Disease-associated repeat instability and mismatch repair.

PubMed

Schmidt, Monika H M; Pearson, Christopher E

2016-02-01

Expanded tandem repeat sequences in DNA are associated with at least 40 human genetic neurological, neurodegenerative, and neuromuscular diseases. Repeat expansion can occur during parent-to-offspring transmission, and arise at variable rates in specific tissues throughout the life of an affected individual. Since the ongoing somatic repeat expansions can affect disease age-of-onset, severity, and progression, targeting somatic expansion holds potential as a therapeutic target. Thus, understanding the factors that regulate this mutation is crucial. DNA repair, in particular mismatch repair (MMR), is the major driving force of disease-associated repeat expansions. In contrast to its anti-mutagenic roles, mammalian MMR curiously drives the expansion mutations of disease-associated (CAG)·(CTG) repeats. Recent advances have broadened our knowledge of both the MMR proteins involved in disease repeat expansions, including: MSH2, MSH3, MSH6, MLH1, PMS2, and MLH3, as well as the types of repeats affected by MMR, now including: (CAG)·(CTG), (CGG)·(CCG), and (GAA)·(TTC) repeats. Mutagenic slipped-DNA structures have been detected in patient tissues, and the size of the slip-out and their junction conformation can determine the involvement of MMR. Furthermore, the formation of other unusual DNA and R-loop structures is proposed to play a key role in MMR-mediated instability. A complex correlation is emerging between tissues showing varying amounts of repeat instability and MMR expression levels. Notably, naturally occurring polymorphic variants of DNA repair genes can have dramatic effects upon the levels of repeat instability, which may explain the variation in disease age-of-onset, progression and severity. An increasing grasp of these factors holds prognostic and therapeutic potential. Copyright © 2015 Elsevier B.V. All rights reserved.

Impact of DNA mismatch repair system alterations on human fertility and related treatments.

PubMed

Hu, Min-hao; Liu, Shu-yuan; Wang, Ning; Wu, Yan; Jin, Fan

2016-01-01

DNA mismatch repair (MMR) is one of the biological pathways, which plays a critical role in DNA homeostasis, primarily by repairing base-pair mismatches and insertion/deletion loops that occur during DNA replication. MMR also takes part in other metabolic pathways and regulates cell cycle arrest. Defects in MMR are associated with genomic instability, predisposition to certain types of cancers and resistance to certain therapeutic drugs. Moreover, genetic and epigenetic alterations in the MMR system demonstrate a significant relationship with human fertility and related treatments, which helps us to understand the etiology and susceptibility of human infertility. Alterations in the MMR system may also influence the health of offspring conceived by assisted reproductive technology in humans. However, further studies are needed to explore the specific mechanisms by which the MMR system may affect human infertility. This review addresses the physiological mechanisms of the MMR system and associations between alterations of the MMR system and human fertility and related treatments, and potential effects on the next generation.

Tissue-specific mismatch repair protein expression: MSH3 is higher than MSH6 in multiple mouse tissues.

PubMed

Tomé, Stéphanie; Simard, Jodie P; Slean, Meghan M; Holt, Ian; Morris, Glenn E; Wojciechowicz, Kamila; te Riele, Hein; Pearson, Christopher E

2013-01-01

Mismatch repair (MMR) proteins have critical roles in the maintenance of genomic stability, both class-switch recombination and somatic hypermutation of immunoglobulin genes and disease-associated trinucleotide repeat expansions. In the genetic absence of MMR, certain tissues are predisposed to mutations and cancer. MMR proteins are involved in various functions including protection from replication-associated and non-mitotic mutations, as well as driving programmed and deleterious mutations, including disease-causing trinucleotide repeat expansions. Here we have assessed the levels of MSH2, MSH3, and MSH6 expression in a large number of murine tissues by transcript analysis and simultaneous Western blotting. We observed that MMR expression patterns varied widely between 14 different tissue types, but did not vary with age (13-84 weeks). MMR protein expression is highest in testis, thymus and spleen and lowest in pancreas, quadriceps and heart, with intermediate levels in liver, kidney, intestine, colon, cortex, striatum and cerebellum. By equalizing antibody signal intensity to represent levels found in mMutSα and mMutSβ purified proteins, we observed that mMSH3 protein levels are greater than mMSH6 levels in the multiple tissues analyzed, with more MSH6 in proliferating tissues. In the intestinal epithelium MSH3 and MSH6 are more highly expressed in the proliferative undifferentiated cells of the crypts than in the differentiated villi cells, as reported for MSH2. This finding correlates with the higher level of MMR expression in highly proliferative mouse tissues such as the spleen and thymus. The relative MMR protein expression levels may explain the functional and tissue-specific reliance upon the roles of each MMR protein. Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.

Selenium compounds activate ATM-dependent DNA damage responses via the mismatch repair protein hMLH1 in colorectal cancer cells

USDA-ARS?s Scientific Manuscript database

Epidemiological and animal studies indicate that selenium supplementation suppresses risk of colorectal and other cancers. The majority of colorectal cancers are characterized by a defective DNA mismatch repair (MMR) process. Here, we have employed the MMR-deficient HCT 116 colorectal cancer cells ...

Analysis of MSH3 in endometrial cancers with defective DNA mismatch repair.

PubMed

Swisher, E M; Mutch, D G; Herzog, T J; Rader, J S; Kowalski, L D; Elbendary, A; Goodfellow, P J

1998-01-01

To clarify the origin of defective mismatch repair (MMR) in sporadic endometrial cancers with microsatellite instability (MSI), a thorough mutation analysis was performed on the human mismatch repair gene MSH3. Twenty-eight MSI-positive endometrial cancers were investigated for mutations in the human mismatch repair gene MSH3 using single-strand conformation variant (SSCV) analysis of all 24 exons. All variants were sequenced. Loss of heterozygosity was investigated at all MSH3 polymorphisms discovered. A subset of tumors were investigated for methylation of the 5' promoter region of MSH3 using Southern blot hybridization. An identical single-base deletion (delta A) predicted to result in a truncated proteins was discovered in six tumors (21.4%). This deletion occurs in a string of eight consecutive adenosine residues (A8). Because simple repeat sequences are unstable in cells with defective MMR, the observed mutation may be an effect, rather than a cause, of MSI. Evidence of inactivation of the second MSH3 allele in tumors with the delta A mutation would strongly support a causal role for these MSH3 mutations. However, there was no evidence of a second mutation, loss of sequences, or methylation of the promoter region in any of the tumors with the delta A mutation. Although the delta A mutation is a frequent event in sporadic MSI-positive endometrial cancers, it may not be causally associated with defective DNA MMR.

Peritumoral granulomatous reaction in endometrial carcinoma: association with DNA mismatch repair protein deficiency, particularly loss of PMS2 expression.

PubMed

Stewart, Colin J R; Pearn, Amy; Pachter, Nicholas; Tan, Adeline

2018-04-30

The observation of peritumoral granulomatous reactions (PGRs) in two endometrial carcinomas (ECs) with a PMS2-deficient/MLH1-intact expression pattern led us to investigate whether PGRs in EC were specifically associated with DNA mismatch repair (MMR) protein deficiency, particularly PMS2 loss. Hysterectomy specimens from 22 MMR protein-intact and 54 MMR protein-deficient ECs were reviewed with specific attention to the presence of a PGR and a tumour-associated lymphoid reaction [including tumour-infiltrating lymphocytes (TILs) and stromal lymphoid infiltrates]. The MMR protein-deficient ECs included 22 cases with combined MLH1/PMS2 loss, 11 with combined MSH2/MSH6 loss, 11 with isolated MSH6 loss, and 10 with PMS2 loss but intact MLH1 staining (including the two 'index' cases). Overall, PGRs were identified in seven of 54 (13%) MMR protein-deficient ECs, five of which showed a PMS2-deficient/MLH1-intact immunophenotype; three of these patients had germline PMS2 mutations and one additional patient had a germline MSH6 mutation. None of the MMR protein-intact tumours showed a PGR. Although five of the seven PGR-positive ECs had a high-grade histological component, six were stage I. Most ECs with PGRs also showed TILs and stromal lymphoid reactions, similarly to MMR protein-deficient ECs in general. MMR protein-deficient ECs, particularly those with PMS2 loss, occasionally show PGRs in addition to stromal lymphoid infiltrates and TILs. Therefore, PGRs could be considered to constitute a histological prompt for consideration of Lynch syndrome. The potential prognostic significance of PGRs in EC requires further study. © 2018 John Wiley & Sons Ltd.

Complete Remission Following Pembrolizumab in a Woman with Mismatch Repair-Deficient Endometrial Cancer and a Germline BRCA1 Mutation.

PubMed

Dizon, Don S; Dias-Santagata, Dora; Bregar, Amy; Sullivan, Laura; Filipi, Jennifer; DiTavi, Elizabeth; Miller, Lucy; Ellisen, Leif; Birrer, Michael; DelCarmen, Marcela

2018-02-22

Endometrial cancer is the most common gynecologic malignancy in the U.S. and, although the majority of cases present at an early stage and can be treated with curative intent, those who present with advanced disease, or develop metastatic or recurrent disease, have a poorer prognosis. A subset of endometrial cancers exhibit mismatch repair (MMR) deficiency. It is now recognized that MMR-deficient cancers are particularly susceptible to programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) inhibitors, and in a landmark judgement in 2017, the U.S. Food and Drug Administration granted accelerated approval to pembrolizumab for these tumors, the first tumor-agnostic approval of a drug. However, less is known about the sensitivity to PD-1 blockade among patients with known mutations in double-strand break DNA repair pathways involving homologous recombination, such as those in BRCA1 or BRCA2 . Here we report a case of a patient with an aggressive somatic MMR-deficient endometrial cancer and a germline BRCA1 who experienced a rapid complete remission to pembrolizumab. Endometrial cancers, and in particular endometrioid carcinomas, should undergo immunohistochemical testing for mismatch repair proteins.Uterine cancers with documented mismatch repair deficiency are candidates for treatment with programmed cell death protein 1 inhibition.Genomic testing of recurrent, advanced, or metastatic tumors may be useful to determine whether patients are candidates for precision therapies. © AlphaMed Press 2018.

The unstructured linker arms of Mlh1-Pms1 are important for interactions with DNA during mismatch repair

PubMed Central

Plys, Aaron J.; Rogacheva, Maria V.; Greene, Eric C.; Alani, Eric

2012-01-01

DNA mismatch repair (MMR) models have proposed that MSH proteins identify DNA polymerase errors while interacting with the DNA replication fork. MLH proteins (primarily Mlh1-Pms1 in baker’s yeast) then survey the genome for lesion-bound MSH proteins. The resulting MSH-MLH complex formed at a DNA lesion initiates downstream steps in repair. MLH proteins act as dimers and contain long (20 – 30 nanometers) unstructured arms that connect two terminal globular domains. These arms can vary between 100 to 300 amino acids in length, are highly divergent between organisms, and are resistant to amino acid substitutions. To test the roles of the linker arms in MMR, we engineered a protease cleavage site into the Mlh1 linker arm domain of baker’s yeast Mlh1-Pms1. Cleavage of the Mlh1 linker arm in vitro resulted in a defect in Mlh1-Pms1 DNA binding activity, and in vivo proteolytic cleavage resulted in a complete defect in MMR. We then generated a series of truncation mutants bearing Mlh1 and Pms1 linker arms of varying lengths. This work revealed that MMR is greatly compromised when portions of the Mlh1 linker are removed, whereas repair is less sensitive to truncation of the Pms1 linker arm. Purified complexes containing truncations in Mlh1 and Pms1 linker arms were analyzed and found to have differential defects in DNA binding that also correlated with the ability to form a ternary complex with Msh2-Msh6 and mismatch DNA. These observations are consistent with the unstructured linker domains of MLH proteins providing distinct interactions with DNA during MMR. PMID:22659005

BRAF/KRAS gene sequencing of sebaceous neoplasms after mismatch repair protein analysis.

PubMed

Cornejo, Kristine M; Hutchinson, Lloyd; Deng, April; Tomaszewicz, Keith; Welch, Matthew; Lyle, Stephen; Dresser, Karen; Cosar, Ediz F

2014-06-01

Sebaceous neoplasms are cutaneous markers for the autosomal-dominant Muir-Torre syndrome (MTS). This phenotypic variant of Lynch syndrome (LS) is caused by germline mutations in DNA mismatch repair (MMR) genes. Microsatellite instability or loss of protein expression suggests a mutation or promoter hypermethylation in 1 of the MMR genes. BRAF gene sequencing may help to distinguish between patients with sporadic and LS-associated colorectal carcinomas with loss of MLH1 expression. LS-associated carcinomas are virtually negative for BRAF mutations, but a subset harbors KRAS mutations. The aim of our study was to test sebaceous neoplasms for V600E BRAF or KRAS mutations to determine if these mutations are associated with somatic or germline MMR defects, analogous to colorectal carcinomas. Over a 4-year period, 32 cases comprising 21 sebaceous adenomas, 3 sebaceomas, and 8 sebaceous carcinomas with sufficient material for testing were collected. MMR immunohistochemistry showed that 7 neoplasms had combined loss of MLH1-PMS2, 16 neoplasms had combined loss of MSH2-MSH6, 2 neoplasms had solitary loss of MSH6, and 7 sebaceous neoplasms had intact protein expression. BRAF/KRAS testing revealed all sebaceous neoplasms contained a wild-type BRAF gene. Two (15%) of 13 patients with MTS were found to harbor a KRAS mutation and loss of MLH1 expression. We conclude that a V600E BRAF mutation may not be helpful in distinguishing sporadic from MTS-associated sebaceous neoplasms. Further studies are needed to determine if KRAS mutations are restricted to patients with MTS or are also present in sporadic sebaceous neoplasms. Copyright © 2014 Elsevier Inc. All rights reserved.

The mismatch repair system protects against intergenerational GAA repeat instability in a Friedreich ataxia mouse model.

PubMed

Ezzatizadeh, Vahid; Pinto, Ricardo Mouro; Sandi, Chiranjeevi; Sandi, Madhavi; Al-Mahdawi, Sahar; Te Riele, Hein; Pook, Mark A

2012-04-01

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by a dynamic GAA repeat expansion mutation within intron 1 of the FXN gene. Studies of mouse models for other trinucleotide repeat (TNR) disorders have revealed an important role of mismatch repair (MMR) proteins in TNR instability. To explore the potential role of MMR proteins on intergenerational GAA repeat instability in FRDA, we have analyzed the transmission of unstable GAA repeat expansions from FXN transgenic mice which have been crossed with mice that are deficient for Msh2, Msh3, Msh6 or Pms2. We find in all cases that absence of parental MMR protein not only maintains transmission of GAA expansions and contractions, but also increases GAA repeat mutability (expansions and/or contractions) in the offspring. This indicates that Msh2, Msh3, Msh6 and Pms2 proteins are not the cause of intergenerational GAA expansions or contractions, but act in their canonical MMR capacity to protect against GAA repeat instability. We further identified differential modes of action for the four MMR proteins. Thus, Msh2 and Msh3 protect against GAA repeat contractions, while Msh6 protects against both GAA repeat expansions and contractions, and Pms2 protects against GAA repeat expansions and also promotes contractions. Furthermore, we detected enhanced occupancy of Msh2 and Msh3 proteins downstream of the FXN expanded GAA repeat, suggesting a model in which Msh2/3 dimers are recruited to this region to repair mismatches that would otherwise produce intergenerational GAA contractions. These findings reveal substantial differences in the intergenerational dynamics of expanded GAA repeat sequences compared with expanded CAG/CTG repeats, where Msh2 and Msh3 are thought to actively promote repeat expansions. Copyright © 2012 Elsevier Inc. All rights reserved.

The mismatch repair system protects against intergenerational GAA repeat instability in a Friedreich ataxia mouse model

PubMed Central

Ezzatizadeh, Vahid; Pinto, Ricardo Mouro; Sandi, Chiranjeevi; Sandi, Madhavi; Al-Mahdawi, Sahar; te Riele, Hein; Pook, Mark A.

2013-01-01

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by a dynamic GAA repeat expansion mutation within intron 1 of the FXN gene. Studies of mouse models for other trinucleotide repeat (TNR) disorders have revealed an important role of mismatch repair (MMR) proteins in TNR instability. To explore the potential role of MMR proteins on intergenerational GAA repeat instability in FRDA, we have analyzed the transmission of unstable GAA repeat expansions from FXN transgenic mice which have been crossed with mice that are deficient for Msh2, Msh3, Msh6 or Pms2. We find in all cases that absence of parental MMR protein not only maintains transmission of GAA expansions and contractions, but also increases GAA repeat mutability (expansions and/or contractions) in the offspring. This indicates that Msh2, Msh3, Msh6 and Pms2 proteins are not the cause of intergenerational GAA expansions or contractions, but act in their canonical MMR capacity to protect against GAA repeat instability. We further identified differential modes of action for the four MMR proteins. Thus, Msh2 and Msh3 protect against GAA repeat contractions, while Msh6 protects against both GAA repeat expansions and contractions, and Pms2 protects against GAA repeat expansions and also promotes contractions. Furthermore, we detected enhanced occupancy of Msh2 and Msh3 proteins downstream of the FXN expanded GAA repeat, suggesting a model in which Msh2/3 dimers are recruited to this region to repair mismatches that would otherwise produce intergenerational GAA contractions. These findings reveal substantial differences in the intergenerational dynamics of expanded GAA repeat sequences compared with expanded CAG/CTG repeats, where Msh2 and Msh3 are thought to actively promote repeat expansions. PMID:22289650

Constitutional Mismatch Repair Deficiency in Israel: High Proportion of Founder Mutations in MMR Genes and Consanguinity.

