OBESITY AND PSYCHO-PATHOLOGICAL SYMPTOMS

AND PSYCHOSOCIAL RELATIONSHIP WITH ANTI

PSYCHOTICS AND DISC1 GENE AS GENETIC MARKER

OF SCHIZOPHRENIA SUSCEPTIBILITY

BY

NORLELAWATI A TALIB

A thesis submitted in fulfilment of the requirement for the

degree of Doctor of Philosophy of Pharmaceutical Sciences

(Pharmacology)

Kulliyyah of Pharmacy

International Islamic University Malaysia

JANUARY 2015

ii

ABSTRACT

Schizophrenia is a chronic debilitating mental disorder with a lifetime risk estimated

at about 1%, regardless of ethnicity. The ideal outcome of schizophrenia treatment is

to improve psycho-social function and quality of life thus limiting the debilitating

condition. This can be achieved by reducing the devastating effects of its psycho-

pathological symptoms. Unfortunately, the introduction of atypical anti-psychotic

drugs is linked to obesity, an issue of concern as it is a co-morbid condition that is

closely related to metabolic syndrome. Additionally, there are substantial evidences

that suggest Disrupted-in-Schizophrenia-1 gene (DISC1) function in

neurodevelopment. This supported DISC1 locus as candidate gene of schizophrenia

and a potential target for treatment of schizophrenia. The current research

encompasses three different sub-studies. Firstly, the study investigated the

relationship between psycho symptoms and psycho-social function and anti-

psychotics in the prediction of psycho-social function among schizophrenia patients.

Secondly, the study assessed the correlation of body mass index with antipsychotics.

Subsequently, the data was compared with Malaysian Adult Nutrition Survey

(MANS). Finally, the study assessed the association of DISC1 gene as genetic

susceptibility marker of schizophrenia. A total of 240 schizophrenia patients and 350

control healthy individuals participated in the study. A cross sectional approach was

adopted for the first and second study and unmatched case-control for the third study.

Methods used include interview, psychiatric scales assessment and genotyping for

DISC1 markers. There was an inverse relationship between psycho-symptoms and

psychosocial functions. Disorganization (DIS) was the only significant predictor to all

dimensions of psycho-social functions. Typical anti-psychotics significantly predicted

social function negatively as compared to Sulpiride (b=-0.152, p=0.028).Obesity was

2-fold greater than the reference population. Body mass index was generally related

to antipsychotic drugs (χ2 = 33.42;p = 0.04).Finally, the study found two SNPs,

rs4658971 and rs1538971 within DISC1 gene that predisposed to schizophrenia

andrs2509382located in chromosome 11 that was associated with schizophrenia in

males. The study concluded four important findings. Firstly, obesity was common

among schizophrenia patients and this could be attributed to anti-psychotics.

Secondly, DIS as predictor to all social dimensions emphasized the importance of

cognition in schizophrenia. Thirdly, Sulpiride predicted better social function than

typical antipsychotics. Lastly, the study provided significant evidence of DISC1 gene

as marker of schizophrenia susceptibility.

iii

خلاصة البحث

% 1مرض انفصام الشخصية هو من الامراض النفسية المزمنة والموهنة والتي تشكل خطرا دائميا بالاصابة بالمرض لحوالي

ان الهدف المثالي لعلاج مرض انفصام الشخصية هو تحسين الوظائف النفسية .من الاشخاص مهما يكن نوع اجناسهم

قلل حالة الوهن والضعف وهذا يمكن الحصول عليه وذلك بتقليل التاثيرات الاجتماعية وتحسين نوعية الحياة وذا ت

ان ظهور ادوية الامراض النفسية الغير قياسي فانه مرتبط ومع الاسف بداء السمنة .المدمرة للاعراض النفسية والمرضية

ل توضح اعتلال في هنالك دلائ, بالاضافة الى ذلك.وهذا يشكل مضاعفات اضافية مرتبطة بشدة مع متلازمة الايض

خلال تطور الجهاز العصبي وذلك يدعم موقع الجين المسؤول عن انفصام 1عمل جين انفصام الشخصية رقم

الدراسة تحتوي على : اولا:هذا البحث الجاري يحتوي على ثلاثة محاور مختلفة.الشخصية مما يجعله هدف لعلاج المرض

تماعية النفسية مع ادوية الحالات النفسية مما يساعد على توقع الوظائف العلاقة مابين الامراض النفسية والوظائف الاج

هذه الدراسة او البحث تخمن العلاقة بين مؤشر كتلة الجسم مع : ثانيا.النفسية الاجتماعية بين مرضى انفصام الشخصية

واخيرا فان هذه الدراسة تربط .ادوية الامراض النفسية وبالتالي فان المعلومات قورنت بالمسح الغذائي الماليزي للبالغين

لقد .مع مؤشر القابلية الجينية للاعتلال لمرض انفصام الشخصية 1العلاقة بين اعتلال جين انفصام الشخصية رقم

شخص سليم كمجموعة سيطرة في هذا البحث ولقد تم 350مريض مصاب بانفصام الشخصية مع 240شارك

