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Genetics and inheritance issues in congenital heart disease

van Engelen, K.

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Download date: 15 Nov 2018

Genetics and inheritance issues in congenital heart disease

Klaartje van Engelen

Genetics and inheritance issues in congenital heart disease

Thesis, University of Amsterdam, Amsterdam, The NetherlandsISBN: 978-90-9027882-7Author: Klaartje van EngelenCover design: Klaartje van Engelen en Eelco Roos, Department of Clinical Genetics, AMC, AmsterdamLayout: Eelco Roos, Department of Clinical Genetics, AMC, AmsterdamPrinted by: Ipskamp Drukkers B.V.

Financial support by the Netherlands Heart Foundation for the publication of this thesis is gratefully acknowledged.

Additional financial support for the printing of this thesis was kindly provided by Chipsoft, Pfizer, Boehringer Ingelheim, the University of Amsterdam and the department of clinical genetics, Academic Medical Center, Amsterdam.

Copyright © 2013, K. van Engelen. All rights reserved. No part of this thesis may be reproduced or transmitted in any form or by any means, without written permission from the author.

Genetics and inheritance issues in congenital heart disease

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van doctoraan de Universiteit van Amsterdamop gezag van de Rector Magnificus

prof. dr. D.C. van den Boomten overstaan van een door het college voor promoties

ingestelde commissie,in het openbaar te verdedigen in de Aula der Universiteit

op vrijdag 29 november 2013, te 13:00 uur

door

Klaartje van Engelen

geboren te Nijmegen

Promotiecommissie

Promotor: Prof. dr. B.J.M. Mulder

Co-promotores: Dr. M.J.H. Baars Dr. A.V. Postma

Overige leden: Prof. dr. N.A. Blom Dr. S. Klaassen Prof. dr. I.M. van Langen Prof. dr. E.J. Meijers-Heijboer Prof. dr. M.C. de Ruiter Dr. E.M.A. Smets Prof. dr. A.H. Zwinderman

Faculteit der Geneeskunde

5

Contents

Chapter 1 Introduction and outline of this thesis

PART I GENES IN CONGENITAL HEART DISEASE

Chapter 2 A novel autosomal dominant condition consisting of congenital heart defects and low atrial rhythm maps to chromosome 9q Eur J Hum Genet 2011;19(7):820-826

Chapter 3 Mutations in the sarcomere gene MYH7 in Ebstein anomaly Circ Cardiovasc Genet 2011;4(1):43-50

Chapter 4 Mutations in the cardiac sodium channel gene SCN5A and congenital heart disease in humans Submitted

PART II CONGENITAL HEART DISEASE WITH ASSOCIATED ABNORMALITIES

Chapter 5 22q11.2 deletion syndrome is under-recognized in adult patients with tetralogy of Fallot and pulmonary atresia Heart 2010;96(8):621-624 Letter to the editor: Screening for 22q11.2 microdeletion in adults with tetralogy of Fallot, and author’s reply Heart 2011;97(10):860

Chapter 6 Bicuspid aortic valve morphology and associated cardiovascular abnormalities in fetal Turner syndrome: a pathology study Submitted

Chapter 7 The ambiguous role of NKX2-5 mutations in thyroid dysgenesis PLoS One 2012;7(12):e52685

Chapter 8 Prevalence of congenital heart defects in neuroblastoma patients: a cohort study and systematic review of literature Eur J Pediatr 2009;168(9):1081-1090

9

35

53

69

89

99

103

121

137

PART III THE PATIENTS’ PERSPECTIVE ON INHERITANCE OF CONGENITAL HEART DISEASE

Chapter 9 Adults with congenital heart disease: patients’ knowledge and concerns about inheritance Am J Med Genet A 2011;155(7):1661-1667

Chapter 10 The value of the clinical geneticist caring for adults with congenital heart disease: diagnostic yield and patients’ perspective Am J Med Genet A 2013;161(7):1628-1637

Chapter 11 Summary and future perspectives

Nederlandse samenvatting

Publicaties

Dankwoord

Curriculum vitae

155

171

189

199

203

209

213

Introduction and outline of this thesis

Chapter 1

10

1

11

1IntroductionCongenital heart disease (CHD) is among the most common birth defects, occurring in approxi-mately 8 per 1,000 live births.1 CHD comprises a wide range of cardiovascular malformations, from complex and critical defects presenting prenatally or in the newborn period, to mild defects that are not detected until adulthood. It leads to significant morbidity and mortality in children as well as adults. Due to improvements in cardiac surgery and medical care, the population of adult CHD patients is growing.2-4 Nowadays approximately 90% of CHD patients reach adulthood.Most CHD occurs sporadically and in a non-syndromic fashion. In a subgroup of CHD a genetic origin can be demonstrated, which include chromosomal abnormalities, copy number variations (CNVs) and single gene defects. In the remaining cases there is significant heritability, which is currently largely unexplained. The majority of non-syndromic CHD is historically believed to be multifactorial in origin, i.e. multiple (unknown) genetic and environmental factors contributing to the CHD. In the last decades, many research projects have focused on the genetic causes of non-syndromic CHD and although significant progress has been made, the contributing genetic defects remain largely unknown. The traditional approaches for gene discovery, including positional cloning strategies, are hampered by the rarity of extended CHD families with clear Mendelian inheritance patterns, incomplete penetrance and variable expression of the genetic defects. Studies in animal models have elucidated many fundamental pathways involved in cardiac development leading to the identification of candidate genes. Although several genes involved in human CHD have been identified by screening these candidate genes in human patients, such candidate gene studies are hypothesis driven - genes that seem to be less obvious in heart development at first thus remain undiscovered. In addition, the non-coding regions of the genome have not been studied extensively. Moreover, due to the heterogeneous genetic and clinical nature of CHD, large cohorts of homogeneous CHD were difficult to realize in the past, hampering candidate gene studies or case-control studies of common variants leading to CHD susceptibility. Despite these pitfalls, there has been progress in the identification of genes and signaling path-ways that are involved in cardiovascular development. Due to new and evolving genetic analysis techniques, knowledge is currently expanding at an increasing pace. This introductory chapter provides an overview of non-syndromic and syndromic causes of CHD. In addition, the implica-tions of CHD genetics for patients and family members are discussed.

Non-syndromic CHDIn the majority of patients, CHD occurs in a non-syndromic fashion. It happens mostly sporadically, although in some families a monogenetic inheritance pattern is present. A genetic cause can be demonstrated in only a small subset of individuals and families with non-syndromic CHD. In some patients, high-penetrance mutations in one of several genes that are known to be involved in heart development can be identified (see below). In addition, more recently de novo CNVs have been shown to contribute to several types of non-syndromic CHD, including tetralogy of Fallot (TOF), left-sided heart defects (bicuspid aortic valve (BAV), aortic coarctation) and several other types of

12

1CHD.5-7 In some of these CNVs known CHD genes are located; e.g. GATA4, which is located on 8p23.1, a region in which recurrent CNVs have been identified in CHD cases.6 It was estimated that 5% to over 10% of sporadic, non-syndromic CHD occurs because of a rare CNV. Penetrance is often incomplete.Single gene defects and CNVs are found in only a minority of non-syndromic CHD, however. The majority of non-syndromic CHD is historically thought to be due to multifactorial inheritance. This implies that there is a cumulative effect of multiple variations in many different genes, each of which contributes only for a small part to a person’s susceptibility to CHD. These genetic susceptibility factors interact with each other as well as with environmental risk factors, which, if a certain threshold is reached, together lead to CHD. Several of such susceptibility variants with low penetrance have been identified through small-scale case-control studies, and recently the first genome-wide association studies (GWAS) in CHD have shown associations for some CHD types. Cordell et al. demonstrated two loci on 12q24 and 13q32 to be associated with TOF.8 In addition, a recent GWAS study of cases with VSD, ASDII, transposition of the great arteries (TGA), conotruncal malformations, and left-sided heart defects showed an association of single nucleotide polymor-phisms in a region on chromosome 4p16 for ASD, but no significant associations were found for the other phenotypes studied.9 A GWAS study in a Han Chinese population identified two risk loci for CHD (including septal defects but also other CHD types) on 1p12 and 4q31.10 Odds ratios for CHD risk in these studies ranged from 1.2 to 1.4. It seems remarkable that no consistent loci were identified in these GWAS studies, however, differences in population ethnicity, patient character-istics and study design may explain these differences. Moreover, subcohorts with specific CHD types might have been too small to detect associations.Environmental factors that have been implied in CHD include maternal disease (e.g. diabetes mellitus, hypercholesterolemia, hyperphenylalaninemia), infectious agents (maternal Rubella), several medications (ACE inhibitors, retinoids) and substance abuse (alcohol, cocaine).11 Use of folic acid during pregnancy has been hypothesized to be protective against CHD in the child, and folate deficiency is suspected to be a CHD risk factor, however evidence for this association remains inconclusive.11 Some common variants in genes encoding enzymes involved in folic acid metabolism were shown to be ‘susceptibility alleles’ for CHD.12,13 A specific variation that has been investigated extensively, is the common variant c.667C>T of the MTHFR gene, which in homozygous state is known to cause lower levels of plasma folate. Interestingly, the largest genetic study and meta-analysis performed so far showed no evidence for a relationship between several subtypes of CHD and the MTHFR c.677C/T genotype (in CHD patients as well as mothers of CHD patients).14

Non-syndromic monogenetic CHDThe proportion of non-syndromic CHD that has a monogenetic cause is not precisely known, but it is presumed to be small. Only few families demonstrate CHD with a clear Mendelian inheritance pattern. In these families, mostly autosomal dominant inheritance is seen. In some CHD families

13

1as well as sporadic CHD, a pathogenic mutation underlying the defect can be identified. In these cases, there usually is extensive genetic as well as clinical heterogeneity; i.e. a particular type of CHD can be caused by mutations in different genes mutations, and mutations in one gene can lead to distinct types and severity of CHD, even within a family. Moreover, penetrance is often incomplete.The first reported single-gene mutation in humans with non-syndromic CHD was in the NKX2-5 gene, encoding a homeobox transcription factor.15 Since then, mutations in a substantial number of other genes have been identified, mostly by positional cloning or candidate gene approaches. Mutations have been found in several steps of the multiple pathways that contribute to heart development, including genes coding for extracellular receptor ligands, membrane receptors and transcription factors. Additionally, mutations in sarcomere protein genes and histone-modifying genes have been identified in patients with (familial) CHD.16 Table 1 provides an overview of a selection of the more well known genes involved in human CHD. A subset of these will be dis-cussed in more detail below.

NKX2-5NKX2-5 belongs to the NK-2 family of homeodomain-containing transcription factors, which are conserved from flies to humans.17 Its role as a transcription regulator during early embryonic heart developmental has been known for many years. Mice haplo-insufficient for nkx2.5 show abnor-malities of the (atrial) septum and valve development as well as hypoplasia of the cardiac conduc-tion system, especially the AV-node.18,19 Analogue to these abnormalities in animal studies, in humans most NKX2-5 mutations have been reported in patients with (familial) atrial septal defects (ASD) and conduction disorders, mainly atrioventricular block.15,20,21 Although NKX2-5 mutations can also lead to other types of CHD, the proportion of patients carrying such a muta-tion is lower in these groups.20,21 The overall mutation detection rate in sporadic CHD is reported to be 2%.21 The mutations that have been identified are spread among the entire coding region of the gene, without genotype-phenotype correlation. Most mutations (were predicted to) lead to a truncated protein (haplo-insufficiency) or impaired protein function.

NOTCH1NOTCH1 encodes a large, single-pass transmembrane receptor that functions in a highly conserved cell-to-cell signaling system involved in multiple developmental processes.22,23 In mammals, there are four Notch receptors (1-4), which interact with their ligands (Delta-like 1, 3 and 4, and Jagged 1 and 2) that are expressed on the surface of adjacent cells.23 Notch1-/- mice were shown to have impaired trabeculation and hypoplastic cardiac cushions. In 2005, Garg et al. performed a genome-wide linkage scan in a large family with bicuspid aortic valve, severe aortic calcification and other CHD, including TOF. A single locus was identified on chromosome 9q34-35 and subse-quently NOTCH1 was identified as the causal gene.24 Following this paper, mutations in NOTCH1 were also identified in patients with other left ventricular outflow tract obstruction, including hypoplastic left heart syndrome and aortic coarctation.25 Functional studies of the mutations identified in

14

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16

1human patients with CHD showed a reduction of the amount of receptor at the cell surface as well as reduced binding of ligands to the receptor.25,26 Mutations in other components of the Notch signaling pathway can also lead to syndromic and non-syndromic CHD. Mutations in JAG1, encoding one of the Notch receptor ligands, cause Alagille syndrome characterized by liver disease, dysmorphic features, vertebral abnormalities and CHD, specifically TOF. JAG1 muta-tions have also been identified in patients with non-syndromic CHD.27 In some patients with Alagille syndrome, mutations in NOTCH2 are found.28

MYH6In addition to mutations in transcription factors and receptor/ligand molecules, mutations in struc-tural proteins can also lead to CHD. An example is the alpha-myosin heavy chain, a cardiac sarcomeric protein, which is encoded by MYH6. It is expressed at high levels in the developing atria, and studies in animal models showed that deficiency of MYH6 during heart development leads to disturbed cardiogenesis and subsequent heart malformations.29,30 In 2005, a missense mutation in MYH6 was shown to cause an autosomal dominant form of ASD with incomplete penetrance.29 More recently, mutations were also identified in small proportions of patients with other CHD, among which tricuspid atresia, VSD and pulmonary stenosis.31,32 These mutations were shown to lead to disturbance of the assembly of normal myofibrils.31 Like mutations in other sarcomeric protein genes, including MYH7 and MYBPC3, mutations in MYH6 have also been identified in patients with hypertrophic and dilated cardiomyopathy.33

Syndromic CHDThere are numerous syndromes in which CHD may occur, and many of these are associated with specific CHD types. These syndromes can be caused by chromosomal abnormalities, including aneuploidies and structural aberrations. Moreover, mutations in genes involved in pathways that are important in the development of multiple organ systems can lead to syndromic CHD. Interest-ingly, mutations in some of the genes involved in syndromic CHD have also been identified in non-syndromic CHD patients, including TBX1, TFAP2B and JAG1.27,34-36 Table 2 provides an over-view of some well-known syndromic forms of CHD. A few of these are discussed in more detail below.

Turner syndromeTurner syndrome is caused by complete or partial monosomy for the X chromosome in all or part of the cells. It occurs in 1 in 2500 female live births. However, the majority of Turner syndrome conceptions do not survive until term. The main clinical features include fetal lymphedema, webbed neck, short stature, gonadal dysgenesis and usually a normal intelligence, with nonverbal learning disability. CHD is present in 20-40% of patients.37,38 However, in Turner syndrome fetuses with cystic hygroma, who include the more severely affected patients with a high mortality in utero, the prevalence of CHD is much higher. CHD in Turner syndrome mostly comprises left-sided heart defects, including bicuspid aortic valve and aortic coarctation. Moreover,

17

1patients are at increased risk of aortic aneurysm and dissection. An association between karyo-type and CHD has been reported; frequency of CHD is higher in patients with 45,X than in those with structural abnormalities of the X-chromosome (including X-chromosome deletions, ring chro-mosome X and isochromosome X), though a higher prevalence of bicuspid aortic valve in patients with ring chromosome X was also reported.37,39 The genetic mechanisms that are implicated in CHD in Turner syndrome are unknown.

22q11.2 microdeletion syndromeThe incidence of the 22q11.2 microdeletion syndrome is estimated to be at least one in 4,000, making it the most common microdeletion syndrome in humans.40,41 Most patients have a specific 3 Mb deletion, but atypical deletions are also found. In past and present times, several diagnostic terms have been assigned to the combination of features associated with microdeletion 22q11.2, including velocardiofacial syndrome, DiGeorge syndrome and CATCH22. These terms represent varying manifestations of the same genetic entity. The features associated with 22q11.2 microdeletion syndrome include CHD, cleft palate, velopharyngeal insufficiency with hypernasal speech, hypocalcaemia, psychiatric disturbances, (mild) dysmorphic facial features and mild to moderate mental retardation (reviewed in Kobrynski and Sullivan41). The presence and severity of clinical features is highly variable, however. This can make it difficult to recognize the syndrome in mildly affected patients, especially in adults.42,43 Three quarter of patients have CHD which typically constitute conotruncal malformations such as interrupted aortic arch type B, truncus arteriosus communis, TOF and pulmonary atresia (PA) with ventricular septal defect (VSD).42,44,45 The frequency of 22q11.2 deletion in children with these particular heart defects ranges from about 10% (TOF) to up to 50% (interrupted aortic arch type B), and screening for the deletion is therefore warranted in all patients with these CHD.In over 90% of patients the deletion has arisen de novo, whereas in the remaining patients it is inherited from one of the parents.46 Individuals with 22q11.2 microdeletion syndrome have a 50% chance of transmitting the deletion to their offspring. The 22q11.2 locus encompasses the TBX1 gene, encoding a transcription factor of the T-box family. Mice with one or two disrupted copies of TBX1 were shown to have CHD that are similar to those in 22q11.2 microdeletion syndrome, implying that haplo-insufficiency of TBX1 is a major cause of heart defects in patients with a 22q11.2 deletion.47 Indeed, mutations in TBX1 have also been identified in human patients with CHD, with and without the other abnormalities seen in 22q11.2 microdeletion syndrome.34,48 More recently, CRKL was also identified as a possible CHD candidate gene residing in the 22q11 locus.49,50

18

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20

1Down syndromeDown syndrome, caused by an extra copy of chromosome 21, is the most common chromosomal abnormality among live born infants and is the most frequent cause of intellectual disability. In over 95% of patients, there is a free copy of chromosome 21 in all cells and in most of the remaining patients, a Robertsonian translocation involving chromosome 21 and another acrocen-tric chromosome leads to the Down syndrome phenotype. The syndrome is characterized by well-defined phenotypic features including characteristic facial features, growth retardation, hypotonia and congenital malformations. About 50% of patients have CHD, most frequently atrioventricular septal defect (AVSD) and VSD.51,52 A CHD “critical region” on chromosome 21 as well as several candidate genes have been proposed, but the genetic basis and pathogenesis of CHD in Down syndrome remain largely unknown.52 Mutations in ALK2 and CRELD1 have been identified in some Down syndrome patients with AVSD.53,54

Williams syndromeWilliams syndrome is caused by a heterozygous deletion of circa 1.5 - 2 Mb on chromosome 7q11.23.55,56 Apart from CHD, which is present in 80% of patients, clinical features include intellectual disability, characteristic facial features (‘elfin facies’), hypercalcemia, connective tissue abnormalities and a friendly and outgoing personality. The most typical CHD in Williams syndrome comprise supravalvar aortic stenosis (SVAS) and peripheral pulmonary artery stenosis, although other CHD have also been described.57 SVAS is related to the absence of one copy of the Elastin (ELN) gene which is located in the deleted locus. Mutations in ELN have been identified in patients with non-syndromic SVAS and associated arteriopathies, although some individuals were described to have a “Williams-like” mouth or connective tissue abnormalities such as inguinal hernias.58,59

Holt-Oram syndromeHolt-Oram syndrome is an autosomal dominant ‘heart-hand syndrome’, occurring in about 1 in 100,000 individuals.60-62 Affected individuals show radial ray malformations, including an abnormal carpal bone and absent, hypoplastic, or triphalangeal thumbs with or without radial dysplasia. The limb abnormalities may be symmetrical or asymmetrical. Three quarter of patients have CHD, most commonly ostium secundum ASD and VSD, as well as progressive conduction disease.61,62 Although rare, other CHD have also been reported.63,64 Lower limb defects or other extracardiac malformations or disease (including intellectual disability) do not belong to Holt-Oram syndrome, thus if these are present Holt-Oram syndrome is unlikely.In 1997, TBX5 was identified as the causal gene in Holt-Oram syndrome.65,66 TBX5 functions as a transcription factor that has an important role in cardiac growth and development (especially in cardiac septation), in the development of a cardiac conduction system.67,68 and in forelimb speci-fication and outgrowth.69 TBX5 can interact with other transcription factors including NKX2-5 and GATA4.70 A pathogenic mutation in TBX5 can be identified in over 70% of patients, mostly but not always through the mechanism of TBX5 haplo-insufficiency. About 85% of affected individuals

21

1have been reported to have Holt-Oram syndrome as the result of a de novo mutation. In a small number of patients with Holt-Oram syndrome, mutations in SALL4 have been demonstrated.71

CHD recurrence risks In only a minority of CHD patients, it is possible to provide a CHD recurrence risk based on known Mendelian inheritance in a family or on figures related to chromosomal abnormalities. When a (ge-netic) cause is not identified in an individual non-syndromic patient, empirical estimates have to be used to provide recurrence risk numbers. Several studies have shown that there is an increased risk of CHD for relatives of CHD patients, mostly in the order of magnitude of 2 to 15% for first-degree relatives of a sporadic patient.72-74 A recent population-based study in Denmark demon-strated that the relative risk for all types of CHD taken together is 3.2 among first degree relatives and 1.8 among second degree relatives.75 Risk numbers vary significantly between different types of CHD, however. For example, the heritability of heterotaxy as well as BAV and other left ventricular outflow tract lesions is estimated to be much higher.75-77 Within families, the relative risk is highest for same-type CHD, but the risk is also increased for dissimilar types.75 If more than one family mem-ber is affected, the recurrence risk for other relatives increases. Moreover, the risk for offspring of women with CHD is higher than for offspring of men with CHD72,73; generally the risk for offspring of female CHD patients is estimated to be about 5-6%, whereas the risk for offspring of male CHD patients is about 2-3%.78 The reasons for this gender difference are unknown; imprinting mecha-nisms or maternal and environmental factors may play a role. Sibling recurrence risks are generally stated to be 2-3%. If two siblings are affected, the recurrence risk increases to 10%.78

Genetic counseling in adult CHDThe population of adult CHD patients is growing, and an increasing number of patients will have children. These patients may have questions regarding the origin and inheritance of their disease and the recurrence risk in (future) offspring. For the individual patient, knowledge about the underlying (genetic) origin of CHD is important because 1) the patient and his or her offspring may be at risk for extracardiac disease (in syndromic cases); 2) an individualized recurrence risk for offspring based on the underlying cause can be established; 3) there may be other relatives for whom genetic or cardiologic examination may be appropriate.79,80

The majority of adults with CHD has not had genetic testing or counseling, and those who did in childhood were probably too young to participate in the counseling process. In general, the knowledge of adult CHD patients about inheritance issues, including pregnancy-related topics and recurrence risk in offspring, has been shown to be low.81-84 Clinical genetic consultation can aid in increasing awareness and knowledge about such issues. It may help patients to understand the genetic basis of their CHD (and extracardiac disease), and the implications for family members including their offspring. By means of a detailed medical history of cardiac and extracardiac disease, extensive family history and physical examination aimed at dysmorphic features the clinical geneticist can gather

22

1information to establish a strategy for genetic testing and come to an etiological diagnosis in an individual patient. Subsequently, recurrence risks targeted to the patient’s specific situation can be provided as accurate as possible. In addition to the establishment of an etiological diagnosis and the estimation of a recurrence risk, genetic counseling involves education about management (of cardiac and extracardiac manifestations), inheritance issues and familial implications. Reproduc-tive options, including prenatal ultrasound investigations, prenatal diagnosis and pre-implantation diagnosis, may also be discussed. Moreover, the psychological implications of the diagnosis and recurrence risk are addressed. As CHD is not uncommon and mostly multifactorial in origin with general recurrence risks applying, at current times it seems not eligible to counsel all adult CHD patients. It is however important to identify the subgroup of patients that is especially likely to benefit from genetic counseling. These patients are summed in Table 3. Physicians caring for adult CHD patients should actively identify those patients and refer them at a low threshold.

Table 3. Adult CHD patients who may benefit from clinical genetic testing and counseling

CHD, congenital heart disease; IAA, interrupted aortic arch; TA, truncus arteriosus; TOF, tetralogy of Fallot; PA, pul-monary atresia; VSD, ventricular septal defect; AAA, aortic arch anomaly.

• Patients desiring having children (preconceptional counseling)

• Patients with extracardiac abnormalities/disease

- Congenital malformations

- Intellectual disability

- Dysmorphic features

- Multisystem involvement (e.g. endocrinologic, hematologic, immunologic or sensorineural disorders)

- Psychiatric disorders

• Patients with CHD with high risk of 22q11.2 deletion (IAA, TA, TOF, PA with VSD, AAA)

• Patients with a family history of CHD

• Any other patient who is interested in learning about the origin and inheritance of their CHD

23

1Outline of this thesisThis thesis focuses on the genetics of non-syndromic and syndromic CHD, the implications it has for (adult) CHD patients and the adult CHD patients’ perspective on inheritance issues. It presents different types of studies, including clinical and genetic studies in families with multiple affected individuals with CHD and in patient cohorts with specific CHD subtypes, a morphologic study of post-mortem hearts, as well as questionnaire studies.

In Part I of this thesis, clinical and genetic studies in non-syndromic CHD are presented. These studies were aimed to the identification of genes that are implied in human CHD. Chapter 2 describes the linkage analysis and subsequent identification of a disease locus in a large family with a novel phenotype, which resembles a mild form of left atrial isomerism. Chapter 3 presents the analysis of the sarcomere protein gene MYH7 in a cohort of patients with Ebstein anomaly. In Chapter 4, the role of mutations in the cardiac sodium channel gene SCN5A in CHD is explored; on the one hand, a cohort of patients with a septal defect and conduction disease was analyzed for SCN5A mutations. On the other hand, a cohort of SCN5A mutation carriers was evaluated for the frequency of CHD.

Part II focuses on CHD in association with other (extracardiac) abnormalities, including syndromic CHD. Chapter 5 is dedicated to the 22q11.2 deletion syndrome. Although it is currently common practice to test children with specific CHD types for the presence of a 22q11.2 deletion, this has not routinely been performed by patients who have already reached adult age. We therefore evalu-ated the prevalence of 22q11.2 microdeletion syndrome in adults with TOF and pulmonary atresia with VSD, which is described in chapter 5. In Chapter 6, we focus on CHD in Turner syndrome; in this syndrome left-sided heart defects including aortic valve abnormalities are especially common. We specifically looked at the morphology of the (bicuspid) aortic valve early in development in a selected group of TS patients with adverse outcome, namely in post-mortem heart specimens of Turner syndrome fetuses.Also in the absence of a recognized syndromic etiology, CHD is associated with several extracardiac malformations or disease. As common developmental pathways may underlie these associations, genes implied associated abnormalities may also be implied in cardiac development and CHD, and vice versa. One of the genes that are already well-established in CHD is NKX2-5. More recently, NKX2-5 was also implicated in human thyroid dysgenesis. In Chapter 7 we studied a specific variant in NKX2-5, which we encountered in two unrelated probands with CHD and which had been identified before in a patient with thyroid dysgenesis. Chapter 8 focuses on childhood cancer: we evaluated whether CHD is more frequent in children with neuroblastoma, as was previously reported.

Whereas Part I and II mainly focus on genetic causes and pathogenetic mechanisms leading to CHD, Part III appraises the clinical implications and patients’ perspectives on genetics and inheritance

24

1of CHD. Chapter 9 reports on the information adult CHD recalled to have received about the inheritance of their CHD, patients’ knowledge about inheritance and their concerns in this regard. Chapter 10 focuses on adults with CHD who consulted a clinical geneticist. We report on the etio-logic diagnoses that are made by the geneticist in these patients. Additionally, patient satisfaction with genetic counseling and reproductive choices are presented.

Finally, in Chapter 11, the major findings presented in this thesis are summarized and future perspectives for research and clinical care are discussed.

25

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81 Kovacs AH, Harrison JL, Colman JM, Sermer M, Siu SC, Silversides CK. Pregnancy and contraception in congenital heart disease: what women are not told. J Am Coll Cardiol 2008;52:577-8

82 Moons P, De VE, Budts W et al. What do adult patients with congenital heart disease know about their disease, treatment, and prevention of complications? A call for structured patient education. Heart 2001;86:74-80

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85 Cinquetti R, Badi I, Campione M et al. Transcriptional deregulation and a missense mutation define ANKRD1 as a candidate gene for total anomalous pulmonary venous return. Hum Mutat 2008;29:468-74

86 Sperling S, Grimm CH, Dunkel I et al. Identification and functional analysis of CITED2 mutations in patients with congenital heart defects. Hum Mutat 2005;26:575-82

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187 De LA, Sarkozy A, Ferese R et al. New mutations in ZFPM2/FOG2 gene in tetralogy of Fallot and

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of several pathway members including FOXH1 is linked to human heart defects and holoprosen-cephaly. Am J Hum Genet 2008;83:18-29

90 Wang B, Yan J, Mi R et al. Forkhead box H1 (FOXH1) sequence variants in ventricular septal defect. Int J Cardiol 2010;145:83-5

91 Wessels MW, Willems PJ. Genetic factors in non-syndromic congenital heart malformations. Clin Genet 2010;78:103-23

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93 Bonnefond A, Sand O, Guerin B et al. GATA6 inactivating mutations are associated with heart defects and, inconsistently, with pancreatic agenesis and diabetes. Diabetologia 2012;55:2845-7

94 Muncke N, Jung C, Rudiger H et al. Missense mutations and gene interruption in PROSIT240, a novel TRAP240-like gene, in patients with congenital heart defect (transposition of the great arter-ies). Circulation 2003;108:2843-50

95 Heathcote K, Braybrook C, Abushaban L et al. Common arterial trunk associated with a homeo-domain mutation of NKX2.6. Hum Mol Genet 2005;14:585-93

96 Griffin HR, Topf A, Glen E et al. Systematic survey of variants in TBX1 in non-syndromic tetralogy of Fallot identifies a novel 57 base pair deletion that reduces transcriptional activity but finds no evidence for association with common variants. Heart 2010;96:1651-5

97 Smemo S, Campos LC, Moskowitz IP, Krieger JE, Pereira AC, Nobrega MA. Regulatory variation in a TBX5 enhancer leads to isolated congenital heart disease. Hum Mol Genet 2012;21:3255-63

98 Basson CT, Huang T, Lin RC et al. Different TBX5 interactions in heart and limb defined by Holt-Oram syndrome mutations. Proc Natl Acad Sci U S A 1999;96:2919-24

99 Kirk EP, Sunde M, Costa MW et al. Mutations in cardiac T-box factor gene TBX20 are associated with diverse cardiac pathologies, including defects of septation and valvulogenesis and cardiomyopathy. Am J Hum Genet 2007;81:280-91

100 Qiao Y, Wanyan H, Xing Q et al. Genetic analysis of the TBX20 gene promoter region in patients with ventricular septal defects. Gene 2012;500:28-31

101 Posch MG, Gramlich M, Sunde M et al. A gain-of-function TBX20 mutation causes congenital atrial septal defects, patent foramen ovale and cardiac valve defects. J Med Genet 2010;47:230-5

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105 Kosaki R, Gebbia M, Kosaki K et al. Left-right axis malformations associated with mutations in ACVR2B, the gene for human activin receptor type IIB. Am J Med Genet 1999;82:70-6

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107 Bamford RN, Roessler E, Burdine RD et al. Loss-of-function mutations in the EGF-CFC gene CFC1 are associated with human left-right laterality defects. Nat Genet 2000;26:365-9

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1108 Selamet Tierney ES, Marans Z, Rutkin MB, Chung WK. Variants of the CFC1 gene in patients with

laterality defects associated with congenital cardiac disease. Cardiol Young 2007;17:268-74109 Robinson SW, Morris CD, Goldmuntz E et al. Missense mutations in CRELD1 are associated with

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121 Dasgupta C, Martinez AM, Zuppan CW, Shah MM, Bailey LL, Fletcher WH. Identification of connexin43 (alpha1) gap junction gene mutations in patients with hypoplastic left heart syndrome by denaturing gradient gel electrophoresis (DGGE). Mutat Res 2001;479:173-86

122 Posch MG, Waldmuller S, Muller M et al. Cardiac alpha-myosin (MYH6) is the predominant sarcomeric disease gene for familial atrial septal defects. PLoS One 2011;6:e28872

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1129 Xin B, Puffenberger E, Tumbush J, Bockoven JR, Wang H. Homozygosity for a novel splice site

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Part IGenes in congenital heart disease

A novel autosomal dominant condition consisting of congenital heart defects and low atrial rhythm

maps to chromosome 9q

van de Meerakker JBA*, van Engelen K*, Mathijssen IB, Lekanne dit Deprez RH, Lam J, Wilde AAM, Baars MJH, Mannens MMAM, Mulder BJM, Moorman AFM, Postma AV

*These authors contributed equally

Eur J Hum Genet 2011;19(7):820-826

Chapter 2

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AbstractCongenital Heart Defects (CHDs) occur mostly sporadic, but familial CHD cases have been reported. Mutations in several genes, including NKX2-5, GATA4 and NOTCH1 were identified in families and patients with CHD, but the mechanisms underlying CHD are largely unknown. We performed genome-wide linkage analysis in a large four-generation family with autosomal dominant CHD (including atrial septal defect type I and II, tetralogy of Fallot and persistent left superior vena cava) and low atrial rhythm, an unique phenotype that has not been described before. We obtained phenotypic information including electrocardiography, echocardiography and DNA of 23 family members. Genome-wide linkage analysis on 12 affected and 5 unaffected individuals and 1 obligate carrier demonstrated significant linkage only to chromosome 9q21-33 with a multipoint maximum LOD score of 4.1 at marker D9S1690, between markers D9S167 and D9S1682. This 48 cм critical interval corresponds to 39 Mb and contains 402 genes. Sequence analysis of nine candidate genes in this region (INVS, TMOD1, TGFBR1, KLF4, IPPK, BARX1, PTCH1, MEGF9 and S1PR3) revealed no mutations, nor were genomic imbalances detected using array CGH. In conclusion, we describe a large family with CHD and low atrial rhythm with a significant LOD score to chromosome 9q. The phenotype is representative of a mild form of left atrial isomerism or a developmental defect of the sinus node and surrounding tissue. Because the mechanisms underlying CHD are largely unknown, this study represents an important step towards the discovery of genes implied in cardiogenesis.

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IntroductionCongenital Heart Defects (CHDs) are among the most common congenital defects, occurring with an incidence of 8/1,000 live-births.1 The aetiology of CHD is generally complex, environmental exposures, chromosomal abnormalities and gene defects can all contribute. Most CHD occur sporadic, but in recent years an increasing number of familial cases with various types of CHD have been reported.2-4 Although mutations in a several genes have been identified in a small subset of patients and families with CHD, e.g. NKX2-5,2 GATA4 3 and NOTCH1,5 the mechanisms underlying human cardiogenesis and CHDs remain largely unknown. Some CHD patients and families also display cardiac arrhythmias, which can occur due to the anatomical defect itself or sometimes due to surgical interventions.6 Moreover, in some patients arrhythmias are the direct consequence of the underlying genetic defect, in absence of any structural defect.2,7 Ectopic atrial rhythms originate when a focus outside the sinus node takes over the pacemaker function. Consequently, the direction of atrial activation may be altered, which can be seen as an abnormal P-wave axis on the electrocardiogram (ECG). The appearance of the P-wave depends on the site of origin of the ectopic rhythm. The P-wave can be decreased in amplitude, or it can be biphasic or negative in leads II, III and AVF. The rhythm is regular and the P-R interval is usually normal or, at times, prolonged.8 Usually, ectopic atrial rhythms are haemodynamically insignifi-cant. They can be isolated and idiopathic, especially in children, but can also co-occur with CHD.9 A P-wave frontal axis oriented in a superior direction, reflecting atrial pacemaker tissue located in the lower part of the atrium, is referred to as low atrial rhythm. This can be present due to sinus node dysfunction in patients with left atrial isomerism, a laterality disorder characterized by bilat-eral left sidedness.9,10 Here we describe a large four-generation family with an autosomal dominant condition, comprising various types of CHD and low atrial rhythm, a unique phenotype not described before within a family. A genome wide linkage analysis yielded linkage to a region of 39 Mb on chromosome 9q with a maximum LOD score of 4.1.

MethodsClinical detailsThis study was approved by the Medical Ethical Committee at the Academic Medical Center in Amsterdam. Written informed consent was obtained from all participants. Subjects were clini-cally evaluated by analysis of medical records, physical examination with attention to syndromic features, cardiologic examination, 12-lead electrocardiogram (ECG) and two-dimensional echo-cardiography. Previously performed ECGs were obtained from the medical records if available. The number of available ECGs ranged from one to 15 in the evaluated persons. One experienced paediatric cardiologist (JL) reviewed the ECGs and echocardiographic images. A total of 23 family members were examined. One patient (II-2) refused cardiologic examination for this study and therefore only previous medical records (including ECGs and echocardiography records) were

38

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obtained. We used Magnetic Resonance Imaging (MRI) for evaluation of situs in the proband (IV-10). Abdominal ultrasound for evaluation of abdominal situs was performed in three individuals and chest X-rays for evaluation of situs of the lungs were available for six individuals. Computed Tomography (CT) of the thorax was performed in patient III-3.Low atrial rhythm was defined as a P-wave frontal axis oriented in a superior direction. We defined the P-wave frontal axis to be horizontal when it was approximately 0°, which is abnormal, though not a true low atrial rhythm. A wandering atrial pacemaker was defined as a change in P-wave axis of 60° or more on subsequent ECGs.

Linkage analysis, mutation screen and array comparative genomic hybridizationGenomic DNA of family members was extracted from peripheral blood according to standard procedures. Linkage analysis was performed using the ABI linkage set v2.5 MD10 set on an ABI 3700 Genetic Analyzer (Applied Biosystems).11 Phenotype, genotype, and pedigree information were combined for multipoint linkage analysis with the use of the easyLinkage software package12 running Simwalk v2.9113 with the assumption of an autosomal dominant pattern of inheritance, a disease-allele frequency of 0.0001 and a penetrance of 0.9. Gene frequency was assumed to be equal between males and females. Both parametric and nonparametric linkage were calculated.PCR amplification of all the coding exons of candidate genes inversin (INVS), transforming growth factor-β receptor 1 (TGFBR1), tropomodulin 1 (TMOD1), Kruppel-like factor 4 (KLF4), inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPPK), BARX homeobox 1 (BARX1), patched homologue 1 (PTCH1), sphingosine 1-phosphate receptor 3 (SIPR3) and multiple epidermal growth factor-like domains 9 (MEGF9) (only exon 12b, as this is specifically expressed in brain and heart14) was performed using primers located in flanking intronic sequences (available on request). Subsequently, PCR products were analysed by direct sequencing, using the BigdyeTerminator v3.1 Kit on an ABI 3700. MLPA probes were designed for INVS and TGFBR1 using MAPD, a probe design suited for multiplex ligation-dependent probe amplification assays.15 The MLPA procedure and analysis were carried out as described.16 Array CGH was performed using 4x180K slides (average 13kbp spacing between probes), AMADID 023363 (Agilent, Santa Clara, CA, USA) according to the manufacturer’s protocol (Oligonucleotide Array-Based CGH for Genomic DNA Analysis V5.0 June 2007) with adaptations. No amplifica-tion or restriction was used on the genomic patient and reference DNA. Fluorescent labelling of gDNA was performed using the CGH labelling kit for Oligo Arrays (Enzo Life Sciences, Inc. Farmingdale, NY, USA) and purified using the MinElute PCR Purification Kit (Qiagen, Valencia, CA, USA). Hydridization was performed according to the manufacturer’s protocol (Oligonucleotide Array-Based CGH for Genomic DNA Analysis V5.0 June 2007) and slides were scanned using an Agilent G2250C 2 µm scanner with Agilent Scan Control software (Version A.8.1.3.) using default settings. Spot intensities were measured with Agilent Feature Extraction software (V10.7), and further data analysis was performed using DNA Analytics software (V4.0.76) with algorithm ADM-2 using a filter of minimal three subsequent clones with a minimal absolute ratio of 0.3.

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lled

sym

bols

thos

e w

ith rh

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/con

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ion

dist

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with

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mal

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40

2

ResultsClinical detailsIn the presented family, several individuals had CHD with or without low atrial rhythm, while others had isolated low atrial rhythm. The pedigree of the family is shown in Figure 1. Table 1 summarizes the clinical features of the family members. The proband (IV-10) was diagnosed during her first year of life with an incomplete atrioventricular septal defect (AVSD), a common atrium and a small communication between the left and right ventricle. At 2.5 years of age she underwent surgical correction including septation of the mono-atrium, leaving the coronary sinus draining into the left atrium. During the surgical procedure, the presence of bilateral left atria with bilateral left atrial appendages was observed. At 33 years of age, she was asymptomatic. MRI at that age showed normal situs of the lungs and abdominal organs. ECG persistently showed left QRS axis deviation and low atrial rhythm. The sister of the proband (IV-9) was asymptomatic. Echocardiography did not reveal any abnor-malities, however, ECG at age 32 showed low atrial rhythm (Figure 2A). The asymptomatic father of IV-9 and IV-10 (III-10) also had normal echocardiography, and low atrial rhythm with bradycardia (45/min) on ECG. The paternal aunt of the proband (III-9) was diagnosed with congenital agenesis or malfunction of the sinus node at infant age because of bradycardia with AV-junctional escape rhythm and low atrial rhythm. At later age, she developed intermittent complete AV dissociation. Echocardiography repeatedly did not show any structural abnormalities. Because of chronic fatigue, a pacemaker was implanted at the age of 39 years. During implantation, the lead could not be placed in a stable position in the right atrial appendage and therefore this appendage had been assumed to be absent. Normal situs of the lungs and abdominal organs were confirmed by chest X-ray and abdominal ultrasound. The 18 year old daughter of III-9 (IV-8) was born with a large incomplete AVSD and ASD II which were surgically corrected at the age of 8 months. At surgery, absence of the coronary sinus and a persistent left superior vena cava (LSVC) connecting directly with the left upper corner of the left atrium were noted. The right superior vena cava was hypoplastic and the brachiocephalic vein was absent. ECGs persistently showed low atrial rhythm. Situs of the lungs and abdomen was normal.One of the paternal uncles of the proband (III-6) had tetralogy of Fallot (TOF) and LSVC draining into the coronary sinus, which was surgically corrected at the age of 27. P-wave frontal axis was horizontal. Normal situs of the lungs and bronchi was present. In his 28 year old daughter (IV-5), a cardiac murmur was noticed shortly after birth and she was followed at a cardiology clinic until the age of 7 years. Echocardiography performed at age 20 showed mildly elevated pulmonary artery pressure, with unknown cause. Low atrial rhythm was also present. The paternal grandfather of the proband (II-2) had a myocardial infarction at age 76. He received a pacemaker at age 84 because of atrial and ventricular arrhythmias with severe bradycardia. P-wave frontal axis was horizontal. He developed atrial fibrillation at age 85. Chest X-rays showed normal situs of the lungs and bronchi.

41

2

His sister (II-1) showed low atrial rhythm on ECG at age 74. She developed atrial fibrillation when she was 77 years old. Echocardiography at this age showed dilated atria and mildly elevated pulmonary artery pressure, with normal left ventricular systolic function and diastolic dysfunction. Ultrasound showed normal abdominal situs. Two children of II-1 (III-1 and III-2) had died in the first months of life: III-1 was said to have had aortic hypoplasia, detected at autopsy. III-2 was said to have died of pneumonia. The daughter of II-1 (III-3) was asymptomatic at age 57. Echocardiography and CT showed a LSVC draining into the coronary sinus (Figure 2B), aberrant right subclavian artery arising from the aorta distal from the left subclavian artery (arteria lusoria) and elevated pulmonary artery pres-sure, while on ECG low atrial rhythm and paroxysmal supraventricular tachycardia were present. Pulmonary situs was normal. In the asymptomatic 28-year old daughter of III-3 (IV-1) echocardiog-raphy showed abnormal interventricular wall movements, but no ventricular septal defect or other structural abnormalities were seen. She did have low atrial rhythm. The son of III-3 (IV-2), who was also asymptomatic at age 25, showed a horizontal P-wave frontal axis on ECG with normal echocardiography.

Figure 2. A. ECG of patient IV-9 at age 32 years with isolated low atrial rhythm. The P-wave is negative in leads II, III and aVF. B. Transverse computed tomography image of individual III-3. Persistent left superior vena cava (arrow). AA, ascending aorta, DA, descending aorta.

A B

42

2

Tabl

e 1.

Clin

ical

det

ails

of t

he fa

mily

Pat

ient

cha

ract

eris

tics

C

linic

al fe

atur

esSt

atus

in li

nkag

e an

alys

is

Sex

/ ag

e

Age

of

diag

nosi

s of

C

HD

and

/or

low

atri

al

rhyt

hm

Age

(yea

rs)

at la

st

avai

labl

e E

CG

Low

atri

al

rhyt

hmR

hyth

m/c

ondu

ctio

n di

stur

banc

esC

onge

nita

l hea

rt de

fect

sN

ucle

ar fa

mily

sc

enar

io

II-1

F / 8

374

yrs

77+

AF

-A

ffect

ed

II-2

M /

8984

yrs

84±

Bra

dyca

rdia

, atri

al/v

entri

cula

r ar

rhyt

hmia

, AF*

-O

blig

ate

carr

ier

II-3

M /

8778

yrs

80-

Bra

dyca

rdia

, jun

ctio

nal e

scap

es, A

F,

com

plet

e R

BB

B-

Exc

lude

d

III-3

F / 5

750

yrs

57+

Par

oxys

mal

SV

TLS

VC

, abe

rran

t rig

ht

subc

lavi

an a

rtery

Affe

cted

III-4

M /

5043

--

-N

ot a

ffect

ed

III-5

M /

4740

yrs

40+

1st d

egre

e AV

blo

ck, i

ncom

plet

e R

BB

B-

Affe

cted

III-6

M /

6110

yrs

36±

Com

plet

e R

BB

BTe

tralo

gy o

f Fal

lot,

LSV

CA

ffect

ed

III-7

M /

5958

-S

inus

bra

dyca

rdia

-N

ot a

ffect

ed

III-8

M /

5453

--

Not

affe

cted

III-9

F / 4

74

yrs

38+

Bra

dyca

rdia

, atri

al a

rres

ts w

ith A

V

junc

tiona

l esc

apes

, int

erm

itten

t co

mpl

ete

AV d

isso

ciat

ion

-**

Affe

cted

III-1

0M

/ 60

52 y

rs52

+B

rady

card

ia-

Affe

cted

III-1

1F

/ 52

45-

--

Exc

lude

d

III-1

2F

/ 50

43-

--

Exc

lude

d

IV-1

F / 2

821

yrs

28+

--

Affe

cted

43

2

IV-2

M /

2524

yrs

24±

--

Exc

lude

d

IV-3

F / 2

32

mon

ths

16+

Inco

mpl

ete

RB

BB

AS

D II

, mem

bran

e in

left

atriu

m, L

SV

C, a

bsen

t br

achi

ocep

halic

vei

nA

ffect

ed

IV-4

F / 1

7bi

rth16

-C

hron

ic S

VT;

atri

al e

xtra

syst

oles

-E

xclu

ded

IV-5

F / 2

84

yrs

28+

Inco

mpl

ete

RB

BB

-†A

ffect

ed

IV-6

M /

3433

--

-N

ot a

ffect

ed

IV-7

F / 3

124

--

-N

ot a

ffect

ed

IV-8

F / 1

8<

0.5

yrs

18+

Inco

mpl

ete

RB

BB

Inco

mpl

ete

AVS

D,

AS

D II

, LS

VC

dra

inin

g in

to le

ft at

rium

, abs

ent

coro

nary

sin

us, a

bsen

t br

achi

ocep

halic

vei

n

Affe

cted

IV-9

F / 3

632

yrs

32+

--

Affe

cted

IV-1

0F

/ 33

< 0.

