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Whole exome sequencing identifies novel mutation in eight Chinese children with isolated tetralogy of Fallot

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Oncotarget. 2017; 8:106976-106988. https://doi.org/10.18632/oncotarget.22202

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Lin Liu _, Hong-Dan Wang, Cun-Ying Cui, Yun-Yun Qin, Tai-Bing Fan, Bang-Tian Peng, Lian-Zhong Zhang and Cheng-Zeng Wang

Abstract

Lin Liu1,*, Hong-Dan Wang2,*, Cun-Ying Cui1, Yun-Yun Qin1, Tai-Bing Fan3, Bang-Tian Peng3, Lian-Zhong Zhang1 and Cheng-Zeng Wang4

1Department of Cardiovascular Ultrasound, Henan Provincial People’s Hospital, Zhengzhou University People’s Hospital, Zhengzhou 450003, China

2Institute of Medical Genetics, Henan Provincial People’s Hospital, Zhengzhou University People’s Hospital, Zhengzhou 450003, China

3Children’s Heart Center, Henan Provincial People’s Hospital, Zhengzhou University People’s Hospital, Zhengzhou 450003, China

4Department of Ultrasound, The Affiliated Cancer Hospital, Zhengzhou University, Zhengzhou 450008, China

*These authors have contributed equally to this work

Correspondence to:

Lin Liu, email: [email protected]

Keywords: tetralogy of Fallot; congenital heart disease; whole exome sequencing

Received: April 05, 2017     Accepted: September 05, 2017     Published: October 31, 2017

ABSTRACT

Background: Tetralogy of Fallot is the most common cyanotic congenital heart disease. However, its pathogenesis remains to be clarified. The purpose of this study was to identify the genetic variants in Tetralogy of Fallot by whole exome sequencing.

Methods: Whole exome sequencing was performed among eight small families with Tetralogy of Fallot. Differential single nucleotide polymorphisms and small InDels were found by alignment within families and between families and then were verified by Sanger sequencing. Tetralogy of Fallot-related genes were determined by analysis using Gene Ontology /pathway, Online Mendelian Inheritance in Man, PubMed and other databases.

Results: A total of sixteen differential single nucleotide polymorphisms loci and eight differential small InDels were discovered. The sixteen differential single nucleotide polymorphisms loci were located on Chr 1, 2, 4, 5, 11, 12, 15, 22 and X. Among the sixteen single nucleotide polymorphisms loci, six has not been reported. The eight differential small InDels were located on Chr 2, 4, 9, 12, 17, 19 and X, whereas of the eight differential small InDels, two has not been reported. Analysis using Gene Ontology /pathway, Online Mendelian Inheritance in Man, PubMed and other databases revealed that PEX5, NACA, ATXN2, CELA1, PCDHB4 and CTBP1 were associated with Tetralogy of Fallot.

Conclusions: Our findings identify PEX5, NACA, ATXN2, CELA1, PCDHB4 and CTBP1 mutations as underlying genetic causes of isolated tetralogy of Fallot.


INTRODUCTION

Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart disease (CHD) with an incidence rate of 1/3600 in live births and 10% in CHD [1]. TOF is characterised by cardiac outflow tract malformation caused by non-uniform separation between truncus arteriosus and bulbus arteriosus during embryo stage. TOF pathological features include ventricular septal defect, aortic overriding, right ventricular outflow tract stenosis or pulmonary artery stenosis and right ventricular hypertrophy. In neonatal period, children with TOF may manifest oxygen deficiency, pneumonia, intractable congestive heart failure and other complications with high early mortality. Despite TOF treatment in children, some complications will still occur because of poor prognosis. TOF brings heavy burden to the family and society. Therefore, to investigate TOF etiology, possible pathogenesis and risk factors for prenatal diagnosis and counselling and prognostic evaluation is of great significance.

Heart development is a complex and orderly process including cardiac tube formation, loop formation, intracardiac separation and vascular connection. Heart development is related to many genes, which express and interact in different spaces and at different times to form a precise regulatory mechanism. Abnormal expression occurring in any of these genes is likely to affect heart development, leading to heart malformation. For TOF, embryonic developmental mechanism is nearly definite. However, the molecular pathogenesis remains to be clarified. TOF is associated with gene mutation [2]. TOF-related genes include NKX2-5 [3], GATA4 [3, 4], and JAG-1 [5, 6]. Approximately 15% of TOF are from 22q11 microdeletion syndrome [7] caused by chromosome 22 long arm 1 zone 1 subzone deletion and manifest extracardiac abnormalities, such as abnormal face, thymic hypoplasia, cleft palate and hypocalcemia. In addition, TOF is also related to chromosomal aneuploidy, and approximately 3% of TOF is from 21-trisomy syndrome [8]. However, the pathogenesis of isolated TOF is still to be elucidated.

Whole exome sequencing (WES) is suitable to high-throughput sequencing for all genomic exon regions. Human exome accounting for approximately 1%–2% of genome contains important information of protein synthesis, which directly reflects gene function. In most diseases, related mutations are located in the exome region. WES may be used to find pathogenic gene and predisposing gene in complex diseases, monogenic diseases and cancer because it can investigate protein encoding information in several individuals; its data are also accurate [9]. The aim of this study was to find TOF-related pathogenic genes through WES technology performed among eight small families with TOF, providing a basis for studying TOF pathogenesis.