لاولى والثانية وتم استخدام الحالات المسيطر عليهاغير المطابقة بالنسبة استخدام دراسة مقطعية عرضية للدراستين ا

ودراسة جينية لمؤشرات اعتلال جين , مقياس تقييم نفسي, للدراسة الثالثة وان الطرق المستخدمة احتوت على مقابلة

وان عدم . ماعيةوجدت علاقة عكسية مابين الاعراض النفسية والوظائف النفسية الاجت.1انفصام الشخصية رقم

استخدام الادوية النفسية المثالية .التنظيم كان المؤشر الوحيد ذو اهمية في كل الاتجهات للوظائف النفسية الاجتماعية

السمنة ) 0.152=بي 0.028= ب(اظهرت بشكل واضح وظائف اجتماعية بصورة سلبية بالمقارنة مع السلبرايد

”× ( ن مؤشر كتلة الجسم كان له علاقة عامة بالادوية النفسيةكانت اكثر بضعفين من المصدر البشري وا

ار اس ضمن 1538971ار اس و4658971,اس ان بي اس2اخيرا وجدت الدراسة )0.054=ب 33.42=

ار اس الموجود في 2509382والذي ادى الى مرض انفصام الشخصية و 1جين اعتلال انفصام الشخصية رقم

داء : اولا:الدراسة استنتجت اربعة حقائق مهمة.رتبط بمرض انفصام الشخصية في الرجالالكروموسوم الحادي عشر والم

عدم : ثانيا.السمنة موجود بكثرة بين مرضى انفصام الشخصية وهذا قد ينسب الى استخدام ادوية الامراض النفسية

دواء السلبرايد يعطي : الثاث.التنظيم كمخمن لكل الاتجاهات الاجتماعية يوضح اهمية الادراك في انفصام الشخصية

الدراسة اعطت ادلة مهمة لاعتلال جين انفصام : اخيرا.دلالة احسن للوظائف الاجتماعية عن الادوية النفسية المثالية

.كمؤشر لقابلية الاصابة بانفصام الشخصية 1الشخصية رقم

iv

APPROVAL PAGE

The thesis of Norlelawati A.Talib has been approved by the following:

_____________________________________

Tariq Abdul Razak

Supervisor

______________________________________

Wan Azizi Wan Sulaiman

Co- Supervisor

____________________________________

Abdul Razak Kasmuri

Internal Examiner

_____________________________________

Rusli Ismail

External Examiner

_____________________________________

Zilfalil Alwi

External Examiner

_____________________________________

El Fatih Abdullahi Abdelsalam

Chairman

v

DECLARATION

I hereby declare that this thesis is the result of my own investigation, except where

otherwise stated. I also declare that it has not been previously or concurrently

submitted as a whole for any other degrees at IIUM or other institutions.

Norlelawati A. Talib

Signature…………………. Date …..................

vi

INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA

DECLARATION OF COPYRIGHT AND AFFIRMATION

OF FAIR USE OF UNPUBLISHED RESEARCH

Copyright ©2014 by International Islamic University Malaysia. All rights reserved.

OBESITY AND PSYCHO-PATHOLOGICAL SYMPTOMS AND

PSYCHOSOCIAL RELATIONSHIP WITH ANTI PSYCHOTICS

AND DISC1 GENE AS GENETIC MARKER OF

SCHIZOPHRENIA SUSCEPTIBILITY

No part of this unpublished research may be reproduced, stored in a retrieval system,

or transmitted, in any form or by any means, electronic, mechanical, photocopying,

recording or otherwise without prior written permission of the copyright holder except

as provided below.

1. Any material contained in or derived from this unpublished research may

be used by others in their writing with due acknowledgement.

2. IIUM or its library will have the right to make and transmit copies (print

or electronic) for institutional and academic purposes.

3. The IIUM library will have the right to make, store in a retrieval system

and supply copies of this unpublished research if requested by other

universities and research libraries.

Affirmed by NorlelawatiA.Talib

……..……..…………… …………………..

Signature Date

vii

ACKNOWLEDGEMENT

In the Name of Allah, The Most Gracious, The Most Merciful. Thanks and gratitude

to Almighty Allah for all the blessings upon without which I would not be able to

complete the study.

I am thankful to my supervisor Dato’ Prof. Dr Tariq Abdul Razak for his

support and guidance throughout the study. I am also thankful to my co-supervisor

Asst. Prof Dr Wan Azizi Wan Sulaiman for the advice and support. I might not be

able to complete this study without the tremendous support given by these two very

considerate people.

My greatest appreciation goes to the Kulliyyah of Pharmacy, International

Islamic University Malaysia (IIUM) which provides me the opportunity to pursue my

PhD at their esteem institution. I am indebted to Assoc. Prof DrTaher and his

administrative team for the tremendous support and understanding. I am also thankful

to Prof. Noriah Mohd Nor, Dr Kausar (just to name a few) and all lecturers in

Kulliyyah of Pharmacy for their support and encouragement.

