# Exome Sequencing Identifies Compound Heterozygous Mutations inSCN5A Associated with Congenital Complete Heart Block in the Thai Population

Authors: Chuphong Thongnak; Pornprot Limprasert; Duangkamol Tangviriyapaiboon; Suchaya Silvilairat; Apichaya Puangpetch; Ekawat Pasomsub; Chonlaphat Sukasem; Wasun Chantratita
Journal: Disease Markers
 (2016)

---

## Abstract

Background. Congenital heart block is characterized by blockage of electrical impulses from the atrioventricular node (AV node) to the ventricles. This blockage can be caused by ion channel impairment that is the result of genetic variation. This study aimed to investigate the possible causative variants in a Thai family with complete heart block by using whole exome sequencing.Methods. Genomic DNA was collected from a family consisting of five family members in three generations in which one of three children in generation III had complete heart block. Whole exome sequencing was performed on one complete heart block affected child and one unaffected sibling. Bioinformatics was used to identify annotated and filtered variants. Candidate variants were validated and the segregation analysis of other family members was performed.Results. This study identified compound heterozygous variants, c.101G>A and c.3832G>A, in theSCN5A gene and c.28730C>T in theTTN gene.Conclusions. Compound heterozygous variants in theSCN5A gene were found in the complete heart block affected child but these two variants were found only in the this affected sibling and were not found in other unaffected family members. Hence, these variants in theSCN5A gene were the most possible disease-causing variants in this family.


---

## Body

## 1. Introduction

Congenital heart block is an uncommon disorder that occurs in about 1 in 20,000 live births [1]. It is characterized by anatomical or functional impairment in the conduction system which is caused by blockage of electrical impulses from atrioventricular node (AV node) to the ventricles [2]. The severity of heart block ranges from first-degree in which electrical impulse to the AV node is slower than normal to third-degree or complete heart block in which electrical impulses from the atrium do not reach ventricles at all [3].The conduction defect can be caused by a defective link between cardiomyocytes or by ion channel impairment that changes action potential shapes [4]. Inherited defects in cardiac conduction have been linked to genetic variants in several genes such asSCN5A,SCN1B,KCNJ2,HCN4, NKX2-5, TBX5, LMNA, andANKB [4–9]. Among these genes,SCN5A has been frequently reported with various phenotypes [10].SCN5A encodes α subunit of the cardiac sodium channel (NaV1.5) which controls the flow of sodium ions into cells that is essential in generation and transmission of electrical impulses [11]. A nonfunctional protein, which is caused by a mutation in theSCN5A gene, reduces entrance of sodium into the cells that results in difficulty producing and transmitting electrical signals resulting in heart block [12].Mutations inSCN5A, which lead to loss or gain of sodium channel function, are associated with a spectrum of cardiac diseases including Brugada syndrome, Long QT syndrome type 3, sick sinus syndrome, and progressive familial heart block [12–16].With the advantages of next-generation sequencing especially whole exome sequencing that can explore the sequence of all exons in a single experiment, sequencing has been used in several studies for comprehensive and unbiased identification of causative variants of diseases in the last decade [17, 18]. Likewise, whole exome sequencing has been used in other cardiovascular related studies to identify disease-causing mutations of familial atrial septal defects [19].This study aims to investigate the possible causative variants in a Thai family with complete heart block by using whole exome sequencing. A combined method of familial data, exome sequencing, bioinformatics, and segregation analysis was able to identify 6 variants in 5 genes in which 2 variants inSCN5A were the most plausible disease-causing variants for heart block.


## 2. Materials and Methods

### 2.1. Subjects

The family in this study had 3 generations from which blood samples for DNA preparations were collected: grandmother (I-2), mother (II-2), and two children (III-2 and III-3). The II-2 was a single mother; therefore, we could not obtain blood for DNA from the father. The index case (III-2) had a third-degree AV block and had undergone cardiac surgery for epicardial pacemaker implantation at the age of 18 months. She was diagnosed with autism according to the DSM-IV at the age of 6 years. The other two siblings had no heart defects but one had learning disability (III-1, no DNA) and the other was autistic (III-3) (Figure1). Extracted DNA samples from III-2 and III-3 were controlled for their quality by measuring DNA concentration using Nanodrop ND1000 and measuring fragmentation of DNA by agarose gel electrophoresis. These 2 samples were prepared for whole exome sequencing. DNA samples from other family members (I-2, II-2) were prepared for segregation analysis. Written informed consent was obtained from adult family members themselves and for all children and all procedures were approved by the Institutional Review Board (MURA2012/02/SN1) of the Faculty of Medicine Ramathibodi Hospital, Mahidol University, and EC48/364-006-3 of the Faculty of Medicine, Prince of Songkla University.Figure 1
Electrocardiography of index case (III-2) shows third-degree atrioventricular block, atrial rate 100/min, ventricular rate 49/min, and ventricular pacing spikes before some QRS complexes.


