Systems genetics analysis identifies calcium-signaling defects as novel cause of congenital heart disease

Abstract

Background: Congenital heart disease (CHD) occurs in almost 1% of newborn children and is considered a multifactorial disorder. CHD may segregate in families due to significant contribution of genetic factors in the disease etiology. The aim of the study was to identify pathophysiological mechanisms in families segregating CHD.

Methods: We used whole exome sequencing to identify rare genetic variants in ninety consenting participants from 32 Danish families with recurrent CHD. We applied a systems biology approach to identify developmental mechanisms influenced by accumulation of rare variants. We used an independent cohort of 714 CHD cases and 4922 controls for replication and performed functional investigations using zebrafish as in vivo model.

Results: We identified 1785 genes, in which rare alleles were shared between affected individuals within a family. These genes were enriched for known cardiac developmental genes, and 218 of these genes were mutated in more than one family. Our analysis revealed a functional cluster, enriched for proteins with a known participation in calcium signaling. Replication in an independent cohort confirmed increased mutation burden of calcium-signaling genes in CHD patients. Functional investigation of zebrafish orthologues of ITPR1, PLCB2, and ADCY2 verified a role in cardiac development and suggests a combinatorial effect of inactivation of these genes.

Conclusions: The study identifies abnormal calcium signaling as a novel pathophysiological mechanism in human CHD and confirms the complex genetic architecture underlying CHD.

Keywords: Calcium signaling; Congenital heart disease; Developmental biology; Genetics; Systems biology; Whole exome sequencing.

Fig. 1

Distribution of candidate disease genes and variants across families. a Overlap between CDGs in pairs of families. b Distribution of CDGs across families. The number of CDGs found in one, two, three, and 4–7 families is shown. c Distribution of alleles in CDGs found in three families (1-2-3, three different alleles; 1-1-2, two different alleles; 1-1-1, same allele found in all three families). d Distribution of alleles in CDGs shared in two families (1-2, different alleles; 1-1, same allele).

Fig. 2

Enrichment of CHD genes in candidate disease genes. Overlap between CDGs and known CHD genes from mouse models (a) and patients (b). The number of overlapping genes is plotted against the number of families the CDGs were found in. c The number of overlapping genes (mouse models) per family. Statistical significance of the overlap is indicated by color code (red colors, significant; gray color, not significant (n.s.))

Fig. 3

Identification of a calcium-signaling network affected by rare mutations identified in CHD families. a Network module of CDGs (blue) and their interaction partners (gray). The module accommodates more CDGs than expected by chance (adjusted p value 0.0033). Proteins are shown as hexagons; protein interactions are shown with lines. b Violin plots of distributions of pLI scores in genes encoding the 27 proteins in the network module (upper, blue), known CHD genes from patients and mouse models (middle, red and pink, respectively), and all 18,225 genes listed in ExAC with a calculated pLI score (lower, gray).***p < 0.001. ns, not significant (Kruskal-Wallis one-way analysis of variance on ranks)

Fig. 4

Distribution of MPC and CADD scores of rare variants in 714 CHD cases and 4922 controls. Protein altering and truncating variants (PAV and PTV) with MAF < 0.001 identified in the genes ADCY2, ADCY5, CACNA1D, CACNA1H, CACNA1I, CACNA1S, GRIA4, ITPR1, NFAT5, and PLCB2 were scored using MPC score [22] (a) or CADD score [21] (b). N_CHD = 136 variants. N_Controls = 982 variants. Difference between median values of controls and cases was determined using a Mann-Whitney rank-sum test. **p < 0.01, *p < 0.05

Fig. 5

Functional validation of candidate genes in zebrafish. a Phenotype of controls and morphants. Single genes and combinations of genes targeted by splicing morpholinos are indicated on the left. Upper panels: gross appearance of 48 hpf zebrafish embryos. Lower panel: examples of cardiac phenotypes of 48 hpf wt, control, and morphant embryos. Hearts were visualized by ISH with a probe against the cardiac marker myl7. b Quantification of phenotypes in wt, controls, morphants, and mRNA rescued morphants. Note the combinatorial effects on cardiac phenotypes when more than one gene is affected. c, d Expression of mef2cb in 10 somite stage zebrafish embryos, analyzed by qRT-PCR (c) and ISH (d). ISH staining intensity was quantified and analyzed using Student’s T test. *p < 0.05, **p < 0.01