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Genetic Diagnostic Yield and Novel Causal Genes of Congenital Heart Disease

Congenital heart disease (CHD) is the most common congenital malformation in fetuses and neonates, which also represents a leading cause of mortality. Although significant progress has been made by emerging advanced technologies in genetic etiology diagnosis, the causative genetic mechanisms behind CHD remain poorly understood and more than half of CHD patients lack a genetic diagnosis. Unlike carefully designed large case-control cohorts by multicenter trials, we designed a reliable strategy to analyze case-only cohorts to utilize clinical samples sufficiently. Combined low-coverage whole-genome sequencing (WGS) and whole-exome sequencing (WES) were simultaneously conducted in a patient-only cohort for identifying genetic etiologies and exploring candidate, or potential causative CHD-related genes. A total of 121 sporadic CHD patients were recruited and 34.71% (95% CI, 26.80 to 43.56) was diagnosed with genetic etiologies by low-coverage WGS and WES. Chromosomal abnormalities and damaging variants of CHD-related genes could explain 24.79% (95% CI, 17.92 to 33.22) and 18.18% (95% CI, 12.26 to 26.06) of CHD patients, separately, and 8.26% (95% CI, 4.39 to 14.70) of them have simultaneously detected two types of variants. Deletion of chromosome 22q11.2 and pathogenic variants of the COL3A1 gene were the most common recurrent variants of chromosomal abnormalities and gene variants, respectively. By in-depth manual interpretation, we identified eight candidate CHD-causing genes. Based on rare disease-causing variants prediction and interaction analysis with definitive CHD association genes, we proposed 86 genes as potential CHD-related genes. Gene Ontology (GO) enrichment analysis of the 86 genes revealed regulation-related processes were significantly enriched and processes response to regulation of muscle adaptation might be one of the underlying molecular mechanisms of CHD. Our findings and results provide new insights into research strategies and underlying mechanisms of CHD.


INTRODUCTION
Congenital heart disease (CHD) is a malformation involving structural and functional defects caused by abnormal development of the heart or thoracic and large blood vessels during embryonic development. It is the most common type of congenital malformation and accounts for about 28% of all various kinds of congenital malformations (Van Der Linde et al., 2011). The incidence of CHD in full-term live births is about 8-10/1,000 (0.8-1%), and the incidence of preterm infants may be 10 times higher (about 8.5%) (Jorgensen et al., 2014;Wang et al., 2018). CHD is a complex and highly heterogeneous disease. Its causative factors include environmental factors and genetic or epigenetic factors, but genetic etiologies are considered to be the most important factor. A review study published by Nicholas S. Diab et al. (2021) estimated that genetic etiologies contribute to 90% of CHD cases, although about 55% of them remain unsolved in genetic diagnosis. Chromosomal aneuploidies, copy number variations (CNVs), and damaging variants of disease-causing genes are the mainly diagnosable genetic etiologies (Williams et al., 2019;Buijtendijk et al., 2020).
