Three-step site-directed mutagenesis screen identifies pathogenic MLH1 variants associated with Lynch syndrome

Abstract

BackgroundInactivating mutations in the MLH1 DNA mismatch repair (MMR) gene underlie 42% of Lynch syndrome (LS) cases. LS is a cancer predisposition causing early onset colorectal and endometrial cancer. Nonsense and frameshift alterations unambiguously cause LS. The phenotype of missense mutations that only alter a single amino acid is often unclear. These variants of uncertain significance (VUS) hinder LS diagnosis and family screening and therefore functional tests are urgently needed. We developed a functional test for MLH1 VUS termed ‘oligonucleotide-directed mutation screening’ (ODMS).MethodsThe MLH1 variant was introduced by oligonucleotide-directed gene modification in mouse embryonic stem cells that were subsequently exposed to the guanine analogue 6-thioguanine to determine whether the variant abrogated MMR.ResutsIn a proof-of-principle analysis, we demonstrate that ODMS can distinguish pathogenic and non-pathogenic MLH1 variants with a sensitivity of >95% and a specificity of >91%. We subsequently applied the screen to 51 MLH1 VUS and identified 31 pathogenic variants.ConclusionODMS is a reliable tool to identify pathogenic MLH1 variants. Implementation in clinical diagnostics will improve clinical care of patients with suspected LS and their relatives.

Endometrial cancer in Lynch syndrome

“…Thus, germline testing should be performed based on MMR‐IHC results or MSI status (Figure 1). 48 Also, many functional assays have been developed to analyze VUS 75,79 . In addition, long‐read sequencing has shown promising results in LS diagnosis because of its advantages in the detection of unknown MMR variants, epigenetic modifications of MMR genes and pseudogenes, such as PMS2 80 …”

“…76 VUS refers to the changes in the sequences of MMR gene that are known to only alter a single amino acid, which is not specifically known to cause LS. 75 VUS accounts for 31%, 28%, 47% and 26% of all MLH1, MSH2, MSH6 and PMS2 mutations, respectively. 77 VUS results in challenges in LS diagnosis, and there is no established management for patients with VUS because its pathological functions are unknown.…”

Extensive trimming of short single-stranded DNA oligonucleotides during replication-coupled gene editing in mammalian cells

“…However, the lack of additional planned and unforeseen mutations at the target site (which is adherent to CRISPR/Cas) makes LMO technology especially useful for direct phenotypic assessment of variants identified in disease-related genes. As a proof-of-concept we have demonstrated this approach for the classification of variants of uncertain clinical significance (VUS) identified in the MMR genes MSH2 [ 9 ], MSH6 [ 10 ] and MLH1 [ 11 ]. While identification of pathogenic MMR gene variants was based on their acquired resistance to a methylating compound, the readout in other VUS screens could be based on more subtle and less binary phenotypic changes that can be assessed by flow cytometry, high-throughput microscopy or integrative single cell analyses [ 43 , 44 ].…”

“…Targeting chromosomal DNA during replication has proven to be highly effective for multiplex genome engineering in simple prokaryotic and eukaryotic model organisms like Escherichia coli [4][5][6] and Saccharomyces cerevisiae [7,8]. In addition, we have demonstrated the applicability of this technology in mammalian cells by setting up screens that enable the classification of Lynch syndrome-associated variants of uncertain clinical significance in MSH2, MSH6 and MLH1 [9][10][11].…”

Predictive functional assay‐based classification of PMS2 variants in Lynch syndrome

“…Predisposing variants in PMS2 confer a low prevalence of cancer due to the protein's genetic redundancy (11%–20% lifetime colorectal cancer risk by age 70) (Senter et al, 2008; ten Broeke et al, 2015). The current clinically focused approach of variant classification, therefore, is often underpowered to classify PMS2 VUS, and 98% of PMS2 missense variants (1490/1521) remain unclassified (Class 3, VUS) in the ClinVar database (Accessed, August 19, 2021) (Landrum et al, 2018). Consequently, personalized healthcare for carriers and affected relatives cannot be implemented.…”