5 yr

s33

+In

com

plet

e R

BB

BIn

com

plet

e AV

SD

, com

mon

at

rium

, bila

tera

l lef

t atri

al

appe

ndag

esA

ffect

ed

Low

atri

al rh

ythm

+ m

eans

that

the

P-w

ave

front

al a

xis

was

orie

nted

sup

erio

rly; L

ow a

trial

rhyt

hm ±

mea

ns th

at th

e P

-wav

e fro

ntal

axi

s w

as h

oriz

onta

l (ap

prox

imat

ely

0°).

AF,

atri

al fi

brill

atio

n; R

BB

B, r

ight

bun

dle

bran

ch b

lock

; AS

D II

, sec

undu

m ty

pe a

trial

sep

tal d

efec

t; AV

SD

, atri

oven

tricu

lar s

epta

l def

ect;

SV

T, s

upra

vent

ricul

ar ta

chyc

ardi

a;

LSV

C, p

ersi

sten

t lef

t sup

erio

r ven

a ca

va; *

Afte

r myo

card

ial i

nfar

ctio

n, **

Sus

pici

on o

f abn

orm

al a

trial

app

enda

ge, †

Sys

tolic

mur

mur

, for

whi

ch c

ardi

olog

ic fo

llow

up

until

the

age

of 7

yea

rs, e

leva

ted

pulm

onar

y ar

tery

pre

ssur

e at

age

20.

44

2

In the youngest son of II-1 (III-5), ECG at age 40 showed low atrial rhythm and 1st degree atrio-ventricular (AV) block. On echocardiography at the same age, a mildly dilated left ventricle was present as well as moderate regurgitation of calcified aortic and tricuspid valves. The 23 year old daughter of III-5 (IV-3) was diagnosed in her first year of life with ASD II, membrane in the left atrium, absent brachiocephalic vein, LSVC draining into the coronary sinus and a small right superior vena cava. ECGs persistently showed low atrial rhythm. Situs of the lungs and bronchi was normal. The other daughter of III-5 (IV-4) was diagnosed with frequent paroxysmal supraventricular tachycardia and atrial extrasystoles with the origin in the lower right atrium at birth, with normal sinus rhythm in between. She was on anti-arrhythmic medication until the current age of 17 years because of persistence of the tachycardia. Echocardiography showed no structural abnormalities and chest X-ray showed normal pulmonary situs. At age 78, the asymptomatic brother of II-1 and II-2 (II-3) was found to have bradycardia with arrests up to 2.6 seconds with junctional escapes, atrial extrasystoles and non sustained ventricular tachy-cardia. He also had paroxysmal atrial fibrillation and complete right bundle branch block. P-wave frontal axis was normal. Echocardiography at age 80 demonstrated left ventricular hypertrophy, dilatation of the aortic root (42 mm) and mild mitral valve prolapse, but no congenital abnormalities.

Seven other family members were evaluated in this study (III-4, III-7, III-8, III-11, III-12, IV-6, IV-7), all with normal echocardiography and ECG. None of the individuals with an abnormal P-wave axis had a wandering pacemaker. Clear dysmorphic features were not present in any of the family members.

Linkage analysis and mutation screenBefore performing a genome-wide linkage we excluded GATA4, TBX5 and NKX2-5 as the disease-causing gene in this family by linkage analysis. In the first linkage scenario, termed nuclear family, subjects were considered affected if they had a structural heart defect and/or low atrial rhythm. Therefore, genome-wide linkage was performed on 12 affected (II-1, III-3, III-5, III-6, III-9, IIII-10, IV-1, IV-3, IV-5, IV-8, IV-9 and IV-10), 5 unaffected individuals (III-4, III-7, III-8, IV-6, IV-7) and 1 obligate carrier (II-2) (Figure 1, Table 1). The analysis demonstrated significant linkage to a single locus on chromosome 9q shared by all affected individuals. This locus was absent from unaffected individuals (Figure 1). The shared locus has a multipoint maximum LOD-score of 4.1 at marker D9S1690, and is delineated by markers D9S167 and D9S1682 based on the haplotypes (Figures 1 and 3). No other loci with a LOD score higher than 1.0 were detected in this family genome wide.To reduce the risk of phenocopies, individuals IV-2, IV-4 and II-3 (and therefore his children III-11 and III-12) were not included in the nuclear family linkage analysis. IV-2 was excluded because he had a horizontal P-wave frontal axis, and although this is abnormal, it is not a clear low atrial rhythm. II-3 an IV-4 had significant rhythm and conduction abnormalities, but normal P-wave frontal axes. In a second linkage scenario, termed extended family, we presumed IV-2, IV-4 and II-3 to be affected and III-11 and III-12 to be unaffected. We found that all affected carried the locus, however III-11 did as well, who had normal echocardiography and ECG. The maximal LOD-score for the

45

2

extended family scenario is 4.45; the locus remained the same. A third linkage scenario, termed affected only, was run in which individuals II-1, III-3, III-5, III-6, III-9, IIII-10, IV-1, IV-3, IV-5, IV-8, IV-9 and IV-10 were included as affected. The maximal LOD-score for this affected only scenario was 3.3 also delineated by markers D9S167 and D9S1682The shared locus comprises a 48 cм critical interval, that corresponds to 39 Mb and contains 402 genes, of which 9 (INVS, TMOD1, TGFBR1, KLF4, IPPK, PTCH1, BARX1, MEGF9 and S1PR3) are known to be, directly or indirectly, involved in development and (left-right) patterning of the heart. Therefore, mutation analysis was performed in these genes in the proband (IV-10). Moreover, 10 conserved non coding sequences within 5kb up- or downstream of candidate genes were also screened. Nonetheless, no mutations were identified by direct sequencing. Since large genomic rearrangements could also potentially underlie the phenotype, we also performed an array comparative genomic hybridization (array CGH) on III-6 and IV-10 using a 180K oligo array with genome-wide coverage. However, no genomic imbalances in or outside the co-segregating locus were detected, besides copy number variations also present in controls (data not shown). Moreover, MLPA analysis of the coding exons of INVS and TGFBR1 did not reveal genomic imbal-ances for these genes.

Figure 3. Graphs of the multipoint parametric (A) and nonparametric, NPL (B) LOD scores of microsatellite markers on chromosome 9 for various analysis scenarios. The area of linkage is enlarged, depicting the position of markers and candidate genes screened by sequencing.

46

2

DiscussionWe describe a large family with an autosomal dominant condition with a wide spectrum of cardio-vascular abnormalities. The phenotype comprises different types of CHD with an abnormal atrial rhythm. Remarkably, several family members have low atrial rhythm without detectable structural heart defects, suggesting that these individuals show a mild manifestation of the familial disorder. We identified a locus at chromosome 9q that co-segregates with this autosomal dominant disorder and we excluded several candidate genes present within the shared 39-Mb disease locus.The presence of bilateral left atrial appendages in the proband in combination with low atrial rhythm, led us to hypothesize that the CHDs in this family might be the result of a defect in the establishment of left-right asymmetry, and might represent a mild or variant expression of left atrial isomerism. Left isomerism is a laterality disorder characterized by bilateral left sidedness, i.e. two morphologic left cardiac atria and atrial appendages, often in association with bilateral bilobed (left) lungs, isomerism of the bronchi, abdominal situs abnormalities, polysplenia and abnormalities of the large systemic veins.17,18 The internal architecture of the heart is often disrupted as well, and different forms of CHD are observed in left atrial isomerism.17-19 Besides CHD, the phenotype in this family is characterized by abnormal P-wave frontal axis on ECG, typically present in left atrial isomerism due to sinus node abnormalities. In normal hearts, the sinus node is located in the right atrium at the junction between the superior caval vein and the right atrial appendage, which results in a P-wave frontal axis orientated leftward and inferiorly.20 Histological studies in hearts with left atrial isomerism demonstrate absence, hypoplasia and/or ectopic location of nodal tissue in a large proportion of patients.21,22 In correspondence with these histologic abnormalities, abnormal atrial rhythms are commonly found in patients with left atrial isomerism,10,23 and a low atrial rhythm with a superior P-wave frontal axis is frequently present.10,23 In this family, four of the five individuals with CHD or isolated LSVC had low atrial rhythm, and one individual had a horizontal P-wave axis. In addition, seven family members without detectable structural heart defects also showed low atrial rhythm, and two had a horizontal P-wave axis. Indeed, rhythm and conduction disturbances have been described in patients with confirmed left isomerism without structural cardiac defects.24,25 Taken together, the spectrum of CHD in this family is compatible with that seen in left isomerism. Importantly, we could not confirm signs of left isomerism outside the heart (e.g. polysplenia) in the investigated family members, though imaging of the abdomen and lungs was not available in all subjects, so we cannot rule out laterality defects in every individual. In conclusion, the phenotype of this family resembles a developmental laterality defect, though affected individuals only show a mild expression, which appears to be restricted to the heart.Alternatively, the familial phenotype might represent an underlying developmental defect of the sinus node, as most family members have an abnormal low atrial rhythm, caused by a dominance of a pacemaker located in the lower part of the right atrium over the normal sinus node. In its earli-est stages, when the heart is no more than a simple tube, a dominant pacemaker activity is already found in the entire systemic inflow region of the heart (both left- and right side). Subsequently, newly added myocardium at the inflow part, which differentiates from t-box transcription factor

47

2

Tbx18 expressing precursor cells, will eventually give rise to the right-sided sinus node, among others.26 The developing sinus node also expresses the t-box transcription factor Tbx3, which acts to repress differentiation into working myocardium by imposing a conduction system phenotype.27 Thus, formation of the sinus nodes requires transcription factors such as Tbx18 and Tbx3, and knockout of these genes leads not only to under- or maldevelopment of the sinus node, but also to various forms of CHD.26,27 Consequently, it is possible that the phenotype seen in the current family is the direct result of developmental defects in the formation of the sinus node, possibly via pathways involving t-box transcription factors.The co-segregating locus on chromosome 9q contains 402 genes (genome build 37), and some of these were selected as candidate genes based on current knowledge, literature and data mining analysis: INVS, S1PR3, TGFBR1, IPPK, BARX1, TMOD1, KLF4, MEGF9 and PTCH1. The coding regions and selected conserved non-coding regions of all these genes were sequenced, but no mutations were found. In addition, we looked for genome-wide genomic imbalances using array CGH, however none were identified. No other genes known to play a role in left-right patterning or cardiac development map to the 9q region. The best option to identify the causative gene or region on the 9q locus would be to fully sequence it, and although advances in next generation sequenc-ing have been tremendous, it is currently far from trivial to sequence our 39 Mb-locus. Moreover, investigation of additional families with a similar phenotype might reduce the region of interest and could lead to the detection of the gene responsible for the phenotype in this family.A concern of our study is the possibility of phenocopies. Ectopic atrial rhythms can be seen idiopathi-cally in healthy individuals, especially in children. We cannot rule out that individuals in our family have P-wave abnormalities due to other reasons than the familial genetic defect. To minimize the risk of phenocopies, we performed linkage analysis on the most affected patients, i.e. patients with low atrial rhythm, and excluded the patient with isolated horizontal P-wave frontal axis as well as the patients with significant rhythm disturbances and normal P-wave frontal axis. Though those patients all carried the disease locus, which implies that the familial disorder shows variable expression. Patient III-11, carrier of the risk allele, had normal echocardiography and ECG at age 45, which also indicates that the disorder is not 100% penetrant. Atrial fibrillation and other atrio-ventricular rhythm disturbances were mainly present in the older individuals in the family, suggesting age-dependant penetrance of these abnormalities. As these abnormalities are com-mon in the general population, it is unclear if they are part of the disease phenotype.In conclusion, we present a large four-generation family in which a condition, comprising of CHDs and low atrial rhythm, inherits as an autosomal dominant trait with variable expression, and which, to the best of our knowledge, has not been described before. A significant genome-wide linkage was demonstrated to a locus on chromosome 9q with a LOD-score of 4.1. Although we did not uncover a causative mutation in this family, the mapping of this locus represents an important step toward the discovery of genes implied in cardiac development. Identification of the disease-causing gene will allow genetic screening and will ultimately provide fundamental insights in human cardiogenesis.

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AcknowledgmentsWe thank all family members for their kind participation. We also thank A. Ilgun, A. Mul, D. van Gent, S. de Jong, F. Salehi and F. Asidah for their excellent technical assistance, S. Romeih, and M. Groenink for the assessment of the MRI and CT images, and H.A.C.M. de Bruin-Bon for performing echocardiographic examinations. We thank dr. R.J. Oostra for helpful discussions.

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scription factor NKX2-5. Science 1998;281:108-113 Garg V, Kathiriya IS, Barnes R et al. GATA4 mutations cause human congenital heart defects and

reveal an interaction with TBX5. Nature 2003;424:443-74 Benson DW, Sharkey A, Fatkin D et al. Reduced penetrance, variable expressivity, and genetic

heterogeneity of familial atrial septal defects. Circulation 1998;97:2043-85 Garg V, Muth AN, Ransom JF et al. Mutations in NOTCH1 cause aortic valve disease. Nature

2005;437:270-46 Balaji S, Harris L. Atrial arrhythmias in congenital heart disease. Cardiol Clin 2002;20:459-687 Postma AV, van de Meerakker JB, Mathijssen IB et al. A gain-of-function TBX5 mutation is associated

with atypical Holt-Oram syndrome and paroxysmal atrial fibrillation. Circ Res 2008;102:1433-428 Fisch C, Knoebel S. Chapter 2: atrial arrhythmias. In: Fisch C, Knoebel S, editors. Electrocardiography

of clinical arrhythmias.Armonk, NY: Futura publishing company, 2000. 9 Walsh EP, Cecchin F. Arrhythmias in adult patients with congenital heart disease. Circulation

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isomerism. Am J Cardiol 1990;65:231-611 Reed PW, Davies JL, Copeman JB et al. Chromosome-specific microsatellite sets for fluorescence-

based, semi-automated genome mapping. Nat Genet 1994;7:390-512 Lindner TH, Hoffmann K. easyLINKAGE: a PERL script for easy and automated two-/multi-point

linkage analyses. Bioinformatics 2005;21:405-713 Sobel E, Lange K. Descent graphs in pedigree analysis: applications to haplotyping, location scores,

and marker-sharing statistics. Am J Hum Genet 1996;58:1323-3714 Uchikawa H, Toyoda M, Nagao K et al. Brain- and heart-specific Patched-1 containing exon 12b is a

dominant negative isoform and is expressed in medulloblastomas. Biochem Biophys Res Commun 2006;349:277-83

15 Zhi J, Hatchwell E. Human MLPA Probe Design (H-MAPD): a probe design tool for both electropho-resis-based and bead-coupled human multiplex ligation-dependent probe amplification assays. BMC Genomics 2008;9:407

16 Koopmann TT, Alders M, Jongbloed RJ et al. Long QT syndrome caused by a large duplication in the KCNH2 (HERG) gene undetectable by current polymerase chain reaction-based exon-scanning methodologies. Heart Rhythm 2006;3:52-5

17 Peeters H, Devriendt K. Human laterality disorders. Eur J Med Genet 2006;49:349-6218 Norgard G, Berg A. Isomerism (Heterotaxia). In: Gatzoulis M, Webb G, Daubeney P, editors. Diagnosis

and management of adult congenital heart disease.Edinburgh: Churchill Livingstone, 2003. 413-21.19 Webber S, Uemura H, Anderson R. Isomerism of the atrial appendages. In: Anderson R, Baker E,

Macartney F, Macartney F, Shinebourne E, Tynan M, editors. Paediatric Cardiology. 2nd ed. New York: Churchill Livingstone, 2002. 813-50.

20 Johnson W, Moller J. Diagnostic Methods. In: Johnson W, Moller J, editors. Pediatric Cardiology. 1 ed. Lippincot, Williams and Wilkins, 2001.

21 Ho SY, Seo JW, Brown NA, Cook AC, Fagg NL, Anderson RH. Morphology of the sinus node in human and mouse hearts with isomerism of the atrial appendages. Br Heart J 1995;74:437-42

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22 Smith A, Ho SY, Anderson RH et al. The diverse cardiac morphology seen in hearts with isomerism of the atrial appendages with reference to the disposition of the specialised conduction system. Cardiol Young 2006;16:437-54

23 Wren C, Macartney FJ, Deanfield JE. Cardiac rhythm in atrial isomerism. Am J Cardiol 1987;59:1156-824 Kakura H, Miyahara K, Sohara H et al. Isolated levocardia associated with absence of inferior vena

cava, lobulated spleen and sick sinus syndrome. A case report. Jpn Heart J 1998;39:235-4125 Wessels MW, De Graaf BM, Cohen-Overbeek TE et al. A new syndrome with noncompaction

cardiomyopathy, bradycardia, pulmonary stenosis, atrial septal defect and heterotaxy with sugges-tive linkage to chromosome 6p. Hum Genet 2008;122:595-603

26 Wiese C, Grieskamp T, Airik R et al. Formation of the sinus node head and differentiation of sinus node myocardium are independently regulated by Tbx18 and Tbx3. Circ Res 2009;104:388-97

27 Bakker ML, Boukens BJ, Mommersteeg MT et al. Transcription factor Tbx3 is required for the speci-fication of the atrioventricular conduction system. Circ Res 2008;102:1340-9

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Mutations in the sarcomere gene MYH7 in Ebstein anomaly

Postma AV, van Engelen K, van de Meerakker JBA, Rahman T, Probst S, Baars MJH, Bauer U, Pickardt T, Sperling SR, Berger F, Moorman AFM, Mulder BJM, Thierfelder L,

Keavney BD, Goodship JA, Klaassen S

Circ Cardiovasc Genet 2011;4(1):43-50

Chapter 3

54

3

Abstract

BackgroundEbstein anomaly is a rare congenital heart malformation characterized by adherence of the septal and posterior leaflets of the tricuspid valve to the underlying myocardium. An association between Ebstein anomaly with left ventricular noncompaction (LVNC) and mutations in MYH7 encoding ß-myosin heavy chain has been shown; here we have screened for MYH7 mutations in a cohort of probands with Ebstein anomaly in a large population-based study.

Methods and ResultsMutational analysis in a cohort of 141 unrelated probands with Ebstein anomaly was performed by next-generation sequencing and direct DNA sequencing of MYH7. Heterozygous mutations were identified in 8 of 141 samples (6%). Seven distinct mutations were found, 5 were novel and 2 were known to cause hypertrophic cardiomyopathy (HCM). All mutations except for 1 3-bp deletion were missense mutations, 1 was a de novo change. Mutation-positive probands and family members showed various congenital heart malformations as well as LVNC. Among 8 mutation-positive probands, 6 had LVNC, whereas among 133 mutation-negative probands, none had LVNC. The frequency of MYH7 mutations was significantly different between probands with and without LVNC accompanying Ebstein anomaly (p<0.0001). LVNC segregated with the MYH7 mutation in the pedigrees of 3 of the probands, 1 of which also included another individual with Ebstein anomaly.

ConclusionsEbstein anomaly is a congenital heart malformation that is associated with mutations in MYH7. MYH7 mutations are predominantly found in Ebstein anomaly associated with LVNC and may warrant genetic testing and family evaluation in this subset of patients.

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IntroductionEbstein anomaly is a rare congenital heart malformation affecting both the tricuspid valve and right ventricle.1 The septal and posterior leaflets of the tricuspid valve are displaced apically and divide the right ventricle into two portions. The effective tricuspid orifice is displaced downward into the right ventricular cavity, at the junction of the inlet and apico-trabecular components of the right ventricle. Tricuspid valve incompetence is the main hemodynamic abnormality in Ebstein malfor-mation. A secundum atrial septal defect (ASDII) is present in more than one-third of patients, and the majority of the remainder have a patent foramen ovale resulting in a right-to-left shunt.2 Abnor-malities of left ventricular morphology and function, as well as other left-sided heart lesions, also occur in Ebstein anomaly,3,4 in one study 18% of patients had left ventricular dysplasia resembling left ventricular noncompaction (LVNC).4

The genetic basis of Ebstein anomaly is largely unresolved. Whilst Ebstein anomaly is more common in patients with a family history of congenital heart disease,5 most cases are sporadic and familial Ebstein anomaly is rare. Mutations in the cardiac transcription factor NKX2.5 are respon-sible for a variety of cardiac structural anomalies including Ebstein anomaly and ASD.6 In 1 LVNC family carrying a mutation in MYH7 encoding the sarcomere gene β-myosin heavy chain (MYH7), 4 individuals had Ebstein anomaly.7

Mutations in sarcomere genes are a major cause of cardiomyopathy. LVNC has recently been classified as a primary cardiomyopathy with a genetic etiology,8 and is morphologically charac-terized by a severely thickened 2-layered myocardium, numerous prominent trabeculations and deep intertrabecular recesses.9 Mutations in 6 sarcomere genes, MYH7, α-cardiac actin (ACTC1), cardiac Troponin T (TNNT2), α-tropomyosin (TPM1), cardiac Troponin I (TNNI3), and cardiac myosin-binding protein C (MYBPC3) have been described in familial or non-familial LVNC.10-14 MYH7 is the most frequent disease gene (13%) in adult patients with LVNC, in the absence of other congenital heart anomalies.12 Interestingly, mutations in ACTC1 have been associated with ASD and cardiomyopathy,15,16 and some individuals have both defects.10 Because a possible association between Ebstein anomaly with LVNC and MYH7 mutations previously was shown, this led us to test the association between Ebstein anomaly and MYH7 mutations in a large cohort. We performed mutational analysis of MYH7 in a cohort of 141 unrelated probands with Ebstein anomaly using both next generation sequencing and direct DNA sequencing. Mutations were identified in 8 of 141 probands (6%), the largest resequencing study of Ebstein anomaly so far. We provide further evidence for a link between structural proteins, cardiomyopathy, and congenital heart malformations.

MethodsClinical EvaluationUnrelated patients were recruited from three sources: (1) CONCOR (National Registry and DNA bank of congenital heart defects), The Netherlands, n=114 (2) National Registry for Congenital

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Heart Defects, Berlin, Germany, n=19, and (3) The Institute of Human Genetics, Newcastle University, United Kingdom, n=8. Informed consent was obtained from all participants according to established guidelines. Probands and available family members were evaluated by history taking, review of medical records, physical examination, 12-lead electrocardiography and transthoracic echocardiography. All probands had a physical exam for dysmorphic features and patients with abnormalities pointing to syndromic features or neuromuscular involvement were excluded. Echo-cardiography in Ebstein anomaly shows apical displacement of the septal leaflet of the tricuspid valve from the insertion of the anterior leaflet of the mitral valve by at least 8 mm/m2 body surface area.3,4 Marked enlargement of the right atrium and atrialized right ventricle may be present as well as varying degrees of regurgitation of the tricuspid valve. The diagnosis of LVNC was made by echocardiography based on the presence of the established criteria by Jenni et al.9 In partially penetrant cases of LVNC, the ratio of noncompacted to compacted myocardium is <2. A diagnosis of LVNC was made irrespective of the presence of heart failure or left ventricular systolic dysfunction.8 Echocardiographic studies were performed/ reviewed by 2 independent observers.

Mutation ScreeningMutation screening was carried out with genomic DNA samples from 141 probands. Fusion primers were designed using Primer3 and IDT primer design portal to amplify MYH7 (Genbank accession number, NM_000257) coding and 5`/3` untranslated regions. 20 ng of genomic DNA was amplified using FastStart HighFidelity enzyme in a total reaction volume of 50 µl. Amplification was performed by initial denaturation at 94°C for 3 minutes followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at the recommended temperatures for 45 seconds, extension at 72°C for 1 minute and a final extension at 72°C for 2 minutes. Amplicons were purified using Solid Phase Reversible Immobilization (SPRI) beads (Beckman Coulter Genomics, England). Amplicon quality was assessed using the DNA 1000 LabChip on an Agilent Bioanalyzer and quantified us-ing Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen). 50 PCR amplicons from 141 patients were pooled at equimolar ratios and sequenced on three GS FLX LR70 PicoTitrePlates. Library immo-bilization and emulsion PCR were performed using GS emPCR kits II and III (Roche). After DNA bead recovery, bead enrichment and sequencing-primer annealing, the DNA beads, Packing beads and Enzyme beads were deposited on a GS FLX PicoTiter Plate and sequenced using GS LR70 Sequencing kit (Roche). GS Reference mapper was used to map sequence reads obtained with reference sequences from the Human genome hg18 assembly (NCBI build 36.1). The average read length was 244bp and average fold coverage of 45X per allele. Putative variants detected by GS Amplicon Variant Analyzer software (Roche) that were supported by both forward and reverse reads or with a variant frequency of >1.0% on either the forward or reverse reads were selected for further analyses by MassARRAY MALDI-TOF (Sequenom) to validate changes and, as the sequencing had been carried out in pooled samples, to identify in which samples they were present. Following this each change was confirmed by Sanger sequencing as previously published.12 When a putative mutation was identified, at least 490 ethnically matched controls

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(980 chromosomes) were screened for the absence of the sequence variation (p<0.0001). The microsatellite marker D14S990 was used to rule out a founder mutation for MYH7.12

Fisher`s exact test was used to analyse non-continuous data, probability values <0.05 were considered significant.The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.

Table 1. Cardiovascular anomalies of probands

ASDII, secundum atrial septal defect; VSD, ventricular septal defect; PFO, patent foramen ovale; CCTGA, congeni-tally corrected transposition of the great arteries.

Phenotype of probands n

Ebstein only 64

ASD II 42

PFO 7

Left ventricular noncompaction (with or without ASD, VSD, PFO, pulmonary artery hypoplasia) 6

CCTGA (with or without ASD, VSD, left ventricular outflow tract obstruction) 5

ASD, VSD 3

Pulmonary valve stenosis 2

VSD 1

VSD, PFO 2

Coarctation of the aorta 2

ASD II, PFO 1

Aneurysm of membraneous septum 1

Pulmonary valve stenosis and VSD 1

Aortic valve stenosis 1

Aortic valve abnormality 1

Partial anomalous pulmonary venous connections, sinus venosus ASD, PFO 1

Hypertrophic cardiomyopathy, left ventricular outflow tract obstruction 1

Total 141

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ResultsA cohort of 141 unrelated Caucasian individuals of western European descent (61 men and 80 women; 138 adults and 3 children; mean age, 46 years; range 4 months to 77 years) with the diagnosis of Ebstein anomaly were investigated. Sixty-four probands had no associated cardiac anomalies, the most common associated cardiac malformation being ASDII (48 probands) and cardiomyopathy (7 probands) (Table 1). Heterozygous mutations were identified in 8 of 141 samples (6%). Seven distinct mutations were found, 5 were novel and 2 were known to cause HCM. Two probands had the same mutation. All mutations except for 1 3-bp deletion are mis-sense mutations. In 6/8 probands with MYH7 mutations LVNC was identified in addition to Ebstein anomaly. One of 8 mutation-positive probands had partially penetrant LVNC (kindred 109.787, III-2, Figure 1), and in 1 other proband (AD) LVNC was uncertain. MYH7 mutations were not reported in the subcohort of 133 probands with Ebstein anomaly without evidence of LVNC. The frequency of MYH7 mutations between those Ebstein patients with LVNC and those without LVNC

Figure 1. Pedigrees of kindreds with MYH7 mutations. A, 110.647 (Tyr283Asp); B, 110.240 (Asn1918Lys); C, 109.787 (Glu1573Lys); and D, 16875 (Tyr283Asp). Filled symbols indicate cardiovascular phenotype by a box in the left rectangle/circle for Ebstein anomaly, in the upper right quadrant for left ventricular noncompaction (LVNC), and in the right lower quadrant for other cardiovascular malformation (CVM). Open symbols, normal cardiovascular phenotype; shaded symbols, uncertain clinical status. Plus signs indicate the presence of a mutation; minus signs, absence of a mutation.

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was significant (p=2 e-07, p<0.0001). Clinical phenotypes were assessed in all available family members of the 8 probands with mutations and familial congenital heart malformations or cardio-myopathy was found in 3 of them. In these kindreds MYH7 mutations segregated with LVNC and there was an additional individual with Ebstein anomaly (110.647). The clinical characteristics of all family members with mutations are presented in Table 2 and described below.

Familial casesKindred 110.647We identified a missense mutation at nucleotide 933 in exon 10, which replaces tyrosine with aspartic acid at residue 283 (designated Tyr283Asp), in the proband (III-6). She had been diagnosed with Ebstein anomaly and ASDII at 29 years of age, which were surgically corrected.

Figure 2. Echocardiographic images of affected individuals. A, Apical 4-chamber view, individual IV:2 of family 110.647 with Ebstein anomaly and LVNC. B, The same patient as in A; color Doppler image showing deep rececesses filled with blood from the left ventricular cavity at end-diastole. C, Modified 4-chamber view, patient III:1 of family 16875, demonstrates the atrialized portion of the right ventricle in between the enlarged right atrium and small right ventricle and the thickened left ventricle. D, The same patient as in C; this color Doppler apical 4-chamber view (at end-diastole) demonstrates the left ventricle with prominent trabeculations and deep recesses. E, End-diastolic, and F, End-systolic still frame, apical 4-chamber view, patient III:4 of family 110.240 with Ebstein anomaly and LVNC. G, End-systolic apical 4-chamber view, individual II:2 with LVNC without Ebstein anomaly of the same family 110.240. H, End-diastolic apical 2-chamber view, individual II:2 of family 110.240.

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At age 49, echocardiography revealed LVNC with abnormal LV diastolic function. Family screening identified Ebstein anomaly and LVNC in the proband’s 24-year-old niece (IV-2) (Figure 2A and 2B). Individuals III-2 and III-3 showed only mild left ventricular apical hypertrabeculation (partially penetrant phenotype). III-10 had been diagnosed with a perimembranous VSD at 18 years. MRI was performed at age 59 because of palpitations and the unexplained sudden death in her 40-year old sister (III-9), and showed marked LVNC with mildly abnormal systolic function. A cardio-verter defibrillator was implanted. Her asymptomatic father (II-4) was subsequently diagnosed with LVNC (Figure 1A).

Kindred 110.240In the proband (III-4) a missense mutation (Asn1918Lys) in exon 39 was found. Ebstein anomaly was established after evaluation of a cardiac murmur at 3 years of age. She has always been asymptomatic despite significant tricuspid regurgitation from the age of 30. Marked LVNC was found at age 39 (Figure 2E and 2F). Her youngest son (IV-4) had a bicuspid aortic valve and aortic coarctation, and echocardiography at age 5 years showed LVNC. The proband’s asymptomatic brother (III-1) had LVNC and LV dilatation with LV dysfunction; her mother (II-2) was also found to have LVNC (Figure 2G and 2H). Echocardiography of III-6 could not rule out cardiomyopathy due to poor imaging quality (Figure 1B). Kindred 109.787 A Glu1573Lys missense mutation in exon 34 was detected in the proband (III-2) and her asympto-matic father. Evaluation of a cardiac murmur in the proband’s first year of life led to the diagnoses of Ebstein anomaly and a small doubly committed subarterial VSD. Echocardiography at age 33 years showed mild hypertrabeculation of the apex, not fulfilling the criteria for LVNC (partially pen-etrant phenotype). The parents of the proband had normal echocardiography (Figure 1C).

Kindred 16875The proband (III-1) and his father (II-2) carried the same Tyr283Asp missense mutation as in kindred 110.647. Haplotype analyses ruled out a founder mutation in these two families. The proband presented with Ebstein anomaly, LVNC and pulmonary hypoplasia as a neonate and had an aorto-pulmonary shunt at the 2nd day of life (Figure 2C, 2D). By family screening, his asymptom-atic father was diagnosed with LVNC. The paternal aunt (II-1) was reported to suffer from heart failure and the paternal grandfather (I-1) had received an implantable cardioverter defibrillator (Figure 1D).

Nonfamilial casesThere were 4 sporadic cases with MYH7 mutations. These included 1 de novo mutation; in 3 probands the parental DNA or clinical information was unavailable. In proband AO, who presented with LVNC and LV diastolic dysfunction a 3-basepair in-frame deletion was detected leading to the

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3

Tabl

e 2.

Clin

ical

cha

ract

eris

ticso

f fam

ily m

embe

rs w

ith M

YH

7 m

utat

ions

LVE

D, l

eft v

entri

cula

r end

dias

tolic

dia

met

er; Z

sco

re, n

orm

al re

fere

nce

rang

e -2

< +

2; E

F/FS

, lef

t ven

tricu

lar e

ject

ion

fract

ion/

fract

iona

l sho

rteni

ng; N

L, n

orm

al li

mits

; NA

, not

av

aila

ble;

AF,

atri

al fi

brill

atio

n; N

SAT

, non

sust

aine

d at

rial t

achy

card

ia; C

HF,

con

gest

ive

hear

t fai

lure

; CV

M, c

ardi

ovas

cula

r mal

form

atio

n; D

CM

, dila

ted

card

iom

yopa

thy;

ICD

, in

traca

rdia

c de

fibril

lato

r; E

PI,

elec

troph

ysio

logi

c in

vest

igat

ion;

CV

I, ce

rebr

ovas

cula

r ins

ult;

AS

DII,

sec

undu

m a

trial

sep

tal d

efec

t; P

FO, p

aten

t for

amen

ova

le; V

SD

, ven

tricu

lar

sept

al d

efec

t; TV

, tric

uspi

d va

lve.

* N

onco

mpa

cted

seg

men

ts: N

one=

0; A

pex=

1; A

pex,

mid

vent

ricul

ar w

all=

2; A

pex,

mid

vent

ricul

ar w

all,

basi

s=3;

† ri

ght v

entri

cula

r inv

olve

-m

ent;

§ pa

rtial

ly p

enet

rant

phe

noty

pe; I

nher

itanc

e of

spo

radi

c m

utat

ions

: Not

test

ed, A

O, B

T, a

nd A

D; d

e no

vo, D

B; I

D, p

roba

nds

are

mar

ked

in b

old.

Echo

card

iogr

aphy

Fam

ilyID

Age

(y)

/Sex

Mut

atio

nSi

te o

f LV

NC

*R

V†

LVED

Z-sc

ore

EF/F

S (%

)Ty

pe C

VMC

ardi

ovas

cula

r co

mpl

icat

ions

110.

647

II-4

78/M

p.Y

283D

2no

-2.8

47/N

AN

one

Non

e

III-2

54/F

p.Y

283D

1§no

-1.9

68/3

6N

one

Non

e

III-3

50/M

p.Y

283D

1§no

-2.2

NA

/43

Non

eN

one

III-6

49/F

p.Y

283D

2ye

s-3

.853

/35

Ebs

tein

, AS

DII

TV re

cons

truct

ion,

AS

D c

losu

re, N

SAT

III-1

049

/Fp.

Y28

3D2

no-2

.065

/35

Per

imem

bran

ous

VS

DP

alpi

tatio

ns, I

CD

IV-2

24/F

p.Y

283D

2ye

s-1

.261

/36

Ebs

tein

Syn

cope

110.

240

II-2

61/F

p.N

1918

K2

no0

NA

/34

Non

eN

one

III-1

43/M

p.N

1918

K2

no1.

845

/23

Non

eN

one

III-4

39/F

p.N

1918

K2

no0.

8N

A/3

0E

bste

inN

one

IV-4

5/M

p.N

1918

K3

no1.

962

/30

Coa

rcta

tion

of th

e ao

rta, B

AVC

oarc

tect

omy

109.

787

II-1

66/M

p.E

1573

K0

no-3

.078

/39

Non

eN

one

III-2

33/F

p.E

1573

K1§

no0.

368

/32

Ebs

tein

, P

erim

embr

anou

s V

SD

Non

e

1687

5II-

242

/Mp.

Y28

3D2

no-1

.455

/32

Non

eN

one

III-1

0.4/

Mp.

Y28

3D2

no1.

547

/23

Ebs

tein

, pul

mon

ary

arte

ry h

ypop

lasi

a,

AS

DII

Sur

gery

with

aor

to-p

ulm

onar

y sh

unt,

CH

F

Spo

radi

c

AO

35/M

p.12

20 D

elE

1no

-2.2

55/3

8E

bste

inN

one

Spo

radi

c

DB

26/F

p.Y

350N

2ye

sN

A65

/NA

Ebs

tein

Non

e

Spo

radi

c B

T59

/Mp.

L390

P2

no-7

.060

/32

Ebs

tein

, PFO

AF,

CV

I, TV

reco

nstru

ctio

n, P

FO

clos

ure

Spo

radi

c

AD

58/F

p.K

1459

N0

no-3

.065

/NA

Ebs

tein

NS

AT, E

PI/

abla

tion

62

3

removal of a glutamic acid at residue 1220 in exon 27 (1220delGlu). In proband DB a tyrosine was substituted by an asparagine at residue 350 in exon 12 which was not present in her unaffected parents and had occurred de novo (Tyr350Asp). This patient presented with biventricular noncom-paction with preserved function. In proband BT, a missense mutation (Leu390Pro) was found in exon 13. Cardiac MRI was undertaken as echocardiography of left ventricular morphology was uninformative due to weight-related imaging difficulties, and revealed extensive LVNC. In proband AD a Lys1459Asn substitution in exon 32 was present. Echocardiography of left ventricle morphology was also uninformative; this patient has not had an MRI.

Genetic and clinical evaluation of the cohort with MYH7 mutationsThree of the 7 distinct mutations reside within the genomic sequence of exon 10 to exon 13 of MYH7, which encode the head region of the molecule (Figure 3A). Four mutations are located throughout the rod domain of the β-myosin heavy chain molecule. All identified missense muta-tions affect amino acids with high degrees of conservation throughout evolution, underscoring the functional importance of these residues (Figure 3B). The Tyr350Asn substitution occurred de novo. Together with the observation that none of the mutations are present in more than 980 chromosomes from a control population of western European descent, our findings strongly support a disease-causing role for these mutations.Mutation-positive probands and family members showed various congenital heart disease including ASDII, ventricular septal defect, coarctation of the aorta, bicuspid aortic valve, and pulmonary artery stenosis/ hypoplasia as well as cardiomyopathy including LVNC (Table 2). All individuals with LVNC, including the 3 partially penetrant cases (kindred 110.647, III-2, III-3; kindred 109.787, III-2), carried a MYH7 mutation. There was only 1 mutation carrier with a normal echocardiogram (kindred 109.787, II-3). All individuals without a MYH7 mutation that were part of kindreds with MYH7 positive individuals (Figure 1) had normal echocardiograms. Of the 18 individuals with MYH7 mutations, 16 had LVNC, whereas 9 had Ebstein anomaly (Table 2).The 8 probands with MYH7 mutations were in New York Heart Association class I at the time of genetic evaluation. Except for the infant with additional pulmonary artery hypoplasia (16875, III-1), congestive heart failure had not been present at initial diagnosis. Severe regurgitation required surgical reconstruction of the tricuspid valve in 2 patients. LVNC was always observed in the left ventricular apex and 2 of 8 probands had biventricular involvement. Left ventricular enddiastolic diameters were not enlarged and left systolic function was preserved in 7 of 8 probands (Table 2). Sustained ventricular tachycardias or sudden cardiac deaths (SCD) were not seen in probands, however in kindred 110.647 there had been 1 SCD. In 2 probands non-sustained atrial tachyar-rhythmias were present.

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Figure 3. A, Distribution of the 7 distinct MYH7 mutations in Ebstein anomaly. The resulting amino acid changes in the ß-myosin heavy chain molecule are depicted. B, Alignment of the regions flanking the mutations in MYH7 showing evolutionary conservation of the mutated residues across species. The residues with the amino acid changes are boxed. Dots identify amino acids identical to the one in the human sequence. Accession numbers (FASTA): Human cardiac β myosin heavy chain, NP_000248; rat cardiac α myosin heavy chain, NP058935; chicken fast skeletal myosin heavy chain, NP_001013414; Danio rerio ventricular myosin heavy chain, AAF00096; Drosophila melanogaster muscle myosin heavy chain NP_723999; Caenorhabditis elegans myosin heavy chain, NP_510092.

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DiscussionComprehensive genetic analyses of 141 unrelated probands with Ebstein anomaly identified 8 disease-associated mutations in the gene encoding β-myosin heavy chain. Mutation-positive probands and family members showed various congenital heart malformations as well as LVNC. Signifi-cant pleiotropy and reduced penetrance were characteristic of MYH7 mutation-positive congenital heart malformations. The LVNC phenotype had a higher penetrance with only 1 mutation carrier having a normal echocardiogram. Mutations in MYH7 can cause hypertrophic cardiomyopathy, dilated cardiomyopathy, restrictive cardiomyopathy, and LVNC.17 This study further broadens the spectrum of phenotypes associated with a defect in a structural protein from cardiomyopathies to congenital heart malformations.The main hemodynamic abnormality in Ebstein anomaly producing symptoms is tricuspid regur-gitation.1 When the tricuspid dysplasia or right ventricular myocardial hypoplasia is severe or associations with other cardiac lesions are present clinical symptoms occur in infancy as seen in the proband III:1 of kindred 16875. In contrast, Ebstein anomaly may be asymptomatic in adolescents and adults,1 as in most of the mutation-positive probands in the present cohort. In such cases supraventricular arrhythmias may be the main clinical problem as in probands BT and AD. The frequency of MYH7 mutations between those patients with LVNC and those without LVNC was significantly different. All 8 MYH7 mutations were found in 8 probands with LVNC or with LVNC being partially penetrant or uncertain. There were no MYH7 mutations in 133 probands without LVNC. Since there was no family screening by echocardiography in the 133 mutation-negative probands the true prevalence of cardiomyopathy or congenital heart malformations in the families of the 133 mutation-negative probands remained unknown. In mutation-positive probands several family members were shown to have congenital heart malformations as well as LVNC, of which some were not known before family screening. In Ebstein anomaly associated with muta-tions in NKX2.5 mutations carriers were also more likely to have a positive history of heart disease in the young.6 Familial Ebstein anomaly was found in 1 kindred (110.647). In general, familial Ebstein anomaly is rare and only a few families with autosomal dominant inheritance have been described.18,19 Several genetic loci for Ebstein anomaly have been reported in humans and in animal models. Chromosomal abnormalities as well as mutations in NKX2.5 were found in patients with Ebstein anomaly.6,20 Andelfinger et al.21 demonstrated linkage between tricuspid valve formation and canine chromosome 9 in a region syntenic to human chromosome 1q12-q23. Of interest, penetrance of Ebstein anomaly in the dog was estimated to be 68%. This may represent the poly-genic or multifactorial inheritance pattern proposed in humans with Ebstein anomaly.A significant number of patients with Ebstein anomaly have morphofunctional abnormalities of the left ventricle which may be explained by increased fibrosis of the left ventricular wall and septum as demonstrated by histological studies.22 Attenhofer Jost et al.4 reviewed 106 consecu-tive patients with Ebstein anomaly and LVNC was identified in 18%. Also, in several other studies Ebstein anomaly was associated with LVNC.4,18,23 In 1 large family with autosomal dominant LVNC and Ebstein anomaly a MYH7 mutation was found.7 Ebstein anomaly in families with autosomal

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inheritance of LVNC18,23 might represent a specific subtype with a Mendelian inheritance pattern. The present study supports the association between Ebstein anomaly with LVNC and MYH7 mutations. Clinical and genetic evaluation is recommended to facilitate the diagnosis of cardio-myopathy and congenital heart disease in probands and their first-degree relatives.14,24

Mutations in MYH7 are a common cause for hypertrophic cardiomyopathy, and well recognised in dilated cardiomyopathy and LVNC. Mutations are distributed throughout the molecule and the relationship between mutation location, cardiomyopathy type, and disease severity is poorly understood.17 The first link between sarcomeric proteins and congenital heart malformations was provided by Ching et al. by identifying a mutation in α-myosin heavy chain (MYH6) through genetic linkage analysis.25 Later, a founder mutation in ACTC1 was identified in two families with autosomal dominant ASD, in the absence of other cardiac anomalies.15 How mutations in sarcomere protein genes could have detrimental effects on cardiac morphogenesis and produce septal defects and valve anomalies should be subject to further investigations. As LVNC is thought to result from altered regulation in cell proliferation, differentiation, and maturation during wall formation,26 arrest in directional growth could account for the association of Ebstein anomaly and LVNC.27,28

ConclusionsEbstein anomaly is within the diverse spectrum of cardiac morphologies associated with mutations in the gene encoding β-myosin heavy chain. MYH7 mutations are predominantly found in Ebstein anomaly associated with LVNC. In the subset of patients with Ebstein anomaly carrying a MYH7 mutation genetic and clinical evaluation of family members is recommended to identify congenital heart malformations and cardiomyopathy.

AcknowledgementsWe thank A. Ilgun and M. Sylva for technical assistance and A. Schalinski for assistance with the recruitment of patients.

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2 Frescura C, Angelini A, Daliento L, Thiene G. Morphological aspects of Ebstein’s anomaly in adults. Thorac Cardiovasc Surg. 2000;48:203-208.

3 Attenhofer Jost CH, Connolly HM, Warnes CA, O’Leary P, Tajik AJ, Pellikka PA, Seward JB. Non-compacted myocardium in Ebstein’s anomaly: initial description in three patients. J Am Soc Echocardiogr. 2004;17:677-680.

4 Attenhofer Jost CH, Connolly HM, O’Leary PW, Warnes CA, Tajik AJ, Seward JB. Left heart lesions in patients with Ebstein anomaly. Mayo Clin Proc. 2005;80:361-368.

5 Correa-Villasenor A, Ferencz C, Neill CA, Wilson PD, Boughman JA, for the Baltimore-Washington Infant Study Group. Ebstein’s malformation of the tricuspid valve: genetic and environmental factors. Teratology. 1994;50:137–147.

6 Benson DW, Silberbach GM, Kavanaugh-McHugh A, Cottrill C, Zhang Y, Riggs S, Smalls O, Johnson MC, Watson MS, Seidman JG, Seidman CE, Plowden J, Kugler JD. Mutations in the cardiac transcription factor NKX2.5 affect diverse cardiac developmental pathways. J Clin In-vest.1999;104:1567–1573.

