Genetic findings of children with congenital heart diseases using chromosome microarray and trio-based whole exome sequencing

doi:10.21203/rs.3.rs-3969530/v1

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Genetic findings of children with congenital heart diseases using chromosome microarray and trio-based whole exome sequencing

https://doi.org/10.21203/rs.3.rs-3969530/v1

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Background

Congenital heart disease (CHD) is the most common type of birth defects. Genetic factors are the important contributor to the etiology of CHD. However, the underlying genetic causes in most individuals remain unclear.

Methods

101 individuals with CHD and their unaffected parents were included in this study. Chromosome microarray analysis (CMA) as a first-tier clinical diagnostic tool was applied for all affected individuals, followed by trio-based whole exome sequencing (WES). The function of the genes involved in the genetic variants in the cohort was analyzed.

Results

We detected aneuploidies in 2 individuals (trisomy 21 and monosomy X), other pathogenic/likely pathogenic copy number variants (CNVs) in 20 individuals, and pathogenic /likely pathogenic SNVs/InDels in 9 individuals. The combined genetic diagnostic yield was 30.7%, including 21.8% with chromosomal abnormalities and 8.9% with sequence-level variants. Nineteen CNVs in 19 individuals were associated with 14 recurrent chromosomal microdeletion/microduplication syndromes, the most common being 22q11.2 deletion syndrome. Pathogenic/likely pathogenic sequence-level variants were identified in nine genes, including GATA6, FLNA, KANSL1, HNRNPK, TRAF7, KAT6A, PKD1L1, RIT1, and SMAD6. The function of the genes involved in the CHD relevant CNVs and SNVs was analyzed indicating enriched genes are mainly associated with development of multiple organs, not only heart, but also brain and endocrine system.

Conclusions

CMA is a first-tier clinical diagnostic test to define the underlying genomic architecture of CHD. Trio-based WES increases the diagnostic yield, and should be part of the diagnostic algorithm. Our study expands the genes interaction networks for genetic study of CHD.

Congenital heart disease (CHD)

Chromosome microarray analysis (CMA)

Copy number variation (CNV)

trio-based whole exome sequencing (trio-WES)

functional analysis

Congenital heart disease (CHD) is the most common type of birth defect with an incidence of approximately 10 per 1,000, ranging from 2.3 to 13.1 in 1,000 live births based on the geographic region, and leading to significant morbidity and mortality in childhood worldwide^1–4. It has been estimated that contribution of genetic factors to the etiology is about 40%, or even up to 90% of CHD cases, including chromosome aneuploidies, copy number variations (CNVs), and single nucleotide variants (SNVs) ^5,6, but so far, the genetic etiology has only been identified in ~ 30% of individuals with CHD indicating that many genetic causes are still undetermined^7–9. Moreover, the pathogenicity of some genetic variants is incompletely understood partly due to lack of supporting segregation data and incomplete penetrance of the phenotypes¹⁰.

Chromosomal microarray analysis as a first-tier clinical diagnostic tool in patients with developmental delay/intellectual disability (DD/ID), autism spectrum disorders (ASD), and multiple congenital anomalies (MCA), has revealed that rare CNVs are important contributors to CHD in 3% − 25% of CHD patients with wide variation depending on testing platform and the cohort characteristics^9,11,12. High-throughput sequencing allows the detection of additional types of pathogenic genetic variations in individuals with CHD^13,14. Patients with CHD have increased risks of extracardiac abnormalities and neurodevelopmental disorders (NDDs), and various genetic abnormalities, including CNVs, have been implicated in both CHD and NDDs^15,16. Therefore, studying genetic abnormalities in patients with CHD is not only uncovering the genetic etiology of CHD, but also contributing to a better understanding of the molecular mechanisms of CHD and related disorders including NDDs^15–17. The contribution of rare genetic variants to CHD is well-established^11–14,18 however the overall diagnostic yield of CNVs and SNVs in affected children is still uncertain. Identifying a genetic diagnosis in patients with CHD can facilitate targeted therapies and interventions, genetic counseling for families, and monitoring of extracardiac manifestations including NDDs.

In this study, we investigated clinically relevant genetic variants in a Chinese cohort of 101 patients with CHD and their non-affected parents using chromosome microarray analysis (CMA) followed by trio-based exome sequencing. We also assessed the clinical implications of genetic testing, including the probability of extracardiac features such as NDDs, and recurrence risks of CHD in family members.