PubMed

Baris, Hagit N; Barnes-Kedar, Inbal; Toledano, Helen; Halpern, Marisa; Hershkovitz, Dov; Lossos, Alexander; Lerer, Israela; Peretz, Tamar; Kariv, Revital; Cohen, Shlomi; Half, Elizabeth E; Magal, Nurit; Drasinover, Valerie; Wimmer, Katharina; Goldberg, Yael; Bercovich, Dani; Levi, Zohar

2016-03-01

Heterozygous germline mutations in any of the mismatch repair (MMR) genes, MLH1, MSH2, MSH6, and PMS2, cause Lynch syndrome (LS), an autosomal dominant cancer predisposition syndrome conferring a high risk of colorectal, endometrial, and other cancers in adulthood. Offspring of couples where both spouses have LS have a 1:4 risk of inheriting biallelic MMR gene mutations. These cause constitutional MMR deficiency (CMMRD) syndrome, a severe recessively inherited cancer syndrome with a broad tumor spectrum including mainly hematological malignancies, brain tumors, and colon cancer in childhood and adolescence. Many CMMRD children also present with café au lait spots and axillary freckling mimicking neurofibromatosis type 1. We describe our experience in seven CMMRD families demonstrating the role and importance of founder mutations and consanguinity on its prevalence. Clinical presentations included brain tumors, colon cancer, lymphoma, and small bowel cancer. In children from two nonconsanguineous Ashkenazi Jewish (AJ) families, the common Ashkenazi founder mutations were detected; these were homozygous in one family and compound heterozygous in the other. In four consanguineous families of various ancestries, different homozygous mutations were identified. In a nonconsanguineous Caucasus/AJ family, lack of PMS2 was demonstrated in tumor and normal tissues; however, mutations were not identified. CMMRD is rare, but, especially in areas where founder mutations for LS and consanguinity are common, pediatricians should be aware of it since they are the first to encounter these children. Early diagnosis will enable tailored cancer surveillance in the entire family and a discussion regarding prenatal genetic diagnosis. © 2015 Wiley Periodicals, Inc.

Pitfalls in molecular analysis for mismatch repair deficiency in a family with biallelic pms2 germline mutations.

PubMed

Leenen, C H M; Geurts-Giele, W R R; Dubbink, H J; Reddingius, R; van den Ouweland, A M; Tops, C M J; van de Klift, H M; Kuipers, E J; van Leerdam, M E; Dinjens, W N M; Wagner, A

2011-12-01

Heterozygous germline mutations in the mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2 cause Lynch syndrome. Biallelic mutations in the MMR genes are associated with a childhood cancer syndrome [constitutional mismatch repair deficiency (CMMR-D)]. This is predominantly characterized by hematological malignancies and tumors of the bowel and brain, often associated with signs of neurofibromatosis type 1 (NF1). Diagnostic strategies for selection of patients for MMR gene analysis include analysis of microsatellite instability (MSI) and immunohistochemical (IHC) analysis of MMR proteins in tumor tissue. We report the clinical characterization and molecular analyses of tumor specimens from a family with biallelic PMS2 germline mutations. This illustrates the pitfalls of present molecular screening strategies. Tumor tissues of five family members were analyzed for MSI and IHC. MSI was observed in only one of the analyzed tissues. However, IHC analysis of brain tumor tissue of the index patient and his sister showed absence of PMS2 expression, and germline mutation analyses showed biallelic mutations in PMS2: p.Ser46IIe and p.Pro246fs. The same heterozygous mutations were confirmed in the father and mother, respectively. These data support the conclusion that in case of a clinical phenotype of CMMR-D, it is advisable to routinely combine MSI analysis with IHC analysis for the expression of MMR proteins. With inconclusive or conflicting results, germline mutation analysis of the MMR genes should be considered after thorough counselling of the patients and/or their relatives. © 2011 John Wiley & Sons A/S.

HNPCC-like cancer predisposition in mice through simultaneous loss of Msh3 and Msh6 mismatch-repair protein functions.

PubMed

de Wind, N; Dekker, M; Claij, N; Jansen, L; van Klink, Y; Radman, M; Riggins, G; van der Valk, M; van't Wout, K; te Riele, H

1999-11-01

Cancer predisposition in hereditary non-polyposis colon cancer (HNPCC) is caused by defects in DNA mismatch repair (MMR). Mismatch recognition is attributed to two heterodimeric protein complexes: MutSalpha (refs 2, 3, 4, 5), a dimer of MutS homologues MSH2 and MSH6; and MutSbeta (refs 2,7), a dimer of MSH2 and MSH3. These complexes have specific and redundant mismatch recognition capacity. Whereas MSH2 deficiency ablates the activity of both dimers, causing strong cancer predisposition in mice and men, loss of MSH3 or MSH6 (also known as GTBP) function causes a partial MMR defect. This may explain the rarity of MSH6 and absence of MSH3 germline mutations in HNPCC families. To test this, we have inactivated the mouse genes Msh3 (formerly Rep3 ) and Msh6 (formerly Gtmbp). Msh6-deficient mice were prone to cancer; most animals developed lymphomas or epithelial tumours originating from the skin and uterus but only rarely from the intestine. Msh3 deficiency did not cause cancer predisposition, but in an Msh6 -deficient background, loss of Msh3 accelerated intestinal tumorigenesis. Lymphomagenesis was not affected. Furthermore, mismatch-directed anti-recombination and sensitivity to methylating agents required Msh2 and Msh6, but not Msh3. Thus, loss of MMR functions specific to Msh2/Msh6 is sufficient for lymphoma development in mice, whereas predisposition to intestinal cancer requires loss of function of both Msh2/Msh6 and Msh2/Msh3.

Clinicopathologic analysis with immunohistochemistry for DNA mismatch repair protein expression in synchronous primary endometrial and ovarian cancers.

PubMed

Kobayashi, Yusuke; Nakamura, Kanako; Nomura, Hiroyuki; Banno, Kouji; Irie, Haruko; Adachi, Masataka; Iida, Miho; Umene, Kiyoko; Nogami, Yuya; Masuda, Kenta; Kisu, Iori; Ueki, Arisa; Yamagami, Wataru; Kataoka, Fumio; Hirasawa, Akira; Tominaga, Eiichiro; Susumu, Nobuyuki; Aoki, Daisuke

2015-03-01

Synchronous primary endometrial and ovarian cancers have been an important topic in clinical medicine because it is sometimes difficult to distinguish whether there are 2 primary tumors or a single primary tumor and an associated metastasis. In addition, although these tumors are recommended for either immunohistochemistry for DNA mismatch repair (MMR) proteins or a microsatellite instability test in the Bethesda guidelines as Lynch syndrome-associated cancers, few studies have completed these analyses. In this study, we characterized the clinicopathologic features and the expression pattern of MMR proteins in synchronous primary endometrial and ovarian cancers. Clinicopathologic features and the expression pattern of MMR proteins (MLH1, MSH2, and MSH6) were characterized and analyzed in 32 synchronous primary endometrial and ovarian cancers. Most synchronous cancers are endometrioid type (endometrioid/endometrioid) (n = 24, 75%), grade 1 (n = 19, 59.4%), and diagnosed as stage I (n = 15, 46.9%) in both endometrium and ovary. It is worth mentioning that 75% of the patients (n = 24) had endometriosis, which was more common (n = 21, 87.5%) in endometrioid/endometrioid cancers, whereas only 3 cases (37.5%) were of different histology (P = 0.018). Loss of expression of at least 1 MMR protein was observed in 17 (53.1%) of the endometrial tumors and in 10 (31.3%) of ovarian tumors. Only 4 cases (12.5%) that had specific MMR protein loss showed the same type of loss for both endometrial and ovarian tumors, in which 3 of the cases were losses in MLH1. One case showed concordant MSH6 protein loss, although the cases did not meet the Amsterdam criteria II. These results suggest that most synchronous primary endometrial ovarian cancers are not hereditary cancers caused by germ line mutations but rather sporadic cancers.

DNA conformations in mismatch repair probed in solution by X-ray scattering from gold nanocrystals

PubMed Central

Hura, Greg L.; Tsai, Chi-Lin; Claridge, Shelley A.; Mendillo, Marc L.; Smith, Jessica M.; Williams, Gareth J.; Mastroianni, Alexander J.; Alivisatos, A. Paul; Putnam, Christopher D.; Kolodner, Richard D.; Tainer, John A.

2013-01-01

DNA metabolism and processing frequently require transient or metastable DNA conformations that are biologically important but challenging to characterize. We use gold nanocrystal labels combined with small angle X-ray scattering to develop, test, and apply a method to follow DNA conformations acting in the Escherichia coli mismatch repair (MMR) system in solution. We developed a neutral PEG linker that allowed gold-labeled DNAs to be flash-cooled and stored without degradation in sample quality. The 1,000-fold increased gold nanocrystal scattering vs. DNA enabled investigations at much lower concentrations than otherwise possible to avoid concentration-dependent tetramerization of the MMR initiation enzyme MutS. We analyzed the correlation scattering functions for the nanocrystals to provide higher resolution interparticle distributions not convoluted by the intraparticle distribution. We determined that mispair-containing DNAs were bent more by MutS than complementary sequence DNA (csDNA), did not promote tetramer formation, and allowed MutS conversion to a sliding clamp conformation that eliminated the DNA bends. Addition of second protein responder MutL did not stabilize the MutS-bent forms of DNA. Thus, DNA distortion is only involved at the earliest mispair recognition steps of MMR: MutL does not trap bent DNA conformations, suggesting migrating MutL or MutS/MutL complexes as a conserved feature of MMR. The results promote a mechanism of mismatch DNA bending followed by straightening in initial MutS and MutL responses in MMR. We demonstrate that small angle X-ray scattering with gold labels is an enabling method to examine protein-induced DNA distortions key to the DNA repair, replication, transcription, and packaging. PMID:24101514

Screening for Lynch syndrome and referral to clinical genetics by selective mismatch repair protein immunohistochemistry testing: an audit and cost analysis.

PubMed

Colling, Richard; Church, David N; Carmichael, Juliet; Murphy, Lucinda; East, James; Risby, Peter; Kerr, Rachel; Chetty, Runjan; Wang, Lai Mun

2015-12-01

Lynch syndrome (LS) accounts for around 3% of colorectal cancers (CRCs) and is caused by germline mutations in mismatch repair (MMR) genes. Recently, screening strategies to identify patients with LS have become popular. We audited CRCs screened with MMR immunohistochemistry (IHC) in 2013. 209 tumours had MMR IHC performed at a cost of £12 540. 47/209 (21%) cases showed IHC loss of expression in at least one MMR protein. 28/44 cases with loss of MLH1 had additional BRAF V600E testing, at a cost of £5040. MMR IHC reduced the number of potential clinical genetics referrals from 209 to 47. BRAF mutation testing, performed in a subset of cases with MLH1 loss, further reduced this to 21. At a cost of £1340 per referral, this model of LS screening for clinical genetics referral had significant potential savings (£234 340) and can be easily implemented in parallel with MMR IHC done for prognostication in CRCs. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Single-molecule multiparameter fluorescence spectroscopy reveals directional MutS binding to mismatched bases in DNA

PubMed Central

Cristóvão, Michele; Sisamakis, Evangelos; Hingorani, Manju M.; Marx, Andreas D.; Jung, Caroline P.; Rothwell, Paul J.; Seidel, Claus A. M.; Friedhoff, Peter

2012-01-01

Mismatch repair (MMR) corrects replication errors such as mismatched bases and loops in DNA. The evolutionarily conserved dimeric MMR protein MutS recognizes mismatches by stacking a phenylalanine of one subunit against one base of the mismatched pair. In all crystal structures of G:T mismatch-bound MutS, phenylalanine is stacked against thymine. To explore whether these structures reflect directional mismatch recognition by MutS, we monitored the orientation of Escherichia coli MutS binding to mismatches by FRET and anisotropy with steady state, pre-steady state and single-molecule multiparameter fluorescence measurements in a solution. The results confirm that specifically bound MutS bends DNA at the mismatch. We found additional MutS–mismatch complexes with distinct conformations that may have functional relevance in MMR. The analysis of individual binding events reveal significant bias in MutS orientation on asymmetric mismatches (G:T versus T:G, A:C versus C:A), but not on symmetric mismatches (G:G). When MutS is blocked from binding a mismatch in the preferred orientation by positioning asymmetric mismatches near the ends of linear DNA substrates, its ability to authorize subsequent steps of MMR, such as MutH endonuclease activation, is almost abolished. These findings shed light on prerequisites for MutS interactions with other MMR proteins for repairing the appropriate DNA strand. PMID:22367846

A mutation in EXO1 defines separable roles in DNA mismatch repair and post-replication repair

PubMed Central

Tran, Phuoc T.; Fey, Julien P.; Erdeniz, Naz; Gellon, Lionel; Boiteux, Serge; Liskay, R. Michael

2007-01-01

Replication forks stall at DNA lesions or as a result of an unfavorable replicative environment. These fork stalling events have been associated with recombination and gross chromosomal rearrangements. Recombination and fork bypass pathways are the mechanisms accountable for restart of stalled forks. An important lesion bypass mechanism is the highly conserved post-replication repair (PRR) pathway that is composed of error-prone translesion and error-free bypass branches. EXO1 codes for a Rad2p family member nuclease that has been implicated in a multitude of eukaryotic DNA metabolic pathways that include DNA repair, recombination, replication, and telomere integrity. In this report, we show EXO1 functions in the MMS2 error-free branch of the PRR pathway independent of the role of EXO1 in DNA mismatch repair (MMR). Consistent with the idea that EXO1 functions independently in two separate pathways, we defined a domain of Exo1p required for PRR distinct from those required for interaction with MMR proteins. We then generated a point mutant exo1 allele that was defective for the function of Exo1p in MMR due to disrupted interaction with Mlh1p, but still functional for PRR. Lastly, by using a compound exo1 mutant that was defective for interaction with Mlh1p and deficient for nuclease activity, we provide further evidence that Exo1p plays both structural and catalytic roles during MMR. PMID:17602897

Role of Cell Cycle Regulation and MLH1, A Key DNA Mismatch Repair Protein, In Adaptive Survival Responses. Final Report

DOE Office of Scientific and Technical Information (OSTI.GOV)

David A. Boothman

1999-08-11

Due to several interesting findings on both adaptive survival responses (ASRs) and DNA mismatch repair (MMR), this grant was separated into two discrete Specific Aim sets (each with their own discrete hypotheses). The described experiments were simultaneously performed.

Immunohistochemical null-phenotype for mismatch repair proteins in colonic carcinoma associated with concurrent MLH1 hypermethylation and MSH2 somatic mutations.

PubMed

Wang, Tao; Stadler, Zsofia K; Zhang, Liying; Weiser, Martin R; Basturk, Olca; Hechtman, Jaclyn F; Vakiani, Efsevia; Saltz, Lenard B; Klimstra, David S; Shia, Jinru

2018-04-01

Microsatellite instability, a well-established driver pathway in colorectal carcinogenesis, can develop in both sporadic and hereditary conditions via different molecular alterations in the DNA mismatch repair (MMR) genes. MMR protein immunohistochemistry (IHC) is currently widely used for the detection of MMR deficiency in solid tumors. The IHC test, however, can show varied staining patterns, posing challenges in the interpretation of the staining results in some cases. Here we report a case of an 80-year-old female with a colonic adenocarcinoma that exhibited an unusual "null" IHC staining pattern with complete loss of all four MMR proteins (MLH1, MSH2, MSH6, and PMS2). This led to subsequent MLH1 methylation testing and next generation sequencing which demonstrated that the loss of all MMR proteins was associated with concurrent promoter hypermethylation of MLH1 and double somatic truncating mutations in MSH2. These molecular findings, in conjunction with the patient's age being 80 years and the fact that the patient had no personal or family cancer history, indicated that the MMR deficiency was highly likely sporadic in nature. Thus, the stringent Lynch syndrome type surveillance programs were not recommended to the patient and her family members. This case illustrates a rare but important scenario where a null IHC phenotype signifies complex underlying molecular alternations that bear clinical management implications, highlighting the need for recognition and awareness of such unusual IHC staining patterns.

FANCJ localization by mismatch repair is vital to maintain genomic integrity after UV irradiation.

PubMed

Guillemette, Shawna; Branagan, Amy; Peng, Min; Dhruva, Aashana; Schärer, Orlando D; Cantor, Sharon B

2014-02-01

Nucleotide excision repair (NER) is critical for the repair of DNA lesions induced by UV radiation, but its contribution in replicating cells is less clear. Here, we show that dual incision by NER endonucleases, including XPF and XPG, promotes the S-phase accumulation of the BRCA1 and Fanconi anemia-associated DNA helicase FANCJ to sites of UV-induced damage. FANCJ promotes replication protein A phosphorylation and the arrest of DNA synthesis following UV irradiation. Interaction defective mutants of FANCJ reveal that BRCA1 binding is not required for FANCJ localization, whereas interaction with the mismatch repair (MMR) protein MLH1 is essential. Correspondingly, we find that FANCJ, its direct interaction with MLH1, and the MMR protein MSH2 function in a common pathway in response to UV irradiation. FANCJ-deficient cells are not sensitive to killing by UV irradiation, yet we find that DNA mutations are significantly enhanced. Thus, we considered that FANCJ deficiency could be associated with skin cancer. Along these lines, in melanoma we found several somatic mutations in FANCJ, some of which were previously identified in hereditary breast cancer and Fanconi anemia. Given that, mutations in XPF can also lead to Fanconi anemia, we propose collaborations between Fanconi anemia, NER, and MMR are necessary to initiate checkpoint activation in replicating human cells to limit genomic instability.

