Experimental Characterization of Genetic Variants Causing Hypertrophic Cardiomyopathy

• Main Author: de Jong, Hannah Naomi
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2022
• Subjects: Design; Mutation; Plasmids
• ISBN: 9798357508973

Hypertrophic cardiomyopathy (HCM) is a common inherited heart condition, affecting one in 500 people. It is most often caused by variants in the genes that encode the cardiac sarcomere, including the gene encoding beta cardiac myosin, MYH7. However, hundreds of such variants exist, and most are very rare. This presents an obstacle to variant pathogenicity assignment, and population genetic studies are limited in their utility because they will never capture every possible variant. In this dissertation, I discuss the diagnostic challenges presented by uncharacterized variation in disease genes like those associated with HCM. I then describe a combination of experimental approaches that provide insight into the pathogenicity of HCM-associated genetic variants.I performed high-throughput mutagenesis MYH7 in induced pluripotent stem cells (iPSCs) using two methods, the in vitro PCR-based strategy POPcode and the direct genomic mutagenesis method CRISPR-X, comparing efficacy and comprehensiveness. To study the impacts of changes to MYH7 copy number, which is altered by some mutagenesis strategies, I both knocked out a copy of MYH7 and inserted a copy of cDNA. I also developed two methods for pooled disease phenotyping of cells differentiated to iPSC-cardiomyocytes (iPSC-CMs). The first sorts cells by size; hypertrophy is the pathognomonic feature of HCM. The second utilizes a fluorescent reporter for a gene, NPPB, that is consistently up-regulated in HCM-affected cardiac tissue. I used both of these methods to sort mutagenized cells and identify variants that may cause cardiomyocyte hypertrophy.Part of the missing heritability of HCM may be explained by variation in non-sarcomeric genes. I also share a study where we identified a novel variant in the Wnt signaling regulator WTIP in a family with HCM, and characterize its phenotypic impacts in multiple models.Together, these strategies can be combined to generate functional phenotypic data across variants in MYH7 and other HCM-associated genes, providing us with a deeper biological understanding of the structure-function relationships of the variant proteins that cause HCM and informing patient diagnosis in the clinic.