan and issues the annual action plan. The Work Plan, is a strategic document, it is developed for the period ending in 2020, and it is the base for the development of annual operational plans, with the following objectives:

• Identifi cation and monitoring of RD. The thematic areas include: classifi cation of RD and establishment of a national registry for RD;

• Improving the capacity for early diagnosis and access to appropriate medical treatment (orphan drugs, rehabilitation). The thematic areas include: screening, guidelines, early diagnosis, treatment and rehabilitation;

• Improving the mechanisms for an integrated approach to RD. The thematic areas include: primary prevention, social services, social inclusion, patient organisations;

• Improving access to information for patients, health and other professionals and for the general public. The thematic areas include: availability of information to the public, network of reference centres, education for health and other professionals.

2.7 Spain

The Rare Diseases Strategy of the National Health System of Spain [ 15 ] (2010) was defi ned following the European Council Recommendation [ 1 ] and the Senate Report [ 15 ], and was supported by all political parties. Given the decentralised health administration (management) of the Autonomous Communities (Regional Governments), the Strategy will act as a set of recommendations for the different regions, which will be in charge of its implementation.

This Strategy defi nes seven lines of action, as follows:

1. Information on RD, available resources, health registers, coding and classifi cation;

2. Prevention and early detection; 3. Healthcare; 4. Therapies: orphan medicinal products, adjuvants and health products, advanced

therapies, rehabilitation; 5. Integrated health and social care; 6. Research; 7. Training.

Appropriate background information has been provided, the general objectives have been established for each of the strategy lines, and for their subsections, and

D. Taruscio et al.

11

specifi c objectives and recommendations have been developed for each of them. Finally, the document defi nes a procedure to systematically monitor and evaluate the Strategy, with guidelines for its planning, dissemination and follow-up. Indicators to evaluate the implementation of the plan have also been defi ned.

2.8 The Netherlands

The main objective of the Netherlands Strategy in the fi eld of Rare Diseases (2012) [ 16 ] is to improve the health of patients suffering from RD.

Since 1995, the Ministry of Health, Welfare and Sport has consulted with rele-vant parties (or stakeholders) in the fi eld of RD, through the Council for Health Research (RGO). These parties included patients, healthcare providers, researchers, health care insurers, pharmaceutical companies, and independent authorities such as the Health Care Insurance Board (CVZ) and the Medicines Evaluation Board (CBG-MEB). A very important component of the strategy was the Steering Committee on Orphan Drugs, which was established in April 2001 and functioned up to the end of 2011. Most tasks of the Steering Committee were subsequently devolved to various stakeholders from this group. Some remaining tasks were assigned to the Netherlands Organisation for Health Research and Development (ZonMw). Despite these changes, the Ministry declared to remain committed to RD and orphan drugs both at national and international level. The current strategy will be partly continued and partly modifi ed in the next few years. Even though the past strategy produced a number of important results, some changes had to be made because of the changing circumstances occurred in the past 10 years.

The policy regarding the steering group was changed in January 2012. Most tasks were devolved to stakeholders in the former steering group and the remaining tasks were entrusted to ZonMw. However, extra funding for the stakeholders is not foreseen in the new strategy, with the exception of the funding of some projects by patient organisations. Nevertheless, concerning the remaining tasks, the Ministry has allocated specifi c funding to ZonMw, which has set up a dedicated Secretariat. Its tasks include collecting and streamlining information from relevant parties or individuals with the aim to help implementing the Ministry’s strategies and policies or to promote policies proposed by various stakeholders. ZonMw will also facilitate a number of projects such as strengthening the patient’s voice, establishing an infor-mation desk for patients and improving medical and social care of RD patients. These projects will be realised in consultation and cooperation with health care providers and patients. The Secretariat will help to formulate a strategy for the years after 2015. Lastly, it will participate in additional relevant international activities.

In summary, the Steering Group was disbanded, but its activities will neverthe-less be undertaken by relevant stakeholders, by a newly installed Secretariat at ZonMw and by FBG. The government will remain involved, but not so closely as in previous years.

National Plans on Rare Diseases

12

Other aspects of the National Strategy are, to a large extent, a continuation of the already existing policies, such as:

• Improving diagnosis and treatment of RD; • Increasing the involvement of patient organisations in developing policies; • Screening for RD; • Creating a specifi c scientifi c research programme; • Improving reimbursement for orphan drugs; • Registration of RD; • Continued participation in European policies and in policy development.

Table 2 shows the conformity among priorities of the “Council Recommendation of 8 June 2009 on an action in the fi eld of rare diseases” (2009/c 151/02) and existing National Plans/strategies in EU Countries (2012).

Table 2 Conformity among priorities of the “Council recommendation of 8 June 2009 on an action in the fi eld of rare diseases” (2009/C 151/02) and existing National Plans/strategies in eu countries (2012) (Modifi ed by Taruscio et al. [ 17 ], p 20)

EU Countries with existing RD National Plan/Strategy

Bul

garia

Cze

ch R

epub

lic

Fra

nce

Gre

ece

Net

herlan

ds

Por

tuga

l

Slo

veni

a

Spa

in

II. Adequate definition, *

(4) Easily accessible and dynamic inventory of RD

III. Research on RD

(6) Identify ongoing research and research

(7) Needs and priorities for basic, clinical

(9) Foster research in the field of RD(10) Research cooperation with third countries

IV. Centres of expertise and (11) Identify appropriate centres of expertise

PRIORITIESof the Council Recommendation on an action in the field of rare diseases

(RD)

I. Plans and strategies in the field of RD

European referencenetworks for RD

codification andinventorying of RD

(1) Establish and implement plans and/or strategies on RD(2) Use a RD common definition of no more than 5 per 10,000 persons(3) Adequate coding, trace and recognition in the national healthcare and reimbursement systems

(5) Specific disease information networks, registries and databases

resources in the national and Communityframeworks

translational and social research and promoteinterdisciplinary cooperative approaches

(8) Foster the participation of national researchers in research projects

(12) Participation of centres of expertise in European reference networks(13) Organise healthcare pathways for patients

VII. Sustainability

(14) Use of information and communication technologies (15) Diffusion and mobility of expertise and knowledge (16) Centres of expertise, based on a multidisciplinary approach to care(17) Gather national expertise and support the pooling of that expertise with EU(18) Consult patients and facilitate access to updated information(19) Promote the activities performed by patient organisations(20) Ensure the long-term sustainability of infrastructures

V. Gathering the expertise on RD at European level

VI. Empowerment of patient organisations

*Only for Orphan drugs

D. Taruscio et al.

13

3 Situation in Eastern European Countries

3.1 Policy Background

While on a European level policy-makers widely agree on the fact that RD should be considered a top public health priority, at national level there are signifi cant dif-ferences in how these policy guidelines are being transposed and implemented [ 18 ]. Eastern European countries (EEC) represent a mosaic of EU and non-EU Member States. However, they all share several important common characteristics, which predispose a number of similarities in their national policies, including the ones in the public health sector [ 19 ]. Moreover, “RD topic” is an exclusively new territory for most of them, resulting in similar backgrounds and challenges for the whole region [ 20 ].

The National Plans for RD are really the crème-de-la-crème of RD-orientated public health activities. By defi nition, a National Plan is an offi cial strategic public health document (1) issued by the government, (2) containing specifi c priorities, objectives, strategies, actions and a timetable for its implementation, and (3) having a dedicated budget [ 3 ]. This triple formula has been considered and recently pro-moted as the most adequate way to start solving the long-lasting problems of the RD community. Indeed, the complexity of RD requires a systematic, multidisciplinary approach. That is why a National Plan is the most comprehensive way to strategically address multilevel problems in a synergetic way, involving the effort of different stakeholders. This idea has been unanimously adopted and has became the core of the EU RD offi cial policy [ 1 ]. EU has taken an even more active role in the promo-tion of National Plans by co-funding RD-orientated European projects, which have greatly contributed in identifying, collecting, analysing and disseminating the best policy models to address relevant RD issues.

All these circumstances are prompting the EEC to start considering RD issues in a systematic and organised manner, by adapting international and European guidelines to fi t adequately their national public health environment. Our analysis is identifi es three key factors, showing how and why the adoption of RD National Plans could be substantially benefi cial for EEC. The three factors are:

1. EU integration and cohesion

In the “United Europe” era, integration and cohesion is the most powerful political factor across Eastern Europe. All national policies should be harmonised with the EU legal base. Health policy is one of the sectors which is not directly infl uenced by the EU, as it is widely believed that healthcare is a nation- specifi c domain, and each country should know best how to deal with its medical needs and resources. Nevertheless, healthcare services cannot be excluded from the overall EU development trends. Within the free mobility of people, goods and services, public health stakeholders have the opportunity to get acquainted with the latest achievements in the EU and to try implementing them in their respective countries.

National Plans on Rare Diseases

14

Therefore, EU is providing a generalised framework for RD policy development, which still leaves enough space for adaptation and fl exibility at Member State level. At the same time, this political programme is detailed enough and contains specifi c recommendations which, despite the national policy variability, has to be taken into consideration [ 18 ]. Member States are advised to take concrete steps in terms of plans and strategies, centres of expertises, registries, patient empowerment, etc. This is a major advantage from an Eastern European perspective, as policy makers avoid wasting valuable resources in trial-error efforts. And, secondly, a cross-country benchmarking is available, so that progress can be closely monitored and managed if outcomes differ signifi cantly [ 19 ].

Of course, the economic aspects of RD policy cannot be ignored. Moreover, the transition economy countries of Eastern Europe have been particularly hit by the recent economic crisis. However, health care funding is a common problem all over the EU, and Member States are exploring different ways of mobilising and allocating resources equitably and effi ciently to satisfy the growing needs and demands for health services. Addressing RD policy should be done in a step-by-step manner. RD policies have been continuously demonised as extremely expensive. However, implementing a National Plan should be considered a very effi cient, cost- effective activity, because it is a process of optimisation, reorganisation and management improvement that can bring a signifi cant added-value for the scarce EEC health budgets.

2. Public awareness

The general RD awareness in Eastern Europe remains low [ 21 ]. In a Eurobarometer study, EEC were consistently placed in the bottom ranking, which inevitably plays a negative role for all RD activities [ 22 ]. This situation has its logical explanation. For decades, RD, and especially RD patients, have been virtually considered as non- existing in Eastern Europe. Their problems and everyday struggle have been hidden from the society’s general perspective. This denial has had its consequences for the medical education and medical services provision. RD have not been specifi cally addressed in the medical schools’ curricula and have been regarded as a minor and insignifi cant area with no practical value. The result is evident: general practitioners are practically unaware of RD [ 22 ]. Medical specialists simply do not have the necessary experience in order to gain some expertise, because no patients have been referred to them. It is not surprising that RD patients in Eastern Europe are suffering most from this indifference, rather than from lack of specifi c policies.

All this used to be a common issue for all EEC. However, the democracy’s return in this region has provided ground for the re-establishment of the active civil soci-ety. In fact, patient organisations have gained an increasingly important role for the effective reversal of the limited RD awareness tendency, even if it has been a slow process. Starting from a very small number of enthusiastic patients, these move-ments have produced highly respected national alliances in almost all EEC, which are nowadays the core of the RD National Plan advocacy platforms. They have used the opportunities of the open democratic society to organise and raise the voice for a more fair and adequate access to health care, as well as to fi ght the discrimination against the RD community.

D. Taruscio et al.

15

The experience of the EEC, which have already started discussing, elaborating, adopting and implementing RD National Plan, shows that the presence of such associations is a must for the overall RD progress. But the organised patient com-munity is not only effective for the establishment of a national policy. They have a role that goes far beyond this: they can raise RD awareness among society. Furthermore, with a well-informed and educated society on their side, RD people can cause signifi cant changes.

Another factor for improving the situation regarding RD awareness and visibility is the accumulation of RD information resources across the region [ 22 ]. Various information centres, online libraries, patient websites and forums have signifi cantly increased the volume of information in national languages [ 23 – 28 ]. There is a major language barrier for both patients and physicians and the dissemination of free online resources is a considerable opportunity to work on this issues. Moreover, these directories serve also as a gathering point for stakeholders to communicate and collaborate too.

3. EU cross-border healthcare directive

This particular EU directive demonstrates the combined strong effect of the above discussed political and social factors on the present day public health policy in the EU. With the upcoming deadline of the Directive’s transposition to the national legislation, it is sure that there will be more and more publicity about RD in Eastern Europe. There is suffi cient pressure for both sides. Patients feel that it is the right time to seek solutions to their legitimate claims and politicians believe it is the time to reorganise their national health systems in order to make them more effective.

3.2 Rare Diseases National Plans in Eastern Europe

For the purposes of the following analysis, the United Nations Statistics Division defi nition of Eastern Europe has been used, covering Belarus, Bulgaria, Czech Republic, Hungary, Moldova, Poland, Romania, Russia, Slovakia, and Ukraine [ 29 ]. Currently, these countries can be grouped into three categories according to the availability of a RD National Plan (offi cial government approval, specifi c agenda and own budget).

• Countries implementing a RD National Plan

Bulgaria and Czech Republic are the only EEC to have offi cially adopted such a political public health document. The Bulgarian National Plan [ 8 ] was approved at the end of 2008 and was offi cially launched in 2009 covering a 5-year term. It was a signifi cant event for the RD community from all over Eastern Europe, because a relatively small and resources-limited Eastern European nation has become the second country after France to start implementing a specifi c RD strategy. Bulgaria and Czech Republic have demonstrated that the National Plan for RD is not an option only for high-income Member States. The adoption and implementation depends

National Plans on Rare Diseases

16

much more on the presence of a core advocacy group rather than on the country’s economic situation.

The Bulgarian National Plan consists of nine priorities, as it is described in the sub-chapter regarding the Bulgarian plan.

The National Plan was supposed to receive an overall budget of 11.3 million Euros for a 5-year implementation period. Nevertheless, the plan has substantially failed to meet the fi nancial expectations of the RD community. Subsequently, a decreased and unbalanced budget has been allocated, whereby the genetic labo-ratory activities were given a higher priority in the use of funds at the expense of the other planned activities. As well, the necessary legislative amendments were consistently abandoned.

Czech Republic followed Bulgaria and, in 2010, a National Strategy for Rare Diseases 2010–2020 [ 9 ] was approved by the government. Its main objectives were to ensure access to high-quality care and the best method of treatment based on equality and solidarity. The strategy was planned to be covered by the existing budgetary chapters and domestic and foreign subsidies.

In August 2012, the Czech government has adopted a 3-year National Plan [ 10 ] to implement corrective measures in accordance with the strategy’s objectives. Eleven priority fi elds have been indicated for specifi c actions in 2012–2014:

– improving awareness; – education; – prevention; – improving screening and diagnosis; – improving the availability and quality of care; – improving the quality of life and social integration; – support for science and research; – harmonisation and development of data collection and biological sampling; – support and strengthening of the role of patient organisations; – inter-ministerial and inter-disciplinary collaboration; – international cooperation.

The plan was meant to be funded from different sources (public health insurance, domestic and foreign subsidies), but no further details were provided. It was only indicated that the Ministry of Health would provide 0.2–0.4 million Euros to set up a National Coordination Centre and to concentrate care in specialised centres [ 7 ].

Without making a direct comparison between the two countries, it is evident that the third criteria for RD National Plan – a dedicated budget for RD may be the EEC’s weakest point when developing RD national plans. RD should not compete against common diseases and, when a RD National Plan is offi cially adopted, government and RD stakeholders should take their respective responsibilities and make sure that the funding for the planned activities is earmarked.

• Countries, offi cially considering a RD National Plan

Several other EEC – Hungary [ 25 ], Poland [ 26 ], Romania [ 27 ], and Slovakia – have initiated the process to adopt a RD National Plan [ 7 ]. Most of them have

D. Taruscio et al.

17

successfully elaborated a draft that was later submitted to the respective policy decision-makers. However, no offi cial approval or budget has been reported for these countries. Instead, they have focused on various short-term programmes to address different aspects of RD, mainly the provision of expensive therapy for specifi c patient subgroups and the establishment of disease-specifi c registries.

It is no doubt that starting RD specifi c programmes and projects is a step forward for these countries. Nevertheless, all these fragmented efforts may not be sustainable in the long-term. Rather, they appear as waste resources because different programmes operate in divergent manners, on various levels and have different, independent objectives to achieve. These can even compete among each other in some specifi c cases. A general guideline from the EU RD recommendation is that no country can manage RD issues on its own efforts. It is a very wide fi eld, where cooperation and pooling of resources are the only way to guarantee progress.

• Countries not offi cially considering a RD National Plan

This category of countries includes Belarus, Moldova, Russia, and Ukraine. It is no surprise, as the EU adhesion factor does not have major role in these countries. However, it does not mean that specifi c RD policies are not emerging in these nations. Patient organisations are steadily growing in this region and are the locomotive for several policy initiations.

In 2012, Russia made the most important RD policy decision thus far. The Government adopted a decree that laid down rules for the creation and functioning of the Federal Registry for RD. Initially, the Registry will include patients suffering from haemophilia, cystic fi brosis, pituitary nanism, Gaucher’s disease, malignancies of lymphatic and blood-forming tissues, multiple sclerosis, as well as transplanted patients [ 30 ]. Health legislation amendments were also offi cially proposed in the National Parliament of Ukraine. These include, in particular: formal defi nition of RD, obligation to the Ministry of Health for the provision of epidemiological data on RD, improvement of prevention of RD, guaranteed access to reliable treatment for RD patients [ 31 ].

It should be recognised that both these moves are patient-driven and use a bottom-up approach. Though these countries are not politically part of the EU, there is an urgent need to strengthen bridges with the local RD stakeholders. This will ensure the sustainability of the RD processes and create effi cient channels for communication and partnership with the prospects of future integration activities.

In summary, our analysis shows that EEC have signifi cantly improved their position on RD policy during the last years. Almost all of the monitored countries have started implementing different activities in the fi eld of RD. Of course, there is still unresolved problems and the ongoing EU integration and cohesion provides an excellent prospective for future solutions.

The National Plan for RD emerges as the best alternative for the EEC. It would provide long-term sustainability and effectiveness for both their national health systems and the different RD stakeholders. That is why it is important to continue the European and international dialogue and partnership on these issues, as EEC could extremely benefi t from such approach towards RD managment.

National Plans on Rare Diseases

18

4 Conclusions

4.1 Overall Rare Diseases Policy Analysis

Apparently, RD national policies and particularly RD National Plans have made a signifi cant leap forward since the Commission’s Communication (2008) and the Council’s Recommendation (2009). RD stakeholders on both European and national level are undoubtedly convinced in RD National Plans’ capacity to pro-vide effective and effi cient solutions of the long-lasting problems in this fi eld. Such strategic public health documents are emerging in a growing number of countries and virtually all EU Member States are working on drafting and adopting RD National Plans.

The review of the existing offi cially approved and acting RD plans shows great consistency with the overall European RD policy framework. Nevertheless, RD plans on country level demonstrate different levels of completeness regarding the various RD action fi elds and, most importantly, different level of political and stakeholders’ commitment towards the plans’ implementation. Our analysis inden-tifi es two stages of crucial importance regarding RD plans: (1) planning and (2) evaluating processes. As a matter of fact, RD National Plan’s overall success heavily depends on the initial planning’s effectiveness and on the fi nal evaluation’s objectiveness.

RD Policy Planning Process

Despite being well supported by various EU policy documents and EU public health projects’ outcomes, RD National Planning process has not produced enough results so far. Several operational and strategic issues remain to be dealt with.

• Operational challenges include:

– the elaboration and inclusion of identifi able and measurable indicators of the plan’s progress;

– a rieliable budgetary framework; – the involvement and empowerment of all RD stakeholders (e.g., patients,

clinicians, etc.) in the plan’s management and monitoring.

In operational terms, most plans currently do not appear to be endowed with adequate funding, which will prove to be crucial for the plan’s overall implementa-tion. National authorities should more carefully deal with this issue, as subsequent changes and funding re-allocation may hinder the implementation process. Multiple stakeholder participation is a must, not only for the planning stage. It should be fi rmly attached to the plan’s management and to the monitoring process. As most plans are a result of bottom-up efforts (excluding the Greek Plan), non-governmental groups (including patient organisations) are often those who ensure plan’s vitality.

D. Taruscio et al.

19

Precise indicators allow better monitoring and evaluation, as well as interregional and international benchmarking.

• Strategic challenges include:

– Long-term sustainability of RD infrastructures and funding; – Availability and mobility of RD expertise and knowledge; – Cross-border cooperation and integration.

In a strategic aspect, most national activities in the EU ignore cross-border coop-eration and integration. Such intentions are included in all plans, but no subsequent steps are undertaken. It is a very important issue, as both material and human resources are very scarce in the RD fi eld and no single country can successfully deal with these problems on its own. In particular, small-size countries may greatly benefi t by cross-border cooperation, as it would give their respective RD community better opportunities for actions and progress.

RD Policy Evaluation Process

Regarding RD National Plans’ interim and fi nal evaluations, currently only France has had this experience with her RD National Plan. Indeed, all EU policy documents have predominantly focused on planning and implementing so far, leaving evaluation to Member States’ consideration. Evaluation should be a core element of each RD National Plan. Generally, plans last only for a specifi c period, but RD achievements should go further and their long-tern sustainability should be guaranteed. Proper identifi cation, interpretation and assessment of the plan’s outcomes will provide a base to undertake corrective actions and to ensure optimal use of their impact.

As an example, the French First National Plan for Rare Diseases (2005–2008) [ 32 ] has been evaluated [ 33 ] by a committee from that Country’s High Council of Public Health (Haut Conseil de la Santé Publique, HCSP). The 10-person committee has been co-chaired by a HCSP member and an external expert (clinician). It consisted of experts in public health, economy and sociology.

The evaluation process has focused on six major topics:

1. Evaluation of the plan’s implementation: progress status of plan’s measures, dif-ference between planned and actually adopted measures, fi nancial balance, mobilisation of stakeholders, organisation of management and monitoring, obstacles encountered during implementation;

2. Impact analysis in terms of equity: access to competent care and better treatment, access to reliable and accurate information on RD, improvements in the quality of medical, paramedical and social care, patient individual fi nancial burden, patient perception of the plan, patient satisfaction;

3. Plan’s effect in terms of improving medical response, both clinical and research: short- and long-term expectations of undertaken actions, improvement of work conditions and effi cacy of medical professionals after the setting of reference centres, plan’s impact on European RD actions, improvements of research efforts

National Plans on Rare Diseases

20

and coordination on national and EU level, integration of French research into international research, outcomes in terms of diagnosis and treatment, international publications;

4. Relevance of objectives: plan’s refl ection of the preparatory work’s conclusions, existence of new elements, leading to formulation of plan’s objectives, fi nancial cost justifi cation;

5. Achievement of plan’s objectives by the stated actions; 6. Use of plan evaluation to prepare future actions (improvement of plan’s relevance

by modifying certain objectives, reconsideration of national and international partnerships, improvements of plan’s system and pillar structures (reference centres, Orphanet, research facilities), elaboration of indicators and evaluation method for the next plan).

5 Final Remarks

In conclusion, EU RD policy has greatly supported Member States in the process of elaboration and adoption of RD National Plans. This series of actions should con-tinue, but a new focus on monitoring and evaluation should be added and further developed. The optimal scenario would include collaboration and expertise sharing at international level, as RD knowledge is steadily growing, but it is till very limited. At national level, RD stakeholders should carefully consider the planning process, as its outcomes would greatly determine the RD National Plan’s overall success.

References

1. EU Council recommendation of 8 June 2009 on an action in the fi eld of rare diseases. Offi cial journal of the European Union (2009/C 151/02)

2. Communication from the Commission to the European Parliament, the Council, the European Economic and Social Committee and the Committee of the Regions on Rare Diseases: Europe’s challenges http://ec.europa.eu/health/ph_threats/non_com/docs/rare_com_en.pdf . Accessed 17 Dec 2012

3. European Project for Rare Diseases National Plans Development (EUROPLAN) http://www.europlanproject.eu/_europlanproject/index.html . Accessed 17 Dec 2012

4. World Bank. What is empowerment? http://siteresources.worldbank.org/INTEMPOWERMENT/Resources/486312-1095094954594/draft2.pdf . Accessed 28 Dec 2012

5. Neuhauser D (2003) The coming third healthcare revolution: personal empowerment. Qual Manag Health Care 12:171–184

6. Aymé S, Kole A, Groft S (2008) Empowerment of patients: lessons from the rare diseases com-munity. Lancet 371:2048–2051

7. Aymé S, Rodwell C (eds) (2012) Report on the State of the Art of Rare Disease Activities in Europe of the European Union Committee of Experts on Rare Diseases, July 2012

8. National Plan for Rare Diseases 2009–2013 (Genetic, congenital malformation and nonheredi-tary disease) [in Bulgarian]. http://www.mh.government.bg/Articles.aspx?lang=bg-BG&pageid=427&categoryid=889 . Accessed 17 Dec 2012

D. Taruscio et al.

21

9. Czech National Strategy for Rare Diseases: 2010–2020 [in Czech]. http://kormoran.vlada.cz/usneseni/usneseni_webtest.nsf/0/9F67CBDF7AB3D010C125773E00446BC7/$FILE/466%20uv100614.0466.pdf . Accessed 17 Dec 2012

10. Czech National Plan for Rare Diseases (in Czech). http://www.mzcr.cz/dokumenty/narodni-akcni- plan-pro-vzacna-onemocneni-na-leta-2012-2014_6713_1.html . Accessed 17 Dec 2012

11. French National Plan for Rare Diseases (2011–2014) [in French]. http://www.sante.gouv.fr/IMG/pdf/Plan_national_maladies_rares.pdf . Accessed 17 Dec 2012

12. Greek National Plan of Action for Rare Diseases (2008–2012) [in Greek]. http://www.ygeianet.gov.gr/HealthMapUploads/Files/SPANIES_PATHISEIS_TELIKO_LOW.pdf . Accessed 17 Dec 2012

13. Portuguese National Programme for Rare Diseases (2008–2015) [in Portuguese]. http://www.portaldasaude.pt/NR/rdonlyres/555DD3B3-45F0-4F74-B633-28889E721BF1/0/i010420.pdf . Accessed 17 Dec 2012

14. Slovenian Work Plan for Rare Diseases [in Slovenian]. http://www.mz.gov.si/fi leadmin/mz.gov.si/pageuploads/redke_bolezni_2012_-_nacrt_dela/Nacrt_dela_na_podrocju_redkih_bolezni.pdf . Accessed 17 Dec 2012

15. Rare Diseases Strategy of the National Health System of Spain [in Spanish]. http://www.msc.es/organizacion/sns/planCalidadSNS/docs/enfermedadesRaras.pdf . Accessed 17 Dec 2012

16. Strategy of The Netherlands in the fi eld of Rare Diseases [in Dutch]. http://www.npzz.nl/2012/08/21/concept-nationaal-plan-zeldzame-ziekten/ . Accessed 17 Dec 2012

17. Taruscio D, Vittozzi L, Stefanov R (2010) National plans and strategies on rare diseases in Europe. In: Posada de la Paz M, Groft SC (eds) Rare diseases epidemiology. Springer Dordrecht Heidelberg London New York

18. Taruscio D, Trama A, Stefanov R (2007) Tackling rare diseases at European level: why do we need a harmonized framework? Folia Med (Plovdiv) 49(1–2):59–67

19. Stefanov R, Taruscio D (2009) Rare diseases and orphan drugs in Eastern European Countries. Ital J Publ Health 6(4):289–293

20. Iskrov G, Miteva-Katrandzhieva T, Stefanov R (2012) Challenges to orphan drugs access in Eastern Europe: the case of Bulgaria. Health Policy 108(1):10–18

21. Miteva TS, Jordanova R, Iskrov G, Stefanov R (2011) General knowledge and awareness on rare diseases among general practitioners in Bulgaria. Georgian Med News 193:16–19

22. European awareness of rare diseases. Special Eurobarometer 361 (2011) 23. Stefanov R (2009) Policy on rare diseases: the case of Bulgaria. In: Proceedings of the 4th

Eastern European conference for rare diseases and orphan drugs, 13–14 June 2009, Plovdiv (Bulgaria). BAPES, pp 40–41

24. Vejvalkova S (2009) Czech Republic. In: Proceedings of the 4th Eastern European con-ference for rare diseases and orphan drugs, 13–14 June 2009, Plovdiv (Bulgaria). BAPES, pp 50–52

25. Sandor J (2009) Hungary. In: Proceedings of the 4th Eastern European conference for rare diseases and orphan drugs, 13–14 June 2009, Plovdiv (Bulgaria). BAPES, pp 55–57

26. Wegrzyn G (2009) Poland. In: Proceedings of the 4th Eastern European conference for rare diseases and orphan drugs, 13–14 June 2009, Plovdiv (Bulgaria). BAPES, pp 64–65

27. Dan D (2009) Romania. In: Proceedings of the 4th Eastern European conference for rare diseases and orphan drugs, 13–14 June 2009, Plovdiv (Bulgaria). BAPES, pp 66–67

28. Sokolov A (2009) Russian Federation. In: Proceedings of the 4th Eastern European con-ference for rare diseases and orphan drugs, 13–14 June 2009, Plovdiv (Bulgaria). BAPES, pp 68–70

29. United Nations Statistics Division. Standard Country and Area Codes Classifi cations (M49) http://unstats.un.org/unsd/methods/m49/m49regin.htm#europe . Accessed 17 Dec 2012

30. Decree on the approval of the Rules of the Federal Registry of persons with hemophilia, cystic fi brosis, pituitary nanism, Gaucher’s disease, malignancies of lymphatic and blood-forming tissues, multiple sclerosis, as well as transplanted patients [in Russian]. http://pravo.gov.ru/proxy/ips/?docbody=&prevDoc=102319042&backlink=1&&nd=102311335 . Accessed 17 Dec 2012

National Plans on Rare Diseases

22

31. Bill on legislative amendments to provide medical care of people with rare diseases [in Ukrainian]. http://w1.c1.rada.gov.ua/pls/zweb2/webproc4_2?id=&pf3516=10383&skl=7 . Accessed 17 Dec 2012

32. French National Plan for Rare Diseases (2005–2008). http://www.sante.gouv.fr/IMG/pdf/French_National_Plan_for_Rare_Diseases.pdf . Accessed 17 Dec 2012

33. Evaluation of French National Plan for Rare Diseases (2005–2008) [in French]. http://www.hcsp.fr/docspdf/avisrapports/hcspr20090317_maladiesRares.pdf . Accessed 17 Dec 2012

D. Taruscio et al.

23M. Özgüç (ed.), Rare Diseases: Integrative PPPM Approach as the Medicine of the Future, Advances in Predictive, Preventive and Personalised Medicine 6,DOI 10.1007/978-94-017-9214-1_2, © Springer Science+Business Media Dordrecht 2015

Abstract For the past 15 years biobanks have been infrastructures that have permitted to ensure the quality of the biological resources used as well as guarantee the rights of stakeholders. Due to their medical and scientifi c expertise, they offer collections of human biological resources that meet with research project require-ments. Because of their secure storage capacities, biobanks are front-line actors in collection management by enabling their development, ensuring their continued existence and their valorisation in relevant research projects. At the cross-roads of patient inclusion, sample and associated data collection and logistical facilities, bio-banks have become a signifi cant partner of translational research and personalised medicine, almost essential in the fi eld of rare diseases research as a result of the diffi culty of obtaining the samples.

Keywords Biobanks • Repositories • Collection • Bioresources • Human samples • Rare diseases • Ethic • Best practice • Personalised medicine

1 Introduction

The development of genetics and molecular biology techniques have allowed for breakthroughs in the knowledge of the physiopathological mechanisms of rare diseases, the improvement of diagnosis, patient care and now the development of personalised medicine. If this new therapeutic approach is studied closely when it comes to cancer [ 1 , 2 ], it is also relevant when applied to rare diseases [ 3 ].

In order to develop the concept of “the right treatment for the right patient at the right time”[ 4 ], studies on personalised medicine need to identify specifi c profi les, whether they be genetic, biological, dependent on the environment or not. The

Biobanking for Rare Diseases – Impact on Personalised Medicine

Jeanne-Hélène di Donato

J.-H. di Donato (*) 3C-R , Castelginest , France e-mail: jhdd@wanadoo.fr

24

identifi cation of these profi les requires the highlighting of correlations between responses (or non-responses) to a treatment and a specifi c characteristic of the person in a given context. The development of this medicine [ 5 ] thus requires great quanti-ties of information to accumulate data and demonstrate the veracity of the link between treatment and response according to a specifi c biomarker. Some of this essential data stemming from the analysis of samples collected from patients [ 6 ], Biobanks have become indispensable partners of this type of research by providing their biological sample management know-how [ 7 ]. Requested for the de novo con-stitution of collections, they can also swiftly supply samples that have already been collected in order to verify the presence or the lack of a biomarker. With regard to rare diseases, where access to numerous samples is problematic and can represent a major hindrance for research, biobanks can set up networks to pool their strength to obtain collections that will represent a suffi cient critical mass for the conduct of studies.

2 Biobank Organisation

2.1 The Advent of a New Trade

Biobanks, repositories or biological resource centres (BRC) [ 8 ], represent the same type of infrastructure dedicated to the management of annotated samples for the purpose of promoting their optimum use in scientifi c research programmes.

The advent of this professionalism stems from the symbiosis of various trends bearing concepts that are now viewed as fundamental: quality and security of the collections, traceability of exchanges, optimisation of the use of bioresources, trans-parency and control of biobanking and respect for regulatory and ethical rules. The importance of the impact of these biobanks on scientifi c research is now perfectly acknowledged [ 9 – 11 ] this is demonstrated by the number of publications on the subject which keeps increasing (PubMed searches on “biobanks*” in the title and summary show 2 articles in 2000, 39 in 2005 and 278 in 2012).

Even if some Biobank have been established by scientists (clinicians or researchers who wished to create a collection for their own research purposes), management responsibilities have now extended [ 12 , 13 ] to legal and ethical com-petences [ 14 ] as well as quality management skills in the sense of the ISO 9001 standard or the French standard NF S 96–900 specifi cally published for the certifi -cation of BRCs [ 15 ]. Biobanks are the guarantors of the best use of biological resources for all stakeholders: the patients and theirs families, clinicians, geneti-cists, researchers, consortiums, etc.

Moreover trust in biobanks is furthered by the legal obligation to obtain an authorisation for their activities which is being implemented in an increasing num-ber of countries [ 16 , 17 ].

J.-H. di Donato

25

2.2 Collection Manager

A collection is defi ned as an “ assemblage, for research purposes, of biological material selected on the basis of clinical or biological characteristics ” [ 18 ]. As such, a collection is a « research device » in itself. It allows the identifi cation of different profi les, the validation of diagnostic hypotheses and the control of the pertinence of the therapeutics. Managed by a BRC, the storage of the collection is not only ensured throughout the fi rst research subject (which can take years) but also beyond to allow for a secondary use. Diffi cult and costly to set up, collections represent a major interest [ 19 ] and must remain available to researchers. Their stor-age by an established facility ensures their continued existence and prevents their loss after the collector has left.

In the case of rare diseases because of the low number of patients, collection constitution poses an added challenge that biobanks, thanks to their profession-alism, manage to overcome by working together as a network. The Italian exam-ple of Telethon Network of Genetic Biobanks (TNGB: www.biobanknetwork.org ) aptly demonstrates the power that can be achieved through federated work with the publication of 248 articles since 2008. And when demand is too high for national supply, international networks can also come into play like EuroBioBank ( www.eurobiobank.org ) with its more than 440,000 samples col-lected by 16 biobanks from 8 countries which has permitted the publication of 173 articles since 2003.

This networking can however only increase research potentialities if the samples that come from different locations are prepared according to harmonised procedures and monitored with the same acuity [ 20 ].

2.3 Infrastructures as Well as Research Partners

Biobanks are not simple sample suppliers. Their medical and scientifi c expertise, which allows them to establish relevant collections, combined with their technical know-how, have gradually given them a genuine and undisputed place in transla-tional research.

Thus they have quite naturally found their place in pluridisciplinary thematic networks or research consortiums to assist in collection constitution and man-agement (RD-CONNECT [ 21 ], IRDiRC [ 22 ], RDRC [ 23 ], Treat-NMD [ 24 ]). Their participation allows for greater responsiveness and the decrease in the time required to access collections which is a bottleneck for research on rare diseases.

Biobanking for Rare Diseases – Impact on Personalised Medicine

26

3 Appropriated Biological Samples for Research on Rare Diseases

Biological samples as elements of the human body are bearers of the intrinsic characteristics of the person. However their use can only produce reliable results if their quality is established. Without this quality assurance, research can not be car-ried out [ 25 ] or the results can be skewed. Yet the quality of samples can be deterio-rated at all stages, from the collection to the preparation and storage through to the transfer. Constant monitoring is essential to satisfy the requirements set forth by researchers.

In the context of rare diseases, which render biological samples even more precious, the structuration of biobanks is the guarantee of the respect of best profes-sional practice and of an optimised organisation of pre analytical phases, of preparation and storage of the biological resources.

3.1 Pre Analytical Phase

During the pre analytical phase there are fundamental points that can infl uence sample quality [ 26 – 28 ]; they involve:

• Sampling or collecting techniques, to obtain the best possible sample in suffi -cient quantity;

• Primary receptacle of packaging must guarantee sample quality (sterilised, DNAse free, quality of the raw material to avoid certain molecule adsorption) and enable secure transport [ 29 ];

• The delays to obtain the samples that can be more or less crucial depending on the nature of the sample and the research that will be carried out; if blood samples for genetic studies do not present a major challenge, research on blood biomark-ers can require shorted shipping delays [ 30 ] especially if dosage must be reiter-ated over a period of time [ 31 ].

• Proper and secure identifi cation of the sample to avoid any identity or annotation mistake;

• Shipping conditions including when dealing with hospital pneumatics [ 32 ].

Incoming sample monitoring is thus a critical stage that biobanks execute according to criteria predefi ned either by the ongoing research project or by the state of the art. This caution allows for the correction of a possible lack of conformity and for the recording of the relevant data thus avoiding the supply of non compliant biological material.

J.-H. di Donato

27

3.2 Sample Preparation

The intervention of biobanks in sample preparation is extremely varied, depending on scientifi c needs as established by professional recommendations as well as the requirements for a specifi c research.

Sample preparation (fi xation, freezing, derivatives preparation, aliquoting) are executed according to standard operating procedures (SOPs) that professional biobank networks (ISBER, BBMRI, ESBB, EuroBioBank) or research consortiums attempt to harmonise to avoid interpretation errors due to preparation biases. Harmonising procedures is “essential for communication and comparability, for assuring quality and avoiding unnecessary duplication” [ 33 , 34 ].

These SOPs are updated thanks to the work accomplished by biobanks to increase sample conformity as expected for a current as well as subsequent use by attempting to anticipate future requirements.

3.3 Sample Conservation

Secure storage on a long term basis is probably at the crux of the matter that pushed biobanks towards professionalisation. Too many faulty or poorly monitored freezers have resulted in the loss of years of work by inducing thawing of samples, too many collections have been lost due to the departure of their investigator, too many sam-ples have been subjected to frequent temperature increases that were detrimental to their quality. This state of affairs often noted, but seldom published represents a substantial scientifi c as well as economic loss.

To certify the appropriate preservation of samples, biobanks have implemented storage logistics that ensure unparalleled optimum secure storage. Premises are watched over and controlled, storage areas are under surveillance 24 h a day with on-call duty protocols that allow for the transfer of samples to backup storage areas when problems occur, effective storage temperatures are continuously recorded to monitor storage quality.

Moreover and when possible, collections are duplicated to prevent their complete disappearance in case of major incident.

Combined with increasingly extensive storage capacities, biobank storage logistics allow the establishment of centralised collections that are all the more important in the case of rare diseases whereupon years can be necessary to obtain a usable collection.

Beyond pure logistics, biobanks play an important part in storage management by monitoring the possible degradation of certain biomarkers over time [ 35 ] and by ensuring the renewal of a collection so that it remains usable under the best conditions.

Biobanking for Rare Diseases – Impact on Personalised Medicine

28

4 Management of Associated Data

Biological material only has real scientifi c value if it is associated with pertinent data. They are what enable the choice of samples both for collection constitution and for their supply.

Thus biobanks must not only ensure the collection and management of the data characterising the samples but also that of the pathology, the patient and his/her environment. The accuracy and the sum of this data is a key element in personalised research. The special position enjoyed by biobanks in the fi eld of human health, often incorporated in hospitals and managed by clinicians, simplifi es the access to patients and their medical records. In some cases they can have access to databases generated by cohorts or the case report forms (CRFs) used in clinical trial research.

Occasionally it may occur that research results can be procured to create added value to the information initially obtained. In the case of rare diseases, knowledge of the mutation identifi ed thanks to research work is a classic new annotation example of samples that can sometimes have been stored for several years with the sole information being the pathology or even the pathology family.

5 Supply of Biological Material

The main objective of any human health biobank is to develop the collections to enhance scientifi c knowledge that will allow better patient care. Their role is not the storing but the sharing of the collections.

To do so, they publish catalogues [ 36 – 39 ] to disseminate information on avail-able collections and they ensure their distribution through procedures that are rec-ognized as guaranteeing the rights of stakeholders. Biobanks ensure compliance with the consent of donors, the possible reservation of the collection involved in a research project and secure the agreement of the initial depositor to protect his/her right of priority if so desired.

The procedures for the provision of samples also take into account the require-ments established by the scientifi c policy of the biobank, the authentifi cation of the requesting researcher, the scientifi c validation of the project by an independent Comity. They also allow to check compliance with legal rules and to manage poten-tial confl icts of interest.

All these stages are concluded with the drafting of a contract or a material trans-fer agreement that will trigger the actual supply of the samples.

6 Ethical Engagement of Biobanks

Because biobanks work with elements of the human body, health data and often- times with information on genetic characteristics in the context of personalised medicine, the weight of ethical rules is important.

J.-H. di Donato

29

Several studies [ 40 ] have demonstrated that patients and their families were not opposed to a use in research, on the condition that they be assured of the ethically and scientifi cally controlled management of their samples. They wish for the optimum use of the collections to further science and obtain results that will be use-ful for future generations. The intervention of a biobank is thus the guarantee of a controlled use of the collections on a long term basis in keeping with the autonomy of the person as expressed in the ever rescindable consent. At the present time rec-ommendations can allow for specifi c consent for biobanks [ 41 ] and projects such as EnCoRe [ 42 ] are attempting to globalise the long term management of consents to enhance their monitoring.

Research activities can thus be performed in the general interest without violating the rights of the individual [ 43 , 44 ].

7 Conclusion

Biobanks, in the same way that other research support infrastructures (genomic and proteomic platforms, databases) have become in 15 years effi cient partners that allow access to great quantities of quality samples and pertinent collections. By applying best practices and setting up security systems, they can guarantee the best possible support for the samples and the collections.

Their professionalisation has transformed the simple task of managing a freezer into social responsibility. Biobanks have thus become the guarantors of an ethical use of biological resources and of the optimisation of the use of the collections to obtain as swiftly as possible therapeutic outcomes that have been long-awaited by patients.

References

1. Oktay MH, Hui P (2012) Molecular pathology as the driving force for personalized oncology. Expert Rev Mol Diagn 12(8):811–813

2. Kalia M (2013) Personalized oncology: recent advances and future challenges. Metabolism 62:S11–S14, Suppl1

3. Palau F (2012) Personalized medicine in rare disease. Personalized Med 9(2):137–141 4. Rugnetta M, Whitney K (2009) Paving the way for personalized medicine. Sci Prog. http://

www.scienceprogress.org/wp-content/uploads/2009/09/personalized_medicine.pdf . Accessed 12 Dec 2012

5. Lehrach H (2012) A revolution in healthcare: challenges and opportunities for personalized medicine. Personalized Med 9(2):105–108

6. Clotworthy M (2012) The application of human tissue for drug discovery and development. Expert Opin Drug Discov 7(7):543–547

7. Yüzbaşioğlu A, Ozgüç M (2012) Biobanking: sample acquisition and quality assurance for ‘omics’ research. N Biotechnol 30(3):339–342

Biobanking for Rare Diseases – Impact on Personalised Medicine

30

8. OECD (2000) Biological resources centres: underpinning the future of life sciences and biotechnology. DSTI/STP/BIO 4 REV1 http://www.oecd.org/sti/biotech/2487422.pdf . Accessed 30 Dec 2012

9. European Strategy Forum in Research Infrastructures (ESFRI) (2012) European research infrastructures with global impact. http://www.copori.eu/_media/ESFRI_Brochure_210912_lowres.pdf . Accessed 30 Dec 2012

10. Meeting Europe’s challenges: the role and importance of biological and medical sciences research infrastructures (2010) http://www.eurobioimaging.eu/sites/default/fi les/BMS%20Strategy%20Paper.pdf . Accessed 30 Dec 2012

11. Meijer I, Mattson P, Nooijen A, Boekholt P, Molas Gallart J, Amat CB (2010) BBMRI: an evaluation strategy for socio-economic impact assessment http://www.technopolis-group.com/resources/downloads/life_sciences/1093_BBMRIfi nalreport_100921.pdf . Accessed 30 Dec 2012

12. OECD best practice guidelines for biological resource centres (2007) http://www.oecd.org/sti/biotechnologypolicies/38777417.pdf . Accessed 30 Dec 2012

13. Gottweis H, Kaye J, Bignami F, Rial-Sebbag E, Lattanzi R, Macek M (2012) Biobanks for Europe – a challenge for governance. doi: 10.2777/68942 . http://www.coe.int/t/dg3/healthbio-ethic/Activities/10_Biobanks/biobanks_for_Europe.pdf . Accessed 30 Dec 2012

14. Martín Uranga A, Martín-Arribas MC, di Donato JH, de la Paz Posada M (2005) Outstanding legal and ethical issues on biobanks. Instituto de Salud Carlos III, Madrid

15. Norme NF S 96–900: Quality of Biological Resources Centres (BRC) – Quality management system of BRC and quality of biological resources. (2011) French document

16. Martín Uranga A, Martín-Arribas MC, di Donato JH, de la Paz Posada M (2005) Outstanding legal and ethical issues on biobanks. Instituto de Salud Carlos III, Madrid, Chapter 6

17. WIKI legal platform of BBMRI http://www.bbmri.eu/index.php?option=com_content&view=article&id=67&Itemid=58 . Accessed 30 Dec 2012

18. OECD best practice guidelines for biological resource centres (2007) Chapter best practice guidelines on human-derived material. http://www.oecd.org/sti/biotechnologypolicies/38777417.pdf . Accessed 30 Dec 2012

19. Lochmüller H, Aymé S, Pampinella F, Melegh B, Kuhn K, Antonarakis SE, Meitinger T (2009) The role of biobanking in rare diseases: European Consensus Expert Group report. Biopreserv Biobank 7(3):155–156

20. Lochmüller H, Schneiderat P (2010) Biobanking in rare disorders. Adv Exp Med Biol 686:105–113

21. RD-CONNECT: an integrated platform connecting databases, registries, biobanks and clinical bioinformatics for rare disease research. http://www.rd-connect.eu/ . Accessed 30 Dec 2012

22. International Rare Disease Research Consortium. http://www.irdirc.org/ . Accessed 30 Dec 2012

23. Rare disorders research consortium of Oregon Health & science university. http://www.ohsu.edu/xd/research/clinical-research/hgi/consortium/ . Accessed 30 Dec 2012

24. Treat-NMD neuromuscular network. http://www.treat-nmd.eu/ . Accessed 30 Dec 2012 25. Moore HM, Kelly AB, Jewell SD, McShane LM, Clark DP, Greenspan R, Hayes DF, Hainaut

P, Kim P, Mansfi eld EA, Potapova O, Riegman P, Rubinstein Y, Seijo E, Somiari S, Watson P, Weier HU, Zhu C, Vaught J (2011) Biospecimen reporting for improved study quality (BRISQ) Cancer Cytopathology. Cancer Cytopathol 119(2):92–102. doi 10.1002/cncy.20147

26. Azimi-Nezhad M, Lambert D, Ottone C, Perrin C, Chapel C, Gaillard G, Pfi ster M, Masson C, Tabone E, Betsou F, Meyronet D, Ungeheuer MN, S-Siest V (2012) Infl uence of pre-analytical Variables on VEGF gene expression and circulating protein concentrations. Biopreserv Bioreposit 10(5):454–461

27. González-Gross M, Breidenassel C, Gómez-Martínez S, Ferrari M, Béghin L, Spinneker A, Díaz LE, Maiani G, Demailly A, Al-Tahan J, Albers U, Wärnberg J, Stoffel-Wagner B, Jiménez-Pavón D, Libersa C, Pietrzik K, Marcos A, Stehle P (2008) Sampling and processing of fresh blood samples within a European multicenter nutritional study: evaluation of bio-marker stability during transport and storage. Int J Obes (Lond) 32(Suppl 5):S66–S75

28. Lehmann S, Roche S, Allory Y, Barthelaix A, Beaudeux JL, Berger F, Betsou F, Borg J, Dupuy A, Garin J, Quillard M, Lizard G, Peoc’h K, Riviere M, Ducoroy P (2009) Preanalytical

J.-H. di Donato

31

guidelines for clinical proteomics investigation of biological fl uids. Ann Biol Clin (Paris) 67(6):629–639, French

29. Stumm MM, Walker MR, Stork C, Hanoteau N, Wagner U, O’Reilly TM (2012) Validation of a postfi xation tissue storage and transport medium to preserve histopathology and molecular pathology analyses (total and phosphoactivated proteins, and FISH). Am J Clin Pathol 137(3):429–436

30. Rai AJ, Vitzthum F (2006) Effects of preanalytical variables on peptide and protein measure-ments in human serum and plasma: implications for clinical proteomics. Expert Rev Proteomics 3:409–426

31. Policepatil SM, Caplan RH, Dolan M (2012) Hypocalcemic myopathy secondary to hypopara-thyroidism. Wisc Med Soc 111(4):173–175

32. Amann G, Zehntner C, Marti F, Colucci G (2012) Effect of acceleration forces during transport through a pneumatic tube system on ROTEM® analysis. Clin Chem Lab Med 50(8):1335–1342

33. OECD best practice guidelines for biological resource centres (2007) Chapter II, p 16. http://www.oecd.org/sti/biotechnologypolicies/38777417.pdf . Accessed 30 Dec 2012

34. Marko-Vaga G, Végvari A, Welinder C, Lindberg H, Rezeli M, Edula G, Svensson KJ, Belting M, Laurell T, Fehniger T (2012) Standardization and utilization of biobank resources in clini-cal protein science with examples of emerging applications. J Proteome Res 11(11):5124–5134

35. Kugler KG, Hackl WO, Mueller L, Fiegl H, Graber A, Pfeiffer R (2011) The impact of sample storage time on estimates of association in biomarker discovery studies. J Clin Bioinform 1:9

36. P3G http://p3g.org/ . Accessed 30 Dec 2012 37. I3-CRB http://www.i3crb.fr/ . Accessed 30 Dec 2012 38. EuroBioBank http://www.eurobiobank.org/ . Accessed 30 Dec 2012 39. TV GSO: Virtuel tumor bank of Canceropole GSO http://www.biobank-gso.org/apex/

f?p=200:1:1682323211602182 . Accessed 30 Dec 2012 40. Clerkin P, Buckley BS, Murphy AW, MacFarlane AE (2012) Patients’ views about the use of

their personal information from general practice medical records in health research: a qualita-tive study in Ireland. Fam Pract. doi: 10.1093/fampra/cms036

41. Kosseim P (2011) Banking for the future: “Informing” consent in the context of biobanks. Paper submitted at the OIV International Seminar on the UNSECO Universal Declaration on Bioethics and Human rights. http://www.priv.gc.ca/media/sp-d/2011/SP-d_20110121_pk_e.asp . Accessed 30 Dec 2012

42. Ensuring Consent & Revocation – A collaborative IT research project being undertaken by UK industry & academia. http://www.encore-project.info/index.html . Accessed 30 Dec 2012

43. Presidential Commission for the study of bioethical issues (2012) Privacy and progress in whole genome sequencing. http://bioethics.gov/cms/sites/default/fi les/PrivacyProgress508.pdf . Accessed 12 Dec 2012

44. Soto C, Tarrant C, Pritchard-Jones K, Dixon-Woods M (2012) Consent to tissue banking for research: qualitative study and recommendations. Arch Dis Child 97:632–636

Biobanking for Rare Diseases – Impact on Personalised Medicine

33M. Özgüç (ed.), Rare Diseases: Integrative PPPM Approach as the Medicine of the Future, Advances in Predictive, Preventive and Personalised Medicine 6,DOI 10.1007/978-94-017-9214-1_3, © Springer Science+Business Media Dordrecht 2015

Abstract Rare diseases are a heavy burden on affected individuals and, collectively, on society. Since most rare diseases have a genetic cause, the identifi cation of dis-ease-causing genes is the fi rst step in the unravelling of pathogenic mechanisms and in the search for specifi c therapeutic options. New technologies based on Next Generation Sequencing (NGS) greatly facilitate the discovery of disease-causing genes, especially when coupled with effi cient data-analysis strategy. Additionally, NGS is slowly entering the clinical arena as a diagnostic tool for rare diseases, dras-tically reducing the time required to arrive at a correct diagnosis.

Keywords Rare diseases • Gene identifi cation • Next generation sequencing • Exome sequencing • Genome sequencing • Filtering strategies • Human genetics

1 Introduction

A rare disease is defi ned by the European Union as a life-threatening or chronically debilitating disease having a prevalence of <1 per 2,000 people. More than 7,000 rare diseases have been described so far, collectively affecting e.g. 30–40 million Europeans and 25 million North Americans [ 1 , 2 ]. Considering the total number of affected people, rare diseases in general are actually common and, thereby, these diseases are an immense burden both on affected individuals and on society [ 3 ].

Emerging Technologies for Gene Identifi cation in Rare Diseases

Filippo Beleggia and Bernd Wollnik

F. Beleggia • B. Wollnik (*) Institute of Human Genetics , University Medical Faculty, University of Cologne , Kerpener Str. 34 , 50931 Cologne , Germany

Center for Molecular Medicine Cologne (CMMC) , University of Cologne , Cologne , Germany

Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD) , University of Cologne , Cologne , Germany e-mail: bwollnik@uni-koeln.de

34

As compared to common diseases, rare diseases used to be unattractive for scientifi c investments by the pharmaceutical industry and possible therapeutic drugs were therefore referred to as “orphan” drugs [ 2 ]. This view has changed in recent years and rare diseases have received much more attention at the scientifi c, political, and societal level. Various national and international initiatives have been started to address the clinical, genetic, and pathophysiological aspects of many rare diseases. Many networks have been formed, bringing together clinicians, geneticists, and molecular biologists with the aim of overcoming the vast heterogeneity of rare dis-ease and of increasing our understanding of the underlying pathogenic mechanisms. The outcome of these studies will help to improve many aspects of patient care. In contrast to common diseases, most rare diseases have symptoms which overlap with one or more other diseases, complicating the diagnostic process and leading to diag-nostic delays. On average, 5–30 years elapse from the fi rst symptoms to the correct diagnosis [ 4 ]. Additionally, before reaching the fi nal diagnosis, 40 % of the patients are initially misdiagnosed [ 5 ]. For these reasons, parents of children with a rare disease often have to go through a medical odyssey before a diagnosis is given and in many cases no clinical or molecular diagnosis can be established at all, even after many years. It is estimated that almost 90 % of all rare diseases have a genetic basis; the identifi cation of disease-associated genes is thus essential (i) to improve molec-ular diagnosis, (ii) to get insights into the underlying pathogenesis, and (iii) to contribute to the development of specifi c therapeutic strategies.

2 The Old-Fashioned Way of Gene Identifi cation

As mentioned above, the identifi cation of disease-associated genes in rare Mendelian disorders is crucial as a fi rst step to gain insight into the molecular pathogenesis of the disease and to improve patient care. In the last decades, two major traditional methods have been successfully used for gene identifi cation: Functional cloning started from the knowledge about the function of a specifi c protein and an educated guess about the disorder that might be caused by dysfunction of the protein caused by mutations in the corresponding gene. A second and more successful method for gene identifi cation was to perform a genome-wide mapping study followed by the positional identifi cation of the disease-associated gene. Many so-called “loci” for rare diseases were mapped for autosomal dominant, autosomal recessive, and X-linked disorders, initially by using panels of microsatellite markers and later on with SNP arrays [ 6 , 7 ]. Although it was very successful and led to the identifi cation of numerous disease-causing genes, this mapping approach had two major disad-vantages: First, the need for large families with many affected individuals in order to get statistically signifi cant LOD score results; and second the fact that the mapped interval was often very large and contained sometimes up to 100 genes. In the absence of an excellent positional candidate, all genes in the linked chromosomal region had to be analyzed by Sanger sequencing, a very time-consuming strategy.

F. Beleggia and B. Wollnik

35

Although these old gene identifi cation approaches could be still used for gene identifi cation studies, the advent of the next generation sequencing technology has dramatically simplifi ed disease gene identifi cation in Mendelian disorders [ 8 , 9 ] and we will describe the use of these powerful novel technologies for gene identifi cation in greater detail.

3 The Future Is Now

3.1 Next Generation Sequencing Technologies

Next generation sequencing (NGS) technologies include different strategies for the simultaneous sequencing of many DNA fragments in parallel. The sequences of all fragments are then aligned to a reference genome or joined together using their overlap to obtain a full-length sequence of the DNA of interest. NGS technologies can thus be used to generate the reference genome of a new species or to compare a DNA sample to an existing reference, identifying specifi c genetic traits as “differences” from the reference. The four main NGS technologies currently avail-able are: Illumina, Roche, IonTorrent and Solid [ 10 ].

In the Illumina sequencing protocol, the DNA to be sequenced is initially frag-mented and each fragment is bound to an adapter sequence which anneals to a complementary adapter primer fi xed onto the sequencing slide. Each fragment is then amplifi ed with a “bridge” technique, creating many identical copies around it, also fi xed to the sequencing slide. These “grooves” of identical DNA strands are then amplifi ed again with fl uorescent nucleotides of different colors, one base at a time. The sequencer recognizes each labeled nucleotide and all grooves are sequenced at the same time.

In the Roche 454 technology, the fragments are fi rst attached to a bead and amplifi ed, before the bead is inserted into a slot in a microtiterplate and sequenced. The 454 utilizes pyrosequencing, a system in which the addition of any nucleotide produces the same fl ash of light, but only one type of nucleotide at a time is added to the plate, so that the light indicates that the correct nucleotide was added.

The Ion Torrent system, from Life Technologies, is similar to the 454, but is based on electrochemical detection. The incorporation of a nucleotide causes the release of a proton, which is detected as a variation in pH.

The SOLiD chemistry is also based on bead amplifi cation, which is followed by “sequencing by ligation”, in which labeled dinucleotide probes are ligated to the fragment and read one by one.

The many small sequences derived from NGS runs, called “reads”, are initially aligned to the reference genome using various algorithms which do not require a perfect match [ 11 , 12 ]. This feature is necessary to align those reads which differ from the reference sequence due to the presence of a polymorphism or mutation. Increasing the tolerance of the alignment algorithms for mismatches results in a

Emerging Technologies for Gene Identifi cation in Rare Diseases

36

higher percentage of reads being successfully aligned and thus in a higher sensitivity, but it also allows more misalignments, which introduce errors. Most commonly, misalignments are due to the presence of pseudogenes, repetitive sequences or errors in the reference genome and are one of the major sources of false positive polymorphisms. Usually, only the initial part of each fragment is actually sequenced, as the risk of sequencing errors increases with the length of the read. Most modern platforms, however, allow the sequencing of both ends of each DNA molecule, creating two reads that are separated by a few hundred base pairs. Since both reads need to be aligned to the reference and their distance is known, this “paired-end” technique greatly diminishes the number of misalignments.

Once the alignment is complete, various tools can be used to improve the quality of the data. If the DNA was amplifi ed, it is possible to identify reads that start exactly at the same position with Picard [ 13 ] or samtools [ 14 ] and to mark them as PCR duplicates, which will be ignored in further analysis. This allows to correct for allele-selective amplifi cation and limits the impact of mutations introduced by the amplifi cation of the DNA.

Present-day alignment programs have diffi culties identifying insertions and deletions, especially when the read covers the indel at one of its ends. In this sce-nario, identifying as “mismatch” the few bases that are at the other side of the indel is usually simpler than actually looking for the exact breakpoints of the insertion or deletion. Two approaches can mitigate this problem, which potentially causes a huge number of false missense calls. The fi rst approach, with samtools, is the calcu-lation of a base-specifi c alignment score, which is low in the case of ambiguous alignments, when the program is unsure whether it should report an indel or a few mismatches. The second approach, with GATK [ 15 ], involves looking for all places where an indel is present and realign all reads that touch it, so that the reads that cover the deletion at their center can be used as a guide to correctly align the reads that cover it at one end.

To avoid false positive calls due to technical errors, it is possible to recalibrate the base quality scores of each read with GATK. The recalibration is based on the comparison between true variations, which are frequent in the general population, and all other variations, which might be false positives. For example, technical false positives occur frequently at the end of a read, but true variations found in a public database are evenly distributed in the reads; so it is possible to lower the quality of any mismatch found at the end of the read. Other variables that can be used in the same way are the specifi c sequences upstream or downstream of a base and the original quality scores.

The next step in the pipeline is the variant calling, which produces a “.vcf” (variant calling format) fi le. Different programs, such as GATK or samtools can be used for this purpose. A .vcf fi le contains information on every variation from the reference that was identifi ed in the patient’s DNA, including the position, how many reads report it and various quality scores which can be used by downstream programs to identify false positives.

The variant calling is followed by the annotation of each variation, in which additional information is searched for in the reference sequence and in additional

F. Beleggia and B. Wollnik

37

databases [ 16 , 17 ]. Common examples are the name of the gene containing the variation, whether or not and in what way it can change the protein sequence, the distance from the nearest exon/intron boundary and thus the probability that it will affect splicing. Once the annotation is complete, the data is ready to be analyzed by fi ltering scripts.

3.2 Whole-Exome Sequencing in Rare Diseases

The “exome” is defi ned as the sum of all exons in the genome. These include regions that are transcribed either into mRNAs or into ncRNAs and account for about 1 % of the genome. According to the data published in the Human Gene Mutation Database (HGMD) [ 18 ], 81 % of all reported disease-causing mutations are con-tained within the exome or in the intronic regions close to exons.

It is therefore theoretically possible to detect the vast majority of disease-causing mutations by sequencing the exons and a small part of the introns surrounding them. Compared to sequencing the genome, this “whole-exome sequencing” is extremely fast and inexpensive; it is routinely used in research for the identifi cation of genes involved in monogenic and complex diseases and it is slowly entering the clinical arena [ 8 , 19 ].

In the most common protocols, such as Illumina’s TruSeq, Roche’s NimbleGen and Agilent’s SureSelect, the genomic DNA is enriched by hybridization with spe-cifi c probes which only bind to target sequences present in the exome. The hybrid-ized molecules are then captured with magnetic streptavidin beads binding to the probes. An amplifi cation step can either precede or follow the enrichment and gen-erates a suffi cient quantity of DNA molecules for the sequencing. As little as 1 ug of DNA can be used for sequencing the entire exome.

3.3 How to Find the Needle in the Haystack

The average whole-exome sequencing run identifi es up to 50,000 loci where the exome of the patient differs from the reference. The biggest challenge in the identi-fi cation of disease-causing genes in Mendelian disorders lies in discriminating the one mutation among all the benign variations and polymorphisms. Different fi lter-ing strategies can be used to discard variations that are unlikely to be pathogenic, with the knowledge that the risk of fi ltering out the actual mutation increases at every step.

Common strategies include the use of publicly available databases of common polymorphisms, including dbSNP [ 20 ], the 1 000 genomes project [ 21 ] and the Exome Variant Server [ 22 ]. Per defi nition, a common polymorphism cannot be the cause of a rare genetic syndrome, but sometimes actual mutations end up in these databases. A “cutoff” approach which only fi lters out polymorphisms for which the frequency in

Emerging Technologies for Gene Identifi cation in Rare Diseases

38

the general population is known greatly decreases the risk of misfi ltering, at the cost of retaining many benign variations. These databases can be used to eliminate more than 90 % of the initial variations from the analysis [ 23 ].

An “in-house” database is extremely useful to fi lter out all reproducible technical errors that are often specifi c to both the sequencing technology and the bioinformat-ics pipeline. It can additionally be used to improve the previous database-based fi l-ters for the detection of common polymorphisms and can also reduce the number of possible mutations by more than 90 %.

The predicted effect of a variation can also be used as a fi lter to retain only variations affecting either the sequence of the protein or the splicing of its exons. The fi rst group includes all missense and nonsense substitutions, insertions and deletions within the exons, while the second group includes the fi rst 10–25 bases of the introns. In whole-exome sequencing, which already selects for protein-coding regions, such fi lters can be used to discard up to 50 % of all variations.

Taken together, these strategies usually yield a manageable number of less than 1,000 variations which need to be analyzed depending on the inheritance mode, family history, predicted severity of the protein change and gene function.

Recessive disorders are caused by homozygous or compound heterozygous variations, which, depending on parental consanguinity, typically account for 5–20 % of the fi ltered total and are thus much easier to identify than dominant muta-tions. Linkage analysis can be extremely powerful in combination with whole-exome sequencing, as concentrating on the variations present in linked regions often leaves only a few possible candidates [ 24 ]. In large families, it is advisable to sequence more patients and look for variations which are shared among them or to sequence healthy relatives to be used as controls to fi lter out “private” variations present in the family. An extreme case is the search for de novo mutations, which are not inherited but newly occur in the offspring. Using a “trio” strategy, in which all variations present in the exome of the parents are fi ltered out from the exome of the affected child, it is often possible to be left with only one or two variations [ 25 ].

In the case of consanguineous parents, it is possible to assume that the mutation is homozygous and has been inherited within a chromosome present in the common ancestor. Successive recombinations alter the ancestral chromosome in each parent, so that they will pass on to the offspring two chromosomes that are not completely homozygous but do present large stretches of homozygosity which are very likely to contain the causative mutation. It is possible to map such regions independently [ 26 ] or using data derived exclusively from whole-exome sequencing, simply by detect-ing areas in which all novel and known polymorphisms are homozygous [ 27 ]. Such mapping allows to discard all variations which are homozygous by chance rather than by common ancestry.

Nonsense, frameshift and canonical splicing variants (within two basepairs from the exon/intron boundary) are the best candidates for a loss-of-function mutation, since they completely alter the sequence of the protein. Variations farther from the exon are less and less likely to actually affect splicing, and missense variations can be completely benign or severely damaging. Different programs are available for predicting the severity of a splicing or missense variation [ 28 – 30 ], but numerous mutations have been described which are predicted to be benign even though they

F. Beleggia and B. Wollnik

39

actually cause a disease. Such prediction algorithms should therefore not be used as fi lters, but as prioritization tools.

The “candidate-gene” approach is based on information available on the gene harboring a variation, which can be very useful in deciding whether it is actually the disease-causing mutation. Expression in the right tissues, compatible function, the phenotype of knock-out animal models, conservation across species and the interac-tion with genes relevant to the pathogenesis are all clues to the involvement of a gene in the disorder. Protein-protein interactions with proteins that have already been described for the disorder are particularly useful, and internet databases, such as STRING [ 31 ], MINT [ 32 ] or I2D [ 33 ], offer this information proteome-wide.

3.4 Example 1: The Homozygosity Mapping Strategy

As an example of gene identifi cation in rare diseases, we performed whole-exome sequencing on a patient affected by Weaver syndrome. Weaver syndrome is an overgrowth syndrome, characterized by pre- and postnatal overgrowth, marked macrocephaly, facial dysmorphism including large ears and micrognathia, mental retardation and an increased incidence of tumors. The family comprised the consan-guineous parents and two affected children, suggesting autosomal recessive inheri-tance, even though the literature describes mainly sporadic and dominant cases [ 34 ].

The workfl ow included enrichment with Agilent SureSelect, sequencing on an Illumina Genome Analyzer, alignment with bwa [ 12 ], PCR duplicates marking with Picard [ 13 ], indel realignment, base-quality recalibration and variant calling with GATK [ 15 ], annotation with Annovar [ 16 ] and an in-house script for the calculation of homozygous regions using stretches of homozygous variations found in the WES data. This pipeline initially produced 44,599 variations. Only 4,298 were not pres-ent in our in-house database, and only 1,980 were also absent from public databases (dbSNP [ 20 ], 1000 genomes [ 21 ], EVS [ 22 ]). Put together, more than 95 % of the variations could be fi ltered out because of their frequency in the general population. Of the remaining variations, only 1,031 were predicted to possibly affect protein sequence, either by directly changing one or more amino acids or by infl uencing the splicing of an exon. Due to the consanguinity and under the assumption that the inheritance was autosomal recessive in this family, only the 67 homozygous varia-tions were considered and additionally fi ltered by the presence of a homozygous region, to arrive at a fi nal number of 16 variations.

Among them, the best candidate was a nonsense mutation in the ubiquitin ligase HACE1, a known tumor suppressor involved in the regulation of cell proliferation. The mutation was shown to segregate with the disease in the family and is predicted to truncate the second half of the protein, including the HECT domain, which is responsible for the ubiquitin ligase activity. Functional analysis revealed an interac-tion between HACE1 and the histone methyltransferase EZH2, a known dominant Weaver syndrome gene. In HACE1-defi cient cells, the half-life and total quantity of EZH2 are increased, suggesting that HACE1 causes Weaver syndrome through the upregulation of EZH2.