The Molecular Origin of the MMR-dependent Apoptosis Pathway from Dynamics Analysis of MutSα-DNA Complexes

PubMed Central

Negureanu, Lacramioara; Salsbury, Freddie R.

2012-01-01

The cellular response to DNA damage signaling by MMR proteins is incompletely understood. It is generally accepted that MMR-dependent apoptosis pathway in response to DNA damage detection is independent of MMR's DNA repair function. In this study we investigate correlated motions in response to the binding of mismatched and PCL DNA fragments by MutSα, as derived from 50 ns molecular dynamics simulations. The protein dynamics in response to the mismatched and damaged DNA recognition suggests that MutSα signals their recognition through independent pathways providing evidence for the molecular origin of the MMR-dependent apoptosis. MSH2 subunit is indicated to play a key role in signaling both mismatched and damaged DNA recognition; localized and collective motions within the protein allow identifying sites on the MSH2 surface possible involved in recruiting proteins responsible for downstream events. Unlike in the mismatch complex, predicted key communication sites specific for the damage recognition are on the list of known cancer causing mutations or deletions. This confirms MSH2's role in signaling DNA-damage induced apoptosis and suggests that defects in MMR alone is sufficient to trigger tumorigenesis, supporting the experimental evidence that MMR-damage response function could protect from the early occurrence of tumors. Identifying these particular communication sites may have implications for the treatment of cancers that are not defective for MMR, but are unable to function optimally for MMR-dependent responses following DNA damage such as the case of resistance to cisplatin. PMID:22712459

[Expression of DNA mismatch repair protein in endometrial carcinomas and its correlation with clinicopathologic features].

PubMed

Bi, R; Tu, X Y; Xiao, Y X; Shan, B E; Wang, H Y; Cai, X; Zhou, X Y; Yang, W T

2016-05-08

To study the expression of mismatch repair protein in a series of endometrial carcinomas and its correlation with clinicopathologic features. The clinical data of 150 consecutive cases of endometrial carcinoma were collected during the period from December, 2014 to August, 2015 in Fudan University Cancer Center. Morphologic features including tumor infiltrating lymphocytes (TIL), peritumoral lymphocytes and tumor heterogeneity were reviewed. Immunohistochemistry for expression of mismatch repair proteins was performed. The correlation with clinicopathologic features was analyzed. Loss of mismatch repair protein expression was observed in 43 cases (28.7%), including loss of MLH1/PMS2 in 27 cases (18%), loss of MSH2/MSH6 in 7 cases (4.7%), loss of MSH6 in 6 cases (4%) and loss of PMS2 in 3 cases (2%). There were 23.3% and 27.1% of mismatch repair protein-deficient endometrial carcinomas in women under and above 50 years of age, respectively, which was not statistically significant. Amongst the 12 cases with family history of tumors, 4 of the 6 mismatch repair protein-deficient cases were under 50 years of age, which was higher than that in the 6 cases with mismatch repair protein expression (P=0.014). The mismatch repair protein-deficient group showed significantly more prominent TIL and peritumoral lymphocytes than protein-expression group (P=0.033 and <0.001). Moreover, there were also significant differences in depth of myometrial invasion and occurrence of synchronous malignancy (2 cases of ovarian clear cell carcinoma and 1 case of colonic carcinoma) between the two groups (P=0.039 and 0.022). However, there were no significant differences in lymph node metastasis, tumor heterogeneity, lower uterine segment involvement and tumor stage between the two groups. Prominent TIL and peritumoral lymphocytes characteristically occur in mismatch repair protein-deficient endometrial carcinomas. Patient age does not significantly correlate with the loss of mismatch repair

Upper tract urothelial carcinomas: frequency of association with mismatch repair protein loss and lynch syndrome.

PubMed

Harper, Holly L; McKenney, Jesse K; Heald, Brandie; Stephenson, Andrew; Campbell, Steven C; Plesec, Thomas; Magi-Galluzzi, Cristina

2017-01-01

Increased risk for upper tract urothelial carcinoma is described in patients with Lynch syndrome, caused by germline mutations in mismatch repair genes. We aimed to identify the frequency of mismatch repair protein loss in upper tract urothelial carcinoma and its potential for identifying an association with Lynch syndrome. We queried our database to identify upper tract urothelial carcinomas. Patients were cross-referenced for history of colorectal carcinoma or other common Lynch syndrome-associated neoplasms to enrich for potential Lynch syndrome cases. Tumor histopathologic characteristics were reviewed and each case was analyzed for loss of mismatch repair proteins, MLH1, MSH2, MSH6, and PMS2, by immunohistochemistry. Of 444 patients with upper tract urothelial carcinoma, a subset of 215 (encompassing 30 with upper tract urothelial carcinoma and another common Lynch syndrome-associated neoplasm) was analyzed for loss of mismatch repair protein expression. Of 30 patients with Lynch syndrome-associated neoplasms, six had documented Lynch syndrome, including two with Muir-Torre syndrome. Mismatch repair protein loss was identified in 7% of total upper tract urothelial carcinomas and 30% of patients with Lynch syndrome-associated neoplasms (including all patients with Lynch syndrome/Muir-Torre syndrome). Of patients without history of Lynch syndrome-associated neoplasms, 5 of 184 (2.7%) had loss of mismatch repair protein expression. Twelve cases with mismatch repair protein loss demonstrated loss of MSH2 and MSH6, and 2 had isolated loss of MSH6. MLH1 and PMS2 expression were consistently retained. Although increased intratumoral lymphocytes, inverted growth, pushing tumor-stromal interface, and lack of nuclear pleomorphism were more commonly seen in cases with mismatch repair protein loss, only intratumoral lymphocytes and presence of pushing borders were statistically significant. MLH1 and PMS2 testing appear to have little utility in upper tract urothelial

Mismatch repair gene MSH3 polymorphism is associated with the risk of sporadic prostate cancer

PubMed Central

Hirata, Hiroshi; Hinoda, Yuji; Kawamoto, Ken; Kikuno, Nobuyuki; Suehiro, Yutaka; Okayama, Naoko; Tanaka, Yuichiro; Dahiya, Rajvir

2014-01-01

Purpose The mismatch repair (MMR) system is a DNA repair mechanism that corrects mispaired bases during DNA replication errors. Cancer cells deficient in the MMR proteins have a 102 –103-fold increase in the mutation rate. Single nucleotide polymorphisms (SNPs) of MMR genes have been shown to cause a reduction in DNA repair activity. We hypothesized that mismatch repair gene polymorphism could be a risk factor for prostate cancer (PC) and that p53 Pro/Pro genotype carriers could influence MSH3 and MSH6 polymorphisms. Material and Methods DNA samples from 110 cases of prostate cancer and healthy controls (n=110) were analyzed by SSCP and PCR-RFLP to determine the genotypic frequency of five different polymorphic loci on two MMR genes (MSH3 and MSH6) and p53 codon72. The chi-square test was applied to compare the genotype frequency between patients and controls. Results A significant increase in the G/A+A/A genotype of MSH3 Pro222Pro was observed in patients compared to controls (OR, 1.87; 95% CI, 1.0–3.5). The frequency of A/G + G/G genotypes of MSH3 exon23 Thr1036Ala also tended to increase in patients (OR, 1.57; 95% CI, 0.92–2.72). Among p53 codon72 Arg/Pro + Pro/Pro carriers, the frequency of the AG + GG genotype of MSH3 exon23 was significantly increased in patients compared to controls (OR = 2.1, 95% CI; 1.05–4.34). Conclusion This is the first report on the association of MSH3 gene polymorphisms in prostate cancer. These results suggest that the MSH3 polymorphism may be a risk factor for prostate cancer. PMID:18355840

Mismatch repair deficiency commonly precedes adenoma formation in Lynch Syndrome-Associated colorectal tumorigenesis.

PubMed

Sekine, Shigeki; Mori, Taisuke; Ogawa, Reiko; Tanaka, Masahiro; Yoshida, Hiroshi; Taniguchi, Hirokazu; Nakajima, Takeshi; Sugano, Kokichi; Yoshida, Teruhiko; Kato, Mamoru; Furukawa, Eisaku; Ochiai, Atsushi; Hiraoka, Nobuyoshi

2017-08-01

Lynch syndrome is a cancer predisposition syndrome caused by germline mutations in mismatch repair (MMR) genes. MMR deficiency is a ubiquitous feature of Lynch syndrome-associated colorectal adenocarcinomas; however, it remains unclear when the MMR-deficient phenotype is acquired during tumorigenesis. To probe this issue, the present study examined genetic alterations and MMR statuses in Lynch syndrome-associated colorectal adenomas and adenocarcinomas, in comparison with sporadic adenomas. Among the Lynch syndrome-associated colorectal tumors, 68 of 86 adenomas (79%) and all adenocarcinomas were MMR-deficient, whereas all the sporadic adenomas were MMR-proficient, as determined by microsatellite instability testing and immunohistochemistry for MMR proteins. Sequencing analyses identified APC or CTNNB1 mutations in the majority of sporadic adenomas (58/84, 69%) and MMR-proficient Lynch syndrome-associated adenomas (13/18, 72%). However, MMR-deficient Lynch syndrome-associated adenomas had less APC or CTNNB1 mutations (25/68, 37%) and frequent frameshift RNF43 mutations involving mononucleotide repeats (45/68, 66%). Furthermore, frameshift mutations affecting repeat sequences constituted 14 of 26 APC mutations (54%) in MMR-deficient adenomas whereas these frameshift mutations were rare in MMR-proficient adenomas in patients with Lynch syndrome (1/12, 8%) and in sporadic adenomas (3/52, 6%). Lynch syndrome-associated adenocarcinomas exhibited mutation profiles similar to those of MMR-deficient adenomas. Considering that WNT pathway activation sufficiently drives colorectal adenoma formation, the distinct mutation profiles of WNT pathway genes in Lynch syndrome-associated adenomas suggest that MMR deficiency commonly precedes adenoma formation.

Use of Single-Cysteine Variants for Trapping Transient States in DNA Mismatch Repair.

PubMed

Friedhoff, Peter; Manelyte, Laura; Giron-Monzon, Luis; Winkler, Ines; Groothuizen, Flora S; Sixma, Titia K

2017-01-01

DNA mismatch repair (MMR) is necessary to prevent incorporation of polymerase errors into the newly synthesized DNA strand, as they would be mutagenic. In humans, errors in MMR cause a predisposition to cancer, called Lynch syndrome. The MMR process is performed by a set of ATPases that transmit, validate, and couple information to identify which DNA strand requires repair. To understand the individual steps in the repair process, it is useful to be able to study these large molecular machines structurally and functionally. However, the steps and states are highly transient; therefore, the methods to capture and enrich them are essential. Here, we describe how single-cysteine variants can be used for specific cross-linking and labeling approaches that allow trapping of relevant transient states. Analysis of these defined states in functional and structural studies is instrumental to elucidate the molecular mechanism of this important DNA MMR process. © 2017 Elsevier Inc. All rights reserved.

Mismatch repair proteins recruited to ultraviolet light-damaged sites lead to degradation of licensing factor Cdt1 in the G1 phase.

PubMed

Tanaka, Miyuki; Takahara, Michiyo; Nukina, Kohei; Hayashi, Akiyo; Sakai, Wataru; Sugasawa, Kaoru; Shiomi, Yasushi; Nishitani, Hideo

2017-04-03

Cdt1 is rapidly degraded by CRL4 Cdt2 E3 ubiquitin ligase after UV (UV) irradiation. Previous reports revealed that the nucleotide excision repair (NER) pathway is responsible for the rapid Cdt1-proteolysis. Here, we show that mismatch repair (MMR) proteins are also involved in the degradation of Cdt1 after UV irradiation in the G1 phase. First, compared with the rapid (within ∼15 min) degradation of Cdt1 in normal fibroblasts, Cdt1 remained stable for ∼30 min in NER-deficient XP-A cells, but was degraded within ∼60 min. The delayed degradation was also dependent on PCNA and CRL4 Cdt2 . The MMR proteins Msh2 and Msh6 were recruited to the UV-damaged sites of XP-A cells in the G1 phase. Depletion of these factors with small interfering RNAs prevented Cdt1 degradation in XP-A cells. Similar to the findings in XP-A cells, depletion of XPA delayed Cdt1 degradation in normal fibroblasts and U2OS cells, and co-depletion of Msh6 further prevented Cdt1 degradation. Furthermore, depletion of Msh6 alone delayed Cdt1 degradation in both cell types. When Cdt1 degradation was attenuated by high Cdt1 expression, repair synthesis at the damaged sites was inhibited. Our findings demonstrate that UV irradiation induces multiple repair pathways that activate CRL4 Cdt2 to degrade its target proteins in the G1 phase of the cell cycle, leading to efficient repair of DNA damage.

DNA Mismatch Repair Deficiency Promotes Genomic Instability in a Subset of Papillary Thyroid Cancers.

PubMed

Javid, Mahsa; Sasanakietkul, Thanyawat; Nicolson, Norman G; Gibson, Courtney E; Callender, Glenda G; Korah, Reju; Carling, Tobias

2018-02-01

Efficient DNA damage repair by MutL-homolog DNA mismatch repair (MMR) enzymes, MLH1, MLH3, PMS1 and PMS2, are required to maintain thyrocyte genomic integrity. We hypothesized that persistent oxidative stress and consequent transcriptional dysregulation observed in thyroid follicles will lead to MMR deficiency and potentiate papillary thyroid tumorigenesis. MMR gene expression was analyzed by targeted microarray in 18 papillary thyroid cancer (PTC), 9 paracarcinoma normal thyroid (PCNT) and 10 normal thyroid (NT) samples. The findings were validated by qRT-PCR, and in follicular thyroid cancers (FTC) and follicular thyroid adenomas (FTA) for comparison. FOXO transcription factor expression was also analyzed. Protein expression was assessed by immunohistochemistry. Genomic integrity was evaluated by whole-exome sequencing-derived read-depth analysis and Mann-Whitney U test. Clinical correlations were assessed using Fisher's exact and t tests. Microarray and qRT-PCR revealed reduced expression of all four MMR genes in PTC compared with PCNT and of PMS2 compared with NT. FTC and FTA showed upregulation in MLH1, MLH3 and PMS2. PMS2 protein expression correlated with the mRNA expression pattern. FOXO1 showed lower expression in PMS2-deficient PTCs (log2-fold change -1.72 vs. -0.55, U = 11, p < 0.05 two-tailed). Rate of LOH, a measure of genomic instability, was higher in PMS2-deficient PTCs (median 3 and 1, respectively; U = 26, p < 0.05 two-tailed). No correlation was noted between MMR deficiency and clinical characteristics. MMR deficiency, potentially promoted by FOXO1 suppression, may explain the etiology for PTC development in some patients. FTC and FTA retain MMR activity and are likely caused by a different tumorigenic pathway.

E. coli mismatch repair enhances AT-to-GC mutagenesis caused by alkylating agents.

PubMed

Nakano, Kota; Yamada, Yoko; Takahashi, Eizo; Arimoto, Sakae; Okamoto, Keinosuke; Negishi, Kazuo; Negishi, Tomoe

2017-03-01

Alkylating agents are known to induce the formation of O 6 -alkylguanine (O 6 -alkG) and O 4 -alkylthymine (O 4 -alkT) in DNA. These lesions have been widely investigated as major sources of mutations. We previously showed that mismatch repair (MMR) facilitates the suppression of GC-to-AT mutations caused by O 6 -methylguanine more efficiently than the suppression of GC-to-AT mutations caused by O 6 -ethylguanine. However, the manner by which O 4 -alkyT lesions are repaired remains unclear. In the present study, we investigated the repair pathway involved in the repair of O 4 -alkT. The E. coli CC106 strain, which harbors Δprolac in its genomic DNA and carries the F'CC106 episome, can be used to detect AT-to-GC reverse-mutation of the gene encoding β-galactosidase. Such AT-to-GC mutations should be induced through the formation of O 4 -alkT at AT base pairs. As expected, an O 6 -alkylguanine-DNA alkyltransferase (AGT) -deficient CC106 strain, which is defective in both ada and agt genes, exhibited elevated mutant frequencies in the presence of methylating agents and ethylating agents. However, in the UvrA-deficient strain, the methylating agents were less mutagenic than in wild-type, while ethylating agents were more mutagenic than in wild-type, as observed with agents that induce O 6 -alkylguanine modifications. Unexpectedly, the mutant frequencies decreased in a MutS-deficient strain, and a similar tendency was observed in MutL- or MutH-deficient strains. Thus, MMR appears to promote mutation at AT base pairs. Similar results were obtained in experiments employing double-mutant strains harboring defects in both MMR and AGT, or MMR and NER. E. coli MMR enhances AT-to-GC mutagenesis, such as that caused by O 4 -alkylthymine. We hypothesize that the MutS protein recognizes the O 4 -alkT:A base pair more efficiently than O 4 -alkT:G. Such a distinction would result in misincorporation of G at the O 4 -alkT site, followed by higher mutation frequencies in wild

Tumor mismatch repair immunohistochemistry and DNA MLH1 methylation testing of patients with endometrial cancer diagnosed at age younger than 60 years optimizes triage for population-level germline mismatch repair gene mutation testing.

