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.

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.

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.

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.

The mismatch repair-dependent DNA damage response: Mechanisms and implications.

An important role for the DNA mismatch repair (MMR) pathway in maintaining genomic stability is embodied in its conservation through evolution and the link between loss of MMR function and tumorigenesis. The latter is evident as inheritance of mutations within the major MMR genes give rise to the cancer predisposition condition, Lynch syndrome. Nonetheless, how MMR loss contributes to tumorigenesis is not completely understood. In addition to preventing the accumulation of mutations, MMR also directs cellular responses, such as cell cycle checkpoint or apoptosis activation, to different forms of DNA damage. Understanding this MMR-dependent DNA damage response may provide insight into the full tumor suppressing capabilities of the MMR pathway. Here, we delve into the proposed mechanisms for the MMR-dependent response to DNA damaging agents. We discuss how these pre-clinical findings extend to the clinical treatment of cancers, emphasizing MMR status as a crucial variable in selection of chemotherapeutic regimens. Also, we discuss how loss of the MMR-dependent damage response could promote tumorigenesis via the establishment of a survival advantage to endogenous levels of stress in MMR-deficient cells.

ATR-Chk1 activation mitigates replication stress caused by mismatch repair-dependent processing of DNA damage.

The mismatch repair pathway (MMR) is essential for removing DNA polymerase errors, thereby maintaining genomic stability. Loss of MMR function increases mutation frequency and is associated with tumorigenesis. However, how MMR is executed at active DNA replication forks is unclear. This has important implications for understanding how MMR repairs O6-methylguanine/thymidine (MeG/T) mismatches created upon exposure to DNA alkylating agents. If MeG/T lesion recognition by MMR initiates mismatch excision, the reinsertion of a mismatched thymidine during resynthesis could initiate futile repair cycles. One consequence of futile repair cycles might be a disruption of overall DNA replication in the affected cell. Herein, we show that in MMR-proficient HeLa cancer cells, treatment with a DNA alkylating agent slows S phase progression, yet cells still progress into the next cell cycle. In the first S phase following treatment, they activate ataxia telangiectasia and Rad3-related (ATR)-Checkpoint Kinase 1 (Chk1) signaling, which limits DNA damage, while inhibition of ATR kinase activity accelerates DNA damage accumulation and sensitivity to the DNA alkylating agent. We also observed that exposure of human embryonic stem cells to alkylation damage severely compromised DNA replication in a MMR-dependent manner. These cells fail to activate the ATR-Chk1 signaling axis, which may limit their ability to handle replication stress. Accordingly, they accumulate double-strand breaks and undergo immediate apoptosis. Our findings implicate the MMR-directed response to alkylation damage as a replication stress inducer, suggesting that repeated MMR processing of mismatches may occur that can disrupt S phase progression.

Mismatch repair defects and Lynch syndrome: The role of the basic scientist in the battle against cancer.

We have currently entered a genomic era of cancer research which may soon lead to a genomic era of cancer treatment. Patient DNA sequencing information may lead to a personalized approach to managing an individual's cancer as well as future cancer risk. The success of this approach, however, begins not necessarily in the clinician's office, but rather at the laboratory bench of the basic scientist. The basic scientist plays a critical role since the DNA sequencing information is of limited use unless one knows the function of the gene that is altered and the manner by which a sequence alteration affects that function. The role of basic science research in aiding the clinical management of a disease is perhaps best exemplified by considering the case of Lynch syndrome, a hereditary disease that predisposes patients to colorectal and other cancers. This review will examine how the diagnosis, treatment and even prevention of Lynch syndrome-associated cancers has benefitted from extensive basic science research on the DNA mismatch repair genes whose alteration underlies this condition.

Milestones of Lynch syndrome: 1895-2015.

Lynch syndrome, which is now recognized as the most common hereditary colorectal cancer condition, is characterized by the predisposition to a spectrum of cancers, primarily colorectal cancer and endometrial cancer. We chronicle over a century of discoveries that revolutionized the diagnosis and clinical management of Lynch syndrome, beginning in 1895 with Warthin's observations of familial cancer clusters, through the clinical era led by Lynch and the genetic era heralded by the discovery of causative mutations in mismatch repair (MMR) genes, to ongoing challenges.