### 2.2. Exome Sequencing and Data Analysis

Whole exome sequencing was performed on family members III-2 and III-3. Samples were prepared following standard SOLID 5500xl (Applied Biosystems, California, USA) protocols for whole exome sequencing. Three micrograms of genomic DNA of each sample were fragmented using Covaris S2 (Covaris, Massachusetts, USA) and were captured for exome sequencing using TargetSeq Exome and Custom Enrichment System (Invitrogen, California, USA). The captured DNA was sequenced with 150 bp paired-end read on the SOLID 5500xl system according to the manufacturer’s protocol. Primary analysis was performed on the sequencing machine using SOLID ICS software; then, raw sequenced data was transferred to the LifeScope Genomic Analysis server for secondary analysis. Sequence reads were mapped to the human reference genome assembly hg19 (GRCh37); then, variants calling for SNV, insertions, and deletions were performed.Tertiary analysis was performed by using Golden Helix SVS software on identified candidate variants. Variants were filtered for minimum genotype quality of 20 and minimum coverage depths of 10 and then the qualified variants were annotated with the UCSC KnownGenes database to remove noncoding and synonymous variants. High frequency variants (minor allele frequencies greater than 2%) were excluded by annotation with allele frequencies from the 1000 Genomes Project Phase 3 [20] and an in-house exome database which consists of 172 Thai individuals. Deleterious protein functions were predicted using dbNSFP that compiled prediction scores from eleven prediction algorithms (SIFT, Polyphen2, LRT, MutationTaster, MutationAssessor, FATHMM, VEST3, CADD, MetaLR, MetaSVM, and PROVEAN) and 4 conservation scores, PhyloP, phastCons, GERP++, and SiPhy, and other related information [21]. Variants that were predicted not to alter protein function by any algorithm were excluded in this step. To narrow down variants, variants were focused on where these were located in the candidate genes list (Table 1). This genes list was created from combined known and suspected genes involved in the cardiovascular system from several sources and the cardiovascular defective candidate genes from the Enlis Genome Research software (Enlis, Berkeley, CA) gene panel. This genes list consists of 359 genes in total. Lastly, variants were filtered by their genotype. A genotype that was only in family member III-2 was indicated as a candidate variant for heart block. The summarized variants filter steps are shown in Figure 3.Table 1
