Search for Rare Copy-Number Variants in Congenital Heart...

DOI: 10.1161/CIRCGENETICS.115.001213

1

Search for Rare Copy-Number Variants in Congenital Heart Defects

Identifies Novel Candidate Genes and a Potential Role for FOXC1 in Patients

with Coarctation of the Aorta

Running title: Sanchez-Castro et al.; Copy-number variations in congenital heart defects

Marta Sanchez-Castro, PhD1-3; Hadja Eldjouzi, AS1-3; Eric Charpentier, MS1-3; Pierre-François

Busson, MS1-3; Quentin Hauet, MD1,4; Pierre Lindenbaum, PhD1-3,5; Béatrice Delasalle-

Guyomarch, MS1-3,5; Adrien Baudry, MS1-3; Olivier Pichon, MS6; Cécile Pascal, MD7; Bruno

Lefort, MD, PhD8; Fanny Bajolle, MD, PhD9,10; Philippe Pezard, MD4†; Jean-Jacques Schott,

PhD1-3,5; Christian Dina, PhD1-3,5; Richard Redon, PhD1-3,5; Véronique Gournay, MD, PhD11;

Damien Bonnet, MD, PhD9,10; Cédric Le Caignec, MD, PhD1-3,6

1INSERM, UMR1087, l’institut du thorax; 2Université de Nantes ; 3CNRS, UMR 6291, Nantes; 4CHU Angers, Service de pédiatrie, Angers; 5CHU Nantes, l’institut du thorax, Service de Cardiologie; 6CHU Nantes, Service de Génétique

Médicale; 7Centre d’échographie de l’ile Gloriette, Nantes; 8CHU de Tours, unité de cardiologie pédiatrique, service de médecine Pédiatrique, Tours; 9Université Paris Descartes, Sorbonne Paris Cité; 10Centre de Référence Malformations Cardiaques Congénitales Complexes-M3C, Hôpital Universitaire Necker-Enfants Malades, AP-HP, Université Paris

Descartes, Paris; 11CHU Nantes, Service de Cardiologie Pédiatrique, Nantes, France†deceased

Correspondence:

Prof. Cédric Le Caignec, MD, PhD

Service de Génétique Médicale

CHU, 9, quai Moncousu

44093 Nantes

France

Tel: +33 240084284

Fax: +33 240083943

E-mail: [email protected]

Journal Subject Terms: Genetics, Developmental biology, Pathophysiology

PhD1-3,5; Christian Dina, PhD1-3,5; Richard Redon, PhD1-3,5; Véronique Gournaaay,y,y, MMMD,D,D, PPPhDhDhD111111;

Damien Bonnet, MD, PhD9,10; Cédric Le Caignec, MD, PhD1-3,6

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Abstract:

Background - Congenital heart defects (CHD) are the most frequent malformations among

newborns and a frequent cause of morbidity and mortality. Although genetic variation

contributes to CHD, their precise molecular bases remain unknown in the majority of patients.

Methods and Results - We analyzed, by high-resolution array comparative genomic

hybridization (aCGH), 316 children with sporadic, non-syndromic CHD, including 76

coarctation of the aorta (CoA), 159 transposition of the great arteries (TGA) and 81 tetralogy of

Fallot (ToF), as well as their unaffected parents. We identified by aCGH, and validated by

quantitative real-time PCR, 71 rare de novo (n=8) or inherited (n=63) copy-number variants

(CNVs) (50 duplications; 21 deletions) in patients. We identified 113 candidate genes for CHD

within these CNVs, including BTRC, CHRNB3, CSRP2BP, ERBB2, ERMARD, GLIS3, PLN,

PTPRJ, RLN3 and TCTE3. No de novo CNVs were identified in patients with TGA in contrast to

CoA and ToF (p=0.002; Fisher’s exact test). A search for transcription factor binding-sites

showed that 93% of the rare CNVs identified in patients with CoA contained at least one gene

with FOXC1 binding-sites. This significant enrichment (p<0.0001; permutation test) was not

observed for the CNVs identified in patients with TGA and ToF. We hypothesize that these

CNVs may alter the expression of genes regulated by FOXC1. Foxc1 belongs to the forkhead

transcription factors family, which plays a critical role in cardiovascular development in mice.

Conclusions - These data suggest that deregulation of FOXC1 or its downstream genes play a

major role in the pathogenesis of CoA in humans.

Key words: congenital heart disease; copy number variant; FOXC1, array CGH

, g , , , , , , ,

PTPRJ,JJ RLN3 and TCTE3. No de novo CNVs were identified in d patients with TGGGA A A ininn cccononontrtrtraasa t t to

CoA and ToF (p=0.002; Fisher’s exact test). A search for transcription factor bindiing-sites

hhowowowededed thahahatt t 9993% %% ooof the rare CNVs identified in paaatiiients with CoA A A conttaiaiainnned at least one gene

wwwithhh FOXC1 bibiinndndiing-g-g sisitetetesss.. ThThThisisis sigigigninin fifificaaant eeennnrichmhmhmennt t t (p(p(p<0<0<0.0.0.000001;; permrmrmuuutattioioionn n teeststst))) wawawas nononottt

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ranscription factorsss fafafamimimilylyly,, whwhwhicicichh h plplplayayayss aaa crcrcritititiciccalala rororolelele iiin cacacardrdrdioioiovavavassscucuculalalarrr dededevevevellolopment in mice.

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Introduction

Congenital heart defects (CHD) are the most common congenital malformations with an

incidence of 0.5-1% of live births 1. They also are the first cause of mortality during the first year

of life of newborns in developed countries 2. Despite therapeutic advances, CHD are associated

with a high proportion of long term morbidity. Among CHD, a large subset involves the outflow

tract. This heterogeneous group of malformations represents 20-30% of the CHD diagnosed in

newborns 3. Transposition of the great arteries (TGA) accounts for 5-7% of all CHD 4 and is one

of the most common cyanotic disorder diagnosed in the neonatal period with a prevalence of 0.2

per 1,000 live births. The most common form of TGA is the dextro-looped type, which consists

in a discordant ventriculo-arterial connection implying that the aorta incorrectly arises from the

right ventricle in an anterior and right-sided position, whereas the pulmonary artery incorrectly

arises from the left ventricle in a posterior and left-sided position. By contrast to the normal heart

in which both outflow tracts and great vessels show a dextral (right-handed) spiralization, the

great vessels in TGA present with a parallel course lacking normal spiralization. Coarctation of

the aorta (CoA) is an outflow tract defect by which the aorta narrows in the area where the

ductus arteriosus inserts. This is a relatively common defect that accounts for around 7% of all

CHD 5. Tetralogy of Fallot (ToF) is defined by a combination of malpositioned aorta that

overrides both ventricles, ventricular septal defect, pulmonary stenosis obstructing the blood

flow into the lungs and right ventricular hypertrophy. ToF is the most common cyanotic

congenital cardiac disease in humans with an occurrence of one per 3,000 live births and

accounts for 10% of all CHD 6.

Although most of the patients undergo successful surgery in developed countries, the risk

of cardiac malformation in their offspring is significantly higher than in the general population 7

n a discordant ventriculo-arterial connection implying that the aorta incorrectly aaariririsess sss frfrfromomom ttthhhe

ight veventntricllee in n ana anterior and right-sided positioonnd , whereas the pulmononary artery incorrectly

ararrisseees from thhhe leleleftftft vvenenentrtrtriciciclelele innn aaa popopoststs erererioioior ananandd d leftt-ssideddd ppposososititi ioonn.n. ddd ByByBy cocoontntntrastt tttooo thththee e nooormrmrmalalal hhheaeae rt

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great vessels in TGAAA prprp ese ent wiwiwitht a pap rallel courssse ee lackinng g g nonn rmal spipipirararalill zation. Coarctation of

hhe e aoaortrtaa (C(C( oAoA))) isis aan n ououtftflolow w trtracact t dedefefectct bybyy wwhihichch tthehe aoaortrtaa nanarrrrowowss inin tthehe aarerea a whwherere e ththe e



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suggesting genetic defects. Despite the high incidence of CHD, the etiology of these

malformations remains largely unknown. About 20% of CHD can be attributed to known causes

such as chromosomal abnormalities, single gene disorders or exposure to teratogens while no

etiology is identified in about 80% of the patients 8. A multifactorial origin associating

environmental and genetic factors seems to be the usual mode of inheritance 2,8. The

identification of new genes involved in non-syndromic forms of CHD would help to better

understand the molecular mechanisms leading to these malformations and to improve genetic

counseling and disease prevention for couples having an affected child as well as adult patients

willing to reproduce.

Array comparative genomic hybridization (aCGH) is a method allowing to detect copy-

number variations (CNVs) (i.e. deletions and duplications) at a genome-wide level. The study of

sporadic patients with non-syndromic CHD by aCGH is an alternative to classic family studies

for the identification of new genes implicated in these pathologies. A few studies have evidenced

rare CNVs in patients with non-syndromic CHD using this method 9–16.

Here we report a study performed in 316 children with non-syndromic CHD and their normal

parents. Our data show a high contribution of rare inherited but also de novo CNVs to human

CHD and suggest a major role of FOXC1 in the pathogenesis of CoA.

Materials and Methods

Patients

Informed consent for genetic analyses was obtained from all individuals participating in the

study. The protocol was approved by the ethics committee of the University Hospital of Nantes

(BRD 09/3A). Children from 468 families (85 with CoA, 291 with TGA and 92 with ToF)

presenting with a sporadic and non-syndromic outflow tract (OFT) defect were referred to the

Array comparative genomic hybridization (aCGH) is a method allowing ttoto dddetettececect t t cococoppyp -

numberer vav riatationsns (CNVs) (i.e. deletions and dupplilicac tions) at a genome-wiw de level. The study of

ppporrradic patiienenentststs wiww tth h h nononon-n-n-syss ndndndrororomimim c c CHCHCHDDD bybyby aCGGGH isiss ananan alalalternrnrnatttivivivee tototo ccclassssicicic fffamamamily y y stststudududieieiesss

fofoor r thththe identificaaatioon ooof nnew gegegenen s immmplllicccateddd iiin thhhesse papapathtthooolooogiess. A fewwwff stuuudidiies haaavevee eeeviviided nnnceeed

are CNVs in patientntntsss wiww th nooon-n-n-sysyndromic CHD usuu ingg thisiss memem tht od 9–16. .

HeHerere wwee rerepopop rtrt aa sstutudydyy pppererfoformrmeded iin n 31316 6 chchilildrdrenen wwitith h nonon-n sysyyndndroromimic c CHCHDD anand d ththeieir r nonormrmalal



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University Hospitals of Nantes, Tours, Angers and Necker Enfants Malades in Paris. In all

included families except one (#417, two siblings with TGA), a single child was affected. A male-

to-female sex ratio bias of 2.2:1 was observed in our series of patients with TGA, which is

similar to that previously published in the literature 17. Both parents were available for 316

families (i.e. 76 with CoA, 159 with TGA and 81 with ToF) which were retained for the study.

Since only one parent was available for the remaining 152 families, the latter were not studied.

Patients with extra-cardiac features, such as learning disability, brain, craniofacial or renal

anomalies, or carrying a clinically recognizable microdeletion/microduplication syndrome or a

monogenic disorder had been excluded from the cohort. The patients for whom one of the

parents or another relative was known to present a CHD were also excluded from the study.

None of the included parents were symptomatic for any heart disease or underwent cardiac

surgery. If a parent reported a symptom that could suggest any CHD, echocardiography was

performed to exclude a minor anomaly.

DNA extraction

DNA from all probands and their normal parents was extracted from whole peripheral blood

using NucleoSpin® Blood XL (Macherey Nagel), illustraTM DNA Extraction Kit BACC2 (GE

Healthcare) or UltraPure™ Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v) (Life

TechnologiesTM) according to manufacturers’ instructions.

Array comparative genomic hybridization (aCGH) analysis

aCGH experiments were performed on 316 family trios using 2*400K Agilent custom-designed

arrays (024825_D_F_20090731). According to our ethical rules and in order to minimize the

detection of unsolicited findings (i.e. detection of genomic imbalances unrelated to CHD), ~800

OMIM genes responsible for X-linked, autosomal dominant or recessive genetic disorders were

parents or another relative was known to present a CHD were also excluded frommm tttheheh sstututudydydy..

None oof f tht e ininclududed parents were symptomatic foor r any heart disease or unu derwent cardiac

uuurgggery. If a papaparererentnn rrepepeporororteteted d d a sysysympmpmptototom m m thtt atatat cccouoo ldd suuuggegegeststst aaanynn CHCHCHD,D,D, eechchchocoo arrdididiogogograraraphphp y y y wawawasss

pepeerffforoo med to excccluuude aa mmminooor anana omalala yy.

DNA extraction

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excluded from the design of the array (list of genes available upon request). Microarrays

contained 300,000 probes located exclusively in exonic sequences with at least one probe in each

exon of ~19,000 genes. In addition, 100,000 probes covered with high-density 297 candidate

genes known or suspected to play a role in heart development in humans or animal models

(Supplementary Table 1). These probes were located in the exonic and intronic sequences, 10 kb

upstream and downstream of the coding regions. Digestion, labelling and hybridization were

performed according to the protocols provided by Agilent. Children’s DNA were hybridized

twice, once with that of the father and once with that of the mother (Figure 1). The arrays were

analyzed with the Agilent scanner and the Feature Extraction software (v.9.1.3). Graphical

overview was obtained using the customized SigFrame software

(https://github.com/lindenb/jvarkit/wiki/SigFrame). All genomic coordinates were based on the

February 2009 assembly of the reference genome (GRCh37/hg19).