Human pluripotent stem cells have a novel mismatch repair-dependent damage response.

Human pluripotent stem cells (PSCs) are presumed to have robust DNA repair pathways to ensure genome stability. PSCs likely need to protect against mutations that would otherwise be propagated throughout all tissues of the developing embryo. How these cells respond to genotoxic stress has only recently begun to be investigated. Although PSCs appear to respond to certain forms of damage more efficiently than somatic cells, some DNA damage response pathways such as the replication stress response may be lacking. Not all DNA repair pathways, including the DNA mismatch repair (MMR) pathway, have been well characterized in PSCs to date. MMR maintains genomic stability by repairing DNA polymerase errors. MMR is also involved in the induction of cell cycle arrest and apoptosis in response to certain exogenous DNA-damaging agents. Here, we examined MMR function in PSCs. We have demonstrated that PSCs contain a robust MMR pathway and are highly sensitive to DNA alkylation damage in an MMR-dependent manner. Interestingly, the nature of this alkylation response differs from that previously reported in somatic cell types. In somatic cells, a permanent G2/M cell cycle arrest is induced in the second cell cycle after DNA damage. The PSCs, however, directly undergo apoptosis in the first cell cycle. This response reveals that PSCs rely on apoptotic cell death as an important defense to avoid mutation accumulation. Our results also suggest an alternative molecular mechanism by which the MMR pathway can induce a response to DNA damage that may have implications for tumorigenesis.

Translating mismatch repair mechanism into cancer care.

The first DNA mismatch repair gene was identified in Escherichia coli nearly fifty years ago. Since then, five decades of basic biomedical research on this important repair pathway have led to an extensive understanding of its molecular mechanism. The significance of this work was clearly highlighted in the early 1990's when mutations in the human homologs of the mismatch repair genes were identified as responsible for Lynch syndrome (also known as hereditary non-polyposis colon cancer), the most common form of hereditary colorectal cancer. Basic science research on mismatch repair in lower organisms directly led researchers to the discovery of this link between defective mismatch repair and cancer and continues to guide clinical decisions today. The knowledge that disrupted mismatch repair function gives rise to the nucleotide-level form of genomic instability called microsatellite instability continues to be an important diagnostic tool for identifying Lynch syndrome patients as well as sporadic cancer patients who suffer from mismatch repairdefective cancers. Today, clinicians are using the information about mismatch repair molecular mechanism to guide decisions about cancer therapy as well to devise new therapies. In this review, we will examine what is known about the molecular function of the human mismatch repair pathway. We will highlight how this information is being used in cancer diagnosis and treatment. We will also discuss strategies being designed to target the 10-15% of colorectal, endometrial, ovarian and other cancers with defective mismatch repair.

Application of a 5-tiered scheme for standardized classification of 2,360 unique mismatch repair gene variants in the InSiGHT locus-specific database.

The clinical classification of hereditary sequence variants identified in disease-related genes directly affects clinical management of patients and their relatives. The International Society for Gastrointestinal Hereditary Tumours (InSiGHT) undertook a collaborative effort to develop, test and apply a standardized classification scheme to constitutional variants in the Lynch syndrome-associated genes MLH1, MSH2, MSH6 and PMS2. Unpublished data submission was encouraged to assist in variant classification and was recognized through microattribution. The scheme was refined by multidisciplinary expert committee review of the clinical and functional data available for variants, applied to 2,360 sequence alterations, and disseminated online. Assessment using validated criteria altered classifications for 66% of 12,006 database entries. Clinical recommendations based on transparent evaluation are now possible for 1,370 variants that were not obviously protein truncating from nomenclature. This large-scale endeavor will facilitate the consistent management of families suspected to have Lynch syndrome and demonstrates the value of multidisciplinary collaboration in the curation and classification of variants in public locus-specific databases.

Determining the functional significance of mismatch repair gene missense variants using biochemical and cellular assays.