Chromosomal aneuploidy is the earliest confirmed cause of CHD and accounts for about 9-18% of CHD, and 28-45% of CHD cases diagnosed by a prenatal diagnosis have aneuploidy abnormalities (Mademont-Soler et al., 2013). The application of next-generation sequencing has extended the molecular insights into sub-chromosomal abnormalities, and CNVs have received increased attention as a causative factor of CHD. Deletion at 22q11.2 in DiGeorge syndrome is one of the most common chromosomal abnormalities associated with CHD and is detected in approximately 2% of all CHD patients (Agergaard et al., 2012;Goldmuntz, 2020). Other recurrently affected regions like 15.q11.2, 1.q21.1, and 7q11.23 have also been detected in CHD (Soemedi et al., 2012;Edwards and Gelb, 2016;Mustafa et al., 2020). Rare genetic deletions contribute to about 4% of the population-attributable risk of sporadic CHD (Soemedi et al., 2012) and likely causal CNVs are presumed in 5.6% of CHD patients (Hartman et al., 2011). Cardiac development is a sophisticated process involving a complex regulatory network. Gene encoding transcription factors, cell signal transduction factors, and chromatin modifiers can all interfere with important cell differentiation patterns in heart development, thereby variants disrupting the structure and function of the heart would cause CHD to occur (Lage et al., 2012;Li et al., 2015). Studies have reported that CHD caused by monogenic damaging variants accounts for about 8-11% of CHD cases (Jin et al., 2017;Zaidi and Brueckner, 2017;Diab et al., 2021). At present, a number of CHD pathogenic genes have been located based on family studies, NKX2.5 (Schott et al., 1998), TBX5 (Zhang et al., 2020), and GATA4 (Li et al., 2018) are the most important CHD pathogenic genes. Moreover, wholeexome sequencing (WES) has been routinely used in cohort studies and predicted about 10% of cases are attributable to de novo mutations (DNMs) in >400 target genes, including dramatic enrichment for damaging mutations in genes encoding chromatin modifiers (Zaidi et al., 2013;Homsy et al., 2015). Genetic etiology diagnosis in CHD patients plays an important role in prenatal diagnosis and also in clinical intervention programs. The discovery of novel diseasecausing genes or potential association genes and genetic patterns of CHD cohorts is important for the underlying molecular mechanisms of CHD. Unlike previous carefully designed large case-control cohorts by multicenter trials, we designed a reliable strategy in this study to analyze a case-only cohort that could utilize clinical samples sufficiently. This study recruited 121 patients with clinically diagnosed CHD and simultaneously performed low-coverage whole-genome sequencing (WGS) and whole-exome sequencing (WES). The genetic data were manually interpreted and thoroughly bioinformatically analyzed to determine genetic diagnostic yield and potential causative gene prediction. Our findings and results would provide new insights into research strategies and the underlying mechanisms of CHD.

Patient's Enrollment and Ethical Conduct of Research
One hundred and twenty-one diagnosed sporadic CHD fetuses or neonates were recruited in Fujian Provincial Maternal and Child Health Hospital from August 2018 to December 2020. The guardians of all involved patients have signed a written informed consent before participating in this project. This study has been approved by the Ethics Committee of the Fujian Maternity and Child Health Hospital (protocol code 2019-147).

Research Strategy and CHD Relative Genes
Low-coverage WGS and WES were combined to explore the genetic etiologies of CHD patients. The low-coverage WGS was designed to identify chromosomal abnormalities including aneuploidies and CNVs, meanwhile, the WES was utilized to detect the single-nucleotide variants (SNVs) and short insertions or deletions variants (InDels). Considering the high genetic heterogeneity of CHD, we deciphered the genetic causing of CHD not only among the previously reported CHD-related genes but also among other possibly candidate CHD genes following ACMG guidelines. For the CHD-related genes, we manually curated a set of 452 unique genes based on two authoritative reviews of recently published research (Williams et al., 2019;Morton et al., 2022), two large cohort genome-wide association studies (Sifrim et al., 2016;Jin et al., 2017), and 29 case reports retrieved in PubMed (Supplementary Table S1). The retrieved rules in PubMed were: "{[congenital heart ( title)] and [(candidate) or (related) or (relative) or (associated) or (association)]} and [(gene) or (variant)]" and results were filtered by "Case Report" and "Humans". There were 64 reports preserved, and then the retained studies were subsequently excluded from chromosome abnormalities manually. Finally, 30 CHD genes from 29 case reports were curated.

Sample Collection and Genomic DNA Extraction
According to the sample condition, cord blood, amniotic fluid, or heart tissues were collected for genomic DNA extraction. Two milliliters of blood were collected in an EDTA tube for cord blood samples, and 20 ml of amniotic fluid was collected and divided into two 15 ml Corning sterilized centrifuge tubes for amniotic fluid samples. Generally, the heart tissue samples were derived from the heart organs of aborted fetuses or muscle tissue from cardiac surgeries. As for the aborted fetuses, we first separated the heart organ and immediately flushed the blood with PBS buffer at 4°C, and then the blood vessels and fat around the heart were cleaned in a sterile environment. Finally, we separated the left and right atrial and ventricle and took more than 100 mg of tissue from each part of the separated tissues. The tissue samples divided from aborted fetuses or cardiac surgeries were all placed in EP tubes and quickly frozen by liquid nitrogen, and subsequently transferred to a −80°C refrigerator for preservation. The genomic DNA (gDNA) was isolated from cord blood or heart tissue utilizing the HiPure Tissue Blood DNA Mini Kit (Magen) and the magnetic bead method amniotic fluid genome extraction kit (NanoMagBio) for amniotic fluid samples. The concentration of gDNA was quantitated using Qubit 2.0 (ThermoFisher Scientific), and the integrity was assessed by agarose gel electrophoresis. The total mass of gDNA was needed more than 1 mg and the integrity of gDNA was required not to be completely degraded.

