Bibliographic record
Abstract
3343 Germline mutations in the mismatch repair (MMR) genes predispose to a common form of colorectal cancer known as hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch Syndrome. Approximately 25% of sequence alterations in the MMR genes are of uncertain pathogenic relevance, which poses difficulties for predictive testing programs offered to familial colorectal cancer patients. We hypothesize that missense alterations in distinct domains of the MMR proteins likely affect their function(s) to varying degrees and this likely contributes to the variable disease phenotype observed in HNPCC. Hence this project aims to characterize the pathogenicity associated with a panel of variants of unknown functional significance, and to elucidate the mechanisms by which they exert their effects. We have selected a panel of unclassified variants identified in HNPCC patients based on their presence in functional domains, evolutionary status and pathogenicity as predicted by computational databases. The variants chosen in hMLH1 include: I19F, A128S, R265C, K618A and L749Q. These variants were created in plasmids containing wild type MLH1 cDNA by site-directed mutagenesis, verified by sequencing and transiently transfected into the colon cancer cell line HCT116, which is deficient for hMLH1. Western blotting assays were carried out to ascertain the expression levels of the proteins. Coimmunoprecipitation experiments were performed to determine if these putative mutations disrupt interactions with partner proteins. In order to differentiate between endogenous and transfected mRNA, we have sub-cloned the wild type and variant MLH1 cDNAs into tagged expression constructs, which will be used in mRNA stability assays following Actinomycin D treatment. Cyto-nuclear fractionation experiments are in progress to ascertain if the variants affect the localization of the MMR proteins. Results indicate that the variants R265C and K618A significantly decrease the overall expression of MLH1 compared to the wild type vector. I19F, A128S and L749Q however, affect the temporal expression profile of MLH1, indicating a possible decrease in the stability of the protein. Coimmunoprecipitation experiments indicate that the ability of the MLH1 L749Q variant to interact with its cognate partner PMS2 is compromised. This is an interesting observation as this variant is located just adjacent to the previously defined PMS2 interaction domain. Preliminary results from cyto-nuclear fractionation studies show no difference between the localization of the variants R265C, L749Q and wild type MLH1. Our results indicate that the majority of unclassified variants investigated in this study perturb the expression and/ or interaction of the MMR proteins. In addition to its clinical utility, these studies will also provide a better understanding of the MMR proteins and the manner in which they contribute to colorectal cancer progression.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.009 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".