7 Budde BS, Binner P, Waldmuller S, Hohne W, Blankenfeldt W, Hassfeld S, Bromsen J, Dermintzo-glou A, Wieczorek M, May E, Kirst E, Selignow C, Rackebrandt K, Muller M, Goody RS, Vosberg HP, Nurnberg P, Scheffold T. Noncompaction of the ventricular myocardium is associated with a de novo mutation in the beta-myosin heavy chain gene. PLoS ONE. 2007;2:e1362.

8 Maron BJ, Towbin JA, Thiene G, Antzelevitch C, Corrado D, Arnett D, Moss AJ, Seidman CE, Young JB; Contemporary definitions and classification of the cardiomyopathies: an American Heart Associa-tion Scientific Statement from the Council on Clinical Cardiology, Heart Failure and Transplantation Committee; Quality of Care and Outcomes Research and Functional Genomics and Translational Biology Interdisciplinary Working Groups; and Council on Epidemiology and Prevention. Circulation. 2006;113:1807–1816.

9 Jenni R, Oechslin E, Schneider J, Attenhofer Jost C, Kaufman PA. Echocardiographic and pathoana-tomical characteristics of isolated left ventricular non-compaction: a step towards classification as a distinct cardiomyopathy. Heart 2001;86:666-671.

10 Monserrat L, Hermida-Prieto M, Fernandez X, Rodriguez I, Dumont C, Cazn L, Cuesta MG, Gonza-lez-Juanatey C, Jesús Peteiro J, Alvarez N, Penas-Lado M, Castro-Beiras A. Mutation in the alpha-cardiac actin gene associated with apical hypertrophic cardiomyopathy, left ventricular non-compac-tion, and septal defects. Eur Heart J. 2007;28:1953-1961.

11 Hoedemaekers YM, Caliskan K, Majoor-Krakauer D, van de Laar I, Michels M, Witsenburg M, ten Cate FJ, Simoons ML, Dooijes D. Cardiac beta-myosin heavy chain defects in two families with non-compaction cardiomyopathy: linking noncompaction to hypertrophic, restrictive, and dilated cardio-myopathies. Eur Heart J. 2007;28:2732-2737.

12 Klaassen S, Probst S, Oechslin E, Gerull B, Krings G, Schuler P, Greutmann M, Hurlimann D, Yegit-basi M, Pons L, Gramlich M, Drenckhahn JD, Heuser A, Berger F, Jenni R, Thierfelder L. Mutations in sarcomere protein genes in left ventricular noncompaction. Circulation. 2008;117:2893-2901.

13 Dellefave LM, Pytel P, Mewborn S, Mora B, Guris DL, Fedson S, Waggoner D, Moskowitz I, McNally EM. Sarcomere mutations in cardiomyopathy with left ventricular hypertrabeculation. Circ Cardio-vasc Genet. 2009;2:442-449.

14 Hoedemaekers YM, Caliskan K, Michels M, Frohn-Mulder I, van der Smagt EJG, Phefferkorn JE, Wessels MW, ten Cate FJ, Sijbrands EJG, Dooijes D, Majoor-Krakauer DF. The importance of ge-netic counseling, DNA diagnostics, and cardiologic family screening in left ventricular noncompaction cardiomyopathy. Circ Cardiovasc Genet. 2010;3:232-239.

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15 Matsson M, Eason J, Bookwalter CS, Klar J, Gustavsson P, Sunnegårdh J, Enell H, Jonzon A, Vikkula M, Gutierrez I, Granados-Riveron J, Pope M, Bu’Lock F, Cox J, Robinson TE, Song F, Brook2 DJ, Marston S, Trybus M, Dahl N. Alpha-cardiac actin mutations produce atrial septal defects Hum Mol Genet. 2008;17:256-265.

16 Mogensen J, Klausen IC, Pedersen AK, Egeblad H, Bross P, Kruse TA, Gregersen N, Hansen PS, Baandrup U, Borglum AD. Alpha-cardiac actin is a novel disease gene in familial hypertrophic car-diomyopathy. J Clin Invest. 1999;103:R39–43.

17 Walsh R, Rutland C, Thomas R, Loughna S. Cardiomyopathy: a systematic review of disease-causing mutations in myosin heavy chain 7 and their phenotypic manifestations. Cardiology. 2010;115:49-60.

18 Sinkovec M, Kozelj M, Podnar T. Familial biventricular myocardial noncompaction associated with Ebstein’s malformation. Int J Cardiol. 2005;102:297-302.

19 Schunkert H, Brockel U, Kromer EP, Elsner D, Jacob HJ, Riegger GA. A large pedigree with valvu-loseptal defects. Am J Cardiol. 1997;80:968-970.

20 Miller MS, Rao PN, Dudovitz RN, Falk RE. Ebstein anomaly and duplication of the distal arm of chro-mosome 15: report of two patients. Am J Medical Genet A. 2005;139A:141-145.

21 Andelfinger G, Wright KN, Lee HS, Siemens LM, Benson DW. Canine tricuspid valve malformation, a model of human Ebstein anomaly, maps to dog chromosome 9. J Med Genet. 2003;40:320-324.

22 Daliento L, Angelini A, HoSY, Frescura C, Turrini P, Baratella MC, Thiene G, Anderson RH. Angio-graphic and morphologic features of the left ventricle in Ebstein malformation. Am J Cardiol. 1997;80: 1051-1059.

23 Bagur RH, Lederlin M, Montaudon M, Latrabe V, Corneloup O, Iriart X, Laurent F. Images in cardiovascular medicine. Ebstein anomaly associated with left ventricular noncompaction. Circula-tion. 2008;118:e662-664.

24 Klaassen S, Focused Review: Ventricular noncompaction: An update. In: Libby P, Bonow RO, Mann DL, Zipes DP, eds. Braunwald`s Heart Disease, A Textbook of Cardiovascular Medicine, Philadel-phia: Saunders Elsevier, 2008, 8th E-dition, Part VIII, Chapter 65

25 Ching YH, Ghosh TK, Cross SJ, Packham EA, Honeyman L, Loughna S, Robinson TE, Dearlove AM, Ribas G, Bonser AJ, Thomas NR, Scotter AJ, Caves LS, Tyrrell GP, Newbury-Ecob RA, Munnich A, Bonnet D, Brook JD. Mutation in myosin heavy chain 6 causes atrial septal defect. Nat Genet. 2005;37:423-428.

26 Chen H, Zhang W, Li D, Cordes TM, Payne RM, Shou W. Analysis of Ventricular Hypertrabeculation and Noncompaction Using Genetically Engineered Mouse Models Pediatr Cardiol. 2009;30:626-63.

27 de Lange FJ, Moorman AFM, Anderson RH, Männer J, Soufan AT, de Gier-de Vries C, Schneider MD, Webb S, van den Hoff MJB, Christoffels VM. Lineage and Morphogenetic Analysis of the Cardiac Valves. Circ Res. 2004;95:645-654.

28 Meilhac SM, Esner M, Kerszberg M, Moss JE, Buckingham ME. Oriented clonal cell growth in the developing mouse myocardium underlies cardiac morphogenesis. J Cell Biol. 2004;164:97-109.

Mutations in the cardiac sodium channel gene SCN5A and congenital heart disease in humans

van Engelen K, Postma AV, Beekman L, Hofman N, Alders M, Misuzawa Y, van der Werf C, Kolder I, Baars MJH, Tan HL, Mulder BJM,

Wilde AAM, Bezzina CR

Submitted

Chapter 4

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AbstractBackgroundGenetic factors contributing to congenital heart disease (CHD) are largely unknown. Recent animal studies have demonstrated that ion channels are involved in heart development. The aim of our study was to explore the association between mutations in the cardiac sodium channel gene SCN5A and CHD in humans.

Methods and resultsFirstly, we reviewed cardiac imaging reports for evidence of CHD in a consecutive cohort of SCN5A mutation carriers. Six of 192 (3.1%) loss-of-function mutation carriers with avail-able imaging reports had CHD, five of which were septal defects. The CHD prevalence in our cohort was significantly higher than reported in the general population (P = 0.002). None of 42 gain-of-function mutation carriers had CHD. Secondly, we sequenced SCN5A in 164 patients with a septal defect and conduction disease and identified a (possibly) pathogenic mutation in six patients (3.7%).

ConclusionsOur results suggest a role for SCN5A loss-of-function mutation in CHD in humans, particularly septal defects. As SCN5A mutations are present in almost 4% of patients with septal defects and conduction disease, we recommend low threshold genetic testing in these patients.

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IntroductionCongenital heart disease (CHD) ranks among the most common human congenital defects, affecting approximately 8 per 1,000 live-births,1 and is a major cause of morbidity and mortality throughout life. Mutations in several genes, including transcription factors, membrane receptors/ligands, sarcomeric proteins and more recently histone-modifying genes, have been found in a small subset of patients, but (genetic) factors contributing to CHD remain unknown in the large majority.2-6 In a substantial proportion of CHD patients, rhythm- and conduction disorders are present. The substrate for these rhythm- and conduction disorders is often complex and deter-mined by several factors, including cellular injury from hypoxia and intracardiac shunting leading to chronic volume and pressure overload, ventricular dysfunction and hypertrophy. In addition, surgical procedures may result in conduction disease by causing direct trauma to conduction tissue and fibrosis at suture lines.7 However, CHD-associated rhythm- and conduction disorders may also arise from the pathophysiological process that underlies the CHD itself. Several genetic alterations are known to cause both structural heart disease and developmental abnormalities of the cardiac conduction system. Examples include mutations in the cardiac transcription factor NKX2-5 leading to secundum type atrial septal defect (ASDII) associated with atrioventricular conduction delay,2,8 and mutations in TBX5 causing Holt-Oram syndrome characterized by upper limb malformations, septal defects and conduction disease.9,10 Several other genes are known to be involved in human conduction disorders without the pres-ence of gross structural heart disease. One such gene is SCN5A, which encodes the α-subunit of the voltage-gated cardiac sodium channel. Opening of the channel allows a rapid influx of Na+ ions, quickly depolarizing the membrane potential of cardiac cells and triggering the action potential. The sodium channel, among other ion channels, thereby plays a crucial role in cardiac excitability and conduction of the cardiac impulse. Mutations in the SCN5A gene are a well estab-lished cause of several arrhythmia syndromes, including Brugada syndrome, Long QT syndrome (LQTS) type 3 and cardiac conduction disease (reviewed in 11). Interestingly, recent animal studies have shown that voltage-gated sodium channels are also involved in embryonic heart develop-ment.12-14 Zebrafish knockouts for SCN5A homologues exhibited developmental abnormalities of the cardiac chamber and perturbed looping.13 These studies led us to speculate that human SCN5A mutation carriers may also be at risk for developmental abnormalities of the heart, including CHD. In this study, we explored the association between SCN5A mutations and CHD in humans, by 1) determination of the prevalence of CHD in a consecutive cohort of SCN5A loss-of-function and gain-of-function mutation carriers, and 2) mutation analysis of SCN5A in patients with septal defects and conduction disease or certain dysrhythmias.

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Patients and methods

Prevalence of congenital heart disease in SCN5A mutation carriersIn this retrospective analysis, we selected all loss-of-function and gain-of-function SCN5A muta-tion carriers (probands and relatives) who were counseled before January 2011 at the clini-cal genetics department of one university hospital (Academic Medical Center Amsterdam). All carriers were of Dutch ancestry (self-reported). Probands had been ascertained because of suspected or confirmed electrical heart disease or sudden unexplained death in the family. SCN5A mutation-carrying relatives had been identified by cascade screening after identification of a mutation in the proband. DNA-analysis had been performed in a diagnostic setting in DNA extracted from peripheral blood lymphocytes or from stored tissue specimens after autopsy. The coding regions of SCN5A were screened using standard procedures as described previously.15 For mutation classification we used a list of mutation-specific features based on in silico analysis using the mutation interpretation software AlaMut (version 1.5). A score was given depending on the outcome of a prediction test for each feature (i.e. Grantham distance, Polyphen, SIFT, evolutionary conservation). Then, depending on the total score and the availability of the variant in ethnically and preferably demographically matched control alleles (data obtained from the literature, genome and exome databases, e.g. 1000 Genomes,16 Exome Variant Server17 and GoNl18 or from own control alleles) mutations were classified as pathogenic, not pathogenic, or as a variant of unknown clinical significance (VUS1, unlikely to be pathogenic; VUS2, uncertain; or VUS3, likely to be pathogenic).Family information (co-segregation) and/or functional analysis are needed to classify a variant as pathogenic. For this, we used strict criteria (details available on request). In the current study, we included patients with VUS3 or a pathogenic mutation. Mutations identified in families with a primary phenotype of Brugada syndrome, (progressive) conduction disease, sick sinus syndrome or atrial standstill were classified as (presumably) loss-of-function mutations, whereas those found in families with LQTS were classified as gain-of-function mutations. Carriers with loss-of-function mutations and those with gain-of-function mutations were analyzed separately.To determine the presence of CHD we reviewed records of heart imaging (echocardiography or cardiac magnetic resonance imaging (CMR), whichever available) or autopsy records. If no records were available within our own university hospital, records were collected from other hospitals. All subjects provided written informed consent for the use of their medical and genetic data for research purposes.

Data analysisPrevalence of CHD in our SCN5A mutation carrier cohort was defined as the proportion of indi-viduals with CHD within the cohort. We compared the prevalence of CHD in our total cohort to the prevalence of CHD in the general population as reported by Marelli et al.19 We chose this control

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population, because it comprised adults as well as children, as our own cohort. Marelli et al. deter-mined the CHD prevalence by use of administrative databases and reported a CHD prevalence of 5.78 per 1,000 individuals of all age groups.19 We also analyzed children and adults separately. The prevalence of CHD in children (aged 17 years or younger) in our cohort was compared to data from EUROCAT Northern Netherlands (1981-2011), a network that registers all congenital anomalies detected before the age of 10 years after report by midwives, general practitioners and specialists, which reported 6.57 CHD per 1,000 live births.20 The prevalence of CHD in adults in our cohort was compared to data from Van der Bom et al., who recently estimated the prevalence of CHD in the general adult population to be 3 per 1,000 individuals.21 Data were compared using the z-test for proportion. P < 0.05 was considered to be significant.

SCN5A mutation analysis in patients with a septal defect and conduction diseasePatients were selected from CONCOR, the national registry database and DNA bank for adults with CHD, described in detail previously.22 In CONCOR, patients are ascertained through their

Male 86 (52)

Age in years, median (range) 42 (20-88)

Septal defect (primary diagnosis)

VSD 62 (38)

Secundum ASD 44 (27)

Primum ASD 26 (16)

ASD - not specified 22 (13)

Complete AVSD 9 (6)

Common atrium 1 (1)

Conduction disease and dysrhythmias *

1st degree AV-block 29 (18)

2nd degree AV-block 4 (2)

Complete AV-block 10 (6)

Complete LBBB 6 (4)

Complete RBBB 85 (52)

Congenital complete heart block 3 (2)

Sinus bradycardia 17 (10)

Sick sinus syndrome 29 (18)

VF 4 (2)

Numbers are n (%), unless otherwise indicated, VSD, ventricular septal defect; ASD, atrial septal defect; AVSD, atrio-ventricular septal defect; LBBB, left bundle branch block; RBBB, right bundle branch block; VF, ventricular fibrillation. * Multiple types of conduction disease/dysrhythmias could be present within one patient.

Table 1. Clinical details of patients with septal defects and conduction disease screened for SCN5A mutations (n = 164)

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cardiologist or advertisements in the local media. Approximately 75% of registered patients origi-nate from tertiary referral centers. Diagnoses, procedures and clinical events are classified with the use of the European Pediatric Cardiac Code short list coding scheme. DNA is isolated from peripheral blood and stored for research purposes. For the current study, we selected all patients with a primary diagnosis of any type of septal defect and conduction disease or certain dysrhyth-mias (as registered in CONCOR); for included diagnoses see Table 1. We chose to study septal defects, as these were the most common type of CHD we encountered in the SCN5A mutation carrier cohort in the first part of our study. Only patients with available DNA were included. Patients with known genetic syndromes and/or intellectual disability were excluded. The entire coding region of SCN5A was PCR-amplified and sequenced by Sanger sequencing using standard methods (GenBank accession number M77235.1). Identified variants were classi-fied as described above.

ResultsPrevalence of congenital heart disease in SCN5A mutation carriersLoss-of-function mutation carriersFourty-five different (presumably) loss-of-function SCN5A mutations (see supplemental material) had been identified in 257 consecutive individuals from 74 families at the clinical genetics department of our university hospital. The predominant phenotype was Brugada syndrome in 59 families, (progressive) conduction disease in 12 families, sick sinus syndrome in two families and atrial standstill in one family. Imaging of the heart or autopsy records were available in 192 of 257 (75%) mutation carriers (51% male, median age at DNA-analysis 44 years (range 0 to 80,5 years): echocardiography in 145 (76%), MRI in 44 (23%) and autopsy records in three (2%) carriers. Median age at cardiac imaging or autopsy was 45 years (range 0 – 77 years). There were no significant differences in age and gender between mutation carriers with and without available imaging. Six of 192 (3.1%) mutation carriers with available imaging/autopsy records showed evidence of CHD: four mutation carriers had VSD, whereas ASDII and tetralogy of Fallot (TOF) were each present in one carrier. Table 2 shows details of these individuals. Two patients carried a p.Phe861TrpfsX9 mutation and two carried a p.Gly1743Glu mutation; those patients were not closely related. One patient was a compound heterozygote for two SCN5A mutations (p.Arg225Trp and p.Trp156X); this patient has been reported previously.23 The prevalence of CHD in our total cohort was significantly higher than the prevalence in the general population as reported by Marelli et al.19 (P = 0.002). The prevalence of CHD in children was significantly higher than reported by EUROCAT20 (P = 0.014) and the prevalence in adults was significantly higher than reported by Van der Bom et al.21 (P = 0.003). If we assume that all SCN5A carriers without available imaging of the heart were free of CHD, the prevalence rates of CHD in our total cohort as well as in children and adults separately would still be higher than in the control populations (P = 0.008, P = 0.044 and P = 0.010, respectively).

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4

Tabl

e 2.

Pat

ient

s w

ith c

onge

nita

l hea

rt di

seas

e fro

m th

e co

hort

of S

CN

5A lo

ss-o

f-fun

ctio

n m

utat

ion

carr

iers

PtSe

xM

utat

ion

Mut

atio

n cl

ass

CH

DA

ge a

t CH

D

dete

ctio

n (y

rs)

Age

at

SCN

5A

mut

atio

n de

tect

ion

(yrs

)

Asc

erta

in-

men

t for

ge

netic

test

-in

g

Surg

ery

for C

HD

Con

duct

ion

dise

ase

1M

c.52

28 G

>Ap.

Gly

1743

Glu

Mut

TOF

02

Rel

ativ

eye

s

2 *

Fc.

5228

G>A

p.G

ly17

43G

lu

Mut

AS

DII

Chi

ldho

od30

Rel

ativ

eno

Sin

us b

rady

car-

dia,

PQ

0.2

8 s,

Q

RS

0.1

2 s

3F

c.25

82_2

583d

elTT

p.P

he86

1Trp

fsX

90

Mut

Mus

cula

r VS

D2

37R

elat

ive

no1s

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2nd

de-

gree

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blo

ck,

QR

S 0

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s

4F

c.25

82_2

583d

elTT

p.P

he86

1Trp

fsX

90

Mut

Mem

bran

eous

V

SD

044

Rel

ativ

eno

RB

BB

, PQ

0.2

0 s,

QR

S 0

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s

5F

c.45

42+2

_3de

lTG

Mut

VS

D2

50P

roba

ndno

RB

BB

, Typ

e 3

ST

elev

atio

n,

PQ

0.2

0 s,

Q

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0.1

2 s

6 **

Mc.

673C

>T +

c.

468G

>A(c

ompo

und

he

tero

zygo

us)

p.A

rg22

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+

p.Tr

p156

XM

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a-ne

ous

VS

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fter b

irth

0 y

Pro

band

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ide

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plex

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chyc

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prol

onge

d co

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s

(pro

gres

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)

Pt,

patie

nt; M

ut, p

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geni

c m

utat

ion;

TO

F, te

tralo

gy o

f Fal

lot;

AS

D, a

trial

sep

tal d

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t; V

SD

, ven

tricu

lar s

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l def

ect;

RB

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ht b

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anch

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Thi

s pa

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was

al

so id

entifi

ed in

the

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NC

OR

coh

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f pat

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. ** T

his

patie

nt w

as re

porte

d pr

evio

usly.

23

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4

Gain-of-function mutation carriersFour different gain-of-function mutations had been identified in 12 families with LQTS (Supple-mental material). These families consisted of 42 mutation carriers. Information regarding the pres-ence of CHD was available in 22 (52%) carriers (48% male, median age at DNA-analysis 37 years (range 0 to 75 years): echocardiography in 20, MRI in one and autopsy records in one carrier. Median age at cardiac imaging or autopsy was 37 years (range 0 – 77 years). None of the muta-tion carriers with or without available imaging showed evidence of CHD.

SCN5A mutation analysis in patients with a septal defect and conduction diseaseIn the CONCOR national registry and DNA bank, DNA of 164 patients with a septal defect and conduction disease or dysrhyhmias was available. Clinical details of these patients are shown in Table 1. Six different (possibly) pathogenic mutations were identified in six (3.7%) patients. Table 3 gives details of these mutations and clinical features of the patients. Coincidentally, the patient found to carry the p.Gly1743Glu mutation had also been identified in our SCN5A mutation carrier cohort from the first part of this study (patient 2). In an additional six patients (3.7%) a rare variant was identified (p.Pro2006Ala in three patients; p.Phe2004Leu in one patient; p.Ala572Asp in two patients). The total proportion of rare variants in our cohort is not different from reports in healthy white controls.24

Spectrum of SCN5A mutations in patients with congenital heart diseaseIn the two cohorts combined, we identified 10 different (possibly) pathogenic mutations in a total of 11 patients with CHD. There were two splice-site mutations (p.Gly1573Gly and c.4542+2_3delTG), one frameshift mutation (p.Phe861TrpfsX90), one nonsense mutation (p.Trp156X) and six missense mutations (p.Gly1743Glu, p.Arg225Trp, p.Val1543Ala, p.Arg1232Glu, p.Gly1420Arg, p.Pro701Leu). The mutations were localized throughout the cardiac sodium channel (Figure 1).

Figure 1. Schematic representation of the α-subunit of SCN5A protein in which the position of the 10 mutations identified in patients with congenital heart disease is shown.

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4

Tabl

e 3.

Cha

ract

eris

tics

of p

atie

nts

with

an

SCN

5A m

utat

ion

from

the

coho

rt of

pat

ient

s w

ith s

epta

l def

ects

and

con

duct

ion

dise

ase

scre

ened

for S

CN

5A m

utat

ions

Pt,

patie

nt; M

ut, p

atho

geni

c m

utat

ion;

VU

S, v

aria

nt o

f unk

now

n si

gnifi

canc

e; A

SD

I, pr

imum

atri

al s

epta

l def

ect;

VS

D, v

entri

cula

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ptal

def

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AS

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sec

undu

m a

trial

sep

tal

defe

ct; I

VC

, inf

erio

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a ca

va; R

BB

B, r

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in th

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(yrs

)M

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ph

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M

utA

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I, V

SD

Con

geni

tal m

itral

re

gurg

itatio

n

1st d

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e

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lock

yes

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44

2 *

F / 3

2c.

5228

G>A

p.G

ly17

43G

luM

utA

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II1s

t deg

ree

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blo

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oB

rs 45

-47

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/ 53

c.36

95 G

>Ap.

Arg

1232

Gln

VU

S3

VS

DS

ick

sinu

s sy

ndro

me

yes

DC

car

-di

over

sion

P

acem

aker

Brs

45

4F

/ 78

c.42

58 G

>Cp.

Gly

1420

Arg

V

US

3A

SD

II,S

econ

dary

pul

mon

ary

hype

rtens

ion

Sin

us b

rady

-ca

rdia

, 1s

t deg

ree

AV

blo

ck,

Atri

al fl

utte

r

yes

Pac

emak

er

Brs

4 5

5M

/ 28

c.21

02 C

>Tp.

Pro

701L

euV

US

2P

erim

embr

anou

s V

SD

Left

ante

rior

hem

iblo

ck,

1st d

egre

e

AV b

lock

yes

B

rs, L

QTS

45

,48

6F

/ 41

c.46

28 T

>Cp.

Val1

543A

laV

US

2C

omm

on a

trium

, Tru

e cl

eft o

f mitr

al

valv

eA

zygo

us c

ontin

uatio

n of

IVC

Pul

mon

ary

arte

rial h

yper

tens

ion

Sup

rava

lvar

pul

mon

ary

trunk

ste

nosi

s

Sic

k si

nus

synd

rom

e,

Com

plet

e R

BB

B1s

t deg

ree

AV

blo

ck

yes

Pac

emak

erP

artia

l situ

s in

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DiscussionIn this study we explored the association between SCN5A mutations and congenital heart dis-ease in humans, with a focus on cardiac septal defects. Key findings of our study are: 1) a CHD prevalence of 3.1% among SCN5A loss-of-function mutation carriers, which is significantly higher than in the general population, 2) the most prevalent types of CHD in SCN5A mutation carriers are septal defects, and are present in five of six patients with CHD, and 3) 3.7% of patients with a septal defect and conduction disease carried a pathogenic mutation in SCN5A. The results of our study suggest a role of SCN5A loss-of-function mutations in human CHD. In humans, there have been incidental reports of CHD in SCN5A mutation carriers with various electrical phenotypes, including Brugada syndrome, (progressive) conduction disease, LQTS and overlapping syndromes.23,25-27 However, as in reports on SCN5A mutation carriers the presence of CHD was not systematically analyzed, the frequency of CHD in these individuals was unknown. In our study, we found CHD in 3.1% of the SCN5A loss-of-function mutation cohort, as opposed to 0.58% that was reported in a general population with a similar age distribution. Although the majority of SCN5A mutation carriers do not have CHD, the significantly increased prevalence of CHD suggests a contribution of SCN5A mutations to the origin of CHD in humans. Interestingly, CHD was only present in families with a phenotype compatible with a loss-of-function mutation. This might be due to the small cohort of patients with gain-of-function mutations (of which the majority carried the same mutation p.Ile1768Val), but it may also reflect a specific pathogenic role for loss-of-function mutations in the development of human CHD. As the CHD-associated mutations that we identified were localized throughout the cardiac sodium channel, no clear structure-func-tion relation could be established. Interestingly, five of six CHDs were septal defects. As VSDs and ASDII are among the most common CHD types in the general population, this might reflect the normal distribution of CHD, but possibly defective SCN5A expression may specifically lead to abnormalities of the cardiac septum. One patient in our cohort had TOF. Chiu et al. analyzed SCN5A (among other genes) in 84 TOF patients and did not identify pathogenic mutations.28

Because of the high frequency of septal defects that we found in our mutation carrier cohort, we proceeded to explore the frequency of SCN5A mutations in a cohort of patients with septal defects and conduction disease and identified a (possibly) pathogenic mutation in six patients (almost 4%), The mutations may have contributed to the conduction abnormalities in these patients, in addition to or instead of factors related to surgical procedures or the heart defect itself. The muta-tions may also have contributed to the septal defect, but this cannot be determined by our study design. Future studies comparing the frequency of mutations in CHD patients with conduction disease to patients with conduction disease only could give insight into this issue.How SCN5A mutations may lead to CHD remains speculative. Ion channels, including the pore-forming α1C subunit of the L-type calcium current and possibly HCN4 (carrier of the lf pace-maker current), have been shown to be involved in the development of the heart in embryonic stages in animal studies.29,30 First evidence for such a role for SCN5A came from studies showing that Scn5a knockout mice die early in embryonic development, their hearts having develop-

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mental defects including small ventricles and reduced trabeculation of the ventricular wall.12 Mice heterozygous for an Scn5a mutation survive and do not show obvious structural abnormalities, however, the presence of CHD such as septal defects has not systematicaly been investigated. More recent studies demonstrated that zebrafish with knockdown of SCN5A homologues have significantly diminished numbers of cardiomyocytes, associated with marked developmental abnormalities of the ventricles and looping abnormalities.13,14 Interestingly, pharmacological blockade of the sodium current in wild-type zebrafish embryos did not lead to cardiac develop-mental abnormalities.13 This suggests that sodium channel expression but not sodium current is required for early heart development and that the developmental role for SCN5A thus seems to be mediated by a non-electrogenic function of the channels.13,14 SCN5A knockout zebrafish showed reduced expression levels of myocardial precursor genes Nkx2-5, Gata4 and Hand2.13,14 These genes all code for cardiac transcription factors that play central roles in cardiac develop-ment. They interact with each other and many other transcription factors, and correct spatiotem-poral presence of transcription factors is crucial for normal development.31-34 Knockout mice for Nkx2-5, Gata4 or Hand2 die during embryonic development due to severe cardiac abnormali-ties35-37 and mice haplo-insufficient for Nkx2-5 or Gata4 have cardiac malformations including septal defects.38,39 In humans, mutations in NKX2-5, GATA4 and HAND2 have been identified in patients with CHD, especially septal defects.2,3,8,40 Thus, possibly, the reduced expression of these core transcription factors may contribute to CHD in SCN5A mutation carriers.

Clinical implicationsIt is common practice to perform cardiac imaging in SCN5A loss-of-function mutation carriers for assessment of ventricular abnormalities. If an association between SCN5A mutations and CHD can be confirmed in future studies, imaging should also focus on (asymptomatic) CHD. Moreover, as we showed that SCN5A mutations are not uncommon in patients with septal defects and conduction disease, we recommend genetic testing at low threshold in these patients because of its clinical implications. In SCN5A mutation carriers arrhythmias can be precipitated by fever and by drugs that affect sodium current, and therefore carriers are advised to prevent and treat fever and to avoid these drugs, regardless of symptom status or electrocardiographic manifesta-tions.41 Patients in whom risk stratification indicates an increased risk for sudden death are treated with ICD implantation. Moreover, as mutations in SCN5A are transmitted in an autosomal domi-nant manner, relatives are also at risk of carrying the familial mutation and cascade family screening is warranted.

Study limitationsOur study on the prevalence of CHD in a cohort of SCN5A mutation carriers is retrospective in design. Overestimation of CHD prevalence could have occurred as a result of ascertainment bias, as SCN5A mutation carriers might be more likely to have cardiac imaging with subsequent detection of (asymptomatic) CHD. Conversely, CHD patients may be more likely to have (asymptomatic)

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conduction disease detected and undergo genetic testing. We believe that this ascertainment bias has not played a major role in our study, as in only one patient (patient 6) the VSD was detected during workup for severe conduction disorders. Four CHD patients were identified as a mutation carrier only after identification of the mutation in a relative, and the fifth was already known with CHD when SCN5A analysis was performed because of symptomatic conduction disease. In contrast, CHD prevalence in our study might be underestimated, as most SCN5A mutation carriers underwent cardiac imaging only at adult age. In these patients, septal defects that have closed spontaneously during childhood (comprising about two thirds of all VSDs 42) were missed. Moreover, patients who died previously due to CHD were missed in our study. Another concern regards the control populations which we used for to estimate the prevalence of CHD in the general population. Reported prevalence estimates of CHD in the general population vary widely according to different studies, and depend on methods of ascertainment, in- and exclu-sion of certain types of heart lesions, and diagnostic tools used for detection of CHD.43 Moreover, CHD prevalence highly depends on the age of the population: prevalence is higher in children than in adults because of early mortality of severe defects and spontaneous closure of mainly VSDs during childhood. Thus, ideally, an age-matched control group with echocardiographic screening should have been used. Because our cohort of SCN5A mutation carriers comprised a wide age range (from young children to old adults), and CHD prevalence studies in such population are not available for the Dutch, we chose to compare our prevalence with published numbers from the general Canadian population. We also analyzed children and adults separately, using control populations that are applicable to the Dutch. In all comparisons, the prevalence of CHD was higher in our cohort than in the control population.

ConclusionsIn conclusion, our results suggest a role for SCN5A in heart development in humans. Loss of sodium channel current may contribute to CHD, particularly septal defects. As the large majority of SCN5A mutation carriers do not have CHD though, this contribution does not seem large. Further studies in human CHD patients may provide more insight into the role of SCN5A in human cardiac development. SCN5A mutations are not uncommon in patients with septal defects and conduction disease; the conduction disease in these patients may be due to the underlying SCN5A mutation in addition to or instead of factors related to the heart defect itself or surgical procedures. We recommend genetic testing at low threshold in these patients because of the malignant clinical implications of carrying such mutation.

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22 van der Velde ET, Vriend JW, Mannens MM, Uiterwaal CS, Brand R, Mulder BJ. CONCOR, an initiative towards a national registry and DNA-bank of patients with congenital heart disease in the Netherlands: rationale, design, and first results. Eur J Epidemiol 2005;20:549-57

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24 Ackerman MJ, Splawski I, Makielski JC et al. Spectrum and prevalence of cardiac sodium channel variants among black, white, Asian, and Hispanic individuals: implications for arrhythmogenic susceptibility and Brugada/long QT syndrome genetic testing. Heart Rhythm 2004;1:600-7

25 Holst AG, Liang B, Jespersen T et al. Sick sinus syndrome, progressive cardiac conduction disease, atrial flutter and ventricular tachycardia caused by a novel SCN5A mutation. Cardiology 2010;115:311-6

26 Lee YS, Baek JS, Kim SY et al. Childhood brugada syndrome in two korean families. Korean Circ J 2010;40:143-7

27 Gewillig M, Witters I, Eyskens B et al. A heterozygous SCN5A point mutation in the domain I-II linker leading to LQT syndrome with 2;1 AV block, ventricular hypertrophy, contraction and relaxation abnormalities. Cardiology in the Young 2002;12

28 Chiu SN, Wu MH, Su MJ et al. Coexisting mutations/polymorphisms of the long QT syndrome genes in patients with repaired Tetralogy of Fallot are associated with the risks of life-threatening events. Hum Genet 2012;131:1295-304

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30 Stieber J, Herrmann S, Feil S et al. The hyperpolarization-activated channel HCN4 is required for the generation of pacemaker action potentials in the embryonic heart. Proc Natl Acad Sci U S A 2003;100:15235-40

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33 Lee Y, Shioi T, Kasahara H et al. The cardiac tissue-restricted homeobox protein Csx/Nkx2.5 physi-cally associates with the zinc finger protein GATA4 and cooperatively activates atrial natriuretic factor gene expression. Mol Cell Biol 1998;18:3120-9

34 Singh MK, Li Y, Li S et al. Gata4 and Gata5 cooperatively regulate cardiac myocyte proliferation in mice. J Biol Chem 2010;285:1765-72

35 Lyons I, Parsons LM, Hartley L et al. Myogenic and morphogenetic defects in the heart tubes of murine embryos lacking the homeo box gene Nkx2-5. Genes Dev 1995;9:1654-66

36 Molkentin JD, Lin Q, Duncan SA, Olson EN. Requirement of the transcription factor GATA4 for heart tube formation and ventral morphogenesis. Genes Dev 1997;11:1061-72

37 Srivastava D, Thomas T, Lin Q, Kirby ML, Brown D, Olson EN. Regulation of cardiac mesodermal and neural crest development by the bHLH transcription factor, dHAND. Nat Genet 1997;16:154-60

38 Biben C, Weber R, Kesteven S et al. Cardiac septal and valvular dysmorphogenesis in mice het-erozygous for mutations in the homeobox gene Nkx2-5. Circ Res 2000;87:888-95

39 Pu WT, Ishiwata T, Juraszek AL, Ma Q, Izumo S. GATA4 is a dosage-sensitive regulator of cardiac morphogenesis. Dev Biol 2004;275:235-44

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41 Postema PG, Wolpert C, Amin AS et al. Drugs and Brugada syndrome patients: review of the literature, recommendations, and an up-to-date website (www.brugadadrugs.org). Heart Rhythm 2009;6:1335-41

42 Warnes CA, Liberthson R, Danielson GK et al. Task force 1: the changing profile of congenital heart disease in adult life. J Am Coll Cardiol 2001;37:1170-5

43 van der Bom T, Zomer AC, Zwinderman AH, Meijboom FJ, Bouma BJ, Mulder BJ. The changing epidemiology of congenital heart disease. Nat Rev Cardiol 2011;8:50-60

44 Bardai A, Amin AS, Blom MT et al. Sudden cardiac arrest associated with use of a non-cardiac drug that reduces cardiac excitability: evidence from bench, bedside, and community. Eur Heart J 2013;34:1506-16

45 Kapplinger JD, Tester DJ, Alders M et al. An international compendium of mutations in the SCN5A-encoded cardiac sodium channel in patients referred for Brugada syndrome genetic testing. Heart Rhythm 2010;7:33-46

46 Meregalli PG, Tan HL, Probst V et al. Type of SCN5A mutation determines clinical severity and degree of conduction slowing in loss-of-function sodium channelopathies. Heart Rhythm 2009;6:341-8

47 Vernooy K, Sicouri S, Dumaine R et al. Genetic and biophysical basis for bupivacaine-induced ST segment elevation and VT/VF. Anesthesia unmasked Brugada syndrome. Heart Rhythm 2006;3:1074-8

48 Kapplinger JD, Tester DJ, Salisbury BA et al. Spectrum and prevalence of mutations from the first 2,500 consecutive unrelated patients referred for the FAMILION long QT syndrome genetic test. Heart Rhythm 2009;6:1297-303

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Supplemental material

Table 1. SCN5A mutations encountered in our cohort

Mutation LOF/GOF* Class n families n patients

p.Ile137_Cys139dup LOF VUS3 1 3

p.Trp156X LOF Pathogenic mutation 2 4

p.Glu161Lys LOF Pathogenic mutation 2 14

p.Thr220Ile LOF VUS3 1 9

p.Arg225Trp LOF Pathogenic mutation 1 3

c.934+1G>A LOF Pathogenic mutation 3 8

p.Asp356Asn LOF Pathogenic mutation 1 1

p.Arg367Cys LOF Pathogenic mutation 3 10

p.Arg367His LOF Pathogenic mutation 1 1

p.Arg376Cys LOF VUS3 1 1

p.Tyr399Cys LOF VUS3 1 1

p.Asp501Gly LOF VUS3 1 1

P.Gly514Cys LOF Pathogenic mutation 1 6

p.Gly752Arg LOF Pathogenic mutation 1 2

p.Tyr774fsX28 LOF Pathogenic mutation 2 4

p.Phe861TrpfsX90 LOF Pathogenic mutation 11 53

p.Arg893His LOF VUS3 1 1

p.Cys915Arg LOF VUS3 1 6

p.Asn927Ser LOF VUS3 2 8

p.Arg965His LOF VUS3 1 1

p.Pro1048Leufs98 LOF Pathogenic mutation 1 4

c.3228+2delT LOF Pathogenic mutation 1 2

p.Glu107Argfs24 LOF Pathogenic mutation 1 3

p.Arg1232Gln LOF VUS3 1 1

p.Gly1262Ser LOF VUS3 1 3

p.Asp1275Asn LOF Pathogenic mutation 1 3

p.Ile1278Asn GOF VUS3 1 5

c.3840+1G>A LOF Pathogenic mutation 1 3

p.Gly1319Val LOF Pathogenic mutation 6 13

p.Leu1346Pro LOF VUS3 1 2

p.Cys1363Tyr LOF VUS3 1 2

p.Leu1373X LOF Pathogenic mutation 1 15

p.Val1405Leu LOF VUS3 2 2

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LOF, loss-of-function; GOF, gain-of-function; VUS, variant of unknown significance. * Proven or presumed by phenotype.

Mutation LOF/GOF* Class n families n patients

p.Gly1420Arg LOF VUS3 2 4

c.4437+5G>A LOF Pathogenic mutation 1 3

p.1507_1509delGlnLysPro GOF Pathogenic mutation 1 3

c.4542+2_3delTG LOF Pathogenic mutation 1 1

p.Gly1573Gly LOF Pathogenic mutation 1 2

p.Cys1575Arg LOF VUS3 1 2

p.Leu1582Pro LOF VUS3 1 9

p.Arg1629Gln LOF VUS3 1 3

p.Arg1635Ile GOF VUS3 1 1

p.Arg1638X LOF Pathogenic mutation 2 6

p.Ile1643Asn LOF VUS3 1 6

p.Ile1660Val LOF VUS3 (low penetrance) 2 2

p.Asp1714Gly LOF Pathogenic mutation 1 1

p.Gly1740Arg LOF Pathogenic mutation 1 1

p.Gly1743Glu LOF Pathogenic mutation 5 29

p.Ile1768Val GOF Pathogenic mutation 9 33

Table 1. Continued.

Part IICongenital heart disease with

associated abnormalities

22q11.2 deletion syndrome is under-recognized in adult patients with tetralogy of Fallot and pulmonary atresia

van Engelen K, Topf A, Keavney BD, Goodship JA, van der Velde ET, Baars MJH, Snijder S, Moorman A, Postma AV, Mulder BJM

Heart 2010;96(8):621-624

Chapter 5

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Abstract

BackgroundThree quarters of patients with 22q11.2 deletion syndrome (22q11.2DS) have congenital heart disease (CHD), typically conotruncal heart defects. Although it is currently common practice to test all children with typical CHD for 22q11.2DS, many adult patients have not been tested in the past and therefore 22q11.2DS might be under-recognized in adults.

ObjectivesTo determine the prevalence of 22q11.2DS in adults with tetralogy of Fallot (TOF) and pulmonary atresia (PA)/ventricular septal defect (VSD) and to assess the level of recognition of the syndrome in adult patients.

MethodsPatients were identified from CONCOR, a nationwide registry for adult patients with CHD. Inclu-sion criteria were diagnosis of TOF or PA/VSD, and availability of DNA. Patients with syndromes other than 22q11.2DS were excluded. Multiplex Ligation-dependent Probe Amplification was used to detect 22q11.2 microdeletions.

Results479 patients with TOF and 79 patients with PA/VSD (56% male, median age 34.7 years) were included and analyzed. Twenty patients were already known to have 22q11.2DS. A 22q11.2 microdeletion was detected in a further 24 patients. Thirty-one patients with TOF (6.5%) had 22q11.2DS, whereas 13 patients with PA/VSD had 22q11.2DS (16.5%). Of all 22q11.2 microdele-tions, 54% (24/44) were unknown before this study.

ConclusionThis study shows that although the prevalence of 22q11.2DS in adults with TOF and PA/VSD is substantial, it is unrecognized in more than half of patients. As the syndrome has important clinical and reproductive implications, a diagnostic test should be considered in all adult patients with TOF and PA/VSD.

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Introduction22q11.2 deletion syndrome (22q11.2DS) is the most common microdeletion syndrome in humans with an estimated prevalence of at least 1 in 4,000.1,2 The features associated with 22q11.2DS include congenital heart disease (CHD), cleft palate, velopharyngeal insufficiency with hypernasal speech, hypocalcaemia, dysmorphic facial features and mild to moderate mental retardation, with a high variability in number and severity of associated features (reviewed in 2). CHD is present in about 75% of patients with 22q11.2DS and typically constitutes conotruncal malformations such as interrupted aortic arch type B, truncus arteriosus communis, tetralogy of Fallot (TOF) and pulmonary atresia (PA) with ventricular septal defect (VSD).3,4 Psychiatric disorders, most notably schizophrenia, develop in up to two thirds of adults with 22q11.2DS.4-6 Individuals with 22q11.2DS have a 50% chance of transmitting the deletion to his or her offspring.In the early 1990s of the last century Fluorescence In Situ Hybridization (FISH) became widely available as a diagnostic test for 22q11.2DS and it is currently common practice to test all children with typical CHD for 22q11.2DS. However, such a diagnostic test was not yet available at the time that many adult patients with CHD were children. Some adult patients will have been tested as the phenotype was defined and a diagnostic test became available, but this was not undertaken in a systematic way. The variable phenotype can make it difficult to recognize the syndrome, which may contribute to under-recognition in adults.5,7 As more and more patients with CHD reach adulthood due to improved surgical techniques,8 adult cardiologists will encounter an increasing number of patients with 22q11.2DS. Recognition of the syndrome is important, as it has significant clinical and reproductive implications for the patient. The aim of this study was to determine the prevalence of 22q11.2DS in adult patients with TOF and PA/VSD and to assess the level of recognition of the syndrome in these patients.

Patients and methodsPatients were selected from the CONCOR national registry database and DNA-bank, which has been described in detail previously.9 In short, CONCOR aims to facilitate research on the aetiology of CHD and its outcome. In August 2009, 10,942 patients with CHD aged ≥ 16 years had been included in the registry. Patients are contacted through their cardiologist or advertisements in the local media. Approximately 75% of registered patients originate from tertiary referral centres. Diagnoses, procedures, and clinical events are classified with the use of the European Paediatric Cardiac Code Short List coding scheme. DNA is isolated from peripheral blood and stored for research purposes. For this study, we selected all patients registered with TOF and PA/VSD for whom DNA was available. Patients with known syndromes associated with CHD, other than 22q11.2DS, were excluded. Other extracardiac symptoms were not considered. We employed Multiplex Ligation-dependent Probe Amplification (MLPA) to detect 22q11.2 deletions, using the SALSA MLPA

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P250-A1 DiGeorge kit (MRC Holland) containing 30 probes in the 22q11 region.10 In the case of detected 22q11.2 deletions, the medical charts of the patients for whom the deletion was not recorded in CONCOR were reviewed, to clarify whether the diagnosis had indeed not been made previously. For statistical analyses, SPSS 16.0 for Windows was used. P values <0.05 were considered statisti-cally significant. The data regarding age are presented as median with range because of a skewed distribution. Comparison of discrete variables (ie, gender and cardiac diagnosis) between patients in whom 22q11.2DS was known and patients in whom 22q11.2DS was not known prior to this study was performed using the Fisher’s exact test. Comparison of continuous variables (ie, age) between groups were made by the Mann-Whitney U test.

ResultsAt 1 January 2007, 993 patients with diagnoses of interest to this study had been registered in the CONCOR registry (total number of registered patients at that moment: 7,503), with DNA available for 577 patients. Fifteen patients were excluded because they had a recognized syndrome other than 22q11.2DS, leaving 562 patients eligible for this study. The main cardiac diagnosis was TOF in 483 of these patients and PA/VSD in 79 patients. MLPA was successful in 479 patients with TOF, and in all 79 patients with PA/VSD. Therefore, analysis was performed on 558 patients (56% male; median age 34.7 years, range 19.8 to 82.8). Twenty patients had already been diagnosed with 22q11.2DS before this study. We detected a 22q11.2 deletion in an additional 24 patients. Therefore, a total of 44 out of 558 (7.9%, 95% Confidence Interval (CI) 5.9 to 10.4) patients had 22q11.2DS. Thirty-one of 479 (6.5%, 95% CI 4.6 to 9.1) patients with TOF had a 22q11.2 deletion, and 13 of 79 (16.5%, 95% CI 9.7 to 26.3) patients with PA/VSD had a 22q11.2 deletion (Table 1). Of the patients with 22q11.2DS, 22 were men and 22 were women. In 24 of 44 (54%) patients with 22q11.2DS, the deletion was not known to be present before this study. Patients with unknown 22q11.2DS were significantly older than the patients with known 22q11.2DS (median age 37.4 and 28.5 years, respectively, P = 0.03). No significant differences were present between patients with known 22q11.2DS and unknown 22q11.2DS with regard to gender or cardiac diagnosis (TOF or PA/VSD).

