Case–control association study of congenital heart disease from a tertiary paediatric cardiac centre from North India

Congenital Heart Defect (CHD) is common structural abnormality occurring at the time of foetal development. Limited information is available on the exact mechanism of CHD pathogenesis. It affects 9 in 1000 live births globally [1]. This corresponds to 17% of the world CHD load from India [2], yet meagre genetic information available for the disease in the country. In early gestation, incidence is even higher as certain CHDs are complex and have been shown to result in foetal demise [3]. Septational abnormalities account for half of the cardiac congenital defects ranging from nonpathological to lethal [4]. Cardiac development is a complex process and requires intricate coordination of several molecular events for eventual normal structure and function of the heart. Any error/s in these steps result in pathogenic remodelling of heart [5]. Chromosomal aneuploidies like Trisomy 21, 18 and 13 are commonly associated with CHDs [6, 7]. Though 80% CHDs are sporadic in origin [8], some familial cases of Atrial Septal Defect (ASD), Ventricular Septal Defect (VSD) and Hypoplastic Left Heart Syndrome (HLHS) are recorded but the inheritance patterns are complex [9, 10]. The recurrence risk in off springs of CHD patients varies from 3- 20% depending on the lesion, with slightly higher recurrence in females [11]. Almost one half of the siblings with recurrent lesions in a family have a different lesion suggesting multifactorial etiology and illusive molecular mechanisms [8, 9, 12].

Technological advances now enable study of these developmental defects, thus closing the gap of knowledge between the morphology and genetics [6]. Novel techniques identify regions in the genome on which transcription factors act, driving their target genes and provide new knowledge on CHD development. The T-box transcription factors (TBX5) gene is reported to interact with Nk2 Homeobox 5 (NKX2.5) and GATA binding protein 4 (GATA4) both transcriptional activator of Natriuretic peptide B, which positively regulates the developing heart. The involvement of several well-established cardiac transcription factors that are expressed in cardiogenic plates such as NKX2.5, GATA4, TBX5, TBX20, Myosin heavy chain 6 (MYH6), Actin alpha cardiac muscle (ACTC1) and Myocyte enhancer factor 2C (MEF2C) have been extensively studied in both human and animal experiments [13-17]. Role of Mutations in transcription factors have been studied for non-syndromic CHDs [18].

Point mutations of cardiac transcription factor genes, single nucleotide polymorphism (SNPs), aneuploidy, and chromosomal copy number variants (CNVs) are directly associated with CHDs. Association of single SNPs seldom lead to complex disease manifestation [19]. There are substantial genetic predispositions to inherited as well as de novo variants with variable effect sizes towards disease risk. Since mutations are rare it requires large numbers to be screened, also same mutations may not be present in all samples. Conventionally a multifactorial inheritance model has been proposed for CHD involving a multitude of susceptibility genes, with low-penetrant common variants or intermediate-penetrant rare variants, superposed on unfavourable environmental factors as causal [15, 20, 21]. Several ethnic or racial differences may also be observed [22]. It is important to investigate genotype–phenotype correlation to provide leads with an opportunity to predict the prognosis.

Limited genetic diagnosis is available for many of the CHDs. Therefore several commercial ventures to sequence genes have been undertaken [23, 24]. These ventures discovered several de novo mutations in the known as well as new genes [6, 12]. Genome wide association studies (GWAS) involve the comparison of genetic variants (known as well as unknown), which can be used to detect genetic risk factors of big and small effect to CHD manifestation [12, 25, 26]. So far more than 500 genes have been estimated with a potential role in the development of CHD [27]. Genetic studies on transcription factors like GATA4, NKX2.5, TBX1and TBX20 have previously shown to identify new mutations in Indian population. [28-33]. Till date limited candidate gene studies have been done in India [34] and only one using exome sequencing [35].

Next generation sequencing (NGS) technologies meet a high standard of evidence and also afford correct predictions in novel datasets. In this study we select association findings from GWAS on CHDs and evaluate in a north Indian cohort. Screening for SNPs and not mutations may reflect better in an association study. Therefore, we chose to assess association of common variants primarily from previous GWAS or meta-analysis studies [26, 36-39] in Caucasians and tested their replicability in our adequately powered study samples.

A total of n = 23 SNPs in Hardy Weinberg Equilibrium (stringent cut off: p > 0.001) were included in the analysis (Supplementary Table 2). Since two SNPs rs2046060 and rs12165908 were monomorphic hence they could not be utilised further for association study. No significant allelic association was seen among cyanotic and acyanotic cases groups. These were still analysed separately vs all controls.

Markers with allelic and genotypic associations are tabulated and presented in Table 1 and 2 for acyanotic, cyanotic and combined categories.

Allelic Association: rs73118372 on chr. 3, rs28711516 and rs735712 on chr. 14 exhibited association for all three categories (acyanotic, cyanotic and combined categories; Table 1). rs73118372 in CRELD1(χ² = 15.7; p < 0.0001); rs28711516 in MYH6(χ² = 11.18; p = 0.00083) and rs735712 in MYH7 (χ² = 10.98; p = 0.0009) showed strong allelic associations. Variant rs11874 in GOSR2 had nominal association (χ² = 3.81; p = 0.051) and rs185531658 an intergenic SNP on Chr 5 [39] showed association on Fisher’s test as allele counts were low (p = 0.043) while rs659366 in UCP2 and rs2388896 intergenic SNP demonstrated a weak trend of association (Table 1).

All associations were mainly driven by similar association in the larger acyanotic group. For the SNP rs659366 the association strengthen (χ² = 5.74; p = 0.017) in the acyanotic group. The cyanotic association was established only for rs73118372, rs28711516 and rs735712.