My appreciation also goes to the previous dean of Kulliyyah of Medicine

(KOM), Prof Dr Fauzi Abdul Rani and Prof. Pakeer Syed Ahamed, the head of Basic

Medical Sciences Department, KOM for the support and encouragement throughout

my study. Thanks to all colleagues in BMS department (Prof Naznin, Dr Zamzila, Dr

Norsidah, Dr Hairul Aini, Dr Zuna…..just to name a few), for their support and

encouragement. Special thanks to Asst. Prof. Dr Norsidah Kuzaifah, the co-researcher

of the study. Her criticism, advice and support are very much appreciated. I am also

indebted to the other co-researchers, Asst. Prof Dr Kartini Abdullah and Assoc. Prof.

Dr Ramli Musa from the Department of Psychiatry, KOM for their guidance and help.

My deepest gratitude to IIUM Endowment fund, Exploratory Research Grant

Scheme (ERGS) and IIUM Research Management Centre for the financial support.

My deepest gratitude also goes to staff of Molecular Research Laboratory; Sr. Zaza,

Sr. Syafini, Sr. Rina and Sr Nadia and Research Assistants; Br Isa, Br Amirul and Sr

Zafirah for their technical help and support. Not to forget, the Head and staff of the

Department of Psychiatry, Hospital Tengku Ampuan Afzan, Kuantan for the support

and help during patients recruitment process.

To my beloved husband, Dr Mohd Aziz bin Husni and my four children

Aisyah, Adham, Asma and Aliyah, thank you for the support and understanding. I

love you all. To my late parents, Allahyarham Talib Bin Hj Mansor and

Allahyarhamah Khalimah Binti Hj Taib, for emphasizing the importance of Iman and

Amal. Glory is only to Allah, in the beginning and the end.

viii

TABLE OF CONTENTS

Abstract .......................................................................................................................... ii

Abstract in Arabic ......................................................................................................... iii

Approval Page ............................................................................................................... iv

Declaration ..................................................................................................................... v

Copyright Page .............................................................................................................. vi

Acknowledgement ....................................................................................................... vii

List of Tables ............................................................................................................... xii

List of Figures ............................................................................................................. xiv

List of Abbreviations ................................................................................................... xv

CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW ........................ 1

1.1 Introduction................................................................................................... 1

1.2 Schizophrenia ............................................................................................... 4

1.2.1 Epidemiology and Burden of Disease ................................................ 4

1.2.2 Etiology of Schizophrenia .................................................................. 5

1.2.2.1 Pathophysiology of Schizophrenia. ......................................... 6

1.2.2.2 Genetic Theory of Schizophrenia ............................................ 9

1.2.3 Manifestation of Schizophrenia ....................................................... 11

1.2.3.1 Psycho-pathological Symptoms in Schizophrenia. ............... 11

1.2.3.2 Psychosocial Effect of Schizophrenia ................................... 12

1.2.3.3 Criteria for Diagnosis of Schizophrenia ................................ 13

1.2.4 Antipsychotic Treatment of Schizophrenia ...................................... 15

1.2.4.1 Mechanism of Action ............................................................ 16

1.2.4.2 Adverse Effects of Anti-psychotics. ...................................... 19

1.3 Psycho-Symptoms and Psycho-Social Function Relationship with

Antipsychotics .................................................................................................. 20

1.3.1 Approach to Antipsychotic Assessment .......................................... 20

1.3.2 Psychosocial Function as Outcome Criteria .................................... 21

1.4 Obesity in Schizophrenia ............................................................................ 22

1.4.1 Body Mass Index ............................................................................. 23

1.4.2 Antipsychotics and Obesity.............................................................. 23

1.4.2.1 Neurotransmitter Roles in Weight Gain ................................ 25

1.5 The Genetic of Schizophrenia .................................................................... 26

1.5.1 Evidence of Genetic Predisposition in Schizophrenia ..................... 26

1.5.2 Genetic Polymorphism as the Etiology of Schizophrenia................ 26

1.5.2.1 Single Nucleotide Polymorphism. ......................................... 27

1.5.2.2 Importance of SNPs ............................................................... 28

1.5.3 Identifying Schizophrenia Susceptibility Gene ................................ 29

1.5.3.1 Genome Wide Scans Studies for Schizophrenia. .................. 30

1.5.4 Candidate Gene of Schizophrenia: DISC1 gene .............................. 35

1.5.4.1 DISC1 Gene ........................................................................... 35

1.5.4.2 The Discovery of DISC Gene ................................................ 35

1.5.4.3 DISC1 as Candidate Gene of Schizophrenia ......................... 36

1.5.4.4 Assumed Function of DISC1 ................................................. 40

1.6 Aims and Significance of The Research ..................................................... 41

ix

1.6.1 Aims of the Research ....................................................................... 41

1.6.2 Significance of the Research ............................................................ 41

1.6.2.1 Psycho-Symptoms and Psycho-Social Function Relationship

with Antipsychotics ........................................................................... 41