Subjects

A total of 101 unrelated children with congenital heart disease (CHD) (male: 57, female: 44, mean age: 9 months, range from 1 day to 9 years old) and their unaffected parents were enrolled in the study between March 2015 and December 2017. All subjects were Han Chinese population in Shandong province and recruited from the department of cardiovascular medicine in Jinan Children’s Hospital (Children’s Hospital Affiliated to Shandong University). The 101 patients were diagnosed by experienced pediatric cardiologists based on medical history, physical examination, color echocardiogram and/or thoracic computed tomography angiography, and subsequently confirmed by cardiac surgery.

The 101 cases were categorized into 3 groups according to the American Heart Association: septal defects, obstructive defects and cyanotic defects. One proband with ventricular septal defect (VSD) had a twin brother who had tetralogy of Fallot (TOF), atrial septal defect (ASD) and umbilical hernia. Except this case, there was no family history of CHD in the cohort including different types of CHD patients: 65 with septal defects, 14 with obstructive defects and 22 with cyanotic defects (Table 1). Based on the absence or presence of extra-cardiac abnormalities including DD and ID, CHD was classified into isolated and non-isolated CHD (Table 1). A total of 75 (74.3%) individuals were diagnosed with isolated CHD, while 26 (25.7%) were found to have non-isolated CHD with at least one extra-cardiac abnormality, such as dysmorphic facial features, other congenital anomalies (hernia, bronchial dysplasia, hypospadias, joint dislocation and cleft palate), intellectual disability (ID) and developmental delay (DD) (Table 1, Supplemental Table S1). Detailed clinical examinations and diagnosis of CHD were performed by experienced pediatricians and cardiovascular specialists.

Table 1

Pathogenic and likely pathogenic variants (CNVs, SNVs/InDels and aneuploidies) identified in different types of CHD
CHD classification		Overall				Isolated CHD				Non-isolated CHD
CHD classification		No. cases	Positive detection rate (%)	No. CNVs & aneuploidy	No. SNVs	No. cases	Positive detection rate (%)	No. CNVs & aneuploidy	No. SNVs	No. cases	Positive detection rate (%)	No. CNVs & aneuploidy(%)	No. SNVs (%)
TOTAL		101	31(30.7)	24(21.8)	10(9.9)	75	11(14.7)	5(6.7)	6(8.0)	26	20(76.9)	19(73.1)	4(15.4)
Septal defects	TOTAL	65	18(27.7)	16	4	47	3(6.4)	2	3	18	15(83.3)	14	3
	VSD	48	11(22.9)	10	3	37	3(8.1)	2	2	11	8(72.7)	8	2
	ASD	9	4(44.4)	3	1	5	0(0.0)	0	1	4	4(100.0)	3	1
	AVSD	3	1(33.3)	1	0	2	0(0.0)	0	0	1	1(100.0)	1	0
	CECD	5	2(40.0)	2	0	3	0(0.0)	0	0	2	2(100.0)	2	0
Obstructive defects	TOTAL	14	5(35.7)	2	4	10	3(30.0)	0	1	4	2(50.0)	2	1
	PS	3	1(33.3)	0	1	3	1(33.3)	0	0	0	0	0	0
	COA	7	2(28.6)	0	2	5	1(20.0)	0	0	2	1(50.0)	0	1
	IAA	3	1(33.3)	2	0	1	0	0	0	2	1(50.0)	2	0
	Cor-t	1	1(100)	0	1	1	1(100)	0	1	0	0	0	0
Cyanotic defects	TOTAL	22	8(36.4)	6	2	19	5(26.3)	3	2	4	3(75.0)	3	0
	TOF	10	5(50.0)	4	1	6	2(33.3)	1	1	4	3(75.0)	3	0
	TGA	1	0	0	0	1	0	0	0	0	0	0	0
	TAPVC	10	3(30.0)	2	1	10	3(30.0)	2	1	0	0	0	0
	PA	1	0	0	0	1	0	0	0	0	0	0	0
VSD: ventricular septal defect; ASD: atrial septal defect; AVSD: atrioventricular septal defect; CECD: complete endocardial cushion defect; PS: pulmonary stenosis; COA: coarctation of the aorta; IAA: interruption of aortic arch; Cor-t: cor triatriatum; TOF: tetralogy of Fallot; TGA: complete transposition of great arteries; TAPVC: total anomalous pulmonary venous connection; PA: pulmonary atresia.

The study was approved by Ethics Committee of Jinan Children’s Hospital (Children’s Hospital Affiliated to Shandong University). Informed written consent was obtained from the patients’ parents. The information of the patients’ and their families was anonymized prior to genotyping and analysis. All the procedures performed in the study were in accordance with the Declaration of Helsinki.