List of genes involved in cardiovascular system defects.
AARS2 ATP6V0A2 CSRP3 FASTKD2 HAND1 LMF1 MYH11 PLN SFTPA1 TGFB2 ABCA1 ATRX CTF1 FBLN5 HAND2 LMNA MYH6 PLOD1 SFTPA2 TGFB3 ABCA3 B3GAT3 CTNNA3 FBN1 HCN4 LPIN1 MYH7 PNPLA3 SFTPB TGFBR1 ABCC6 BAG3 DES FBN2 HERG LPL MYL2 PPARA SFTPC TGFBR2 ABCC9 BCOR DHCR24 FGD1 HFE LRP5 MYL3 PPARG SFTPD THEMIS ABCG5 BMPR2 DHCR7 FGFR2 HOXA1 LTBP4 MYLK PPP1R17 SGCD TLL1 ABCG8 BRAF DLL3 FHL1 HRAS MAP2K1 MYLK2 PRDM16 SGCG TMEM43 ACADVL CACNA1B DMD FHL2 IGBP1 MAP2K2 MYO6 PRKAB2 SHOC2 TMEM70 ACTA1 CACNA1C DMPK FKTN ILK MED12 MYOCD PRKAG2 SKI TMPO ACTA2 CACNA1D DNAH11 FLNA IRX4 MEF2C MYOM1 PRKAR1A SLC25A3 TNNC1 ACTC CACNA2D1 DNAH5 FLNB JAG1 MHY11 MYOT PRKG1 SLC25A4 TNNI3 ACTC1 CACNB2 DNAI1 FOXC2 JPH2 MIB1 MYOZ2 PSEN1 SLC2A10 TNNT2 ACTN2 CALM1 DNAJC19 FOXH1 JUP MID1 MYPN PSEN2 SLMAP TNXB ACVR2B CALM2 DNM1L FOXRED1 KCNA5 MKKS NEBL PTPN11 SMAD3 TOPBP1 ACVRL1 CALR3 DOLK FRYL KCND3 MKS1 NEXN PTRF SMAD4 TPM1 ADCK3 CASQ2 DPP6 FXN KCNE1 MOG1 NF1 RAF1 SMAD9 TPM2 ADRB1 CAV1 DSC2 GAA KCNE1L MRPL3 NIPBL RAI1 SNTA1 TRDN AGL CAV3 DSG2 GATA4 KCNE2 MTND1 NKX2.5 RANGRF SNX3 TRIM63 AKAP9 CBL DSP GATA5 KCNE3 MTND5 NKX2.6 RASA1 SOS1 TRPM4 AKT3 CBS DTNA GATA6 KCNH2 MTND6 NKX2-5 RBM10 SOX2 TSFM ALMS1 CFC1 EFEMP2 GATAD1 KCNJ2 MTTD NODAL RBM20 SOX7 TTN ALPK3 CHD7 EIF2AK4 GATT6 KCNJ5 MTTG NOS1AP RET SPEG TTR ANGPTL3 CHST14 ELMOD2 GDF1 KCNJ8 MTTH NOTCH1 RPL4 SPRED1 TWIST1 ANGPTL4 COA5 ELN GDF2 KCNK3 MTTI NOTCH2 RPSA SURF1 TXNRD2 ANK2 COL18A1 EMD GJA1 KCNQ1 MTTK NOTCH3 RYR1 SYNE1 UQCRB ANKRD1 COL1A1 ENG GJA5 KRAS MTTL1 NPC1 RYR2 SYNE2 USF1 ANO5 COL1A2 EPHX2 GLA LAMA4 MTTL2 NPHP3 SALL1 TAZ VCL APOA1 COL2A1 ESCO2 GLB1 LAMP2 MTTM NPPA SALL4 TBX1 VCP APOA2 COL3A1 EVC GLI3 LBR MTTP NRAS SCN1B TBX20 VHL APOA5 COL4A1 EVC2 GNAI2 LCAT MTTQ NSDHL SCN2B TBX3 XK APOB COL5A1 EYA1 GPC3 LDB3 MTTS1 NUBPL SCN3B TBX5 ZASP APOC2 COL5A2 EYA4 GPD1L LDLR MTTS2 PCSK9 SCN4B TCAP ZFPM2 APOE CREBBP FANCA GPIHBP1 LDLRAP1 MUC5B PDLIM3 SCN5A TCTN3 ZIC3 ARHGAP31 CRELD1 FANCC GSN LEFTY2 MYBPC3 PEX7 SCO2 TERC ZMPSTE24 ARX CRYAB FANCD2 GUSB LIPC MYCN PKP2 SDHA TERT ZNF469 ATP5E CSF2RA FANCE HADH LIPI MYF6 PKP4 SEMA5A TFAP2BAdditionally, pathogenic variants which were related to other diseases followed were explored in The American College of Medical Genetics and Genomics (ACMG) recommendations for reporting of incidental findings in clinical exome and genome sequencing [22]. Known pathogenic variants were identified by using The Human Gene Mutation Database (HGMD) [23].