CNVs detection

Rare CNVs were selected according to the following criteria: 1) CNVs containing at least part of

an exon; 2) CNVs absent or with a frequency lower than 1% from the CNV consensus reference

set version 2.1 (42 million probes study 18, WTCCC study (http://www.wtccc.org.uk/), 1000

genomes project (http://www.1000genomes.org/) and DDD controls project

(http://www.ddduk.org/), integrating a number of high-quality copy-number variants studies; 3)

CNVs present less than 4 times in the Database of Genomic Variants

(http://dgv.tcag.ca/dgv/app/home) 19.

Quantitative real-time PCR (qPCR) validation

The CNVs fulfilling selection criteria were subjected to validation by qPCR. At least one pair of

primers was designed in each selected CNV (Supplementary Table 2). All qPCR reactions were

overview was obtained using the customized SigFrame software

httpps:////gigig thubub.comom/lindenb/jvarkit/wiki/SigFramee))).. All genomic coordinnata es were based on the

FeFeFebrbrbruary 20000999 asasassess mbmbmblylyly ooof f f tht e e e rererefefefererer ncncnce ee geeenononomem (((GGGRChChCh373737/h/h/hg1199)9)..

CNCNCNVVVs detectionnn

Rare CNVs were selellecececteted accococordrdrding g to the followiwiwingng criteeririria:aa 1) ) CNVsVss ccconoo taining at least part of

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®Premix Ex TaqTM (Takara Bio Inc., Shiga, Japan). qPCR conditions

comprised an initial denaturation at 95°C for 4 min, followed by 40 cycles at 95°C for 15 sec,

60°C for 10 sec and 72°C for 10 sec. qPCR reactions were carried out in a LightCycler®480

System (Roche Diagnostics GmbH, Roche Applied Science, Mannheim, Germany).

Amplification products were analyzed using LightCycler®480 software version 1.5.0 (Roche

Diagnostics GmbH, Roche Applied Science, Mannheim, Germany). The ,

as previously described, the

ALB and TNNI3K genes for normalization. The validated CNVs were uploaded in the LOVD

v.3.0 Leiden Open Variation Database (http://www.lovd.nl/) (Supplementary data: Accession

numbers).

Transcription Factor Binding Sites (TFBS) enrichment analyses

Sixty-nine out of 71 qPCR-validated CNVs identified in affected children were used for TFBS

enrichment analyses. Two validated CNVs (7.9 Mb and 14.5 Mb) were removed from the

analyses because they could induce bias due to their large size. To identify the predicted

transcription factors binding the genes included in the 69 CNVs present in the 316 patients and in

each subgroup of CHD (i.e. CoA, TGA and ToF), we used the HMR Conserved TFBS track of

UCSC which contains the location and score of TFBS conserved in the human/mouse/rat

alignment 20. A TFBS was retained when it was partially or entirely included in one of the 69

rare CNVs. The score and threshold were computed with the Transfac Matrix Database (v7.0)

created by Biobase (http://www.biobase-international.com/). Over-representation of the TFBS in

the observed data (i.e. 69 rare CNVs in the patients) was assessed through random permutations.

We simulated 10,000 datasets with 69 chromosomal regions randomly picked on the genome (a

v.3.0 Leiden Open Variation Database (http://www.lovd.nl/) (Supplementary dattta:a: AAAccccccesesessisisiononon

numberers)s)).

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chromosomal region had a chance to be picked as location for a CNV proportional to its length).

This assignation was repeated until the CNV was positioned in a region spanning at least one

gene and where no polymorphic CNV exists. This procedure allowed mimicking the original

experiment and provided a distribution of any enrichment statistics which was really tailored for

this experiment. Therefore, random regions were equivalent in size to the 69 CNVs, and 90% of

their length had to overlap real rare CNVs. For each permutation, the number of random regions

overlapping at least one binding site for the selected transcription factor was recorded, and the p-

value was computed as the number of permutations where this number exceeded the number

observed in the real CNVs, divided by the total number of permutations. We used the rare

validated CNVs (n=78) present in unaffected parents but absent in affected children as a negative

control group.

Results

Rare CNVs detected in affected children

The 316 family trios (affected children and both unaffected parents) retained and analyzed by

aCGH led to the identification of 152 rare CNVs that fulfilled selection criteria. They were 38 in

trios with CoA, 72 in trios with TGA and 42 in trios with ToF. qPCR analysis performed in the

children and their parents showed that 71 CNVs (50 duplications and 21 deletions) were present

in affected children (15 in CoA, 29 in TGA and 27 in ToF) (Supplementary Table 3). Moreover,

78 CNVs were present in one of the parents (Supplementary Table 4) and 3 CNVs were aCGH

false-positive results. The size of the CNVs ranged between 2.9 kb and 1.8 Mb except for two

large CNVs of 7.9 Mb and 14.5 Mb respectively. After exclusion of frequent CNVs, at least one

of the 71 rare CNVs potentially related to CHD was retrieved in 65 (20.6%) patients. Six patients

carried two rare CNVs. Among the 71 CNVs identified, 63 (88.7%) were inherited from one of

validated CNVs (n=78) present in unaffected parents but absent in affected childdrerer n nn asass aaa nnnegegegatative

control l grgrg oup.pp

RReR ssusults

Raree CCCNVNVNVs deettete tctctededd iiin n afa fectttededed childrereennn

The 316 family triososs (((afafaffefefecctctededed ccchihihildldldrereren nn ananand dd boboboththth uuunananaffffffeeecteeeddd papaparererentntnts)s)s) rrretetetaiaiainenened dd aanand analyzed by



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the parents and 8 (11.3%) were de novo CNVs, not observed in either parent. Three de novo

CNVs (4.1%; 1 duplication and 2 deletions) were observed in 76 patients with CoA and 5 (6.6%;

2 duplications and 3 deletions) in 81 patients with ToF (Table 1). No de novo CNV was

identified in the 159 patients with TGA. Fisher’s exact test showed a highly significant

difference (p=0.0028) in the proportion of de novo CNVs across the three groups of CHD [0 out

of 159 (group of TGA) vs 3 out of 76 (group of CoA) vs 5 out of 81 (group of ToF)].

Comparison between the group of TGA and the groups of CoA and ToF together (0 out of 159 vs

8 out of 157) also led to a significant difference (p=0.0034) (Table 1). Duplications (70.5%) were

more frequent than deletions (29.5%). Among the inherited CNVs, 30 CNVs were inherited from

the father, 32 from the mother, and 1 from both parents, showing no parental bias of transmission

(Supplementary Table 3). No CNV responsible for a known syndromic disorder, such as the

22q11.2 microdeletion syndrome, was identified.

Genomic distribution of the CNVs

The 71 rare CNVs appeared to be distributed all over the genome (Supplementary Figure 1 and

Supplementary Table 3). No recurrent CNVs were identified but partially overlapping CNVs

were observed for three genomic regions, respectively at 10q24.32, 11p11.2 and 20p11.23. An

overlapping duplication at 10q24.32 was identified in two unrelated children with TGA (patients

#351 and #222). In both these cases, the CNVs were inherited and included a portion of the

BTRC gene (Figure 2A,B). The second overlapping region at 11p11.2 was observed in two

unrelated children presenting with two different forms of CHD. One patient presented with CoA

(patient #174) and carried a paternally inherited duplication including the entire coding sequence

of the PTPRJ gene. The other patient presented with ToF (patient #153) and carried a paternally

inherited deletion including a portion of the PTPRJ gene (Figure 2C,D). The third overlapping

he father, 32 from the mother, and 1 from both parents, showing no parental biaasss ofofof ttrararansnsnsmimimisss iion

Suppppplelemem ntara y y TaTable 3). No CNV responsible for r a a known syndromic didisorder, such as the

22222qq1q11.2 micrrrodododeleleletee iooonn n sysysyndndndromememe, , wawaw s ss idididennntititifififiedee .

GGeGennonomic distribububutionnn ooof theee CNCC Vsss

The 71 rare CNVs aaappppppeaeared totoo bbbe distributed all ovovovere the gggenenenomo e (S( upupupplplpleme entary Figure 1 and

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region at 20p11.23 was detected in two unrelated children presenting with two different forms of

CHD. One patient presented with ToF (patient #42) and carried a de novo CNV duplication. The

other patient presented with TGA (patient #341) and carried a maternally inherited duplication.

Both CNVs included the ZNF133, POLR3F, RBBP9, and DZANK1 entire gene coding sequences

(Figure 2E,F).

In one family (Figure 3A), we identified a homozygous deletion in the affected child. The

deletion was inherited from both consanguineous parents, each being heterozygous, and

contained a portion of the CHRNB3 gene (Figure 3B). In another family (Figure 3C), we

identified a rare duplication in two siblings. The duplication was inherited from a phenotypically

normal mother and contained a portion of the TCTE3 gene and the ERMARD gene (Figure 3D).

Finally, we identified a 1q21 duplication in a patient with ToF (patient #43). The

duplication was inherited from a phenotypically normal mother.

Transcription Factor Binding Sites (TFBS) enrichment for FOXC1

The list of predicted TFBS was downloaded from UCSC

(http://hgdownload.soe.ucsc.edu/goldenPath/hg19/database/tfbsConsSites.txt.gz). An

overrepresentation of FOXC1 binding sites (p<0.0001), estimated through the permutation

procedure described in the Methods section, was observed for 54 of the 69 CNVs retained for

TFBS searches (Supplementary Table 5). Fifty-four out of 69 CNVs (77%) contained at least one

FOXC1 binding site. Considering each group of CHD, a significant enrichment of FOXC1

binding sites was only observed for the CNVs identified in the group of patients with CoA and

not in the groups of patients with TGA or ToF (at least one FOXC1 binding site was identified in

14/15 CoA CNVs (93%; p<0.0001) versus 20/29 (69%; p=0.023) in TGA and 19/25 (76%;

p=0.057) in ToF).

normal mother and contained a portion of the TCTE3 gene and the ERMARD gennene (((FiFiFigggururureee 333DD)D).

FiFinallly,y wwe e identified a 1q21 duplication in n a a patient with ToF (papap tient #43). The

dududuplplplication waww s s s inininheeriririteteted d d frfrfromomm aaa ppphehehenononotytytypiiicacacalllllly nooorrmrmalall mmmototothehh r..

TrTrTranananscription FaFaFactorrr BBBindiiingngng Siteseses (TFTFTFBSSS) enriiichmememenntnt fffor FOXOXOXC1

The list of predicted d d TFTFTFBS wwasasas ddowo nloao ded frommm UUCSC

hhttttp:p:p ////hghggdodownwnloloadad.s.soeoe.u.ucscsc.c.ededu/u/gogog ldldenenPaPathth/h/hg1g1g 9/9/dadatatababasese//tftfbsbsCoConsnsSiSitetes.s.txtxt.t.gzgzg ).).) AAn n



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By contrast, the rare validated CNVs (n=78) present in unaffected parents but absent in

affected children used as a negative control group did not show any significant enrichment in

FOXC1 binding sites (p=0.294), comforting the specificity of FOXC1 overrepresentation in

CHD.

Discussion

This study reports a family trio-based study performed to identify rare CNVs in patients with

sporadic, non-syndromic cardiac outflow tract defects of three different types, respectively CoA,

TGA and ToF. The family trio design allowed us to identify 8 (11.3%) de novo rare CNVs and

63 (88.7%) inherited ones. De novo CNVs were significantly more frequent in patients with CoA

(4.1%) and ToF (6.6%) compared to patients with TGA (no CNV in 159 patients) (p=0.002).

This difference indicates that novel genetic events are less frequent in the pathogenesis of TGA

than in that of ToF and CoA. Thus, TGA could result from a genetic predisposition related to a

number of low-impact, mostly inherited, variants associated to environmental factors. The

frequency of de novo CNVs identified in patients with ToF (6.6%) is broadly similar to

previously reported frequencies 11,21 considering the differences in the arrays and analysis

pipelines between the studies. For example, Greenway et al. 11 reported 10% of de novo CNVs in

their ToF patients’ cohort which is slightly more than what we observed. This slight difference

might also be due to the fact that some of the de novo CNVs described by Greenway et al. 11 are

usually considered as syndromic such as the 22q11.2 microdeletion syndrome. Our stringent

clinical selection criteria may explain the absence of detection of such genomic disorders in our

cohort.

The proportion of inherited CNVs that we detected was similar between the three types of

CHD. The fact that a majority of the rare CNVs were inherited from a phenotypically healthy

63 (88.7%) inherited ones. d De novo CNVs were significantly more frequent in paaatititienenentststs wwwititith h h CoCoC A

4.1%) and ToF (6.6%) compared to patients with TGA (no CNV in 159 patients) (p( =0.002).

Thhhisiss differenennccce indicates that novel genetic events arrre less frequenennt t t in the pathogegg nesis of TGA

hhhannn in that of ToooFFF anddd CCCoA... TTThus, TGGGAAA couuulddd resususult froroom m m aa a gggeneeetiiic preeediiisppoosssition rereelal ttteddd to d a

numbmbbererr ooofff lllow-iiimpapactctt, momostlyly iiinhnhnheriteddd, vvavaririiana tss aassssocociaiaiateteted d tototo envvviririrononmeme tntntal fffacacctototors. ThThThe e

frequency of de novvooo CNCNCNVsVsVs iiidededentntntifififieieieddd ininin papapatititienenentststs wwwititithhh TooFFF (6(6(6 66.6%)%)%) isisis brbrbroaoaoadldldlyyy sssimilar to

1111 2211



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parent suggests that they contribute to the CHD but are not sufficient by themselves to cause the

disease. Such variable expressivity and incomplete penetrance are again observed in such

genomic disorders as the 22q11.2 microdeletion syndrome that can be inherited from a “healthy”

parent.

Point mutations or CNVs involving binding sites located in regulatory regions of genes

may cause developmental defects, such as SHH and polydactyly or PAX6 and aniridia 22.