Chromosome Microarray Analysis

Peripheral blood samples were obtained from all subjects. Genomic DNA was extracted using a TIANGEN Blood DNA Mini Kit (TIANGEN, BEIJING, China). Two CMA platforms of Affymetrix CytoScan HD Arrays (Affymetrix, Santa Clara, CA) and Illumina HumanCytoSNP-12 array (Illumina, San Diego, CA) were applied for detection of CNVs. Genomic DNA was digested, amplified, and hybridized to the arrays following the protocols of Affymetrix or Illumina. The arrays were scanned on Cytoscan or iSan System, respectively.

Primary data were analyzed with Affymetrix Chromosome Analysis Suite (ChAS) or Illumina GenomeStudio software. The reporting threshold was set at 50 kb (markers ≥ 20) for deletions and 100 kb (markers ≥ 50) for duplications. Secondary analysis was performed and 1679 Chinese subjects from multiple sources were compared according to Liu et al¹⁹. Frequency of prioritized CNVs was computed against the aforementioned controls in Liu et al¹⁹. CNVs with > 50% reciprocal overlap were deemed identical²⁰. All CNVs were classified as pathogenic, likely pathogenic, variants of uncertain significance (VUS), likely benign, and benign according to the American College of Medical Genetics and Genomics guidelines^21–23.

Whole exome sequencing and data analysis

To further assess the causal role of sequence variants, trio-based whole exome sequencing with xGen Exome Hyb Panel (IDT, USA) on the Illumina NovaSeq 6000 platform was applied for the patients and their parents. In brief, genomic DNA was extracted using same kit as descripted above. A total of 1.5 µg genomic DNA was used to shear by KAPA Frag Enzyme (KAPA Inc, MA, USA) to generate average size of 300bp libraries and then purified using Agencourt AMPure XP kit (Beckman Coulter Inc, CA, USA). After ligation with the paired-end adaptor and enriched by PCR, the DNA samples were hybridized with xGen Exome Hyb Panel and the targeted molecules were captured using Dynabeads MyOne Streptavidin T1 (Life Technologies, CA, USA), and then sequenced on an Illumina NovaSeq 6000 (Illumina Inc, CA, USA). Enrichment, sequencing kits and sequencing system were supplied by Illumina Inc (Illumina Inc, CA, USA).

Preliminary data analysis included read alignment, variant calling, and annotation. After quality control of raw reads with low quality sequences (quality score < 20) removed, sequencing reads were aligned to the human reference genome (GRCh37/hg19) using Sentieon (Sentieon Inc, Shanghai) and the Burrows-Wheeler Aligner^24,25. Variant calling and annotation were performed according to the Broad Institute’s GATK (v4.0) and Annovar. The median coverage was 100 X with 98% of the target bases being covered ≥ 10 X. Variants were filtered out when their minor allele frequency > 0.5% in public databases including 1000 Genomes Project (https://www.internationalgenome.org/) and gnomAD (https://gnomad.broadinstitute.org). ClinVar (http://www.ncbi.nlm.nih.gov/clinvar) and the Human Gene Mutation Database (http://www.hgmd.cf.ac.uk) were used to search for previously reported variant-disease associations. In silico analysis of the variants was carried out using PolyPhen-2, SIFT and Mutation Taster to predict potentially disrupted protein function. The variants identified using this procedure were classified according to the 2015 American College of Medical Genetics and Genomics (ACMG) guidelines²⁶.

Validation of gene variants

Real-time qPCR with SYBR Green chemistry were utilized to verify potentially clinical relevant CNVs in cases and their parents, as described before²⁷. Sanger sequencing was used to validate pathogenic and likely pathogenic variants identified by whole exome sequencing. Sanger validation primer sets were designed using Primer Premier v5.0 software. PCR amplification was performed with AmpliTaq Gold® 360 DNA Polymerase (Applied Biosystems). PCR products were further purified and sequenced using an ABI Prism 3700 automated sequencer (Applied Biosystems, Foster City, CA).

Gene functional analysis

Different bioinformatic software packages were used to analyze the function and pathway of the genes involved in the CNV and SNV loci. The two gene lists were generated from the CNV and SNV loci: (1) all genes involved in the CNVs in Supplemental Table S2; (2) all genes involved in the SNVs in Supplemental Table S3; (3) OMIM genes filtered from Table S2 and S3 (Supplemental Table S4). Lists were then imported separately into Enrichr²⁸ and results were exported. The Cytoscape²⁹ with ClueGo³⁰ and Cluepedia³¹ were utilized to visualize the pathway of the implicated genes using an overlap coefficient cutoff of 0.05. Associated gene clusters were labeled with circle layout.