With the discovery that the hereditary cancer susceptibility disease Lynch syndrome (LS) is caused by deleterious germline mutations in the DNA mismatch repair (MMR) genes nearly 20 years ago, genetic testing can now be used to diagnose this disorder in patients. A definitive diagnosis of LS can direct how clinicians manage the disease as well as prevent future cancers for the patient and their families. A challenge emerges, however, when a germline missense variant is identified in a MMR gene in a suspected LS patient. The significance of a single amino acid change in these large repair proteins is not immediately obvious resulting in them being designated variants of uncertain significance (VUS). One important strategy for resolving this uncertainty is to determine whether the variant results in a non-functional protein. The ability to reconstitute the MMR reaction in vitro has provided an important experimental tool for studying the functional consequences of VUS. However, beyond this repair assay, a number of other experimental methods have been developed that allow us to test the effect of a VUS on discrete biochemical steps or other aspects of MMR function. Here, we describe some of these assays along with the challenges of using such assays to determine the functional consequences of MMR VUS which, in turn, can provide valuable insight into their clinical significance. With increased gene sequencing in patients, the number of identified VUS has expanded dramatically exacerbating this problem for clinicians. However, basic science research laboratories around the world continue to expand our knowledge of the overall MMR molecular mechanism providing new opportunities to understand the functional significance, and therefore pathogenic significance, of VUS.

Pathological assessment of mismatch repair gene variants in Lynch syndrome: past, present, and future.

Lynch syndrome (LS) is caused by germline mutations in DNA mismatch repair (MMR) genes and is the most prevalent hereditary colorectal cancer syndrome. A significant proportion of variants identified in MMR and other common cancer susceptibility genes are missense or noncoding changes whose consequences for pathogenicity cannot be easily interpreted. Such variants are designated as "variants of uncertain significance" (VUS). Management of LS can be significantly improved by identifying individuals who carry a pathogenic variant and thus benefit from screening, preventive, and therapeutic measures. Also, identifying family members that do not carry the variant is important so they can be released from the intensive surveillance. Determining which genetic variants are pathogenic and which are neutral is a major challenge in clinical genetics. The profound mechanistic knowledge on the genetics and biochemistry of MMR enables the development and use of targeted assays to evaluate the pathogenicity of variants found in suspected patients with LS. We describe different approaches for the functional analysis of MMR gene VUS and propose development of a validated diagnostic framework. Furthermore, we call attention to common misconceptions about functional assays and endorse development of an integrated approach comprising validated assays for diagnosis of VUS in patients suspected of LS.

The predicted truncation from a cancer-associated variant of the MSH2 initiation codon alters activity of the MSH2-MSH6 mismatch repair complex.

Lynch syndrome (LS) is caused by germline mutations in DNA mismatch repair (MMR) genes. MMR recognizes and repairs DNA mismatches and small insertion/deletion loops. Carriers of MMR gene variants have a high risk of developing colorectal, endometrial, ovarian, and other extracolonic carcinomas. We report on an ovarian cancer patient who carries a germline MSH2 c.1A>C variant which alters the translation initiation codon. Mutations affecting the MSH2 start codon have been described previously for LS-related malignancies. However, the patients often lack a clear family history indicative of LS and their tumors often fail to display microsatellite instability, a hallmark feature of LS. Therefore, the pathogenicity of start codon variants remains undefined. Loss of the MSH2 start codon has been predicted to result in a truncated protein translated from a downstream in-frame AUG that would lack the first 25 amino acids. We therefore purified recombinant MSH2(NΔ25)-MSH6 and MSH2(NΔ25)-MSH3 to examine their DNA lesion recognition and adenosine nucleotide processing functions in vitro. We found that the MSH2(NΔ25) mutant confers distinct biochemical defects on MSH2-MSH6, but does not have a significant effect on MSH2-MSH3. We confirmed that expression of the MSH2 c.1A>C cDNA results in the production of multiple protein products in human cells that may include the truncated and full-length forms of MSH2. An in vivo MMR assay revealed a slight reduction in MMR efficiency in these cells. These data suggest that mutation of the MSH2 initiation codon, while not a strong, high-risk disease allele, may have a moderate impact on disease phenotype.

Aberrant crypt foci as predictors of colorectal neoplasia on repeat colonoscopy.