Regarding CHD, Smemo et al. published an elegant study showing that regulatory variation in a

TBX5 enhancer leads to isolated congenital heart disease 23. We also previously showed that

deletions upstream of SOX9 containing regulatory elements are likely responsible for isolated

congenital heart defects 24. We performed here a computational approach to search for an

enrichment of binding-sites of transcription factor genes within our rare CNVs dataset that might

have altered the expression of genes and thus contributed to the CHD. Our TFBS approach led us

to identify a significant enrichment of FOXC1 binding sites in the rare CNVs present in affected

children (Supplementary Table 5). The strongest enrichment was observed for CoA (p<0.0001)

compared to TGA (p=0.023) and ToF (p=0.057). No enrichment in FOXC1 binding sites was

observed in the rare CNVs identified in unaffected parents and it was absent in children. FOXC1

belongs to the forkhead family of transcription factors and plays an essential role in the

regulation of embryonic development in different model organisms 25. It is notably involved in

cardiovascular development and in particular in the morphogenesis of the cardiac outflow tract

26–28. Human FOXC1 heterozygous mutations are responsible for the Axenfeld-Rieger syndrome,

a developmental disorder affecting structures in the anterior segment of the eye. Mutations in

FOXC1 have been identified in a few patients presenting CHD in addition to Axenfeld-Rieger

syndrome 29,30. Interestingly, a de novo deletion of ~45 kb including FOXC1 has been reported in

congenital heart defects 2424. We performed here a computational approach to searccch hh fofof rrr ananan

enrichmement oof f bindnding-sites of transcription factorr gggene es within our rare CCNVs dataset that might

hhhavavaveee altered thththe e e exexexprresesessisis ononon oof f gegegenenenesss anananddd thhususus cccontrrribbbutededed ttto o o ththt e CHCHCHD.DD OuOuOurrr TFBFBBSSS apapapprpp oaoaoachchch lllededed usr

ooo iddedentify a signnniffficannnt enriccchmhmment ofofo FFFOOOXCC1C1 binndiiinggg sssititites innn theee y rrrare CCCNVNVNVsss pppresennnt t inin affffectttedd

children (Supplemeentntntarara y yy Tablblleee 555).). The strongegg st eeenrnrichmenenent t t waw s obseseervrvrvedee for CoA (p<0.0001)

cocompmpparareded toto TGTGA A (p(p(p=0=0.0.02323) ) ) anand d ToToFF (p(p(p=0=0.0.05757).).) NoNo enenririchchmementnt iin n FOFOXCXC11 bibindndining g g sisitetess wawass



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a patient with atrial septal defect in addition to bilateral congenital glaucoma, partial aniridia and

club feet 9. A recent study 31 has reported mutations in FOXC1 that affect gene transactivation in

patients presenting with non-syndromic TOF. Taken together, these previously published data

and our results strongly suggest that a dysregulation of FOXC1 or its downstream regulated

genes may contribute to the pathogenesis of CHD and in particular CoA. Nevertheless, further

experimental analyses of putative sites need to be performed to strength this conclusion.

We compared our data to CNVs from patients with CHD downloaded from DECIPHER

(https://decipher.sanger.ac.uk/) and ISCA (https://www.iscaconsortium.org/) public databases

and from the existing literature. After exclusion of the patients with the largest CNVs, for whom

genotype-phenotype correlations were not consistent, we identified seven patients from the

public databases and the literature carrying a CNV partially overlapping with a CNV identified

in four patients of our cohort (Table 2). (i) Three patients from the public databases presenting

with patent ductus arteriosus (PDA) or CoA (DECIPHER 1578, ISCA nssv706487, ISCA

nssv706596) carried deletions of variable sizes including GLIS3. These deletions partially

overlapped with a duplication identified in one of our patients (#437). GLIS3 plays a role in the

regulation of a variety of cellular processes during development 32 such as cell migration. (ii)

Two duplications, one identified in a patient with TGA (DECIPHER 250627) and one in a

patient with ToF from the literature 15,overlapped with the duplication identified in one of our

TGA patients (#172). All three duplications included the entire PLN coding sequence gene. PLN

is a membrane protein that regulates the Ca2+ pump in cardiac and skeletal muscle cells.

Mutations in this gene cause dilated cardiomyopathy or arrhythmogenic right ventricular

cardiomyopathy 33. (iii) A deletion present in a patient with dextrocardia (ISCA nssv580437)

overlapped with a duplication identified in one of our TGA patients (patient 335). Both genomic

genotype-phenotype correlations were not consistent, we identified seven patienttsts fffrorr m m m thththe e e

public ddata abasases aandn the literature carrying a CNVV pppartially overlappinggg wiw th a CNV identified

nnn fofofour patiennntststs oooff f ouur r r cococohohohortrr (T(T(Tababablelele 222).).). (i(( ) ThThThrerr e pppatttienntststs fffrororom mm ththhe pupupublbllicicic datababbasasaseseses prpp esesesenenentititingngng

wiwiwithhh patent ductususus arteeeriiiosuss s (P(PPDA) ) oro CCCoA (DDDECCCIPPHPHERERER 15757578, IIISCCA nnssssv77070666487, ISSSCCAA

nssv706596) carried d d dededeletionnsss ofofo vvariablb e sizes innnclcc udu ing GLGLGLIS3. Thesesseee dedd letions partially

ovovererlalappppppeded wwitith h a a duduplplp icicatatioion n ididenentitififieded iin n onone e ofof oourur pppatatieientnts s (#(#( 43437)7)).. GGLILIS3S3 plplp ayayys s a a rorolele iin n ththe e



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imbalances included the entire RLN3 gene. RLN3 plays a role in regulating blood pressure,

controlling heart rate and releasing oxytocin and vasopressin. Moreover, relaxins stimulate

angiogenesis via the induction of vascular endothelial growth factor 34. (iv) Finally, a duplication

including the entire ERBB2 coding sequence gene was identified in a patient with total

anomalous pulmonary venous return (TAPVR) (ISCA nssv 578742) and in a de novo duplication

in one of our ToF patients (#188). ERBB2 encodes a member of the epidermal growth factor

(EGF) receptor family. ErbB2 signaling is essential for heart development and function in mice

35. In one patient with ToF (#43), we identified 1q21 duplication. Recurrent deletions and

duplications at this locus have been associated with both syndromic and non-syndromic forms of

CHD, including ToF 11,21,36. Since the GJA5 mutant mice exhibit a wide range of CHD, among

them conotruncal defects 37, the gene appears to be a good candidate for the cardiac

malformations, although no point mutations have been identified in patients yet.

Most of the rare CNVs identified in our study show a genome-wide distribution and a

single-occurrence (Supplementary Figure 1). Only three chromosomal regions with a variable

number of copies were identified in more than one patient (Supplementary Figure 1): at

10q24.32 in two patients with TGA (#351 and 222), 11p11.2 in one patient with CoA (#174) and

one ToF patient (#153) and at 20p11.23 in one TGA patient (#341) and one ToF patient (#42).

These CNVs encompass several genes among which BTRC, PTPRJ, and CSRP2BP are good

candidates for CHD. BTRC has been related to the Wnt signaling pathway 38 which regulates

diverse cellular processes, such as gene transcription and cell proliferation, migration, polarity,

and division 39. Wnt2 and Wnt11 mutations are responsible for CHD in mice 40,41. PTPRJ is

critical for embryonic heart development and vasculogenesis in mice 42. CSRP2BP is a

component of the ATAC complex, a complex with histone acetyltransferase activity on histones

CHD, including ToF 11,21,36. Since the GJA5 mutant mice exhibit a wide range offf CCCHDHDHD,,, amamamonono g

hem ccononotruuncn alal ddefects 37, the gene appears to bee a a good candidate forr ttheh cardiac

mmmalflflformationnns, , , alalalthtt ououughghgh nnno o o poooininint tt mumum tatatatititionononsss hahahave bbbeeeen idididenenentititifif eddd iin n n papap tititienenents yyyetetet...

Most of tttheee rarrre CNVVVsss identiiifif eeed in ououourr studududy shshshooow aa genomomome-wiiiddde dddisisstrtt ibutttioioon n ananand a d

ingle-occurrence (SuSuSupppppplemeentntntaraa y y Figguru e 1).) Onlylyy tthree cchrhrhromomo osomalall rrregee ions with a variable

nunumbmberer oof f cocopipip eses wwerere e ididenentitififieded iin n momorere tthahan n onone e papap titienent t (S(S( upuppplplp ememenentataryryy FiFigggurure e 11):):) atat



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15

H3 and H4. Of note, mutations in histone-modifying genes have recently been related to CHD 43.

Moreover, the double-histone-acetyltransferase complex ATAC is essential for mammalian

development 44. Thereby, these genes are strong candidates for CHD in our patients.

Two additional rare CNVs were of special interest (Figure 3). A 3.7 kb homozygous

deletion encompassing a portion of CHRNB3 was detected in a child with TGA (# 342) (Figure

3A,B) issued from consanguineous heterozygous parents. CHRNB3 belongs to the nicotinic

acetylcholine family of receptors expressed in the neural tube during embryonic development 45.

Neural tube signals are critical during heart formation and differentiation in chicken and quail

embryos 46. The second CNV of interest was observed in two TGA siblings (patient #417). Both

children carried a duplication including ERMARD, which was inherited from their

phenotypically normal mother (Figure 3C,D). The exact function of ERMARD remains unknown

but it has been recently shown to play a major role in the control of neuronal migration.

Haploinsufficiency of ERMARD causes periventricular nodular heterotopia 47. Since

periventricular nodular heterotopia has been related to cardiovascular defects, it is conceivable

that duplication of ERMARD plays a role in CHD 48.

By combining genes present in de novo CNVs, in overlapping CNVs (between our

patients or public databases), and from TFBS analyses

(http://hgdownload.soe.ucsc.edu/goldenPath/hg19/database/tfbsConsSites.txt.gz), we identified a

set of 113 candidate genes for CHD (Supplementary Table 6) and a short-list of 10 top-candidate

genes (Table 3). Developmental anomalies resulting in conotruncal defects have been associated

with distinct changes in gene expression 49, describing a pattern of expression of

developmentally important networks. This supports the hypothesis that converging and

accumulating rare genomic and epigenetic variants may disrupt regulatory networks during heart

children carried a duplication including d ERMARD, which was inherited from theeiirir

phenottypypypicalllyly nonormr al mother (Figure 3C,D). The e exe act function of ERMAMARD remains unknown

bububut iiit has beeeennn rererececec ntnttlylyly ssshohohownwnwn ttto o o plplplayayay a a a mamamajojojorrr roleee innn thhhe e e cococontntn rool l l ofofof nnneueueurororonann l mimimigrgrgratatatioioi n.n.n.

HaHaHaplplploinsufficiennncyyy off EEERMAAARRDRD caususu esss ppperivvveeentricccuuularrr nnnooduuulaar hhetetetere otopppiiia 4447. SiS nceee

periventricular noduuulalalar rr heteroootototopipip a a has been relatttedede to cardrddioioiovavascular dddefefefects, it is conceivable

hhatat ddupupplilicacatitionon oof f ERERMAMARDRD plplp ayayyss a a rorolele iin n CHCHDD 484848. .



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development, ultimately leading to CHD 12,13,50. Since we excluded the non-coding regions of

our custom array, we were not able to detect chromosomal imbalances involving regulatory

elements, which is a limitation of our study. According to the multifactorial origin of CHD,

environmental factors during embryo development may also be considered as contributing

factors to CHD in addition to genetic variations. Further work needs to be done to determine

more precisely the origin of non-syndromic CHD and consequently helping in their diagnosis

and management.

Acknowledgments: We acknowledge the Genomics platform of Nantes (Biogenouest Genomics) core facility for its technical support and use of the bioresources of the Necker Imagine DNA biobank (BB-033-00065). We thank Martine Le Cunff and Marie-France Le Cunff for technical support, Marie C. Béné for her critical reading of the manuscript and Dr Eleni Giannoulatou from the Victor Chang Cardiac Research Institute for statistical expertise.This study makes use of data generated by the DECIPHER Consortium.

Funding Sources: C.L.C and M.S.C. were supported by grants from «Projet Hospitalier de Recherche Clinique (PHRC) Interregional (2008)», the Société Française de Cardiologie / Fédération Française de Cardiologie (2009) and Translational Research of Région des Pays de la Loire (2009). M.S.C. was also supported by École nationale supérieure des mines de Nantes and Genavie enterprise foundation. F.B. was supported by grants (platform CARREG) from the Société Française de Cardiologie / Fédération Française de Cardiologie (2012). P.-F. Busson was supported by grant from «Agence Nationale de la Recherche» ANR-13-MONU-0013.

Conflict of Interest Disclosures: None.

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49499. BiBB ttel DC,, Butututler MGMGMG, KKiKibibibiryeva a a NNN, MMMarrrshhhall JAJAJA, ChChCheen J,, Loffflaaand GKGKGK, eetet aaal. Geeneneeeexprprpresesessisisiononon iiinnn cacacarrrdididiacacac tttisisssususues fffrororom m inininfaf ntntntss wiwiwiththth iiidididiopopopatatathihihic c c cococonononotrtrt ununncacacalll dededefefeectctc s.s BMBMBMC C C MeMeMeddd Genomics. 2011;4:11..

