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

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.

Functional interrogation of Lynch syndrome-associated MSH2 missense variants via CRISPR-Cas9 gene editing in human embryonic stem cells.

Lynch syndrome (LS) predisposes patients to cancer and is caused by germline mutations in the DNA mismatch repair (MMR) genes. Identifying the deleterious mutation, such as a frameshift or nonsense mutation, is important for confirming an LS diagnosis. However, discovery of a missense variant is often inconclusive. The effects of these variants of uncertain significance (VUS) on disease pathogenesis are unclear, though understanding their impact on protein function can help determine their significance. Laboratory functional studies performed to date have been limited by their artificial nature. We report here an in-cellulo functional assay in which we engineered site-specific MSH2 VUS using clustered regularly interspaced short palindromic repeats-Cas9 gene editing in human embryonic stem cells. This approach introduces the variant into the endogenous MSH2 loci, while simultaneously eliminating the wild-type gene. We characterized the impact of the variants on cellular MMR functions including DNA damage response signaling and the repair of DNA microsatellites. We classified the MMR functional capability of eight of 10 VUS providing valuable information for determining their likelihood of being bona fide pathogenic LS variants. This human cell-based assay system for functional testing of MMR gene VUS will facilitate the identification of high-risk LS patients.

Fidelity of end joining in mammalian episomes and the impact of Metnase on joint processing.

BACKGROUND: Double Stranded Breaks (DSBs) are the most serious form of DNA damage and are repaired via homologous recombination repair (HRR) or non-homologous end joining (NHEJ). NHEJ predominates in mammalian cells at most stages of the cell cycle, and it is viewed as 'error-prone', although this notion has not been sufficiently challenged due to shortcomings of many current systems. Multi-copy episomes provide a large pool of genetic material where repair can be studied, as repaired plasmids can be back-cloned into bacteria and characterized for sequence alterations. Here, we used EBV-based episomes carrying 3 resistance marker genes in repair studies where a single DSB is generated with virally-encoded HO endonuclease cleaving rapidly at high efficiency for a brief time post-infection. We employed PCR and Southern blot to follow the kinetics of repair and formation of processing intermediates, and replica plating to screen for plasmids with altered joints resulting in loss of chloramphenicol resistance. Further, we employed this system to study the role of Metnase. Metnase is only found in humans and primates and is a key component of the NHEJ pathway, but its function is not fully characterized in intact cells. RESULTS: We found that repair of episomes by end-joining was highly accurate in 293 T cells that lack Metnase. Less than 10% of the rescued plasmids showed deletions. Instead, HEK293 cells (that do express Metnase) or 293 T transfected with Metnase revealed a large number of rescued plasmids with altered repaired joint, typically in the form of large deletions. Moreover, quantitative PCR and Southern blotting revealed less accurately repaired plasmids in Metnase expressing cells. CONCLUSIONS: Our careful re-examination of fidelity of NHEJ repair in mammalian cells carrying a 3' cohesive overhang at the ends revealed that the repair is efficient and highly accurate, and predominant over HRR. However, the background of the cells is important in establishing accuracy; with human cells perhaps surprisingly much more prone to generate deletions at the repaired junctions, if/when Metnase is abundantly expressed.

Functional and phenotypic consequences of an unusual inversion in MSH2.

Lynch syndrome is an autosomal dominant disorder that usually results from a pathogenic germline variant in one of four genes (MSH2, MSH6, MLH1, PMS2) involved in DNA mismatch repair. Carriers of such variants are at risk of developing numerous cancers during adulthood. Here we report on a family suspected of having Lynch syndrome due to a history of endometrial adenocarcinoma, ovarian clear cell carcinoma, and adenocarcinoma of the duodenum in whom we identified a germline 29 nucleotide in-frame inversion in exon 3 of MSH2. We further show that this variant is almost completely absent at the protein level, and that the associated cancers have complete loss of MSH2 and MSH6 expression by immunohistochemistry. Functional investigation of this inversion in a laboratory setting revealed a resultant abnormal protein function. Thus, we have identified an unusual, small germline inversion in a mismatch repair gene that does not lead to a premature stop codon yet appears likely to be causal for the observed cancers.

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

Lynch syndrome (LS) is the most common hereditary form of colon cancer, resulting from a germline mutation in a DNA mismatch repair (MMR) gene. Loss of MMR in cells establishes a mutator phenotype, which may underlie its link to cancer. Acquired downstream mutations that provide the cell a selective advantage would contribute to tumorigenesis. It is unclear, however, whether loss of MMR has other consequences that would directly result in a selective advantage. We found that knockout of the MMR gene MSH2 results in an immediate survival advantage in human stem cells grown under standard cell culture conditions. This advantage results, in part, from an MMR-dependent response to oxidative stress. We also found that loss of MMR gives rise to enhanced formation and growth of human colonic organoids. These results suggest that loss of MMR may affect cells in ways beyond just increasing mutation frequency that could influence tumorigenesis.

Enhanced CRISPR-based DNA demethylation by Casilio-ME-mediated RNA-guided coupling of methylcytosine oxidation and DNA repair pathways.

Here we develop a methylation editing toolbox, Casilio-ME, that enables not only RNA-guided methylcytosine editing by targeting TET1 to genomic sites, but also by co-delivering TET1 and protein factors that couple methylcytosine oxidation to DNA repair activities, and/or promote TET1 to achieve enhanced activation of methylation-silenced genes. Delivery of TET1 activity by Casilio-ME1 robustly alters the CpG methylation landscape of promoter regions and activates methylation-silenced genes. We augment Casilio-ME1 to simultaneously deliver the TET1-catalytic domain and GADD45A (Casilio-ME2) or NEIL2 (Casilio-ME3) to streamline removal of oxidized cytosine intermediates to enhance activation of targeted genes. Using two-in-one effectors or modular effectors, Casilio-ME2 and Casilio-ME3 remarkably boost gene activation and methylcytosine demethylation of targeted loci. We expand the toolbox to enable a stable and expression-inducible system for broader application of the Casilio-ME platforms. This work establishes a platform for editing DNA methylation to enable research investigations interrogating DNA methylomes.

Phenothiazine Inhibitors of TLKs Affect Double-Strand Break Repair and DNA Damage Response Recovery and Potentiate Tumor Killing with Radiomimetic Therapy.

The Tousled-like kinases (TLKs) are involved in chromatin assembly, DNA repair, and transcription. Two TLK genes exist in humans, and their expression is often dysregulated in cancer. TLKs phosphorylate Asf1 and Rad9, regulating double-strand break (DSB) repair and the DNA damage response (DDR). TLKs maintain genomic stability and are important therapeutic intervention targets. We identified specific inhibitors of TLKs from several compound libraries, some of which belong to the family of phenothiazine antipsychotics. The inhibitors prevented the TLK-mediated phosphorylation of Rad9(S328) and impaired checkpoint recovery and DSB repair. The inhibitor thioridazine (THD) potentiated tumor killing with chemotherapy and also had activity alone. Staining for γ-H2AX revealed few positive cells in untreated tumors, but large numbers in mice treated with low doxorubicin or THD alone, possibly the result of the accumulation of DSBs that are not promptly repaired as they may occur in the harsh tumor growth environment.