Anne-Marie Atanga
Research Mentor(s): Adam Helms
Research Mentor School/College/Department: Department of Cardiology
Presentation Date: 08/03/2022
Presentation Type: Oral Presentation
Poster Number: N/A
Session: Session I: 12:30 – 1:20pm
Room: League Ballroom
Authors: Anne-Marie Atanga, Joshua Meisner, Adam Helms, Sabrina Friedline
Abstract
Hypertrophic cardiomyopathy (HCM) is the leading cause of sudden death in young adults and athletes and occurs in ~1/500 adults. The most common genetic cause of HCM are pathogenic variants in the MYBPC3 gene which encodes for a protein that regulates myosin binding. Heterozygous loss of function variants results in autosomal dominant familial HCM. Despite the clinical significance and prevalence, there are currently no reliable mouse model that accurately represents MYBPC3 related HCM. Haploinsufficiency is compensated by protein degradation and homozygous loss of function results in early onset dilated cardiomyopathy. We recently identified a human hypomorphic MYBPC3 variant due to partial alternative splicing. We hypothesized that creation of transgenic knock in of the identified partial splice variant could achieve a moderate reduction of MYBPC3 protein levels sufficient to develop young adult onset of hypertrophic cardiomyopathy. Method: transgenic knock in of the c.442G>A variant in humanized exon 4 of MYBPC3 was generated using CRISPR/Cas9 and homology directed repair (Mybpc3 v1). Non-HDR CRISPR directed 61bp deletion frame shift mice (KO) were used for comparison to homozygous/heterozygous loss of function variants followed by backcrossing to C57bl6. After initial mybpc3 v1 phenotyping, additional transgenic mouse lines were generated using CRISPR directed HDR with four distinct c.442G>A variants (Mybpc3 v6-v9). These were designed to weaken the alternative splice acceptor site and/or strengthen the endogenous splice acceptor site. Results: RNA and MYBPC3 amplicon sequencing of homozygous Mybpc3 v1 mice demonstrated 97% alternative splicing. Quantitative proteomics of homozygous Mybpc3 v1 mice demonstrated 5% residual MYBPC3 protein, with mice developing dilated cardiomyopathy with severely depressed ventricular function by 2 weeks of age compared to littermate wild type and het mybpc3 v1 controls. Given the extent of mybpc3 protein level decrease with some degree of alternative splicing, we hypothesized manipulation of endogenous and alternative splice acceptor sites in exon 4 could produce the goal partial reduction of 50% Mybpc3 protein, which has been demonstrated in human HCM cardiac tissue. Genotyping has confirmed generation of mybpc3 v6-v9 knock in. Mice are currently being back crossed for determining development of early onset HCM. If successful, these mice will fill the critical gap of a pre-clinic animal model for targeted therapies including gene replacement therapy for MYBPC3 related HCM.