Low Coverage Whole Genome Sequencing, Analysis, and Interpretation
The sequencing libraries were performed using MGIEasy Universal DNA Library Prep Set (MGITech) following the standard operating procedures with a total of 50 ng qualified gDNA input. Constructed libraries with sample index were circularized by splint oligo and based on the rolling circle amplification (RCA) mechanism to generate DNA nanoballs for sequencing. More than 0.1X whole-genome coverage raw reads in 35bp plus 10bp (index) were generated for each sample on the MGISEQ-2000 sequencer platform (MGITech). The alignment was conducted by SOAP2 (-v 2 -r 1 -s 25) and the CNVs were detected based on PSCC (population-scale CNV calling) algorithm with default parameters (Li et al., 2014). High-credible CNVs with p-values all significant in multiplex statistics test were retained for database annotation and pathogenicity prediction using in-house unpublished software. The annotated databases include disease databases (Decipher, ClinVar), health control individuals database (DGV), and generelated diseases or syndromes in the structural variant regions in Human Gene Mutation Database (HGMD), and Online Mendelian Inheritance in Man (OMIM). The principle of the unpublished software for pathogenicity prediction was implemented following the ACMG guidelines (Riggs et al., 2020). Comprehensive pathogenicity interpretations were conducted on each variant based on all the previously annotated information. Uncertain significant CNVs with deletion or duplication chromosomal regions containing curated 452 CHD genes were proposed as candidate genetic etiologies of CHD patients, while likely pathogenic and pathogenic CNVs were considered as definitive genetic etiologies of CHD patients.

Rare Disease Causal Variants Predict and Analysis of Candidate Disease Genes
KGGSeq software is an efficient and comprehensive piece of software that could successfully narrow down whole exome variants to very small numbers of candidate variants in a proband cohort (Li et al., 2012). In this study, we utilized KGGSeq software to narrow down the disease-causing variants based on a 121 CHD cohort and STRING software to conduct functional protein association networks among the candidate disease genes, which are genes from manually interpreted WES and KGGSeq analysis. The bioinformatic pipelines are briefly summarized as follows. First, variants of all 121 samples in "gvcf" format were merged into a single cohort file by the CombineGVCFs module (-A QualByDepth -A RMSMappingQuality -A MappingQualityRankSumTest -A ReadPosRankSumTest -A FisherStrand -A StrandOddsRatio -A Coverage) of GATK v4.1.4.0. Then the joint genotyping was performed by the GATK4 GenotypeGVCFs tool (-A QualByDepth -A RMSMappingQuality -A MappingQualityRankSumTest -A ReadPosRankSumTest -A FisherStrand -A StrandOddsRatio -A Coverage --allow-old-rms-mapping-quality-annotation-data) and the variant quality score recalibration of SNPs and InDels was conducted by the GATK4 VariantRecalibrator module (SNP: -resource:hapmap, known = false, training = true, truth = true, prior = 15.0 hapmap_3.3.hg19.sites.vcf -resource:omni, known = false, training = true, truth = false, prior = 12.0 1000G_omni2.5.hg19.sites.vcf -resource:1000G,known = false, training = true, truth = false, prior = 10.0 1000G_phase1. snps.high_confidence.hg19.sites.vcf -resource:dbsnp, known = true, training = false, truth = false, prior = 2.0 dbSNP_ b151_GRCh37p13.vcf -an DP -an QD -an FS -an SOR -an MQ -an MQRankSum -an ReadPosRankSum -mode SNP; InDel: -resource:mills, known = true, training = true, truth = true, prior = 12.0 Mills_and_1000G_gold_standard.indels.hg19. sites.vcf -resource:1000G,known = false, training = true, truth = false, prior = 10.0 1000G_phase1.indels.hg19.sites.vcf -an DP -an QD -an FS -an SOR -an MQ -an MQRankSum -an ReadPosRankSum -mode INDEL) and ApplyVQSR module (--truth-sensitivity-filter-level 99.0), separately. Subsequently, KGGSeq software was utilized to filter variants based on both genotype and variant level qualities and isolate the most promising disease causal candidate variants. Finally, genes with rare disease causal variants were selected for enrichment analysis by STRING software (version 11.5). Meanwhile, we combined the genes with rare disease causal variants and curated 452 CHD genes for potential CHD-related gene analysis. Genes that have interactions with at least 2 curated CHD genes and where the interaction confidence is no less than 0.9 were retained as potential CHD association genes. Enrichment analysis was also conducted on selected potential CHD association genes.