DiscussionIn this study we found that 6.5% of adult patients with TOF and 16.5% of adult patients with PA/VSD had 22q11.2DS and, importantly, that more than half of the patients with 22q11.2DS had not been diagnosed before this study, reflecting the under-recognition of the syndrome. Two smaller studies reported on the prevalence of 22q11.2DS among adults with TOF and PA/VSD. Beauchesne et al. found prevalence rates of 3.8% in adults with TOF and 8.7% in PA/VSD,

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5which have overlapping confidence intervals with the rates in our study.7 In another study that included 377 adult patients with TOF, only the patients with TOF plus additional features suggestive of 22q11.2DS (n = 103) were screened and a prevalence of 29.1% was found, which translated to a minimum prevalence of 6.6% in the whole group.11 The prevalence data in our study and these two other studies in adult patients are lower than generally reported in paediatric studies (TOF 6 - 26%, PA/VSD 24 - 46%).12-20 This might be explained by the cardiac and extracardiac complications associated with 22q11.2DS, decreasing survival into adulthood. Differences in ascertainment may also contribute. We used a national registry, which provided the opportunity to review a group of patients with CHD without knowledge of additional characteristics of the patients. However, most patients (75%) in the CONCOR registry originate from tertiary referral centres, which might have lead to an overrepresentation of patients with complex heart defects and extra-cardiac disorders and therefore to an overestimated prevalence of 22q11.2DS.In our study, less than half of the patients with 22q11.2DS had been diagnosed before this study, reflecting under-recognition of the syndrome in adult patients. The highly variable phenotype of the syndrome and difficulties in recognizing the associated manifestations of the syndrome may contribute to the under-recognition in adult patients.7 The facial features (Box 1) are often subtle if present at all in adults.21 In addition, the lack of awareness of the high prevalence and lack of knowledge among adult cardiologists about the availability of molecular testing may play a role. With regard to recognition of the presence of 22q11.2DS, no differences in gender and cardiac diagnosis of the patients were present. However, patients in whom 22q11.2DS was not known to be present before this study were significantly older than the patients with known 22q11.2DS. Some of the younger patients in this study may have routinely been tested when they were still children or adolescents because of the availability of a test. In addition, the syndrome might be easier to recognize in younger patients.Nevertheless, recognition of 22q11.2DS has important implications in directing immediate and long-term clinical management. Patients with TOF and 22q11.2DS more frequently have addi-tional cardiovascular abnormalities, including right aortic arch, aberrant right subclavian artery

Table 1. Prevalence of 22q11.2 deletion syndrome in patients with TOF and PA/VSD

TOF, tetralogy of Fallot; PA/VSD, pulmonary atresia with ventricular septal defect.

Main cardiac diagnosis

Totaln of patients

with 22q11.2DS

% of patients with 22q11.2DS

(95% CI)

n of patients with

unknown 22q11.2DS

% of patients with unknown 22q11.2DS

(95% CI)

TOF 479 31 6.5 (4.6 - 9.1) 18 58 (41 - 74)

PA/VSD 79 13 16.5 (9.7 - 26.3) 6 46 (23 - 71)

Total 558 44 7.9 (5.9 - 10.4) 24 54 (40 - 68)

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and major aortopulmonary collateral arteries, than patients with TOF without a deletion.12,14,17,22 The post-operative morbidity and mortality of 22q11.2DS patients is higher than in other patients due to the presence of these associated cardiac anomalies as well as the extracardiac anomalies of the syndrome.12,14,23 In addition, individuals with 22q11.2DS who survive childhood have an increased risk of sudden death and diminished life expectancy which cannot be attributed to a single factor.24 Many of the associated extracardiac conditions are treatable once detected, emphasiz-ing the importance of follow-up in a coordinated multidisciplinary setting.5,25 In children, features requiring attention besides CHD are velopharyngeal insufficiency and feeding difficulties, immu-nodeficiency, hypocalcaemia and developmental delay, among others.2 Specific problems in adolescents and adults are autoimmune and endocrinologic disorders, including thyroid dysfunction.5 In addition, psychiatric disorders are reported in up to 58% of patients, with schizophrenia being especially common (18-24%),4-6 although other disorders such as anxiety and mood disorders and attention deficit disorders also occur frequently.5,26,27 Hypocalcaemia due to hypoparathyroidism, common in the neonatal period, may also occur in adulthood,5,26 and renal abnormalities may lead to complications in adults as well. Recurrent respiratory tract infections are often present.5,26 If treating physicians are aware of these problems, early intervention can take place, which may significantly reduce morbidity. Another important issue is the heredity of 22q11.2DS; the deletion arises as a de novo event in approximately 90% of patients, but an affected individual has a

Typically associated CHD*

Interrupted aortic arch (type B)

PA/VSD

TOF

Isolated arch anomalies

Truncus arteriosus

Extracardiac features

Mental retardation/learning disability

Psychiatric history

Typical facial features**

Hypernasal speech

History of cleft palate

Hypocalcaemia

Family history of CHD or extracardiac features

Box 1. Features that should raise clinical suspicion of 22q11.2 deletion syndrome in adults 5,21,26

CHD, congenital heart disease; TOF, tetralogy of Fallot; PA/VSD, pulmonary atresia with ventricular septal defect. * Especially in the presence of additional cardiovascular abnormalities such as right aortic arch, aberrant right subclavian artery and major aortopulmonary collateral arteries ** Facial features are often absent or subtle in adults, but include: long face, prominent nasal root, full nasal tip, small mouth and chin, squared off external ears, narrow palpebral fissures.

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50% chance of transmitting the deletion to his or her offspring. Appropriate (preconception) genetic counselling is important for these patients.Nonetheless, the majority of patients in our study were found to be negative for 22q11.2DS. Several other genetic disorders are known to underlie TOF and PA/VSD, a subset of patients having other known cytogenetic abnormalities leading to syndromic CHD, e.g. trisomy 21 (Down syndrome). Other syndromic patients have single gene disorders such as Holt-Oram syndrome, caused by mutations in TBX5. Patients with known syndromes were excluded from our study. The greater part of patients with TOF and PA/VSD have isolated, non syndromic TOF. Recently, Greenway et al. identified copy number variations (CNVs) other than 22q11.2 deletions at 9 loci in 15 of 512 patients with isolated TOF, although their significance has yet to be determined.28 Moreover, they also found 22q11.2 deletions in 2 patients with isolated TOF, as well as in 1 of 2,265 control subjects. Mutations in several genes, including the transcription factor genes NKX2.5,29 and GATA4,30 and transmembrane receptor gene NOTCH131 and its ligand JAG1,32 are also known to be implied in a small subset of isolated TOF. Such single gene mutations and CNVs may underlie the heart defects in some of our patients.It remains controversial which adults should be screened for 22q11.2DS.7,13,33 Given the high prevalence, the variable phenotype and the difficulties in recognizing the often subtle features, we believe that testing for the deletion should be performed in all adults with selected conotruncal heart defects, including TOF, PA/VSD, interrupted aortic arch (type B), isolated aortic arch anoma-lies and truncus arteriosus communis, also in patients who apparently do not have extracardiac features. As this might not be feasible due to practical and/or financial reasons, at the very least, testing should be performed in patients with extracardiac features of the syndrome, as well as in patients with additional heart defects, such as major aortopulmonary collateral arteries, right aortic arch and aberrant right subclavian artery. Clinical features that should raise the suspicion of 22q11.2DS are listed in Box 1. Fung et al.11 determined a model for the prediction of the pres-ence of 22q11.2DS in adults with CHD in a brief encounter with the patient. A combination of three out of four features (global dysmorphic face, voice abnormalities, learning difficulties and age < 30 years) yielded the highest sensitivity and discriminant ability. Education about the syndrome for physicians managing adult patients with CHD would contribute to a greater awareness and recognition of the syndrome.In conclusion, this study shows that although the prevalence of 22q11.2DS in adult patients with TOF and PA/VSD is substantial (6.5 and 16.5%, respectively), it is unrecognized in more than half of patients. This reinforces the need for cardiologists’ awareness of the syndrome. As the syndrome has important implications for surveillance and reproduction, screening should be considered in these patients, especially in the presence of extracardiac symptoms of the 22q11.2DS.

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Reference List

1 Goodship J, Cross I, LiLing J, Wren C. A population study of chromosome 22q11 deletions in infancy. Arch Dis Child 1998;79:348-51

2 Kobrynski LJ, Sullivan KE. Velocardiofacial syndrome, DiGeorge syndrome: the chromosome 22q11.2 deletion syndromes. Lancet 2007;370:1443-52

3 Donald-McGinn DM, Kirschner R, Goldmuntz E et al. The Philadelphia story: the 22q11.2 deletion: report on 250 patients. Genet Couns 1999;10:11-24

4 Ryan AK, Goodship JA, Wilson DI et al. Spectrum of clinical features associated with interstitial chro-mosome 22q11 deletions: a European collaborative study. J Med Genet 1997;34:798-804

5 Bassett AS, Chow EW, Husted J et al. Clinical features of 78 adults with 22q11 Deletion Syndrome. Am J Med Genet A 2005;138:307-13

6 Murphy KC, Jones LA, Owen MJ. High rates of schizophrenia in adults with velo-cardio-facial syndrome. Arch Gen Psychiatry 1999;56:940-5

7 Beauchesne LM, Warnes CA, Connolly HM et al. Prevalence and clinical manifestations of 22q11.2 microdeletion in adults with selected conotruncal anomalies. J Am Coll Cardiol 2005;45:595-8

8 Marelli AJ, Mackie AS, Ionescu-Ittu R, Rahme E, Pilote L. Congenital heart disease in the general population: changing prevalence and age distribution. Circulation 2007;115:163-72

9 van der Velde ET, Vriend JW, Mannens MM, Uiterwaal CS, Brand R, Mulder BJ. CONCOR, an initiative towards a national registry and DNA-bank of patients with congenital heart disease in the Netherlands: rationale, design, and first results. Eur J Epidemiol 2005;20:549-57

10 Jalali GR, Vorstman JA, Errami A et al. Detailed analysis of 22q11.2 with a high density MLPA probe set. Hum Mutat 2008;29:433-40

11 Fung WL, Chow EW, Webb GD, Gatzoulis MA, Bassett AS. Extracardiac features predicting 22q11.2 Deletion Syndrome in adult congenital heart disease. Int J Cardiol 2008;131:51-8

12 Ziolkowska L, Kawalec W, Turska-Kmiec A et al. Chromosome 22q11.2 microdeletion in children with conotruncal heart defects: frequency, associated cardiovascular anomalies, and outcome following cardiac surgery. Eur J Pediatr 2008;167:1135-40

13 Goldmuntz E, Clark BJ, Mitchell LE et al. Frequency of 22q11 deletions in patients with conotruncal defects. J Am Coll Cardiol 1998;32:492-8

14 Chessa M, Butera G, Bonhoeffer P et al. Relation of genotype 22q11 deletion to phenotype of pulmo-nary vessels in tetralogy of Fallot and pulmonary atresia-ventricular septal defect. Heart 1998;79:186-90

15 Iserin L, de LP, Viot G et al. Prevalence of the microdeletion 22q11 in newborn infants with congenital conotruncal cardiac anomalies. Eur J Pediatr 1998;157:881-4

16 Worthington S, Bower C, Harrop K, Loh J, Walpole I. 22q11 deletions in patients with conotruncal heart defects. J Paediatr Child Health 1998;34:438-43

17 Lammer EJ, Chak JS, Iovannisci DM et al. Chromosomal abnormalities among children born with conotruncal cardiac defects. Birth Defects Res A Clin Mol Teratol 2009;85:30-5

18 Frohn-Mulder IM, Wesby SE, Bouwhuis C et al. Chromosome 22q11 deletions in patients with selected outflow tract malformations. Genet Couns 1999;10:35-41

19 Digilio MC, Marino B, Grazioli S, Agostino D, Giannotti A, Dallapiccola B. Comparison of occurrence of genetic syndromes in ventricular septal defect with pulmonic stenosis (classic tetralogy of Fallot) versus ventricular septal defect with pulmonic atresia. Am J Cardiol 1996;77:1375-6

20 Lu JH, Chung MY, Hwang B, Chien HP. Prevalence and parental origin in Tetralogy of Fallot associ-ated with chromosome 22q11 microdeletion. Pediatrics 1999;104:87-90

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21 Lin AE, Basson CT, Goldmuntz E et al. Adults with genetic syndromes and cardiovascular abnormali-ties: clinical history and management. Genet Med 2008;10:469-94

22 Marino B, Digilio MC, Toscano A et al. Anatomic patterns of conotruncal defects associated with dele-tion 22q11. Genet Med 2001;3:45-8

23 Anaclerio S, Di C, V, Michielon G et al. Conotruncal heart defects: impact of genetic syndromes on immediate operative mortality. Ital Heart J 2004;5:624-8

24 Bassett AS, Chow EW, Husted J et al. Premature death in adults with 22q11.2 deletion syndrome. J Med Genet 2009;46:324-30

25 Greenhalgh KL, Aligianis IA, Bromilow G et al. 22q11 deletion: a multisystem disorder requiring multidisciplinary input. Arch Dis Child 2003;88:523-4

26 Cohen E, Chow EW, Weksberg R, Bassett AS. Phenotype of adults with the 22q11 deletion syndrome: A review. Am J Med Genet 1999;86:359-65

27 Bassett AS, Chow EW. Schizophrenia and 22q11.2 deletion syndrome. Curr Psychiatry Rep 2008;10:148-57

28 Greenway SC, Pereira AC, Lin JC et al. De novo copy number variants identify new genes and loci in isolated sporadic tetralogy of Fallot. Nat Genet 2009;41:931-5

29 Schott JJ, Benson DW, Basson CT et al. Congenital heart disease caused by mutations in the tran-scription factor NKX2-5. Science 1998;281:108-11

30 Tomita-Mitchell A, Maslen CL, Morris CD, Garg V, Goldmuntz E. GATA4 sequence variants in patients with congenital heart disease. J Med Genet 2007;44:779-83

31 Garg V, Muth AN, Ransom JF et al. Mutations in NOTCH1 cause aortic valve disease. Nature 2005;437:270-4

32 Eldadah ZA, Hamosh A, Biery NJ et al. Familial Tetralogy of Fallot caused by mutation in the jagged1 gene. Hum Mol Genet 2001;10:163-9

33 Digilio MC, Marino B, Giannotti A, Mingarelli R, Dallapiccola B. Guidelines for 22q11 deletion screening of patients with conotruncal defects. J Am Coll Cardiol 1999;33:1746-8

Letter to the editor: Screening for 22q11.2 microdeletion in adults

with tetralogy of Fallot, and author’s reply

van Engelen K, Baars MJH, Postma AV, Mulder BJM

Heart 2011;97(10):860

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Letter to the editorWe read with interest the paper by van Engelen et al,1 analysing the prevalence of 22q11.2 deletion syndrome (22q11.2DS) in adults with tetralogy of Fallot (TOF) and with pulmonary atresia (PA)/ventricular septal defect (VSD). We agree with the fact that many adult patients with TOF have not been tested for 22q11.2DS in the past, and awareness of the syndrome is needed among clinicians who care for adults with congenital heart defects. Nevertheless, we disagree to introduce as general practice the large-scale screening of all TOF patients, irrespective of their clinical phenotype. Personal experience in paediatric patients with 22q11.2DS has evidenced that the deletion is virtually never found in nonsyndromic patients with conotruncal defects.2 In addition, it has been evidenced that distinct subtypes of conotruncal heart defects are likely to be found in association with 22q11.2DS. In regard to TOF, patients with 22q11.2DS often have right/cervical aortic arch with/without aberrant left subclavian artery, hypoplasia or absence of the infundibular septum, absence of the pulmonary valve and hypoplasia and discontinuity of the pulmonary arteries.2 Among the children with TOF and PA, 35% carry a 22q11.2DS, and distinctive recognizable patterns of congenital heart defects include major aorto-pulmonary collateral arteries, sometimes with discontinuity of the pulmonary arteries.2 The review of the literature about clinical character-istics of adults with 22q11.2DS shows that extracardiac anomalies can help clinician to suspect 22q11.2DS.3 Particularly, previous series reported that facial anomalies can be detected in 99-100% of the cases, ranging from subtle to characteristic. Additional signs of evidence include hypernasal speech (90%), intellectual disability of any degree and/or learning difficulties (93-97%). The rare occurrence of extremely mild clinical expression of 22q11.2DS in a parent of an affected child can now be explained with the molecular mechanisms of genetic compensation (presence of a 22q11.2 deletion on one chromosome and 22q11.2 duplication on the other allele of chromo-some 22).4 In conclusion, the search for 22q11.2DS is important in adult patients with TOF and with PA/VSD, since recognition of the syndrome has clinical and reproductive implications, but genetic testing, in our opinion, should be reserved to patients with associated ‘classic’ or ‘subtle’ extracardiac anomalies and to those with distinct anatomic cardiac subtypes.

Reference List

1 van Engelen K, Topf A, Keavney BD et al. 22q11.2 Deletion Syndrome is under-recognised in adult patients with tetralogy of Fallot and pulmonary atresia. Heart 2010;96:621-4

2 Marino B, Digilio MC, Toscano A et al. Anatomic patterns of conotruncal defects associated with dele-tion 22q11. Genet Med 2001;3:45-8

3 Fung WL, Chow EW, Webb GD, Gatzoulis MA, Bassett AS. Extracardiac features predicting 22q11.2 Deletion Syndrome in adult congenital heart disease. Int J Cardiol 2008;131:51-8

4 Carelle-Calmels N, Saugier-Veber P, Girard-Lemaire F et al. Genetic compensation in a human genomic disorder. N Engl J Med 2009;360:1211-6

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Author’s replyWe thank Digilio et al. for their interest in our paper,1 showing that 22q11.2 deletion syndrome (22q11.2DS) is under-recognized in adults with tetralogy of Fallot (TOF) and with pulmonary atresia (PA)/ventricular septal defect (VSD).2 Digilio et al. disagree with our recommendation to consider genetic testing for the syndrome in all adults with TOF and PA/VSD. Rather they propose to reserve this for patients with associated ‘classic’ or ‘subtle’ extracardiac anomalies and to those with distinct anatomic cardiac subtypes. We recognize that the issue of testing for 22q11.2DS is controversial. We surely agree with Digilio et al. that specific additional cardiac anomalies are often present in patients with TOF and PA/VSD and 22q11.2DS, such as right/cervical aortic arch, hypoplasia or absence of the infundibular septum and major aortopulmonary collateral arteries. The presence of these abnormalities as well as extracardiac features including hypernasal speech, intellectual disability and specific facial features may indisputably help the clinician to suspect 22q11.2DS, and should prompt genetic testing. However, because these additional features may be present only in a subtle manner, they may remain undetected or unrecognized as part of the syndrome. In an adult population with undetected 22q11.2DS one can expect a bias towards those patients with more subtle features, as patients who exhibit clear features will probably have been diagnosed at an earlier stage. Although in retrospect the majority of patients show (subtle) facial features of the syndrome, physi-cians, including geneticists, do not always reliably recognize these facial features.3 Moreover, the facial features in adult patients are known to be often more subtle than in children or absent.4 For these reasons, identifying adult patients who might carry the deletion may prove difficult. In clinical practice, given the high prevalence and the relevance of detecting the deletion in terms of clinical and reproductive issues, to our opinion genetic testing should not be reserved for TOF and PA/VSD patients with associated anomalies. Greater awareness of and more experience with the syndrome among physicians may eventually lead to a screening strategy as proposed by Digilio et al. Hopefully the current discussion will contribute to such greater awareness.

Reference List

1 Digilio MC, Marino B, Dallapiccola B. Screening for 22q11.2 microdeletion in adults with tetralogy of Fallot. Heart 2011;97:860

2 van Engelen K, Topf A, Keavney BD et al. 22q11.2 Deletion Syndrome is under-recognised in adult patients with tetralogy of Fallot and pulmonary atresia. Heart 2010;96:621-4

3 Becker DB, Pilgram T, Marty-Grames L, Govier DP, Marsh JL, Kane AA. Accuracy in identification of patients with 22q11.2 deletion by likely care providers using facial photographs. Plast Reconstr Surg 2004;114:1367-72

4 Lin AE, Basson CT, Goldmuntz E et al. Adults with genetic syndromes and cardiovascular abnormali-ties: clinical history and management. Genet Med 2008;10:469-94

Bicuspid aortic valve morphology and associated cardiovascular abnormalites in fetal Turner syndrome:

a pathology study

van Engelen K, Bartelings MM, Gittenberger-de Groot AC, Baars MJH, Postma AV, Bijlsma EK, Mulder BJM, Jongbloed MRM

Submitted

Chapter 6

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AbstractBackground Bicuspid aortic valve (BAV) is common in Turner syndrome (TS). In adult TS, 82-95% of BAVs have fusion of the right and left coronary leaflets. Data in fetal stages are scarce. We studied fetal TS hearts, with the purpose of gaining insight in aortic valve morphology and associated cardio-vascular abnormalities in a selected cohort with adverse outcome early in development.

Methods and results We studied post-mortem heart specimens of 36 TS fetuses and one TS newborn. BAV was present in 28 (76%) hearts. BAVs showed fusion of the right and left coronary leaflet (Type 1 BAV) in 61% and fusion of the right coronary and noncoronary leaflet (Type 2 BAV) in 39%. There were no significant differences in occurrence of additional cardiovascular abnormalities between hearts with Type 1 and Type 2 BAV. However, all hearts with Type 2 BAV showed ascending aorta hypo-plasia and tubular hypoplasia of the B segment, as opposed to only 55% and 64% of hearts with Type 1 BAV, respectively.

ConclusionThe vast majority of TS fetuses shows abnormal aortic valve morphology, with BAV present in 76%. The proportion of Type 2 BAV seems higher in TS fetuses than in adults. Fetal TS hearts with Type 2 BAV were all associated with severe aortic pathology, possibly contributing to a worse prognosis of Type 2 BAV than Type 1 BAV in TS.

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IntroductionBicuspid aortic valve (BAV) is the most common congenital heart defect with a prevalence of about 0.5-2% in the general population.1,2 It may lead to significant morbidity and mortality due to complications such as aortic valve stenosis, regurgitation and aortic aneurysm and dissection.3-5 BAV may occur as an isolated anomaly, as part of more complex congenital heart disease (CHD) and in the context of several genetic syndromes. One such genetic syndrome with particularly high frequency of BAV is Turner syndrome (TS). TS is caused by complete or partial monosomy for the X chromosome in all or part of the cells. It occurs in 1 in 2500 female live births, but only a small minority of fetuses with 45,X survives to term.6,7 Apart from the main features of fetal lymphedema, short stature and gonadal dysgenesis, CHD is present in 20-40% of patients and mostly comprises left-sided heart defects. BAV has been reported to occur in about 12-20% of children and adults with Turner syndrome,8-11 though a recent study using MRI found a prevalence rate as high as 40%.12 In TS fetuses with cystic hygroma, who include the more severely affected patients with a high mortality in utero, the prevalence of CHD is much higher, and BAV has been reported in 40-85%.13-16

In the general population, BAVs with different morphology types can be distinguished. The majority (65-80%) of BAV cases are due to fusion of the right and left coronary leaflets, whereas in 10-30% the right coronary and noncoronary leaflets are fused and in a small minority (2-3%) there is fusion of the left coronary and noncoronary leaflet.17-21 Recent animal studies have suggested that BAVs with different morphology types are of distinct etiologic origin.22 Interestingly, several studies have reported that BAV morphology type has prognostic significance in terms of valve disease, aortic dilatation and necessity for intervention.17,19-21,23 In TS, 82-95% of BAVs in adults have been reported to be due to fusion of the right and left coronary leaflets.24,25 In contrast, although data on BAV leaflet orientation in prenatal TS are scarce, a larger proportion (3 of 11) of TS fetuses with BAV had fusion of the noncoronary leaflet with either the right or left coronary leaflet in one small study.16 This might indicate a worse prognosis with a higher risk of fetal demise in BAVs with fusion of the noncoronary leaflet with the right or left coronary leaflet, possibly due to association with additional CHD. We performed a pathology study of hearts in a larger group of fetuses with TS. This gave us the unique opportunity to study cardiac morphology early in development in TS patients at the most severe end of the spectrum, We especially focused on BAV morphology and explored the association of distinct morphology types with other CHD in this selected group of TS with adverse outcome.

MethodsStudy populationThis study was undertaken in accordance with the local ethics committee and the Dutch regulation for the proper use of human tissue for medical research purposes. We included all TS fetuses

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(n = 36) and newborns (n = 1, age of death 2 weeks) from the Leiden collection of malformed hearts (Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands). This collection includes hearts preserved in ethanol and glycerin, dating from the nineteen fifties to the current era. Birth date of the included cases ranged from February 1993 to May 2012. Thirty-four fetuses and the newborn case had a non-mosaic 45,X karyotype confirmed in cells obtained by chorion villus biopsy (n = 20), amniotic punction (n = 13) or in fibroblasts (n = 2). In the two remaining fetuses no information regarding karyotype was available, though as they had clinical features (strongly suggestive) of TS (cystic hygroma, hydrops, intestinal malrotation) they were included in this study. In 31 fetuses the reason for ascertainment was known; these all had cystic hygroma or nuchal translucency >P95. Delivery was spontaneous or instigated because of intra-uterine death in six (17%) fetal cases, while in 26 (72%) termination of pregnancy of a living fetus was opted for; in four (11%) cases this information was unavailable. The gestation period ranged from 11 to 22 weeks (mean 17 ± 3 weeks), with exclusion of the neonatal case.

Anatomical studiesThe hearts of 25 of 37 (68%) cases were still available. Two experienced observers (MMB and KvE) investigated the hearts in consensus under a dissection microscope. Cardiac morphology was assessed using sequential segmental analysis, performed as completely as possible. For the hearts that were not available anymore (n = 12), details of cardiac anatomy were derived from records of previous autopsies. All of these autopsies had been performed by one of the current observers (MMB). Details of BAV morphology subtype were not available in autopsy reports; these hearts were included only in the study on the general cardiac morphology.Hypoplastic left heart (HLH) was considered to be present when the apex was formed by the right ventricle and the left ventricle was severely hypoplastic (less than one third of the right ventricle). Hearts with the apex formed by the right ventricle with the left ventricular length being larger than one third of the base-apex distance of the right ventricle (in the absence of right-sided abnor-malities) are referred to as borderline left ventricle (BLV).26 In some hearts, the lumen of the left ventricle was smaller than expected, but the apex was nevertheless formed by the left ventricle. We refer to these hearts as ‘suggestive HLH’, as these may evolve to HLH later in pregnancy. The ascending aorta was considered hypoplastic if the aortic orifice diameter was smaller than expected as compared to the pulmonary orifice diameter. Due to the small size of the hearts as well as the different preservation methods, it was not possible to measure dimensions validly. Tubular hypoplasia was defined as a narrowed (often elongated) aortic arch segment. Aortic coarctation was defined as a localized obstructive ridge within the arch consisting of a constriction of the aortic wall and/or a shelf protruding into the aortic lumen.27 Polyvalvular disease was defined as the presence of two or more abnormal or dysplastic cardiac valves.28 The frequencies of occurrence of associated cardiovascular abnormalities in the different aortic valve morphology subgroups were compared.

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BAV classification and terminologyWe defined BAV morphology by the orientation of the leaflets with respect to each other, the fusion of the leaflets and the presence and position of a raphe (Figure 1). The orientation of the leaflets was either anterior-posterior (AP) or left-right (LR). AP-BAV corresponds with BAV with fusion of the right coronary leaflet and left coronary leaflet; this is referred to as Type 1 BAV. LR-BAV can be either due to fusion of the right coronary leaflet and noncoronary leaflet, referred to as Type 2 BAV, or due to fusion of the left coronary and noncoronary leaflet, referred to as Type 3 BAV. Of note, although the term ‘fusion’ may suggest a developmental anlage of three leaflets two of which fuse together somewhere during development, there is no evidence of such a mechanism - the term ‘fusion’ as used in this paper should not be presumed to imply such mechanism. A raphe

Figure 1. Schematic representation of aortic valve morphology in 36 TS fetuses and 1 TS newborn (as viewed from above with the left coronary sinus on the right side). Numbers of cases with tricuspid aortic valve, bicuspid aortic valve, unicuspid aortic valve and aortic atresia are indicated. BAVs are divided into BAV with anterior-posterior leaflet orientation and BAV with left-right leaflet orientation. These groups are further subdivided into Type 1, Type 2 and Type 3 and with respect to the presence and position of a raphe (dashed line). *Numbers and proportions describing BAV morphology refer to cases that could be reinvestigated for BAV type (n = 18). TAV, tricuspid aortic valve, BAV, bicuspid aortic valve; UAV, unicuspid aortic valve; AV, aortic valve; PV, pulmonary valve; NCC, noncoronary cusp; LCC, left coronary cusp; RCC, right coronary cusp; LCA, left coronary artery; RCA, right coronary artery.

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was defined as a ridge located in the fused area of two leaflets, representing a malformed commissure between these leaflets. Raphes could be complete, i.e. extending to the leaflet edge, or incomplete, i.e. present in the proximal portion of the leaflet but not extending to the leaflet edge.

Statistical analysisFor comparison of the frequency of occurrence of cardiac abnormalities between hearts with tricuspid aortic valves and hearts with abnormal aortic valve morphology, as well as between different BAV morphology types, we performed contingency table analysis (Fisher’s exact test). P-values < 0.05 were considered statistically significant.

ResultsGeneral cardiovascular morphologyDetails about the cardiac morphology of the 37 cases are provided in Table 1. Two of 37 hearts showed completely normal morphology. Atrial situs as well as atrioventricular and ventriculo-arterial connections were normal in all hearts. Table 2 provides an overview of observed cardiovascular abnormalities. The most frequent anomaly was an abnormal aortic valve morphology, which was present in 32 of 37 (86%) hearts: BAV was observed in 28 (76%) hearts, unicommissural aortic valve in 2 (5%) and aortic atresia in 2 (5%) hearts. Ascending aorta hypoplasia, tubular hypoplasia of the aortic segment between the left common carotid artery and left subclavian artery (the aortic B segment) and persistent left superior caval vein (PLSCV) were present in more than half of the cases (Table 2). We did not observe aortic coarctation in any of the fetal cases that could be investigated for this abnormality (n = 32), though a juxtaductal coarctation was found in the neonatal heart. Coronary artery branching was normal in all cases in which this could be fully assessed (n = 19). The left and right coronary ostiae were located above the sinotubular junction in four and five of these hearts, respectively (Table 1).

Normal versus abnormal aortic valve morphology and associated cardio-vascular abnormalitiesIn five hearts (14%), the aortic valve was normal tricuspid. In these hearts, significantly less additional cardiovascular abnormalities were present as compared to hearts with abnormal aortic valve morphology: in none of the hearts with tricuspid aortic valve, there was evidence of (suggestive) HLH or BLV (as opposed to 17 of 32 hearts (53%) with abnormal aortic valve, P = 0.036), ascending aorta hypoplasia (abnormal aortic valve: 23 of 32 (72%), P = 0.005) or tubular hypoplasia (abnormal aortic valve: 23 of 30 (77%), P = 0.007). In addition, none of the hearts with tricuspid aortic valve showed polyvalvular disease (abnormal aortic valve: 10 of 28 (36%), P = 0.142) or PLSCV (abnormal aortic valve: 19 of 30 (63%), P = 0.013). In all three cases with tricuspid aortic valve with assessable coronary arteries, both coronary orifices were

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situated normally below the sinotubular junction. In two of the five hearts with tricuspid aortic valve the right subclavian artery originated abnormally from the aorta distal to the left subclavian artery and showed a retroesophageal course (arteria lusoria).

BAV morphology type and associated cardiovascular abnormalitiesEighteen hearts with BAV were still available, of which 11 (61%) showed Type 1 BAV and seven (39%) Type 2 BAV. There were no hearts with Type 3 BAV (Figure 1, Figure 2A-D). A raphe was identified in 14 of 18 (78%) BAVs (eight of 11 Type 1 BAVs and six of seven Type 2 BAVs). All raphes, except for one in Type 2 BAV, were incomplete.

Figure 2. Bicuspid aortic valves with different morphology types and associated abnormalities. A and B: Type 1 bicuspid aortic valve (case 10418 (A) and case 8690 (B)). In both valves, a raphe could not be identified. C and D: Type 2 bicuspid aortic valve (case 9457 (C) and case 9023 (D) In C, the valve is closed. E: tubular hypoplasia (arrow) of the aortic segment between the left common carotid artery and the left subclavian artery, the aortic B segment (case 9023). F: aberrant left vertebral artery (VA) originating directly from the aortic arch, between the left common carotid artery and the left subclavian artery (case 9457). G: right subclavian artery originating abnormally from the aorta distal to the left subclavian artery and showing a retroesophageal course (arteria lusoria, AL), case 8337. PT, pulmonary trunk; AA, aorta ascendens; BA, brachiocephalic artery; LCCA, left common carotid artery; LSA, left subclavian artery; AD, aorta descendens.

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+0at

resi

ana

3nd

/ nd

ndnd

-H

LH +

+M

itral

val

ve

sten

osis

9330

45,X

-20

+5B

AVnd

ndnd

/ nd

n +

+n

-nd

9361

45,X

-20

+6B

AVnd

ndnd

/ nd

n +

+H

LH -

nd

9372

45,X

-17

+4B

AVnd

ndnd

/ nd

n +

+H

LH +

-

9441

45,X

-17

+0B

AVnd

ndnd

/ nd

AL

+ +

n -

nd

9554

45,X

-u

BAV

ndnd

nd /

ndnd

ndnd

HLH

- -

Mitr

al v

alve

at

resi

a

1020

045

,X -

13+4

BAV

ndnd

nd /

ndA

L +

-H

LH +

-

1057

445

,X -

18+5

BAV

ndnd

nd /

ndn

+ +

sHLH

- +

TAP

VR

1057

545

,X -

15+3

BAV

ndnd

nd /

ndA

L -

-n

- -

1062

845

,X -

14+3

BAV

ndnd

nd /

ndn

+ +

n -

-

1071

845

,X -

15+0

BAV

ndnd

nd /

ndn

+ +

HLH

+ +

Bic

uspi

d P

V,

hypo

plas

ia le

ft pu

lmon

ary

arte

ry

8270

45,X

+20

+3B

AV1

1be

low

/ be

low

AL

- -

sHLH

+ -

8722

u +

13+0

BAV

10

belo

w /

belo

wn

+ +

n -

-S

ecun

dum

AS

D

8960

45,X

+20

+0B

AV1

0on

/ on

n +

-n

- -

Pre

mat

urel

y cl

osed

FO

9407

45,X

+19

+3B

AV1

1be

low

/ ab

ove

AL

+ +

n -

+

1031

545

,X +

16+3

BAV

11

belo

w /

belo

wn

+ -

BLV

+ -

1041

845

,X +

14+3

BAV

10

nd /

ndn

+ +

BLV

+ -

111

6u,

unk

now

n; n

a, n

ot a

pplic

able

; nd

, not

det

erm

ined

; AV,

aor

tic v

alve

; BAV

, bic

uspi

d ao

rtic

valv

e; A

P, B

AV w

ith a

nter

ior-

post

erio

r orie

ntat

ion

of th

e le

aflet

s; L

R, B

AV w

ith le

ft-rig

ht o

rien-

tatio

n of

the

leafl

ets;

TAV

, tric

uspi

d ao

rtic

valv

e; U

AV, u

nico

mm

isur

al a

ortic

val

ve; R

CA

, rig

ht c

oron

ary

arte

ry; L

CA

, lef

t cor

onar

y ar

tery

; AL,

arte

ria lu

soria

; AVA

, abe

rran

t lef

t ver

tebr

al

arte

ry; B

A-L

CC

A, c

omm

on o

stiu

m o

f bra

chio

ceph

alic

arte

ry a

nd le

ft co

mm

on c

arot

id a

rtery

; PLS

CV,

per

sist

ent l

eft s

uper

ior c

aval

vei

n; L

V, le

ft ve

ntric

le; H

LH, h

ypop

last

ic le

ft he

art;

BLV

, bo

rder

line

left

vent

ricle

; sH

LH, s

ugge

stiv

e hy

popl

astic

left

hear

t; LV

H, l

eft v

entri

cula

r hyp

ertro

phy;

PV

D, p

olyv

alvu

lar d

isea

se; A

SD

, atri

al s

epta

l def

ect;

FO, f

orm

ane

oval

e; T

AP

VR

, tot

al

anom

alou

s pu

lmon

ary

veno

us re

turn

; PV,

pul

mon

ary

valv

e. *

Pos

ition

of o

stiu

m o

f RC

A an

d LC

A in

rela

tion

to th

e si

notu

bula

r jun

ctio

n.

1058

045

,X +

20+5

BAV

11

on /

abov

eA

L -

-n

- -

1095

745

,X +

14+0

BAV

11

abov

e / o

nn

+ +

n -

+

1149

445

,X +

16+6

BAV

11

belo

w /

belo

wB

A-L

CC

A -

+n

- +

1154

545

,X +

14+0

BAV

11

belo

w /

belo

wn

+ +

n -

-

9244

45,X

+15

+0B

AV1

1nd

/ on

AL

- -

BLV

- -

8223

45,X

+21

+3B

AV2

1be

low

/ be

low

AVA

+ -

n -

-A

SD

sin

us c

oro-

nariu

s ty

pe

8568

45,X

+11

+0B

AV2

1on

/ nd

n +

+B

LV -

-

8856

45,X

+18

+3B

AV2

1be

low

/ be

low

n +

-n

+ -

9023

u +

19+0

BAV

20

belo

w /

belo

wn

+ +

sHLH

+ +

9457

45,X

+17

+5B

AV2

1be

low

/ be

low

AVA

+ +

n -

+

9458

45,X

+16

+5B

AV2

1be

low

/ be

low

n +

+B

LV +

+

1189

845

,X +

14+0

BAV

21

nd /

ndn

+nd

BLV

-nd

8337

45,X

+12

+0TA

Vna

0n

/ nA

L -

-n

- -

9162

45,X

+18

+3TA

Vna

0n

/ nn

- -

n -

-

1002

445

,X +

20+5

TAV

na0

n / n

AL

- -

n +

-

1009

745

,X -

13+3

TAV

na0

nd /

ndn

- -

n -

-

1014

645

,X -

13+0

TAV

na0

nd /

ndnd

nd -

n +

-

8422

45,X

+P

ost

UAV

na2

belo

w /

belo

wn

- +

HLH

+ -

Aor

tic c

oarc

tatio

n

1092

045

,X +

14+1

UAV

na2

n / n

n -

+n

- -

112

6

Table 2. Cardiovascular abnormalities of 36 Turner syndrome fetuses and one postnatal case

* n represents the number of observed cases / the number of hearts that could be assessed for this particular abnor-mality. HLH, hypoplastic left heart; BA, brachiocephalic artery; LCCA, left common carotid artery; PLSCV, persistent left superior caval vein; ASD, atrial septal defect; TAPVR, total anomalous pulmonary venous return; FO, foramen ovale.

n * %

Aortic valve morphology

Tricuspid 5 / 37 14

Unicommissural 2 / 37 5

Bicuspid 28 / 37 76

Atresia 2 / 37 5

HLH 8 / 37 22

Borderline/suggestive HLH 9 / 37 24

Left ventricular hypertrophy 14 / 37 38

Ascending aorta hypoplasia 23 / 37 62

Tubular hypoplasia aortic B segment 23 / 34 68

Aortic coarctation 1 / 31 3

Aortic branching pattern

Normal 23 / 35 66

Arteria lusoria 9 / 35 26

Aberrant left vertebral artery 2 / 35 6

Common ostium BA and LCCA 1 / 35 3

PLSCV 19 / 35 54

Polyvalvular disease 10 / 33 30

Other structural malformations

Secundum ASD 1 / 37 3

ASD sinus coronarius type 1 / 37 3

Mitral valve atresia 1 / 37 3

TAPVR 1 / 37 3

Bicuspid pulmonary valve 1 / 37 3

Prematurely closed FO 1 / 37 3

113

6There were no significant differences in the general occurrence of additional cardiovascular abnor-malities between hearts with Type 1 or Type 2 BAV (Table 3). Remarkably, all seven hearts with Type 2 BAV showed ascending aorta hypoplasia, as opposed to 55% (6/11) of hearts with Type 1 BAV. In addition, all hearts with type 2 BAV showed tubular hypoplasia of the aortic B segment, compared to 64% (7/11) of cases with Type 1 BAV. In two hearts with Type 2 BAV, the aortic B segment was almost atretic. Arteria lusoria was found in four of 11 (36%) Type 1 BAV cases but in none of Type 2 BAV cases. In contrast, an aberrant left vertebral artery originating directly from the aortic arch was present in two of seven (29%) Type 2 BAV cases but in none of Type 1 BAV cases.

Table 3. BAV morphology type and associated cardiovascular abnormalities (n = 18)

* n represents the number of observed cases / the number of hearts that could be assessed for this particular abnor-mality. HLH, hypoplastic left heart; BA, brachiocephalic artery; LCCA, left common carotid artery; PLSCV, persistent left superior caval vein.

Type 1 BAV (n = 11) Type 2 BAV (n = 7)

n* % n* % P-value

HLH 0 / 11 0 0 / 7 0

Borderline/suggestive HLH 4 / 11 36 4 / 7 57 0.630

Left ventricular hypertrophy 3 / 11 27 3 / 7 43 0.627

Ascending aorta hypoplasia 6 / 11 55 7 / 7 100 0.101

Tubular hypoplasia B segment 7 / 11 64 7 / 7 100 0.119

Aortic coarctation 0 / 10 0 0 / 5 0

Aortic branching pattern

Normal 6 / 11 55 5 / 7 71 0.637

Arteria lusoria 4 / 11 36 0 / 7 0 0.119

Aberrant left vertebral artery 0 / 11 0 2 / 7 29 0.137

Common ostium BA and LCCA 1 / 11 9 0 / 7 0 1.000

PLSCV 6 / 11 55 4 / 6 67 1.000

Polyvalvular disease 3 / 11 26 3 / 6 50 0.600

114

6

DiscussionWe performed a pathological study focused on the morphology of the aortic valve and its associa-tion with other CHD in prenatal hearts of a selected group of TS patients ascertained for cystic hygroma or NT >P95. We were able to include the hearts of 36 TS fetuses and one TS newborn, which is a unique number taking into account the scarcity of such specimens. Key findings of our study are: 1) abnormal aortic valve morphology in the vast majority (86%) of hearts, with BAV in 76%, 2) the majority (61%) of BAVs showing fusion of the right and left coronary leaflets (Type 1 BAV), 3) a higher percentage of BAVs shows fusion of the right coronary and noncoronary leaflets (Type 2 BAV) as compared to reports in adults with TS, and 4) an association of all cases with Type 2 BAV with hypoplasia of the ascending aorta and tubular hypoplasia as compared to a lower association in Type 1 BAV.The high number of aortic valve and other left sided heart defects as found in our fetal TS popula-tion is in accordance with previous studies.13-16 We encountered both Type 1 BAV and Type 2 BAV. Interestingly, recent animal studies have shown that BAVs with distinct morphology types result from different underlying genotypic variants. Fernandez et al.22 demonstrated that a well estab-lished Syrian hamster model has Type 1 BAV, resulting from abnormal septation of the outflow tract, possibly by abnormal neural crest cell behavior. In contrast, a mice model lacking endothe-lium specific nitric oxide synthase (Nos3) has Type 2 BAV, originating from abnormal formation of the outflow tract cushions which is probably caused by altered nitric oxide dependent endothelial to mesenchymal transition.22 In humans, it has been shown however, that there is only 76% concordance of BAV type within families with multiple affected individuals with BAV.23 The TS fetuses in our study shared the same genetic abnormality (complete non-mosaic monosomy X) though we encountered both Type 1 BAV and Type 2 BAV. This implies that the lack of one sex-chromosome can lead to different BAV morphologies, and that other factors have a role in the eventual BAV morphology type. Environmental events, hemodynamic factors,13 modifier genes and epigenetic mechanisms may all be involved. Although BAV and aortic coarctation have been linked to deletion of the X chromosome short arm in TS,29 no X-linked genes that have a definite role in human BAV have been identified. In the general BAV population, aortic coarctation and other left sided heart defects are more frequent in Type 1 BAV.19 As clear aortic coarctation, defined as a localized obstructive ridge within the arch, was macroscopically present in only one patient in our study, our results are not informative on this subject. We did not find significant associations between BAV type and other cardiovascular abnormalities, but the absolute numbers in our population were quite small. Particularly interesting, ascending aorta hypoplasia and tubular hypoplasia of the aortic B segment were both present in all Type 2 BAVs but in only half and two-thirds of Type 1 BAVs, respectively. This might reflect a flow related association between Type 2 BAV and aortic arch abnormalities (i.e. less flow due to greater obstruction in Type 2 BAV, leading to more severe hypoplasia of distal parts of the aorta), but it may also result from a common developmental origin. The aortic B segment derives from the embryological left fourth pharyngeal arch artery, which is derived from neural crest cells.30 This

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6

suggests a developmental origin related to deficiency of a left sided contribution of neural crest cells in Type 2 BAVs, or a deficient interaction between neural crest and the surrounding second heart field, as has been suggested.31 Accordingly, an aberrant left vertebral artery was only present in Type 2 BAV. Although the exact developmental background of vertebral artery aberrance has not been resolved, it is considered an abnormal persistence of the lower intervertebral arteries and occurs more often on the left.32 In contrast, we found a right arteria lusoria, which is associated with a deficiency of the right fourth pharyngeal arch artery, only in combination with Type 1 BAV.30 Fernandez et al. suggested that particularly Type 1 BAV results from abnormal neural crest cell behavior.22 Our data may indicate that also the sidedness of neural crest contributions may be of relevance, as was shown previously for second heart field contributions to the outflow tract.33 Further study in larger, unselected (non-TS) cohorts is required to support this hypothesis. The proportions of Type 1 (61%) and Type 2 BAV (39%) in our fetal TS population are similar to those reported in the general BAV population.17-21 Interestingly, in the general TS population, 82-95% of BAVs were reported to be of Type 1 and 5-18% of Type 2.24,25 In our fetal BAV cases, the proportion of Type 2 BAV was thus higher. This difference might be explained by differences in assessment method (pathology versus echocardiography/MRI), though this seems unlikely. We speculate that in TS, Type 2 BAV has a worse prognosis than Type 1 BAV, leading to a higher rate of fetal death. The occurrence of severe aortic pathology in 100% of Type 2 BAVs, compared to a lower association with Type 1 BAVs, might be an explanation for the worse prognosis. In the general BAV population, an age-dependent association of BAV type with prognosis has been reported: aortic valve pathology (stenosis and regurgitation) is associated most with Type 2 BAV in children,19,20 but with Type 1 BAV in adults.23 Possibly, this age-related association might thus go back into fetal life, with a worse prognosis of Type 2 BAV even in utero. It would be interesting to test this hypothesis in fetal hearts of non-TS patients. However, as fetal post-mortem heart speci-mens of cases with isolated, nonsyndromic BAVs are virtually absent, this might prove difficult to realize. With improving prenatal imaging techniques such as 3D ultrasound or MRI, prospec-tive studies on BAV morphology, associated cardiovascular abnormalities and outcome in living fetuses may become possible. If indeed there is a worse prognosis of Type 2 BAV in utero, these improved techniques may become particularly relevant for risk stratification (by determination of BAV morphology in early pregnancies) of fetuses with unfavorable outcome, thus making BAV morphology type a prognostic marker.