RESULTS

Clinical features

All the eight TOF children manifested cyanosis and rough systolic-ejection murmurs at the left sternal border between the second and fourth ribs. Echocardiography showed right ventricular enlargement, right ventricular anterior wall thickening, right ventricular outflow tract and pulmonary artery stenosis, ventricular septal defect and aortic overriding (Figure 1). The echocardiographic results of eight TOF children in the right ventricle, right ventricular anterior wall, right ventricular outflow tract, main pulmonary artery, left pulmonary artery, right pulmonary artery, ventricular septal defect and left ventricular ejection fraction (Table 1). The eight TOF children displayed no other malformations, and their parents showed no abnormalities.

Figure 1:

Figure 1: (A) Ventricular septal defect and aortic overriding shown in the left ventricular long-axis view of transthoracic echocardiography. (B) Pulmonary artery stenosis shown in the aorta short-axis view of transthoracic echocardiography.

LA: left atrium; LV: left ventricle; AO: aorta; VSD: ventricular septal defect; RA: right atrium; RV: right ventricle; PA: pulmonary artery; RVOT: right ventricular outflow tract.

Table 1: Results of echocardiogram in the 8 children with tetralogy of Fallot

ID

Gender

Age

RV
(mm)

RVAW
(mm)

RVOT
(mm)

MPA
(mm)

LPA
(mm)

RPA
(mm)

VSD
(mm)

LVEF
(%)

1

female

3months

8.0

5.0

5.4

5.6

4.3

4.8

9.5

73

2

male

8months27days

12.0

6.0

5.7

5.3

4.3

5.3

10.2

68

3

male

1years11months

13.9

6.8

7.2

10.5

6.6

6.7

13.5

72

4

female

2months24days

11.8

4.1

6.0

15.3

9.3

7.7

11.5

57

5

female

6months

11.1

6.1

9.0

11.8

7.6

7.6

12.9

67

6

male

8months2days

13.0

6.3

7.0

6.4

4.2

4.4

13.5

72

7

female

11months26days

13.4

5.9

5.8

12.4

6.7

7.1

13.1

69

8

male

6months11days

10.3

5.0

6.1

9.0

4.3

5.7

13.2

74

RV: right ventricle; RVAW: right ventricular anterior wall; RVOT: Right ventricular outflow tract; MPA: main pulmonary artery; LPA: left pulmonary artery; RPA: right pulmonary artery; VSD: ventricular septal defect; LVEF: left ventricular ejection fraction.

Mutation detection

A total of sixteen differential single nucleotide polymorphisms (SNPs) loci and eight differentially small InDels were discovered by sequencing all exons in the eight small families and alignment of SNPs and small InDels within families and between families (Table 2 and Table 3).

Table 2: SNPs identified by whole exome sequencing

Chr

Position

ID

Ref

Alt

Mutation

Symbol

Amino
acids

SIFT

PolyPhen

chr12

56717682

rs745387506

T

G

missense

NACA

H/P

-

unknown(0)

chr5

141123595

-

T

A

missense

PCDHB4

S/T

tolerated (1)

benign(0)

chr12

7190555

-

G

C

missense

PEX5

A/P

tolerated (0.22)

benign(0.067)

chr2

240042663

-

C

T

missense

PRR21

R/H

tolerated (0.56)

unknown(0)

chr4

1237208

-

A

T

stop_gained

CTBP1

C/*

-

-

chrX

37010556

rs61999275

G

C

missense

FAM47C

A/P

tolerated(1)

benign(0)

chr1

26282352

rs6667693

C

A

missense

UBXN11

G/C

tolerated (0.05)

unknown(0)

chr1

86368686

rs75376884

T

C

missense

ODF2L

M/V

tolerated (0.14)

benign(0.001)

chr4

87614868

-

G

A

missense

DSPP

D/N

-

unknown(0)

chr4

87614877

rs150132251

A

G

missense

DSPP

N/D

-

unknown(0)

chr4

88008301

rs117078377

G

A

missense

PKD2

A/T

tolerated(0.3)

benign(0.395)

chr11

71527593

rs199903176

A

G

missense

KRTAP5-7

Y/C

tolerated (0.22)

unknown(0)

chr12

51346625

rs117443541

T

G

missense

CELA1

Y/S

tolerated(0.13)

benign(0.045)

chr15

88856792

rs12899191

A

G

missense

ACAN

T/A

tolerated(0.56)

benign(0.084)

chrX

104250509

rs9697856

T

G

missense

ESX1

T/P

tolerated (0.54)

unknown(0)

chr22

11068057

-

A

T

missense

BAGE5

R/W

tolerated (0.12)

damaging(0.675)

Chr: chromosome; Ref: reference sequence base; Alt: alternative base identified; SIFT: sorting intolerant from tolerant.

Table 3: Small InDels identified by whole exome sequencing

Chr

Position

ID

Ref

Alt

Mutation

Symbol

Amino
acids

SIFT

PolyPhen

chr2

104856098

rs775150602

AGCC

A

inframe_deletion

POU3F3

A/-

-

-

chr12

111598972

rs769170503

C

CTGT

inframe_insertion

ATXN2

Q/QQ

-

-

chr4

13632182

.

CT

C

splice_acceptor_variant&non_coding_transcript_variant

-

-

chr9

136327352

.