Statistical analysis

Statistical analysis was performed with SPSS 26 software. Chi-square test or two-sided Fisher’s test was used to compare the positive rates of between Isolated and non-isolated CHD as well as the three CHD subgroups. A difference was considered statistically significant when the p value was less than 0.05 and the confidence interval was 95%.

General diagnostic yield

We analyzed samples from 101 Han Chinese patients with CHD. The overall genetic diagnostic yield was 30.7% (31/101 probands), including 21.8% (22/101) probands with chromosomal abnormalities and 8.9% (9/101) probands with SNV variants. The detection rates in different subgroups of CHD were summarized in Table 1.

Diagnostic yield of CMA

Using CMA, we identified 22 pathogenic/ likely pathogenic CNVs (including 2 aneuploidies) in 20 individuals with CHD, and 5 VUS in 5 individuals (Table 2, Table S2). The 2 aneuploidies included one monosomy X in a 4-month old girl (ID: J005) and one trisomy 21 in an 11 days old boy (ID: J143) (Table S1). When only considering pathogenic/likely pathogenic CNVs and aneuploidies, a stringent diagnostic yield of CMA for this CHD cohort was 21.8% (22/101).

Table 2

Pathogenic and likely pathogenic CNVs identified in the CHD patients by CMA
Sample ID	Cytoband	Size (kb)	CNV type	Parent of origin	Genetic syndrome
J016	1q21.1	414	deletion	paternal	1q21.1 deletion syndrome
J016	18p11.32p11.31	4810	deletion	maternal	18p deletion syndrome
J132	3p26.3	846	deletion	paternal	3p deletion syndrome
J003	7q11.23	1424	deletion	de novo	Williams-Beuren syndrome
**J153	7p22.3	1800	deletion	de novo
**J153	7q33q36.3	23500	duplication	de novo
J217	8q24.11q24.22	14200	deletion	de novo	Langer-Giedion syndrome
J009	9q33.1	71112	deletion	maternal
J180	11q14.1q25	51900	duplication	de novo	11q partial trisomy
*J144	13q14.2q22.1	27706	deletion	de novo	Interstitial deletion 13q
J109	13q34	3256	deletion	de novo	13q deletion syndrome
J199	15q11.2	546	deletion	paternal	15q11.2 microdeletion syndrome
J106	15q11.2	512	deletion	paternal	15q11.2 microdeletion syndrome
J177	16p13.12p13.11	1530	deletion	de novo	16p13.11 microdeletion syndrome
J150	16q11.2q21	13100	duplication	de novo	16q partial trisomy
J179	17p11.2	3530	deletion	de novo	Smith-Magenis syndrome
J128	17q23.1q23.2	2177	duplication	paternal	17q duplication syndrome
J194	22q11.21	2560	deletion	de novo	22q11.2 deletion syndrome
J202	22q11.21	2570	deletion	maternal	22q11.2 deletion syndrome
J173	22q11.21	2580	deletion	de novo	22q11.2 deletion syndrome
J131	22q11.21	2882	deletion	de novo	22q11.2 deletion syndrome
J201	22q11.21	3050	deletion	de novo	22q11.2 deletion syndrome
*this subject has a mosaic deletion (1–2) of chr13:47,645,885 − 75,352,286.
*this subject has a de novo* deletion of chr7:43,360-1,813,034, immediately adjacent to a de novo duplication of chr7:1,813,405-3,642,604 (VUS not shown) and then another de novo duplication of chr7:135,539,873 − 159,119,707.

The 22 pathogenic/likely pathogenic CNVs included 18 (81.8%) deletions and 4 (19.2%) duplications (Table 2). Of the 22 CNVs, 14 (63.6%) were de novo, 5 (22.7%) paternally and 3 (13.6%) maternally inherited. The size of the CNVs varied from 414 kb to 71.1Mb. The majority of CNVs (16/22, 72.7%) were smaller than 10 Mb in size, which were submicroscopic CNVs and would likely not have been identified by conventional karyotyping. Nineteen CNVs in 19 individuals were associated with 14 recurrent chromosomal microdeletion/ microduplication syndromes, including five classic A-D deletions at 22q11.2 (4 de novo and 1 maternally inherited) diagnosed as 22q11.2 deletion syndrome /DiGeorge syndrome, a 1.4 Mb deletion at 7q11.23 in a 4-month-old boy with supravalvar aortic stenosis, atrial septal defect, craniofacial anomalies and developmental delay diagnosed as Williams-Beuren syndrome, a 3.53 Mb deletion at 17p11.2 in a 1-year-1-month boy with TOF and DD diagnosed as Smith-Magenis syndrome, and one deletion at 8q24 associated with Langer-Giedion syndrome (Table 2).