PubMed

Buchanan, Daniel D; Tan, Yen Y; Walsh, Michael D; Clendenning, Mark; Metcalf, Alexander M; Ferguson, Kaltin; Arnold, Sven T; Thompson, Bryony A; Lose, Felicity A; Parsons, Michael T; Walters, Rhiannon J; Pearson, Sally-Ann; Cummings, Margaret; Oehler, Martin K; Blomfield, Penelope B; Quinn, Michael A; Kirk, Judy A; Stewart, Colin J; Obermair, Andreas; Young, Joanne P; Webb, Penelope M; Spurdle, Amanda B

2014-01-10

Clinicopathologic data from a population-based endometrial cancer cohort, unselected for age or family history, were analyzed to determine the optimal scheme for identification of patients with germline mismatch repair (MMR) gene mutations. Endometrial cancers from 702 patients recruited into the Australian National Endometrial Cancer Study (ANECS) were tested for MMR protein expression using immunohistochemistry (IHC) and for MLH1 gene promoter methylation in MLH1-deficient cases. MMR mutation testing was performed on germline DNA of patients with MMR-protein deficient tumors. Prediction of germline mutation status was compared for combinations of tumor characteristics, age at diagnosis, and various clinical criteria (Amsterdam, Bethesda, Society of Gynecologic Oncology, ANECS). Tumor MMR-protein deficiency was detected in 170 (24%) of 702 cases. Germline testing of 158 MMR-deficient cases identified 22 truncating mutations (3% of all cases) and four unclassified variants. Tumor MLH1 methylation was detected in 99 (89%) of 111 cases demonstrating MLH1/PMS2 IHC loss; all were germline MLH1 mutation negative. A combination of MMR IHC plus MLH1 methylation testing in women younger than 60 years of age at diagnosis provided the highest positive predictive value for the identification of mutation carriers at 46% versus ≤ 41% for any other criteria considered. Population-level identification of patients with MMR mutation-positive endometrial cancer is optimized by stepwise testing for tumor MMR IHC loss in patients younger than 60 years, tumor MLH1 methylation in individuals with MLH1 IHC loss, and germline mutations in patients exhibiting loss of MSH6, MSH2, or PMS2 or loss of MLH1/PMS2 with absence of MLH1 methylation.

Binding of insertion/deletion DNA mismatches by the heterodimer of yeast mismatch repair proteins MSH2 and MSH3.

PubMed

Habraken, Y; Sung, P; Prakash, L; Prakash, S

1996-09-01

DNA-mismatch repair removes mismatches from the newly replicated DNA strand. In humans, mutations in the mismatch repair genes hMSH2, hMLH1, hPMS1 and hPMS2 result in hereditary non-polyposis colorectal cancer (HNPCC) [1-8]. The hMSH2 (MSH for MutS homologue) protein forms a complex with a 160 kDa protein, and this heterodimer, hMutSalpha, has high affinity for a G/T mismatch [9,10]. Cell lines in which the 160 kDa subunit of hMutSalpha is mutated are specifically defective in the repair of base-base and single-nucleotide insertion/deletion mismatches [9,11]. Genetic studies in S. cerevisiae have suggested that MSH2 functions with either MSH3 or MSH6 in mismatch repair, and, in the absence of the latter two genes, MSH2 is inactive [12,13]. MSH6 encodes the yeast counterpart of the 160 kDa subunit of hMutSalpha [12,13]. As in humans, yeast MSH6 forms a complex with MSH2, and the MSH2-MSH6 heterodimer binds a G/T mismatch [14]. Here, we find that MSH2 and MSH3 form another stable heterodimer, and we purify this heterodimer to near homogeneity. We show that MSH2-MSH3 has low affinity for a G/T mismatch but binds to insertion/deletion mismatches with high specificity, unlike MSH2-MSH6.

Immunotherapy holds the key to cancer treatment and prevention in constitutional mismatch repair deficiency (CMMRD) syndrome.

PubMed

Westdorp, Harm; Kolders, Sigrid; Hoogerbrugge, Nicoline; de Vries, I Jolanda M; Jongmans, Marjolijn C J; Schreibelt, Gerty

2017-09-10

Monoallelic germline mutations in one of the DNA mismatch repair (MMR) genes cause Lynch syndrome, with a high lifetime risks of colorectal and endometrial cancer at adult age. Less well known, is the constitutional mismatch repair deficiency (CMMRD) syndrome caused by biallelic germline mutations in MMR genes. This syndrome is characterized by the development of childhood cancer. Patients with CMMRD are at extremely high risk of developing multiple cancers including hematological, brain and intestinal tumors. Mutations in MMR genes impair DNA repair and therefore most tumors of patients with CMMRD are hypermutated. These mutations lead to changes in the translational reading frame, which consequently result in neoantigen formation. Neoantigens are recognized as foreign by the immune system and can induce specific immune responses. The growing evidence on the clinical efficacy of immunotherapies, such as immune checkpoint inhibitors, offers the prospect for treatment of patients with CMMRD. Combining neoantigen-based vaccination strategies and immune checkpoint inhibitors could be an effective way to conquer CMMRD-related tumors. Neoantigen-based vaccines might also be a preventive treatment option in healthy biallelic MMR mutation carriers. Future studies need to reveal the safety and efficacy of immunotherapies for patients with CMMRD. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

Evaluating Mismatch Repair Deficiency in Pancreatic Adenocarcinoma: Challenges and Recommendations.

PubMed

Hu, Zishuo I; Shia, Jinru; Stadler, Zsofia K; Varghese, Anna M; Capanu, Marinela; Salo-Mullen, Erin; Lowery, Maeve A; Diaz, Luis A; Mandelker, Diana; Yu, Kenneth H; Zervoudakis, Alice; Kelsen, David P; Iacobuzio-Donahue, Christine A; Klimstra, David S; Saltz, Leonard B; Sahin, Ibrahim H; O'Reilly, Eileen M

2018-03-15

Purpose: Immune checkpoint inhibition has been shown to generate profound and durable responses in mismatch repair deficient (MMR-D) solid tumors and has elicited interest in detection tools and strategies to guide therapeutic decision-making. Herein we address questions on the appropriate screening, detection methods, patient selection, and initiation of therapy for MMR-D pancreatic ductal adenocarcinoma (PDAC) and assess the utility of next-generation sequencing (NGS) in providing additional prognostic and predictive information for MMR-D PDAC. Experimental Design: Archival and prospectively acquired samples and matched normal DNA from N = 833 PDAC cases were analyzed using a hybridization capture-based, NGS assay designed to perform targeted deep sequencing of all exons and selected introns of 341 to 468 cancer-associated genes. A computational program using NGS data derived the MSI status from the tumor-normal paired genome sequencing data. Available germline testing, IHC, and microsatellite instability (MSI) PCR results were reviewed to assess and confirm MMR-D and MSI status. Results: MMR-D in PDAC is a rare event among PDAC patients (7/833), occurring at a frequency of 0.8%. Loss of MMR protein expression by IHC, high mutational load, and elevated MSIsensor scores were correlated with MMR-D PDAC. All 7 MMR-D PDAC patients in the study were found to have Lynch syndrome. Four (57%) of the MMR-D patients treated with immune checkpoint blockade had treatment benefit (1 complete response, 2 partial responses, 1 stable disease). Conclusions: An integrated approach of germline testing and somatic analyses of tumor tissues in advanced PDAC using NGS may help guide future development of immune and molecularly directed therapies in PDAC patients. Clin Cancer Res; 24(6); 1326-36. ©2018 AACR . ©2018 American Association for Cancer Research.

Mismatch repair gene MSH3 polymorphism is associated with the risk of sporadic prostate cancer.

PubMed

Hirata, Hiroshi; Hinoda, Yuji; Kawamoto, Ken; Kikuno, Nobuyuki; Suehiro, Yutaka; Okayama, Naoko; Tanaka, Yuichiro; Dahiya, Rajvir

2008-05-01

The mismatch repair system is a DNA repair mechanism that corrects mispaired bases during DNA replication errors. Cancer cells deficient in MMR proteins have a 10(2) to 10(3)-fold increase in the mutation rate. Single nucleotide polymorphisms of mismatch repair genes have been shown to cause a decrease in DNA repair activity. We hypothesized that mismatch repair gene polymorphism could be a risk factor for prostate cancer and p53 Pro/Pro genotype carriers could influence MSH3 and MSH6 polymorphisms. DNA samples from 110 patients with prostate cancer and 110 healthy controls were analyzed by single strand conformational polymorphism and polymerase chain reaction-restriction fragment length polymorphism to determine the genotypic frequency of 5 polymorphic loci on 2 MMR genes (MSH3 and MSH6) and p53 codon72. The chi-square test was applied to compare genotype frequency between patients and controls. A significant increase in the G/A+A/A genotype of MSH3 Pro222Pro was observed in patients compared to controls (OR 1.87, 95% CI 1.0-3.5). The frequency of A/G + G/G genotypes of MSH3 exon23 Thr1036Ala also tended to increase in patients (OR 1.57, 95% CI 0.92-2.72). In p53 codon72 Arg/Pro + Pro/Pro carriers the frequency of the AG + GG genotype of MSH3 exon23 was significantly increased in patients compared to controls (OR 2.1, 95% CI 1.05-4.34). To our knowledge this is the first report of the association of MSH3 gene polymorphisms in prostate cancer. These results suggest that the MSH3 polymorphism may be a risk factor for prostate cancer.

Activation of Saccharomyces cerevisiae Mlh1-Pms1 Endonuclease in a Reconstituted Mismatch Repair System.

PubMed

Smith, Catherine E; Bowen, Nikki; Graham, William J; Goellner, Eva M; Srivatsan, Anjana; Kolodner, Richard D

2015-08-28

Previous studies reported the reconstitution of an Mlh1-Pms1-independent 5' nick-directed mismatch repair (MMR) reaction using Saccharomyces cerevisiae proteins. Here we describe the reconstitution of a mispair-dependent Mlh1-Pms1 endonuclease activation reaction requiring Msh2-Msh6 (or Msh2-Msh3), proliferating cell nuclear antigen (PCNA), and replication factor C (RFC) and a reconstituted Mlh1-Pms1-dependent 3' nick-directed MMR reaction requiring Msh2-Msh6 (or Msh2-Msh3), exonuclease 1 (Exo1), replication protein A (RPA), RFC, PCNA, and DNA polymerase δ. Both reactions required Mg(2+) and Mn(2+) for optimal activity. The MMR reaction also required two reaction stages in which the first stage required incubation of Mlh1-Pms1 with substrate DNA, with or without Msh2-Msh6 (or Msh2-Msh3), PCNA, and RFC but did not require nicking of the substrate, followed by a second stage in which other proteins were added. Analysis of different mutant proteins demonstrated that both reactions required a functional Mlh1-Pms1 endonuclease active site, as well as mispair recognition and Mlh1-Pms1 recruitment by Msh2-Msh6 but not sliding clamp formation. Mutant Mlh1-Pms1 and PCNA proteins that were defective for Exo1-independent but not Exo1-dependent MMR in vivo were partially defective in the Mlh1-Pms1 endonuclease and MMR reactions, suggesting that both reactions reflect the activation of Mlh1-Pms1 seen in Exo1-independent MMR in vivo. The availability of this reconstituted MMR reaction should now make it possible to better study both Exo1-independent and Exo1-dependent MMR. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Activation of Saccharomyces cerevisiae Mlh1-Pms1 Endonuclease in a Reconstituted Mismatch Repair System*

PubMed Central

Smith, Catherine E.; Bowen, Nikki; Graham, William J.; Goellner, Eva M.; Srivatsan, Anjana; Kolodner, Richard D.

2015-01-01

Previous studies reported the reconstitution of an Mlh1-Pms1-independent 5′ nick-directed mismatch repair (MMR) reaction using Saccharomyces cerevisiae proteins. Here we describe the reconstitution of a mispair-dependent Mlh1-Pms1 endonuclease activation reaction requiring Msh2-Msh6 (or Msh2-Msh3), proliferating cell nuclear antigen (PCNA), and replication factor C (RFC) and a reconstituted Mlh1-Pms1-dependent 3′ nick-directed MMR reaction requiring Msh2-Msh6 (or Msh2-Msh3), exonuclease 1 (Exo1), replication protein A (RPA), RFC, PCNA, and DNA polymerase δ. Both reactions required Mg2+ and Mn2+ for optimal activity. The MMR reaction also required two reaction stages in which the first stage required incubation of Mlh1-Pms1 with substrate DNA, with or without Msh2-Msh6 (or Msh2-Msh3), PCNA, and RFC but did not require nicking of the substrate, followed by a second stage in which other proteins were added. Analysis of different mutant proteins demonstrated that both reactions required a functional Mlh1-Pms1 endonuclease active site, as well as mispair recognition and Mlh1-Pms1 recruitment by Msh2-Msh6 but not sliding clamp formation. Mutant Mlh1-Pms1 and PCNA proteins that were defective for Exo1-independent but not Exo1-dependent MMR in vivo were partially defective in the Mlh1-Pms1 endonuclease and MMR reactions, suggesting that both reactions reflect the activation of Mlh1-Pms1 seen in Exo1-independent MMR in vivo. The availability of this reconstituted MMR reaction should now make it possible to better study both Exo1-independent and Exo1-dependent MMR. PMID:26170454

Mismatch repair mRNA and protein expression in intestinal adenocarcinoma in sika deer (Cervus nippon) resembling heritable non-polyposis colorectal cancer in man.

PubMed

Jahns, H; Browne, J A

2015-01-01

Intestinal adenocarcinomas seen in an inbred herd of farmed sika deer (Cervus nippon) morphologically resembled human hereditary non-polyposis colorectal cancer (HNPCC). Features common to both included multiple de novo sites of tumourigenesis in the proximal colon, sessile and non-polyposis mucosal changes, the frequent finding of mucinous type adenocarcinoma, lymphocyte infiltration into the neoplastic tubules and Crohn's-like lymphoid follicles at the deep margin of the tumour. HNPCC is defined by a germline mutation of mismatch repair (MMR) genes resulting in their inactivation and loss of expression. To test the hypothesis that similar MMR gene inactivation occurs in the deer tumours, the expression of the four most important MMR genes, MSH2, MLH1, MSH6 and PMS2, was examined at the mRNA level by reverse transcriptase polymerase chain reaction (n = 12) and at the protein level by immunohistochemistry (n = 40) in tumour and control tissues. All four genes were expressed equally in normal and neoplastic tissues, so MMR gene inactivation could not be implicated in the carcinogenesis of this tumour in sika deer. Copyright © 2014 Elsevier Ltd. All rights reserved.

Clinicopathologic implications of DNA mismatch repair status in endometrial carcinomas.

PubMed

Shikama, Ayumi; Minaguchi, Takeo; Matsumoto, Koji; Akiyama-Abe, Azusa; Nakamura, Yuko; Michikami, Hiroo; Nakao, Sari; Sakurai, Manabu; Ochi, Hiroyuki; Onuki, Mamiko; Satoh, Toyomi; Oki, Akinori; Yoshikawa, Hiroyuki

2016-02-01

Endometrial carcinoma is the most common malignancy in women with Lynch syndrome caused by mismatch repair (MMR) deficiency. We investigated the clinicopathologic significance of deficient MMR and Lynch syndrome presumed by MMR analyses in unselected endometrial carcinomas. We analyzed immunohistochemistry of MMR proteins (MLH1/MSH2/MSH6/PMS2) and MLH1 promoter methylation in primary endometrial carcinomas from 221 consecutive patients. Based on these results, tumors were categorized as sporadic or probable Lynch syndrome (PLS). Clinicopathologic variables and prognosis were compared according to MMR status and sporadic/PLS classification. Deficient MMR showed only trends towards favorable overall survival (OS) compared with intact MMR (p=0.13), whereas PLS showed significantly better OS than sporadic (p=0.038). Sporadic was significantly associated with older age, obesity, deep myometrial invasion, and advanced stage (p=0.008, 0.01, 0.02 and 0.03), while PLS was significantly associated with early stage and Lynch syndrome-associated multiple cancer (p=0.04 and 0.001). The trend towards favorable OS of PLS was stronger in advanced stage than in early stage (hazard ratio, 0.044 [95% CI 0-25.6] vs. 0.49 [0.063-3.8]). In the subset receiving adjuvant therapies, PLS showed trends towards favorable disease-free survival compared to sporadic by contrast with patients receiving no adjuvant therapies showing no such trend (hazard ratio, 0.045 [95% CI 0-20.3] vs. 0.81 [0.095-7.0]). The current findings suggest that analyzing MMR status and searching for Lynch syndrome may identify a subset of patients with favorable survival and high sensitivity to adjuvant therapies, providing novel and useful implications for formulating the precision medicine in endometrial carcinoma. Copyright © 2015 Elsevier Inc. All rights reserved.

Mismatch repair deficiency does not enhance ENU mutagenesis in the zebrafish germ line.

PubMed

Feitsma, Harma; de Bruijn, Ewart; van de Belt, Jose; Nijman, Isaac J; Cuppen, Edwin

2008-07-01

S(N)1-type alkylating agents such as N-ethyl-N-nitrosourea (ENU) are very potent mutagens. They act by transferring their alkyl group to DNA bases, which, upon mispairing during replication, can cause single base pair mutations in the next replication cycle. As DNA mismatch repair (MMR) proteins are involved in the recognition of alkylation damage, we hypothesized that ENU-induced mutation rates could be increased in a MMR-deficient background, which would be beneficial for mutagenesis approaches. We applied a standard ENU mutagenesis protocol to adult zebrafish deficient in the MMR gene msh6 and heterozygous controls to study the effect of MMR on ENU-induced DNA damage. Dose-dependent lethality was found to be similar for homozygous and heterozygous mutants, indicating that there is no difference in ENU resistance. Mutation discovery by high-throughput dideoxy resequencing of genomic targets in outcrossed progeny of the mutagenized fish did also not reveal any differences in germ line mutation frequency. These results may indicate that the maximum mutation load for zebrafish has been reached with the currently used, highly optimized ENU mutagenesis protocol. Alternatively, the MMR system in the zebrafish germ line may be saturated very rapidly, thereby having a limited effect on high-dose ENU mutagenesis.

