Samantha Nichols
Pronouns: she/her/hers
Research Mentor(s): Andrea Thompson
Research Mentor School/College/Department: Department of Internal Medicine/Cardiovascular Division / Medicine
Program: CG
Session: Session 1 (9:00am – 9:50am)
Authors: Samantha Nichols, Ulla Lilienthal , Andrea Thompson
Abstract
Hypertrophic Cardiomyopathy (HCM) is a genetic disease involving sarcomeres deinfed as hypertrophy of the myocardium, this can lead to heart failure, arrhythmia, left ventricular outflow tract obstruction, and even sudden cardiac death. Pathogenic loss-of-function variants in myosin binding protein C (MYBPC3) gene account for ~50% of familial HCM. Still, most missense variants of MYBPC3 are classified as variants of uncertain significance (VUS). We hypothesize that we will be able to expand the pool of known pathogenic variants in MYBPC3 by evaluating MYBPC3 missense VUS for protein misfolding as one mechanism of loss of function. MyBP-C, the protein encoded by MYBPC3, is made up of a series of fibronectin and immunoglobulin subdomains. We plan to test individual subdomains starting with the immunoglobulin subdomain C3 using landing pad technology in HEK293T cells (Human Embryonic Kidney cells).HEK293T Landing Pad (LP) cells contain the AttB site that expresses blue fluorescent protein (BFP) under a doxycycline-inducible promoter. We can inactivate the BFP and express our protein of interest, a green fluorescent protein (GFP) labeled MyBP-C C3 subdomain, when the promoter-less plasmid and the Bxb1 enzyme are transfected into cells using fugene6 and incubated for 48 hours. This allows one copy of MyBP-C or one MyBP-C VUS to be introduced per cell. After transfection, the cells were treated with doxycycline to induce expression and incubated for 24 hours after which they were analyzed with flow cytometry for data collection. These cells will also express mCherry independently to control for variation in promoter activity as an intracellular control. If the variant leads to misfolding of the C3 subdomain, a reduced GFP/mCherry signal will be observed. If the variant does not affect folding, no change in GFP/mCherry signal will be observed. We have demonstrated that cells expressing two variants in C3, that were predicted computationally to cause misfolding, exhibit a lower GFP signal than cells expressing wild type. The next step is to create a library containing all possible single amino acid MyBP-C C3 subdomain missense variants. Expressing this library in the landing pad cells and sorting using flow cytometry and next generation sequence will allow us to rapidly identify missense variants that lead to MyBP-C loss of function by causing subdomain misfolding. This would allow us to expand the pool of known pathogenic variants, enabling clinicians to provide patients with improved prognostic information and guidance regarding at-risk relatives.