Emerging Technologies for Gene Identifi cation in Rare Diseases

40

3.5 Example 2: The Pooling Strategy

In the case of large families, with many affected individuals, it is possible to sequence all affected family members and look for shared variations. Compared to the simple one-exome strategy of our fi rst example, this method is much more powerful but also slower and very expensive. However, if the parents are consan-guineous and recessive inheritance is expected, a simpler strategy is to pool equal quantities of DNA from each patient and sequence the pooled DNA only once. In this scenario only the variations which are homozygous in all patients will be pres-ent in 100 % of the sequencing reads.

Recently, we used this strategy in a case of otofaciocervical syndrome (OFCS), a rare disorder characterized by facial anomalies, preauricular fi stulas, hearing loss, skeletal abnormalities and mild intellectual disability [ 35 ]. OFCS is part of a spec-trum of branchial-arch disorders, which also includes branchiootorenal syndrome (BORS), branchiootic syndrome (BOS) and branchiooculofacial syndrome (BOFS). The known disease-causing genes for these disorders form the so-called EYA-DACH- SIX-PAX pathway, involved in organ development.

Using the same sequencing and bioinformatic procedure as for example 1, we detected 45,609 variations. Only 1,059 were neither present in our in-house data-base, nor in dbSNP, in the EVS or in the 1000 genome project. Of these, 610 pos-sibly affected the sequence or splicing of a protein and only 10 were possibly homozygous. Due to the pooling strategy, however, only one of the variations was present in truly 100 % of the sequencing reads. The single remaining variation was a missense in the highly conserved DNA-binding “paired box” domain of PAX1, which impairs the transcriptional activity of PAX1. The fi nding of a PAX1 mutation as the cause of OFCS adds PAX1 to the EYA-DACH-SIX-PAX pathway and strength-ens the genetic link between branchial arch spectrum disorders.

3.6 Whole-Genome Sequencing in Rare Diseases

While whole-exome sequencing represents the most cost-effective solution, some of its limitations can be overcome by whole-genome sequencing. The obvious advantage of the latter is that it might identify mutations outside of the exome, including intronic and regulatory regions. Additionally, it can be used to detect structural changes, such as large deletions, insertions, duplications and transloca-tions, which account for about 10 % of all reported mutations [ 18 ]. This is achieved by looking at split reads, which cover the breakpoints of these rearrangements and are usually not aligned in standard bioinformatics pipelines. In the case of a large deletion, for example, the beginning and end of such reads show the sequence upstream and downstream of the missing region, while for a translocation they show sequences from different chromosomes [ 36 ]. In paired-end sequencing, the distance between the paired reads, or their mapping to different chromosomes, can also be used to detect structural variations [ 37 , 38 ].

F. Beleggia and B. Wollnik

41

4 Future Applications

4.1 Exome/Genome Sequencing for Everybody?

The described new applications of NGS technologies represent a major breakthrough for gene identifi cation studies in rare diseases but can also be applied for effi cient molecular diagnostic purposes. The so-called “panel diagnostics” is frequently applied in heterogeneous disorders (e.g. testing of all HCM-associated genes in a patient with a familial form of hypertrophic cardiomyopathy), but it can only be used in cases where a specifi c clinical diagnosis exists. In a large number of patients, however, e.g. with a congenital syndrome, a clinical diagnosis cannot be estab-lished in the neonatal period or in early childhood. We are strongly convinced that in these cases, diagnostic whole-exome sequencing will be the method of choice in the near future. Some of the larger genomics centers worldwide have already initi-ated programs for neonatal applications of exome sequencing [ 39 , 19 ]. Preliminary results show that in patients with unsolved clinical diagnosis, a molecular diagnosis can be established in 20–25 % of the cases in a routine setting [ 19 ]. We believe that these numbers can be further increased if a scientifi c workup of WES data is per-formed whenever the initial routine analysis does not come up with a convincing mutation(s). In our experience, gene identifi cation studies using WES in a research context lead to the identifi cation of the causative mutations (mainly novel genes) within a couple of weeks of data analysis in approximately 25 % of the cases, and within 1–3 years for another 50 %. The main key to the “late success” is substantial re-analysis, re-fi ltering, re-evaluation, and testing of various unusual variants and patterns of inheritance.

Even though we currently prefer the use of WES for gene identifi cation, some researchers have already started to apply whole-genome sequencing (WGS) [ 40 , 41 ]. Since most of the causative mutations in individuals with rare diseases are located within the coding regions of the genes or affect positions very close to the splice sites, WES application seems to have – at the moment – an advantage over WGS, because it goes with a better coverage of the target regions and produces a smaller number of total variants. Nevertheless, mutations could be located within large intronic sequences, promoter regions, long-range regulatory elements, or might be present as structural variation within or outside the genes. To detect these types of changes, WGS could be applied as a second step, if WES cannot fi nd the causative mutations. Moreover, further technological developments and decreasing costs might give rise to a shift towards WGS in rare disease gene identifi cation studies.

It is important to note that in our view, current applications of WES/WGS should have a clear and restricted clinical indication and should not be applied to every-body! The authors believe that they should be used for the identifi cation of the genetic causes of clinically relevant phenotypes and symptoms and that they should not be applied without a specifi c medical indication.

Emerging Technologies for Gene Identifi cation in Rare Diseases

42

4.2 All Genes Identifi ed, What’s Next?

Interestingly, little information is available about unsuccessful WES-based gene identifi cation studies in monogenic diseases. Surely, even the best strategy does not necessarily lead to success as demonstrated by an estimated success rate of about 60 % [ 23 ]. Still, successful application of NGS-based gene identifi cation studies will continuously improve thanks to advancements in both technologies and bioinformatics strategies and will lead to the identifi cation of the associated genes for almost all described 7,000 rare disorders in the next couple of years. The question remains how the fi eld of molecular and human genetics will move forward after all these genes have been identifi ed? We have no doubt that many new questions will arise concerning the genetics of rare diseases, opening new areas of genetic investigations such as: What are the genetic modifi ers of pheno-typic variability in monogenic diseases? How frequent are digenic and poly-genic inheritance in rare disorders? What is the impact of epigenetic variation on phenotypes? What is the exact role of somatic mosaicism? All these ques-tions will be addressed and we expect novel fascinating fi ndings to underline the complexity of genetic inheritance even in monogenic and rare disorders. Moreover, we will spend much more time elucidating the molecular pathogen-esis of rare diseases using detailed functional studies in vitro and in vivo. It will not be surprising if these functional studies will point towards only a few fi nal common pathways altered in many different disease entities and overlapping disease spectra.

Will the elucidation of the molecular pathogenesis of all these rare disease lead to the design of novel therapeutic strategies? This question is not easy to answer and a translational link towards improved therapies might be hard to establish, even with the knowledge about the underlying pathogenesis. Nevertheless, clues as to advanced therapies might come up for selected disorders once the disease-causing mechanisms are known. An interesting example is our recent identifi ca-tion of autosomal recessive mutations in WNT1 in patients with osteogenesis imperfecta [ 42 ]. In that study, we identifi ed homozygous mutations in fi ve families with autosomal recessive osteogenesis imperfect (OI). It is interesting to note that bisphosphonate treatment, which is the common therapy in OI patients, did not ameliorate the bone phenotype in these patients. Moreover, we found an additional causative heterozygous mutation in WNT1 in a family with an autosomal domi-nantly inherited form of early-onset osteoporosis. Functional analysis investigat-ing the consequences of WNT1 mutations at the protein level indicated a reduced potential to activate the LRP5-mediated canonical WNT signaling cascade in an in vitro system. Therefore, as a potential therapeutic option for WNT1-related bone fragility, we proposed the use of specifi c drugs enhancing WNT signaling, such as antibodies neutralizing the WNT antagonist sclerostin, which is currently under investigation in clinical trials [ 43 ].

F. Beleggia and B. Wollnik

43

References

1. Joppi R, Bertele’ V, Garattini S (2013) Orphan drugs, orphan diseases. The fi rst decade of orphan drug legislation in the EU. Eur J Clin Pharmacol 69(4):1009–1024

2. Melnikova I (2012) Rare diseases and orphan drugs. Nat Rev Drug Discov 11(4):267–268 3. Wästfelt M, Fadeel B, Henter JI (2006) A journey of hope: lessons learned from studies on rare

diseases and orphan drugs. J Intern Med 260(1):1–10 4. Tambuyzer E (2010) Rare diseases, orphan drugs and their regulation: questions and miscon-

ceptions. Nat Rev Drug Discov 9(12):921–929 5. Schieppati A, Henter JI, Daina E, Aperia A (2008) Why rare diseases are an important medical

and social issue. Lancet 371(9629):2039–2041 6. Collins FS (1995) Positional cloning moves from perditional to traditional. Nat Genet

9(4):347–350 7. Collins FS (1992) Positional cloning: let’s not call it reverse anymore. Nat Genet 1(1):3–6 8. Bamshad MJ, Ng SB, Bigham AW, Tabor HK, Emond MJ, Nickerson DA, Shendure J (2011)

Exome sequencing as a tool for Mendelian disease gene discovery. Nat Rev Genet 12(11):745–755

9. Rabbani B, Mahdieh N, Hosomichi K, Nakaoka H, Inoue I (2012) Next-generation sequenc-ing: impact of exome sequencing in characterizing Mendelian disorders. J Hum Genet 57(10):621–632

10. Liu L, Li Y, Li S, Hu N, He Y, Pong R, Lin D, Lu L, Law M (2012) Comparison of next- generation sequencing systems. J Biomed Biotechnol 2012:251364

11. Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-effi cient align-ment of short DNA sequences to the human genome. Genome Biol 10(3):R25

12. Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows-Wheeler trans-form. Bioinformatics 25(14):1754–1760

13. Homer N, Tibbetts K, Wysoker A, Fennell T, McCowan M (2009) Picard. http://picard.source-forge.net . Accessed 25 June 2014

14. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, 1000 Genome Project Data Processing Subgroup (2009) The sequence alignment/map format and SAMtools. Bioinformatics 25(16):2078–2079

15. DePristo MA, Banks E, Poplin R, Garimella KV, Maguire JR, Hartl C, Philippakis AA, del Angel G, Rivas MA, Hanna M, McKenna A, Fennell TJ, Kernytsky AM, Sivachenko AY, Cibulskis K, Gabriel SB, Altshuler D, Daly MJ (2011) A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet 43(5):491–498

16. Wang K, Li M, Hakonarson H (2010) ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res 38(16):e164

17. Cingolani P, Platts A, Wang le L, Coon M, Nguyen T, Wang L, Land SJ, Lu X, Ruden DM (2012) A program for annotating and predicting the effects of single nucleotide polymor-phisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly (Austin) 6(2):80–92

18. Stenson PD, Mort M, Ball EV, Shaw K, Phillips A, Cooper DN (2014) The human gene muta-tion database: building a comprehensive mutation repository for clinical and molecular genet-ics, diagnostic testing and personalized genomic medicine. Hum Genet 133(1):1–9

19. Yang Y, Muzny DM, Reid JG, Bainbridge MN, Willis A, Ward PA, Braxton A, Beuten J, Xia F, Niu Z, Hardison M, Person R, Bekheirnia MR, Leduc MS, Kirby A, Pham P, Scull J, Wang M, Ding Y, Plon SE, Lupski JR, Beaudet al, Gibbs RA, Eng CM (2013) Clinical whole-exome sequencing for the diagnosis of Mendelian disorders. N Engl J Med 369(16):1502–1511

20. Sherry ST, Ward MH, Kholodov M, Baker J, Phan L, Smigielski EM, Sirotkin K (2001) dbSNP: the NCBI database of genetic variation. Nucleic Acids Res 29(1):308–311

21. Abecasis GR, Auton A, Brooks LD, DePristo MA, Durbin RM, Handsaker RE, Kang HM, Marth GT, McVean GA (2012) An integrated map of genetic variation from 1,092 human genomes. Nature 491(7422):56–65

Emerging Technologies for Gene Identifi cation in Rare Diseases

44

22. NHLBI GO Exome Sequencing Project (2011) Exome variant server. http://evs.gs.washington.edu/EVS . Accessed 25 June 2014

23. Gilissen C, Hoischen A, Brunner HG, Veltman JA (2012) Disease gene identifi cation strategies for exome sequencing. Eur J Hum Genet 20(5):490–497

24. Schreml J, Durmaz B, Cogulu O, Keupp K, Beleggia F, Pohl E, Milz E, Coker M, Ucar SK, Nürnberg G, Nürnberg P, Kuhn J, Ozkinay F (2014) The missing “link”: an autosomal reces-sive short stature syndrome caused by a hypofunctional XYLT1 mutation. Hum Genet 133(1):29–39

25. Vissers LE, de Ligt J, Gilissen C, Janssen I, Steehouwer M, de Vries P, van Lier B, Arts P, Wieskamp N, del Rosario M, van Bon BW, Hoischen A, de Vries BB, Brunner HG, Veltman JA (2010) A de novo paradigm for mental retardation. Nat Genet 42(12):1109–1112

26. Walsh T, Shahin H, Elkan-Miller T, Lee MK, Thornton AM, Roeb W, Abu Rayyan A, Loulus S, Avraham KB, King MC, Kanaan M (2010) Whole exome sequencing and homozygosity mapping identify mutation in the cell polarity protein GPSM2 as the cause of nonsyndromic hearing loss DFNB82. Am J Hum Genet 87(1):90–94

27. Becker J, Semler O, Gilissen C, Li Y, Bolz HJ, Giunta C, Bergmann C, Rohrbach M, Koerber F, Zimmermann K, de Vries P, Wirth B, Schoenau E, Wollnik B, Veltman JA, Hoischen A, Netzer C (2011) Exome sequencing identifi es truncating mutations in human SERPINF1 in autosomal-recessive osteogenesis imperfecta. Am J Hum Genet 88(3):362–371

28. Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, Kondrashov AS, Sunyaev SR (2010) A method and server for predicting damaging missense mutations. Nat Methods 7(4):248–249

29. Schwarz JM, Rödelsperger C, Schuelke M, Seelow D (2010) MutationTaster evaluates dis-ease-causing potential of sequence alterations. Nat Methods 7(8):575–576

30. Desmet FO, Hamroun D, Lalande M, Collod-Beroud G, Claustres M, Beroud C (2009) Human Splicing Finder: an online bioinformatics tool to predict splicing signals. Nucleic Acids Res 37(9):e67

31. Jensen LJ, Kuhn M, Stark M, Chaffron S, Creevey C, Muller J, Doerks T, Julien P, Roth A, Simonovic M, Bork P, von Mering C (2009) STRING 8-a global view on proteins and their functional interactions in 630 organisms. Nucleic Acids Res 37(Database issue):D412–D416

32. Licata L, Briganti L, Peluso D, Perfetto L, Iannuccelli M, Galeota E, Sacco F, Palma A, Nardozza AP, Santonico E, Castagnoli L, Cesareni G (2012) MINT, the molecular interaction database: 2012 update. Nucleic Acids Res 40(Database issue):D857–D861

33. Brown KR, Jurisica I (2005) Online predicted human interaction database. Bioinformatics 21(9):2076–2082

34. Opitz JM, Weaver DW, Reynolds JF Jr (1998) The syndromes of Sotos and Weaver: reports and review. Am J Med Genet 79(4):294–304

35. Pohl E, Aykut A, Beleggia F, Karaca E, Durmaz B, Keupp K, Arslan E, Onay MP, Yigit G, Ozkinay F, Wollnik B (2013) A hypofunctional PAX1 mutation causes autosomal recessively inherited otofaciocervical syndrome. Hum Genet 132(11):1311–1320

36. Ye K, Schulz MH, Long Q, Apweiler R, Ning Z (2009) Pindel: a pattern growth approach to detect break points of large deletions and medium sized insertions from paired-end short reads. Bioinformatics 25(21):2865–2871

37. Hormozdiari F, Hajirasouliha I, Dao P, Hach F, Yorukoglu D, Alkan C, Eichler EE, Sahinalp SC (2010) Next-generation VariationHunter: combinatorial algorithms for transposon inser-tion discovery. Bioinformatics 26(12):350–357

38. Chen K, Wallis JW, McLellan MD, Larson DE, Kalicki JM, Pohl CS, McGrath SD, Wendl MC, Zhang Q, Locke DP, Shi X, Fulton RS, Ley TJ, Wilson RK, Ding L, Mardis ER (2009) BreakDancer: an algorithm for high-resolution mapping of genomic structural variation. Nat Methods 6(9):677–681

39. Neveling K, Feenstra I, Gilissen C, Hoefsloot LH, Kamsteeg EJ, Mensenkamp AR, Rodenburg RJ, Yntema HG, Spruijt L, Vermeer S, Rinne T, van Gassen KL, Bodmer D, Lugtenberg D, de Reuver R, Buijsman W, Derks RC, Wieskamp N, van den Heuvel B, Ligtenberg MJ, Kremer

F. Beleggia and B. Wollnik

45

H, Koolen DA, van de Warrenburg BP, Cremers FP, Marcelis CL, Smeitink JA, Wortmann SB, van Zelst-Stams WA, Veltman JA, Brunner HG, Scheffer H, Nelen MR (2013) A post-hoc comparison of the utility of Sanger sequencing and exome sequencing for the diagnosis of heterogeneous diseases. Hum Mutat 34(12):1721–1726

40. Veeramah KR, O’Brien JE, Meisler MH, Cheng X, Dib-Hajj SD, Waxman SG, Talwar D, Girirajan S, Eichler EE, Restifo LL, Erickson RP, Hammer MF (2012) De novo pathogenic SCN8A mutation identifi ed by whole-genome sequencing of a family quartet affected by infantile epileptic encephalopathy and SUDEP. Am J Hum Genet 90(3):502–510

41. Jiang YH, Yuen RK, Jin X, Wang M, Chen N, Wu X, Ju J, Mei J, Shi Y, He M, Wang G, Liang J, Wang Z, Cao D, Carter MT, Chrysler C, Drmic IE, Howe JL, Lau L, Marshall CR, Merico D, Nalpathamkalam T, Thiruvahindrapuram B, Thompson A, Uddin M, Walker S, Luo J, Anagnostou E, Zwaigenbaum L, Ring RH, Wang J, Lajonchere C, Wang J, Shih A, Szatmari P, Yang H, Dawson G, Li Y, Scherer SW (2013) Detection of clinically relevant genetic variants in autism spectrum disorder by whole-genome sequencing. Am J Hum Genet 93(2):249–263

42. Keupp K, Beleggia F, Kayserili H, Barnes AM, Steiner M, Semler O, Fischer B, Yigit G, Janda CY, Becker J, Breer S, Altunoglu U, Grünhagen J, Krawitz P, Hecht J, Schinke T, Makareeva E, Lausch E, Cankaya T, Caparrós-Martín JA, Lapunzina P, Temtamy S, Aglan M, Zabel B, Eysel P, Koerber F, Leikin S, Garcia KC, Netzer C, Schönau E, Ruiz-Perez VL, Mundlos S, Amling M, Kornak U, Marini J, Wollnik B (2013) Mutations in WNT1 cause different forms of bone fragility. Am J Hum Genet 92(4):565–574

43. Rachner TD, Khosla S, Hofbauer LC (2011) Osteoporosis: now and the future. Lancet 377(9773):1276–1287

Emerging Technologies for Gene Identifi cation in Rare Diseases

47M. Özgüç (ed.), Rare Diseases: Integrative PPPM Approach as the Medicine of the Future, Advances in Predictive, Preventive and Personalised Medicine 6,DOI 10.1007/978-94-017-9214-1_4, © Springer Science+Business Media Dordrecht 2015

Abstract Hearing loss is the most common sensory disorder around the world. Genetic factors account for at least 50 % of congenital or prelingual onset deafness. While the clinical phenotype, deafness, is shared and similar across different individuals, the substantial genetic heterogeneity makes the genetic etiology in individual cases rare. Identifi cation of genetic causes has been central to a growing body of knowledge related to diagnostic tools, genetic testing and clinical categori-zation systems. We will provide an overview of the current state of knowledge and future directions that are guiding the concept of personalized medicine for the deaf individuals.

Keywords Comprehensive genetic testing • Deafness • Genetic heterogeneity • GJB2 • Hearing loss • Personalized medicine

1 Introduction

In developed countries, approximately 1/500 newborns present with bilateral, mod-erate-profound hearing loss [ 1 ]. More than 50 % of congenital deafness is attributed to genetic causes. However, despite the substantial genetic contribution to hearing loss, the majority of the genetic etiologies of individual cases remain rare.

The genetic heterogeneity of hearing loss has been central to the paradox between deafness as a common, phenotypically to some extent homogeneous condition, and the rarity of its single gene causes. To date, more than 100 loci/genes have been

Personalized Medicine for Hereditary Deafness

Jessica Ordóñez , Oscar Diaz-Horta , and Mustafa Tekin

J. Ordóñez • O. Diaz-Horta • M. Tekin (*) Dr. John T. Macdonald Foundation Department of Human Genetics and John P. Hussman Institute for Human Genomics , University of Miami Miller School of Medicine , 1501 NW 10th Avenue, BRB-610 (M-860) , Miami , FL 33136 , USA e-mail: mtekin@med.miami.edu

48

associated with deafness. All 22 autosomes and both sex chromosomes have been reported to contain deafness causing mutations in single genes [ 2 ]. Both, recessive and dominant variants have been described. Two modifi ers have also been reported so far. Several mutations in mitochondrial genes can also cause both syndromic and non-syndromic deafness.

The evolution of technological and diagnostic tools has built an understanding of the genetic heterogeneity of deafness that is now translational to clinic. Reliable audiometry and physiologic hearing testing have led to universal hearing screening in the United States and in many developed countries. Emerging genotype- phenotype correlations are guiding genetic counseling. With technological advances, the medical practitioner is becoming able to provide cost-effective, multi-gene testing in the clinic. The unprecedented progress over the last 15 years has paved the way for the concept of personalized medicine for the deaf individuals. We will provide a snapshot of the technological and clinical progress in hearing loss genet-ics that has led to a comprehensive medical evaluation of deafness.

2 The Evaluation for Genetic Causes of Hearing Loss in 2013

2.1 Diagnosis of Hearing Loss Today

Most cases of hearing loss in most developed countries are diagnosed neonatally; this has occurred via technological advancements in audiology and public health campaigns. Although there was awareness about the importance of a neonatal diag-nosis of hearing loss since the 1960s [ 3 ] and hospitals engaged in individual efforts for neonatal screening, it was not until the 1990s when consensus statements sup-ported neonatal hearing screen for all newborns [ 4 ]. As of April, 2013, 35/51 states in the U.S. have legislation or rule requiring universal hearing screening. Screening is universally offered but not yet required in ten states and offered to select popula-tions or by request in six states [ 5 ]. The recommendations for universal hearing screen are evidence-based, recognizing that an early diagnosis and intervention for hearing loss can signifi cantly advance speech development in those who are deaf or hard of hearing [ 6 , 7 ].

The implementation of highly sensitive automated hearing screening has facilitated the success of universal screening programs in the U.S. Automated Brain Stem Response (ABR) and Evoked Otoacoustic Emissions (EOAEs) have been used since the 1990s for newborn hearing screening. Using clicks as stimuli, ABR measures the response of the auditory brain stem to sound. In contrast, EOAEs measures the vibration produced by the cochlear outer hair cells in response to sound. Both testing techniques are physiologic, meaning that they assess the physi-cal response of different parts of the auditory system to sound. When newborns are

J. Ordóñez et al.

49

screened by either of these technologies, they can “pass” the screen, which signifi cantly lowers the probability of hearing loss in the child. Alternatively, new-borns can get a “refer” result, which grants audiology referral and further diagnostic work-up. A “refer” result is generally followed by one or more of types of audiom-etry testing, which elicit the child’s processing of auditory information through behavioral responses.

2.2 Hearing Loss Clinical Categorization

Hearing loss today has several classifi cation systems that refl ect the diagnostic, genetic and technological progress in the fi eld. Age of onset, type and severity of hearing loss and the presence of systemic fi ndings are important categories for clinical evaluation. According to age of onset, hearing loss can be classifi ed as pre- lingual, or present before speech development. Hearing loss can also be post-lingual, or present after speech-development. According to type, hearing loss can be conductive (defects in the external and middle ear), sensorineural (malfunction of the cochlea or the inner ear), mixed (a combination of conductive and sensorineural hearing loss) or central auditory dysfunction (dysfunction of the auditory nerve, auditory brainstem of cerebral cortex). According to severity based on the pure tone average of the hearing thresholds between 0.5 and 4 KHz, hearing loss can be mild (20–40 dB HL), moderate (41–70 dB HL), severe (71–95 dB HL) and profound (>95 dB HL) [ 8 ].

The presence or absence of systemic fi ndings distinguishes syndromic from non- syndromic hearing loss. Approximately 70 % of prelingual deafness is non- syndromic (Fig. 1 ). Non-syndromic and syndromic deafness are both caused by mutations in a large number, and sometimes overlapping, genes.

3 Non-syndromic Hearing Loss

No additional clinical or laboratory fi ndings are seen in non-syndromic hearing loss. Approximately three-fourths of non-syndromic hearing loss follows autosomal recessive inheritance while the remaining one fourth is autosomal dominant or X-linked.

Connexins are transmembrane proteins involved in cochlear homeostasis with an important role in deafness, particularly the autosomal recessive non-syndromic type. The discovery of Connexin 26, encoded by GJB2 , in 1997 was a landmark in the genetics of deafness [ 19 ]. In contrast to all other types of deafness, signifi cant proportion of autosomal recessive non-syndromic hearing loss in some populations is explained by mutations in GJB 2. In individuals of European descent, the carrier rate for one mutation, c.35delG, approximates 1/50 [ 20 , 21 ]. The c.167delT mutation is the most common variant among Ashkenazi Jews [ 22 ] and the c.235delC is the

Personalized Medicine for Hereditary Deafness

50

most common mutation among East Asians [ 23 , 24 ]. GJB2 genotyping has also guided clinical predictions of deafness severity. Homozygous status for inactivating mutations in GJB2 is frequently associated with severe-to-profound hearing loss. A homozygous status for non-inactivating mutations is associated with mild hearing loss in more than 50 % of patients [ 25 ]. In contrast, deafness severity can be quite varied in those who are compound heterozygous for a non-inactivating and an acti-vating mutation [ 25 ].

Connexin 30, encoded by GJB6 , also plays an important role in deafness. Two large deletions (∆ GJB6 -D13S1830 and ∆ GJB6 -D13S1854) account for the majority of GJB6 -associated deafness [ 26 , 27 ]. Approximately 2 % of individuals with a point mutation in GJB2 will have a common deletion in GJB6 [ 27 ]. In summary, GJB2 and GJB6 are the most common genes implicated in autosomal recessive non- syndromic hearing loss and their associated allelic heterogeneity has been suc-cessfully utilized for genotype-phenotype correlations that are guiding counseling. However, to date, multiple mutations in over 40 other genes have been associated with autosomal recessive non-syndromic hearing loss [ 28 ], which leaves a signifi -cant body of knowledge yet untapped for future translational applications.

>400 syndromes19%

GJB214%

MYO�5A5%

Unknown non-syndromicdeafness genes

20%

>60 recognized non-syndromic deafness genes

25%

TMC�,TMPRSS�,OTOF,MYO�A,PCDH��,TECTA

6%

Pendredand EVA

5%

Ushersyndrome

6%

Fig. 1 Distribution of genetic causes in congenital/prelingual-onset hearing loss. Frequencies were compiled from [ 9 – 18 ] and our unpublished data. EVA enlarged vestibular aqueduct

J. Ordóñez et al.

51

Non-syndromic hearing loss can also be mitochondrial in nature. Mutations in the MT-TS1 and MT-RNR1 are associated with hearing loss of maternal transmission associated with interesting environmental contributions. The MT-RNR1 , m.1555G > A mutation is the most common cause of non-syndromic mitochondrial hearing loss. This mutation occurs as a homoplasmic variant associated with bilateral, severe-profound hearing loss in some cases exposed to aminoglycosidase ototoxicity [ 29 ]. Penetrance and age of onset can be variable and delayed in those individuals with the m.1555G > A mutation and no aminoglycoside exposure [ 30 , 31 ]. In contrast, muta-tions in the MT-TS1 gene are heteroplasmic variants not associated with aminogly-coside otoxicity. Individuals with MT-TS1 mutations can have hearing loss of variable severity and age of onset with characteristic progression. While mutations in MT-TS1 and MT-RNR1 are typically associated with non- syndromic, mitochon-drial hearing loss, the m.7445A > G mutation in MT-TS1 has been seen in individuals with palmoplantar keratoderma [ 32 ].

X-linked hearing loss can be mixed (conductive and sensorineural) in some cases [ 33 ]. Mutations in PRPS1 , POU3F4 and SMPX are associated with X-linked non- syndromic hearing loss [ 2 ]. Mutations in POU3F4 cause sensorineural hearing loss with a conductive component due to stapedial fi xation. Age of onset and hear-ing loss severity show wide variation, although progression is virtually always seen.

In contrast to autosomal recessive non-syndromic deafness, no single gene accounts for a large part of autosomal dominant non-syndromic deafness [ 34 ]. The tremendous genetic heterogeneity of autosomal dominant non-syndromic hearing loss is a signifi cant feature of this hearing loss type. There are 65 mapped loci for autosomal dominant deafness with genes identifi ed for only 25 [ 2 ]. However, among the identifi ed genes, mutations may be more common among TECTA , WFS1 , KCNQ4 , COCH , and GJB2 than in other mapped genes. Non-syndromic autosomal dominant hearing loss tends to be progressive; it is also generally post-lingual and affects the high frequencies, although some exceptions exist partially according to gene involved.

Another signifi cant feature of autosomal dominant non-syndromic hearing loss is the existing genotype-phenotype correlations. “Cookie-bite” shaped audiograms are generally associated with TECTA mutations although specifi c audiogram profi les and rate of progression will depend on the location of the mutation in the protein domain [ 35 , 36 ]. Low-frequency hearing loss is associated with WFS1 mutations and characteristic rate of progression in the high frequencies is common in audio-grams of individuals with COCH mutations [ 36 , 37 ]. Furthermore, such correlations have been harvested to create computational algorithms with clinical use. AudioGene is a publically available prediction tool for autosomal dominant non- syndromic genes. A patient’s audiogram is analyzed through a computational clustering system for defi ned autosomal dominant genes to provide the three most likely genes that may bear a mutation. The prediction accuracy has been estimated at 70 % [ 34 ]. Overall, autosomal dominant non-syndromic hearing loss is an outstanding example of the juncture of audiology, computational science and genetics to create transla-tional tools able to grasp genetic heterogeneity.

Personalized Medicine for Hereditary Deafness

52

4 Syndromic Hearing Loss

Syndromic hearing loss is associated with additional fi ndings, often involving ophthalmological, renal, vertebral, and endocrine systems. More than 400 genetic syndromes associated with hearing loss have been described [ 38 ]. Syndromic hear-ing loss accounts for approximately 30 % of prelingual hearing loss although it has an overall smaller contribution to generalized deafness.

The most common autosomal recessive syndromic form of deafness is Usher syndrome, characterized by the association of hearing loss with retinitis pigmentosa and mutations in ten described genes including MYO7A as the most common [ 2 ]. There are three recognized types of Usher syndrome distinguished by age of onset and presence or absence of vestibular fi ndings. Other common autosomal recessive forms of deafness include Pendred syndrome, associated with enlarged vestibular aqueducts and goiter. Jervell and Lange-Nielsen syndrome is also an autosomal recessive disorder where congenital deafness is associated with prolonged QT interval on EKGs. Affected individuals may be at risk for syncope and sudden cardiac death. Mutations in the KCNQ1 or KCNE1 have been implicated in the majority of cases.

Waardenburg syndrome constitutes a relatively common type of autosomal dominant syndromic deafness. The characteristic features of this group of condi-tions are pigmentary anomalies in the skin, hair and eyes (skin hypopigmentation, white forelock and heterochromia iridum, respectively) with hearing loss of variable degree. There are four major types of Waardenburg syndrome distinguished by the presence of additional features including dystopia canthorum, limb anomalies and Hirschsprung disease. Mutations in seven different genes have been found to be causative.

Branchio-oto-renal syndrome (BOR) is another relatively common cause of autosomal dominant syndromic hearing loss; it is caused by mutations in EYA1 , SIX1/5 , and other yet unidentifi ed genes. The characteristic features are branchial clefts and fi stulae, renal anomalies and external ear malformations. Hearing loss can be conductive, sensorineural or mixed of variable degree. Other more rare syn-dromic causes of hearing loss include Neurofi bromatosis type 2 characterized by bilateral vestibular schwannomas and Stickler syndrome, where hearing loss is accompanied by cleft palate, osteoarthritis and severe myopia. Overall, syndromic hearing loss cases are rare and exhibit markedly varied clinical signs, inheritance patterns and genes involved.

4.1 The Clinical Genetics Evaluation Today

The current clinical evaluation of hearing loss refl ects the multiple hearing loss clinical classifi cation systems. Professional societies and advocacy groups including the American Academy of Pediatrics, the American College of Medical Genetics,

J. Ordóñez et al.

53

the American Academy of Audiology, the Joint Committee on Infant Hearing and the U.S. Preventive Services Taskforce have issued professional statements guiding the role of different specialties in the diagnosis, medical management and follow-up of hearing loss [ 39 – 42 ].

A multidisciplinary setting combining audiology, ENT, genetics, and social work services is the ideal setting for the medical evaluation of the newly diagnosed patient with congenital hearing loss. The clinical genetics evaluation plays a central role in the etiology, risk assessment, prognosis, and management recommendations for the deaf patient. The initial clinical genetics visit for a new diagnosis of congenital deafness typically encompasses the following:

• Medical history : There is close attention to environmental risk factors for hearing loss such as prenatal CMV or other teratogenic exposures, ototoxicity, and prenatal ultrasound fi ndings.

• Family history : A three to four generation pedigree is obtained with attention to hearing loss inheritance patterns as well as hearing loss type, progression, age of onset, and syndromic vs. non-syndromic presentation in other affected relatives. Hearing status of fi rst degree relatives, consanguinity, ethnicity and country of origin are also assessed.

• Physical examination : There is focus on identifying characteristics that may suggest syndromic deafness such as external ear anomalies, pigmentation changes, craniofacial and skeletal fi ndings and dysmorphic features.

• Systemic surveillance : This usually entails EKG and ophthalmological evalua-tion, kidney ultrasound, and imaging studies to assess for inner ear anomalies. If not already in place, the patient is referred to early intervention programs to assess developmental status and need for therapies and school accommodations.

• Genetic counseling and genetic testing : The clinical classifi cation systems for hearing loss are reviewed. The family is educated about suspected inheritance patterns and recurrence risks given the history. A single-gene vs. multi-gene approach to genetic testing is evaluated. The family is engaged in discussion about possible outcomes of genetic testing including positive, negative results and variants of uncertain signifi cance. Potential implications of genetic test results on medical management and risk assessment are addressed and a follow- up plan for results disclosure is established.

Genetic testing and completion of systemic surveillance recommendations usually follow the initial clinical evaluation. Genetic testing may lead to the identi-fi cation of positive results, or a causative mutation. If such is the case, information related to inheritance patterns, genotype-phenotype correlations and the syndromic vs. non-syndromic nature of hearing loss is reviewed with the family and the patient is referred to other specialists as needed. Hearing loss management strategies are reviewed with the family, including availability of cochlear implantation, amplifi ca-tion systems, hearing aids, sign language and assistive communication technology. A signifi cant portion of the session is also dedicated to address recurrence risks, risk assessment for family members, and follow up genetics plan.

Personalized Medicine for Hereditary Deafness

54

Genetic testing may also identify one or several variants of uncertain (VUS). Variants may be suspected benign polymorphisms, true variants or suspected delete-rious mutations. VUS identifi cation usually leads to extensive discussions about molecular and clinical evidence that may support interpretation of test results. Information about recurrence risks as well as management and follow-up recom-mendations is reviewed based on existing personal, family history and VUS litera-ture. The VUS result is usually followed by a recommendation for parental testing to determine the de novo vs. inherited nature of the variant(s) as well as cis vs. trans confi guration if the case is autosomal recessive.

Genetic testing may also reveal negative results or no underlying etiology. If such is the case, it is important to review genetic disorders that had been previ-ously excluded as well as the residual risk for syndromic deafness and the need for continued follow up. There is discussion about empirical recurrence risk fi gures and family history-based recurrence risk assessment. These types of cases are usually addressed with a customized, multidisciplinary follow-up plan. Regardless of genetic test results, the family is referred to local deafness advocacy groups and national support foundations for further resources and psychosocial assistance that may be necessary.

5 The Evolution of the Genetic Evaluation of Hearing Loss

The evolution of technological tools has gone at par with the clinical evaluation approach in the hearing loss clinic. Gene mutations causing deafness were initially documented in the 1990s paving the way for genetic testing. Linkage analysis using microsatellite markers in affected families was the fi rst tool identifying new loci and genes. Mutations in GJB2 were the fi rst identifi ed genetic association with autosomal recessive non-syndromic hearing loss [ 19 ]. Subsequently, GJB2 was shown to be the most common cause of isolated deafness in different populations [ 14 , 43 , 44 ]. As new deafness genes were identifi ed, sequential gene screening was implemented for genetic testing. Although it remains common to pursue a single-gene, sequential testing approach in the clinic, this is costly, time-intensive and possibly emotionally taxing for the patient and the family. Furthermore, the now known genetic heterogeneity of hearing loss has limited the genetic diagnostic yield of this strategy for many affected individuals.

Multi-gene genetic testing has emerged as a clinical testing approach for hearing loss. There are currently fi ve laboratories in the U.S. offering multi-gene hearing loss panels [ 45 ]. Panels vary in their testing approach, technologies and number of genes tested. Some panels assess GJB2 , GJB6 and mitochondrial mutations by traditional technologies such as multiplex PCR and bidirectional sequencing. Other panels go beyond, including 20–60 genes associated with common syndromic and non-syndromic hearing loss [ 46 , 47 ] using targeted genome enrichment fol-lowed by mass parallel sequencing (MPS) [ 48 ]. This technology allows an extremely high coverage (average number of sequencing reads that align to each base within

J. Ordóñez et al.

55

the sample DNA) of selected deafness genes and lower costs. Multi-gene testing is also able to identify syndromic forms of deafness that may be indistinguishable from non-syndromic forms during infancy and early childhood. Retinitis pigmen-tosa and goiter may not appear until adolescence for individuals with Usher and Pendred syndromes, respectively. Multi-gene testing can be cost-effective and suc-cessful in early identifi cation of syndromic disorders that may lead to important changes in medical management, education and counseling. However, the weakness of this approach resides on the limited amount of genes to be interrogated. This is relevant when a minimum of 34 % of deafness genes are yet to be discovered [ 49 ]. In addition, with the current pace of gene discovery, costume capture kits for deaf-ness should be frequently updated.

Whole-exome enrichment followed by mass parallel sequencing (WES) is also an important approach to deafness genetic screening [ 50 ]. In contrast to custom capture enrichment, in WES most of the whole coding DNA sequence can be interrogated allowing screening of mutations in known deafness genes but also gene discovery. Lower coverage and higher prices remain as the main limitations of WES. A recent study shows that WES effi ciently detects rare mutations in known deafness genes [ 50 ]. An advantage of whole-exome or whole genome sequencing is that it is possible to search for causative variants in genes not previously associated with deafness, as exemplifi ed by recent discoveries of mutations in OTOGL [ 51 ] and SLITRK6 [ 52 ]. These instances demonstrate the promise in next-generation DNA sequencing technology to bring to completion the list of deafness genes in near future.

6 Future Directions

The genetic heterogeneity of deafness complicates the diagnosis of individual cases and approach to genetic testing. Yet these challenges have propelled progress towards personalized medicine for the deaf individual. There are ongoing efforts to adapt the next-generation DNA sequencing tools to an epidemiological scale that may serve for newborn screening programs in the future [ 53 , 54 ]. This approach may supplement the current newborn hearing screening programs by diagnosing delayed-onset childhood hearing loss cases at birth. The migration of this new tech-nology to newborn screening will certainly raise some diffi culties and limitations related to detection rates and incidence of variants of uncertain signifi cance, for instance. This may lead in parallel to changes in newborn screening algorithms and increased complexity of genetic counseling. However, migration of high throughput DNA sequencing tools to newborn screening may bring important benefi ts in genetic diagnosis and treatment, risk assessment, as well as educational and occupational interventions. Finding the detailed pathogenic mechanisms of hearing loss increases hopes for treatment by correcting the underlying pathologies. Only through multidisciplinary approaches between genetics, audiology, ENT, bioinfor-matics, and molecular biology we may grasp the genetic heterogeneity of deafness and make it translational in medicine.

Personalized Medicine for Hereditary Deafness

56

References

1. Morton CC, Nance WE (2006) Newborn hearing screening – a silent revolution. N Engl J Med 354:2151–2164

2. Hereditary Hearing Loss. In: Hereditary hearing loss homepage. http://hereditaryhearingloss.org/ . Accessed 15 Oct 2012

3. Downs MP, Sterritt GM (1964) Identifi cation audiometry for neonates: a preliminary report. J Audit Res 4:69–80

4. The National Institutes of Health (NIH) (2012) Consensus Development Program: early iden-tifi cation of hearing impairment in infants and young children

5. National Newborn Screening and Global Resource Center. In: National Newborn Screening and Global Resource Center. http://genes-r-us.uthscsa.edu/reports

6. Yoshinaga-Itano C, Sedey AL, Coulter DK, Mehl AL (1998) Language of early- and later- identifi ed children with hearing loss. Pediatrics 102:1161–1171

7. Moeller MP (2000) Early intervention and language development in children who are deaf and hard of hearing. Pediatrics 106:E43

8. Mazzoli M, Van Camp G, Newton V, Giarbini N, Declau F, Parving A (2003) Recommendations for the description of genetic and audiological data for families with nonsyndromic hereditary hearing impairment. Audiol Med 1:148–150

9. Van Camp G, Willems PJ, Smith RJ (1997) Nonsyndromic hearing impairment: unparalleled heterogeneity. Am J Hum Genet 60:758–764

10. Hutchin T, Coy NN, Conlon H, Telford E, Bromelow K, Blaydon D, Taylor G, Coghill E, Brown S, Trembath R, Liu XZ, Bitner-Glindzicz M, Mueller R (2005) Assessment of the genetic causes of recessive childhood non-syndromic deafness in the UK – implications for genetic testing. Clin Genet 68:506–512

11. Lench N, Houseman M, Newton V, Van Camp G, Mueller R (1998) Connexin-26 mutations in sporadic non-syndromal sensorineural deafness. Lancet 351:415

12. Marazita ML, Ploughman LM, Rawlings B, Remington E, Arnos KS, Nance WE (1993) Genetic epidemiological studies of early-onset deafness in the U.S. school-age population. Am J Med Genet 46:486–491

13. Kokotas H, Petersen MB, Willems PJ (2007) Mitochondrial deafness. Clin Genet 71:379–391

14. Duman D, Sirmaci A, Cengiz FB, Ozdag H, Tekin M (2011) Screening of 38 genes identifi es mutations in 62 % of families with nonsyndromic deafness in Turkey. Genet Test Mol Biomarkers 15:29–33

15. Coyle B, Coffey R, Armour JA, Gausden E, Hochberg Z, Grossman A, Britton K, Pembrey M, Reardon W, Trembath R (1996) Pendred syndrome (goitre and sensorineural hearing loss) maps to chromosome 7 in the region containing the nonsyndromic deafness gene DFNB4. Nat Genet 12:421–423

16. Park H-J, Shaukat S, Liu X-Z, Hahn SH, Naz S, Ghosh M, Kim H-N, Moon S-K, Abe S, Tukamoto K, Riazuddin S, Kabra M, Erdenetungalag R, Radnaabazar J, Khan S, Pandya A, Usami S-I, Nance WE, Wilcox ER, Riazuddin S, Griffi th AJ (2003) Origins and frequencies of SLC26A4 (PDS) mutations in east and south Asians: global implications for the epidemiology of deafness. J Med Genet 40:242–248

17. Möller CG, Kimberling WJ, Davenport SL, Priluck I, White V, Biscone-Halterman K, Odkvist LM, Brookhouser PE, Lund G, Grissom TJ (1989) Usher syndrome: an otoneurologic study. Laryngoscope 99:73–79

18. Kimberling WJ, Hildebrand MS, Shearer AE, Jensen ML, Halder JA, Trzupek K, Cohn ES, Weleber RG, Stone EM, Smith RJH (2010) Frequency of Usher syndrome in two pediatric populations: implications for genetic screening of deaf and hard of hearing children. Genet Med 12:512–516

J. Ordóñez et al.

57

19. Kelsell DP, Dunlop J, Stevens HP, Lench NJ, Liang JN, Parry G, Mueller RF, Leigh IM (1997) Connexin 26 mutations in hereditary non-syndromic sensorineural deafness. Nature 387:80–83

20. Scott DA, Kraft ML, Carmi R, Ramesh A, Elbedour K, Yairi Y, Srisailapathy CR, Rosengren SS, Markham AF, Mueller RF, Lench NJ, Van Camp G, Smith RJ, Sheffi eld VC (1998) Identifi cation of mutations in the connexin 26 gene that cause autosomal recessive nonsyn-dromic hearing loss. Hum Mutat 11:387–394

21. Gasparini P, Rabionet R, Barbujani G, Melçhionda S, Petersen M, Brøndum-Nielsen K, Metspalu A, Oitmaa E, Pisano M, Fortina P, Zelante L, Estivill X (2000) High carrier fre-quency of the 35delG deafness mutation in European populations. Genetic Analysis Consortium of GJB2 35delG. Eur J Hum Genet 8:19–23

22. Morell RJ, Kim HJ, Hood LJ, Goforth L, Friderici K, Fisher R, Van Camp G, Berlin CI, Oddoux C, Ostrer H, Keats B, Friedman TB (1998) Mutations in the connexin 26 gene (GJB2) among Ashkenazi Jews with nonsyndromic recessive deafness. N Engl J Med 339:1500–1505

23. Abe S, Usami S, Shinkawa H, Kelley PM, Kimberling WJ (2000) Prevalent connexin 26 gene (GJB2) mutations in Japanese. J Med Genet 37:41–43

24. Kudo T, Ikeda K, Kure S, Matsubara Y, Oshima T, Ki W, Kawase T, Narisawa K, Takasaka T (2000) Novel mutations in the connexin 26 gene (GJB2) responsible for childhood deafness in the Japanese population. Am J Med Genet 90:141–145

25. Snoeckx RL, Huygen PLM, Feldmann D, Marlin S, Denoyelle F, Waligora J, Mueller- Malesinska M, Pollak A, Ploski R, Murgia A, Orzan E, Castorina P, Ambrosetti U, Nowakowska-Szyrwinska E, Bal J, Wiszniewski W, Janecke AR, Nekahm-Heis D, Seeman P, Bendova O, Kenna MA, Frangulov A, Rehm HL, Tekin M, Incesulu A, Dahl H-HM, du Sart D, Jenkins L, Lucas D, Bitner-Glindzicz M, Avraham KB, Brownstein Z, del Castillo I, Moreno F, Blin N, Pfi ster M, Sziklai I, Toth T, Kelley PM, Cohn ES, Van Maldergem L, Hilbert P, Roux A-F, Mondain M, Hoefsloot LH, Cremers CWRJ, Löppönen T, Löppönen H, Parving A, Gronskov K, Schrijver I, Roberson J, Gualandi F, Martini A, Lina-Granade G, Pallares- Ruiz N, Correia C, Fialho G, Cryns K, Hilgert N, Van de Heyning P, Nishimura CJ, Smith RJH, Van Camp G (2005) GJB2 mutations and degree of hearing loss: a multicenter study. Am J Hum Genet 77:945–957

26. Del Castillo I, Moreno-Pelayo MA, Del Castillo FJ, Brownstein Z, Marlin S, Adina Q, Cockburn DJ, Pandya A, Siemering KR, Chamberlin GP, Ballana E, Wuyts W, Maciel-Guerra AT, Alvarez A, Villamar M, Shohat M, Abeliovich D, Dahl H-HM, Estivill X, Gasparini P, Hutchin T, Nance WE, Sartorato EL, Smith RJH, Van Camp G, Avraham KB, Petit C, Moreno F (2003) Prevalence and evolutionary origins of the del(GJB6-D13S1830) mutation in the DFNB1 locus in hearing-impaired subjects: a multicenter study. Am J Hum Genet 73:1452–1458

27. del Castillo FJ, Rodríguez-Ballesteros M, Alvarez A, Hutchin T, Leonardi E, de Oliveira CA, Azaiez H, Brownstein Z, Avenarius MR, Marlin S, Pandya A, Shahin H, Siemering KR, Weil D, Wuyts W, Aguirre LA, Martín Y, Moreno-Pelayo MA, Villamar M, Avraham KB, Dahl H-HM, Kanaan M, Nance WE, Petit C, Smith RJH, Van Camp G, Sartorato EL, Murgia A, Moreno F, del Castillo I (2005) A novel deletion involving the connexin-30 gene, del(GJB6- d13s1854), found in trans with mutations in the GJB2 gene (connexin-26) in subjects with DFNB1 non-syndromic hearing impairment. J Med Genet 42:588–594

28. Duman D, Tekin M (2012) Autosomal recessive nonsyndromic deafness genes: a review. Front Biosci (Landmark Ed) 17:2213–2236

29. Estivill X, Govea N, Barceló E, Badenas C, Romero E, Moral L, Scozzri R, D’Urbano L, Zeviani M, Torroni A (1998) Familial progressive sensorineural deafness is mainly due to the mtDNA A1555G mutation and is enhanced by treatment of aminoglycosides. Am J Hum Genet 62:27–35

30. Bykhovskaya Y, Yang H, Taylor K, Hang T, Tun RY, Estivill X, Casano RA, Majamaa K, Shohat M, Fischel-Ghodsian N (2001) Modifi er locus for mitochondrial DNA disease: linkage

Personalized Medicine for Hereditary Deafness

58

and linkage disequilibrium mapping of a nuclear modifi er gene for maternally inherited deafness. Genet Med 3:177–180

31. Ballana E, Morales E, Rabionet R, Montserrat B, Ventayol M, Bravo O, Gasparini P, Estivill X (2006) Mitochondrial 12S rRNA gene mutations affect RNA secondary structure and lead to variable penetrance in hearing impairment. Biochem Biophys Res Commun 341:950–957

32. Sevior KB, Hatamochi A, Stewart IA, Bykhovskaya Y, Allen-Powell DR, Fischel-Ghodsian N, Maw MA (1998) Mitochondrial A7445G mutation in two pedigrees with palmoplantar kerato-derma and deafness. Am J Med Genet 75:179–185

33. Weegerink NJD, Huygen PLM, Schraders M, Kremer H, Pennings RJE, Kunst HPM (2011) Variable degrees of hearing impairment in a Dutch DFNX4 (DFN6) family. Hear Res 282:167–177

34. Taylor KR, Deluca AP, Shearer AE, Hildebrand MS, Black-Ziegelbein EA, Anand VN, Sloan CM, Eppsteiner RW, Scheetz TE, Huygen PLM, Smith RJH, Braun TA, Casavant TL (2013) AudioGene: predicting hearing loss genotypes from phenotypes to guide genetic screening. Hum Mutat 34:539–545

35. Bahmad F, O’Malley J, Tranebjaerg L, Merchant SN (2008) Histopathology of nonsyndromic autosomal dominant midfrequency sensorineural hearing loss. Otol Neurotol 29:601–606

36. Hildebrand MS, Morín M, Meyer NC, Mayo F, Modamio-Hoybjor S, Mencía A, Olavarrieta L, Morales-Angulo C, Nishimura CJ, Workman H, DeLuca AP, del Castillo I, Taylor KR, Tompkins B, Goodman CW, Schrauwen I, Wesemael MV, Lachlan K, Shearer AE, Braun TA, Huygen PLM, Kremer H, Van Camp G, Moreno F, Casavant TL, Smith RJH, Moreno-Pelayo MA (2011) DFNA8/12 caused by TECTA mutations is the most identifi ed subtype of nonsyn-dromic autosomal dominant hearing loss. Hum Mutat 32:825–834

37. Hildebrand MS, DeLuca AP, Taylor KR, Hoskinson DP, Hur IA, Tack D, McMordie SJ, Huygen PLM, Casavant TL, Smith RJH (2009) A contemporary review of AudioGene audio-profi ling: a machine-based candidate gene prediction tool for autosomal dominant nonsyn-dromic hearing loss. Laryngoscope 119:2211–2215

38. Toriello HV, Reardon W, Gorlin RJ (2004) Hereditary hearing loss and its syndromes. Oxford University Press, Oxford/New York

39. Joint Committee on Infant Hearing, American Academy of Audiology, American Academy of Pediatrics, American Speech-Language-Hearing Association, Directors of Speech and Hearing Programs in State Health and Welfare Agencies (2000) Year 2000 position statement: princi-ples and guidelines for early hearing detection and intervention programs. Joint Committee on Infant Hearing, American Academy of Audiology, American Academy of Pediatrics, American Speech-Language-Hearing Association, and Directors of Speech and Hearing Programs in State Health and Welfare Agencies. Pediatrics 106:798–817

40. ACMG (2002) Genetics evaluation guidelines for the etiologic diagnosis of congenital hearing loss. Genetic evaluation of congenital hearing loss expert panel. ACMG statement. Genet Med 4:162–171

41. American Academy of Pediatrics, Joint Committee on Infant Hearing (2007) Year 2007 position statement: principles and guidelines for early hearing detection and intervention pro-grams. Pediatrics 120:898–921

42. US Preventive Services Task Force (2008) Universal screening for hearing loss in newborns: US Preventive Services Task Force recommendation statement. Pediatrics 122:143–148

43. Lucotte G, Mercier G (2001) Meta-analysis of GJB2 mutation 35delG frequencies in Europe. Genet Test 5:149–152

44. Liu XZ, Xia XJ, Ke XM, Ouyang XM, Du LL, Liu YH, Angeli S, Telischi FF, Nance WE, Balkany T, Xu LR (2002) The prevalence of connexin 26 (GJB2) mutations in the Chinese population. Hum Genet 111:394–397

45. (2009) GeneTests. In: GeneTests. http://www.genetests.org/ . Accessed 10 Apr 2012 46. Hearing Loss Gene Tests. In: LMM hearing loss PCPGM. http://pcpgm.partners.org/lmm/

tests/hearing-loss . Accessed 17 Oct 2012

J. Ordóñez et al.

59

47. (2012) Cincinnati Children’s. In: Cincinnati Children’s Hospital Medical Center. http://www.cincinnatichildrens.org/default/ . Accessed 9 Jan 2014

48. Shearer AE, DeLuca AP, Hildebrand MS, Taylor KR, Gurrola J 2nd, Scherer S, Scheetz TE, Smith RJH (2010) Comprehensive genetic testing for hereditary hearing loss using massively parallel sequencing. Proc Natl Acad Sci U S A 107:21104–21109

49. Shearer AE, Smith RJH (2012) Genetics: advances in genetic testing for deafness. Curr Opin Pediatr 24:679–686

50. Diaz-Horta O, Duman D, Foster J 2nd, Sırmacı A, Gonzalez M, Mahdieh N, Fotouhi N, Bonyadi M, Cengiz FB, Menendez I, Ulloa RH, Edwards YJK, Züchner S, Blanton S, Tekin M (2012) Whole-exome sequencing effi ciently detects rare mutations in autosomal recessive nonsyndromic hearing loss. PLoS One 7:e50628

51. Yariz KO, Duman D, Seco CZ, Dallman J, Huang M, Peters TA, Sirmaci A, Lu N, Schraders M, Skromne I, Oostrik J, Diaz-Horta O, Young JI, Tokgoz-Yilmaz S, Konukseven O, Shahin H, Hetterschijt L, Kanaan M, Oonk AMM, Edwards YJK, Li H, Atalay S, Blanton S, Desmidt AA, Liu X-Z, Pennings RJE, Lu Z, Chen Z-Y, Kremer H, Tekin M (2012) Mutations in OTOGL, encoding the inner ear protein otogelin-like, cause moderate sensorineural hearing loss. Am J Hum Genet 91:872–882

52. Tekin M, Chioza BA, Matsumoto Y, Diaz-Horta O, Cross HE, Duman D, Kokotas H, Moore- Barton HL, Sakoori K, Ota M, Odaka YS, Foster J 2nd, Cengiz FB, Tokgoz-Yilmaz S, Tekeli O, Grigoriadou M, Petersen MB, Sreekantan-Nair A, Gurtz K, Xia X-J, Pandya A, Patton MA, Young JI, Aruga J, Crosby AH (2013) SLITRK6 mutations cause myopia and deafness in humans and mice. J Clin Invest 123:2094–2102

53. De Keulenaer S, Hellemans J, Lefever S, Renard J-P, De Schrijver J, Van de Voorde H, Tabatabaiefar MA, Van Nieuwerburgh F, Flamez D, Pattyn F, Scharlaken B, Deforce D, Bekaert S, Van Criekinge W, Vandesompele J, Van Camp G, Coucke P (2012) Molecular diag-nostics for congenital hearing loss including 15 deafness genes using a next generation sequencing platform. BMC Med Genomics 5:17

54. Tang W, Qian D, Ahmad S, Mattox D, Todd NW, Han H, Huang S, Li Y, Wang Y, Li H, Lin X (2012) A low-cost exon capture method suitable for large-scale screening of genetic deafness by the massively-parallel sequencing approach. Genet Test Mol Biomarkers 16:536–542

Personalized Medicine for Hereditary Deafness

61M. Özgüç (ed.), Rare Diseases: Integrative PPPM Approach as the Medicine of the Future, Advances in Predictive, Preventive and Personalised Medicine 6,DOI 10.1007/978-94-017-9214-1_5, © Springer Science+Business Media Dordrecht 2015

Abstract Mitochondrial diseases (prevalence 1:5,000) represent a heterogeneous group of multisystemic disorders which often affects skeletal muscle and nervous system and is mostly due to dysfunction of the mitochondrial respiratory chain. The tissues with the highest energy expenditure are the most vulnerable. The dis-orders are caused either by mutations of mtDNA, or nDNA. A special group of the mitochondrial disorders is the defect of intergenomic communication disturbancy which affects the mtDNA quantitatively (mtDNA depletion) or qualitatively (mul-tiple mtDNA deletion). Owing to the unequal distribution of mitochondria in the different tissues and the co-existence of mutant and wild type mtDNA, these disor-ders may present with a huge variety of symptoms, making diagnosis diffi cult. In every year about ten new disease-causing genes are discovered. In the diagnosis of the mitochondrial disorders the muscle histology and the molecular biology play the most important role. Identifi cation of the disease causing mutations may help to select the most effective therapy. E.g. ketogenic diet is effective mostly in pyru-vate dehydrogenase defi ciency, valproate should be avoided in cases with special SNPs in the POLG1 gene. In summary: the molecular stratifi cation of the mito-chondrial disorders is very important in the predictive and preventive treatment of this rare disease group.

Keywords Mitochondrial disease • mtDNA disorders • Intergenomial communication • Gene therapy • Heteroplasmy • Supplementation therapy • Sirtuin • EPI-743 • Predictive, preventive and personalized medicine

Mitochondrial Diseases

Maria Judit Molnar and Klara Pentelenyi

M. J. Molnar (*) • K. Pentelenyi Institute of Genomic Medicine and Rare Disorders , Semmelweis University , Budapest , Hungary e-mail: molnarmj@gmail.com; pentelenyi.klara@gmail.com

62

Mitochondrial dysfunction was fi rst described in a patient who was losing weight despite normal thyroid function [ 1 ]. The disease affects multiple organs in varying location and severity. Clinical heterogenity make mitochondrial disorders diffi cult to diagnose, patients typically see multiple specialists and have several parallel diagnosis.

1 Prevalence

Combining the results of the epidemiological data on childhood and adult mitochon-drial disease suggests that the minimum prevalence is at least 1 in 5,000 and could be much higher. The fi rst population based study of all mitochondrial disorders esti-mated a mtDNA mutation prevalence of 12.48/100,000, and a disease prevalence of 6.57/100,000 [ 2 ]. It is hard to determine the true prevalence in childhood, because of the varying age of onset and the wide spectrum of clinical manifestation (easy to confuse with other diseases: similar symptoms). Dual genomic interplay complicate the aspect: nuclear DNA mutation may induce further mtDNA mutations. A prospec-tive American study guesses the prevalence 1:200 harboring pathogen mutation with risk for developing mitochondrial disease [ 3 ].

2 Manifestation of Mitochondrial Diseases

Mitochondrial cytopathies represent a heterogeneous group of multisystem disorders which often affects skeletal muscle and nervous system and is mostly due to dys-function of the mitochondrial respiratory chain [ 4 , 5 ]. A variety of organs may be affected by mitochondrial dysfunction. The most oxidative tissues (brain, retina, muscle and kidney) are the most vulnerable to OXPHOS defects. It was described in association with neurodegenerative diseases, diabetes, deafness, visual-, heart-, liver-, kidney-problems, stroke, migraine, infertility and pharmaceutical toxicity [ 6 ].

The disorders are caused either by mutations of the maternally inherited mito-chondrial genome, or by nuclear DNA mutations. Today more than 200 different disease-causing mutations of mitochondrial DNA (mtDNA) are known and due to the increased knowledge about nuclear genetics during the last few years, further 100 nuclear mutations are being described. 70–85 % of mitochondrial diseases evolve due to nuclear mutation, not mtDNA mutation. Owing to the unequal distribution of mitochondria in the different tissues and the co-existence of mutant and wild type mtDNA in these organelles, these disorders may present with a huge variety of symptoms, even if the same mutation is involved [ 7 ]. Children often have severe disease in the background with nDNA mutation, causing respiratory chain defect, crashing mitochondrium integrity [ 8 ].

Mitochondria besides their fundamental role in the cellular energy metabolism, seem to contribute to the pathogenesis of many degenerative diseases, to aging and cancer.

M.J. Molnar and K. Pentelenyi

63

3 Genetic Motives of Mitochondrial Diseases

The mitochondrial disease may be the result of the defect of the mtDNA or of the genes in the nuclear genome which are responsible for the proper mitochondrial function. The mtDNA is inherited mostly maternally, the nuclear genes are inher-ited autosomal dominant, recessive or X linked manner. The most important characteristic of the mtDNA [ 5 ]:

• the mitochondrial genome is a 16,569 base pairs long circular DNA, • it consists 37 genes that encode 13 proteins, 22 tRNAs, and 2 rRNAs. • The mitochondrial genome is not able to independently produce all of the pro-

teins needed for functionality; thus, mitochondria rely heavily on imported nuclear gene products.

• In one mitochondrion there are multiple copies of its mtDNA. A cell contains several thousand copies of its mitochondrial genome (polyplasmy). The wild type and mutated mtDNA are present at the same time in a cell (heteroplasmy)

• To develop a clinical sign a certain amount of mutated mtDNA has to be present in a tissue (threshold effect).

Nuclear DNA encodes ~1,000 mitochondrial proteins, OXPHOS proteins (74) and factors, forming the respiratory chain complexes; enzymes; mitochondrial membrane proteins and transporters; factors for mtDNA replication-transcription- translation and ribosomal proteins [ 9 ]. Proteins, encoded in nDNA are synthesized in the cytoplasm and then imported into the mitochondrion via specifi c transport systems.

4 Frequent mtDNA Diseases

The most common mitochondrial disease due to mtDNA mutations is LHON (Leber hereditary optic neuropathy) with homoplasmic mtDNA mutations G11778A (69 %), G3460A (13 %), T14484C (14 %) and other substitutions. Men are involved more than women, the painless visual loss is beginning in young adulthood with optic atrophy. The subsequent prevalent phenotype is MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like syndrome) with ophthalmo-plegia externa, diabetes mellitus, hearing loss, early onset stroke like symptoms, migraine, and cognitive dysfunction due to mutations in the mitochondrial tRNA Leu gene (A3243G 80 %, T3271C 7 %, A3260G and A3252G 5–5 %). MERRF (myoclonic epilepsy with ragged-red fi bers) evolves due to mtDNA tRNA Lys mutations as A8344G (80 %), T8356C, G8363A and G8361A (10 %), causing myoclonus epilepsy, ataxia, dementia, neuropathy, myopathy [ 10 ]. The A8993C or T substitution may result in NARP (neuropathy, ataxia, retinitis pigmentosa) syndrome or maternally inherited Leigh disease. The ratio of heteroplasmy is in many cases very high in these patients. Usually in mtDNA disorders the higher ratio of heteroplasmy is associated with more severe clinical phenotype.

Mitochondrial Diseases

64

5 Nuclear Encoded Mitochondrial Disease Groups

As far as we know presently more than 200 nuclear-encoded genes are implicated in mitochondrial diseases. In every year about 10 new disease-causing genes are discovered [ 11 ]. There are fi ve groups of nDNA mutations causing diseases:

1. Mutations in genes encoding respiratory chain proteins causing principally Leigh-syndrome.

2. Defects of mitochondrial dynamics: fusion genes are in the inner membrane OPA1 (optic atrophy1), in the outer membrane MFN2 (mitofusin2). Their muta-tions causing optic atrophy and CMT2A respectively. Fission gene is DLPA1 (dynamin-like protein1).

3. Defects of mitochondrial protein synthesis: mutations in tRNA modifying enzymes (PUS1, TRMU), elongation factors (TUFM, TSFM) or mitochondrial aminoacyl tRNA synthetases (RARS2, DARS2, YARS2).

4. Defects of intergenomial communication (see the next topic). 5. Defects in lipid milieu/transporter carriers of the inner mitochondrial membrane,

like TAZ gene (taffazin transacylase ) catalyzing cardiolipin maturation, or DDP1 (TIMM8A) causing Mohr-Tranebjaerg syndrome [ 8 ].

6 Defect of Intergenomial Communication

Cross-talk between nuclear and mitochondrial genomes is crucial for mitochondrial biogenesis and function, and the two genomes are probably subjected to co- evolutionary processes. The defect of intergenomic signaling can affect mtDNA quantitatively (mtDNA depletion) and qualitatively (multiple mtDNA deletion). Nuclear genes are needed for mitochondrial DNA replication and repair (POLG, Twinkle), for mitochondrium biogenesis and for the maintenance of nucleotide pool. Proper balance of the mitochondrial deoxynucleotide pools is essential in the maintenance of mtDNA copy number. Defects in these genes lead to depletion of mtDNA. The enzymes which are responsible for maintaining the nucleotide, deoxy-guanosine pool may located in the mitochondrium : deoxyguanozin kinase – coding gene DGUOK, thymidin kinase 2 – coding gene TK2, nucleoside diphosphate kinase and SUCLA succynil CoA ligase – coding genes NDPK, SUCLG1,2, SUCLA2; in the cytosol: thymidin phosphorylase – coding gene TYMP, thymidin kinase 1 – coding gene TK1, thymidilate synthase – coding gene TYMS, ribonucleotide reductase – coding gene RRM2B; and in the mitochondrial membrane: mitochondrial inner membrane protein – coding gene MPV17.

The most frequent gene, affected in intergenomial communication disturbances is POLG (polymerase gamma). More than 80 mutations are described in this gene, associated with several phenotype as adPEO, Alpers, MIRAS (Mitochondrial Recessive Ataxia Syndrome), SANDO (Sensory Ataxic Neuropathy with Dysarthria and Ophthalmoparesis), MEMSA (Myoclonic epilepsy myopathy sensory ataxia), MCHS (myocerebrohepatopathy spectrum), causing multiplex deletions. Mutations

M.J. Molnar and K. Pentelenyi

65

in Twinkle provoke multiplex deletion with adPEO phenotype. In these disorders a secondary mtDNA deletion is present.

In the background of MDS (Mitochondrial Depletion Syndrome) is the nucleotide pool impaired. The myopathic form is due to mutations mostly in ANT1 (responsible for ADP/ATP balance), TK2 (in charge of pyrimidine biosynthesis), or RRM2B, POLG. Hepatocerebral form evolves due to mutated DGUOK (in charge of purin biosynthesis), POLG or Twinkle. The encephalomyopathy usually is due to by mutated SUCLA and RRM2B genes [ 12 ]. The MNGIE (Mitochondrial NeuroGastrointestinal Encephalomyopathy) caused by mutations in TYMP (thymidine phosphorylase) which leads to mtDNA depletion and deletion.

7 Diagnostic Principles [ 5 ]

The most important laboratory parameters are resting serum lactate and pyruvate, which are frequently increased. The ratio of lactate/pyruvate is increased in many cases. Serum CK levels are either normal or slightly elevated. Serum lactate increases during slight exercise in mitochondrial patients and 30 min after the exercise will not decline to the baseline. EMG is normal, neurogenic or myogenic, or not specifi c. The muscle biopsy always displays the characteristic ragged red or ragged blue fi ber pathology. The ragged red fi bers usually do not have cytochrome C oxidase (COX) activity. The ultrastructural analysis of the muscle reveals aberrant, enlarged mitochondria usually with paracristallin inclusions or abnormally organized cristae. Biochemical investigations detect the reduced activity of the affected enzyme. Searching for mtDNA mutation genetic testing recommended on postmitotic tissue (e.g. muscle biopsy specimen). In the routine diagnostic the mtDNA mutation hotspots are screened. In many cases the whole mtDNA is sequenced. The mutations in the nuclear genes nowadays are searched based on the clinical and imaging phenotype. In the close future the whole exome analysis by next generation sequencing will improve the diagnostic of the mitochondrial disorders due to nuclear gene mutations.

8 Therapeutic Principles [ 13 ]

Despite identifying new clinical trials in these days, currently no clear evidence support-ing the use of any intervention in mitochondrial disorders. However there are different strategies to improve the mitochondrial function in these disorders. These strategies are: (a) gene shifting, (b) pharmacotherapy, (c) dietary therapies, (d) supplementation therapy and (e) other treatments.

