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

Abstract

PURPOSE:Functional assays provide important evidence for classifying the disease significance of germline variants in the DNA mismatch repair genes. We sought to develop a cell-based approach for testing the function of variants of uncertain significance (VUS) in the MLH1 gene. METHODS:Using CRISPR gene editing, we knocked-in MLH1 VUS into the endogenous MLH1 loci in human embryonic stem cells. We examined their impact at the RNA and protein level, including their ability to maintain stability of microsatellite sequences and instigate a DNA damage response. We calibrated these assays by testing well-established pathogenic and benign control variants. RESULTS:Five VUS resulted in functionally abnormal protein, 15 VUS resulted in functionally normal protein, and one VUS showed mixed results. Furthermore, we converted the functional outputs into a single odds in favor of pathogenicity score for each VUS. CONCLUSION:Our CRISPR-based functional assay successfully models phenotypes observed in patients in a cellular context. Using this approach, we generated evidence for or against pathogenicity for utilization by variant classification expert panels. Ultimately, this information will assist in proper diagnosis and disease management for suspected Lynch syndrome patients.

Aberrant cytoplasmic localization of MLH1 characterizes a sub-clonal breast cancer cell population that seeds recurrence

“…To identify the mechanism by which MLH1 G55V confers endocrine therapy resistance, we conducted RNAseq analysis of isogenic MCF7 WT and MCF7 G55V cells at baseline and after fulvestrant treatment. We included vehicle- and fulvestrant-treated MCF7 shMLH1 and MCF7 E717* cells as additional controls to identify pathways uniquely dysregulated by MLH1 G55V . Pathway analysis identified cell cycle dysregulation (Fig S4A), specifically G1/S checkpoint activity (Fig 4A), as a distinguishing feature of the MCF7 G55V cellular response to fulvestrant treatment (FDR<0.05).…”

“…(A) Gene set enrichment analysis of RNAseq data comparing MCF7 G55V to MCF7 shMLH1, MCF7 WT and MCF7 E717 * cells after treatment with 1 µM fulvestrant for 36h with bar graphs depicting the significance of individual cell cycle component pathways (expressed as 1-FDR for visibility). Associated Reactome analyses are presented in Fig S4A.…”

“…Despite the importance of MLH1 loss as a driver of ER+ breast cancer outcome, little is known about the pathogenicity of somatic mutations in MLH1. The majority of MLH1 mutations in ER+ patient tumors, are missense and non-recurrent making determination of variant pathogenicity burdensome [14][15][16][17][18][19] . Nevertheless, understanding how MLH1 mutations impact endocrine treatment resistance could significantly improve the predictive value of identifying these mutations through routine clinical diagnostics, for ER+ breast, and other, cancer patients 18 .…”

Loss of mismatch repair promotes a direct selective advantage in human stem cells

“…Restoration of MSH2 significantly reduced the growth advantage; KI hESCs grew less than the parental MSH2 KO2 line ( Figure 1 D). We also examined whether this growth advantage was specific to MSH2 loss or loss of MMR function in general, by utilizing cells in which the MLH1 (MIM: 120436 ) gene was knocked out by CRISPR gene editing ( Rath et al., 2022 ). We saw a similar growth advantage in MLH1 KO cells, much like when MSH2 was lost ( Figure 1 E), suggesting that the advantage was due to loss of MMR function.…”

Mismatch repair gene specifications to the ACMG/AMP classification criteria: Consensus recommendations from the InSiGHT ClinGen Hereditary Colorectal Cancer / Polyposis Variant Curation Expert Panel

“…As per the Brnich et al recommendation, at least 11 controls are required to reach a Moderate level evidence and a rigorous statistical analysis to reach a Strong level of evidence. There are currently three assays recognized with calibrated odds of pathogenicity that reach a Strong level of evidence (PS3), namely, the Complete in vitro MMR Assay (CIMRA) for MLH1, MSH2, MSH6, and PMS2 (Drost, 2019), (Drost, 2020), (Rayner, 2022), a deep mutational scan of MSH2 (Scott, 2022) (Jia, 2021), and a cell-based assay for MLH1 (Rath A, 2022). These assays employed rigorous statistical analysis calibrating the level of in vitro MMR activity to the corresponding probability of pathogenicity.…”