Variants of Uncertain Significance in Lynch Syndrome: Screening for Pathogenicity
Introduction.1-4 Lynch Syndrome (LS) is a heritable form of cancer, predominant in colorectal as well as endometrial cancers. Treatment for LS is very different from other forms of colorectal cancer. Listed as autosomal dominant, LS is caused by the mutation in one allele for any of 4 genes (MSH2, MSH6, MLH1, and PMS2) encoding for Mismatch Repair (MMR) proteins which function to check for errors in DNA Microsatellite areas common for mutations. Not all mutations necessarily will lead to a pathogenic phenotype, however, and the term Variants of Uncertain Significance (VUS) is applied to such missense mutations that have a variable effect on protein function. Various screening techniques are being developed to help determine which VUS are pathogenic and which VUS are non-pathogenic. Methods. One screen employs oligonucleotide-directed mutagenesis (OGM) to mutate mouse embryonic stem cells with LS with a specific VUS, and exposes these cells to Thiopurine 6-thioguanine (6TG) (a mutagen).6 Cells that survive would have loss of MMR function, in theory, and indicate that the specific mutation or VUS is pathogenic. A similar screening technique employs CRISPR-Cas9 as its mutagen, and Methylnitronitrosoguanidine as its mutagenic filter.7 Another technique simulates protein stability on the basis of the .8 Lastly, targeted next generation sequencing (TNGS) shows promise for diagnosing LS quickly and efficiently.9 Results. OGM studied 26 VUS of unknown pathogenicity. 8 of these were deemed pathogenic, with 18 being non-pathogenic (not surviving 6TG). OGM screening had a self-reported specificity of 90% and sensitivity of 85%. The CRISPR-Cas9 screen was similar both in concept and in specificity/sensitivity to that of OGM, but did have the additional benefit of using human stem cell lines (humans and mice have 86% concurrence in MMR genes). The thermodynamic simulation was able to screen over 10,000 distinct mutations, although likely none of these would be considered VUS: as this simply tells the likelihood of complete collapse of the MMR protein, not the more subtle effects common in VUS. TNGS studied 243 colorectal cancer biopsies, and had a specificity of 98% and sensitivity of 91% against conventional diagnostics of immunohistochemistry. Conclusions. Screening is needed to pave the way for diagnosing LS in patients with mutations in the VUS category. Such screens include OGM, CRISPR-Cas9, and thermodynamic simulation, and could improve upon the already promising TNGS. Diagnosing LS is critical in that its treatment is very different from that of typical colorectal cancer.
- Sinicrope FA. Lynch Syndrome-Associated Colorectal Cancer. The New England Journal of Medicine. 2018;379:764-773. DOI: 10.1056/NEJMcp1714533.
- Nguyen LH, Goel A, Chung DC. Pathways of Colorectal Carcinogenesis. Gastroenterology. 2020; 158:291-302.
- Yurgelun MB, Hampel H. Recent advances in Lynch Syndrome: diagnosis, treatment, and cancer prevention. American Society of Clinical Oncology. 2018.
- Boland CR, Lynch HT. The History of Lynch Syndrome. Fam Cancer. 2013; 12(2): 145-157. doi:10.1007/s10689-013-9637-8.
- D’Arcy BM, Blount J, Prakash A. Biochemical and structural characterization of two variants of uncertain significance in the PMS2 gene. Human Mutation. DOI: 10.1002/humu.23708.
- Houlleberghs H, Goverde A, et. al. Suspected Lynch syndrome associated MSH6 variants: A functional assay to determine their pathogenicity. PLOS Genetics. 2017.
- Rath A, Mishra A, et. al. Functional interrogation of Lynch syndrome-associated MSH2 missense variants via CRISPR-Cas9 gene editing in human embryonic stem cells. Human Mutation. 2018.
- Abildgaard AB, Stein A, et. Al. Computational and cellular studies reveal structural destabilization and degradation of MLH1 variants in Lynch syndrome. Cancer Biology. 2019;8:e49138.
- Nowak JA, Yurgelun MB, et. al. Detection of mismatch repair deficiency and microsatellite instability in colorectal adenocarcinoma by targeted next-generation sequencing. J Mol Diagn. 2017, 19: 84-91.