1.6.2.2 Obesity in Schizophrenia ....................................................... 42

1.6.2.3 DISC-1 Gene as Genetic Markers of Schizophrenia ............. 42

1.7 Objectives of The Research ........................................................................ 44

1.7.1 Specific Objectives........................................................................... 44

1.7.1.1 Psycho-symptoms and Psychosocial Function Relationship

with Antipsychotics ........................................................................... 44

1.7.1.2 Assessment of BMI in Schizophrenia ................................... 44

1.7.1.3 DISC-1 Gene as Genetic Marker of Schizophrenia .............. 44

1.8 Research Hypothesis ................................................................................... 45

1.9 Study Flow .................................................................................................. 45

1.9.1 Ethics Approval ................................................................................ 45

1.9.2 Study sequence ................................................................................. 45

CHAPTER 2: PHYSCHO-SYMPTOMS AND PSYCHO-SOCIAL FUNCTION

RELATIONSHIP ....................................................................................................... 48

2.1 Introduction................................................................................................. 48

2.2 Objectives ................................................................................................... 49

2.3 Methods ...................................................................................................... 50

2.3.1 Study Design and Subjects ............................................................... 50

2.3.2 Inclusion and Exclusion Criteria ...................................................... 50

2.3.3 Collection of Socio-Demography and Medical Data ....................... 51

2.3.4 Assessment of Psycho-Symptoms and Psycho-Social Function ...... 52

2.3.4.1 Positive and Negative Syndrome Scale ................................. 53

2.3.4.2 Personal and Social Performance Scale ................................ 55

2.3.5 Statistical analyses ........................................................................... 58

2.4 Results ........................................................................................................ 59

2.4.1 Socio Demographic and General Clinical-Psychosocial

Characteristic............................................................................................. 59

2.4.2 Psycho- symptoms and Psycho-social Functions Relationship ....... 62

2.4.3 Social Demographic and Psycho-Social Function Relationship ...... 63

2.4.4 Association of Antipsychotic and Psycho-Social Function ............. 63

2.4.5 Prediction of Psycho-Social Function .............................................. 64

2.4.6 Anti-psychotics prediction on psycho-social function ..................... 66

2.5 Discussion ................................................................................................... 69

2.5.1 Psycho-symptoms and Psycho-Social Function Relationship. ........ 69

2.5.2 Socio Demographic Status and Psycho-Social Functions

Relationship .............................................................................................. 70

2.5.3 Prediction of Psycho-Social Function .............................................. 71

2.5.4 Anti-psychotics Prediction on Psycho-Social Function ................... 72

2.6 Conclusion .................................................................................................. 73

CHAPTER 3: OBESITY IN SCHIZOPHRENIA .................................................. 74

3.1 Introduction................................................................................................. 74

3.2 Objectives ................................................................................................... 75

3.3 Methods ...................................................................................................... 76

x

3.3.1 Study Design and Subjects ............................................................... 76

3.3.2 Demographic and Clinical Data Collections .................................... 76

3.3.3 Body Mass Index ............................................................................. 76

3.3.4 Control Subjects to Represent Malaysian Healthy Adults ............... 77

3.3.5 Statistical Analysis ........................................................................... 78

3.4 Results ........................................................................................................ 79

3.4.1 General and BMI Characteristics of the Study Participants............. 79

3.4.2 Association of Body Mass Index Status with the Study Variables .. 81

3.4.3 Association of Body Mass Index with the Status of Employment and

Education................................................................................................... 81

3.4.4 Prevalence and Comparison of Body Mass Index Status Between

Schizophrenia Patients and the Malaysian Adult Nutrition Survey

(MANS) Data ............................................................................................ 82

3.4.5 Association between Body Mass Index Status and Anti-Psychotic

Treatment .................................................................................................. 85

3.5 Discussion ................................................................................................... 87

3.5.1 Characteristic of Body Mass Index among the Study Participants. . 87

3.5.2 Relationship of BMI with the Status of Employment and Education

................................................................................................................... 88

3.5.3 Prevalence of Obesity and Overweight in the Study Participants ... 88

3.5.4 Comparison of BMI Status between Schizophrenia Patients and the

Malaysian Adult Nutrition Survey (MANS) Data .................................... 90

3.5.5 Relationship between BMI Distribution and Anti-psychotic ........... 91

3.6 Conclusion .................................................................................................. 92

CHAPTER 4: DISC-1 GENE AS GENETIC MARKERS OF SCHIZOPHRENIA

SUSCEPTIBILITY .................................................................................................... 93

4.1 Introduction................................................................................................. 93

4.2 Objectives ................................................................................................... 95

4.3 Methods ...................................................................................................... 95

4.3.1 Study Subjects .................................................................................. 95

4.3.2 Extraction of Human Genomic DNA from White Blood Cells ....... 96

4.3.3 Selection of SNPs for Mapping of DISC1 Gene Markers. .............. 97

4.3.4 Marker Assay Design and Genotyping ............................................ 97

4.3.5 Designing Assay Primer ................................................................... 98

4.3.6 PCR ................................................................................................ 100

4.3.6.1 Restriction of PCR Amplified DNA to Distinguish between

SNP Alleles ..................................................................................... 101