(a) Gene therapy , gene shifting targets the decrease of the ratio of heteroplasmy: The following techniques may help to decrease the ratio of heteroplasmy (Some techniques may act only in certain tissues)

• Converting mutated mtDNA genes into normal nDNA genes (allotopic expression)

Mitochondrial Diseases

66

• Importing cognate genes from other species (xenotopic expression) • Correcting mtDNA mutations by importing specifi c restriction endonucleases • Inducing muscle regeneration

(b) Pharmacological therapy Dichloracetate can reduce serum and tissue lactate level, but it may cause peri-pheral neuropathy. Dexpramipexole may inhibit calcium-induced permeability. A clinical trial is going on by EPI-743 ( para-benzoquinone, which is the cofactor of the NADPH quinone oxidase 1 (NQO1)). The symptoms of children with Leigh syndrome improved after 180 days follow up. There are many preclinical experiments e.g. to clear the altered mtDNA by mitoTALENs (nuclease target-ing the mitochondrium), or sirtuins – (SIRT 1–7: histon deacetylase modifi ers) infl uencing the metabolic enzymes by the peroxisomal proliferator (PPAR). The PPAR signaling modifi es the gluconeogenesis, fatty acid oxidation, fat cell differentiation, cell survival and ubiquitination. Resveratrol and bezafi brate (PPAR agonist) can induce fatty acid oxidation and improve mitochondrial biogenesis, ATP synthesis. The resveratrol is SIRT1 agonist, antioxidant, inhibits the apoptosis. Phase 2 clinical trials will start soon by SIRT-1 agonists.

(c) Dietary therapy The fasting is not recommended for patients with mitochondrial disease. There are no general dietary recommendations in mitochondrial disorders. In some forms (e.g. in pyruvate dehydrogenase defi ciency) ketogenic diet may help to improve the symptoms, but it is not recommended in disorders with altered fatty acid oxidation.

(d) Supplementation therapy with coenzyme Q10, carnitine and antioxidants (Vitamin C, K3, E). In Friedreich ataxia idebenone had positive effect on the cardiomyopathy, but there was no change in the ataxia. Ribofl avin provides fl avin precursors to complex I and II. Niacin increases NAD/NADH + pool. Succinate donates electrons to complex II. Creatin monohydrate is good for myopathies, to improve muscle phosphocreatine content. N-Acetylcysteine may help as glutathione precursor. L-Arginine, L-Citrulline promotes the endo-thelial relaxation. This is the reason why is recommended to use to alleviate the stroke like symptoms in mitochondrial disorders.

(e) Other treatments In some cases surgical therapy may correct some deformity, e.g. ptosis.

Acknowledgment MJM and KP was supported by the grant KTIA_AIK_12-1-2013-0017.

References

1. Luft R, Ikkos D, Palmieri G, Ernster L, Afzelius B (1962) A case of severe hypermetabolism of nonthyroid origin with a defect in the maintenance of mitochondrial respiratory control: a correlated clinical, biochemical, and morphological study. J Clin Invest 41:1776–1804

2. Schaefer AM, Taylor RW, Turnbull DM, Chinnery PF (2004) The epidemiology of mitochondrial disorders – past, present and future. Biochim Biophys Acta 1659:115–120

M.J. Molnar and K. Pentelenyi

67

3. Saneto RP, Sedensky MM (2013) Mitochondrial disease in childhood: mtDNA encoded. Neurotherapeutics 10:199–211. doi: 10.1007/s13311-012-0167-0

4. DiMauro S, Hirano M (2005) Mitochondrial encephalomyopathies: an update. Neuromuscul Disord 15:276–286

5. Shoubridge E, Molnar MJ (2002) Structural and molecular basis of skeletal muscle diseases. ISN Neuropathology Press, Basel

6. Calvo S, Jain M, Xie X, Sheth SA, Chang B, Goldberger OA, Spinazzola A, Zeviani M, Carr SA, Mootha VK (2006) Systematic identifi cation of human mitochondrial disease genes through integrative genomics. Nat Genet 38:576–582

7. Molnar MJ (2010) Further mitochondrial cytopathies. In: Kazazian HH Jr, Klein G, Moser HW, Orkin SH, Roizman B, Thakker V, Watkins H (eds) Encyclopedia of molecular medicine. Wiley, New York

8. Goldstein A, Bhatia P, Vento JM (2013) Mitochondrial disease in childhood: nuclear encoded. Neurotherapeutics 10:212–226. doi: 10.1007/s13311-013-0185-6

9. Wong CL-J (2010) Molecular genetics of mitochondrial disorders. Dev Disabil Res Rev 16:154–162. doi: 10.1002/ddrr.104

10. Cohen BH (2013) Neuromuscular and systematic presentations in adults: diagnosed beyond MERRF and MELAS. Neurotherapeutics 10:227–242. doi: 10.1007/s13311-013-0188-3

11. Calvo SE, Compton AG, Hershman SG, Lim SC, Lieber DS, Tucker EJ, Laskowski A, Garone C, Liu S, Jaffe DB, Christodoulou J, Fletcher JM, Bruno DL, Goldblatt J, Dimauro S, Thorburn DR, Mootha VK (2012) Molecular diagnosis of infantile mitochondrial disease with targeted next-generation sequencing. Sci Transl Med 4:118ra10. doi: 10.1126/scitranslmed.3003310

12. Mao CC, Holt IJ (2009) Clinical and molecular aspects of diseases of mitochondrial DNA instability. Chang Gung Med J 32:354–369

13. Goldstein A, Wolfe LA (2013) The elusive magic pill: fi nding effective therapies for mitochon-drial disorders. Neurotherapeutics 10:320–328. doi: 10.1007/s13311-012-0175-0

Mitochondrial Diseases

69M. Özgüç (ed.), Rare Diseases: Integrative PPPM Approach as the Medicine of the Future, Advances in Predictive, Preventive and Personalised Medicine 6,DOI 10.1007/978-94-017-9214-1_6, © Springer Science+Business Media Dordrecht 2015

Abstract Mendelian disorders are diseases which occur due to a mutation in the DNA sequence of a single gene. However, as we learn more about these inherited diseases, it is clear that there can be a vast spectrum of associated phenotypes. Gaucher disease is an example of a “simple” monogenic disorder with complex features. It results from the defi ciency of the recessively inherited enzyme gluco-cerebrosidase, and is the most common lysosomal storage disorder. One of the chief clinical challenges facing geneticists and medical practitioners is to assess how adequately one can use genotype data to predict phenotypes. The ability to make such predictions is an essential tenet of individualized medicine and has implications for prenatal decision making. By understanding the limitations of genotype- phenotype correlation in monogenic disorders, we can gain insights that will help us to better understand the complexity in interpreting genetic data in multigene disorders. Factors including genetic modifi ers, gene-gene interaction, reduced penetrance, imprinting, processed and non-processed pseudogenes, regu-latory polymorphisms, epigenetics and the abundant number of private mutations, provide challenges for those seeking to understand genetic contributions to dis-tinct phenotypes. Through a careful evaluation of one specifi c Mendelian disor-der, Gaucher disease, we can learn lessons directly applicable to other diseases, both rare and common.

Keywords Gaucher disease • Glucocerebrosidase • Mendelian disorder • Genotype- phenotype correlation • Genetic modifi ers • Parkinson disease • Neurodegeneration

Complexity of Genotype-Phenotype Correlations in Mendelian Disorders: Lessons from Gaucher Disease

Nima Moaven , Nahid Tayebi , Ehud Goldin , and Ellen Sidransky

N. Moaven • N. Tayebi • E. Goldin • E. Sidransky (*) Section on Molecular Neurogenetics, Medical Genetics Branch , National Human Genome Research Institute (NHGRI), National Institutes of Health (NIH) , Building 35, Room 1E623, 35 Convent Dr., MSC , Bethesda , MD 20892-3708 , USA e-mail: sidranse@mail.nih.gov

70

Abbreviations

OMIM Online Mendelian Inheritance in Man PAS Periodic acid-Schiff ICGG International Collaborative Gaucher Group LSD Lysosomal Storage Disorder LIMP Lysosomal Integral Membrane Protein AMRF Action Myoclonus-Renal Failure CMT Charcot Marie Tooth MRI Magnetic Resonance Imaging HGMD Human Gene Mutation Database GWAS Genome Wide Association Studies CVS Chorionic Villus Sampling AVN Avascular necrosis FDA Food and Drug Administration ERT Enzyme Replacement Therapy SRT Substrate Reduction Therapy CNS Central Nervous System

1 Introduction

Mendelian disorders, defects resulting from specifi c mutations in the DNA sequence of a single gene, are recognized by their classic segregation patterns, which include autosomal dominant, autosomal recessive, co-dominant and sex-linked modes of inheritance (See Glossary of Genetic Terms). It is increasingly apparent that these monogenic or “simple” single gene disorders are more complicated that initially appreciated. In fact, complex phenotypes can result from the contributions of intricate patterns of penetrance, variable expressivity, pleiotrophy and imprinting, likely infl uenced by factors such as genetic and allelic heterogeneity, environmental exposure, genetic modifi ers and epigenetics [ 1 – 3 ]. Today, over 13,000 genes known to be associated with Mendelian disorders have been identifi ed, as detailed in the database the Online Mendelian Inheritance in Man (OMIM, http://www.ncbi.nlm.nih.gov/Omim/mimstats.html ). While collectively they affect only a small fraction of the world’s population, studies of the genetic variability associated with mono-genic disorders provide an anchor for understanding the contribution of individual genes to the etiology of complex traits.

Initially, many of the genes associated with Mendelian disorders were identifi ed though techniques such as linkage studies and positional cloning. More recently, the successful pursuit of genes implicated in monogenic disorders has benefi ted from the improved accuracy, speed and cost of technologies that map the sequence of DNA. In addition to their use in monogenic disorders, these genomic strategies have been widely employed to identify genetic variants associated with complex disorders in large patient cohorts. However, it is now thought that common genetic variants

N. Moaven et al.

71

explain less than 5–10 % of phenotypic variation in most disorders [ 3 , 4 ]. Copy number variants, insertions and deletions of sequences into an individual’s genome, may also account for a part of the heritability of common diseases, highlighting the contribution of rare variants to common multifactorial traits. The impact of rare variants already identifi ed as the basis of specifi c Mendelian disorders to both related and unrelated complex disorders still remains to be determined [ 5 , 6 ].

While genome-wide approaches continue to be developed and improved, there is a resurgence in the need to focus on rare Mendelian disorders, which provide a unique means to approach a challenging dilemma currently relevant to both com-mon and rare disorders: phenotypic variability [ 7 ]. This clinical variation encom-passes observations of phenotypic differences encountered in individuals with a monogenetic disorder, as well as the mixed penetrance observed in carriers of the same genetic mutation. In this chapter, we will describe insights gained from the study of one such Mendelian disorder, Gaucher disease.

2 Overview of Gaucher Disease

2.1 History of Gaucher Disease

In 1982, Phillipe Charles Ernest Gaucher described a medical phenotype in his medical doctoral thesis [ 8 ] which, 20 years later, was named Gaucher disease by Dr. Nathan Brill, who fi rst recognized that this was a familial disorder [ 9 ]. Gaucher observed large unusual-appearing cells in the patient’s spleen, a histological feature currently referred to as “Gaucher cells,” a pathologic hallmark of this disorder [ 10 ]. Dr. Roscoe Brady described the enzymatic defect underlying Gaucher disease in 1965 [ 11 ], and 27 years later, in 1991, enzyme replacement therapy (ERT) was successfully developed as a therapy for Gaucher disease [ 12 ].

2.2 Gaucher Disease: A Simple Disorder with Complex Features

Gaucher disease is an autosomal recessive Mendelian disorder resulting from muta-tions on both alleles of the GBA gene. GBA encodes for an important lysosomal enzyme, glucocerebrosidase (GCase, E.C. 3.2.1.45), that catalyzes the breakdown of glucosylceramide to glucose and ceramide [ 11 ]. As a result, glucosylceramide accumulates in the mononuclear phagocyte system, primarily within macrophages. This accumulation leads to macrophages loaded with lipid-engorged lysosomes [ 13 ]. Using light microscopy, Periodic acid-Schiff (PAS) positive stored material can be observed in Gaucher macrophages, giving rise to their “wrinkled tissue paper”-like cytoplasm. As a result of the presence of undegraded lipid within vastly

Complexity of Genotype-Phenotype Correlations in Mendelian Disorders…

72

enlarged lysosomes, the nuclei of these macrophages often appear displaced. On electron microscopy, the cytoplasm of Gaucher cells display frequent irregular, elongated and tubular structures that distort the lysosome [ 14 ].

Single mutations in GBA result in wide phenotypic diversity. There are both non- neurologic and neuronopathic types of Gaucher disease, with a spectrum of diverse clinical symptoms, ranging from fetal death to elderly seniors who have no disease symptoms [ 15 ]. Classically, based on the absence of and the rate of progression of the neurological manifestations, Gaucher disease is divided into three types (see Sect. 2.3 ). However, Gaucher clinics examining affected individuals are confronted with many different and atypical manifestations. Comprehending the range and mechanisms of phenotypic heterogeneity in Gaucher disease, as well as other monogenic disorders, is a major challenge in the fi eld of human genetics. It has been long known that there is limited association between the patient’s phenotype and the residual enzymatic activity, or the amount of lipid stored in the macrophages [ 16 ]. When the gene was fi rst identifi ed, there was great excitement that at last the pheno-typic viability associated with this disorder might be explained by the severity of the particular gene mutation. However, this turned out to be far more complicated than initially anticipated, and currently research is focused on identifying the roles of modifi ers and other elements that lead to the clinical variation observed.

2.3 Types of Gaucher Disease

Gaucher disease is generally classifi ed into three main subtypes. Gaucher type 1 (MIM # 230800) is defi ned as the subtype lacking any associated neurological mani-festations. Gaucher type 2 (MIM # 230900), and Gaucher type 3 (MIM # 2301000) are characterized by central nervous system involvement. While patients with type 2 Gaucher disease generally have acute neurologic progression with severe neurode-generation and early death, patients with type 3 Gaucher disease have a more chronic course, with slowing or looping of the horizontal saccadic eye-movements as the most common neurologic manifestation [ 13 , 17 ]. The OMIM database also lists two other subtypes of Gaucher disease: perinatal lethal Gaucher disease (MIM #608013), a severe form of type 2 Gaucher disease, and Gaucher disease type 3C (MIM #231005), patients with cardiovascular calcifi cations or fi brosis [ 18 ]. Table 1 summarizes the main characteristics of the three major subtypes of Gaucher disease.

2.4 Clinical Manifestations of Gaucher Disease

A wide range of symptoms is encountered among patients with Gaucher disease. Affected individuals can be asymptomatic, especially those who are homozygous for mutation N370S (c.1226A > G). On the other hand, patients can develop signifi cant visceral manifestations, anemia and thrombocytopenia, skeletal involvement,

N. Moaven et al.

73

Parkinsonism, myoclonic epilepsy, malignancy, and pulmonary hypertension. The International Collaborative Gaucher Group (ICGG) analyzed 1,474 children with type 1 Gaucher disease, and found that the most common clinical manifestations observed were moderate to severe splenomegaly (in 97 %), thrombocytopenia (in 91 %), and hepatomegaly (in 89 %) [ 18 ]. However, it must be noted that the reg-istry only includes those cases that have come to medical attention and may not include many of the milder patients or asymptomatic individuals. Table 2 lists symp-toms that may be suggestive of Gaucher disease.

Visceral Involvement

Splenomegaly and hepatomegaly are commonly seen among patients with Gaucher disease. Enlarged spleens can be huge, weighing as much as 5–10 kg. In fact, among Ashkenazi Jews, Gaucher disease is the most common cause of splenomegaly, while malignancy is a more common cause of splenomegaly in other populations. The spleen size is usually followed by periodic MRI evaluations or ultrasonography [ 13 , 18 , 19 ].

Skeletal Involvement

Bone disease is a signifi cant cause of morbidity in Gaucher disease. On skeletal X-rays, signs of osteonecrosis, bone remodeling failure, osteopenia, and bone marrow infi ltration are common [ 20 , 21 ]. As a result of the local bone marrow

Table 1 Description of the three types of Gaucher disease

Type Associated features

Type 1 (non- neuronopathic)

Panethnic disorder, although more common among Ashkenazi Jews. Presents at any age. Associated with clinical heterogeneity. Many asymptomatic individuals. Wide range of symptom severity. Bone disease is a frequent cause of morbidity. Treated with enzyme replacement therapy

Hepatosplenomegaly, anemia, and thrombocytopenia are common Type 2 (acute

neuronopathic) Rare, panethnic disorder. Can present prenatally, at birth, or in the fi rst

year of life. Developmental delay. Rapidly progressive neurological deterioration. Early death within days to years. Enzyme replacement therapy does not reverse or halt neurological progression

Type 3 (chronic neuronopathic)

Includes several different phenotypes. Certain subtypes more prevalent in different ethnicities. Includes several different phenotypes with variable age of onset and longevity. Accompanied by a specifi c disorder of horizontal saccadic eye movements Subgroup develops cardiac valve calcifi cations, hydrocephalus and other abnormalities. Some patients develop myoclonic epilepsy. Visceral, but not neurological involvement responds to enzyme replacement therapy. Associated with distinct learning disabilities in some patients

Complexity of Genotype-Phenotype Correlations in Mendelian Disorders…

74

infi ltration by Gaucher cells, the shaft of the distal end of the femur fails to retain its normal tubular shape, resulting in a radiographic fi nding known as the “Erlenmeyer fl ask deformity” of the distal femur. The “Erlenmeyer fl ask defor-mity” is reported in up to 59 % of patients; however, there is no link between symptoms and the existence or severity of other skeletal manifestations [ 22 ]. The presence of the Gaucher cells can also result in infl ammation in bone. “Bone crises” characterized by severe sudden bone pain, are another relatively common bone manifestation, often requiring aggressive pain management and hydration. Low bone density is frequently observed, as well as pathological fractures [ 23 ]. Bisphosphonates, as a monotherapy or combined with ERT, have been used for the treatment of osteopenia or osteoporosis in patients with Gaucher disease [ 24 ].

Hematopoietic Involvement

Anemia, coagulopathies, thrombocytopenia and neutropenia or pancytopenia, are the most common hematological manifestations of Gaucher disease. The cytopenia is due both to degradation of cells secondary to hypersplenism and decreased pro-duction due to bone marrow infi ltration. Severe thrombocytopenia, defi ned as plate-let counts of less than 60,000/μl, was reported in 15 % of the patients enrolled in the ICGG. Patients with Gaucher disease are more prone to bruising and bleeding [ 25 ]. Often nose bleeds, heavier menstrual fl ow, and bleeding during dental procedures are observed [ 26 – 28 ].

Table 2 Symptoms suggestive of Gaucher disease

Symptom Frequency Age

Painless splenomegaly +++ All ages Thrombocytopenia +++ All ages Anemia +++ All ages Bone infarcts/fractures ++ All ages Coagulopathy + All ages Parkinsonism + Adulthood Pulmonary hypertension + Adulthood Cardiac valves + All- associated with D409H Slowed horizontal saccades ++ All- neuronopathic forms Myoclonic epilepsy + All- neuronopathic forms Learning defi cits + All- neuronopathic forms Failure to thrive ++ Infancy- all forms Opisthotonus + Infancy- type 2 Congenital ichthyosis + Infancy- type 2 Hydrops fetalis + Infancy- type 2

+++ = very frequent, ++ = moderately frequent, + = rare

N. Moaven et al.

75

Gaucher Disease and Parkinsonism

Over the past decade there has been increased awareness of an association between glucocerebrosidase mutations and the development of Parkinsonism. This was fi rst observed in patients with Gaucher disease who developed Parkinsonian manifesta-tions. Such patients exhibit a range of symptoms, including some with rapidly pro-gressive or early onset disease, while others have symptoms characteristic of sporadic Parkinson disease. However, it is important to note that the majority of patients with Gaucher disease do not develop Parkinson disease, so mutations in GBA appear to be a risk factor.

Furthermore, large studies have concluded that heterozygosity for a mutation in GBA is also a risk factor for developing Parkinsonism. Subjects with Parkinson disease are over fi ve times more likely to carry a mutation in this gene [ 29 – 32 ]. For subjects with the diagnosis of Dementia with Lewy bodies, the odds ratio for carry-ing a GBA mutation is actually over eight [ 33 ]. Multiple studies indicate that the age of onset of Parkinsonian manifestation in patients carrying a GBA mutation is roughly 5 years earlier than in those without mutations [ 32 ].

The observation that heterozygosity for a Mendelian disorder can impact the development of common complex diseases is not limited to the case of Gaucher disease and Parkinson disease, but is also seen in other diseases. Examples of other disorders include the association of glycerol kinase mutations and diabetes [ 34 ], the identifi cation of mutations in methyltetrahydrofolate reductase in atherothrombotic disease [ 35 ] and, that TREM2 mutations, resulting in the rare disorder Nasu-Hokala disease, are an important risk factor for Alzheimer disease [ 36 ].

Myoclonic Epilepsy

Among patients with type 3 Gaucher disease, there are cases that develop myo-clonic epilepsy [ 37 – 39 ]. This association between glucocerebrosidase mutations and myoclonic epilepsy is not well understood. Myoclonic epilepsy has been asso-ciated with several of the lysosomal storage disorders (LSDs) [ 40 ]. However, the mechanism by which the lysosomal enzymes are traffi cked distinguishes Gaucher disease from many of the other LSDs. Glucocerebrosidase utilizes the transport protein lysosomal integral membrane protein 2 (LIMP-2) for delivery to the lyso-some (Fig. 1 ), unlike the majority of other lysosomal enzymes, which use the man-nose 6-phosphate receptor [ 41 ]. Mutations in one or both alleles of SCARB2 , the gene that encodes LIMP-2, have been identifi ed in individuals with inherited myo-clonic epilepsy, including action myoclonus-renal failure (AMRF) [ 42 , 43 ]. The link between Gaucher disease and SCARB2 opens new avenues for investigation.

Malignancy in Patients with Gaucher Disease

There are many reports of the development of specifi c malignancies in association with Gaucher disease. Patients developing Gaucher manifestations later in life seem to be at higher risk of cancer, especially hematologic malignancies. This is

Complexity of Genotype-Phenotype Correlations in Mendelian Disorders…

76

particularly true of multiple myeloma [ 18 , 44 ]. Macrophages, the primary cells affected in Gaucher disease, play a crucial role in immunologic responses and infl ammation. There is also evidence of increased infl ammatory markers and B cell-related cancers in patients with Gaucher disease; however, the connection between Gaucher disease and cancer is still not well understood [ 45 ]. Recently, it has been proposed that biallelic mutations in the MSH6 gene can be a modifi er for the cancer phenotype in patients with Gaucher disease [ 46 ].

2.5 Epidemiology of Gaucher Disease

The three types of Gaucher disease are observed in all races, with an overall frequency estimated at 1 in 40,000–75,000 live births [ 47 ]. However, Gaucher disease type 1, the most common type, accounts for approximately 94 % of cases in the United States and Europe [ 48 ]. Type 1 is more prevalent among Ashkenazi Jews, where the carrier frequency is around 1 in 12–1 in 15 [ 13 ]. Types 2 and 3 tend to be far rarer, with a reported incidence of less than 1 in 100,000 births [ 49 ].

Fig. 1 Schematic representation of the glucocerebrosidase traffi cking pathway from the rough Endoplasmic Reticulum to the lysosome

N. Moaven et al.

77

2.6 Atypical Inheritance

Instances of unusual disease transmission have been observed in Gaucher disease. One child with type 3 Gaucher disease was noted to also have manifestations of Charcot Marie Tooth (CMT) disease, inherited from the father in an autosomal dominant family. It was discovered that a portion of chromosome 1q21 encom-passes both a CMT gene and GBA . In this proband, both mutations in GBA and CMT1 were present in the homozygous form, and were shown to result from unipa-rental disomy of the paternal allele [ 50 ].

In two interesting families of infants who died from type 2 Gaucher disease, two mutations were identifi ed in each proband, but no mutations were detected in their respective mothers. Thus it was concluded that the second allele likely resulted from a maternal germline mutation. In both cases, the de novo mutant allele was L444P, a known “hotspot” for mutation. Since such germline mutations or mosiacism are not generally associated with autosomal recessive disorders, these cases have signifi cant consequences for understanding molecular diagnostics and genetic counseling in recessive disorder [ 51 ].

3 The GBA Gene

3.1 The GBA Gene at a Glance

The gene encoding glucocerebrosidase, GBA (OMIM #606463), consisting of 11 exons and 10 introns is localized on the long arm of chromosome 1 at 1q21. GBA is located in a gene-rich region, which includes seven genes and two pseu-dogenes within 85 kb. In close proximity, directly downstream of GBA is a highly homologous pseudogene ( GBAP ) that includes the same number of exons and introns [ 52 – 54 ] and shares 97 % homology in the coding regions. As will be discussed in more detail in Sect. 3.3 , recombination between GBA and GBAP has led to different mutations. To date at least 400 mutations in GBA have been listed in the Human Gene Mutation Database (HGMD), http://www.hgmd.cf.ac.uk/ac/gene.php?gene=GBA .

3.2 GBA Mutations

Mutant GBA alleles arise via different mechanisms. They include missense mutations (most common), nonsense mutations, small insertions or deletions that may result in frameshifts or in-frame alterations, splice junction mutations, and complex alleles carrying more than one mutation in cis. Although not the

Complexity of Genotype-Phenotype Correlations in Mendelian Disorders…

78

standard convention, GBA mutations are commonly described based on the position in the nucleotide sequence of the cDNA, where the alanine of the fi rst residue is designated as position 1 of the mature protein [ 54 ]. For instance, N370S mutation is the change of asparagine at the 370th amino acid to serine, and 84GG (c.84insG) is the insertion of a second guanine at the 84th nucleotide of the cDNA sequence.

It is not always easy to detect mutant GBA alleles because of the highly homolo-gous pseudogene sequence. Amplifi cation primers must be designed to distinguish the functional gene from the pseudogene. The fi rst two GBA mutations described were L444P (c.1448T > C) and N370S [ 50 ]. Four mutated GBA alleles (N370S, L444P, 84GG and IVS2 + 1 g > a) account for approximately 90 % of mutant alleles among Ashkenazi Jewish patients; however, in non-Jewish populations, these mutant alleles make up only around 50–60 % of mutations [ 55 ].

The same GBA mutations resulting in Gaucher disease have recently been dis-covered to be risk factors for the development of Parkinson disease and dementia with Lewy bodies. Parkinsonism occurs more commonly among both Gaucher homozygotes and heterozygotes [ 56 , 57 ]. In autopsy studies, glucocerebrosidase was detected in 32–90 % of the Lewy bodies in brain tissue samples from subjects with Parkinsonism who carry a GBA mutation, further supporting the link between GBA and Parkinsonism [ 32 ]. In addition, other studies suggest that GBA may play a role in immune regulation where the accumulation of substrates due to mutant GBA can result in an extensive immune dysregulation [ 58 ].

3.3 The GBA Pseudogene

Approximately 16 kb downstream of GBA is a 5.7 kb highly homologous pseu-dogene which has the same pattern of exons and introns as GBA . While gener-ally, rearrangement takes place between non-sister chromatids of homologous chromosomes, the high sequence homology between GBA and PGBA renders this sequence prone to cross-over, generating different recombinant alleles. These “complex” alleles in the homozygous state are generally associated with severe and lethal forms of Gaucher disease. Recombination events can introduce deletions, duplications, inversions, and gene fusions [ 59 , 60 ]. The recombinant alleles can be formed in at least three different ways. Two mechanisms of recom-bination involve gene conversion and a double unequal cross over, where PGBA functions as the sequence donor. As a result, a segment of the pseudogene is surrounded on both sides by the GBA sequence. The third mechanism occurs when a single non-homologous crossover causes a major rearrangement in the locus encompassing both GBA and PGBA [ 61 ]. Such recombination events occurring at the GBA gene region can be considered one source of the complexity encountered in Gaucher disease [ 62 ]. Moreover, the presence of these recombi-nant alleles can lead to mistakes in genotyping if the laboratory is not familiar with these mutations.

N. Moaven et al.

79

3.4 Phenotypic Variation in Gaucher Disease Is Not Always Explained by Genotype

Knowledge of genotype-phenotype correlation can assist genetic counselors to advise at-risk couples [ 63 ]. Also, genotype-phenotype studies are crucial for understanding the foundation of clinical variation within a specifi c disease. Studies have confi rmed that in Gaucher disease and in other monogenic disorders, there is genotypic hetero-geneity among clinically similar patients. Likewise, patients with the same genotype can have many different phenotypes, making predictions diffi cult [ 1 , 64 – 66 ]. The phe-notypic diversity seen in many monogenic disorders may be due to heterogeneity of mutations, phenotypic heterogeneity due to gene interactions with alleles at different loci, genomic imprinting, and random X-chromosome inactivation [ 67 – 69 ].

In order to understand the complexity of genotype-phenotype correlation in Gaucher disease, investigators fi rst focused on improving the genotyping methods. As discussed in Sect. 3.3 due to the presence of the PGBA, simple PCR-based screening is inadequate and can lead to erroneous genotypes. By designing appro-priate GBA specifi c primers and sequencing all GBA exons, many novel mutations and several polymorphisms have been identifi ed [ 37 , 70 – 72 ].

Initially, there was hope that point mutation knock-in mouse models would help to explain phenotypic heterogeneity in Gaucher disease. However, evalua-tions of different models generated with the same point mutations encountered in patients with Gaucher disease did not result in the anticipated phenotypes [ 67 ]. Neonatal mortality was reported in a mouse homozygous for the “mild” N370S mutation [ 73 ]. On the other hand, inbreeding of mice homozygous for the more “severe” L444P resulted in mice with few symptoms [ 74 ]. These dif-ferences suggest the possible role of PGBA in disease pathogenesis, as murine gba does not have a pseudogene .

Mutations have been categorized into three types: null, severe, and mild, based on the severity of the associated phenotype. Null mutations completely prevent the pro-duction of glucocerebrosidase, and are considered to be lethal. For example, muta-tion 84GG is considered to be a null mutation, and there have been no reported live born homozygotes. Severe mutations such as recombinant alleles result in unstable enzyme with very low catalytic activity. In the homozygous state, these mutations are usually associated with the neuronopathic types of Gaucher disease. Mild mutations result in enzyme with low catalytic activity, but nearly normal stability, and are asso-ciated with the non-neuronopathic form of the disease [ 71 – 77 ]. For example, the mutant enzyme resulting from an N370S mutation has a reduced ability to interact with the GCase activator Saposin C, (Sect. 4 ) and cannot bind effi ciently to anionic phospholipid-containing membranes [ 78 ].

To better establish the degree of clinical correlation among patients with the same genotype, subsets of patients sharing the same mutations have been evaluated. Studies reveal that patients that are homozygous for the D409H (c.1342G > C) mutation or patients carrying this mutation coupled with a null allele generally develop a unique phenotype with involvement of the cardiac valves [ 79 – 81 ].

Complexity of Genotype-Phenotype Correlations in Mendelian Disorders…

80

Mutations N188S (c.680A > G), G377S (c.1246G > A), and V394L (c.1297G > T) on one allele, together with a second null allele are often seen in patients with myo-clonic epilepsy [ 38 , 82 , 83 ]. Previously, homozygosity for L444P was associated with type 2 and 3 Gaucher disease [ 84 , 85 ]; however, recent studies reveal that in type 2 Gaucher disease, usually at least one L444P allele also includes other pseu-dogene sequences in the form of a recombinant allele. Homozygosity for L444P usually is associated with type 3 Gaucher disease or an intermediate phenotype in children [ 86 , 87 ]. Homozygosity for N370S is the most common genotype in Ashkenazi Jews with Gaucher disease. Importantly, mutation N370S is not encoun-tered in patients with neuronopathic forms of Gaucher disease [ 18 ].

4 The Role of Modifi ers in Gaucher Disease

A modifi er gene is defi ned as any gene that affects the phenotypic expression of a target gene. This can include genes in other loci that interact with the specifi c gene at the RNA or protein level or contiguous genes, located in close proximity to the primary gene [ 88 , 89 ]. It has become increasingly clear that genetic modifi ers impact the phenotypes associated with different Mendelian disorders [ 90 , 91 ]. However, identifying these factors remains a challenge. Genes involved in the meta-bolic pathway of the target genes, and genes involved in post translational process-ing, chaperoning, protein traffi cking, and proteasome function are all potential genetic modifi ers that could have an impact on the phenotype [ 66 ]. Discordant phe-notypes in patients with the same genotype and twin pairs showing divergent phe-notypes implicate the role of modifi ers in Gaucher disease [ 66 , 90 ]. RNAi screens, genome wide association studies (GWAS) and candidate gene approaches have been recently employed in order to identify genetic modifi er [ 91 , 96 ].

Metaxin, SCARB2 , saposin C, the Vitamin D receptor, CLN8, and MSH6 are among the candidate genes that have been explored as genetic modifi ers in patients with GBA mutations.

Metaxin ( MTX ) has been recognized as part of the pre-protein import complex in the outer mitochondrial membrane [ 92 ]. It is located directly downstream of the PGBA with a pseudogene (pMTX) located between GBA and PGBA. Since recom-bination between PGBA and GBA or MTX and MTXP can introduce different mutant alleles [ 60 ] it has been hypothesized that MTX could act as a modifi er for GBA, but there is not any hard evidence for this.

The SCARB2 gene (OMIM ID: 602257), encoding for the transport protein LIMP-2 has been discussed in section “ Myoclonic epilepsy ”. A case study of two brothers with congruent mutations, but different phenotypes confi rmed that SCARB2 can serve as a modifi er for Gaucher disease [ 93 ].

Saposin C (Sap C) is one of four proteins encoded by the gene for prosaposin ( PSAP ) (OMIM 176801). Sap C is known to increase glucocerebrosidase activity, working in concert to break down glucocerebroside. Its defi ciency results in a rare disorder that mimics neuronopathic Gaucher disease (OMIM 610539), although patients have normal glucocerebrosidase activity [ 94 ].

N. Moaven et al.

81

Other studies have explored whether mutations or polymorphisms in different candidate genes including the Vitamin D receptor, ceroid-lipofuscinosis neuronal 8 ( CLN8), and MSH6 could function as modifi ers for Gaucher disease. Polymorphisms in the Vitamin D receptor are hypothesized to be an independently sorting modifi er corresponding to the severity of bone mineral density and bone involvement in type 1 Gaucher disease [ 95 ]. A recent GWAS conducted among individuals with Gaucher disease suggested that CLN8 could be a modifi er gene for Gaucher disease. CLN8 encodes a transmembrane protein which has a role in lipid synthesis, transport, or sensing. CLN8 might serve as a protective sphingolipid sensor in glycosphingolipid traffi cking [ 96 ]. Mutations in the MSH6 gene, encoding the mutS homolog 6, leads to a constitutional mismatch repair defi ciency syndrome and enhances the risk for malignancy. Genome analysis through exome capture and parallel sequencing has identifi ed MSH6 as a genetic modifi er, contributing to malignancies in some indi-viduals with Gaucher disease [ 46 ].

5 Diagnosis, and Treatment of Gaucher Disease

5.1 Diagnosis

Early diagnosis of Gaucher disease can be crucial, both for genetic counseling, and to avoid complications of the disease. Testing can be performed prenatally, during cho-rionic villus sampling or amniocentesis. In some instances, early treatment may help to prevent failure-to-thrive in children, complications of splenomegaly or bony involvement such as avascular necrosis of head of the femur. The “gold standard” still used for the diagnosis of Gaucher disease is low enzymatic activity of glucocerebro-sidase in peripheral blood leukocytes or other nucleated cells, compared to levels in normal control samples taken on the same day [ 97 ]. Historically, bone marrow exami-nation was used to make this diagnosis, revealing the presence of lipid- engorged, PAS positive macrophages known as “Gaucher cells”. This procedure is no longer encour-aged because the test is unnecessarily invasive, and because Gaucher-like cells can be observed in other diseases like lymphoblastic leukemia, Hodgkin’s disease, thalas-semia, and multiple myeloma [ 98 ]. More recently, DNA analysis has been used for diagnosis. However, screening for specifi c common mutation has limited utility in the non-Ashkenazi Jewish population, as many mutations can be rare or private. Gene sequencing, while cumbersome, is far more accurate [ 99 ].

5.2 Treatment

There is no cure for Gaucher disease, but there are currently two different FDA approved treatment strategies: Enzyme Replacement Therapy (ERT) and Substrate Reduction Therapy (SRT). Before these two treatments were widely available, in

Complexity of Genotype-Phenotype Correlations in Mendelian Disorders…

82

some cases splenectomies or bone marrow transplantation were performed. Other treatments currently under development for Gaucher disease include gene therapy and chemical chaperone therapy.

Enzyme Replacement Therapy

The goal of ERT is to replace the defi cient enzyme, and facilitate the breakdown of stored sphingolipids. The enzyme is infused intravenously, usually weekly or every other week. It is helpful for most patients with hematologic or visceral involvement, successfully increasing hemoglobin levels in 4–6 months, platelet counts, and growth velocity in children. However, it has some drawbacks: since the enzyme does not cross the blood brain barrier, it does not alter CNS manifestations. Moreover, the treatment is extremely costly [ 97 ].

Substrate Reduction Therapy

With SRT, the goal is to reduce the load of the substrate glucosylceramide to a level where residual enzymatic activity is adequate to prevent the accumulation of lipids inside cells. This has largely been approached by using iminosugar derivatives such as NB-DNJ. However, this treatment also has had little utility for neuronopathic Gaucher disease, and many patients fi nd the drug diffi cult to tolerate. Other improved forms of SRT are under development [ 97 , 100 ].

Chemical Chaperone Therapy

Glucocerebrosidase is synthesized in the endoplasmic reticulum. It is then glycosyl-ated, folded, and attains its functional tertiary structure in the lysosome. However, many mutated forms of the enzyme are misfolded and degraded, and thus never reach the lysosome. The goal of chemical chaperone therapy is to bind to the active site and enhance folding, allowing delivery of the enzyme to the lysosome. Several iminosugar derivatives have been considered as potential pharmacological chaper-ones. More recently screens of large libraries of small molecules have been per-formed, identifying several new lead compounds that may prove to be good chaperones, including some compounds that are non-inhibitory. Studies suggest that chemical chaperones may partially correct the enzyme defi ciency in Gaucher disease and other LSDs [ 101 , 102 ].

6 Conclusions

Gaucher disease is a prime example of a recessive Mendelian disorder characterized by vast clinical variability. Over 350 different mutations have been identifi ed in GBA , a relatively small gene. While some of the phenotypes can be explained by the

N. Moaven et al.

83

associated genotype, patients with the identical mutations can manifest with very different symptoms and disease presentations. One lesson from the genotype- phenotype studies is to treasure your exceptions. Efforts at understanding these exceptions led to a better understanding of the complexity of the GBA gene locus and the need for careful genotyping. Moreover, studies of genotype-phenotype correla-tion resulted in the identifi cation of new disease phenotypes, which have contributed to new insights into the role of glucocerebrosidase in human. For example, the appre-ciation of neonatal lethal Gaucher disease with ichthyosis contributed to our under-standing of the role of glucocerebrosidase in the maintenance of the epidermal barrier. The identifi cation of patients with Parkinson disease led to a recognition of the importance of lysosomal pathways in the pathogenesis of Parkinsonism.

In the new era of individualized medicine and whole exome or genome sequenc-ing, we are likely to discover many unanticipated phenotypes associated with genes assigned to specifi c disorders. At fi rst, it will not be clear what is coincidental and what changes indicate a broadening of the disease phenotype. Proteins will likely have different roles in different target organs or pathways. We are only just begin-ning to appreciate the vast complexity of our genetic architecture, and great care must be taken before drawing conclusions regarding the contribution of individual genes to phenotype.

Acknowledgements This work was supported by the Intramural Research Programs of the National Human Genome Research Institute and the National Institutes of Health. We acknowl-edge the assistance of Julia Fekecs in the preparation of the fi gure.

Glossary of Genetic Terms

Allelic Heterogeneity Different mutated alleles in a same gene can result in the same phenotype or symptom of a trait or a disorder.

Autosomal Dominant Autosomal dominant disorders occur through the inheritance of a single copy of a mutated gene found on an autosomal chromosome (non-sex chromosome). The single defective allele is suffi cient to result in the phenotype.

Autosomal Recessive For an autosomal recessive disorder to occur, both copies (alleles) of the gene must be mutated. If only one allele is mutated, the product normal allele is considered to be suffi cient to protect the individual from having the disorder, but such individual is considered to be a carrier of the condition.

Co-Dominant Co-dominant inheritance occurs when both alleles are expressed, and contribute to a phenotype.

Epigenetics Epigenetics results from changes in the regulation of the expression of a gene without an alteration in the genetic structure. A common epigenetic modifi cation is methylation, where a methyl group binds to segments of DNA and turns off the gene so that no transcription results.

Exome The exome includes all of the coding exons of genes. This accounts for 1.5 % (50 Mb) of the human genome. Whole exome sequencing is used to screen all of a patient’s coding regions to identify mutations in genes.

Complexity of Genotype-Phenotype Correlations in Mendelian Disorders…

84

Genotype A specifi c set of alleles inherited at a locus, or the two alleles inherited for a particular gene.

Genome-Wide Association Study (GWAS) An approach to compare genetic variant markers across the complete DNA sequence of a group of patients or with those of appropriate controls to in order to identify genetic associations with recognizable traits or a disease. The markers are usually Single Nucleotide Polymorphism (SNP).

Inherited Diseases Diseases caused by mutations in genes or chromosomal abnor-malities. A genetic disorder may or may not be a heritable disorder . Some genetic disorders are passed down from the parents’ genes, but others are almost always caused by new mutations or changes in DNA packaging.

Imprinting Maternal and/or paternal chromosomes are uniquely modifi ed and lead to different expression of a certain gene or genes.

Monogenic Disorder These disorders are the result of a single mutated gene that can be passed on to subsequent generations in several ways (recessive, dominant, X-linked and co-dominant).

Mutation An alteration in the native sequence of a gene. A mutation may be dis-ease-causing or a benign, normal variant. Mutations can be introduced during cell division by many factors such as radiation, mutagenic chemicals, or from infection by viruses. De novo mutations are new changes in a gene that occur in a germ cell (egg or sperm). Private mutations are mutations that are found in single families or isolated populations.

Penetrance A condition (most commonly inherited in an autosomal dominant manner) is said to have complete penetrance if clinical symptoms are present in all individuals who have the disease-causing mutation, and to have reduced or incomplete penetrance if clinical symptoms are not always present in all indi-viduals who have the disease-causing mutation.

Phenotype The entire clinical, biochemical and physiological presentation of an individual determined both by a particular genotype and environmental infl uences.

Pleiotropy Several unrelated physical symptoms caused by a single mutant allele or both alleles.

Polymorphisms Natural variations in the DNA sequence of a gene or chromosome that have no adverse effects on the individual, and occur with high frequency in the general population. Polymorphisms involve one of two or more variants of a particular DNA sequence. The most common type of polymorphism is called a single nucleotide polymorphism, or SNP.

Pseudogene An incomplete copy of a gene which it does not have essential DNA sequence segments necessary for being a functional gene. A non- processed pseu-dogene includes most introns and exons of the gene. Integration of the cDNA (reverse transcription of an mRNA) of a gene into the genomic sequence results in a processed pseudogene and can occur during the course of evolution.

Recombinant Allele The result of the exchange of a segment of sister chromatid DNA between two homologous chromosomes during meiosis by a cross- over event, resulting to a new combination of genetic material in the offspring. This phenomenon is an important cause of the genetic variation seen among offspring.

N. Moaven et al.

85

RNA Interference (RNAi) An evolutionary process where small, double stranded RNA (dsRNA, 21–23 nucleotides) molecules inhibit or silence the expression or activity of a gene.

Sex-Linked Traits The traits or the disorders that their responsible genes are located on the sex chromosome (X or Y). Most of the genes are located on Y chromosome (one of the smallest chromosome) are also present on X chromo-some. Therefore, the majority of sex-linked traits or disorders are X-linked. More than 1,000 human X-linked genes are known.

Variable Expressivity Individuals with the same mutation, even within a family, may demonstrate variation in clinical features (type and severity) of a genetic disorder.

References

1. Dipple KM, McCabe ER (2000) Phenotypes of patients with “simple” Mendelian disorders are complex traits: thresholds, modifi ers, and systems dynamics. Am J Hum Genet 66(6):1729–1735

2. Dermitzakis ET, Clark AG (2009) Genetics. Life after GWA studies. Science 326(5950):239–240

3. Manolio TA (2010) Genomewide association studies and assessment of the risk of disease. N Engl J Med 363(2):166–176

4. Antonarakis SE, Chakravarti A, Cohen JC, Hardy J (2010) Mendelian disorders and multifac-torial traits: the big divide or one for all? Nat Rev Genet 11(5):380–384

5. Arora P, Newton-Cheh C (2010) Blood pressure and human genetic variation in the general population. Curr Opin Cardiol 25(3):229–237

6. Cox TM (2003) Future perspectives for glycolipid research in medicine. Philos Trans R Soc Lond B Biol Sci 358(1433):967–973

7. McClellan J, King MC (2010) Genetic heterogeneity in human disease. Cell 141(2):210–217

8. Gaucher PCE (1882) De l’epithelioma primitif de la rate, hypertrophie idiopathique de la rate sans leucemie. Thesis, University of Paris, Paris

9. Brill NE, Mandlebaum FS (1913) Large-cell splenomegaly (Gaucher’s disease): a clinical and pathological study. Am J Med Sci 146(6):863–882

10. Boven LA, van Meurs M, Boot RG, Mehta A, Boon L, Aerts JM, Laman JD (2004) Gaucher cells demonstrate a distinct macrophage phenotype and resemble alternatively activated mac-rophages. Am J Clin Pathol 122:359–369

11. Brady RO (1966) The sphingolipidoses. N Engl J Med 275(6):312–318 12. Barton NW, Brady RO, Dambrosia JM, Di Bisceglie AM, Doppelt SH, Hill SC, Mankin HJ,

Murray GJ, Parker RI, Argoff CE, Grewal RP, Yu KT (1991) Replacement therapy for inher-ited enzyme defi ciency–macrophage-targeted glucocerebrosidase for Gaucher’s disease. N Engl J Med 324(21):1464–1470

13. Beutler E, Grabowski G (2001) Gaucher disease. In: Scriver CR, Beaudet al, Sly WS, Valle D (eds) The metabolic and molecular bases of inherited disease, 8th edn. McGraw-Hill, New York, pp 3635–3668

14. Fisher ER, Reidbord H (1962) Gaucher’s disease: pathogenetic considerations based on elec-tron microscopic and histochemical observations. Am J Pathol 41(6):679–692

15. Sidransky E (2012) Gaucher disease: insights from a rare Mendelian disorder. Discov Med 14(77):273–281

16. Martin BM, Sidransky E, Ginns EI (1989) Gaucher’s disease: advances and challenges. Adv Pediatr 36:277–306

Complexity of Genotype-Phenotype Correlations in Mendelian Disorders…

86

17. Goker-Alpan O, Hruska KS, Orvisky E, Kishnani PS, Stubblefi eld BK, Schiffmann R, Sidransky E (2005) Divergent phenotypes in Gaucher disease implicate the role of modifi ers. J Med Genet 42(6):e37

18. Mistry PK, Weinthal JA, Weinreb NJ (2012) Disease state awareness in Gaucher disease: a Q&A expert roundtable discussion. Clin Adv Hematol Oncol 10(6 Suppl 8):1–16

19. Zimran A, Altarescu G, Rudensky B, Abrahamov A, Elstein D (2005) Survey of hematologi-cal aspects of Gaucher disease. Hematology 10(2):151–156

20. Mankin HJ, Rosenthal DI, Xavier R (2001) Gaucher disease. New approaches to an ancient disease. J Bone Joint Surg Am 83-A(5):748–762

21. Maas M, Poll LW, Terk MR (2002) Imaging and quantifying skeletal involvement in Gaucher disease. Br J Radiol 75(Suppl 1):A13–A24

22. Goker-Alpan O (2011) Therapeutic approaches to bone pathology in Gaucher disease: past, present and future. Mol Genet Metab 104(4):438–447

23. Mikosch P, Hughes D (2010) An overview on bone manifestations in Gaucher disease. Wien Med Wochenschr 160(23–24):609–624

24. Cox TM, Aerts JM, Belmatoug N, Cappellini MD, vom Dahl S, Goldblatt J, Grabowski GA, Hollak CE, Hwu P, Maas M, Martins AM, Mistry PK, Pastores GM, Tylki-Szymanska A, Yee J, Weinreb N (2008) Management of non-neuronopathic Gaucher disease with special refer-ence to pregnancy, splenectomy, bisphosphonate therapy, use of biomarkers and bone disease monitoring. J Inherit Metab Dis 31(3):319–336

25. Hughes DA, Pastores GM (2013) Haematological manifestations and complications of Gaucher disease. Curr Opin Hematol 20(1):41–47

26. Lewis S (2001) Gaucher’s disease. Nose bleeds and bruising. Lancet 358 Suppl:S30 27. Zimran A, Morris E, Mengel E, Kaplan P, Belmatoug N, Hughes DA, Malinova V, Heitner R,

Sobreira E, Mrsić M, Granovsky-Grisaru S, Amato D, vom Dahl S (2009) The female Gaucher patient: the impact of enzyme replacement therapy around key reproductive events (menstruation, pregnancy and menopause). Blood Cells Mol Dis 43(3):264–288

28. Givol N, Goldstein G, Peleg O, Shenkman B, Zimran A, Elstein D, Kenet G (2012) Thrombocytopenia and bleeding in dental procedures of patients with Gaucher disease. Haemophilia 18(1):117–121

29. Sidransky E, Nalls MA, Aasly JO, Aharon-Peretz J, Annesi G, Barbosa ER, Bar-Shira A, Berg D, Bras J, Brice A, Chen CM, Clark LN, Condroyer C, De Marco EV, Dürr A, Eblan MJ, Fahn S, Farrer MJ, Fung HC, Gan-Or Z, Gasser T, Gershoni-Baruch R, Giladi N, Griffi th A, Gurevich T, Januario C, Kropp P, Lang AE, Lee-Chen GJ, Lesage S, Marder K, Mata IF, Mirelman A, Mitsui J, Mizuta I, Nicoletti G, Oliveira C, Ottman R, Orr-Urtreger A, Pereira LV, Quattrone A, Rogaeva E, Rolfs A, Rosenbaum H, Rozenberg R, Samii A, Samaddar T, Schulte C, Sharma M, Singleton A, Spitz M, Tan EK, Tayebi N, Toda T, Troiano AR, Tsuji S, Wittstock M, Wolfsberg TG, Wu YR, Zabetian CP, Zhao Y, Ziegler SG (2009) Multicenter analysis of glucocerebrosidase mutations in Parkinson’s disease. N Engl J Med 361(17):1651–1661

30. Yap TL, Gruschus JM, Velayati A, Westbroek W, Goldin E, Moaven N, Sidransky E, Lee JC (2011) Alpha-synuclein interacts with Glucocerebrosidase providing a molecular link between Parkinson and Gaucher diseases. J Biol Chem 286(32):28080–28088

31. Goker-Alpan O, Stubblefi eld BK, Giasson BI, Sidransky E (2010) Glucocerebrosidase is pres-ent in α-synuclein inclusions in Lewy body disorders. Acta Neuropathol 120(5):641–649

32. Sidransky E, Lopez G (2012) The link between the GBA gene and Parkinsonism. Lancet Neurol 11(11):986–998

33. Nalls MA, Duran R, Lopez G, Kurzawa-Akanbi M, McKeith IG, Chinnery PF, Morris CM, Theuns J, Crosiers D, Cras P, Engelborghs S, De Deyn PP, Van Broeckhoven C, Mann DM, Snowden J, Pickering-Brown S, Halliwell N, Davidson Y, Gibbons L, Harris J, Sheerin UM, Bras J, Hardy J, Clark L, Marder K, Honig LS, Berg D, Maetzler W, Brockmann K, Gasser T, Novellino F, Quattrone A, Annesi G, De Marco EV, Rogaeva E, Masellis M, Black SE, Bilbao JM, Foroud T, Ghetti B, Nichols WC, Pankratz N, Halliday G, Lesage S, Klebe S, Durr A, Duyckaerts C, Brice A, Giasson BI, Trojanowski JQ, Hurtig HI, Tayebi N, Landazabal C,

N. Moaven et al.

87

Knight MA, Keller M, Singleton AB, Wolfsberg TG, Sidransky E (2013) A multicenter study of glucocerebrosidase mutations in dementia with Lewy bodies. JAMA Neurol 70:727–735

34. Stepanian SV, Huyn ST, McCabe ER, Dipple KM (2003) Characterization of the human glycerol kinase promoter: identifi cation of a functional HNF-4alpha binding site and evi-dence for transcriptional activation. Mol Genet Metab 80(4):412–418

35. Kluijtmans LA, Whitehead AS (2001) Methylenetetrahydrofolate reductase genotypes and predisposition to atherothrombotic disease; evidence that all three MTHFR C677T genotypes confer different levels of risk. Eur Heart J 22(4):294–299

36. Jonsson T, Stefansson H, Steinberg S, Jonsdottir I, Jonsson PV, Snaedal J, Bjornsson S, Huttenlocher J, Levey AI, Lah JJ, Rujescu D, Hampel H, Giegling I, Andreassen OA, Engedal K, Ulstein I, Djurovic S, Ibrahim-Verbaas C, Hofman A, Ikram MA, van Duijn CM, Thorsteinsdottir U, Kong A, Stefansson K (2013) Variant of TREM2 associated with the risk of Alzheimer’s disease. N Engl J Med 368(2):107–116

37. Park JK, Tayebi N, Stubblefi eld BK, LaMarca ME, MacKenzie JJ, Stone DL, Sidransky E (2002) The E326K mutation and Gaucher disease: mutation or polymorphism? Clin Genet 61(1):32–34

38. Tajima A, Ohashi T, Hamano S, Higurashi N, Ida H (2010) Gaucher disease patient with myoclonus epilepsy and a novel mutation. Pediatr Neurol 42(1):65–68

39. Verghese J, Goldberg RF, Desnick RJ, Grace ME, Goldman JE, Lee SC, Dickson DW, Rapin I (2000) Myoclonus from selective dentate nucleus degeneration in type 3 Gaucher disease. Arch Neurol 57:389–395

40. de Siqueira LF (2010) Progressive myoclonic epilepsies: review of clinical, molecular and therapeutic aspects. J Neurol 257:1612–1619

41. Blanz J, Groth J, Zachos C, Wehling C, Saftig P, Schwake M (2010) Disease-causing muta-tions within the lysosomal integral membrane protein type 2 (LIMP-2) reveal the nature of binding to its ligand beta-glucocerebrosidase. Hum Mol Genet 19(4):563–572

42. Dardis A, Filocamo M, Grossi S, Ciana G, Franceschetti S, Dominissini S, Rubboli G, Di Rocco M, Bembi B (2009) Biochemical and molecular fi ndings in a patient with myoclonic epilepsy due to a mistarget of the beta-glucosidase enzyme. Mol Genet Metab 97(4):309–311

43. Balreira A, Gaspar P, Caiola D, Chaves J, Beirao I, Lima JL, Azevedo JE, Miranda MC (2008) A nonsense mutation in the LIMP-2 gene associated with progressive myoclonic epi-lepsy and nephrotic syndrome. Hum Mol Genet 17(14):2238–2243

44. Choy FY, Campbell TN (2011) Gaucher disease and cancer: concept and controversy. Int J Cell Biol 2011:150450

45. Allen MJ, Myer BJ, Khokher AM, Rushton N, Cox TM (1997) Pro-infl ammatory cytokines and the pathogenesis of Gaucher’s disease: increased release of interleukin-6 and interleukin- 10. QJM 90(1):19–25

46. Lo SM, Choi M, Liu J, Jain D, Boot RG, Kallemeijn WW, Aerts JM, Pashankar F, Kupfer GM, Mane S, Lifton RP, Mistry PK (2012) Phenotype diversity in type 1 Gaucher disease: discovering the genetic basis of Gaucher disease/hematologic malignancy phenotype by indi-vidual genome analysis. Blood 119(20):4731–4740

47. Meikle PJ, Hopwood JJ, Clague AE, Carey WF (1999) Prevalence of lysosomal storage dis-orders. JAMA 281(3):249–254

48. Fairley C, Zimran A, Phillips M, Cizmarik M, Yee J, Weinreb N, Packman S (2008) Phenotypic heterogeneity of N370S homozygotes with type I Gaucher disease: an analysis of 798 patients from the ICGG Gaucher Registry. J Inherit Metab Dis 31(6):738–744

49. Mehta A (2006) Epidemiology and natural history of Gaucher’s disease. Eur J Intern Med 17(Suppl):S2–S5

50. Benko WS, Hruska KS, Nagan N, Goker-Alpan O, Hart PS, Schiffmann R, Sidransky E (2008) Uniparental disomy of chromosome 1 causing concurrent Charcot-Marie-Tooth and Gaucher disease Type 3. Neurology 70(12):976–978

51. Saranjam H, Chopra SS, Levy H, Stubblefi eld BK, Maniwang E, Cohen IJ, Baris H, Sidransky E, Tayebi N (2013) A germline or de novo mutation in two families with Gaucher disease: implications for recessive disorders. Eur J Hum Genet 21(1):115–117

Complexity of Genotype-Phenotype Correlations in Mendelian Disorders…

88

52. Barneveld RA, Keijzer W, Tegelaers FP, Ginns EI, Geurts van Kessel A, Brady RO, Barranger JA, Tager JM, Galjaard H, Westerveld A, Reuser AJ (1993) Assignment of the gene coding for human beta-glucocerebrosidase to the region q21-q31 of chromosome 1 using monoclo-nal antibodies. Hum Genet 64:227–231

53. Winfi eld SL, Tayebi N, Martin BM, Ginns EI, Sidransky E (1997) Identifi cation of three additional genes contiguous to the glucocerebrosidase locus on chromosome 1q21: implica-tions for Gaucher disease. Genome Res 7:1020–1026

54. Hruska KS, LaMarca ME, Scott CR, Sidransky E (2008) Gaucher disease: mutation and polymorphism spectrum in the glucocerebrosidase gene (GBA). Hum Mutat 29(5):567–583

55. Alfonso P, Aznarez S, Giralt M, Pocovi M, Giraldo P (2007) Mutation analysis and genotype/phenotype relationships of Gaucher disease patients in Spain. J Hum Genet 52:391–396

56. Tayebi N, Walker J, Stubblefi eld B, Orvisky E, LaMarca ME, Wong K, Rosenbaum H, Schiffmann R, Bembi B, Sidransky E (2003) Gaucher disease with parkinsonian manifesta-tions: does glucocerebrosidase defi ciency contribute to a vulnerability to parkinsonism? Mol Genet Metab 79(2):104–109

57. Velayati A, Yu WH, Sidransky E (2010) The role of glucocerebrosidase mutations in Parkinson disease and Lewy body disorders. Curr Neurol Neurosci Rep 10(3):190–198

58. Liu J, Halene S, Yang M, Iqbal J, Yang R, Mehal WZ, Chuang WL, Jain D, Yuen T, Sun L, Zaidi M, Mistry PK (2012) Gaucher disease gene GBA functions in immune regulation. Proc Natl Acad Sci U S A 109(25):10018–10023

59. Beutler E, West C (2002) Polymorphisms in glucosylceramide (glucocerebroside) synthase and the Gaucher disease phenotype. Isr Med Assoc J 4(11):986–988

60. Velayati A, Knight MA, Stubblefi eld BK, Sidransky E, Tayebi N (2011) Identifi cation of recombinant alleles using quantitative real-time PCR implications for Gaucher disease. J Mol Diagn 13(4):401–405

61. Martínez-Arias R, Comas D, Mateu E, Bertranpetit J (2001) Glucocerebrosidase pseudogene variation and Gaucher disease: recognizing pseudogene tracts in GBA alleles. Hum Mutat 17(3):191–198

62. Tayebi N, Stubblefi eld BK, Park JK, Orvisky E, Walker JM, LaMarca ME, Sidransky E (2003) Reciprocal and nonreciprocal recombination at the glucocerebrosidase gene region: implications for complexity in Gaucher disease. Am J Hum Genet 72(3):519–534

63. Grabowski GA (1997) Gaucher disease: gene frequencies and genotype/phenotype correla-tions. Genet Test 1(1):5–12

64. Grabowski GA (2000) Gaucher disease: considerations in prenatal diagnosis. Prenat Diagn 20(1):60–62

65. Scriver CR, Waters PJ (1999) Monogenic traits are not simple: lessons from phenylketonuria. Trends Genet 15(7):267–272

66. Sidransky E (2004) Gaucher disease: complexity in a “simple” disorder. Mol Genet Metab 83(1–2):6–15

67. Wolf U (1991) Identical mutations and phenotypic variation. Hum Genet 100(3–4):305–321 68. Summers KM (1996) Relationship between genotype and phenotype in monogenic diseases:

relevance to polygenic diseases. Hum Mutat 7(4):283–293 69. Todd JA (1999) From genome to aetiology in a multifactorial disease, type 1 diabetes.

Bioessays 21(2):164–174 70. Orvisky E, Park JK, Parker A, Walker JM, Martin BM, Stubblefi eld BK, Uyama E, Tayebi N,

Sidransky E (2002) The identifi cation of eight novel glucocerebrosidase (GBA) mutations in patients with Gaucher disease. Hum Mutat 19(4):458–459

71. Koprivica V, Stone DL, Park JK, Callahan M, Frisch A, Cohen IJ, Tayebi N, Sidransky E (2000) Analysis and classifi cation of 304 mutant alleles in patients with type 1 and type 3 Gaucher disease. Am J Hum Genet 66(6):1777–1786

72. Walker JM, Lwin A, Tayebi N, LaMarca ME, Orvisky E, Sidransky E (2003) Glucocerebrosidase mutation T369M appears to be another polymorphism. Clin Genet 63(3):237–238

73. Xu YH, Quinn B, Witte D, Grabowski GA (2003) Viable mouse models of acid beta- glucosidase defi ciency: the defect in Gaucher disease. Am J Pathol 163(5):2093–2101

N. Moaven et al.

89

74. Liu Y, Suzuki K, Reed JD, Grinberg A, Westphal H, Hoffmann A, Döring T, Sandhoff K, Proia RL (1998) Mice with type 2 and 3 Gaucher disease point mutations generated by a single insertion mutagenesis procedure. Proc Natl Acad Sci U S A 95(5):2503–2508

75. Gan-Or Z, Giladi N, Orr-Urtreger A (2009) Differential phenotype in Parkinson’s disease patients with severe versus mild GBA mutations. Brain 132(Pt 10):e125

76. Gieselmann V (2005) What can cell biology tell us about heterogeneity in lysosomal storage diseases? Acta Paediatr Suppl 94(447):80–86, discussion 79

77. Mao R, O’Brien JF, Rao S, Schmitt E, Roa B, Feldman GL, Spence WC, Snow K (2001) Identifi cation of a 55-bp deletion in the glucocerebrosidase gene in Gaucher disease: phenotypic presentation and implications for mutation detection assays. Mol Genet Metab 72(3):248–253

78. Salvioli R, Tatti M, Scarpa S, Moavero SM, Ciaffoni F, Felicetti F, Kaneski CR, Brady RO, Vaccaro AM (2005) The N370S (Asn370 > Ser) mutation affects the capacity of glucosylce-ramidase to interact with anionic phospholipid-containing membranes and saposin C. Biochem J 390(Pt 1):95–103

79. Pasmanik-Chor M, Laadan S, Elroy-Stein O, Zimran A, Abrahamov A, Gatt S, Horowitz M (1996) The glucocerebrosidase D409H mutation in Gaucher disease. Biochem Mol Med 59(2):125–133

80. Uyama E, Uchino M, Ida H, Eto Y, Owada M (1997) D409H/D409H genotype in Gaucher- like disease. J Med Genet 34(2):175

81. Chabás A, Cormand B, Grinberg D, Burguera JM, Balcells S, Merino JL, Mate I, Sobrino JA, Gonzàlez-Duarte R, Vilageliu L (1995) Unusual expression of Gaucher’s disease: cardiovascular calcifi cations in three sibs homozygous for the D409H mutation. J Med Genet 32(9):740–742

82. Park JK, Orvisky E, Tayebi N, Kaneski C, Lamarca ME, Stubblefi eld BK, Martin BM, Schiffmann R, Sidransky E (2003) Myoclonic epilepsy in Gaucher disease: genotype- phenotype insights from a rare patient subgroup. Pediatr Res 53(3):387–395

83. Kowarz L, Goker-Alpan O, Banerjee-Basu S, LaMarca ME, Kinlaw L, Schiffmann R, Baxevanis AD, Sidransky E (2005) Gaucher mutation N188S is associated with myoclonic epilepsy. Hum Mutat 26(3):271–3; author reply 274–5

84. Goker-Alpan O, Schiffmann R, Park JK, Stubblefi eld BK, Tayebi N, Sidransky E (2003) Phenotypic continuum in neuronopathic Gaucher disease: an intermediate phenotype between type 2 and type 3. J Pediatr 143(2):273–276

85. Latham TE, Theophilus BD, Grabowski GA, Smith FI (1991) Heterogeneity of mutations in the acid beta-glucosidase gene of Gaucher disease patients. DNA Cell Biol 10(1):15–21

86. Grabowski GA, Horowitz M (1997) Gaucher’s disease: molecular, genetic and enzymologi-cal aspects. Baillieres Clin Haematol 10(4):635–656

87. Panicker LM, Miller D, Park TS, Patel B, Azevedo JL, Awad O, Masood MA, Veenstra TD, Goldin E, Stubblefi eld BK, Tayebi N, Polumuri SK, Vogel SN, Sidransky E, Zambidis ET, Feldman RA (2012) Induced pluripotent stem cell model recapitulates pathologic hallmarks of Gaucher disease. Proc Natl Acad Sci U S A 109(44):18054–18059

88. Romeo G, McKusick VA (1994) Phenotypic diversity, allelic series and modifi er genes. Nat Genet 7(4):451–453

89. Latham T, Grabowski GA, Theophilus BD, Smith FI (1990) Complex alleles of the acid beta- glucosidase gene in Gaucher disease. Am J Hum Genet 47(1):79–86

90. Lachmann RH, Grant IR, Halsall D, Cox TM (2004) Twin pairs showing discordance of phenotype in adult Gaucher’s disease. QJM 97(4):199–204

91. Kissler S (2011) From genome-wide association studies to etiology: probing autoimmunity genes by RNAi. Trends Mol Med 17(11):634–640

92. Armstrong LC, Komiya T, Bergman BE, Mihara K, Bornstein P (1997) Metaxin is a compo-nent of a preprotein import complex in the outer membrane of the mammalian mitochon-drion. J Biol Chem 272(10):6510–6518

93. Velayati A, DePaolo J, Gupta N, Choi JH, Moaven N, Westbroek W, Goker-Alpan O, Goldin E, Stubblefi eld BK, Kolodny E, Tayebi N, Sidransky E (2011) A mutation in SCARB2 is a modifi er in Gaucher disease. Hum Mutat 32(11):1232–1238

Complexity of Genotype-Phenotype Correlations in Mendelian Disorders…

90

94. Tamargo RJ, Velayati A, Goldin E, Sidransky E (2012) The role of saposin C in Gaucher disease. Mol Genet Metab 106(3):257–263

95. Greenwood A, Elstein D, Zimran A, Altarescu G (2010) Effect of vitamin D receptor (VDR) genotypes on the risk for osteoporosis in type 1 Gaucher disease. Clin Rheumatol 29(9):1037–1041

96. Zhang CK, Stein PB, Liu J, Wang Z, Yang R, Cho JH, Gregersen PK, Aerts JM, Zhao H, Pastores GM, Mistry PK (2012) Genome-wide association study of N370S homozygous Gaucher disease reveals the candidacy of CLN8 gene as a genetic modifi er contributing to extreme phenotypic variation. Am J Hematol 87(4):377–383

97. Zimran A (2011) How I treat Gaucher disease. Blood 118(6):1463–1471 98. Saroha V, Gupta P, Singh M, Singh T (2009) Pseudogaucher cells obscuring multiple

myeloma: a case report. Cases J 2:9147 99. Sidransky E, Bottler A, Stubblefi eld B, Ginns EI (1994) DNA mutational analysis of type 1

and type 3 Gaucher patients: how well do mutations predict phenotype? Hum Mutat 3(1):25–28

100. McEachern KA, Fung J, Komarnitsky S, Siegel CS, Chuang WL, Hutto E, Shayman JA, Grabowski GA, Aerts JM, Cheng SH, Copeland DP, Marshall J (2007) A specifi c and potent inhibitor of glucosylceramide synthase for substrate reduction therapy of Gaucher disease. Mol Genet Metab 91(3):259–267

101. Benito JM, García Fernández JM, Ortiz Mellet C (2011) Pharmacological chaperone therapy for Gaucher disease: a patent review. Expert Opin Ther Pat 21(6):885–903

102. Goldin E, Zheng W, Motabar O, Southall N, Choi JH, Marugan J, Austin CP, Sidransky E (2012) High throughput screening for small molecule therapy for Gaucher disease using patient tissue as the source of mutant glucocerebrosidase. PLoS One 7(1):e29861

N. Moaven et al.

91M. Özgüç (ed.), Rare Diseases: Integrative PPPM Approach as the Medicine of the Future, Advances in Predictive, Preventive and Personalised Medicine 6,DOI 10.1007/978-94-017-9214-1_7, © Springer Science+Business Media Dordrecht 2015

Abstract Lysosomal storage disorders are inherited metabolic disorders resulting from progressive accumulation of non-recycled compounds that build-up in the lysosomes before expanding to most of body tissues and organs. Impaired enzyme activity, molecular traffi cking and transport of these proteins resulting from genetic mutations constitute the main pathogenic mechanisms. Clinical manifestations include storage signs and symptoms such as enlarged liver and spleen, coarse fea-tures, skeletal deformities and many of them are associated with a neurodegenera-tive course. Clinical suspicion can be supported by detection of accumulation of abnormal compounds such as mucopolysaccharides, oligosaccharides, sialic acid and free cholesterol in body fl uids and tissues, confi rmed by enzymatic assays and molecular testing allowing also prenatal diagnosis and genetic counseling. Management of lysosomal storage disorders can be symptomatic but also specifi c for some of them with two main treatment modalities: hematopoietic stem cell transplantation and enzyme replacement therapy. Despite recent progress in the fi eld, access of these therapies to key organs such as the brain and bone remain chal-lenging and may be addressed in the near future by original or complementary approaches including molecular chaperones, substrate inhibitors and gene therapy. From this perspective, medical awareness and early detection constitute the corner-stones for early intervention and hope for a better outcome.

