title: Oligonucleotide-directed mutagenesis screen to identify pathogenic Lynch syndrome-associated MSH2 DNA mismatch repair gene variants.
abstract: Single-stranded DNA oligonucleotides can achieve targeted base-pair substitution with modest efficiency but high precision. We show that "oligo targeting" can be used effectively to study missense mutations in DNA mismatch repair (MMR) genes. Inherited inactivating mutations in DNA MMR genes are causative for the cancer predisposition Lynch syndrome (LS). Although overtly deleterious mutations in MMR genes can clearly be ascribed as the cause of LS, the functional implications of missense mutations are often unclear. We developed a genetic screen to determine the pathogenicity of these variants of uncertain significance (VUS), focusing on mutator S homolog 2 (MSH2). VUS were introduced into the endogenous Msh2 gene of mouse embryonic stem cells by oligo targeting. Subsequent selection for MMR-deficient cells using the guanine analog 6-thioguanine allowed the detection of MMR-abrogating VUS. The screen was able to distinguish weak and strong pathogenic variants from polymorphisms and was used to investigate 59 Msh2 VUS. Nineteen of the 59 VUS were identified as pathogenic. Functional assays revealed that 14 of the 19 detected variants fully abrogated MMR activity and that five of the detected variants attenuated MMR activity. Implementation of the screen in clinical practice allows proper counseling of mutation carriers and treatment of their tumors.
authors.affiliation: Division of Biological Stress Response, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands;
authors.forename: Hellen
authors.initials: H
authors.lastname: Houlleberghs
authors.affiliation: Division of Biological Stress Response, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands;
authors.forename: Marleen
authors.initials: M
authors.lastname: Dekker
authors.affiliation: Division of Biological Stress Response, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands;
authors.forename: Hildo
authors.initials: H
authors.lastname: Lantermans
authors.affiliation: Division of Biological Stress Response, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands;
authors.forename: Roos
authors.initials: R
authors.lastname: Kleinendorst
authors.affiliation: Department of Pathology, Erasmus Medical Center, 3015 CN Rotterdam, The Netherlands;
authors.forename: Hendrikus Jan
authors.initials: HJ
authors.lastname: Dubbink
authors.affiliation: Department of Clinical Genetics, Erasmus Medical Center, 3015 CN Rotterdam, The Netherlands;
authors.forename: Robert M W
authors.initials: RM
authors.lastname: Hofstra
authors.affiliation: Family Cancer Clinic, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.
authors.forename: Senno
authors.initials: S
authors.lastname: Verhoef
authors.affiliation: Division of Biological Stress Response, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands; h.t.riele@nki.nl.
authors.forename: Hein
authors.initials: H
authors.lastname: Te Riele
mesh_terms: D003123:Colorectal Neoplasms, Hereditary Nonpolyposis / Q000235:genetics*; D053843:DNA Mismatch Repair; D006801:Humans; D051718:MutS Homolog 2 Protein / Q000235:genetics*; D016296:Mutagenesis; D009841:Oligonucleotides / Q000235:genetics*
chemical_list: D009841:Oligonucleotides; C497172:MSH2 protein, human; D051718:MutS Homolog 2 Protein
keywords: DNA mismatch repair; Lynch syndrome; MSH2; site-directed mutagenesis; variants of uncertain significance