5050.. LiLi YY,,, KlKlenena a NTNT, , , GaGabrbrieiel l GCGC, , , LiLiu u X,X,, KKimim AAJ,J,, LLememkeke KK,,, etet aal.l. GlGlobobalal gggeneneteticic aananalylyysisis s inin mmiciceeununveveililss cecentntrarall rorolele fforor ccililiaia iinn cocongngenenititalal hheaeartrt ddisiseaeasese NaNatuturere 20201515;5;52121:5:52020 5–52424



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Table 1: Inheritance pattern of patients’ qPCR validated CNVs

Number of patients

de novo CNVs inherited CNVs

number percentage† number percentage†

85 CoA 76 3 4.1* 12 18.7

291 TGA 159 0 0 29 22.3

92 ToF 81 5 6.6* 22 37.3

Total 316 8 2.6 63 24.9

*p=0.002, Fisher’s exact test †de novo and inherited CNVs percentages were calculated regarding the total number of trios, for each type of CHD, submitted to CGH array †de novo a d e ted CNVs pe ce tages we e ca cu ated ega d g t e tota u bere o t os,for each type of CHD, submitted to CGH array



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Table 2: Comparison between CNVs from this study data and ISCA and DECIPHER databases

Present study ISCA/DECIPHER public databases

Trio ID

Type of

CHD

Parental inheritance

Deletion/duplication

(coordinates hg19)

Chr. Region

CNVsize

(Mb)ID Type of

CHDParental

inheritance

Deletion/duplication

(coordinates hg19)

CNVsize

(Mb)

Candidate gene

437 CoA fatherDuplication

(chr9:3781683-4161396)

9p24.2 0.38 1578 PDA unknownDeletion

(chr9:10190-11351967)

11.34 GLIS3

nssv706487 CoA unknownDeletion

(chr9:204104-11298187)

11.1

nssv706596 CoA unknownDeletion

(chr9:204193-16897578)

16.7

335 TGA fatherDuplication

(chr19:14138589-14159806)

19p13.12 0.021 nssv580437 Dextrocardia unknownDeletion

(chr19:13644739-14369645)

0.7 RLN3

172 TGA motherDuplication

(chr6:118771397-119031236)

6q22.31 0.26

250627 TGA UnknownDuplication

(chr6:118692303-119537523)

0.85

PLN

Bittel DC et al. 2014 ToF Unknown

Duplication(chr6:118842120-

119121565)0.28

188 ToF de novoDuplication

(chr17:37813254-38033098)

17q12 0.22 nssv578742 TAPVR unknownDuplication

(chr17:37356126-43706945)

6.4 ERBB2

CoA father (chr9:3781683-4161396)

9p24.2 0.38 1578 PDA unknown (chr9:10019191900-113555191919676767)))

11.34

nssv706487 CoA unknownDeletitiiononon

(chr9:204104-11298187)

11.1

nsssv70665966 CoCooAAA uununkknknowowwnnnDeDeeleleletitiono

(((chchchr9r9r9:2204040419191933-11168975787878)

161616 7.7.7

TGGAAA fafafathththeererDuplliicaaation

(c(c( hrhrhr191919:1:1:14141383838585 9-1441515989 06)

19191 p1p1p 3.3.3.1112 0.02020 111 nsnsnssvsvsv585880040437377 DeDextxtxtrrorocacac rdrdrdiaiai ununknknknowowwnnnDDeD letiononn

((chhr1r1r19:9:9:131313646464447473999---14369645)

0.0.0 777

TGTGAA ththDu lpliicatiion

(( hh 66 111187877171393977 66 2222 3311 00 2626

252506062727 TGTGAA UnU knk ownDDDuplication

(chrh 6:118692303-1111119595953737375252523)3)3)

0.85



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Table 3: List of the 10 top-candidate genes for CHD

Gene Trio ID Type of CHD

Deletion/ duplication

Chr. region (hg19)

Length (Kb) Start End Parental

inheritance

Frequency in general population

BTRC 351 TGA duplication 10q24.32 152,533 103190059 103342592 Paternal 0%

222 TGA duplication 10q24.32 26,002 103291033 103317035 Maternal doubleton to 1%

CHRNB3 342 TGA deletion 8p11.21 3,712 42583985 42587697 De novo 0%

CSRP2BP 341 TGA duplication 20p11.23 343,15 18162446 18505596 Maternal 0%

42 ToF duplication 20p12.2-p11.1 14587,372 11247299 25834671 De novo 0%

DZANK1 341 TGA duplication 20p11.23 343,15 18162446 18505596 Maternal 0%

42 ToF duplication 20p12.2-p11.1 14587,372 11247299 25834671 De novo 0%

ERBB2 188 ToF duplication 17q12 219,844 37813254 38033098 De novo 0%

ERMARD 417 TGA duplication 6q27 62,207 170140381 170202588 Maternal 0%

GLIS3 437 CoA duplication 9p24.2 379,713 3781683 4161396 Paternal 0%

OVOL2 42 ToF duplication 20p12.2-p11.1 14587,372 11247299 25834671 De novo 0%

PLN 172 TGA duplication 6q22.31 259,839 118771397 119031236 Maternal doubleton to 1%

PTPRJ 174 CoA duplication 11p11.2 477,368 47870014 48347382 Paternal 0%

153 ToF deletion 11p11.2 99,596 47993091 48092687 Paternal 0%

RLN3 335 TGA duplication 19p13.12 21,217 14138589 14159806 Paternal 0%

TCTE3 417 TGA duplication 6q27 62,207 170140381 170202588 Maternal 0%

222 TGA duplication 10q24.32 26,002 103291033 103317035 MaMaMatetet rnnnalalal doubletototo 111%

NB3 342 TGA deletion 8p11.21 3,712 42583985 42587697 DeDeDe nnnovovvooo 0%0%0%

P2BP 341 TGA duplication 20p11.23 343,15 18162446 18505596 Maternal 0%

4222 TToToF duplication 20p12.2-p11.1 1444587,372 112472999999 25558383834671 De novo 0%

NNNK111 341 TGTGTGAA dududuplplplici atattioioionnn 202020 1p1p11.232323 34443,15155 181818162424244666 181818505050559666 MaMaMatetet rnnnalalal 0%0%0%

42 TToF duplicccatattioi n 20p1112..2-p11.111 1444587,37222 111112472229999 2583334667771 Dee nooovo 0%%%

B222 1888 TTooF duplicccataa ioioion n 17171 q1q1q 2 221219,848444 3737781132225444 38803033303 989898 Deee nnnooovo 0%%%

MARD 414 7 TGA duplicata ion 6q27 62,207 170140381 170202588 Maternal 0%

S3 437 CoAAA dududuplplplicicicataa ioiionnn 9p9p9p242424.2.2.2 3737379,9,9,717171333 373737818181686868333 4144 616161393939666 Paternal 0%

LL22 4242 ToToFF duduplplp icicatatioion n 2020p1p1p 2.2.2-2 p1p1p 1.1.11 141458587,7,,373722 1111242472729999 2525838346467171 DeDe nnovovoo 0%0%

dd bbll



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Figures Legends:

Figure 1: Workflow of CNV detection. A cohort of 316 family trios (76 CoA, 159 TGA and 81

ToF) was selected for aCGH. Children’s DNA was labeled in green (Cy5) and parents’ DNA in

red (Cy3). A mix of labeled DNAs (affected child - unaffected father or affected child -

unaffected mother) was co-hybridized on custom 400K array. A total of 12,019 CNVs was

obtained from analysis of the 316 trios. Graphical overview was obtained using the customized

SigFrame software (https://github.com/lindenb/jvarkit/wiki/SigFrame). After exclusion of

polymorphisms and intronic regions, 152 rare CNVs were identified. After qPCR analysis, 149

CNVs were validated and 3 were considered aCGH false-positive results (<2%). Seventy-one out

of 149 rare qPCR-validated CNVs were present in children with CHD. Eight CNVs were de

novo and 63 inherited from an unaffected parent.

Figure 2: Overlapping anomalies identified in the patients. (A) Pedigrees of families #351 and

#222 showing overlapping duplications at 10q24.32 in two unrelated children with TGA. (B)

Both CNVs were inherited and included a portion of the BTRC gene. (C) Pedigrees of families

#174 and #153 showing overlapping anomalies at 11p11.2 in two unrelated children with two

different forms of CHD. (D) One patient presented with CoA (Family 174) and carried a

paternally inherited duplication including the entire coding sequence of the PTPRJ gene. The

other patient presented with ToF (Family 153) and carried a paternally inherited deletion

including a portion of the PTPRJ gene (E) Pedigrees of families #42 and #341 showing

overlapping anomalies at 20p11.23 in two unrelated children presenting with two different forms

of CHD. (F) One patient presented with ToF (Family 42) and carried a de novo CNV

CNVs were validated and 3 were considered aCGH false-positive results (<2%). SeSeSeveveentntntyyy-o-o-onenen ou

of 149 rrarre qPqPq CRR-validated CNVs were present inn chc ildren with CHD. EiEighg t CNVs were de

nonoovvovo and 63 iniinheheheriririteed d d frfrfromomom anan uuunananaffffffececcteteted dd papaparererentn .

Figure 2: Overlapppinining g g ana ommalalalieieies identit fied in thhe e e papatientsss. (A(A( )) Pedigrgrreeeeeess of families #351 and

#2#22222 sshohowiwingngg ooveverlrlapapppipip ngngg ddupupplilicacatitionons s atat 110q0qq2424.3.322 inin ttwowo uunrnrelelatateded cchihildldreren n wiwithth TTGAGA.. (((B)B))



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25

duplication. The other patient presented with TGA (Family #341) and carried a maternally

inherited duplication. Both CNVs included the ZNF133, POLR3F, RBBP9, and DZANK1 entire

gene coding sequences Blue horizontal bars indicate duplications, red horizontal bars indicate

deletions; +/+: individual with two wild-type alleles; +/del, +/dupl: individual with heterozygous

deletion or duplication, respectively; the white symbols indicate phenotypically normal

individuals; the whole-black and black-and-white squared symbols indicate affected children; the

arrows indicate probands.

Figure 3: Example of rare CNVs with strong candidate genes for CHD. (A) Pedigree of family

#342 with a child presenting with TGA and carrying a rare homozygous deletion inherited from

both consanguineous parents. (B) The deletion includes a portion of the CHRNB3 gene. (C)

Pedigree of family #417 with two children presenting with TGA and two unaffected parents. (D)

The duplication containing the ERMARD gene and part of the TCTE3 gene was identified in both

children and was maternally inherited. Blue horizontal bars indicate duplications, red horizontal

bars indicate deletions.

#342 with a child presenting with TGA and carrying a rare homozygous deletionnn iinhnhnheereritititededed fffrror m

both cononsas ngngguiu neeouo s parents. (B) The deletion inclcludu es a portion of the CHC RNB3 gene. (C)

PPPededediiigree of fffamamamililily yy #4#44171717 wwwititith twtwtwo o o chchchililldrdrdrenee ppprereresentttinnng wiwiwiththth TGTGTGA A A ananand dd twtwwooo unuu affffefefectctctededed pparararenenentststs..dd (D(D(D)

ThThThe ee dud plication cooontaiiiniiing thhhe e e ERMAMAMARDRDRD genenene anddd ppparrrtt t ofofof thehehe TCCCTETETE3 geeene wwwasasas idenntntiififieeed in bbbooth

children and was mamaateteternrnr allyyy iiinhnhnherited. Blue horizzzonono tal barsrsrs iiindndicate duduuplplplications, red horizontal

babarsrs iindndicicatate e dedeleletitionons.s.



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Véronique Gournay, Damien Bonnet and Cédric Le CaignecBruno Lefort, Fanny Bajolle, Philippe Pezard, Jean-Jacques Schott, Christian Dina, Richard Redon,Pierre Lindenbaum, Béatrice Delasalle-Guyomarch, Adrien Baudry, Olivier Pichon, Cécile Pascal, Marta Sanchez-Castro, Hadja Eldjouzi, Eric Charpentier, Pierre-François Busson, Quentin Hauet,

Genes and a Potential Role for FOXC1 in Patients with Coarctation of the AortaSearch for Rare Copy-Number Variants in Congenital Heart Defects Identifies Novel Candidate

Print ISSN: 1942-325X. Online ISSN: 1942-3268 Copyright © 2015 American Heart Association, Inc. All rights reserved.

TX 75231is published by the American Heart Association, 7272 Greenville Avenue, Dallas,Circulation: Cardiovascular Genetics

published online December 7, 2015;Circ Cardiovasc Genet.

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SUPPLEMENTAL MATERIAL

Accession Numbers

The LOVD Online gene-centered collection and display of DNA variations

(http://www.lovd.nl/) accession numbers for the 71 rare genomic variants reported in

this paper are: #0000061112, #0000061117, #0000061118, #0000061119,

#0000061586, #0000061587, #0000061588, #0000061589, #0000061590,

#0000061591, #0000061592, #0000061593, #0000061594, #0000061595,

#0000061596, #0000061597, #0000061598, #0000061599, #0000061600,

#0000061601, #0000061603, #0000061604, #0000061605, #0000061606,

#0000061608, #0000061609, #0000061610, #0000061611, #0000061612,

#0000061613, #0000061614, #0000061615, #0000061616, #0000061617,

#0000061618, #0000061619, #0000061620, #0000061621, #0000061622,

#0000061623, #0000061624, #0000061625, #0000061626, #0000061628,

#0000061630, #0000061631, #0000061634, #0000061636, #0000061637,

#0000061638, #0000061639, #0000061640, #0000061641, #0000061642,

#0000061643, #0000061644, #0000061645, #0000061646, #0000061647,

#0000061648, #0000061649, #0000061650, #0000061651, #0000061652,

#0000061653, #0000061654, #0000061655, #0000061656, #0000061657,

#0000061658, #0000061659.

http://www.lovd.nl/

Supplementary Figure 1. Distribution of the 71 validated CNVs identified in our series of

316 affected children. CoA are represented by circles, TGA by triangles and ToF by squares.

Filled geometric figures represent de novo rearrangements and empty geometric figures

represent inherited rearrangements. Geometric figures colored in red refer to deletions and

those colored in blue refer to duplications. Idiographica version 2.2 (July 2013)

(http://www.ncrna.org/idiographica/) was used to create our own ideogram. Three

chromosomal regions with a variable copy-number were identified in more than one patient:

at 10q24.32 in two patients with TGA (patients 351 and 222), 11p11.2 in one patient with

CoA (case 174) and one patient with ToF (patient 153) and 20p11.23 in one patient with TGA

(case 341) and one patient with ToF (patient 42).

http://www.ncrna.org/idiographica/

Supplementary Table 1. List of 297 candidate genes known or suspected to play a role in

heart development in humans or animal models. 100,000 probes covered with a high-density

the exonic and intronic sequences and 10 kb upstream and downstream of the coding regions

of those genes. Seven out of the 297 candidate genes were present in the CNVs identified in

the affected patients.

