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Oligonucleotide-directed mutagenesis screen to identify pathogenic Lynch syndrome-associated MSH2 DNA mismatch repair gene variants

Significance The advances in whole-genome sequencing have greatly facilitated the identification of mismatch repair (MMR) gene mutations in suspected Lynch syndrome (LS) cases. The bottleneck in diagnosing LS patients now lies in determining whether MMR gene variants are pathogenic or polymorphic. In some cases the mutation is clearly deleterious for gene function. However, many suspected LS patients carry single missense mutations in one of the MMR genes, and the functional implications of these missense mutations are often elusive. To help clinicians diagnose and treat patients, we present a site-specific mutagenesis screen that takes advantage of oligonucleotide-directed gene-modification and is capable of detecting pathogenic mutator S homolog 2 (MSH2) variants in three simple steps. 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.