Alyssa Bashir
Pronouns: she/her/hers
Research Mentor(s): Shelby Hemker
Research Mentor School/College/Department: Human Genetics / Medicine
Program: UROPF
Session: Session 3 (11:00am – 11:50am)
Authors: Alyssa Bashir, Shelby Hemker
Abstract
MUTYH is a DNA repair gene that helps to prevent carcinogenesis. Thus, MUTYH loss of function variants result in a disease that causes an increased risk of cancer. Currently, most reported variants are of unknown significance (VUSs), largely due to the disease being autosomal recessive and most patients present as compound heterozygotes, making it difficult for physicians to diagnose disease and thus determine those individuals’ cancer risk. To address this gap in diagnostic potential, we are creating high-throughput functional assays to test many VUSs at once. This method utilizes knock-in of MUTYH cDNA into cells lacking endogenous MUTYH expression; however, our current construct results in overexpression, which has been found to skew these functional assay results. In order to counteract this problem, multiple approaches were taken. Firstly, the promoter was analyzed to determine if any sequence changes could be made to down regulate expression. Through a thorough literature search on the history of the promoter and its sequence, it was discovered that no further changes could be made to reduce expression. Next, potassium bromate, a reactive oxygen species (ROS), was used to treat cells with knocked-in MUTYH to create more DNA damage lesions for MUTYH to repair, thus reducing the amount of free MUTYH able to act in the functional assay. However, this assay did not work as planned, which we suspect is due to potassium bromate activating alternative repair pathways within the cells and therefore skewing the results and not having the desired effect on MUTYH. Finally, we are currently exploring the use of upstream open reading frames (uORFs) to reduce the amount of translation of MUTYH cDNA. If successful, the uORF-mediated down regulation of knock-in MUTYH will be used for the high-throughput functional assays to determine the pathogenicity of various MUTYH variants.