Gene name Mapping (hg19) Gene name Mapping (hg19)

MTHFR chr1:11845786-11866115 NOTCH1 chr9:139388895-139440238

NPPA chr1:11905768-11907840 ITGB1 chr10:33189245-33247293

NPPB chr1:11917520-11918992 NRP1 chr10:33466418-33623833

ECE1 chr1:21543739-21672034 CDC2 chr10:62538219-62554604

HSPG2 chr1:22148736-22263750 NODAL chr10:72191691-72201465

WNT4 chr1:22443797-22469519 NRG3 chr10:83635069-84746935

ID3 chr1:23884408-23886322 BMPR1A chr10:88516395-88684945

GJA4 chr1:35258598-35261348 PTEN chr10:89623194-89728532

PTCH2 chr1:45288086-45308616 HHEX chr10:94449680-94455408

TGFBR3 chr1:92145901-92351787 NKX2-3 chr10:101292689-101296280

VCAM1 chr1:101185296-101204601 LBX1 chr10:102986732-102988717

RBM15 chr1:110881944-110889303 FGF8 chr10:103529886-103535827

NOTCH2 chr1:120454175-120612276 FGFR2 chr10:123237843-123357972

GJA5 chr1:147228331-147245484 HRAS chr11:532241-535550

GJA5 chr1:147228331-147245484 TEAD1 chr11:12695968-12966299

SHC1 chr1:154934773-154946959 SOX6 chr11:15987995-16497935

VANGL2 chr1:160370366-160398464 MYOD1 chr11:17741109-17743678

PBX1 chr1:164528801-164821045 CSRP3 chr11:19203577-19223589

RXRG chr1:165370349-165414430 WT1 chr11:32409324-32457087

TNNT2 chr1:201328141-201346805 NR1H3 chr11:47270448-47290401

CSRP1 chr1:201452659-201476387 MYBPC3 chr11:47352956-47374253

PROX1 chr1:214161859-214209762 PTPRJ chr11:48002109-48192394

CENPF chr1:214776531-214837914 FGF19 chr11:69513005-69519106

TGFB2 chr1:218519390-218617959 FGF4 chr11:69587796-69590171

LEFTY1 chr1:226073981-226076836 WNT11 chr11:75897369-75917574

LEFTY2 chr1:226124302-226128920 SLN chr11:107578100-107582787

MIXL1 chr1:226411382-226413513 ETS1 chr11:128328655-128457453

WNT3A chr1:228194751-228248961 CCND2 chr12:4382901-4414521

MTR chr1:236958580-237067281 NTF3 chr12:5541279-5604465

ID2 chr2:8822112-8824583 KCNJ8 chr12:21917888-21927747

MYCN chr2:16080682-16087129 KRAS chr12:25358179-25403854

OSR1 chr2:19551245-19558372 COL2A1 chr12:48366747-48398285

SOS1 chr2:39208689-39347604 WNT1 chr12:49372235-49376395

SLC8A1 chr2:40339285-40739575 ERBB3 chr12:56473891-56497128

MEIS1 chr2:66662531-66799891 STAT6 chr12:57489192-57505161

BMP10 chr2:69092612-69098649 CDK4 chr12:58142002-58146164

TGFB3 chr2:75640686-75664337 LRRC10 chr12:70002344-70004942

SMYD1 chr2:88367381-88412902 NR1H4 chr12:100867678-100957643

GLI2 chr2:121554866-121750229 ATP2A2 chr12:110719031-110788897

CFC1B chr2:131278666-131285565 MYL2 chr12:111348623-111358404

CFC1 chr2:131278835-131357082 MYL2 chr12:111348623-111358404

ACVR1 chr2:158592958-158732374 PTPN11 chr12:112856535-112947717

DLX2 chr2:172964165-172967478 TBX5 chr12:114791734-114846247

TTN chr2:179390717-179672150 TBX3 chr12:115108058-115121969

ITGA4 chr2:182321618-182402468 MED13L chr12:116396382-116714991

CALCRL chr2:188207848-188313021 NOS1 chr12:117650978-117799582

COL3A1 chr2:189839098-189877472 IFT88 chr13:21141207-21265576

CASP8 chr2:202098165-202152434 FGF9 chr13:22245214-22278640

BMPR2 chr2:203241049-203432474 FLT1 chr13:28874482-29069265

ERBB4 chr2:212240441-213403352 SMAD9 chr13:37422206-37494409

WNT6 chr2:219724545-219738954 APEX1 chr14:20923289-20925926

PAX3 chr2:223064606-223163700 NDRG2 chr14:21484921-21493935

OXTR chr3:8792094-8811300 MYH6 chr14:23851198-23877482

CRELD1 chr3:9975505-9987090 MYH7 chr14:23881946-23904870

RAF1 chr3:12625099-12705700 NFATC4 chr14:24836144-24848810

TGFBR2 chr3:30647993-30735633 NKX2-8 chr14:37049215-37051786

ACVR2B chr3:38495789-38534633 SIP1 chr14:39583487-39606177

MYL3 chr3:46899356-46904973 BMP4 chr14:54416454-54423554

FLNB chr3:57994126-58157977 MNAT1 chr14:61201469-61435398

FOXP1 chr3:71004735-71633140 HIF1A chr14:62162118-62214977

NPHP3 chr3:132399453-132441276 MTHFD1 chr14:64854758-64926725

SOX14 chr3:137483578-137484396 SLC8A3 chr14:70510933-70655787

ATR chr3:142168076-142297668 DPF3 chr14:73136659-73360809

SHOX2 chr3:157813799-157823936 PSEN1 chr14:73603142-73690398

PRKCI chr3:169940219-170023770 LGMN chr14:93170154-93215012

DVL3 chr3:183873283-183891314 RTF1 chr15:41709301-41775760

ECE2 chr3:183967444-184010819 FBN1 chr15:48700504-48937918

FGFR3 chr4:1795038-1810599 ALDH1A2 chr15:58245626-58357906

MSX1 chr4:4861391-4865660 SMAD6 chr15:66994673-67074335

EVC2 chr4:5564151-5710294 STRA6 chr15:74471809-74501371

EVC chr4:5712923-5816031 NRG4 chr15:76235843-76304785

NKX3-2 chr4:13542453-13546114 CHRNA3 chr15:78887651-78913322

RBPJ chr4:26321331-26433278 MESP1 chr15:90293099-90294540

PDGFRA chr4:55095263-55164412 NR2F2 chr15:96869156-96883490

PITX2 chr4:111538579-111558508 IGF1R chr15:99192760-99507759

BBS7 chr4:122745634-122791642 MKL2 chr16:14165195-14360630

FGF2 chr4:123747862-123819390 DOC2A chr16:30016834-30022401

GAB1 chr4:144257982-144395717 TBX6 chr16:30097116-30103205

SMAD1 chr4:146402950-146480325 IRX5 chr16:54965110-54968395

EDNRA chr4:148401906-148466106 MMP2 chr16:55513080-55540586

TLL1 chr4:166794409-167024993 NDRG4 chr16:58497548-58547523

HAND2 chr4:174447651-174451378 NFATC3 chr16:68119374-68260837

CASP3 chr4:185548849-185570629 CDH1 chr16:68771194-68869444

IRX4 chr5:1877540-1882880 FOXC2 chr16:86600856-86602535

MTRR chr5:7869216-7901235 FOXC2 chr16:86600856-86602535

ISL1 chr5:50678957-50690563 ZFPM1 chr16:88520013-88601574

HMGCR chr5:74632992-74657924 CYBA chr16:88709696-88717457

DHFR chr5:79922044-79950800 SNAI3 chr16:88744089-88752882

VCAN chr5:82767529-82877800 DVL2 chr17:7128660-7137863

MEF2C chr5:88014058-88199869 DVL2 chr17:7128660-7137863

KIF3A chr5:132028322-132073265 MYH10 chr17:8377529-8534036

SMAD5 chr5:135468535-135518422 MAPK7 chr17:19281033-19286856

NRG2 chr5:139227259-139422879 NOS2 chr17:26083792-26127555

ADAM19 chr5:156904311-157002768 ADAP2 chr17:29248753-29286211

FGF18 chr5:170846666-170884164 NF1 chr17:29421994-29704695

NKX2-5 chr5:172659137-172662262 MED1 chr17:37560537-37607527

MSX2 chr5:174151574-174157902 ERBB2 chr17:37844392-37884915

FOXC1 chr6:1610680-1614129 HDAC5 chr17:42154120-42201014

BMP6 chr6:7727010-7881961 FZD2 chr17:42634924-42636907

EDN1 chr6:12290528-12297427 HEXIM1 chr17:43224683-43229468

JARID2 chr6:15246526-15522253 MYL4 chr17:45286427-45301045

SOX4 chr6:21593971-21598849 HOXB6 chr17:46673098-46682334

PBX2 chr6:32152509-32157963 DLX3 chr17:48067368-48072588

RXRB chr6:33161364-33168432 NOG chr17:54671059-54672951

SRF chr6:43138919-43149244 TBX2 chr17:59477256-59486827

TFAP2B chr6:50786438-50815326 MAP3K3 chr17:61699800-61773670

BMP5 chr6:55620236-55740375 PRKCA chr17:64298925-64806862

TBX18 chr6:85444156-85473899 SOX9 chr17:70117160-70122560

PLN chr6:118869441-118881587 JMJD6 chr17:74708913-74722881

GJA1 chr6:121756744-121770873 GATA6 chr18:19749415-19782227

HEY2 chr6:126070731-126082415 DTNA chr18:32073253-32471808

EYA4 chr6:133562494-133853258 SMAD2 chr18:45359465-45457515

CITED2 chr6:139693396-139695785 SMAD7 chr18:46446222-46477081

MAP3K7IP2 chr6:149639062-149732747 NFATC1 chr18:77155771-77289323

SOD2 chr6:160100148-160114353 INSR chr19:7112265-7294011

TWIST1 chr7:19155090-19157295 SMARCA4 chr19:11071597-11172958

TBX20 chr7:35242041-35293242 GDF1 chr19:18979360-19006953

GLI3 chr7:42000549-42276618 MEGF8 chr19:42829760-42882921

MYL7 chr7:44178462-44180916 SLC8A2 chr19:47931279-47975307

ELN chr7:73442426-73484236 NR1H2 chr19:50879684-50886267

SEMA3C chr7:80371853-80548667 TNNI3 chr19:55663135-55669100

SEMA3D chr7:84624871-84751247 BMP2 chr20:6748744-6760910

SRI chr7:87834431-87856308 JAG1 chr20:10618331-10654694

KRIT1 chr7:91828282-91875414 OVOL2 chr20:18004795-18038521

CAV1 chr7:116164838-116201230 ID1 chr20:30193091-30194313

BRAF chr7:140433812-140624564 KIF3B chr20:30865466-30922811

NOS3 chr7:150688143-150711686 IFT52 chr20:42219578-42275862

SMARCD3 chr7:150936058-150974231 SNAI1 chr20:48599526-48605420

SHH chr7:155595557-155604967 SALL4 chr20:50400582-50419048

GATA4 chr8:11561716-11617509 BMP7 chr20:55743808-55841707

DLC1 chr8:12940871-13372395 GATA5 chr20:61038552-61051026

NKX3-1 chr8:23536227-23540450 ADAMTS1 chr21:28208605-28217728

NKX2-6 chr8:23559964-23563922 TBX1 chr22:19744225-19771112

NRG1 chr8:31497267-32622558 TXNRD2 chr22:19863039-19929359

FGFR1 chr8:38268655-38326352 CHEK2 chr22:29083730-29137822

SNAI2 chr8:49830240-49833988 CBY1 chr22:39052657-39069855

SOX17 chr8:55370494-55373456 MAP3K7IP1 chr22:39795758-39833132

CHD7 chr8:61591338-61779467 BCOR chrX:39910498-40036582

ZFPM2 chr8:106331146-106816765 CITED1 chrX:71521489-71527037

ZFPM2 chr8:106331146-106816765 APLN chrX:128779324-128788914

FOXH1 chr8:145699116-145701718 ZIC3 chrX:136648345-136654259

TEK chr9:27109146-27230172 FLNA chrX:153576899-153603006

BARX1 chr9:96713908-96717608 TAZ chrX:153639876-153650061

PTCH1 chr9:98205263-98279247 HSA-MIR-1-1 chr20:61151513-61151583

TGFBR1 chr9:101867411-101916473 HSA-MIR-1-2 chr18:19408965-19409049

INVS chr9:102861510-103063426 HSA-MIR-133A-1 chr18:19405659-19405746

PBX3 chr9:128509616-128729653 HSA-MIR-133A-2 chr20:61162119-61162220

ENG chr9:130577290-130617047 HSA-MIR-133B chr6:52013721-52013839

RXRA chr9:137218315-137332432

Supplementary Table 2. List of primers used for qPCR validation to confirm the rare CNVs

identified by aCGH. All primers were designed with Primer3-PCR primer design tool v.0.4.0.