Clinical Characteristics of Cohort
There were 121 cases of sporadic CHD diagnosed on fetal or neonatal echocardiogram. Fetal gender was identified with sexual chromosome data distribution which was based on low coverage whole-genome sequencing analysis. In our sporadic CHD cohort, male patients are much more than female patients (57.02% vs. 42.98%) ( Table 1). The sample types included in our cohort were tissue, cord blood, and amniotic fluid, which accounted for 56.20, 34.71, and 9.09% of samples, respectively ( Table 1). Ultrasound- based clinical diagnosis records were summarized in Supplementary Table S2, and the primary subtypes of CHD were extracted as core clinical features to judge the subgroup of samples. We defined the single CHD subtype as an isolated CHD subgroup and combined CHD subtypes or syndromic forms of CHD as a non-isolated CHD subgroup.  (Table 3). Del (15q11.2) was also a recurring CNV that was identified in two CHD samples, accounting for 1.65% [2/121, 95%CI (0.08%-6.20%)] ( Table 3).
The remaining 16 CNVs were detected once in CHD samples ( Table 3).

WES Reveals Genetic Etiology in 22 CHD Samples and Identified 8 Candidate CHD-Associated Genes
Data quality of WES was also recorded in Supplementary Table S3

Total Diagnostic Yield of Genetic Etiology in Congenital Heart Disease by Combined WES and Low Coverage WGS
The methodology and processes of genetic diagnosis were summarized in Figure 1. The landscape of the distribution of genetic etiologies and clinical features in our cohort (N = 121) was demonstrated in Figure 2.

Rare Disease-Causing Variants Predicted by KGGSeq and Enrichment Analysis of Potential CHD-Related Genes
A total of 1,133 variants were predicted as rare disease-causing variants by KGGSeq which involved 886 genes (Supplementary  (2) 19S5253078R, 20S2200077, 20B10556839, 19B4152086, 19B4152090, 19B4152105, 19B4152110, 19D5253044, 19S5253038R, 19S5253106, 19S5347586, 19S5347592 20B10556837, 19B4152096, 19B4152103, 19B4152105, 19B4152086, 19D5253226, 19S5253093, 19S5253099, 20S10556850, 20S10556862, 19B5347543-1, 19S5253089R, 20S10556845, 20S2200077, and 20S2200078.  AD 3 AD, Autosomal dominant; LP, likely pathogenic; P, pathogenic. Note: Genotype level 3 means the molecular basis for the disorder is known and at least a mutation has been found in the gene. Table S7). Among the predicted 886 genes, 36 of them were contained in the curated 452 genes, and then we combined the remaining 850 genes with the curated 452 CHD genes for protein interaction analysis which aimed to narrow down the targeted potential CHD-related genes. Ultimately, 86 genes that have interactions with at least two curated CHD genes and where the interaction confidence is no less than 0.9 were retained as potential CHD-related genes. Among the 86 potential CHD-related genes, FN1 has the most associations with definitive CHD genes (13), and genes IRS1, and CCT5 both have the second most interactions with 11 definitive CHD genes (Table S8 and Figures  3A-C). The enrichment analysis of the gene set with 886 genes or 86 potential CHD-related genes was performed using STRING software, and the statistically significant terms with a false discovery rate (FDR) < 0.05 were collected in Supplementary Table S9. Fourteen biological processes by the Gene Ontology resource ( Figure 3D) and two KEGG pathways were enriched in the 886 gene set. Multicellular organismal processes (FDR = 1.14E-06), developmental processes (FDR = 0.00036), and system development (FDR = 0.00036) are the top three biological processes, and the two KEGG pathways were ECM-receptor interaction (FDR = 0.0002) and protein digestion and absorption (FDR = 0.021). The 86 potential CHD-related gene set was enriched in 255 biological processes