4.3.6.2 Agarose Gel Electrophoresis. .............................................. 101

4.3.7 Statistical Analysis ......................................................................... 102

4.4 Results ...................................................................................................... 103

4.4.1 Markers Selected for Genotyping .................................................. 103

4.4.2 Markers Assay Design ................................................................... 105

4.4.3 PCR Optimization .......................................................................... 107

4.4.4 Determining the Polymorphic Markers and Genotyping Assay. ... 109

4.4.5 Case-control Populations ............................................................... 112

4.4.6 Association of DISC-1 Markers with Schizophrenia ..................... 112

4.4.7 Association of DISC-1 markers with Schizophrenia based on Gender

................................................................................................................. 116

xi

4.4.8 Association of DISC-1 Markers with Schizophrenia based on the

Racial Differences ................................................................................... 119

4.5 Discussion ................................................................................................. 121

4.6 Conclusion ................................................................................................ 125

CHAPTER 5: SUMMARY AND FINAL CONCLUSION .................................. 126

5.1 Summary of The Study ............................................................................. 126

5.2 Conclusion of The Study .......................................................................... 128

5.2.1 With Regards to the Relationship between Psycho-symptoms and

Psycho-social Functions .......................................................................... 128

5.2.2 With Regards to Prediction of Psychosocial Functions ................. 129

5.2.3 With Regards to BMI Distribution and Obesity in Schizophrenia 129

5.2.4 With Regards to Genetic Markers of DISC1 in Schizophrenia ..... 129

5.3 Study Limitations and Recommendations ................................................ 130

BIBLIOGRAPHY .................................................................................................... 132

APPENDIX 1: LIST OF WORK PRESENTED ....................................................... 152

APPENDIX 2: LIST OF PUBLICATIONS .............................................................. 155

APPENDIX 3: ETHICAL APPROVAL ................................................................... 157

APPENDIX 4: STUDY INFORMATION SHEET (MALAY) ................................. 158

APPENDIX 5: STUDY INFORMATION SHEET (ENGLISH) .............................. 160

APPENDIX 6: CONSENT FORM (MALAY) .......................................................... 162

APPENDIX 7: CONSENT FORM (ENGLISH) ....................................................... 163

APPENDIX 8: PSYCHIARIST ASSESSMENT FORM .......................................... 164

APPENDIX 9: PATIENT DEMOGRAPHY FORM ............................................... 167

APPENDIX 10: PANSS RATING FORM ................................................................ 170

APPENDIX 11: PSP SCALE FORM ........................................................................ 171

APPENDIX 12: DATA OF BMI IN MALAYSIAN ADULT NUTRITION

SURVERY ................................................................................................................. 172

APPENDIX 13: PREVALENCE OF OBESITY IN MALAYSIAN ADULT

NUTRITION SURVERY .......................................................................................... 173

xii

LIST OF TABLES

Table No. Page No.

1.1 Three diagnostics criteria of DSM-IV-TR 15

1.2 Antipsychotics and mechanism of action 18

1.3 Details of some of the genome wide scan case-control

association studies in recent years

34

1.4 Case control study of DISC1 gene susceptibility in

schizophrenia

39

2.1 Components of the psycho-pathological symptoms in

Positive and Negative Syndrome Scale (PANSS)

54

2.2 Scoring model for the PANSS five-factor model 55

2.3 The PSP domains and scoring guides 57

2.4 Socio-demographic and general clinical-psychosocial

data of the subjects

61

2.5 Correlation of PSP total score and its dimensions with

five sub-domains of PANSS and the specific G6 score

63

2.6 PANSS five factors domains prediction on psycho-social

functions as evaluated by PSP sub-dimensions

65

2.7 Social demographic data prediction on psycho-social

functions as evaluated by PSP sub-dimensions

66

2.8 Anti-psychotic prediction on psycho-social functions as

evaluated by PSP total score

68

3.1 Classification of BMI 77

3.2 Demographic data of the study population 80

3.3 Prevalence and comparison of body mass index status

between schizophrenia patients and the Malaysian Adult

Nutrition Survey (MANS) data

84

3.4 Comparison of schizophrenia patients and Malaysian

Adult Nutrition Survey (MANS) data stratified by obesity

severity

85

xiii

Table No. Page No.

3.5 Association between antipsychotic treatment and body

mass index status

86

4.1 Recipe of PCR conditions 100

4.2 RFLP- PCR thermal cycling conditions 101

4.3 Oligonucleotide primers for genotyping of the selected

SNPs

106

4.4 Information of the selected SNPs and the genotyping

assays

108

4.5 Gender and racial distribution of the case-control cohort 112

4.6 Genotype and allele frequencies of four SNPs in the

DISC1 Gene in schizophrenia patients and controls that

do not conform to Hardy-Weinberg equilibrium

114

4.7 Genotype and allele frequencies of nine SNPs in the

DISC1 Gene in schizophrenia patients and controls that

conform to Hardy-Weinberg equilibrium

115

4.8 Association of DISC1 gene with schizophrenia in males 117

4.9 Association of DISC1 gene with schizophrenia in females 118

4.10 Alleles and genotypes frequency of the DISC-1 markers

and their association with schizophrenia among the

Malay

120

xiv

LIST OF FIGURES

Figure No. Page No.

