Affordable Access

deepdyve-link
Publisher Website

Functional interrogation of Lynch syndrome-associated MSH2 missense variants via CRISPR-Cas9 gene editing in human embryonic stem cells.

Authors
  • Rath, Abhijit1
  • Mishra, Akriti2
  • Ferreira, Victoria Duque3
  • Hu, Chaoran4, 5
  • Omerza, Gregory6
  • Kelly, Kevin6
  • Hesse, Andrew6
  • Reddi, Honey V6
  • Grady, James P5
  • Heinen, Christopher D1
  • 1 Center for Molecular Oncology and Institute for Systems Genomics, UConn Health, Farmington, Connecticut.
  • 2 Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut.
  • 3 University of St. Joseph, West Hartford, Connecticut.
  • 4 Department of Statistics, University of Connecticut, Storrs, Connecticut.
  • 5 Connecticut Institute for Clinical and Translational Science, UConn Health, Farmington, Connecticut.
  • 6 Clinical Genomics Laboratory, The Jackson Laboratory for Genomic Medicine, Farmington, Connecticut.
Type
Published Article
Journal
Human Mutation
Publisher
Wiley (John Wiley & Sons)
Publication Date
Nov 01, 2019
Volume
40
Issue
11
Pages
2044–2056
Identifiers
DOI: 10.1002/humu.23848
PMID: 31237724
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

Lynch syndrome (LS) predisposes patients to cancer and is caused by germline mutations in the DNA mismatch repair (MMR) genes. Identifying the deleterious mutation, such as a frameshift or nonsense mutation, is important for confirming an LS diagnosis. However, discovery of a missense variant is often inconclusive. The effects of these variants of uncertain significance (VUS) on disease pathogenesis are unclear, though understanding their impact on protein function can help determine their significance. Laboratory functional studies performed to date have been limited by their artificial nature. We report here an in-cellulo functional assay in which we engineered site-specific MSH2 VUS using clustered regularly interspaced short palindromic repeats-Cas9 gene editing in human embryonic stem cells. This approach introduces the variant into the endogenous MSH2 loci, while simultaneously eliminating the wild-type gene. We characterized the impact of the variants on cellular MMR functions including DNA damage response signaling and the repair of DNA microsatellites. We classified the MMR functional capability of eight of 10 VUS providing valuable information for determining their likelihood of being bona fide pathogenic LS variants. This human cell-based assay system for functional testing of MMR gene VUS will facilitate the identification of high-risk LS patients. © 2019 Wiley Periodicals, Inc.

Report this publication

Statistics

Seen <100 times