Keywords Lysosomal storage disease • Enzyme replacement therapy • Muco-polysaccharidosis type I • Mucopolysaccharidosis type II • Mucopolysaccharidosis type VI • Gaucher disease • Pompe disease • Fabry disease • Personalized medicine

Enzyme Replacement Therapy in Lysosomal Storage Diseases

Vassili Valayannopoulos

V. Valayannopoulos (*) Reference Center for Inherited Metabolic Disease in Children and Adults (MaMEA) and IMAGINE Institute , Necker-Enfants Malades Hospital and Paris Descartes University , 149, Rue de Sèvres, 75743 Paris Cedex 15 , France e-mail: vassili.valaya@nck.aphp.fr; vassilival@gmail.com

92

Abbreviations

(rh)GAA (recombinant) a-glucosidase CNS Central nervous system CRIM Cross-reactive immunological material ERT Enzyme replacement therapy FVC Forced vital capacity GAG Glycosaminoglycans GSD Glycogen storage disease HSCT Hematopoietic stem-cell transplantation i.v. Intravenous IAR Infusion-associated reaction LSD Lysosomal storage disease MPS Mucopolysaccharidosis MRI Magnetic resonance imaging U Units

1 Introduction

Lysosomes are cell organelles responsible for the recycling of cellular compounds operating in an acidic environment. Inborn errors of metabolism secondary to lysosomal enzyme defi cits are a heterogeneous group characterized by a progres-sive accumulation of non-digested macromolecules responsible for an increase of the size of the organelles, provoking cellular dysfunction that leads to clinical manifestations.

Lysosomal storage diseases (LSD) have been previously classifi ed according to the accumulated substrate (mucopolysaccharidoses, glycoproteinoses, mucolipidoses, sphingolipidoses). The current classifi cation takes into account various pathophysi-ological mechanisms other than isolated enzymopathies. The diagnosis of these diseases can be confi rmed easily in most cases by immunoenzymatic techniques and molecular biology. Some of them could be accessible to neonatal screening. On the molecular level, a great heterogeneity exists in spite of similarities with respect to clinical and biochemical phenotype and enzyme activity.

Even though these enzymatic defi cits result in an accumulation of pathological substrates, the underlying mechanisms responsible for the pathogenesis of the disease are not entirely known. Nevertheless the distribution of the accumulated material determines the affected organs. More particularly, in the central nervous system (CNS), neurons are often involved owing to the accumulation of storage material and their incapacity for renewal. LSD can be responsible for mental retardation or for a neurodegenerative course in the CNS.

However, more attenuated phenotypes, generally of late onset, have been identifi ed associated with residual enzyme activity. These patients may display a

V. Valayannopoulos

93

non- neuronopathic phenotype. Similarly in several LSD the genotype may be associated with a neurological phenotype.

Hematopoietic stem cell transplantation was the fi rst therapy demonstrating effi cacy especially on the neurological involvement in various LSD, such as mucopolysaccharidoses type I, VI, and VII, mannosidoses, Krabbe’s disease, and metachromatic leukodystrophy [ 1 ].

Enzyme replacement therapy was proposed three decades ago for lysosomal storage disorders, based on the discovery that the storage is caused by defi ciencies of lysosomal degradative enzymes and the realization that the lysosomal interior is functionally contiguous with the extracellular space.

The earliest attempts at enzyme replacement were ineffective owing to an inad-equate understanding of receptor-mediated endocytosis and insuffi cient sources of highly purifi ed enzymes with appropriate markers for targeted uptake. These prob-lems were fi rst solved for Gaucher disease, a defi ciency of glucocerebrosidase that leads to storage predominantly within macrophages. An effective enzyme replace-ment therapy was developed for type I or non-neuronopathic Gaucher disease using highly purifi ed placenta-derived glucocerebrosidase. Sequential deglycosylation of the purifi ed enzyme was used to enhance uptake through mannose receptors of affected tissue macrophages. Effective enzyme replacement therapy in other lyso-somal storage disorders has been more diffi cult to develop at that time, primarily because adequate production of properly processed, purifi ed enzymes requires the creation of recombinant sources. This has been particularly true for the mucopoly-saccharidoses, even though correction of the metabolic defect was achieved in cul-tured cells nearly 30 years ago.

Enzyme replacement therapy is now available for Gaucher disease, Fabry dis-ease, mucopolysaccharidoses type I, type II, and type VI, Pompe disease, and on a clinical trial basis for metachromatic leukodystrophy (MLD), Morquio disease (MPS IV), acid lipase defi ciency (Wolman and Cholesterol Ester Storage Disease) and Sanfi lippo type A disease (MPS IIIA).

2 The Mucopolysaccharidoses

The mucopolysaccharidoses (MPSs) are lysosomal storage disorders caused by the accumulation of sulphated carbohydrate polymers in the lysosomes leading to a cascade of multisystemic disease manifestations. The sulphated polymers are com-posed of a central core protein attached to disaccharide branches deriving from sulphate monosaccharides or glycosaminoglycans (GAGs). The primary storage products are: dermatan sulphate, chiefl y a constituent of conjunctive tissues; hepa-ran sulphate, chiefl y a constituent of cellular membranes; and keratan sulphate and chondroitin sulphate, found abundantly in the cartilages and in the cornea. GAG excretion in urine allows screening for MPSs quantitatively (elevated urinary GAG) and qualitatively (characteristic profi le of sulphate derivatives) [ 2 , 3 ].

Enzyme Replacement Therapy in Lysosomal Storage Diseases

94

Catabolic enzymes responsible for GAG degradation are defective in MPS disorders. Eleven enzymatic defi cits are known to be responsible for seven different diseases (MPS I, II, III, IV, VI, VII and IX). All MPS disorders are progressive, multivisceral diseases that involve the musculoskeletal system (bones and joints), heart, lungs, eyes (cornea, retina and optic nerves), liver and spleen, and in some of the diseases, the CNS [ 2 , 3 ].

During the last several decades, the outlook for patients with MPS disorders has improved considerably, with better understanding of their pathogenesis and natural history, advances in supportive care and fi nally, the availability of disease-specifi c treatments for some of the disorders. Table 1 summarizes current disease-specifi c treatment options for all of the MPS disorders. The two primary treatment modalities are enzyme replacement therapy (ERT) and hematopoietic stem cell transplantation (HSCT), both of which offer substantial benefi t but do not cure the disease.

Due to the progressive nature of these diseases, early diagnosis and early thera-peutic intervention is of major importance. Early treatment is supported by the pathophysiological mechanisms: disease progression is associated with organ damage that occurs through multiple, complex secondary pathways involving GAGs, rather than just GAG accumulation. This secondary damage is often irre-versible. Clinical evidence also points to improved outcome with early interven-tion for MPS I and VI. Sibling case studies of MPS I, II and VI demonstrate much better outcome for younger siblings diagnosed at birth and started on ERT in the fi rst 6 months of life [ 4 – 7 ].

Earlier transplant is also associated with better outcome (lower mortality and morbidity [ 8 ] improved cognitive status [ 9 , 10 ] and a lower incidence of carpal tun-nel syndrome [ 11 ] in children with MPS I).

MPS disorders are best managed by a multidisciplinary team coordinated by a physician with experience in the treatment of these complex disorders. Both sup-portive and disease-specifi c treatments, if available, are important. Anesthetic-risk management due to upper airway obstruction should always be considered before programmed surgery in patients affected with MPS [ 12 ].

Regular follow-up is essential to monitor disease progression and response to treatment [ 13 , 14 ]. It is also important to be aware of the considerable psychosocial burden of these chronic, debilitating and progressive conditions. Family and indi-vidual counselling can be helpful. Additionally, patient societies may provide invaluable networking opportunities for patients and families to share information and connect with others experiencing the same challenges.

The treatment regimen for ERT involves intravenous (i.v.) infusions of the recombinant human enzyme weekly. ERT is a life-long therapy, and each infusion takes 1–4 h depending on the enzyme and the dose. There is a potential for severe infusion reactions; life-threatening anaphylaxis has occurred in some patients receiving ERT. Most infusions are given in a hospital setting because of this risk, but home infusions are reported to be feasible and safe for some patients. The feasibility of home therapy for any MPS patient should be based on a risk-benefi t evaluation by the treating physician, the patient and the patient’s care giver.

V. Valayannopoulos

Tabl

e 1

Cur

rent

ther

apie

s fo

r th

e M

PS d

isor

ders

a

MPS

I (

Hur

ler,

Hur

ler-

Sche

ie, S

chei

e)

MPS

II

(Hun

ter)

M

PS I

II (

Sanfi

lipp

o Sy

ndro

me

type

s A

-D)

MPS

IV

(M

orqu

io

type

s A

and

B)

MPS

VI

(Mar

otea

ux-L

amy)

M

PS V

II (

Sly)

Defi

cie

nt

lyso

som

al

enzy

me

α-L

-idu

roni

dase

Id

uron

ate

sulf

atas

e A

: Hep

aran

N-s

ulfa

tase

A

: Gal

acto

se

6-su

lfat

ase

B:

β-ga

lact

osid

ase

Ary

lsul

fata

se B

ß-

Glu

curo

nida

se

B:α

-N a

ceyt

ylgl

ucos

amin

idas

e C

: Ace

tyl-

CoA

: α-g

luco

sam

inid

e ac

yltr

ansf

eras

e D

: N –

acet

ylgl

ucos

amin

e-6-

sulf

atas

e Su

bstr

ate

accu

mul

ated

D

erm

atan

sul

fate

, he

para

n su

lfat

e D

erm

atan

sul

fate

, he

para

n su

lfat

e H

epar

an s

ulfa

te

Ker

atan

sul

fate

D

erm

atan

sul

fate

D

erm

atan

sul

fate

, he

para

n su

lfat

e C

ogni

tive

sta

tus

Var

ies

from

sev

ere

to n

o im

pair

men

t V

arie

s fr

om s

ever

e to

no

impa

irm

ent

Impa

ired

N

orm

al

Nor

mal

M

ildly

impa

ired

Inhe

rita

nce

Aut

osom

al r

eces

sive

X

-lin

ked

rece

ssiv

e (m

ost p

atie

nts

are

mal

e)

Aut

osom

al r

eces

sive

A

utos

omal

re

cess

ive

Aut

osom

al

rece

ssiv

e A

utos

omal

rec

essi

ve

Est

imat

ed

inci

denc

e (v

arie

s w

/pop

ulat

ion)

~1:1

00,0

00

~1:1

00,0

00

~1:2

5,00

0–75

,000

1:

40,0

00–2

00,0

00

1:24

0,00

0–30

0,00

0 ?

Ava

ilabl

e tr

eatm

ents

E

nzym

e re

plac

emen

t th

erap

y

Lar

onid

ase

(Ald

uraz

yme ®

),

som

atic

ben

efi ts

Idur

sulf

ase

(Ela

pras

e ® )

som

atic

be

nefi t

s

Clin

ical

tria

ls u

nder

way

for

type

A

and

in d

evel

opm

ent f

or ty

pe B

C

linic

al tr

ials

un

derw

ay

Gal

sulf

ase

(Nag

lazy

me ®

),

som

atic

be

nefi t

s

In d

evel

opm

ent

Hem

atop

oiet

ic

stem

cel

l tr

ansp

lant

atio

n

Rec

omm

ende

d fo

r H

urle

r pa

tient

s be

fore

age

2; c

an

pres

erve

cog

nitio

n

Litt

le d

ata,

mix

ed

resu

lts,

neur

ocog

nitiv

e be

nefi t

unc

lear

Litt

le d

ata,

mix

ed r

esul

ts,

neur

ocog

nitiv

e be

nefi t

unc

lear

L

ittle

dat

a, m

ixed

re

sults

L

ittle

dat

a, m

ixed

re

sults

L

ittle

dat

a, m

ixed

re

sults

, ne

uroc

ogni

tive

bene

fi t u

ncle

ar

a MPS

IX

, hya

luro

nida

se d

efi c

ienc

y, is

not

incl

uded

as

it is

ext

rem

ely

rare

. Alth

ough

the

mol

ecul

ar d

efec

t has

bee

n el

ucid

ated

, the

re a

re n

o cu

rren

t the

rapi

es

96

Initially, up to half of patients treated with ERT experience mild to moderate infusion-associated reactions (IARs) such as headache, fl ushing, fever and/or rash. These reactions usually can be managed by pre-treatment with anti-pyretics and/or anti-histamines and may decrease with time. The development of IARs generally coincides with an immune response to the enzyme protein and tends to occur more frequently as dosage increases [ 15 ].

3 ERT for MPS I (Hurler, Hurler-Scheie and Scheie Syndrome)

Laronidase (recombinant human a-L-iduronidase; Genzyme Corporation, Cambridge, MA and BioMarin Pharmaceutical, Inc., Novato, CA, USA) was the fi rst ERT approved for treatment of an MPS disorder and has been available in the USA and Europe since 2003. Four clinical trials have been conducted, encompass-ing patients of all phenotypes and an age range of 0.8–43 years [ 15 – 19 ]. Clinical benefi ts noted in the drug label include increased distance walked in the 6-min walk test, improved per cent predicted forced vital capacity (FVC), decreased liver vol-ume and decreased (but not normalized) urinary GAG levels. Additional benefi ts experienced by the majority of patients in the pivotal randomized placebo-controlled trial and extension, include stabilized or improved joint range of motion, stabilized or decreased sleep apnea, decreased left ventricular hypertrophy and improved quality of life [ 15 – 19 ].

A dose optimization study found that the labelled dose [0.58 mg/kg (100 U)/kg/week] appeared to offer the most favorable risk-benefi t ratio, but that a double dose every 2 weeks could be an acceptable alternative regimen for patients who have diffi culty receiving weekly infusions [ 15 ]. Approximately half of all patients experi-ence at least one IAR and >90 % of patients develop antibodies to laronidase. Life-threatening anaphylactic reactions have occurred in a small number of patients.

Rare disease registries, such as the MPS I Registry [ 20 ] will increasingly be a resource for long-term outcome analyses through longitudinal data. Finally, case reports and case series can provide valuable data by articulating benefi ts or drawbacks for patients and families not captured by trial end points.

Two case series providing data from siblings who began treatment at different ages suggest that initiation of laronidase treatment in infancy, before the development of signifi cant disease manifestations, may improve outcome with respect to musculo-skeletal disease [ 6 ], cardiac valve disease [ 6 ] and brain MRI abnormalities [ 5 ].

In a dog model of MPS, treatment with intrathecal enzyme replacement therapy by monthly or quarterly treatment regimens with laronidase achieved supranormal iduronidase enzyme levels in the brain, spinal cord, and meninges; total brain gly-cosaminoglycan storage was normalized; and spinal meningeal glycosaminoglycan storage was reduced by 58–70 %. This successful use of enzyme therapy via the CSF represents a potentially useful approach for MPS I and for other lysosomal storage disorders [ 21 ].

V. Valayannopoulos

97

4 ERT for MPS II (Hunter Syndrome)

Idursulfase (Shire Human Genetic Therapies, Inc., Cambridge, MA, USA), a recombinant form of human iduronate-2-sulfatase, has been commercially available since 2006. Four clinical trials of idursulfase have been conducted in patients with MPS II, encompassing an age range of 5–53 years [ 22 – 26 ].

No patient in the trials had baseline cognitive impairment. Benefi ts noted in the drug label are improved walking capacity, along with decreased liver and spleen volume and reduction (but not normalization) of urinary GAG levels [ 22 ]. In the pivotal trial, there was also a statistically signifi cant improvement in a composite end point combining walking and respiratory benefi ts as measured by changes in per cent predicted pulmonary forced vital capacity (FVC) [ 22 ]. IARs occurred in over half of clinical trial participants and antibodies developed in 50 % [ 22 , 23 , 26 ].

An analysis of 124 MPS II patients <6 years of age from the Hunter Outcome Survey who were treated with idursulfase identifi ed no new safety concerns [ 25 ]. Life-threatening anaphylactic reactions have occurred in some patients during idur-sulfase infusions as well as biphasic anaphylactic reactions [ 23 ].

The current experience with Elaprase ® confi rmed these data, but also showed the absence of cognitive or neurological improvement in treated patients [ 27 , 28 ]. A safety and dose ranging study of idursulfase (intrathecal) administration via an intrathecal drug delivery device in pediatric patients with Hunter syndrome who have CNS involvement and are receiving treatment with Elaprase ® and a similar phase I-II intrathecal study with recombinant heparan-N-sulfatase in MPSIIIA have been completed recently.

5 ERT for MPS VI (Maroteaux-Lamy)

Galsufase (Biomarin, Novato, CA, USA), a recombinant form of human arylsulpha-tase B, has been available since 2005. Three clinical trials of galsulfase have been conducted in patients with severe disease manifestations ranging in age from 5 to 29 years [ 29 – 33 ]. Clinical benefi ts noted in the drug label are improvements in walking and stair climbing capacity and reductions (but not normalization) of urinary GAG excretion. Additional analyses of combined data from all three trials also found pulmonary benefi t and improvements in growth [ 29 – 35 ].

In the clinical trials, over half of the patients experienced at least one IAR and 16 % of patients experienced an IAR that was judged anaphylactoid (allergic type reactions that recurred during multiple infusions) [ 33 ]. Almost all patients developed antibodies to galsulfase [ 36 ]. A sibling case-control study suggested that early, pre- symptomatic intervention with galsulfase in infancy may improve outcome with respect to development of scoliosis, joint movement, cardiac valve disease and facial morphology [ 4 ].

Enzyme Replacement Therapy in Lysosomal Storage Diseases

98

6 Enzyme Replacement Therapy Under Development for Other Mucopolysaccharidoses

Currently (August 2013), a phase III double-blind, randomized clinical trial has been completed in order to evaluate the effi cacy and safety of BMN 110 a recombinant N-acetylgalactosamine-6-sulfatase in patients with Mucopolysaccharidosis IVA (Morquio A Syndrome). The extension phase of this trial is actually ongoing.

7 Gaucher Disease

Gaucher disease is due to defi ciency of glucocerebrosidase or exceptionally its acti-vator. Its prevalence is about 1: 60 000 in the general population but it can reach 1: 1000 in the Ashkenazi Jewish population. There is a great clinical variability in the age of onset and clinical severity. However three clinical forms can be distinguished: Type I is hematological, involving 95 % of all patients; type II is an acute neurologi-cal form (1 %); type III a subacute neurological form (5 %).

Type I Gaucher disease is a visceral, hematological and bone disorder present-ing with hepatosplenomegaly, anemia, leukopenia, and thrombopenia. Bone manifestations include acute pain crises, osteonecrosis, and pathological frac-tures. More rarely, lung interstitial disease and cardiomyopathy may occur. The acute neurological form (Gaucher type II) is the most severe. The onset occurs between 3 and 6 months with progressive bulbar involvement (stridor, squint, and swallowing diffi culties). Pyramidal tract involvement (opisthotonus, head retrofl exion, spasticity, and trismus) and cognitive impairment may or may not be present. Pyramidal involvement is invariably associated with cognitive impair-ment and is a poor prognostic sign. The subacute neurological type (Gaucher type III) associates signs of Gaucher type I with a late-onset neurological impair-ment and a progressive course. Horizontal ophthalmoplegia is frequently met in association with cerebellar ataxia, progressive spasticity, myoclonous epilepsy, and parkinsonism [ 37 ].

High-risk genotypes associated with neuronopathic Gaucher disease include L444P/L444P, D409H/D409H, or L444P/D409H mutations. Genotyping should be confi rmed by direct DNA sequencing, particularly in cases where L444P alleles are suspected [ 38 ].

Enzyme replacement therapy by imiglucerase (Ceredase ® then Cerezyme ® ) modifi ed in a dramatic way the natural history of the disease. It is currently admin-istered every 2 weeks with doses varying between 15 and 60 U/kg/infusion. With this treatment the volume of the liver and spleen decreases quickly and is stabilized thereafter; anemia and thrombopenia are corrected; the painful crises are amended and the general state improves. Follow-up of clinical features and biological biomarkers (chitotriosidase, acid phosphatase, angiotensin-converting enzyme) permits the evaluation of treatment effi cacy and adjustment of enzyme doses.

V. Valayannopoulos

99

In both non-neuronopathic and neuronopathic Gaucher disease, enzyme replacement therapy has demonstrated an excellent safety profi le. There is clear evidence in most patients that enzyme replacement therapy ameliorates systemic involvement (skeletal deterioration, visceromegaly, hematological abnormalities) in non- neuronopathic as well as chronic neuronopathic Gaucher disease, enhancing quality of life [ 39 ]. However, there is no evidence that enzyme replacement therapy has reversed, stabilized, or slowed the progression of neurological involvement even when used in high doses (up to 120 U/kg/2 weeks) [ 40 ].

Substrate reduction therapy has been used as a second intention therapy in Gaucher disease. Miglustat (Zavesca ® , Actelion, Switzerland), an inhibitor of the synthesis of sphingolipids, is used as a daily oral therapy (100 mg/1.73 m 2 of body surface three times daily). The reported effi cacy of this treatment is slower com-pared to enzyme replacement therapy; thus it is indicated in patients presenting a stable clinical condition under enzyme replacement therapy. Because of its structure, miglustat has a large tissue distribution, including brain and cerebrospinal fl uid. This offers a rationale to test miglustat in the treatment of neuronopathic forms of Gaucher disease. However, except from single case reports, no large-scale effi cacy has been reported so far [ 41 ]. Moreover, adverse events may occur frequently: they include diarrhea responsible for weight loss, tremor, and more rarely, peripheral neuropathy that requires a careful monitoring by electromyography.

Other therapies involving small molecules such as pharmacological chaperones (e.g., AT3375) or a new ceramide analog for a new oral substrate reduction therapy (eliglustat) are currently being studied in clinical trials.

Two biosimilar agents, velaglucerase-alfa (VPRIV ® ) and taliglucerase-alfa (Elelyso ® ), are now approved for treatment of Gaucher type I disease. Although none of these drugs have shown effects on the CNS signs of the disease, they are believed to reduce the cost of production and thus the high price of these therapies, which remains a burden in most countries [ 42 , 43 ].

8 Fabry Disease

Fabry disease is a rare X-linked disorder caused by defi cient activity of the lyso-somal enzyme a-galactosidase A. Progressive accumulation in lysosomes of the undegraded glycosphingolipids leads to a multisystem disease with dermatological, ocular, renal, cardiac, and neurological manifestations. Peripheral nerve involve-ment, neuropathic pain, and chronic acroparesthesiae, are frequent and early-onset signs revealing the disease [ 44 ]. They are due to the involvement of small nerve fi bers, thus explaining the normality of electromyography. Cochleovestibular and autonomic nervous system involvement is frequent. Besides rare aseptic meningitis, central nervous system involvement is essentially represented by cerebrovascular events (stroke, transient ischemic attack). Affecting essentially the posterior circula-tion, their etiologies have to be clarifi ed: progressive stenosis of small vessels with globotriasocylceramide deposits, arterial remodeling, endothelial dysfunction,

Enzyme Replacement Therapy in Lysosomal Storage Diseases

100

prothrombotic state, cerebral hypoperfusion consecutive to dysautonomy, and cardiac embolism [ 45 ]. Magnetic resonance imaging (MRI) shows numerous silent lesions, increasing with age, mainly in small perforant arteries (periventricular white matter, brainstem, cerebellum, and basal ganglia).

Outside the US, enzyme replacement therapy with agalsidase alfa 0.2 mg/kg every other week (EOW) (Replagal ® ) and agalsidase beta 1.0 mg/kg EOW (Fabrazyme ® ) is available for the treatment of patients with Fabry disease, while agalsidase beta 1.0 mg/kg EOW is the only approved drug in the US. The effi cacy of enzyme replacement therapy in Fabry disease has been measured against a variety of end-points, the majority of which were subclinical parameters rather than clinical out-comes. Plasma levels of GL-3 together with accumulation in the kidney, heart, and skin were the most commonly studied endpoints, followed by renal endpoints of proteinuria and glomerular fi ltration rate, whereas cardiac and neurological end-points such as stroke and transient ischemic attack were not commonly studied [ 46 ]. However, it has been demonstrated that patients with Fabry disease have elevated cerebral blood fl ow velocities that signifi cantly improved with enzyme replacement therapy [ 47 ]. Also enzyme replacement therapy signifi cantly improves function of C, A-d, and A-b nerve fi bres and intradermal vibration receptors in Fabry neuropa-thy and is effective in improving pain-related quality of life.

Small molecule therapies such as pharmacological chaperones (AT1001) are currently studied in phase II–III clinical trials.

9 Pompe Disease

Pompe disease, also known as glycogen storage disease type II (GSD II) or acid maltase defi ciency, is an autosomal recessive neuromuscular disorder caused by mutations in the gene that encodes the lysosomal hydrolase acid a-glucosidase (GAA) [ 48 ]. The defi ciency of this enzyme results in lysosomal glycogen accumu-lation in multiple tissues, responsible for cellular dysfunction, particularly in skeletal and cardiac muscle.

Epidemiological studies have estimated the combined incidence of all clinical forms around 1 for 40,000 live births. GSD II encompasses a broad spectrum of phenotypes that range from the severe infantile-onset form to the more slowly progressive late-onset form [ 49 ].

The infantile form is characterized by generalized hypotonia, muscle weakness, and hypertrophic cardiomyopathy leading to death during the fi rst year of life due to cardiac and/or respiratory failure. Late onset forms present with progressive muscle weakness mainly involving proximal muscle and diaphragm and resulting in prema-ture death from respiratory failure.

The different clinical forms of GSD II are due to a large variety of mutations on the GAA gene. Among them, some common mutations have been reported: the mutations c.24811102_2646131del (exon 18 deletion) and c.525delT are usually found in infantile forms, whereas the mutation c.-32-13 T > G in intron 1 is gener-ally associated with late-onset forms of the disease.

V. Valayannopoulos

101

Nutrition (high-protein and low-carbohydrate diet) and exercise therapy have been used in patients with late-onset GSD II in order to slow muscle deterioration, but this approach is only palliative.

In Pompe disease, large-scale production of recombinant human acid a- glucosidase (rhGAA) was obtained in Chinese hamster ovary (CHO) cells and in transgenic rabbit milk [ 50 – 52 ].

Administration of rhGAA of both origins led to an increase of GAA activity in muscle, heart, and liver. However, these studies demonstrated an effi cient glycogen clearance in cardiac muscle and liver while a modest effect was observed in skeletal muscle [ 53 , 54 ].

Clinical studies based on the administration of rhGAA in classical infantile Pompe patients showed a prominent effect on cardiac hypertrophy, motor skill improvement as well as substantial life-span increase [ 55 – 58 ].

Administration of recombinant enzyme in late-onset patients results in a mild improvement of motor and respiratory functions, but ERT effi cacy in these patients needs to be evaluated at long-term [ 59 , 60 ]. These studies demonstrated that the outcome is more robust if treatment starts early in the course of the disease.

Even if CHO-derived rhGAA (alglucosidase alfa, Myozyme ® /Lumizyme ® , Genzyme, Cambridge, MA, USA) was approved in the USA, Europe, and Canada in 2006 and subsequently in numerous countries, becoming the standard treatment for Pompe disease, some drawbacks appeared:

1. A major limitation to ERT is the requirement for i.v. injection of a particularly high dose of recombinant enzyme (20 mg/kg, every 2 weeks), compared with other ERT for lysosomal storage diseases [ 61 ]. The rhGAA is poorly targeted to muscle and mainly trapped in liver leading to 80 % loss of the administered enzyme [ 52 ].

2. An ineffective response of type II skeletal muscle fi bers to ERT was clearly described in GAA-KO mice [ 52 ], due to the dysregulation of the autophagic pathway in glycolytic type II myofi bers leading to rhGAA retention in autopha-gosomes and mistargeting to lysosomes [ 62 , 63 ]. In humans, the situation remains unclear but both type I and type IIA muscle fi bers seem able to respond to ERT in infantile forms of Pompe disease [ 64 ].

3. Repeated infusions of high amount of exogenous enzyme often lead to the induc-tion of an immune response, especially in cross-reactive immunological material (CRIM)-negative patients with infantile forms [ 65 ].

4. The potential contribution of a neural defi cit resulting from glycogen storage into the central nervous system (CNS) has been highlighted in GAA-defi cient mice and patients. Although glycogen is normally absent from neurons, several reports demonstrated glycogen accumulation in spinal cord, sensory ganglia, and brain leading to degeneration of axons [ 66 , 67 ]. Progressive phrenic nerve injury could contribute to respiratory insuffi ciency in late-onset GSD II patients. Contribution of neuronal glycogen storage to the pathology is not completely elucidated, but it is clear that delivery of the therapeutic protein to the CNS is unlikely due to the blood-brain barrier impermeability.

Enzyme Replacement Therapy in Lysosomal Storage Diseases

102

Patients who are unable to produce native enzyme due to deleterious mutations are named CRIM-negative patients. They are prone to develop a sustained immune response to recombinant enzyme, with a particularly high titer of anti-GAA antibodies. Although the role of this antibody response on ERT effi ciency is unclear, it is speculated that CRIM status infl uences ERT outcome in infants with Pompe disease [ 65 ]. The induction of immune tolerance to the therapeutic enzyme would greatly enhance the benefi t of ERT in these patients [ 68 ].

In a patient with an infantile form of Pompe disease, the simultaneous injection of anti-CD20 monoclonal antibody, MTX, and gamma immunoglobulin has demon-strated its capacity to drastically reduce the humoral immune response consecutive to ERT [ 69 ].

Finally, it can be recommended to determine by Western-blot the CRIM status of each patient before the fi rst infusion of recombinant enzyme. This step is essential to optimize the treatment by increasing doses and/or combining ERT with induction of immune tolerance in patients CRIM-negative and/or having a poor response.

10 Financial and Ethical Considerations

Despite indisputable improvement for most of these treatments in patients’ condi-tions and quality of life, some major drawbacks still exist namely their high fi nan-cial cost that can account for several thousands of euros per patient and per year. In most of the developed countries this costs are usually covered by health insurance national or private policies. However in less fi nancially developed country, lack of reimbursement represents a major obstacle for granting access to these treatments for most affected patients. It is possible that the arrival of bio-similar therapies infl uences the currents costs of these molecules.

Ethical questions may arise when patients are irreversibly affected whether to start or pursue treatment. The latter occurs quite frequently in patients with neuro-logical complications (such as MPS I and MPS II) where ERT is not expected to have benefi cial effects.

Some of these patients (namely in MPS II) may display severe behavioral prob-lems making a weekly infusion technically diffi cult and may require sedation. Also it may be diffi cult for patients and families to undergo weekly or every-other-weekly infusions especially with respect to school attendance, professional and family organization as in some countries home therapy may not be available.

National or local expert committees and treatment guideline are needed to address the above-mentioned questions with respect to treatment initiation and interruption and to defi ne the best practice for these rare diseases for specialists and general practitioners.

It is also noteworthy that ERT must always be associated with symptomatic treat-ments such as physical therapy but also support for patients and families. In the latter patients associations may play a crucial role.

V. Valayannopoulos

103

11 Conclusions

Enzyme replacement therapies are changing the natural history of most lysosomal storage diseases by alleviating many signs and symptoms and by improving the patients’ quality of life. The over all tolerance profi le is good with the exception of a few anaphylactoid reactions occurring in general at the onset of treatment and generally improving over time. However generation of anti-recombinant protein antibodies may in some cases reduce treatment effi cacy. The drawbacks of these therapies include the need for frequent i.v. infusions and their high cost.

New therapies including recombinant enzymes and other strategies (substrate reduction therapies, chaperones, etc.) and in a near future gene therapy may change the devastating course of lysosomal storage disease and address unmet needs such as the central nervous system and the bone.

References

1. Boelens JJ (2006) Trends in haematopoietic cell transplantation for inborn errors of metabolism. J Inherit Metab Dis 29:413–420

2. Piraud M, Boyer S, Mathieu M, Maire I (1993) Diagnosis of mucopolysaccharidoses in a clinically selected population by urinary glycosaminoglycan analysis: a study of 2,000 urine samples. Clin Chim Acta 221:171–181

3. Neufeld E, Muenzer J (2001) The mucopolysaccharidoses. In: Scriver CR, Sly WS, Childs B, Beaudet al, Valle D, Kinzler KW, Vogelstein B (eds) The metabolic and molecular bases of inherited disease, 8th edn. McGraw-Hill, Health Professions Division, New York

4. McGill JJ, Inwood AC, Coman DJ, Lipke ML, de Lore D, Swiedler SJ, Hopwood JJ (2010) Enzyme replacement therapy for mucopolysaccharidosis VI from 8 weeks of age – a sibling control study. Clin Genet 77:492–498

5. Wang RY, Cambray-Forker EJ, Ohanian K, Karlin DS, Covault KK, Schwartz PH, Abdenur JE (2009) Treatment reduces or stabilizes brain imaging abnormalities in patients with MPS I and II. Mol Genet Metab 98:406–411

6. Gabrielli O, Clarke LA, Bruni S, Coppa GV (2010) Enzyme-replacement therapy in a 5-month- old boy with attenuated presymptomatic MPS I: 5-year follow-up. Pediatrics 125:e183–e187

7. Tylki-Szymanska A, Jurecka A, Zuber Z, Rozdzynska A, Marucha J, Czartoryska B (2012) Enzyme replacement therapy for mucopolysaccharidosis II from 3 months of age: a 3-year follow-up. Acta Paediatr 101:e42–e47

8. Prasad VK, Kurtzberg J (2010) Transplant outcomes in mucopolysaccharidoses. Semin Hematol 47:59–69

9. Peters C, Balthazor M, Shapiro EG, King RJ, Kollman C, Hegland JD, Henslee-Downey J, Trigg ME, Cowan MJ, Sanders J, Bunin N, Weinstein H, Lenarsky C, Falk P, Harris R, Bowen T, Williams TE, Grayson GH, Warkentin P, Sender L, Cool VA, Crittenden M, Packman S, Kaplan P, Lockman LA, Anderson J, Krivit W, Dusenbery K, Wagner J (1996) Outcome of unrelated donor bone marrow transplantation in 40 children with Hurler syndrome. Blood 87:4894–4902

10. Peters C, Shapiro EG, Anderson J, Henslee-Downey PJ, Klemperer MR, Cowan MJ, Saunders EF, deAlarcon PA, Twist C, Nachman JB, Hale GA, Harris RE, Rozans MK, Kurtzberg J, Grayson GH, Williams TE, Lenarsky C, Wagner JE, Krivit W (1998) Hurler syndrome: II. Outcome of HLA-genotypically identical sibling and HLA-haploidentical related donor bone marrow transplantation in fi fty-four children The Storage Disease Collaborative Study Group. Blood 91:2601–2608

Enzyme Replacement Therapy in Lysosomal Storage Diseases

104

11. Khanna G, Van Heest AE, Agel J, Bjoraker K, Grewal S, Abel S, Krivit W, Peters C, Orchard PJ (2007) Analysis of factors affecting development of carpal tunnel syndrome in patients with Hurler syndrome after hematopoietic cell transplantation. Bone Marrow Transplant 39:331–334

12. Walker RW, Darowski M, Morris P, Wraith JE (1994) Anaesthesia and mucopolysaccharidoses. A review of airway problems in children. Anaesthesia 49:1078–1084

13. Muenzer J, Wraith JE, Clarke LA, International Consensus Panel on Management and Treatment of Mucopolysaccharidosis I (2009) Mucopolysaccharidosis I: management and treatment guidelines. Pediatrics 123:19–29

14. Valayannopoulos V, Nicely H, Harmatz P, Turbeville S (2010) Mucopolysaccharidosis VI. Orphanet J Rare Dis 5:5

15. Giugliani R, Rojas VM, Martins AM, Valadares ER, Clarke JTR, Góes JEC, Kakkis ED, Worden MA, Sidman M, Cox GF (2009) A dose-optimization trial of laronidase (Aldurazyme) in patients with mucopolysaccharidosis I. Mol Genet Metab 96:13–19

16. Clarke LA, Wraith JE, Beck M, Kolodny EH, Pastores GM, Muenzer J, Rapoport DM, Berger KI, Sidman M, Kakkis ED, Cox GF (2009) Long-term effi cacy and safety of laronidase in the treatment of mucopolysaccharidosis I. Pediatrics 123:229–240

17. Kakkis ED, Muenzer J, Tiller GE, Waber L, Belmont J, Passage M, Izykowski B, Phillips J, Doroshow R, Walot I, Hoft R, Neufeld EF (2001) Enzyme-replacement therapy in mucopoly-saccharidosis I. N Engl J Med 344:182–188

18. Wraith JE, Beck M, Lane R, van der Ploeg A, Shapiro E, Xue Y, Kakkis ED, Guffon N (2007) Enzyme replacement therapy in patients who have mucopolysaccharidosis I and are younger than 5 years: results of a multinational study of recombinant human alpha-L-iduronidase (laronidase). Pediatrics 120:e37–e46

19. Wraith JE, Clarke LA, Beck M, Kolodny EH, Pastores GM, Muenzer J, Rapoport DM, Berger KI, Swiedler SJ, Kakkis ED, Braakman T, Chadbourne E, Walton-Bowen K, Cox GF (2004) Enzyme replacement therapy for mucopolysaccharidosis I: a randomized, double-blinded, placebo-controlled, multinational study of recombinant human alpha-L-iduronidase (laronidase). J Pediatr 144:581–588

20. Pastores GM, Arn P, Beck M, Clarke JTR, Guffon N, Kaplan P, Muenzer J, Norato DYJ, Shapiro E, Thomas J, Viskochil D, Wraith JE (2007) The MPS I registry: design, methodology, and early fi ndings of a global disease registry for monitoring patients with Mucopolysaccharidosis Type I. Mol Genet Metab 91:37–47

21. Dickson P, McEntee M, Vogler C, Le S, Levy B, Peinovich M, Hanson S, Passage M, Kakkis E (2007) Intrathecal enzyme replacement therapy: successful treatment of brain disease via the cerebrospinal fl uid. Mol Genet Metab 91:61–68

22. Muenzer J, Wraith JE, Beck M, Giugliani R, Harmatz P, Eng CM, Vellodi A, Martin R, Ramaswami U, Gucsavas-Calikoglu M, Vijayaraghavan S, Wendt S, Wendt S, Puga AC, Puga A, Ulbrich B, Shinawi M, Cleary M, Piper D, Conway AM, Conway AM, Kimura A (2006) A phase II/III clinical study of enzyme replacement therapy with idursulfase in mucopolysac-charidosis II (Hunter syndrome). Genet Med 8:465–473

23. Muenzer J, Gucsavas-Calikoglu M, McCandless SE, Schuetz TJ, Kimura A (2007) A phase I/II clinical trial of enzyme replacement therapy in mucopolysaccharidosis II (Hunter syn-drome). Mol Genet Metab 90:329–337

24. Muenzer J, Beck M, Eng CM, Escolar ML, Giugliani R, Guffon NH, Harmatz P, Kamin W, Kampmann C, Koseoglu ST, Link B, Martin RA, Molter DW, Muñoz Rojas MV, Ogilvie JW, Parini R, Ramaswami U, Scarpa M, Schwartz IV, Wood RE, Wraith E (2009) Multidisciplinary management of Hunter syndrome. Pediatrics 124:e1228–e1239

25. Muenzer J, Beck M, Eng CM, Giugliani R, Harmatz P, Martin R, Ramaswami U, Vellodi A, Wraith JE, Cleary M, Gucsavas-Calikoglu M, Puga AC, Shinawi M, Ulbrich B, Vijayaraghavan S, Wendt S, Conway AM, Rossi A, Whiteman DAH, Kimura A (2011) Long-term, open- labeled extension study of idursulfase in the treatment of Hunter syndrome. Genet Med 13:95–101

26. Okuyama T, Tanaka A, Suzuki Y, Ida H, Tanaka T, Cox GF, Eto Y, Orii T (2010) Japan Elaprase Treatment (JET) study: idursulfase enzyme replacement therapy in adult patients with attenu-ated Hunter syndrome (Mucopolysaccharidosis II, MPS II). Mol Genet Metab 99:18–25

V. Valayannopoulos

105

27. Wraith JE (2008) Enzyme replacement therapy with idursulfase in patients with mucopolysac-charidosis type II. Acta Paediatr Suppl 97:76–78

28. Wraith JE, Scarpa M, Beck M, Bodamer OA, De Meirleir L, Guffon N, Meldgaard Lund A, Malm G, Van der Ploeg AT, Zeman J (2008) Mucopolysaccharidosis type II (Hunter syn-drome): a clinical review and recommendations for treatment in the era of enzyme replacement therapy. Eur J Pediatr 167:267–277

29. Harmatz P, Whitley CB, Waber L, Pais R, Steiner R, Plecko B, Kaplan P, Simon J, Butensky E, Hopwood JJ (2004) Enzyme replacement therapy in mucopolysaccharidosis VI (Maroteaux- Lamy syndrome). J Pediatr 144:574–580

30. Harmatz P, Ketteridge D, Giugliani R, Guffon N, Teles EL, Miranda MCS, Yu Z-F, Swiedler SJ, Hopwood JJ, MPS VI Study Group (2005) Direct comparison of measures of endurance, mobility, and joint function during enzyme-replacement therapy of mucopolysaccharidosis VI (Maroteaux-Lamy syndrome): results after 48 weeks in a phase 2 open-label clinical study of recombinant human N-acetylgalactosamine 4-sulfatase. Pediatrics 115:e681–689

31. Harmatz P, Kramer WG, Hopwood JJ, Simon J, Butensky E, Swiedler SJ, Mucopolysaccharidosis VI Study Group (2005) Pharmacokinetic profi le of recombinant human N-acetylgalactosamine 4-sulphatase enzyme replacement therapy in patients with mucopolysaccharidosis VI (Maroteaux-Lamy syndrome): a phase I/II study. Acta Paediatr Suppl 94:61–68; discussion 57

32. Harmatz P, Giugliani R, Schwartz I, Guffon N, Teles EL, Miranda MCS, Wraith JE, Beck M, Arash L, Scarpa M, Yu Z-F, Wittes J, Berger KI, Newman MS, Lowe AM, Kakkis E, Swiedler SJ, MPS VI Phase 3 Study Group (2006) Enzyme replacement therapy for mucopolysacchari-dosis VI: a phase 3, randomized, double-blind, placebo-controlled, multinational study of recombinant human N-acetylgalactosamine 4-sulfatase (recombinant human arylsulfatase B or rhASB) and follow-on, open-label extension study. J Pediatr 148:533–539

33. Harmatz P, Giugliani R, Schwartz IVD, Guffon N, Teles EL, Miranda MCS, Wraith JE, Beck M, Arash L, Scarpa M, Ketteridge D, Hopwood JJ, Plecko B, Steiner R, Whitley CB, Kaplan P, Yu Z-F, Swiedler SJ, Decker C, MPS VI Study Group (2008) Long-term follow-up of endur-ance and safety outcomes during enzyme replacement therapy for mucopolysaccharidosis VI: fi nal results of three clinical studies of recombinant human N-acetylgalactosamine 4-sulfatase. Mol Genet Metab 94:469–475

34. Harmatz P, Yu Z-F, Giugliani R, Schwartz IVD, Guffon N, Teles EL, Miranda MCS, Wraith JE, Beck M, Arash L, Scarpa M, Ketteridge D, Hopwood JJ, Plecko B, Steiner R, Whitley CB, Kaplan P, Swiedler SJ, Hardy K, Berger KI, Decker C (2010) Enzyme replacement therapy for mucopolysaccharidosis VI: evaluation of long-term pulmonary function in patients treated with recombinant human N-acetylgalactosamine 4-sulfatase. J Inherit Metab Dis 33:51–60

35. Decker C, Yu Z-F, Giugliani R, Schwartz IVD, Guffon N, Teles EL, Miranda MCS, Wraith JE, Beck M, Arash L, Scarpa M, Ketteridge D, Hopwood JJ, Plecko B, Steiner R, Whitley CB, Kaplan P, Swiedler SJ, Conrad S, Harmatz P (2010) Enzyme replacement therapy for muco-polysaccharidosis VI: growth and pubertal development in patients treated with recombinant human N-acetylgalactosamine 4-sulfatase. J Pediatr Rehabil Med 3:89–100

36. Giugliani R, Harmatz P, Wraith JE (2007) Management guidelines for mucopolysaccharidosis VI. Pediatrics 120:405–418

37. Erikson A, Bembi B, Schiffmann R (1997) Neuronopathic forms of Gaucher’s disease. Baillieres Clin Haematol 10:711–723

38. Hruska KS, LaMarca ME, Scott CR, Sidransky E (2008) Gaucher disease: mutation and poly-morphism spectrum in the glucocerebrosidase gene (GBA). Hum Mutat 29:567–583

39. Martins AM, Valadares ER, Porta G, Coelho J, Semionato Filho J, Pianovski MAD, Kerstenetzky MS, de Montoril MFP, Aranda PC, Pires RF, Mota RMV, Bortolheiro TC, Brazilian Study Group on Gaucher Disease and other Lysosomal Storage Diseases (2009) Recommendations on diagnosis, treatment, and monitoring for Gaucher disease. J Pediatr 155:S10–18

40. Vellodi A, Tylki-Szymanska A, Davies EH, Kolodny E, Bembi B, Collin-Histed T, Mengel E, Erikson A, Schiffmann R, European Working Group on Gaucher Disease (2009) Management of neuronopathic Gaucher disease: revised recommendations. J Inherit Metab Dis 32:660–664

Enzyme Replacement Therapy in Lysosomal Storage Diseases

106

41. Schiffmann R, Fitzgibbon EJ, Harris C, DeVile C, Davies EH, Abel L, van Schaik IN, Benko W, Timmons M, Ries M, Vellodi A (2008) Randomized, controlled trial of miglustat in Gaucher’s disease type 3. Ann Neurol 64:514–522

42. van Dussen L, Zimran A, Akkerman EM, Aerts JMFG, Petakov M, Elstein D, Rosenbaum H, Aviezer D, Brill-Almon E, Chertkoff R, Maas M, Hollak CEM (2013) Taliglucerase alfa leads to favorable bone marrow responses in patients with type I Gaucher disease. Blood Cells Mol Dis 50:206–211

43. Gonzalez DE, Turkia HB, Lukina EA, Kisinovsky I, Dridi M-FB, Elstein D, Zahrieh D, Crombez E, Bhirangi K, Barton NW, Zimran A (2013) Enzyme replacement therapy with velaglucerase alfa in Gaucher disease: results from a randomized, double-blind, multinational, Phase 3 study. Am J Hematol 88:166–171

44. Clavelou P, Besson G, Elziere C, Ferrier A, Pinard J-M, Hermier M, Artigou JY, Germain DP (2006) Neurological aspects of Fabry’s disease. Rev Neurol (Paris) 162:569–580

45. Moore DF, Kaneski CR, Askari H, Schiffmann R (2007) The cerebral vasculopathy of Fabry disease. J Neurol Sci 257:258–263

46. Schaefer RM, Tylki-Szymańska A, Hilz MJ (2009) Enzyme replacement therapy for Fabry disease: a systematic review of available evidence. Drugs 69:2179–2205

47. Moore DF, Altarescu G, Ling GSF, Jeffries N, Frei KP, Weibel T, Charria-Ortiz G, Ferri R, Arai AE, Brady RO, Schiffmann R (2002) Elevated cerebral blood fl ow velocities in Fabry disease with reversal after enzyme replacement. Stroke 33:525–531

48. Hirschhorn R, Reuser AJ (2001) Glycogen storage disease type II; acid-glucosidase (acid maltase) defi ciency. In: Scriver CR, Sly WS, Childs B, Beaudet al, Valle D, Kinzler KW, Vogelstein B (eds) The metabolic and molecular bases of inherited disease, 8th edn. McGraw-Hill, Health Professions Division, New York

49. ACMG Work Group on Management of Pompe Disease, Kishnani PS, Steiner RD, Bali D, Berger K, Byrne BJ, Case LE, Case L, Crowley JF, Downs S, Howell RR, Kravitz RM, Mackey J, Marsden D, Martins AM, Millington DS, Nicolino M, O’Grady G, Patterson MC, Rapoport DM, Slonim A, Spencer CT, Tifft CJ, Watson MS (2006) Pompe disease diagnosis and man-agement guideline. Genet Med 8:267–288

50. Bijvoet AG, Van Hirtum H, Kroos MA, Van de Kamp EH, Schoneveld O, Visser P, Brakenhoff JP, Weggeman M, van Corven EJ, Van der Ploeg AT, Reuser AJ (1999) Human acid alpha- glucosidase from rabbit milk has therapeutic effect in mice with glycogen storage disease type II. Hum Mol Genet 8:2145–2153

51. Van Hove JL, Yang HW, Wu JY, Brady RO, Chen YT (1996) High-level production of recom-binant human lysosomal acid alpha-glucosidase in Chinese hamster ovary cells which targets to heart muscle and corrects glycogen accumulation in fi broblasts from patients with Pompe disease. Proc Natl Acad Sci U S A 93:65–70

52. Raben N, Danon M, Gilbert AL, Dwivedi S, Collins B, Thurberg BL, Mattaliano RJ, Nagaraju K, Plotz PH (2003) Enzyme replacement therapy in the mouse model of Pompe disease. Mol Genet Metab 80:159–169

53. Raben N, Fukuda T, Gilbert AL, de Jong D, Thurberg BL, Mattaliano RJ, Meikle P, Hopwood JJ, Nagashima K, Nagaraju K, Plotz PH (2005) Replacing acid alpha-glucosidase in Pompe disease: recombinant and transgenic enzymes are equipotent, but neither completely clears glycogen from type II muscle fi bers. Mol Ther 11:48–56

54. Hawes ML, Kennedy W, O’Callaghan MW, Thurberg BL (2007) Differential muscular glyco-gen clearance after enzyme replacement therapy in a mouse model of Pompe disease. Mol Genet Metab 91:343–351

55. Kishnani PS, Nicolino M, Voit T, Rogers RC, Tsai AC-H, Waterson J, Herman GE, Amalfi tano A, Thurberg BL, Richards S, Davison M, Corzo D, Chen YT (2006) Chinese hamster ovary cell-derived recombinant human acid alpha-glucosidase in infantile-onset Pompe disease. J Pediatr 149:89–97

56. Van den Hout JM, Kamphoven JH, Winkel LP, Arts WF, De Klerk JB, Loonen MC, Vulto AG, Cromme-Dijkhuis A, Weisglas-Kuperus N, Hop W, Van Hirtum H, Van Diggelen OP, Boer M, Kroos MA, Van Doorn PA, Van der Voort E, Sibbles B, Van Corven EJ, Brakenhoff JP,

V. Valayannopoulos

107

Van Hove J, Smeitink JA, de Jong G, Reuser AJ, Van der Ploeg AT (2004) Long-term intravenous treatment of Pompe disease with recombinant human alpha- glucosidase from milk. Pediatrics 113:e448–e457

57. Nicolino M, Byrne B, Wraith JE, Leslie N, Mandel H, Freyer DR, Arnold GL, Pivnick EK, Ottinger CJ, Robinson PH, Loo J-CA, Smitka M, Jardine P, Tatò L, Chabrol B, McCandless S, Kimura S, Mehta L, Bali D, Skrinar A, Morgan C, Rangachari L, Corzo D, Kishnani PS (2009) Clinical outcomes after long-term treatment with alglucosidase alfa in infants and children with advanced Pompe disease. Genet Med 11:210–219

58. Chen L-R, Chen C-A, Chiu S-N, Chien Y-H, Lee N-C, Lin M-T, Hwu W-L, Wang J-K, Wu M-H (2009) Reversal of cardiac dysfunction after enzyme replacement in patients with infantile- onset Pompe disease. J Pediatr 155:271–275.e2

59. Strothotte S, Strigl-Pill N, Grunert B, Kornblum C, Eger K, Wessig C, Deschauer M, Breunig F, Glocker FX, Vielhaber S, Brejova A, Hilz M, Reiners K, Müller-Felber W, Mengel E, Spranger M, Schoser B (2010) Enzyme replacement therapy with alglucosidase alfa in 44 patients with late-onset glycogen storage disease type 2: 12-month results of an observational clinical trial. J Neurol 257:91–97

60. van der Ploeg AT, Clemens PR, Corzo D, Escolar DM, Florence J, Groeneveld GJ, Herson S, Kishnani PS, Laforet P, Lake SL, Lange DJ, Leshner RT, Mayhew JE, Morgan C, Nozaki K, Park DJ, Pestronk A, Rosenbloom B, Skrinar A, van Capelle CI, van der Beek NA, Wasserstein M, Zivkovic SA (2010) A randomized study of alglucosidase alfa in late-onset Pompe’s disease. N Engl J Med 362:1396–1406

61. Desnick RJ (2004) Enzyme replacement and enhancement therapies for lysosomal diseases. J Inherit Metab Dis 27:385–410

62. Fukuda T, Ahearn M, Roberts A, Mattaliano RJ, Zaal K, Ralston E, Plotz PH, Raben N (2006) Autophagy and mistargeting of therapeutic enzyme in skeletal muscle in Pompe disease. Mol Ther 14:831–839

63. Cardone M, Porto C, Tarallo A, Vicinanza M, Rossi B, Polishchuk E, Donaudy F, Andria G, De Matteis MA, Parenti G (2008) Abnormal mannose-6-phosphate receptor traffi cking impairs recombinant alpha-glucosidase uptake in Pompe disease fi broblasts. Pathogenetics 1:6

64. Drost MR, Schaart G, van Dijk P, van Capelle CI, van der Vusse GJ, Delhaas T, van der Ploeg AT, Reuser AJ (2008) Both type 1 and type 2a muscle fi bers can respond to enzyme therapy in Pompe disease. Muscle Nerve 37:251–255

65. Kishnani PS, Goldenberg PC, DeArmey SL, Heller J, Benjamin D, Young S, Bali D, Smith SA, Li JS, Mandel H, Koeberl D, Rosenberg A, Chen Y-T (2010) Cross-reactive immunologic material status affects treatment outcomes in Pompe disease infants. Mol Genet Metab 99:26–33

66. Sidman RL, Taksir T, Fidler J, Zhao M, Dodge JC, Passini MA, Raben N, Thurberg BL, Cheng SH, Shihabuddin LS (2008) Temporal neuropathologic and behavioral phenotype of 6neo/6neo Pompe disease mice. J Neuropathol Exp Neurol 67:803–818

67. DeRuisseau LR, Fuller DD, Qiu K, DeRuisseau KC, Donnelly WH Jr, Mah C, Reier PJ, Byrne BJ (2009) Neural defi cits contribute to respiratory insuffi ciency in Pompe disease. Proc Natl Acad Sci U S A 106:9419–9424

68. Koeberl DD, Kishnani PS (2009) Immunomodulatory gene therapy in lysosomal storage disorders. Curr Gene Ther 9:503–510

69. Mendelsohn NJ, Messinger YH, Rosenberg AS, Kishnani PS (2009) Elimination of antibodies to recombinant enzyme in Pompe’s disease. N Engl J Med 360:194–195

Enzyme Replacement Therapy in Lysosomal Storage Diseases

109M. Özgüç (ed.), Rare Diseases: Integrative PPPM Approach as the Medicine of the Future, Advances in Predictive, Preventive and Personalised Medicine 6,DOI 10.1007/978-94-017-9214-1_8, © Springer Science+Business Media Dordrecht 2015

Abstract According to IARC fi gures ‘rare and less common’ cancers comprise more than a third of all cancer diagnoses as a group. However, advances in molecular biology have resulted in novel ways to classify cancers based upon genetic alterations and not just anatomical location, and this revised classifi cation is at the heart of any move toward more personalized healthcare. It is now increasingly accepted that cancer should be thought of as many hundreds of more rare subtypes, each of which will have specifi c therapeutic options. We have selected colorectal carcinoma to illustrate the concept that each cancer is ‘rare’, and demonstrate why this is important for delivering on the concept of Predictive, Preventive and Personalized Medicine (PPPM) for cancer in terms of prediction of who will get the disease, how it will behave and how to prevent it.

Keywords Cancer • Colorectal • Molecular classifi cation • Histopathology • Targeted therapies • Predictive, preventive and personalized medicine

Rare Cancers

Nikolajs Zeps and Chris Hemmings

N. Zeps (*) Bendat Family Comprehensive Cancer Centre , St John of God HealthCare , 12 Salvado Road , Subiaco , WA 6008 , Australia

School of Surgery , University of Western Australia , Crawley , Australia e-mail: nik.zeps@sjog.org.au

C. Hemmings School of Surgery , University of Western Australia , Crawley , Australia

Department of Anatomic Pathology , St John of God Pathology , Subiaco , Australia

110

1 Introduction

1.1 Cancer as a ‘Rare’ Disease

According to the World Health Organization (WHO) cancer is the leading cause of death worldwide, accounting for some 7.6 million deaths in 2008 [ 1 ]. This would appear to rule out the inclusion of cancer in a book on rare diseases, however it is broadly understood that cancer is not a single disease but is comprised of many hundreds of individual disorders linked only by the fact that they involve the uncon-trolled proliferation of our own cells, arising from alterations in our genes or how those genes are regulated.

When considering rare types of cancer, it is necessary to start with some sort of defi nition of what constitutes “rare”. One widely accepted suggestion is those malig-nancies with an annual incidence of <6/100,000 population. Using this defi nition, EU-Commission supported project RARECARE have listed 186 rare cancers [ 2 ], which together affect more than four million Europeans and account for 22 % of all cancer diagnoses, including all cancers diagnosed in children. Cancers are conven-tionally grouped according to organ of origin, and this has been the basis for deter-mining their clinical management for the last 100 years. This anatomically based classifi cation has largely arisen on the basis of surgical convenience, rather than a biologically meaningful distinction between tumor types. But increasing knowledge of the molecular biology of different tumors, coupled with the resulting proliferation of systemic therapies for unresectable disease, may bring us to the point where in the not-too-distant future we are more likely to refer to a patient with a “KRAS cancer” (or “BRAF” or “EGFR”) rather than “lung” or “breast” or “colon cancer”.

Numerically the “Top 5” cancers (lung, breast, colorectal, stomach and prostate) account for 48.3 % of all cancer diagnoses, and 45.5 % of all cancer deaths [ 1 ] The next 5 most prevalent cancers comprise another 19.7 % of all cancer diagnoses. However, an 11th category referred to as “other and unspecifi ed” accounts for 34.8 % of all diagnoses and an almost equivalent amount of cancer deaths (34.3 %). This catch-all grouping comprises many of the so-called ‘rare and less common’ cancers, but is essentially meaningless in telling us anything informative about them or for planning their management. In fact, whilst breaking up cancer statistics by organ or tissue of origin is convenient, it says very little about the nature of any cancers and in an era of personalized medicine is becoming increasingly anachro-nistic. Over the last 50 years there has been a dramatic increase in our understanding of the molecular basis of cancer, and each broad category of cancer is now recog-nized to be comprised of unique types of cancer that in many instances have more similarities with cancers from other anatomical sites than they do with their “neigh-bours” (Fig. 1 ).

With the advent of monoclonal antibodies and small molecule inhibitors that have been designed to specifi cally target key proteins and pathways involved in cancer, there is a greater necessity to defi ne cancer in a more sophisticated manner,

N. Zeps and C. Hemmings

111

to ensure that the correct treatment is given to the right person and in the optimal dose. In addition to our molecular understanding of cancer, healthcare has also become increasingly sophisticated with respect to treating a person within their spe-cifi c context, that is, taking into account their social, familial and environmental status, as well as their co-morbidities and their own highly personal status with respect to how their disease will affect them and how they wish to be managed. We can therefore regard cancer as the embodiment of a disease suited to the personal-ized approach.

In this chapter we will explore the theme of predictive, preventive and personal-ized medicine (PPPM) with respect to colorectal cancer – one of the commonest cancers if classifi ed solely on the basis of anatomic location, rather than molecular biology. In so doing we will illustrate why it is time to rethink the way we classify cancer, what this means for management, and how taking this new approach has the potential to benefi t all cancer patients.

Lum

inal

ALu

min

al B

HER2

Normal-like

Basal

Breas

t

Colore

ctal

NSCLC

Prosta

te

Mela

nom

a

anti-

EGFR

Resp

onsi

veRa

s w

t

MMREGFR

TMPR

SS2-

erg

BRAF

mut

ated

CIMP

BRCA

EML-ALK

Ovaria

n

GIST

c-ki

t mut

ated

ER

+E

R -

BRCA

ness

HER2

Gastri

c

PDAC

Abl-like KinaseDependant

EGFR dependent

HER2 Amplified

ALK inhibitorresponsive

DDR Defective

BRAF mutant

EstrogenDependent

COLONNSCLC SCCHN

BREAST

OVARYBREAST Pa

GASTRICBREAST Pa

NSCLC NHL NB

CML GIST RCC

MELANOMA

NOVEL mutant

a

b

Fig. 1 A molecular taxonomy for cancer. ( a ) Cancers of different organs subclassifi ed by molecular phenotype. ( b ) Molecular phenotypes subclassifi ed by organ of origin may be advantageous in molecularly diverse and less common cancers

Rare Cancers

112

2 Colorectal Cancer

In the International Statistical Classifi cation of Diseases and related health prob-lems (10th Revision) [ 3 ] codes C15–C26 refer to malignant neoplasms of digestive organs that includes those cancers considered more common, such as the colon (C18), and those that are rarer, such as those arising in the pancreas (C25). Each of these can be further subcategorized such that the colon has 7 subcategories defi ned by anatomical location. For instance, C18.2 refers to the Ascending colon, which can be further subcategorized as ileocolic, right colic, or middle colic – a distinction which no doubt has surgical signifi cance but which tells us nothing about the patient’s prognosis. Within each of the subcategories, pathological features that are the basis of staging systems can further defi ne the disease type. Staging for colorec-tal cancer has its origins in the system fi rst proposed by the pathologist Cuthbert Dukes in 1930 [ 4 ] who understood that there are signifi cantly different outcomes for patients based upon the extent of their disease at diagnosis. The most widely used staging system for CRC is the “TNM” system employed by the American Joint Committee on Cancer (AJCC) and the Union for International Cancer Control (UICC), which is essentially a summation of the rate of growth of the tumor (T), the number of lymph nodes involved (N) and the degree of metastatic spread (M) [ 5 ] (Tab les 1 and 2 ).

Each of these staging parameters is clinically meaningful, since treatment options will differ as stage increases. For instance, a T1N0M0 cancer will require conserva-tive surgical treatment without chemotherapy, whereas a T3N1M1 will require com-plex multidisciplinary intervention and will have a considerably worse prognosis. Similarly, a locally advanced adenocarcinoma arising in the rectum will typically be treated with neoadjuvant chemoradiotherapy, whereas an (at least histologically) apparently identical tumor arising in the descending colon will be referred for “up front” surgery. Thus, if we apply such categorizations it is easy to see how the third most common cancer type, “colorectal”, can be broken down into a larger number of diagnostically and prognostically relevant subcategories with far fewer cases in each than under the overall umbrella category. But the “splitting” doesn’t end with anatomic location and stage at diagnosis: the list of rare cancers referred to above includes tumors with distinctive histology (squamous cell carcinoma of the colon and rectum, and basaloid carcinoma of the rectum), but it is now recognized that multiple genetically distinct types of carcinoma can arise within the colorectum and in these only subtle differences in histology are apparent, if at all.

Cancer is accepted to be a disease arising from dysregulation of genes involved in normal cell growth and repair either through alteration(s) in genetic or epigenetic sequence [ 6 ]. In colorectal cancers there are three well-established pathways; Chromosomal Instability (CIN), Microsatellite Instability (MSI) and the CpG Island Methylator Phenotype (CIMP), the latter being most closely related to cancers with MSI. About 5 % of all colorectal cancers are thought to have a hereditary basis and a great deal of what we know about cancer biology has been enabled through the study of these. The familial nature of the disease has permitted linkage analysis to

N. Zeps and C. Hemmings

113

locate the underlying genetic mutations in six separate forms of hereditary colorec-tal cancer, each of which are relatively rare forms of cancer if we consider them as separate diseases linked only by the organ in which they arise. It is likely that other types of hereditary colorectal cancer exist, but the molecular alterations associated with them have not yet been identifi ed. We describe these heritable forms of colorec-tal cancer and compare them with those that arise sporadically, to illustrate how knowledge of the underlying biology allows clinicians to select personalized pre-vention and treatment strategies for all of the subtypes of colorectal cancer.

3 Familial Adenomatous Polyposis (FAP)

Approximately 0.5–1.0 % of all colorectal cancers arise in people who have the autosomal dominant syndrome known as Familial Adenomotous Polyposis (FAP), which is a cancer-associated genetic signature caused by germline mutations in the

Table 1 Anatomical staging parameters for colorectal cancer, derived from AJCC (7th Edition) recommendations

Primary tumor (T) Tx Primary tumor cannot be assessed T0 No evidence of primary tumor Tis Carcinoma in situ: intraepithelial/invasion of lamina propria T1 Tumor invades submucosa T2 Tumor invades muscularis propria T3 Tumor invades through the muscularis propria into pericolorectal

tissues T4a Tumor penetrates to the surface of the visceral peritoneum T4b Tumor directly invades or is adherent to other organs or structures Regional lymph nodes (N) Nx Regional lymph nodes cannot be assessed N0 No regional lymph node metastasis N1 Metastasis in 1–3 regional lymph nodes

N1a Metastasis in one regional lymph node N1b Metastasis in 2–3 regional lymph nodes N1c Tumor deposit(s) in the subserosa, mesentery, or non-

peritonealized pericolic/perirectal tissues, without regional nodal metastases

N2 Metastasis in 4 or more regional lymph nodes N2a Metastasis in 4–6 regional lymph nodes N2b Metastasis in 7 or more regional lymph nodes

Distant metastasis (M) M0 No distant metastasis M1 Distant metastasis

M1a Metastasis confi ned to one organ or site (eg. liver, lung, ovary, nonregional node)

M1b Metastases in more than one organ/site, or the peritoneum

Rare Cancers

114

Adenomatous Polyposis Coli (APC) gene found on chromosome 5q21 [ 6 – 8 ]. If the 5-year prevalence of bowel cancer is estimated to be 3.26 million [ 1 ] and the world population in 2008 was 6 billion, the 5-year prevalence of colorectal cancer was approximately 0.054 % of the total world population. Therefore one may calculate that less than 0.0003 % of the population has colon cancer due to APC mutations, satisfying the defi nition of a rare disease. Indeed, FAP accounts for just 750–1,500 cases of colon cancer per year in the US (based upon 0.5–1.0 % of the nearly 150,000 people diagnosed each year) [ 9 ].

Given the relative rarity of the disease, it is remarkable that FAP was recognized as a discrete entity as early as it was. Surgery for bowel cancers was only pioneered with any success in the 1880s and even then a pathologist would have examined very few of the resected specimens. However, as pathology as a discipline became more integrated into clinical practice the presence of colorectal cancers presenting at a relatively young age in people with a very large number of polyps were increasingly being reported. It was a young pathologist, Cuthbert Dukes, working at St Mark’s Hospital in London who fi rst published a paper in 1930 noting the hereditary compo-nent of the disease [ 4 ]. In a later paper Dukes wrote that he was not the fi rst to notice that there were two distinct types of polyposis that arose in the bowel [ 10 ], but that Erdmann and Morris [ 11 ] had made the distinction between familial and acquired polyposis. Notably, Lockhart-Mummery [ 12 ], a surgical colleague of Dukes and one who had been an early advocate of the use of the sigmoidoscope and also family trees

Table 2 Colorectal stage grouping, according to AJCC 7th Edition (refer to Table 1 for staging parameters)

N. Zeps and C. Hemmings

115

to defi ne pedigree, had provided early data that led Dukes to pursue his investigations further. This early example of linkage analysis depends upon comprehensive and reliable genealogies with suffi cient members and generations to enable confi rmation of hereditary traits. Although FAP is relatively rare, the early age of onset and the presence of a large number of precancerous polyps that defi ne it, gave it a special place in colorectal cancer research. It is interesting to note that Dukes expressed the opinion that surgery was the only useful treatment for FAP, either with curative intent for those who had developed cancer, or as prophylaxis for those at risk.

It would be another 50 years until the precise genetic locus of FAP was identifi ed [ 7 , 8 ] and since that time an enormous amount of unrelated research using cell lines and animal models has revealed how the APC gene functions in our cells and how its loss leads to cancer. It is important to note, however, that mutations in APC are only observed in 85 % of all colon cancers that have the classic FAP phenotype of >100 colonic adenomata. In these non-APC cases the condition must arise from other genetic mutations, and these are discussed further below .