Dependence of the cytotoxicity of DNA-damaging agents on the mismatch repair status of human cells.

PubMed

Papouli, Efterpi; Cejka, Petr; Jiricny, Josef

2004-05-15

Mismatch repair (MMR) deficiency was reported to increase resistance of mammalian cells to killing by several genotoxic substances. However, although MMR-deficient cells are approximately 100-fold more resistant to killing by S(N)1 type methylating agents than MMR-proficient controls, the sensitivity differences reported for the other agents were typically <2-fold. To test whether these differences were linked to factors other than MMR status, we studied the cytotoxicities of mitomycin C, chloroethylcyclohexyl nitrosourea, melphalan, psoralen-UVA, etoposide, camptothecin, ionizing radiation, and cis-dichlorodiaminoplatinum (cisplatin) in a strictly isogenic system. We now report that MMR deficiency reproducibly desensitized cells solely to cisplatin.

Modified bases enable high-efficiency oligonucleotide-mediated allelic replacement via mismatch repair evasion

PubMed Central

Wang, Harris H.; Xu, George; Vonner, Ashley J.; Church, George

2011-01-01

Genome engineering using single-stranded oligonucleotides is an efficient method for generating small chromosomal and episomal modifications in a variety of host organisms. The efficiency of this allelic replacement strategy is highly dependent on avoidance of the endogenous mismatch repair (MMR) machinery. However, global MMR inactivation generally results in significant accumulation of undesired background mutations. Here, we present a novel strategy using oligos containing chemically modified bases (2′-Fluoro-Uridine, 5-Methyl-deoxyCytidine, 2,6-Diaminopurine or Iso-deoxyGuanosine) in place of the standard T, C, A or G to avoid mismatch detection and repair, which we tested in Escherichia coli. This strategy increases transient allelic-replacement efficiencies by up to 20-fold, while maintaining a 100-fold lower background mutation level. We further show that the mismatched bases between the full length oligo and the chromosome are often not incorporated at the target site, probably due to nuclease activity at the 5′ and 3′ termini of the oligo. These results further elucidate the mechanism of oligo-mediated allelic replacement (OMAR) and enable improved methodologies for efficient, large-scale engineering of genomes. PMID:21609953

Genetic and clinical determinants of constitutional mismatch repair deficiency syndrome: report from the constitutional mismatch repair deficiency consortium.

PubMed

Bakry, Doua; Aronson, Melyssa; Durno, Carol; Rimawi, Hala; Farah, Roula; Alharbi, Qasim Kholaif; Alharbi, Musa; Shamvil, Ashraf; Ben-Shachar, Shay; Mistry, Matthew; Constantini, Shlomi; Dvir, Rina; Qaddoumi, Ibrahim; Gallinger, Steven; Lerner-Ellis, Jordan; Pollett, Aaron; Stephens, Derek; Kelies, Steve; Chao, Elizabeth; Malkin, David; Bouffet, Eric; Hawkins, Cynthia; Tabori, Uri

2014-03-01

Constitutional mismatch repair deficiency (CMMRD) is a devastating cancer predisposition syndrome for which data regarding clinical manifestations, molecular screening tools and management are limited. We established an international CMMRD consortium and collected comprehensive clinical and genetic data. Molecular diagnosis of tumour and germline biospecimens was performed. A surveillance protocol was developed and implemented. Overall, 22/23 (96%) of children with CMMRD developed 40 different tumours. While childhood CMMRD related tumours were observed in all families, Lynch related tumours in adults were observed in only 2/14 families (p=0.0007). All children with CMMRD had café-au-lait spots and 11/14 came from consanguineous families. Brain tumours were the most common cancers reported (48%) followed by gastrointestinal (32%) and haematological malignancies (15%). Importantly, 12 (30%) of these were low grade and resectable cancers. Tumour immunohistochemistry was 100% sensitive and specific in diagnosing mismatch repair (MMR) deficiency of the corresponding gene while microsatellite instability was neither sensitive nor specific as a diagnostic tool (p<0.0001). Furthermore, screening of normal tissue by immunohistochemistry correlated with genetic confirmation of CMMRD. The surveillance protocol detected 39 lesions which included asymptomatic malignant gliomas and gastrointestinal carcinomas. All tumours were amenable to complete resection and all patients undergoing surveillance are alive. CMMRD is a highly penetrant syndrome where family history of cancer may not be contributory. Screening tumours and normal tissues using immunohistochemistry for abnormal expression of MMR gene products may help in diagnosis and early implementation of surveillance for these children. Copyright © 2013 Elsevier Ltd. All rights reserved.

Relationship among mismatch repair deficiency, CDX2 loss, p53 and E-cadherin in colon carcinoma and suitability of using a double panel of mismatch repair proteins by immunohistochemistry.

PubMed

Sayar, Ilyas; Akbas, Emin Murat; Isik, Arda; Gokce, Aysun; Peker, Kemal; Demirtas, Levent; Gürbüzel, Mehmet

2015-09-01

Biomarkers such as mismatch repair proteins, CDX2, p53, and E-cadherin are blamed for colon cancers, but the relationships of these biomarkers with each other and with pathological risk factors in colon carcinoma are still not clear. The aim of this study was to evaluate the association of these biomarkers with each other by using immunohistochemical staining and to compare their expression with pathological risk factors for colonic adenocarcinoma. We also aimed to study the usability of a double panel of mismatch repair proteins. One hundred and eleven cases with colonic adenocarcinoma were examined. There was a statistically significant relationship between tumor histological differentiation and perineural invasion, vascular invasion, mismatch repair deficiency, p53, CDX2, and E-cadherin (p < 0.05). PMS2 and MSH6 loss covered 100% of cases with mismatch repair deficiency. Mismatch repair deficiency was correlated with CDX2 loss and E-cadherin expression (p < 0.05). It was also observed that cases with PMS2 loss covered all the cases with CDX2 loss. In conclusion, this double panel may be used instead of a quadruple panel for detecting mismatch repair deficiency. Association of CDX2 and PMS2 in the present study is necessary to conduct further genetic and pathological studies focusing on these two markers together.

Efficient engineering of chromosomal ribosome binding site libraries in mismatch repair proficient Escherichia coli.

PubMed

Oesterle, Sabine; Gerngross, Daniel; Schmitt, Steven; Roberts, Tania Michelle; Panke, Sven

2017-09-26

Multiplexed gene expression optimization via modulation of gene translation efficiency through ribosome binding site (RBS) engineering is a valuable approach for optimizing artificial properties in bacteria, ranging from genetic circuits to production pathways. Established algorithms design smart RBS-libraries based on a single partially-degenerate sequence that efficiently samples the entire space of translation initiation rates. However, the sequence space that is accessible when integrating the library by CRISPR/Cas9-based genome editing is severely restricted by DNA mismatch repair (MMR) systems. MMR efficiency depends on the type and length of the mismatch and thus effectively removes potential library members from the pool. Rather than working in MMR-deficient strains, which accumulate off-target mutations, or depending on temporary MMR inactivation, which requires additional steps, we eliminate this limitation by developing a pre-selection rule of genome-library-optimized-sequences (GLOS) that enables introducing large functional diversity into MMR-proficient strains with sequences that are no longer subject to MMR-processing. We implement several GLOS-libraries in Escherichia coli and show that GLOS-libraries indeed retain diversity during genome editing and that such libraries can be used in complex genome editing operations such as concomitant deletions. We argue that this approach allows for stable and efficient fine tuning of chromosomal functions with minimal effort.

Tumor Budding and PDC Grade Are Stage Independent Predictors of Clinical Outcome in Mismatch Repair Deficient Colorectal Cancer.

PubMed

Ryan, Éanna; Khaw, Yi Ling; Creavin, Ben; Geraghty, Robert; Ryan, Elizabeth J; Gibbons, David; Hanly, Ann; Martin, Sean T; O'Connell, P Ronan; Winter, Desmond C; Sheahan, Kieran

2018-01-01

Mismatch repair deficient (dMMR) colorectal cancer (CRC) despite its association with poor histologic grade often has improved prognosis compared with MMR proficient CRC. Tumor budding and poorly differentiated clusters (PDCs) may predict metastatic potential of colorectal adenocarcinoma (CRC). In addition, their assessment may be more reproducible than the evaluation of other histopathologic parameters. Therefore, we wished to determine their potential as prognostic indicators in a cohort of dMMR CRC patients relative to histologic grade. We investigated the predictive value of conventional WHO grade, budding, PDC grade and other histopathologic parameters on the presence of lymph node metastasis (LNM) and clinical outcome in 238 dMMR CRCs. MMR status was determined by immunohistochemistry for the mismatch repair proteins hMLH1, hMSH2, hMSH6, and hPMS2. Tumor budding and PDCs were highly correlated (r=0.701; P<0.000). Both budding and PDC grade were associated with WHO grade, perineural invasion, lympho-vascular invasion, and extramural vascular invasion, and the presence of LNM in dMMR CRC (P<0.009). Independent predictors of LNM were PDC grade (odds ratio, 4.12; 95% confidence interval [CI], 1.69-10.04; P=0.011) and EMVI (odds ratio, 3.81; 95% CI, 1.56-9.19; P<0.000). Only pTstage (hazard ratio [HR], 4.11; 95% CI, 1.48-11.36; P=0.007) and tumor budding (HR, 2.99; 95% CI, 1.72-5.19; P<0.000) were independently associated with worse disease-free survival (DFS). If tumor budding was excluded from the model, PDC grade became significant for DFS (HR, 2.34; 95% CI, 1.34-4.09; P=0.003). WHO Grade does not independently correlate with clinical outcome in dMMR CRC. PDC grade and extramural vascular invasion are independent predictors of LNM. Tumor budding and pTstage are the best predictors of DFS. If tumor budding cannot be assessed, PDC grade may be used as a prognostic surrogate.

Mlh2 Is an Accessory Factor for DNA Mismatch Repair in Saccharomyces cerevisiae

PubMed Central

Srivatsan, Anjana; Bowen, Nikki; Gries, Kerstin; Desai, Arshad; Putnam, Christopher D.; Kolodner, Richard D.

2014-01-01

In Saccharomyces cerevisiae, the essential mismatch repair (MMR) endonuclease Mlh1-Pms1 forms foci promoted by Msh2-Msh6 or Msh2-Msh3 in response to mispaired bases. Here we analyzed the Mlh1-Mlh2 complex, whose role in MMR has been unclear. Mlh1-Mlh2 formed foci that often colocalized with and had a longer lifetime than Mlh1-Pms1 foci. Mlh1-Mlh2 foci were similar to Mlh1-Pms1 foci: they required mispair recognition by Msh2-Msh6, increased in response to increased mispairs or downstream defects in MMR, and formed after induction of DNA damage by phleomycin but not double-stranded breaks by I-SceI. Mlh1-Mlh2 could be recruited to mispair-containing DNA in vitro by either Msh2-Msh6 or Msh2-Msh3. Deletion of MLH2 caused a synergistic increase in mutation rate in combination with deletion of MSH6 or reduced expression of Pms1. Phylogenetic analysis demonstrated that the S. cerevisiae Mlh2 protein and the mammalian PMS1 protein are homologs. These results support a hypothesis that Mlh1-Mlh2 is a non-essential accessory factor that acts to enhance the activity of Mlh1-Pms1. PMID:24811092

Clinicopathological characteristics of patients with upper urinary tract urothelial cancer with loss of immunohistochemical expression of the DNA mismatch repair proteins in universal screening.

PubMed

Urakami, Shinji; Inoshita, Naoko; Oka, Suguru; Miyama, Yu; Nomura, Sachio; Arai, Masami; Sakaguchi, Kazushige; Kurosawa, Kazuhiro; Okaneya, Toshikazu

2018-02-01

To assess the detection rate of putative Lynch syndrome-associated upper urinary tract urothelial cancer among all upper urinary tract urothelial cancers and to examine its clinicopathological characteristics. A total of 143 patients with upper urinary tract urothelial cancer who had received total nephroureterectomy were immunohistochemically stained for the expression of mismatch repair proteins MLH1, PMS2, MSH2 and MSH6. For all suspected mismatch repair-deficient cases, MMR genetic testing was recommended and clinicopathological features were examined. Loss of mismatch repair proteins was found in seven patients (5%) who were thus categorized as putative Lynch syndrome-associated upper urinary tract urothelial cancer. Five of these patients showed dual loss of MSH2/MSH6. Two patients were confirmed to be MSH2 germline mutation carriers. Histologically, all seven tumors were low-grade atypical urothelial carcinoma and showed its unique histological features, such as an inverted papilloma-like growth pattern and a villous to papillary structure with mild stratification of tumor cells. Six tumors had no invasion of the muscularis propria. No recurrence or cancer-related deaths were reported in these seven patients. Just three patients met the revised Amsterdam criteria. This is the first report that universally examined mismatch repair immunohistochemical screening for upper urinary tract urothelial cancers. The prevalence (5%) of putative Lynch syndrome-associated upper urinary tract urothelial cancers is much higher than we had expected. We ascertained that putative Lynch syndrome-associated upper urinary tract urothelial cancers were clinically in the early stage and histologically classified into low-grade malignancy with its characteristic pathological features. The clinicopathological characteristics that we found in the present study could become additional possible markers in the diagnosis of Lynch syndrome-associated upper urinary tract urothelial cancers

Influence of oxidized purine processing on strand directionality of mismatch repair.

PubMed

Repmann, Simone; Olivera-Harris, Maite; Jiricny, Josef

2015-04-17

Replicative DNA polymerases are high fidelity enzymes that misincorporate nucleotides into nascent DNA with a frequency lower than [1/10(5)], and this precision is improved to about [1/10(7)] by their proofreading activity. Because this fidelity is insufficient to replicate most genomes without error, nature evolved postreplicative mismatch repair (MMR), which improves the fidelity of DNA replication by up to 3 orders of magnitude through correcting biosynthetic errors that escaped proofreading. MMR must be able to recognize non-Watson-Crick base pairs and excise the misincorporated nucleotides from the nascent DNA strand, which carries by definition the erroneous genetic information. In eukaryotes, MMR is believed to be directed to the nascent strand by preexisting discontinuities such as gaps between Okazaki fragments in the lagging strand or breaks in the leading strand generated by the mismatch-activated endonuclease of the MutL homologs PMS1 in yeast and PMS2 in vertebrates. We recently demonstrated that the eukaryotic MMR machinery can make use also of strand breaks arising during excision of uracils or ribonucleotides from DNA. We now show that intermediates of MutY homolog-dependent excision of adenines mispaired with 8-oxoguanine (G(O)) also act as MMR initiation sites in extracts of human cells or Xenopus laevis eggs. Unexpectedly, G(O)/C pairs were not processed in these extracts and failed to affect MMR directionality, but extracts supplemented with exogenous 8-oxoguanine DNA glycosylase (OGG1) did so. Because OGG1-mediated excision of G(O) might misdirect MMR to the template strand, our findings suggest that OGG1 activity might be inhibited during MMR. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Combined mismatch repair and POLE/POLD1 defects explain unresolved suspected Lynch syndrome cancers

PubMed Central

Jansen, Anne ML; van Wezel, Tom; van den Akker, Brendy EWM; Ventayol Garcia, Marina; Ruano, Dina; Tops, Carli MJ; Wagner, Anja; Letteboer, Tom GW; Gómez-García, Encarna B; Devilee, Peter; Wijnen, Juul T; Hes, Frederik J; Morreau, Hans

2016-01-01

Many suspected Lynch Syndrome (sLS) patients who lack mismatch repair (MMR) germline gene variants and MLH1 or MSH2 hypermethylation are currently explained by somatic MMR gene variants or, occasionally, by germline POLE variants. To further investigate unexplained sLS patients, we analyzed leukocyte and tumor DNA of 62 sLS patients using gene panel sequencing including the POLE, POLD1 and MMR genes. Forty tumors showed either one, two or more somatic MMR variants predicted to affect function. Nine sLS tumors showed a likely ultramutated phenotype and were found to carry germline (n=2) or somatic variants (n=7) in the POLE/POLD1 exonuclease domain (EDM). Six of these POLE/POLD1-EDM mutated tumors also carried somatic MMR variants. Our findings suggest that faulty proofreading may result in loss of MMR and thereby in microsatellite instability. PMID:26648449

Clinicopathologic Significance of Mismatch Repair Defects in Endometrial Cancer: An NRG Oncology/Gynecologic Oncology Group Study

PubMed Central

McMeekin, D. Scott; Tritchler, David L.; Cohn, David E.; Mutch, David G.; Lankes, Heather A.; Geller, Melissa A.; Powell, Matthew A.; Backes, Floor J.; Landrum, Lisa M.; Zaino, Richard; Broaddus, Russell D.; Ramirez, Nilsa; Gao, Feng; Ali, Shamshad; Darcy, Kathleen M.; Pearl, Michael L.; DiSilvestro, Paul A.; Lele, Shashikant B.