according to the Gene Ontology resource ( Figure 3E) and 12 KEGG pathways. The 255 biological processes contained 11 of 14 items of the 886 gene set, which included the top three biological processes mentioned earlier, and the 12 KEGG pathways contained all the two pathways identified in the 886 gene set. The biological processes of 86 potential CHD-related genes were mainly enriched in regulation-related processes, accounting for 51.37% (131/255) of the total significantly enriched biological processes, and the top ten biological processes are listed in Figure 3E. In addition, the bubble plot of biological processes reflected that multiple responses to regulation of muscle adaptation have an extremely high gene ratio (observed number of genes in GO term/expected number of genes in GO term) ( Figure 3E). Gene MYOG, a muscle-specific transcription factor that can induce myogenesis in a variety of cell types in tissue culture, is present in all of these processes, as shown in Figure 3E. Among the enriched 12 KEGG pathways, in concordance with the 886 gene set, the ECM-receptor interaction has the most significant FDR (1.80E-10) and three pathways have the equally second most significant FDR (5.14E-05), which include the PI3K-Akt signaling pathway, focal adhesion and dilated cardiomyopathy (Supplementary Table S9). In addition to dilated cardiomyopathy, hypertrophic cardiomyopathy is another significantly enriched cardiac lesion, and its FDR was 0.0024. The gene ratio of dilated cardiomyopathy and hypertrophic cardiomyopathy was 16.60 and 12.88, separately (Supplementary Table S9). In the enrichment results of the molecular functions of 86 potential CHD-related genes, we found the most enriched items were binding functions, which accounted for 68.75% (11/16) of the total significantly enriched items, and the remaining were molecular actively-related functions (3/16) and extracellular matrix structure functions (2/16) (Supplementary Table S9).  (Zaidi and Brueckner, 2017;Wang et al., 2018;Diab et al., 2021;Morton et al., 2022). Meanwhile, damaging variants of definitive or candidate CHD-associated genes accounted for 18.18% [22/121, 95%CI (12.26%-26.06%)] of the samples, much higher than the previously reported 8%-11% (Jin et al., 2017;Zaidi and Brueckner, 2017;Diab et al., 2021). We speculate this high diagnostic yield was caused by two factors, on one hand, a relatively comprehensive CHD-related gene set was manually curated which explained the genetic etiology for 16 samples (13.22%), on the other hand, the in-depth manual interpretation, we additionally identified eight candidate CHDassociated genes which elevated the diagnostic yield by 4.96% (6/ 121). Multiple studies consistently estimated that pathogenic variants in at least 400 genes contribute to CHD (Zaidi et al., 2013;Homsy et al., 2015;Jin et al., 2017;Diab et al., 2021), and with the application of next-generation sequencing in large cohort studies, novel CHD-related genes will continue to be identified, which means the elevation of the explainable ratio of CHD. Aneuploidies were the earliest identified genetic etiology of CHD and most commonly are trisomy 21, 18, 13, and monosomy X (Hartman et al., 2011). Deletion of chromosome 22q11.2 is considered one of the most frequent genetic etiologies of CHD and is detected in approximately 1.9% of all CHD patients (Agergaard et al., 2012). Results of our cohort reflected that deletion 22q11.2 is the most frequent chromosomal abnormality, which explained genetic etiologies in 4.96% (6/ 121) of all CHD samples. While trisomy 18, 21, monosomy X, and deletion 15q11.2 are both recurrently detected in two samples, which occupied 1.65% of all CHD samples separately. Deletion 15q11.2 was first linked to CHD by Soemedi