APC plays a critical role in regulating cell-cell adhesion, cell migration, chromo-somal segregation and apoptosis [ 13 ], and works in close concert with several other intercellular proteins. It is part of the WNT-signaling pathway that is frequently dysregulated in colorectal cancers and as such its identifi cation was a key milestone in understanding colorectal tumorigenesis. In the cell, APC forms a complex with glycogen synthase kinase 3-ß (GSK-3ß) and axin, which in turn can then bind to ß-catenin in the cytoplasm. GSK-3ß phosphorylates ß-catenin, which targets it for ubiquitination and thus degradation, preventing its translocation to the nucleus where it acts as a transcription factor for proliferation genes. APC is therefore vital in regulating ß-catenin function as a nuclear transcription factor. APC is defi ned within the class of ‘tumor-suppressor’ genes, so-called because their loss permits cells to grow in the uncontrolled manner observed in tumors.

We can further refi ne FAP into the specifi c mutations that occur within the APC gene. 95 % are nonsense/frameshift alterations leading to premature stop codons. However, mutations at the 5′ end of the gene may lead to Attenuated FAP (aFAP), a clinically distinct form of APC-associated cancer characterized by less than 100 polyps (though this number may vary widely within families). Sporadic mutations in APC are more commonly seen within a mutation cluster region (aa1286–1513), which affects axin binding. Such mutations appear to occur early in sporadic dis-ease and are present even in microscopic adenomas.

However, loss of APC is not suffi cient in itself to cause colorectal cancer and it is now widely accepted that additional mutations are required in what is known as the ‘multi-step’ theory of cancer put forward by Vogelstein and Kinzler, who have been instrumental in defi ning many of the molecular pathways underlying CRC [ 14 ]. In brief, this revolutionary concept provided a model that satisfi ed many of the observations based upon study of oncogene and tumor suppressor mutations in diseases like CRC. That is, a series of mutations appeared to be required to drive transformation processes that ultimately resulted in a carcinoma. The importance of this concept is discussed further in this chapter in the context of sporadic cancers.

Rare Cancers

116

3.1 APC Gene Mutations and Other Rare Cancers

Whilst it was not initially believed that people with FAP also had a greater predis-position to other cancer types, studies have demonstrated that even those that don’t develop colorectal cancer may develop other types of cancer. Such APC-related cancers are themselves considered to be rare cancers and include hepatoblastoma, a form of papillary thyroid carcinoma and medulloblastoma. In addition, adrenal masses including pheochromocytoma and adrenal cortical carcinoma may also arise. Duodenal and peri-ampullary carcinoma are seen later in life in those FAP carriers who survive longer due to prophylactic or curative surgery for a previous FAP tumor. The emergence of some of these other forms of ‘late-onset’ FAP cancers appears to be related to curative treatment for ‘early-onset’ FAP that results in car-riers living longer than they previously would have, and therefore having more time to develop a second disease.

MutY Human Homologue (MYH)- Associated Polyposis (MAP)

Routine testing of people with polyposis revealed that some patients do not carry APC mutations. Furthermore, the typical pattern of autosomal dominance was not observed in some families; rather their pedigrees indicated an autosomal recessive inheritance. Genetic analysis of the polyps themselves also indicated that the pattern of mutations present differed from those in people with APC gene mutations [ 15 ]. Like Lynch Syndrome, the pathogenic mutation that underlies this disease occurs in a gene responsible for DNA repair. The mutY human homologue (MYH) gene is one of a family of three proteins involved in base-excision-repair (BER), along with OGG1 and MTH. However, to date only mutations in MYH appear to cause polyposis.

The role of BER proteins is to repair oxidative damage to DNA and loss of MYH function leads to an accumulation of transversions in the genome (often including the APC gene [ 16 ]). Thus the end result of MYH mutation that leads to colorectal cancer is similar in many respects to FAP, and it is therefore only logical that this disease be known as MAP. Interestingly, tumors that arise in MAP do not have the widespread chromosomal instability that is typical of FAP and many sporadic tumors, nor do they have the microsatellite instability that defi nes Lynch Syndrome. In this way they are a discrete subtype of colorectal cancer, but their relatively recent discovery and the small numbers of patients affected means that there are little data on patient outcomes. It is also very diffi cult to accurately quantify the prevalence of this syndrome as the recessive nature of the mutations means that MAP may be under-diagnosed in apparently sporadic cases, and it might actually be much more common than the currently reported incidence of 0.4–3 % [ 17 ]. As it has been reviewed for 7,225 individuals in a Swiss cohort, MYH was more frequently observed in people presenting with attenuated polyposis with a frequency similar to APC [ 18 ].

MYH mutation has a very high penetrance, with 100 % of homozygotes or compound heterozygotes developing colorectal cancer by the age of 60 [ 19 ]. The

N. Zeps and C. Hemmings

117

lifetime risk in heterozygous carriers is unclear, but some estimates suggest a modest increase in risk of 1.6 relative to the general population [ 20 ], suggesting that environmental and other genetic factors may also infl uence risk for any individual. As with FAP and Lynch Syndrome, affected individuals can undergo increased surveillance and consider prophylactic surgery, depending on the type of mutation carried (for instance it would be more usual to recommend colectomy for a homozygote than a heterozygote).

Other Polyposis Syndromes

There are a number of other rare familial polyposis syndromes that predispose to colorectal cancer and for which the molecular basis has now been elucidated. Juvenile Polyposis is characterized by the presence of multiple colorectal polyps with a distinctive histologic appearance and arises in patients with mutations in the PTEN gene on chromosome 10q, which are inherited in an autosomal dominant pattern. In the absence of screening, more than half of mutation carriers will develop colorectal cancer in their lifetime. Cowden Syndrome is also associated with PTEN mutations with an autosomal dominant pattern of inheritance and high penetrance, but is characterized phenotypically by polyps with differing histologic appearances. Affected individuals display a characteristic phenotype including spinal abnormali-ties, adenoid facies and a tendency to low intelligence, and are at risk of various malignancies including cancers of breast, endometrium, urinary tract and thyroid, as well as colorectal carcinoma and melanoma. A third polyposis syndrome, also showing autosomal dominant inheritance and high penetrance, is Peutz-Jegher syn-drome. This syndrome is again associated with characteristic colorectal polyps, as well as other distinctive phenotypic features such as circumoral pigmentation. Peutz-Jegher syndrome arises through mutations in the LKBI gene on chromosome 19p. Up to 90 % of mutation carriers will develop some kind of cancer in their life-time; approximately 40 % will develop colorectal cancer but other malignancies include carcinoma of the esophagus, stomach, small intestine, pancreas, breast, thyroid, lung or prostate, and sex cord stromal tumors of the gonads.

Recently a syndrome of serrated polyposis has also been recognized. Whereas a subset of sporadic colon cancers arise via BRAF mutation and CpG island hyper-methylation (described in more detail below), some individuals develop multiple colonic polyps which also harbour these molecular genetic changes and which pre-dispose affected individuals to right-sided colonic carcinoma, which often occurs at a younger age than in the general population. Histologically these polyps resemble the hyperplastic polyps commonly seen in the distal colon, but have a predilection for the proximal colon and exhibit subtle histologic differences recognizable as Sessile Serrated Adenoma (SSA, also variously called sessile serrated polyp or ‘Type I” ser-rated adenoma). Disturbingly, it appears that the progression of such polyps to inva-sive cancer may occur at a faster rate than the conventional adenoma- carcinoma sequence, and this suggests that these individuals need more frequent endoscopic surveillance, and/or consideration of prophylactic right hemicolectomy (Fig . 2 ).

Rare Cancers

118

Lynch Syndrome (Including Hereditary Non-polyposis Colon Cancer)

As with FAP, clinicians in the late nineteenth and early twentieth century began to recognize another familial cancer syndrome resulting in an increased risk of developing gastric, endometrial and colorectal cancers. Like Duke, Aldred Scott Warthin 1 was a pathologist who had taken a keen interest in bowel cancers. A chance visit by a young seamstress afraid of developing cancer revealed that she belonged to a family with an astonishingly high incidence of cancer [ 21 ]. Warthin’s work was carried on by his protégé Carl Weller who published follow up data on Warthin’s ‘Family G’ in 1937, reporting that 41 out of 174 members of this family developed neoplasms by the age of 25. Henry Lynch also followed several of these families and his landmark paper in 1966 [ 22 ] identifi ed this as an inheritable disease distinct from FAP and became known as “Lynch Syndrome” (LS) by those in the fi eld, whereas Lynch himself called the disease hereditary non-polyposis colorectal can-cer (HNPCC), because these patients did not develop multiple colorectal polyps [ 23 ]. It is now widely accepted that the term ‘Lynch syndrome’ should be used for the autosomal dominant disease caused by a germline mutation in a DNA Mismatch Repair (MMR) gene that includes HNPCC [ 24 ]. Estimates of the incidence of LS vary and it is reported as being responsible for between 1 and 3 % of all colon cancers [ 25 ], signifi cantly more than FAP. Three percent of all colorectal cancer equates to 4,500 individuals per year in the US, or just 0.0014 % of the population. Like FAP, this would appear to make it a clear candidate for being considered a rare cancer type.

Tumors arising in LS appear to arise through the classical adenoma-carcinoma route [ 26 ], are notable for their preponderance in the right or proximal colon

1 Warthin has Warthin’s tumor or Warthin tumour named after him. It is also known as papillary cystadenoma lymphomatosum , monomorphic adenoma or adenolymphoma, and is a type of benign tumor of the salivary glands.

Fig. 2 ( a ) This 23 year-old man presented acutely with an obstructing, locally advanced (pT4a) carcinoma of the transverse colon (H&E, original magnifi cation x40). ( b ) Examination of the resection specimen revealed multiple polyps showing histologic features of sessile serrated adenoma (H&E, original magnifi cation x100)

N. Zeps and C. Hemmings

119

(about twice as prevalent as sporadic tumors, with 60–70 % rather than 30 % of cases being proximal) [ 27 ], and having a mucinous and poorly differentiated histo-logical appearance, with prominent tumor-infi ltrating lymphocytes [ 28 ]. Similar to FAP, investigation into the underlying genetic basis of LS has revealed a great deal useful to our comprehension of the biology of both normal and cancerous cells. Investigations in the 1990s identifi ed that the genetic changes observed in LS bore a striking resemblance to those seen in bacteria and yeast that had defective DNA MMR genes. This results in the accumulation of a large number of changes in so-called DNA microsatellites, a genetic defect now known as microsatellite instabil-ity (MSI). These microsatellites comprise stretches of DNA where either single nucleotides (mononucleotides) or units of two or more nucleotides (di-, tri-, tetra-, penta-nucleotides, etc.) are repeated [ 29 ]. MSI may be detected using a routine test comprising of fi ve mononucleotide markers (BAT25, BAT26, NR21, NR22, and NR24 ), [ 30 , 31 ], the presence of which can be defi ned as High (MSI- H) or low (MSI-L). In all instances MSI-H is present in confi rmed LS patients. The cause of MSI-H in LS was initially described as being due to mutations in genes specifi c regions of DNA on Chromosomes 2p22-21 [ 32 ] and 3p22.3 [ 33 ]. It was subsequently discov-ered that these regions are the location of the human homologues of MSH2 and MLH1 genes, respectively, both of which encode proteins involved in DNA mis-match repair. Two other MMR genes which are mutated in LS have been identifi ed, namely MSH6 and PMS2. The frequency of mutations differs for each of the MMR; MLH1 and MSH2 account for approximately 90 % of cases [ 34 ], whereas in an Australian study MSH6 mutations comprised 10.3 % of cases and PMS2 just 1.9 % [ 35 ]. The penetrance also differs for each MMR gene; and the average cumulative risk of colorectal cancer to age 70 years (95 % confi dence intervals) for MLH1 and MSH2 mutation carriers were estimated to be 34 % (25–50 %) and 47 % (36–60 %) respectively, for males and 36 % (25–51 %) and 37 % (27–50 %), respec-tively, for females suggesting that even within carriers there is considerable heterogeneity of effect [ 36 ]. Variations can occur throughout the genes that alter amino acid (AA) sequence. Not all of these are pathogenic and about a third do not lead to dysfunction. However, mutations occurring in either the ATP-binding and hydrolysis regions, or the region involved in binding with other MMR genes, can impair MMR function. Like APC, mutations in MMR genes don’t in themselves directly lead to uncontrolled cellular proliferation, and it is the identifi cation of genes that are mutated as a result of MSI that underlie cancer, that are important. The genes affected vary and these are described further in the section on sporadic colorectal cancers, below.

3.2 Other Organ Specifi c Cancers Related to HNPCC

Additional extracolonic cancers include ovary, stomach, small bowel, pancreas, hepa-tobiliary tract, upper uroepithelial tract, brain (Turcot variant) and sebaceous adeno-mas/carcinomas (Muir-Torre variant). It is beyond the scope of this chapter to describe

Rare Cancers

120

all of these here, but they serve to illustrate the broad heterogeneity of cancer subtypes based upon the underlying mutation rather than the anatomical subtype.

3.3 Sporadic Colorectal Cancers

Sporadic CRC has an annual incidence of about 0.05 % of the population in the US, bringing it close to the 1 in 2000 threshold to be classifi ed as a rare disease [ 37 ]. However, improved survival through screening for at-risk populations and better treatments means that the prevalence measured over 5 or 10 years places colorectal cancers (CRC) outside of the defi nition of rare and less common cancers. Research into the underlying genetic causes of CRC over the last 30 years has established that there are a number of specifi c gene alterations that appear to be quite discrete between different CRC, and it is therefore incorrect to think of even sporadic colorectal cancer as a single disease. These molecular genetic alterations may be grouped into those that affect the WNT-signaling pathway (including APC, which is mutated in 80 % of sporadic CRC) [ 38 ], the EGFR-signaling pathway (including RAS and BRAF), other MAPK pathway members (including PDGF, IGF and PI3K), and those that involve mutations in other genes such as p53 or TGF-ß. Approximately 15 % of CRC also exhibit MSI without evidence of germline mutations in any of the known mismatch repair genes. In sporadic cases of MSI there is typically silencing of MLH-1 through hypermethylation [ 39 ] and this is most often associated with the CpG Island Methylator Phenotype (CIMP). Such adult-onset loss of MLH1 function is associated with BRAF mutation, which is rarely observed in patients with germline MMR mutations [ 40 ]. Thus whilst there may be some similar mechanisms that underpin the development of colorectal cancer in familial and sporadic cases, their pathophysiology differ suffi ciently to distinguish them as separate diseases.

The development and use of next generation sequencing technology has permit-ted comprehensive analysis of all genetic alterations present in cancer, and The Cancer Genome Atlas Network recently published its fi ndings based upon 276 colorectal cancer cases [ 41 ]. It broadly defi nes two groups: those that have a large number of mutations (which comprise about 16 % of cases) and those that don’t (which are in the majority). This analysis was able to further separate hypermutated tumors into several additional categories: those with MSI (23/30) and those without; those with MSI that had MMR gene dysfunction both through hypermethylation associated with (17/19) and without (2/19) CIMP; and those that had neither MSI, CIMP or MLH1 methylation but had somatic mutations in other MMR genes, or had POLE aberrations that are rare in non-hypermutated tumors. In a follow up to this work, hypermutated CRCs with MLH1 silencing had signifi cantly reduced levels of WNT signaling and increased BRAF signaling, relative to other types of hypermu-tated CRCs [ 42 ].

Not surprisingly the hyper-mutated and non hyper-mutated colorectal cancers had different mutation profi les, but many of the genes mutated were common to

N. Zeps and C. Hemmings

121

both, and it was only the frequency of mutation that varied. Most of the genes iden-tifi ed in this comprehensive analysis were well known already, but this analysis also revealed that there were alterations in the targets of ß-catenin, TCF/LEF transcrip-tion factors, that suggested more than a passive role in mediating the WNT-signaling pathway. This work also identifi ed the IGF/PI3K pathway as having frequent altera-tions in CRC, including amplifi cation of IGF2. They found IGF2 overexpression to be highly correlated with activating alterations in PI3K signaling pathways, sug-gesting that this pathway may be a useful target for inhibition for CRC therapeutic strategies.

The Cancer Genome Atlas (TCGA) authors noted that the although the non- hypermutated CRC were indistinguishable on the basis of whether they arose in the colon or rectum, those arising in the right colon were more likely to be hypermeth-ylated [ 41 ]. It is important to note that the left/right distinction has been suggested for many years [ 43 ] before the genetics were elucidated, and it has been demon-strated that right-sided cancers confer a worse prognosis [ 44 ]. Methylation of right- sided tumors is more common in females and the proportion increases with age [ 45 ]. It is not clear why right- and left-sided tumors should behave differently but there are embryological differences (the right colon arises from the midgut and the left from the hind gut), and natural variation in environmental exposure. Given the methylation status it is not surprising that there is a signifi cantly increased amount of MSI in right sided CRC arising from silencing of MLH1 and this would seem to run counter to the worse prognosis observed, as MSI-H CRC appear less likely to metastasise [ 46 , 47 ]. However, the reasons underlying the survival difference are not yet understood, and at any rate this does not currently alter clinical management.

3.4 Personalized Treatment of Colorectal Cancer: Subgroup Matters

To facilitate successful intervention (either in terms of prevention or treatment), any classifi cation of colorectal cancer subtypes has to matter clinically. This is certainly the case for prevention, where knowing that colorectal cancers arise in certain families has led to development of specifi c prevention strategies for individuals identifi ed as at-risk. The identifi cation of an APC gene mutation through genetic testing by a familial cancer service allows individuals and other family members that are identi-fi ed as carriers to undertake regular colonoscopic screening (perhaps from as early as 12 years), and to consider prophylactic colectomy in some cases. In FAP, polyps tend to arise earlier in the left hand side of the colon and the rectum, and such screening has revealed that over 90 % of those who carry germline mutations in FAP will develop polyps between 25 and 30 years of age. Where FAP is identifi ed it is now usual to recommend prophylatic colectomy, often with preservation of the distal rectum and construction of a pouch, which is still at risk of neoplasia and will require lifelong surveillance. Such surgery carries a high risk of morbidity and has to be balanced with the risk of developing cancer. As such it is very much an individual decision to be

Rare Cancers

122

made by an adult carrier of FAP. There is a spectrum of risk associated with the differ-ent APC gene mutations that can present, e.g. a mutation in codon 1309 which is associated with a severe phenotype; however, environmental and other person specifi c genetic factors affect how the gene mutations affect an individual. At present there is no clinically useful way to utilize such information, but it is hoped that advances in DNA sequencing technologies may permit the collection of such information in an affordable and timely manner so that it may be used in clinical management.

The vast majority of colorectal cancers will arise at random, through largely non-preventable causes. Epidemiological studies have not yet identifi ed any clinically useful factors to prevent sporadic cancers besides very general, population- based interventions that would have broad utility (such as controlling obesity, taking regu-lar exercise and minimizing exposure to alcohol and tobacco). Therefore for the majority of the population, screening for colorectal cancer has to be based upon the likely incidence of cancer arising at a specifi c age, balanced with the cost of implementing such a scheme. Several countries (including Australia, New Zealand and the UK) have now introduced national colorectal screening programs. In Australia fecal occult blood testing (FOBT), with colonoscopic examination of those cases returning a positive result, is currently offered to older adults (at 50, 55 and 65 years of age), and the introduction of rescreening at 2-yearly intervals is cur-rently being rolled out. However, as syndromic cancers tend to arise at younger ages, identifying these at-risk individuals for earlier intervention remains a priority.

3.5 Chemotherapy

Chemotherapeutic approaches to treat cancer have only been around since the 1950s, with the fi rst use of anti-folates conducted by Sidney Farber for childhood leukemias. Chemotherapy for colon cancer was fi rst developed for advanced dis-ease, where it has led to statistically signifi cant improvements in disease free sur-vival (DFS) [ 48 ]. Adjuvant therapy has been in use since clinical trials showed a survival advantage for stage III CRC of adjuvant 5-FU based chemotherapy [ 49 ]. A number of additional agents have been used in combination with 5-FU including irinotecan (topoisomerase inhibitor) and leucovorin (Folinic acid), which enhances the action of 5-FU through inhibition of thymidylate synthase. Addition of oxalipla-tin has improved incrementally DFS but not Overall Survival (OS) [ 50 ]. It is disap-pointing that none of the genetic mutations identifi ed in familial or sporadic colorectal cancers have yet proven useful in determining who should, or should not receive cytotoxic chemotherapy. Laboratory based investigations have suggested that tumors with MSI arising from MMR defects, either by mutation or methylation, are more resistant to the effects of cytotoxic drugs [ 51 , 52 ]. Examination of MSI as a predictive factor for response to 5-FU based chemotherapy suggests that it is asso-ciated with resistance, as predicted by in vitro studies [ 53 ]. Some have suggested that the presence or absence of MSI-H should be used to determine which Stage II

N. Zeps and C. Hemmings

123

CRC patients should receive chemotherapy [ 54 ] but as yet this is not used clini-cally, and definitive clinical trials are urgently needed. For the reasons pre-sented in this chapter, patient stratifi cation based upon molecular and pathophysiological markers will need to be employed to ensure that the results are not skewed by an unknown confounding factor. Interestingly it seems that MMR defi ciency may result in improved responsiveness to drugs other than 5-FU, such as the topoisomerase inhibitors [ 55 ], though again this stratifi cation is not presently used in clinical practice, nor is it to substratify patients in clinical trials. In a simi-lar vein there are confl icting reports of the utility of the CpG Island Methylator Phenotype (CIMP) as either a prognostic or predictive marker but at present there is no consensus about a possible clinical application [ 56 ].

The lack of such markers of responsiveness represents a signifi cant hole in our knowledge and there is an urgent need to identify not only those patients who are likely to respond to various drugs, but also those who will not benefi t, so that the use of unnecessary, expensive and potentially harmful cytotoxics can be avoided. It is also important to identify those patients who do not require adjuvant therapy at all, but to date no such prognostic markers are reliable enough to be used clinically.

3.6 Targeted Therapies

The advent of monoclonal antibodies and small molecule inhibitors to some of the oncogenic proteins identifi ed in sporadic cancers, such as the EGFR-mediated pathway, has heralded a new era in the treatment of colorectal cancer. Early trials such as CO17 which examined the use of Cetuximab in advanced colorectal cancer showed a signifi cant but modest benefi t in patients with chemotherapy-refractive CRC [ 57 ]. However, when patients were further classifi ed into those that had mutant or wild type KRAS [ 58 ], the benefi t was shown to be restricted to those patients who did not harbor a KRAS mutation. This trial highlighted the necessity to provide more personalized approaches to treatment, since the use of cetuximab would not have been supported without the companion biomarker. Trials for other such tar-geted therapies such as panitumumab and bevacizumab have not shown as much promise in colorectal cancer, although again the absence of suffi ciently sophisti-cated biomarker assays may underlie failure to identify which patients do derive signifi cant advantage from their use. However, the story has been complicated by the fi nding that whereas most patients with KRAS mutations do not benefi t from EGFR blockade, those patients with one particular mutation (G13D) may in fact have a positive response [ 59 ]. Presently these patients are not eligible for treatment through government-funded mechanisms, as there is as yet no evidence to support its use in these cases. ICECREAM 2 is a trial conducted by the Australasian

2 (Australian Clinical trial Register ACTRN12612000901808).

Rare Cancers

124

Gastro- intestinal Trials group (AGITG) that seeks to provide the Level 2 supporting evidence of a benefi t that is required to extend routine use to patients.

A further complexity is the presence of subclones of cancer cells with differ-ing molecular genetic profi les arising within the same tumor, each having a potentially different response to treatment. For example, SISH analysis has revealed a portion of colorectal cancers in which foci within the tumor demon-strate EGFR amplifi cation, whereas the rest of the tumor is diploid [ 60 ]. In our own clinical practice we have observed one patient harbouring both G12D and G13D mutations in KRAS; treatment with cetuximab would theoretically be con-traindicated by the fi rst but recommended by the second, and it is unclear which should prevail.

3.7 Additional Considerations for Personalizing Therapy

As described above colorectal tumors arise from multiple pathways, but it is also important to recognize that these occur within the context of the individual in whom they arise. That is to say that every person has their own unique genetic background which infl uences their phenotype. In carcinoma it is apparent that not only do factors in the stroma modulate the effects of mutations occurring in the epithelial cells, but mutations occurring in the stroma itself can also infl uence the interplay between stromal elements and tumor cells [ 61 ]. A number of studies have demonstrated that isolated stromal cells from carcinomata can support the growth of malignant epithelium more effectively than can normal stromal cells, for exam-ple, fi broblasts isolated from prostatic carcinoma can stimulate signifi cantly increased growth of prostatic epithelial cells in culture [ 62 ]. Furthermore, gene expression analysis of stroma derived from human breast cancers showed that genes associated with desmoid- type fi bromatosis defi ned a distinct group of tumors, whereas another subset was defi ned by the expression of genes associated with solitary fi brous tumor. These two groups of breast tumors, defi ned on the basis of gene expression in the stroma (rather than the tumor epithelium) correlated with clinical outcome [ 63 , 64 ].

In addition, there is emerging evidence that the immune system of the person is also important in tumor surveillance and therefore outcome. There are confl icting reports about whether or not tumor infi ltration by infl ammatory cells is good or bad in terms of prognosis, and it appears that the type of infl ammatory cell as well as the type of immune cells is relevant [ 65 ]. At present the stroma and immune cells are considered ‘contaminants’ when performing genomic profi ling, but it is becom-ing obvious that they too must be analyzed in the context of making a diagnosis and determining any therapy, if we are to be sure to deliver a truly personalized approach. Such a consideration further underlines how every cancer is as unique as the person in whom it arises, and that our present system of classifi cation requires an overhaul.

N. Zeps and C. Hemmings

125

3.8 Social and Political Reasons for Defi ning “Rare” in Cancer?

Creating categories and defi nitions are useful insofar as they serve a specifi c pur-pose. The fi ve most common cancer groups benefi t from being defi ned as such, in terms of both clinical management and political advocacy, which has led to signifi -cant investment in prevention and care measures as well as key support for those diagnosed. Breast cancer has become raised in our consciousness through myriad campaigns using pink as a signature, and more recently prostate cancer has become associated with men wearing a moustache for the month of November (“Movember”). However, patients with rare cancers also deserve expert care and optimal treatment and support, and, until recently there were no support or advo-cacy groups for many of these diseases. There are now a variety of organizations that aim to highlight the plight of people diagnosed with cancers that fall outside of the most numerous categories. In Australia Rare Cancers Australia [ 66 ] has a mission to improve awareness, support and treatment for the 42,000 people diag-nosed with Rare and Less Common (RLC) cancers each year (which equates to almost 35 % of all cancers diagnosed in Australia). Rare Cancers Europe [ 67 ] has broadly similar aims, as does the Rare Cancer Alliance [ 68 ] in the United States of America. There is a potential problem for some of these organizations if all cancers were regarded as ‘rare’ and we are mindful of these issues in our proposal here. Nevertheless, the biological reality of cancer should be separated from socio-political drives to ensure that sufferers of malignancies all receive the appropriate care and support that they deserve.

3.9 What Does the Future Hold?

The advent of ‘omic’ technologies is challenging the current practice of defi ning can-cer on the basis of anatomical location, and has already led to revisions in not only the way we categorize cancer but also how we treat it. Programs such as the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA) have formally integrated the work of many cancer researchers worldwide, and to date have profi led 1,000’s of tumors for gene mutations/variations, transcriptional profi les, methylation patterns and protein expression. The next and even more challenging steps will be to use this information to classify cancers in a manner that will improve on current staging systems. However, to make signifi cant improvements the classifi ca-tion has to be clinically useful and it is not enough to just describe the disease better, if outcomes do not change. While the technical challenge of creating tools to analyze genetic variation has largely been met, the more diffi cult task ahead is making biologi-cal sense of the growing body of information to improve healthcare.

One problem of delivering personalized healthcare for cancer will be that the cur-rent approach taken by clinical trials will have to be completely rethought. At present

Rare Cancers

126

trials often take a long time to recruit suffi cient numbers of eligible patients. If each trial now has to also stratify patients on the basis of their molecular profi le, the clas-sical approach to trials will simply not work. It is important to keep abreast of these changes to enable trials to be designed that logically allocate patients to a treatment regime based on the underlying biological cause, with intent to reduce variation and increase the statistical signifi cance of outcomes.

As the cost of molecular testing becomes lower it will become feasible that all new diagnoses of cancer will be subject to broad molecular profi ling that is cur-rently reserved only for specifi c circumstances and is often only done at the patient’s own expense. Such testing is done on an ad hoc basis and this has raised issues for pathology practices in regard to availability of residual material for testing. Whilst pathology practices typically retain diagnostic material for various lengths of time (depending on jurisdiction), the emergence of research biobanks in the last decade has contributed to the depletion of archived pathology tissue peripheral to diagnos-tic practice. Such access may need to be strictly limited in the future, as more mate-rial from ever smaller biopsies will be required for diagnostic testing and mutational analysis as part of personalized medicine. This will require a rethink of how we structure biobanks, and will need to include mechanisms to enable the use of all current and emerging diagnostic information for research purposes.

4 Conclusion

Studying cancers that fall into so-called ‘rare’ categories is a powerful way to iden-tify the underlying molecular basis of cancer. There is a need to rethink the way we diagnose and treat cancer so that each patient is regarded as having a ‘rare’ disease that is unique to them; indeed in time we may come to the conclusion that there is no such thing as a “common” cancer! This change in perspective may enable us to truly apply the principles of personalized medicine to ensure that each patient receives optimal care. Ultimately we would wish to make all cancer a rare disease through prevention; that goal can only be met through strategies that recognize the complexity of the disease and identify which factors can be modifi ed to impede tumor development.

Acknowledgement A very special thank you to Dr. Elizabeth Thomas for her assistance with the proofreading and referencing of this document and her helpful comments.

References

1. Ferlay J et al (2008) GLOBOCAN 2008 v2.0, Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 10. 2008. Available from: http://globocan.iarc.fr

2. Gatta G (2007) Surveillance of rare cancers in Europe. 2007 [cited 2013]. Available from: http://www.rarecare.eu/rarecancers/rarecancers.asp

N. Zeps and C. Hemmings

127

3. Organization, W.H. International Classifi cation of Diseases. 2010. Available from: http://apps.who.int/classifi cations/icd10/browse/2010/en

4. Dukes CE (1930) The hereditary factor in polyposis intestine, or multiple adenomata. Cancer Rev 5:241–251

5. Edge SB, Compton CC (2010) The American Joint Committee on Cancer: the 7th edition of the AJCC cancer staging manual and the future of TNM. Ann Surg Oncol 17(6):1471–1474

6. Weinberg RA (2008) Chapter 1 – Cancer: a genetic disorder. In: John M et al (eds) The molec-ular basis of cancer, 3rd edn. W.B. Saunders, Philadelphia, pp 3–16

7. Bodmer WF, Bailey CJ, Bodmer J, Bussey HJ, Ellis A, Gorman P, Lucibello FC, Murday VA, Rider SH, Scambler P, Sheer D, Solomon E, Spurr NK (1987) Localization of the gene for familial adenomatous polyposis on chromosome 5. Nature 328(6131):614–616

8. Kinzler KW, Nilbert MC, Su LK, Vogelstein B, Bryan TM, Levy DB, Smith KJ, Preisinger AC, Hedge P, McKechnie D, Finniear R, Matrkam A, Groffen J, Boguski MS, Altschul SF, Horii A, Ando H, Miyoshi Y, Miki Y, Nishisho I, Nakamura Y (1991) Identifi cation of FAP locus genes from chromosome 5q21. Science 253(5020):661–665

9. National Cancer Institute (NCI) (2013) Surveillance and epidemiology and end results (SEER) 1973–2011 DATA. http://seer.cancer.gov/data/

10. Dukes CE (1947) Familial intestinal polyposis. J Clin Pathol 1(1):34–38 11. Erdmann JF, Morris JH (1925) Polyposis of the colon; a survey of the subject. J Surg Gynecol

Obstet 40:460–468 12. Lockhart-Mummery P (1925) Cancer and heredity. Lancet 205(5296):427–429 13. Nathke I (2006) Cytoskeleton out of the cupboard: colon cancer and cytoskeletal changes

induced by loss of APC. Nat Rev Cancer 6(12):967–974 14. Vogelstein B, Kinzler KW (1993) The multistep nature of cancer. Trends Genet 9(4):138–141 15. Al-Tassan N, Chmiel NH, Maynard J, Fleming N, Livingston AL, Williams GT, Hodges AK,

Davies DR, David SS, Sampson JR, Cheadle JP (2002) Inherited variants of MYH associated with somatic G:C → T:A mutations in colorectal tumors. Nat Genet 30(2):227–232

16. Russo MT, De Luca G, Degan P, Bignami M (2007) Different DNA repair strategies to combat the threat from 8-oxoguanine. Mutat Res 614(1–2):69–76

17. Enholm S, Hienonen T, Suomalainen A, Lipton L, Tomlinson I, Kärjä V, Eskelinen M, Mecklin JP, Karhu A, Järvinen HJ, Aaltonen LA (2003) Proportion and phenotype of MYH-associated colorectal neoplasia in a population-based series of Finnish colorectal cancer patients. Am J Pathol 163(3):827–832

18. Russell AM, Zhang J, Luz J, Hutter P, Chappuis PO, Berthod CR, Maillet P, Mueller H, Heinimann K (2006) Prevalence of MYH germline mutations in Swiss APC mutation-negative polyposis patients. Int J Cancer 118(8):1937–1940

19. Farrington SM, Tenesa A, Barnetson R, Wiltshire A, Prendergast J, Porteous M, Campbell H, Dunlop MG (2005) Germline susceptibility to colorectal cancer due to base-excision repair gene defects. Am J Hum Genet 77(1):112–119

20. Tenesa A, Farrington SM, Dunlop MG (2005) Re: association between biallelic and monoal-lelic germline MYH gene mutations and colorectal cancer risk. J Natl Cancer Inst 97(4):320–321, author reply 321–322

21. Warthin A (1913) Heredity with reference to carcinoma: as shown by the study of the cases examined in the pathological laboratory of the University of Michigan, 1895–1913. Arch Intern Med XII(5):546–555

22. Lynch HT, Shaw MW, Magnuson CW, Larsen AL, Krush AJ (1966) Hereditary factors incancer: study of two large midwestern kindreds. Arch Intern Med 117(2):206–212

23. Schuelke GS, Kimberling WJ, Albano WA, Lynch JF, Biscone KA, Lipkin ML, Deschner EE, Mikol YB, Sandberg AA, Elston RC, Bailey-Wilson JE, Danes BS (1985) Hereditary nonpol-yposis colorectal cancer (Lynch syndromes I and II). II. Biomarker studies. Cancer 56(4):939–951

24. Boland CR (2005) Evolution of the nomenclature for the hereditary colorectal cancer syn-dromes. Fam Cancer 4(3):211–218

Rare Cancers

128

25. Hampel H, Frankel WL, Martin E, Arnold M, Khanduja K, Kuebler P, Clendenning M, Sotamaa K, Prior T, Westman JA, Panescu J, Fix D, Lockman J, LaJeunesse J, Comeras I, de la Chapelle A (2008) Feasibility of screening for Lynch syndrome among patients with colorectal cancer. J Clin Oncol 26(35):5783–5788

26. Mecklin JP, Aarnio M, Läärä E, Kairaluoma MV, Pylvänäinen K, Peltomäki P, Aaltonen LA, Järvinen HJ (2007) Development of colorectal tumors in colonoscopic surveillance in Lynch syndrome. Gastroenterology 133(4):1093–1098

27. Gaglia P, Atkin WS, Whitelaw S, Talbot IC, Williams CB, Northover JM, Hodgson SV (1995) Variables associated with the risk of colorectal adenomas in asymptomatic patients with a fam-ily history of colorectal cancer. Gut 36(3):385–390

28. Jass JR, Smyrk TC, Stewart SM, Lane MR, Lanspa SJ, Lynch HT (1994) Pathology of heredi-tary non-polyposis colorectal cancer. Anticancer Res 14(4B):1631–1634

29. de la Chapelle A, Hampel H (2010) Clinical relevance of microsatellite instability in colorectal cancer. J Clin Oncol 28(20):3380–3387

30. Suraweera N, Duval A, Reperant M, Vaury C, Furlan D, Leroy K, Seruca R, Iacopetta B, Hamelin R (2002) Evaluation of tumor microsatellite instability using fi ve quasimonomorphic mononucleotide repeats and pentaplex PCR. Gastroenterology 123(6):1804–1811

31. Iacopetta B, Grieu F, Amanuel B (2010) Microsatellite instability in colorectal cancer. Asia Pac J Clin Oncol 6(4):260–269

32. Fishel R, Lescoe MK, Rao MR, Copeland NG, Jenkins NA, Garber J, Kane M, Kolodner R (1993) The human mutator gene homolog MSH2 and its association with hereditary nonpol-yposis colon cancer. Cell 75(5):1027–1038

33. Bronner CE, Baker SM, Morrison PT, Warren G, Smith LG, Lescoe MK, Kane M, Earabino C, Lipford J, Lindblom A, Tannengard P, Bollag RJ, Godwin AR, Ward DC, Nordenskjold M, Fishel R, Kolodner R, Liskay RM (1994) Mutation in the DNA mismatch repair gene homologue hMLH1 is associated with hereditary non-polyposis colon cancer. Nature 368(6468):258–261

34. Roncari B, Pedroni M, Maffei S, Di Gregorio C, Ponti G, Scarselli A, Losi L, Benatti P, Roncucci L, De Gaetani C, Camellini L, Lucci-Cordisco E, Tricarico R, Genuardi M, Ponz de Leon M (2007) Frequency of constitutional MSH6 mutations in a consecutive series of fami-lies with clinical suspicion of HNPCC. Clin Genet 72(3):230–237

35. Talseth-Palmer BA, McPhillips M, Groombridge C, Spigelman A, Scott RJ (2010) MSH6 and PMS2 mutation positive Australian Lynch syndrome families: novel mutations, cancer risk and age of diagnosis of colorectal cancer. Hered Cancer Clin Prac 8(1):5

36. Dowty JG, Win AK, Buchanan DD, Lindor NM, Macrae FA, Clendenning M, Antill YC, Thibodeau SN, Casey G, Gallinger S, Marchand LL, Newcomb PA, Haile RW, Young GP, James PA, Giles GG, Gunawardena SR, Leggett BA, Gattas M, Boussioutas A, Ahnen DJ, Baron JA, Parry S, Goldblatt J, Young JP, Hopper JL, Jenkins MA (2013) Cancer risks for MLH1 and MSH2 mutation carriers. Hum Mutat 34(3):490–497

37. Rare Diseases Act, in P.L. 107–280 (H.R. 4013), O.o.L.P.a. Analysis, Editor 2002, National Institutes of Health Department of Health and Human Services: USA

38. Kinzler KW, Vogelstein B (1996) Lessons from hereditary colorectal cancer. Cell 87(2):159–170

39. Nicolaides NC, Papadopoulos N, Liu B, Wei YF, Carter KC, Ruben SM, Rosen CA, Haseltine WA, Fleischmann RD, Fraser CM, Adams MD, Venter JC, Dunlop MG, Hamilton SR, Petersen GM, de la Chapelle A, Vogelstein B, Kinzler KW (1994) Mutations of two PMS homologues in hereditary nonpolyposis colon cancer. Nature 371(6492):75–80

40. Parsons MT, Buchanan DD, Thompson B, Young JP, Spurdle AB (2012) Correlation of tumour BRAF mutations and MLH1 methylation with germline mismatch repair (MMR) gene muta-tion status: a literature review assessing utility of tumour features for MMR variant classifi ca-tion. J Med Genet 49(3):151–157

41. Cancer Genome Atlas Network (2012) Comprehensive molecular characterization of human colon and rectal cancer. Nature 487(7407):330–337

N. Zeps and C. Hemmings

129

42. Donehower LA, Creighton CJ, Schultz N, Shinbrot E, Chang K, Gunaratne PH, Muzny D, Sander C, Hamilton SR, Gibbs RA, Wheeler D (2013) MLH1-silenced and non-silenced sub-groups of hypermutated colorectal carcinomas have distinct mutational landscapes. J Pathol 229(1):99–110

43. Iacopetta B (2002) Are there two sides to colorectal cancer? Int J Cancer 101(5):403–408 44. Meguid RA, Slidell MB, Wolfgang CL, Chang DC, Ahuja N (2008) Is there a difference in

survival between right- versus left-sided colon cancers? Ann Surg Oncol 15(9):2388–2394 45. Kakar S, Burgart LJ, Thibodeau SN, Rabe KG, Petersen GM, Goldberg RM, Lindor NM

(2003) Frequency of loss of hMLH1 expression in colorectal carcinoma increases with advanc-ing age. Cancer 97(6):1421–1427

46. Malesci A, Laghi L, Bianchi P, Delconte G, Randolph A, Torri V, Carnaghi C, Doci R, Rosati R, Montorsi M, Roncalli M, Gennari L, Santoro A (2007) Reduced likelihood of metastases in patients with microsatellite-unstable colorectal cancer. Clin Cancer Res 13(13):3831–3839

47. Popat S, Hubner R, Houlston RS (2005) Systematic review of microsatellite instability and colorectal cancer prognosis. J Clin Oncol 23(3):609–618

48. Board RE, Valle JW (2007) Metastatic colorectal cancer: current systemic treatment options. Drugs 67(13):1851–1867

49. O’Connell MJ, Laurie JA, Kahn M, Fitzgibbons RJ Jr, Erlichman C, Shepherd L, Moertel CG, Kocha WI, Pazdur R, Wieand HS, Rubin J, Vukov AM, Donohue JH, Krook JE, Figueredo A (1998) Prospectively randomized trial of postoperative adjuvant chemotherapy in patients with high-risk colon cancer. J Clin Oncol 16(1):295–300

50. Yothers G, O’Connell MJ, Allegra CJ, Kuebler JP, Colangelo LH, Petrelli NJ, Wolmark N (2011) Oxaliplatin as adjuvant therapy for colon cancer: updated results of NSABP C-07 Trial, including survival and subset analyses. J Clin Oncol 29(28):3768–3774

51. Aebi S, Kurdi-Haidar B, Gordon R, Cenni B, Zheng H, Fink D, Christen RD, Boland CR, Koi M, Fishel R, Howell SB (1996) Loss of DNA mismatch repair in acquired resistance to Cisplatin. Cancer Res 56(13):3087–3090

52. Carethers JM, Chauhan DP, Fink D, Nebel S, Bresalier RS, Howell SB, Boland CR (1999) Mismatch repair profi ciency and in vitro response to 5-fl uorouracil. Gastroenterology 117(1):123–131

53. Ribic CM, Sargent DJ, Moore MJ, Thibodeau SN, French AJ, Goldberg RM, Hamilton SR, Laurent-Puig P, Gryfe R, Shepherd LE, Tu D, Redston M, Gallinger S (2003) Tumor microsatellite- instability status as a predictor of benefi t from fl uorouracil-based adjuvant che-motherapy for colon cancer. N Engl J Med 349(3):247–257

54. Sargent DJ, Marsoni S, Monges G, Thibodeau SN, Labianca R, Hamilton SR, French AJ, Kabat B, Foster NR, Torri V, Ribic C, Grothey A, Moore M, Zaniboni A, Seitz JF, Sinicrope F, Gallinger S (2010) Defective mismatch repair as a predictive marker for lack of effi cacy of fl uorouracil-based adjuvant therapy in colon cancer. J Clin Oncol 28(20):3219–3226

55. Jacob S, Aguado M, Fallik D, Praz F (2001) The role of the DNA mismatch repair system in the cytotoxicity of the topoisomerase inhibitors camptothecin and etoposide to human colorec-tal cancer cells. Cancer Res 61(17):6555–6562

56. Marzouk O, Schofi eld J (2011) Review of histopathological and molecular prognostic features in colorectal cancer. Cancers 3(2):2767–2810

57. Jonker DJ, O’Callaghan CJ, Karapetis CS, Zalcberg JR, Tu D, Au HJ, Berry SR, Krahn M, Price T, Simes RJ, Tebbutt NC, van Hazel G, Wierzbicki R, Langer C, Moore MJ (2007) Cetuximab for the treatment of colorectal cancer. N Engl J Med 357(20):2040–2048

58. Karapetis CS, Khambata-Ford S, Jonker DJ, O’Callaghan CJ, Tu D, Tebbutt NC, Simes RJ, Chalchal H, Shapiro JD, Robitaille S, Price TJ, Shepherd L, Au HJ, Langer C, Moore MJ, Zalcberg JR (2008) K-ras mutations and benefi t from cetuximab in advanced colorectal cancer. N Engl J Med 359(17):1757–1765

59. De Roock W, Jonker DJ, Di Nicolantonio F, Sartore-Bianchi A, Tu D, Siena S, Lamba S, Arena S, Frattini M, Piessevaux H, Van Cutsem E, O’Callaghan CJ, Khambata-Ford S, Zalcberg JR, Simes J, Karapetis CS, Bardelli A, Tejpar S (2010) Association of kras p.g13d

Rare Cancers

130

mutation with outcome in patients with chemotherapy-refractory metastatic colorectal cancer treated with cetuximab. JAMA 304(16):1812–1820

60. Hemmings C, Broomfi eld A, Bean E, Whitehead M, Yip D (2009) Immunohistochemical expression of EGFR in colorectal carcinoma correlates with high but not low level gene ampli-fi cation, as demonstrated by CISH. Pathology 41(4):356–360

61. Hemmings C (2013) Is carcinoma a mesenchymal disease? The role of the stromal microenvi-ronment in carcinogenesis. Pathology 45(4):371–381

62. Olumi AF, Grossfeld GD, Hayward SW, Carroll PR, Tlsty TD, Cunha GR (1999) Carcinoma- associated fi broblasts direct tumor progression of initiated human prostatic epithelium. Cancer Res 59(19):5002–5011

63. West RB, Nuyten DS, Subramanian S, Nielsen TO, Corless CL, Rubin BP, Montgomery K, Zhu S, Patel R, Hernandez-Boussard T, Goldblum JR, Brown PO, van de Vijver M, van de Rijn M (2005) Determination of stromal signatures in breast carcinoma. PLoS Biol 3(6):e187

64. Beck AH, Espinosa I, Gilks CB, van de Rijn M, West RB (2008) The fi bromatosis signature defi nes a robust stromal response in breast carcinoma. Lab Invest 88(6):591–601

65. West NR, Kost SE, Martin SD, Milne K, Deleeuw RJ, Nelson BH, Watson PH (2013) Tumour- infi ltrating FOXP3(+) lymphocytes are associated with cytotoxic immune responses and good clinical outcome in oestrogen receptor-negative breast cancer. Br J Cancer 108(1):155–162

66. Australia, R.C. Rare Cancers Australia. 2012 [cited 2013]. Available from: http://www.rare-cancers.org.au

67. Europe, R.C. Rare Cancers Europe. 2012 [cited 2013]. Available from: http://www.rarecancers.org.au

68. Alliance, R.C. Rare Cancer Alliance. 2011 [cited 2013]. Available from: http://www.rare- cancer.org/rare-diseases.php

N. Zeps and C. Hemmings

131M. Özgüç (ed.), Rare Diseases: Integrative PPPM Approach as the Medicine of the Future, Advances in Predictive, Preventive and Personalised Medicine 6,DOI 10.1007/978-94-017-9214-1_9, © Springer Science+Business Media Dordrecht 2015

Abstract Gene therapy has always found a home in rare diseases and the approval of Glybera ® for lipoprotein lipase defi ciency has marked a major milestone in gene therapy and has brought this type of therapy even closer to clinical practice. There is little closer to the personalisation of medicine than the ability to repair or restore the function of a person’s own faulty genes, the core principle of gene therapy. It is therefore reasonable to argue that gene therapy is the ultimate personalised medicine. At the same time, advances in scientifi c understanding and technological ability to analyse the human genome imply that gene therapy could also be used to prevent the development of disease. Using examples from the AAV-based gene therapy fi eld as is applied to rare diseases, the fi rst section of this chapter aims to illustrate how gene therapy aligns to the principles of PPPM whereas the second part of the chapter intends to provide an in depth review of the developments in the AAV fi eld that underpin the use of these viruses as gene therapy delivery systems.

PPPM-Related Keywords Gene therapy • Adeno-associated viral vectors • Rare disease prevention • Rare disease personalised treatment • Alipogene tiparvovec • Glybera® • Predictive, preventive and personalised medicine

1 Introduction

Alipogene tiparvovec (Glybera®, uniQure), the fi rst gene therapy to be approved in the West, received approval by the European Commission in November 2012 for the treatment patients with Lipoprotein Lipase Defi ciency (LPLD) [ 1 ]. Its success has brought full circle the translation of gene therapy, from pre- to clinical research and

Adeno-Associated Virus Gene Therapy and Its Application to the Prevention and Personalised Treatment of Rare Diseases

Konstantina Grosios , Harald Petry , and Jacek Lubelski

K. Grosios (*) • H. Petry • J. Lubelski uniQure B.V , Meibergdreef 61 , 1105 BA Amsterdam , The Netherlands e-mail: n.grosios@uniqure.com

132

now to clinical practice. Gene therapy as a whole has come a long way since the approval of the protocol for the fi rst gene therapy clinical trial in the USA in 1990 which was conducted by the NIH in two children with Adenosine Deaminase Severe Combined Immunodefi ciency (ADA-SCID) [ 2 ]. More than two decades later, clini-cal trials involving over 40 patients have demonstrated that retroviral-mediated hae-matopoietic autologous cell gene therapy can correct this disease with a long term effi cacy of more than 70 % and with 100 % survival [ 3 ]. Similar success has been demonstrated for another form of SCID disorder, SCID-X1 which was the fi rst genetic disorder to be successfully treated using a defective γc Moloney retrovirus–derived vector and ex vivo infection of CD34+ cells [ 4 , 5 ], and it is now evident that SCID-X1 patients can be cured using gene therapy [ 6 ]. It appears that safety con-cerns are also being dealt with as four out of the fi ve patients that have developed leukaemia during these studies have been successfully treated [ 7 ].

The European Association for Predictive, Preventive and Personalised Medicine (EPMA) defi nes Predictive, Preventive and Personalised Medicine (PPPM) as “a new philosophy in healthcare aimed at application of innovative biotechnologies in the prediction of human pathologies, the development of timely prevention and individualized therapy planning” [ 8 ]. EPMA asserts that it is the consideration and progress in all three Ps together that is crucial in driving the development of new medical and biomedical applications. It could be argued that by its nature gene therapy which targets a person’s own genetic defects, is the ultimate personalised medicine. Similarly knowledge of genetic defects with the potential to cause disease will allow gene therapy to be used as preventive medicine. Furthermore, develop-ment of novel gene therapies shares concepts and can utilise strategies applicable to predictive medicine, especially in terms of technological tools for screening and diagnosis. The 2012 EPMA White Paper highlights the fact that PPPM has a key role to play in advancing diagnosis and treatment of rare diseases and is itself pro-moting multi-national and cross-sectoral initiatives in this area [ 9 ]. In this chapter the relevance of PPPM to gene therapy for rare diseases will be discussed together with an introduction to adeno-associated vector (AAV) technology and its future.

The three central features to any gene therapy product are the transgene (genetic defect to be rectifi ed), the target tissues (the site of target gene expression) and the vector (the vehicle that delivers the transgene). So far, vector technology has been the rate limiting step in progressing gene therapy to real clinical practice [ 10 , 11 ]. One type of vector that has long been considered to be the most suitable and safe vector for long-term transgene expression is AAV [ 12 – 14 ]. Its ability to transduce post-mitotic tissues, low immunogenicity and persistence as episomal concatamers are its key characteristics. The history of AAV has recently been marked by a major milestone. The fi rst ever, in the Western world, approval of a gene therapy product is an AAV-based therapy [ 1 ]. Alipogene tiparvovec is an adeno-associated virus (AAV) vector of serotype 1 encoding a naturally occurring gain of function variant of LPL, LPL S447X that is associated with lower plasma TG levels and incidence of cardiovascular disease [ 15 ].

Using examples from the AAV-based gene therapy fi eld as is applied to rare dis-eases, the fi rst section of this chapter aims to illustrate how gene therapy aligns to

K. Grosios et al.

133

the principles of PPPM whereas the second part of the chapter intends to provide an in depth review of the developments in the AAV fi eld that underpin the use of these viruses as gene therapy delivery systems.

2 Gene Therapy and PPPM

2.1 Gene Therapy as Personalised Medicine

Technological advances in performing rapid, low cost and accurate sequencing of the human genome and increasing efforts in generating tools to analyse and align this information to the context of health and disease is enabling progress in medicine. Identifying the gene(s) responsible for a disease, through prenatal or other screening or through standard diagnostic procedures, means that there may be opportunities to intervene as early as possible in life or in the disease patho-genesis process. Even though our understanding of, and experience with gene therapy is still evolving it is easy to envisage that gene therapy based strategies can be used to replace a defective gene and/or restore its function even before any symptoms of the disease appear. Gene therapy is inherently personalised and alipogene tiparvovec is a good example of this. The gene sequence for lipopro-tein lipase was published in 1987 [ 16 ] and the fi rst mutations in the gene where characterised 2 years later [ 17 ] with many more subsequently [ 18 ]. These muta-tions give rise to defi ciency in lipoprotein lipase (LPL), an enzyme that is secreted by adipocytes and smooth muscle cells and which mediates catabolism of tri-glyceride-rich glycoproteins. Defi ciency in the enzyme results in inability to clear lipids (triglycerides) from the blood causing the ultra-rare autosomal reces-sive inherited disease, Lipoprotein Lipase Defi ciency (LPLD). LPLD manifests in a series of complications, including episodes of abdominal pain and poten-tially fatal pancreatitis [ 19 ]. Alipogene tiparvovec introduces a healthy LPL gene into the body, which results in the production of functional LPL thus restoring production of the enzyme. Furthermore, it is approved for patients who are genetically diagnosed and confi rmed to have LPLD.

The case is not dissimilar for the approximately 1,800 gene therapy clinical trials ongoing currently [ 7 ]. They all aim to replace a body’s own defective single gene, as is the case in monogenic disorders, or address a central genetic malfunc-tion driving complex diseases such as cancer, cardiovascular, neurological or oth-ers. Interestingly, the number of gene therapy trials for monogenic diseases, the majority of rare diseases, has been increasing over the last decade to 8.7 % of all gene therapy trials in 2012. Currently the Journal of Gene Medicine Clinical Trial website indicates that just under half of all gene therapy trials conducted in mono-genic diseases employ an AAV-based approach and diseases being targeted include, muscle diseases such as spinal muscle atrophy and various muscular dystrophy disorders, eye disease including, Leber Congenital Amaurosis and

Adeno-Associated Virus Gene Therapy and Its Application to the Prevention…

134

Choroideremia, various CNS and neuronal disorders including Sanfi lippo B, Metachromatic leukodystrophy and Tay–Sachs disease, as well as diseases affecting the liver such as Haemophilia B and Acute Intermittent Porphyria ( www.abedia.com/wiley/index.html ).

2.2 Gene Therapy and Biomarkers

Biomarkers, a central concepts and tool of PPPM, are integral part in the develop-ment of new gene therapy medicinal products, like any other drug product. Conventional drug development categorises biomarkers into, (a) biomarkers that indicate the drug is hitting the target (or proof of mechanism biomarkers), (b) bio-markers indicating that once the target has been hit there is a consequence in patho-physiology (or proof of principle biomarkers) and (c) biomarkers indicating that the previous two steps affect the clinical status of the patient (proof of concept biomark-ers) [ 20 ]. The same concepts can be applied to human gene therapy (Fig. 1 ), the development path of which entails some distinct and many similar challenges. Whereas the notion of proof of principle and proof of concept biomarkers remains the same for gene therapy as in conventional therapies, in the case of proof of mech-anism biomarker there is a clear distinction. In gene therapy proof of mechanism would entail confi rmation that the viral vector is transducing the appropriate target organ and is doing so effi ciently. Vector tropism and the use of tissue specifi c pro-moters in the expression cassette of gene therapy vectors intend to ensure this hap-pens. Numerous evidence has been collected to provide information about the tropism, or preference to transduce, of viruses used in gene therapy, including natu-rally occurring and modifi ed AAVs [ 11 , 21 – 23 ]. However proof of mechanism is not as straight forward to demonstrate in clinical trials, especially for diseases affecting internal organs. During the clinical development programme of alipogene

Fig. 1 Biomarkers in gene therapy – during the development stages of new gene therapies biomarkers can be categorised following a similar paradigm the has been for conventional therapies

K. Grosios et al.

135

tiparvovec it was possible to obtain muscle biopsies from patients and confi rm AAV1 transduction of this tissue [ 24 ]. In contrast, in diseases affecting, for example, the liver or brain obtaining tissue biopsies from patients would be more complicated or even impossible. Non-invasive imaging has been used extensively to follow the delivery and assess the transduction ability of AAVs in various animals models but this can only at present be achieved using reporter genes which would not be feasible to use in human clinical trials [ 25 – 29 ].

A challenge often encountered in the non-gene therapy arena is the strength of the correlation between biomarkers and clinical outcome or in fact whether there is associative, correlative or even causal effect [ 30 , 20 , 31 ]. Gene therapy trialing is not dissimilar in this respect. In a now considered landmark study in Haemophilia B proof of concept was possibly less complicated to establish than in other gene therapy trials [ 32 ]. This was due to the manifestations of the disease as well as the fact that Haemophilia B is one of the most well characterised rare diseases where proof of principle and proof of concept biomarkers (plasma Factor IX levels and recombinant protein consumption) correlate very well with the disease outcome (need for prophylaxis as an indication of the severity of disease) [ 33 ]. In this study, six patients treated with an AAV8-FIX gene therapy demonstrated persistent FIX plasma levels between 1 and 8 % of normal levels, converting from severe to moder-ate disease phenotype and were either without the need for prophylaxis or had sig-nifi cantly reduced need for prophylactic treatment for between 6 and 16 months following gene therapy administration (at the time of the report) [ 32 ]. Whether this will be a long-/life-lasting effect still remains to be seen. On the other hand, demon-strating a correlation between biomarkers and clinical outcome is not as unproblem-atic in more complex or rare conditions where the pathophysiology and clinical picture is more intricate. In rare diseases in particular this is even more of an issue due to the very low number of patients. For example, the use of triglycerides and postprandial chylomicrons was extensively investigated during the trials of alipo-gene tiparvovec. Clinical data indicated that in half of the LPLD patients treated with alipogene tiparvovec there was a reduction of at least 40 % in fasting plasma triglyceride concentrations in the blood between 3 and 12 weeks which was associ-ated with sustained improvements in the postprandial metabolism of newly formed, large/buoyant chylomicrons and a clinically relevant reduction in the frequency of acute pancreatitis [ 34 , 15 ]. However work still continues, after the approval of the drug, in order to validate the link of these biomarkers to the clinical features of the disease [ 35 ]. Further use of this product and collection of data through a dedicated patient registry will provide the means to fully evaluate them in order to enable extending the use of the product to less severely affected patients and help the devel-opment of newer and improved products for the disease.

The concept of stratifi ed medicine is integral to the context of PPPM. It is widely accepted that patient stratifi cation using various biomarkers or diagnostic tools is becoming a key enabler in conducting clinical trials and managing health and disease [ 36 ]. Even for highly prevalent diseases though such as cancer and cardiovascular disease new molecular characterisation means that diseases (and patients) are strati-fi ed into increasingly smaller segments. This in turn has implications in terms of,

Adeno-Associated Virus Gene Therapy and Its Application to the Prevention…

136

designing appropriately powered clinical studies in small patient populations, the need for placebo or control randomised clinical trials and ethical consequences as well as implications in the regulatory path to drug approval, market and patient access and commercial impact [ 37 , 38 ]. These are all issues that gene therapy for rare diseases has been facing since its inception and in this case gene therapy is paving the way and could potentially provide benefi t for other forms of advanced therapies.

2.3 Gene Therapy and Prevention

A number of attempts are being made to apply AAV gene therapy in prevention. In cardiovascular disease for example, even though surgical revascularisation (bypass surgery) has dramatically improved clinical outcomes the problem of vein graft disease still remains [ 39 ]. Restoring the production of molecules such as endothelial nitric oxide synthase and prostaglandin or inhibiting infl ammatory reactions by using AAV-mediated gene transfer may help restore normal vascular function and prevent vein graft disease [ 40 , 41 ]. There are however two key limiting factors that need to be overcome, namely, the lack of AAV tropism for endothelium and smooth muscle cells and the delayed onset in AAV vector gene expression. The former obstacle is being addressed by phage display approaches used to develop AAV capsids with modifi ed tropism to be able effi ciently transduce such cell types [ 42 ].

An even more obvious application of gene therapy in preventive medicine is its potential to be applied in utero as a means of restoring or rectifying a genetic defect at the molecular level as early in life as possible. A key limitation of AAV in this arena is the fact that these viruses are only able to transduce post-mitotic tissues. However, intrauterine gene therapy is only at its early stages and there are still unknown or unproven aspects worth exploring. There have been a number of pre- clinical studies showing effi cacy of AAV intrauterine gene therapies in animal models of, for example, Haemophilia B, Wilson’s disease and hypophosphatasia [ 43 – 46 ].

Continuous progress in gene therapy relies on progress being made in PPPM and its introduction to clinical practice, and vice versa. A key aspect of this is the development of advanced molecular diagnostics which is also a core element of PPPM [ 47 ]. For many rare inherited disorders, diagnosis is often a lengthy and complicated process which in itself is a big emotional and fi nancial burden for patients, their families and healthcare systems [ 48 ]. At the same time, even when diagnosis is made there is often no treatment or cure available. Here is where AAV, and other, gene therapy can have a major impact by potentially offering new therapeutic options.

Since vector technology has been the main determinant of progress in gene therapy so far, in considering the future of AAV gene therapy for rare diseases it is important to understand the basics of AAV technology, the current state of the art and future directions. This is what we attempt to do in the second part of this chapter.

K. Grosios et al.

137

3 AAV Vector Technology

3.1 AAV Structure and Organisation

AAV belongs to the genus Dependovirus , family: Parvoviridae and was fi rst noted around 1966 as contamination of adenovirus preparation [ 49 ]. One of its advantages in comparison to other viral delivery systems is its excellent safety profi le, which is related to the fact that AAV up to date has not been associated with any human disease and is known to elicit rather mild immune response. First reports describing molecular cloning of AAV, which started in depth analysis of AAV sequence, its genes and proteins dates back to early 1980s [ 50 , 51 ]. Multiple AAV serotypes have been iso-lated to date revealing uniformed compact genetic organisation in an exceptionally small capsid having a diameter of ~22 nm. AAV DNA is packaged in an icosahedral capsid as a single stranded molecule which contains overlapping open reading frames encoding proteins responsible for DNA replication, packaging and capsid formation (Fig. 2 ). The genes are fl anked by inverted terminal repeats (ITRs) which

Fig. 2 Genetic organisation of AAV . AAV genome is composed of two open reading frames located between inverted terminal repeats (ITRs): rep gene , encodes four non-structural regulatory proteins i.e. Rep78, Rep68, Rep52 and Rep40 and a cap gene encoding three capsid proteins VP1, VP2 and VP3. Rep proteins are expressed from two separate promoters, p5 that drives expression of Rep 78, Rep68 and p19 that promotes Rep52 and Rep40. The Rep68 and Rep40 are spliced variants of their bigger counterparts. The expression of capsid proteins VP1, VP2 and VP3 is driven by p40 promoter. Two transcripts are being formed due to alternative splicing. First one result in predominant production of VP1 and the second splice variant where ATG of VP1 is removed result in production of VP2 and VP3. VP2 is initiated from an alternative start codon i.e. ACG and VP3 is translated from downstream ATG start site. The use of alternative splicing and non-standard translational start codon (ACG) results in 1:1:10 stoichiometry of VP1:VP2:VP3 proteins in assembled capsids

Adeno-Associated Virus Gene Therapy and Its Application to the Prevention…

138

are the only genetic elements required in cis for DNA cassette to be replicated and packaged. Figure 2 shows the compactness of the AAV gene organisation where eight proteins are encoded by overlapping orf’s encompassed on approximately 4.7 kbp long DNA molecule.

Inverted terminal repeats (ITR’s) – For the fi rst time AAV inverted terminal repeats were shown by electron microscopy by the group of Kenneth Berns in the 1970s [ 52 – 55 ]. Subsequently their full sequence details were disclosed by the same group [ 56 ] who also used the chemical sequencing reaction of Maxam and Gilbert [ 57 ] to revile the DNA sequence of ITRs. Inverted terminal repeats are ~145 bp long and contain regions of complementary, partially palindromic sequence which when folded on its own will from a T-shaped hairpin structure. They can adopt two orientations, fl ip and fl op, which differ by the confi guration of their arms [ 50 ]. Mutagenesis studies have shown that ITRs are indispensable for replication and packaging and that they are cleaved in a unique sequence termed terminal resolution site ( trs ) [ 58 – 69 ].

Rep enzymes – The AAV genome encodes the rep gene, expression of which results in a family of four transcripts initiated from two promoters, p5 and p19. The expression from p5 results in generation of two major Rep proteins. This includes Rep78 and its splice variant Rep68 (Fig. 2 ). The minor Rep proteins are generated from promoter p19 and are termed Rep52 and rep40 (Fig. 2 ). Minor Reps are smaller version of major Reps but their functions differ. Major Reps are involved in the genome replication process were they bind non-covalently to RBE elements of ITRs and resolve replication intermediates by cleaving ITRs at the terminal resolution site ( trs ) [ 70 ]. Furthermore, major Rep’s possess a number of biochemical activities, which include ATPase, nicking and helicase activity [ 58 , 70 – 72 ]. Both major and minor Rep’s are involved in the encapsidation reaction of previously replicated ITR fl anked DNA into preformed capsids. It has been shown that Rep78 binds cova-lently to 3′ ITR and performs single stranded DNA packaging in concert with Rep52. The minor Rep with its ATPase and helicase activity, actively unwind dou-ble stranded DNA replication intermediates in 3′–5′ direction preparing them for an active transport to the preformed capsids [ 73 – 76 ].

Structural proteins (Cap) – The capsid is formed by three viral proteins, termed VP1, VP2, VP3. To date there are nine different capsid structures of various AAV serotypes deposited in Protein Data Bank ( www.rcsb.org ). Structural studies indicate that the AAV capsid displays molecular stoichiometry of 1:1:10 of VP1:VP2:VP3, respectively, amounting to 60 molecules in total. Expression of cap gene is driven by p40 promoter and results in mRNA which is translated into three viral proteins by combination of leaky ribosomal scanning and splicing (Fig. 2 ). It has been demonstrated that the assembly of capsid can occur spontaneously and that it is possible to make particles consisting of only VP3 or its combination with the other two VP’s. The correct stoichiometry of the three VP’s has been shown to be important for potency of the vector [ 77 ]. In particular low incorporation of VP1 can have profound impact on ability of the vector to deliver its cargo to nucleus [ 77 ]. This is likely to be related to the fact that the unique N-terminal part of VP1 contains

K. Grosios et al.

139

an enzymatic domain termed phospholipase A 2 (PLA 2 ). This domain is under normal conditions buried inside of the viral particle. At the time the virus is taken up by the cell and entrapped in the endosome where it is exposed to low pH, the N-terminal part of VP1 is released. This leads to exposure of the PLA 2 domain which is able to hydrolase specifi cally the 2-acyl ester ( sn-2 ) bond of phospholipid substrates, resulting in release of lysophospholipids and free fatty acid allowing, in turn, endosomal escape of AAV [ 78 , 79 ].

3.2 AAV Manufacturing

Developments of AAV Production Systems: A Historical Perspective

Molecular cloning of AAV in early 1980s paved the way for establishing fi rst protocols for AAV based vector generation. Since then recombinant AAV (rAAV) production systems have gone through incremental developments (Fig. 3 ) driven by various rationales, such as: safety, yields per cell, scalability, impurities profi le, simplicity, modularity, robustness and fi nally commercial and regulatory requirements.

AAV production methods can be classifi ed into two main groups based on the host organisms used. Mammalian cell lines, historically the fi rst, and insect cells. Very recently attempts are being made to adopt Saccharomyces cerevisiae for the same purpose. All the protocols for generation of rAAV contain the same basic parts. This includes, (a) a production organism which provides the production envi-ronment; (b) the DNA cassette of interest which is fl anked by ITRs; (c) Rep proteins which have regulatory role during replication and active role during packaging

Fig. 3 A historical perspective of rAAV evolution – major milestones in the development of various rAAV production systems for gene therapy applications

Adeno-Associated Virus Gene Therapy and Its Application to the Prevention…

140

reaction; (d) Cap proteins which form capsid; and (e) Viral proteins which provide helper function that are needed due to the fact that AAV is an dependovirus and as such does not encode for all the functions necessary for its life cycle but relies on the helper virus instead.

Mammalian Cell Based Production Systems

Transfection/transduction protocols – The initial discovery, which paved the road towards rAAV generation, involved the molecular cloning of AAV into a bacterial plasmids, transfection of which into adenovirus transduced human cell line resulted in excision of the AAV genome, leading to generation of virus offspring [ 80 , 50 ]. Soon after, the potential of AAV as a gene transfer vehicle was recognised and reported by Muzyczka and colleagues who substituted the neomycin resistant gene for the AAV cap gene and used this recombinant vector for successful gene transfer to murine and human cell cultures [ 81 ]. Subsequently the fi rst protocols for produc-tion or recombinant adeno-associated virus based vectors were reported [ 82 , 83 ].