LimitationsAs the aim of our study was to investigate BAV morphology and associated cardiovascular abnor-malities in fetal life in a selected group of TS patients with a severe phenotype, prevalence rates of cardiovascular abnormalities can by no means be applied to the general TS population. Although the inclusion of 36 TS fetuses and one TS newborn is a large number taking into account the rarity of these kind of specimens, the absolute number is quite small. Moreover, as this study regarded

116

6

very small and sometimes macerated specimens it was not possible to assess all cardiovascular structures in every heart or to measure dimensions validly. Data on the hearts that were not avail-able anymore were derived from the previous autopsy reports, which may have lead to differences in assessment between hearts that were still available and hearts that were not. However, as all previous autopsies had been performed by one of the current investigators, we believe these differences were minimal. Indeed, we did not encounter important differences by comparison of the abnormalities in every newly assessed heart and its previous autopsy report.

ConclusionsThe vast majority (86%) of TS fetuses with cystic hygroma has abnormal aortic valve morphology, with BAV present in 76%. Different BAV morphology types are present. This implies that other factors than haplo-insufficiency of genes on the sex-chromosomes contribute to the eventual BAV type, such as other (epi)genetic, hemodynamic and environmental factors. We speculate that the occurrence of different cardiovascular malformations in different BAV types might reflect a sided-ness in disturbed neural crest contribution in Type 1 versus Type 2 BAV during development. Moreover, it is remarkable that the proportion of Type 2 BAV seems higher in fetuses than in adults with TS. As hearts with Type 2 BAV all had severe pathology of the aorta, Type 2 BAV may have a worse prognosis than Type 1 BAV, leading to a lower survival in utero. The numbers in our study were small, however, and with increasing applications of advanced imaging modalities such as (3D)fetal echocardiography, it may become possible to prospectively study BAV morphology in early (TS) pregnancies and further explore associations between BAV type, additional cardiovas-cular malformations and pregnancy outcome.

AcknowledgementsThe authors express their gratitude to Jan Leeflang, Lambertus Wisse and Marjolein van Mespel for technical assistance.

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2 Hoffman JI, Kaplan S. The incidence of congenital heart disease. J Am Coll Cardiol 2002;39:1890-9003 Roberts WC, Ko JM. Frequency by decades of unicuspid, bicuspid, and tricuspid aortic valves in

adults having isolated aortic valve replacement for aortic stenosis, with or without associated aortic regurgitation. Circulation 2005;111:920-5

4 Fedak PW, Verma S, David TE, Leask RL, Weisel RD, Butany J. Clinical and pathophysiological implications of a bicuspid aortic valve. Circulation 2002;106:900-4

5 Luijendijk P, Stevens AW, de Bruin-Bon RH et al. Rates and determinants of progressive aortic valve dysfunction in aortic coarctation. Int J Cardiol 2012:-doi: 10.1016/j.ijcard.2012.07.028

6 Hook EB, Warburton D. The distribution of chromosomal genotypes associated with Turner’s syndrome: livebirth prevalence rates and evidence for diminished fetal mortality and severity in geno-types associated with structural X abnormalities or mosaicism. Hum Genet 1983;64:24-7

7 Byrne J, Warburton D, Kline J, Blanc W, Stein Z. Morphology of early fetal deaths and their chromo-somal characteristics. Teratology 1985;32:297-315

8 Sybert VP. Cardiovascular malformations and complications in Turner syndrome. Pediatrics 1998;101:E11

9 Mazzanti L, Cacciari E. Congenital heart disease in patients with Turner’s syndrome. Italian Study Group for Turner Syndrome (ISGTS). J Pediatr 1998;133:688-92

10 Volkl TM, Degenhardt K, Koch A, Simm D, Dorr HG, Singer H. Cardiovascular anomalies in children and young adults with Ullrich-Turner syndrome the Erlangen experience. Clin Cardiol 2005;28:88-92

11 Loscalzo ML, Van PL, Ho VB et al. Association between fetal lymphedema and congenital cardiovas-cular defects in Turner syndrome. Pediatrics 2005;115:732-5

12 Kim HK, Gottliebson W, Hor K et al. Cardiovascular anomalies in Turner syndrome: spectrum, preva-lence, and cardiac MRI findings in a pediatric and young adult population. AJR Am J Roentgenol 2011;196:454-60

13 Clark EB. Neck web and congenital heart defects: a pathogenic association in 45 X-O Turner syndrome? Teratology 1984;29:355-61

14 Surerus E, Huggon IC, Allan LD. Turner’s syndrome in fetal life. Ultrasound Obstet Gynecol 2003;22:264-7

15 Barr M, Jr., Oman-Ganes L. Turner syndrome morphology and morphometrics: Cardiac hypoplasia as a cause of midgestation death. Teratology 2002;66:65-72

16 Miyabara S, Nakayama M, Suzumori K, Yonemitsu N, Sugihara H. Developmental analysis of cardio-vascular system of 45,X fetuses with cystic hygroma. Am J Med Genet 1997;68:135-41

17 Schaefer BM, Lewin MB, Stout KK et al. The bicuspid aortic valve: an integrated phenotypic clas-sification of leaflet morphology and aortic root shape. Heart 2008;94:1634-8

18 Sievers HH, Schmidtke C. A classification system for the bicuspid aortic valve from 304 surgical specimens. J Thorac Cardiovasc Surg 2007;133:1226-33

19 Fernandes SM, Sanders SP, Khairy P et al. Morphology of bicuspid aortic valve in children and adolescents. J Am Coll Cardiol 2004;44:1648-51

20 Fernandes SM, Khairy P, Sanders SP, Colan SD. Bicuspid aortic valve morphology and interventions in the young. J Am Coll Cardiol 2007;49:2211-4

21 Jassal DS, Bhagirath KM, Tam JW et al. Association of Bicuspid aortic valve morphology and aortic root dimensions: a substudy of the aortic stenosis progression observation measuring effects of rosuvastatin (ASTRONOMER) study. Echocardiography 2010;27:174-9

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22 Fernandez B, Duran AC, Fernandez-Gallego T et al. Bicuspid aortic valves with different spatial orientations of the leaflets are distinct etiological entities. J Am Coll Cardiol 2009;54:2312-8

23 Calloway TJ, Martin LJ, Zhang X, Tandon A, Benson DW, Hinton RB. Risk factors for aortic valve disease in bicuspid aortic valve: a family-based study. Am J Med Genet A 2011;155A:1015-20

24 Sachdev V, Matura LA, Sidenko S et al. Aortic valve disease in Turner syndrome. J Am Coll Cardiol 2008;51:1904-9

25 Mortensen KH, Hjerrild BE, Stochholm K et al. Dilation of the ascending aorta in Turner syndrome - a prospective cardiovascular magnetic resonance study. J Cardiovasc Magn Reson 2011;13:24

26 Mahtab EA, Gittenberger-de Groot AC, Vicente-Steijn R et al. Disturbed myocardial connexin 43 and N-cadherin expressions in hypoplastic left heart syndrome and borderline left ventricle. J Thorac Cardio-vasc Surg 2012:-doi: 10.1016/j.jtcvs.2012.02.011

27 Elzenga NJ, Gittenberger-de Groot AC, Oppenheimer-Dekker A. Coarctation and other obstructive aortic arch anomalies: their relationship to the ductus arteriosus. Int J Cardiol 1986;13:289-308

28 Bartram U, Bartelings MM, Kramer HH, Gittenberger-de Groot AC. Congenital polyvalvular disease: a review. Pediatr Cardiol 2001;22:93-101

29 Bondy C, Bakalov VK, Cheng C, Olivieri L, Rosing DR, Arai AE. Bicuspid aortic valve and aortic co-arctation are linked to deletion of the X chromosome short arm in Turner syndrome. J Med Genet 2013

30 Molin DG, Poelmann RE, DeRuiter MC, Azhar M, Doetschman T, Gittenberger-de Groot AC. Trans-forming growth factor beta-SMAD2 signaling regulates aortic arch innervation and development. Circ Res 2004;95:1109-17

31 Rochais F, Mesbah K, Kelly RG. Signaling pathways controlling second heart field development. Circ Res 2009;104:933-42

32 Chen CJ, Wang LJ, Wong YC. Abnormal origin of the vertebral artery from the common carotid ar-tery. AJNR Am J Neuroradiol 1998;19:1414-6

33 Scherptong RW, Jongbloed MR, Wisse LJ et al. Morphogenesis of outflow tract rotation during cardiac development: the pulmonary push concept. Dev Dyn 2012;241:1413-22

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The ambiguous role of NKX2-5 mutations in thyroid dysgenesis

van Engelen K, Mommersteeg MTM, Baars MJH, Lam J, Ilgun A, van Trotsenburg ASP, Smets AMJB, Christoffels VM, Mulder BJM, Postma AV

PLoS One 2012;7(12):e52685

Chapter 7

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7

AbstractNKX2-5 is a homeodomain-containing transcription factor implied in both heart and thyroid development. Numerous mutations in NKX2-5 have been reported in individuals with congenital heart disease (CHD), but recently a select few have been associated with thyroid dysgenesis, among which the p.A119S variation. We sequenced NKX2-5 in 303 sporadic CHD patients and 38 families with at least two individuals with CHD. The p.A119S variation was identified in two unrelated patients: one was found in the proband of a family with four affected individuals with CHD and the other in a sporadic CHD patient. Clinical evaluation of heart and thyroid showed that the mutation did not segregate with CHD in the familial case, nor did any of the seven mutation carriers have thyroid abnormalities. We tested the functional consequences of the p.A119S variation in a cellular context by performing transactivation assays with promoters relevant for both heart and thyroid development in rat heart derived H10 cells and HELA cells. There was no difference between wildtype NKX2-5 and p.A119S NKX2-5 in activation of the investigated promoters in both cell lines. Additionally, we reviewed the current literature on the topic, showing that there is no clear evidence for a major pathogenic role of NKX2-5 mutations in thyroid dysgenesis. In conclusion, our study demonstrates that p.A119S does not cause CHD or TD and that it is a rare variation that behaves equal to wildtype NKX2-5. Furthermore, given the wealth of published evidence, we suggest that NKX2-5 mutations do not play a major path-ogenic role in thyroid dysgenesis, and that genetic testing of NKX2-5 in TD is not warranted.

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7

IntroductionPersistent congenital hypothyroidism of thyroidal origin is a relatively common disorder, occurring in about 1/2500 live births.1 In 85% of cases it is caused by thyroid dysgenesis (TD), consisting of agenesis, hypoplasia or ectopia of the thyroid gland.2 TD is a heterogeneous disorder that occurs mostly sporadically, though 2% of cases are reported as familial.3 The patho-genesis of TD is largely unknown; possible roles for environmental, genetic and epige-netic factors have been suggested, and in a minority of humans with TD mutations in NKX2-1,4 FOXE1,5 PAX8,6 and TSHR 7 have been identified. Additionally, in a recent study mutations in NKX2-5 were reported in a small proportion of patients with persistent congenital hypothyroidism.8

NKX2-5 encodes a homeodomain-containing transcription factor that is expressed during thyroid development (for review see 9), but it is mainly known to play a crucial role in heart development.10 NKX2-5 mutations have been found in a subset of patients with congenital heart disease (CHD), mostly septal defects.11,12 As CHD is overrepresented among children with TD and vice versa, a developmental association between the cardiac and thyroid systems has been suggested.13-15 We screened families and individual patients with CHD for mutations in NKX2-5. In this paper we focus on the p.A119S variation, which we found in two probands. Dentice et al. reported this as a causative mutation in a child with ectopic thyroid gland, with functional studies showing a dominant negative effect of the mutation.8 We evaluated the heart and the thyroid gland in our two families with a total of seven p.A119S carriers and we performed follow-up functional studies. Additionally, we discuss existing literature on the connection between NKX2-5 mutations and TD.

MethodsEthics statementThis study was approved by the Medical Ethical Committee of the Academic Medical Center in Amsterdam. Written informed consent was obtained from all participants.

Patients and clinical evaluationDNA from 303 patients with primum atrial septal defect (ASDI, n = 271) or secundum atrial septal defect (ASDII, n = 32) was extracted from CONCOR, a nationwide registry and DNA bank for adult patients with CHD, described in detail elsewhere.16 Additionally, probands of 38 families with multiple (at least two) affected patients with several forms of CHD, identified at the depart-ments of clinical genetics or cardiology of the AMC, were included. In this study, we focused on patients who were found to carry the p.A119S NKX2-5 variation. Probands with this variation, as well as their available family members, were clinically evaluated. Medical records were analyzed and all individuals underwent physical examination with attention to syndromic features. Cardio-logic examination consisted of a 12-lead electrocardiogram (ECG) and two-dimensional echocardiography, which were assessed by a cardiologist who was blinded for the mutational status.

124

7

Thyroid ultrasound and thyroid function analysis were used to investigate the thyroid gland. TSH and free T4 were measured by time-resolved fluoroimmunoassay (Delfia, hTSH Delfia Ulta resp. FT4 Delfia , Perkin Elmer, Turku, Finland), detection limits: 0.01 mU/L for TSH and 2 pmol/L for free T4, total assay variation: 4 - 5% for TSH and 6 - 7% for free T4. Thyreoglobulin and anti-TPO were measured by chemiluminescence immunoassay (LUMI-test Tg resp. anti-TPO, BRAHMS, Berlin, Germany), detection limits: 1 pmol/L for Tg and 30 kU/L for anti-TPO, total assay variation: 7 - 13 % for Tg and 8-12 % for anti-TPO. The results of the ultrasound were ana-lyzed by a radiologist who was blinded for mutational status as well as cardiologic status.

Mutation analysisGenomic DNA of CHD patients as well as relatives of patients carrying the A119S variation was extracted from peripheral blood according to standard procedures. Coding regions and intron–exon boundaries of NKX2-5 (NM_004387.3) were analyzed using direct sequence analysis on an ABI3730xl capillary sequencer using Big-Dye Terminator v3.1 (Applied Biosystems). Data were analyzed using Codoncode analysis software (v3.1, CodonCode Corporation). In the proband with the p.A119S variation who had aortic coarctation and bicuspid aortic valve, sequence analysis of the NOTCH1 gene was also performed.

Plasmid constructs and transfections Human clones for NKX2-5 and TBX5 were obtained from the IMAGE consortium.17 The human clones were in the following vectors: pCMVSport6-hNKX2-5 and pcDNA3.1-hTBX5. Promoter construct for ANF-luc is as described before,18 the promoters for Dio2, Tg and TPO were cloned from their appropriate species, as described before 8, and subcloned into pGL3 basic expression vectors (Promega). Expression and promoter constructs were all sequence veri-fied. pCMVSport6-hNKX2-5 mutants (p.A119S, p.N188K) were constructed using site-directed mutagenesis (Strategene). Transfections were performed using polyethylenimine (25 kDa, linear, Brunschwick).

EMSA, Probe AnnealingRadioactive Electrophoretic Mobility Shift Assay (EMSA) was performed using the following wildtype sequence as probes: 5’-TCTGCTCTTCTCACACCTTTGAAGTGGGGGCCTCTT and its complementary oligo (5’-GCCTCAAGAGGCCCCCACTTCAAAGGTGTG), as described before.18 The specific conditions were as follows: bandshift buffer (BB) (10 mM Tris pH 7.9, 10% glycerol, 50 mM NaCl, 0.5 mM EDTA); non-specific competitor Spermidine 3-HCl (Sigma, S2501) at a concentration of 1 µg/µl; prepared according manufacturers instruction. First 5.0 µg crude nuclear cell extracts were pre-incubated for 5 min at +15 to +25 °C in a reaction containing 14 µl BB, 1 µl of the non-specific competitor spermidine, 1 µg BSA, 1 mM DTT and supplemented with H20 up to 20 μl. Input was corrected for Nkx2.5 expression and total amount protein was kept constant at 5.0 µg by addition of empty vector nuclear extracts. Then 2 µl of labelled Nkx-specific

125

7

probe (30000 c.p.m) was added. Complexes were allowed to form for 20-25 min at +15 to +25 °C. The samples were loaded on 6%-TBE polyacrylamide gel which was prerunned at RT for 30 min at 25 V. Complexes were separated at 4 V/cm at RT for 60 min. Gels were dried unfixed on Whatman 3MM and exposed for autoradiography.

Luciferase assay Neonatal rat heart myocytes, immortalized with a temperature-sensitive SV40 antigen (H10 cells,19 were grown in standard 12-wells plates in DMEM supplemented with 10% FCS (Gibco-BRL) and glutamine. HeLa cells were grown according to standard culturing conditions.20 700 ng Nppa/TPO/Dio2/Tg-luciferase constructs were co-transfected with 1 ng of cmv-renilla vector, as normali-zation control (Promega), together with appropriate combinations of expression constructs (pCMVSport6-hNKX2-5, pcDNA3.1-hTBX5) up to 900 ng. Measurements were performed on a Glomax 20/20 luminometer. Triplo transfection experiments were repeated at least three times for each condition, data were corrected for intersession variation as described.21 Statistical analysis was performed using two-tailed t-test, P < 0.05 was considered significant.

Nuclear localization Cos7 cells 22 were seeded in standard 12-wells plates and transfected with 500 ng WT or p.A119S NKX2.5. 24 h post-transfection, cells were fixed in 2% paraformaldehyde, permeabilized using 0.3% Triton X-100, and incubated with rabbit anti-Nkx2-5 (Santra cruz) and DAPI (Sigma).

ResultsMutational analysisWe identified a total of three missense NKX2-5 variations in our cohort of 341 CHD patients: a p.C270Y variant in a patient with ASDI and cleft mitral valve, and twice the p.A119S variant in separate probands (see below). We will only discuss the results of the p.A119S variant, as the other variation is outside the scope of this study. The nucleotide change from G to T at codon 119 in NKX2-5 was identified in two patients. This results in the substitution of an alanine for a serine leading to p.A119S. This variation was present once in a proband from one of the 38 families tested and once in a proband from 303 sporadic patients with ASD. The p.A119S variation was not found in 200 local controls. Data from the NHLBI exome sequencing project shows that it is a very rare variation with a minor allele frequency of 0.001% (7/6470 individuals, rs1378526).23 Alignment of the amino acids of proteins of different species shows that this position is conserved up to rat Nkx2-5 (Figure 1A). However, chicken Nkx2-5 protein actually has a serine at this position, though the surrounding amino acids differ. The pedigrees of the families with the p.A119S variation are shown in Figure 1B. Table 1 summarizes the clinical features of both families. Analysis of NOTCH1 in the proband of family 1, who had aortic coarctation and bicuspid aortic valve, did not show a pathogenic mutation.

126

7Figure 1. Family pedigrees, amino acid alignments, nuclear localization of NKX2-5 protein and electro mobility shift assay. A. Pedigrees of the two families with the p.A119S NKX2-5 mutation. Individuals with congenital heart defects are indicated with a filled black symbol, while individuals with normal echocar-diography are indicated with a white symbol. Grey symbols represent individuals that have not been evaluated clinically. A slash denotes a deceased individual; the proband is indicated by an arrow. None of the evaluated family members showed thyroid abnormalities. Heterozygous carriers of p.A119S are represented by +/- and non-carriers by -/-. B. Multiple alignments of amino acids of the region surrounding p.A119 for various species. C. Nuclear localization of either wildtype or p.A119S NKX2-5 protein in COS7 cells. Nuclei are stained in green, red represents either the wildtype or mutant protein, orange indicates nuclei that are positive for wildtype or mutant protein. D. Electro mobility shift assay in cos7 cells, using the published nkx2.5 binding site [26]. Wildtype nkx2.5 protein and p.A119S Nkx2.5 protein bind equally well, – is untransfected control.

127

7

Patie

nts

A11

9SH

eart

Thy

roid

Rem

arks

Se

x / a

ge a

t ev

alua

tion

(yea

rs)

C

HD

ECG

Posi

tion

in n

eck

Asp

ect

Volu

me

left

lobe

(m

l)

Volu

me

right

lo

be (m

l)

TSH

(m

E/L)

Free

T4

(pm

ol/L

)

Thyr

eo-

glob

ulin

(p

mol

/L)

Ant

i-TPO

an

ti-bo

dies

Fam

ily 1

I-1M

/ 81

+C

oA,

BAV

Atria

l fib

rilla

tion

Nor

mal

H

om9.

59.

02.

2014

.54

Neg

II-1

F / 5

1+

VSD

Nor

mal

N

orm

al

Hom

4.6

3.3

3.60

12.7

9N

eg

II-2

F / 4

9 -

Non

eLo

w

volta

ges

Nor

mal

H

om3.

73.

91.

6012

.35

Neg

II-3

M /

48+

Non

eN

orm

al

Nor

mal

H

om5.

07.

52.

1014

.46

ND

II-4

M /

10 d

ays

ND

CoA

ND

ND

ND

ND

ND

ND

ND

ND

ND

Dec

ease

d at

age

10

day

s

III-1

F / 2

4-

VSD

Rig

ht a

xis

devi

atio

nN

orm

al

Hom

2,9

31.

7013

.69

Neg

III-2

F / 2

1-

Non

eN

orm

al

Nor

mal

H

om4.

64.

81.

5013

.46

Neg

III-4

F / 2

0+

Non

eN

orm

al

Nor

mal

H

om3.

06.

01.

4015

.36

Neg

III-5

F / 1

2+

Non

eN

orm

al

Nor

mal

H

om2.

63.

5N

DN

DN

DN

D

Fam

ily 2

I-1M

/ 60

-N

one

ND

Nor

mal

H

om4.

45.

91.

8016

.24

Neg

I-2F

/ 62

+N

one

repo

rted

ND

ND

ND

ND

ND

ND

ND

ND

ND

II-1

M /

35-

Non

eN

DN

orm

al

Hom

6.7

8.9

1.50

14.2

6N

eg

II-2

M /

31+

ASD

I, AS

DII

RBB

BN

orm

al

Hom

5.6

5.0

1.40

17.0

2Po

s

(6

20 k

U/l)

Hom

ogen

eous

so

lid n

odul

e in

le

ft th

yroi

d lo

be,

diam

eter

6 m

m

ND

, not

det

erm

ined

; neg

, neg

ativ

e; p

os, p

ositi

ve; C

HD

, con

geni

tal h

eart

dise

ase;

CoA

, aor

tic c

oarc

tatio

n; B

AV, b

icus

pid

aorti

c va

lve;

VS

D, v

entri

cula

r sep

tal d

efec

t; A

SD

I, os

tium

prim

um

atria

l sep

tal d

efec

t; A

SD

II, o

stiu

m s

ecun

dum

atri

al s

epta

l def

ect;

RB

BB

, rig

ht b

undl

e br

anch

blo

ck. A

nti-T

PO

ant

ibod

ies

‘neg

ativ

e’ m

eans

val

ue <

50

kU/l,

Hom

; Hom

ogen

eous

.

Tabl

e 1.

Clin

ical

det

ails

of t

he fa

mily

mem

bers

128

7

Family phenotypesFamily 1The p.A119S variation was found in the proband (I-1), who had an aortic coarctation and bicuspid aortic valve diagnosed at age 45 years. The coarctation was surgically corrected at age 45 years and an artificial aortic valve was implanted at age 73 years because of severe calci-fication and stenosis. The proband’s youngest son (II-4) died 20 days after birth, post-mortem pathology revealing aortic coarctation. The oldest daughter of the proband (II-1) was born with a ventricular septal defect (VSD) that closed spontaneously during childhood. Her oldest daughter (III-1) also had a VSD which was surgically closed when she was 7 months old. Echocar-diography was normal in the other family members. Normal location, volume and structure of the thyroid gland were shown by ultrasound in all investigated family members. Thyroid func-tion was also normal in all individuals. The p.A119S variation did not segregate with the cardiac defects within the family, as III-1 is affected but she does not have the mutation and several family members without any evidence of CHD carried the mutation (II-3, III-4, III-5).

Family 2The p.A119S variation was also found in a sporadic patient with ASDI with cleft mitral valve as well as small ASDII, for which a surgical correction took place at the age of 5 years. Thyroid ultrasound showed normal location and volume of the gland, but a small nodule was present in the left lobe. Additionally, anti-TPO antibodies were positive (620 kU/l) with normal thyroid function tests. These abnormalities are frequent in the general population,24, 25 and we therefore do not consider them to fall outside the range of expected findings. The proband’s mother was found to carry the p.A119S variation and the father and brother did not. The mother was not available for clinical evaluation, though she did not have a history of cardiac or thyroid disorders. The proband’s father had a myocardial infarction at age 55. He did not have CHD. Echocardiography in the proband’s brother did not show CHD either. Thyroid evaluation of the proband’s father and brother was also normal.

Normal sub cellular distribution of the NKX2-5 p.A119S protein To be able to regulate transcription and exert its function, the NKX2-5 protein needs to be present in the nucleus. The localization of the p.A119S NKX2-5 protein was assessed by transfecting it into rat heart-derived cells (H10) and COS7 cells. The localization of mutant and WT proteins was visualized with an antibody against Nkx2-5. Figure 1C shows that both the wildtype and p.A119S NKX2-5 protein localize exclusively inside the nucleus, indicating that the process of nuclear import is not affected by the variation.

No functional defect in DNA binding of p.A119S Nkx2.5The transcription factor Nkx2.5 activates its target genes by binding to the DNA. To test whether the DNA binding capacity of p.A119S Nkx2.5 was altered, we used an electrophoretic mobility shift assay (EMSA).18 A fragment of the Nppa promoter was used, a well characterized promoter

129

7

relevant in heart development, containing a functional Nkx2.5 binding element.26 As shown in figure 1D, both wildtype and p.A119S Nkx2.5 protein bind equally well, indicating that there is no difference in DNA binding capacity between p.A119S and wildtype Nkx2.5 protein.

No difference in promoter activations of wildtype NKX2-5 or p.A119S NKX2-5To test the functional consequences of the p.A119S variation in a relevant cellular context, we used reporter assays in which the proximal NPPA promoter (-270 to +1),26 and the Dio2, Tg and TPO8 promoters involved in thyroid gland function, were fused to a luciferase reporter. These promoters all contain functional binding sites for NKX2-5. We also tested NKX2-5 in combination with the TBX5 transcription factor as TBX5 synergizes with NKX2-5 in the activation of the NPPA promoter.26 These transactivation assays were performed in both H10 cells and HELA cells, as used in the original publication of Dentice et al. on the possible connection between p.A119S and TD.8 As a negative control we also used the p.N188K NKX2-5 mutant, reported as causative in a family with five affected presenting with atrial septal defects, Ebsteins anomaly and abnormal AV conduction.27 No thyroid abnormalities were reported for this mutation. p.N188K introduces a mutation in the homedomain of Nkx2.5, an element conserved in all members of the Nkx protein family and known to directly contact adenine in the major groove of DNA. The p.N188K mutation leads to a complete loss-of-function in DNA binding 28 and can there-fore serve as a negative control.In the H10 cells, the wildtype NKX2-5 and the p.A119S protein both significantly activated the NPPA promoter driven reporter. When transfected together with TBX5 both wildtype NKX2-5 and p.A119S NKX2-5 also activated the reporter construct synergistically (Figure 2A). There was no difference between wildtype NKX2-5 and p.A119S NKX2-5 in the activation of the NPPA promoter construct for any condition tested. Likewise, we observed no difference in activa-tion of the Dio2, Tg or TPO promoter constructs between wildtype and p.A119S in H10 cells. We repeated all experiments in HELA cells, and found stronger activation of all constructs in comparison to the H10 cells. However, once again, we observed no difference in activation of any promoter tested between wildtype NKX2-5 and p.A119S NKX2-5 (Figure 2B).

DiscussionIn our population of 303 patients with ASD and 38 probands from families with CHD, we found the NKX2-5 p.A119S variation in two patients. The variation did not segregate with CHD in the familial case, nor were any signs of TD present in the seven mutation carriers. Furthermore, functional studies showed no difference between wildtype and p.A119S protein in activation of four different promoters in either H10 cells or HELA cells. Taken together, our results strongly suggest that the p.A119S variation behaves similar to wild type NKX2-5 and that it has no discernible pathogenic role in either CHD or TD.NKX2-5 belongs to the NK-2 family of homeodomain-containing transcription factors, which are

130

7

conserved from flies to humans.10 Its role as a transcription regulator during early embryonic heart developmental has been known for many years, and mutations in NKX2-5 are found in patients with CHD.10-12 NKX2-5 has also been shown to be required for thyroid development in animal studies8,29 The link between thyroid development and NKX2-5 was highlighted by a recent publication of Dentice et al.,8 who reported three variations in NKX2-5 in four of the 241 patients with persistent congenital hypothyroidism studied, amongst them the p.A119S variation. They performed functional studies and showed a reduced DNA binding capacity and reduced transac-tivation properties with a dominant negative effect for p.A119S in comparison to wildtype Nkx2-5. The p.A119S mutation identified by Dentice et al. occurred in a girl with an ectopic thyroid gland. Her mother, who also carried the mutation, had auto-immune hypothyroidism but no evidence of TD and both were without evidence of CHD. Our molecular testing of the p.A119S variation in both rat heart derived (H10) cells and HELA cells showed no difference in transactivation of any

Figure 2. Relative activation of the Nppa, Dio2, Tg and TPO promoters in combination with wildtype NKX2-5, p.A119S NKX2-5, p.N188K NKX2-5 or TBX5. A. In H10 cells. B. In HELA cells. Significant differ-ences between vector and condition tested are marked with *, P < 0.05. # denotes a significant difference between conditions tested with and without TBX5, P < 0.05. Error bars represent standard deviation (SD). Each condition has been tested at least in three independent triplicate experiments.

131

7

of the four promoters tested (NPPA, Tg, Dio2, TPO), which is in contrast to the results obtained by Dentice et al. Nevertheless, the results of our functional studies are in agreement with our clinical data as the mutation did not segregate with CHD in our familial case and none of our seven mutation carriers had thyroid disease. Moreover, the A119S variation is present in the general population at a low rate (0.001%) and classified as a SNP.23 In general, we conclude that we cannot uphold the results obtained by Dentice et al., and it is unclear why the molecular results between the two studies are different, as the same proteins, promoters and cell lines were used. One difference is the fact that we did include a negative control (p.N188K) to show that our assay is robust and no confounding variables acted on the experiment.Given the above, an important question is to what extent NKX2-5 mutations are involved in the pathogenesis of TD. In addition to p.A119S, three other NKX2-5 variations have been reported in literature thus far to be associated with TD: p.R25C, p.S265R and p.R161P.8, 30 The p.R25C variation has been identified in several patients with CHD, none of whom were reported to have TD.31 Moreover, this variation is present in 1% of the general population as a SNP (rs2893667),23 making it unlikely that it plays any pathogenic role in TD. In contrast, both the p.S265R and the p.R161P variation have not been reported in the general population. The p.S265R variation was reported in a girl with TD who also carried a mutation in the PAX8 promoter region and the mutant protein was shown to have a reduced function.30 However, as the girl’s healthy brother, father and grandmother also carried the NKX2-5 variation and the PAX8 mutation may have accounted for TD in the girl, there is no direct evidence that the p.S265R variation causes TD. The p.R161P NKX2-5 variation was found in a TD patient; however her father also carried the mutation but had no TD or CHD. Taken together, none of the four currently published NKX2-5 variations have been demonstrated to segregate with a phenotype of TD within a family. Although incomplete penetrance cannot be totally excluded, there is no strong genetic evidence of a clear pathogenic effect of the mutations. To gain further insight into the role of NKX2-5 mutations in TD, a cohort of TD patients can be investigated for mutations in NKX2-5. However, the NKX2-5 gene has been analyzed in over 460 congenital hypothyroidism patients to date, but no additional mutations were identified (Table 2)14,15,32-35 Interestingly, 51 of these patients also had CHD14,33 Furthermore, none of the more than 150 CHD patients with a demonstrated NKX2-5 mutation were reported to have thyroid problems.12

Although there is a lack of evidence for a strong pathogenic effect of NKX2-5 mutations in human TD, Nkx2-5 has been shown to be involved in thyroid development. Evidence for this comes from studies using wildtype Nkx2-5 mice, showing Nkx2-5 expression in the thyroid primordium up to E11.5.8 Moreover, Nkx2-5 knockout mice demonstrate thyroid bud hypoplasia.8,28 Although these studies suggest that absence of Nkx2-5 could lead to (a form of) TD, one should keep in mind that these observations are based on Nkx2-5 null mice, which die around E9-10.36 In contrast, heterozygous knockout mice are viable and are not reported to have TD.37 This suggests that the loss of one Nkx2-5 allele is tolerated, perhaps by compensation during development by paralogue genes such as NKX2-1, which activates the same promoter regions as NKX2-5.

132

7

Tabl

e 2.

Stu

dies

ana

lyzi

ng N

KX

2-5

in p

atie

nts

with

per

sist

ent c

onge

nita

l hyp

othy

roid

ism

Aut

hor

Type

of p

atie

nts

N o

f pa

tient

sN

of m

utat

ion

carr

iers

(%

)R

emar

ks

Den

tice

et a

l., 2

0068

Per

sist

ent C

H (a

thyr

eosi

s 53

; thy

roid

ect

opy

98;

thyr

oid

hypo

plas

ia 1

5; 7

5 C

H w

ithou

t goi

ter)

241

4 (1

,7)

Two

mut

atio

ns (p

.A11

9S a

nd p

.R25

C),

pres

ent i

n 3/

4 pa

tient

s, h

ave

been

repo

rted

as a

SN

P.23

Al T

aji e

t al.,

200

733P

ersi

sten

t prim

ary

non-

auto

-imm

une,

non

-goi

tre

hypo

thyr

oidi

sm a

nd C

HD

150

(0)

Ram

os e

t al.,

200

915Th

yroi

d hy

popl

asia

or a

thyr

eosi

s35

0 (0

)

Can

gul e

t al.,

200

932P

rimar

y no

n-au

to-im

mun

e, n

on-g

oitre

hyp

othy

roid

-is

m, f

rom

con

sang

uine

ous

fam

ilies

90

(0)

In a

n ad

ditio

nal 1

30 p

atie

nts

from

con

sang

uine

ous

fam

ilies

link

age

to th

e N

KX

2.5

locu

s w

as a

ssum

ed to

be

exc

lude

d be

caus

e he

tero

zygo

sity

for t

he g

ene

was

de

tect

ed (n

o m

utat

iona

l ana

lysi

s pe

rform

ed).

Nar

umi e

t al.,

201

035

Per

man

ent p

rimar

y C

H d

iagn

osed

by

neon

atal

sc

reen

ing

(thyr

oid

ecto

py 3

7; th

yroi

d ap

lasi

a 6;

th

yroi

d hy

popl

asia

8; o

ther

51)

10

20

(0)

Pas

seri

et a

l., 2

01114

CH

D a

nd n

on-a

utoi

mm

une

CH

(nor

mal

thyr

oid

volu

me

35; h

emia

gene

sis

1)

360

(0)

Her

man

ns e

t al.,

201

130nm

nm1

Cas

e re

port:

the

p.S

265R

var

iatio

n w

as id

entifi

ed in

a

girl

with

thyr

oid

dysg

enes

is w

ho a

lso

carr

ied

a m

utat

ion

in th

e PA

X8

prom

oter

regi

on.

Bru

st e

t al.,

201

234Th

yroi

d dy

sgen

esis

(thy

roid

ect

opy

13; h

ypop

lasi

a 11

; ath

yreo

sis

3)

270

(0)

CH

D, c

onge

nita

l hea

rt di

seas

e; C

H, C

onge

nita

l Hyp

othy

roid

ism

; nm

, not

men

tione

d.

133

7

Altogether, given the wealth of published evidence, we believe that NKX2-5 mutations do not play a major pathogenic role in TD. A role of NKX2-5 as a genetic modifier cannot entirely be excluded though. To our opinion, there is currently not enough evidence to warrant routine genetic testing for NKX2-5 mutations in TD patients, and vice versa, to evaluate the thyroid in individuals carrying an NKX2-5 mutation.In conclusion, the results of our study demonstrate that p.A119S does not cause CHD or TD and that it is a rare variation that behaves equal to wildtype NKX2-5. Furthermore, given the lack of clear evidence of pathogenicity of the reported NKX2-5 mutations, the high amounts of patients with TD without an NKX2-5 mutation and the absence of TD in NKX2-5 mutation carriers, we suggest that NKX2-5 mutations do not play a major pathogenic role in thyroid dysgenesis and that genetic testing for NKX2-5 in TD is not warranted. A role of NKX2-5 as a genetic modifier cannot entirely be excluded.

AcknowledgementsWe are grateful to the patients and family members for their kind participation. We also thank E. Endert (Laboratory for Endocrinology, Academic Medical Center) for the performance of thyroid function analyses.

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Reference List

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2. van Vliet G. Development of the thyroid gland: lessons from congenitally hypothyroid mice and men. Clin Genet 2003;63:445-55

3. Castanet M, Polak M, Bonaiti-Pellie C, Lyonnet S, Czernichow P, Leger J. Nineteen years of national screening for congenital hypothyroidism: familial cases with thyroid dysgenesis suggest the involvement of genetic factors. J Clin Endocrinol Metab 2001;86:2009-14

4. Krude H, Schutz B, Biebermann H et al. Choreoathetosis, hypothyroidism, and pulmonary alterations due to human NKX2-1 haploinsufficiency. J Clin Invest 2002;109:475-80

5. Clifton-Bligh RJ, Wentworth JM, Heinz P et al. Mutation of the gene encoding human TTF-2 associated with thyroid agenesis, cleft palate and choanal atresia. Nat Genet 1998;19:399-401

6. Macchia PE, Lapi P, Krude H et al. PAX8 mutations associated with congenital hypothyroidism caused by thyroid dysgenesis. Nat Genet 1998;19:83-6

7. Sunthornthepvarakui T, Gottschalk ME, Hayashi Y, Refetoff S. Brief report: resistance to thyrotropin caused by mutations in the thyrotropin-receptor gene. N Engl J Med 1995;332:155-60

8. Dentice M, Cordeddu V, Rosica A et al. Missense mutation in the transcription factor NKX2-5: a novel molecular event in the pathogenesis of thyroid dysgenesis. J Clin Endocrinol Metab 2006;91:1428-33

9. Fagman H, Nilsson M. Morphogenetics of early thyroid development. J Mol Endocrinol 2011;46:R33-R4210. Bartlett H, Veenstra GJ, Weeks DL. Examining the cardiac NK-2 genes in early heart development.

Pediatr Cardiol 2010;31:335-4111. Schott JJ, Benson DW, Basson CT et al. Congenital heart disease caused by mutations in the transcrip-

tion factor NKX2-5. Science 1998;281:108-1112. Reamon-Buettner SM, Borlak J. NKX2-5: an update on this hypermutable homeodomain protein and

its role in human congenital heart disease (CHD). Hum Mutat 2010;31:1185-9413. Olivieri A, Stazi MA, Mastroiacovo P et al. A population-based study on the frequency of additional

congenital malformations in infants with congenital hypothyroidism: data from the Italian Registry for Congenital Hypothyroidism (1991-1998). J Clin Endocrinol Metab 2002;87:557-62

14. Passeri E, Frigerio M, De FT et al. Increased Risk for Non-Autoimmune Hypothyroidism in Young Patients with Congenital Heart Defects. J Clin Endocrinol Metab 2011:E1115-E1119

15. Ramos HE, Nesi-Franca S, Boldarine VT et al. Clinical and molecular analysis of thyroid hypoplasia: a population-based approach in southern Brazil. Thyroid 2009;19:61-8

16. van der Velde ET, Vriend JW, Mannens MM, Uiterwaal CS, Brand R, Mulder BJ. CONCOR, an initiative towards a national registry and DNA-bank of patients with congenital heart disease in the Netherlands: rationale, design, and first results. Eur J Epidemiol. 2005;20:549-57

17. Lennon G, Auffray C, Polymeropoulos M, Soares MB. The I.M.A.G.E. Consortium: an integrated molecular analysis of genomes and their expression. Genomics 1996;33:151-2

18. Postma AV, van de Meerakker JB, Mathijssen IB et al. A gain-of-function TBX5 mutation is associated with atypical Holt-Oram syndrome and paroxysmal atrial fibrillation. Circ Res 2008;102:1433-42

19. Jahn L, Sadoshima J, Greene A, Parker C, Morgan KG, Izumo S. Conditional differentiation of heart- and smooth muscle-derived cells transformed by a temperature-sensitive mutant of SV40 T antigen. J Cell Sci 1996;109 ( Pt 2):397-407

20. Gey GO, Coffman WD, Kubicek MT. Tissue culture studies of the proliferative capacity of cervical carcinoma and normal epithelium. Cancer Res 1952;12:264-5

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21. Ruijter JM, Thygesen HH, Schoneveld OJ, Das AT, Berkhout B, Lamers WH. Factor correction as a tool to eliminate between-session variation in replicate experiments: application to molecular biology and retrovirology. Retrovirology 2006;3:2

22. Gluzman Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell 1981;23:175-82

23. Tennessen JA, Bigham AW, O’Connor TD et al. Evolution and Functional Impact of Rare Coding Variation from Deep Sequencing of Human Exomes Science 2012;337:64-9

24. Dean DS, Gharib H. Epidemiology of thyroid nodules. Best.Pract.Res Clin Endocrinol Metab 2008;22:901-11

25. Spencer CA, Hollowell JG, Kazarosyan M, Braverman LE. National Health and Nutrition Examination Survey III thyroid-stimulating hormone (TSH)-thyroperoxidase antibody relationships demonstrate that TSH upper reference limits may be skewed by occult thyroid dysfunction. J Clin Endocrinol Metab 2007;92:4236-40

26. Habets PE, Moorman AF, Clout DE et al. Cooperative action of Tbx2 and Nkx2.5 inhibits ANF expression in the atrioventricular canal: implications for cardiac chamber formation. Genes Dev 2002;16:1234-46

27. Benson DW, Silberbach GM, Kavanaugh-McHugh A et al. Mutations in the cardiac transcription factor NKX2.5 affect diverse cardiac developmental pathways. J Clin Invest 1999;104:1567-73

28. Kasahara H, Lee B, Schott JJ et al. Loss of function and inhibitory effects of human CSX/NKX2.5 homeoprotein mutations associated with congenital heart disease. J Clin Invest 2000;106:299-308

29. Lints TJ, Parsons LM, Hartley L, Lyons I, Harvey RP. Nkx-2.5: a novel murine homeobox gene expressed in early heart progenitor cells and their myogenic descendants. Development 1993;119:419-31

30. Hermanns P, Grasberger H, Refetoff S, Pohlenz J. Mutations in the NKX2.5 Gene and the PAX8 Promoter in a Girl with Thyroid Dysgenesis. J Clin Endocrinol Metab 2011;96:E977-E981

31. Beffagna G, Cecchetto A, Dal BL et al. R25C mutation in the NKX2.5 gene in Italian patients affected with non-syndromic and syndromic congenital heart disease. J Cardiovasc Med (Hagerstown ) 2012

32. Cangul H, Morgan NV, Forman JR et al. Novel TSHR mutations in consanguineous families with congenital nongoitrous hypothyroidism. Clin Endocrinol (Oxf) 2010;73:671-7

33. Al Taji E, Biebermann H, Limanova Z et al. Screening for mutations in transcription factors in a Czech cohort of 170 patients with congenital and early-onset hypothyroidism: identification of a novel PAX8 mutation in dominantly inherited early-onset non-autoimmune hypothyroidism. Eur J Endocrinol 2007;156:521-9

34. Brust ES, Beltrao CB, Chammas MC, Watanabe T, Sapienza MT, Marui S. Absence of mutations in PAX8, NKX2.5, and TSH receptor genes in patients with thyroid dysgenesis. Arq Bras Endocrinol Metabol 2012;56:173-7

35. Narumi S, Muroya K, Asakura Y, Adachi M, Hasegawa T. Transcription factor mutations and congenital hypothyroidism: systematic genetic screening of a population-based cohort of Japanese patients. J Clin Endocrinol Metab 2010;95:1981-5

36. Lyons I, Parsons LM, Hartley L et al. Myogenic and morphogenetic defects in the heart tubes of murine embryos lacking the homeo box gene Nkx2-5. Genes Dev 1995;9:1654-66

37. Biben C, Weber R, Kesteven S et al. Cardiac septal and valvular dysmorphogenesis in mice heterozy-gous for mutations in the homeobox gene Nkx2-5. Circ Res 2000;87:888-95

Prevalence of congenital heart defects in neuroblastoma patients: a cohort study and systematic

review of literature

van Engelen K, Merks JHM, Lam J, Kremer LCM, Backes M, Baars MJH, van der Pal HJH, Postma AV, Versteeg R, Caron HN, Mulder BJM

Eur J Pediatr 2009;168(9):1081-1090

Chapter 8

138

8

AbstractData on the prevalence of congenital heart defects (CHD) in neuroblastoma patients are incon-sistent. If CHD are more common in neuroblastoma patients than in the general population, cardiac screening might be warranted. In this study we used echocardiography to determine the prevalence of CHD in a single centre cohort of surviving neuroblastoma patients. In addition, we performed a systematic review of the literature. Echocardiography was performed in 119 of 133 patients (89.5%). Only 2 patients (1.7%) had CHD. The prevalence of CHD was not significantly different from a previously published control group of 192 leukaemia patients examined by echo-cardiography (P = 0.49). Literature search revealed 17 studies, showing prevalence rates of CHD in neuroblastoma patients ranging from 0 to 20%. Prevalence was less than 3.6% in the majority of studies. Most studies lacked information on validity. We conclude that current evidence does not support standard cardiac screening in all patients with neuroblastoma.