R. et al. and was found in 0.53% (12/2,256) of CHD samples (Soemedi et al., 2012). In addition to the recurrent chromosomal abnormalities, protein-truncating variants of eight genes were also recurrently identified in our cohort. Gene COL3A1 (3.31%, 4/121) is the most frequent causative gene, followed by MED13L, KDR, ANK3 (2.48%, 3/121), and SMAD6, NIPBL, ATP2C1, and APC (1.65%, 2/121). A large cohort GWES study by Sheng C. J. et al. in 2017 showed a totally different recurrently gene pattern that variants in gene GDF1 account for~5% of severe CHD in Ashkenazim, variants in gene MYH6 accounting for FIGURE 1 | Methodological workflows of genetic etiological diagnosis and CHD-related gene prediction. Each case had conducted low-coverage WGS and WES. The unique reads mean depth of whole-genome by low-coverage WGS was 0.37X. A total of 204 CNVs were identified in 95 samples, and the ACMG classifications of these 204 CNVs were demonstrated in a stacked bar chart. Six aneuploidy variants, seven LP|P CNVs, and eight candidates CHD pathogenic VUS CNVs were proposed as CHD causative CNVs. These 21 causative CNVs all explained 30 samples and some of the causative CNVs were diagnosed repeatedly in multiple samples ( Table 3). The mean depth in the capture region of WES was 171.04X. Total 724 P|LP variants were identified in 268 genes involving 100 samples and their functional effect was shown in a stacked bar chart. Among these 724 P|LP variants there were 94 variants belonging to 33 definitive CHD genes and 8 variants belonging to candidate CHD-related genes. Combined with low-coverage WGS and WES, there were 42 samples identified with genetic etiology with a 34.71% diagnostic yield. We also explored the potential CHD association genes by KGGSeq software based on WES data. A total of 1,133 variants on 886 genes were predicted as rare diseasecausing. 36 of the 886 were definitive CHD-related genes. The left 850 genes were further narrowed down based on interactions with curated CHD genes. Finally, 86 genes were proposed as CHD-related genes. Abbreviations: UR, unique reads; LP|P, likely pathogenic or pathogenic; CR, capture region. ~11% of Shone complex, and variants in gene FLT4 accounting for 2.3% of Tetralogy of Fallot (TOF) (Jin et al., 2017). Another large cohort GWES study by Sifrim et al. (2016) demonstrated that genes NOTCH1 (0.47%, 4/847), FBN2 (0.24%, 2/847), SOS1 (0.24%, 2/847), NOTCH2 (0.24%, 2/847) recurrently detected protein-truncating variants in non-syndromic CHD cases, while protein-truncating variants in gene NSD1, KMT2A, and ADNP were detected in 0.77% (4/518) syndromic CHD cases separately, and followed by gene CHD7, KMT2D, and ANKRD11 in 0.58% (3/518) and gene MED13L in 0.39% (2/ 518) syndromic CHD cases. Multiple studies have shown that different CHD subtypes or cohorts have different patterns of recurrence of chromosomal abnormalities or CHD-associated genes, and by the analysis of the odds ratio of identifiable genetic etiology in gender, latest ultrasound periods, and clinical features, we found female, fetuses, and isolated CHD patients have a higher risk of genetic factors than male, neonates, and non-isolated CHD. The recurrence of monosomy X (in 3 cases) in our cohort might be one of the factors in high OR in female CHD cases. Fetuses diagnosed with CHD included cases not surviving to term in which chromosomal abnormalities were considered major causes (~60% of spontaneous miscarriage) (Daniely et al., 1998;Li et al., 2020), and the latest follow-up time of isolated CHD cases were focused on the fetal period (78.43%, 40/51). The aforementioned factors and the characteristics of our cohort led to the OR assessment results which reflect the genetic profile of an unselected sporadic CHD cohort.