Gene name Forward Reverse

ALB GCTGTCATCTCTTGTGGGCTGT AAACTCATGGGAGCTGCTGGTT

TNNI3KF TCACTTCTCTGCTTCACAGTGG TGTTTTCACAGGTGCCAAAG

PLB1 GCATCCTTTGCCTACAGCTC AGGGCTATTTTCCCCTTGAA

PPP1CB GGTCTCTCACAAGGCTCCAA AATGAAGCATCCCACGTAGC

MAD1L1 TTCCATGGTTGCTTTCCTTC TGAGCTCCAATGTGCGTTAG

FTSJ2 TTGAAGAATGCGTGAATTGC AACCTGGGCAACATAGCAAC

NT5C2 AAGAGTGGGGGAAGGAGAAA ATCGACCATGGCAGTCCTAC

CNNM2 AGCAGGTGGAGAGTCCAGAA ATTCAAGGTCAGCCACAAGG

CCDC76 TGGCTGCTTAAGCTGTTGTG ACCTGCTGTTTCAGGTGCTT

SASS6 CAAGCTGGTCCTTATCCTTGA GGAGCTACAGCGGACTAAGC

CDC2L5.1 CAATTGGGTGGCGAGTAGAT ATTTATCCACGCAGCGTTTT

CDC2L5.2 TCTGAAAGTGTGGAGGCAAA GAGCACTGCTTTCACCCATC

PTPRJ.1 GGGAATGGTGACTGAAATGG TCGCAAAACACCTCCCTAAC

PTPRJ.2 TGTGTCCTTCCTTCCCTTTG TCCAACTAACCCACCCTCTG

ATM ATTGGTTTGAGTGCCCTTTG CCTGTTGCATGACAGCATCT

KDELC2 GGCTTTTCCAAGCTCCTTTT GGTAGAGACAGCCGAGAGGA

CPNE8.1 TTGTTCGCACAAAATTGCAT CAAACTTGGCCATTTGCATT

CPNE8.2 TGCATGGCTAAACTGCTCAC CAGGGTCCTATGGTTGCACT

GABRG3.1 AGTGCCTAGACCCCCAGATT TATGGTGACTGCATCGTGGT

GABRG3.2 TTTCCACCATGTCCTTGTGA CGTCAATTACGGTCGGTTTT

NFATC3.1 TGCTCCATCCCACAAACATA GCTGATAGGAAGACCCCACA

NFATC3.2 AACACCAACAGATGCCCACT ACACCTGCCCCTCTGTAATG

MYO19.1 AGGGTCCTATTGGGAAATGG CCAATGGACCTTCTCTTGGA

MYO19.2 CACTGTTGAGCCTGTCTGGA TGTGTGACTCTGGGCAGTTC

MAD1L1 AAAACCCAGGAGAGCTGTGA TTGTCCTGCATCACAGAAGG

FTSJ2 TCAGGAGCTCAGGCTACGTT GCCAGCAGGAAAGAGTCATC

LMO7.1 CCCCTGCTGATGTGATTTTT CTGTCCTGCAGCATTTCCTT

LMO7.2 AATCTGGGCTGAAAATGTGC AGCCAAATCCCAAGGAGACT

MGAM.2 CCTTCAGTGGAAATGCTGCT CCCCCTTCTTCATGCTTACA

XRN1.1 CAGGGTTGGAAGGAAAACAA TGATTTGAGGTTCCTCTACCG

XRN1.2 TGTCTGCTCTGCCATGTTTC TGCAGGAGCTTCCTTTCCTA

CENPP_OGN CTGCTGCACTCAGTCTGCTT CCTGGGAAATCACCAAAATG

CENPP_OMD CAAACCAGGAGGCATTGATT TCCAAACTGCAGACAAATGC

KANK1.1 ATCCCTTCAGTGGCTTCGTA GCATGTCCTTCTCTGCTTCC

KANK1.2 GGCTTGCCTCATTCATCTTC TGGGTTTCTCAAACCTCCAG

SDC1.1 GGGAGCTGTTATTGGGAACA TTGTTCTTGGGGGTTTTTGA

SDC1.2 TCAGGCTATGAGGGAGGCTA CAGAAAAGCTTTCCCAGGTG

NR2C1.1 GCTTTCCTGAAGGTAGGCTTT CAGCAGAGGGGTAATCACCTT

NR2C1.2 CCTGCCCAGTCACTTCAAA GCCAGAACACAAGACACCAA

KLHL12 GGGGGAATCTGTTGCCTTAT GCAGAAAGGCGATAATGCTT

ADIPOR1 AGGACCTGCTGGAAGATTCA CTATCGCTGAGGGCTTTGTC

DYDC1.1 TGAGTTAAACAGGCCCCAAG GACTGAGGGCAGGGTGTATG

DYDC1.2 ATCCTGGCCTAGCCTCATTT TGGAGCACCTAACTTGAGCA

NME6.1 TCCCATTGAAGGGTGAGAAG GTCTGAGTGGTGGGTCCTGT

NME6.2 CCTCCAGAATCAGTGGATGG ATGTAGGGAAGGCTGGGAGT

LTN1.1 TCCCCTCTATTGAGCCAATTT CATTTGGCTTCTCTGGGTGT

LTN1.2 AGGCTGGCTCTTCTTCTTCA TTCTGCTCTTGAGCACGACTT

08-oct CAATTTCTGTTGCTCCACCTC TTGGTGGGGTACTGGAATGT

ETS1.1 AGCAGAGTGGCTGGAGAGAG TGCTTAGGCCAACTCCATCT

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=search&term=29960

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=search&term=29960

ETS1.2 AGGCTGTATGGGCACATTTC ACACCTCCTGCCTGTACTGC

EML4.1 ATGGGAGTTTTGTGGGACAT TGTCCTTCTCCCAATTCCTG

EML4.2 TTATGAAGAGCCATGCAACG CACTGCCCATCCTGTTACCT

C1orf25.1 CTGTCCACCACCACTTTCAA ATGGGATTACAGTGGGCAAA

C1orf25.2 ATTCTGGAGGTGACGACGAA GCCCCCATTAACCTCTTTGT

NEXN AAAACTTGGCAAGGGTGATG CCTGCTTTCTCCTGTTCCAT

COCH CCCAAAGTGGTGGTGGTATT GAACCATCCCCAGTTCTTCA

TTC19 CCACCGTCAGTCTGGAAGTT CCCTGGCTGCTGTACTTACC

CDK13.1 CCTGAGCTACCAGGAGGAGA CCAGAATCACTGCTGGTCAA

DNAJC2 CCACCTCCACCAGTTGATTT GCCCTAGCCAGGAAGAGAGT

TMEM38B.1 TCATGACTTTTTGGCAGATGA CAATTTGGCAAAACACAACAA

TMEM38B.2 GTTTGGGATGGAAGGAGACA AATGGGTGATTTTTGGCACT

SH2D4B.1 GGGACAGCCCTTGACCTTAT AGAAGGCTCTTTCCAACACG

TMEM184B.1 GAGGGAACCTGTTCATTCCA CCCAGATTATCCCTGGGTTT

PTPRN2.1 GCTCAATGCCCAGATTTGTT ACTCCCCTGACCTCACCTTT

PTPRN2(2).1 GGTCCCCAAGGGAGATGTAT CAATTCCCTGCCAGTTGTTT

SLN.1 GCCTACTGGGATCAACCAAA TCTAGGCACATCCTGGACCT

RASA2.1 TGAAGCTGTTTTGTGGTGTGA ACCAGGCTTGATGAGAGGAA

PDE8A.1 TGACGTCAACATGGGTTTGT CAGGCACATTTCAGGCACTA

PGAM1 TGCTTCACTGTGGGCTTCTA GGCCCCCACCATACTTATTT

TM2D3 GGCTTTTTCTAAGCCCTGCT CTGGGAATATGGACGCTGAT

PRKCD CCATGAAAACATTGCTGTGG CTGTTACTCCGAGGCTCCAG

RNF115 TAATGGGCTCCATCTGCTCT TGAAGCTACCTCACCGTTCC

GLIS3.1 CTTGCCACACACACAAATCC GGTTTTACGCCCACAAAATG

KIAA1432 TGGGAATGACCGACACTTCT TGGGATAGGCAAAAGGGATT

WDR72.1 CTTTCAGGGCAAACTGTGCT AGGTTCCTGTTTTGCTGTCG

PRPF4B GCAGGTCTCCACTTTTGAATG TCCTCTCTGGTTCTCGTCGT

ADH1B.1 TACGCTCCATGCAAAGACTG ACCTGCTTCACTCTGGGAAA

TDRD3.2 GCCTGCATCATGAACACTTG TGCAGTGGCATAAACTTTGAA

TRPM1 AGGCTGGGGAAAGCATCTAT CCTGTGGCTCTTTCCAGAAG

PVT1.1 ATCCCTTCTGAATGGCACAC AGGAAACATGGCAAGACTGG

CIITA CCGACACAGACACCATCAAC GCAAAGAACTCTTGCCCTTG

VPS13B.1 TCAGCCTCAGAAACGAACAA GGTGGGGAGACAAAGGAAAT

FMR1-AS1 CAAGGACCCTGTAGGGACTG CATAGAGGCCCTGCACCTTA

CAMSAP1L1.1 ACTGGAATTCACGTTCCTTCA TGGTGTTGCCAGTTCGAATA

PRKACB.1 TGGGAGAATCCAACTCAGGT GGCAAAATTCCCATTTTAAGC

CSPP1 AAACCGAAACCTTCCAAACA TCTAAGGGACCACCATGCTT

ARSB TGACACCCACTGGTGAAAGA GCCATTTAAATCTGGGGTGA

CSMD1.1 TAATGTTCCCCCTGGATCTG GCCCTTTCCAATCGGTTAGT

FBN1.1 TGGGGTCCAGTAAATCCGTA GTCCTTCCAGAGGACCACAA

WDR33.1 GCTGCAAGACACTGCATGTTA GAAAGAGGCATGGAGCAGAG

PLEK.1 GGAGGCGAATGGAGTACAAA TAGCCCCTGCTTTTTCCTTT

BTRC.1 GCACCAAATCAACTGCTCAA CACTACCAGCCTGTCCCTGT

PLN_C6orf204 CTGTTCCCATAAACTGGGTGA CCAAAGTCAGCGAAATCTGTT

FAM150A CTTCCCACCCACAGACATTT TTCACCAGAATGCAGCAAAC

NDUFAF1 GCCTCACCTACCCTTGGAAT AAGACGATTGGAGGCAGAAG

TNFRSF19 TGGTAGCAAAAGGACCTTGG GGGTATCCCATTTTCTTGGAA

NEUROG2 GCCCGTCTGAATGAAGGATA GCCTTAGTGGGTTCCCTTTC

ICA1.1 TGCCGAAAAGTCTCCACTTC AGATTGGCAGAATGTCAAGGA

AGBL2 GGCATCCAATTGGGAGTACA TGAACTCACCTTGCGAACTG

