Researchers from the Department of Human Genetics at Michigan Medicine, and a team of collaborators have reported a method for screening so-called genetic variants of uncertain significance in the hopes of identifying those mutations that could cause colorectal cancer. The findings were featured in the paper, ‘Massively parallel functional testing of MSH2 missense variants conferring Lynch syndrome risk’, published in The American Journal of Human Genetics
The team, led by Dr Jacob Kitzman, used a genetic condition called Lynch syndrome (hereditary non-polyposis colorectal cancer). Like BRCA1, a gene known to cause certain breast cancers, there are a handful of genes behind Lynch syndrome that have been well described.
As most mutations are rare in the human population, it can be difficult to tell if any particular one is problematic, and studying one variant in a lab at a time takes a lot of time - often too much to be useful for making clinical decisions. Using a technique called deep mutational scanning, the research team set out to measure the impact of mutations in the gene MSH2, which when mutated, is one major cause of Lynch syndrome.
“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," explained Kitzman. "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.”
Using CRISPR-Cas technology, they deleted 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 cell carried a unique MSH2 mutation. This population of cells was 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, added 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. Until now, geneticists could not be sure whether these are benign or pathogenic."
The hope is that, with other patient-specific information, some of these variants may be able to be reclassified, and those people notified that they should undergo more intense screening.
"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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