ACCCCCTC

A

TF_binding_site_variant&TFBS_ablation

GPSM1

-

-

-

chr17

75616380

rs371699907

CAGG

C

inframe_deletion

MYO15B

QE/Q

-

-

chr19

9251064

rs201985790

CT

C

frameshift_variant

OR7E24

F/X

-

-

chr19

51694076

rs10689461

C

CAG

splice_acceptor_variant&non_coding_transcript_variant

SPACA6P

-

-

-

chrX

19345745

rs776856509

TC

TCC, T

frameshift_variant

PDHA1

S/X

-

-

Ten of the sixteen differential SNPs were reported, namely, Chr1 (rs6667693, UBXN11), Chr1 (rs75376884, ODF2L), Chr4 (rs150132251, DSPP), Chr4 (rs117078377, PKD2), Chr11 (rs199903176, KRTAP5-7), Chr12 (rs745387506, NACA), Chr12 (rs117443541, CELA1), Chr15 (rs12899191, ACAN), ChrX (rs61999275, FAM47C) and ChrX rs9697856, ESX1). Six of the 16 differential SNPs were not reported, namely, Chr 2 (PRR21), Chr 4 (CTBP1), Chr 4 (DSPP), Chr 5 (PCDHB4), Chr12 (PEX5) and Chr22 (BAGE5).

Six of the eight differentially small InDels were reported, namely, Chr2 (rs775150602, POU3F3), Chr12 (rs769170503, ATXN2), Chr17 (rs371699907, MYO15B), Chr19 (rs201985790, OR7E24), Chr19 (rs10689461, SPACA6P) and ChrX (rs776856509, PDHA1). Two of the eight differentially small InDels were not reported, namely, Chr4 (position: 13632182) and Chr9 (GPSM1).

SNPs and small InDel-related Online Mendelian Inheritance in Man (OMIM) genes and clinical phenotypes are shown in Table 4.

Table 4: SNPs- and small InDels-related OMIM genes and clinical phenotypes

Gene

Chromosome

OMIM

Gene description

Function

NACA

Chr12

601234

gene encodes a protein that associates with BTF3 to form NAC

skeletal development

PCDHB4

Chr5

606330

a member of the protocadherin beta gene cluster

cell-cell neural connections

PEX5

Chr12

600414

peroxisomal biogenesis factor 5

Peroxisome biogenesis disorder 2A, 2B;
Rhizomelic chondrodysplasia punctata, type 5

PRR21

Chr2

-

-

-

CTBP1

Chr4

602618

gene encodes a protein that binds to the C-terminus of adenovirus E1A proteins

transcriptional repressor and cellular proliferation

FAM47C

ChrX

-

-

-

UBXN11

Chr1

609151

a protein with a divergent C-terminal UBX domain

affect the actin cytoskeleton and alter cell shape

ODF2L

Chr1

-

-

-

DSPP

Chr4

125485

a member of the small integrin-binding ligand N-linked glycoprotein family of proteins

Deafness, autosomal dominant 39, with dentinogenesis;
Dentin dysplasia, type II;
Dentinogenesis imperfecta, Shields type II, III

PKD2

Chr4

173910

a member of the polycystin protein family

Polycystic kidney disease 2

KRTAP5-7

Chr11

-

-

-

CELA1

Chr12

130120

Elastases form a subfamily of serine proteases that hydrolyze many proteins in addition to elastin

evolutionarily silenced in pancreatic acinar cells

ACAN

Chr15

155760

a member of the aggrecan/versican proteoglycan family

Osteochondritis dissecans, short stature, and early-onset osteoarthritis;
Spondyloepimetaphyseal dysplasia, aggrecan type;
Spondyloepiphyseal dysplasia, Kimberley type

ESX1

ChrX

300154

gene encodes a dual-function 65 kDa protein that undergoes proteolytic cleavage to produce a 45 kDa N-terminal fragment with a paired-like homeodomain and a 20 kDa C-terminal fragment with a proline-rich domain

placental development and spermatogenesis

BAGE5

Chr22

-

-

-

POU3F3

Chr2

602480

a member of the class III POU family of transcription factors

expressed in the central nervous system

ATXN2

Chr12

601517

gene belongs to a group of genes that is associated with microsatellite-expansion diseases, a class of neurological and neuromuscular disorders caused by expansion of short stretches of repetitive DNA

Spinocerebellar ataxia 2;
susceptibility to Amyotrophic lateral sclerosis;
susceptibility to Parkinson disease, late-onset

GPSM1

Chr9

609491

gene encodes a receptor-independent activator of G protein signaling,

influence the basal activity of G-protein signaling systems

MYO15B

Chr17

-

-

-

OR7E24

Chr19

-

-

-

SPACA6P

Chr19

-

-

-

PDHA1

ChrX

300502

a nuclear-encoded mitochondrial multienzyme complex that catalyzes the overall conversion of pyruvate to acetyl-CoA and CO(2), and provides the primary link between glycolysis and the tricarboxylic acid cycle

Pyruvate dehydrogenase E1-alpha deficiency

We discovered fourteen SNPs- and Small InDels-related OMIM genes, including: NACA, PCDHB4, PEX5, CTBP1, UBXN11, DSPP, PKD2, CELA1, ACAN, ESX1, POU3F3, ATXN2, GPSM1, PDHA1. PEX5 (OMIM:600414) is associated with Peroxisome biogenesis disorder 2A, 2B. NACA (OMIM:601234) is associated with skeletal development. ATXN2 (OMIM:601517) is associated with Spinocerebellar ataxia 2, susceptibility to Amyotrophic lateral sclerosis, susceptibility to Parkinson disease and late-onset. CELA1 (OMIM:130120) is associated with evolutionarily silenced in pancreatic acinar cells. PCDHB4 (OMIM:606330) cell-cell neural connections is associated with CTBP1(OMIM:602618) transcriptional repressor and cellular proliferation.

Results verified by Sanger sequencing

We designed twenty-four pairs of primers to verify sixteen differential SNPs loci and eight differentially small InDels. The results of Sanger sequencing are consistent with that of WES (Figure 2).

