Dissecting the mechanisms of repeat expansion in the Fragile X-related disorders

Presenter's Name(s)

Cai McCannFollow

Conference Year

January 2020

Abstract

Repeat expansion diseases, including Fragile X Syndrome (FXS), Huntington’s Disease, and other diseases, are characterized by distinct DNA mutations of large numbers of trinucleotide or triplet repeats that destabilize genomes. Specifically, FXS is the most common form of inherited intellectual disability (ID) and is associated with autism. While many people remain undiagnosed, estimates are about 1.4 per 10,000 males and 0.9 per 10,000 females presenting FXS (CDC 2019). FXS is caused by unstable CGG/CCG-repeat expansion in the 5’ UTR of the Fragile X Mental Retardation 1 (FMR1) gene on the X chromosome. To date, repeat expansion diseases are thought to arise from anomalous DNA processing and pathways (Zhao & Usdin 2015). Even so, we lack a complete understanding of the underlying mechanisms causing expansions and subsequent diseases.

This project is focused specifically on elucidating the mechanistic function and downstream effects linking FXS full mutation repeat expansion. We ask, do DNA repair proteins from the RAD54 and RAD52 family contribute to the repeat expansion mechanism? To investigate, we will use a two-pronged approach involving the following: (1) small-molecule inhibition of repair proteins, RAD52 and RAD54, within a dose-response curve of varying drug concentrations, (-) - Epigallocatechin and B02; and (2) CRISPR-Cas9 induced double knockouts in proteins of the RAD54 family.

This investigation revealed how EGC and B02 treatment may slow expansion. We additionally identified multiple, promising repeat-matched Rad54 mutant cell lines possessing successful Rad54 knockout using genomic PCR screening, repeat analysis, and Western blotting. Further examination of other small molecule inhibitors and frequencies of CGG-repeat expansion in both approaches may contribute to the evidence towards this work. Investigating the genetic variation and mechanisms in FXS will increase our understanding of Repeat Expansion disease mutations and be relevant to clinical approaches concerning FXS incidence.

Primary Faculty Mentor Name

Lori Stevens

Secondary Mentor Name

Carson Miller, Bruce Hayward, Grace Kim

Status

Undergraduate

Student College

College of Arts and Sciences

Program/Major

Biology

Primary Research Category

Biological Sciences

Secondary Research Category

Health Sciences

Abstract only.

Share

COinS
 

Dissecting the mechanisms of repeat expansion in the Fragile X-related disorders

Repeat expansion diseases, including Fragile X Syndrome (FXS), Huntington’s Disease, and other diseases, are characterized by distinct DNA mutations of large numbers of trinucleotide or triplet repeats that destabilize genomes. Specifically, FXS is the most common form of inherited intellectual disability (ID) and is associated with autism. While many people remain undiagnosed, estimates are about 1.4 per 10,000 males and 0.9 per 10,000 females presenting FXS (CDC 2019). FXS is caused by unstable CGG/CCG-repeat expansion in the 5’ UTR of the Fragile X Mental Retardation 1 (FMR1) gene on the X chromosome. To date, repeat expansion diseases are thought to arise from anomalous DNA processing and pathways (Zhao & Usdin 2015). Even so, we lack a complete understanding of the underlying mechanisms causing expansions and subsequent diseases.

This project is focused specifically on elucidating the mechanistic function and downstream effects linking FXS full mutation repeat expansion. We ask, do DNA repair proteins from the RAD54 and RAD52 family contribute to the repeat expansion mechanism? To investigate, we will use a two-pronged approach involving the following: (1) small-molecule inhibition of repair proteins, RAD52 and RAD54, within a dose-response curve of varying drug concentrations, (-) - Epigallocatechin and B02; and (2) CRISPR-Cas9 induced double knockouts in proteins of the RAD54 family.

This investigation revealed how EGC and B02 treatment may slow expansion. We additionally identified multiple, promising repeat-matched Rad54 mutant cell lines possessing successful Rad54 knockout using genomic PCR screening, repeat analysis, and Western blotting. Further examination of other small molecule inhibitors and frequencies of CGG-repeat expansion in both approaches may contribute to the evidence towards this work. Investigating the genetic variation and mechanisms in FXS will increase our understanding of Repeat Expansion disease mutations and be relevant to clinical approaches concerning FXS incidence.