Transfection/transduction protocols in mammalian cells consist of transfecting cells with two different plasmids, one carrying AAV genes responsible for capsid formations ( cap ) and involved in replication/packaging ( rep ) and a second (vec) harboring expression cassette of interest fl anked by inverted terminal repeats (ITRs). Subsequently host cells, which provide the molecular machinery and appropriate environment needed for vector generation, are transduced with a helper virus, ade-novirus; a natural helper of AAV (Fig. 4a ). There are a number of variable compo-nents of this system including, the host cell type and helper virus choice. Helper virus functions can be effi ciently delivered not only by adenovirus but also by Herpes Simplex virus [ 84 ]. Furthermore, various human cell lines can be used for generation of rAAV, which include HEK 293, HeLa and A549. This transfection/transduction based protocols are however rather ineffi cient and can only generate ~10 2 AAV particles per producer cell [ 85 ]. Another drawback is the use of a patho-genic helper virus which requires selective purifi cation steps to ensure viral clear-ance of the produced vector. The transfection step, use of adherent cells and potentially pathogenic human viruses as helpers make this basic protocol not well suited for industrial applications.

Transfection protocols – The elimination of the necessity to use helper virus became possible after identifying a subset of genes which were required to support generation of rAAV and experiments demonstrating that transfecting adenovirus DNA would suffi ce to provide the required helper functions [ 86 ]. Subsequent exper-iments concentrated at removing parts of adenoviral genome such as replication origin and packaging signals preventing generation of infectious adenovirus during vector production [ 87 ]. In order to simplify the protocol and skip the cumbersome preparation step of non-infectious adenovirus DNA a number of groups simultane-ously reported generation of a helper plasmid that carried all adenovirus helper genes needed for rAAV production [ 88 – 91 ]. This three plasmid protocol resulted in rAAV yields ranging from 10 2 up to 10 4 vector genomes per cell [ 85 , 92 ] (Fig. 4b ).

K. Grosios et al.

141

Fig. 4 Comparison of mammalian cells based rAAV production systems . The various rAAV mammalian cell based production systems are shown and classifi ed based on the route of introduction of the required genetic material needed for rAAV production into the cells ( a – d )

Adeno-Associated Virus Gene Therapy and Its Application to the Prevention…

142

In parallel a number of other advancements was made which greatly improved the yields per cell of rAAV production elevating it close to the wild type levels [ 87 ]. Li and colleagues [ 93 ] studied the role of regulation of AAV genes and reported that elevated expression of Rep had an immediate negative effect on rAAV yields. Whereas lowering of Rep78/68 expression via mutation of initiator ATG to ACG resulted in up regulation of cap which was related to increase in rAAV yields [ 93 ]. Soon after a three plasmid transfection system was reported and it was followed by a two plasmid system where adenoviral helper genes E2A , VAI and E4 genes where combined with AAV rep and cap genes on one plasmid whereas the expression cas-sette fl anked by ITR’s was supplied by a separate plasmid [ 91 , 94 ] (Fig. 4b ).

Another milestone in rAAV development was marked by cross-packaging of expression cassettes fl anked by AAV serotype 2 ITRs with AAV2 Rep into capsid shells from various serotypes [ 95 ]. The possibility to use one Rep and its cognate ITRs for packaging DNA to capsids originating from various serotypes greatly added to the modularity of the system and to the standardisation of production methods.

High fl exibility and modularity provides great advantage to the transfection based rAAV production systems. The time needed for preparation of new rAAV vectors for instance harboring many variants of a given expression cassette is rela-tively short. This feature is for the large part responsible for the high popularity of the transfection systems and its suitability for preclinical studies. However the use of transfection methodologies and adherently cultured cells limits the scale up and usefulness of this system for industrial medical applications. A comparison of theo-retical yields of rAAV produced in suspension and adherently grown cells assuming constant yield per cell (10 4 ) has shown that in order to generate 10 15 particles con-taining vector genomes a rather modest 50 L bioreactor scale is needed for suspen-sion grown inset cells. Whereas in order to generate the same amount of particles in adherently grown cell cultures, an area of 1,000,000 cm 2 or 5,700 × 15 cm tissue culture plates would be required [ 96 ]. Last but not least use of bacterial plasmids in a mammalian host results in generation of DNA impurities which inadvertently may be packaged into rAAV during production. These impurities may carry expression units with promoters that can be expressed in patient cells and as such carry a poten-tial safety risk.

Stable cell lines for rAAV production – one of the main obstacles that prevented plasmid transfection based rAAV production systems from being developed for medical applications is the limitation of production capacity. As described above, the obtained yields are too low and production scale up is limited by space, labor and costs. The use of stable cell lines carrying all of rep , cap and the transgene cas-settes or some of these components provides an alternative. These cells usually need to be transduced with wild type helper virus or a hybrid helper virus (Fig. 4c ). The latter can also serve as a vehicle to deliver rep/cap or transgene. It has been demon-strated that high expression of rep in mammalian cells is deleterious for cell prolif-eration and results in cytotoxicity [ 97 , 98 ]. This made it diffi cult to select clones which would produce high levels of rAAV for which high Rep expression is required, which in turns interferes with the selection procedure due to negative

K. Grosios et al.

143

infl uence of Rep on cell proliferation. Nevertheless, fi rst stable integrant harboring Rep protein emerged which were under an inducible promoter capable of assem-bling viral particles upon providing Cap and transgene cassette in trans [ 97 ]. Subsequently producer cell lines [ 99 ], containing all the needed AAV genes and packaging cell lines [ 100 – 102 ] containing rep and cap genes were reported (Fig. 4c ). The obtained yields were rather low, not exceeding 10 2 vector genomes per cell [ 99 ]. Fan et al. [ 103 ] demonstrated that the lack of replication of rep and cap during production resulted in too low expression of the genes, which in turn caused the low yields [ 103 ]. In order to circumvent this problem and yet generate stable cell lines, with integrated components for rAAV production, different strategies were undertaken. Inducible amplifi cation of rep and cap integration loci linked to SV40 origin of replication were fi rst described [ 104 ]. The amplifi cation of rep and cap genes resulted in boost of their expression and resulted in production levels approaching 10 4 of vector genomes/cell. Similar amplifi cation of rep and cap genes was noted in HeLa [ 105 ] and A549 [ 106 ] cell lines upon transduction of replicative adenovirus and resulted in signifi cant yields of rAAV. All the above mentioned stable cell lines needed replication competent adenovirus infection until a helper-virus- free inducible AAV producer cell line was constructed [ 107 ]. This cell line contained all the AAV elements needed for its production including inducible adenovirus E1A and E1B genes and generated rAAV upon induction of integrated adenoviral genes and transduction with replication incompetent adenovirus with deleted genes E1A, E1B and E3 genes [ 107 ]. Stable producer cell lines proved to be slightly more effi cient in production of rAAV (10 4 –10 6 vector genomes per cell) than transfection based protocols [ 108 ]. They appear to be also more amenable for scale up due to elimination of transfection step but they still require transduction with the helper virus such as adenovirus or herpes simplex virus, albeit in order to minimalise potential risks replication incompetent helpers are being used [ 108 ]. Farson and colleagues [ 109 ] demonstrated that stable producer cell lines can be adapted to suspension grown in serum free conditions. This represents another step towards industrial mammalian based rAAV production system [ 109 ]. One of the limitations however of stable cell line production protocols is their lack of fl exibility. Yuan et al. [ 110 ] reported a simplifi ed gateway cloning protocol for generation of stable rAAV producer cell lines with additional control of rep expression, termed dual silencing switch. The introduction of the dual silencing switch tighten expres-sion of rep and in turn reduced greatly its unwanted effects on cell growth resulting in improved stability of these cell lines [ 110 ].

Transduction – In early 1980s it became apparent that AAV replication and pack-aging can be supported not only by adenovirus but also by other viruses e.g. herpes simplex virus [ 84 ]. Subsequently a subset of four genes which consisted of a helixase- primase complex and the major DNA-binding protein were identifi ed as required to support rAAV generation [ 111 ]. These discoveries opened up the way to use herpes simplex as a vehicle to deliver genes needed for rAAV production to the mammalian cells. Previous attempts to use adenovirus for the same purpose had proven to be unsuccessful. This was associated mainly with Rep exerting negative infl uence on adenovirus replication [ 112 ]. The full helper capabilities of herpes

Adeno-Associated Virus Gene Therapy and Its Application to the Prevention…

144

simplex made it a good candidate for generation of HSV/AAV hybrids. Conway et al. [ 113 ] reported the generation of an HSV-1 amplicon expressing rep and cap genes. This amplicon was generated by co-transfection of plasmids containing rep , cap , HSV replication origin and packaging signals with wild type HSV. HSV pro-vided all the helper function needed in trans for generation of HSV based particles fi lled with amplicon containing rep and cap genes termed HSV-RC. HSV-RC was subsequently used for amplifi cation and packaging of rAAV DNA from parvoviral cell lines [ 113 , 114 ]. The amplicon strategy presented a number of disadvantages such as, contamination with wild type HSV helper of HSV-RC stocks or the hetero-geneity of the amplicon itself. The second generation of HSV based rAAV produc-tion system was based on true HSV/AAV hybrids where rep and cap genes were integrated to the rHSV genome; such hybrids were subsequently used for rescue of parvoviral cell lines [ 115 ]. The HSV/AAV hybrid system does not rely on transfec-tion and as such is potentially scalable. It has been demonstrated to support genera-tion of rAAV with yields of up to 3 × 10 3 after optimisation [ 116 ]. The next step for HSV based production systems represented elimination of the necessity of parvovi-rus cell line generation. This was achieved by construction of two pairs of HSV/AAV hybrids, carrying either rep and cap genes or an ITR fl anked cassette of inter-est [ 117 , 118 ] (Fig. 4d ). Finally, use of suspension grown BHK cells transduced with replication incompetent HSV/AAV hybrids (ICP-27 defi cient) showed genera-tion of up to 8 × 10 4 viral genomes per cell demonstrating the amenability of this system for industrial large scale applications [ 119 ].

3.3 Insect Cells Based Production Systems

Transduction protocols – The Baculovirus Expression Vector System (BEVS) has been widely and successfully used for expression of variety of heterologous pro-teins [ 120 ]. This includes generation of high quantity of protein for mechanistic, structural, enzymatic research but also for production of therapeutic biologics, e.g. Flublock (seasonal infl uenza vaccine, Protein Science Corporation), Diamyd (therapeutic vaccine for type 1 diabetes; Diamyd Medical AB), Cervarix TM (GlaxoSmithKline) and, as already discussed Glybera® (gene therapy for lipopro-tein lipase defi ciency; uniQure).

The ground breaking idea to use baculoviruses as helper and delivery vectors for genes needed to generate rAAV in insect cells was coined by Urabe, Ding and Kotin in 2002 [ 121 ]. Before that time there were a number of reports using BEVS to express the individual components of AAV system. As early as in 1991 Owens et al. [ 122 ] reported BEVS driven expression of functional Rep78 and Rep68 [ 122 ]. A year later Ruffi ng and colleagues demonstrated generation of AAV virus like parti-cles (VLPs) using insect cells as host for production. They expressed the AAV viral proteins VP1, VP2 and VP3 from three separate baculoviruses demonstrating that these proteins could assemble in particles that resemble AAV [ 123 ]. Subsequently Bac-AAV hybrid systems intended for transduction of mammalian cells were reported.

K. Grosios et al.

145

These hybrids contained rep gene and an expression cassette fl anked by ITRs cloned into the baculovirus backbone [ 124 ]. Importantly it was noticed that the Rep protein which was notoriously known for inhibiting replication of a number of viruses including adenovirus did not appear to inhibit propagation of baculoviruses [ 124 ]. Expression of the separate components of rAAV generation system and lack of neg-ative infl uence of Rep on the amplifi cation of Autographa californica multicapsid nucleopolyhedrovirus (AcmNVP), one of most studied baculoviruses, make BEVS an interesting candidate for rAAV production.

After the Urabe et al. [ 121 ] publication it became evident that suspension grown insect cells have potential to provide an alternative or even improved system for rAAV generation, having a number of advantages as compared to competitor systems: (1) it is modular and fl exible, (2) generates high yields of rAAV, (3) it is scalable due to use of suspension grown cells and elimination of transfection step, (4) comprises of well characterised baculoviruses which are not human pathogens and are well suited as a helper virus for rAAV production, (5) the use of serum free media and insect cells as production host increase the safety of the system.

Three baculovirus confi guration (the original system) – The initial system described by Urabe et al. [ 121 ] consists of three baculoviruses, namely Bac-Rep, Bac-cap and Bac-vec, co-infection of which into insect cells e.g. SF9 resulted in generation of rAAV (Fig. 5a ). The properties of such produced rAAV, i.e. physical and molecular characteristic including potency, did not differ signifi cantly from the rAAV generated in mammalian cells [ 121 ]. In order to accomplish effi cient genera-tion of AAV vectors in insect cells the AAV proteins needed for the process had to be expressed at appropriate levels. This required a number of adaptations of operons encoding for Rep and Cap proteins. Wild type AAV expresses large Rep78 to small Rep52 from two distinct promoters p5 and p19 respectively and splicing of the two messengers results in generation of Rep68 and Rep52 variants. Urabe and colleagues constructed a DNA cassette in which expression of Rep78 was driven by the partially deleted promoter for the immediate-early 1 gene ( ΔIE-1 ) whereas Rep52 expression was controlled by a strong polyhedrin promoter ( polh ) (Fig. 5a ). The spliced variants of large and small Reps were not observed in insect cells which likely relates to the difference in splicing processes between mammalian and insect cells.

Another technical challenge to be overcome was related to the expression of the three major viral proteins (VP’s). Wild type AAV expresses VP1, 2 and 3 from p40 promoter. Arising messenger RNA is spliced into two species: one responsible for VP1 expression whereas the second expresses both VP2 and VP3 via a “leaky ribo-somal scanning mechanism” where the protein is initiated from non-canonical start i.e. ACG, is occasionally missed by the ribosome complex which than proceeds further until it fi nds the canonical start of VP3. Due to the differences in splicing machinery between vertebrate and insect cells the above described mechanism did not result in generation of proper capsids in insect cells. Urabe et al. [ 121 ] decided to introduce a modifi cation of translational start of VP1 which was similar to these found in the VP2 in such a way that the translational start of VP1 was changed to

Adeno-Associated Virus Gene Therapy and Its Application to the Prevention…

146

Fig. 5 Comparison of insect cells based rAAV production systems. The various rAAV insect cell based production systems are shown and classifi ed based on the number of baculovirus construct used

K. Grosios et al.

147

ACG and the initiation context, which consists of nine nucleotides proceeding VP1, was changed to those proceeding VP2. These genetic alterations resulted in expres-sion of the three VPs in the correct stoichiometry that could properly assemble into capsids. The transgene cassette on the other hand was similar to what was previ-ously described for mammalian based systems, fl anked by ITRs as the only in trans required elements for replication and packaging (Fig. 5a ).

Five baculovirus confi guration : One of the reported drawbacks of the system described by Urabe et al. [ 121 ] was the stability of the baculovirus construct where the design of Bac-rep included rep78 and rep52 in head-to-head orientation. This created a large palindromic region due to the fact that the exact sequence of rep52 is a part of the rep78 open reading frame. Thereby, this construct was prone to degradation during serial passages due to homologues recombination between identical fragments [ 77 ]. Zolotukhin et al. [ 77 ] proposed modifi cations to the original system to alleviate the genetic instability and to allow easy incorporation of new AAV serotypes (Fig. 5b ) [ 77 ]. In order to reduce the genetic instability and improve yields the authors re-cloned the large rep78 and small rep52 into two sepa-rate baculoviruses and in order to adopt the other AAV serotypes (e.g. AAV5 and AAV8) tried to mimic the changes introduced by Urabe et al., to produce AAV2. These attempts met with limited success; the produced vectors had low infectivity due to low incorporation of VP1. Exchange of N-terminal domain harboring PLA 2 domain of various AAV serotypes for that of AAV2 increased incorporation of VP1 into capsids resulting in elevation of PLA 2 activity of the produced particles. Although composition of the capsid had been improved it was still different from the wild type AAV5 or AAV8 generated in 293 cells. Further increase in VP1 expression and final incorporation into the capsid was achieved by the incor-poration of another baculovirus construct which harbored the AAV2 VP1 gene (Baqc-VP-AAV2-107). The VP1 in this construct was driven by polh promoter and contained the wild type AUG translational start, which in insect cells results in production of primarily VP1. In order to regulate the expression of the gene a ribo-switch element – toyocamycin regulated HH Rz – was used. This element induces the degradation of the mRNA which is blocked by addition of toyocamycin [ 77 ] (Fig. 5b ). The proposed modifi cation improved the stability of the baculoviruses and tuned the expression of required proteins but expanded the number of baculo-viruses to be used to four or fi ve.

Three baculovirus confi guration: artifi cial intron: Chen et al. [ 125 ] reported a three baculovirus system where different principle is used to ensure expression of the components needed for rAAV production (Fig. 5c ) [ 125 ]. Rep and cap cassettes contain an artifi cial intron with a polh promoter. This ensures that the overlapping genes coding for rep78 and rep52 are adequately expressed. More specifi cally, the expression of rep78 is driven by the p10 promoter, whereas the expression of rep52 (a part of the rep78 coding frame) is promoted by polH , which is upstream to rep52 but does not interfere with rep78 coding frame. Thereby, it is fl anked by donor and acceptor sites and forms an artifi cial intron which is spliced out during maturation of rep78 mRNA. The same philosophy is applied to the cap expression cassette, where VP1 is driven from polh promoter and VP2 and VP3 are promoted by polh

Adeno-Associated Virus Gene Therapy and Its Application to the Prevention…

148

present within the artifi cial intron. This genetic construction ensures adequate expression of the require proteins but also alleviate the instability problem reported in the past [ 125 ] (Fig. 5c ).

Two baculovirus confi guration (Consolidated system) : in order to decreased the genetic instability [ 126 ] of the initial Bac-rep construct disclosed by Urabe et al., the same group proposed to reconfi gure the three baculovirus system into two bacu-lovirus consolidated system, with rep and cap genes positioned on one baculovirus (Fig. 5d ). Both large and small reps were expressed from one transcript controlled by the polh promoter by means of “leak ribosomal scanning”. The initiation triplet of rep78 was changed to non-canonical CTG whereas the wild type ATG was pre-served in rep52 . This allowed expression of both proteins at the same level. The Cap gene was cloned in a head-to-head orientation with respect to the rep gene. Confi guration of Cap was similar to that initially proposed by Urabe et al., with a modifi ed promoter changed to p10 . The baculovirus containing cassette of interest fl anked by two ITR’s remained unchanged [ 127 , 128 ] (Fig. 5d ). Use of the consoli-dated baculovirus system as well as alleviating the instability problem of Bac-rep also allowed use of lower MOI [ 127 , 129 ].

Insect cell producer cell line – Aslanidi et al. [ 130 ] constructed stably trans-formed insect cell lines harboring helper genes required for rAAV generation. This system consisted of two elements i.e. an insect cell line containing integrated copies of rep and cap genes, which are evoked and amplifi ed by infection of baculovirus containing expression cassette of interest [ 130 ] (Fig. 5e ). The amplifi cation and expression of the rep and cap genes upon transduction of the cell line with baculo-virus is a result of the molecular design, where rep and cap genes are under control of three different genetic elements i.e. polH promoter, hr2 region of baculovirus and RBE (rep binding elements) derived from AAV genome. Baculovirus hr elements were described previously to act as transcriptional enhancers (Fig. 5e ). The authors hypothesised that combination of binding of transcriptional factors from BEV (IE- 1) and host cell (SP1) to genetic elements such as hr2-0.9 and p19 (promoters driving expression of rep52 in wild type AAV), respectively results in expression of both rep78 and rep52 . This activation is only possible upon in trans supply of BEV regu-latory protein(s) and thereby the cassettes are completely shut off when these are not present. This feature aids the stable integration of both cassettes to insect cells genome. Another part of the system, namely RBE element, appears to be required for boosting expression of both rep and cap . This seems to occur via rescue and replication of the genetic elements adjacent to RBE. The rescue and amplifi cation capability of Rep(s) has been previously shown in HeLa cells [ 130 ].

Baculoviruses and mammalian cells – Yu-chen Hu et al. [ 131 ] constructed three baculoviruses, namely Bac-vec, Bac-rep/cap and Bac-hlper [ 131 ]. The latter one contained adenovirus helper genes needed for rAAV generation in mammalian cells. These baculoviruses were used for production of rAAV upon co-infection of HEK- 293 cells grown in a packed-bed reactor. These authors reported that the obtained yields were comparable or superior to those obtained with other production systems, reaching ~3.8 × 10 4 vg per cell [ 131 ].

K. Grosios et al.

149

In addition to all the designs described above, signifi cant development efforts were and are being conducted in order to improve the production process and to defi ne optimal production conditions especially with regards to up-scaling. The BEV system is well suited for this and volumes of 10 L and 40 L in stirred tank bioreactors (STBR) [ 132 ] and 5 L and 25 L in Wave TM bioreactors [ 132 ] have been reported. Recently, Cecchini et al. [ 133 ] reported successful up-scaling of rAAV production using the BEV system up to 200 L with a single-use stirred-tank bioreac-tor with a paddle-drive agitator [ 133 ]. Process parameters such as multiplicity of infection were investigated and it was demonstrated that the 1:1 stoichiometry of baculoviruses during the time of infection is detrimental for high rAAV yields [ 88 ]. Higher load of Bac-cap resulted in overproduction of capsids which were not fi lled with transgene. It appeared that the concentration of the Bac-vec had much milder infl uence on the rAAV yields that the Bac-rep/Bac-cap couple [ 134 ]. Besides the relative amount of added baculovirus the absolute MOI has been studied, conclud-ing that low MOI (0.03) can be successfully used without compromising rAAV yields and quality [ 126 ]. The relation between baculovirus MOI, cell density and rAAV yields were also reported by Mena et al. [ 135 ] indicating that low MOI/high cell density improves vector yields [ 135 ]. The temperature of production of rAAV was shown by Aucoin et al. [ 136 ] to be important for the infectivity of the fi nal product, with the optimum being 30 °C [ 136 ].

3.4 Future Challenges

At the beginning of the twenty-fi rst century clinical experience with AAV-based gene therapy products was limited to a couple of trials in cystic fi brosis and Haemophilia B [ 137 – 139 ]. Just over a decade later, a survey of all gene therapy tri-als indicated that there are close to 100 clinical studies using AAV vectors ongoing or completed, representing approximately 5 % of all gene therapy trials [ 7 ]. As major progress is being made we should not however be complacent and forget tragic events in the history of AAV gene therapy, as there is still a lot to discover [ 140 ]. The approval of alipogene tiparvovec and successful clinical results to date that have been shown for Haemophilia B and Leber’s congenital amaurosis are very promising [ 32 , 141 ]. Now however that the fi rst hurdle has been overcome, there are still some old and new challenges ahead. AAVs are non-integrating vectors there-fore expression is gradually lost when injected into a growing animal or a dividing tissue. This has implications in terms of considering developing gene therapies for children and with regards to the sustainability of long term expression in adults. The latter raises issues of re-administration of the same or an alternative/different vector to resurrect lost transgene expression and address the cause of the disease. Close to this is the ability of vectors to avoid immune recognition and eliciting self- eliminating immune responses. These are all issues that the gene therapy research community is now engaged in addressing.

Adeno-Associated Virus Gene Therapy and Its Application to the Prevention…

150

In terms of AAV technology, the approval of alipogene tiparvovec has provided full manufacturing and regulatory validation of BEVS made AAV therapy products for commercial use. AAV gene therapy as a personalised medicine approach could provide hope for many rare (as well as non-rare) diseases for which there is currently no effective treatment. Furthermore it offers the potential to not only treat or revert but also to prevent disease enabling a paradigm shift from reactive to preventive medicine. In that it only reinforces the principles of PPPM. It has the potential to completely transform medicine and how we deal with disease and health. Gene therapy should be considered an essential element of PPPM and current advances in both can help underpin further research and accelerate the development of novel gene therapy medicinal products.

References

1. Buning H (2013) Gene therapy enters the pharma market: the short story of a long journey. EMBO Mol Med 5(1):1–3. doi: 10.1002/emmm.201202291

2. Blaese RM, Culver KW, Miller AD, Carter CS, Fleisher T, Clerici M, Shearer G, Chang L, Chiang Y, Tolstoshev P, Greenblatt JJ, Rosenberg SA, Klein H, Berger M, Mullen CA, Ramsey WJ, Muul L, Morgan RA, Anderson WF (1995) T lymphocyte-directed gene therapy for ADA- SCID: initial trial results after 4 years. Science 270(5235):475–480

3. Rivat C, Santilli G, Gaspar HB, Thrasher AJ (2012) Gene therapy for primary immunodefi -ciencies. Hum Gene Ther 23(7):668–675. doi: 10.1089/hum.2012.116

4. Cavazzana-Calvo M, Hacein-Bey S, de Saint BG, Gross F, Yvon E, Nusbaum P, Selz F, Hue C, Certain S, Casanova JL, Bousso P, Deist FL, Fischer A (2000) Gene therapy of human severe combined immunodefi ciency (SCID)-X1 disease. Science 288(5466):669–672

5. Hacein-Bey-Abina S, Fischer A, Cavazzana-Calvo M (2002) Gene therapy of X-linked severe combined immunodefi ciency. Int J Hematol 76(4):295–298

6. Cavazzana-Calvo M, Fischer A, Hacein-Bey-Abina S, Aiuti A (2012) Gene therapy for primary immunodefi ciencies: part 1. Curr Opin Immunol 24(5):580–584. doi: 10.1016/j.coi.2012.08.008

7. Ginn SL, Alexander IE, Edelstein ML, Abedi MR, Wixon J (2013) Gene therapy clinical tri-als worldwide to 2012 – an update. J Gene Med 15(2):65–77. doi: 10.1002/jgm.2698

8. Golubnitschaja O (2010) Time for new guidelines in advanced diabetes care: paradigm change from delayed interventional approach to predictive, preventive & personalized medicine. EPMA J 1(1):3–12. doi: 10.1007/s13167-010-0014-5

9. Golubnitschaja O, Costigliola V, EPMA (2012) General report & recommendations in predictive, preventive and personalised medicine 2012: white paper of the European Association for Predictive, Preventive and Personalised Medicine. EPMA J 3(1):14. doi: 10.1186/1878-5085-3-14

10. Thomas CE, Ehrhardt A, Kay MA (2003) Progress and problems with the use of viral vectors for gene therapy. Nat Rev Genet 4(5):346–358. doi: 10.1038/nrg1066

11. Asokan A, Schaffer DV, Samulski RJ (2012) The AAV vector toolkit: poised at the clinical crossroads. Mol Ther Journal Am Soc Gene Ther 20(4):699–708. doi: 10.1038/mt.2011.287

12. Buning H, Perabo L, Coutelle O, Quadt-Humme S, Hallek M (2008) Recent developments in adeno-associated virus vector technology. J Gene Med 10(7):717–733. doi: 10.1002/jgm.1205

13. Ortolano S, Spuch C, Navarro C (2012) Present and future of adeno associated virus based gene therapy approaches. Recent Pat Endocr Metab Immune Drug Discov 6(1):47–66

K. Grosios et al.

151

14. Mingozzi F, High KA (2011) Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges. Nat Rev Genet 12(5):341–355. doi: 10.1038/nrg2988

15. Gaudet D, Methot J, Dery S, Brisson D, Essiembre C, Tremblay G, Tremblay K, de Wal J, Twisk J, van den Bulk N, Sier-Ferreira V, van Deventer S (2013) Effi cacy and long-term safety of alipogene tiparvovec (AAV1-LPLS447X) gene therapy for lipoprotein lipase defi -ciency: an open-label trial. Gene Ther 20(4):361–369. doi: 10.1038/gt.2012.43

16. Wion KL, Kirchgessner TG, Lusis AJ, Schotz MC, Lawn RM (1987) Human lipoprotein lipase complementary DNA sequence. Science 235(4796):1638–1641

17. Langlois S, Deeb S, Brunzell JD, Kastelein JJ, Hayden MR (1989) A major insertion accounts for a signifi cant proportion of mutations underlying human lipoprotein lipase defi ciency. Proc Natl Acad Sci U S A 86(3):948–952

18. Rahalkar AR, Giffen F, Har B, Ho J, Morrison KM, Hill J, Wang J, Hegele RA, Joy T (2009) Novel LPL mutations associated with lipoprotein lipase defi ciency: two case reports and a literature review. Can J Physiol Pharmacol 87(3):151–160. doi: 10.1139/y09-005

19. Santamarina-Fojo S (1998) The familial chylomicronemia syndrome. Endocrinol Metab Clin North Am 27(3):551–567, viii

20. Frank R, Hargreaves R (2003) Clinical biomarkers in drug discovery and development. Nat Rev Drug Discov 2(7):566–580. doi: 10.1038/nrd1130

21. Agbandje-McKenna M, Kleinschmidt J (2011) AAV capsid structure and cell interactions. Methods Mol Biol 807:47–92. doi: 10.1007/978-1-61779-370-7_3 , Clifton, NJ

22. Howard DB, Powers K, Wang Y, Harvey BK (2008) Tropism and toxicity of adeno- associated viral vector serotypes 1, 2, 5, 6, 7, 8, and 9 in rat neurons and glia in vitro. Virology 372(1):24–34. doi: 10.1016/j.virol.2007.10.007

23. Taymans JM, Vandenberghe LH, Haute CV, Thiry I, Deroose CM, Mortelmans L, Wilson JM, Debyser Z, Baekelandt V (2007) Comparative analysis of adeno-associated viral vector serotypes 1, 2, 5, 7, and 8 in mouse brain. Hum Gene Ther 18(3):195–206. doi: 10.1089/hum.2006.178

24. Stroes ES, Nierman MC, Meulenberg JJ, Franssen R, Twisk J, Henny CP, Maas MM, Zwinderman AH, Ross C, Aronica E, High KA, Levi MM, Hayden MR, Kastelein JJ, Kuivenhoven JA (2008) Intramuscular administration of AAV1-lipoprotein lipase S447X lowers triglycerides in lipoprotein lipase-defi cient patients. Arterioscler Thromb Vasc Biol 28(12):2303–2304. doi: 10.1161/ATVBAHA.108.175620

25. Salegio EA, Samaranch L, Kells AP, Forsayeth J, Bankiewicz K (2012) Guided delivery of adeno-associated viral vectors into the primate brain. Adv Drug Deliv Rev 64(7):598–604. doi: 10.1016/j.addr.2011.10.005

26. Pike LS, Tannous BA, Deliolanis NC, Hsich G, Morse D, Tung CH, Sena-Esteves M, Breakefi eld XO (2011) Imaging gene delivery in a mouse model of congenital neuronal ceroid lipofuscinosis. Gene Ther 18(12):1173–1178. doi: 10.1038/gt.2011.118

27. Yin L, Greenberg K, Hunter JJ, Dalkara D, Kolstad KD, Masella BD, Wolfe R, Visel M, Stone D, Libby RT, Diloreto D Jr, Schaffer D, Flannery J, Williams DR, Merigan WH (2011) Intravitreal injection of AAV2 transduces macaque inner retina. Invest Ophthalmol Vis Sci 52(5):2775–2783. doi: 10.1167/iovs.10-6250

28. Wang Z, Storb R, Lee D, Kushmerick MJ, Chu B, Berger C, Arnett A, Allen J, Chamberlain JS, Riddell SR, Tapscott SJ (2010) Immune responses to AAV in canine muscle monitored by cellular assays and noninvasive imaging. Mol Ther 18(3):617–624. doi: 10.1038/mt.2009.294

29. Tarantal AF, Lee CC (2010) Long-term luciferase expression monitored by bioluminescence imaging after adeno-associated virus-mediated fetal gene delivery in rhesus monkeys (Macaca mulatta). Hum Gene Ther 21(2):143–148. doi: 10.1089/hum.2009.126

30. Kelloff GJ, Sigman CC (2012) Cancer biomarkers: selecting the right drug for the right patient. Nat Rev Drug Discov 11(3):201–214. doi: 10.1038/nrd3651

31. Zwierzina H (2008) Biomarkers in drug development. Ann Oncol Off J Eur Soc Med Oncol/ESMO 19(Suppl 5):v33–v37. doi: 10.1093/annonc/mdn309

Adeno-Associated Virus Gene Therapy and Its Application to the Prevention…

152

32. Nathwani AC, Tuddenham EG, Rangarajan S, Rosales C, McIntosh J, Linch DC, Chowdary P, Riddell A, Pie AJ, Harrington C, O’Beirne J, Smith K, Pasi J, Glader B, Rustagi P, Ng CY, Kay MA, Zhou J, Spence Y, Morton CL, Allay J, Coleman J, Sleep S, Cunningham JM, Srivastava D, Basner-Tschakarjan E, Mingozzi F, High KA, Gray JT, Reiss UM, Nienhuis AW, Davidoff AM (2011) Adenovirus-associated virus vector-mediated gene transfer in hemo-philia B. N Engl J Med 365(25):2357–2365. doi: 10.1056/NEJMoa1108046

33. O’Mahony B, Noone D, Giangrande PL, Prihodova L (2013) Haemophilia care in Europe – a survey of 35 countries. Haemophilia 19(4):e239–e247. doi: 10.1111/hae.12125

34. Carpentier AC, Frisch F, Labbe SM, Gagnon R, de Wal J, Greentree S, Petry H, Twisk J, Brisson D, Gaudet D (2012) Effect of alipogene tiparvovec (AAV1-LPL(S447X)) on post-prandial chylomicron metabolism in lipoprotein lipase-defi cient patients. J Clin Endocrinol Metab 97(5):1635–1644. doi: 10.1210/jc.2011-3002

35. Bryant LM, Christopher DM, Giles AR, Hinderer C, Rodriguez JL, Smith JB, Traxler EA, Tycko J, Wojno AP, Wilson JM (2013) Lessons learned from the clinical development and market authorization of glybera. Hum Gene Therapy Clin Dev 24(2):55–64. doi: 10.1089/humc.2013.087

36. Trusheim MR, Berndt ER, Douglas FL (2007) Stratifi ed medicine: strategic and economic implications of combining drugs and clinical biomarkers. Nat Rev Drug Discov 6(4):287–293. doi: 10.1038/nrd2251

37. Griggs RC, Batshaw M, Dunkle M, Gopal-Srivastava R, Kaye E, Krischer J, Nguyen T, Paulus K, Merkel PA, Rare Diseases Clinical Research N (2009) Clinical research for rare disease: opportunities, challenges, and solutions. Mol Genet Metab 96(1):20–26. doi: 10.1016/j.ymgme.2008.10.003

38. Heemstra HE, van Weely S, Buller HA, Leufkens HG, de Vrueh RL (2009) Translation of rare disease research into orphan drug development: disease matters. Drug Discov Today 14(23–24):1166–1173. doi: 10.1016/j.drudis.2009.09.008

39. Harskamp RE, Lopes RD, Baisden CE, de Winter RJ, Alexander JH (2013) Saphenous vein graft failure after coronary artery bypass surgery: pathophysiology, management, and future directions. Ann Surg 257(5):824–833. doi: 10.1097/SLA.0b013e318288c38d

40. Zhang X, Zhuang J, Wu H, Chen Z, Su J, Chen S, Chen J (2010) Inhibitory effects of calcito-nin gene-related peptides on experimental vein graft disease. Ann Thorac Surg 90(1):117–123. doi: 10.1016/j.athoracsur.2010.03.063

41. Maeda Y, Shimada K, Ikeda U (2004) Gene transfer of nitric oxide synthase via the use of adeno-associated virus vectors. Methods Mol Biol 279:213–224. doi: 10.1385/1-59259- 807- 2:213 , Clifton, NJ

42. Work LM, Buning H, Hunt E, Nicklin SA, Denby L, Britton N, Leike K, Odenthal M, Drebber U, Hallek M, Baker AH (2006) Vascular bed-targeted in vivo gene delivery using tropism-modifi ed adeno-associated viruses. Mol Ther J Am Soc Gene Ther 13(4):683–693. doi: 10.1016/j.ymthe.2005.11.013

43. Sabatino DE, Mackenzie TC, Peranteau W, Edmonson S, Campagnoli C, Liu YL, Flake AW, High KA (2007) Persistent expression of hF.IX After tolerance induction by in utero or neo-natal administration of AAV-1-F.IX in hemophilia B mice. Mol Ther 15(9):1677–1685. doi: 10.1038/sj.mt.6300219

44. Mattar CN, Nathwani AC, Waddington SN, Dighe N, Kaeppel C, Nowrouzi A, McIntosh J, Johana NB, Ogden B, Fisk NM, Davidoff AM, David A, Peebles D, Valentine MB, Appelt JU, von Kalle C, Schmidt M, Biswas A, Choolani M, Chan JK (2011) Stable human FIX expression after 0.9G intrauterine gene transfer of self-complementary adeno-associated viral vector 5 and 8 in macaques. Mol Ther 19(11):1950–1960. doi: 10.1038/mt.2011.107

45. Roybal JL, Endo M, Radu A, Gray L, Todorow CA, Zoltick PW, Lutsenko S, Flake AW (2012) Early gestational gene transfer with targeted ATP7B expression in the liver improves phenotype in a murine model of Wilson’s disease. Gene Ther 19(11):1085–1094. doi: 10.1038/gt.2011.186

46. Sugano H, Matsumoto T, Miyake K, Watanabe A, Iijima O, Migita M, Narisawa S, Millan JL, Fukunaga Y, Shimada T (2012) Successful gene therapy in utero for lethal murine hypophos-phatasia. Hum Gene Ther 23(4):399–406. doi: 10.1089/hum.2011.148

K. Grosios et al.

153

47. Golubnitschaja O, Watson ID, Topic E, Sandberg S, Ferrari M, Costigliola V (2013) Position paper of the EPMA and EFLM: a global vision of the consolidated promotion of an integrative medical approach to advance health care. EPMA J 4(1):12. doi: 10.1186/1878-5085-4-12

48. Schieppati A, Henter JI, Daina E, Aperia A (2008) Why rare diseases are an important medical and social issue. Lancet 371(9629):2039–2041. doi: 10.1016/S0140-6736(08)60872-7

49. Atchison RW, Casto BC, Hammon WM (1966) Electron microscopy of adenovirus- associated virus (AAV) in cell cultures. Virology 29(2):353–357. doi: http://dx.doi.org/10.1016/0042-6822(66)90045-6

50. Samulski RJ, Berns KI, Tan M, Muzyczka N (1982) Cloning of adeno-associated virus into pBR322: rescue of intact virus from the recombinant plasmid in human cells. Proc Natl Acad Sci U S A 79(6):2077–2081

51. Senapathy P, Carter BJ (1984) Molecular cloning of adeno-associated virus variant genomes and generation of infectious virus by recombination in mammalian cells. J Biol Chem 259(7):4661–4666

52. Fife KH, Berns KI, Murray K (1977) Structure and nucleotide sequence of the terminal regions of adeno-associated virus DNA. Virology 78(2):475–487. doi: http://dx.doi.org/10.1016/0042-6822(77)90124-6

53. Berns KI, Kort J, FIFE KH, Grogan EW, Spear I (1975) Study of the fi ne structure of adeno- associated virus DNA with bacterial restriction endonucleases. J Virol 16(3):712–719

54. Berns KI, Kelly Jr TJ (1974) Visualization of the inverted terminal repetition in adeno- associated virus DNA. J Mol Biol 82(2):267–271. doi: http://dx.doi.org/10.1016/0022-2836(74)90344-1

55. Gerry HW, Kelly Jr TJ, Berns KI (1973) Arrangement of nucleotide sequences in adeno- associated virus DNA. J Mol Biol 79(2):207–225. doi: http://dx.doi.org/10.1016/0022-2836(73)90001-6

56. Lusby E, Fife KH, Berns KI (1980) Nucleotide sequence of the inverted terminal repetition in adeno-associated virus DNA. J Virol 34(2):402–409

57. Maxam AM, Gilbert W (1977) A new method for sequencing DNA. Proc Natl Acad Sci U S A 74(2):560–564

58. Ryan JH, Zolotukhin S, Muzyczka N (1996) Sequence requirements for binding of Rep68 to the adeno-associated virus terminal repeats. J Virol 70(3):1542–1553

59. McCarty DM, Ryan JH, Zolotukhin S, Zhou X, Muzyczka N (1994) Interaction of the adeno- associated virus Rep protein with a sequence within the A palindrome of the viral terminal repeat. J Virol 68(8):4998–5006

60. Chiorini JA, Afi one S, Kotin RM (1999) Adeno-associated virus (AAV) type 5 Rep protein cleaves a unique terminal resolution site compared with other AAV serotypes. J Virol 73(5):4293–4298

61. Wu J, Davis MD, Owens RA (2001) A Rep recognition sequence is necessary but not suffi -cient for nicking of DNA by adeno-associated virus type-2 Rep proteins. Arch Biochem Biophys 389(2):271–277. doi: 10.1006/abbi.2001.2348

62. Davis MD, Wu J, Owens RA (2000) Mutational analysis of adeno-associated virus type 2 Rep68 protein endonuclease activity on partially single-stranded substrates. J Virol 74(6):2936–2942

63. Wu J, Davis MD, Owens RA (1999) Factors affecting the terminal resolution site endonucle-ase, helicase, and ATPase activities of adeno-associated virus type 2 Rep proteins. J Virol 73(10):8235–8244

64. Davis MD, Wonderling RS, Walker SL, Owens RA (1999) Analysis of the effects of charge cluster mutations in adeno-associated virus Rep68 protein in vitro. J Virol 73(3):2084–2093

65. Xiao X, Xiao W, Li J, Samulski RJ (1997) A novel 165-base-pair terminal repeat sequence is the sole cis requirement for the adeno-associated virus life cycle. J Virol 71(2):941–948

66. Wang XS, Ponnazhagan S, Srivastava A (1996) Rescue and replication of adeno-associated virus type 2 as well as vector DNA sequences from recombinant plasmids containing deletions in the viral inverted terminal repeats: selective encapsidation of viral genomes in progeny virions. J Virol 70(3):1668–1677

67. Wang XS, Ponnazhagan S, Srivastava A (1995) Rescue and replication signals of the adeno- associated virus 2 genome. J Mol Biol 250(5):573–580. doi: 10.1006/jmbi.1995.0398

Adeno-Associated Virus Gene Therapy and Its Application to the Prevention…

154

68. Wang XS, Srivastava A (1997) A novel terminal resolution-like site in the adeno-associated virus type 2 genome. J Virol 71(2):1140–1146

69. Wang XS, Qing K, Ponnazhagan S, Srivastava A (1997) Adeno-associated virus type 2 DNA replication in vivo: mutation analyses of the D sequence in viral inverted terminal repeats. J Virol 71(4):3077–3082

70. Chiorini JA, Weitzman MD, Owens RA, Urcelay E, Safer B, Kotin RM (1994) Biologically active Rep proteins of adeno-associated virus type 2 produced as fusion proteins in Escherichia coli. J Virol 68(2):797–804

71. Zhou X, Zolotukhin I, Im DS, Muzyczka N (1999) Biochemical characterization of adeno- associated virus rep68 DNA helicase and ATPase activities. J Virol 73(2):1580–1590

72. Brister JR, Muzyczka N (2000) Mechanism of Rep-mediated adeno-associated virus origin nicking. J Virol 74(17):7762–7771

73. Smith RH, Kotin RM (1998) The Rep52 gene product of adeno-associated virus is a DNA helicase with 3′-to-5′ polarity. J Virol 72(6):4874–4881

74. Im DS, Muzyczka N (1992) Partial purifi cation of adeno-associated virus Rep78, Rep52, and Rep40 and their biochemical characterization. J Virol 66(2):1119–1128

75. Collaco RF, Kalman-Maltese V, Smith AD, Dignam JD, Trempe JP (2003) A biochemical characterization of the adeno-associated virus Rep40 helicase. J Biol Chem 278(36):34011–34017. doi: 10.1074/jbc.M301537200

76. Dignam SS, Collaco RF, Bieszczad J, Needham P, Trempe JP, Dignam JD (2007) Coupled ATP and DNA binding of adeno-associated virus Rep40 helicase. Biochemistry 46(2):568–576. doi: 10.1021/bi061762v

77. Kohlbrenner E, Aslanidi G, Nash K, Shklyaev S, Campbell-Thompson M, Byrne BJ, Snyder RO, Muzyczka N, Warrington KH Jr, Zolotukhin S (2005) Successful production of pseudo-typed rAAV vectors using a modifi ed baculovirus expression system. Mol Ther 12(6):1217–1225. doi: 10.1016/j.ymthe.2005.08.018

78. Popa-Wagner R, Porwal M, Kann M, Reuss M, Weimer M, Florin L, Kleinschmidt JA (2012) Impact of VP1-specifi c protein sequence motifs on adeno-associated virus type 2 intracellu-lar traffi cking and nuclear entry. J Virol 86(17):9163–9174. doi: 10.1128/JVI.00282-12

79. Girod A, Wobus CE, Zadori Z, Ried M, Leike K, Tijssen P, Kleinschmidt JA, Hallek M (2002) The VP1 capsid protein of adeno-associated virus type 2 is carrying a phospholipase A2 domain required for virus infectivity. J Gen Virol 83(Pt 5):973–978

80. Laughlin CA, Tratschin JD, Coon H, Carter BJ (1983) Cloning of infectious adeno- associated virus genomes in bacterial plasmids. Gene 23(1):65–73

81. Hermonat PL, Muzyczka N (1984) Use of adeno-associated virus as a mammalian DNA cloning vector: transduction of neomycin resistance into mammalian tissue culture cells. Proc Natl Acad Sci 81(20):6466–6470

82. Samulski RJ, Chang LS, Shenk T (1989) Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression. J Virol 63(9):3822–3828

83. McLaughlin SK, Collis P, Hermonat PL, Muzyczka N (1988) Adeno-associated virus general transduction vectors: analysis of proviral structures. J Virol 62(6):1963–1973

84. Buller RM, Janik JE, Sebring ED, Rose JA (1981) Herpes simplex virus types 1 and 2 com-pletely help adenovirus-associated virus replication. J Virol 40(1):241–247

85. Grimm D, Kleinschmidt JA (1999) Progress in adeno-associated virus type 2 vector production: promises and prospects for clinical use. Hum Gene Ther 10(15):2445–2450. doi: 10.1089/10430349950016799

86. Ferrari FK, Samulski T, Shenk T, Samulski RJ (1996) Second-strand synthesis is a rate- limiting step for effi cient transduction by recombinant adeno-associated virus vectors. J Virol 70(5):3227–3234

87. Ferrari FK, Xiao X, McCarty D, Samulski RJ (1997) New developments in the generation of Ad-free, high-titer rAAV gene therapy vectors. Nat Med 3(11):1295–1297

88. Xiao X, Li J, Samulski RJ (1998) Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus. J Virol 72(3):2224–2232

K. Grosios et al.

155

89. Salvetti A, Oreve S, Chadeuf G, Favre D, Cherel Y, Champion-Arnaud P, David-Ameline J, Moullier P (1998) Factors infl uencing recombinant adeno-associated virus production. Hum Gene Ther 9(5):695–706. doi: 10.1089/hum.1998.9.5-695

90. Matsushita T, Elliger S, Elliger C, Podsakoff G, Villarreal L, Kurtzman GJ, Iwaki Y, Colosi P (1998) Adeno-associated virus vectors can be effi ciently produced without helper virus. Gene Ther 5(7):938–945. doi: 10.1038/sj.gt.3300680

91. Grimm D, Kern A, Rittner K, Kleinschmidt JA (1998) Novel tools for production and purifi -cation of recombinant adenoassociated virus vectors. Hum Gene Ther 9(18):2745–2760. doi: 10.1089/hum.1998.9.18-2745

92. Galibert L, Merten OW (2011) Latest developments in the large-scale production of adeno- associated virus vectors in insect cells toward the treatment of neuromuscular diseases. J Invertebr Pathol 107(Suppl):S80–S93. doi: 10.1016/j.jip.2011.05.008

93. Li J, Samulski RJ, Xiao X (1997) Role for highly regulated rep gene expression in adeno- associated virus vector production. J Virol 71(7):5236–5243

94. Collaco RF, Cao X, Trempe JP (1999) A helper virus-free packaging system for recombinant adeno-associated virus vectors. Gene 238(2):397–405

95. Rabinowitz JE, Rolling F, Li C, Conrath H, Xiao W, Xiao X, Samulski RJ (2002) Cross- packaging of a single adeno-associated virus (AAV) type 2 vector genome into multiple AAV serotypes enables transduction with broad specifi city. J Virol 76(2):791–801

96. Cecchini S, Negrete A, Kotin RM (2008) Toward exascale production of recombinant adeno- associated virus for gene transfer applications. Gene Ther 15(11):823–830. doi: 10.1038/gt.2008.61

97. Yang Q, Chen F, Trempe JP (1994) Characterization of cell lines that inducibly express the adeno-associated virus Rep proteins. J Virol 68(8):4847–4856

98. Caillet-Fauquet P, Perros M, Brandenburger A, Spegelaere P, Rommelaere J (1990) Programmed killing of human cells by means of an inducible clone of parvoviral genes encoding non-structural proteins. EMBO J 9(9):2989–2995

99. Clark KR, Voulgaropoulou F, Fraley DM, Johnson PR (1995) Cell lines for the production of recombinant adeno-associated virus. Hum Gene Ther 6(10):1329–1341. doi: 10.1089/hum.1995.6.10-1329

100. Flotte TR, Barraza-Ortiz X, Solow R, Afi one SA, Carter BJ, Guggino WB (1995) An improved system for packaging recombinant adeno-associated virus vectors capable of in vivo transduction. Gene Ther 2(1):29–37

101. Tamayose K, Hirai Y, Shimada T (1996) A new strategy for large-scale preparation of high- titer recombinant adeno-associated virus vectors by using packaging cell lines and sulfonated cellulose column chromatography. Hum Gene Ther 7(4):507–513. doi: 10.1089/hum.1996.7.4-507

102. Gao GP, Qu G, Faust LZ, Engdahl RK, Xiao W, Hughes JV, Zoltick PW, Wilson JM (1998) High-titer adeno-associated viral vectors from a Rep/Cap cell line and hybrid shuttle virus. Hum Gene Ther 9(16):2353–2362. doi: 10.1089/hum.1998.9.16-2353

103. Fan PD, Dong JY (1997) Replication of rep-cap genes is essential for the high-effi ciency production of recombinant AAV. Hum Gene Ther 8(1):87–98. doi: 10.1089/hum.1997.8.1-87

104. Inoue N, Russell DW (1998) Packaging cells based on inducible gene amplifi cation for the production of adeno-associated virus vectors. J Virol 72(9):7024–7031

105. Chadeuf G, Favre D, Tessier J, Provost N, Nony P, Kleinschmidt J, Moullier P, Salvetti A (2000) Effi cient recombinant adeno-associated virus production by a stable rep-cap HeLa cell line correlates with adenovirus-induced amplifi cation of the integrated rep-cap genome. J Gene Med 2(4):260–268

106. Gao GP, Lu F, Sanmiguel JC, Tran PT, Abbas Z, Lynd KS, Marsh J, Spinner NB, Wilson JM (2002) Rep/Cap gene amplifi cation and high-yield production of AAV in an A549 cell line expressing Rep/Cap. Mol Ther 5(5 Pt 1):644–649. doi: 10.1006/mthe.2001.0591

107. Qiao C, Li J, Skold A, Zhang X, Xiao X (2002) Feasibility of generating adeno-associated virus packaging cell lines containing inducible adenovirus helper genes. J Virol 76(4):1904–1913

Adeno-Associated Virus Gene Therapy and Its Application to the Prevention…

156

108. Blouin V, Brument N, Toublanc E, Raimbaud I, Moullier P, Salvetti A (2004) Improving rAAV production and purifi cation: towards the defi nition of a scaleable process. J Gene Med 6(Suppl 1):S223–S228. doi: 10.1002/jgm.505

109. Farson D, Harding TC, Tao L, Liu J, Powell S, Vimal V, Yendluri S, Koprivnikar K, Ho K, Twitty C, Husak P, Lin A, Snyder RO, Donahue BA (2004) Development and characteriza-tion of a cell line for large-scale, serum-free production of recombinant adeno-associated viral vectors. J Gene Med 6(12):1369–1381. doi: 10.1002/jgm.622

110. E1A and E1B Yuan Z, Qiao C, Hu P, Li J, Xiao X (2011) A versatile adeno-associated virus vector producer cell line method for scalable vector production of different serotypes. Hum Gene Ther 22(5):613–624. doi: 10.1089/hum.2010.241

111. Weindler FW, Heilbronn R (1991) A subset of herpes simplex virus replication genes provides helper functions for productive adeno-associated virus replication. J Virol 65(5):2476–2483

112. Weitzman MD, Fisher KJ, Wilson JM (1996) Recruitment of wild-type and recombinant adeno-associated virus into adenovirus replication centers. J Virol 70(3):1845–1854

113. Conway JE, Zolotukhin S, Muzyczka N, Hayward GS, Byrne BJ (1997) Recombinant adeno- associated virus type 2 replication and packaging is entirely supported by a herpes simplex virus type 1 amplicon expressing Rep and Cap. J Virol 71(11):8780–8789

114. Clement N, Knop DR, Byrne BJ (2009) Large-scale adeno-associated viral vector production using a herpesvirus-based system enables manufacturing for clinical studies. Hum Gene Ther 20(8):796–806. doi: 10.1089/hum.2009.094

115. Conway JE, Rhys CM, Zolotukhin I, Zolotukhin S, Muzyczka N, Hayward GS, Byrne BJ (1999) High-titer recombinant adeno-associated virus production utilizing a recombinant herpes simplex virus type I vector expressing AAV-2 Rep and Cap. Gene Ther 6(6):986–993. doi: 10.1038/sj.gt.3300937

116. Feudner E, de Alwis M, Thrasher AJ, Ali RR, Fauser S (2001) Optimization of recombinant adeno-associated virus production using an herpes simplex virus amplicon system. J Virol Methods 96(2):97–105

117. Booth MJ, Mistry A, Li X, Thrasher A, Coffi n RS (2004) Transfection-free and scalable recombinant AAV vector production using HSV/AAV hybrids. Gene Ther 11(10):829–837. doi: 10.1038/sj.gt.3302226

118. Kang W, Wang L, Harrell H, Liu J, Thomas DL, Mayfi eld TL, Scotti MM, Ye GJ, Veres G, Knop DR (2009) An effi cient rHSV-based complementation system for the production of multiple rAAV vector serotypes. Gene Ther 16(2):229–239. doi: 10.1038/gt.2008.158

119. Thomas DL, Wang L, Niamke J, Liu J, Kang W, Scotti MM, Ye GJ, Veres G, Knop DR (2009) Scalable recombinant adeno-associated virus production using recombinant herpes simplex virus type 1 coinfection of suspension-adapted mammalian cells. Hum Gene Ther 20(8):861–870. doi: 10.1089/hum.2009.004

120. van Oers MM (2011) Opportunities and challenges for the baculovirus expression system. J Invertebr Pathol 107(Suppl):S3–S15. doi: 10.1016/j.jip.2011.05.001

121. Urabe M, Ding C, Kotin RM (2002) Insect cells as a factory to produce adeno-associated virus type 2 vectors. Hum Gene Ther 13(16):1935–1943. doi: 10.1089/10430340260355347

122. Owens RA, Trempe JP, Chejanovsky N, Carter BJ (1991) Adeno-associated virus rep pro-teins produced in insect and mammalian expression systems: wild-type and dominant- negative mutant proteins bind to the viral replication origin. Virology 184(1):14–22

123. Ruffi ng M, Zentgraf H, Kleinschmidt JA (1992) Assembly of viruslike particles by recombinant structural proteins of adeno-associated virus type 2 in insect cells. J Virol 66(12):6922–6930

124. Palombo F, Monciotti A, Recchia A, Cortese R, Ciliberto G, La Monica N (1998) Site- specifi c integration in mammalian cells mediated by a new hybrid baculovirus-adeno- associated virus vector. J Virol 72(6):5025–5034

125. Chen H (2008) Intron splicing-mediated expression of AAV Rep and Cap genes and produc-tion of AAV vectors in insect cells. Mol Ther J Am Soc Gene Ther 16(5):924–930. doi: 10.1038/mt.2008.35

126. Negrete A, Yang LC, Mendez AF, Levy JR, Kotin RM (2007) Economized large-scale production of high yield of rAAV for gene therapy applications exploiting baculovirus expression system. J Gene Med 9(11):938–948. doi: 10.1002/jgm.1092

K. Grosios et al.

157

127. Negrete A, Kotin RM (2009) Production of recombinant adeno-associated vectors using two bioreactor confi gurations at different scales. J Virol Methods 145(2):155–161. doi: 10.1016/j.jviromet.2007.05.020

128. Smith RH, Levy JR, Kotin RM (2009) A simplifi ed baculovirus-AAV expression vector sys-tem coupled with one-step affi nity purifi cation yields high-titer rAAV stocks from insect cells. Mol Ther 17(11):1888–1896. doi: 10.1038/mt.2009.128

129. Virag T, Cecchini S, Kotin RM (2009) Producing recombinant adeno-associated virus in foster cells: overcoming production limitations using a baculovirus-insect cell expression strategy. Hum Gene Ther 20(8):807–817. doi: 10.1089/hum.2009.092

130. Aslanidi G, Lamb K, Zolotukhin S (2009) An inducible system for highly effi cient produc-tion of recombinant adeno-associated virus (rAAV) vectors in insect Sf9 cells. Proc Natl Acad Sci U S A 106(13):5059–5064. doi: 10.1073/pnas.0810614106

131. Huang KS, Lo WH, Chung YC, Lai YK, Chen CY, Chou ST, Hu YC (2007) Combination of baculovirus-mediated gene delivery and packed-bed reactor for scalable production of adeno- associated virus. Hum Gene Ther 18(11):1161–1170. doi: 10.1089/hum.2007.107

132. Meghrous J, Aucoin MG, Jacob D, Chahal PS, Arcand N, Kamen AA (2005) Production of recombinant adeno-associated viral vectors using a baculovirus/insect cell suspension culture system: from shake fl asks to a 20-L bioreactor. Biotechnol Prog 21(1):154–160. doi: 10.1021/bp049802e

133. Cecchini S, Virag T, Kotin RM (2011) Reproducible high yields of recombinant adeno- associated virus produced using invertebrate cells in 0.02- to 200-liter cultures. Hum Gene Ther 22(8):1021–1030. doi: 10.1089/hum.2010.250

134. Aucoin MG, Perrier M, Kamen AA (2006) Production of adeno-associated viral vectors in insect cells using triple infection: optimization of baculovirus concentration ratios. Biotechnol Bioeng 95(6):1081–1092. doi: 10.1002/bit.21069

135. Mena JA, Aucoin MG, Montes J, Chahal PS, Kamen AA (2010) Improving adeno-associated vector yield in high density insect cell cultures. J Gene Med 12(2):157–167. doi: 10.1002/jgm.1420

136. Aucoin MG, Perrier M, Kamen AA (2007) Improving AAV vector yield in insect cells by modulating the temperature after infection. Biotechnol Bioeng 97(6):1501–1509. doi: 10.1002/bit.21364

137. Wagner JA, Moran ML, Messner AH, Daifuku R, Conrad CK, Reynolds T, Guggino WB, Moss RB, Carter BJ, Wine JJ, Flotte TR, Gardner P (1998) A phase I/II study of tgAAV-CF for the treatment of chronic sinusitis in patients with cystic fi brosis. Hum Gene Ther 9(6):889–909. doi: 10.1089/hum.1998.9.6-889

138. Aitken ML, Moss RB, Waltz DA, Dovey ME, Tonelli MR, McNamara SC, Gibson RL, Ramsey BW, Carter BJ, Reynolds TC (2001) A phase I study of aerosolized administration of tgAAVCF to cystic fi brosis subjects with mild lung disease. Hum Gene Ther 12(15):1907–1916. doi: 10.1089/104303401753153956

139. Kay MA, Manno CS, Ragni MV, Larson PJ, Couto LB, McClelland A, Glader B, Chew AJ, Tai SJ, Herzog RW, Arruda V, Johnson F, Scallan C, Skarsgard E, Flake AW, High KA (2000) Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector. Nat Genet 24(3):257–261. doi: 10.1038/73464

140. Yarborough M, Sharp RR (2009) Public trust and research a decade later: what have we learned since Jesse Gelsinger’s death? Mol Genet Metab 97(1):4–5. doi: 10.1016/j.ymgme.2009.02.002

141. Maguire AM, Simonelli F, Pierce EA, Pugh EN Jr, Mingozzi F, Bennicelli J, Banfi S, Marshall KA, Testa F, Surace EM, Rossi S, Lyubarsky A, Arruda VR, Konkle B, Stone E, Sun J, Jacobs J, Dell’Osso L, Hertle R, Ma JX, Redmond TM, Zhu X, Hauck B, Zelenaia O, Shindler KS, Maguire MG, Wright JF, Volpe NJ, McDonnell JW, Auricchio A, High KA, Bennett J (2008) Safety and effi cacy of gene transfer for Leber’s congenital amaurosis. N Engl J Med 358(21):2240–2248. doi: 10.1056/NEJMoa0802315

Adeno-Associated Virus Gene Therapy and Its Application to the Prevention…

159M. Özgüç (ed.), Rare Diseases: Integrative PPPM Approach as the Medicine of the Future, Advances in Predictive, Preventive and Personalised Medicine 6,DOI 10.1007/978-94-017-9214-1_10, © Springer Science+Business Media Dordrecht 2015

Abstract Pluripotent cells including induced pluripotent stem (iPS) cells are regarded as a powerful source for cell therapy, because these cells function both by direct cell replacement and also by paracrine effects. Advantage of iPS cells is also their unlimited availability. In this chapter we characterize the pluripotent state of cells starting from embryonic stem (ES) cells and comparing them with iPS cells. We also describe different ways of using iPS cells: replacement of damaged cells and cell replacement in combination with gene therapy. We summarize recent achievements in these areas and conclude that although the developments are highly promising, there are still potential risks of adverse effects, which need more fundamental research before iPS cell therapy will become a routine clinical practice. One more promising area of iPS cell technology is derivation of these cells from patients with genetic or other disorders and use of them as a “human cell model of human disease” to understand the mechanisms of the disease and to possibly fi nd new chemicals to correct the defective pathways. This approach has already led to discoveries of new routes to medical treatments and potentially will form a new and effi cient basis for personalized therapy of patients.

Keywords Induced pluripotency • iPS cells • Embryonic stem cells • Cell therapy • Gene therapy • Models of human disease • Personalized medicine

Abbreviations

6-OHDA 6-hydroxydopamine ADA-SCID Adenosine deaminase defi ciency-related severe combined

immunodefi ciency

Induced Pluripotency for the Study of Disease Mechanisms and Cell Therapy

Toivo Maimets

T. Maimets (*) Institute of Molecular and Cell Biology , University of Tartu , 23 Riia str. , 51010 Tartu , Estonia e-mail: Toivo.Maimets@ut.ee

160

ALS Amyotrophic lateral sclerosis BMD Becker muscular dystrophy DA neurons Dopaminergic neurons DMD Duchenne muscular dystrophy DS Down syndrome ES cells Embryonic stem cells FA Fanconi anemia FACS Fluorescence-activated cell sorting FD Familial dysautonomia FDA Food and Drug Administration FXS Fragile X syndrome GABA Gamma aminobutyric acid GD Gaucher disease HD Huntington disease ICM Inner Cell Mass iPS cells Induced pluripotent stem cells JDM Juvenile-onset type 1 diabetes mellitus mRNA – micro RNA NCAM Neural cell adhesion molecule NT Nuclear transfer PD Parkinson’s Disease PGD Preimplantation genetic diagnosis ROS Reactive Oxygen Species RTT Rett syndrome SBDS Shwachman-Bodian-Diamond syndrome SCNT Somatic Cell Nuclear Transfer SMA Spinal muscular atrophy TALEN Transcription activator–like effector nuclease T1D Type 1 diabetes X-CGD X-linked chronic granulomatous disease ZFN Zinc-fi nger nucleases

1 Introduction

The Nobel Prize in Physiology or Medicine 2012 was awarded jointly to Sir John B. Gurdon and Shinya Yamanaka “for the discovery that mature cells can be repro-grammed to become pluripotent.” For decades since the fi rst half of the twentieth century the prevailing view was that in an adult organism most of the cells are irrevers-ibly locked into their differentiated status. Although it was clear that almost all cells (with some very rare exceptions) contain the same amount of DNA, the development of an organism and cellular differentiation were seen as unidirectional processes, dur-ing which most of the genes are permanently “shut off”, so that only the genes specifi c for a particular cell type (in addition to “housekeeping” genes) remain actively tran-scribed. Conrad Hal Waddington (1905–1975) used a metaphor of “epigenetic land-scape of mountains and valleys” to describe differentiation and development [ 1 ].

T. Maimets

161

Undifferentiated cells were represented as marbles, which reside on a mountain top. When differentiation starts, they move down into energetically more stable valleys, where they eventually end up as differentiated cells. The underlying assumption was that reversal of the cells into less differentiated states was as diffi cult as making the marble move up the hill until the top of the mountain by itself.

Although during 1950s there were also others questioning this paradigm, John B. Gurdon was the fi rst to experimentally demonstrate that nuclei from differenti-ated cells can be reprogrammed to fully immature, pluripotent state, so that they can give rise to all cells of an adult organism. He took an egg of frog Xenopus laevis and destroyed its nucleus by UV-irradiation. Then he injected into it another nucleus from a fully differentiated tadpole intestinal epithelium cell. The zygote started to develop and gave rise to a normal tadpole and eventually a mature frog. It had been proven that a differentiated nucleus can be reverted into undifferenti-ated pluripotent sate [ 2 ].

Gurdon’s discovery was a major paradigm shift, which decades later lead to cloning numerous mammals using basically the same technology (called Somatic Cell Nuclear Transfer, SCNT) and also to the discovery of these “factors”, which are present in egg cells and cause the developmental reversal of any mature cell.

2 Pluripotency and ES Cells

Pluripotency is defi ned as the capacity of individual cells to initiate all lineages of the mature organism in response to signals from the embryo or cell culture environ-ment [ 3 ]. It usually refers to a stem cell that has the potential to differentiate into any of the three germ layers: endoderm (which gives interior stomach lining, gastroin-testinal tract and lungs), mesoderm (muscle, bone, blood, urogenital tract), or ecto-derm (epidermal tissues and nervous system).

The property of cell pluripotency was fi rst demonstrated by Hans Driesch in 1891, when he separated the two cells of a sea urchin blastocyst and observed the development of two complete sea urchins [ 4 ].

A zygote and the cells of a developing embryo after the fi rst cell divisions are called totipotent, because they can give rise to any fetal or adult cell type as well as to some extraembryonic tissues, such as parts of placenta. Four to fi ve days after conception the human embryo passes the fi rst differentiation of embryonic cells. In this blastocyst stage the embryo is a ball of 0.7 mm diameter covered with trophec-toderm cells, which form the extraembryonic tissues. Inside this ball there is a clump of cells called Inner Cell Mass (ICM), which give rise to all 250–300 cell types of a future human being – therefore they are pluripotent (but not totipotent any more). Further differentiation of these cells during ontogenesis leads to further spe-cialization of cells – oligopotent (e.g. hematopoetic stem cells) and fully differenti-ated cells (e.g. neurons or, more drastically, erythrocytes).

ICM cells can be isolated from the blastocyst and cultivated ex vivo as an Embryonic Stem (ES) cell line. In 1981 the ES cells were fi rst derived from mouse embryos independently by two groups, Martin Evans and Matthew Kaufman [ 5 ] and

Induced Pluripotency for the Study of Disease Mechanisms and Cell Therapy

162

Gail R. Martin [ 6 ]. In 1998 researchers led by James Thomson at the University of Wisconsin-Madison fi rst developed a technique to isolate and grow human embry-onic stem cells in cell culture [ 7 ]. The ES cells are distinguished by two distinctive properties: (1) these cells replicate indefi nitely and (2) are truly pluripotent.

The ability of ES cells to give rise to all cell types of an adult has created a hope to use them as “spare parts” for illnesses connected with cell degradation – Parkinson’s disease (PD), Alzheimer disease, type 1 diabetes, several forms of can-cer etc. In 2009 Phase I clinical trials for transplantation of oligodendrocytes derived from human ES cells into spinal cord-injured individuals received approval from the U.S. Food and Drug Administration (FDA), marking it the world’s fi rst human ES cell human trial. The study leading to this scientifi c advancement was conducted by Hans Keirstead and colleagues [ 8 ] at the University of California, Irvine and sup-ported by Geron Corporation of Menlo Park, CA. In November 2011, however, Geron announced it was dropping out of stem cell research for fi nancial reasons and is looking for new partners for these developments.

There are two different ways to look at the pluripotent state of the cell, as described by Armstrong et al. [ 9 ]. First, pluripotency can be described as a “ground state”, where the role of pluripotency factors is to inhibit differentiation and thereby maintain this ground state [ 3 , 10 ]. This is a prevailing view today. Loh and Lim [ 11 ] have challenged this view recently and present an alternative view of pluripotency factors, which maintain pluripotency by acting as mutually antagonistic lineage specifi ers. As long as all factors are present and correct, this results in a metastable state that is pluripotency. Thus, by Loh and Lim, pluripotency is an inherently pre-carious condition in which rival lineage specifi ers continually compete to specify differentiation along mutually exclusive lineages.

How to test the pluripotency of a particular cell? The ultimate test is to see, whether it can form an organism, which is able to reproduce (contains both somatic and germ cells). In animals, this can be done by tetraploid complementation assay. Tetraploid complementation is a technique, where two mammalian embryos are combined to form a new embryo [ 12 ]. Normal mammalian somatic cells are diploid: each chromo-some is present in duplicate. An embryo at two-cell stage is treated with electrical current, so the cells fuse and form a tetraploid cell. This cell will continue to divide and all its daughter cells will also be tetraploid. Such a tetraploid embryo can develop normally to the blastocyst stage and will implant in the wall of the uterus. The tetra-ploid cells can form the extra-embryonic tissue, but usually not the tissues of the fetus. Such a tetraploid embryo can now be combined with normal diploid embryonic stem cells (ES) from a different organism, either by direct injection of ES cells into tetra-ploid embryos or aggregation of ES cells with 4-cell stage tetraploid embryo. The embryo will then develop normally; the fetus is exclusively derived from the ES cells, while the extra-embryonic tissues are derived from the tetraploid cells.