2016-01-01

Purpose The clinicopathologic significance of mismatch repair (MMR) defects in endometrioid endometrial cancer (EEC) has not been definitively established. We undertook tumor typing to classify MMR defects to determine if MMR status is prognostic or predictive. Methods Primary EECs from NRG/GOG0210 patients were assessed for microsatellite instability (MSI), MLH1 methylation, and MMR protein expression. Each tumor was assigned to one of four MMR classes: normal, epigenetic defect, probable mutation (MMR defect not attributable to MLH1 methylation), or MSI-low. The relationships between MMR classes and clinicopathologic variables were assessed using contingency table tests and Cox proportional hazard models. Results A total of 1,024 tumors were assigned to MMR classes. Epigenetic and probable mutations in MMR were significantly associated with higher grade and more frequent lymphovascular space invasion. Epigenetic defects were more common in patients with higher International Federation of Gynecology and Obstetrics stage. Overall, there were no differences in outcomes. Progression-free survival was, however, worse for women whose tumors had epigenetic MMR defects compared with the MMR normal group (hazard ratio, 1.37; P < .05; 95% CI, 1.00 to 1.86). An exploratory analysis of interaction between MMR status and adjuvant therapy showed a trend toward improved progression-free survival for probable MMR mutation cases. Conclusion MMR defects in EECs are associated with a number of well-established poor prognostic indicators. Women with tumors that had MMR defects were likely to have higher-grade cancers and more frequent lymphovascular space invasion. Surprisingly, outcomes in these patients were similar to patients with MMR normal tumors, suggesting that MMR defects may counteract the effects of negative prognostic factors. Altered immune surveillance of MMR-deficient tumors, and other host/tumor interactions, is likely to determine outcomes for patients with MMR

Clinicopathologic Significance of Mismatch Repair Defects in Endometrial Cancer: An NRG Oncology/Gynecologic Oncology Group Study.

PubMed

McMeekin, D Scott; Tritchler, David L; Cohn, David E; Mutch, David G; Lankes, Heather A; Geller, Melissa A; Powell, Matthew A; Backes, Floor J; Landrum, Lisa M; Zaino, Richard; Broaddus, Russell D; Ramirez, Nilsa; Gao, Feng; Ali, Shamshad; Darcy, Kathleen M; Pearl, Michael L; DiSilvestro, Paul A; Lele, Shashikant B; Goodfellow, Paul J

2016-09-01

The clinicopathologic significance of mismatch repair (MMR) defects in endometrioid endometrial cancer (EEC) has not been definitively established. We undertook tumor typing to classify MMR defects to determine if MMR status is prognostic or predictive. Primary EECs from NRG/GOG0210 patients were assessed for microsatellite instability (MSI), MLH1 methylation, and MMR protein expression. Each tumor was assigned to one of four MMR classes: normal, epigenetic defect, probable mutation (MMR defect not attributable to MLH1 methylation), or MSI-low. The relationships between MMR classes and clinicopathologic variables were assessed using contingency table tests and Cox proportional hazard models. A total of 1,024 tumors were assigned to MMR classes. Epigenetic and probable mutations in MMR were significantly associated with higher grade and more frequent lymphovascular space invasion. Epigenetic defects were more common in patients with higher International Federation of Gynecology and Obstetrics stage. Overall, there were no differences in outcomes. Progression-free survival was, however, worse for women whose tumors had epigenetic MMR defects compared with the MMR normal group (hazard ratio, 1.37; P < .05; 95% CI, 1.00 to 1.86). An exploratory analysis of interaction between MMR status and adjuvant therapy showed a trend toward improved progression-free survival for probable MMR mutation cases. MMR defects in EECs are associated with a number of well-established poor prognostic indicators. Women with tumors that had MMR defects were likely to have higher-grade cancers and more frequent lymphovascular space invasion. Surprisingly, outcomes in these patients were similar to patients with MMR normal tumors, suggesting that MMR defects may counteract the effects of negative prognostic factors. Altered immune surveillance of MMR-deficient tumors, and other host/tumor interactions, is likely to determine outcomes for patients with MMR-deficient tumors. © 2016 by American

Microsatellites in the Eukaryotic DNA Mismatch Repair Genes as Modulators of Evolutionary Mutation Rate

NASA Technical Reports Server (NTRS)

Chang, Dong Kyung; Metzgar, David; Wills, Christopher; Boland, C. Richard

2003-01-01

All "minor" components of the human DNA mismatch repair (MMR) system-MSH3, MSH6, PMS2, and the recently discovered MLH3-contain mononucleotide microsatellites in their coding sequences. This intriguing finding contrasts with the situation found in the major components of the DNA MMR system-MSH2 and MLH1-and, in fact, most human genes. Although eukaryotic genomes are rich in microsatellites, non-triplet microsatellites are rare in coding regions. The recurring presence of exonal mononucleotide repeat sequences within a single family of human genes would therefore be considered exceptional.

Mismatch repair status in the prediction of benefit from adjuvant fluorouracil chemotherapy in colorectal cancer

PubMed Central

Jover, R; Zapater, P; Castells, A; Llor, X; Andreu, M; Cubiella, J; Piñol, V; Xicola, R M; Bujanda, L; Reñé, J M; Clofent, J; Bessa, X; Morillas, J D; Nicolás‐Pérez, D; Payá, A; Alenda, C

2006-01-01

Aim Some retrospective studies have shown a lack of benefit of 5‐fluorouracil (5‐FU) adjuvant chemotherapy in patients with mismatch repair (MMR) deficient colorectal cancer. Our aim was to assess if this molecular marker can predict benefit from 5‐FU adjuvant chemotherapy. A second objective was to determine if MMR status influences short term survival. Methods We included 754 patients with a median follow up of 728.5 days (range 1–1097). A total of 260 patients with stage II or III tumours received 5‐FU adjuvant chemotherapy, according to standard clinical criteria and irrespective of their MMR status. A tumour was considered MMR deficient when either BAT‐26 showed instability or there was loss of MLH1 or MSH2 protein expression. Results At the end of the follow up period, 206 patients died and 120 presented with tumour recurrence. Sixty six (8.8%) patients had MMR deficient tumours. There were no significant differences in overall survival (MMR competent 72.1%; MMR deficient 78.8%; p = 0.3) or disease free survival (MMR competent 61.3%; MMR deficient 72.3%; p = 0.08). In patients with stage II and III tumours, benefit from 5‐FU adjuvant chemotherapy was restricted to patients with MMR competent tumours (overall survival: chemotherapy 87.1%; non‐chemotherapy 73.5%; log rank, p = 0.00001). Patients with MMR deficient tumours did not benefit from adjuvant chemotherapy (overall survival: chemotherapy 89.5%; non‐chemotherapy 82.4%; log rank, p = 0.4). Conclusions Benefit from 5‐FU adjuvant chemotherapy depends on the MMR status of tumours in patients with colorectal cancer. 5‐FU adjuvant chemotherapy improves survival in patients with MMR competent tumours but this benefit from chemotherapy cannot be extended to patients with MMR deficient tumours. PMID:16299036

Inactivation of DNA mismatch repair by variants of uncertain significance in the PMS2 gene.

PubMed

Drost, Mark; Koppejan, Hester; de Wind, Niels

2013-11-01

Lynch syndrome (LS) is a common cancer predisposition caused by an inactivating mutation in one of four DNA mismatch repair (MMR) genes. Frequently a variant of uncertain significance (VUS), rather than an obviously pathogenic mutation, is identified in one of these genes. The inability to define pathogenicity of such variants precludes targeted healthcare. Here, we have modified a cell-free assay to test VUS in the MMR gene PMS2 for functional activity. We have analyzed nearly all VUS in PMS2 found thus far and describe loss of MMR activity for five, suggesting the applicability of the assay for diagnosis of LS. © 2013 WILEY PERIODICALS, INC.

Inhibition of colorectal cancer genomic copy number alterations and chromosomal fragile site tumor suppressor FHIT and WWOX deletions by DNA mismatch repair

PubMed Central

Gelincik, Ozkan; Blecua, Pedro; Edelmann, Winfried; Kucherlapati, Raju; Zhou, Kathy; Jasin, Maria; Gümüş, Zeynep H.; Lipkin, Steven M.

2017-01-01

Homologous recombination (HR) enables precise DNA repair after DNA double strand breaks (DSBs) using identical sequence templates, whereas homeologous recombination (HeR) uses only partially homologous sequences. Homeologous recombination introduces mutations through gene conversion and genomic deletions through single-strand annealing (SSA). DNA mismatch repair (MMR) inhibits HeR, but the roles of mammalian MMR MutL homologues (MLH1, PMS2 and MLH3) proteins in HeR suppression are poorly characterized. Here, we demonstrate that mouse embryonic fibroblasts (MEFs) carrying Mlh1, Pms2, and Mlh3 mutations have higher HeR rates, by using 7,863 uniquely mapping paired direct repeat sequences (DRs) in the mouse genome as endogenous gene conversion and SSA reporters. Additionally, when DSBs are induced by gamma-radiation, Mlh1, Pms2 and Mlh3 mutant MEFs have higher DR copy number alterations (CNAs), including DR CNA hotspots previously identified in mouse MMR-deficient colorectal cancer (dMMR CRC). Analysis of The Cancer Genome Atlas CRC data revealed that dMMR CRCs have higher genome-wide DR HeR rates than MMR proficient CRCs, and that dMMR CRCs have deletion hotspots in tumor suppressors FHIT/WWOX at chromosomal fragile sites FRA3B and FRA16D (which have elevated DSB rates) flanked by paired homologous DRs and inverted repeats (IR). Overall, these data provide novel insights into the MMR-dependent HeR inhibition mechanism and its role in tumor suppression. PMID:29069730

Constitutional mismatch repair-deficiency syndrome: have we so far seen only the tip of an iceberg?

PubMed

Wimmer, Katharina; Etzler, Julia

2008-09-01

Heterozygous mutations in one of the mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2 cause the dominant adult cancer syndrome termed Lynch syndrome or hereditary non-polyposis colorectal cancer. During the past 10 years, some 35 reports have delineated the phenotype of patients with biallelic inheritance of mutations in one of these MMR genes. The patients suffer from a condition that is characterised by the development of childhood cancers, mainly haematological malignancies and/or brain tumours, as well as early-onset colorectal cancers. Almost all patients also show signs reminiscent of neurofibromatosis type 1, mainly café au lait spots. Alluding to the underlying mechanism, this condition may be termed as "constitutional mismatch repair-deficiency (CMMR-D) syndrome". To give an overview of the current knowledge and its implications of this recessively inherited cancer syndrome we summarise here the genetic, clinical and pathological findings of the so far 78 reported patients of 46 families suffering from this syndrome.

Involvement of the DNA mismatch repair system in cisplatin sensitivity of testicular germ cell tumours.

PubMed

Rudolph, Christiane; Melau, Cecilie; Nielsen, John E; Vile Jensen, Kristina; Liu, Dekang; Pena-Diaz, Javier; Rajpert-De Meyts, Ewa; Rasmussen, Lene Juel; Jørgensen, Anne

2017-08-01

Testicular germ cell tumours (TGCT) are highly sensitive to cisplatin-based chemotherapy, but patients with tumours containing differentiated teratoma components are less responsive to this treatment. The cisplatin sensitivity in TGCT has previously been linked to the embryonic phenotype in the majority of tumours, although the underlying mechanism largely remains to be elucidated. The aim of this study was to investigate the role of the DNA mismatch repair (MMR) system in the cisplatin sensitivity of TGCT. The expression pattern of key MMR proteins, including MSH2, MSH6, MLH1 and PMS2, were investigated during testis development and in the pathogenesis of TGCT, including germ cell neoplasia in situ (GCNIS). The TGCT-derived cell line NTera2 was differentiated using retinoic acid (10 μM, 6 days) after which MMR protein expression and activity, as well as cisplatin sensitivity, were investigated in both undifferentiated and differentiated cells. Finally, the expression of MSH2 was knocked down by siRNA in NTera2 cells after which the effect on cisplatin sensitivity was examined. MMR proteins were expressed in proliferating cells in the testes, while in malignant germ cells MMR protein expression was found to coincide with the expression of the pluripotency factor OCT4, with no or low expression in the more differentiated yolk sac tumours, choriocarcinomas and teratomas. In differentiated NTera2 cells we found a significantly (p < 0.05) lower expression of the MMR and pluripotency factors, as well as a reduced MMR activity and cisplatin sensitivity, compared to undifferentiated NTera2 cells. Also, we found that partial knockdown of MSH2 expression in undifferentiated NTera2 cells resulted in a significantly (p < 0.001) reduced cisplatin sensitivity. This study reports, for the first time, expression of the MMR system in fetal gonocytes, from which GCNIS cells are derived. Our findings in primary TGCT specimens and TGCT-derived cells suggest that a reduced

Frameshift mutational target gene analysis identifies similarities and differences in constitutional mismatch repair-deficiency and Lynch syndrome.

PubMed

Maletzki, Claudia; Huehns, Maja; Bauer, Ingrid; Ripperger, Tim; Mork, Maureen M; Vilar, Eduardo; Klöcking, Sabine; Zettl, Heike; Prall, Friedrich; Linnebacher, Michael

2017-07-01

Mismatch-repair deficient (MMR-D) malignancies include Lynch Syndrome (LS), which is secondary to germline mutations in one of the MMR genes, and the rare childhood-form of constitutional mismatch repair-deficiency (CMMR-D); caused by bi-allelic MMR gene mutations. A hallmark of LS-associated cancers is microsatellite instability (MSI), characterized by coding frameshift mutations (cFSM) in target genes. By contrast, tumors arising in CMMR-D patients are thought to display a somatic mutation pattern differing from LS. This study has the main goal to identify cFSM in MSI target genes relevant in CMMR-D and to compare the spectrum of common somatic mutations, including alterations in DNA polymerases POLE and D1 between LS and CMMR-D. CMMR-D-associated tumors harbored more somatic mutations compared to LS cases, especially in the TP53 gene and in POLE and POLD1, where novel mutations were additionally identified. Strikingly, MSI in classical mononucleotide markers BAT40 and CAT25 was frequent in CMMR-D cases. MSI-target gene analysis revealed mutations in CMMR-D-associated tumors, some of them known to be frequently hit in LS, such as RNaseT2, HT001, and TGFβR2. Our results imply a general role for these cFSM as potential new drivers of MMR-D tumorigenesis. © 2017 Wiley Periodicals, Inc.

Saccharomyces cerevisiae MSH2-MSH3 and MSH2-MSH6 complexes display distinct requirements for DNA binding Domain I in mismatch recognition.

PubMed Central

Lee, Susan D.; Surtees, Jennifer A.; Alani, Eric

2007-01-01

In eukaryotic mismatch repair (MMR) MSH2-MSH6 initiates the repair of base-base and small insertion/deletion mismatches while MSH2-MSH3 repairs larger insertion/deletion mismatches. In this study we showed that the msh2Δ1 mutation, containing a complete deletion of the conserved mismatch recognition Domain I of MSH2, conferred a separation of function phenotype with respect to MSH2-MSH3 and MSH2-MSH6 functions. Strains bearing the msh2Δ1 mutation were nearly wild-type in MSH2-MSH6-mediated MMR and in suppressing recombination between DNA sequences predicted to form mismatches recognized by MSH2-MSH6. However, these strains were completely defective in MSH2-MSH3-mediated MMR and recombination functions. This information encouraged us to analyze the contributions of Domain I to the mismatch binding specificity of MSH2-MSH3 in genetic and biochemical assays. We found that Domain I in MSH2 contributed a non-specific DNA binding activity while Domain I of MSH3 appeared important for mismatch binding specificity and for suppressing non-specific DNA-binding. These observations reveal distinct requirements for the MSH2 DNA binding Domain I in the repair of DNA mismatches and suggest that the binding of MSH2-MSH3 to mismatch DNA involves protein-DNA contacts that appear very different from those required for MSH2-MSH6 mismatch binding. PMID:17157869

Saccharomyces cerevisiae MSH2-MSH3 and MSH2-MSH6 complexes display distinct requirements for DNA binding domain I in mismatch recognition.

PubMed

Lee, Susan D; Surtees, Jennifer A; Alani, Eric

2007-02-09

In eukaryotic mismatch repair (MMR) MSH2-MSH6 initiates the repair of base-base and small insertion/deletion mismatches while MSH2-MSH3 repairs larger insertion/deletion mismatches. Here, we show that the msh2Delta1 mutation, containing a complete deletion of the conserved mismatch recognition domain I of MSH2, conferred a separation of function phenotype with respect to MSH2-MSH3 and MSH2-MSH6 functions. Strains bearing the msh2Delta1 mutation were nearly wild-type in MSH2-MSH6-mediated MMR and in suppressing recombination between DNA sequences predicted to form mismatches recognized by MSH2-MSH6. However, these strains were completely defective in MSH2-MSH3-mediated MMR and recombination functions. This information encouraged us to analyze the contributions of domain I to the mismatch binding specificity of MSH2-MSH3 in genetic and biochemical assays. We found that domain I in MSH2 contributed a non-specific DNA binding activity while domain I of MSH3 appeared important for mismatch binding specificity and for suppressing non-specific DNA binding. These observations reveal distinct requirements for the MSH2 DNA binding domain I in the repair of DNA mismatches and suggest that the binding of MSH2-MSH3 to mismatch DNA involves protein-DNA contacts that appear very different from those required for MSH2-MSH6 mismatch binding.

Risk of colorectal cancer for people with a mutation in both a MUTYH and a DNA mismatch repair gene.

