Abstract EC.08: Novel Candidate Variant In The Gene Coding For Receptor Tyrosine-protein Kinase ErbB-2 Is Associated With Left Ventricular Outflow Tract Obstruction Defects In Human

Abstract

Congenital heart defects (CHD) are the most common congenital malformations. Both genetic and environmental factors, such as blood flow during development, are known to contribute to disease development. The genetic predisposition is likely in most cases oligogenic, and currently, robust evidence for few predisposing genes for monogenic forms exist. Here we show evidence for a rare variant in the ERBB2 gene as potentially causal for CHD. ERBB2 is known to be essential for cardiac development in animal models, but so far human data are lacking. We studied a Finnish cohort of left ventricular outflow tract obstruction (LVOTO) patients and their families and identified a variant in ERBB2 (R569C) that segregated with disease in three unrelated families with multiple affected members. We performed functional studies, and show that the mutated receptor is functional, as NRG1 stimulation phosphorylates the receptor. However, a protein-protein interaction assay showed that the mutated receptor is mainly located in ER/mitochondria, whereas the wild type ERBB2 is located at the plasma membrane. This was confirmed by a flow cytometry analysis showing decreased receptor presence in the plasma membrane of patient derived cells. Zebrafish embryos expressing the R599C mutant allele demonstrated altered myocardium structure and reduced contractility compared to the wild-type allele or a mock control. Finally, we differentiated induced pluripotent stem cells (iPSCs) from a patient with the variant into cardiomyocytes. Single-cell RNA sequencing demonstrated reduced expression of genes related to heart development, muscle contraction and response to oxidative stress in patient cells compared to healthy controls. As endothelial cells (EC) are important during cardiac development, we also differentiated patient and healthy control iPSCs into ECs. Transcriptomic analysis showed significant differences in the expression of angiogenesis and vasculature development related genes in the patient cells. In summary, ERBB2 R599C variant segregates with CHD patients in three unrelated families with LVOTO defects. The variant influences location of the protein and causes compromised heart function in zebrafish embryos. We propose ERBB2 as a new candidate gene for CHD.