Identification of Rare Variants in Right Ventricular Outflow Tract Obstruction Congenital Heart Disease by Whole-Exome Sequencing

Abstract

Background: Pulmonary atresia (PA) is a kind of congenital heart disease characterized by right ventricular outflow tract obstruction. It is divided into PA with intact ventricular septum (PA/IVS) whose favorable form is pulmonary valvular stenosis (PS), and PA with ventricular septal defect (PA/VSD) whose favorable form is tetralogy of Fallot (TOF). Due to limitations in genetics etiology, whole-exome sequencing (WES) was utilized to identify new variants associated with the diseases.

Methods: The data from PS-PA/IVS (n = 74), TOF-PA/VSD (n = 100), and 100 controls were obtained. The common sites between PS and PA/IVS, PA/VSD and TOF, were compared. The novel rare damage variants, and candidate genes were identified by gene-based burden analysis. Finally, the enrichment analysis of differential genes was conducted between case and control groups.

Results: Seventeen rare damage variants located in seven genes were predicted to be associated with the PS through burden analysis. Enrichment analysis identified that the Wnt and cadherin signaling pathways were relevant to PS-PA/IVS.

Conclusion: This study put forth seven candidate genes (APC, PPP1R12A, PCK2, SOS2, TNR, MED13, and TIAM1), resulting in PS-PA/IVS. The Wnt and cadherin signaling pathways were identified to be related to PS-PA/IVS by enrichment analysis. This study provides new evidence for exploring the genetic mechanism of PS-PA/IVS.

Keywords: congenital heart disease; pulmonary atresia with intact ventricular septum; pulmonary valvular stenosis; right ventricular outflow tract obstruction; single nucleotide polymorphism; whole-exome sequencing.

Figure 1

The flow chart showed the different steps taken during whole-exome sequencing analysis. After variant calling, annotation and screening, variants were filtered by gene-based burden analysis. Candidate genes were collected by gene expression analysis. FDR, false discovery rate; MAF, minor allele frequency; PA/IVS, pulmonary atresia with intact ventricular septum; PA/VSD, pulmonary atresia with ventricular septal defect; PS, Pulmonary valvular stenosis; SNP, single nucleotide polymorphism; TOF, tetralogy of Fallot; WES, whole-exome sequencing.

Figure 2

The comparisons of the variants among four disease groups. The localization of selected variants (A), variants type (B), the function of exonic variants (C), and SNV classes (D) of four diseases. (E) The comparisons of the variants between PA/IVS and PS, TOF and PA/VSD.

Figure 3

The PS-associated genes were identified by gene-based burden analysis. (A) The known genes related to the PS phenotypes were searched and summarized. (B) Genes associated with PS phenotypes were shown after excluding the genes related to the syndromes. (C) The association between genes tested and disease phenotype was predicted. (D) The score of genes tested was listed by Phenolyzer (http://phenolyzer.wglab.org/).

Figure 4

Enrichment analysis of differential genes between PS-PA/IVS and control groups. Top 20 of disease (A), ontology (B), and pathway (C) enrichment were listed.

Figure 5

Mutation validation of seven genes with 19 SNPs. Only two SNPs (APC chr5:112170769 and PPP1R12A chr12:80203601) were not validated successfully. The yellow arrows represent the sites of SNPs.