In many individuals suspected of the common cancer predisposition Lynch syndrome, variants of unclear significance (VUS), rather than an obviously pathogenic mutations, are identified in one of the ...DNA mismatch repair (MMR) genes. The uncertainty of whether such VUS inactivate MMR, and therefore are pathogenic, precludes targeted healthcare for both carriers and their relatives. To facilitate the identification of pathogenic VUS, we have developed an in cellulo genetic screen-based procedure for the large-scale mutagenization, identification, and cataloging of residues of MMR genes critical for MMR gene function. When a residue identified as mutated in an individual suspected of Lynch syndrome is listed as critical in such a reverse diagnosis catalog, there is a high probability that the corresponding human VUS is pathogenic. To investigate the applicability of this approach, we have generated and validated a prototypic reverse diagnosis catalog for the MMR gene MutS Homolog 2 (Msh2) by mutagenizing, identifying, and cataloging 26 deleterious mutations in 23 amino acids. Extensive in vivo and in vitro analysis of mutants listed in the catalog revealed both recessive and dominant-negative phenotypes. Nearly half of these critical residues match with VUS previously identified in individuals suspected of Lynch syndrome. This aids in the assignment of pathogenicity to these human VUS and validates the approach described here as a diagnostic tool. In a wider perspective, this work provides a model for the translation of personalized genomics into targeted healthcare.
Abstract
The prevalent cancer predisposition Lynch syndrome (LS, OMIM #120435) is caused by an inherited heterozygous defect in any of the four core DNA mismatch repair (MMR) genes MSH2, MSH6, MLH1 ...or PMS2. MMR repairs errors by the replicative DNA polymerases in all proliferating tissues. Its deficiency, following somatic loss of the wild-type copy, results in a spontaneous mutator phenotype that underlies the rapid development of, predominantly, colorectal cancer (CRC) in LS. Here, we have addressed the hypothesis that aberrant responses of intestinal stem cells to diet-derived mutagens may be causally involved in the restricted cancer tropism of LS. To test this we have generated a panel of isogenic mouse embryonic stem (mES) cells with heterozygous or homozygous disruption of multiple MMR genes and investigated their responses to the common dietary mutagen and carcinogen 2-amino-1-methyl-6-phenylimidazo4,5-bpyridine (PhIP). Our data reveal that PhIP can inactivate the wild-type allele of heterozygous mES cells via the induction of either loss of heterozygosity (LOH) or intragenic mutations. Moreover, while protective DNA damage signaling (DDS) is compromised, PhIP induces more mutations in Msh2, Mlh1, Msh6 or Pms2-deficient mES cells than in wild-type cells. Combined with their spontaneous mutator phenotypes, this results in a compound hypermutator phenotype. Together, these results indicate that dietary mutagens may promote CRC development in LS at multiple levels, providing a rationale for dietary modifications in the management of LS.
A dietary mutagen induces loss of the wild-type allele in mismatch repair gene-heterozygous cells. This results in impaired DNA damage signaling and in compound hypermutagenesis. These findings provide insights into the etiology of colorectal carcinogenesis in Lynch syndrome.
Graphical Abstract
Graphical Abstract
ABSTRACT
Lynch syndrome (LS) is a common cancer predisposition caused by an inactivating mutation in one of four DNA mismatch repair (MMR) genes. Frequently a variant of uncertain significance (VUS), ...rather than an obviously pathogenic mutation, is identified in one of these genes. The inability to define pathogenicity of such variants precludes targeted healthcare. Here, we have modified a cell‐free assay to test VUS in the MMR gene PMS2 for functional activity. We have analyzed nearly all VUS in PMS2 found thus far and describe loss of MMR activity for five, suggesting the applicability of the assay for diagnosis of LS.
The cancer predisposition Lynch syndrome is diagnosed by the identification of a disruptive mutation in one of the DNA mismatch repair genes. Unfortunately, the identification of a variant of uncertain significance frequently precludes diagnosis, which interferes with clinical management. Here we use a rapid and cell‐free assay to test functional activity of nearly all variants identified to date in PMS2, revealing loss of activity for five variants. This assay may greatly facilitate the diagnostic assessment of PMS2 variants.
The large majority of germline alterations identified in the DNA mismatch repair (MMR) gene PMS2, a low‐penetrance gene for the cancer predisposition Lynch syndrome, represent variants of uncertain ...significance (VUS). The inability to classify most VUS interferes with personalized healthcare. The complete in vitro MMR activity (CIMRA) assay, that only requires sequence information on the VUS, provides a functional analysis‐based quantitative tool to improve the classification of VUS in MMR proteins. To derive a formula that translates CIMRA assay results into the odds of pathogenicity (OddsPath) for VUS in PMS2 we used a set of clinically classified PMS2 variants supplemented by inactivating variants that were generated by an in cellulo genetic screen, as proxies for cancer‐predisposing variants. Validation of this OddsPath revealed high predictive values for benign and predisposing PMS2 VUS. We conclude that the OddsPath provides an integral metric that, following the other, higher penetrance, MMR proteins MSH2, MSH6 and MLH1 can be incorporated as strong evidence type into the upcoming criteria for MMR gene VUS classification of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP).