1.1 Comparison of a Selected Set of Relatively Well-Established

Risk Factors for Schizophrenia

6

1.2 Hypotheses of schizophrenia 7

1.3 Schizophrenia in an evolutionary frameworks 10

1.4 Historical approaches of anti-psychotic outcome criteria 21

1.5 Genome wide meta-analysis result 32

1.6 The DISC1 (Chr1: 231,762,561-232,177,018) from

Ensemble Genome Browser on Human(GRCh37/hg19)

Assembly

36

1.7 Connection between psycho-symptom, psycho-social

function, obesity, DISC1 and anti-psychotics.

43

1.8 The major steps of the research flow 47

2.1 Scoring guides of PSP 58

2.2 Correlation between PSP total score and PANSS total score,

Positive, Negative, Disorganization, Excitement and

Emotion sub-domains

62

3.1 Distribution of body mass index status according to

employment status and education level

82

4.1

Designing primers and determination of restriction enzyme

in 2 SNPS

99

4.2 DISC1 haplotypes view 104

4.3 PCR optimization results 107

4.4 PCR Optimization results 109

4.5 PCR Optimization results 110

4.6 PCR-RFLP successful results

111

xv

LIST OF ABBREVIATIONS

µL Microlitre

µM Micromolar

AKTI V-akt Murine Thymoma Viral Oncogene Homolog-1

BMI Body mass index

CI Confidence interval

CNV Copy number variants

COMT Catechol-O-Methyltransferase

DAOA D-amino-acid oxidase activator

DIS Disorganization

DISC1 Disrupted in schizophrenia 1

DNA Double stranded Nucleic Acid

DTNBPI Dysbindin

EDTA Ethylenediaminetetraacetic Acid

EMO Emotion

EXC Excitement

fRFLP Forced restriction fragment polymorphism

GAF Global assessment of functioning scale.

GWAS Genome Wide Association Scan

HTAA Hospital TengkuAmpuanAfzan, Kuantan.

HWE Hardy Weinberg Equilibrium

ICD International classification of disease

LD Linkage disequilibrium

MAF Minor allele frequency

MANS Malaysian Adult Nutrition Survey

MgCl2 Magnesium chloride

mL Mililitre

mM Milimolar

NEG Negative

NRG1 Neuregulin 1

OR Odds ratio

PANSS Positive and Negative syndrome scale

PCR Polymerase chain reaction

POS Positive

PPP3CC Protein phosphatase-3 Catalytic Subunit Calcineurin

Isoform

PSP Psychosocial and performance scale

RFLP Restriction fragments length polymorphism

RFLP Restriction fragment polymorphism

RGS1 Regulator of G-protein Signaling 4

SNP Single nucleotide polymorphism

SOFAS

Social and occupational functioning assessment scale

TBE Tris Boric EDTA

WHO World Health Organisation

µL Microlitre

xvi

µM Micromolar

AKTI V-akt Murine Thymoma Viral Oncogene Homolog-1

BMI Body mass index

CI Confidence interval

1

CHAPTER ONE

INTRODUCTION AND LITERATURE REVIEW

1.1 INTRODUCTION

Schizophrenia is a psychotic disorder, the causes of which are still largely unknown.

It involves a complex set of disturbances of thinking, perception, affect and social

behaviour. No society or culture is free from schizophrenia (Jablensky, 2000). This

illness is a serious public health problem (Colton & Manderscheid, 2006; Jablensky,

2000; Jones, Macias, Barreira, Fisher, Hargreaves, & Harding, 2004). There is no

specific cause of Schizophrenia (National Institute of Mental Health, United States,

2009). The disease is considered a complex disease with an estimated heritability at

up to 80% (McGuffin, Owen, & Gottesman, 2002). Evidence suggesting the role of

genetics in schizophrenia is mainly derived from twins and adoption studies.

Schizophrenia traits among monozygotic twins who theoretically share 100%

sequence similarity was around 40-75% (Folstein, 1996; Franzek & Beckmann, 1998).

The incidence was much lower at around 4%-15% among dizygotic twins who

theoretically share 50% sequence similarity (Cardno, Marshall, Coid, Macdonald,

Ribchester, Davies, Venturi, Jones, Lewis, Sham, Gottesman, Farmer, McGuffin,

Reveley, & Murray, 1999; Onstad, Skre, Torgersen, & Kringlen, 1991).