There were 3 cases who harbored more than one clinically relevant CNVs, of which a 1-year-5-month boy with IAA and DD (ID: J016) was detected a 414 kb paternally inherited deletion at 1q21.1 and a 4.8 Mb maternally inherited deletion at 18p11.32p11.31 that related to two known syndromes of 1q21.1 deletion syndrome and 18p deletion syndrome; a 2-month-7-day boy with VSD and pulmonary atresia (PA)(ID: J109), was identified with a 3.3Mb de novo deletion at 13q34 and a 1.6Mb paternally inherited duplication at 19p13.3; a 3-month-17-day boy with VSD and congenital hypospadias(ID: J153) harboring a de novo deletion at 7p22.3, and 2 de novo duplications at 7p22.3p22.2 and 7q33q36.3. The phenotypes of above cases were considered combined effects from the CNVs (Table 2, table S6).

Incremental yield of WES

Apart from 22 individuals with CHD and pathogenic, likely pathogenic CNVs and aneuploidies, the remaining 79 patients were analyzed through WES. Among them, 2 patients (J007, J104) and their fathers were found to be not biologically related and 1 patient (J046) and both his parents were found to be biologically unrelated. Thus, we obtained the sequencing data from 76 trio-based and 3 probands-only WES. Single nucleotide variations (SNVs) and small insertion and deletions (InDels) were detected based on comparison with the human reference sequence (hg19) .

We identified ten pathogenic/likely pathogenic variants in nine genes from nine CHD patients. Five of the variants have been previously reported in the literature, and five were novel, including seven de novo, one paternally inherited and two compound heterozygous variants (Table 3). The seven de novo variants consisted of five missense variants in five CHD genes, GATA6, HNRNPK, TRAF7, KAT6A and RIT1, and two loss-of-function variants in KANSL1 and FLNA. We identified 2 compound heterozygous loss-of-function variants in PKD1L1 and 1 paternally inherited loss-of-function variant in SMAD6 (Table 3). The 5 novel variants were identified in FLNA (exon 8–9 duplication), HNRNPK (c.1171C > G), KAT6A (c.2044C > A) and PKD1L1 (c.5451C > G, c.1071delT). The incremental yield of WES was 8.9% (9/101), resulting in a combined molecular diagnostic rate of 30.7% (31/101). Additionally, we found 6 potentially relevant variants which were classified as variants of uncertain significance (VUS) according to the ACMG guidelines (Table 3, Table S7).

Table 3

Pathogenic and likely pathogenic SNVs and InDels identified in the CHD patients by trio-based WES
Sample ID	Clinical phenotypes	Gene	Transcript: variant	Zygosity	Inheritance: Phenotype in OMIM	Parent of origin	Pathogenicity	Novel variant
J057	COA, VSD, ASD; DD	GATA6	NM_005257.4: c.1366C > T, p.(R456C)	het	AD: Atrial septal defect 9, Atrioventricular septal defect 5, Pancreatic agenesis and congenital heart defects, Tetralogy of Fallot	de novo	P(PS2_VeryStrong + PM5 + PM2_Supporting + PP3)	No
J102	VSD, PDA, PAH; respiratory failure, umbilical hernia	FLNA	NM_001456.3: exon 8–9 dup (487bp) (chrX:153594371–153594858)	het	XL: periventricular heterotopia, cardiac valvular dystrophy, FG syndrome	de novo	P(PVS1 + PS2 + PM2_Supporting)	Yes
J104	ASD; DD, ID	KANSL1	NM_001193466.1: c.1042C > T, p.(R348X)	het	AD: Koolen-De Vries syndrome	de novo	P(PVS1 + PS2 + PM2_Supporting)	No
J118	Cor triatrium	HNRNPK	NM_002140.3: c.1171C > G, p.(Q391E)	het	AD: Au-Kline syndrome	de novo	LP(PVS1 + PM2_Supporting)	Yes
J145	VSD, COA, PDA; DD	TRAF7	NM_032271.2: c.1570C > T, p.(R524W)	het	AD: TRAF7 syndrome; Cardiac, facial, and digital anomalies with developmental delay	de novo	LP(PS2 + PM2_Supporting + PP3)	No
J159	TOF	KAT6A	NM_001099412.1: c.2044C > A, p.(P682T)	het	AD: Arboleda-Tham syndrome	de novo	LP(PS2 + PM2_Supporting + PP3)	Yes
J172	VSD, ASD, cor triatriatum	PKD1L1	NM_138295.3: c.5451C > G, p.(Y1817X)	CH	AR: Autosomal visceral heterotaxy-8	paternal	P(PVS1 + PM2_Supporting + PM3)	Yes
J172	VSD, ASD, cor triatriatum	PKD1L1	NM_138295.3: c.1071delT, p.(H357Ifs*22)	CH	AR: Autosomal visceral heterotaxy-8	maternal	LP(PVS1 + PM2_Supporting)	Yes
J184	ASD, PS	RIT1	NM_006912.5: c.170C > G, p.(A57G)	het	AD: Noonan syndrome 8	de novo	P(PS2 + PS3 + PS4 + PM2_Supporting + PP3)	No
J200	TAPVC	SMAD6	NM_005585.4: c.269dupC, p.(R91Efs*30)	het	AD: Aortic valve disease 2	paternal	P(PVS1 + PS4_Supporting + PM2_Supporting)	No

