A calibrated cell-based functional assay to aid classification of MLH1 DNA mismatch repair gene variants

Hum Mutat. 2022 Dec;43(12):2295-2307. doi: 10.1002/humu.24462. Epub 2022 Sep 12.


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.

Keywords: CRISPR gene editing; DNA damage response; Lynch syndrome; functional assay; microsatellite instability; mismatch repair; variant of uncertain significance.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Colorectal Neoplasms, Hereditary Nonpolyposis* / diagnosis
  • Colorectal Neoplasms, Hereditary Nonpolyposis* / genetics
  • Colorectal Neoplasms, Hereditary Nonpolyposis* / pathology
  • DNA Mismatch Repair* / genetics
  • Germ-Line Mutation / genetics
  • Humans
  • Microsatellite Instability
  • Mismatch Repair Endonuclease PMS2 / genetics
  • MutL Protein Homolog 1 / genetics


  • MutL Protein Homolog 1
  • Mismatch Repair Endonuclease PMS2
  • MLH1 protein, human