SORBS2.1 CCCCTCAATGATTTCCCATA AGCAGGAAGGGAAAGGAGAA

EPHA5.1 GGCACAATTGGTTCCACTG CCCTGGCAGGATCTCATTTA

ACSL6.1 TGATTACGTGGGCCTCTTTC TTTGGGTTCTGGATCTGGAG

SGK196.2 CGTTTGGACTATGGCGATTT CTCCAGCTCAGGTCTCCATC

VPS33A TGCAAACCAATGATTCAGGA TTTGGCCTGATTGCACAGTA

IQCG AATCTGCAGCTCGGTATTGG CGCTGACTGAACTGAATGGA

ESYT2 GTGTGGATGGAGCTGTGTTG CCTCTAGGTGCTCCATCTCG

ZMYM4 CTCTCGCATTGAGGAAGAGC TTCAGAGTCCTGGTCCGTCT

TRIM45 GGCATGCTTCCTAGCAGAAC GATCCAGCCAAATGTGTCCT

PEX1 AACCCTCATGAGTTCCCAAA GCGCTTTCCAAGAAAATGAC

PNPLA4.1 CGCTGCAAATGATAGGTTGA AGAAAGCCTTGGAACGATGA

RLN3 GCTGGAAGTCTGAGGACAGG CGGAAGAAAAACCAACTTGC

POLR3F CCACGAAGGTGGTGAGATTT TGCTTTCAAGGAGATTTCGTG

CHRNB3.1 CTGAGTCCCCAGTAGGGTCA TTGTGGTCTGTCCATTCCTG

TNFRSF10A.1 AGGAAGCACAGGGCTACAGA GCCCACAGTGTAGTCCAGGT

ACAD10 TCACTGATGTGCCTCGAAAG AGACTCCAGGCTTCTTGCAG

FAM47E AGACCAAACCAAGCCATGAT TCCCCTTCTGCAATTACCTG

CCNI.1 ATTCAGGTGGCAGGAAAATG GGAAAGTTGGTGAGAGTGCTG

KCTD7.1 GAGTGTCCGCTCCTCAACTC GCAGTGCAGCAGGTCATAAA

RERG.1 ATTCCCAGATGAACCGTTTG AGGGTTGTGTGGAGCATTTC

DAOA.1 CTGTTGCAGCAAAGGAGACA ACTCTGCATAGGGCTGAGGA

PIK3C3.1 CTGAAGGCCACTCTCAAACC TGCCTGGATGTTGACAAGAA

ERAP1.1 TCTGTACGCACGGCTGATAG CTGGTCCCTGTTTCCCTGTA

GFPT1 TCAGCTTTTGCCAAGATATGC TGCCTGTGATGGTGGAACTA

CDR2.1 TTTTGAATGGACCCTTGAGC TCCCCTTAGGAGTCCAGGAT

ATG5.1 CATGAAAAACCACAGGCTGA TTGGCTATATCCTGGCTTGG

VIP.1 TGTTGCAAAATGCATTAGCTG TCGGAATAGCTTCATGGTGA

ARNTL2 ACGAAAGCTGGTTTGCAAGT TTGAGTTGGGTAAGCATCCA

TNRC6B.1 CCCTTCTTCCAGGTGTGTGT ACGGGATCTGACTGGTTCTG

NFE2L3 CTAAAGCCCCAACCAGTTCA TCAGGCTGTGATGAAAGCAA

CHEK2.1 TCCGAAAGTGTTTCTTGCTG ATTCAACAGCCCTCTGATGC

TCTE3 TTAATTTTGCCAGGGCACTC GCCTTTCTCTCTTCCCCTCT

WDR41 AAAAGCAGGAGTTGCTTGGA GCCAGCCAACCTGTTAAAAA

RAP1GAP2.1 TAAGGAGAACGCCTTGTTGG AAGGTCCTCCCTACCCAGAA

PRDM5.2 GGAAGCCTCCATGTCAGAAA CACAGTCCACTGCTGTCCTG

EXOSC9_CCNA2 ACACAGACCACCAGTGCAAA CCAGGGTTCTCAGAATGGAA

ASAP2 TCAAGATCCTGCCTCTACGG TCAGAGCTGGGCTATGGACT

TBX20.1 TATGCCATTCCCAAACATCA TATTTGAGGGCACGTGGAAT

VPS54 AACAACTTGCAGTGCACCAG GGCCATTGAATGAGAGGAAA

RAB11FIP5.1 AGGTGCGACACACACTTCTG GGTGGCAGCTGTTTTGTTTT

IKZF3 TGCCACATTGCTTGCTAATC CCTTTTCTCCCCATGTACCA

PVT1.1 TGCTCTGACGTCCTCATTTG ATCTGGGAGCCCGTTATTCT

MORC1 TGCTGCAAAGCAGACAGAGT ATCCCCCAGTATTCCAGGTC

DSP ACGAAGACCACCATCAAGGA GCTGTCATTGAGCCTGACAA

ENPP2 AGCCAAGGCCTGGATAAAAT ATCAGCAAATCTTCCCCAAA

EPCAM.1 TGAGTTCCATGGCAGATCAC TCCACTGGAGTATGCCTTCA

ODZ1.1 TACCAATGAGGCCCCACTAC AGGGCTTTTTACCCAGAGGA

SFXN1 GAACAGGGCAAAGAGAGCAC CGGCAAGGATCATGAAGAAT

DGS4.1 TCATTGTCCAAGAGGCACAG TCACGTTCCCTTTTGAGCTT

ZFP106.1 CCATGCTGTCCCAGAGAAGT CCAGGCATACCCCTAGCATA

HIPK3 AAGGCATTGCTTGTGGAGAG GCACACCCAGGCAGTGTAG

KCNK12 ATGGGAGGAGGATTGAGGTT AGAGGAGACATGGGTGGTTG

NUP85 GACCTGTTTCTGGGAGGTGA GCCACTCTAGAAACGCAAGG

MGAT5 ATTGCTTTGGGATCATCTGC CAAGACAGTGTGCCCTCTCA

TRAP1 GTGAGCATGTGAGCTCCTGA CCAGAGCAGGTGTGAACTGA

NASP.1 TAACCGGGATATGCAAGAGC GCCCTTTCCTAATCCAAAGG

PTPRJ(2).2 GACGGTCAAAGCAGAAGAGG CGTCCATTTCACTGGACCTT

RAD18 ATTTGGCAAAGTGGGAGTTG CTTTAGCTGGGTGCCTCTTG

ACCN1.1 AACTGGCTTCAGGTTGATCG AAACACTCACCCCTGTCAGC

BCMO1 TTCTTGGCACTGAGGGAGTT TTCCCAGGAAAGGTATGTGC

EVC.1 GGCATCACATGGACTGAGTG CAGACTGCTGGAAGGAGAGG

CSRP2BP GGCTTTCTTGACAGGAGCAC GAAGGAGATGGGCTTGTCAG

PCNT TAGTGCCTGTTCCCTCTTGG CACAGACCACCTGGAAACCT

GJA5 TACAGAGACCAGGCCAATCC AGCAGGGGCAAGGAAATAGT

GPR83 TGCTGCCTTGTCTCATTCTG GGATCCCCAGATGGATTTTT

DYDC1 GCCTAACTGACCCTGAGCTG TGGAGTGGTCTTGGGAAAAC

BIRC6 GTGCGATTCAATCCAAACCT AAAGAAACCCATGCCCTCTT

NCAM2 TTACATTGTGCTCGGATTGC ATCGCATTTGCATGTGTTGT

CGNL1 GCAACCCTATGACCCTGAAA GTTTGGGAAAGCAAGTCCAA

SPATA6 CAGGTGGAATGAAATGTTGCT AAATATGCCCCTTTGGTGGT

GOLPH3L CCACCAAATGGGAGAAAATG ATTTGGGGAGAGCAACTTCA

EIF3B TTGGATGTACCACAGGCTGA TACTTCCGGAATGGATCTCG

TTC3 CGTACCGTTCTGGGATCTGT ATCCCGTTCCAAGGGATTAG

ARPC4 GGCTTCTTTTCCCTGAGACC ATTAATGGTGCCTGGACTGC

NUP54 CAGGGCCAGGGAATAAAAAG TTCACTGCGTTTCTTCAGGTT

TJP1 TCGCTACAGCACTCATCTGG CAACAGCATCCTTCCACCTT

STBD1 CCCCTGGGAGAACAAGTGTA CTCCACAGTCCAGGCCATAC

PSMD13 CACAGATGCAGCCAGACCTA TCACTACCCCTCCAGACAGG

DEFB119 TGACCCAGCCCTAGTGACTT TCAGGTTGCTTAGGGTCCAC

TNN ATCAAAGGATGTGGCCTGAG CTCTCTTTCAGTGGGCCAAG

GMDS ACAGCCAAACCATCATGACA TGTCAAAGCCCAACAAGTCA

MAP3K4 CGTTTGATGTCTGGGGAGTT GCCCACATGACACATTTCAG

EXOSC1 TGGGGATTACTAGGGCTTCA TGGCCAGAGTGGCTCTTATT

TARSL2 GGGCAAAAGTGTCCTTTTCA GACTTCACGGAGGTGCTCTC

PRKCD CCTAGGTCCTGTTCGCTCAC GTCCATGGAGTCGATGAGGT

FMR1-AS1 GACAGGACGCATGACTGCTA ACTCGCCTTTCCTCAACTCA

DSG3 ATGAGCATTTGGCAGTCACA TCCCCTTTCCACAAATTATCC

ETS1 GTGGGCAGGCAACTATCAAT AAATGACTTTGTGGCCTTGG

TBX20 CCCCATCATAACCACATGTCT TGGCTGCTGTTTTCCTTACC

ECI2 TATGACTGGCAACGCTGAAG TGATTGCAGTGGTCAATGGT

Supplementary Table 3. List of 71 validated rare CNVs (50 duplications and 21 deletions) in

children with outflow tract defect. CNV sizes ranged from 2.9 kb to 1.8 Mb, except for two

large CNVs of 7.9 Mb (Family 42) and 14.5 Mb (Family 149) (highlighted). Sixty-three of 71

CNVs (16 deletions; 47 duplications) were inherited from one of the parents, 8/71 CNVs (5

deletions; 3 duplications) were de novo. The frequencies of these CNVs in the general

population varies from 0% to 1% in some cases (according to the following databases: 42

million probes study (Conrad et al., 2010), WTCCC study http://www.wtccc.org.uk/, 1000

genomes project http://www.1000genomes.org/ and DDD controls project

http://www.ddduk.org/).