Diagram of the Sanger sequencing for two mutations.

Figure 2: Diagram of the Sanger sequencing for two mutations. (A) chr4:13632182 (CT→C). (B) chr1:86368686 (T→C).

The verification of sixteen differential SNPs sites included NACA (rs745387506, T>G), FAM47C (rs61999275, G>C), UBXN11 (rs6667693, C>A), ODF2L (rs75376884, T>C), DSPP (rs150132251, A>G), PKD2 (rs117078377, G>A), KRTAP5-7 (rs199903176, A>G), CELA1 (rs117443541, T>G), ACAN (rs12899191, A>G), ESX1 (rs9697856, T>G), PCDHB4 (chr5, 141123595, T>G), PEX5 (chr12, 7190555, G>C), PRR21 (chr2, 240042663, C>T), CTBP1 (chr4, 1237208, A>T), DSPP (chr4, 87614868, G>A) and BAGE5 (chr22, 11068057, A>T) were investigated.

The verification of eight differentially small InDels sites included AGCC (rs775150602, AGCC>A), ATXN2 (rs769170503, C>CTGT), MYO15B (rs371699907, CAGG>C), OR7E24 (rs201985790, CT>C), SPACA6P (rs10689461, C>CAG), PDHA1 (rs776856509, TC>TCCT), GPSM1 (chr9, 136327352, ACCCCCTC>A), and (chr4, 13632182, CT>T) were investigated.

Gene ontology (GO)/pathway analysis

GO analysis indicated the percentages of 22 genes enriched in GO term (Figure 3). Growth was related to CHD, and it was associated with some genes including PEX5, NACA, ATXN2 and CELA1 (Table 5). PEX5, ATXN2 and CELA1 are responsible for multicellular organism growth (GO:0035264). PEX5, ATXN2, NACA and CELA1 are responsible for developmental growth (GO:0048589). PEX5, ATXN2 and NACA are responsible for regulation of developmental growth (GO:0048638) and growth regulation (GO:0040008). PEX5 and ATXN2 are responsible for regulation of multicellular organism growth (GO:0040014). PEX5 and NACA are responsible for positive regulation of developmental growth (GO:0048639) and positive growth regulation (GO:0045927). PEX5 is responsible for positive regulation of multicellular organism growth (GO:0040018). ATXN2 is responsible for negative regulation of multicellular organism growth (GO:0040015), epidermal growth-factor receptor binding (GO:0005154), negative regulation of developmental growth (GO:0048640), growth-factor receptor binding (GO:0070851) and negative growth regulation (GO:0045926).

GO analysis of 22 genes.

Figure 3: GO analysis of 22 genes.

Table 5: Genes involved by growth and possibly associated with CHD in GO term

Gene

Term

Database

ID

PEX5:5830|ATXN2:6311|CELA1:1990

multicellular organism growth

Biological Process

GO:0035264

PEX5:5830|ATXN2:6311|NACA:4666|CELA1:1990

developmental growth

Biological Process

GO:0048589

PEX5:5830|ATXN2:6311|NACA:4666

regulation of developmental growth

Biological Process

GO:0048638

PEX5:5830|ATXN2:6311

regulation of multicellular organism growth

Biological Process

GO:0040014

PEX5:5830|NACA:4666

positive regulation of developmental growth

Biological Process

GO:0048639

PEX5:5830|ATXN2:6311|NACA:4666|CELA1:1990

growth

Biological Process

GO:0040007

PEX5:5830|NACA:4666

positive regulation of growth

Biological Process

GO:0045927

PEX5:5830|ATXN2:6311|NACA:4666

regulation of growth

Biological Process

GO:0040008

PEX5:5830

positive regulation of multicellular organism growth

Biological Process

GO:0040018

ATXN2:6311

negative regulation of multicellular organism growth

Biological Process

GO:0040015

ATXN2:6311

epidermal growth factor receptor binding

Molecular Function

GO:0005154

ATXN2:6311

negative regulation of developmental growth

Biological Process

GO:0048640

ATXN2:6311

growth factor receptor binding

Molecular Function

GO:0070851

ATXN2:6311

negative regulation of growth

Biological Process

GO:0045926

Repression of Wnt target genes and Notch signalling were related to CHD, and was associated with PCDHB4 and CTBP1 genes (Table 6). Pathway analysis showed that twenty-two genes were enriched at the top fifty of pathway term (Figure 4). Genes involved in the Wnt (CTBP1 and PCDHB4) and Notch (CTBP1) signalling are important for pathway analysis in cardiac development. The mutation sites in the six genes were as follows: PEX5 (A60P), NACA (H1283P), ATXN2 (Q21QQ), CELA1 (Y5S), PCDHB4 (S533T) and CTBP1 (C35*). The transcript number of ATXN2 (Q21QQ) is ENST00000608853.5, and the transcript number of CTBP1 (C35*) is ENST00000629223.1.

Pathway analysis of 22 genes.

Figure 4: Pathway analysis of 22 genes.