### 2.3. Variant Validations and Segregation Analysis

Sanger sequencing was used to validate the candidate variants found in whole exome sequencing and segregation analyses were performed on the family members. Primers were designed using the Primer3 version 0.4.0 web-based tool [24]. Sequencing reactions were performed using Applied Biosystems 3130 DNA Analyzer (Life Technologies, Carlsbad, CA, USA).


## 2.1. Subjects

The family in this study had 3 generations from which blood samples for DNA preparations were collected: grandmother (I-2), mother (II-2), and two children (III-2 and III-3). The II-2 was a single mother; therefore, we could not obtain blood for DNA from the father. The index case (III-2) had a third-degree AV block and had undergone cardiac surgery for epicardial pacemaker implantation at the age of 18 months. She was diagnosed with autism according to the DSM-IV at the age of 6 years. The other two siblings had no heart defects but one had learning disability (III-1, no DNA) and the other was autistic (III-3) (Figure1). Extracted DNA samples from III-2 and III-3 were controlled for their quality by measuring DNA concentration using Nanodrop ND1000 and measuring fragmentation of DNA by agarose gel electrophoresis. These 2 samples were prepared for whole exome sequencing. DNA samples from other family members (I-2, II-2) were prepared for segregation analysis. Written informed consent was obtained from adult family members themselves and for all children and all procedures were approved by the Institutional Review Board (MURA2012/02/SN1) of the Faculty of Medicine Ramathibodi Hospital, Mahidol University, and EC48/364-006-3 of the Faculty of Medicine, Prince of Songkla University.Figure 1
Electrocardiography of index case (III-2) shows third-degree atrioventricular block, atrial rate 100/min, ventricular rate 49/min, and ventricular pacing spikes before some QRS complexes.