Trio IDType

of CHD

Gain

LossRegion

Length

(Kb)Start End

Parental

inheritanceRefSeq Genes in the region

Frequency in general

population

212 CoA Gain 2p32.2 524,815 28796008 29320823 De novoPLB1, PPP1CB, SPDYA, TRMT61B,

WDR43, FAM179A, C2orf71, CLIP40%

163 CoA Loss 7p22.3 19,527 2255641 2275168 De novo MAD1L1, FTSJ2 1 singleton observation

6 CoA Loss 10q24.32 28,683 104828409 104857092 De novo CNNM2, NT5C2 0%

179 CoA Gain 7p14.1 109,041 40027276 40136317 Paternal CDK13 0%

174 CoA Gain 11p11.2 477,368 47870014 48347382 PaternalNUP160, PTPRJ, OR4B1, OR4X2, OR4X1,

OR4S1, OR4C30%

44 CoA Gain 16q22.1 123,877 68170552 68294429 Maternal NFATC3, ESRP2, MIR6773, PLA2G15 0%

59 CoA Loss 7q34 35,204 141705389 141740593 Paternal MGAM 0%

23 CoA Gain 2p24.1 47,889 20403619 20451508 Paternal SDC1, PUM2 0%

90 CoA Gain 1p31.1 75,025 78308983 78384008 Paternal FAM73A, NEXN-AS1, NEXN 0%

77 CoA Loss 13q22.2 17,101 76391357 76408458 Maternal LMO7 0%

77 CoA Gain 22q13.1 17,5 38609792 38627292 Paternal MAFF, TMEM184B 0%

211 CoA Loss 3q23 112,858 141272742 141385600 Maternal RASA2 0%

226 CoA Gain 15q25.3 76,111 85606120 85682231 Maternal PDE8A 1 singleton observation

437 CoA Gain 9p24.2 379,713 3781683 4161396 Paternal GLIS3, GLIS3-AS1 0%

437 CoA Gain 9p24.1 231,015 5534835 5765850 Paternal PDCD1LG2, KIAA1432 0%

255 TGA Gain 4q23 15,292 100226779 100242071 Maternal ADH1B 0%

194 TGA Gain15q13.2-

q13.31207,316 31196707 32404023 Maternal

FAN1, MTMR10, TRPM1, MIR211, RP11-

16E12.2, KLF13, OTUD7A, CHRNA7doubleton to 1%

231 TGA Loss 4q27 82,199 121616153 121698352 Paternal PRDM5 0%

252 TGA Loss 8q22.2 198,691 100205095 100403786 Maternal VPS13B 0%

210 TGA Gain 15q21.1 16,788 48883414 48900202 Paternal FBN1 0%

196 TGA Loss 2q14.3 7,523 128476795 128484318 Maternal WDR33 0%

196 TGA Loss 8p23.2 250,895 2975904 3226799 Paternal CSMD1 0%

265 TGA Gain 2p25.1 124,867 9525380 9650247 Paternal ASAP2, ITGB1BP1, CPSF3, IAH1, ADAM17 0%

277 TGA Gain 3q29 241,257 197566221 197807478 PaternalLRCH3, IQCG, RPL35A, LMLN,

ANKRD18DPdoubleton to 1%

335 TGA Gain 19p13.12 21,217 14138589 14159806 Paternal RLN3, IL27RA 0%

335 TGA Gain Xp22.31 56,998 7866965 7923963 Paternal PNPLA4 0%

351 TGA Gain 10q24.32 152,533 103190059 103342592 Paternal BTRC, POLL 0%

358 TGA Gain 13q33.2 1647,761 105161812 106809573 Maternal DAOA-AS1, DAOA, LINC00343 0%

222 TGA Gain 10q24.32 26,002 103291033 103317035 Maternal BTRC doubleton to 1%

172 TGA Gain 6q22.31 259,839 118771397 119031236 Maternal CEP85L, BRD7P3, PLN doubleton to 1%

172 TGA Gain 8q11.23 438,514 53161037 53599551 Maternal ST18, FAM150A, RB1CC1 0%

297 TGA Gain 4q27 4,621 122735142 122739763 Maternal EXOSC9, CCNA2 0%

165 TGA Loss 15q15.1 25,534 41663852 41689386 Paternal NUSAP1, NDUFAF1 0%

http://www.wtccc.org.uk/

http://www.1000genomes.org/

http://www.ddduk.org/

284 TGA Gain 4q35.1 66,434 186558702 186625136 Paternal SORBS2 1 singleton observation

302 TGA Gain 5q31.1 64,839 131247533 131312372 Paternal ACSL6, LOC728637 0%

341 TGA Gain 20p11.23 343,15 18162446 18505596 MaternalCSRP2BP, ZNF133, LINC00851, DZANK1,

POLR3F, MIR3192, RBBP9, SEC23B0%

356 TGA Gain 16p12.2 74,101 22357397 22431498 Maternal CDR2, PRN3P3 doubleton to 1%

417 TGA Gain 6q27 62,207 170140381 170202588 Maternal TCTE3, ERMARD, LINC00242, LINC00574 0%

393 TGA Gain 5q15 36,42 96107398 96143818 Maternal CAST, ERAP1 doubleton to 1%

294 TGA Gain13q12.11-

q12.121280,378 23235248 24515626 Maternal

BASP1P1, SGCG, SACS, SACS-AS1,

LINC00327, TNFRSF19, MIPEP,

C1QTNF9B-AS1, ANKRD20A19P

0%

295 TGA Gain 7q36.3 279,466 158331982 158611448 Maternal PTPRN2, MIR5707, NCAPG2, ESYT2 0

329 TGA Gain12q24.12-

q24.13126,387 112182542 112308929 Paternal

ACAD10, ALDH2, MIR6761, MAPKAPK5-

AS1, MPKAPK5doubleton to 1%

342 TGA Loss 8p11.21 3,712 42583985 42587697 De novo CHRNB3 0%

42 ToF Gain20p12.2-

p11.114587,372 11247299 25834671 De novo

LOC339593, LINC00687, BTBD3, RP5-

1069C8.2, LOC102606466,

LOC100505515, SPTLC3, ISM1, ISM1-

AS1, TASP1, ESF1, NDUFAF5, SEL1L2,

MACROD2, FLRT3, MACROD2-IT1,

MACROD2-AS1, KIF16B, SNRPB2, OTOR,

PCSK2, BFSP1, DSTN, RRBP1, BANF2,

SNX5, SNORD17, MGME1, OVOL2,

CSRP2BP, ZNF133, LINC00851, DZANK1,

POLR3F, MIR3192, RBBP9, SEC23B,

LINC00493, DTD1, RP11-379J5.5,

LINC00652, LOC100270804, C20orf78,

SCP2D1, SLC24A3, RP5-1027G4.3, RIN2,

NAA20, CRNKL1, C20orf26, INSM1,

RALGAPA2, KIZ, RP4-777D9.2, XRN2,

NKX2-4, NKX2-2, LOC101929625,

LOC1019608, PAX1, RP11-125P18.1, RP5-

828K20.2, RP5-1004I9.1, LINC00261,

FOXA2, RP4-788L20.3, SSTR4, THBD,

CD93, LINC00656, NXT1, RP3-322G13.5,

GZF1, NAPB, CSTL1, CST11, CST8,

CST13P, CST9L, CST9, CST3, CST4,

CST1, CST2, CST5, GGTLC1, FLJ33581,

SYNDIG1, CST7, APMAP, ACSS1, VSX1,

RP4-738P15.1, LOC101926889, ENTPD6,

PYGB, ABHD12, GINS1, NINL, NANP,

ZNF337, FAM182B, LOC101926935

0%

188 ToF Gain 17q12 219,844 37813254 38033098 De novoSTARD3, TCAP, PNMT, PGAP3, ERBB2,

MIR4728, MIEN1, GRB7, IKZF3, ZPBP20%

24 ToF Loss 17q12 741,259 31856936 32598195 De novoASIC2, AA06, RP11-215E13.1, CCL2,

CCL70%

11 ToF Loss 7p22.3 19,967 2400442 2420409 De novo EIF3B 0%

515 ToF Loss 11p15.5 127,12 193816 320936 De novo

LOC653486, SCGB1C1, ODF3, BETIL1,

RIC8A, MIR6743, SIRT3, PSMD13, NLRP6,

ATHL1, IFITM5, IFITM2, IFITM1, IFITM3

doubleton to 1%

205 ToF Gain 2p14 457,802 64107729 64565531 Paternal UGP2, VPS54, PELI1, LINC00309 doubleton to 1%

218 ToF Loss 2p13.2 56,753 73285631 73342384 Paternal SFXN5, RAB11FIP5 1 singleton observation

188 ToF Loss 8q24.21 2,879 129108394 129111273 Maternal PVT1 0%

193 ToF Gain 3q13.13 277,172 108626684 108903856 Paternal GUCA1C, MORC1, FLJ22763, LINC00488 1 singleton observation

167 ToF Gain 8q24.12 551,841 120628587 121180428 MaternalENPP2, TAF2, DSCC1, DEPTOR,

COL14A10

184 ToF Gain Xq25 361,263 123509834 123871097 Maternal TENM1 0%

148 ToF Gain 11p13 187,151 33191299 33378450 Paternal CSTF3-AS1, HIPK3 0%

146 ToF Gain 2p21-p16.3 398,949 47537252 47936201 MaternalAC073283.4, EPCAM, MIR559, MSH2,

KCNK120%

149 ToF Loss 2q21.2-q22.2 7962,887 134275082 142237969 Maternal

NCKAP5, MIR3679, MGAT5, TMEM163,

ACMSD, CCNT2-AS1, CCNT2, MAP3K19,

RAB3GAP1, ZRANB3, R3HDM1, MIR128-

1, UBXN4, LCT, LOC100507600, MCM6,

DARS, AC093391.2, CXCR4, THSD7B,

HNMT, SPOPL, NXPH2, YY1P2, LRP1B

0%

153 ToF Gain 1p34.1 12,36 46072235 46084595 Paternal NASP 0%

5 ToF Gain 3p25.3 132,049 8919991 9052040 Paternal RAD18, SRGAP3 0%

29 ToF Loss 16q23.2 211,72 81134814 81346534 Paternal PKD1L2, BCMO1 0%

36 ToF Gain 21q22.3 165,777 47690060 47855837 Maternal MCM3AP, YBEY, C21orf58, PCNT 0%

43 ToF Gain 1q21.2-q22.2 2090,029 145761268 147851297 Maternal

NBPF20, NBPF10, PDZK1, GPR89A,

NBPF25P, GPR89C, PDZK1P, NBPF11,

NBPF12, LOC728989, NBPF13P, PRKAB2,

PDIA3P1, FMO5, CHD1L, LINC00624,

BCL9, ACP6, GJA5, GJA8, GPR89B,

PDZK1P1, NBPF8, NBPF11, MIR5087

0%

84 ToF Gain 2p22.3 234,773 32815822 33050595 Maternal BIRC6, TTC27, MIR4765, LINC00486 doubleton to 1%

93 ToF Loss 21q21.1 1570,572 22115159 23685731 PaternalLINC00320, NCAM2, RNU6-67P,

LINC00317, AP000475.2, LINC003080%

106 ToF Gain 15q21.3 64,885 57689066 57753951 Paternal CGNL1 doubleton to 1%

217 ToF Gain 4q21.1 68,634 77033621 77102255 Maternal ART3, NUP54, SCARB2 doubleton to 1%

214 ToF Gain 15q13.1 1013,794 29212452 30226246 Maternal APBA2, FAM189A1, NDNL2, TJP1 0%

153 ToF Loss 11p11.2 99,596 47993091 48092687 Paternal PTPRJ 0%

53 ToF Gain 1q21.3 358,831 150413398 150772229 Maternal

RPRD2, TARS2, MIR6878, ECM1,

LINC00568, ADAMTSL4, MIR4257,

ADAMSTL4-AS1, MCL1, ENSA, GOLPH3L,

HORMAD1, CTSS, CTSK

0%

158 ToF Gain 18q12.1 112,124 28937235 29049359 Maternal DSG1, RP11-534N16.1, DSG4, DSG3 0%

161 TGA Loss 6p25.2-p25.1 1020,47 3983732 5004202 MaternalPRPF4B, FAM217A, C6orf201, ECI2, RP3-

400B16.1, CDYL, RPP400%

Supplementary Table 4. List of 78 validated rare CNVs (60 duplications and 18 deletions) in

unaffected parents having children with outflow tract defect. The frequencies of these CNVs

in the general population varies from 0% to 1% in some cases (according to the following

databases: 42 million probes study (Conrad et al., 2010), WTCCC study

http://www.wtccc.org.uk/, 1000 genomes project http://www.1000genomes.org/ and DDD

controls project http://www.ddduk.org/). When a CNV was found just once it was indicated

as a “singleton observation” and “doubleton observation” when a CNV was detected twice.

Trio ID Mapping (hg19)

Size (kb)

CNV’s Carrier

Type of CHD

Deletion/ duplication

Freq. in general population

100 chr1:100568471-100643738 75,267 Father CoA Duplication 0

100 chr17:34391756-34916684 574,928 Mother CoA Duplication 0



37 chr15:27188490-27611215 513,574 Mother CoA Duplication 1 singleton observation

50 chr9:95094453-95218885 124,432 Mother CoA Deletion 0

64 chr1:202860213-202920327 60,114 Father CoA Duplication 1 singleton observation


89 chr11:128346199-128350280 4,082 Mother CoA Duplication doubleton to 1%

90 chr14:30839437-31496524 657,087 Mother CoA Duplication 1 singleton observation

110 chr17:15719243-16055277 336,034 Father CoA Deletion doubleton to 1%


108 chr15:53997262-54015036 17,774 Mother CoA Deletion doubleton to 1%


226 chr10:99190389-99202998 12,609 Father and Mother CoA Duplication 0

226 chr15:102161841-102255214 93,373 Father CoA Duplication doubleton to 1%

190 chr11:107578575-107582633 4,058 Mother CoA Deletion 1 singleton observation






231 chr17:2861035-2995389 134,354 Father TGA Duplication 2 singleton observation

253 chrX:146990967-147063193 72,227 Mother TGA Duplication 0

177 chr1:200797675-200842926 45,251 Mother TGA Duplication doubleton to 1%

192 chr1:84610179-84663541 53,362 Mother TGA Duplication 0


249 chr5:78137626-78427922 290,296 Father TGA Duplication 0

243 chr8:2999966-3166063 166,097 Mother TGA Deletion 0




334 chrX:7810316-8139238 328,922 Mother TGA Duplication 0




http://www.wtccc.org.uk/

http://www.1000genomes.org/

http://www.ddduk.org/


312 chrX:6967019-8434412 1417,393 Father TGA Duplication 0



364 chr18:39623688-39664323 40,635 Father TGA Duplication doubleton to 1%









332 chr8:23049143-23082641 33,498 Father TGA Deletion 0


317 chr6:153073418-153080745 7,327 Father TGA Deletion 0

317 chr12:27450610-27788481 337,871 Mother TGA Deletion doubleton to 1%









410 chr6:1914752-2483597 568,845 Father TGA Duplication doubleton to 1%




58 chr10:82095853-82116463 20,61 Mother TOF Duplication 0


171 chr2:47596450-47613798 17,348 Father TOF Deletion 1 singleton observation


151 chr15:42705102-42720236 15,134 Mother TOF Deletion 0

146 chr17:73178996-73328908 149,912 Father TOF Duplication 0

122 chr16:3704209-3716095 11,886 Father TOF Deletion doubleton to 1%






516 chr20:29845480-30152020 306,54 Mother TOF Duplication doubleton to 1%

Supplementary Table 5. List of genes included in our CNVs predicted to have binding sites

for the FOXC1 transcription factor, 54/69 of the validated CNVs (77%) identified in affected

children contain at least one FOXC1 binding site as detected by TFBS.

Trio CNV TFBS

ID chr start end FOXC1 Binding Sites 212 chr2 28649512 29174327 PPP1CB, WDR43, FAM179A

163 chr7 2222167 2241694 near to MAD1L1

6 chr10 104818399 104847082 CNNM2

179 chr7 39993801 40102842 CDK13

174 chr11 47826590 48303958 PTPRJ, near to NUP160

44 chr16 66728053 66851930 NFATC3,ESRP2

59 chr7 141351858 141387062 MGAM

23 chr2 20267100 20314989 PUM2

77 chr13 75289358 75306459 LMO7

77 chr22 36939738 36957238 MAFF, TMEM184B

211 chr3 142755432 142868290 RASA2

226 chr15 83407124 83483235 PDE8A

437 chr9 3771683 4151396 GLIS3

437 chr9 5524835 5755850 KIAA1432

194 chr15 28983999 30191315 FAN1, MTMR10, LOC283710, KLF13

231 chr4 121835603 121917802 PRDM5

252 chr8 100274271 100472962 VPS13B

265 chr2 9442831 9567698 ASAP2, ADAM17

277 chr3 199050618 199291875 LRCH3

335 chr19 13999589 14020806 RLN3

351 chr10 103180049 103332582 BTRC

358 chr13 103959813 105607574 LINC00343, near to DAOA

172 chr6 118878090 119137929 CEP85L

172 chr8 53323590 53762104 ST18, RB1CC1

165 chr15 39451144 39476678 NDUFAF1

284 chr4 186795696 186862130 SORBS2

302 chr5 131275432 131340271 ACSL6

341 chr20 18110446 18453596 CSRP2BP, DZANK1, SEC23B

356 chr16 22264898 22338999 CDR2

393 chr5 96133154 96169574 ERAP1

294 chr13 22133248 23413626 SACS, TNFRSF19, MIPEP, C1QTNF9B-AS1, C1QTNF9B

295 chr7 158024743 158304209 PTPRN2, NCAPG2, ESYT2

329 chr12 110666925 110793312 MAPKAPK5

188 chr17 35066780 35286624 ERBB2, GRB7, IKZF3

24 chr17 28881049 29622308 ASIC2

205 chr2 63961233 64419035 PELI1

218 chr2 73139139 73195892 RAB11FIP5

193 chr3 110109374 110386546 MORC1

167 chr8 120697768 121249609 ENPP2, TAF2, DEPTOR

184 chrX 123337515 123698778 ODZ1

148 chr11 33147875 33335026 HIPK3

146 chr2 47390756 47789705 KCNK12

5 chr3 8894991 9027040 RAD18, SRGAP3

36 chr21 46514488 46680265 YBEY, PCNT

43 chr1 144472625 146317921 GPR89A, GPR89C, NBPF10, NBPF20, NBPF25P, LOC728989, FMO5, CHD1L, BCL9, ACP6, GPR89C,GPR89B

84 chr2 32669326 32904099 BIRC6, TTC27

93 chr21 21037030 22607602 NCAM2

106 chr15 55476358 55541243 CGNL1

217 chr4 77252645 77321279 NUP54

214 chr15 26999744 28013538 APBA2, FAM189A1, TJP1

53 chr1 148680022 149038853 GOLPH3L, HORMAD1, CTSS, CTSK

158 chr18 27191233 27303357 LOC101927718, near to DSG4

161 chr6 3928731 4949201 PRPF4B, C6orf201, CDYL

Supplementary Table 6. List of 113 candidate genes for CHD,

CANDIDATE GENE LIST

ACP6 DZANK1 LMO7 PPP1CB

ACSL6 EIF3B LOC101927718 PRDM5

ADAM17 ENPP2 LOC283710 PRPF4B

APBA2 ERAP1 LOC728989 PTPRJ

ASAP ERBB2 LRCH3 PTPRN2

ASIC2/ACCN1 ERMARD MAD1L1 PUM2

BCL9 ESRP2 MAFF RAB11FIP5

BIRC6 ESYT2 MAPKAPK5 RAD18

BTRC FAM179A MGAM RASA2

C1QTNF9B FAM189A1 MIPEP RB1CC

C1QTNF9B-AS1 FAN1 MORC1 RB1CC1

C6orf201 FMO5 MTMR10 RBBP9

CDK13 FOXC1 NBPF10 RIC8A

CDR2 FTSJ2 NBPF20 RLN3

CDYL GLIS3 NBPF25P SACS

CEP85L GOLPH3L NCAM2 SEC23B

CGNL1 GPR89A NCAPG2 SORBS2

CHD1L GPR89B NDUFAF1 SRGAP3

CHRNA7 GPR89C NFATC3 ST18

CHRNB3 GPR89C NFATC3 TAF2

CNNM2 GRB7 NUP160 TCAP

CSRP2BP HIPK3 NUP54 TCTE3

CTSK HORMAD1 ODZ1 TJP1

CTSS IKZF3 PCNT TMEM184B

CTSS KCNK12 PDE8A TNFRSF19

DAOA KIAA1432 PELI1 TTC27

DEPTOR KLF13 PLN VPS13B

DSG4 LINC00343 POLR3F YBEY

ZNF133

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