Table 6: Genes involved by pathway and possibly associated with CHD

Gene

Term

Database

ID

CTBP1:1487

repression of WNT target genes

Reactome

REACT_264567

CTBP1:1487

AXIN mutants destabilize the destruction complex, activating WNT signaling

Reactome

REACT_264496

CTBP1:1487

deletions in the AXIN genes in hepatocellular carcinoma result in elevated WNT signaling

Reactome

REACT_264286

CTBP1:1487

WNT signaling pathway

KEGG PATHWAY

hsa04310

CTBP1:1487

TCF dependent signaling in response to WNT

Reactome

REACT_264596

CTBP1:1487

RNF mutants show enhanced WNT signaling and proliferation

Reactome

REACT_264378

CTBP1:1487

Signaling by WNT

Reactome

REACT_11045

CTBP1:1487

Notch signaling pathway

KEGG PATHWAY

hsa04330

PCDHB4:56131|CTBP1:1487

WNT signaling pathway

PANTHER

P00057

Variant analysis

By alignment of protein sequences of PEX5, NACA, ATXN2, CELA1, PCDHB4 and CTBP1, S5333T of PCDHB4 was highly evolutionarily conserved in diverse species including Homo sapiens, Pan paniscus, Pongo abelii and gorilla (Figure 5a). In addition, Y5S of CELA1 was highly evolutionarily conserved in diverse species including H. sapiens, gorilla, Saimiri boliviensis, Papio anubis, Peromyscus maniculatus, Tapirus bairdii, Eptesicus fuscus and Sus scrofa (Figure 5b). The highly evolutionarily conserved protein sequences were not found in PEX5, NACA, ATXN2 and CTBP1 in other species.

Figure 5:

Figure 5: (A) Alignment of multiple PCDHB4 protein sequences in different species reveals that the S533T amino acid is located in the highly conserved amino acid region in different species. (B) Alignment of multiple CELA1 protein sequences in different species reveals that the Y33T amino acid is located in the highly conserved amino acid region in different species.

DISCUSSION

TOF as the most common cyanotic CHD is characterised by right ventricular outflow tract obstruction, pulmonary stenosis, ventricular septal defect and aortic overriding caused by non-uniform separation between the truncus arteriosus and bulbus arteriosus at embryo stage. Currently, surgical repair is usually performed in the first month after birth to relieve the stenosis of the right outflow tract and close ventricular septal defect, enabling an exclusive ejection of oxygenated blood via the left ventricle. Cumulative survival and event-free survival were 72% and 25%, respectively, 40 years after follow-up [12].

TOF etiology still needs to be completely clarified because of TOF complexity attributed by both genetic and nongenetic effectors. Next-generation sequencing technology, such as WES with rapid, high throughput and cost-effective features, can meet the requirements for medical studies. WES has been used to obtain information about genetic alterations and potential predispositions possibly associated with TOF occurrence.

Eight small families were tested through WES, and differential SNP loci and InDels were found. From the comparison within each family and among the families, sixteen differential SNPs loci and eight differential Small InDels were screened out. Corresponding genes exist in the database, wherein ten of the sixteen differential SNPs have been reported and six of the sixteen differential SNPs have not been reported. Among the eight Small InDels, seven small Indels have corresponding genes in the database and one Small InDels does not have. Six of the eight differential Small InDels have been reported, whereas two of them have not. Among them, no gene in the database corresponds to the mutation of chr4, position: 13632182. It might be located among the genes or among noncoding regions, and the function requires further study. Go/Pathway analysis of the sixteen differential SNPs loci and eight differential Small InDels were conducted and PEX5, NACA, ATXN2, CELA1, PCDHB4 and CTBP1 were finally screened out and were associated with TOF.

Similar to some genes found to be related to TOF by detection of chromosome karyotype, gene copy number variation and de novo mutation (Table 7), Greenway et al. [13] found four genes (PRKAB2, CHD1L, BCL9 and GJA5) with the highest expression in the right ventricular outflow tract, which is malformed in TOF. Bittel et al. [14] included both idiopathic TOF patients and three patients, which harbour the syndromic 22q11.2 deletion. The expression of the genes A2BP1, VEGF, NPPA and NPPB located in the 22q11.2 region was half-reduced among syndromic patients as expected, whereas none of these genes was differentially expressed in any of the idiopathic TOF subjects. Notch pathway was also suppressed in patients with 22q11.2 deletions. Sharma et al. [15, 16] found that TOF expression of the majority of the genes associated with Wnt and Notch signalling pathways was significantly reduced. Their results also confirmed an upregulated expression of VEGF and proteins of the extracellular matrix reported in previous studies. Feiner et al. [17] found a novel locus at 1q32.2 in two unrelated TOF who displayed loss of copy number variations overlapping the PLXNA2 gene. Knockout mice deficient for PLXNA2 display CHD including TOF [18]. Amarillo et al. [19] found a 3.76 Mb de novo contiguous gain of 9q34.2-q34.3 by chromosome microarray analysis and then also confirmed it by karyotype analysis and fluorescence in situ hybridization. This duplicated interval disrupted retinoid X receptor alpha (OMIM #180245) at intron 1. To our knowledge, PEX5, PCDHB4, CTBP1, NACA, ATXN2 and CELA1 are novel SNPs associated with TOF, which have not been reported.

Table 7: Summary of genes with a potential relevance to TOF

References

Subjects

Platform

Genes

Conclusions

Steven C et al
(2009)

114 TOF Trios
398 TOF

Affymetrix Human Genome Wide SNP Array 6.0

PRKAB2, CHDIL, BCL9, GJA5

potential TOF candidate gene

Douglas C Bittel et al
(2011)

19 TOF

Applied Microarrays Inc. CodeLink
Human Whole Genome Bioarray

A2BP1, VEGF, NPPA, NPPB,

potential relevance for TOF

Amy Rodemoyer et al
(2011)

16 TOF

Affymetrix Human Exon 1.0 ST v2

NEFH, SST, TF, KRT6A, KRT7,
NTRK2, MYL2, PPBP, NRGN,
APOC3

dysregulated network in TOF

Goodship et al
(2012)