Because of the complexity of this technology and the fact that it cannot be used for human organism, surrogate assays for pluripotency are often used. They use either the ability of pluripotent cells to form teratomas (benign tumors containing tissues of all three germ layers) in experimental animals or to express certain pluri-potency markers (e.g. proteins Nanog, Oct or others, see below).

T. Maimets

163

Human embryonic stem cells allow investigators to explore early human development through in vitro differentiation, which recapitulates aspects of normal gastrulation and tissue formation. Embryos shown to carry genetic diseases by virtue of preimplantation genetic diagnosis (PGD; genetic analysis of single blastomeres obtained by embryo biopsy) can yield stem cell lines that model single gene disor-ders [ 13 ], but the use of ES cells for this purpose has been quite limited [ 14 ].

Another strategy for producing autologous, patient-derived pluripotent stem cells is somatic cell nuclear transfer (SCNT) described above. In a proof of principle experiment, NT-ES cells generated from mice with genetic immunodefi ciency were used to combine gene and cell therapy to repair the genetic defect [ 15 ]. To date, NT has not proven successful in the human, and given the paucity of human oocytes, is destined to have limited utility.

Therefore, another technology originally developed by Yamanaka has opened totally new possibilities to produce patient-derived pluripotent cells both for study-ing the mechanism of disease as well as working out methods for therapy.

3 Induced Pluripotency

Shinya Yamanaka had studied the factors, which were important for keeping the ES cells pluripotent. In parallel with Austin Smith [ 16 ], he discovered a transcription fac-tor Nanog as one of the central proteins in maintaining cell pluripotency [ 17 ]. ES cells were known earlier to induce pluripotency in somatic cell nuclei after fusions includ-ing ES and somatic cells [ 18 ]. Hence, the ES cells express all the factors needed for nuclear reprogramming. He selected 24 transcription factors known to be expressed specifi cally in ES cells and using retroviruses introduced their genes into differenti-ated fi broblasts. Few of the cells developed indeed into cells resembling ES cells. One-by-one reduction of the genes ended up with just four genes – Myc, Oct3/4, Sox2 and Klf4 – which were able to change mouse embryonic fi broblasts into pluripotent cells [ 19 ]. These cells were named as Induced Pluripotent Stem (iPS) cells and numer-ous laboratories have since then used different mixtures of genes, proteins and low-molecular weight compounds to create iPS cells from different organisms. The fi rst human iPS cells were in parallel made by Yamanaka using the same four factors [ 20 ] and by Thomson, who used combination of genes for proteins Lin28, Nanog, Oct4 and Sox2 [ 21 ]. Yamanaka had made a real breakthrough showing that, in addition to nuclei, also entire differentiated somatic cells can be reprogrammed to pluripotency and it takes only a small number of factors to accomplish it.

The fi rst transcription factor combinations were ineffi cient with less than 0.1 % of cells converted into iPS cells. Also, the use of strong oncogenes like Myc and retro-viruses, which are randomly inserted into the genome, were clear obstacles to use this technology for medical purposes. Therefore, over the last 6 years, extensive research has been done using other methods of transcription factor delivery: transposons, adenoviruses, different plasmids, proteins microRNAs and modifi ed mRNAs (see [ 22 ] for recent review). In addition, several co-factors have been found, which

Induced Pluripotency for the Study of Disease Mechanisms and Cell Therapy

164

improve the outcome. For example, it is known that protein p53 actively pushes the pluripotent cells towards differentiation [ 23 ], therefore using reagents that reduce p53 levels increases the effi ciency of reprogramming [ 24 , 25 ].

In many aspects iPS cells are similar to natural stem cells, such as ES cells, but the full extent of this similarity is still under study. Differences between iPS- and blastocyst-derived embryonic stem cells have been reported for gene expression, DNA methylation and differentiation potential. In addition, reprogramming to iPS cells seems to compromise genomic integrity, introducing de novo mutations and copy number variations. It is clear now that iPS cells are not identical to ES cells [ 26 ]. It is possible, however, that with more advanced technologies the differences between these two cell types will decrease. In addition, it seems that for many appli-cations of iPS cells this full identity is not actually needed.

It has been assumed that use of autologous iPS cells for grafting would be less prone to immune rejection, which would make them especially valuable for cell therapy. However, this assumption has been also questioned [ 27 ]. This work showed that abnormal gene expression in some cells differentiated from iPS cells can induce T-cell-dependent immune response in syngeneic recipients, which may thus pose an unexpected hurdle toward potential clinical use of iPS cells.

There are three principal ways, how iPS cells can be used for medical purposes (Fig. 1 ). First, patient-specifi c cells can be used to replace the cells damaged or lost

PATIENT

Skin biopsy

Reprogramming

iPS cell lines

Reprogrammingfactors (e.g.Oct4,Sox2, Klf4, Myc)

In vitro differentiation

Gene therapy

Disease-specific phenotypeassay (either with iPS or

their differentiated progeny

Large-scale therapeuticscreen

Drug development

A

BC

Fig. 1 Using iPS cells for medical purposes. iPS cells are produced by taking patient cells (e.g. by skin biopsy) and reprogramming them by known cocktails of transcription factors or other com-pounds. High-quality iPSCs are selected, differentiated into mature cell types and re-introduced into the patient (A). This can also be combined gene therapy to replace the parts of DNA known to carry mutations (B). The iPS cells (or their differentiated progeny) can be also used to model human diseases, if exhibiting a disease-specifi c phenotype that is readily detected by cellular and/or molecular assays (C). High throughput screens based on such assays can be carried out to discover therapeutics, which correct the disease phenotypes. Hits from these screens are candidates for lead optimization by medicinal chemistry, and then for further preclinical studies

T. Maimets

165

during many diseases (A). This can be also combined with gene therapy, where before re-introduction of derived autologous iPS cells parts of their DNA are changed, for example to correct inherited genetic lesions (B). In addition, iPS cells can be used to model human disease to understand the disease mechanisms and to fi nd possible ways of intervention to correct abnormal developments (C).

4 Replacement of Damaged Cells

A proof of principle comes from Jaenisch lab, who demonstrated that mouse iPS cells could be effi ciently differentiated into neural precursor cells, giving rise to neuronal and glial cell types in culture [ 28 ]. Upon transplantation into the fetal mouse brain, the cells migrated into various brain regions and differentiated into glia and neurons, including glutamatergic, GABA-ergic, and catecholaminergic subtypes. Electrophysiological recordings and morphological analysis demon-strated that the grafted neurons had mature neuronal activity and were functionally integrated in the host brain. Furthermore, iPS cells were induced to differentiate into dopamine neurons of midbrain character and were able to improve behavior in a rat model of Parkinson’s disease upon transplantation into the adult brain. These results demonstrated the therapeutic potential of directly reprogrammed fi broblasts for neuronal cell replacement in the animal model.

Hargus and others [ 29 ] differentiated iPS cells from patients with Parkinson’s disease (PD) into dopaminergic (DA) neurons and showed that these DA neurons can be transplanted without signs of neurodegeneration into the adult rodent stria-tum. The PD patient iPS (PDiPS) cell-derived DA neurons survived at high num-bers, showed arborization, and mediated functional effects in an animal model of PD as determined by reduction of amphetamine- and apomorphine-induced rota-tional asymmetry, but only a few DA neurons projected into the host striatum at 16 weeks after transplantation. They also applied FACS for the neural cell adhesion molecule NCAM on differentiated PDiPS cells before transplantation, which resulted in surviving DA neurons with functional effects on amphetamine-induced rotational asymmetry in a 6-OHDA animal model of PD. They found that PDiPS cell-derived non-DA neurons send axons along white matter tracts into specifi c close and remote gray matter target areas in the adult brain. These data demon-strated proof of principle of survival and functional effects of PDiPS cell-derived DA neurons in an animal model of PD.

Induced pluripotent stem cells have been generated from patients with type 1 diabetes (T1D) by reprogramming their adult fi broblasts with three transcription factors (OCT4, SOX2, KLF4) [ 30 ]. T1D-specifi c iPS cells could be differenti-ated into insulin-producing cells, which gives a chance to develop cell replace-ment therapy.

Induced Pluripotency for the Study of Disease Mechanisms and Cell Therapy

166

5 Autologous Use of iPS Cells in Combination with Gene Therapy

Several experiments on lab animals have shown that it is possible to treat monogenic diseases using iPS cells in combination with gene therapy. For example, Jaenisch group has demonstrated that it was possible to treat of sickle cell anemia mouse model with iPS cells generated from autologous skin [ 31 ]. Using a humanized sickle cell anemia mouse model, they showed that mice can be rescued after transplantation with hematopoietic progenitors obtained in vitro from autologous iPS cells. This was achieved after correction of the human sickle hemoglobin allele by gene-specifi c targeting. That was a ground-breaking paper to provide proof of principle for using transcription factor-induced reprogramming combined with gene and cell therapy for disease treatment in mice.

A big obstacle to use gene therapy in combination with iPS cells in humans is the fact that the effi ciency of homologous recombination in human iPS cells (as well as in ES cells) is very low. Furthermore, the cloning of targeted cells is very compli-cated as these cells grow very poorly when plated as single cells (a practice needed to select rare targeted clones).

A promising gene-editing technology is using zinc-fi nger nucleases (ZFN), which introduce targeted double stranded DNA breaks and increases signifi cantly the rate of homologous recombination. This approach has been successfully used to correct a point mutation A53T in the α-synuclein gene, suggesting the possibility that genetic defects causing familial Parkinson disease could be repaired before cell-replacement therapy for PD [ 32 ]. A homologous recombination-based approach using Cre-LoxP system and ZFN to precisely correct the sickle cell disease muta-tion in patient-derived iPS cells with 2 mutated β-globin alleles (β(s)/β(s)) has been also recently reported [ 33 ].

In addition, by using (ZFN)-mediated genome editing, sets of isogenic disease and control human pluripotent stem cells have been generated, that differ exclu-sively at either of two susceptibility variants for Parkinson’s disease by modifying the underlying point mutations in the α-synuclein gene [ 32 ].

iPS cells have been derived from a patient with X-linked chronic granulomatous disease (X-CGD), a defect of neutrophil microbicidal reactive oxygen species (ROS) generation resulting from gp91(phox) defi ciency [ 33 ]. Mature neutrophils differentiated from X-CGD iPS cells lack Reactive Oxygen Species (ROS) produc-tion, reproducing the pathognomonic CGD cellular phenotype. ZFN-mediated gene targeting of a single-copy gp91(phox) therapeutic minigene into one allele of the “safe harbor” AAVS1 locus in X-CGD iPS cells without off-target inserts resulted in sustained expression of gp91(phox) and substantially restored neutrophil ROS production.

Although ZFN method stimulates homologous recombination rates signifi cantly, its levels are still not high enough and it is too time-consuming to be adaptable for clinical settings. Therefore, other technologies are being actively explored: tran-scription activator–like effector nucleases (TALENs) (which are more modular than

T. Maimets

167

ZFNs), adeno-associated viruses (which are effi cient at targeting several human somatic cell types), gutless adenoviral vectors that allow high-level gene transfer and large cargos of longer homology arms for effi cient homologous recombination, and bacterial artifi cial chromosome-based plasmid vectors with extremely large homology arms (see [ 22 ] for references).

Li and others have recently reported a protocol for targeted removal of human trisomy, which can alter cellular phenotypes and produce congenital abnormalities such as Down syndrome (DS). They generated induced pluripotent stem cells from DS fi broblasts and introduced a TKNEO transgene (containing genes for thymidine kinase and neomycin resistance) into one copy of chromosome 21 by gene targeting [ 34 ]. When selecting against TKNEO , spontaneous chromosome loss was the most common cause for survival, with a frequency of ~10 −4 , while point mutations, epigenetic silenc-ing, and TKNEO deletions occurred at lower frequencies in this unbiased comparison of inactivating mutations. The derived, disomic cells proliferated faster and produced more endothelia in vivo than their otherwise isogenic trisomic counterparts.

Although the number of papers describing the use of iPS in combination with gene therapy is rapidly accumulating, it is clear that there are still many unknown factors connected with safety and effi cacy, which need much more fundamental research. Any cell therapy must ultimately be superior in safety and effi cacy to any drug therapy, and establishing such utility will require large-scale and painstaking prospective trials to be conducted over many years. Thus, despite huge promise, cell therapy as the standard of care for many diseases is but at a distant horizon.

6 iPS Cells to Model Human Disease

A more imminent use of iPS in human medicine is to derive iPS cells from patients with genetic or other disorders and then use them as a “human cell model of human disease” to understand the mechanisms of the disease and to possibly fi nd chemicals to correct the defective pathways.

This “human cell model” can replace transgenic “mouse models” of human dis-ease, which often are of very limited use because of big differences between human and mouse metabolism and the fact that the same types of mutations in human and mouse respective genes do not necessarily cause the same disease phenotype. For example, a mouse model for trisomy 21 (Down syndrome or DS) critical region on distal human chromosome 21 fails to recapitulate the human cranial abnormalities commonly associated with trisomy 21 [ 35 ]. Orthologous segments to human chro-mosome 21 are present on mouse chromosomes 10 and 17 and distal human chro-mosome 21 corresponds to mouse chromosome 16, whereas trisomy 16 in the mouse is lethal [ 36 ]. Thus, a true murine equivalent of human trisomy 21 does not exist. Also, murine strains carrying the same genetic defi ciencies as the human bone marrow failure disease Fanconi anemia demonstrate DNA repair defects consistent with the human condition (e.g. [ 37 ], yet none develop the spontaneous bone marrow failure that is the hallmark of the human disease.

Induced Pluripotency for the Study of Disease Mechanisms and Cell Therapy

168

A long list of iPS models for different diseases has emerged including more than 60 diseases today and the list is expanding rapidly (see Table 1 in [ 38 ]).

The fi rst disease-specifi c iPS cells were derived from patients with familial amy-otrophic lateral sclerosis (ALS) and a number of genetic diseases [ 14 , 39 ]. ALS is the most common degenerative disease of the motor neuron system, accompanied by loss of many types of neurons. Dimos and others created iPS cells from an 82-year-old woman diagnosed with a familial form of amyotrophic lateral sclerosis by transducing skin fi broblasts with four transcription factors (Yamanaka factors). Then they turned iPS cells into embryoid bodies which, after exposure to sonic hedgehog and retinoic acid, generated both motor neurons and astrocytes – the cells destroyed in ALS [ 39 ].

In the same year Park and others created iPS cells from patients with a variety of genetic diseases with either Mendelian or complex inheritance. The list included adenosine deaminase defi ciency-related severe combined immunodefi ciency (ADA- SCID), Shwachman-Bodian-Diamond syndrome (SBDS), Gaucher disease (GD) type III, Duchenne (DMD) and Becker muscular dystrophy (BMD), Parkinson disease (PD), Huntington disease (HD), juvenile-onset type 1 diabetes mellitus (JDM), Down syndrome (DS)/trisomy 21 and the carrier state of Lesch-Nyhan syndrome [ 14 ]. These authors, as well as Dimos et al. [ 39 ] used mostly retroviral vectors to transduce the patient skin cells. However, because of stochastic genomic integration these vectors have usually been found not suitable for patient treatment, therefore other methods for reprogramming cells into iPS cell state are being actively looked for.

To achieve a suitable iPS cell model for human disease, a critical point is that either the iPS cells themselves or their more differentiated progeny must express relevant cellular or molecular phenotype. The best candidates for this approach are therefore monogenic diseases affecting a specifi c cell type, which can be easily derived from pluripotent stem cells. Examples here are neurological diseases spinal muscular atrophy (SMA) and Rett syndrome; metabolic diseases such as α1-antitrypsin defi ciency, familial hypercholesterolemia and glycogen storage dis-ease type 1A; cardiovascular diseases such as Timothy syndrome and type 1 and 2 Long QT syndrome (see Table 1 [ 38 ]).

Most of the iPS cells obtained from these patients express an observable pheno-type upon differentiation. For example, iPS cells derived from a child with SMA maintained the disease genotype and generated motor neurons that showed selective defi cits compared to those derived from the child’s unaffected mother [ 40 ].

iPS cells from Rett syndrome (RTT) patients were able to undergo X-inactivation and generate functional neurons. Neurons derived from RTT-iPS cells had fewer synapses, reduced spine density, smaller soma size, altered calcium signaling and electrophysiological defects when compared to controls [ 41 ]. These authors also used RTT neurons to test the effects of drugs in rescuing synaptic defects.

Recently it was demonstrated that human iPS cells derived from Gaucher disease (an autosomal recessive disorder caused by mutations in the acid β-glucocerebrosidase gene) patients can effectively recapitulate pathologic hallmarks of the disease and can be a valuable tool for understanding molecular mechanisms and developing therapeutic approaches for this disease [ 42 ].

T. Maimets

169

However, in addition to these success stories there are also studies, when iPS cells have been generated from patients, but no phenotype could be observed. It could be explained with the lack of either suitable differentiation protocols or spe-cifi c assays for the molecular defect [ 38 ], but this is certainly not always the case. For example, iPS cells derived from sporadic Parkinson’s disease patients could be effi ciently differentiated into dopaminergic neurons, but the resulting cells did not show signifi cant differences in various in vitro assays, when compared to control iPS cells [ 29 , 43 ]. In their paper Soldner et al. [ 43 ], however, were able to derive transgene-free iPS cells using Cre-recombinase excisable viruses.

iPS cells or their differentiated progeny with robust phenotype can be used to screen possible therapeutic compounds to correct the disease. One of the fi rst stud-ies here is on Familial dysautonomia (FD). FD is a rare but fatal peripheral neuropa-thy, caused by a point mutation in the IKBKAP gene involved in transcriptional elongation. The disease is characterized by the depletion of autonomic and sensory neurons due to mutations in the IκB kinase complex associated protein IKBKAP gene. This causes a tissue-specifi c splicing defect and lowers the level of corre-sponding protein. Lee et al. [ 44 ] derived iPS cells from FD patient and differentiated them into neural precursor cells, which exhibited three FD-associated phenotypes: defective IKBKAP splicing, decreased rate of neurogenesis and reduced migration. Screening a number of therapeutic compounds resulted in a compound called kine-tin, which was able to partially reverse the aberrant splicing and the defects of neu-rogenesis and migration.

The list of disease-specifi c derived iPS cells used to test candidate therapeutic compounds is growing rapidly. Examples here are SMA-specifi c iPS cells, where motor neuron survival defects could be partially corrected by valproic acid and tobramycin [ 40 ] and RTT-iPS neurons, where IGF1 treatment increased synapse formation ability [ 41 ]. These and other studies provide good evidence that iPS cells can be effectively used to discover new disease-specifi c therapeutic agents.

7 Limitations of iPS Cell-Based Disease Models

Two recent reviews [ 38 , 45 ] also list several limitations for using iPS cells as tools for understanding disease mechanisms and fi nd possible drugs. First, certain genetic lesions inhibit or even preclude the derivation of iPS cells from patients by interfer-ing with the reprogramming process itself. For example, it has been diffi cult to derive iPS cells from patients with Fanconi anemia (FA), which is a recessive syn-drome characterized by progressive fatal bone marrow failure and chromosomal instability. FA cells have inactivating mutations in a signaling pathway, which is critical for maintaining genomic integrity and protecting cells from the DNA dam-age caused by cross-linking agents. It has recently been shown that reprogramming leads to activation of the FA pathway, increased DNA double-strand breaks and senescence. Defects in the FA DNA-repair pathway decreased the reprogramming effi ciency of both murine and human primary cells. FA pathway complementation,

Induced Pluripotency for the Study of Disease Mechanisms and Cell Therapy

170

however, reduced senescence and restored the reprogramming effi ciency of somatic FA cells to normal levels [ 46 ].

Second, there is evidence indicating that several epigenetic disorders are not reset by somatic cell reprogramming. An example here is Fragile X syndrome (FXS), which is caused by silencing of the FMR1 gene due to CGG triplet expan-sion, which results in aberrant DNA methylation and accumulation of repressive histone marks. In iPS cells derived from FXS patients the FMR1 gene still remained inactive [ 47 ]. Therefore, Fragile X iPS cells could not model the silencing of the FMR1 gene occurring during development.

The third obstacle to use iPS cell-derived models comes from clone-to-clone variability of iPS cell clones produced. iPS cell lines have been generated from multiple patients with FXS and differentiated into post-mitotic neurons and glia [ 48 ]. It came out that clones from reprogrammed FXS patient fi broblast lines exhibit variation with respect to the predominant CGG-repeat length in the FMR1 gene. In two cases, iPS cell clones contained predominant CGG-repeat lengths shorter than measured in corresponding input population of fi broblasts. In another instance, reprogramming a mosaic patient having both normal and pre-mutation length CGG repeats resulted in genetically matched iPS cell clonal lines differing in FMR1 pro-moter CpG methylation and FMRP expression.

A major reason for such a cell-to-cell variability is the lack of robust in vitro dif-ferentiation protocols, so the existing procedures create a mixture of diverse cell types. One improvement here could be to introduce reporter or selection genes under the control of lineage- or cell-type-specifi c promoters, which allow the iden-tifi cation and selection of specifi c cell types [ 45 ].

The situation is even more sophisticated when modeling complex genetic dis-eases or late-onset diseases that have large environmental components. In these cases it is diffi cult to derive differentiation protocols for disease-relevant cell types.

8 Conclusions

Pluripotent stem cells including iPS cells are regarded as a powerful source for cell therapy because these cells function both by direct cell replacement and also by paracrine effects. Unlimited availability of the cells is another advantage for iPS cells. However, effi cient differentiation technologies should be developed in paral-lel for applying these cells in the clinic because of the potential risk of unwanted side effects such as tumor formation. Therefore, more fundamental research on these issues is desperately needed before iPS cell therapy can become a routine clinical practice.

A more imminent use of iPS in human medicine is to derive iPS cells from patients with genetic or other disorders and then use them as a “human cell model of human disease” to understand the mechanisms of the disease and to possibly fi nd chemicals to correct the defective pathways. This approach has already led to dis-covery of new approaches to medical treatments and potentially will form a new and effi cient basis for personalized therapy of patients.

T. Maimets

171

References

1. Waddington CH (1957) The strategy of the genes: a discussion of some aspects of theoretical biology. Allen & Unwin, London

2. Gurdon J (1962) The developmental capacity of nuclei taken from intestinal epithelium cells of feeding tadpoles. J Embryol Exp Morphol 10:622–640

3. Wray J, Kalkan T, Smith AG (2010) The ground state of pluripotency. Biochem Soc Trans 38:1027–1032

4. Driesch H (1891) Entwicklungsmechanische Studien I. Der Wert der ersten beiden Furchungszellen in der Echinodermenentwickelung Experimentelle Erzeugung von Teil und Doppelbildungen. Ztschr f Wiss Zool 53:160–183

5. Evans M, Kaufman M (1981) Establishment in culture of pluripotent cells from mouse embryos. Nature 292:154–156

6. Martin GR (1981) Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A 78:7634–7638

7. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM (1998) Embryonic stem cell lines derived from human blastocysts. Science 282:1145–1147

8. Keirstead HS, Nistor G, Bernal G, Totoiu M, Cloutier F, Sharp K, Steward O (2005) Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury. J Neurosci 25:4694–4705

9. Armstrong L, Lako M, Buckley N, Lappin TRJ, Murphy MJ, Nolta JA, Pittenger M, Stojkovic M (2012) Editorial: our top 10 developments in stem cell biology over the last 30 years. Stem Cells 30:2–9

10. Jaenisch R, Young R (2008) Stem cells, the molecular circuitry of pluripotency and nuclear reprogramming. Cell 132:567–582

11. Loh KM, Lim B (2011) A precarious balance: pluripotency factors as lineage specifi ers. Cell Stem Cell 8:363–369

12. Kaufman MH, Webb S (1990) Postimplantation development of tetraploid mouse embryos produced by electrofusion. Development 110:1121–1132

13. Verlinsky Y, Strelchenko N, Kukharenko V, Rechitsky S, Verlinsky O, Galat V, Kuliev A (2005) Human embryonic stem cell lines with genetic disorders. Reprod Biomed Online 10:105–110

14. Park I-H, Arora N, Huo H, Maherali N, Ahfeldt T, Shimamura A, Lensch MW, Cowan C, Hochedlinger K, Daley GQ (2008) Disease-specifi c induced pluripotent stem cells. Cell 134:877–886

15. Rideout WM 3rd, Hochedlinger K, Kyba M, Daley GQ, Jaenisch R (2002) Correction of a genetic defect by nuclear transplantation and combined cell and gene therapy. Cell 109:17–27

16. Chambers I, Colby D, Robertson M, Nichols J, Lee S, Tweedie S, Smith A (2003) Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113:643–655

17. Mitsui K, Tokuzawa Y, Itoh H, Segawa K, Murakami M, Takahashi K, Maruyama M, Maeda M, Yamanaka S (2003) The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 113:631–642

18. Tada M, Takahama Y, Abe K, Nakatsuji N, Tada T (2001) Nuclear reprogramming of somatic cells by in vitro hybridization with ES cells. Curr Biol 11:1553–1558

19. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fi broblast cultures by defi ned factors. Cell 126:663–676

20. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S (2007) Induction of pluripotent stem cells from adult human fi broblasts by defi ned factors. Cell 131:861–872

21. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science 318:1917–1920

Induced Pluripotency for the Study of Disease Mechanisms and Cell Therapy

172

22. Mali P, Cheng L (2012) Concise review: Human cell engineering: cellular reprogramming and genome editing. Stem Cells 30:75–81

23. Maimets T, Neganova I, Armstrong L, Lako M (2008) Activation of p53 by nutlin leads to rapid differentiation of human embryonic stem cells. Oncogene 27:5277–5287

24. Kawamura T, Suzuki J, Wang YV, Menendez S, Morera LB, Raya A, Wahl GM, Izpisúa Belmonte JC (2009) Linking the p53 tumour suppressor pathway to somatic cell reprogram-ming. Nature 460:1140–1144

25. Hong H, Takahashi K, Ichisaka T, Aoi T, Kanagawa O, Nakagawa M, Okita K, Yamanaka S (2009) Suppression of induced pluripotent stem cell generation by the p53-p21 pathway. Nature 460:1132–1135

26. Lowry WE (2012) Does transcription factor induced pluripotency accurately mimic embryo derived pluripotency? Curr Opin Genet Dev 22:429–434

27. Zhao T, Zhang Z-N, Rong Z, Xu Y (2011) Immunogenicity of induced pluripotent stem cells. Nature 474:212–215

28. Wernig M, Zhao J-P, Pruszak J, Hedlund E, Fu D, Soldner F, Broccoli V, Constantine-Paton M, Isacson O, Jaenisch R (2008) Neurons derived from reprogrammed fi broblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson’s disease. Proc Natl Acad Sci U S A 105:5856–5861

29. Hargus G, Cooper O, Deleidi M, Levy A, Lee K, Marlow E, Yow A, Soldner F, Hockemeyer D, Hallett PJ, Osborn T, Jaenisch R, Isacson O (2010) Differentiated Parkinson patient-derived induced pluripotent stem cells grow in the adult rodent brain and reduce motor asymmetry in Parkinsonian rats. Proc Natl Acad Sci U S A 107:15921–15926

30. Maehr R, Chen S, Snitow M, Ludwig T, Yagasaki L, Goland R, Leibel RL, Melton DA (2009) Generation of pluripotent stem cells from patients with type 1 diabetes. Proc Natl Acad Sci U S A 106:15768–15773

31. Hanna J, Wernig M, Markoulaki S, Sun C-W, Meissner A, Cassady JP, Beard C, Brambrink T, Wu L-C, Townes TM, Jaenisch R (2007) Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science 318:1920–1923

32. Soldner F, Laganière J, Cheng AW, Hockemeyer D, Gao Q, Alagappan R, Khurana V, Golbe LI, Myers RH, Lindquist S, Zhang L, Guschin D, Fong LK, Vu BJ, Meng X, Urnov FD, Rebar EJ, Gregory PD, Zhang HS, Jaenisch R (2011) Generation of isogenic pluripotent stem cells differing exclusively at two early onset Parkinson point mutations. Cell 146:318–331

33. Zou J, Sweeney CL, Chou B-K, Choi U, Pan J, Wang H, Dowey SN, Cheng L, Malech HL (2011) Oxidase-defi cient neutrophils from X-linked chronic granulomatous disease iPS cells: functional correction by zinc fi nger nuclease-mediated safe harbor targeting. Blood 117:5561–5572

34. Li LB, Chang K-H, Wang P-R, Hirata RK, Papayannopoulou T, Russell DW (2012) Trisomy correction in Down syndrome induced pluripotent stem cells. Cell Stem Cell 11:615–619

35. Olson LE, Richtsmeier JT, Leszl J, Reeves RH (2004) A chromosome 21 critical region does not cause specifi c Down syndrome phenotypes. Science 306:687–690

36. Nelson DL, Gibbs RA (2004) Genetics. The critical region in trisomy 21. Science 306:619–621

37. Chen M, Tomkins DJ, Auerbach W, McKerlie C, Youssoufi an H, Liu L, Gan O, Carreau M, Auerbach A, Groves T, Guidos CJ, Freedman MH, Cross J, Percy DH, Dick JE, Joyner AL, Buchwald M (1996) Inactivation of Fac in mice produces inducible chromosomal instability and reduced fertility reminiscent of Fanconi anaemia. Nat Genet 12:448–451

38. Onder TT, Daley GQ (2012) New lessons learned from disease modeling with induced plu-ripotent stem cells. Curr Opin Genet Dev 22:500–508

39. Dimos JT, Rodolfa KT, Niakan KK, Weisenthal LM, Mitsumoto H, Chung W, Croft GF, Saphier G, Leibel R, Goland R, Wichterle H, Henderson CE, Eggan K (2008) Induced pluripo-tent stem cells generated from patients with ALS can be differentiated into motor neurons. Science 321:1218–1221

40. Ebert AD, Yu J, Rose FF Jr, Mattis VB, Lorson CL, Thomson JA, Svendsen CN (2009) Induced pluripotent stem cells from a spinal muscular atrophy patient. Nature 457:277–280

T. Maimets

173

41. Marchetto MCN, Carromeu C, Acab A, Yu D, Yeo GW, Mu Y, Chen G, Gage FH, Muotri AR (2010) A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells. Cell 143:527–539

42. Panicker LM, Miller D, Park TS, Patel B, Azevedo JL, Awad O, Masood MA, Veenstra TD, Goldin E, Stubblefi eld BK, Tayebi N, Polumuri SK, Vogel SN, Sidransky E, Zambidis ET, Feldman RA (2012) Induced pluripotent stem cell model recapitulates pathologic hallmarks of Gaucher disease. Proc Natl Acad Sci U S A 109:18054–18059

43. Soldner F, Hockemeyer D, Beard C, Gao Q, Bell GW, Cook EG, Hargus G, Blak A, Cooper O, Mitalipova M, Isacson O, Jaenisch R (2009) Parkinson’s disease patient-derived induced plu-ripotent stem cells free of viral reprogramming factors. Cell 136:964–977

44. Lee G, Papapetrou EP, Kim H, Chambers SM, Tomishima MJ, Fasano CA, Ganat YM, Menon J, Shimizu F, Viale A, Tabar V, Sadelain M, Studer L (2009) Modelling pathogenesis and treat-ment of familial dysautonomia using patient-specifi c iPSCs. Nature 461:402–406

45. Soldner F, Jaenisch R (2012) Medicine. iPSC disease modeling. Science 338:1155–1156 46. Müller LUW, Milsom MD, Harris CE, Vyas R, Brumme KM, Parmar K, Moreau LA,

Schambach A, Park I-H, London WB, Strait K, Schlaeger T, Devine AL, Grassman E, D’Andrea A, Daley GQ, Williams DA (2012) Overcoming reprogramming resistance of Fanconi anemia cells. Blood 119:5449–5457

47. Urbach A, Bar-Nur O, Daley GQ, Benvenisty N (2010) Differential modeling of fragile X syndrome by human embryonic stem cells and induced pluripotent stem cells. Cell Stem Cell 6:407–411

48. Sheridan SD, Theriault KM, Reis SA, Zhou F, Madison JM, Daheron L, Loring JF, Haggarty SJ (2011) Epigenetic characterization of the FMR1 gene and aberrant neurodevelopment in human induced pluripotent stem cell models of fragile X syndrome. PLoS One 6:e26203

Induced Pluripotency for the Study of Disease Mechanisms and Cell Therapy

175M. Özgüç (ed.), Rare Diseases: Integrative PPPM Approach as the Medicine of the Future, Advances in Predictive, Preventive and Personalised Medicine 6,DOI 10.1007/978-94-017-9214-1, © Springer Science+Business Media Dordrecht 2015

A Aaltonen, L.A. , 116, 118 Aarnio, M. , 118 Aasly, J.O. , 75 Abbas, Z. , 143 Abdenur, J.E. , 94, 96 Abe, K. , 163 Abe, S. , 50 Abecasis, G.R. , 36, 37, 39 Abedi, M.R. , 132, 133, 149 Abel, L. , 99 Abel, S. , 94 Abeliovich, D. , 50 Abrahamov, A. , 73, 79 Abu Rayyan, A. , 38 Acab, A. , 168, 169 Adams, M.D. , 120 Adina, Q. , 50 Adzhubei, I.A. , 38 Aebi, S. , 122 Aerts, J.M.F.G. , 71, 74, 76, 81, 82, 99 Afi one, S.A. , 138, 143 Afzelius, B. , 62 Agbandje-McKenna, M. , 134 Agel, J. , 94 Aglan, M. , 42 Aguado, M. , 123 Aguirre, L.A. , 50 Aharon-Peretz, J. , 75 Ahearn, M. , 101 Ahfeldt, T. , 163, 168 Ahmad, S. , 55 Ahnen, D.J. , 119 Ahuja, N. , 121 Aitken, M.L. , 149 Aiuti, A. , 132

Akkerman, E.M. , 99 Alagappan, R. , 166 Albano, W.A. , 118 Albers, U. , 26 Alexander, I.E. , 132, 133, 149 Alexander, J.H. , 136 Alfonso, P. , 78 Ali, R.R. , 144 Alkan, C. , 40 Allay, J. , 135, 149 Allegra, C.J. , 122 Allen, J. , 135 Allen, M.J. , 76 Allen-Powell, D.R. , 51 Allory, Y. , 26 Al-Tahan, J. , 26 Altarescu, G. , 73, 81, 100 Al-Tassan, N. , 116 Altschul, S.F. , 114, 115 Altshuler, D. , 36, 39 Altunoglu, U. , 42 Alvarez, A. , 50 Amalfi tano, A. , 101 Amann, G. , 26 Amanuel, B. , 119 Amat, C.B. , 24 Amato, D. , 74 Ambrosetti, U. , 50 Amling, M. , 42 Anagnostou, E. , 41 Anand, V.N. , 51 Anderson, J. , 94 Anderson, W.F. , 132 Ando, H. , 114, 115 Andreassen, O.A. , 75 Andria, G. , 101

Author Index

176

Angeli, S. , 54 Annesi, G. , 75 Antill, Y.C. , 119 Antonarakis, S.E. , 25, 71 Antosiewicz-Bourget, J. , 163 Aoi, T. , 164 Aperia, A. , 34, 136 Appelt, J.U. , 136 Apweiler, R. , 40 Arai, A.E. , 100 Aranda, P.C. , 99 Arash, L. , 97 Arcand, N. , 149 Arena, S. , 123 Argoff, C.E. , 71 Armour, J.A. , 50 Armstrong, L. , 162, 164 Armstrong, L.C. , 80 Arn, P. , 96 Arnett, A. , 135 Arnold, G.L. , 101 Arnold, M. , 118 Arnos, K.S. , 50 Aronica, E. , 135 Arora, N. , 163, 168 Arora, P. , 71 Arruda, V.R. , 149 Arslan, E. , 40 Artigou, J.Y. , 99 Arts, P. , 38 Arts, W.F.M. , 101 Aruga, J. , 55 Askari, H. , 100 Aslanidi, G. , 138, 147, 148 Asokan, A. , 132, 134 Atalay, S. , 55 Atchison, R.W. , 137 Atkin, W.S. , 119 Au, H.J. , 123 Aucoin, M.G. , 149 Auerbach, A. , 167 Auerbach, W. , 167 Auricchio, A. , 149 Austin, C.P. , 82 Auton, A. , 37, 39 Avenarius, M.R. , 50 Aviezer, D. , 99 Avraham, K.B. , 38, 50 Awad, O. , 80, 168 Aykut, A. , 40 Aymé, S. , 6, 16, 25 Azaiez, H. , 50 Azevedo, J.E. , 75 Azevedo, J.L. , 80, 168

Azimi-Nezhad, M. , 26 Aznarez, S. , 78

B Badenas, C. , 51 Baekelandt, V. , 134 Bahmad, F. , 51 Bailey, C.J. , 114, 115 Bailey-Wilson, J.E. , 118 Bainbridge, M.N. , 37, 41 Baisden, C.E. , 136 Baker, A.H. , 136 Baker, J. , 37, 39 Baker, S.M. , 119 Bal, J. , 50 Balcells, S. , 79 Bali, D. , 100–102 Balkany, T. , 54 Ball, E.V. , 37, 40 Ballana, E. , 50, 51 Balreira, A. , 75 Balthazor, M. , 94 Bamshad, M.J. , 35, 37 Banerjee-Basu, S. , 80 Banfi , S. , 149 Bankiewicz, K. , 135 Banks, E. , 36, 39 Barbosa, E.R. , 75 Barbujani, G. , 49 Barceló, E. , 51 Bardelli, A. , 123 Baris, H. , 77 Barnes, A.M. , 42 Barnetson, R. , 116 Barneveld, R.A. , 77 Bar-Nur, O. , 170 Baron, J.A. , 119 Barranger, J.A. , 77 Barraza-Ortiz, X. , 143 Bar-Shira, A. , 75 Barthelaix, A. , 26 Barton, N.W. , 71, 99 Basner-Tschakarjan, E. , 135, 149 Batshaw, M. , 136 Baxevanis, A.D. , 80 Bean, E. , 124 Beard, C. , 166, 169 Beaudet, A.L , 37, 41 Beaudeux, J.L. , 26 Beck, A.H. , 124 Beck, M. , 96, 97 Becker, J. , 38, 42 Béghin, L. , 26

Author Index

177

Beiraom, I. , 75 Bekaert, S. , 55 Bekheirnia, M.R. , 37, 41 Beleggia, F. , 33–42 Bell, G.W. , 169 Belmatoug, N. , 74 Belmont, J. , 96 Belting, M. , 27 Bembi, B. , 75, 78, 98, 99 Benatti, P. , 119 Bendova, O. , 50 Benito, J.M. , 82 Benjamin, D. , 101, 102 Benko, W.S. , 77, 78, 99 Bennett, J. , 149 Bennicelli, J. , 149 Benvenisty, N. , 170 Berg, D. , 75 Berger, C. , 135 Berger, F. , 26 Berger, K.I. , 96, 97, 100 Berger, M. , 132 Bergman, B.E. , 80 Bergmann, C. , 38 Berlin, C.I. , 49 Bernal, G. , 162 Berndt, E.R. , 135 Berns, K.I. , 137, 138, 140 Beroud, C. , 38 Berry, S.R. , 123 Bertele, V. , 33 Berthod, C.R. , 116 Bertranpetit, J. , 78 Besson, G. , 99 Betsou, F. , 26 Beuten, J. , 37, 41 Beutler, E. , 71–73, 76, 78 Bhatia, P. , 62, 64 Bhirangi, K. , 99 Bianchi, P. , 121 Bieszczad, J. , 138 Bigham, A.W. , 35, 37 Bignami, F. , 24 Bignami, M. , 116 Bijvoet, A.G. , 101 Bilbao, J.M. , 75 Biscone, K.A. , 118 Biscone-Halterman, K. , 50 Biswas, A. , 136 Bitner-Glindzicz, M. , 50 Bjoraker, K. , 94 Bjornsson, S. , 75 Black, S.E. , 75 Black-Ziegelbein, E.A. , 51

Blaese, R.M. , 132 Blak, A. , 169 Blanton, S. , 55 Blanz, J. , 75 Blaydon, D. , 50 Blin, N. , 50 Blouin, V. , 143 Board, R.E. , 122 Bodamer, O.A. , 97 Bodmer, D. , 41 Bodmer, J. , 114, 115 Bodmer, W.F. , 114, 115 Boekholt, P. , 24 Boelens, J.J. , 93 Boer, M. , 101 Boguski, M.S. , 114, 115 Boland, C.R. , 118, 122 Bollag, R.J. , 119 Bolz, H.J. , 38 Bonyadi, M. , 55 Boon, L. , 71 Boot, R.G. , 71, 76, 81 Booth, M.J. , 144 Borg, J. , 26 Bork, P. , 38, 39 Bornstein, P. , 80 Bortolheiro, T.C. , 99 Bottler, A. , 81 Boussioutas, A. , 119 Bousso, P. , 132 Boven, L.A. , 71 Bowen, T. , 94 Boyer, S. , 93, 94 Braakman, T. , 96 Brady, R.O. , 71, 77, 79, 100, 101 Brakenhoff, J.P. , 101 Brambrink, T. , 166 Brandenburger, A. , 142 Bras, J. , 75 Braun, T.A. , 51 Bravo, O. , 51 Braxton, A. , 37, 41 Breakefi eld, X.O. , 135 Breer, S. , 42 Breidenassel, C. , 26 Brejova, A. , 101 Bresalier, R.S. , 122 Breunig, F. , 101 Brice, A. , 75 Briganti, L. , 39 Brill, N.E. , 71 Brill-Almon, E. , 99 Brisson, D. , 132, 135 Brister, J.R. , 138

Author Index

178

Britton, K. , 50 Britton, N. , 136 Broccoli, V. , 165 Brockmann, K. , 75 Bromelow, K. , 50 Brøndum-Nielsen, K. , 49 Bronner, C.E. , 119 Brookhouser, P.E. , 50 Brooks, L.D. , 37, 39 Broomfi eld, A. , 124 Brown, K.R. , 39 Brown, P.O. , 124 Brown, S. , 50 Brownstein, Z. , 50 Brument, N. , 143 Brumme, K.M. , 170 Bruni, S. , 94, 96 Brunner, H.G. , 38, 41, 42 Bruno, D.L. , 64 Brunzell, J.D. , 133 Bryan, T.M. , 114, 115 Bryant, L.M. , 135 Buchanan, D.D. , 119, 120 Buchwald, M. , 167 Buckley, B.S. , 29 Buckley, N. , 162 Buijsman, W. , 41 Buller, H.A. , 136 Buller, R.M. , 140, 143 Bunin, N. , 94 Buning, H. , 131, 132, 136 Burgart, L.J. , 121 Burguera, J.M. , 79 Bussey, H.J. , 114, 115 Butensky, E. , 97 Bykhovskaya, Y. , 51 Byrne, B.J. , 100, 101, 138, 144, 147

C Caillet-Fauquet, P. , 142 Caiola, D. , 75 Callahan, M. , 79 Calvo, S.E. , 62, 64 Cambray-Forker, E.J. , 94, 96 Camellini, L. , 119 Campagnoli, C. , 136 Campbell, H. , 116 Campbell, T.N. , 76 Campbell-Thompson, M. , 138, 147 Cankaya, T. , 42 Cao, D. , 41 Cao, X. , 142 Caparrós-Martín, J.A. , 42

Caplan, R.H. , 26 Cappellini, M.D. , 74 Cardone, M. , 101 Carethers, J.M. , 122 Carey, W.F. , 76 Carmi, R. , 49 Carnaghi, C. , 121 Carpentier, A.C. , 135 Carr, S.A. , 62 Carreau, M. , 167 Carroll, P.R. , 124 Carromeu, C. , 168, 169 Carter, B.J. , 137, 140, 143, 144, 149 Carter, C.S. , 132 Carter, K.C. , 120 Carter, M.T. , 41 Casano, R.A. , 51 Casanova, J.L. , 132 Casavant, T.L. , 51 Case, L.E. , 100 Casey, G. , 119 Cassady, J.P. , 166 Castagnoli, L. , 39 Casto, B.C. , 137 Castorina, P. , 50 Cavazzana-Calvo, M. , 132 Cecchini, S. , 142, 148, 149 Cengiz, F.B. , 50, 54, 55 Cenni, B. , 122 Certain, S. , 132 Cesareni, G. , 39 Chabás, A. , 79 Chabrol, B. , 101 Chadbourne, E. , 96 Chadeuf, G. , 140, 143 Chaffron, S. , 39 Chahal, P.S. , 149 Chakravarti, A. , 71 Chalchal, H. , 123 Chamberlain, J.S. , 135 Chamberlin, G.P. , 50 Chambers, I. , 163 Chambers, S.M. , 169 Champion-Arnaud, P. , 140 Chan, J.K. , 136 Chang, B. , 62 Chang, D.C. , 121 Chang, K. , 120 Chang, K.-H. , 167 Chang, L. , 132 Chang, L.S. , 140 Chapel, C. , 26 Chappuis, P.O. , 116 Charria-Ortiz, G. , 100

Author Index

179

Chauhan, D.P. , 122 Chaves, J. , 75 Cheadle, J.P. , 116 Chejanovsky, N. , 144 Chen, C.-A. , 101 Chen, C.M. , 75 Chen, C.Y. , 148 Chen, F. , 142, 143 Chen, G. , 168, 169 Chen, H. , 147, 148 Chen, J. , 136 Chen, K. , 40 Chen, L.-R. , 101 Chen, M. , 167 Chen, N. , 41 Chen, S. , 136, 165 Chen, Y.-T. , 101, 102 Chen, Z. , 136 Chen, Z.-Y. , 55 Cheng, A.W. , 166 Cheng, L. , 163, 166, 167 Cheng, S.H. , 82, 101 Cheng, X. , 41 Cherel, Y. , 140 Chertkoff, R. , 99 Chew, A.J. , 149 Chiang, Y. , 132 Chien, Y.-H. , 101 Chinnery, P.F. , 62, 75 Chiorini, J.A. , 138 Chioza, B.A. , 55 Chiu, S.-N. , 101 Chmiel, N.H. , 116 Cho, J.H. , 81 Choi, J.H. , 80, 82 Choi, M. , 76, 81 Choi, U. , 166 Choolani, M. , 136 Chopra, S.S. , 77 Chou, B.-K. , 166 Chou, S.T. , 148 Chowdary, P. , 135, 149 Choy, F.Y. , 76 Christen, R.D. , 122 Christodoulou, J. , 64 Christopher, D.M. , 135 Chrysler, C. , 41 Chu, B. , 135 Chuang, W.L. , 78, 82 Chung, W. , 168 Chung, Y.C. , 148 Ciaffoni, F. , 79 Ciana, G. , 75 Cibulskis, K. , 36, 39

Ciliberto, G. , 145 Cingolani, P. , 37 Cizmarik, M. , 76 Clague, A.E. , 76 Clark, A.G. , 70 Clark, K.R. , 143 Clark, L.N. , 75 Clarke, J.T.R. , 96 Clarke, L.A. , 94, 96 Claustres, M. , 38 Clavelou, P. , 99 Cleary, M. , 97 Clemens, P.R. , 101 Clement, N. , 144 Clendenning, M. , 118, 119 Clerici, M. , 132 Clerkin, P. , 29 Clotworthy, M. , 23 Cloutier, F. , 162 Cockburn, D.J. , 50 Coelho, J. , 99 Coffey, R. , 50 Coffi n, R.S. , 144 Cogulu, O. , 38 Cohen, B.H. , 63 Cohen, I.J. , 77, 79 Cohen, J.C. , 71 Cohn, E.S. , 50 Coker, M. , 38 Colangelo, L.H. , 122 Colby, D. , 163 Coleman, J. , 135, 149 Collaco, R.F. , 138, 142 Collin-Histed, T. , 99 Collins, B. , 101 Collins, F.S. , 34 Collis, P. , 140 Collod-Beroud, G. , 38 Colosi, P. , 140 Colucci, G. , 26 Coman, D.J. , 94, 97 Comas, D. , 78 Comeras, I. , 118 Compton, A.G. , 64 Compton, C.C. , 112 Condroyer, C. , 75 Conlon, H. , 50 Conrad, C.K. , 149 Conrad, S. , 97 Conrath, H. , 142 Constantine-Paton, M. , 165 Conway, A.M. , 97 Conway, J.E. , 144 Cook, E.G. , 169

Author Index

180

Cool, V.A. , 94 Coon, H. , 140 Coon, M. , 37 Cooper, D.N. , 37, 40 Cooper, O. , 165, 169 Copeland, D.P. , 82 Copeland, N.G. , 119 Coppa, G.V. , 94, 96 Corless, C.L. , 124 Cormand, B. , 79 Correia, C. , 50 Cortese, R. , 145 Corzo, D. , 101 Costigliola, V. , 132, 136 Coucke, P. , 55 Coulter, D.K. , 48 Coutelle, O. , 132 Couto, L.B. , 149 Covault, K.K. , 94, 96 Cowan, C. , 163, 168 Cowan, M.J. , 94 Cox, G.F. , 96, 97 Cox, T.M. , 71, 74, 76, 80 Coy, N.N. , 50 Coyle, B. , 50 Cras, P. , 75 Creevey, C. , 39 Creighton, C.J. , 120 Cremers, C.W.R.J. , 50 Cremers, F.P. , 41 Crittenden, M. , 94 Croft, G.F. , 168 Crombez, E. , 99 Cromme-Dijkhuis, A. , 101 Crosby, A.H. , 55 Crosiers, D. , 75 Cross, H.E. , 55 Cross, J. , 167 Crowley, J.F. , 100 Cryns, K. , 50 Culver, K.W. , 132 Cunha, G.R. , 124 Cunningham, J.M. , 135, 149 Czartoryska, B. , 94

D Daheron, L. , 170 Dahl, H.-H.M. , 50 Daifuku, R. , 149 Daina, E. , 34, 136 Daley, G.Q. , 163, 168–170 Dalkara, D. , 135 Dallman, J. , 55

Daly, M.J. , 36, 39 Dambrosia, J.M. , 71 Dan, D. , 15, 16 D’Andrea, A. , 170 Danes, B.S. , 118 Danon, M. , 101 Dao, P. , 40 Dardis, A. , 75 Darowski, M. , 94 Davenport, S.L. , 50 David, A. , 136 David, S.S. , 116 David-Ameline, J. , 140 Davidoff, A.M. , 135, 136, 149 Davidson, Y. , 75 Davies, D.R. , 116 Davies, E.H. , 99 Davis, M.D. , 138 Davison, M. , 101 Dawson, G. , 41 de Alwis, M. , 144 De Deyn, P.P. , 75 De Gaetani, C. , 119 de Jong, D. , 101 de Jong, G. , 101 De Keulenaer, S. , 55 De Klerk, J.B.C. , 101 de la Chapelle, A. , 118–120 de la Paz Posada, M. , 24 de Ligt, J. , 38 de Lore, D. , 94, 97 De Luca, G. , 116 De Marco, E.V. , 75 De Matteis, M.A. , 101 De Meirleir, L. , 97 de Montoril, M.F.P. , 99 de Oliveira, C.A. , 50 de Reuver, R. , 41 De Roock, W. , 123 de Saint, B.G. , 132 De Santis, M. , 1–20 De Schrijver, J. , 55 de Siqueira, L.F. , 75 de Vries, B.B. , 38 de Vries, P. , 38 de Vrueh, R.L. , 136 de Wal, J. , 132, 135 de Winter, R.J. , 136 deAlarcon, P.A. , 94 DeArmey, S.L. , 101, 102 Debyser, Z. , 134 Decker, C. , 97 Declau, F. , 49 Deeb, S. , 133

Author Index

181

Deforce, D. , 55 Degan, P. , 116 Deist, F.L. , 132 del Angel, G. , 36, 39 Del Castillo, F.J. , 50 del Castillo, I. , 50, 51 del Rosario, M. , 38 Delconte, G. , 121 Deleeuw, R.J. , 124 Deleidi, M. , 165, 169 Delhaas, T. , 101 Deliolanis, N.C. , 135 Dell’Osso, L. , 149 DeLuca, A.P. , 51, 54 Demailly, A. , 26 Denby, L. , 136 Denoyelle, F. , 50 DePaolo, J. , 80 DePristo, M.A. , 36, 37, 39 Derks, R.C. , 41 Dermitzakis, E.T. , 70 Deroose, C.M. , 134 DeRuisseau, K.C. , 101 DeRuisseau, L.R. , 101 Dery, S. , 132, 135 Deschauer, M. , 101 Deschner, E.E. , 118 Desmet, F.O. , 38 Desmidt, A.A. , 55 Desnick, R.J. , 75, 101 DeVile, C. , 99 Devine, A.L. , 170 Di Bisceglie, A.M. , 71 di Donato, J.H. , 23–29 Di Gregorio, C. , 119 Di Nicolantonio, F. , 123 Di Rocco, M. , 75 Díaz, L.E. , 26 Diaz-Horta, O. , 47–55 Dib-Hajj, S.D. , 41 Dick, J.E. , 167 Dickson, D.W. , 75 Dickson, P. , 96 Dighe, N. , 136 Dignam, J.D. , 138 Dignam, S.S. , 138 Diloreto, D. Jr. , 135 DiMauro, S. , 62, 64 Dimos, J.T. , 168 Ding, C. , 144, 145, 147 Ding, L. , 40 Ding, Y. , 37, 41 Dipple, K.M. , 70, 75, 79 Dixon-Woods, M. , 29

Djurovic, S. , 75 Doci, R. , 121 Dodge, J.C. , 101 Doerks, T. , 39 Dolan, M. , 26 Dominissini, S. , 75 Donahue, B.A. , 143 Donaudy, F. , 101 Donehower, L.A. , 120 Dong, J.Y. , 143 Donnelly, W.H. Jr. , 101 Donohue, J.H. , 122 Doppelt, S.H. , 71 Döring, T. , 79 Doroshow, R. , 96 Douglas, F.L. , 135 Dovey, M.E. , 149 Dowey, S.N. , 166 Downs, M.P. , 48 Downs, S. , 100 Dowty, J.G. , 119 Drebber, U. , 136 Dridi, M.-F.B. , 99 Driesch, H. , 161 Drmic, I.E. , 41 Drost, M.R. , 101 Du, L.L. , 54 du Sart, D. , 50 Ducoroy, P. , 26 Dukes, C.E. , 112, 114 Duman, D. , 50, 54, 55 Dunkle, M. , 136 Dunlop, J. , 49, 54 Dunlop, M.G. , 116, 117, 120 Dupuy, A. , 26 Duran, R. , 75 D’Urbano, L. , 51 Durbin, R.M. , 35–37, 39 Durmaz, B. , 38, 40 Dürr, A. , 75 Dusenbery, K. , 94 Duval, A. , 119 Duyckaerts, C. , 75 Dwivedi, S. , 101

E Earabino, C. , 119 Ebert, A.D. , 168, 169 Eblan, M.J. , 75 Edelstein, M.L. , 132, 133, 149 Edge, S.B. , 112 Edmonson, S. , 136 Edula, G. , 27

Author Index

182

Edwards, Y.J.K. , 55 Eger, K. , 101 Eggan, K. , 168 Ehrhardt, A. , 132 Eichler, E.E. , 40, 41 Elbedour, K. , 49 Elkan-Miller, T. , 38 Elliger, C. , 140 Elliger, S. , 140 Ellis, A. , 114, 115 Elroy-Stein, O. , 79 Elstein, D. , 73, 74, 81, 99 Elston, R.C. , 118 Elziere, C. , 99 Emond, M.J. , 35, 37 Endo, M. , 136 Eng, C.M. , 37, 41, 97 Engdahl, R.K. , 143 Engedal, K. , 75 Engelborghs, S. , 75 Enholm, S. , 116 Eppsteiner, R.W. , 51 Erdenetungalag, R. , 50 Erdmann, J.F. , 114 Erickson, R.P. , 41 Erikson, A. , 98, 99 Erlichman, C. , 122 Ernster, L. , 62 Escolar, D.M. , 101 Escolar, M.L. , 97 Eskelinen, M. , 116 Espinosa, I. , 124 Essiembre, C. , 132, 135 Estivill, X. , 49–51 Eto, Y. , 79, 97 Evans, M. , 161 Eysel, P. , 42

F Fadeel, B. , 33 Fahn, S. , 75 Fairley, C. , 76 Falk, P. , 94 Fallik, D. , 123 Fan, P.D. , 143 Farrer, M.J. , 75 Farrington, S.M. , 116, 117 Farson, D. , 143 Fasano, C.A. , 169 Fauser, S. , 144 Faust, L.Z. , 143 Favre, D. , 140, 143 Feenstra, I. , 41

Fehniger, T. , 27 Feldman, G.L. , 79 Feldman, R.A. , 80, 168 Feldmann, D. , 50 Felicetti, F. , 79 Fennell, T. , 36, 39 Fennell, T.J. , 36, 39 Ferlay, J. , 110, 114 Ferrari, F.K. , 140, 142 Ferrari, M. , 26, 136 Ferrelli, R. , 1–20 Ferri, R. , 100 Ferrier, A. , 99 Feudner, E. , 144 Fialho, G. , 50 Fidler, J. , 101 Fiegl, H. , 27 Fife, K.H. , 138 Figueredo, A. , 122 Filocamo, M. , 75 Fink, D. , 122 Finniear, R. , 114, 115 Fischel-Ghodsian, N. , 51 Fischer, A. , 132 Fischer, B. , 42 Fishel, R. , 119, 122 Fisher, E.R. , 72 Fisher, K.J. , 143 Fisher, R. , 49 Fisk, N.M. , 136 Fitzgibbon, E.J. , 99 Fitzgibbons, R.J. Jr. , 122 Fix, D. , 118 Flake, A.W. , 136, 149 Flamez, D. , 55 Flannery, J. , 135 Fleischmann, R.D. , 120 Fleisher, T. , 132 Fleming, N. , 116 Fletcher, J.M. , 64 Florence, J. , 101 Florin, L. , 139 Flotte, T.R. , 143, 149 Fong, L.K. , 166 Foroud, T. , 75 Forsayeth, J. , 135 Fortina, P. , 49 Foster, J. II , 55 Foster, N.R. , 123 Fotouhi, N. , 55 Fraley, D.M. , 143 Franceschetti, S. , 75 Frane, J.L. , 163 Frangulov, A. , 50

Author Index

183

Frank, R. , 134, 135 Frankel, W.L. , 118 Franssen, R. , 135 Fraser, C.M. , 120 Frattini, M. , 123 Frazzica, R.G. , 1–20 Freedman, M.H. , 167 Frei, K.P. , 100 French, A.J. , 122, 123 Freyer, D.R. , 101 Friderici, K. , 49 Friedman, T.B. , 49 Frisch, A. , 79 Frisch, F. , 135 Fu, D. , 165 Fukuda, T. , 101 Fukunaga, Y. , 136 Fuller, D.D. , 101 Fulton, R.S. , 40 Fung, H.C. , 75 Fung, J. , 82 Furlan, D. , 119

G Gabriel, S.B. , 36, 39 Gabrielli, O. , 94, 96 Gage, F.H. , 168, 169 Gaglia, P. , 119 Gagnon, R. , 135 Gaillard, G. , 26 Galat, V. , 163 Galeota, E. , 39 Galibert, L. , 140 Galjaard, H. , 77 Gallinger, S. , 119, 122, 123 Gan, O. , 167 Ganat, Y.M. , 169 Gan-Or, Z. , 75, 79 Gao, G.P. , 143 Gao, Q. , 166, 169 Garattini, S. , 33 Garber, J. , 119 García Fernández, J.M. , 82 Garcia, K.C. , 42 Gardner, P. , 149 Garimella, K.V. , 36, 39 Garin, J. , 26 Garone, C. , 64 Gaspar, H.B. , 132 Gaspar, P. , 75 Gasparini, P. , 49–51 Gasser, T. , 75 Gatt, S. , 79

Gatta, G. , 110 Gattas, M. , 119 Gaucher, P.C.E. , 71 Gaudet, D. , 132, 135 Gausden, E. , 50 Gennari, L. , 121 Gentile, A.E. , 1–20 Genuardi, M. , 119 Gerasimova, A. , 38 Germain, D.P. , 99 Gerry, H.W. , 138 Gershoni-Baruch, R. , 75 Geurts van Kessel, A. , 77 Ghetti, B. , 75 Ghosh, M. , 50 Giangrande, P.L. , 135 Giarbini, N. , 49 Giasson, B.I. , 75 Gibbons, L. , 75 Gibbs, R.A. , 37, 41, 120, 167 Gibson, R.L. , 149 Giegling, I. , 75 Gieselmann, V. , 79 Giffen, F. , 133 Giladi, N. , 75, 79 Gilbert, A.L. , 101 Gilbert, W. , 138 Giles, A.R. , 135 Giles, G.G. , 119 Gilissen, C. , 38, 41, 42 Gilks, C.B. , 124 Ginn, S.L. , 132, 133, 149 Ginns, E.I. , 72, 77, 81 Giraldo, P. , 78 Giralt, M. , 78 Girirajan, S. , 41 Girod, A. , 139 Giugliani, R. , 96, 97 Giunta, C. , 38 Givol, N. , 74 Glader, B. , 135, 149 Glocker, F.X. , 101 Godwin, A.R. , 119 Góes, J.E.C. , 96 Goforth, L. , 49 Goker-Alpan, O. , 72, 74, 75, 77, 78, 80 Goland, R. , 165, 168 Golbe, L.I. , 166 Goldberg, R.F. , 75 Goldberg, R.M. , 121, 122 Goldberger, O.A. , 62 Goldblatt, J. , 64, 74, 119 Goldblum, J.R. , 124 Goldenberg, P.C. , 101, 102

Author Index

184

Goldin, E. , 69–85, 168 Goldman, J.E. , 75 Goldstein, A. , 62, 64–66 Goldstein, G. , 74 Golubnitschaja, O. , 132, 136 Gómez-Martínez, S. , 26 Gonzalez, D.E. , 99 Gonzalez, M. , 55 Gonzàlez-Duarte, R. , 79 González-Gross, M. , 26 Goodman, C.W. , 51 Gopal-Srivastava, R. , 136 Gordon, R. , 122 Gorlin, R.J. , 52 Gorman, P. , 114, 115 Gottweis, H. , 24 Govea, N. , 51 Graber, A. , 27 Grabowski, G.A. , 71–74, 76, 79, 80, 82 Grace, M.E. , 75 Granovsky-Grisaru, S. , 74 Grant, I.R. , 80 Grassman, E. , 170 Gray, J.T. , 135, 149 Gray, L. , 136 Grayson, G.H. , 94 Greenberg, K. , 135 Greenblatt, J.J. , 132 Greentree, S. , 135 Greenwood, A. , 81 Gregersen, P.K. , 81 Gregory, P.D. , 166 Grewal, R.P. , 71 Grewal, S. , 94 Grieu, F. , 119 Griffi th, A.J. , 50, 75 Griggs, R.C. , 136 Grigoriadou, M. , 55 Grimm, D. , 140, 142 Grinberg, A. , 79 Grinberg, D. , 79 Grissom, T.J. , 50 Groeneveld, G.J. , 101 Groffen, J. , 114, 115 Groft, S. , 6 Grogan, E.W. , 138 Gronskov, K. , 50 Groombridge, C. , 119 Grosios, K. , 131–150 Gross, F. , 132 Grossfeld, G.D. , 124 Grossi, S. , 75 Grossman, A. , 50 Groth, J. , 75

Grothey, A. , 123 Groves, T. , 167 Grunert, B. , 101 Grünhagen, J. , 42 Gruschus, J.M. , 75 Gryfe, R. , 122 Gualandi, F. , 50 Gucsavas-Calikoglu, M. , 97 Guffon, N.H. , 96, 97 Guggino, W.B. , 143, 149 Guidos, C.J. , 167 Gunaratne, P.H. , 120 Gunawardena, S.R. , 119 Gupta, N. , 80 Gupta, P. , 81 Gurdon, J. , 161 Gurevich, T. , 75 Gurrola, J. II. , 54 Gurtz, K. , 55 Guschin, D. , 166

H Hacein-Bey, S. , 132 Hacein-Bey-Abina, S. , 132 Hach, F. , 40 Hackl, W.O. , 27 Haggarty, S.J. , 170 Hahn, S.H. , 50 Haile, R.W. , 119 Hajirasouliha, I. , 40 Hakonarson, H. , 37, 39 Halder, J.A. , 50 Hale, G.A. , 94 Halene, S. , 78 Hallek, M. , 132, 136, 139 Hallett, P.J. , 165, 169 Halliday, G. , 75 Halliwell, N. , 75 Halsall, D. , 80 Hamano, S. , 75, 80 Hamelin, R. , 119 Hamilton, S.R. , 120, 122, 123 Hammer, M.F. , 41 Hammon, W.M. , 137 Hampel, H. , 75, 118, 119 Hamroun, D. , 38 Han, H. , 55 Handsaker, B. , 36 Handsaker, R.E. , 37, 39 Hang, T. , 51 Hanna, J. , 166 Hanna, M. , 36, 39 Hanoteau, N. , 26

Author Index

185

Hanson, S. , 96 Har, B. , 133 Harding, T.C. , 143 Hardison, M. , 37, 41 Hardy, J. , 71, 75 Hardy, K. , 97 Hargreaves, R. , 134, 135 Hargus, G. , 165, 169 Harmatz, P. , 94, 97 Harrell, H. , 144 Harrington, C. , 135, 149 Harris, C.E. , 99, 170 Harris, J. , 75 Harris, R.E. , 94 Harskamp, R.E. , 136 Hart, P.S. , 77, 78 Hartl, C. , 36, 39 Harvey, B.K. , 134 Haseltine, W.A. , 120 Hatamochi, A. , 51 Hauck, B. , 149 Haute, C.V. , 134 Hawes, M.L. , 101 Hayden, M.R. , 133, 135 Hayward, G.S. , 144 Hayward, S.W. , 124 He, M. , 41 He, Y. , 35 Hecht, J. , 42 Hedge, P. , 114, 115 Hedlund, E. , 165 Heemstra, H.E. , 136 Hegele, R.A. , 133 Hegland, J.D. , 94 Heilbronn, R. , 143 Heinimann, K. , 116 Heitner, R. , 74 Hellemans, J. , 55 Heller, J. , 101, 102 Hemmings, C. , 109–126 Henderson, C.E. , 168 Henny, C.P. , 135 Henslee-Downey, P.J. , 94 Henter, J.I. , 33, 34, 136 Herman, G.E. , 101 Hermier, M. , 99 Hermonat, P.L. , 140 Hernandez-Boussard, T. , 124 Hershman, S.G. , 64 Herson, S. , 101 Hertle, R. , 149 Herzog, R.W. , 149 Hetterschijt, L. , 55 Hienonen, T. , 116

High, K.A. , 132, 135, 136, 149 Higurashi, N. , 75, 80 Hilbert, P. , 50 Hildebrand, M.S. , 50, 51, 54 Hilgert, N. , 50 Hill, J. , 133 Hill, S.C. , 71 Hilz, M.J. , 100, 101 Hinderer, C. , 135 Hirai, Y. , 143 Hirano, M. , 62 Hirata, R.K. , 167 Hirschhorn, R. , 100 Ho, J. , 133 Ho, K. , 143 Hochberg, Z. , 50 Hochedlinger, K. , 163, 168 Hockemeyer, D. , 165, 166, 169 Hodges, A.K. , 116 Hodgson, S.V. , 119 Hoefsloot, L.H. , 41, 50 Hofbauer, L.C. , 42 Hoffmann, A. , 79 Hofman, A. , 75 Hoft, R. , 96 Hoischen, A. , 38, 42 Hollak, C.E.M. , 74, 99 Holt, I.J. , 65 Homer, N. , 36, 39 Hong, H. , 164 Honig, L.S. , 75 Hood, L.J. , 49 Hop, W. , 101 Hopper, J.L. , 119 Hopwood, J.J. , 76, 94, 97, 101 Horii, A. , 114, 115 Hormozdiari, F. , 40 Horowitz, M. , 79, 80 Hoskinson, D.P. , 51 Hosomichi, K. , 35 Houlston, R.S. , 121 Houseman, M. , 50 Howard, D.B. , 134 Howe, J.L. , 41 Howell, R.R. , 100 Howell, S.B. , 122 Hruska, K.S. , 72, 77, 78, 98 Hsich, G. , 135 Hu, N. , 35 Hu, P. , 143 Hu, Y.C. , 148 Huang, K.S. , 148 Huang, M. , 55 Huang, S. , 55

Author Index

186

Hubner, R. , 121 Hue, C. , 132 Hughes, D.A. , 74 Hughes, J.V. , 143 Hui, P. , 23 Hunt, E. , 136 Hunter, J.J. , 135 Huo, H. , 163, 168 Hur, I.A. , 51 Hurtig, H.I. , 75 Husak, P. , 143 Hutchin, T. , 50 Huttenlocher, J. , 75 Hutter, P. , 116 Hutto, E. , 82 Huygen, P.L.M. , 50, 51 Huyn, S.T. , 75 Hwu, P. , 74 Hwu, W.-L. , 101