OBJECTIVE: To estimate the risk for colorectal neoplasia detected on repeat colonoscopy in relation to aberrant crypt foci (ACF) frequency reported during the previous baseline examination. METHODS: From July 2003 until December 2008, patients had a colonoscopy with an ACF study using a magnifying colonoscope. The distal 20 cm section of colon was sprayed with Methylene Blue to ascertain the ACF frequency, the independent variable. Patients were categorized into low and high ACF count using the median as the cut point. Data collected from consenting patients included age, gender, height, weight, ethnicity, smoking history, family history of colorectal cancer (CRC), and personal history of colorectal neoplasia. A follow-up colonoscopy was performed at an interval as dictated by clinical surveillance guidelines. The main outcome was surveillance detected advanced colorectal neoplasia (SDAN) detected on repeat colonoscopy. Logistic Regression was used to calculate risk of SDAN on repeat colonoscopy in relation to baseline ACF count. RESULTS: 74 patients had a baseline ACF exam and a repeat surveillance colonoscopy. The median ACF was six and thus a high ACF count was >6 ACF and a low ACF count was ≤6 ACF. Patients diagnosed with SDAN were more likely to have had a high ACF number at baseline compared to patients without these lesions at follow-up (adjusted odds ratio = 12.27; 95% confidence interval: 2.00-75.25) controlling for age, sex, smoking, history of prior adenoma, family history of colon cancer, obesity, and time interval to surveillance exam. A sub analysis of our results demonstrated that this relationship was observed in 48 patients who were undergoing a surveillance colonoscopy for a previous adenoma and not those receiving surveillance for a family history of neoplasia. CONCLUSIONS: Increased number of ACF in the distal colorectum was independently associated with substantial risk for future advanced neoplasia. This relationship was observed in patients undergoing surveillance for previous adenomas. Thus, ACF may serve as potential biomarkers in patients with adenomas to help identify patients who may need additional surveillance.

Human MSH2 (hMSH2) protein controls ATP processing by hMSH2-hMSH6.

The mechanics of hMSH2-hMSH6 ATP binding and hydrolysis are critical to several proposed mechanisms for mismatch repair (MMR), which in turn rely on the detailed coordination of ATP processing between the individual hMSH2 and hMSH6 subunits. Here we show that hMSH2-hMSH6 is strictly controlled by hMSH2 and magnesium in a complex with ADP (hMSH2(magnesium-ADP)-hMSH6). Destabilization of magnesium results in ADP release from hMSH2 that allows high affinity ATP binding by hMSH6, which then enhances ATP binding by hMSH2. Both subunits must be ATP-bound to efficiently form a stable hMSH2-hMSH6 hydrolysis-independent sliding clamp required for MMR. In the presence of magnesium, the ATP-bound sliding clamps remain on the DNA for ∼8 min. These results suggest a precise stepwise kinetic mechanism for hMSH2-hMSH6 functions that appears to mimic G protein switches, severely constrains models for MMR, and may partially explain the MSH2 allele frequency in Lynch syndrome or hereditary nonpolyposis colorectal cancer.

DNA mismatch repair proteins are required for efficient herpes simplex virus 1 replication.

Herpes simplex virus 1 (HSV-1) is a double-stranded DNA virus that replicates in the nucleus of its human host cell and is known to interact with many cellular DNA repair proteins. In this study, we examined the role of cellular mismatch repair (MMR) proteins in the virus life cycle. Both MSH2 and MLH1 are required for efficient replication of HSV-1 in normal human cells and are localized to viral replication compartments. In addition, a previously reported interaction between MSH6 and ICP8 was confirmed by coimmunoprecipitation and extended to show that UL12 is also present in this complex. We also report for the first time that MLH1 associates with ND10 nuclear bodies and that like other ND10 proteins, MLH1 is recruited to the incoming genome. Knockdown of MLH1 inhibits immediate-early viral gene expression. MSH2, on the other hand, which is generally thought to play a role in mismatch repair at a step prior to that of MLH1, is not recruited to incoming genomes and appears to act at a later step in the viral life cycle. Silencing of MSH2 appears to inhibit early gene expression. Thus, both MLH1 and MSH2 are required but appear to participate in distinct events in the virus life cycle. The observation that MLH1 plays an earlier role in HSV-1 infection than does MSH2 is surprising and may indicate a novel function for MLH1 distinct from its known MSH2-dependent role in mismatch repair.