DISCUSSION
By in-depth interpretation, we identified and proposed eight candidate CHD association genes: SYNE2, MYLK, PKP2, TRPM4, MIB1, TCAP, SON, and DSP. They all have defined molecular pathogenic mechanisms associated with the abnormal cardiac phenotype and detected protein-truncating variants that are considered pathogenic or likely pathogenic variants according to ACMG rules. Gene SYNE2 is linked to Emery-Dreifuss muscular dystrophy (#612999) and its clinical manifestations of proximal muscle weakness, as well as cardiac involvements such as arrhythmias, dilated cardiomyopathy, or heart failure (Heller et al., 2020). Gene MYLK is linked to familial thoracic aortic aneurysm (#613780), which manifests clinically as cardiovascular system abnormalities (Wang et al., 2010;Isselbacher et al., 2016). Gene PKP2 was found to be responsible for arrhythmogenic right ventricular dysplasia 9 (#609040) (Gerull et al., 2004). Gene TRPM4 is associated with progressive familial heart block, type IB (#604559) (Liu et al., 2010). Gene MIB1 is the causative gene of left ventricular noncompaction 7 (#615092) (Luxán et al., 2013). Gene TCAP is the causative gene of cardiomyopathy, hypertrophic, 25 (#607487) (Hayashi et al., 2004;Bos et al., 2006). Gene SON is associated with ZTTK syndrome (#617140), which is a severe multisystem developmental disorder and has congenital defects of the heart in some patients (Kim et al., 2016;Takenouchi et al., 2016). The gene DSP causes arrhythmogenic right ventricular dysplasia 8 (#607450) (Yang et al., 2006;Christensen et al., 2010) and dilated cardiomyopathy (#615821) (Norgett et al., 2006;Boulé et al., 2012;Boyden et al., 2016). In this study, 86 genes were proposed as potential CHDrelated genes. The enrichment analysis of these 86 genes displayed that except pathways of cardiac lesions were significantly enriched, including dilated cardiomyopathy and hypertrophic cardiomyopathy, other pathways like ECMreceptor interaction, PI3K-Akt signaling pathway, and Focal adhesion were the top significance of FDR. A research study published by Chen and Jiang (2019) revealed that downregulated differentially expressed genes in arrhythmogenic right ventricular cardiomyopathy (ARVC) of males compared to females were mainly enriched in the "ECM-receptor interaction" and "protein digestion and absorption" pathways. The "protein digestion and absorption" pathway was also enriched in our results with FDR = 0.0316. In a study by Yu et al. (2022) proved that gene PDTLN1 caused cardiac developmental defects in zebrafish via suppressing the PI3K/AKT signaling pathway, and another study by Zhao et al. (2021) discovered that a high level of melatonin could inhibit growth by inducing apoptosis and cell cycle arrest via PI3K-AKT signaling pathway, thereby interfering with embryonic heart development. In a review by Liu et al. (2019) summarized recent research progresses of actin cytoskeleton in the deployment process of mouse second heart field (SHF) progenitor cells and revealed that actin cytoskeleton played a significant role in mouse SHF development. Regulation of actin cytoskeleton pathway was also enriched in our results with FDR = 0.0022. According to our GO enrichment results, the biological processes of 86 potential CHD-related genes were mainly enriched in regulation-related processes, accounting for 51.37% (131/255) of all significant terms. Generally, the heart is the first functional organ to be developed in vertebrate embryos, and this process is strictly controlled by a gene regulatory network (Saliba et al., 2020). Notably, multiple responses to regulation of muscle adaptation have an extremely high gene ratio in our GO enrichment results, although no relevant studies have been reported so far. Our findings may provide insights for further CHD pathogenic mechanism research. SNVs in the noncoding regions, particularly those regulating gene expressions, were missed due to insufficient sequencing depth in our study, in which low-coverage WGS was used to detect chromosomal abnormities. A study published by Sweeney et al. (2021) revealed that whole-genome sequencing with about 40-fold diagnosed genetic etiologies in 46% of CHD infants. We can anticipate that a high depth of WGS combined with other omics technologies, such as transcriptome or metabolome, will lead to the discovery of new genetic etiologies and novel insights into the pathogenesis of CHD.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study have been deposited into CNGB Sequence Archive (CNSA) of China National GeneBank DataBase (CNGBdb) with accession number CNP0003160.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Ethics Committee of the Fujian Maternity and Child Health Hospital. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.