PubMed

Win, Aung Ko; Reece, Jeanette C; Buchanan, Daniel D; Clendenning, Mark; Young, Joanne P; Cleary, Sean P; Kim, Hyeja; Cotterchio, Michelle; Dowty, James G; MacInnis, Robert J; Tucker, Katherine M; Winship, Ingrid M; Macrae, Finlay A; Burnett, Terrilea; Le Marchand, Loïc; Casey, Graham; Haile, Robert W; Newcomb, Polly A; Thibodeau, Stephen N; Lindor, Noralane M; Hopper, John L; Gallinger, Steven; Jenkins, Mark A

2015-12-01

The base excision repair protein, MUTYH, functionally interacts with the DNA mismatch repair (MMR) system. As genetic testing moves from testing one gene at a time, to gene panel and whole exome next generation sequencing approaches, understandin g the risk associated with co-existence of germline mutations in these genes will be important for clinical interpretation and management. From the Colon Cancer Family Registry, we identified 10 carriers who had both a MUTYH mutation (6 with c.1187G>A p.(Gly396Asp), 3 with c.821G>A p.(Arg274Gln), and 1 with c.536A>G p.(Tyr179Cys)) and a MMR gene mutation (3 in MLH1, 6 in MSH2, and 1 in PMS2), 375 carriers of a single (monoallelic) MUTYH mutation alone, and 469 carriers of a MMR gene mutation alone. Of the 10 carriers of both gene mutations, 8 were diagnosed with colorectal cancer. Using a weighted cohort analysis, we estimated that risk of colorectal cancer for carriers of both a MUTYH and a MMR gene mutation was substantially higher than that for carriers of a MUTYH mutation alone [hazard ratio (HR) 21.5, 95% confidence interval (CI) 9.19-50.1; p < 0.001], but not different from that for carriers of a MMR gene mutation alone (HR 1.94, 95% CI 0.63-5.99; p = 0.25). Within the limited power of this study, there was no evidence that a monoallelic MUTYH gene mutation confers additional risk of colorectal cancer for carriers of a MMR gene mutation alone. Our finding suggests MUTYH mutation testing in MMR gene mutation carriers is not clinically informative.

Germline PMS2 mutation screened by mismatch repair protein immunohistochemistry of colorectal cancer in Japan.

PubMed

Sugano, Kokichi; Nakajima, Takeshi; Sekine, Shigeki; Taniguchi, Hirokazu; Saito, Shinya; Takahashi, Masahiro; Ushiama, Mineko; Sakamoto, Hiromi; Yoshida, Teruhiko

2016-11-01

Germline PMS2 gene mutations were detected by RT-PCR/direct sequencing of total RNA extracted from puromycin-treated peripheral blood lymphocytes (PBL) and multiplex ligation-dependent probe amplification (MLPA) analyses of Japanese patients with colorectal cancer (CRC) fulfilling either the revised Bethesda Guidelines or being an age at disease onset of younger than 70 years, and screened by mismatch repair protein immunohistochemistry of formalin-fixed paraffin embedded sections. Of the 501 subjects examined, 7 (1.40%) showed the downregulated expression of the PMS2 protein alone and were referred to the genetic counseling clinic. Germline PMS2 mutations were detected in 6 (85.7%), including 3 nonsense and 1 frameshift mutations by RT-PCR/direct sequencing and 2 genomic deletions by MLPA. No mutations were identified in the other MMR genes (i.e. MSH2, MLH1 and MSH6). The prevalence of the downregulated expression of the PMS2 protein alone was 1.40% among the subjects examined and IHC results predicted the presence of PMS2 germline mutations. RT-PCR from puromycin-treated PBL and MLPA may be employed as the first screening step to detect PMS2 mutations without pseudogene interference, followed by the long-range PCR/nested PCR validation using genomic DNA. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

DNA mismatch repair gene MLH1 induces apoptosis in prostate cancer cells.

PubMed

Fukuhara, Shinichiro; Chang, Inik; Mitsui, Yozo; Chiyomaru, Takeshi; Yamamura, Soichiro; Majid, Shahana; Saini, Sharanjot; Hirata, Hiroshi; Deng, Guoren; Gill, Ankurpreet; Wong, Darryn K; Shiina, Hiroaki; Nonomura, Norio; Dahiya, Rajvir; Tanaka, Yuichiro

2014-11-30

Mismatch repair (MMR) enzymes have been shown to be deficient in prostate cancer (PCa). MMR can influence the regulation of tumor development in various cancers but their role on PCa has not been investigated. The aim of the present study was to determine the functional effects of the mutL-homolog 1 (MLH1) gene on growth of PCa cells. The DU145 cell line has been established as MLH1-deficient and thus, this cell line was utilized to determine effects of MLH1 by gene expression. Lack of MLH1 protein expression was confirmed by Western blotting in DU145 cells whereas levels were high in normal PWR-1E and RWPE-1 prostatic cells. MLH1-expressing stable transfectant DU145 cells were then created to characterize the effects this MMR gene has on various growth properties. Expression of MLH1 resulted in decreased cell proliferation, migration and invasion properties. Lack of cell growth in vivo also indicated a tumor suppressive effect by MLH1. Interestingly, MLH1 caused an increase in apoptosis along with phosphorylated c-Abl, and treatment with MLH1 siRNAs countered this effect. Furthermore, inhibition of c-Abl with STI571 also abrogated the effect on apoptosis caused by MLH1. These results demonstrate MLH1 protects against PCa development by inducing c-Abl-mediated apoptosis.

DNA mismatch repair gene MLH1 induces apoptosis in prostate cancer cells

PubMed Central

Mitsui, Yozo; Chiyomaru, Takeshi; Yamamura, Soichiro; Majid, Shahana; Saini, Sharanjot; Hirata, Hiroshi; Deng, Guoren; Gill, Ankurpreet; Wong, Darryn K.; Shiina, Hiroaki; Nonomura, Norio; Dahiya, Rajvir; Tanaka, Yuichiro

2014-01-01

Mismatch repair (MMR) enzymes have been shown to be deficient in prostate cancer (PCa). MMR can influence the regulation of tumor development in various cancers but their role on PCa has not been investigated. The aim of the present study was to determine the functional effects of the mutL-homolog 1 (MLH1) gene on growth of PCa cells. The DU145 cell line has been established as MLH1-deficient and thus, this cell line was utilized to determine effects of MLH1 by gene expression. Lack of MLH1 protein expression was confirmed by Western blotting in DU145 cells whereas levels were high in normal PWR-1E and RWPE-1 prostatic cells. MLH1-expressing stable transfectant DU145 cells were then created to characterize the effects this MMR gene has on various growth properties. Expression of MLH1 resulted in decreased cell proliferation, migration and invasion properties. Lack of cell growth in vivo also indicated a tumor suppressive effect by MLH1. Interestingly, MLH1 caused an increase in apoptosis along with phosphorylated c-Abl, and treatment with MLH1 siRNAs countered this effect. Furthermore, inhibition of c-Abl with STI571 also abrogated the effect on apoptosis caused by MLH1. These results demonstrate MLH1 protects against PCa development by inducing c-Abl-mediated apoptosis. PMID:25526032

DNA mismatch repair and oligonucleotide end-protection promote base-pair substitution distal from a CRISPR/Cas9-induced DNA break

PubMed Central

Harmsen, Tim; Klaasen, Sjoerd; van de Vrugt, Henri; te Riele, Hein

2018-01-01

Abstract Single-stranded oligodeoxyribonucleotide (ssODN)-mediated repair of CRISPR/Cas9-induced DNA double-strand breaks (DSB) can effectively be used to introduce small genomic alterations in a defined locus. Here, we reveal DNA mismatch repair (MMR) activity is crucial for efficient nucleotide substitution distal from the Cas9-induced DNA break when the substitution is instructed by the 3′ half of the ssODN. Furthermore, protecting the ssODN 3′ end with phosphorothioate linkages enhances MMR-dependent gene editing events. Our findings can be exploited to optimize efficiencies of nucleotide substitutions distal from the DSB and imply that oligonucleotide-mediated gene editing is effectuated by templated break repair. PMID:29447381

Calibration of Multiple In Silico Tools for Predicting Pathogenicity of Mismatch Repair Gene Missense Substitutions

PubMed Central

Thompson, Bryony A.; Greenblatt, Marc S.; Vallee, Maxime P.; Herkert, Johanna C.; Tessereau, Chloe; Young, Erin L.; Adzhubey, Ivan A.; Li, Biao; Bell, Russell; Feng, Bingjian; Mooney, Sean D.; Radivojac, Predrag; Sunyaev, Shamil R.; Frebourg, Thierry; Hofstra, Robert M.W.; Sijmons, Rolf H.; Boucher, Ken; Thomas, Alun; Goldgar, David E.; Spurdle, Amanda B.; Tavtigian, Sean V.

2015-01-01

Classification of rare missense substitutions observed during genetic testing for patient management is a considerable problem in clinical genetics. The Bayesian integrated evaluation of unclassified variants is a solution originally developed for BRCA1/2. Here, we take a step toward an analogous system for the mismatch repair (MMR) genes (MLH1, MSH2, MSH6, and PMS2) that confer colon cancer susceptibility in Lynch syndrome by calibrating in silico tools to estimate prior probabilities of pathogenicity for MMR gene missense substitutions. A qualitative five-class classification system was developed and applied to 143 MMR missense variants. This identified 74 missense substitutions suitable for calibration. These substitutions were scored using six different in silico tools (Align-Grantham Variation Grantham Deviation, multivariate analysis of protein polymorphisms [MAPP], Mut-Pred, PolyPhen-2.1, Sorting Intolerant From Tolerant, and Xvar), using curated MMR multiple sequence alignments where possible. The output from each tool was calibrated by regression against the classifications of the 74 missense substitutions; these calibrated outputs are interpretable as prior probabilities of pathogenicity. MAPP was the most accurate tool and MAPP + PolyPhen-2.1 provided the best-combined model (R2 = 0.62 and area under receiver operating characteristic = 0.93). The MAPP + PolyPhen-2.1 output is sufficiently predictive to feed as a continuous variable into the quantitative Bayesian integrated evaluation for clinical classification of MMR gene missense substitutions. PMID:22949387

DNA Mismatch Binding and Antiproliferative Activity of Rhodium Metalloinsertors

PubMed Central

Ernst, Russell J.; Song, Hang; Barton, Jacqueline K.

2009-01-01

Deficiencies in mismatch repair (MMR) are associated with carcinogenesis. Rhodium metalloinsertors bind to DNA base mismatches with high specificity and inhibit cellular proliferation preferentially in MMR-deficient cells versus MMR-proficient cells. A family of chrysenequinone diimine complexes of rhodium with varying ancillary ligands that serve as DNA metalloinsertors has been synthesized, and both DNA mismatch binding affinities and antiproliferative activities against the human colorectal carcinoma cell lines HCT116N and HCT116O, an isogenic model system for MMR deficiency, have been determined. DNA photocleavage experiments reveal that all complexes bind to the mismatch sites with high specificities; DNA binding affinities to oligonucleotides containing single base CA and CC mismatches, obtained through photocleavage titration or competition, vary from 104 to 108 M−1 for the series of complexes. Significantly, binding affinities are found to be inversely related to ancillary ligand size and directly related to differential inhibition of the HCT116 cell lines. The observed trend in binding affinity is consistent with the metalloinsertion mode where the complex binds from the minor groove with ejection of mismatched base pairs. The correlation between binding affinity and targeting of the MMR-deficient cell line suggests that rhodium metalloinsertors exert their selective biological effects on MMR-deficient cells through mismatch binding in vivo. PMID:19175313

Mismatch repair proteins, meiosis, and mice: understanding the complexities of mammalian meiosis.

PubMed

Svetlanov, Anton; Cohen, Paula E

2004-05-15

Mammalian meiosis differs from that seen in lower eukaryotes in several respects, not least of which is the added complexity of dealing with chromosomal interactions across a much larger genome (12 MB over 16 chromosome pairs in Saccharomyces cerevisiae compared to 2500 MB over 19 autosome pairs in Mus musculus). Thus, the recombination machinery, while being highly conserved through eukaryotes, has evolved to accommodate such issues to preserve genome integrity and to ensure propagation of the species. One group of highly conserved meiotic regulators is the DNA mismatch repair protein family that, as their name implies, were first identified as proteins that act to repair DNA mismatches that arise primarily during DNA replication. Their function in ensuring chromosomal integrity has also translated into a critical role for this family in meiotic recombination in most sexually reproducing organisms. In mice, targeted deletion of certain family members results in severe consequences for meiotic progression and infertility. This review will focus on the studies involving these mutant mouse models, with occasional comparison to the function of these proteins in other organisms.

Mismatch repair genes Mlh1 and Mlh3 modify CAG instability in Huntington's disease mice: genome-wide and candidate approaches.

PubMed

Pinto, Ricardo Mouro; Dragileva, Ella; Kirby, Andrew; Lloret, Alejandro; Lopez, Edith; St Claire, Jason; Panigrahi, Gagan B; Hou, Caixia; Holloway, Kim; Gillis, Tammy; Guide, Jolene R; Cohen, Paula E; Li, Guo-Min; Pearson, Christopher E; Daly, Mark J; Wheeler, Vanessa C

2013-10-01

The Huntington's disease gene (HTT) CAG repeat mutation undergoes somatic expansion that correlates with pathogenesis. Modifiers of somatic expansion may therefore provide routes for therapies targeting the underlying mutation, an approach that is likely applicable to other trinucleotide repeat diseases. Huntington's disease Hdh(Q111) mice exhibit higher levels of somatic HTT CAG expansion on a C57BL/6 genetic background (B6.Hdh(Q111) ) than on a 129 background (129.Hdh(Q111) ). Linkage mapping in (B6x129).Hdh(Q111) F2 intercross animals identified a single quantitative trait locus underlying the strain-specific difference in expansion in the striatum, implicating mismatch repair (MMR) gene Mlh1 as the most likely candidate modifier. Crossing B6.Hdh(Q111) mice onto an Mlh1 null background demonstrated that Mlh1 is essential for somatic CAG expansions and that it is an enhancer of nuclear huntingtin accumulation in striatal neurons. Hdh(Q111) somatic expansion was also abolished in mice deficient in the Mlh3 gene, implicating MutLγ (MLH1-MLH3) complex as a key driver of somatic expansion. Strikingly, Mlh1 and Mlh3 genes encoding MMR effector proteins were as critical to somatic expansion as Msh2 and Msh3 genes encoding DNA mismatch recognition complex MutSβ (MSH2-MSH3). The Mlh1 locus is highly polymorphic between B6 and 129 strains. While we were unable to detect any difference in base-base mismatch or short slipped-repeat repair activity between B6 and 129 MLH1 variants, repair efficiency was MLH1 dose-dependent. MLH1 mRNA and protein levels were significantly decreased in 129 mice compared to B6 mice, consistent with a dose-sensitive MLH1-dependent DNA repair mechanism underlying the somatic expansion difference between these strains. Together, these data identify Mlh1 and Mlh3 as novel critical genetic modifiers of HTT CAG instability, point to Mlh1 genetic variation as the likely source of the instability difference in B6 and 129 strains and suggest that MLH1

Mismatch Repair Genes Mlh1 and Mlh3 Modify CAG Instability in Huntington's Disease Mice: Genome-Wide and Candidate Approaches

PubMed Central

Pinto, Ricardo Mouro; Dragileva, Ella; Kirby, Andrew; Lloret, Alejandro; Lopez, Edith; St. Claire, Jason; Panigrahi, Gagan B.; Hou, Caixia; Holloway, Kim; Gillis, Tammy; Guide, Jolene R.; Cohen, Paula E.; Li, Guo-Min; Pearson, Christopher E.; Daly, Mark J.; Wheeler, Vanessa C.

2013-01-01

The Huntington's disease gene (HTT) CAG repeat mutation undergoes somatic expansion that correlates with pathogenesis. Modifiers of somatic expansion may therefore provide routes for therapies targeting the underlying mutation, an approach that is likely applicable to other trinucleotide repeat diseases. Huntington's disease HdhQ111 mice exhibit higher levels of somatic HTT CAG expansion on a C57BL/6 genetic background (B6.HdhQ111) than on a 129 background (129.HdhQ111). Linkage mapping in (B6x129).HdhQ111 F2 intercross animals identified a single quantitative trait locus underlying the strain-specific difference in expansion in the striatum, implicating mismatch repair (MMR) gene Mlh1 as the most likely candidate modifier. Crossing B6.HdhQ111 mice onto an Mlh1 null background demonstrated that Mlh1 is essential for somatic CAG expansions and that it is an enhancer of nuclear huntingtin accumulation in striatal neurons. HdhQ111 somatic expansion was also abolished in mice deficient in the Mlh3 gene, implicating MutLγ (MLH1–MLH3) complex as a key driver of somatic expansion. Strikingly, Mlh1 and Mlh3 genes encoding MMR effector proteins were as critical to somatic expansion as Msh2 and Msh3 genes encoding DNA mismatch recognition complex MutSβ (MSH2–MSH3). The Mlh1 locus is highly polymorphic between B6 and 129 strains. While we were unable to detect any difference in base-base mismatch or short slipped-repeat repair activity between B6 and 129 MLH1 variants, repair efficiency was MLH1 dose-dependent. MLH1 mRNA and protein levels were significantly decreased in 129 mice compared to B6 mice, consistent with a dose-sensitive MLH1-dependent DNA repair mechanism underlying the somatic expansion difference between these strains. Together, these data identify Mlh1 and Mlh3 as novel critical genetic modifiers of HTT CAG instability, point to Mlh1 genetic variation as the likely source of the instability difference in B6 and 129 strains and suggest that MLH1 protein

Risk of colorectal cancer for people with a mutation in both a MUTYH and a DNA mismatch repair gene

PubMed Central

Win, Aung Ko; Reece, Jeanette C.; Buchanan, Daniel D.; Clendenning, Mark; Young, Joanne P.; Cleary, Sean P.; Kim, Hyeja; Cotterchio, Michelle; Dowty, James G.; MacInnis, Robert J.; Tucker, Katherine M.; Winship, Ingrid M.; Macrae, Finlay A.; Burnett, Terrilea; Le Marchand, Loïc; Casey, Graham; Haile, Robert W.; Newcomb, Polly A.; Thibodeau, Stephen N.; Lindor, Noralane M.; Hopper, John L.; Gallinger, Steven; Jenkins, Mark A.