362 TOF

SEQUENOM MALDI-TOF

PTPN11

risk allele for TOF

Candice K et al
(2012)

433 TOF

Affymetrix Genome-Wide Human
SNP Array 6.0

GJA5, PLXNA2

potential TOF candidate gene

Rachel Soemedi et al
(2012)

283 TOF Trios

Illumina 660W SNP Array 6.0

GJA5, HAND2, EDIL3, CNOT6

potential TOF candidate gene

Heather J.Cordell et al
(2013)

798 TOF

Illumina 600W QUAD array

PTPN11, GPC5, NRP1

risk allele for TOF

Marcel Grunert et al
(2014)

22 TOF

NimbleGen sequence capture 365K array
Genome Sequencer FLX
Illumina Genome Analyzer

deleterious SNPs in BARX1, BCCIP, DAG1, EDN1, FANCL, FANCM, FMR1, FOXK1, HCN2, MYOM2, PEX6, ROCK1, TCEB3, TP53BP2, TTN, WBSCR16

imbalance of functional networks in TOF

Tan ZP et al
(2015)

1 TOF

Illumina Hiseq2000

SCN5A

a possible cause of TOF

LaHaye S et al
(2016)

2 TOF

Illumina Hiseq2000

MYBPC3, SOS1

uncertain significance

Our study aimed to identify other monogenic causes of TOF. GO analysis indicated that PEX5 was related to multicellular organism growth (GO:0035264), developmental growth (GO:0048589), regulation of developmental growth (GO:0048638), regulation of multicellular organism growth (GO:0040014), positive regulation of developmental growth (GO:0048639), positive regulation of growth (GO:0045927), growth (GO:0040007) and positive regulation of multicellular organism growth. In the 26 cases with TOF, Grunert M et al. [20] found deleterious SNPs in 16 genes, including BARX1, BCCIP, DAG1, EDN1, FANCL, FANCM, FMR1, FOXK1, HCN2, MYOM2, PEX6, ROCK1, TCEB3, TP53BP2, TTN and WBSCR16 which resulted in the imbalance of function networks in TOF. Huffnagel IC et al.[21] found that two of the 21 genetically proven Rhizomelic chondrodysplasia punctata patients suffered from TOF and showed PEX7 mutations. Plasmalogens were not detected in cardiac tissue of PEX7 knock-out mice, which is a model for RCDP type 1. The above-mentioned PEX5, PEX6 and PEX7 are all PEX family gene named Peroxins (PEXs) which are proteins essential for the assembly of functional peroxisomes. Therefore, PEX5 might participate in TOF growth and development process. PCDHB4 is located on Chr5q31.3. This gene is a member of the protocadherin beta gene cluster and is one of the three related and tandemly linked gene clusters at chromosome 5. Their specific functions are unknown but they most likely play a critical role in the establishment and function of specific cell–cell neural connections. Alazami AM et al. [22] performed WES on 143 multiplex consanguineous families, in which known disease genes had been excluded by autozygosity mapping. Patients, whose clinical phenotype is featured by global developmental delay and brain atrophy, have mutation in PCDHB4 (NM_021908:c.489T>G:p.Y163X) based on the candidate gene analysis, and the function of which participated in the growth and development process. Base on our research, Go analysis indicated that PCDHB4 (P00057) was related to WNT signaling pathway. Thus, PCDHB4 (P00057) may be a potential TOF candidate gene. NACA is located on Chr12q13.3. The protein encoded by NACA is associated with basic transcription factor 3, which forms the nascent polypeptide-associated complex. In this study, GO analysis indicated that NACA was related to positive regulation of developmental growth (GO:0048639) and positive regulation of growth (GO:0045927). Thus, NACA may be a potential TOF candidate gene. CELA1 is located on Chr12q13.13. This gene forms a subfamily of serine proteases that hydrolyze many proteins in addition to elastin. Humans possess six elastase genes, which encode the structurally similar elastases 1, 2, 2A, 2B, 3A and 3B. Unlike other elastases, pancreatic elastase 1 is not expressed in the pancreas. GO analysis indicated that CELA1 was related to multicellular organism growth (GO:0035264), developmental growth (GO:0048589) and growth (GO:0040007). Thus, CELA1 may have a potential relevance to TOF. ATXN2 is located on Chr12q24.12 with a transcript number of ENST00000608853.5. This gene belongs to a group of genes that are associated with microsatellite-expansion diseases, a class of neurological and neuromuscular disorders caused by expansion of short stretches of repetitive deoxyribonucleic acid (DNA). Genome-wide association studies indicate that loss-of-function mutations in this gene may be associated with susceptibility to type I diabetes, obesity and hypertension. Alternative splicing leads to multiple transcript variants. GO analysis indicated that ATXN2 was related to negative regulation of multicellular organism growth (GO:0040015), epidermal growth-factor receptor binding (GO:005154), negative regulation of developmental growth (GO:0048640), growth-factor receptor binding (GO:0070851) and negative regulation of growth (GO:0045926). Thus, ATXN2 may have a potential relevance to TOF. CTBP1 is located on Chr4p16.12 with a transcript number of ENST00000629223.1. This gene encodes a protein that binds to the C-terminus of adenovirus E1A proteins. This phosphoprotein is a transcriptional repressor and may play a role in cellular proliferation. This protein and the product of a second closely related gene, CTBP1, can dimerise. Both proteins can also interact with a polycomb group protein complex, which participates in regulation of gene expression during development. Alternative splicing of transcripts from this gene results in multiple transcript variants. GO analysis indicated that CTBP1 was related to Wnt signalling pathway and Notch signalling pathway. Thus, CTBP1 may be a potential TOF candidate gene.

