Pathogenic variants in the mismatch repair genes are associated with an elevated lifetime risk of colorectal cancer (CRC). We previously identified two variants of uncertain significance (VUS) in the MLH1 gene, c.696_698del, p.(Cys233del) and c.1919C > G, p.(Pro640Arg), in Danish families with numerous occurrences of CRC.
To reclassify the variants we collected clinical data, initiated tumor and co-segregation analysis, and performed RNA splicing analysis, subcellular localization, and protein stability studies.
The functional analysis revealed that the c.696_698del, p.(Cys233del) variant had an effect at the RNA level, on subcellular localization, and on protein stability, while the c.1919C > G, p.(Pro640Arg) variant showed decreased expression in localization studies and decreased protein stability. These results suggest both variants disrupt DNA mismatch repair.
By applying all collected data and functional results we propose to reclassify the c.696_698del, p.(Cys233del) and the c.1919C > G, p.(Pro640Arg) variants as likely pathogenic (class 4) using MMR gene-specific ACMG/AMP guidelines. Consequently, the two MLH1 variants can now be used for risk assessment of variant carriers, while family members without the variants can be excluded from intensified cancer surveillance and follow population recommendations.
© 2024 The Author(s). Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.

Molecular classification according to The Cancer Genome Atlas (TCGA) improves endometrial endometrioid carcinoma (EEC) prognostication and has specific treatment implications; however, original data were skewed towards low-grade and low-stage tumours. Herein, we molecularly classify EECs metastatic at the time of diagnosis or with subsequently documented recurrent/metastatic disease to examine correlation with clinical outcomes.
TCGA categories include POLE-mutated, microsatellite instability (MSI), p53 abnormal (p53 abnl) and no specific molecular profile (NSMP). POLE targeted sequencing at exons 9, 11, 13 and 14 and immunohistochemistry (IHC) for PMS2, MSH6 and p53 were performed to establish molecular classification.
The distribution in our cohort of 141 EECs was similar to that generally reported in EEC, with nine POLE-mutated (6%), 45 MSI (32%), 16 p53 abnl (11%) and 71 NSMP (50%), with similar distributions between low- and high-stage cohorts. We demonstrate that when stratified by molecular subtype, disease-specific survival from the time of high-stage (stages III-IV) presentation or time of recurrence in low-stage (stages I-II) disease among metastatic and/or recurrent EEC is strongly associated with TCGA classification (high-stage P = 0.02, low-stage P = 0.017). Discordant molecular classification between primary and metastatic/recurrent tumours occurred in four of 105 (3.8%) patients, two related to PMS2/MSH6 IHC and two related to p53 IHC.
We demonstrate that molecular classification is prognostically relevant not only at the time of diagnosis, but also at the time of recurrence and in the metastatic setting. Rare subclonal alterations occur and suggest a role for confirming TCGA classification in recurrent/metastatic tumours.
© 2024 John Wiley & Sons Ltd.

Polyploidy, a significant catalyst for speciation and evolutionary processes in both plant and animal kingdoms, has been recognized for a long time. However, the exact molecular mechanism that leads to polyploid formation, especially in vertebrates, is not fully understood. Our study aimed to elucidate this phenomenon using the zebrafish model. We successfully achieved an effective knockout of the cyclin N-terminal domain containing 1 (cntd1) using CRISPR/Cas9 technology. This resulted in impaired formation of meiotic crossovers, leading to cell-cycle arrest during meiotic metaphase and triggering apoptosis of spermatocytes in the testes. Despite these defects, the mutant (cntd1-/-) males were still able to produce a limited amount of sperm with normal ploidy and function. Interestingly, in the mutant females, it was the ploidy not the capacity of egg production that was altered. This resulted in the production of haploid, aneuploid, and unreduced gametes. This alteration enabled us to successfully obtain triploid and tetraploid zebrafish from cntd1-/- and cntd1-/-/- females, respectively. Furthermore, the tetraploid-heterozygous zebrafish produced reduced-diploid gametes and yielded all-triploid or all-tetraploid offspring when crossed with wild-type (WT) or tetraploid zebrafish, respectively. Collectively, our findings provide direct evidence supporting the crucial role of meiotic crossover defects in the process of polyploidization. This is particularly evident in the generation of unreduced eggs in fish and, potentially, other vertebrate species.
© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.

Although immunohistochemistry (IHC) for mismatch repair (MMR) proteins (MMR IHC) is used to identify DNA MMR status, universal screening of all patients with colorectal cancer (CRC) using a combination of both MMR IHC and genetic testing for the BRAFV600E mutation is limited in Japan. This study aimed to better understand the histopathological characteristics of CRCs, which exhibit both deficient mismatch repair (dMMR) and BRAFV600E mutation. MMR IHC of formalin-fixed paraffin-embedded tissues from tumor areas obtained from 651 patients with CRC who underwent surgical resection at Hamamatsu University Hospital (Hamamatsu, Japan) between August 2016 and March 2022 were used to evaluate MMR status, which was determined by staining for the expression of 4 MMR proteins (MLH1, MSH2, PMS2, and MSH6). All dMMR tumors were additionally evaluated for BRAFV600 mutation status via Sanger sequencing. Patient clinical characteristics (age, sex, tumor location, size, and tumor pathology) were then classified using their dMMR and BRAFV600 mutation statuses. Among the 651 patients with CRC, 58 carried tumors with dMMR, of which 52 were deficiency in MLH1 (dMLH1). Interestingly, all 16 medullary carcinomas that were analyzed showed characteristics corresponding to the presence of both dMLH1 and BRAFV600E mutation (P = .01). These results suggest that colorectal medullary carcinomas can be diagnosed based on their unique characteristics of harboring the BRAFV600E mutation and exhibiting dMLH1 expression.
Copyright © 2023 the Author(s). Published by Wolters Kluwer Health, Inc.

Functional assays provide important evidence for classifying the disease significance of germline variants in DNA mismatch repair genes. Numerous laboratories, including our own, have developed functional assays to study mismatch repair gene variants. However, previous assays are limited due to the model system employed, the manner of gene expression, or the environment in which function is assessed. Here, we developed a human cell-based approach for testing the function of variants of uncertain significance (VUS) in the MLH1 gene. Using clustered regularly interspaced short palindromic repeats gene editing, we knocked in MLH1 VUS into the endogenous MLH1 loci in human embryonic stem cells. We examined their impact on RNA and protein, including their ability to prevent microsatellite instability and instigate a DNA damage response. A statistical clustering analysis determined the range of functions associated with known pathogenic or benign variants, and linear regression was performed using existing odds in favor of pathogenicity scores for these control variants to calibrate our functional assay results. By converting the functional outputs into a single odds in favor of pathogenicity score, variant classification expert panels can use these results to readily reassess these VUS. Ultimately, this information will guide proper diagnosis and disease management for suspected Lynch syndrome patients.
© 2022 Wiley Periodicals LLC.