Subgroup analysis

The overall detection rate of pathogenic/likely pathogenic variants in three CHD categories ranged from 27.7% in individuals with septal defects, 35.7% in individuals with obstructive defects and 36.4% in individuals with cyanotic defects, though without statistical significance (Table 1).

We also compared the detection rates between individuals with isolated CHD and those with CHD and extra cardiac features (non-isolated CHD). In the 26 cases with extracardiac phenotypes, we identified 20 genetic diagnoses (detection rate of 76.9%), including 1 trisomy 21, 1 monosomy X, 17 CNVs and 2 SNVs/InDels. In the 75 individuals with apparently isolated CHD, we detected 5 CNVs and 6 SNVs (detection rate of 14.7%; Table 1). The overall detection rate in individuals with non-isolated CHD was significantly higher than in individuals with isolated CHD (p = 2.0x10^− 4). The highest yield was 83.3% in septal defects with extracardiac features (n = 18 individuals), while the lowest detective rate was 6.4% in Septal defects of isolated CHD (n = 47 individuals), indicating a statistically significant difference (p = 1.0x10^− 5).

Gene enrichment and functional analysis

The function and pathway of the genes involved in the CNVs and SNVs were analyzed and summarized in Table S2 – S4. Totally 77 gene sets were significantly enriched (corrected P < 0.05, Table S5), including positive regulation of cellular protein catabolic process, very-lwo-density lipoprotein particle remodeling, positive regulation of early endosome to late endosome transport, cardiac muscle contraction, heart contraction, regulation of organ morphogenesis, and so on. Gene pathway analysis demonstrated that the genes implicated in CNV loci are mainly associated with development of multiple organs. The associated genes were circled layout as several gene clusters as cardiac development, muscle and organism development, cerebral development, and VLDL particle remodeling (Fig. 1).

In this study, we investigated the prevalence of clinically relevant CNVs and SNVs/Indels in an unselected cohort of 101 patients with CHD, using high resolution chromosome microarray analysis (CMA) and exome sequencing. The general prevalence of chromosomal abnormalities in the patients with CHD was 21.8% (22/101) including 2 individuals with aneuploidies and 20 individuals with other pathogenic/likely pathogenic CNVs, which is consistent with a recent Chinese study identifying pathogenic/likely pathogenic CNVs in 24/104 (23.1%) patients with CHD³². The highest yield was obtained for clinically relevant CNVs of less than 10 Mb, confirming an important role for CMA in clinical settings. We then performed trio-based whole exome sequencing (WES) for non-positive samples from 79 cases and their parents. In total, we identified likely pathogenic SNVs/InDels in 9 CHD-related genes in 9 individuals, which incremented the yield to 30.7%.

Aneuploidies were the earliest identified genetic causes of CHD³³. In this study, 26 individuals classified as having non-isolated CHD had at least one extracardiac defect, such as facial anomalies, congenital diaphragmatocele, hip dislocation, tracheal malformation, exomphalos, funnel chest, hypospadia, cleft palate, DD and ID. Our study identified two aneuploidies: one trisomy 21 and one monosomy X (Turner syndrome) in two individuals with non-isolated CHD, accounting for 11.1% (2/18) of all genetic causes in 26 patients with non-isolated CHD, matching a recent study which identified two patients with aneuploidies of chromosomes 19 and 21 in 104 patients with CHD³². Fourteen of these individuals were found to harbor 17 CNVs. We re-assessed the clinical presentations of the 14 patients and compared with cases previously reported in the literature. Ten of the 14 patients were retrospectively found to have recognizable features of the identified genetic syndromes, for example, five with 22q11.2 deletion syndrome (DiGeorge syndrome) and characteristic facial features (4/5), TOF/VSD (5/5), hypocalcemia (2/5), immunodeficiency (2/5), and DD (1/5). 22q11.2 deletions are among the most frequent genetic causes in individuals with CHD and present with a variety of clinical features. About 64% of individuals with 22q11.2 deletion syndrome have heart defects and > 90% are de novo^34,35.