## 2.2. Exome Sequencing and Data Analysis

Whole exome sequencing was performed on family members III-2 and III-3. Samples were prepared following standard SOLID 5500xl (Applied Biosystems, California, USA) protocols for whole exome sequencing. Three micrograms of genomic DNA of each sample were fragmented using Covaris S2 (Covaris, Massachusetts, USA) and were captured for exome sequencing using TargetSeq Exome and Custom Enrichment System (Invitrogen, California, USA). The captured DNA was sequenced with 150 bp paired-end read on the SOLID 5500xl system according to the manufacturer’s protocol. Primary analysis was performed on the sequencing machine using SOLID ICS software; then, raw sequenced data was transferred to the LifeScope Genomic Analysis server for secondary analysis. Sequence reads were mapped to the human reference genome assembly hg19 (GRCh37); then, variants calling for SNV, insertions, and deletions were performed.Tertiary analysis was performed by using Golden Helix SVS software on identified candidate variants. Variants were filtered for minimum genotype quality of 20 and minimum coverage depths of 10 and then the qualified variants were annotated with the UCSC KnownGenes database to remove noncoding and synonymous variants. High frequency variants (minor allele frequencies greater than 2%) were excluded by annotation with allele frequencies from the 1000 Genomes Project Phase 3 [20] and an in-house exome database which consists of 172 Thai individuals. Deleterious protein functions were predicted using dbNSFP that compiled prediction scores from eleven prediction algorithms (SIFT, Polyphen2, LRT, MutationTaster, MutationAssessor, FATHMM, VEST3, CADD, MetaLR, MetaSVM, and PROVEAN) and 4 conservation scores, PhyloP, phastCons, GERP++, and SiPhy, and other related information [21]. Variants that were predicted not to alter protein function by any algorithm were excluded in this step. To narrow down variants, variants were focused on where these were located in the candidate genes list (Table 1). This genes list was created from combined known and suspected genes involved in the cardiovascular system from several sources and the cardiovascular defective candidate genes from the Enlis Genome Research software (Enlis, Berkeley, CA) gene panel. This genes list consists of 359 genes in total. Lastly, variants were filtered by their genotype. A genotype that was only in family member III-2 was indicated as a candidate variant for heart block. The summarized variants filter steps are shown in Figure 3.Table 1
List of genes involved in cardiovascular system defects.
