Whole-exome sequencing and an iPSC-derived cardiomyocyte model provides a powerful platform for gene discovery in left ventricular hypertrophy

Abstract

Rationale: Left ventricular hypertrophy (LVH) is a heritable predictor of cardiovascular disease, particularly in blacks.

Objective: Determine the feasibility of combining evidence from two distinct but complementary experimental approaches to identify novel genetic predictors of increased LV mass.

Methods: Whole-exome sequencing (WES) was conducted in seven African-American sibling trios ascertained on high average familial LV mass indexed to height (LVMHT) using Illumina HiSeq technology. Identified missense or nonsense (MS/NS) mutations were examined for association with LVMHT using linear mixed models adjusted for age, sex, body weight, and familial relationship. To functionally assess WES findings, human induced pluripotent stem cell-derived cardiomyocytes (induced pluripotent stem cell-CM) were stimulated to induce hypertrophy; mRNA sequencing (RNA-seq) was used to determine gene expression differences associated with hypertrophy onset. Statistically significant findings under both experimental approaches identified LVH candidate genes. Candidate genes were further prioritized by seven supportive criteria that included additional association tests (two criteria), regional linkage evidence in the larger HyperGEN cohort (one criterion), and publically available gene and variant based annotations (four criteria).

Results: WES reads covered 91% of the target capture region (of size 37.2 MB) with an average coverage of 65×. WES identified 31,426 MS/NS mutations among the 21 individuals. A total of 295 MS/NS variants in 265 genes were associated with LVMHT with q-value <0.25. Of the 265 WES genes, 44 were differentially expressed (P < 0.05) in hypertrophied cells. Among the 44 candidate genes identified, 5, including HLA-B, HTT, MTSS1, SLC5A12, and THBS1, met 3 of 7 supporting criteria. THBS1 encodes an adhesive glycoprotein that promotes matrix preservation in pressure-overload LVH. THBS1 gene expression was 34% higher in hypertrophied cells (P = 0.0003) and a predicted conserved and damaging NS variant in exon 13 (A2099G) was significantly associated with LVHMT (P = 4 × 10(-6)).

Conclusion: Combining evidence from cutting-edge genetic and cellular experiments can enable identification of novel LVH risk loci.

Keywords: cardiomyocyte; exome; genomics; hypertrophy; left ventricular mass.

Figure 1

(A) Relative cell size of iCell cardiomyocytes under isoproterenol (ISO) hypertrophic stimulation versus control cells. (B) Photographs of iCell cardiomyocytes after hypertrophic stimulation versus controls.