One individual with craniofacial anomalies, supravalvar aortic stenosis, atrial septal defect and DD, carrying a de novo 1.42 Mb deletion at 7q11.23 including ELN (130160) and 31 other genes, was diagnosed with Williams-Beuren syndrome, which is well-known and another common recognizable syndrome in CHD cohorts³⁶. Other rare chromosome syndromes identified in this cohort included Smith-Magenis syndrome in an individual with TOF and DD (de novo deletion of 3.53 Mb at 17p11.2)³⁷; a 1-month-12-day boy with feeding problems, mild dysmorphic features, CAVCD and DD with Langer-Giedion syndrome (de novo deletion of 14.2 Mb at 8q24.11q24.22)³⁸; a male patient with PA, VSD and DD harboring a 846 kb deletion at 3p26.3 consistent with 3p deletion syndrome³⁹; one 51.9 Mb partial trisomy at 11q14.1q25 in a 6-month girl with atrial septal defect, dysmorphic features, and dislocation of hip joint, matching 11q partial trisomy syndrome^40,41.

Interestingly, we detected 3 rare de novo CNVs at chromosome 7 in one 3-month boy with complex phenotypes of multiple systems such as VSD, ASD, pulmonary arterial hypertension, tricuspid regurgitation, agenesis of corpus callosum, scoliosis, microphallus, hypotonia, hypospadias, and DD. The 3 CNVs include a 1.8 Mb deletion at 7p22.3 encompassing 36 genes and a major OMIM gene of FAM20C (#611060). There are 6 individuals with overlapping deletions (from 1.12 Mb to 2.32 Mb) and a variety of phenotypes mainly including DD, ID, cognitive impairment and seizures in the DECIPHER database. The second CNVs, a 1.8 Mb duplication at 7p22.2 involving 19 genes has been associated with developmental delay, mild ID, asthma, myopia, dysmorphic features in the literature⁴². There are additional 21 cases with overlapping duplications (from 198.88 kb to 2.23 Mb) and a variety of phenotypes mainly including abnormal heart morphology, cerebellar vermis hypoplasia, renal hypoplasia, cognitive impairment, scoliosis, strabismus, and ID in the DECIPHER database. The third CNV is a 23.5 Mb duplication at 7q33q36.3 encompassing over 100 genes, overlapping reported however smaller microduplications of 507 kb, 730 kb and 1.35 Mb from 7q36.1 to 7q36.3. The 507 kb duplication at 7q36.3 was found in a 20w6d fetus with multiple congenital anomalies like cleft lip/palate, prominent cavum septum pellucidum, right-sided heart position, absent right radius and thumb, fixed right forearm, and scoliosis⁴³; the 730 kb duplication at 7q36.3 was found in four individuals from a three-generation family with agenesis of the corpus callosum and mild ID, macrocephaly⁴⁴; the 1.35 Mb duplication at 7q36.1q36.2 was found in a 22 month old child presenting with hypotonia, respiratory distress, feeding difficulties, cardiovascular malformation, growth failure with microcephaly, short stature, sensorineural hearing loss, myopia, cryptorchidism, hypospadias, microphallus, distinctive facial features and DD⁴⁵. Furthermore, from the DECIPHER database additional 109 cases with duplications within 7q33q36.3 presented some overlapping manifestations, such as corpus callosum dysplasia, scoliosis, muscular hypotonia and microphallus, macrocephaly, cardiac defects, TOF, and ID. Likely, the 3 de novo CNVs could be a more complex structural rearrangement, such as a ring chromosome (often mosaic) which contribute to the phenotype of this individual, but more follow-up work is needed.

Through WES, we identified ten pathogenic and likely pathogenic variants in nine individuals, including eight genes with dominant variants (seven de novo, one paternally inherited) and two recessive (compound heterozygous) variants. Five variants were novel (have to our knowledge not been reported before), including 2 de novo missense variants in HNRNPK and KAT6A, one de novo predicted loss-of-function variant in FLNA, and two compound heterozygous loss-of-function variants in PKD1L1 (Table 3):. Most genes (including FLNA, KANSL1, HNRNPK, TRAF7, KAT6A, PKD1L1, and RIT1) have previously been associated with syndromic CHD^46–52. However, the 4 genes of HNRNPK, KAT6A, PKD1L1, and RIT1 were found in probands with isolated CHD. The first reason should be phenotypic heterogeneity of the syndromes and the second one might be clinically unrecognized associated features especially in young patients. Our findings provide further evidence supporting an important role of these genes in CHD.

