Classification of a VUS in <i>MLH1</i> using a combination of family segregation studies and protein biochemistry.
Bibliographic record
Abstract
342 Background: The identification of variants of unknown clinical significance (VUS) presents a challenge for diagnostic laboratories and clinicians working with families with Lynch syndrome (LS). Although a variety of strategies exist to attempt clarification, data from multiple sources is often required before a VUS is re-classified to pathogenic or benign. Methods: We report on the investigation of 2 families with a variant in the MMR gene MLH1, c.2038T>C (p.Cys680Arg). In addition to segregation analysis, and review of published literature, the c-terminal domain of MLH1was cloned, the p.Cys680Arg variant was generated using site-directed mutagenesis and protein expression was induced. In addition, this point mutation was modelled onto the crystal structure of the dimerization domain of MLH1. Results: Both families fit Amsterdam criteria for a diagnosis of LS. All available tumours exhibited MLH1 deficiency and microsatellite instability and all living affected individuals available were positive for MLH1 p.Cys680Arg variant. Protein studies confirmed that p.Cys680 resides in a tight hydrophobic cavity and the p.Cys680Arg mutation disrupts the folding of the C-terminal domain of MLH1. In two families this variant segregated with disease in a total of 12 individuals (>10 meiosis and >1000:1 odds in favor of linkage) and 3 had an MLH1 deficient tumour, supporting a pathogenic role for this variant. Inspection of the crystal structure of MLH1 suggested that the Cys680Arg mutation should severely alter the folding of the dimerization domain. As opposed to the dimerization domain of MLH1 that can be readily produced recombinantly and is stable in solution, the same domain of MLH1 encoding the Cys680Arg mutation was expressed as inclusion bodies, confirming that this mutation causes misfolding of the dimerization domain of MLH1. Conclusions: Our data confirms that the p.Cys680Arg mutation destabilizes the dimerization domain of MLH1, and presumably the DNA mismatch repair proficiency of individuals carrying this mutation. Based on this evidence and family history data, the MLH1 C680R missense variant is classified as pathogenic. This approach may prove useful in the classification of other variants.
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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 | 0.001 | 0.003 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.004 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.001 | 0.000 |
| Insufficient payload (model declined to judge) | 0.002 | 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".