NOTCH2, ATIC, MRI1, SLC6A13, ATP13A2 Genetic Variations are Associated with Ventricular Septal Defect in the Chinese Tibetan Population Through Whole-Exome Sequencing

Figures & data

Table 1 Sample Information of Enrolled Patients

Sample Number	Gender	Age	Diagnostic Result	Echocardiogram Report
VSD-1	Female	2.9 years	CHD	CHD: After VSD operation, left atrial enlargement, CDFI: no shunt color beams at the level of atrium, ventricle, and great vessels, tricuspid regurgitation (small amount)
VSD-2	Male	7 months	CHD	CHD: Tricuspid valve atresia, VSD (perimembranous type 7 mm) ventricular level left-to-right shunt, ASD (central type 11 mm) atrial level left-to-right shunt, consider: complete endocardial cushion defect may be pulmonary valve stenosis
VSD-3	Female	3.7 years	CHD	CHD: After right ventricular double outlet + VSD + PAH surgery: VSD (30 mm) ventricular level bidirectional shunt, ASD (12 mm) atrial level left to right, PAH (80 mmHg)
VSD-4	Male	10 years	CHD	CHD: After VSD, CDFI: no shunting color beams at the ventricular level, PAH (43 mmHg), tricuspid regurgitation (moderate volume)
VSD-5	Male	7 months	CHD	CHD: VSD (membranous 6 mm), CDFI: left-to-right shunt at ventricular level, PAH (46 mmHg), tricuspid regurgitation (small amount)
VSD-6	Female	6 years	CHD	VSD (membranous 5.6 mm), CDFI: left-to-right shunt at ventricular level, patent foramen ovale (3 mm), CDFI: left-to-right shunt at atrial level, PAH (90 mmHg), tricuspid regurgitation, three cuspid regurgitation (moderate-large)
VSD-7	Male	6.11 years	CHD	CHD: VSD (membranous 3 mm), CDFI: left-to-right shunt at ventricular level
VSD-8	Female	4 years	CHD	Postoperative VSD: no shunting color beam at ventricular level, PAH (38 mmHg), tricuspid regurgitation (small amount)
VSD-9	Female	11 years	CHD	CHD: After VSD + tricuspid valvuloplasty, right atrium and right ventricle enlargement, tricuspid valve depression deformity? (Tricuspid septal valve is 20 mm away from atrioventricular valve), PAH (50 mmHg), tricuspid regurgitation (moderate volume)
VSD-10	Female	4.7 years	CHD	CHD: Postoperative VSD: no shunt color beam at the ventricular level, tricuspid regurgitation (small amount)
VSD-11	Female	2.2 years	CHD	CHD: VSD (membranous 4 mm), CDFI: left-to-right shunt at ventricular level
VSD-12	Male	3.9 years	CHD	Postoperative VSD: no shunt color beam at the ventricular level, tricuspid regurgitation (small amount)
VSD-13	Male	9 months	CHD	CHD: VSD (membranous 5 mm), CDFI: left-to-right shunt at ventricular level, PAH (40mmHg), tricuspid regurgitation (small amount)
VSD-14	Female	4.5 years	CHD	CHD: Postoperative VSD: no shunting color beam at ventricular level, tricuspid regurgitation (small amount)
VSD-15	Female	2.9 years	CHD	CHD: VSD (membrane 7 mm), CDFI: left-to-right shunt at ventricular level, mitral insufficiency, mitral regurgitation (moderate volume), PAH (80 mmHg), tricuspid regurgitation (moderate volume), Pulmonary regurgitation (small amount)
VSD-16	Female	2.1 years	CHD	CHD: VSD (membranous 4 mm), CDFI: left-to-right shunt at ventricular level
VSD-17	Male	4.9 years	CHD	CHD: After VSD: ventricular level residual small bundle shunt (2 mm), tricuspid regurgitation (small amount)
VSD-18	Female	2.3 years	CHD	CHD: After VSD surgery, no shunting color beams were seen at the ventricular level
VSD-19	Female	1.5 months	CHD	CHD: VSD (membranous 4 mm), CDFI: left-to-right shunt at ventricular level, patent foramen ovale (2 mm), CDFI: left-to-right shunt at atrial level, mitral regurgitation (small amount), PAH (36 mmHg), tricuspid regurgitation (small amount)
VSD-20	Female	8 years	CHD	CHD: After VSD surgery, no shunt color beams were seen at the ventricular level, PAH (44 mmHg), tricuspid regurgitation (small amount)

Abbreviations: CHD, Congenital heart disease; VSD, ventricular septal defect; CDFI, color Doppler flow imaging; ASD, atrial septal defect; PAH, Pulmonary Arterial Hypertension.

Table 2 Number of SNV/InDel Loci for Different Genotypes

Sample	Het_SNV	Hom_SNV	Novel_SNV	Het_InDel	Hom_InDel	Novel_InDel	Het_SNV/Hom_SNV
VSD-1	54830	37066	1842	7967	3742	978	1.479253224
VSD-2	53015	38341	1887	7802	3849	950	1.382723455
VSD-3	54107	36634	1912	7779	3648	1067	1.476961293
VSD-4	56339	39923	2109	9221	4365	1199	1.411191544
VSD-5	55212	39111	2057	8074	4087	985	1.411674465
VSD-6	57515	38627	1945	9196	4154	1142	1.488984389
VSD-7	55699	38855	1878	8547	4018	1026	1.433509201
VSD-8	56486	38165	1990	8131	4036	944	1.480047164
VSD-9	53427	37515	1737	6976	3663	768	1.42415034
VSD-10	54386	37075	1919	7492	3688	803	1.466918409
VSD-11	53930	35809	1798	6756	3459	708	1.506045966
VSD-12	52216	37604	1718	6647	3674	787	1.388575683
VSD-13	49489	35433	1756	5805	3307	637	1.396692349
VSD-14	51391	36103	1804	6370	3417	759	1.423455115
VSD-15	53257	36629	1707	7047	3645	799	1.453957247
VSD-16	51707	36832	1694	6563	3498	749	1.403860773
VSD-17	51174	36150	1762	5935	3537	643	1.41560166
VSD-18	55913	38715	2073	9230	4058	1082	1.444220586
VSD-19	55254	37057	1876	7933	3711	893	1.491054322
VSD-20	54505	37566	1831	7660	3858	966	1.45091306

