CRISPR gene editing, cardiac disease modelling and transcriptomics interpretation of a novel GATA4 patient genetic variant

Over 400 million people worldwide live with a rare disease. Whole exome and whole genome sequencing often identifies potential disease causative genetic variants that are novel, which must be classified as variants of uncertain significance (VUS). Functional laboratory assays must be performed to prove disease causation, creating major delays in patient diagnostics. Here we investigate a GATA4 (p.Arg283Cys) VUS in a with congenital heart disease. The variant was CRISPR gene edited into inducible pluripotent stem cells, followed by cardio-myocyte differentiation. Genetic variant and healthy cells were similar at the level of cardiomy-ocyte cell marker expression of troponin T (cTNNT), and GATA4 protein expression. Strik-ingly, transcriptomics profiling identified differences in differentiation consistent with the pa-tient’s disease characteristics, and elucidated changes in calcium signaling and adrenergic sig-naling in cardiomyocytes. Altered action potential changes in GATA4,p.Arg283Cys (GA-TA4_HDR) cardiomyocytes were indicative of cardiac abnormalities. Our work provides strong molecular evidence for classifying the GATA4 p.Arg283Cys variant as pathogenic. Furthermore, we demonstrate the combined utility of iPSCs, CRISPR gene edit-ing and differentiation into cardiomyocytes in assessing variants associated with cardiac pheno-types. Comparative gene expression profiling analysis of RNA-seq data for GATA4_WT and SNV KOLF-2 iPSCs and their derived cardiomyocytes. GATA4 CRISPR gene editing in KOLF-2C. Genomic DNA representation of GATA4 in clones with introduced GATA4 c.847C>T mutation.