AARS2 ATP6V0A2 CSRP3 FASTKD2 HAND1 LMF1 MYH11 PLN SFTPA1 TGFB2 ABCA1 ATRX CTF1 FBLN5 HAND2 LMNA MYH6 PLOD1 SFTPA2 TGFB3 ABCA3 B3GAT3 CTNNA3 FBN1 HCN4 LPIN1 MYH7 PNPLA3 SFTPB TGFBR1 ABCC6 BAG3 DES FBN2 HERG LPL MYL2 PPARA SFTPC TGFBR2 ABCC9 BCOR DHCR24 FGD1 HFE LRP5 MYL3 PPARG SFTPD THEMIS ABCG5 BMPR2 DHCR7 FGFR2 HOXA1 LTBP4 MYLK PPP1R17 SGCD TLL1 ABCG8 BRAF DLL3 FHL1 HRAS MAP2K1 MYLK2 PRDM16 SGCG TMEM43 ACADVL CACNA1B DMD FHL2 IGBP1 MAP2K2 MYO6 PRKAB2 SHOC2 TMEM70 ACTA1 CACNA1C DMPK FKTN ILK MED12 MYOCD PRKAG2 SKI TMPO ACTA2 CACNA1D DNAH11 FLNA IRX4 MEF2C MYOM1 PRKAR1A SLC25A3 TNNC1 ACTC CACNA2D1 DNAH5 FLNB JAG1 MHY11 MYOT PRKG1 SLC25A4 TNNI3 ACTC1 CACNB2 DNAI1 FOXC2 JPH2 MIB1 MYOZ2 PSEN1 SLC2A10 TNNT2 ACTN2 CALM1 DNAJC19 FOXH1 JUP MID1 MYPN PSEN2 SLMAP TNXB ACVR2B CALM2 DNM1L FOXRED1 KCNA5 MKKS NEBL PTPN11 SMAD3 TOPBP1 ACVRL1 CALR3 DOLK FRYL KCND3 MKS1 NEXN PTRF SMAD4 TPM1 ADCK3 CASQ2 DPP6 FXN KCNE1 MOG1 NF1 RAF1 SMAD9 TPM2 ADRB1 CAV1 DSC2 GAA KCNE1L MRPL3 NIPBL RAI1 SNTA1 TRDN AGL CAV3 DSG2 GATA4 KCNE2 MTND1 NKX2.5 RANGRF SNX3 TRIM63 AKAP9 CBL DSP GATA5 KCNE3 MTND5 NKX2.6 RASA1 SOS1 TRPM4 AKT3 CBS DTNA GATA6 KCNH2 MTND6 NKX2-5 RBM10 SOX2 TSFM ALMS1 CFC1 EFEMP2 GATAD1 KCNJ2 MTTD NODAL RBM20 SOX7 TTN ALPK3 CHD7 EIF2AK4 GATT6 KCNJ5 MTTG NOS1AP RET SPEG TTR ANGPTL3 CHST14 ELMOD2 GDF1 KCNJ8 MTTH NOTCH1 RPL4 SPRED1 TWIST1 ANGPTL4 COA5 ELN GDF2 KCNK3 MTTI NOTCH2 RPSA SURF1 TXNRD2 ANK2 COL18A1 EMD GJA1 KCNQ1 MTTK NOTCH3 RYR1 SYNE1 UQCRB ANKRD1 COL1A1 ENG GJA5 KRAS MTTL1 NPC1 RYR2 SYNE2 USF1 ANO5 COL1A2 EPHX2 GLA LAMA4 MTTL2 NPHP3 SALL1 TAZ VCL APOA1 COL2A1 ESCO2 GLB1 LAMP2 MTTM NPPA SALL4 TBX1 VCP APOA2 COL3A1 EVC GLI3 LBR MTTP NRAS SCN1B TBX20 VHL APOA5 COL4A1 EVC2 GNAI2 LCAT MTTQ NSDHL SCN2B TBX3 XK APOB COL5A1 EYA1 GPC3 LDB3 MTTS1 NUBPL SCN3B TBX5 ZASP APOC2 COL5A2 EYA4 GPD1L LDLR MTTS2 PCSK9 SCN4B TCAP ZFPM2 APOE CREBBP FANCA GPIHBP1 LDLRAP1 MUC5B PDLIM3 SCN5A TCTN3 ZIC3 ARHGAP31 CRELD1 FANCC GSN LEFTY2 MYBPC3 PEX7 SCO2 TERC ZMPSTE24 ARX CRYAB FANCD2 GUSB LIPC MYCN PKP2 SDHA TERT ZNF469 ATP5E CSF2RA FANCE HADH LIPI MYF6 PKP4 SEMA5A TFAP2BAdditionally, pathogenic variants which were related to other diseases followed were explored in The American College of Medical Genetics and Genomics (ACMG) recommendations for reporting of incidental findings in clinical exome and genome sequencing [22]. Known pathogenic variants were identified by using The Human Gene Mutation Database (HGMD) [23].


## 2.3. Variant Validations and Segregation Analysis

Sanger sequencing was used to validate the candidate variants found in whole exome sequencing and segregation analyses were performed on the family members. Primers were designed using the Primer3 version 0.4.0 web-based tool [24]. Sequencing reactions were performed using Applied Biosystems 3130 DNA Analyzer (Life Technologies, Carlsbad, CA, USA).