139

8

IntroductionNeuroblastoma is an embryonal cancer of the postganglionic sympathetic nervous system, which mostly arises in the adrenal gland. It is the most common extracranial solid tumour in children, comprising 8 to 10% of all childhood cancers. It affects about 1 per 100,000 children under the age of 15 years.1

Several case reports have been published of patients with coexisting neuroblastoma and congenital heart defects (CHD).2-6 Some studies of patients with neuroblastoma suggest a higher prevalence of CHD in neuroblastoma patients than in the general population,7-11 however, others did not find an association between neuroblastoma and CHD.12-18 An association between neuroblastoma and CHD is considered to be plausible as neuroblastoma originates from embryonal neural crest-derived cells,1 and neural crest-derived cells are essential in cardiogenesis as well.19 Neural crest cells play an important role in the septation of the outflow tract of the heart and in the formation of the conotruncal part of the ventricular septum.19 Abnormal development or migration of neural crest cells, possibly due to an underlying genetic defect, has been postulated as a mechanism that could contribute to both conditions.3,6,7,20 Indeed, neural crest derived CHD have been reported to be more frequent in neuroblastoma patients than is expected when considering the normal distri-bution of subtypes of CHD.7,20

An association between neuroblastoma and CHD might have clinical consequences: if neuroblas-toma patients have a higher risk of CHD, cardiac screening might be indicated for all neuroblas-toma patients. Early detection of CHD could be important for the patient, in terms of bacterial endo-carditis prophylaxis, choice of anti-cancer treatment and possible need for treatment of the CHD. To assess the prevalence of CHD in neuroblastoma patients we conducted an echocardiographic study in a large single centre cohort of consecutive neuroblastoma patients. In addition, we systematically searched and critically appraised the literature to evaluate the existing evidence regarding the prevalence of CHD in neuroblastoma patients.

Materials and methodsPatientsThe study group consisted of all patients diagnosed with neuroblastoma or ganglioneuroblastoma at the Emma Children’s Hospital, Academic Medical Center (AMC), the Netherlands, between June 1966 and September 2006, who were eligible for echocardiography, i.e. all living patients (surviving patients and patients more recently diagnosed with neuroblastoma). The Emma Children’s Hospital-AMC is one of the five paediatric oncology centres in the Netherlands with a constant referral region. To minimize referral bias, we did not include patients who had been referred from another Dutch paediatric oncology centre to the Emma Children’s Hospital-AMC for specialized cancer treatment. Patients referred from other countries were also excluded. We placed special emphasis on making the cohort as complete as possible to minimize bias by selective follow up. Patients who were deceased were not included in the echocardiography study; however, we reviewed medical charts of these patients to look for CHD.

140

8

MethodsOne experienced paediatric cardiologist (JL) reviewed and performed all echocardiograms at the same laboratory in the AMC. First, we retrospectively analysed the echocardiographic images of patients who underwent echocardiography in the past before the start of this study. If the images were extensive enough to diagnose or exclude CHD, we used these in this study. If the images were not sufficient, or if echocardiographic data were not available, patients were invited to participate in this study. In the vast majority of patients, echocardiography was prospectively performed especially for this study. In those patients in whom accurate echocardiography could not be obtained we reviewed medical charts for evidence of CHD. Standard diagnostic definitions were used for CHD. Patent foramen ovale (PFO) at any age and atrial septal defect (ASD) or patent ductus arteriosus (PDA) at less than two months of age were regarded to be normal stages of cardiovascular development and therefore where not considered to be CHD.Neuroblastoma staging was performed according to the International Neuroblastoma Staging System (INSS) criteria.21 In patients who had been diagnosed with neuroblastoma before the introduction of the INSS, we converted the staging system that had been used into the INSS. MYCN oncogene amplification was analyzed by southern blot analysis of tumour cells.

Data analysisWe compared the prevalence of CHD in our cohort of neuroblastoma patients who had echocardi-ography with the prevalence of CHD in the ALL cohort as presented by George et al.7 This cohort consisted of 192 children diagnosed with ALL in one hospital in Boston, USA, between 1990 and 2000, who all had had echocardiography screening. We also compared the prevalence of CHD in our cohort to data from EUROCAT Northern Netherlands (1981-2005), a network for the registra-tion of congenital anomalies (EUropean Registration Of Congenital Anomalies).22 Children with congenital anomalies are registered in EUROCAT since 1981, after report by midwives, general practitioners and specialists. We estimated prevalence of CHD in patients as a proportion. Signifi-cance of our findings was determined by use of two-tailed P-values calculated by comparing the proportion of patients with CHD in our cohort to the proportion of patients with CHD in the control group (Fisher exact test).

ResultsBetween June 1966 and September 2006, 385 patients had been diagnosed with (ganglio)neuro-blastoma at the Emma Children’s Hospital-AMC. Sixty-seven patients were excluded from this study because they had been referred from other centres. One-hundred-eighty-five patients had deceased by the time of the start of this study. The study group therefore consisted of 133 living patients. Inclusion and exclusion of patients is illustrated in Figure 1.In 34 of 133 patients an echocardiogram had been performed in the past. The images of 17 of 34 patients were extensive enough for confirming or ruling out the presence of CHD and therefore

141

8were analyzed retrospectively. The 17 patients with inaccurate echocardiography as well as 99 patients without echocardiography were approached to undergo cardiac evaluation. We obtained echocardiography in 104 of these 116 patients. In the remaining 12 patients echocardiography could not be performed: three patients had moved abroad, two patients were untraceable and seven patients refused to undergo echocardiography. Two additional patients, children aged 12 and 24 months, were eventually excluded because echocardiography of sufficient quality could not be obtained due to unrest during the performance of the echocardiogram. Altogether, accurate echocardiography was available in 119 of the 133 eligible patients (89.5%), of which 102 were obtained prospectively and 17 retrospectively. Characteristics of these 119 patients are given in Table 1. Two of 119 patients with echocardiography had CHD (1.7%, 95% CI 0.20-5.94). One patient was a 19-year-old male with a ventricular septal defect (VSD) at birth, which had spontaneously closed when he was 1 year old. In the other patient, a 19-year-old woman, echocardiography revealed a persistent left superior cava. Both patients were asymptomatic. No CHD was detected in any of the remaining 117 patients. In one patient, a 3-year-old boy with psychomotor retardation, hyper-trophic cardiomyopathy was present, which had already been diagnosed before the start of this study and pre-existed before treatment for the neuroblastoma. The cause of the cardiomyopathy

Figure 1. Flow chart of inclusion and exclusion of patients.

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8

is unknown. In the control group of ALL patients, seven of 192 (3.6%) patients were reported to have CHD.7 The proportion of CHD in our cohort of surviving neuroblastoma patients (2 of 119, 1.7%) did not differ significantly from the proportion of CHD in this control group (7 of 192, 3.6%) (P = 0.49). EUROCAT Northern Netherlands reported 2,526 patients with CHD in 404,790 live births between January 1981 and January 2006, which corresponds to a prevalence rate of 62.4 per 10,000 (0.62%). The proportion of CHD in our neuroblastoma cohort was not significantly higher than the proportion of CHD reported by EUROCAT (P = 0.17).Review of the medical charts of the 14 surviving patients in whom echocardiography could not be obtained (n = 12) or echocardiography was inaccurate (n = 2), did not reveal evidence of CHD. Medical records were still available of 176 of 185 deceased patients (95.1%). Review of these records showed CHD in 1 patient (coarctation of the aorta and bicuspid aortic valve). In the remaining 175 patients no evidence of CHD was observed. When we considered all survivors and non-survivors together, echocardiography or medical records were available in 309 patients (133 survivors and 176 non-survivors). If we assumed that all patients without evidence of CHD have normal hearts, three of 309 patients (1.0%, 95% CI 0.20-2.81) had CHD.

Figure 2. Publications identified for study and exclusions.

Table 1. Characteristics of the study group (n = 119)

Male (%) 51 (42.9)

Median age at diagnosis of neuroblastoma in years (range) 0.8 (0.0 - 10.5)

Tumour stage (INSS) I – III (%) 74 (62.2)

IV (%) 45 (37.8)

Tumour MYCN amplification yes (%) 4 (3.4)

unknown (%) 49 (41.2)

Median age at echocardiography in years (range) 15,7 (0.8 – 41.4)

143

8Systematic review of literature, methods and resultsTo evaluate the existing evidence regarding the prevalence of CHD in neuroblastoma patients, we searched the electronic databases of PubMed (January 1966 to December 2007) and Embase (1980 to December 2007), as well as references of eligible papers. The main search terms were neuroblastoma and congenital heart defects. We collected all studies of neuroblastoma patient series that reported on the proportion of CHD. The study cohort should at least include 20 neuro-blastoma patients. The number of 20 was arbitrarily chosen, as we estimated that smaller cohorts or case series might introduce uncontrollable bias. Information about study design, study group and results were abstracted by two independent reviewers (KVE and JHM). The two independent reviewers also critically appraised internal and external validity of each study. The validity assess-ment was based on the guidelines proposed by Hayden et al.23 Details about the search strategy and validity assessment can be obtained from the authors.The literature search identified 515 unique articles. Reasons for exclusion are detailed in Figure 2. Seventeen articles were eligible for our review. Table 2 presents descriptive characteristics and results of all studies, including the present one. The studies differed largely in terms of patient characteristics and methodology. The study of George et al.7 was the only study in which patients had undergone echocardiography; the authors reviewed the echocardiographic images that had been recorded previously. In all other articles, it was not mentioned which cardiac assessment

Figure 3. Overview of prevalence rates (%) of CHD in the selected studies with 95% Confidence Interval. The prevalence rate in the control group (George et al.7) is also shown.

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8

Tabl

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Des

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(100

) 12

08 (1

00)

58 (1

00)

323

(>95

)

NB

sta

ge

nm

nm

Sta

ge 1

, 23.

4%; 2

, 9.

2%; 3

, 24.

1%; 4

, 31

.9%

; 4s,

11.

3%

nm

nm

nm

Con

trol p

atie

nts

Age

- and

sex

mat

ched

de

sign

ated

con

trols

from

ge

nera

l pra

ctiti

oner

list

s (n

=

555)

-

Live

birt

hs in

Brit

ish

Col

umbi

a H

ealth

S

urve

illan

ce R

egis

try

1979

-198

8 (n

=

419,

646)

- -

-

Met

hod

of re

view

of c

ardi

ac

stat

us

Par

enta

l int

ervi

ew,

verif

icat

ion

of in

form

atio

n in

obs

tetri

c re

cord

s an

d ge

nera

l pra

ctiti

oner

s'

reco

rds

Rev

iew

of r

ecor

ds fr

om

birth

def

ects

regi

stry

af

ter l

inka

ge w

ith c

ance

r re

gist

ry

Pat

ient

s: re

view

of

char

ts

Con

trols

: dat

a in

re

gist

ry

Info

rmat

ion

from

hos

pita

ls a

nd

fam

ily d

octo

rs, p

osta

l qu

estio

nnai

re to

fam

ily d

octo

rs (i

n pa

rt of

pat

ient

s)

Rev

iew

of d

ata

in

com

pute

rized

aut

opsy

da

taba

nk

Can

cer r

egis

try d

ata

Res

ults

N o

f pat

ient

s w

ith C

HD

(%)

1 (2

.9)

1 (2

.9)

6 (4

.3)

7 (0

.6)

2 (3

.5)

2 (0

.6)

N o

f con

trols

with

CH

D (%

) 0

(0)

- E

xpec

ted

in p

atie

nt

coho

rt: 1

.75

- -

-

Sig

nific

ance

nm

-

P <

0.0

1 -

- -

Tabl

e 2.

Con

tinue

d

146

8N

B, n

euro

blas

tom

a; C

HD

, con

geni

tal h

eart

defe

cts;

nm

, not

men

tione

d.

* N

euro

blas

tom

a w

as d

etec

ted

inci

dent

ally

at a

utop

sy in

all

patie

nts

with

CH

D.

** In

6 p

atie

nts

neur

obla

stom

a w

as d

etec

ted

inci

dent

ally,

in 3

cas

es d

urin

g ev

alua

tion

of C

HD

. †

In a

ll pa

tient

s w

ith C

HD

neu

robl

asto

ma

was

det

ecte

d at

scr

eeni

ng o

f urin

e sa

mpl

es.

‡ O

dds

ratio

for n

euro

blas

tom

a ris

k in

pat

ient

s w

ith C

HD

.

Tabl

e 2.

Con

tinue

d

Stud

y G

eorg

e et

al,

2004

7 M

eneg

aux

et a

l, 20

05.11

Ya

nai e

t al.,

200

624

Cho

w e

t al.,

200

710

Mun

zer e

t al.,

200

718

Cur

rent

stu

dy

Cou

ntry

and

tim

e pe

riod

US

A, 1

990-

2000

U

SA

/Can

ada,

199

2-19

94

Japa

n, 1

990-

2002

U

SA

, 198

0-20

04

Fran

ce, 2

003-

2004

N

ethe

rland

s, 1

966-

2006

Des

ign

and

setti

ng

Sin

gle

cent

re re

trosp

ectiv

e co

hort

stud

y

Mul

ticen

tre (1

39

hosp

itals

) ret

rosp

ectiv

e co

hort

stud

y

Two-

cent

re

retro

spec

tive

patie

nt

serie

s

Mul

ticen

tre (n

atio

nal c

ance

r re

gist

ry) r

etro

spec

tive

coho

rt st

udy

Mul

ti-ce

ntre

(Nat

iona

l R

egis

try o

f chi

ldho

od

solid

tum

ours

) re

trosp

ectiv

e co

hort

stud

y

Sin

gle

cent

re

retro

spec

tive/

pros

pect

ive

coho

rt st

udy

Patie

nts

and

met

hods

NB

pat

ient

s P

atie

nts

new

ly d

iagn

osed

w

ith N

B (n

=15

8) **

Pat

ient

s (<

19 y

ears

) ne

wly

dia

gnos

ed w

ith

NB

(n =

741

)

Pat

ient

s ne

wly

di

agno

sed

with

NB

(n

= nm

) †

Pat

ient

s (<

20 y

rs) n

ewly

di

agno

sed

with

NB

and

bor

n in

W

ashi

ngto

n (n

= 2

40)

Pat

ient

s (<

15 y

ears

) ne

wly

dia

gnos

ed w

ith

NB

, sur

vivi

ng a

nd n

ot

term

inal

ly il

l (n

= 23

5)

Pat

ient

s ne

wly

dia

gnos

ed

with

NB

, aliv

e at

tim

e of

st

udy

(n =

133

)

N o

f pat

ient

s an

alyz

ed (%

) 70

(44)

53

8 (7

3)

156

(% u

ncle

ar)

240

(100

) 19

1 (7

5)

119

(89.

5)

NB

sta

ge

INS

S s

tage

1-3

, 31%

; st

age

4, 6

9%

nm

nm

Loca

lized

, 12.

9%; r

egio

nal,

20%

; di

stan

t met

asta

tic, 4

0.8%

; un

spec

ified

, 26.

3%

nm

INS

S s

tage

I-3,

62.

2%; s

tage

4,

37.

8%

Con

trol p

atie

nts

Con

secu

tive

patie

nts

with

ac

ute

lym

phob

last

ic

leuk

aem

ia fr

om th

e sa

me

cent

re, s

ame

perio

d

(n =

192

)

Age

mat

ched

con

trols

se

lect

ed th

roug

h a

rand

om d

igit

dial

ling

met

hod

(n

= 5

04)

- B

irth

certi

ficat

es o

f age

mat

ched

co

ntro

ls fr

om t

he s

ame

area

(n =

2,

400)

Age

and

sex

mat

ched

co

ntro

ls s

elec

ted

thro

ugh

a ra

ndom

dig

it di

alin

g m

etho

d (n

=

1681

)

Con

secu

tive

patie

nts

with

ac

ute

lym

phob

last

ic

leuk

aem

ia o

ntro

l gro

up

pres

ente

d by

Geo

rge

et a

l.11

(n =

192

)

Met

hod

of re

view

of c

ardi

ac

stat

us

Ret

rosp

ectiv

e re

view

of

echo

card

iogr

aphi

c re

ports

S

tand

ardi

zed

tele

phon

e in

terv

iew

with

mot

her

Rev

iew

of c

harts

Rev

iew

of b

irth

certi

ficat

es a

nd

hosp

ital d

isch

arge

reco

rds

data

base

afte

r lin

kage

with

ca

ncer

regi

stry

Sta

ndar

dize

d te

leph

one

inte

rvie

w w

ith m

othe

r

Pro

spec

tive

echo

card

iogr

aphy

in 1

02

patie

nts;

revi

ew o

f ec

hoca

rdio

grap

hy im

ages

in

17 p

atie

nts

Res

ults

N o

f pat

ient

s w

ith C

HD

(%)

14 (2

0.0)

15

(2.0

) 4

(2.6

) 5

(2.1

) 0

(0)

2 (1

.68)

N o

f con

trols

with

CH

D (%

) 7

(3.6

) 3

(0.6

) -

9 (0

.4)

5 (0

.3)

7 (3

.6)

Sig

nific

ance

P

= 0

.000

1 O

dds

ratio

4.2

7

(95%

CI 1

.22

- 15.

0) ‡

-

Odd

s ra

tio 5

.84

(9

5% C

I 1.9

3-17

.66)

‡

Odd

s ra

tio 0

‡

P =

0.4

9

N

B, n

euro

blas

tom

a; C

HD

, con

geni

tal h

eart

defe

cts;

nm

, not

men

tione

d.

* N

euro

blas

tom

a w

as d

etec

ted

inci

dent

ally

at a

utop

sy in

all

patie

nts

with

CH

D

** In

6 p

atie

nts

neur

obla

stom

a w

as d

etec

ted

inci

dent

ally

, in

3 ca

ses

durin

g ev

alua

tion

of C

HD

†

In a

ll pa

tient

s w

ith C

HD

neu

robl

asto

ma

was

det

ecte

d at

scr

eeni

ng o

f urin

e sa

mpl

es

‡ O

dds

ratio

for n

euro

blas

tom

a ris

k in

pat

ient

s w

ith C

HD

Tabl

e 2.

Con

tinue

d

147

8

was used. The reported prevalence rates of CHD in neuroblastoma patients ranged from 0 to 20%, although the majority of the studies (14 out of 17) found a prevalence of less than 3.6%.2,10-

18,24-27 In the echocardiographic review study, the prevalence was 20%. Figure 3 is an illustration of the prevalence rates of CHD with 95% confidence interval of proportion as calculated by us. A statistically higher prevalence of CHD in patients as compared to controls was present in six of eight studies that included a control group.7-11,27 However, all studies lacked information on different items of validity. In conclusion, several studies have evaluated the prevalence of CHD in neuroblastoma patients, with little information on validity. Echocardiography had been performed in only one study. The majority of the studies reported a prevalence of less than 3.6%, however, a prevalence of 20% was found in the echocardiographic review study.

DiscussionIn this single centre cohort of consecutive neuroblastoma patients, the prevalence of CHD was 1.7%, which was not significantly different from two control groups.7 The prevalence of CHD found in our study was much lower than the prevalence of 20% reported in the only other study in which echocardiography was used for detection of CHD, the study by George et al.7 Both studies have some limitations, however. In both studies it was not possible to evaluate the patients who died before echocardiographic assessment and this might have led to under-estimation of prevalence of CHD in both studies. George et al.7 did not perform echocar-diography in all surviving neuroblastoma patients. Instead, the authors retrospectively analysed echocardiographic records, and echocardiography was available in only 43% of neuroblastoma patients. This might have led to an over-estimation of the prevalence of CHD, since patients with CHD are more likely to have had echocardiography. In our study, we prospectively performed echocardiography in almost all eligible patients, regardless of symptoms or need for specific treat-ment. Another explanation for the difference in outcome of our study and the study by George et al. might be that patients in our study were older at time of echocardiography. Because some types of CHD can resolve spontaneously over time, such as spontaneous closure of septal defects,28 CHD might have been missed in our study. One patient in our study indeed had a VSD that had spontaneously closed. However, most CHD do not disappear over time and would have been detected if present. An additional explanation for the high prevalence rate found by George et al. as compared to our study could be referral bias in their tertiary care centre study.7 An important potential source of bias that may have led to overestimation of CHD in the study of George et al. as well as in other studies and case reports, is surveillance bias: patients with neuroblastoma are investigated thoroughly, during which (asymptomatic) CHD might be detected. Likewise, neuro-blastoma can be detected during workup of CHD patients. It is well known that lower stage neuro-blastoma can regress spontaneously over time, and therefore these neuroblastoma might never have come into clinical attention without the thorough investigations after detection of CHD. This

148

8

may artificially increase the difference between prevalence of CHD in neuroblastoma patients and controls. Indeed, in the study of George et al.7 neuroblastoma was detected during workup for CHD in three patients. George et al. found a relatively high proportion of patients with neural crest derived CHD. Five of 14 (36%) patients with CHD had neural crest derived CHD, which is more than expected when considering the normal distribution of CHD.13 This seems in favour of a possible association between the two conditions, with its source in the common neural crest origin. However, numbers were very small in the study of George et al.The 16 other studies on CHD in neuroblastoma patients reviewed here, showed prevalence rates much lower than the 20% reported by George et al. In these studies it was not mentioned how patients were evaluated for the presence of CHD, but it is unlikely that the majority of patients underwent echocardiography or other imaging techniques. CHD might thus have been missed. In all of these studies information was lacking on other items of validity as well. The validity of a study addresses the issue of whether the researcher actually measures what is said to be measured. It concerns the extent to which the results of a study can be interpreted adequately and the extent to which we can trust these results. A lack of information on validity items might lead to invalid results; the studies reviewed here might have been subject to various types of bias. For example, a well defined definition of CHD was not given in the studies, which led to difficulties in interpretation of what exactly had been considered as CHD. The use of a non-representative sample of patients from the original study group may have led to either over- or under-estimation of the true preva-lence of CHD, depending on whether patients with a higher or a lower risk profile were selected for the study. In addition, cancer registries, birth certificates and medical charts might not contain all medical information and therefore CHD could be underreported. In studies were data from these registries served as control data, the significance of a higher prevalence of CHD in neuroblastoma patients might have been overestimated.8,22,29 In studies based on parental interviews, recall bias may have led to overestimation of CHD in neuroblastoma patients. In addition, studies in tertiary care centres might have been subject to referral bias. Non-accuracy of the control group can be a source of bias as well. We used the consecutive ALL cohort presented by George et al. as a control group.7 We chose this control group because all consecutive children in this cohort had undergone echocardiography, like the patients in our study group. A concern might be that the control group comprises patients from another country. We therefore also compared the prevalence of CHD in our cohort to data from EUROCAT Northern Netherlands, with non-significant results. As mentioned above, use of data from a health registry for comparison may lead to bias as well. However, because data in health registries might be prone to underrepresentation of anomalies in the general population,8,22,29 this would imply that the significance of the difference is even less than described.In conclusion, although several studies have addressed the prevalence of CHD in neuroblastoma patients, only the present study and the study of George et al.7 used adequate methodology and had reasonable validity to determine the prevalence of CHD in these patients. However, the results

149

8

differed largely between these two studies. The difference may in part be explained by differences in methodology and patient characteristics, but may be due to chance as well. Therefore, the asso-ciation between neuroblastoma and CHD remains unclear. To confirm or reject the true existence of such an association, further research in a large and complete cohort of neuroblastoma patients is needed. Our study and systematic review have shown, however, that clear evidence of an association between neuroblastoma and CHD is lacking. Standard cardiac screening in all patients with neuroblastoma is therefore not supported by current evidence.

AcknowledgementsWe thank E. Leclercq for her support in the construction of the search strategies and J.C. Vis for help in the arrangement and performance of the cardiologic examinations.

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Reference List

1 Brodeur GM, Maris JM. Neuroblastoma. In: Pizzo PA, Poplack DG, editors. Principles and practice of pediatric oncology. 5th Edition ed. Philadelphia: Lippincott Williams and Wilkinson, 2006. 933-70.

2 Friedman DM, Dunnigan A, Magid MS. Coarctation of the aorta associated with neuroblastoma. Pediatr Cardiol 1998;19:480-1

3 Bellah R, D’Andrea A, Darillis E, Fellows KE. The association of congenital neuroblastoma and congenital heart disease. Is there a common embryologic basis? Pediatr Radiol 1989;19:119-21

4 Faingold R, Babyn PS, Yoo SJ, Dipchand AI, Weitzman S. Neuroblastoma with atypical metastases to cardiac and skeletal muscles: MRI features. Pediatr Radiol 2003;33:584-6

5 McElhinney DB, Reddy VM, Feuerstein BG, Marx GR, Hanley FL. Intraoperative discovery of neuro-blastoma in an infant with pulmonary atresia. Ann Thorac Surg 1997;64:1827-9

6 Rosti L, Lin AE, Frigiola A. Neuroblastoma and congenital cardiovascular malformations. Pediatrics 1996;97:258-61

7 George RE, Lipshultz SE, Lipsitz SR, Colan SD, Diller L. Association between congenital cardiovas-cular malformations and neuroblastoma. J Pediatr 2004;144:444-8

8 Foulkes WD, Buu PN, Filiatrault D, Leclerc JM, Narod SA. Excess of congenital abnormalities in French-Canadian children with neuroblastoma: a case series study from Montreal. Med Pediatr On-col 1997;29:272-9

9 de la Monte SM, Hutchins GM, Moore GW. Peripheral neuroblastic tumors and congenital heart disease. Possible role of hypoxic states in tumor induction. Am J Pediatr Hematol Oncol 1985;7:109-16

10 Chow EJ, Friedman DL, Mueller BA. Maternal and perinatal characteristics in relation to neuroblas-toma. Cancer 2007;109:983-92

11 Menegaux F, Olshan AF, Reitnauer PJ, Blatt J, Cohn SL. Positive association between congenital anomalies and risk of neuroblastoma. Pediatr Blood Cancer 2005;45:649-55

12 Miller RW, Fraumeni JF, Jr., Hill JA. Neuroblastoma: epidemiologic approach to its origin. Am J Dis Child 1968;115:253-61

13 Miller RW. Childhood cancer and congenital defects. A study of U.S. death certificates during the period 1960-1966. Pediatr Res 1969;3:389-97

14 Berry CL, Keeling J, Hilton C. Coincidence of congenital malformation and embryonic tumours of childhood. Arch Dis Child 1970;45:229-31

15 Narod SA, Hawkins MM, Robertson CM, Stiller CA. Congenital anomalies and childhood cancer in Great Britain. Am J Hum Genet 1997;60:474-85

16 Neglia JP, Smithson WA, Gunderson P, King FL, Singher LJ, Robison LL. Prenatal and perinatal risk factors for neuroblastoma. A case-control study. Cancer 1988;61:2202-6

17 Nakissa N, Constine LS, Rubin P, Strohl R. Birth defects in three common pediatric malignancies; Wilms’ tumor, neuroblastoma and Ewing’s sarcoma. Oncology 1985;42:358-63

18 Munzer C, Menegaux F, Lacour B et al. Birth-related characteristics, congenital malformation, maternal reproductive history and neuroblastoma: The ESCALE study (SFCE). Int J Cancer 2007;122:2315-21

19 Hutson MR, Kirby ML. Neural crest and cardiovascular development: a 20-year perspective. Birth Defects Res C Embryo Today 2003;69:2-13

20 Holzer R, Franklin RC. Congenital heart disease and neuroblastoma: just coincidence? Arch Dis Child 2002;87:61-4

21 Brodeur GM, Pritchard J, Berthold F et al. Revisions of the international criteria for neuroblastoma diagnosis, staging, and response to treatment. J Clin Oncol 1993;11:1466-77

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22 EUROCAT. Northern Netherlands (2007) Prevalence of congenital malformations in the Northern Netherlands, 1981-2005. Updated 2007, June 30th. Available via EUROCAT. http://www.rug.nl/umcg/faculteit/disciplinegroepen/medischegenetica/eurocat. Accessed 15 Dec 2007. EUROCAT 2007.

23 Hayden JA, Cote P, Bombardier C. Evaluation of the quality of prognosis studies in systematic reviews. Ann Intern Med 2006;144:427-37

24 Yanai T, Hasegawa D, Kosaka Y et al. Congenital cardiovascular malformations are complicated in neuroblastomas identified by mass screening but not by clinical examination in Japan [letter]. J Pediatr 2006;149:145-6

25 Nishi M, Miyake H, Takeda T, Hatae Y. Congenital malformations and childhood cancer. Med Pediatr Oncol 2000;34:250-4

26 Mili F, Lynch CF, Khoury MJ, Flanders WD, Edmonds LD. Risk of childhood cancer for infants with birth defects. II. A record-linkage study, Iowa, 1983-1989. Am J Epidemiol 1993;137:639-44

27 Mann JR, Dodd HE, Draper GJ et al. Congenital abnormalities in children with cancer and their rela-tives: results from a case-control study (IRESCC). Br J Cancer 1993;68:357-63

28 Perloff JK. Therapeutics of nature--the invisible sutures of “spontaneous closure”. Am Heart J 1971;82:581-5

29 Larsen H, Nielsen GL, Bendsen J, Flint C, Olsen J, Sorensen HT. Predictive value and completeness of the registration of congenital abnormalities in three Danish population-based registries. Scandina-vian Journal of Public Health 2003;31:12-6

Part IIIThe patients’ perspective on inheritance

of congenital heart disease

Adults with congenital heart disease: patients’ knowledge and concerns about inheritance

van Engelen K, Baars MJH, van Rongen LT, van der Velde ET, Mulder BJM, Smets EMA

Am J Med Genet A 2011;155(7):1661-1667

Chapter 9

156

9

AbstractWith recent advances in medical and surgical management, most patients with congenital heart disease (CHD) survive to reproductive age. Current guidelines recommend counseling about inheritance and transmission of CHD to offspring. We evaluated whether adult CHD patients recalled having received information about the inheritance of their CHD, patients’ knowledge about inheritance and their concerns in this regard. A questionnaire was sent to 486 non-syndromic CHD patients aged 20 to 45 years. We received 332 useful questionnaires (response rate 68%). One-third (33%) of patients recalled receiving information about inheritance of CHD from their cardiologist, and 13% had consulted a clinical geneticist. Eight percent of patients who were considering having children estimated the recurrence risk for their own offspring to be 1% or lower, whereas one fourth (25%) estimated it to be higher than 10%. According to our classification, 44% estimated the recurrence risk in a correct range of magnitude. Additional information about inher-itance of CHD was desired by 41% of patients. Forty-two percent of patients considering having children reported concerns about transmitting CHD to offspring. We conclude that a substantial proportion of adult CHD patients lacks knowledge and desires more information about inheritance, indicating a need for better patient education. Current guidelines and/or their implementation do not seem to meet the needs of these patients. A dedicated program of counseling for adults with CHD has to be developed to optimize knowledge and satisfaction with information provision and to reduce or manage concerns regarding inheritance of CHD.

157

9

IntroductionCongenital heart disease (CHD) affects approximately 6 to 8 per 1,000 live-births.1 With medical and surgical advances, most children survive to adulthood, significantly increasing the number of adult patients with CHD.2,3 Reproduction and inheritance, including transmission of CHD to offspring, are important issues in this population. Current guidelines for care for adults with CHD recommend counseling about these topics.4-7

The recurrence risk for CHD in offspring largely depends on the etiology of the CHD in the parent. CHD mostly occurs as an isolated anomaly, but may also be part of a large number of specific syndromes or chromosomal abnormalities, or as a consequence of teratogenic exposure.8,9 Many syndromes with CHD are monogenic, including Noonan syndrome, Alagille syndrome and Holt-Oram syndrome, or caused by a chromosomal abnormality or contiguous gene microdeletion, such as 22q11 deletion syndrome. The recurrence risk for offspring in an individual with syndromic CHD depends on the specific syndrome and can be up to 50%. Although single gene defects and copy number variations have also been described in non-syndromic CHD,10,11 the majority of non-syn-dromic CHD is historically believed to be multifactorial in origin, with multiple genetic and environ-mental factors interacting to produce CHD.8,12 Offspring of patients with non-syndromic CHD have an increased risk of CHD, for which empirical estimations are available. Overall, the recurrence risk for children of males with non-syndromic CHD is estimated to be around 2-3%, whereas the risk for children of females with non-syndromic CHD is estimated to be about 5-6.5%.9,13,14 In the presence of a positive family history of CHD the recurrence risk may be higher and occasionally, non-syndromic CHD can be inherited as a simple Mendelian trait.14 Little is known about CHD patients’ knowledge about inheritance, the concerns they have about transmission of CHD to offspring and the consequences thereof for reproductive choices and use of prenatal screening. Only a few studies have briefly addressed inheritance issues, generally concluding that patients lack significant knowledge about inheritance of their CHD.15-18 To evaluate the current guidelines and optimize care for adult CHD patients regarding inheritance issues, we feel that an assessment of knowledge, concerns and desire for information of adult patients with CHD is needed. The aims of our study were to assess 1) whether adult patients with non-syndromic CHD recall receiving information about the inheritance of their CHD, 2) the patients’ level of knowledge and concerns regarding transmission of CHD to offspring, 3) their desire for more information, and 4) if level of knowledge, concerns and desire for more information can be predicted from clinical and demographic factors.

MethodsPatients and study designIn this cross-sectional survey study we randomly derived 486 patients from one university hospital from the CONCOR registry, the Dutch national registry for adult patients with CHD, described in detail previously.19 The inclusion criterion was age between 20 and 45 years on September 1, 2009,

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assuming reproduction issues to be most relevant for individuals this age. Exclusion criteria were 1) any kind of known syndrome or developmental disability, and 2) residence outside The Nether-lands. After receipt of the questionnaire, patients stating to have an additional cardiac disorder with a specific genetic cause (e.g. hypertrophic cardiomyopathy) were excluded. Cardiac diagnoses of remaining patients were divided into 13 categories of main diagnoses and into three groups of complexity level.2 All patients were sent a questionnaire. After four weeks, we sent a reminder to non-responders to encourage participation. After another four weeks, we phoned the non-responders to establish receipt of the questionnaire and once again request participation. The Medical Ethics Committee (MEC) of the Academic Medical Center stated that formal approval for this study was not required, as the study does not fall within the range of the Dutch Medical Research Involving Human Subjects Act.

QuestionnaireWe designed a questionnaire covering seven domains including 1) basic demographic (age, gen-der, level of education, etc) and clinical characteristics (comorbidities, family history of CHD etc), 2) information about inheritance patients recalled receiving from health care providers, 3) knowledge about recurrence risks of CHD for own offspring, 4) general knowledge about inheritance of CHD, 5) concerns regarding transmission of CHD to offspring, 6) satisfaction with knowledge and coun-seling needs, and 7) pregnancy-related actions including prenatal screening.Information received from health care providers was assessed using multiple choice questions and statements (response options: very much, some, none, do not remember) (see Table 2). Patients’ notions about the recurrence risk of CHD for their own children were evaluated by having patients estimate the risk on a scale from 0% to 100%. As a rough screening tool for evaluation of the estimations we broadly classified the estimations into four groups including 1) correct range of magnitude, 2) too low, 3) slightly too high, 4) far too high. To classify each individual we used empirical literature data; in females: an estimation of 4-8% was considered a correct range of magnitude; 0-3% too low; 9-13% slightly too high; >13% far too high. In males: 1-5% correct range of magnitude; 0% too low; 6-10% slightly too high; >10% far too high. We accounted for the reported family history of CHD and roughly estimated the recurrence risk, adjusting the accuracy of the answer accordingly. The type of CHD was not taken into account. General knowledge about the inheritance of CHD was evaluated using 10 questions. There were four multiple choice questions regarding the magnitude of the risk of CHD for offspring in different situations (e.g. risk of CHD for offspring of a healthy couple, risk of CHD in offspring of a male with CHD), and six statements related to factors potentially influencing the magnitude of the recurrence risk (e.g. gender of parent with CHD, family history of CHD, number of surgical procedures in the parent with CHD). We assigned one point for each correct answer and zero points if the answer was false or no answer was given, resulting in an individual knowledge score ranging from 0 to 10 points.Concerns about transmission of CHD to offspring were assessed with six statements (response

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options: totally agree, somewhat agree, do not agree/do not disagree, somewhat do not agree, totally do not agree). Counseling needs were evaluated with yes/no questions. Pregnancy-related actions were evaluated by yes/no questions and multiple choice questions. The face validity of the questionnaire was evaluated by three genetic counselors and one clinical psychologist. We did a pilot involving 10 patients visiting the cardiology outpatient clinic and adapted the questionnaire according to their remarks.

n %

Age (years) 31.9 ± 7.3

Male 171 52

Having children 119 36

Considering having (additional) children 211 64

Level of education

Low 39 12

Medium 146 44

High 147 44

CHD type

Ventricular septal defect 47 14

Tetralogy of Fallot 45 14

Bicuspid aortic valve 17 5

Atrial septal defect, primum type 15 5

Atrial septal defect, secundum type 38 11

Pulmonary valve stenosis 31 9

Subvalvar aortic stenosis 7 2

CCTGA 6 2

Aortic coarctation 43 13

Aortic valve stenosis 15 5

Transposition of the great arteries 11 3

Patent ductus arteriosus 4 1

Other 53 16

CHD complexity

Mild 164 49

Moderate 122 37

Severe 46 14

Positive family history for CHD (reported) 70 21

Table 1. Characteristics of the study population (n = 332)

CHD, congenital heart disease; CCTGA, congenitally corrected transposition of the great arteries.

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Data analysisFor statistical analyses, SPSS (version 17.0) for Windows was used. Statistical significance was set at P < 0.05. Descriptive data are presented as mean with standard deviation as they were normally distributed. For comparison of discrete variables between two groups (e.g. those informed and those not informed by health care providers) we used the Chi-square or Fisher’s exact test and for comparing continuous variables we used the unpaired Student’s T-tests (data were normally distributed). To identify clinical and demographic predictors for estimation of recur-rence risk in the correct range of magnitude, having concerns about transmission of CHD and desire for more information we used multivariate logistic regression (dichotomous outcome varia-bles), while for identification of predictors for a higher knowledge score we used multivariate linear regression (continuous outcome variable). Predictor variables were gender, age, having children, considering having children, education level, complexity of CHD, comorbidities, family history of CHD, reporting to have received information from the cardiologist, reporting to have consulted a clinical geneticist and general knowledge score. We included variables in the multivariate regres-sion analyses if univariate analysis showed P-value < 0.1.

ResultsSampleThree hundred thirty-six (69%) of 486 patients returned the questionnaire. We excluded four patients after receipt of the questionnaires because two patients had not completed most of the items, and two patients reported additional genetic cardiac disorders (1 patient with hypertrophic cardiomyopathy and 1 patient with arrhythmogenic right ventricular cardiomyopathy). We analyzed total of 332 (68%) useful questionnaires. There were no statistically significant differ-ences in gender, age and complexity of CHD between responders and non-responders. Charac-teristics of the included patients (mean age 31.9 ± 7.3 years, 52% male) are shown in Table 1. Of the 119 patients with children (total of 233 children), five patients reported having a child with CHD (2% of all children). Comorbidities were reported by 23% of patients. Ten (3%) patients reported additional malformations that suggested an underlying syndrome.

Information provisionOne-third (33%) of the patients reported receiving information about inheritance from their cardi-ologist or specialized nurse, while 14% did not remember if they received any information. State-ments about the received information and patients’ answers are shown in Table 2. Patients who reported receiving information were significantly older (P = 0.001), more often had children (P = 0.011) and less often considered having (additional) children (P = 0.007). The information was stated to be provided before a first pregnancy by 80%, during the first pregnancy by 12% and after having the first child by 8% of patients. Two thirds (67%) of patients reported having asked for information, while in 33% the information was said to be provided on the cardiologist’ or nurse’s initiative.

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A minority (13%) of patients reported having consulted a clinical geneticist. Table 2 shows state-ments and patients’ answers about the information received from the geneticist. Patients who stated having consulted a geneticist were older (P = 0.049), more often reported a positive family history of CHD (P = 0.023) or additional congenital malformations suggestive of an underlying syndrome (P = 0.028), a low level of education (P = 0.002) and more often had children (P = 0.009). Over half (54%) of the patients said that the cardiologist had taken the initiative for the referral, while 46% of patients reported having requested referral themselves. Most patients (72%) stated having consulted the geneticist before the first pregnancy, 14% during the first pregnancy and 14% after having the first child. Three percent of patients reported receiving information about inheritance of CHD from someone other than the cardiologist or geneticist, such as a general practitioner, and 22% said they searched for information using the internet or through patient organizations.

KnowledgeKnowledge about recurrence risk for offspring. Among the patients who were considering having (additional) children (n = 211), 67% estimated the recurrence risk to be 2-10%. One fourth (25%) estimated the recurrence risk higher than 10%, of which 8% higher than 50%. The remaining 8% of patients thought that the recurrence risk would be 0%.

Table 2. Reportedly received information about inheritance of CHD

CHD, congenital heart disease. * Patients who stated ‘yes’ to the general question ‘did you receive information from the cardiologist or specialized nurse about inheritance of CHD?’ are included. ** Patients who stated ‘yes’ to the general question ‘did you consult a clinical geneticist for your CHD?’ are included.

Reportedly received information from cardiologist or nurse (n = 110)*

Very much (%)

Some (%) No (%)Do not

remember (%)

I received general information about inheritance of my CHD 32 56 5 7

I received information about the recurrence risk of CHD in my children

28 36 20 16

The information was sufficient 31 46 11 12

The information was clear 29 46 12 13

Reportedly received information from clinical geneticist (n = 42)**

Very much (%)

Some (%) No (%)Do not

remember (%)

I received general information about inheritance of my CHD 67 26 0 7

I received information about the recurrence risk of CHD in my children

60 29 5 6

The information was sufficient 57 33 2 8

The information was clear 60 29 2 9

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According to our classification, which included reported family history, 44% estimated the recur-rence risk of CHD for offspring in the correct range of magnitude, 15% too low, 18% a little too high and 23% far too high. Multivariate regression analysis showed that male gender (P = 0.002) and higher general knowledge score (P = 0.013) were independent predictors for estimating the recurrence risk in the correct range of magnitude (Table 3).

General knowledge. The risk of having a child with CHD for a healthy couple was answered correctly by 53%, for a man with CHD by 30%, for a woman with CHD by 35% and for a healthy couple with a previous child with CHD by 24%. The majority of patients knew that the recurrence risk is higher if more than one relative has CHD (69%), and that the number of surgical interventions in the parent does not influ-ence the recurrence risk (70%). About half of the patients answered correctly that the cause of the CHD in the parent might influence the recurrence risk, and that the severity of a particular type of CHD in the parent does not (46% and 44% respectively). Twelve percent knew that the recurrence risk for children of women with CHD is generally higher than the recurrence risk for children of men with CHD and 40% knew that diseases in the mother other than CHD may contribute to CHD.The mean knowledge score was 4.2 (±1.9; range 0-9). A high education level was the only inde-pendent predictor for a higher knowledge score, explaining 14% of the variance (P < 0.001, Table 4).

Concerns Of the patients who were considering having children, 42% had concerns about future children having CHD. Eighty percent found it important that their future children would be screened for CHD during pregnancy and 93% after delivery. One out of five patients (21%) assumed feelings of guilt if their child would have CHD. Of the five patients who did have a child with CHD, two felt guilty to some extent, three did not. Of all women, 21% had concerns about having a miscarriage due to their CHD. Multivariate regression analysis showed that having children (P = 0.031) and having estimated the recurrence risk too high according to our classification (P = 0.041) were inde-pendent predictors for concerns about transmitting the CHD to future offspring (Table 5).

Satisfaction with knowledge and counseling needsPersonal knowledge about inheritance aspects of their CHD was considered insufficient by 68% of patients, and 41% desired more information. In the multivariate analysis, considering having children (P = 0.001) was an independent predictor for desire for information, while patients who reported receiving information from their cardiologist or a clinical geneticist less often desired more information (P = 0.03 and P = 0.04, respectively) (Table 6). Of patients who said not to have consulted a clinical geneticist, 38% would like to do so.

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OR 95% CI P-value

Male gender 2.63 1.43 - 4.76 0.002

Severe complexity of CHD 0.60 0.34 - 1.09 0.095

Knowledge score 1.20 1.04 - 1.39 0.013

Table 3. Multivariate logistic regression results for variables determining estimation of recurrence risk in correct range of magnitude (n = 211)*

* Only patients considering having (additional) children are included.

β 95% CI P-value

Having children 0.38 - 0.02 - 0.77 0.060

High education level 1.29 0.91 - 1.67 < 0.001

Reportedly having received information from cardiologist

0.30 - 0.11 - 0.70 0.147

Table 4. Multivariate linear regression results for variables determining knowledge score (n = 332)

OR 95% CI P-value

Having children 2.55 1.09 - 5.96 0.031

Reportedly having consulted clinical geneticist

1.92 0.74 - 4.96 0.177

Too high estimation of recurrence risk 1.90 1.03 - 3.54 0.041

Table 5. Multivariate logistic regression results for variables determining having concerns about transmit-ting CHD to offspring (n = 211)*

* Only patients considering having (additional) children are included.

OR 95% CI P-value

Age 1.00 0.96 - 1.05 0.926

Having children 0.84 0.44 - 1.58 0.577

Considering having children 3.32 1.61 - 6.84 0.001

Reportedly having received information from cardiologist

0.56 0.33 - 0.96 0.034

Reportedly having consulted clinical geneticist

0.52 0.19 - 0.95 0.038

Table 6. Multivariate logistic regression results for variables determining desire for more information about inheritance of CHD (n = 332)

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Pregnancy-related actionsPast events Of the patients who had children (n = 119), 78% had used folic acid related to their pregnancy (in case of male patients; their female partner). Ten percent of patients did not know folic acid is advantageous. During pregnancy, 80% of patients underwent prenatal screening: nuchal translu-cency measurement in 28%, combination test (nuchal translucency measurement in combination with maternal serum markers) in 20%, ‘regular’ ultrasound screening in 33% and advanced ultra-sound screening in 79% of patients.

Anticipated actions in the event of increased risk of CHD. Of patients considering having children, 74% would consider advanced ultrasound screening during pregnancy in case of a 5% risk of a child having CHD, and slightly more patients (76%) in case of a 50% risk. Fifteen percent would consider giving up having children if the risk of CHD would be strongly increased (50%), as opposed to 4% in case of a slightly (5%) increased risk.