Three‐step classification of Lynch syndrome‐associated Variants of Uncertain Significance (VUSs) in any of the DNA mismatch repair genes. (1) Protein VUSs are synthesized in vitro, based on sequence information provided by the clinical geneticist. (2) Their functional activity are assayed and quantified. (3) The odds of pathogenicity are calculated and incorporated in the final classification according to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology BS3/PS3 criteria
Pathogenic
proofreading domain mutations are found in many malignancies where they are associated with ultramutation and favorable prognosis. The extent to which this prognosis depends on their ...sensitivity to adjuvant treatment is unknown, as is the optimal therapy for advanced-staged or recurrent
-mutant cancers.
We examined the recurrence-free survival of women with
-mutant and
wild-type endometrial cancers (EC) in the observation arm of the randomized PORTEC-1 endometrial cancer trial (
= 245 patients with stage I endometrial cancer for analysis). Sensitivity to radiotherapy and selected chemotherapeutics was compared between
-mutant mouse-derived embryonic stem (mES) cells, generated using CRISPR-Cas9 (
mutations D275A/E275A, and cancer-associated P286R, S297F, V411L) and isogenic wild-type cell lines.
In the observation arm of the PORTEC-1 trial (
= 245), women with
-mutant endometrial cancers (
= 16) had an improved recurrence-free survival (10-year recurrence-free survival 100% vs. 80.1% for
wild-type; HR, 0.143; 95% confidence interval, 0.001-0.996;
= 0.049).
mutations did not increase sensitivity to radiotherapy nor to chemotherapeutics in mES cells. In contrast,
-mutant cells displayed significantly increased sensitivity to cytarabine and fludarabine (IC
P286R-mutant vs. wild-type: 0.05 vs. 0.17 μmol/L for cytarabine, 4.62 vs. 11.1 μmol/L for fludarabine;
< 0.001 for both comparisons).
The favorable prognosis of
-mutant cancers cannot be explained by increased sensitivity to currently used adjuvant treatments. These results support studies exploring minimization of adjuvant therapy for early-stage
-mutant cancers, including endometrial and colorectal cancers. Conversely,
mutations result in hypersensitivity to nucleoside analogues, suggesting the use of these compounds as a potentially effective targeted treatment for advanced-stage
-mutant cancers.
.
To enhance classification of variants of uncertain significance (VUS) in the DNA mismatch repair (MMR) genes in the cancer predisposition Lynch syndrome, we developed the cell-free in vitro MMR ...activity (CIMRA) assay. Here, we calibrate and validate the assay, enabling its integration with in silico and clinical data.
Two sets of previously classified MLH1 and MSH2 variants were selected from a curated MMR gene database, and their biochemical activity determined by the CIMRA assay. The assay was calibrated by regression analysis followed by symmetric cross-validation and Bayesian integration with in silico predictions of pathogenicity. CIMRA assay reproducibility was assessed in four laboratories.
Concordance between the training runs met our prespecified validation criterion. The CIMRA assay alone correctly classified 65% of variants, with only 3% discordant classification. Bayesian integration with in silico predictions of pathogenicity increased the proportion of correctly classified variants to 87%, without changing the discordance rate. Interlaboratory results were highly reproducible.
The CIMRA assay accurately predicts pathogenic and benign MMR gene variants. Quantitative combination of assay results with in silico analysis correctly classified the majority of variants. Using this calibration, CIMRA assay results can be integrated into the diagnostic algorithm for MMR gene variants.
We describe a family severely affected by colorectal cancer (CRC) where whole‐exome sequencing identified the coinheritance of the germline variants encoding MSH6 p.Thr1100Met and MUTYH p.Tyr179Cys ...in, at least, three CRC patients diagnosed before 60 years of age. Digenic inheritance of monoallelic MSH6 variants of uncertain significance and MUTYH variants has been suggested to predispose to Lynch syndrome‐associated cancers; however, cosegregation with disease in the familial setting has not yet been established. The identification of individuals carrying multiple potential cancer risk variants is expected to rise with the increased application of whole‐genome sequencing and large multigene panel testing in clinical genetic counseling of familial cancer patients. Here we demonstrate the coinheritance of monoallelic variants in MSH6 and MUTYH consistent with cosegregation with CRC, further supporting a role for digenic inheritance in cancer predisposition.
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