## 3. Results

A family with complete heart block in only one of the 3rd generation family members was explored. By whole exome sequencing, a total of 99,834 variants in family members III-2 and III-3 were detected with an average depth over 60x coverage. After removal of low quality, noncoding and synonymous variants, 14,284 variants remained. Subsequently, variants were reduced by a filtering pipeline that included variants with minor allele frequencies, inheritance models, and a candidate gene list which reduced the number of variants to 36 variants in 28 genes. Finally, variants were prioritized and selected as candidate variants by annotated information from the deleterious protein function prediction database that resulted in 21 heart defective candidate variants in 18 genes. A list of candidate variants is shown in Table2. All 21 candidate variants were then investigated for validation and segregation analysis. Six variants successfully passed this step while the other variants were dropped for 2 reasons: (1) a discordance between whole exome sequencing and Sanger sequencing or (2) variants found in the affected child being found in other unaffected family members except for two or more variants that were found in the same gene which indicated compound heterozygous inheritance. These 6 variants in 5 genes were identified as candidate variants for heart defects in this family. The list of final candidate variants is shown in Table 3.Table 2
Candidate variants from whole exome sequencing.
Position Gene Classification Transcript HGVS coding dbSNP Chr1: 13036736 PRAMEF22 Nonsyn SNV NM_001100631 c.808T>A rs202011965 Chr2: 179402104 TTN Nonsyn SNV NM_003319 c.72635G>A — Chr2: 179542464 TTN Nonsyn SNV NM_133378 c.30443C>T — Chr2: 179549988 TTN Nonsyn SNV NM_133378 c.28730C>T rs146400809 Chr2: 203395591 BMPR2 Nonsyn SNV NM_001204 c.1042G>A rs201067849 Chr3: 38607905 SCN5A Nonsyn SNV NM_000335 c.3832G>A rs199473341 Chr3: 38674698 SCN5A Nonsyn SNV NM_000335 c.101G>A rs199473046 Chr3: 132438619 NPHP3 Nonsyn SNV NM_153240 c.449C>T rs142663818 Chr7: 42064927 GLI3 Nonsyn SNV NM_000168 c.1292A>G — Chr9: 34506694 DNAI1 Nonsyn SNV NM_012144 c.1133A>T — Chr9: 97080945 FAM22F Del NM_017561 c.2071_2073delTCT rs150455117 Chr11: 1267969 MUC5B Nonsyn SNV NM_002458 c.9859A>C — Chr11: 1271591 MUC5B Nonsyn SNV NM_002458 c.13481A>C rs201038498 Chr11: 47356616 MYBPC3 Unknown NM_000256 c.2883C>T — Chr11: 126143258 FOXRED1 Frameshift Del NM_017547 c.445delC — Chr12: 58177067 TSFM Splicing NM_001172695 c.231+1_231+2delGT — Chr12: 112892433 PTPN11 Stop-gain NM_002834 c.591T>G rs76982592 Chr16: 1245007 CACNA1H Nonsyn SNV NM_001005407 c.335T>C — Chr16: 71061495 HYDIN Stop-loss NM_017558 c.3052T>C rs146649547 Chr16: 89815152 FANCA Nonsyn SNV NM_000135 c.3263C>T rs17233497 Chr21: 35821734 KCNE1 Nonsyn SNV NM_001127670 c.199C>T rs199473645Table 3
List of final candidate variants.
Genomic coordinates G e n o t y p e ∗ AA change Gene MAF Functional prediction III-2 III-3 II2 I2 1kG (ASN) Thai SIFT Polyphen 2 HumVar Mutation taster Chr2: 179549988 A/G G/G G/G G/G p.Pro9577Leu TTN 0.01 0 Damaging Probably damaging Disease causing Chr3: 38607905 C/T C/T C/C C/C p.Val1278Ile SCN5A 0 0 Damaging Probably damaging Disease causing Chr3: 38674698 C/T C/C C/T C/C p.Arg34His SCN5A 0 0 Damaging Possibly damaging Disease causing Chr3: 132438619 A/G G/G G/G G/G p.Ala150Val NPHP3 0.02 0.0067 Damaging Probably damaging Disease causing Chr9: 34506694 A/T A/A A/A A/A p.Tyr378Phe DNAI1 0 0 Damaging Benign Disease causing Chr11: 1271591 A/G A/A A/A A/A p.Lys4494Thr MUC5B 0 0.0067 Damaging Benign Polymorphism G   ∗ e n o t y p e s in this table are FWD genotype while genotypes in HGVS are REV genotype.Two nonsynonymous missense variants in theSCN5A gene in this study, c.101G>A and c.3832G>A (NM_000335), were likely to be present in a compound heterozygous fashion because 2 heterozygous variants were found in same gene in the affected case but only one or none of them were found in unaffected family members. Heterozygous c.101G>A was found in III-2 (index) and II-2 (mother) while heterozygous c.3832G>A was found in III-2 (index) and III-3 (brother) (Figure 2). Chromatograms of both variants in all available subjects are shown in Figure 4.Figure 2
The lineage of the family with complete heart block indicated by the dark symbol, females by circles, and males by squares. Letters (a) and (b) indicate genotypes of 2 variants in theSCN5A gene for c.101G>A and c.3832G>A. The homozygous reference genotypes are indicated by (−/−) and heterozygous alternate genotypes by (−/+). The genotypes of II-1 are presumed genotypes which are inferred from his children.Figure 3
Filtering procedure of variants obtained by whole exome sequencing 2nd data analysis.Figure 4
Chromatograms of 2 heterozygous missense variants inSCN5A gene: c.101G>A (a) and c.3832G>A (b). Letters in [] indicate complementary (FWD) alleles.
(a)
SCN5A  c.101G>A [C>T] (b)
SCN5A  c.3832G>A [C>T]All other 4 variants were nonsynonymous missense variants in which their genotypes were heterozygous. These variants consisted of c.28730C>T (NM_133378) in theTTN gene, c.449C>T (NM_153240) in theNPHP3 gene, c.1133A>T (NM_012144) in theDNAI1 gene, and c.13481A>C (NM_002458) in theMUC5B gene.TTN encoded Titin or connectin, a giant muscle protein, is expressed in the cardiac and skeletal muscles.NPHP3 encodes a protein that is required for normal ciliary development.DNAI1 encodes a member of the dynein intermediate chain family. Lastly,MUC5B encodes a protein member of the mucin family.Finally, incidental findings in the whole exome data in this family following ACMG recommendations were explored. Aside from variants in theSCN5A gene that were assigned in the ACMG panel as known/expected pathogenic variants for Romano-Ward Long QT syndromes Types 1, 2, and 3 and Brugada syndrome, which were key variants in this family, a variant in theMYBPC3 gene that was assigned in the ACMG panel for hypertrophic/dilated cardiomyopathy was explored and validated.


