Thousands of Lynch Syndrome-Causing Variants Uncovered via Deep Mutational Scanning

Colon polyps, illustration
Colon polyps, illustration. [Source: Sebastian Kaulitzki/Science Photo Library/Getty Images]

Researchers have identified  the majority (94%) of MSH2 variants that could cause Lynch syndrome, a type of inherited cancer. This work has provided a functional effect map to help resolve questions about many variants of uncertain significance (VUS). Using deep mutational scanning, they conduced a massively parallel screen in human cells to identify loss-of-function missense variants in that gene. MSH2 is a DNA mismatch repair factor (MMR), and variants are a major cause of Lynch syndrome. Previously, nearly 90% of missense variants seen in MSH2 were VUS.

“The key advance is rather than doing one mutation at a time, we did it in a pooled format which allowed us to test about 18,000 mutations in a single batch,” says Jacob Kitzman, PhD., senior author of the paper and an assistant professor in the Department of Human Genetics at Michigan Medicine. The group’s paper came out last month in American Journal of Human Genetics. 

The researchers hope that their work will improve diagnosis and treatment of colon cancer, by reducing the number of VUS, and can be used as a model for further functional assessment and classification of variants in other diseases.

Colorectal cancer is the third most common type of malignancy and Lynch Syndrome, also called hereditary non-polyposis colorectal cancer,  is one of the most common heritable forms of cancer. Patients with a family history of this type of cancer are often recommended for screening, but nearly 90% of clinically observed missense variants have been classified as VUS.  In this study, Kitzman and his colleagues at Michigan Medicine, set out to systematically resolve the functional status of as many of these mutations at one time.

A handful of genes behind Lynch syndrome have been well described. However, “there’s a whole universe of possible genetic variants that can occur in genes associated with Lynch syndrome that we basically know nothing about,” says Kitzman.

As the researchers note in their paper: “Most affected individuals inherit a single loss-of-function variant in one of four MMR factors (MSH2, MLH1, MSH6, PMS2), followed by a somatic “second hit” inactivating the remaining functional copy. Further, they write that, “Missense changes together comprise 20%–30% of Lynch syndrome variants and are particularly challenging to interpret: their functional impacts may range from minimal to profound.”

Studies of gene function are often used to support variant classification, but these are impractical in the clinical setting. They also use a variety of techniques so that results may vary depending on the model system employed.

Using a technique called deep mutational scanning, the Michigan Medicine team set out to measure the impact of mutations in MSH2. They used CRISPR-Cas technology to delete the normal copy of MSH2 from human cells, and replaced it with library of every possible mutation in the MSH2 gene. This created a mix of cells where each one carried a unique MSH2 mutation. Those cells were then treated with a drug known as 6-thioguanine, a chemotherapy that killed only the cells that had a functional variant of MSH2.

The counterintuitive idea, notes Kitzman, is that the surviving cells are the ones without functioning MSH2–which are the ones with mutations that are most likely to be disease-causing.

“We were basically trying to sit down and make the mutations we could so they could serve as a reference for ones that are newly seen or are amongst the thousands of variants of unknown significance identified in people from clinical testing,” says Kitzman. “Until now, geneticists could not be sure whether these are benign or pathogenic.”

The team’s hope is that, with other patient-specific information, some of these variants may be able to be reclassified, and it will be clearer which patients need to undergo more intense screening.

Says Kitzman, “One of the next areas that will need some focus in the field of human genetics is to create these sorts of maps for many different genes where there is a clinical connection, so we can be more predictive when variants are found in an individual.”

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