This study has some limitations, and the sample size is small. In the future, the number of samples will be further increased and an in-depth research will be conducted. The identified mutation sites have not been verified based on large TOF sample (n>50). Thus, whether the identified mutation sites may occur in a large group of TOF people has not been confirmed. Moreover, further study is necessary to verify relevant function genes and accordingly illustrate the pathogenesis of TOF.

In summary, TOF pathogenesis is complex because TOF is associated with more than one genetic variant. Eight small families were observed through WES and found that TOF-related genes were PEX5, NACA, ATXN2, CELA1, PCDHB4 and CTBP1, whose potential function might participate in TOF growth and development process. Although none of the three variants were predicted to be highly influential, they are in genes belonging to networks involved in relevant developmental pathways. Further study on the potential developmental mechanisms will be conducted based on these results.

MATERIALS AND METHODS

Subjects

A total of eight small families whose Chinese children received surgical TOF treatment at the Children’s Heart Center of Henan Provincial People’s Hospital between January 2016 and June 2016 were enrolled in this study. The eight children ageing 3–11 months included four male babies and four female babies. All manifested isolated TOF. Their parents were normal. All the eight children were diagnosed with TOF by echocardiogram, clinical symptoms and signs and intraoperative findings.

Methods

DNA extraction

In each member of the eight small families including 24 individuals, 600 μl peripheral blood was collected to extract genomic DNA using genomic DNA Extraction Kit (Qiage, Germany). The remaining blood samples were stored at −80°C.

WES and mutation screening

WES and subsequent variant annotation were performed on genomic DNA derived from the eight TOF children and their parents. Paired-end libraries were prepared according to the manufacturer’s protocols (Agilent). Exome libraries for the eight families were constructed using Agilent SureSelectXT Target Enrichment System according to Illumina Paired End Sequencing Protocol (Agilent Technologies, CA, USA). Capturing of whole exon was carried out according to the protocol of Agilent’s Sure Select Human All 1 UTRs 71 MB v4 kit. The flow cells were sequenced as paired-end 150 base pair reads on an Illumina HiSeq ×10 platform to a minimum depth of 50× targeted region coverage using TruSeq SBS sequencing kit version 3 and HiSeq data collection version 2.0.12.0 software (Illumina, Inc., San Diego, CA, USA).

The raw sequence reads we obtained were aligned to the human genome reference sequence (hg19) using Burrows–Wheeler Aligner (BWA) with standard parameters [10]. The BWA-aligned reads were statistically calculated using PICARD software to exclude polymerase chain reaction duplicates. Regional realignment and quality score recalibration were carried out using Genome Analysis Toolkit [11] with recommended parameters, which included local realignment of the sequences around InDels, base quality score recalibration, variant calling and variant quality score recalibration.

Variant detection in the eight TOF children by Sanger sequencing

BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and ABI 3130 Genetic Analyzer were used to detect variants among the eight TOF children.

Ethics statement

Approval was obtained from local ethics committees of Henan Provincial People’s Hospital, Zhengzhou University People’s Hospital. Informed consents were provided to all the participants of this study.

Abbreviations

TOF: tetralogy of Fallot; CHD: congenital heart disease; WES: whole-exome sequencing; SNPs: single nucleotide polymorphisms; CNVs: copy number variations; DNA: deoxyribonucleic acid; BWA: Burrows–Wheeler Aligner; GATK: Genome Analysis Toolkit; BTF3: basic transcription factor 3; NAC: nascent polypeptide-associated complex ; SRP: signal recognition particle.

Author contributions

Lin Liu designed the experiments, recruited patients, collected samples, analysed the data, performed statistics and wrote the manuscript. Hong-dan Wang designed the experiments, recruited the patients, collected samples, analysed the data, performed statistics and wrote the manuscript. Cun-ying Cui recruited the patients, collected the samples, analysed the data and wrote the manuscript. Yun-yun Qin collected samples, analysed the data and wrote the manuscript. Tai-bing Fan collected the samples. Bang-tian Peng collected the samples. Lian-zhong Zhang performed the statistics. Cheng-zeng Wang designed the experiments, recruited patients and critically reviewed the manuscript.

ACKNOWLEDGMENTS

The authors are grateful to the patients and families who participated in this research. Team members who supported the subject recruitment and sequencing are as follows: Yan-ze Li (CapitalBio Technology), Qian Zhang, Hao-ju Dong, Shu-bo Song, Yan-wei Zhang, Jia-yong Zheng, Feng Ai, Dan-qing Huang and Juan Zhang.

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

FUNDING

This study was supported by National Natural Science Foundation of China (81401419), Program of Science and Technology of Henan Province (172102310003) and Program of Advanced Study to go Abroad of Henan Provincial Health System (2016047).

REFERENCES

1. Bédard E, McCarthy KP, Dimopoulos K, Giannakoulas G, Gatzoulis MA, Ho SY. Structural abnormalities of the pulmonary trunk in tetralogy of Fallot and potential clinical implications: a morphological study. J Am Coll Cardiol. 2009; 54:1883–90.

2. Beauchesne LM, Warnes CA, Connolly HM, Ammash NM, Grogan M, Jalal SM, Michels VV. Prevalence and clinical manifestations of 22q11.2 microdeletion in adults with selected conotruncal anomalies. J Am Coll Cardiol. 2005; 45:595–98.

3. Goldmuntz E, Geiger E, Benson DW. NKX2.5 mutations in patients with tetralogy of Fallot. Circulation. 2001; 104:2565–68.

