FUNCTIONAL VALIDATION OF NON-CODING REGULATORY VARIANTS ASSOCIATED WITH CHILDHOOD CARDIOMYOPATHY

Cardiomyopathy (CMP) is a genetic disease of the heart muscle that causes heart failure and sudden cardiac death in children. Our goal was to define the missing genetic etiology of childhood onset CMP by identifying the role of functionally active genomic variants in regulatory elements of CMP genes.

A total of 225 unrelated CMP families underwent whole-genome sequencing to explore coding and non-coding variants in 133 known CMP genes. 37% harboured pathogenic coding variants in known genes. An additional 20% cases harboured a high burden of rare, high-risk variants in promoters and enhancers that were predicted to alter transcription of CMP genes. To evaluate the functional impact of non-coding variants, functional assays were conducted on high-risk Tier 1 regulatory variants of 7 genes: BRAF, DSP, DTNA, FKTN, LARGE1, PRKAG2, TGFB3. Target gene and protein expression was measured in patient LV myocardium. Promoter variants of BRAF, FKTN and LARGE1 were associated with downregulation of mRNA expression and protein expression (p < 0.05 vs controls without the variant). Promoter variant in DSP and enhancer variants in PRKAG2 and TGFB3 were associated with upregulation of mRNA and protein expression (p < 0.05 vs controls). The activity of a luciferase reporter gene was down- or upregulated under the effect of the variant promoter/enhancer sequences compared to wild-type control in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). High-throughput massively parallel reporter assay in hiPSC-CMs was used to test the effect of prioritized regulatory variants on target gene expression. Of 54 variants tested, 29 (54%) variants were associated with a significant up- or down-regulation of target gene transcription compared to the reference allele (False discovery rate < 0.05).

High risk variants in regulatory elements of CMP genes that affect target gene expression may contribute to childhood CMP and represent an important advance in our understanding of the genomic etiology of CMP.