It is postulated that many genes contribute to Schizophrenia susceptibility. It

has been hypothesized that the combination of susceptible genes and environmental

stressor lead to disruption of several hypothetical pathways implicated in

Schizophrenia (Lang, Puls, Muller, Strutz-Seebohm, & Gallinat, 2007). On the basis

of strong familial predisposition to schizophrenia, searching for genetic entities that

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contribute to the susceptibility to schizophrenia is sensible. Efforts have been made by

many studies to identify the candidate genes for schizophrenia. As any other complex

diseases, genetic variability are believed to be responsible to individual susceptibility

to the disease. Among the many candidate genes studied, theDisrupted-in-

Schizophrenia-1 gene (DISC1) locus has remained a gene that has shown encouraging

positive association (Hennah, Tuulio-Henriksson, Paunio, Ekelund, Varilo, Partonen,

Cannon, Lonnqvist, & Peltonen, 2005; Qu, Tang, Yue, Ruan, Lu, Liu, Zhang, Han,

Zhang, Wang, & Zhang, 2007).

However, many studies reported conflicting associations between DISC1 gene

and schizophrenia. This warrants further work that is it applies to specific populations.

Review on the functional studies of DISC1 revealed diverse role of DISC1in

neurodevelopment, cytoskeletal function and cAMP signaling (Chubb, Bradshaw,

Soares, Porteous, & Millar, 2008). Several genes that interact with DISC1 have also

been defined as independent genetic susceptibility factors for psychiatric illness

(Chubb et al., 2008). The sites for targeting therapy aiming towards DISC-1 related

pathology werealso recently discussed (Hikida, Gamo, & Sawa, 2012). Therefore, the

DISC1 gene markers for schizophrenia susceptibility relevant to local population

should be identified and clarified. This might be helpful in future, in making stand or

opinion on therapy targeting to this gene and its products.

With regards to the management of schizophrenia, the progress of

pharmacotherapy in the last 50 years has been well documented. The assessment of

the effectiveness of antipsychotic has changed in parallel to the introduction of newer

anti-psychotics. With the introduction of antipsychotic in the late1950’s, the

management aim of schizophrenia has significantly changed. At that time, the aim was

rather simple which was to control aggression, self-harm and to enable better personal

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hygiene. Further inventions of newer antipsychotic drugs, refinement of the drugs

delivery system and optimization of therapy have altered the treatment goals to more

specific objectives such as a reduction of certain psycho-pathological symptoms and

side effects control of the drugs. In chronic debilitating diseases such as

schizophrenia, the ideal outcome of treatment is to improve psycho-social function

and quality of life of the patients. This can be done by reducing the devastating effects

of psycho-pathological symptoms. The earlier treatment that emphasized on treating

the negative symptoms for improvement of quality of life in schizophrenia is not

completely fruitful. It became clear that additional symptoms of schizophrenia such as

depressive and cognitive deficits significantly influence the psycho-social function

and quality of life of schizophrenia patients (Lysaker & Davis, 2004). Recovery from

schizophrenia is considered successful once the patient is accepted by the community,

and is socially and financially independent. Understanding the basic relationship

between psycho-pathological symptoms and psychosocial performance as well as the

effect of anti-psychotics on psycho-social performance, could bring tremendous

advantage in the management of schizophrenia.

Among the most discussed side effects of the second generation anti-

psychotics are obesity and development of metabolic syndrome. The link between

antipsychotics, schizophrenia and obesity is not something that is entirely new or

suddenly appeared after the introduction of second generation anti-psychotics. Earlier

reports before 1960 had suggested that chlorpromazine and reserpine were strongly

associated with obesity. Subsequently, the second-generation antipsychotic drugs have

also been associated with the likelihood of gaining weight (Allison, Mentore, Heo,

Chandler, Cappelleri, Infante, & Weiden, 1999; Goudie, Cooper, & Halford, 2005).

Conflicting to this notion however, it was noted that although themean body mass

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index (BMI) was significantly higher among schizophrenia patients than non-

schizophrenia participants between the year 1987 and 1996, the incremental trend of

the BMI index was not specific to the atypical antipsychotic drugs introduced at that

time (Homel, Casey, & Allison, 2002). Investigations on the prevalence of obesity

among our own schizophrenia patients may give a clearer picture on the severity of

the condition (obesity) within the local context. Furthermore, the relationship

between obesity and the antipsychotics used can be determined. The findings may

help in the current and future management of schizophrenia.

1.2 SCHIZOPHRENIA

1.2.1 Epidemiology and Burden of Disease

The prevalence of schizophrenia varies in different populations. It is reportedly as

high as 7 in 1000 population in the United States to as low as 1.4 in 1000 population

in some parts of Indonesia (Jablensky, 2000). The lifetime risk of having this

disabling chronic mental disorder is estimated at about 1%, regardless of ethnicity.

Schizophrenia often begins in early adulthood, affecting men more than women. The

relative risk for males compared to females is 1.42 (95% CI: 1.31-1.51) (Aleman,

Kahn, & Selten, 2003). In Malaysia, the National Mental Health registry has 7351

notified cases of Schizophrenia between the year 2003 and 2005 in which 3714 were

new cases. Among the 7351 cases notified, two third were within the reproductive

age (20– 40 years old), 70% of them were unemployed and males constituted 62% of

the cases(Aziz, Salina, Abdul Kadir, Badiah, Cheah, Nor Hayati, Ruzanna, Sharifah

Suziah, & Chee, 2008).

