Rare variants in CAPN2 increase risk for isolated hypoplastic left heart syndrome

Abstract

Hypoplastic left heart syndrome (HLHS) is a severe congenital heart defect (CHD) characterized by hypoplasia of the left ventricle and aorta along with stenosis or atresia of the aortic and mitral valves. HLHS represents only ∼4%-8% of all CHDs but accounts for ∼25% of deaths. HLHS is an isolated defect (i.e., iHLHS) in 70% of families, the vast majority of which are simplex. Despite intense investigation, the genetic basis of iHLHS remains largely unknown. We performed exome sequencing on 331 families with iHLHS aggregated from four independent cohorts. A Mendelian-model-based analysis demonstrated that iHLHS was not due to single, large-effect alleles in genes previously reported to underlie iHLHS or CHD in >90% of families in this cohort. Gene-based association testing identified increased risk for iHLHS associated with variation in CAPN2 (p = 1.8 × 10^-5), encoding a protein involved in functional adhesion. Functional validation studies in a vertebrate animal model (Xenopus laevis) confirmed CAPN2 is essential for cardiac ventricle morphogenesis and that in vivo loss of calpain function causes hypoplastic ventricle phenotypes and suggest that human CAPN2^707C>T and CAPN2^1112C>T variants, each found in multiple individuals with iHLHS, are hypomorphic alleles. Collectively, our findings show that iHLHS is typically not a Mendelian condition, demonstrate that CAPN2 variants increase risk of iHLHS, and identify a novel pathway involved in HLHS pathogenesis.

Keywords: Xenopus; association; congenital heart defect; development; exome sequencing; frog; oligogenic.

Figure 1

Discovery association tests (A–D) Comparison of SKAT-O (A and B) and burden (C and D) approaches to gene-based rare variant aggregate association analysis. Manhattan (B and D) and QQ (A and C) plots demonstrate that CAPN2 is the most significantly associated gene tested, regardless of approach. A low median λ value for the SKAT-O QQ plot (A) suggests genomic deflation in this approach.

Figure 2

CAPN2 variants identified in individuals with isolated hypoplastic left heart syndrome (iHLHS) Gene diagram of CAPN2 (GenBank: NM_001748.4) with locations of variants identified in individuals with iHLHS. Black dots represent variants present in the association subset of the cohort, blue dots represent variants identified in samples excluded from the association analysis due to lack of ancestry-matched controls.

Figure 3

CRISPR-Cas9-mediated functional studies of CAPN2 variants in vivo Xenopus laevis embryos were injected with capn2 single guide RNA (sgRNA) alone (control; A), capn2 sgRNA plus Cas9 protein (to elicit capn2 indels and loss of Capn2 function; B), or capn2 sgRNA/Cas9 co-injected with WT capn2 mRNA (C), mutant capn2 mRNA engineered to contain the orthologous human CAPN2^707C>T variant (707 mRNA; D), or mutant capn2 mRNA engineered to contain the orthologous human CAPN2^1112C>T variant (1112 mRNA; E). Injected F0 embryos were allowed to develop through cardiogenesis and immunostained for Troponin T (red) to highlight the outflow tract (oft) and ventricle (v) of the heart (top row); optical sectioning was used to visualize the internal ventricular chamber (arrows, bottom row). In contrast to controls (A), embryos with CRISPR-Cas9-mediated loss of function of Capn2 (B) develop a hypoplastic ventricle and reduced ventricular chamber. Normal phenotype (C) is restored by co-injecting exogenous WT capn2 mRNA, confirming specificity of the phenotype. Co-injection of 707 mRNA can also restore normal ventricle phenotypes (D), although the frequency of rescue was not statistically significant (F), indicating that the CAPN2^707C>T variant may be a mildly hypomorphic allele. Co-injection of 1112 mRNA is unable to rescue the HLHS-like phenotype at all (E and F), indicating the CAPN2^1112C>T variant may represent a null allele with respect to function necessary for normal ventricular development. (F) Results were quantified from three independent trials (n = 14–57 embryos per condition, per experiment); error bars indicate standard deviation. Significance of differences between the percentage of HLHS-like phenotypes in each condition is noted. Scale bars, 100 μm.