Suspected Lynch syndrome associated MSH6 variants: A functional assay to determine their pathogenicity

PLoS Genetics
Hellen HoulleberghsHein te Riele

Abstract

Lynch syndrome (LS) is a hereditary cancer predisposition caused by inactivating mutations in DNA mismatch repair (MMR) genes. Mutations in the MSH6 DNA MMR gene account for approximately 18% of LS cases. Many LS-associated sequence variants are nonsense and frameshift mutations that clearly abrogate MMR activity. However, missense mutations whose functional implications are unclear are also frequently seen in suspected-LS patients. To conclusively diagnose LS and enroll patients in appropriate surveillance programs to reduce morbidity as well as mortality, the functional consequences of these variants of uncertain clinical significance (VUS) must be defined. We present an oligonucleotide-directed mutagenesis screen for the identification of pathogenic MSH6 VUS. In the screen, the MSH6 variant of interest is introduced into mouse embryonic stem cells by site-directed mutagenesis. Subsequent selection for MMR-deficient cells using the DNA damaging agent 6-thioguanine (6TG) allows the identification of MMR abrogating VUS because solely MMR-deficient cells survive 6TG exposure. We demonstrate the efficacy of the genetic screen, investigate the phenotype of 26 MSH6 VUS and compare our screening results to clinical data from suspect...Continue Reading

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Citations

May 22, 2020·Indian Journal of Dermatology, Venereology and Leprology·Sarah AlsukaitFahad Alsaif
Apr 10, 2019·Journal of the Endocrine Society·Ravinder Jeet KaurIrina Bancos
Sep 12, 2020·Cancer Communications·Teng-Jia JiangFeng Wang
Mar 19, 2019·Oncology Letters·Ivana KašubováLukáš Plank
Jan 23, 2020·Genetics in Medicine : Official Journal of the American College of Medical Genetics·Mark DrostNiels de Wind
Aug 28, 2021·International Journal of Molecular Sciences·Jane H FrederiksenThomas V O Hansen

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Methods Mentioned

BETA
PCR
gene modification
transfection

Software Mentioned

NNSPLICE
InSiGHT
PolyPhen
Human Splicing Finder
GeneSplicer
duplo

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