Globally, schizophrenia with other neuropsychiatric illnesses accounted to

almost one third of the cases of Years Lost due to Disability in adults aged 15 years

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and above [World Health Organisation (WHO), 2004]. The overall health care cost for

Schizophrenia in the United States in 2002 was estimated to be USD 62.7 billion (Wu,

Birnbaum, Shi, Ball, Kessler, Moulis, & Aggarwal, 2005).In Malaysia, there is no

specific data on the health care cost for Schizophrenia. However, we assume by

combining the health care cost with the loss of productivity due to unemployment, the

cost for Schizophrenia in Malaysia could also reach billions of ringgit.

1.2.2 Etiology of Schizophrenia

Epidemiological research from the last century has identified a set of risk factors for

schizophrenia. The risks are categorized as prenatal and antenatal risk factors (R.

Murray, Jones, Suser, Os, & Cannon, 2003). Having a first-degree family history of

schizophrenia is associated with an odds ratio of almost ten (Sullivan, 2005). The

substantial support for family history in schizophrenia susceptibility suggests that

familial determinants can provide clues on the etiology of schizophrenia. The familial

determinants could be related to life-style and/orenvironmental exposure and/or the

heritable genetic susceptibility. Other risk factor and odd ratios to develop

schizophrenia are as illustrated in Figure 1.1.

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Figure 1.1: Comparison of a Selected Set of Relatively Well-Established Risk Factors

for Schizophrenia (abbreviations: CNS: central nervous system; depr: depression; Rh:

Rhesus). A ten-fold likeliness for schizophrenia in persons with first degree family

history of schizophrenia*.Source: The Genetics of Schizophrenia (Sullivan, 2005).

1.2.2.1 Pathophysiology of Schizophrenia.

As any other complex diseases, schizophrenia is thought to arise due to multiple

predisposing factors, both genetic and non genetic. Evidence presented by two

breakthrough papers published more than 20 and 10 years ago respectively, on the

important of combined effects of genetic and non-genetic factors for schizophrenia

have remained solid until the present day (Kiberstis & Roberts, 2002; Lander &

Schork, 1994).

There are several theories on the pathophysiology of schizophrenia. The

theories involve a numbers of risk factors that present early in life. Among the risk

factors include maternal malnutrition, maternal infection and delivery complications

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(D. A. Lewis & Levitt, 2002).Interestingly, both advanced paternal age and young

paternal age at conception are associated with increased risk of schizophrenia(Miller,

Messias, Miettunen, Alaraisanen, Jarvelin, Koponen, Rasanen, Isohanni, &

Kirkpatrick, 2011). To date there is no single theory of schizophrenia that prevails

over the rest. Lakhan and Viera (2009) in their extensive review of pathophysiology of

schizophrenia had listed more than fifteen pathophysiological models of

schizophrenia. Some of the models include the disruption of neurotransmitter

pathways of dopamine and glutamine, the famous neurodevelopment hypotheses,

cognitive deficiency, genetic inheritance and abnormalities in several molecular

pathways such as DISC-1 (Lang et al., 2007)(Figure 1.2).

Figure 1.2: Hypothesis of schizophrenia based on molecular mechanism of

schizophrenia. Figure constructed based on a review article by Lang et al., (2007)

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The early hypothesis on the disruption of dopamine neurotransmitter pathway

in schizophrenia is based primarily on pharmacologic evidence. This hypothesis has

dominated theories on schizophrenia for more than 50 years. In the late 1960s, Van

Rossum hypothesized that hyperactive dopamine transmission is the cause for the

disorder (P. Seeman, 1987). As most of the clinically effective anti-psychotic drugs,

either typical anti-psychotics or atypical anti-psychotics, block the dopamine D2

receptors, the hypothesis seems most plausible and established. Additionally,

supporting evidence also came from a number of observational studies that found

psychotic manifestations in participants treated with an amphetamine. Amphetamine

is a dopamine agonist. These manifestation is relieved upon treatment with dopamine-

D2 receptors blocking agents (Meltzer & Stahl, 1976).

The neurodevelopment hypotheses of schizophrenia proposed that brain

abnormalities arise early in life and remain dormant until the pruning of neural

connections (R. M. Murray & Lewis, 1987). There was also suggestion that linked the

neurodevelopment hypothesis with a dysfunction at the glutaminergic

neurotransmitter pathway. It is also reported that shifts of the NMDA receptor subunit

result in the transition from juvenile to “adult” neural processing in many brain

regions (Dumas, 2005; Henson, Roberts, Perez-Otano, & Philpot, 2010).

Unraveling the pathogenesis of schizophrenia is critical for efforts at the

prevention of schizophrenia. Additionally, deciphering the pathophysiology of

schizophrenia is essential for the identification of novel targets for future therapeutic

intervention (David A. Lewis, 2002).Both pathogenesis and pathophysiology

knowledge of schizophrenia are important to understand better this extremely complex

mental disorder.