Functional interrogation of Lynch syndrome‐associated<i>MSH2</i>missense variants via CRISPR‐Cas9 gene editing in human embryonic stem cells

Rath, Abhijit; Mishra, Akriti; Ferreira, Victoria Duque; Hu, Chaoran; Omerza, Gregory; Kelly, Kevin; Hesse, Andrew; Reddi, Honey V.; Grady, James; Heinen, Christopher D.

doi:10.1002/humu.23848

Cited by 29 publications

(27 citation statements)

References 76 publications

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“…We sought to validate these pooled measurements by comparison to traditional, low-throughput functional studies. We collated a set of 184 MSH2 missense variants previously characterized as functionally deleterious or neutral by individual cell-based 15 , 19 , 45 , 48 , 49 , 50 , 51 , 52 and/or biochemical assays, 53 , 54 discarding four variants where multiple studies disagreed and eight predicted to impact splicing, an effect not measured by this approach (SpliceAI 32 score > 0.2; Tables S4 and S5 ). Our LoF scores agreed with these earlier reports for the great majority of variants covered (158/165, 95.8%; Figure 3 A).…”

Section: Resultsmentioning

confidence: 99%

Massively parallel functional testing of MSH2 missense variants conferring Lynch syndrome risk

Jia

Burugula

Chen

et al. 2021

The American Journal of Human Genetics

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The lack of functional evidence for the majority of missense variants limits their clinical interpretability and poses a key barrier to the broad utility of carrier screening. In Lynch syndrome (LS), one of the most highly prevalent cancer syndromes, nearly 90% of clinically observed missense variants are deemed ''variants of uncertain significance'' (VUS). To systematically resolve their functional status, we performed a massively parallel screen in human cells to identify loss-of-function missense variants in the key DNA mismatch repair factor MSH2. The resulting functional effect map is substantially complete, covering 94% of the 17,746 possible variants, and is highly concordant (96%) with existing functional data and expert clinicians' interpretations. The large majority (89%) of missense variants were functionally neutral, perhaps unexpectedly in light of its evolutionary conservation. These data provide ready-to-use functional evidence to resolve the $1,300 extant missense VUSs in MSH2 and may facilitate the prospective classification of newly discovered variants in the clinic.

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Section: Resultsmentioning

confidence: 99%

Massively parallel functional testing of MSH2 missense variants conferring Lynch syndrome risk

Jia

Burugula

Chen

et al. 2021

The American Journal of Human Genetics

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“…However, this specific approach would have to be adapted to account for the fact that unlike BRCA1, the MMR genes are not essential ( Blomen et al, 2015 ). In this regard, an assay based on gene editing of human embryonic stem cells and assessment of both DNA damage response and microsatellite repair was recently developed, holding great promise for the study of variant-induced splicing changes and missense alterations in Lynch syndrome ( Rath et al, 2019 ).…”

Section: Resultsmentioning

confidence: 99%

Contribution of mRNA Splicing to Mismatch Repair Gene Sequence Variant Interpretation

et al. 2020

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Functional assays that assess mRNA splicing can be used in interpretation of the clinical significance of sequence variants, including the Lynch syndrome-associated mismatch repair (MMR) genes. The purpose of this study was to investigate the contribution of splicing assay data to the classification of MMR gene sequence variants. We assayed mRNA splicing for 24 sequence variants in MLH1, MSH2, and MSH6, including 12 missense variants that were also assessed using a cell-free in vitro MMR activity (CIMRA) assay. Multifactorial likelihood analysis was conducted for each variant, combining CIMRA outputs and clinical data where available. We collated these results with existing public data to provide a dataset of splicing assay results for a total of 671 MMR gene sequence variants (328 missense/in-frame indel), and published and unpublished repair activity measurements for 154 of these variants. There were 241 variants for which a splicing aberration was detected: 92 complete impact, 33 incomplete impact, and 116 where it was not possible to determine complete versus incomplete splicing impact. Splicing results mostly aided in the interpretation of intronic (72%) and silent (92%) variants and were the least useful for missense substitutions/in-frame indels (10%). MMR protein functional activity assays were more useful in the analysis of these exonic variants but by design they were not able to detect clinically important splicing aberrations identified by parallel mRNA assays. The development of high throughput assays that can quantitatively assess impact on mRNA transcript expression and protein function in parallel will streamline classification of MMR gene sequence variants.

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“…Ser-723 is a highly conserved amino acid and the variant is predicted to be disease causing by several in silico prediction tools. Several groups have evaluated this variant using in vitro MMR activity assays, yeast assays or murine or human embryonic stem cells; all studies indicate that the variant interrupts normal mismatch repair function and is pathogenic (Gammie et al, 2007;Drost et al, 2012;Houlleberghs et al, 2016;Rath et al, 2019). Based on a suggested functional effect in four studies, in combination with our data with co-segregation in two individuals with early onset CRC, we consider the MSH2 c.2168C>T variant likely pathogenic.…”

Section: Pathogenic and Likely Pathogenic High-risk Variantsmentioning

confidence: 87%

New Pathogenic Germline Variants in Very Early Onset and Familial Colorectal Cancer Patients

et al. 2020

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A genetic diagnosis facilitates personalized cancer treatment and clinical care of relatives at risk, however, although 25% of colorectal cancer cases are familial, around 95% of the families are genetically unresolved. In this study, we performed gene panel analysis on germline DNA of 32 established or candidate colorectal cancer predisposing genes in 149 individuals from either families with an accumulation of colorectal cancers or families with only one sporadic case of very early onset colorectal cancer (≤40 years at diagnosis). We identified pathogenic or likely pathogenic genetic variants in 10.1% of the participants in genes such as APC, POLE, MSH2 or PMS2. The MSH2 variant, c.2168C>T, p.(Ser723Phe) was previously described as a variant of unknown significance, but we have now reclassified it to be likely pathogenic. The POLE variant, c.1089C>A, p.(Asn363Lys) was identified in a patient with three metachronous colorectal cancers from age 28 and turned out to be de novo. One pathogenic PMS2 variant was novel. We also identified a number of highly interesting variants of unknown significance in APC, BUB1, TP53 and RPS20. The RPS20 variant is novel and was found in a large Amsterdam I positive family with a multi tumor phenotype including 12 cases of CRC from as early as age 24. This variant was found to segregate with cancer in the family and multiple in silico tools predict it to be pathogenic. Our data further support the shift from phenotypic-based cancer panels to large panels including all established genes involved in hereditary cancer syndromes or (targeted) whole genome sequencing. Additionally, identification of a likely disease-predisposing variant in RPS20 expands the phenotypic spectrum of RPS20-related cancers and emphasize that this gene is relevant to include in colorectal cancer gene panels.

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Functional interrogation of Lynch syndrome‐associatedMSH2missense variants via CRISPR‐Cas9 gene editing in human embryonic stem cells

Cited by 29 publications

References 76 publications

Massively parallel functional testing of MSH2 missense variants conferring Lynch syndrome risk

Massively parallel functional testing of MSH2 missense variants conferring Lynch syndrome risk

Contribution of mRNA Splicing to Mismatch Repair Gene Sequence Variant Interpretation

New Pathogenic Germline Variants in Very Early Onset and Familial Colorectal Cancer Patients

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