Abbreviations: SNV, single nucleotide variations; InDel, insertion-deletion; VSD, ventricular septal defect; Het_SNV, SNV of heterozygous genotype; Hom_SNV, SNV of homozygous genotype; Novel_SNV, new (not annotated by dbSNP) SNV.

Figure 1 Distribution ratio of SNV/InDel sites in different regions.

Figure 2 Distribution ratio of SNV/InDel sites for different functional types.

Table 3 Predicted Gene Frequencies and Harmful Prediction Tables

Gene	First Priority	Freq_Alt (1000g)	ExAC03_EAS	esp6500	gnomAD_exome_EAS	SIFT Pred	POLYPhen V2 Pred	MutationTaster Pred	Cadd Raw	Dann
NOTCH2	First1	0.00159744	0	0.0002	0	D	D	D	2.971	0.995
ATIC	First1	0.000199681	0	0.000077	0.00005437	D	D	D	4.162	0.999
MRI1	First1	0.000399361	0.0001	0.000077	0.00005452	D	D	D	4.39	1
SLC6A13	First1	0.000199681	0.0003	0.000077	0.0002	D	D	D	5.478	0.999
ATP13A2	First1	0.000199681	0.0005	0.000077	0.0004	D	D	D	4.313	0.999

Abbreviations: First Priority, the best considered gene; Freq_Alt (1000g), the frequency of non-reference alleles in the 1000 Genome Project; ExAC03_EAS, allele frequency data of 65,000 exons in East Asia; esp6500, from ESP (NHLBI GO Exome Sequencing Project) Frequencies in 6500 samples of whole exome sequencing; gnomAD_exome_EAS, gnomAD data in East Asia; SIFT Pred, POLYPhen V2 Pred, Mutation Taster Pred, predicting the effect of gene variation on function, protein hazard prediction; D, pathogenic; Cadd Raw and Dann, SNV deleterious score (the higher the value, the more deleterious the mutation is).

Table 4 Genetic Information for Predicted Genes

SNV NO.	Gene	Chrs	SNP ID	Gene Region	Function	Predicted Protein Variants	Genetic Model	SNP Function
SNV10061	NOTCH2	chr1	rs141935585	Exonic	Nonsynonymous SNV	c.1396C>A:p.Gln466Lys:exon8	AD	TF binding, DNase peak
SNV01363	ATIC	chr2	rs367615313	Exonic	Nonsynonymous SNV	c.235C>T:p.Arg79Cys:exon4	AR	TF binding, DNase peak
SNV08935	MRI1	chr19	rs141094096	Exonic	Nonsynonymous SNV	c.629G>A:p.Arg210Gln:exon4	–	TF binding, DNase peak
SNV13971	SLC6A13	chr12	rs377179026	Exonic	Nonsynonymous SNV	c.1138G>A:p.Gly380Arg:exon10	–	TF binding, DNase peak
SNV01397	ATP13A2	chr1	rs371445094	Exonic	Nonsynonymous SNV	c.1363C>T:p.Arg455Trp:exon15	AR	TF binding, any motif, DNase peak

Abbreviations: SNV NO., single nucleotide variation number; Chrs, chromosome number; SNP ID, the number of the locus in the dbSNP of NCBI; Gene Region, the gene region where the variation is located; Function, gene function; Predicted Protein Variants, Using snpEFF for mutation annotation, the amino acid changes of the protein caused by the gene predicted from the reference sequence; Genetic model, Gene inheritance model; nonsynonymous SNV, missense mutation; AD, autosomal dominant; AR, autosomal recessive; TF binding, Transcription factor binding.

Table 5 Predicting Gene Disease Functional Annotation

Gene	InterVar	HGMD	HGMD_gene	HPO	MGI
NOTCH2	Uncertain significance	–	Atrioventricular septal defect; Bicuspid aortic valve; PAH	VSD; ASD; TOF; Peripheral pulmonary artery stenosis; Patent ductus arteriosus	Cardiovascular system phenotype; Respiratory system phenotype; Hematopoietic system phenotype
ATIC	Uncertain significance	–	Left ventricular obstruction with extracardiac anomalies and Neurodevelopmental disorder; Transposition of the great arteries with extra-cardiac anomalies and neurodevelopmental disorder	ASD	-
MRI1	Uncertain significance	DM	-	-	-
SLC6A13	Uncertain significance	-	-	-	-
ATP13A2	Uncertain significance	-	-	-	-

Abbreviations: InterVar, Mutation clinical interpretation information according to the American College of Medical Genetics and Genomics (ACMG) scoring rules; HGMD, Human Gene Mutation Database; HPO, Human Phenotype Ontology; MGI, Mouse Genome Informatics; DM, Pathogenic mutation; PAH, Pulmonary Arterial Hypertension; VSD, ventricular septal defect; ASD, atrial septal defect; TOF, tetralogy of fallot.