DiscussionIn this study addressing inheritance issues in adult patients with CHD, we found the majority of patients having no recollection of being informed about inheritance. The patients’ general knowl-edge about inheritance of CHD is limited, and more importantly, many seem to have imperfect knowledge about the recurrence risk for their offspring. Almost half of patients desire more infor-mation about inheritance of CHD. Current guidelines for care for adult CHD patients recommend counseling about genetics and recurrence risks of CHD in offspring, some 5,6 more explicitly and extensively than others.4,7 In our study, only one third of patients stated to have received information about inheritance of their CHD from their cardiologist or nurse practitioner, a minority finding the information very clear and sufficient. Although some patients may not recall having received information, this nevertheless suggests that most patients either did not receive any information or information provision was insufficient to be remembered. Patients who reported not receiving information were younger and childless, suggesting that cardiologists may not have considered inheritance issues as relevant for these patients yet. A clinical geneticist had been consulted by only 13% of all patients. Although patients with a family history of CHD or additional congenital malformations suggestive of an underlying syndrome were more likely to have consulted a geneticist, most of these had not seen a geneticist. This is recommended as an underlying syndrome may have clinical consequences for the patient and may significantly increase recurrence risks (e.g., in certain syndromes, as high as 50%), and offspring may not only being at risk for CHD but also for the non-cardiac features of the syndrome.5,8,13,20 Additionally, a family history of CHD may imply a specific underlying genetic cause and/or a higher recurrence risk.8,13

The patients’ lack of knowledge about inheritance in our study is consistent with other studies,

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showing gaps in knowledge about inheritance as well as other topics regarding CHD, including complications, endocarditis prophylaxis, pregnancy risks and contraception.15-17,21-24 While the topics we addressed to evaluate the overall knowledge about inheritance of CHD were general and not necessarily relevant for all patients, adequate knowledge of recurrence risk is relevant to patients. In our study, eight percent of patients estimated the recurrence risk to be 0%, which is similar to or lower than the frequency in the general population, while one fourth estimated the risk higher than 10%. According to our broad classification, only 44% of patients estimated the recurrence risk in the correct range of magnitude, the risk often estimated too high. We want to stress though that an individual’s recurrence risk can only be estimated accurately after an in-depth work up has been performed, including review of medical history, physical examination, extensive family history, cardiac evaluation of family members and possibly genetic analyses. As we did not perform such work-up, the recurrence risks and classification as used by us represent only a rough estimate. We also did not take into account type of CHD, though some types of CHD have been reported to have higher recurrence rates than others (e.g. left sided obstructive heart defects were reported to have relatively high heritability).25 We chose this strategy as we did not want to stress current knowledge about specific recurrence risks of different CHD types, but this may also have led to a non-optimal classification of recurrence risk by us. Several factors may contribute to lack of knowledge, including physicians not providing the information, providing inaccurate information, providing information in an inadequate manner and patients not retaining the information. In our study, patients who stated to be informed did not estimate the recurrence risk more often in the correct range of magnitude nor had a higher knowl-edge score than patients who stated not to be informed, suggesting that patients indeed do not receive or recall the provided information correctly. Lack of continuity of care and coordinated services as well as conflicting information from different health care providers may also contribute to non-optimal knowledge. Men more often estimated the recurrence risk in the correct range of magnitude than women, which might be explained by notable gender based differences in care provision. Generally, women are more likely to receive care from multiple health care disciplines (e.g., cardiology, gynecology) and may be more likely to receive specific information about repro-ductive issues (e.g., on the adverse effects of pregnancy for themselves, risks of medication use and cardiologic follow-up during pregnancy). These differences might lead to difficulties in recalling the received information, the possibility of receiving conflicting information from different health care providers, or confusion about how the different kinds of information fit together.Inadequate knowledge about recurrence risk in offspring may falsely reassure or scare patients,13

and may hamper patients from making informed choices regarding family planning and prenatal screening. As in many patients the actual recurrence risk may be lower than estimated by them-selves, counseling about the recurrence risk may therefore prevent or allay concerns in some patients. Concerns were reported by 42% of patients who considered having children, which is comparable to other studies.18,26 A gender difference was not present, in contrast to the study of Reid et al, who found women to be more concerned about transmission of CHD.18

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Most patients were not satisfied with their own knowledge level and almost half of all patients indicated a desire for more information about inheritance, mainly patients who were considering having children. Patients who reported having received information were more satisfied with their level of knowledge and less often desired additional information, suggesting that information provision meets patients’ needs. The gaps in patients’ knowledge however imply that the current guidelines for the care of adults with CHD and/or the way they are implemented do not meet the goal of having patients knowledgeable about inheritance of CHD. As we learn more about the needs of this population, including the complex and often difficult to disentangle concerns about CHD recurrence and potential burdens of guilt, the value of a coordinated and focused care team becomes increasingly clear. Therefore, a dedicated counseling program for adults with CHD has to be developed to optimize knowledge and satisfaction with information provision and to manage concerns regarding transmission of CHD. This program should include inheritance issues to be discussed at an early age, at least onward from the first meeting with the adult cardiologist after transition from pediatric to adult care, and to be repeatedly addressed. Counseling about inheritance issues by specialized nurses in adult CHD clinics could also be incorporated in the program. Preferably, additional written material should be provided.5 Moreover, health care pro-viders should take the lead in information provision and not wait for patients to ask questions. Unfortunately, previous studies have shown that many health care providers, including cardiologists, do not have sufficient knowledge about genetics,27,28 and this should be optimized for adequate information provision. Close collaboration with clinical geneticists should also be part of the program, as they play a significant role in the clinical and genetic assessment of patients, aiming at an accurate etiologic diagnosis and providing accurate counseling regarding recurrence risk and prenatal diagnosis and screening options.13 Use of folic acid from 4 weeks before until 8 weeks after conception might reduce the risk of CHD in offspring,29,30 which should be explained to patients. The optimal timing and manner to provide the necessary information about inheritance of CHD has to be evaluated in further studies.

LimitationsAlthough the response rate is satisfactory and no differences in gender, age and complexity of CHD emerged between responders and non-responders, we cannot exclude the possibility that patients who were most motivated or concerned about transmission of CHD did return the ques-tionnaire. Recall bias may have been introduced as some items of the questionnaire referred to events in the distant past. As we did not check medical charts, no data regarding the exact nature of the provided information about inheritance was available. As stated before, recurrence risks were roughly classified as ‘correct range of magnitude’, ‘too low’ or ‘too high’ based on empirical lit-erature data and the provided information by patients, however the actual recurrence risks may be different due to several facts we could not deduce from the questionnaire. Finally, making gen-eralizations about our data may be difficult because they were collected at one tertiary care centre.

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ConclusionsThe substantial proportion of adult CHD patients lacking knowledge and desiring more information about inheritance of CHD indicates a need for better patient education on these topics. Current guidelines and/or their implementation do not seem to meet the needs of these patients. A dedi-cated program of counseling for adults with CHD has to be developed to optimize knowledge and satisfaction with information provision and to reduce or manage concerns regarding inheritance of CHD. The optimal timing and manner to provide this education has to be evaluated in future studies.

AcknowledgementsThe authors would like to thank all patients for their participation.

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disease in adult life. J Am Coll Cardiol 2001;37:1170-53 Engelfriet P, Boersma E, Oechslin E et al. The spectrum of adult congenital heart disease in Europe:

morbidity and mortality in a 5 year follow-up period. The Euro Heart Survey on adult congenital heart disease. Eur Heart J 2005;26:2325-33

4 Baumgartner H, Bonhoeffer P, De Groot NM et al. ESC Guidelines for the management of grown-up congenital heart disease (new version 2010): The Task Force on the Management of Grown-up Congenital Heart Disease of the European Society of Cardiology (ESC). Eur Heart J 2010;31:2915-57

5 Warnes CA, Williams RG, Bashore TM et al. ACC/AHA 2008 guidelines for the management of adults with congenital heart disease: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Writing Committee to Develop Guidelines on the Management of Adults With Congenital Heart Disease). Developed in Collaboration With the American Society of Echocardiography, Heart Rhythm Society, International Society for Adult Congenital Heart Disease, Society for Cardiovascular Angiography and Interventions, and Society of Thoracic Surgeons. J Am Coll Cardiol 2008;52:e1-121

6 Marelli A, Beauchesne L, Mital S, Therrien J, Silversides CK. Canadian Cardiovascular Society 2009 Consensus Conference on the management of adults with congenital heart disease: introduction. Can J Cardiol 2010;26:e65-e69

7 Oakley C, Child A, Jung B, Presbitero P, Tornos P. Expert consensus document on management of cardiovascular diseases during pregnancy. Task Force on the Management of Cardiovascular Diseases During Pregnancy of the European Society of Cardiology. Eur Heart J 2003;24:761-81

8 Pierpont ME, Basson CT, Benson DW, Jr. et al. Genetic basis for congenital heart defects: current knowledge: a scientific statement from the American Heart Association Congenital Cardiac Defects Committee, Council on Cardiovascular Disease in the Young: endorsed by the American Academy of Pediatrics. Circulation 2007;115:3015-38

9 Firth HV, Hurst JA, Hall JG. Congenital Heart Disease. In: Firth HV, Hurst JA, Hall JG, editors. Desk reference clinical genetics.Oxford: Oxford University Press, 2006. 84-7.

10 Greenway SC, Pereira AC, Lin JC et al. De novo copy number variants identify new genes and loci in isolated sporadic tetralogy of Fallot. Nat Genet 2009;41:931-5

11 Garg V, Muth AN, Ransom JF et al. Mutations in NOTCH1 cause aortic valve disease. Nature 2005;437:270-4

12 Wessels MW, Willems PJ. Genetic factors in non-syndromic congenital heart malformations. Clin Genet 2010;78:103-23

13 Bernier FP, Spaetgens R. The geneticist’s role in adult congenital heart disease. Cardiol Clin 2006;24:557-vi

14 Harper P. Cardiovascular and respiratory disorders. In: Koster J, Burrows S, editors. Practical genetic counseling. sixth edition ed. London: Arnold, Hodder Headline Group, 2004. 265-79.

15 Kovacs AH, Harrison JL, Colman JM, Sermer M, Siu SC, Silversides CK. Pregnancy and contraception in congenital heart disease: what women are not told. J Am Coll Cardiol 2008;52:577-8

16 Moons P, De VE, Budts W et al. What do adult patients with congenital heart disease know about their disease, treatment, and prevention of complications? A call for structured patient education. Heart 2001;86:74-80

17 Uzark K, VonBargen-Mazza P, Messiter E. Health education needs of adolescents with congenital heart disease. J Pediatr Health Care 1989;3:137-43

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18 Reid GJ, Siu SC, McCrindle BW, Irvine MJ, Webb GD. Sexual behavior and reproductive concerns among adolescents and young adults with congenital heart disease. Int J Cardiol 2008;125:332-8

19 van der Velde ET, Vriend JW, Mannens MM, Uiterwaal CS, Brand R, Mulder BJ. CONCOR, an initia-tive towards a national registry and DNA-bank of patients with congenital heart disease in the Nether-lands: rationale, design, and first results. Eur J Epidemiol 2005;20:549-57

20 van Engelen K, Topf A, Keavney BD et al. 22q11.2 Deletion Syndrome is under-recognised in adult patients with tetralogy of Fallot and pulmonary atresia. Heart 2010;96:621-4

21 Dore A, de GP, Mercier LA. Transition of care to adult congenital heart centres: what do patients know about their heart condition? Can J Cardiol 2002;18:141-6

22 Cetta F, Warnes CA. Adults with congenital heart disease: patient knowledge of endocarditis prophy-laxis. Mayo Clin Proc 1995;70:50-4

23 Kantoch MJ, Collins-Nakai RL, Medwid S, Ungstad E, Taylor DA. Adult patients’ knowledge about their congenital heart disease. Can J Cardiol 1997;13:641-5

24 Ferencz C, Wiegmann FL, Jr., Dunning RE. Medical knowledge of young persons with heart disease. J Sch Health 1980;50:133-6

25 Hinton RB, Jr., Martin LJ, Tabangin ME, Mazwi ML, Cripe LH, Benson DW. Hypoplastic left heart syndrome is heritable. J Am Coll Cardiol 2007;50:1590-5

26 Doucet SB. The young adult’s perceptions of the effect of congenital heart disease on his life style. Nurs Pap 1981;13:3-16

27 Baars MJ, Henneman L, Ten Kate LP. Deficiency of knowledge of genetics and genetic tests among general practitioners, gynecologists, and pediatricians: a global problem. Genet Med 2005;7:605-10

28 van Langen IM, Birnie E, Leschot NJ, Bonsel GJ, Wilde AA. Genetic knowledge and counselling skills of Dutch cardiologists: sufficient for the genomics era? Eur Heart J 2003;24:560-6

29 Jenkins KJ, Correa A, Feinstein JA et al. Noninherited risk factors and congenital cardiovascular defects: current knowledge: a scientific statement from the American Heart Association Council on Cardiovascular Disease in the Young: endorsed by the American Academy of Pediatrics. Circulation 2007;115:2995-3014

30 Bazzano LA. Folic acid supplementation and cardiovascular disease: the state of the art. Am J Med Sci 2009;338:48-9

The value of the clinical geneticist caring for adults with congenital heart disease: diagnostic yield

and patients’ perspective

van Engelen K, Baars MJH, Felix JP, Postma AV, Mulder BJM, Smets EMA

Am J Med Genet A 2013;161(7):1628-1637

Chapter 10

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AbstractFor adult patients with congenital heart disease (CHD), knowledge about the origin and inheri- tance of their CHD is important. Clinical geneticists may play a significant role in their care. We explored the diagnostic yield of clinical genetic consultation of adult CHD patients, patients’ motivations for the consultation, implications for reproductive decisions, patients’ evaluation of the impact of provided information and satisfaction with counseling. Chart review was performed on all adult patients referred for CHD to our clinical genetics department between 2000 and 2011 (n = 90). Additionally, a questionnaire was sent to those patients referred between July 2006 and 2011 (n = 64), of which 46 useful questionnaires were returned (72% response). Of patients without an etiological diagnosis at referral (n = 83), 17 (20%) were eventually diagnosed with syndromic CHD, 6 (7%) with nonsyndromic monogenetic CHD and 45 (54%) with nonsyndromic multifactorial CHD. The diagnosis remained undetermined in 15 (18%) patients. Half of patients who returned the questionnaire had purposefully postponed having children until after genetic consultation and 13% had changed their mind about having children or not after the consultation. Counseling was valued positively. In this study, we showed the added value of clinical genetic consultation in the care for adult CHD patients: it improves diagnostics by establishing an etiological diagnosis and associated recurrence risk in a substantial proportion of patients and leads to more informed reproductive decisions. With new genetic testing technologies an etiological diagnosis may be established in an increasing number of patients in the near future.

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IntroductionCongenital heart disease (CHD) is the most common major congenital anomaly, affecting approximately 8 per 1,000 births.1 Due to improved medical and surgical techniques, the popula-tion of adult CHD patients is growing.2,3 These patients may have questions regarding the origin and inheritance of their disease and the recurrence risk in (future) offspring. For the individual patient, knowledge about the underlying (genetic) origin of CHD is important because 1) the patient and his or her offspring may be at risk for extracardiac disease (in syndromic cases); 2) an individualized recurrence risk for offspring based on the underlying cause can be established; 3) there may be other relatives for whom genetic or cardiologic examination may be appropriate.4 In the majority of patients, CHD occurs as an isolated anomaly with a supposedly multifactorial origin, i.e. several (unknown) genetic and environmental factors contribute to the CHD. The recur-rence risks in offspring differ between various types of nonsyndromic CHD although they are generally low; allover these are estimated at 2-3% for children of males and 5-6% for children of females.5-7 These risk numbers are mainly derived from observations in offspring of large groups of CHD patients. Yet, if there is a specific (genetic) etiology for the CHD in an individual patient, the recurrence risk may differ significantly from these empirical numbers. For example, in patients with monogenetic disease, CHD is inherited as a simple Mendelian trait.8-13 Moreover, CHD can occur in the context of a specific syndrome or a chromosomal abnormality, such as Down syndrome, Noonan syndrome and 22q11.2 deletion syndrome.14,15 Recurrence risks depend on the specific syndrome but may be up to 50%. The clinical geneticist has an important role in establishing an accurate etiological diagnosis in an individual CHD patient, and in providing a personalized recurrence risk for CHD in offspring and other relatives. In addition, counseling regarding reproductive choices, prenatal investigations and possible follow up for syndromic features are part of the clinical geneticist’s tasks.4,7 However, little is known about the results of clinical genetic investigations of adult CHD patients and patients’ opinions about the counseling process. In this study, we explored the diagnostic yield of clinical genetic consultation of adult CHD patients, patients’ motivations for the consultation, implications for reproductive decisions, patients’ evaluation of the impact of the provided information and satisfaction with counseling.

MethodsPatients and study designAll adult patients (age ≥ 18 years) who were referred for CHD to the clinical genetics department of the Academic Medical Center between January 2000 and June 2011 were included (Figure 1). Patients who were seen solely for research purposes were not included. Patient characteristics (gender, age, family history, number of children) and medical data (CHD type, extracardiac malfor-mations or disease, reason for referral, genetic analyses) were collected by means of chart review. The eventual etiological diagnosis as made by the geneticist (including KvE and MJHB)

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10at moment of consultation was also derived from the medical charts. We divided the etiological diagnoses into five categories: 1) syndromic CHD, specified; if a specific syndrome had been diagnosed, 2) syndromic CHD, unspecified; if the geneticist had thought the CHD to be syndromic, but had not been able to establish a specific syndrome diagnosis, 3) nonsyndromic CHD, mono-genetic; when the geneticist had considered the CHD to be nonsyndromic and monogenetic; this was based on the presence of a single gene mutation and/or a family pedigree compatible with monogenetic disease, 4) nonsyndromic CHD, multifactorial; if according to the geneticist there were insufficient indications for a syndrome, the pedigree was not suggestive of monogenetic disease and no single gene mutation was identified, or 5) undetermined diagnosis. In the latter case the geneticist had not been able to determine if the patient had a) syndromic versus nonsyn-dromic CHD or b) monogenetic versus multifactorial CHD.In cases with undetermined diagnosis and those with unspecified syndromic CHD we verified if patients had had complete genetic evaluation as compared to current standards, as many patients

Figure 1. Flow chart of patient inclusion. CHD, congenital heart disease.

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10

were counseled many years ago, before the availability of particular diagnostic genetic tests. We sent a questionnaire to the study patients who consulted the geneticist between July 2006 and June 2011. We assumed that patients who had consulted the geneticist before this date might have significant difficulties in recalling details about the counseling. Patients with intellectual disability were also excluded from the questionnaire. Three weeks after mailing of the question-naire, a reminder was send to non-responders. After another three weeks, non-responders were phoned to establish receipt of the questionnaire and to request participation one more time. As the study does not fall within the range of the Dutch Medical Research Involving Human Subjects Act, no formal ethical approval was required for this study according to the local ethics committee.

QuestionnaireThe questionnaire was developed for the purpose of this study. Motivations for referral as well as reproductive decisions were assessed using respectively four and three multiple choice ques-tions. Impact of the provided information was evaluated by means of one question, assessing if the patient had experienced the information as reassuring, alarming or neither reassuring nor alarming. Patients’ satisfaction with counseling was evaluated through eight statements that assessed satisfaction on a visual analogue scale, ranging from 0 ‘not at all’ via 5 ‘neutral’ to 10 ‘completely’ (Table 1). Scores were summated and a mean overall satisfaction score was calcu-lated. Internal consistency (Cronbach’s alpha) was 0.88.Face validity of the questionnaire was evaluated by three clinical geneticists and a psychologist. We asked six patients to participate in a pilot study and adapted the questionnaire according to their remarks.

Mean score (± SD)The information was sufficient 7.4 ± 1.7

I understood everything 8.1 ± 1.5

The opportunity to ask questions was sufficient 8.3 ± 1.6

My questions have been answered sufficiently 7.7 ± 1.7

The emotional support was sufficient 6.4 ± 2.1

In general, I was content with the conversations with the geneticist 7.7 ± 1.6

The consultation was useful 7.8 ± 1.8

I would recommend other patients consulting a clinical geneticist 8.0 ± 1.8

Overall satisfaction score 7.7 ± 1.3

Table 1. Satisfaction with genetic counseling (n = 46)

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10

All patients

(n = 90)Patients who received and re-turned questionnaire (n = 46)

n (%) n (%)

Age (years) ± SD 30.5 ± 8.5 30.3 ± 7.1

Male 39 (43) 15 (33)

CHD type

Atrial septal defect, type I 3 (3) 3 (7)

Atrial septal defect, type II 8 (9) 5 (11)

Ventricular septal defect 8 (9) 5 (11)

Tetralogy of Fallot 12 (13) 5 (11)

Bicuspid aortic valve 7 (8) 2 (4)

Pulmonary valve stenosis 11 (12) 5 (11)

Subvalvar aortic stenosis 2 (2) 1 (2)

Aortic coarctation 7 (8) 5 (11)

Dextro-TGA 8 (9) 3 (7)

Levo-TGA 1 (1) 0

Patent ductus arteriosus 2 (2) 1 (2)

Laterality disorder 3 (3) 0

Other 8 (9) 5 (11)

CHD complexity *

Mild 26 (29) 15 (33)

Moderate 43 (48) 24 (52)

Severe 21 (23) 7 (15)

Referring physician

Cardiologist 71 (79) 39 (85)

Other physician ** 12 (13) 5 (11)

Self-referral 7 (8) 2 (4)

Extracardiac malformations 34 (38) 17 (37)

Positive family history for CHD † 19 (21) 11 (26)

Having children ‡ 20 (22) 8 (17)

Pregnant *** 9 (10) 4 (9)

Desiring having (additional) children 27 (60)

Education level

Low 4 (9)

Medium 22 (48)

High 20 (44)

Table 2. Characteristics of the study population

CHD, congenital heart disease; TGA, transposition of the great arteries. * According to Warnes et al., 2001. ** E.g. gynaecologist, general practitioner. † 1st or 2nd degree. ‡ At time of consultation. *** Pregnancy of the patient or the patient’s partner (in case of male patients) at time of consultation.

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Data analysisData were analyzed using SPSS (version 17.0) statistical software. Statistical significance was set at P < 0.05. For comparison of discrete variables between two groups (e.g. patients with nonsyn-dromic multifactorial CHD vs. patients with other diagnoses) we used Chi-square or Fisher’s exact test and for comparing continuous variables we used the unpaired Student’s T-tests (data were normally distributed).

ResultsSample Ninety patients were included in the chart review (Figure 1). Characteristics of these patients (mean age 30.5 ± 8.5 years, 43% male) are shown in Table 2 (left panel). At first consultation, 22% already had at least one child and 10% were pregnant (or their partner was, in case of male patients).Sixty-four (71%) of the 90 included patients were sent a questionnaire. Forty-six questionnaires were returned (response rate 72%). Patients’ characteristics (mean age 30.3 ± 7.1 years, 33% male) are shown in Table 2 (right panel). There were no statistically significant differences in gender, age and complexity of CHD between responders and non-responders.

Reasons for referralAt referral, seven patients (8%) had already been diagnosed with a specific syndrome in the past, while 83 (92%) did not have an etiologic diagnosis for their CHD (Table 3). Patients diagnosed with a specific syndrome in the past were referred for counseling regarding the syndrome in general (n = 2) or regarding the recurrence risk in offspring and reproductive choices such as prenatal- and pre-implantation genetic diagnosis (n = 5). Of the 83 patients without an etiological diagnosis at referral, 10 were suspected of a specific syndrome by the referring physician and were mainly referred for syndrome diagnosis. Of the remaining 73 patients, the indicated reason for referral was counseling about the inheritance and/or recurrence risk in offspring in 68 patients, and deter-mination of the cause of the CHD in five patients.

Diagnostic yield of clinical genetic consultationPatients already known with a specific syndrome (n = 7)Seven patients were already known having a specific syndrome at referral (for syndrome diag-noses see Table 4) Genetic analysis had been performed in all but one patient with Noonan syndrome, confirming the diagnosis in five patients. All syndromes were autosomal dominant, except for OFCD syndrome, which shows X-linked inheritance.

Patients without etiological diagnosis at referral (n = 83)Of patients without an etiological diagnosis at referral (n = 83), 17 (20%) were eventually diag-nosed with syndromic CHD, 6 (7%) with nonsyndromic monogenetic CHD and 45 (54%) with

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10

D

iagn

oses

mad

e by

the

clin

ical

gen

etic

ist

Synd

rom

ic C

HD

(n =

24)

Non

synd

rom

ic C

HD

(n =

51)

Dia

gnos

is u

ndet

erm

ined

(n =

15)

nSp

ecifi

edU

nspe

cifie

dM

onog

enet

icM

ultif

acto

rial

Synd

rom

ic

vs.

nons

yndr

omic

Mon

ogen

etic

vs

. m

ultif

acto

rial

Pat

ient

s al

read

y kn

own

with

spe

cific

syn

drom

e7

7-

--

--

Pat

ient

s w

ith s

uspi

cion

of

spec

ific

synd

rom

e10

9-

-1

--

Pat

ient

s w

ithou

t sus

pici

on

of s

peci

fic s

yndr

ome

73

- W

ith e

xtra

card

iac

mal

form

atio

ns17

15

1*6

4-

- W

ithou

t ext

raca

rdia

c

m

alfo

rmat

ions

562

-5*

**

38-

11

Tabl

e 3.

Pat

ient

s re

ferr

ed fo

r CH

D a

nd d

iagn

oses

mad

e by

the

clin

ical

gen

etic

ist (

n =

90)

CH

D, c

onge

nita

l hea

rt di

seas

e.

* In

all

mon

ogen

etic

cas

es, a

utos

omal

dom

inan

t inh

erita

nce

was

ass

umed

. **

In 1

of t

hese

cas

es, a

pro

babl

y pa

thog

enic

mut

atio

n w

as id

entifi

ed in

ELN

. In

the

othe

r five

cas

es w

ith a

dia

gnos

is o

f mon

ogen

etic

CH

D, a

t lea

st fo

ur fa

mily

mem

bers

w

ere

affe

cted

with

CH

D.

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10

nonsyndromic multifactorial CHD. The diagnosis remained undetermined in 15 (18%) patients. Thus, a diagnosis other than the most common nonsyndromic multifactorial CHD was assigned to a total of 23 (27%) patients (17 syndromic and 6 nonsyndromic monogenetic). Table 3 provides an overview of diagnoses assigned by the clinical geneticist.

Patients suspected of a specific syndromeIn all 10 patients suspected of a specific syndrome by the referring physician, genetic testing for that specific syndrome was performed. In five of these the syndrome was genetically confirmed, while in four patients the syndrome was diagnosed on clinical grounds (see Table 4). The 10th patient was a 22-year old woman with interrupted aortic arch and low-normal intelligence, because of which she had been suspected of 22q11.2 deletion syndrome. She did not have the deletion and was eventually diagnosed with nonsyndromic, multifactorial CHD, as her intelligence level was similar to that of her relatives. All diagnosed syndromes show autosomal dominant inheritance.

Patients without suspicion of a specific syndromeOf the 73 patients referred without suspicion of a specific syndrome, 17 (23%) had extracardiac malformations or disease (e.g. hypoplastic thumb, scoliosis, absent pectoralis major muscle, hematological abnormalities or deafness, among others). Conventional karyotyping had been per-formed in 48 (66%) patients, all with normal results, while array-CGH was done in 3 (4%) patients, with a deletion of 16p11.2 identified in a man with subvalvular aortic stenosis, situs inversus and mild intellectual disability. Fifty-one (70%) patients were tested for 22q11.2 deletion syndrome (FISH or MLPA). One deletion was found in a 27-year old woman with tetralogy of Fallot and no other malformations, who was tested because of mild dysmorphic features. Analysis of one or more genes involved in syndromic CHD was performed in 15 (21%) of patients. In one patient, a 31-year old woman with complete AVSD without extracardiac abnormalities, a pathogenic muta-tion was found in PTPN11, confirming the diagnosis of Noonan syndrome. The PTPN11 gene had been investigated in this woman because she showed mild facial features suggestive of Noonan syndrome. Analysis of at least one gene involved in non-syndromic CHD had been performed in 27 (37%) patients. Only one probably pathogenic mutation was found in the Elastin-gene (ELN) in a 26-year old woman with familial supravalvular aortic stenosis. A total of eight (11%) of the 73 patients without syndrome suspicion were nevertheless diagnosed with syndromic CHD (Table 3 and 4): three received a specific syndrome diagnosis while in the other five unspecified syndromic CHD was diagnosed. In four (5%) patients, the geneticist had not been able to distinguish between syndromic or nonsyndromic CHD. The majority (84%) of patients without syndrome suspicion was indeed diagnosed with nonsyndromic CHD. Seven of these had extracardiac malformations or disease, but these had been believed to be coincidental as they comprised minor or typically familial non CHD-related abnormalities, e.g. autosomal dominant hearing loss. Nonsyndromic monogenetic CHD was diagnosed in six patients, one of which was based on the previously mentioned identification of a probably pathogenic mutation in ELN. In

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10

Type

of p

atie

nt a

t ref

erra

l

Patie

nts

alre

ady

know

n w

ith

spec

ific

synd

rom

en

= 7

Patie

nts

with

su

spic

ion

of s

peci

fic

synd

rom

en

= 10

Patie

nts

with

out

susp

icio

n of

spe

cific

sy

ndro

me

n =

73

n w

ith d

iagn

osis

of s

yndr

omic

CH

D7

98

Mai

n fe

atur

esIn

herit

-an

ceM

ain

gene

s/lo

ciin

volv

ed

Noo

nan

synd

rom

e4

(2)

3 (1

)1

(1)

Valv

ular

PS

, sho

rt st

atur

e, d

istin

tctiv

e fa

cial

fe

atur

esA

DP

TPN

11, S

OS

1, R

AF1

, K

RA

S, N

RA

S, B

RA

F,

MA

P2K

1, S

HO

C2,

CB

L

Hol

t-Ora

m s

yndr

ome

2 (2

)2

(2)

-A

SD

, VS

D, c

ondu

ctio

n di

seas

e, u

pper

lim

b ab

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aliti

esA

DTB

X5,

SA

LL4

OFC

D s

yndr

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1 (1

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icro

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ia, A

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, VS

D, d

istin

ctiv

e fa

cial

fe

atur

es, d

enta

l abn

orm

aliti

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-l D

BC

OR

22q1

1 de

letio

n sy

ndro

me

-1

(1)

1 (1

)TO

F, v

elop

hary

ngea

l ins

uffic

ienc

y/cl

eft p

alat

e,

psyc

hiat

ric d

isor

ders

, hyp

ocal

cem

ia, d

istin

tctiv

e fa

cial

feat

ures

, int

elle

ctua

l dis

abili

tyA

DM

icro

dele

tion

22q1

1.2

LEO

PAR

D s

yndr

ome

-1

(1)

-

Lent

igin

es, E

CG

con

duct

ion

abno

rmal

ities

, oc

ular

hyp

erte

loris

m, p

ulm

onic

ste

nosi

s, a

b-no

rmal

gen

italia

, ret

arda

tion

of g

row

th, s

enso

ri-ne

ural

dea

fnes

s

AD

PTP

N11

, RA

F1, B

RA

F

CH

AR

GE

syn

drom

e-

1 (0

)-

Col

obom

a, h

eart

defe

cts,

cho

anal

atre

sia,

re

tard

ed g

row

th a

nd d

evel

opm

ent,

geni

tal

abno

rmal

ities

, ear

ano

mal

ies

AD

CH

D7

Cay

ler s

yndr

ome

-1

(0)

-H

ypop

lasi

a m

uscu

lus

depr

esso

r ang

uli o

ris,

feat

ures

of 2

2q11

del

etio

n sy

ndro

me

AD

Mic

rode

letio

n 22

q11.

2

16p1

1 de

letio

n sy

ndro

me

--

1 (1

)In

telle

ctua

l dis

abili

ty, a

utis

m s

pect

rum

dis

orde

rA

DM

icro

dele

tion

16p1

1.2

Uns

peci

fied

synd

rom

ic C

HD

--

5

Tabl

e 4.

Syn

drom

ic d

iagn

oses

mad

e by

the

clin

ical

gen

etic

ist i

n ad

ult C

HD

pat

ient

s

N o

f pat

ient

s in

who

m th

e sy

ndro

me

diag

nosi

s w

as g

enet

ical

ly c

onfir

med

is m

entio

ned

betw

een

brac

kets

. CH

D, c

onge

nita

l hea

rt di

seas

e; P

S, p

ulm

onar

y st

enos

is; T

OF,

tetra

logy

of F

allo

t; A

SD

II, a

trial

sep

tal d

efec

t typ

e; V

SD

, ven

tricu

lar s

epta

l def

ect;

OFC

D, o

culo

faci

ocar

diod

enta

l; A

D, a

utos

omal

dom

inan

t; X

-l D

, X-li

nked

dom

inan

t.

181

10

the other five patients no genetic mutation was identified, though as at least three close relatives were also affected with CHD they had been diagnosed with monogenetic CHD on a clinical basis. Autosomal dominant inheritance was assumed in all monogenetic cases, with possibly incomplete penetrance in five. A definitive diagnosis could not be determined in 15 (18%) patients. Three of these patients had declined genetic testing. Six (50%) of the remaining 12 patients with an unde-termined diagnosis and three of five (60%) of patients with unspecified syndromic CHD had had incomplete genetic evaluation as compared to current standards, as particular tests had not been available at time of the genetic consultation.

Patients’ motivations for clinical genetic consultationMost patients (80%) had learned about the possibility of clinical genetic consultation from their cardiologist. The majority of patients (78%) initiated the consultation to learn about the recurrence risk in (future) offspring. Sixty-five percent wanted to learn about the heredity of their CHD in general and 26% wanted to know the cause of their CHD. One patient stated that the only reason for consultation was her cardiologist finding it to be useful; she was eventually diagnosed with LEOPARD syndrome (acronym for lentigines, ECG conduction abnormalities, ocular hyper-telorism, pulmonic stenosis, abnormal genitalia, retardation of growth, and sensorineural deafness).

Patients’ reproductive decisionsForty-nine percent of patients had purposefully postponed having children until after the clinical genetic consultation, while another 49% had not and 2% could not remember. There were no significant differences in gender and age between those who had postponed having children and those who had not. While 40% of patients without extracardiac malformations had postponed pregnancy, 67% of patients with at least one such malformation stated so. This difference did not reach statistical significance however (P = 0.129).

Table 5. Adult CHD patients who may benefit from clinical genetic testing and counseling

CHD, congenital heart disease; IAA, interrupted aortic arch; TA, truncus arteriosus; TOF, tetralogy of Fallot; PA, pulmonary atresia; VSD, ventricular septal defect; AAA, aortic arch anomaly.

Patients with a family history of CHD

Patients with extracardiac abnormalities/disease

- Congenital malformations

- Intellectual disability

- Dysmorphic features

- Multisystem involvement (e.g. endocrinologic, hematologic, immunologic or sensorineural disorders)

- Psychiatric disorders

Patients with CHD with high risk of 22q11.2 deletion (IAA, TA, TOF, PA with VSD, AAA)

Patients desiring having children (preconceptional counseling)

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10

Nine patients (20%) had considered giving up having children before clinical genetic consultation. After the consultation, five of these had changed their mind; two of them had been diagnosed with nonsyndromic multifactorial CHD, two with nonsyndromic multifactorial or monogenic CHD and one with unspecified syndromic CHD. Three patients who still contemplated not having children had been diagnosed with multifactorial nonsyndromic CHD, although two of them had extracar-diac malformations (vaginal septum in one patient, hypospadia and cryptorchidism in one patient). The ninth patient did not answer this question.In contrast, one female patient with tetralogy of Fallot (diagnosed with nonsyndromic multifactorial CHD) indicated that she had not considered giving up having children before the clinical genetic consultation, but afterwards she did.

Impact of the information and satisfaction with counselingThe majority of patients (59%) had perceived the information given by the geneticist as reas-suring, while 28% found the information neither reassuring nor alarming. Only one patient considered the information alarming: a woman with familial subvalvular aortic stenosis diagnosed as nonsyndromic monogenic CHD with a recurrence risk of up to 50%. There was no difference in perception between patients diagnosed with nonsyndromic multifactorial CHD and patients with other diagnoses.Mean satisfaction scores are shown in Table 1. In general, the counseling was valued positively. Satisfaction with emotional support had the lowest mean score of all items. Men and women were equally satisfied on all items (mean overall score 7.59 vs. 7.78, P = 0.64). Patients desiring having (additional) children at the moment of consultation scored higher on all items than patients without pregnancy wish, statistical significance being reached for items 4, 5, 6, 7 and the overall satisfaction score (8.3 vs. 7.1, P = 0.002). Patients diagnosed with nonsyndromic multifactorial CHD scored higher than patients with other diagnoses (mean overall satisfaction score 8.2 vs. 7.3, P = 0.038).

DiscussionThis study evaluating clinical genetic care in adult CHD demonstrates that the diagnostic yield of clinical genetic consultation is substantial, 20% of patients being diagnosed with syndromic CHD and 7% with nonsyndromic monogenetic CHD. Etiological diagnoses other than the most common nonsyndromic multifactorial CHD were thus made in more than a quarter of patients. Most patients were not alarmed by the information given by the geneticist, and the counseling was valued positively. CHD can occur in a syndromic or nonsyndromic form. Although most syndrome diagnoses are made during childhood, some syndromic adult CHD patients may not have been identified as not all patients have been evaluated genetically as a child because of limited availability of genetic diagnostics at that time.14,16 In our study, genetic consultation had led to 17 (20%) patients being newly diagnosed with syndromic CHD, of whom 12 with a specific syndrome diagnosis. As nine of

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10

these had been referred specifically with suspicion of the eventually diagnosed syndrome, refer-ring physicians thus had correct considerations in these patients. The remainder of patients had not been suspected of a syndrome by the referring physician, showing that syndromic features are not always easy to recognize in adults. The syndrome diagnosis had important clinical implications for the patients in our study. Firstly, with this diagnosis, the geneticist was able to educate them about their risk for extracardiac mani-festations and follow up hereon, such as screening for hypocalcaemia and thyroid dysfunction in 22q11.2 deletion syndrome.16-18 Secondly, these patients could be informed about the 50% recurrence risk, offspring not only being at risk for CHD but also of the other manifestations of the syndrome. Moreover, in patients with a genetic diagnosis, prenatal- and even pre-implantation genetic diagnostics are possible, giving patients the opportunity to prevent the birth of an affected child. This also applies to the patients who were already known with a syndrome at time of referral, particularly for those in whom the diagnosis was confirmed by identification of a causative muta-tion. Genetic consultation may thus also be useful for patients with a known syndrome, especially if they have a pregnancy wish.There were also clinical implications in the group of patients with a diagnosis of nonsyndromic CHD. Seven percent had been diagnosed with monogenetic CHD: not only could these patients be educated about the higher recurrence risks (up to 50%), cardiologic screening in other relatives could also be recommended, as these are also at risk of having (asymptomatic) CHD. Laboratory genetic analysis did not contribute to the diagnosis in the majority of nonsyndromic CHD cases, as only one probably pathogenic mutation had been found in a nonsyndromic CHD gene in 27 patients tested. However, mutations in nonsyndromic CHD genes have been shown to be rare and not all can be identified with the current knowledge and technology.9,19,20

In about one fifth (18%) of patients the diagnosis remained undetermined after clinical genetic evaluation, and in an additional 6% the syndromic CHD was unspecified. As ongoing research demonstrates that the genetic contributions to CHD are greater than previously suspected 15 and advanced genetic analysis techniques such as high resolution array CGH and next generation sequencing are increasingly becoming available in clinical practice, it may become possible to provide an etiologic diagnosis in a growing number of patients in the near future. Indeed, in our study 50% of patients with an undetermined diagnosis and 60% of patients with unspecified syndromic CHD had incomplete genetic evaluation as to current standards and possibili-ties. Additionally, in 32% of patients with a specific syndrome, the diagnosis was not genetically confirmed. It is crucial that patients as well as referring physicians are informed that the field of clinical genetics is evolving rapidly and that periodic reassessment through adulthood can be beneficial. In some centers, including our own, it is common practice to explicitly state this in a letter to the patient. Although often standard practice in children, a follow-up visit is less commonly scheduled beforehand in adults. In selected patients, this more directive approach could ensure adequate follow up and up-to-date genetic assessment. Previous studies have shown that adult CHD patients have insufficient knowledge about several

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10

topics, including the origin and inheritance of their CHD.21-24 Knowledge about recurrence risk is essential for making informed choices regarding family planning and prenatal screening.7 In our study, six patients (13%) changed their mind about having children or not after clinical genetic consultation. Although their reasons are unknown, genetic consultation does seem to influence some patients’ reproductive decisions. Patients were satisfied with the clinical genetic counseling, specifically patients with a child-wish. Apparently, their needs are met the most. Patients with a diagnosis other than nonsyndromic multifactorial CHD were overall less satisfied. In these patients, the diagnosis was not always clear-cut, possibly making the message more difficult to comprehend, leading to less contentment with the consultation. Interestingly, the provided information was just as reassuring to patients diagnosed with nonsyn-dromic multifactorial CHD as to patients with other diagnoses, whereas the latter were told higher recurrence risks and often a risk for extracardiac disease. Possibly, patients with other diagnoses had been more pessimistic and/or uncertain about recurrence risks and prenatal screening options before the consultation than patients with nonsyndromic multifactorial CHD. The informa-tion that was provided may have reduced uncertainty, thereby providing reassurance. Although clinical genetic consultation may have several implications for adult CHD patients, only a minority consults a clinical geneticist.21 Table 5 shows adult CHD patients for whom genetic counseling may be especially beneficial. Of note, the best timing of clinical genetic consultation is before pregnancy,25 in our study only two thirds of patients were indeed counseled before. As a substantial proportion of patients had postponed pregnancy until after the clinical genetic consulta-tion, physicians caring for adult CHD patients should not delay in referring these patients, particu-larly because the process of genetic testing can take long. The implications of clinical genetic consultation for adult CHD patients’ decisions regarding reproductive issues such as prenatal screening, and prenatal and pre-implantation genetic diagnosis deserves further research.

LimitationsOur study regards a subset of adult CHD patients, namely those referred to the clinical geneticist. The population in our study is therefore not representative of the general adult CHD population. Patients with syndromes including CHD who were referred by other physicians than the cardi-ologist were not included, as they were not referred for CHD as main reason. Furthermore, our study is retrospective in design and we chose to adopt the diagnosis as made by the geneticist at the moment of consultation. As there is no standardization between geneticists, the criteria used to assign a particular diagnosis may have differed between geneticists. This may have particularly been the case in the differentiation between nonsyndromic multifactorial versus syndromic CHD in patients with additional malformations. By review of the medical charts, we indeed felt that there were some patients who might have had syndromic CHD rather than nonsyndromic CHD with coincidental extracardiac abnormalities. Additionally, as there was possible incomplete pene-trance in the families of patients diagnosed with monogenetic CHD, oligogenic inheritance cannot be excluded in these families. Moreover, our study contains patients who consulted the geneticist

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up to 12 years ago and differences in diagnostic strategies through the years may possibly have lead to different diagnoses. This is also reflected by the large proportion of patients who received incomplete genetic evaluation as compared to current standards. For the above-mentioned reasons, our study cannot provide exact proportions of etiological diagnoses in adult CHD patients. Although patients counseled before July 2006 were excluded from the questionnaire, recall bias may have been introduced. In addition, the study sample is relatively small. Finally, as data were collected at one clinical genetics department, making generalizations may be difficult.

ConclusionsWe showed the added value of clinical genetic consultation in the care for adult CHD patients: it improves diagnostics by establishing an etiological diagnosis and associated recurrence risk in a substantial proportion of patients and leads to more informed reproductive decisions. As a large proportion of patients who consulted the clinical geneticist in the past had incomplete examina-tions as compared to current standards, periodic re-evaluation is likely to increase the proportion of patients with an etiological diagnosis. Moreover, as possibilities for diagnostic genetic testing are expanding rapidly, in the near future an etiological diagnosis may be established in even more patients. Clinical geneticist should inform physicians caring for adult CHD patients as well as the patients themselves about the increasing diagnostic possibilities over the years and offer repeated and up-to-date genetic evaluation. Future studies may focus on the choices adult CHD patients make regarding reproduction, prenatal screening, and prenatal and pre-implantation genetic diagnosis.

AcknowledgementsWe would like to thank all patients for their kind participation.

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1 Dolk H, Loane M, Garne E. Congenital heart defects in Europe: prevalence and perinatal mortality, 2000 to 2005. Circulation 2011;123:841-9

2 Engelfriet P, Boersma E, Oechslin E et al. The spectrum of adult congenital heart disease in Europe: morbidity and mortality in a 5 year follow-up period. The Euro Heart Survey on adult congenital heart disease. Eur Heart J 2005;26:2325-33

3 Marelli AJ, Mackie AS, Ionescu-Ittu R, Rahme E, Pilote L. Congenital heart disease in the general population: changing prevalence and age distribution. Circulation 2007;115:163-72

4 Burchill L, Greenway S, Silversides CK, Mital S. Genetic counseling in the adult with congenital heart disease: what is the role? Curr Cardiol Rep 2011;13:347-55

5 Firth HV, Hurst JA, Hall JG. Congenital Heart Disease. In: Firth HV, Hurst JA, Hall JG, editors. Desk reference clinical genetics.Oxford: Oxford University Press, 2006. 84-7.

6 Meijer JM, Pieper PG, Drenthen W et al. Pregnancy, fertility, and recurrence risk in corrected tetral-ogy of Fallot. Heart 2005;91:801-5

7 Bernier FP, Spaetgens R. The geneticist’s role in adult congenital heart disease. Cardiol Clin 2006;24:557-vi

8 Garg V, Muth AN, Ransom JF et al. Mutations in NOTCH1 cause aortic valve disease. Nature 2005;437:270-4

9 Wessels MW, Willems PJ. Genetic factors in non-syndromic congenital heart malformations. Clin Genet 2010;78:103-23

10 Schott JJ, Benson DW, Basson CT et al. Congenital heart disease caused by mutations in the tran-scription factor NKX2-5. Science 1998;281:108-11

11 Joziasse IC, van de Smagt JJ, Smith K et al. Genes in congenital heart disease: atrioventricular valve formation. Basic Res Cardiol 2008;103:216-27

12 Postma AV, van Engelen K, van de Meerakker J et al. Mutations in the sarcomere gene MYH7 in Ebstein anomaly. Circ Cardiovasc Genet 2011;4:43-50

13 van de Meerakker JB, van Engelen K, Mathijssen IB et al. A novel autosomal dominant condition consisting of congenital heart defects and low atrial rhythm maps to chromosome 9q. Eur J Hum Genet 2011;19:820-6

14 van Engelen K, Topf A, Keavney BD et al. 22q11.2 Deletion Syndrome is under-recognised in adult patients with tetralogy of Fallot and pulmonary atresia. Heart 2010;96:621-4

15 Pierpont ME, Basson CT, Benson DW, Jr. et al. Genetic basis for congenital heart defects: current knowledge: a scientific statement from the American Heart Association Congenital Cardiac Defects Committee, Council on Cardiovascular Disease in the Young: endorsed by the American Academy of Pediatrics. Circulation 2007;115:3015-38

16 Beauchesne LM, Warnes CA, Connolly HM et al. Prevalence and clinical manifestations of 22q11.2 microdeletion in adults with selected conotruncal anomalies. J Am Coll Cardiol 2005;45:595-8

17 Bassett AS, Chow EW, Husted J et al. Clinical features of 78 adults with 22q11 Deletion Syndrome. Am J Med Genet A 2005;138:307-13

18 Cohen E, Chow EW, Weksberg R, Bassett AS. Phenotype of adults with the 22q11 deletion syndrome: A review. Am J Med Genet 1999;86:359-65

19 Butler TL, Esposito G, Blue GM et al. GATA4 mutations in 357 unrelated patients with congenital heart malformation. Genet Test Mol Biomarkers 2010;14:797-802

20 Smith KA, Joziasse IC, Chocron S et al. Dominant-negative ALK2 allele associates with congenital heart defects. Circulation 2009;119:3062-9

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21 van Engelen K, Baars MJ, van Rongen LT, van der Velde ET, Mulder BJ, Smets EM. Adults with congenital heart disease: patients’ knowledge and concerns about inheritance. Am J Med Genet A 2011;155A:1661-7

22 Kovacs AH, Harrison JL, Colman JM, Sermer M, Siu SC, Silversides CK. Pregnancy and contracep-tion in congenital heart disease: what women are not told. J Am Coll Cardiol 2008;52:577-8

23 Moons P, De VE, Budts W et al. What do adult patients with congenital heart disease know about their disease, treatment, and prevention of complications? A call for structured patient education. Heart 2001;86:74-80

24 Dore A, de GP, Mercier LA. Transition of care to adult congenital heart centres: what do patients know about their heart condition? Can J Cardiol 2002;18:141-6

25 Aalfs CM, Mollema ED, Oort FJ, de Haes JC, Leschot NJ, Smets EM. Genetic counseling for familial conditions during pregnancy: an analysis of patient characteristics. Clin Genet 2004;66:112-21

Summary and future perspectives

Chapter 11

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SummaryCongenital heart disease (CHD) is among the most common birth defects, occurring in approxi-mately 8 per 1,000 live births. It leads to significant morbidity and mortality in children as well as adults. Due to improvements in cardiac surgery and medical care, nowadays approximately 90% of CHD patients reach adulthood. Although the causes of CHD are largely unknown, there has been significant progress in the identification of genes and signaling pathways that are involved in cardiovascular development and CHD. The increasing knowledge enables physicians caring for CHD patients to inform patients about the origin of their CHD, the inheritance pattern and possible risk for extracardiac disease. This thesis focuses on the genetics of non-syndromic and syndromic CHD, the implications it has for (adult) patients as well as the adult CHD patients’ perspective on inheritance issues.