4. Salazar M, Consoli F, Villegas V, Caicedo V, Maddaloni V, Daniele P, Caianiello G, Pachón S, Nuñez F, Limongelli G, Pacileo G, Marino B, Bernal JE, et al. Search of somatic GATA4 and NKX2.5 gene mutations in sporadic septal heart defects. Eur J Med Genet. 2011; 54:306–09.

5. Eldadah ZA, Hamosh A, Biery NJ, Montgomery RA, Duke M, Elkins R, Dietz HC. Familial Tetralogy of Fallot caused by mutation in the jagged1 gene. Hum Mol Genet. 2001; 10:163–69.

6. Bauer RC, Laney AO, Smith R, Gerfen J, Morrissette JJ, Woyciechowski S, Garbarini J, Loomes KM, Krantz ID, Urban Z, Gelb BD, Goldmuntz E, Spinner NB. Jagged1 (JAG1) mutations in patients with tetralogy of Fallot or pulmonic stenosis. Hum Mutat. 2010; 31:594–601.

7. Lu JH, Chung MY, Betau H, Chien HP, Lu JK. Molecular characterization of tetralogy of Fallot within Digeorge critical region of the chromosome 22. Pediatr Cardiol. 2001; 22:279–84.

8. Stoll C, Dott B, Alembik Y, Roth MP. Associated congenital anomalies among cases with Down syndrome. Eur J Med Genet. 2015; 58:674–80.

9. Ng SB, Turner EH, Robertson PD, Flygare SD, Bigham AW, Lee C, Shaffer T, Wong M, Bhattacharjee A, Eichler EE, Bamshad M, Nickerson DA, Shendure J. Targeted capture and massively parallel sequencing of 12 human exomes. Nature. 2009; 461:272–76.

10. Li H, Durbin R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics. 2010; 26:589–95.

11. DePristo MA, Banks E, Poplin R, Garimella KV, Maguire JR, Hartl C, Philippakis AA, del Angel G, Rivas MA, Hanna M, McKenna A, Fennell TJ, Kernytsky AM, et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet. 2011; 43:491–98.

12. Cuypers JA, Menting ME, Konings EE, Opić P, Utens EM, Helbing WA, Witsenburg M, van den Bosch AE, Ouhlous M, van Domburg RT, Rizopoulos D, Meijboom FJ, Boersma E, et al. Unnatural history of tetralogy of Fallot: prospective follow-up of 40 years after surgical correction. Circulation. 2014; 130:1944–53.

13. Greenway SC, Pereira AC, Lin JC, DePalma SR, Israel SJ, Mesquita SM, Ergul E, Conta JH, Korn JM, McCarroll SA, Gorham JM, Gabriel S, Altshuler DM, et al. De novo copy number variants identify new genes and loci in isolated sporadic tetralogy of Fallot. Nat Genet. 2009; 41:931–35.

14. Bittel DC, Butler MG, Kibiryeva N, Marshall JA, Chen J, Lofland GK, O’Brien JE Jr. Gene expression in cardiac tissues from infants with idiopathic conotruncal defects. BMC Med Genomics. 2011; 4:1–10.

15. Sharma HS, Peters TH, Moorhouse MJ, van der Spek PJ, Bogers AJ. DNA microarray analysis for human congenital heart disease. Cell Biochem Biophys. 2006; 44:1–9.

16. Peters TH, Sharma V, Yilmaz E, Mooi WJ, Bogers AJ, Sharma HS. DNA microarray and quantitative analysis reveal enhanced myocardial VEGF expression with stunted angiogenesis in human tetralogy of Fallot. Cell Biochem Biophys. 2013; 67:305–16.

17. Feiner L, Webber AL, Brown CB, Lu MM, Jia L, Feinstein P, Mombaerts P, Epstein JA, Raper JA. Targeted disruption of semaphorin 3C leads to persistent truncus arteriosus and aortic arch interruption. Development. 2001; 128:3061–70.

18. Brown CB, Feiner L, Lu MM, Li J, Ma X, Webber AL, Jia L, Raper JA, Epstein JA. PlexinA2 and semaphorin signaling during cardiac neural crest development. Development. 2001; 128:3071–80.

19. Amarillo IE, O’Connor S, Lee CK, Willing M, Wambach JA. De novo 9q gain in an infant with tetralogy of Fallot with absent pulmonary valve: patient report and review of congenital heart disease in 9q duplication syndrome. Am J Med Genet A. 2015; 167A:2966–74.

20. Grunert M, Dorn C, Schueler M, Dunkel I, Schlesinger J, Mebus S, Alexi-Meskishvili V, Perrot A, Wassilew K, Timmermann B, Hetzer R, Berger F, Sperling SR. Rare and private variations in neural crest, apoptosis and sarcomere genes define the polygenic background of isolated Tetralogy of Fallot. Hum Mol Genet. 2014; 23:3115–28.

21. Huffnagel IC, Clur SA, Bams-Mengerink AM, Blom NA, Wanders RJ, Waterham HR, Poll-The BT. Rhizomelic chondrodysplasia punctata and cardiac pathology. J Med Genet. 2013; 50:419–24.

22. Alazami AM, Patel N, Shamseldin HE, Anazi S, Al-Dosari MS, Alzahrani F, Hijazi H, Alshammari M, Aldahmesh MA, Salih MA, Faqeih E, Alhashem A, Bashiri FA, et al. Accelerating novel candidate gene discovery in neurogenetic disorders via whole-exome sequencing of prescreened multiplex consanguineous families. Cell Reports. 2015; 10:148–61.


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