I Iacopetta, B. , 119, 121 Iannuccelli, M. , 39 Ibrahim-Verbaas, C. , 75 Ichisaka, T. , 163, 164 Ida, H. , 75, 79, 80, 97 Iijima, O. , 136 Ikeda, K. , 50 Ikeda, U. , 136 Ikkos, D. , 62 Ikram, M.A. , 75 Im, D.S. , 138 Incesulu, A. , 50 Inoue, I. , 35 Inoue, N. , 143 Inwood, A.C. , 94, 97 Iqbal, J. , 78 Isacson, O. , 165, 169 Iskrov, G. , 1–20 Itoh, H. , 163 Itskovitz-Eldor, J. , 162 Iwaki, Y. , 140 Izpisúa Belmonte, J.C. , 164 Izykowski, B. , 96

J Jacob, D. , 149 Jacob, S. , 123 Jacobs, J. , 149 Jaenisch, R. , 162, 163, 165, 166, 169, 170 Jaffe, D.B. , 64 Jain, D. , 76, 78, 81

Jain, M. , 62 James, P.A. , 119 Janda, C.Y. , 42 Janecke, A.R. , 50 Janik, J.E. , 140, 143 Janssen, I. , 38 Januario, C. , 75 Jardine, P. , 101 Järvinen, H.J. , 116, 118 Jass, J.R. , 119 Jeffries, N. , 100 Jenkins, L. , 50 Jenkins, M.A. , 119 Jenkins, N.A. , 119 Jensen, L.J. , 39 Jensen, M.L. , 50 Jewell, S. , 26 Jiang, Y.H. , 41 Jiménez-Pavón, D. , 26 Jin, X. , 41 Johana, N.B. , 136 Johnson, F. , 149 Johnson, P.R. , 143 Jones, J.M. , 162 Jonker, D.J. , 123 Jonsdottir, G.A. , 163 Jonsdottir, I. , 75 Jonsson, P.V. , 75 Jonsson, T. , 75 Joppi, R. , 33 Jordanova, R. , 14 Joy, T. , 133 Joyner, A.L. , 167 Ju, J. , 41 Julien, P. , 39 Jurecka, A. , 94 Jurisica, I. , 39

K Kabat, B. , 123 Kabra, M. , 50 Kaeppel, C. , 136 Kahn, M. , 122 Kairaluoma, M.V. , 118 Kakar, S. , 121 Kakkis, E.D. , 96, 97 Kalia, M. , 23 Kalicki, J.M. , 40 Kalkan, T. , 161, 162 Kallemeijn, W.W. , 76, 81 Kalman-Maltese, V. , 138 Kamen, A.A. , 149 Kamin, W. , 97

Author Index

187

Kamphoven, J.H.J. , 101 Kampmann, C. , 97 Kamsteeg, E.J. , 41 Kanaan, M. , 38, 50, 55 Kanagawa, O. , 164 Kane, M. , 119 Kaneski, C.R. , 79, 80, 100 Kang, H.M. , 37, 39 Kang, W. , 144 Kann, M. , 139 Kaplan, P. , 74, 94, 96, 97 Karaca, E. , 40 Karapetis, C.S. , 123 Karhu, A. , 116 Kärjä, V. , 116 Karlin, D.S. , 94, 96 Kastelein, J.J. , 133, 135 Kaufman, M.H. , 161, 162 Kawamura, T. , 164 Kawase, T. , 50 Kay, M.A. , 132, 135, 149 Kaye, E. , 136 Kaye, J. , 24 Kayserili, H. , 42 Ke, X.M. , 54 Keats, B. , 49 Keijzer, W. , 77 Keirstead, H.S. , 162 Keller, M. , 75 Kelley, P.M. , 50 Kelloff, G.J. , 135 Kells, A.P. , 135 Kelly, A. , 26 Kelly, T.J. Jr. , 138 Kelsell, D.P. , 49, 54 Kenet, G. , 74 Kenna, M.A. , 50 Kennedy, W. , 101 Kern, A. , 140, 142 Kernytsky, A.M. , 36, 39 Kerstenetzky, M.S. , 99 Ketteridge, D. , 97 Keupp, K. , 38, 40, 42 Khambata-Ford, S. , 123 Khan, S. , 50 Khanduja, K. , 118 Khanna, G. , 94 Khokher, A.M. , 76 Kholodov, M. , 37, 39 Khosla, S. , 42 Khurana, V. , 166 Ki, W. , 50 Kim, H. , 169 Kim, H.J. , 49

Kim, H.-N. , 50 Kimberling, W.J. , 50, 118 Kimura, A. , 97 Kimura, S. , 101 King, M.C. , 38, 71 King, R.J. , 94 Kinlaw, L. , 80 Kinzler, K.W. , 114, 115, 120 Kirby, A. , 37, 41 Kirchgessner, T.G. , 133 Kishnani, P.S. , 72, 100–102 Kisinovsky, I. , 99 Kissler, S. , 80 Klebe, S. , 75 Klein, H. , 132 Kleinschmidt, J.A. , 134, 139, 140, 142–144 Klemperer, M.R. , 94 Kluijtmans, L.A. , 75 Knight, M.A. , 75, 78, 80 Knop, D.R. , 144 Kocha, W.I. , 122 Koeberl, D.D. , 101, 102 Koerber, F. , 38, 42 Kohlbrenner, E. , 138, 147 Koi, M. , 122 Kokotas, H. , 50, 55 Kole, A. , 6 Kollman, C. , 94 Kolodner, R. , 119 Kolodny, E.H. , 80, 96, 99 Kolstad, K.D. , 135 Komarnitsky, S. , 82 Komiya, T. , 80 Kondrashov, A.S. , 38 Kong, A. , 75 Konkle, B. , 149 Konukseven, O. , 55 Koolen, D.A. , 41 Koprivica, V. , 79 Koprivnikar, K. , 143 Kornak, U. , 42 Kornblum, C. , 101 Kort, J. , 138 Koseoglu, S.T. , 97 Kosseim, P. , 29 Kost, S.E. , 124 Kotin, R.M. , 138, 142, 144, 145,

147–149 Kowarz, L. , 80 Kraft, M.L. , 49 Krahn, M. , 123 Kramer, W.G. , 97 Kravitz, R.M. , 100 Krawitz, P. , 42

Author Index

188

Kremer, H. , 41, 51, 55 Krischer, J. , 136 Krivit, W. , 94 Krook, J.E. , 122 Kroos, M.A. , 101 Kropp, P. , 75 Krush, A.J. , 118 Kudo, T. , 50 Kuebler, J.P. , 122 Kuebler, P. , 118 Kugler, K.G. , 27 Kuhn, J. , 38 Kuhn, K. , 25 Kuhn, M. , 39 Kuivenhoven, J.A. , 135 Kukharenko, V. , 163 Kuliev, A. , 163 Kunst, H.P.M. , 51 Kupfer, G.M. , 76, 81 Kurdi-Haidar, B. , 122 Kure, S. , 50 Kurtzberg, J. , 94 Kurtzman, G.J. , 140 Kurzawa-Akanbi, M. , 75 Kushmerick, M.J. , 135 Kyba, M. , 163

L La Monica, N. , 145 Laadan, S. , 79 Läärä, E. , 118 Labbe, S.M. , 135 Labianca, R. , 123 Lachlan, K. , 51 Lachmann, R.H. , 80 Laforet, P. , 101 Laganière, J. , 166 Laghi, L. , 121 Lah, J.J. , 75 Lai, Y.K. , 148 LaJeunesse, J. , 118 Lajonchere, C. , 41 Lake, S.L. , 101 Lako, M. , 162, 164 Lalande, M. , 38 Laman, J.D. , 71 LaMarca, M.E. , 75, 77–80, 98 Lamb, K. , 148 Lamba, S. , 123 Lambert, D. , 26 Land, S.J. , 37 Landazabal, C. , 75

Lane, M.R. , 119 Lane, R. , 96 Lang, A.E. , 75 Lange, D.J. , 101 Langer, C. , 123 Langlois, S. , 133 Langmead, B. , 35 Lanspa, S.J. , 119 Lappin, T.R.J. , 162 Lapunzina, P. , 42 Larsen, A.L. , 118 Larson, D.E. , 40 Larson, P.J. , 149 Laskowski, A. , 64 Latham, T.E. , 80 Lattanzi, R. , 24 Lau, L. , 41 Laughlin, C.A. , 140 Laurell, T. , 27 Laurent-Puig, P. , 122 Laurie, J.A. , 122 Lausch, E. , 42 Law, M. , 35 Lawn, R.M. , 133 Le, S. , 96 Leduc, M.S. , 37, 41 Lee, C.C. , 135 Lee, D. , 135 Lee, G. , 169 Lee, J.C. , 75 Lee, K. , 165, 169 Lee, M.K. , 38 Lee, N.-C. , 101 Lee, S. , 163 Lee, S.C. , 75 Lee-Chen, G.J. , 75 Lefever, S. , 55 Leggett, B.A. , 119 Lehmann, S. , 26 Lehrach, H. , 23 Leibel, R.L. , 165, 168 Leigh, I.M. , 49, 54 Leike, K. , 136, 139 Leikin, S. , 42 Lenarsky, C. , 94 Lench, N.J. , 49, 50, 54 Lensch, M.W. , 163, 168 Leonardi, E. , 50 Leroy, K. , 119 Lesage, S. , 75 Lescoe, M.K. , 119 Leshner, R.T. , 101 Leslie, N. , 101

Author Index

189

Leszl, J. , 167 Leufkens, H.G. , 136 Levey, A.I. , 75 Levi, M.M. , 135 Levy, A. , 165, 169 Levy, B. , 96 Levy, D.B. , 114, 115 Levy, H. , 77 Levy, J.R. , 148, 149 Lewis, S. , 74 Ley, T.J. , 40 Li, C. , 142 Li, H. , 35, 36, 39, 55 Li, J. , 138, 140, 142, 143, 149 Li, J.S. , 101, 102 Li, L.B. , 167 Li, M. , 37, 39 Li, S. , 35 Li, X. , 144 Li, Y. , 35, 38, 41, 55 Liang, J. , 41 Liang, J.N. , 49, 54 Libby, R.T. , 135 Libersa, C. , 26 Licata, L. , 39 Lieber, D.S. , 64 Lifton, R.P. , 76, 81 Ligtenberg, M.J. , 41 Lim, B. , 162 Lim, S.C. , 64 Lima, J.L. , 75 Lin, A. , 143 Lin, D. , 35 Lin, M.-T. , 101 Lin, X. , 55 Lina-Granade, G. , 50 Linch, D.C. , 135, 149 Lindberg, H. , 27 Lindblom, A. , 119 Lindor, N.M. , 119, 121 Lindquist, S. , 166 Ling, G.S.F. , 100 Link, B. , 97 Lipford, J. , 119 Lipke, M.L. , 94, 97 Lipkin, M.L. , 118 Lipton, L. , 116 Liskay, R.M. , 119 Liu, B. , 120 Liu, J. , 76, 78, 81, 143, 144 Liu, L. , 35, 167 Liu, S. , 64 Liu, X.-Z. , 50, 54, 55

Liu, Y. , 79 Liu, Y.H. , 54 Liu, Y.L. , 136 Livingston, A.L. , 116 Lizard, G. , 26 Lo, S.M. , 76, 81 Lo, W.H. , 148 Lochmüller, H. , 25 Locke, D.P. , 40 Lockhart-Mummery, P. , 114 Lockman, J. , 118 Lockman, L.A. , 94 Loh, K.M. , 162 London, W.B. , 170 Long, Q. , 40 Loo, J.-C.A. , 101 Loonen, M.C.B. , 101 Lopes, R.D. , 136 Lopez, G. , 75, 78 Löppönen, H. , 50 Löppönen, T. , 50 Loring, J.F. , 170 Lorson, C.L. , 168, 169 Losi, L. , 119 Loulus, S. , 38 Lowe, A.M. , 97 Lowry, W.E. , 164 Lu, F. , 143 Lu, L. , 35 Lu, N. , 55 Lu, X. , 37 Lu, Z. , 55 Lubelski, J. , 131–150 Lucas, D. , 50 Lucci-Cordisco, E. , 119 Lucibello, F.C. , 114, 115 Lucotte, G. , 54 Ludwig, T. , 165 Luft, R. , 62 Lugtenberg, D. , 41 Lukina, E.A. , 99 Lund, G. , 50 Luo, J. , 41 Lupski, J.R. , 37, 41 Lusby, E. , 138 Lusis, A.J. , 133 Lutsenko, S. , 136 Luz, J. , 116 Lwin, A. , 79 Lynch, H.T. , 118, 119 Lynch, J.F. , 118 Lynd, K.S. , 143 Lyubarsky, A. , 149

Author Index

190

M Ma, J.X. , 149 Maas, M.M. , 73, 74, 99, 135 Macek, M. , 24 MacFarlane, A.E. , 29 Maciel-Guerra, A.T. , 50 MacKenzie, J.J. , 75, 79 Mackenzie, T.C. , 136 Mackey, J. , 100 Macrae, F.A. , 119 Madison, J.M. , 170 Maeda, M. , 163 Maeda, Y. , 136 Maehr, R. , 165 Maetzler, W. , 75 Maffei, S. , 119 Magnuson, C.W. , 118 Maguire, A.M. , 149 Maguire, J.R. , 36, 39 Maguire, M.G. , 149 Mah, C. , 101 Mahdieh, N. , 35, 55 Maherali, N. , 163, 168 Maiani, G. , 26 Maillet, P. , 116 Maimets, T. , 159–170 Maire, I. , 93, 94 Majamaa, K. , 51 Makareeva, E. , 42 Malech, H.L. , 166 Malesci, A. , 121 Mali, P. , 163, 167 Malinova, V. , 74 Malm, G. , 97 Mandel, H. , 101, 102 Mandlebaum, F.S. , 71 Mane, S. , 76, 81 Maniwang, E. , 77 Mankin, H.J. , 71, 73 Mann, D.M. , 75 Manno, C.S. , 149 Manolio, T.A. , 70 Mao, C.C. , 65 Mao, R. , 79 Marazita, M.L. , 50 Marcelis, C.L. , 41 Marchand, L.L. , 119 Marchetto, M.C.N. , 168, 169 Marcos, A. , 26 Marder, K. , 75 Mardis, E.R. , 40 Marini, J. , 42 Markham, A.F. , 49 Markoulaki, S. , 166

Marko-Vaga, G. , 27 Marlin, S. , 50 Marlow, E. , 165, 169 Marsden, D. , 100 Marsh, J. , 143 Marshall, C.R. , 41 Marshall, J. , 82 Marshall, K.A. , 149 Marshall, V.S. , 162 Marsoni, S. , 123 Marth, G.T. , 36, 37, 39 Marti, F. , 26 Martin, B.M. , 72, 77, 79, 80 Martin, E. , 118 Martin, G.R. , 162 Martin, R.A. , 97 Martin, S.D. , 124 Martín Uranga, A. , 24 Martín, Y. , 50 Martín-Arribas, M.C. , 24 Martínez-Arias, R. , 78 Martini, A. , 50 Martins, A.M. , 74, 96, 99, 100 Marucha, J. , 94 Marugan, J. , 82 Maruyama, M. , 163 Marzouk, O. , 123 Masella, B.D. , 135 Masellis, M. , 75 Masood, M.A. , 80, 168 Masson, C. , 26 Mata, I.F. , 75 Mate, I. , 79 Mateu, E. , 78 Mathieu, M. , 93, 94 Matrkam, A. , 114, 115 Matsubara, Y. , 50 Matsumoto, T. , 136 Matsumoto, Y. , 55 Matsushita, T. , 140 Mattaliano, R.J. , 101 Mattar, C.N. , 136 Mattis, V.B. , 168, 169 Mattox, D. , 55 Mattson, P. , 24 Maw, M.A. , 51 Maxam, A.M. , 138 Mayfi eld, T.L. , 144 Mayhew, J.E. , 101 Maynard, J. , 116 Mayo, F. , 51 Mazzoli, M. , 49 McCabe, E.R. , 70, 75, 79 McCandless, S.E. , 97, 101

Author Index

191

McCarty, D.M. , 138, 140, 142 McClellan, J. , 71 McClelland, A. , 149 McCowan, M. , 36, 39 McDonnell, J.W. , 149 McEachern, K.A. , 82 McEntee, M. , 96 McGill, J.J. , 94, 97 McGrath, S.D. , 40 McIntosh, J. , 135, 136, 149 McKechnie, D. , 114, 115 McKeith, I.G. , 75 McKenna, A. , 36, 39 McKerlie, C. , 167 McKusick, V.A. , 80 McLaughlin, S.K. , 140 McLellan, M.D. , 40 McMordie, S.J. , 51 McNamara, S.C. , 149 McPhillips, M. , 119 McVean, G.A. , 37, 39 Mecklin, J.P. , 116, 118 Meghrous, J. , 149 Meguid, R.A. , 121 Mehal, W.Z. , 78 Mehl, A.L. , 48 Mehta, A. , 71, 76 Mehta, L. , 101 Mei, J. , 41 Meijer, I. , 24 Meikle, P.J. , 76, 101 Meisler, M.H. , 41 Meissner, A. , 166 Meitinger, T. , 25 Melçhionda, S. , 49 Meldgaard Lund, A. , 97 Melegh, B. , 25 Melnikova, I. , 33, 34 Melton, D.A. , 165 Mena, J.A. , 149 Mencía, A. , 51 Mendelsohn, N.J. , 102 Mendez, A.F. , 148, 149 Menendez, I. , 55 Menendez, S. , 164 Meng, X. , 166 Mengel, E. , 74, 99, 101 Menon, J. , 169 Mensenkamp, A.R. , 41 Merchant, S.N. , 51 Mercier, G. , 54 Merico, D. , 41 Merigan, W.H. , 135 Merino, J.L. , 79

Merkel, P.A. , 136 Merten, O.W. , 140 Messinger, Y.H. , 102 Messner, A.H. , 149 Methot, J. , 132, 135 Metspalu, A. , 49 Meulenberg, J.J. , 135 Meyer, N.C. , 51 Meyronet, D. , 26 Migita, M. , 136 Mihara, K. , 80 Miki, Y. , 114, 115 Mikol, Y.B. , 118 Mikosch, P. , 74 Millan, J.L. , 136 Miller, A.D. , 132 Miller, D. , 80, 168 Millington, D.S. , 100 Milne, K. , 124 Milsom, M.D. , 170 Milz, E. , 38 Mingozzi, F. , 132, 135, 149 Miranda, M.C.S. , 75, 97 Mirelman, A. , 75 Mistry, A. , 144 Mistry, P.K. , 72–74, 76, 78, 80, 81 Mitalipova, M. , 169 Miteva, Ts. , 14 Miteva-Katrandzhieva, T. , 13 Mitsui, J. , 75 Mitsui, K. , 163 Mitsumoto, H. , 168 Miyake, K. , 136 Miyoshi, Y. , 114, 115 Mizuta, I. , 75 Moaven, N. , 69–85 Moavero, S.M. , 79 Modamio-Hoybjor, S. , 51 Moeller, M.P. , 48 Moertel, C.G. , 122 Molas Gallart, J. , 24 Möller, C.G. , 50 Molnar, M.J. , 61–66 Molter, D.W. , 97 Monciotti, A. , 145 Mondain, M. , 50 Monges, G. , 123 Montes, J. , 149 Montgomery, K. , 124 Montorsi, M. , 121 Montserrat, B. , 51 Moon, S.-K. , 50 Moore, D.F. , 100 Moore, H. , 26

Author Index

192

Moore, M.J. , 122, 123 Moore-Barton, H.L. , 55 Mootha, V.K. , 62, 64 Moral, L. , 51 Morales, E. , 51 Morales-Angulo, C. , 51 Moran, M.L. , 149 Moreau, L.A. , 170 Morell, R.J. , 49 Moreno, F. , 50, 51 Moreno-Pelayo, M.A. , 50, 51 Morera, L.B. , 164 Morgan, C. , 101 Morgan, R.A. , 132 Morín, M. , 51 Morris, C.M. , 75 Morris, E. , 74 Morris, J.H. , 114 Morris, P. , 94 Morrison, K.M. , 133 Morrison, P.T. , 119 Morse, D. , 135 Mort, M. , 37, 40 Mortelmans, L. , 134 Morton, C.C. , 47 Morton, C.L. , 135, 149 Moss, R.B. , 149 Mota, R.M.V. , 99 Motabar, O. , 82 Moullier, P. , 140, 143 Mrsić, M. , 74 Mu, Y. , 168, 169 Mueller, H. , 116 Mueller, L. , 27 Mueller, R.F. , 49, 50, 54 Mueller-Malesinska, M. , 50 Muenzer, J. , 93, 94, 96, 97 Mullen, C.A. , 132 Muller, J. , 39 Müller, L.U.W. , 170 Müller-Felber, W. , 101 Mundlos, S. , 42 Muñoz Rojas, M.V. , 97 Muotri, A.R. , 168, 169 Murakami, M. , 163 Murday, V.A. , 114, 115 Murgia, A. , 50 Murphy, A.W. , 29 Murphy, M.J. , 162 Murray, G.J. , 71 Murray, K. , 138 Muul, L. , 132 Muzny, D.M. , 37, 41, 120 Muzyczka, N. , 137, 138, 140, 144, 147

Myer, B.J. , 76 Myers, R.H. , 166

N Nachman, J.B. , 94 Nagan, N. , 77, 78 Nagaraju, K. , 101 Nagashima, K. , 101 Nakagawa, M. , 164 Nakamura, Y. , 114, 115 Nakaoka, H. , 35 Nakatsuji, N. , 163 Nalls, M.A. , 75 Nalpathamkalam, T. , 41 Nance, W.E. , 47, 50, 54 Nardozza, A.P. , 39 Narisawa, K. , 50 Narisawa, S. , 136 Narita, M. , 163 Nash, K. , 138, 147 Nathke, I. , 115 Nathwani, A.C. , 135, 136, 149 Navarro, C. , 132 Naz, S. , 50 Nebel, S. , 122 Needham, P. , 138 Neganova, I. , 164 Negrete, A. , 142, 148, 149 Nekahm-Heis, D. , 50 Nelen, M.R. , 41 Nelson, B.H. , 124 Nelson, D.L. , 167 Netzer, C. , 38, 42 Neufeld, E.F. , 93, 94, 96 Neveling, K. , 41 Newcomb, P.A. , 119 Newman, M.S. , 97 Newton, V. , 49, 50 Newton-Cheh, C. , 71 Ng, C.Y. , 135, 149 Ng, S.B. , 35, 37 Nguyen, T. , 37, 136 Niakan, K.K. , 168 Niamke, J. , 144 Nicely, H. , 94 Nichols, J. , 163 Nichols, W.C. , 75 Nickerson, D.A. , 35, 37 Nicklin, S.A. , 136 Nicolaides, N.C. , 120 Nicoletti, G. , 75 Nicolino, M. , 100, 101 Nie, J. , 163

Author Index

193

Nielsen, T.O. , 124 Nienhuis, A.W. , 135, 149 Nierman, M.C. , 135 Nilbert, M.C. , 114, 115 Ning, Z. , 40 Nishimura, C.J. , 50, 51 Nishisho, I. , 114, 115 Nistor, G. , 162 Niu, Z. , 37, 41 Nolta, J.A. , 162 Nony, P. , 143 Nooijen, A. , 24 Noone, D. , 135 Norato, D.Y.J. , 96 Nordenskjold, M. , 119 Northover, J.M. , 119 Novellino, F. , 75 Nowakowska-Szyrwinska, E. , 50 Nowrouzi, A. , 136 Nozaki, K. , 101 Nürnberg, G. , 38 Nürnberg, P. , 38 Nusbaum, P. , 132 Nuyten, D.S. , 124

O O’Beirne, J. , 135, 149 O’Brien, J.E. , 41 O’Brien, J.F. , 79 O’Callaghan, C.J. , 123 O’Callaghan, M.W. , 101 O’Connell, M.J. , 122 Odaka, Y.S. , 55 Oddoux, C. , 49 Odenthal, M. , 136 Odkvist, L.M. , 50 Ogden, B. , 136 Ogilvie, J.W. , 97 O’Grady, G. , 100 Ohanian, K. , 94, 96 Ohashi, T. , 75, 80 Ohnuki, M. , 163 Oitmaa, E. , 49 Okita, K. , 164 Oktay, M.H. , 23 Okuyama, T. , 97 Olavarrieta, L. , 51 Oliveira, C. , 75 Olson, L.E. , 167 Olumi, A.F. , 124 O’Mahony, B. , 135 O’Malley, J. , 51 Onay, M.P. , 40

Onder, T.T. , 168, 169 Oonk, A.M.M. , 55 Oostrik, J. , 55 Opitz, J.M. , 39 Orchard, P.J. , 94 Ordóñez, J. , 47–55 O’Reilly, T.M. , 26 Oreve, S. , 140 Orii, T. , 97 Orr-Urtreger, A. , 75, 79 Ortiz Mellet, C. , 82 Ortolano, S. , 132 Orvisky, E. , 72, 78–80 Orzan, E. , 50 Osborn, T. , 165, 169 Oshima, T. , 50 Ostrer, H. , 49 Ota, M. , 55 Ottinger, C.J. , 101 Ottman, R. , 75 Ottone, C. , 26 Ouyang, X.M. , 54 Owada, M. , 79 Owens, R.A. , 138, 144 Ozdag, H. , 50, 54 Özgüç, M. , 23 Ozkinay, F. , 38, 40

P Packman, S. , 76, 94 Pais, R. , 97 Palau, F. , 23 Pallares-Ruiz, N. , 50 Palma, A. , 39 Palmieri, G. , 62 Palombo, F. , 145 Pampinella, F. , 25 Pan, J. , 166 Pandya, A. , 50, 55 Panescu, J. , 118 Panicker, L.M. , 80, 168 Pankratz, N. , 75 Papadopoulos, N. , 120 Papapetrou, E.P. , 169 Papayannopoulou, T. , 167 Parenti, G. , 101 Parini, R. , 97 Park, D.J. , 101 Park, H.-J. , 50 Park, I.-H. , 170 Park. I.-H. , 163, 168 Park, J.K. , 75, 78–80 Park, T.S. , 80, 168

Author Index

194

Parker, A. , 79 Parker, R.I. , 71 Parmar, K. , 170 Parry, G. , 49, 54 Parry, S. , 119 Parsons, M.T. , 120 Parving, A. , 49, 50 Pashankar, F. , 76, 81 Pasi, J. , 135, 149 Pasmanik-Chor, M. , 79 Passage, M. , 96 Passini, M.A. , 101 Pastores, G.M. , 74, 81, 96 Patel, B. , 80, 168 Patel, R. , 124 Patterson, M.C. , 100 Patton, M.A. , 55 Pattyn, F. , 55 Paulus, K. , 136 Pazdur, R. , 122 Pedroni, M. , 119 Peebles, D. , 136 Peinovich, M. , 96 Peleg, O. , 74 Peltomäki, P. , 118 Peluso, D. , 39 Pembrey, M. , 50 Pennings, R.J.E. , 51, 55 Pentelenyi, K. , 61–66 Peoc’h, K. , 26 Perabo, L. , 132 Peranteau, W. , 136 Percy, D.H. , 167 Pereira, L.V. , 75 Perfetto, L. , 39 Perrier, M. , 149 Perrin, C. , 26 Perros, M. , 142 Person, R. , 37, 41 Peshkin, L. , 38 Pestronk, A. , 101 Petakov, M. , 99 Peters, C. , 94 Peters, T.A. , 55 Petersen, G.M. , 120, 121 Petersen, M.B. , 49, 50, 55 Petit, C. , 50 Petrelli, N.J. , 122 Petry, H. , 131–150 Pfeiffer, R. , 27 Pfi ster, M. , 26, 50 Pham, P. , 37, 41 Phan, L. , 37, 39 Philippakis, A.A. , 36, 39

Phillips, A. , 37, 40 Phillips, J. , 96 Phillips, M. , 76 Pianovski, M.A.D. , 99 Pickering-Brown, S. , 75 Pie, A.J. , 135, 149 Pierce, E.A. , 149 Piessevaux, H. , 123 Pietrzik, K. , 26 Pike, L.S. , 135 Pinard, J.-M. , 99 Piper, D. , 97 Piraud, M. , 93, 94 Pires, R.F. , 99 Pisano, M. , 49 Pittenger, M. , 162 Pivnick, E.K. , 101 Platts, A. , 37 Plecko, B. , 97 Plon, S.E. , 37, 41 Ploski, R. , 50 Plotz, P.H. , 101 Ploughman, L.M. , 50 Pocovi, M. , 78 Podsakoff, G. , 140 Pohl, C.S. , 40 Pohl, E. , 38, 40 Policepatil, S.M. , 26 Polishchuk, E. , 101 Poll, L.W. , 73 Pollak, A. , 50 Polumuri, S.K. , 80, 168 Pong, R. , 35 Ponnazhagan, S. , 138 Ponti, G. , 119 Ponz de Leon, M. , 119 Pop, M. , 35 Popat, S. , 121 Popa-Wagner, R. , 139 Porta, G. , 99 Porteous, M. , 116 Porto, C. , 101 Porwal, M. , 139 Powell, S. , 143 Powers, K. , 134 Prasad, V.K. , 94 Praz, F. , 123 Preisinger, A.C. , 114, 115 Prendergast, J. , 116 Price, T.J. , 123 Prihodova, L. , 135 Priluck, I. , 50 Prior, T. , 118 Pritchard-Jones, K. , 29

Author Index

195

Proia, R.L. , 79 Provost, N. , 143 Pruszak, J. , 165 Puga, A.C. , 97 Pugh, E.N. Jr. , 149 Pylvänäinen, K. , 118

Q Qian, D. , 55 Qiao, C. , 143 Qing, K. , 138 Qiu, K. , 101 Qu, G. , 143 Quadt-Humme, S. , 132 Quattrone, A. , 75 Quillard, M. , 26 Quinn, B. , 79

R Rabbani, B. , 35 Rabe, K.G. , 121 Raben, N. , 101 Rabinowitz, J.E. , 142 Rabionet, R. , 49, 51 Rachner, T.D. , 42 Radnaabazar, J. , 50 Radu, A. , 136 Ragni, M.V. , 149 Rahalkar, A.R. , 133 Rai, A.J. , 26 Raimbaud, I. , 143 Ralston, E. , 101 Ramaswami, U. , 97 Ramensky, V.E. , 38 Ramesh, A. , 49 Ramsey, B.W. , 149 Ramsey, W.J. , 132 Randolph, A. , 121 Rangachari, L. , 101 Rangarajan, S. , 135, 149 Rao, M.R. , 119 Rao, S. , 79 Rapin, I. , 75 Rapoport, D.M. , 96, 100 Rawlings, B. , 50 Raya, A. , 164 Reardon, W. , 50, 52 Rebar, E.J. , 166 Recchia, A. , 145 Rechitsky, S. , 163 Redmond, T.M. , 149 Redston, M. , 122

Reed, J.D. , 79 Reeves, R.H. , 167 Rehm, H.L. , 50 Reid, J.G. , 37, 41 Reidbord, H. , 72 Reier, P.J. , 101 Reiners, K. , 101 Reis, S.A. , 170 Reiss, U.M. , 135, 149 Remington, E. , 50 Renard, J-P. , 55 Reperant, M. , 119 Restifo, L.L. , 41 Reuser, A.J. , 77, 100, 101 Reuss, M. , 139 Reynolds, J.F. Jr , 39 Reynolds, T.C. , 149 Rezeli, M. , 27 Rhys, C.M. , 144 Rial-Sebbag, E. , 24 Riazuddin, S. , 50 Ribic, C.M. , 122, 123 Richards, S. , 101 Richtsmeier, J.T. , 167 Riddell, A. , 135, 149 Riddell, S.R. , 135 Rideout, W.M. III , 163 Rider, S.H. , 114, 115 Ried, M. , 139 Ries, M. , 99 Ring, R.H. , 41 Rinne, T. , 41 Rittner, K. , 140, 142 Rivas, M.A. , 36, 39 Rivat, C. , 132 Riviere, M. , 26 Roa, B. , 79 Roberson, J. , 50 Roberts, A. , 101 Robertson, M. , 163 Robinson, P.H. , 101 Robitaille, S. , 123 Roche, S. , 26 Rödelsperger, C. , 38 Rodenburg, R.J. , 41 Rodolfa, K.T. , 168 Rodriguez, J.L. , 135 Rodríguez-Ballesteros, M. , 50 Rodwell, C. , 16 Roeb, W. , 38 Rogaeva, E. , 75 Rogers, R.C. , 101 Rohrbach, M. , 38 Rojas, V.M. , 96

Author Index

196

Rolfs, A. , 75 Rolling, F. , 142 Romeo, G. , 80 Romero, E. , 51 Rommelaere, J. , 142 Roncalli, M. , 121 Roncari, B. , 119 Roncucci, L. , 119 Rong, Z. , 164 Rosales, C. , 135, 149 Rosati, R. , 121 Rose, F.F. Jr. , 168, 169 Rose, J.A. , 140, 143 Rosen, C.A. , 120 Rosenbaum, H. , 75, 78, 99 Rosenberg, A.S. , 101, 102 Rosenberg, S.A. , 132 Rosenbloom, B. , 101 Rosengren, S.S. , 49 Rosenthal, D.I. , 73 Ross, C. , 135 Rossi, A. , 97 Rossi, B. , 101 Rossi, S. , 149 Roth, A. , 39 Roux, A.-F. , 50 Roybal, J.L. , 136 Rozans, M.K. , 94 Rozdzynska, A. , 94 Rozenberg, R. , 75 Ruan, J. , 36 Rubboli, G. , 75 Ruben, S.M. , 120 Rubin, B.P. , 124 Rubin, J. , 122 Ruden, D.M. , 37 Rudensky, B. , 73 Ruffi ng, M. , 144 Rugnetta, M. , 23 Ruiz-Perez, V.L. , 42 Rujescu, D. , 75 Ruotti, V. , 163 Rushton, N. , 76 Russell, A.M. , 116 Russell, D.W. ,

143, 167 Russo, M.T. , 116 Rustagi, P. , 135, 149 Ryan, J.H. , 138

S Sabatino, D.E. , 136 Sacco, F. , 39

Sadelain, M. , 169 Safer, B. , 138 Saftig, P. , 75 Sahinalp, S.C. , 40 Sakoori, K. , 55 Salegio, E.A. , 135 Salvetti, A. , 140, 143 Salvioli, R. , 79 Salzberg, S.L. , 35 Samaddar, T. , 75 Samaranch, L. , 135 Samii, A. , 75 Sampson, J.R. , 116 Samulski, R.J. , 132, 134, 137, 138, 140, 142, 149 Samulski, T. , 140 Sandberg, A.A. , 118 Sandberg, S. , 136 Sander, C. , 120 Sanders, J. , 94 Sandhoff, K. , 79 Sandor, J. , 15, 16 Saneto, R.P. , 62 Sanmiguel, J.C. , 143 Santamarina-Fojo, S. , 133 Santilli, G. , 132 Santonico, E. , 39 Santoro, A. , 121 Saphier, G. , 168 Saranjam, H. , 77 Sargent, D.J. , 122, 123 Saroha, V. , 81 Sartorato, E.L. , 50 Sartore-Bianchi, A. , 123 Saunders, E.F. , 94 Scallan, C. , 149 Scambler, P. , 114, 115 Scarpa, M. , 97 Scarpa, S. , 79 Scarselli, A. , 119 Schaart, G. , 101 Schaefer, A.M. , 62 Schaefer, R.M. , 100 Schaffer, D.V. , 132, 134, 135 Schambach, A. , 170 Scharlaken, B. , 55 Scheetz, T.E. , 51, 54 Scheffer, H. , 41 Scherer, S.W. , 41, 54 Schieppati, A. , 34, 136 Schiffmann, R. , 72, 77, 78, 80, 98–100 Schinke, T. , 42 Schlaeger, T. , 170 Schmidt, M. , 136 Schmidt, S. , 38

Author Index

197

Schmitt, E. , 79 Schneiderat, P. , 25 Schoenau, E. , 38 Schofi eld, J. , 123 Schönau, E. , 42 Schoneveld, O. , 101 Schoser, B. , 101 Schotz, M.C. , 133 Schraders, M. , 51, 55 Schrauwen, I. , 51 Schreml, J. , 38 Schrijver, I. , 50 Schuelke, G.S. , 118 Schuelke, M. , 38 Schuetz, T.J. , 97 Schulte, C. , 75 Schultz, N. , 120 Schulz, M.H. , 40 Schwake, M. , 75 Schwartz, I.V.D. , 97 Schwartz, P.H. , 94, 96 Schwarz, J.M. , 38 Scott, C.R. , 77, 78, 98 Scott, D.A. , 49 Scott, R.J. , 119 Scotti, M.M. , 144 Scozzri, R. , 51 Scriver, C.R. , 79 Scull, J. , 37, 41 Sebring, E.D. , 140, 143 Seco, C.Z. , 55 Sedensky, M.M. , 62 Sedey, A.L. , 48 Seelow, D. , 38 Seeman, P. , 50 Segawa, K. , 163 Seitz, J.F. , 123 Selz, F. , 132 Semionato Filho, J. , 99 Semler, O. , 38, 42 Sena-Esteves, M. , 135 Senapathy, P. , 137 Sender, L. , 94 Seruca, R. , 119 Sevior, K.B. , 51 Shahin, H. , 38, 50, 55 Shapiro, E.G. , 94, 96 Shapiro, J.D. , 123 Shapiro, S.S. , 162 Sharma, M. , 75 Sharp, K. , 162 Sharp, R.R. , 149 Shaukat, S. , 50 Shaw, K. , 37, 40

Shaw, M.W. , 118 Shayman, J.A. , 82 Shearer, A.E. , 50, 51, 54, 55 Shearer, G. , 132 Sheer, D. , 114, 115 Sheerin, U.M. , 75 Sheffi eld, V.C. , 49 Shendure, J. , 35, 37 Shenk, T. , 140 Shenkman, B. , 74 Shepherd, L.E. , 122, 123 Sheridan, S.D. , 170 Sherry, S.T. , 37, 39 Sheth, S.A. , 62 Shi, X. , 40 Shi, Y. , 41 Shih, A. , 41 Shihabuddin, L.S. , 101 Shimada, K. , 136 Shimada, T. , 136, 143 Shimamura, A. , 163, 168 Shimizu, F. , 169 Shinawi, M. , 97 Shinbrot, E. , 120 Shindler, K.S. , 149 Shinkawa, H. , 50 Shklyaev, S. , 138, 147 Shohat, M. , 50, 51 Shoubridge, E. , 62, 63, 65 Sibbles, B. , 101 Sidman, M. , 96 Sidman, R.L. , 101 Sidransky, E. , 69–85, 98, 168 Siegel, C.S. , 82 Siemering, K.R. , 50 Siena, S. , 123 Sier-Ferreira, V. , 132, 135 Sigman, C.C. , 135 Simes, J. , 123 Simes, R.J. , 123 Simon, J. , 97 Simonelli, F. , 149 Simonovic, M. , 39 Singh, M. , 81 Singh, T. , 81 Singleton, A.B. , 75 Sinicrope, F. , 123 Sirmaci, A. , 50, 54, 55 Sirotkin, K. , 37, 39 Sivachenko, A.Y. , 36, 39 Skarsgard, E. , 149 Skold, A. , 143 Skrinar, A. , 101 Skromne, I. , 55

Author Index

198

Sleep, S. , 135, 149 Slidell, M.B. , 121 Sloan, C.M. , 51 Slonim, A. , 100 Slukvin, I.I. , 163 Smeitink, J.A. , 41, 101 Smigielski, E.M. , 37, 39 Smith, A. , 163 Smith, A.D. , 138 Smith, A.G. , 161, 162 Smith, F.I. , 80 Smith, J.B. , 135 Smith, K. , 135, 149 Smith, K.J. , 114, 115 Smith, L.G. , 119 Smith, R.H. , 138, 148 Smith, R.J.H. , 49–51, 54, 55 Smith, S.A. , 101, 102 Smitka, M. , 101 Smuga-Otto, K. , 163 Smyrk, T.C. , 119 Snaedal, J. , 75 Snitow, M. , 165 Snoeckx, R.L. , 50 Snow, K. , 79 Snowden, J. , 75 Snyder, R.O. , 138, 143, 147 Sobreira, E. , 74 Sobrino, J.A. , 79 Sokolov, A. , 15 Soldner, F. , 165, 166, 169, 170 Solomon, E. , 114, 115 Solow, R. , 143 Sotamaa, K. , 118 Soto, C. , 29 Southall. N. , 82 Spear, I. , 138 Spegelaere, P. , 142 Spence, W.C. , 79 Spence, Y. , 135, 149 Spencer, C.T. , 100 Spigelman, A. , 119 Spinazzola, A. , 62 Spinneker, A. , 26 Spinner, N.B. , 143 Spitz, M. , 75 Spranger, M. , 101 Spruijt, L. , 41 Spuch, C. , 132 Spurdle, A.B. , 120 Spurr, N.K. , 114, 115 Sreekantan-Nair, A. , 55 Srisailapathy, C.R. , 49 Srivastava, A. , 138

Srivastava, D. , 135, 149 S-Siest, V. , 26 Stark, M. , 39 Steehouwer, M. , 38 Stefanov, R. , 1–20 Stefansson, H. , 75 Stefansson, K. , 75 Stehle, P. , 26 Stein, P.B. , 81 Steinberg, S. , 75 Steiner, M. , 42 Steiner, R.D. , 97, 100 Stenson, P.D. , 37, 40 Stepanian, S.V. , 75 Sterritt, G.M. , 48 Stevens, H.P. , 49, 54 Steward, O. , 162 Stewart, I.A. , 51 Stewart, R. , 163 Stewart, S.M. , 119 Stoffel-Wagner, B. , 26 Stojkovic, M. , 162 Stone, D. , 135 Stone, D.L. , 75, 79 Stone, E. , 149 Stone, E.M. , 50 Storb, R. , 135 Stork, C. , 26 Strait, K. , 170 Strelchenko, N. , 163 Strigl-Pill, N. , 101 Stroes, E.S. , 135 Strothotte, S. , 101 Stubblefi eld, B.K. , 72, 75, 77–81, 168 Studer, L. , 169 Stumm, M.M. , 26 Su, J. , 136 Su, L.K. , 114, 115 Subramanian, S. , 124 Sugano, H. , 136 Summers, K.M. , 79 Sun, C.-W. , 166 Sun, J. , 149 Sun, L. , 78 Sunyaev, S.R. , 38 Suomalainen, A. , 116 Surace, E.M. , 149 Suraweera, N. , 119 Suzuki, J. , 164 Suzuki, K. , 79 Suzuki, Y. , 97 Svendsen, C.N. , 168, 169 Svensson, K.J. , 27 Sweeney, C.L. , 166

Author Index

199

Swiedler, S.J. , 94, 96, 97 Swiergiel, J.J. , 162 Szatmari, P. , 41 Sziklai, I. , 50

T Tabar, V. , 169 Tabatabaiefar, M.A. , 55 Tabone, E. , 26 Tabor, H.K. , 35, 37 Tack, D. , 51 Tada, M. , 163 Tada, T. , 163 Tager, J.M. , 77 Tai, S.J. , 149 Tajima, A. , 75, 80 Takahama, Y. , 163 Takahashi, K. , 163, 164 Takasaka, T. , 50 Taksir, T. , 101 Talbot, I.C. , 119 Talseth-Palmer, B.A. , 119 Talwar, D. , 41 Tamargo, R.J. , 80 Tamayose, K. , 143 Tambuyzer, E. , 34 Tan, E.K. , 75 Tan, M. , 137, 138, 140 Tanabe, K. , 163 Tanaka, A. , 97 Tanaka, T. , 97 Tang, W. , 55 Tannengard, P. , 119 Tannous, B.A. , 135 Tao, L. , 143 Tapscott, S.J. , 135 Tarallo, A. , 101 Tarantal, A.F. , 135 Tarrant, C. , 29 Taruscio, D. , 1–20 Tatò, L. , 101 Tatti, M. , 79 Tayebi, N. , 69–85, 168 Taylor, G. , 50 Taylor, K.R. , 51, 54 Taylor, R.W. , 62 Taymans, J.M. , 134 Tebbutt, N.C. , 123 Tegelaers, F.P. , 77 Tejpar, S. , 123 Tekeli, O. , 55 Tekin, M. , 47–55 Teles, E.L. , 97

Telford, E. , 50 Telischi, F.F. , 54 Temtamy, S. , 42 Tenesa, A. , 116, 117 Terk, M.R. , 73 Tessier, J. , 143 Testa, F. , 149 Theophilus, B.D. , 80 Theriault, K.M. , 170 Theuns, J. , 75 Thibodeau, S.N. , 119, 121–123 Thiruvahindrapuram, B. , 41 Thiry, I. , 134 Thomas, C.E. , 132 Thomas, D.L. , 144 Thomas, J. , 96 Thompson, A. , 41 Thompson, B. , 120 Thomson, J.A. , 162, 163, 168, 169 Thorburn, D.R. , 64 Thornton, A.M. , 38 Thorsteinsdottir, U. , 75 Thrasher, A. , 144 Thrasher, A.J. , 132, 144 Thurberg, B.L. , 101 Tian, S. , 163 Tibbetts, K. , 36, 39 Tifft, C.J. , 100 Tijssen, P. , 139 Tiller, G.E. , 96 Timmons, M. , 99 Tlsty, T.D. , 124 Toda, T. , 75 Todd, J.A. , 79 Todd, N.W. , 55 Todorow, C.A. , 136 Tokgoz-Yilmaz, S. , 55 Tokuzawa, Y. , 163 Tolstoshev, P. , 132 Tomishima, M.J. , 169 Tomkins, D.J. , 167 Tomlinson, I. , 116 Tomoda, K. , 163 Tompkins, B. , 51 Tonelli, M.R. , 149 Topic, E. , 136 Toriello, H.V. , 52 Torri, V. , 121, 123 Torroni, A. , 51 Toth, T. , 50 Totoiu, M. , 162 Toublanc, E. , 143 Townes, T.M. , 166 Trama, A. , 13, 14

Author Index

200

Tran, P.T. , 143 Tranebjaerg, L. , 51 Trapnell, C. , 35 Tratschin, J.D. , 140 Traxler, E.A. , 135 Trembath, R. , 50 Tremblay, G. , 132, 135 Tremblay, K. , 132, 135 Trempe, J.P. , 138, 142–144 Tricarico, R. , 119 Trigg, M.E. , 94 Troiano, A.R. , 75 Trojanowski, J.Q. , 75 Trusheim, M.R. , 135 Trzupek, K. , 50 Tsai, A.C.-H. , 101 Tsuji, S. , 75 Tu, D. , 122, 123 Tucker, E.J. , 64 Tuddenham, E.G. , 135, 149 Tukamoto, K. , 50 Tun, R.Y. , 51 Tung, C.H. , 135 Turbeville, S. , 94 Turkia, H.B. , 99 Turnbull, D.M. , 62 Tweedie, S. , 163 Twisk, J. , 132, 135 Twist, C. , 94 Twitty, C. , 143 Tycko, J. , 135 Tylki-Szymanska, A. , 74, 94, 99, 100

U Ucar, S.K. , 38 Uchino, M. , 79 Uddin, M. , 41 Ulbrich, B. , 97 Ulloa, R.H. , 55 Ulstein, I. , 75 Ungeheuer, M.N. , 26 Urabe, M. , 144, 145, 147 Urbach, A. , 170 Urcelay, E. , 138 Urnov, F.D. , 166 Usami, S.-I. , 50 Uyama, E. , 79

V Vaccaro, A.M. , 79 Valadares, E.R. , 96, 99 Valayannopoulos, V. , 91–103

Valentine, M.B. , 136 Valle, J.W. , 122 van Bon, B.W. , 38 van Broeckhoven, C. , 75 van Camp, G. , 49–51, 55 van Capelle, C.I. , 101 van Corven, E.J. , 101 van Criekinge, W. , 55 van Cutsem, E. , 123 van de, Heyning, P. , 50 van de Kamp, E.H. , 101 van de Rijn, M. , 124 van de Vijver, M. , 124 van de, Voorde, H. , 55 van de Warrenburg, B.P. , 41 van den Bulk, N. , 132, 135 van den Heuvel, B. , 41 van den Hout, J.M.P. , 101 van der Beek, N.A. , 101 van der Ploeg, A.T. , 96, 97, 101 van der Voort, E. , 101 van der Vusse, G.J. , 101 van Deventer, S. , 132, 135 van Diggelen, O.P. , 101 van Dijk, P. , 101 van Doorn, P.A. , 101 van Duijn, C.M. , 75 van Dussen, L. , 99 van Gassen, K.L. , 41 van Hazel, G. , 123 van Heest, A.E. , 94 van Hirtum, H. , 101 van Hove, J.L. , 101 van Lier, B. , 38 van Maldergem, L. , 50 van Meurs, M. , 71 van Nieuwerburgh, F. , 55 van Oers, M.M. , 144 van Schaik, I.N. , 99 van Weely, S. , 136 van Zelst-Stams, W.A. , 41 Vandenberghe, L.H. , 134 Vandesompele, J. , 55 Vaury, C. , 119 Veenstra, T.D. , 80, 168 Veeramah, K.R. , 41 Végvari, A. , 27 Vejvalkova, S. , 15 Velayati, A. , 75, 78, 80 Vellodi, A. , 97, 99 Veltman, J.A. , 38, 41, 42 Ventayol, M. , 51 Venter, J.C. , 120 Vento, J.M. , 62, 64

Author Index

201

Veres, G. , 144 Verghese, J. , 75 Verlinsky, O. , 163 Verlinsky, Y. , 163 Vermeer, S. , 41 Viale, A. , 169 Vicinanza, M. , 101 Vielhaber, S. , 101 Vijayaraghavan, S. , 97 Vilageliu, L. , 79 Villamar, M. , 50 Villarreal, L. , 140 Vimal, V. , 143 Virag, T. , 148, 149 Visel, M. , 135 Viskochil, D. , 96 Visser, P. , 101 Vissers, L.E. , 38 Vittozzi, L. , 12 Vitzthum, F. , 26 Vodyanik, M.A. , 163 Vogel, S.N. , 80, 168 Vogelstein, B. , 114, 115, 120 Vogler, C. , 96 Voit, T. , 101 Volpe, N.J. , 149 vom Dahl, S. , 74 von Kalle, C. , 136 von Mering, C. , 39 Voulgaropoulou, F. , 143 Vu, B.J. , 166 Vukov, A.M. , 122 Vulto, A.G. , 101 Vyas, R. , 170

W Waber, L. , 96, 97 Waddington, C.H. , 160 Waddington, S.N. , 136 Wagner, J.A. , 94, 149 Wagner, J.E. , 94 Wagner, U. , 26 Wahl, G.M. , 164 Waknitz, M.A. , 162 Waligora, J. , 50 Walker, J.M. , 78, 79 Walker, M.R. , 26 Walker, R.W. , 94 Walker, S. , 41 Walker, S.L. , 138 Wallis, J.W. , 40 Walot, I. , 96 Walsh, T. , 38

Walton-Bowen, K. , 96 Waltz, D.A. , 149 Wang, G. , 41 Wang, H. , 166 Wang, J. , 41, 133 Wang, J.-K. , 101 Wang, K. , 37, 39 Wang, L. , 37, 144 Wang le, L. , 37 Wang, M. , 37, 41 Wang, P.-R. , 167 Wang, R.Y. , 94, 96 Wang, X.S. , 138 Wang, Y. , 55, 134 Wang, Y.V. , 164 Wang, Z. , 41, 81, 135 Ward, D.C. , 119 Ward, M.H. , 37, 39 Ward, P.A. , 37, 41 Warkentin, P. , 94 Wärnberg, J. , 26 Warren, G. , 119 Warrington, K.H. Jr. , 138, 147 Warthin, A. , 118 Wasserstein, M. , 101 Wästfelt, M. , 33 Watanabe, A. , 136 Waters, P.J. , 79 Waterson, J. , 101 Watson, I.D. , 136 Watson, M.S. , 100 Watson, P.H. , 124 Waxman, S.G. , 41 Weaver, D.W. , 39 Webb, S. , 162 Weegerink, N.J.D. , 51 Weggeman, M. , 101 Wegrzyn, G. , 15, 16 Wehling, C. , 75 Wei, Y.F. , 120 Weibel, T. , 100 Weil, D. , 50 Weimer, M. , 139 Weinberg, R.A. , 112, 114 Weindler, F.W. , 143 Weinreb, N. , 74, 76 Weinreb, N.J. , 72, 73, 76, 80 Weinstein, H. , 94 Weinthal, J.A. , 72, 73, 76, 80 Weisenthal, L.M. , 168 Weisglas-Kuperus, N. , 101 Weitzman, M.D. , 138, 143 Weleber, R.G. , 50 Welinder, C. , 27

Author Index

202

Wendl, M.C. , 40 Wendt, S. , 97 Wernig, M. , 165, 166 Wesemael, M.V. , 51 Wessig, C. , 101 West, C. , 78 West, N.R. , 124 West, R.B. , 124 Westbroek, W. , 75, 80 Westerveld, A. , 77 Westman, J.A. , 118 Westphal, H. , 79 Wheeler, D. , 120 White, V. , 50 Whitehead, A.S. , 75 Whitehead, M. , 124 Whitelaw, S. , 119 Whiteman, D.A.H. , 97 Whitley, C.B. , 97 Whitney, K. , 23 Wichterle, H. , 168 Wieand, H.S. , 122 Wierzbicki, R. , 123 Wieskamp, N. , 38, 41 Wilcox, E.R. , 50 Willems, P.J. , 50 Williams, C.B. , 119 Williams, D.A. , 170 Williams, D.R. , 135 Williams, G.T. , 116 Williams, T.E. , 94 Willis, A. , 37, 41 Wilson, J.M. , 134, 135, 143 Wilson, R.K. , 40 Wiltshire, A. , 116 Win, A.K. , 119 Wine, J.J. , 149 Winfi eld, S.L. , 77 Winkel, L.P.F. , 101 Wion, K.L. , 133 Wirth, B. , 38 Wiszniewski, W. , 50 Witte, D. , 79 Wittes, J. , 97 Wittstock, M. , 75 Wixon, J. , 132, 133, 149 Wobus, C.E. , 139 Wojno, A.P. , 135 Wolf, U. , 79 Wolfe, L.A. , 65–66 Wolfe, R. , 135 Wolfgang, C.L. , 121 Wolfsberg, T.G. , 75 Wollnik, B. , 33–42

Wolmark, N. , 122 Wonderling, R.S. , 138 Wong, C.L.-J. , 63 Wong, K. , 78 Wood, R.E. , 97 Worden, M.A. , 96 Work, L.M. , 136 Workman, H. , 51 Wortmann, S.B. , 41 Wraith, E. , 97 Wraith, J.E. , 94, 96, 97, 101 Wray, J. , 161, 162 Wright, J.F. , 149 Wu, H. , 136 Wu, J. , 138 Wu, J.Y. , 101 Wu, L.-C. , 166 Wu, M.-H. , 101 Wu, X. , 41 Wu, Y.R. , 75 Wuyts, W. , 50 Wysoker, A. , 36, 39

X Xavier, R. , 73 Xia, F. , 37, 41 Xia, X.-J. , 55 Xiao, W. , 138, 142, 143 Xiao, X. , 138, 140, 142, 143, 149 Xie, X. , 62 Xu, L.R. , 54 Xu, Y. , 164 Xu, Y.H. , 79 Xue, Y. , 96

Y Yagasaki, L. , 165 Yairi, Y. , 49 Yamanaka, S. , 163, 164 Yang, H. , 41, 51 Yang, H.W. , 101 Yang, L.C. , 148, 149 Yang, M. , 78 Yang, Q. , 142, 143 Yang, R. , 78, 81 Yang, Y. , 37, 41 Yap, T.L. , 75 Yarborough, M. , 149 Yariz, K.O. , 55 Ye, G.J. , 144 Ye, K. , 40 Yee, J. , 74, 76

Author Index

203

Yendluri, S. , 143 Yeo, G.W. , 168, 169 Yigit, G. , 40, 42 Yin, L. , 135 Yip, D. , 124 Yntema, H.G. , 41 Yorukoglu, D. , 40 Yoshinaga-Itano, C. , 48 Yothers, G. , 122 Young, G.P. , 119 Young, J.I. , 55 Young, J.P. , 119, 120 Young, R. , 162 Young, S. , 101, 102 Youssoufi an, H. , 167 Yow, A. , 165, 169 Yu, D. , 168, 169 Yu, J. , 163, 168, 169 Yu, K.T. , 71 Yu, W.H. , 78 Yu, Z.-F. , 97 Yuan, Z. , 143 Yuen, R.K. , 41 Yuen, T. , 78 Yüzbaşioğlu, A. , 23 Yvon, E. , 132

Z Zaal, K. , 101 Zabel, B. , 42 Zabetian, C.P. , 75 Zachos, C. , 75 Zadori, Z. , 139 Zahrieh, D. , 99 Zaidi, M. , 78 Zalcberg, J.R. , 123 Zambidis, E.T. , 80, 168 Zaniboni, A. , 123 Zehntner, C. , 26

Zelante, L. , 49 Zelenaia, O. , 149 Zeman, J. , 97 Zentgraf, H. , 144 Zeps, N. , 109–126 Zeviani, M. , 51, 62 Zhang, C.K. , 81 Zhang, H.S. , 166 Zhang, J. , 116 Zhang, L. , 166 Zhang, Q. , 40 Zhang, X. , 136, 143 Zhang, Z.-N. , 164 Zhao, H. , 81 Zhao, J.-P. , 165 Zhao, M. , 101 Zhao, T. , 164 Zhao, Y. , 75 Zheng, H. , 122 Zheng, W. , 82 Zhou, F. , 170 Zhou, J. , 135, 149 Zhou, X. , 138 Zhu, S. , 124 Zhu, X. , 149 Zhuang, J. , 136 Ziegler, S.G. , 75 Zimmermann, K. , 38 Zimran, A. , 73, 74, 76, 79, 81,

82, 99 Zivkovic, S.A. , 101 Zolotukhin, I. , 138, 144 Zolotukhin, S. , 138, 144, 147, 148 Zoltick, P.W. , 136, 143 Zou, J. , 166 Zuber, Z. , 94 Züchner, S. , 55 Zwaigenbaum, L. , 41 Zwierzina, H. , 135 Zwinderman, A.H. , 135

Author Index

205M. Özgüç (ed.), Rare Diseases: Integrative PPPM Approach as the Medicine of the Future, Advances in Predictive, Preventive and Personalised Medicine 6,DOI 10.1007/978-94-017-9214-1, © Springer Science+Business Media Dordrecht 2015

A Abnormalities , 40, 73, 96, 99, 117, 167 Accuracy , 28, 51, 70 Adeno-associated viral vectors , 131–150, 167 Alipogene tiparvovec , 131–133, 135, 149, 150 Amyotrophic lateral sclerosis (ALS) , 168 Anemia , 72–74, 98, 166, 167, 169 Ashkenazi Jewish , 78, 98 Assay , 123, 162, 164, 169

B Best practice , 2–4, 29, 102 Biobanks , 23–29, 126 Biomarker , 24, 27, 98, 123, 134–136 Bioresources , 24 Blood , 26, 81, 82, 100, 122, 133, 135, 161 Brain , 48, 62, 78, 82, 96, 99, 101, 119,

135, 165 Breast , 110, 117, 124, 125

C Cancer , 23, 62, 76, 109–126, 133, 135, 162 Cardiomyopathy , 41, 66, 98 Cardiovascular , 72, 133 Cardiovascular disease , 132, 135, 136, 168 Cell therapy , 159–170 Challenge , 3, 13, 18, 19, 25, 26, 37, 55, 72, 80,

94, 125, 134, 135, 145, 149–150 Chemotherapy , 112, 122–123 Collection , 6, 7, 9, 16, 24–29, 122, 135 Colon , 110, 112, 114, 115, 117–119, 121, 122 Colorectal , 110–124 Complication , 81, 102, 133 Concept , 23, 24, 48, 115, 132, 134, 135, 161

Conservation , 27, 39 Cooperation , 2–4, 7–9, 11, 16, 17, 19 Cost , 20, 38, 41, 55, 70, 99, 102, 103, 122,

126, 133, 142 Council’s Recommendation , 2, 10, 12 Counseling/counselling , 6, 48, 50, 53, 55,

77, 81, 94 Criteria , 2, 7, 16, 26

D Data , 6, 7, 9, 16, 17, 24, 26, 28, 36–39, 41, 50,

62, 95–97, 115, 116, 118, 135, 138, 165 Database , 28, 29, 36–39, 70, 72, 77 Deafness , 47–55, 62 Defi nition , 3, 13, 15, 17, 37, 110, 114, 120, 125 Dementia , 63, 75, 78 Dental , 74 Diabetes mellitus , 63, 168 Disease mechanisms , 159–170 Dissemination , 8, 11, 15 Drug , 3, 10–12, 34, 42, 82, 96, 97, 99, 100,

122, 123, 134–136, 162, 167–169

E Education , 3, 7, 10, 14, 16, 55 Embryonic stem cells (ES cell) , 160–164, 166 Emerging technologies , 33–42 Environment/Environmental , 13, 23, 28, 51,

53, 70, 84, 92, 111, 117, 121, 122, 140, 170

Enzymatic/enzyme activity , 72, 81, 82, 92 Enzyme replacement therapy (ERT) , 70, 71,

73, 74, 81, 82, 91–103 Epidemiology , 8, 76

Subject Index

206

Epigenetic , 42, 70, 83, 112, 160, 167, 170 Ethic , 24, 28–29, 102, 136 EU collaboration , 2, 4, 7 EU-Commission , 1, 3, 4, 7, 110, 131 EU policy , 2, 3, 6, 13–15, 19, 20 EUROPLAN , 4–7 Exome sequencing , 37–40, 83

F Fabry disease , 93, 99–100 Familial , 41, 71, 111–126, 166, 168, 169 Filtering strategies , 37 Framework , 3, 4, 6, 14, 18

G Gastrointestinal , 124, 161 Gaucher disease (GD) , 69–85, 93, 98–99,

160, 168 Gene

identifi cation , 33–42 therapy , 66–67, 82, 91, 103, 131–150,

164–167 Genetic heterogeneity , 47, 48, 51, 54, 55, 79 Genetic modifi ers , 42, 70, 71, 80, 81 Genetic testing , 53, 54 Genome sequencing , 41, 83 Genotype-phenotype correlation , 48, 50, 51,

53, 69–85 Glucocerebrosidase , 71, 75–78, 80–83, 93,

98, 168 Glybera ® , 131, 144 Government , 3, 10, 11, 13, 15–17 Guideline , 3, 10, 11, 13, 17, 102

H Healthcare provider , 11 Health/healthcare/health care , 2–4, 6–11,

13–19, 28, 48, 102, 110–112, 125, 132, 133, 135, 136, 150

Health policy , 1, 2, 13, 15 Hearing loss , 40, 47–55, 63 Hereditary deafness , 47–55 Heteroplasmy , 63, 65 Histopathology , 112, 117, 118 Human genetics , 37, 42, 72, 97 Human samples , 24–28 Hunter syndrome , 97 Hurler, Hurler-Scheie and Scheie syndrome ,

95, 96 Hypertension , 73, 74

I Imaging , 53, 65, 100, 135 Impact , 19, 23–29, 36, 42, 71, 75, 80, 136, 138 Implementation , 3, 4, 6, 9–11, 13, 15, 16, 18,

19, 48 Inadequate , 79 Incidence , 39, 55, 76, 94, 95, 100, 110, 116,

118, 120, 122, 132 Induced pluripotency , 159–170 Infl ammation , 74, 76 Infrastructure , 19, 24, 25, 29 Innovation , 4, 9 Insurance , 11, 16, 102 Intergenomial communication , 64–65 Intervention , 9, 27, 29, 48, 53, 55, 65, 94, 97,

112, 121, 122, 165 iPS cells , 160, 163–170

L Laboratory , 6, 49, 54, 65, 78, 122, 163 Lung , 94, 98, 110, 113, 117, 161 Lynch syndrome , 116–119 Lysosomal storage disease (LSD) , 75, 82,

91–103

M Magnetic resonance imaging (MRI) ,

73, 96, 100 Malignancy , 17, 73, 75–76, 81, 117, 125 Mammalian cells , 139–145, 148 Mechanism , 6, 9, 10, 23, 34, 42, 55, 72, 75,

77, 78, 92, 94, 120, 123, 126, 134, 145, 159–170

Mendelian disorder , 34, 35, 37, 69–85 Mental retardation , 39, 92 Metabolic , 66, 93, 168 Metabolism , 62, 92, 135, 167 Migraine , 62, 63 Mitochondrial disease , 61–66 Model , 13, 39, 79, 96, 115, 135, 136, 163–170 Models of human disease , 167–169 Molecular classifi cation , 110, 124 Morbidity , 2, 73, 94, 121 Mortality , 2, 79, 94 MRI. See Magnetic resonance

imaging (MRI) mtDNA disorders , 63 Mucopolysaccharidosis

type I , 93–96, 102 type II , 93–95, 97, 102 type VI , 93–95, 97

Subject Index

207

Mutations , 28, 34, 48, 62, 70, 98, 113, 133, 164

Myoclonic epilepsy , 63, 64, 73–75, 80 Myopathy , 63, 64, 66

N National plans , 1–20 Network , 3, 4, 8–10, 24, 25, 27, 34, 120 Neurodegeneration , 165 Next generation sequencing (NGS) , 35–37, 41,

42, 65, 120 Nutrition , 101

O Organism , 139, 160–163 Organs , 40, 62, 83, 92, 94, 110–113,

119–120, 134

P Panethnic disorder , 73 Paradigm , 134, 150, 161 Parkinson disease , 75, 78, 83, 166, 168 Patient , 3, 23, 34, 50, 62, 70, 92,

110, 131, 163 Patient organisation , 4, 6, 7, 10–12, 14, 16–18 Personalised medicine , 23–29 Personalizing therapy , 124 Perspective , 14, 126, 139–140 Pharmaceutical , 8, 11, 34, 62, 96 Pharmacological , 66, 82, 99, 100 Pharmacotherapy , 65 Phenotypic , 71, 72, 79–80, 117 Philosophy , 132, 147 Policies , 2–4, 6, 11–15, 17–20, 28, 102 Polyposis , 113–126 Pompe disease , 93, 100–102 Population , 3, 9, 36, 38, 39, 49, 54, 62, 70, 73,

78, 81, 84, 95, 98, 110, 114, 117, 118, 120, 122, 136, 170

Prediction , 39, 50, 51, 79, 132 Predisposition , 116 Preparation , 26, 27, 137, 140, 142 Prevalence , 2, 33, 62, 98, 114, 116, 120 Prevention , 2, 6, 7, 10, 16, 17, 121, 125,

126, 131–150 Professionals , 3, 7–10, 19, 27, 52, 53, 102 Program/programme , 6, 8, 9, 12, 14, 17,

24, 36, 38, 41, 48, 53, 55, 122, 125, 134

Prospective , 17, 167

Prostate , 110, 117, 125 Protocol , 8, 27, 35, 37, 132, 139, 140, 143,

144, 167, 169, 170

Q Quality of life/ care , 2, 4, 6–8, 16, 96, 99,

100, 102, 103

R Rare cancer , 109–126 Rare diseases , 1–20, 23–29, 33–42, 102,

109–126, 131–150 personalized treatment , 131–150 prevention , 131–150

Recognition , 3, 6, 8, 83, 149 Recommendation , 2–4, 8, 10–12, 14, 17, 18,

27, 48, 53, 54, 66, 113 Rehabilitation , 2, 6, 9, 10 Reimbursement , 12, 102 Remodeling/remodelling , 73, 99 Repositories , 24 Requirement , 26, 27, 101, 139 Research , 3, 7–12, 16, 19, 20, 24–29, 37, 41,

72, 115, 120, 125, 126, 131, 144, 149, 150, 162, 163, 167, 170

Retinitis pigmentosa , 52, 55, 63 Risk , 36–38, 52–54, 62, 75, 78, 81, 94, 115,

117–119, 121, 122, 142, 143, 170 Risk assessment , 53–55

S Sanfi lippo syndrome , 93, 95, 134 Screening , 3, 4, 6, 7, 9, 10, 12, 16, 48, 54,

55, 79, 81, 92, 93, 117, 120–122, 132, 133, 169

Services , 2, 9, 10, 13, 14, 53, 121 Sirtuin , 66 Sporadic , 39, 75, 115–117, 119–123, 169 Stakeholders , 2, 4, 11, 13, 15–20, 24, 28 Standard/Standardization , 24, 27, 40, 78, 81,

101, 133, 142, 167 Stem cells , 93–95, 161–168, 170 Stomach , 110, 117, 119, 161 Strategy/Strategic , 2–4, 6–16, 18, 19, 34, 35,

37–40, 42, 53, 54, 65, 70, 81, 103, 113, 121, 126, 132, 133, 143, 144, 163

Stroke , 62, 63, 66, 99, 100 Supplementation therapy , 65, 66 Symptom , 34, 41, 62, 63, 66, 72–75, 79, 83,

84, 103, 133

Subject Index

208

T Target , 7, 37, 41, 65, 80, 83,

110, 115, 121, 132, 134, 165

Targeted therapies , 123–124 Technique , 23, 26, 35, 36, 48, 65,

70, 92, 162 The Cancer Genome Atlas (TCGA) ,

120, 121, 125 Tool , 3, 8, 36, 39, 48, 51, 54, 55, 125,

132–135, 168, 169 Treatment , 2, 6–10, 12, 16, 17, 19, 20,

23, 24, 42, 55, 65, 66, 74, 81–82, 94–103, 111–113, 115, 116, 120–126, 131–150, 166, 168–170

U Ultrasound , 53

V Validation , 25, 28, 150 Virus gene therapy , 131–150

W Warthin’s tumor , 118 World Health Organization (WHO) , 7, 110

X X-linked disorders , 34, 99

Subject Index