## 4. Discussion

Although a congenital heart defect was found in this autistic patient, this was unlike the Timothy syndrome which is a rare disorder that affects heart, nervous system, and fingers/toes. Syndactyly, the webbing of fingers and toes, one of Timothy syndrome signs, was not found in this case. Moreover, by direct sequencing and whole exome sequencing, variants in theCACNA1C gene, which are the common causes of both classical and atypical Timothy syndrome, were not detected [25]. Therefore, it was inferred that heart block and autism in this family are a coincidence.SCN5A, a cardiac sodium channel gene, is important in generation and transmission of electrical impulses by its role in controlling the flow of sodium ions into cells [11]. Several variants in theSCN5A that are located in four homologous domains of alpha subunit of NaV1.5, including 6 segments of each domain and linkers, N-terminal, and C-terminal, have been reported to be associated with various phenotypes of cardiac diseases [26]. The mechanism ofSCN5A mutations associated with cardiac conduction disease has been explained by loss of function in NaV1.5 channels. Loss of NaV1.5 function leads to a reduction of inward sodium flow in the cells that results in difficulty producing and transmitting electrical signals. Conduction defects, however, could occur apart from transmission of inward sodium. The mutations onSCN5A, p.R219H, p.R222Q, and p.R225W which express alternative pathways, through a cation leak through NaV1.5, have been reported to be associated with mixed arrhythmias and dilated cardiomyopathy [27, 28]. In this study, 2 missense variants in theSCN5A gene in a patient who had complete heart block (III-2) were identified. The first variant, c.101G>A, results in replacement of arginine by histidine (p.R34H) in exon 2 whereas the second variant, c.3832G>A, results in replacement of valine by isoleucine (p.V1278I) in exon 21. Both variants were rare variants in which alternate alleles were not found in the Asian population from 1000 genome databases and the Thai population from the Thai in-house exome database. Functional prediction results show that both variants were predicted to alter protein function which indicates that these variants have a high possibility to damage function of the sodium channel and cause the conduction defect in this case (Table 3).c.101G>A (p.R34H) is a novel variant which is located in the N-terminal cytoplasmic domain of the sodium channel alpha subunit (Figure5). A variant (c.80G>A) in the same region, the N-terminal cytoplasmic domain of sodium channel, has been reported to be associated with Brugada syndrome by Priori et al. [29]. Makita et al. have reported a mutation, which resulted in a stop codon (p.Q55X), presented in a Brugada syndrome affected patient with 1st-degree AV block history [30].Figure 5
Schematic of the transmembrane topology of theSCN5A protein. The location of the variants R34H (c.101G>A) and V1278I (c.3832G>A) is shown.c.3832G>A (p.V1278I) has been reported as a “disease causing mutation” of dilated cardiomyopathy in the HGMD database [31–33]. This variant is located in the S3 transmembrane segment of domain III (DIII) of the sodium channel alpha subunit. McNair et al. have reported that heterozygous c.3823G>A variant, which is located near c.3832G>A, was associated with dilated conduction disorder, cardiomyopathy, and arrhythmia [34]. In the same region, an association between atrial standstill and p.D1275N with polymorphisms in other gap junction protein, connexin40, has been reported by Groenewegen et al. [35]. Electrophysiological studies in xenopus oocytes showed that the c.3823G>A mutation results in an activation curve shift of the sodium channel conductance [35]. This activation curve shift toward more positive voltages may result in reduced excitability of myocytes. According to studies of Gosselin-Badaroudine et al. and Moreau et al., this electrical disturbance could be the result from positive charge leakages of mutated NaV1.5 channels that could lead to a Na+ leak into cardiac myocytes [27, 28].Results from segregation analysis indicated that both variants in theSCN5A gene were most possibly inherited in a compound heterozygous manner. Heterozygous c.101G>A was present in family members III-2 (index) and II-2 but was absent in family members I-2 and III-3. Likewise, heterozygous c.3832G>A was present in family members III-2 (index) and III-3 but was absent in family members I-2 and II-2 while only III-2 was the heart block affected family member. Although most of reported variants in theSCN5A gene were autosomal dominant, however, variants that were inherited in compound heterozygous individuals, similar to the current case finding, have been previously reported.For instance, Bezzina et al. described compound heterozygous inheritance of 2 variants in theSCN5A gene which was associated with severe cardiac conduction disturbances and degenerative changes in the conduction system [36]. A nonsense p.W156X, which is located in the S1-S2 linker of domain I, was inherited from the father and a missense p.R225W, which was located in S4 segment of domain I, was inherited from the mother [36].Benson et al. studied compound heterozygous variants inSCN5A in three families with congenital sick sinus syndrome [13]. A heterozygous missense p. P1298L, which is located in S4 segment of domain III, and p.G1408R, which is located in S5-S6 linker of domain III, were found in three siblings of the first family. Heterozygous missense p.T220I and p.R1623X were found in an individual of the second family. These two variants were located in the S4 segment of domain I and S4 segment of domain IV. The third family presented heterozygous deletions p.delF1617 in S3-S4 linker of domain IV and the missense p.R1632H in S4 segment of domain IV [13].Beside the variants inSCN5A gene, 4 heterozygous missense variants inTTN,NPHP3,DNAI1, andMUC5B genes were found in the present case. All variants were found in only the index case, family member III-2. Among these 4 genes, TTN was the most likely cardiac disease associated gene while lack of evidence forNPHP3,DNAI1, andMUC5B genes existed. Mutations inTTN have been reported in about 18% of sporadic dilated cardiomyopathies and 25% of familial autosomal dominant cardiomyopathies and rarely caused hypertrophic cardiomyopathies [37].Since this study only focused on the coding variants in the exon by whole sequencing, noncoding variants and structural variants were not explored. Moreover, it should be noted that novel variants in the study were found by the bioinformatics process so determination of biological functions will be needed in further studies.


## 5. Conclusion

In conclusion, this study demonstrated the potential of whole exome sequencing and a bioinformatics pipeline to identify the possible causative variants of complete heart block in a Thai family. The investigation found compound heterozygous variants in theSCN5A, cardiac sodium channel subunit gene, of which one was a novel variant and another one was a known pathogenic variant. Additionally, a heterozygous missense variant in theTTN, titin or connectin gene, has also been identified.



---

*Source: 3684965-2016-11-28.xml*