50% of tested SNPs were substantially associated in either allelic, genotypic or sub-phenotypes of north Indian CHD cohort validating their strong correlation with disease manifestation. Burden of CHD is overall heavy in India [2] and is prominent in north India. Several genetic determinants of this complex developmental disorder have been reported based on conventional candidate genes and contemporary GWAS but mostly in Caucasian populations. However it is very poorly investigated in the ethnically distinct Indian population. This study was an attempt to test the association of Caucasian findings prior to performing a hypothesis-free approach in the study cohort. Of the 23 SNPs which were successfully genotyped in the modest sized study cohort, 11 SNPs showing allelic or genotypic or association with the CHD sub-phenotypes (Table 1, 2, 3) in a trans ethnic population was noteworthy and reiterates the functional relevance of the associated genes/pathways in CHD pathogenesis.

Of the seven associations observed, four SNPs namely rs73118372 (missense variant) Chr 3; rs659366 (promoter region) on Chr 11; rs735712 (synonymous variant) and rs28711516 (missense variant) on Chr 14; are of functional relevance. Strongest allelic association was observed for rs73118372, rs28711516 and rs735712.

The variant rs73118372(c.1136 T > C) in Exon 9 of CRELD1 is associated with Downs syndrome [47]. CRELD1 is involved in the formation of atrioventricular cushion [48] and disrupts existing exon splicing, thus altering the protein configuration and making it unstable. It was associated for all cyanotic, acyanotic and combined categories, and with ASD and TOF sub-phenotypes. Intronic SNP rs1975649 (SYNPR; Intron2) also on Chr3 exhibited strong association both in allelic and genotypic categories and was also associated with ASD. Cardiac myosin is the molecular motor that powers heart contraction, a property essential for heart function. It also plays a pivotal role in muscle regulation, development, and mechanotransduction [49]. The α-MYHC (MYH6) is expressed in atrial muscle and the β-MYHC (MYH7) in skeletal slow-twitch muscle and have arisen through a tandem gene duplication event [50] on Chr. 14. The duplication event is not evident in the genomes of other vertebrates (e.g. birds, fish, amphibia) [51]. MYH6, MYH7 and MYH7B are associated with R amplitude [52]. Heterozygous pathogenic variants in MHY7 have been associated with septal defects or Ebstein anomaly [48] and of MYH6 with HLHS and cardiac conduction [53, 54]. MYH6 is associated with non-syndromic coarctation of the aorta [38, 50] and also presented in families of Shone complex [54]. Previous reports on MYH6 rs28711516 (c.166G > A; p.G56R) associations with atrial fibrillation [53] and sporadic dilated cardiomyopathy [55] and a GWAS study from south India [35] warrant further investigation of this gene. MYH7 mutations have been reported for Indian families [56, 57]. Exon 12 variant rs735712 (c.1062C > T; p.G354G) has previously been reported in dilated cardiomyopathies in Indian population [58]. Previous linkage study using microarray identified rs1055061in HOMEZ, a ubiquitously expressed transcription factor on the same locus, in 83 consanguineous CHD families from India [59]. In present study, synonymous variant rs3746446 in MYH7B was associated only with ASD phenotype. This SNP showed strong association with congenital cardiovascular left-sided lesions [38]along with rs12045807, a SNP not associated in our study.

Allelic and genotypic association for combined as well as the acyanotic group for promoter variant rs659366 in UCP2, having a role in reactive oxygen species (ROS) pathway [60, 61] is already been reported to be associated with maternal diabetes in CHD offsprings [62] and with dietary factors in Asian populations [60]. It was also associated with ASD subtype. rs185531658, a SNP with the strongest association in 4034 patients of CHD [39], was nominally associated both with our combined data and in acyanotic group.

ELN rs2071307 showed mild association with AVSD and PKD1L2 rs55788414 with TGA subtype. GOSR2 rs11874, a promoter SNP, was detected in patients with anomalies of thoracic arteries and veins, and may affect the expression of GOSR2 [39]. It showed a marginal association in our population for combined and acyanotic categories. Missense variant rs35761929 in JAG1, involved in Notch cell signalling was associated with ASD subtype in present study and was previously found in ten exome sequenced families from India [38]. During development, the notch pathway regulates embryonic cells destiny to be part of the heart, liver, eyes, ears, and spinal column. The Jagged-1 protein continues to play a role throughout life in the development of new blood cells. These four markers reported are too small in number to make a conclusive statement on its role based on the present results and warrant replication in larger sample set.

Findings in the study were predominantly from the coding region and a few from the intergenic region. The CRELD1, MYH6 and MYH7 interact with each other during development of the myosin filament, an active and essential component of the heart tissue. Only one of the strongly associated common variants per gene was tested in this study. There may be more rare and common variants associated from these genes and additional association studies are essential to estimate polygenic risk score to make a significant genotype- phenotype risk prediction for CHD.

This is a first study testing association of Caucasian GWAS SNPs in a north Indian population. All the SNPs studied have been previously shown to have a strong role in the development of CHD. 11 out of 21 SNPs were associated in the study cohort and highlighted the role of CRELD1, MYH6 and MYH7 in non-syndromic CHD. Strong association of markers from Chr3 and Chr14, and with ASD, VSD and TOF sub-phenotypes were notable in this study and warrant replication in independent CHD cohorts of north Indian origin. This may help uncover the mechanism of disease manifestation by a complex landscape of events influenced by variations in genetic, environmental and demographic patterns.