Investigating The Effect Of Patient Missense Variants In CHD7 Using An Induced Pluripotent Stem Cell Model

Congenital heart disease (CHD) is a phenotypically diverse and genetically heterogenous disease. While monogenic causes of familial CHD and damaging de novo variants are known contributors to CHD risk, novel variants of uncertain significance (VUS) detected among CHD probands remain under characterized. In particular, the effect of missense variants in CHD7 (chromodomain helicase DNA binding protein 7), remain poorly explored. CHD7 is an established CHD gene with variants associated with CHARGE syndrome (Coloboma, Heart defects, Atresia of the choanae, Retardation of growth and development, Genital/urinary abnormalities, and Ear abnormalities) and incomplete syndromic phenotypes. We hypothesized that some CHD7 missense variants in genetically engineered human-induced pluripotent stem cells (iPSCs) perturb essential cardiac developmental pathways. CHD7 is highly expressed in iPSCs and during differentiation to iPSC-cardiomyocytes. Out of seventy rare patient missense variants in CHD7 reported by the Pediatric Cardiac Genetics Consortium (PCGC), six were chosen for genomic editing. To investigate the functional effects of missense variants in CHD7, we first predicted pathogenicity using AlphaFold, an algorithm which models variant impact on the three-dimensional structure of proteins. We then introduced six patient missense variants (Q161R, R286G, I1500V, T262A, T894A, L2299P) into an iPSC line using adenine base editors. Heterozygous and homozygous loss-of-function CHD7 lines were generated for comparison. RNA expression profiles of edited iPSCs were analyzed using single cell gene expression with multiplexing to assess the impact of the missense variants on transcriptional profiles. Bulk RNA sequencing was performed in parallel to better characterize transcriptional changes. These results further our understanding of how CHD7 missense variants may contribute to CHD risk. In the future, these approaches could be applied to a broader list of genes, as rare patient missense variants in definitive and candidate CHD genes require further investigation to determine pathogenicity. Developing iPSC models to investigate functional effects of missense variants can aid in understanding the complex genetic architecture of CHD.