FLNA, located on chromosome Xq28, encodes an actin - binding protein (filamin A) expressed in virtually every tissue, and mutations in the FLNA gene cause X-linked filaminopathies including cardiovascular malformations, such as X-linked cardiac valvular dystrophy (CVDPX; OMIM#314400), X-linked periventricular nodular heterotopia (PVNH1; OMIM#300049), FG syndrome-2 (FGS2; OMIM#300321)^53,54. We identified an exon 8–9 duplication in the FLNA gene in a 7-month- old boy (J102) presenting with VSD, PDA, PAH, respiratory failure and umbilical hernia. He died at 10 months of respiratory insufficiency and heart failure. The intragene duplication was predicted to cause loss-of-function of the FLNA gene. Other individuals with loss-of-function variants in the FLNA gene presented with periventricular nodular heterotopia, heart defects, interstitial lung disease, respiratory failure, and/or early death, further indicating an association of FLNA deficiency and congenital malformations of the brain, heart and lungs^55,56.

Function and pathway of the genes involved in clinically relevant CNVs and SNVs in cases with CHD showed that most of these variants were associated with development of organism cardiovascular, cardiac muscle, cardiac conduction, and brain. The identification of the pathway provided further information of interactions between gene-gene/protein - protein which demonstrated most of genes affect development of multiple organs. We divided the genes in to several gene clusters by labeling associated gene clusters with circle layout, such as cardiac development, regulation of muscle and organism development, cerebral development, VLDL particle remodeling. Subclusters were manually annotated. Genes interact with each other to constitute the complex network participating in multiple regulatory circuits in development of brain, heart, embryo, and multiple organs. It is known that TBX1 is the critical transcription factor required for pharyngeal and cardiovascular development. The mutations in TBX1 gene are associated with several anomalies of thymus, palatal anomalies and cardiovascular defects in 22q11.2 deletion syndrome demonstrating an important role in development of organism. It not only interacts with SHH affecting SHH signaling during morphogenesis, but regulated the BMP and TGF-β pathway by binding Smad7 during development⁵⁷. The pathway analysis of TBX1 showed that it directly linked to SHH and other sub-networks implicating development of endocrine system, brain, embryo, heart, and VLDL particle remolding through protein-protein interactions.

We compared the detection rates of clinically relevant genetic variants in 75 individuals with isolated CHD and 26 individuals with non-isolated CHD, combining the results of CMA and trio-WES. The combined genetic diagnostic yield was significantly higher in patients with non-isolated CHD (76.9%) than in patients with isolated CHD (14.7%; p = 2.0x10^− 4). In contrast, no significant difference was found regarding the detection rate in the different subgroups of CHD (27.7% in septal defects vs 35.7% in obstructive defects vs 36.4% in cyanotic defects, p = 0.221). As the number of cases in each subgroup was small in this study, investigations of larger samples are needed for further verification.

In this study, we performed sequential genetic testing using CMA followed by trio-WES in a cohort of unselected CHD patients in order to obtain reliable diagnostic yields. Our data and functional analysis indicates that trio-WES increments the diagnostic yield in patients with extracardiac features but also in those with apparently “isolated” CHD, thus expand the genes interaction networks for genetic study of CHD. While the sample size was small, further studies with large samples are warranted to find new genes and variants, and to assess the clinical spectrum and recurrence risks in affected individuals and their families.

Conflicts of Interest

S.W.S. is on the Scientific Advisory Committees of Deep Genomics, and is a Highly Cited Academic Advisor for the King Abdulaziz University. The other authors declare no conflicts of interest.

Author Contribution

This study was conceived and designed by YL and SWS. The experiments were conducted by GZ, XY, HZ and YW. Data analyzed by RG, CD, MZ, MR, RD, SWS and YL. RG, CD and ZG contributed to the clinical diagnosis of the patients. The paper was written by YL and revised by MZ, MR and SWS. All authors reviewed the manuscript.

Acknowledgments

This work was supported by Jinan City Talent Program Grant (2015515002). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors are grateful to the patients and their parents for their contribution to the work. We would like to thank The Centre for Applied Genomics at the Hospital for Sick Children and the University of Toronto McLaughlin Centre. S.W.S. holds the Northbridge Chair in Paediatric Research at the Hospital for Sick Children and University of Toronto.

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Genetic findings of children with congenital heart diseases using chromosome microarray and trio-based whole exome sequencing

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Introduction

Methods

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Chromosome Microarray Analysis

Whole exome sequencing and data analysis

Validation of gene variants

Gene functional analysis

Statistical analysis

Results

General diagnostic yield

Diagnostic yield of CMA

Incremental yield of WES

Subgroup analysis

Gene enrichment and functional analysis

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Conflicts of Interest

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