In Chapter 1, the current knowledge about genetic causes of CHD is summarized. An overview of causes of common syndromic and non-syndromic CHD is given. Additionally, implications for genetic counseling of CHD patients are discussed, mainly focused on the adult with CHD.

Part I of this thesis comprises studies searching for the genetic causes of non-syndromic CHD, using different strategies. In Chapter 2, traditional genome-wide linkage analysis was used to unravel the genetic cause of a unique autosomal dominant phenotype in a large four-generation family. Affected individuals presented with CHD (including atrial septal defect type I and II, tetralogy of Fallot and persistent left superior vena cava) and/or low atrial rhythm. This phenotype seems to represent a mild or variant expression of left atrial isomerism or a developmental defect of the sinus node and surrounding tissue. A 39-Mb locus at chromosome 9q was identified that co-segregated with this autosomal dominant disorder. By means of direct sequencing we analyzed several candidate genes within this locus, but no mutations were identified. The causa-tive defect has not been identified yet. Chapter 3 describes the comprehensive genetic analysis of a cohort of unrelated probands with Ebstein anomaly. Using next generation sequencing and direct DNA sequencing, we found a disease-associated mutation in MYH7 in 8%. MYH7 encodes the sarcomere protein beta-myosin heavy chain. Mutation positive probands and family members showed left ventricular noncompaction as well as various CHD, including Ebstein anomaly and ventricular septal defect. The penetrance of the left ventricular noncompaction was high, whereas there was significant pleiotropy and reduced penetrance regarding the observed CHD. With this study we showed that there is an association between Ebstein anomaly with left ventricular noncompaction and mutations in MYH7. Chapter 4 focuses on the possible role of sodium channels in CHD. The SCN5A gene encodes the alpha-subunit of the cardiac sodium channel. Mutations in SCN5A underlie several arrhythmia syndromes, including Brugada syndrome and Long QT syndrome. In animal studies, deficiency of scn5a was shown to lead to developmental abnormalities of the heart, seemingly due to a non-electrogenic function of the channel. We explored the role of SCN5A mutations in human

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CHD. In the first part of this study, we determined the proportion of CHD in a consecutive cohort of SCN5A loss-of-function mutation carriers and in a cohort of gain-of-function mutation carriers. The proportion of CHD in the loss-of-function mutation cohort was 3.1%, which was significantly higher than reported in the general population. Observed CHD were mainly septal defects. There were no CHD in the gain-of-function mutation cohort. In the second part of this study, we sequenced SCN5A in a cohort of patients with a septal defect and conduction disease and identified a (possibly) pathogenic mutation in 3.7% of patients. Our results suggest a role for SCN5A loss-of-function mutations in CHD in humans, particularly septal defects.

Part II of this thesis describes studies focuses on CHD in association with other (extracardiac) abnormalities, including syndromic as well as non-syndromic CHD. In Chapter 5, we showed that 22q11.2 deletion syndrome is highly under-recognized in adults with TOF or pulmonary atresia and ventricular septal defect. We investigated patients with these CHD types registered in CONCOR, a nationwide registry and DNA-bank for adult patients with CHD. Using Multiplex liga-tion dependent probe amplification, we identified a 22q11.2 deletion in 6.5% of tetralogy of Fallot patients and 16.5% of pulmonary atresia with ventricular septal defect patients. More than half of the 22q11.2 deletions were unknown before this study; these patients had thus not been diag-nosed with the syndrome. As the syndrome may have important clinical and reproductive implica-tions, we propose that a diagnostic test should be considered in all adult patients with tetralogy of Fallot and pulmonary atresia with ventricular septal defect. Chapter 6 focuses on the morphology of the bicuspid aortic valve (BAV) in fetuses with Turner syndrome (monosomy X). We studied 37 post-mortem heart specimens of Turner syndrome fetuses and concluded that the vast majority shows abnormal aortic valve morphology. BAV was present in 75%. Different BAV morphology types were present; both BAV with fusion of the right and left coronary leaflets (Type 1 BAV) and BAV with fusion of the right coronary and noncoronary leaflets (Type 2 BAV) were encountered. Type 2 BAV seemed to be overrepresented in our fetal Turner syndrome population as compared to reports in adult Turner syndrome patients; this might imply a worse prognosis of Type 2 BAV than Type 1 BAV, possibly due to associations with additional cardiovascular abnormalities.In Chapter 7, we describe clinical and functional studies of the p.A119S variant in NKX2-5. Muta-tions in NKX2-5 are a well-known cause of human CHD; more recently mutations in this gene were also identified in patients with thyroid dysgenesis. The p.A119S variant had been described as causal for thyroid dysgenesis; we encountered this variant in two probands with CHD. In this study, we showed that p.A119S is a rare variant that behaves equal to wildtype NKX2-5 and does not cause CHD or thyroid dysgenesis. Chapter 8 focuses on the previously suggested association between CHD and neuroblastoma, with conflicting evidence in previous literature. We assessed this association by conducting an echocardiographic study in a large single center cohort of consecutive neuroblastoma patients. We did not find a higher prevalence of CHD compared to the control groups and conclude that,

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based on the results of our study as well as an extensive systematic review of the existing litera-ture, clear evidence of an association between neuroblastoma and CHD is lacking. Part III includes studies on the role of the clinical geneticist and the adult patients’ perspective on genetics and inheritance of CHD. Chapter 9 describes the results of a questionnaire study in almost 500 adults with CHD. The majority of adult CHD patients was shown to lack knowledge about inheritance issues and over 40% desires more information about this topic. Almost half of patients has concerns regarding inheritance issues. A dedicated counseling program for adults with CHD may address these topics and thus improve patient care. Chapter 10 regards adult CHD patients who consulted a clinical geneticist. We performed a retro-spective study and showed that an etiological diagnosis other than the most common ‘multifacto-rial’ CHD is provided in more than a quarter of these patients. Moreover, patients value the clinical genetics consultation as positive. These latter two studies demonstrate the added value of the clinical geneticist in the care for adults with CHD.

Finally, Chapter 11 discusses future perspectives regarding research and clinical care in the field of CHD genetics.

Future perspectivesResearch perspectivesAlthough significant progress has been made in the identification of genes and pathways involved in cardiogenesis and (human) CHD, the majority of CHD is unexplained so far. As yet, it remains a challenge to identify additional genes, pathways and mechanisms that are involved in human CHD. However, new genetic analysis techniques are radically changing our way of investigating CHD causes. Classical approaches for gene identification, such as positional cloning strategies, have proven to be difficult to realize due to the lack of extended CHD families with Mendelian inheritance. They will be complemented or replaced by contemporary strategies, which have already yielded promising results. It is increasingly recognized that copy number variations (CNVs) underlie CHD in a subset of patients.1-3 The identification of recurrent CNVs provides opportunities for the discovery of new genes and pathways involved in human CHD by recognition of candidate genes within these regions. Moreover, newer approaches using next generation sequencing can identify large numbers of variations per exome or genome. The decreasing costs of such technologies make that these are becoming available at lower thresholds, and these strategies may yield exciting new insights. Such studies bring huge amounts of data, however, and we still have to learn a lot about its analysis and interpretation. From these data, CHD causing mutations have to be identified by focusing on rare variants that occur in genes involved in heart development and are predicted to have deleterious consequences on protein functions. Moreover, whole genome sequencing can

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also address the large component of the genome that does not code for proteins, but is nonethe-less biologically functional. For example, a homozygous mutation in a cardiac enhancer of TBX5 (located 90 kb downstream) was identified in a CHD patient.4 The potential role of such non-coding variants in CHD has not been explored extensively but should undoubtedly be subject of future studies. New data from next generation sequencing studies have to be combined with data from systems biology approaches, by constructing molecular networks and thus elucidating pathways and interaction of molecules involved in heart development.5 For example, a recent bioinformatics study of rare CNVs identified in 2500 human CHD cases found that the genes in these CNVs were significantly enriched for involvement in the Wnt signaling pathway, thereby providing the first evidence that this pathway is involved in human CHD.2 Additional advancements and opportunities for further research in CHD may arise from initiatives such as CONCOR (CONgeni-tal CORvitia), CHD-GENES (Congenital Heart Disease Genetic Network Study) and CHeartED (Congenital Heart and Environment/Epidemiological Database).6-8 These initiatives provide large collections of homogeneous clinical CHD cases, with extensive clinical data as well as DNA and sometimes tissue of affected patients. Such large collections, particularly for the rarer malforma-tions, were lacking in the past and therefore hampered genetic studies. Now these large collec-tions are available, genetic studies such as genome-wide association studies (GWAS) become possible. This likely increases our knowledge about common genetic variations influencing the risk of CHD. Although GWAS have been performed widely in various complex diseases, only few were done in CHD phenotypes to date. These few studies provided evidence that common variants contribute to at least TOF and septal defects.9-11 Further GWAS studies will surely follow, in different subtypes of CHD.There are several other interesting fields related to CHD origin that require further study. One of these is the biological impact of gene-environment interactions, such as interactions between variants in genes involved in glucose metabolism and nutrition during pregnancy. Other fields that need further exploration are epigenetic mechanisms, since chromatin-remodeling and histone-modifying factors regulating gene expression to control cardiovascular development have already been identified.12,13 Moreover, recently de novo mutations in histone-modifying genes were iden-tified in CHD patients.14 The role of microRNAs also warrants further study, as animal studies suggested that deficiency of certain microRNAs leads to decreased levels of cardiac transcription factors15 and to CHD. In addition, studies in heart tissue of human CHD patients showed micro-RNA dysregulation.16 The relative importance of genetic factors is increasingly recognized in the origin of CHD. Due to rapid molecular advances, the large group of CHD patients historically assigned to have a ‘multifactorial’ origin might prove to be much more genetically heterogeneous. It will remain a major challenge to combine the knowledge of the different fields of investigation and to answer the question of how genetic, epigenetic and environmental factors interact with each other within individual patients to eventually lead to CHD.

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Clinical implicationsThe increase in availability of next generation sequencing facilities in a research setting as well as the growing knowledge about genetic mechanisms will undoubtedly augment our knowledge about heart development and the origins of CHD. These innovations can also be expected to change clinical care for CHD patients in the (near) future. The above mentioned next generation sequencing facilities are not only used in a research setting but are also becoming more and more available in daily clinical practice at decreasing costs. Thus, panels of candidate genes or even total exomes or whole genomes may be analyzed in individual syndromic or non-syndromic CHD patients in a clinical setting. The goal of such analysis would be to identify mutations and variants contributing to CHD in that particular patient. However, clinical utility and relevance to patient management will need to be demonstrated before testing will become routinely available and implemented in care for CHD patients. It is likely that only a small subset of patients have CHD caused by single (or even a few) mutations. Consequently, next generation sequencing in an individual patient will probably identify many rare variants of which the isolated or combined significance cannot easily be predicted. However, as we increasingly gain experience with next generation sequencing in clinical care, it is likely that we will be able to inform more patients and their families about the causes, inheritance and recurrence risk of CHD in their particular situation. This not only helps patients who simply ‘need to know’ the cause of their CHD; it may also influ-ence reproductive choices of CHD patients and their relatives. In combination with continuously improving screening and diagnostic possibilities before and during a pregnancy, such as prenatal- and pre-implantation genetic diagnosis and advanced fetal echocardiography, patients will have more influence on the risk of having a child affected with CHD and/or associated abnormalities. Another opportunity for improvements in patient care could come from future studies on gene-environment interactions. It would be a major achievement if we could identify patients who are particularly susceptible for developing CHD in certain environmental settings (for example, the use of certain drugs during pregnancy could lead to CHD in patients with a certain genetic predisposi-tion). This would enable personalized measures to decrease the risk of CHD in offspring. For the longer term, one may speculate that increasing insights in mechanisms of heart development will lead to future therapeutic measures for attenuation of CHD, such as intra-uterine molecular inter-ventions including supplementation of deficient molecules or epigenetic manipulations.

The ultimate challenge for the coming years is to find ways to analyze and interpret the large amount of data generated by renewed technologies, to apply this knowledge to individual patients and to develop strategies for prevention, treatment or repair of CHD.

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Reference List

1 Greenway SC, Pereira AC, Lin JC et al. De novo copy number variants identify new genes and loci in isolated sporadic tetralogy of Fallot. Nat Genet 2009;41:931-5

2 Soemedi R, Wilson IJ, Bentham J et al. Contribution of global rare copy-number variants to the risk of sporadic congenital heart disease. Am J Hum Genet 2012;91:489-501

3 Hitz MP, Lemieux-Perreault LP, Marshall C et al. Rare copy number variants contribute to congenital left-sided heart disease. PLoS Genet 2012;8:e1002903

4 Smemo S, Campos LC, Moskowitz IP, Krieger JE, Pereira AC, Nobrega MA. Regulatory variation in a TBX5 enhancer leads to isolated congenital heart disease. Hum Mol Genet 2012;21:3255-63

5 Sperling SR. Systems biology approaches to heart development and congenital heart disease. Cardiovasc Res 2011;91:269-78

6 van der Velde ET, Vriend JW, Mannens MM, Uiterwaal CS, Brand R, Mulder BJ. CONCOR, an initiative towards a national registry and DNA-bank of patients with congenital heart disease in the Netherlands: rationale, design, and first results. Eur J Epidemiol 2005;20:549-57

7 Pediatric Cardiac Genomics Consortium. The Congenital Heart Disease Genetic Network Study: rationale, design, and early results. Circ Res 2013;112:698-706

8 CHeartED (Congenital Heart and Environment/Epidemiological Database). http://www.chearted.eu. 20139 Cordell HJ, Topf A, Mamasoula C et al. Genome-wide association study identifies loci on 12q24 and

13q32 associated with Tetralogy of Fallot. Hum Mol Genet 2013;22:1473-8110 Cordell HJ, Bentham J, Topf A et al. Genome-wide association study of multiple congenital heart

disease phenotypes identifies a susceptibility locus for atrial septal defect at chromosome 4p16. Nat Genet 2013;45:822-4

11 Hu Z, Shi Y, Mo X et al. A genome-wide association study identifies two risk loci for congenital heart malformations in Han Chinese populations. Nat Genet 2013;45:818-21

12 Bruneau BG. Chromatin remodeling in heart development. Curr Opin Genet Dev 2010;20:505-1113 Chang CP, Bruneau BG. Epigenetics and cardiovascular development. Annu Rev Physiol 2012;74:41-6814 Zaidi S, Choi M, Wakimoto H et al. De novo mutations in histone-modifying genes in congenital heart

disease. Nature 2013;498:220-315 Zhao Y, Ransom JF, Li A et al. Dysregulation of cardiogenesis, cardiac conduction, and cell cycle in

mice lacking miRNA-1-2. Cell 2007;129:303-1716 O’Brien JE, Jr., Kibiryeva N, Zhou XG et al. Noncoding RNA expression in myocardium from infants

with tetralogy of Fallot. Circ Cardiovasc Genet 2012;5:279-86

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Nederlandse samenvattingAangeboren hartafwijkingen zijn één van de meest voorkomende aangeboren aandoeningen. Ongeveer acht van de duizend levendgeborenen hebben een aangeboren hartafwijking. Aange-boren hartafwijkingen kunnen leiden tot significante morbiditeit en mortaliteit bij zowel kinderen als volwassenen. Dankzij vooruitgang in medische voorzieningen en cardiothoracale chirurgie bereikt 90% van de patiënten nu de volwassen leeftijd. Hoewel de oorzaken van aangeboren hartafwijkingen voor een belangrijk deel onbekend zijn, is er de laatste decennia veel vooruitgang geboekt met de identificatie van genen en signaaltransductie pathways die betrokken zijn bij de ontwikkeling van het hart en het ontstaan van aangeboren hartafwijkingen. Deze toenemende kennis over de genetische basis maakt het mogelijk om patiënten te informeren over de oorzaak van hun hartafwijking, het overervingspatroon en het eventuele risico op extracardiale aandoeningen. Dit proefschrift richt zich op de genetica van syndromale en niet-syndromale aangeboren hartafwijkingen, de implicaties hiervan voor (volwassen) patiënten en hun visie op erfelijkheidskwesties. In Hoofdstuk 1 wordt de huidige kennis over genetische oorzaken van aangeboren hartaf-wijkingen samengevat. Er wordt een overzicht gegeven van veel voorkomende oorzaken van syndromale en van niet-syndromale aangeboren hartafwijkingen. Ook worden de implicaties voor genetische counseling van patiënten met een aangeboren hartafwijking besproken, met name gericht op volwassenen.

Deel I van dit proefschrift beschrijft studies naar genetische oorzaken van niet-syndromale aange-boren hartafwijkingen. In deze studies werden verschillende technieken gebruikt. In Hoofdstuk 2 verrichtten wij genoomwijde linkage analyse (koppelingsonderzoek) om de oorzaak te vinden van een niet eerder beschreven autosomaal dominant fenotype binnen een grote familie. Aangedane familieleden hadden aangeboren hartafwijkingen (waaronder ostium primum en secundum atrium septum defect, tetralogie van Fallot en persisterende linker vena cava superior) en/of een laag atriaal ritme. Dit nieuwe fenotype lijkt een milde uiting te zijn van links isomerisme, of is mogelijk een ontwikkelingsstoornis van de sinusknoop en omliggend weefsel. Wij identificeerden een locus van 39 Mb op chromosoom 9q, dat segregeerde met deze aandoening. Enkele kandidaatgenen binnen dit locus werden door middel van sequencing onderzocht, maar er kon geen mutatie worden aangetoond. Het onderliggende genetische defect is dan ook nog niet geïdentificeerd. Hoofdstuk 3 beschrijft de genetische analyse van een patiëntencohort met Ebstein anomalie. Door middel van next generation sequencing en Sanger sequencing vonden wij een pathogene mutatie in het MYH7-gen bij 8% van de onderzochte patiënten. Dit gen codeert voor het sarcomeereiwit beta-myosin heavy chain. Patiënten met een MYH7-mutatie en hun aangedane familieleden hadden noncompactie van de linker ventrikel en verschillende typen aangeboren hartafwijkingen, waaronder Ebstein anomalie en ventrikel septum defect. De penetrantie van noncompactie was hoog, terwijl er significante pleiotropie en verminderde penetrantie was wat

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betreft de aangeboren hartafwijkingen. Met deze studie hebben wij kunnen aantonen dat er een associatie bestaat tussen Ebstein anomalie, noncompactie van de linker ventrikel en mutaties in MYH7. Hoofdstuk 4 richt zich op een mogelijke rol van natriumkanalen in het ontstaan van aangeboren hartafwijkingen. Het SCNA-gen codeert voor de alfa-subunit van het cardiale natriumkanaal. Mutaties in SCN5A kunnen leiden tot verschillende erfelijke aritmiesyndromen, waaronder Brugada syndroom en Lange QT syndroom. In dierstudies is aangetoond dat het ontbreken van scn5a daarnaast leidt tot stoornissen in de ontwikkeling van het hart. Wij verkenden of er een verband is tussen mutaties in SCN5A en aangeboren hartafwijkingen bij mensen. In het eerste deel van dit onderzoek bepaalden wij de frequentie van aangeboren hartafwijkingen in een cohort van SCN5A loss-of-function mutatiedragers en in een cohort van SCN5A gain-of-function mutatiedragers. Van de loss-of-function mutatiedragers had 3,1% een aangeboren hartafwijking. Dit is een hoger percentage dan in de algemene bevolking. De aangeboren hartafwijkingen die wij zagen waren met name septumdefecten. Van de gain-of-function mutatiedragers had niemand een aangeboren hartafwijking. In het tweede deel van dit onderzoek verrichtten wij Sanger sequencing van SCN5A in een patiëntencohort met septumdefecten en geleidingsstoornissen. Hierbij werd een (mogelijk) pathogene mutatie aangetoond in 3,7% van de patiënten. De resultaten van deze studie suggereren dat SCN5A mutaties kunnen bijdragen aan aangeboren hartafwijkingen bij mensen, met name septumdefecten.

Deel II van dit proefschrift gaat over aangeboren hartafwijkingen geassocieerd met andere (extracardiale) afwijkingen, in zowel syndromale als niet-syndromale vorm. In Hoofdstuk 5 lieten wij zien dat het 22q11.2 deletie syndroom vaak niet herkend wordt bij volwassenen met tetralogie van Fallot of pulmonalisatresie met ventrikel septumdefect. Wij onderzochten hiertoe patiënten met deze hartaandoeningen die geregistreerd stonden in CONCOR, de landelijke registratie en DNA-bank voor volwassenen met aangeboren hartafwijkingen. Door middel van multiplex liga-tion probe amplificaton vonden wij een 22q11.2 deletie bij 6,5% van de tetralogie van Fallot patiënten en 16,5% van de pulmonalisatresie met ventrikel septumdefect patiënten. Meer dan de helft van de aangetoonde 22q11.2 deleties bleek nog niet bekend voordat deze studie werd gedaan. Deze patiënten waren dus niet gediagnosticeerd met dit syndroom. Omdat het 22q11.2 deletie syndroom belangrijke klinische en reproductieve consequenties kan hebben, zijn wij van mening dat een diagnostische test naar het 22q11.2 deletie syndroom overwogen moet worden bij alle patiënten met tetralogie van Fallot en pulmonalisatresie met ventrikelseptumdefect.Hoofdstuk 6 beschrijft een pathomorfologische studie van de bicuspide aortaklep (BAV) in foetussen met Turner syndroom (monosomie X). Wij bestudeerden 37 post-mortem hartpre-paraten van foetussen met Turner syndroom en zagen een abnormale aortaklep morfologie in de overgrote meerderheid. De aortaklep was bicuspide in 75% van de onderzochte harten. Wij observeerden verschillende BAV subtypen; zowel BAV met fusie van het rechter- en linker coro-naire klepblad (Type 1 BAV), als BAV met fusie van het rechter- en non-coronaire klepblad (Type 2 BAV) kwamen voor. In onze foetale populatie zagen wij relatief veel Type 2 BAVs in vergelijking

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met eerdere studies in volwassenen met Turner syndroom. Dit zou kunnen wijzen op een slech-tere prognose met een hogere kans op foetaal overlijden bij Type 2 BAV dan Type 1 BAV, mogelijk veroorzaakt door een associatie met bijkomende cardiovasculaire afwijkingen.In Hoofdstuk 7 wordt klinisch en functioneel onderzoek van de p.A119S variant in het NKX2-5 gen beschreven. Mutaties in NKX2-5 zijn een bekende oorzaak van aangeboren hartafwijkingen; meer recent werden mutaties in dit gen ook gerapporteerd bij patiënten met schildklierdysgenesie. De p.A119S variant was eerder beschreven als oorzaak van schildklierdysgenesie; wij vonden deze variant bij twee indexpatiënten met een aangeboren hartafwijking. Met onze studies konden wij aantonen dat p.A119S een zeldzame variant is, die zich gedraagt als wild type NKX2-5 en die geen oorzaak is van aangeboren hartafwijkingen of schildklierdysgenesie.Hoofdstuk 8 richt zich op de eerder gesuggereerde associatie tussen aangeboren hartafwijkingen en neuroblastoom. De beschikbare literatuur hierover is niet eenduidig. Wij onderzochten deze mogelijke associatie door het verrichten van een echocardiografische studie in een cohort van neuroblastoompatiënten. Wij vonden geen hogere prevalentie aangeboren hartafwijkingen in de neuroblastoompatiënten dan in de controlegroepen. Op basis van deze resultaten en de resul-taten van een systematische review van de literatuur, concludeerden wij dat er onvoldoende bewijs is voor een associatie tussen aangeboren hartafwijkingen en neuroblastoom.

Deel III beschrijft studies naar de rol van de klinisch geneticus en de visie van volwassenen met een aangeboren hartafwijking op genetica en erfelijkheid. In Hoofdstuk 9 worden resultaten van een vragenlijstonderzoek onder volwassen patiënten met een aangeboren hartafwijking beschreven. Een merendeel van de patiënten ontbrak het aan kennis over erfelijkheid en ruim 40% gaf aan behoefte te hebben aan meer informatie over erfelijkheidskwesties. Bijna de helft van de patiënten maakt zich zorgen over erfelijkheid van hun hartaandoening. Een gericht counselingprogramma zou deze onderwerpen bespreekbaar kunnen maken en hiermee de zorg voor volwassenen met een aangeboren hartafwijking kunnen verbeteren. Hoofdstuk 10 richt zich op volwassenen met een aangeboren hartafwijking die een klinisch geneticus hebben geconsulteerd. Wij deden een retrospectief statusonderzoek en lieten zien dat in deze patiëntengroep een etiologische diagnose anders dan de meest voorkomende ‘multifactoriële’ aangeboren hartafwijking gesteld kon worden bij ruim een kwart van de patiënten. De patiënten beoordeelden het bezoek aan de klinisch geneticus als positief. Deze laatste twee hoofdstukken demonstreren de toegevoegde waarde van de klinisch geneticus in de zorg voor volwassenen met een aangeboren hartafwijking.

Hoofdstuk 11 behandelt tot slot wetenschappelijke en klinische perspectieven op het gebied van genetica van aangeboren hartafwijkingen.

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Publicaties

Mutations in the cardiac sodium channel gene SCN5A and congenital heart disease in humans.van Engelen K, Postma AV, Beekman L, Hofman N, Alders M, Misuzawa Y, van der Werf C, Kolder I,

Baars MJ, Tan HL, Mulder BJ, Wilde AA, Bezzina CR

Submitted.

Bicuspid aortic valve morphology and associated cardiovascular abnormalities in fetal Turner syndrome: a pathomorphological study. van Engelen K, Bartelings MM, Gittenberger-de Groot AC, Baars MJ, Postma AV, Bijlsma EK,

Mulder BJ, Jongbloed MR.

Submitted.

A Mutation in the Kozak Sequence of GATA4 Hampers Translation and Associates with Atrial Septal Defect Type II. Mohan RA, van Engelen K, Stefanovic S, Barnett P, Ilgun A, Baars MJ, Bouma BJ, Mulder BJ,

Christoffels VM, Postma AV.

Submitted.

Association between C677T polymorphism of methylene tetrahydrofolate reductase and congenital heart disease: meta-analysis of 7,697 cases and 13,125 controls.Mamasoula C, Prentice RR, Pierscionek T, Hall D, Pangilinan F, Mills JL, Druschel C, Pass K, Russell MW,

Töpf A, Zelenika D, Bentham J, Cosgrove C, Bhattacharya S, Granados Riveron J, Setchfield K, Brook JD,

Bu’Lock FA, Thornborough C, O’Sullivan J, Stuart AG, Parsons J, Blue G, Winlaw D, Postma AV, Mulder

BJM, Zwinderman AH, van Engelen K, Moorman AFM, Rauch A, Gewillig M, Breckpot J, Devriendt K,

Rahman TJ, Palomino Doza J, Tan HL, Santibanez-Koref MF, Lathrop GM, Farrall M, Goodship JA, Cordell

HJ, Brody LC, Keavney BD.

Circ Cardiovasc Genet. 2013 Aug 1;6(4):347-53.

Ebstein anomaly associated with left ventricular noncompaction: an autosomal dominant condition that can be caused by mutations in MYH7. Vermeer AM, van Engelen K, Postma AV, Baars MJ, Christiaans I, de Haij S, Klaassen S, Mulder BJ,

Keavney B.

Am J Med Genet C Semin Med Genet. 2013 Aug;163C(3):178-84.

The value of the clinical geneticist caring for adults with congenital heart disease: diagnostic yield and patients’ perspective. van Engelen K, Baars MJ, Felix JP, Postma AV, Mulder BJ, Smets EM.

Am J Med Genet A 2013;161(7):1628-37.

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Genome-wide association study of multiple congenital heart disease phenotypes identifies a sus-ceptibility locus for atrial septal defect at chromosome 4p16. Cordell HJ, Bentham J, Topf A, Zelenika D, Heath S, Mamasoula C, Cosgrove C, Blue G, Granados

-Riveron J, Setchfield K, Thornborough C, Breckpot J, Soemedi R, Martin R, Rahman TJ, Hall D, van

Engelen K, Moorman AF, Zwinderman AH, Barnett P, Koopmann TT, Adriaens ME, Varro A, George AL

Jr, Dos Remedios C, Bishopric NH, Bezzina CR, O’Sullivan J, Gewillig M, Bu’lock FA, Winlaw D,

Bhattacharya S, Devriendt K, Brook JD, Mulder BJ, Mital S, Postma AV, Lathrop GM, Farrall M,

Goodship JA, Keavney BD.

Nat Genet. 2013 Jul;45(7):822-4.

Genome-wide association study identifies loci on 12q24 and 13q32 associated with tetralogy of Fallot. Cordell HJ, Töpf A, Mamasoula C, Postma AV, Bentham J, Zelenika D, Heath S, Blue G, Cosgrove C,

Granados Riveron J, Darlay R, Soemedi R, Wilson IJ, Ayers KL, Rahman TJ, Hall D, Mulder BJ, Zwinderman

AH, van Engelen K, Brook JD, Setchfield K, Bu’Lock FA, Thornborough C, O’Sullivan J, Stuart AG, Parsons

J, Bhattacharya S, Winlaw D, Mital S, Gewillig M, Breckpot J, Devriendt K, Moorman AF, Rauch A, Lathrop

GM, Keavney BD, Goodship JA.

Hum Mol Genet 2013;22(7):1473-81.

The ambiguous role of NKX2-5 mutations in thyroid dysgenesis.van Engelen K, Mommersteeg MT, Baars MJ, Lam J, Ilgun A, van Trotsenburg AS, Smets AM, Christoffels

VM, Mulder BJ, Postma AV.

PLoS One 2012;7(12):e52685.

Breakpoint mapping of 13 large parkin deletions/duplications reveals an exon 4 deletion and an exon 7 duplication as founder mutations. Elfferich P, Verleun-Mooijman MC, Maat-Kievit JA, van de Warrenburg BP, Abdo WF, Eshuis SA, Leenders KL,

Hovestadt A, Zijlmans JC, Stroy JP, van Swieten JC, Boon AJ, van Engelen K, Verschuuren-Bemelmans CC,

Lesnik-Oberstein SA, Tassorelli C, Lopiano L, Bonifati V, Dooijes D, van Minkelen R.

Neurogenetics 2011;12(4):263-71.

Adults with congenital heart disease: patients’ knowledge and concerns about inheritance. van Engelen K, Baars MJ, van Rongen LT, van der Velde ET, Mulder BJ, Smets EM.

Am J Med Genet A 2011;155(7):1661-7.

Screening for 22q11.2 microdeletion in adults with tetralogy of Fallot – Author’s Reply. van Engelen K, Baars MJ, Postma AV, Mulder BJ.

Heart 2011;97(10):860.

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Ebstein’s anomaly may be caused by mutations in the sarcomere protein gene MYH7.van Engelen K, Postma AV, van de Meerakker JB, Roos-Hesselink JW, Helderman-van den Enden AT,

Vliegen HW, Rahman T, Baars MJ, Sels JW, Bauer U, Pickardt T, Sperling SR, Moorman AF, Keavney B,

Goodship J, Klaassen S, Mulder BJ.

Neth Heart J. 2013 Mar;21(3):113-7.

A novel autosomal dominant condition consisting of congenital heart defects and low atrial rhythm maps to chromosome 9q. van de Meerakker JB*, van Engelen K*, Mathijssen IB, Lekanne dit Deprez RH, Lam J, Wilde AA, Baars MJ,

Mannens MM, Mulder BJ, Moorman AF, Postma AV.

Eur J Hum Genet 2011;19(7):820-6.

Mutations in the sarcomere gene MYH7 in Ebstein anomaly. Postma AV, van Engelen K, van de Meerakker J, Rahman T, Probst S, Baars MJ, Bauer U, Pickardt T,

Sperling SR, Berger F, Moorman AF, Mulder BJ, Thierfelder L, Keavney B, Goodship J, Klaassen S.

Circ Cardiovasc Genet 2011;4(1):43-50.

Connective tissue disorders and smooth muscle disorders in cardiology.van Engelen K, Mulder BJ.

Book chapter in: Clinical Cardiogenetics. Baars HF, van der Smagt JJ, Doevendans PAFM, editors.

London: Springer-Verlag; 2011;263-282.

Nonsense mutations in CABC1/ADCK3 cause progressive cerebellar ataxia and atrophy. Gerards M, van den Bosch B, Calis C, Schoonderwoerd K, van Engelen K, Tijssen M, de Coo R,

van der Kooi A, Smeets H.

Mitochondrion 2010;10(5):510-5.

Haploinsufficiency of TAB2 causes congenital heart defects in humans.Thienpont B, Zhang L, Postma AV, Breckpot J, Tranchevent LC, Van Loo P, Møllgård K,

Tommerup N, Bache I, Tümer Z, van Engelen K, Menten B, Mortier G, Waggoner D, Gewillig M, Moreau Y,

Devriendt K, Larsen LA.

Am J Hum Genet 2010;86(6):839-49.

22q11.2 deletion syndrome is under-recognised in adult patients with tetralogy of Fallot and pulmonary atresia.van Engelen K, Topf A, Keavney BD, Goodship JA, van der Velde ET, Baars MJ, Snijder S, Moorman AF,

Postma AV, Mulder BJ.

Heart 2010;96(8):621-4.

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Cardiovascular disorders among persons with Down syndrome. Vis JC, van Engelen K, Bouma BJ, Bilardo CM, Blom NA, Mulder BJM.

Int Rev Res Ment Ret 2010;39:165-94.

Risk stratification for sudden cardiac death in hypertrophic cardiomyopathy: systematic review of clinical risk markers. Christiaans I, van Engelen K, van Langen IM, Birnie E, Bonsel GJ, Elliott PM, Wilde AA.

Europace 2010;12(3):313-21.

Marfan syndrome masked by Down syndrome? Vis JC, van Engelen K, Timmermans J, Hamel BC, Mulder BJ.

Neth Heart J 2009 Sep;17(9):345-8.

Prevalence of congenital heart defects in neuroblastoma patients: a cohort study and systematic review of literature. van Engelen K, Merks JH, Lam J, Kremer LC, Backes M, Baars MJ, van der Pal HJ, Postma AV, Versteeg R,

Caron HN, Mulder BJ.

Eur J Pediatr 2009;168(9):1081-90.

Spontaneous baroreflex sensitivity in (pre)adolescents.Dietrich A, Riese H, van Roon AM, van Engelen K, Ormel J, Neeleman J, Rosmalen JG.

J Hypertens 2006;24(2):345-52.

* These authors contributed equally

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DankwoordMet veel plezier heb ik de afgelopen jaren de opleiding tot klinisch geneticus gecombineerd met promotieonderzoek op de afdeling cardiologie in het AMC. En nu is mijn proefschrift klaar! Bij mijn onderzoek en bij het schrijven van het proefschrift ben ik geholpen en gesteund door velen. Een aantal hiervan wil ik in het bijzonder bedanken.

Als eerste gaat mijn dank uit naar alle patiënten die hebben meegewerkt aan de verschillende onderzoeken.

Veel dank ben ik verschuldigd aan mijn promotor prof. Dr. B.J.M. Mulder en copromotoren dr. M.J.H. Baars en dr. A.V. Postma.

Beste Barbara, jouw gedrevenheid, enthousiasme en praktische instelling zijn een enorme stimulans. Wat heb ik veel van je geleerd! Kleine en grotere hobbels op de onderzoeksweg wist jij altijd weer glad te strijken. Je gaf mij de ruimte om het onderzoek op mijn eigen manier zodanig in te vullen dat een combinatie met mijn opleiding mogelijk was. En ook al wist ik bij de start van mijn promotie-traject nog niet hoe mijn proefschrift er uit zou komen te zien, jij hebt altijd het vertrouwen gehad dat het goed zou komen. Maar ook je gezelligheid tijdens werk en congressen typeren jou. Ik hoop nog veel mooie verhalen van je te horen!

Beste Marieke en Alex, dank jullie wel voor de goede en leuke begeleiding. Marieke, jouw ‘hoppetop’ instelling heeft mij erg geholpen bij het opzetten van de onderzoeken en het afronden van artikelen. Het was fijn om jou erbij te hebben vanuit de klinisch genetische hoek. Alex, bij jou kon - en kan- ik altijd binnenlopen voor overleg. Ik heb veel van je geleerd over de moleculaire kant van de genetica. We hebben nog een aantal projectjes lopen en ik weet zeker dat we ook daar iets moois van gaan maken!

De andere leden van mijn promotiecommissie: prof. dr. E.J. Meijers-Heijboer, prof. dr. I.M. Van Langen, prof. dr. A.H. Zwinderman, dr. E.M.A. Smets, dr. S. Klaassen, prof. dr. N.A. Blom en prof. dr. M.C. De Ruiter. Hartelijk dank voor het beoordelen van mijn proefschrift en de bereidheid zitting te nemen in mijn promotiecommissie. Met de meesten van jullie heb ik ook samengewerkt op het gebied van onderzoek of patiëntenzorg. Ik hoop dit in de toekomst nog vaker te kunnen doen.

Prof. dr. A.F.M. Moorman, Antoon, bedankt voor de mogelijkheid om onderzoek te doen binnen HeartRepair. Ik heb veel geleerd van de samenwerking met de andere Wp’s. HeartRepair colleagues from Berlin and Newcastle, in particular Judith Goodship, Bernard Keavney, Sabine Klaassen, Thomas Pickardt and Ulrike Bauer – thank you for the fruitful collaboration.

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Congenitale onderzoekers: jullie kwamen, promoveerden en gingen weer. Nu is het ook mijn beurt! Michiel, Mariëlle, Jeroen, Gijs, Paul, Zeliha, Piet, Mark, Teun, Annelieke, Romy, Alexander, Carla, Teodora, Dounya, Carianne en Maayke (en Ali en Saskia horen er ook een beetje bij): ondanks de frustraties die het doen van onderzoek kan opleveren en ondanks de ‘beperkte’ ruimte en het gebrek aan daglicht op onze kamers, ging ik altijd met plezier naar B2. Gewoon dankzij een geweldige groep collega’s. Ik hoop dat jullie (ook straks als cardioloog!) nog even aan mij en mijn telefoongesprekken terugdenken als een patiënt het even moeilijk heeft…

Ook alle andere onderzoekers van de afdeling Cardiologie wil ik bedanken voor heel veel gezelligheid!

Lia, Sylvia en Irene: zonder jullie geen CONCOR en dus geen onderzoek! Ik denk met plezier terug aan de koffiemomentjes. Ik kom snel weer een kopje drinken! En Enno, Maurice en Wybo, bedankt voor het uitvoeren van de vele query’s en analyses in de afgelopen jaren.

Anita, Regina en Lieve, bedankt voor jullie ondersteuning.

Al mijn collega’s van de afdeling Klinische Genetica in het AMC, assistenten, stafleden, genetisch consulenten, secretariaat en psychosociaal medewerkers: bedankt voor de leuke jaren tijdens mijn opleiding. Ik kan me geen leuker vak voorstellen dan klinisch geneticus en ik hoop dat ik ook na mijn opleiding met jullie kan blijven werken. Cora, bedankt voor de ruimte om de opleiding te combineren met onderzoek en voor jouw altijd aanwezige persoonlijke interesse. Inge, dank je voor het opstarten van het familieonderzoek en het verzamelen van veel onderzoekswaardige families.

De mensen van de echokamer, in het bijzonder Rianne, wil ik bedanken voor het maken van vele echo’s in het kader van mijn onderzoek.

Jan Lam, hartelijk dank voor de prettige samenwerking. Ik vind het bewonderenswaardig hoe jij je al je patiënten nog weet te herinneren, ook al zijn ze inmiddels volwassen geworden. Ook alle andere cardiologen die mij hebben geholpen bij het beoordelen van echo’s, ECG’s en andere zaken, bedankt.

Mijn studenten Lotte en Joyce, bedankt voor jullie hulp bij het statusonderzoek en het versturen, invoeren en analyseren van vele vragenlijsten. Ik ben benieuwd wat jullie straks gaan doen!

Alle medeauteurs wil ik bedanken voor hun grote en kleine maar altijd waardevolle bijdragen.

Mijn begeleiders en collega’s in Leiden: Margot, Monique en Rebecca. Ik heb het erg gezellig gevonden bij jullie. Margot, ik heb veel van je geleerd over het beoordelen van de hartpreparaten. En nergens werd zo goed voor mij gezorgd als bij jullie! Bedankt voor alle kopjes thee en biscuitjes.

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Eelco, dank voor je enthousiaste werk aan de lay-out van mijn proefschrift.

Fleur en Michiel, mijn paranimfen, erg fijn dat jullie naast mij staan op deze bijzondere dag! Fleur, we zitten een beetje in hetzelfde schuitje. Ik hoop dat we elkaar nog vaak zien als klinisch geneticus, waar dan ook in Nederland! Michiel, wat een leuke tijd heb ik met jou gehad. Een waterglijbaan zal nooit meer hetzelfde zijn.

Mijn vriendinnen, inmiddels door het hele land. De laatste tijd heb ik jullie veel te weinig gezien. Dat gaan we wat mij betreft snel inhalen (weekendje St. Tropez?).

Mijn schoonfamilie, Koen en Mieke, Rohan, Esther, Lucas en Louise, vanaf dag één voelde ik mij welkom bij jullie en dat is nooit veranderd. Dank voor de interesse, aandacht, steun en afleiding. Ik ben blij dat ik jullie ‘erbij’ gekregen heb.

Bart en Marcella, leuk om te zien dat mijn kleine broer ook al best groot is. We zien elkaar niet zo vaak, maar het is altijd goed. Ik ben blij dat jij mijn broer bent en dat jullie samen zijn.

Lieve ouders, Conny en Rini, ik ben dankbaar voor de onbezorgde jeugd die jullie mij gegeven hebben. Jullie hebben mij altijd mijn eigen keuzes laten maken en altijd achter mij gestaan. En ook al koos ik ervoor om geen Latijn in mijn vakkenpakket te nemen, ik ben toch nog redelijk terecht gekomen ;) Dank jullie wel voor alles.

Ylvo en Synne, jullie zijn het allerbelangrijkste. Synne, er is niets heerlijkers dan thuis te komen en een dikke kus van jou te krijgen. Wat geniet ik van jou! En Ylvo, wat fijn dat jij er bent. Dank je voor je liefde, steun, humor en relativeringsvermogen. Bij het maken van dit boekje, maar eigenlijk bij alles. Door jou weet ik dat het soms gewoon goed genoeg is. En waar het uiteindelijk om gaat. Hvjhv +1.

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Curriculum vitaeKlaartje van Engelen werd op 16 augustus 1978 geboren in Nijmegen. Na het VWO op de Nijmeegse Scholengemeenschap Groenewoud begon zij in 1996 met haar studie geneeskunde aan de Rijksuniversiteit Groningen. Ter verbreding van haar horizon onderbrak zij haar studie tweemaal; gedurende een halfjaar woonde en werkte zij in Israël op een kibboets en negen maanden reisde zij rond en deed zij vrijwilligerswerk in Midden-Amerika. Klaartje behaalde haar doctoraal examen in 2002 en haar artsendiploma (cum laude) in 2004. Aansluitend werkte zij gedurende acht maanden als arts-assistent op de afdeling kindergeneeskunde van het Medisch Centrum Leeuwarden. Tijdens haar werk op deze afdeling werd haar interesse voor de klinische genetica gewekt. Daarom startte zij, na een zeilreis van zeven maanden, in 2006 als arts-assistent op de afdeling klinische genetica van het Academisch Medisch Centrum in Amsterdam. In 2007 begon zij aan de opleiding tot klinisch geneticus. Zij combineerde deze opleiding met wetenschappelijk onderzoek op de afdeling cardiologie van het AMC, met dit proefschrift als resultaat. Momenteel doorloopt zij de laatste fase van haar specialisatie tot klinisch geneticus.Klaartje woont samen met haar man en dochter in Amsterdam.

Stellingen behorend bij het proefschrift:

Genetics and inheritance issues in congenital heart disease

Het 22q11.2 deletie syndroom wordt onvoldoende onderkend in volwassenen met tetralogie van Fallot en pulmonalisatresie met ventrikelseptumdefect. (dit proefschrift)

Ebstein anomalie met noncompactie cardiomyopathie kan worden veroorzaakt door mu-taties in MYH7. (dit proefschrift)

Er is onvoldoende bewijs dat mutaties in NKX2-5 een rol spelen bij het ontstaan van schildklier dysgenesie. (dit proefschrift)

Ook mensen met geleidingsstoornissen in het kader van een septumdefect kunnen drag-er zijn van een mutatie in SCN5A. (dit proefschrift)

Veel volwassenen met een aangeboren hartafwijking zijn niet op de hoogte van het herhalingsrisico op een aangeboren hartafwijking bij hun toekomstige kinderen. (dit proefschrift)

Erfelijkheid zit niet altijd ‘in de genen’.

Het menselijk genoom: kennis van de zaak, einde van het vermaak?

0,5 fte + 0,5 fte = 1,0 fte gaat niet op voor een arts-onderzoeker.

Men kan enkel goed zien met het hart. Het wezenlijke is onzichtbaar voor de ogen. (An-toine de Saint-Exupéry, De Kleine Prins)

Klaar is klaar.

Klaartje van Engelen,29 november 2013