Analysis of FHOD3 Isoform Expression in Hypertrophic Cardiomyopathy
Supervised by Dr. Gordon Huggins, Tufts University School of Medicine
Background
Hypertrophic Cardiomyopathy (HCM) is a life long heart condition distinguished by an abnormal thickening of heart muscle. Current studies estimate that 1 in 500 people in the United States are affected with HCM. Of those ⅔ contain the obstructive type, where the thickening of the heart muscle compromises blood flow to the body. The high prevalence of HCM in the population and its downstream effects on patients leaves the condition as one valuable to understand from a genetic and mechanistic perspective. HCM is primarily considered a result of abnormalities within the cardiac sarcomere, often due to deleterious genetic mutations. The Huggins Laboratory is studying the gene Formin 2 Homology Domain 3 (FHOD3). The gene is highly expressed in heart muscle where it is responsible for actin filament elongation and formation of the molecular motor of the heart, the cardiac sarcomere. Previous research has found that FHOD3L is an essential myocardial sarcomere protein and that without it, a functional heart cannot form. The FHOD3 gene transcript is processed into two isoforms: a short version FHOD3S and a long version, FHOD3L, which contains an extra 8 amino acid residues that promotes its localization to the myocardial sarcomere. The Huggins Lab performed whole-exome sequencing of the Tufts Hypertrophic Cardiomyopathy Cohort and found the single nucleotide polymorphism (SNP) rs144071785 in the FHOD3 gene to be substantially more highly present in patients with HCM, arguing that this variant may contribute to or cause HCM. The SNP affects only FHOD3L isoform and causes a two basepair deletion leading to a frameshift that is predicted to result in an early stop codon. Premature stop codons have been identified as contributing to HCM in other genes through a mechanism called non-sense mediated decay.
Research Question:
We hypothesize that the truncated FHOD3L transcript would be degraded by regulatory cell mechanisms creating an imbalance between FHOD3S and FHOD3L. Currently, no literature investigating this mutation is present, leaving the door open for extensive research on the mutation's effect on myocardial cell phenotype. The guiding question is to study whether an over abundance of one FHOD3 isoform could promote the formation of heterodimers leading to a diminished level of active FHOD3L in cardiomyocytes.
Methods:
The proposed project has two aims: First, to confirm the existence of the rs144071785 mutation, and to understand how different ratios of the two FHOD3 isoforms impact myocardial cell phenotypes. Aim one would utilize SNP genotyping methods to verify the existence of the rs144071785 mutation. The two base pair deletion is a -AG deletion within a repeating segment of six AG nucleotides. GWAS experiments are more likely to produce false positives in repeating elements so validation of the mutation is imperative to this study. I will also study human heart tissue from the Cardiovascular Tissue Bank, which Dr Huggins directs, to confirm an imbalance of the two transcripts. Aim two would test if different ratios of the FHOD3L to FHOD3S proteins in cells create HCM phenotypes. Normally, FHOD3L and FHOD3S proteins would homodimerize, activating each isoform's ability to nucleate and elongate actin filaments. However, it is possible that an over abundance of one isoform could promote the formation of heterodimers leading to a diminished level of active FHOD3L in cardiomyocytes. To test this hypothesis, I will transfect AC16 cells, an immortalized line of cardiomyocytes, with FHOD3-HALOtag fusions of both isomers and then induce varying ratios of FHOD3 isoforms to simulate the effect of the rs144071785 mutation. The HALO-tag enables the precise mapping of the protein localization and measurement of heterodimers in the AC16 cells.
Potenital Impact:
Verifying a SNP that is present in 7.34% of patients affected by HMC would be the discovery of the most prevalent pathogenic variant associated with the disease. This could reshape the current clinical guidelines used to evaluate those at risk for HCM. Additionally, studying the effect of different ratios of FHOD3 isoforms on cardiomyocytes would lay the groundwork for future research on isoform imbalance and could ultimately change the way we understand the molecular mechanisms of FHOD3 and its role in HCM.