2015-01-01

The base excision repair protein, MUTYH, functionally interacts with the DNA mismatch repair (MMR) system. As genetic testing moves from testing one gene at a time, to gene panel and whole exome next generation sequencing approaches, understanding the risk associated with co-existence of germline mutations in these genes will be important for clinical interpretation and management. From the Colon Cancer Family Registry, we identified 10 carriers who had both a MUTYH mutation (6 with c.1187G>A p.(Gly396Asp), 3 with c.821G>A p.(Arg274Gln), and 1 with c.536A>G p.(Tyr179Cys)) and a MMR gene mutation (3 in MLH1, 6 in MSH2, and 1 in PMS2), 375 carriers of a single (monoallelic) MUTYH mutation alone, and 469 carriers of a MMR gene mutation alone. Of the 10 carriers of both gene mutations, 8 were diagnosed with colorectal cancer. Using a weighted cohort analysis, we estimated that risk of colorectal cancer for carriers of both a MUTYH and a MMR gene mutation was substantially higher than that for carriers of a MUTYH mutation alone [hazard ratio (HR) 21.5, 95 % confidence interval (CI) 9.19–50.1; p < 0.001], but not different from that for carriers of a MMR gene mutation alone (HR 1.94, 95 % CI 0.63–5.99; p = 0.25). Within the limited power of this study, there was no evidence that a monoallelic MUTYH gene mutation confers additional risk of colorectal cancer for carriers of a MMR gene mutation alone. Our finding suggests MUTYH mutation testing in MMR gene mutation carriers is not clinically informative. PMID:26202870

Distinct pathways for repairing mutagenic lesions induced by methylating and ethylating agents

PubMed Central

Negishi, Tomoe

2013-01-01

DNA alkylation damage can be repaired by nucleotide excision repair (NER), base excision repair (BER) or by direct removal of alkyl groups from modified bases by O 6-alkylguanine DNA alkyltransferase (AGT; E.C. 2.1.1.63). DNA mismatch repair (MMR) is also likely involved in this repair. We have investigated alkylation-induced mutagenesis in a series of NER- or AGT-deficient Escherichia coli strains, alone or in combination with defects in the MutS, MutL or MutH components of MMR. All strains used contained the Fʹprolac from strain CC102 (FʹCC102) episome capable of detecting specifically lac GC to AT reverse mutations resulting from O 6-alkylguanine. The results showed the repair of O 6-methylguanine to be performed by AGT ≫ MMR > NER in order of importance, whereas the repair of O 6-ethylguanine followed the order NER > AGT > MMR. Studies with double mutants showed that in the absence of AGT or NER repair pathways, the lack of MutS protein generally increased mutant frequencies for both methylating and ethylating agents, suggesting a repair or mutation avoidance role for this protein. However, lack of MutL or MutH protein did not increase alkylation-induced mutagenesis under these conditions and, in fact, reduced mutagenesis by the N-alkyl-N-nitrosoureas MNU and ENU. The combined results suggest that little or no alkylation damage is actually corrected by the mutHLS MMR system; instead, an as yet unspecified interaction of MutS protein with alkylated DNA may promote the involvement of a repair system other than MMR to avoid a mutagenic outcome. Furthermore, both mutagenic and antimutagenic effects of MMR were detected, revealing a dual function of the MMR system in alkylation-exposed cells. PMID:23446177

Warthin tumors do not have microsatellite instability and express normal DNA mismatch repair proteins.

PubMed

Hunt, Jennifer L

2006-01-01

Warthin tumors are controversial entities with a poorly understood etiology. Although some investigators have suggested a neoplastic origin, others have supported a developmental anomaly. A recent study described the absence of staining for hMLH1 and hMSH2 proteins in the epithelial component of Warthin tumors, suggesting that they arise secondary to defects in the DNA mismatch repair system. To determine if Warthin tumors exhibit evidence of DNA mismatch repair defects. Immunostains for hMLH1 and hMSH2 were performed using a standard approach. Microdissection of the epithelial component was followed by DNA extraction from the tissue fragments. Polymerase chain reaction and capillary electrophoresis analyses were performed for the following 5 National Cancer Institute-recommended microsatellites: D2s123, D5s346, D17s250, BAT25, and BAT26. Twelve patients with Warthin tumors were included. The immunostains for hMLH1 and hMSH2 showed preserved expression in the nuclei of the epithelial component of all Warthin tumors. No microsatellite instability was detected, and no loss of heterozygosity was seen. These results are not concordant with previously reported results showing loss of expression of the hMLH1 and hMSH2 DNA mismatch repair enzymes in the epithelial component of Warthin tumors. Furthermore, no microsatellite instability was detected in the 5 loci tested for each tumor in this series. These data demonstrate that Warthin tumors do not have evidence of DNA mismatch repair defects at the genomic or protein expression level.

DNA mismatch repair deficiency and hereditary syndromes in Latino patients with colorectal cancer.

PubMed

Ricker, Charité N; Hanna, Diana L; Peng, Cheng; Nguyen, Nathalie T; Stern, Mariana C; Schmit, Stephanie L; Idos, Greg E; Patel, Ravi; Tsai, Steven; Ramirez, Veronica; Lin, Sonia; Shamasunadara, Vinay; Barzi, Afsaneh; Lenz, Heinz-Josef; Figueiredo, Jane C

2017-10-01

The landscape of hereditary syndromes and clinicopathologic characteristics among US Latino/Hispanic individuals with colorectal cancer (CRC) remains poorly understood. A total of 265 patients with CRC who were enrolled in the Hispanic Colorectal Cancer Study were included in the current study. Information regarding CRC risk factors was elicited through interviews, and treatment and survival data were abstracted from clinical charts. Tumor studies and germline genetic testing results were collected from medical records or performed using standard molecular methods. The mean age of the patients at the time of diagnosis was 53.7 years (standard deviation, 10.3 years), and 48.3% were female. Overall, 21.2% of patients reported a first-degree or second-degree relative with CRC; 3.4% met Amsterdam I/II criteria. With respect to Bethesda guidelines, 38.5% of patients met at least 1 criterion. Of the 161 individuals who had immunohistochemistry and/or microsatellite instability testing performed, 21 (13.0%) had mismatch repair (MMR)-deficient (dMMR) tumors. dMMR tumors were associated with female sex (61.9%), earlier age at the time of diagnosis (50.4 ± 12.4 years), proximal location (61.9%), and first-degree (23.8%) or second-degree (9.5%) family history of CRC. Among individuals with dMMR tumors, 13 (61.9%) had a germline MMR mutation (MutL homolog 1 [MLH1] in 6 patients; MutS homolog 2 [MSH2] in 4 patients; MutS homolog 6 [MHS6] in 2 patients; and PMS1 homolog 2, mismatch repair system component [PMS2] in 1 patient). The authors identified 2 additional MLH1 mutation carriers by genetic testing who had not received immunohistochemistry/microsatellite instability testing. In total, 5.7% of the entire cohort were confirmed to have Lynch syndrome. In addition, 6 individuals (2.3%) had a polyposis phenotype. The percentage of dMMR tumors noted among Latino individuals (13%) is similar to estimates in non-Hispanic white individuals. In the current study, the majority of

Mismatch DNA repair mRNA expression profiles in oral melanin pigmentation lesion and hamartomatous polyp of a child with Peutz-Jeghers syndrome.

PubMed

Vageli, Dimitra P; Doukas, Sotirios G; Markou, Andreas

2013-10-01

Mismatch DNA repair (MMR) mRNA expression analysis was performed on a biopsy of oral mucosa melanin pigmentation lesion, a hamartomatous polyp and peripheral blood derived from a 12-year-old child with Peutz-Jeghers Syndrome (PJS). We present a deficient MMR system, in a PJS patient, which demonstrated low mRNA levels of hMSH6 and hPMS2 and an increasing MMR deficiency from the non-dysplastic lesion to hamartomatous polyp of PJS with a high risk of cancer. Copyright © 2013 Wiley Periodicals, Inc.

Mutational signatures of DNA mismatch repair deficiency in C. elegans and human cancers.

PubMed

Meier, Bettina; Volkova, Nadezda V; Hong, Ye; Schofield, Pieta; Campbell, Peter J; Gerstung, Moritz; Gartner, Anton

2018-05-01

Throughout their lifetime, cells are subject to extrinsic and intrinsic mutational processes leaving behind characteristic signatures in the genome. DNA mismatch repair (MMR) deficiency leads to hypermutation and is found in different cancer types. Although it is possible to associate mutational signatures extracted from human cancers with possible mutational processes, the exact causation is often unknown. Here, we use C. elegans genome sequencing of pms-2 and mlh-1 knockouts to reveal the mutational patterns linked to C. elegans MMR deficiency and their dependency on endogenous replication errors and errors caused by deletion of the polymerase ε subunit pole-4 Signature extraction from 215 human colorectal and 289 gastric adenocarcinomas revealed three MMR-associated signatures, one of which closely resembles the C. elegans MMR spectrum and strongly discriminates microsatellite stable and unstable tumors (AUC = 98%). A characteristic difference between human and C. elegans MMR deficiency is the lack of elevated levels of N C G > NTG mutations in C. elegans, likely caused by the absence of cytosine (CpG) methylation in worms . The other two human MMR signatures may reflect the interaction between MMR deficiency and other mutagenic processes, but their exact cause remains unknown. In summary, combining information from genetically defined models and cancer samples allows for better aligning mutational signatures to causal mutagenic processes. © 2018 Meier et al.; Published by Cold Spring Harbor Laboratory Press.

Spectrum of mismatch repair gene mutations and clinical presentation of Hispanic individuals with Lynch syndrome.

PubMed

Sunga, Annette Y; Ricker, Charité; Espenschied, Carin R; Castillo, Danielle; Melas, Marilena; Herzog, Josef; Bannon, Sarah; Cruz-Correa, Marcia; Lynch, Patrick; Solomon, Ilana; Gruber, Stephen B; Weitzel, Jeffrey N

2017-04-01

Lynch syndrome (LS), the most common hereditary colorectal cancer syndrome, is caused by mismatch repair (MMR) gene mutations. However, data about MMR mutations in Hispanics are limited. This study aims to describe the spectrum of MMR mutations in Hispanics with LS and explore ancestral origins. This case series involved an IRB-approved retrospective chart review of self-identified Hispanic patients (n = 397) seen for genetic cancer risk assessment at four collaborating academic institutions in California, Texas, and Puerto Rico who were evaluated by MMR genotyping and/or tumor analysis. A literature review was conducted for all mutations identified. Of those who underwent clinical genetic testing (n = 176), 71 had MMR gene mutations. Nine mutations were observed more than once. One third (3/9) of recurrent mutations and two additional mutations (seen only once) were previously reported in Spain, confirming the influence of Spanish ancestry on MMR mutations in Hispanic populations. The recurrent mutations identified (n = 9) included both previously reported mutations as well as unique mutations not in the literature. This is the largest report of Hispanic MMR mutations in North America; however, a larger sample and haplotype analyses are needed to better understand recurrent MMR mutations in Hispanic populations. Copyright © 2017. Published by Elsevier Inc.

Role of the mismatch repair gene, Msh6, in suppressing genome instability and radiation-induced mutations

PubMed Central

Barrera-Oro, Julio; Liu, Tzu-Yang; Gorden, Erin; Kucherlapati, Raju; Shao, Changshun; Tischfield, Jay A

2008-01-01

Mismatch repair (MMR) is critical for preserving genomic integrity. Failure of this system can accelerate somatic mutation and increase the risk of developing cancer. MSH6, in complex with MSH2, is the MMR protein that mediates DNA repair through the recognition of 1- and 2-bp mismatches. To evaluate the effects of MSH6 deficiency on genomic stability we compared the frequency of in vivo loss of heterozygosity (LOH) between MSH6-proficient and deficient, 129S2 x C57BL/6 F1 hybrid mice that were heterozygous for our reporter gene Aprt. We recovered mutant cells that had functionally lost APRT protein activity and categorized the spectrum of mutations responsible for the LOH events. We also measured the mutant frequency at the X-linked gene, Hprt, as a second reporter for point mutation. In Msh6−/−Aprt+/− mice, mutation frequency at Aprt was elevated in both T cells and fibroblasts by 2.5-fold and 5.7-fold, respectively, over Msh6+/+Aprt+/− littermate controls. While a modest increase in mitotic recombination (MR) was observed in MSH6-deficient fibroblasts compared to wild type controls, point mutation was the predominant mechanism leading to APRT deficiency in both cell types. Base substitution, consisting of multiple types of transitions, accounted for all of the point mutations identified within the Aprt coding region. We also assessed the role of MSH6 in preventing mutations caused by a common environmental mutagen, ionizing radiation (IR). In Msh6−/−Aprt+/− mice, 4 Gy of X-irradiation induced a significant increase in point mutations at both Aprt and Hprt in T cells, but not in fibroblasts. These findings indicate that MutSα reduces spontaneous and IR-induced mutation in a cell-type dependant manner. PMID:18538799

Simple detection of germline microsatellite instability for diagnosis of constitutional mismatch repair cancer syndrome.

PubMed

Ingham, Danielle; Diggle, Christine P; Berry, Ian; Bristow, Claire A; Hayward, Bruce E; Rahman, Nazneen; Markham, Alexander F; Sheridan, Eamonn G; Bonthron, David T; Carr, Ian M

2013-06-01

Heterozygous mutations in DNA mismatch repair (MMR) genes result in predisposition to colorectal cancer (hereditary nonpolyposis colorectal cancer or Lynch syndrome). Patients with biallelic mutations in these genes, however, present earlier, with constitutional mismatch repair deficiency cancer syndrome (CMMRD), which is characterized by a spectrum of rare childhood malignancies and café-au-lait skin patches. The hallmark of MMR deficiency, microsatellite instability (MSI), is readily detectable in tumor DNA in Lynch syndrome, but is also present in constitutional DNA of CMMRD patients. However, detection of constitutional or germline MSI (gMSI) has hitherto relied on technically difficult assays that are not routinely applicable for clinical diagnosis. Consequently, we have developed a simple high-throughput screening methodology to detect gMSI in CMMRD patients based on the presence of stutter peaks flanking a dinucleotide repeat allele when amplified from patient blood DNA samples. Using the three different microsatellite markers, the gMSI ratio was determined in a cohort of normal individuals and 10 CMMRD patients, with biallelic germline mutations in PMS2 (seven patients), MSH2 (one patient), or MSH6 (two patients). Subjects with either PMS2 or MSH2 mutations were easily identified; however, this measure was not altered in patients with CMMRD due to MSH6 mutation. © 2013 Wiley Periodicals, Inc.

DNA mismatch repair proteins MLH1 and PMS2 can be imported to the nucleus by a classical nuclear import pathway.

PubMed

de Barros, Andrea C; Takeda, Agnes A S; Dreyer, Thiago R; Velazquez-Campoy, Adrian; Kobe, Boštjan; Fontes, Marcos R M

2018-03-01

MLH1 and PMS2 proteins form the MutLα heterodimer, which plays a major role in DNA mismatch repair (MMR) in humans. Mutations in MMR-related proteins are associated with cancer, especially with colon cancer. The N-terminal region of MutLα comprises the N-termini of PMS2 and MLH1 and, similarly, the C-terminal region of MutLα is composed by the C-termini of PMS2 and MLH1, and the two are connected by linker region. The nuclear localization sequences (NLSs) necessary for the nuclear transport of the two proteins are found in this linker region. However, the exact NLS sequences have been controversial, with different sequences reported, particularly for MLH1. The individual components are not imported efficiently, presumably due to their C-termini masking their NLSs. In order to gain insights into the nuclear transport of these proteins, we solved the crystal structures of importin-α bound to peptides corresponding to the supposed NLSs of MLH1 and PMS2 and performed isothermal titration calorimetry to study their binding affinities. Both putative MLH1 and PMS2 NLSs can bind to importin-α as monopartite NLSs, which is in agreement with some previous studies. However, MLH1-NLS has the highest affinity measured by a natural NLS peptide, suggesting a major role of MLH1 protein in nuclear import compared to PMS2. Finally, the role of MLH1 and PMS2 in the nuclear transport of the MutLα heterodimer is discussed. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Clinicopathologic Comparison of Lynch Syndrome-associated and "Lynch-like" Endometrial Carcinomas Identified on Universal Screening Using Mismatch Repair Protein Immunohistochemistry.

PubMed

Mills, Anne M; Sloan, Emily A; Thomas, Martha; Modesitt, Susan C; Stoler, Mark H; Atkins, Kristen A; Moskaluk, Christopher A

2016-02-01

Expanded testing for Lynch syndrome (LS) is increasingly recommended for patients with endometrial carcinomas, and immunohistochemistry (IHC) for tumor loss of mismatch-repair (MMR) protein expression is the most common primary screen. This has led to the recognition of MMR-IHC-deficient cases without identifiable mutations on directed germline sequencing. The clinical implications of such "Lynch-like" (LL) cancers are unclear. We here report the clinicopathologic features of putative familial endometrial carcinoma identified on universal MMR-IHC screening with attention to cases with discordant IHC and germline results. The files of the University of Virginia Pathology Department were retrospectively searched for all MMR-deficient endometrial carcinomas identified on screening. Cases were categorized as likely sporadic (MLH1/PMS2 loss, evidence of MLH1 promoter hypermethylation) or putative LS (PLS) (loss of MSH2/MSH6, MSH6, or PMS2). PLS cases were further subdivided into LS and LL groups on the basis of the presence or absence of a confirmatory mutation by germline testing, and the clinicopathologic features of these cases were compared. A deficiency of ≥1 MMR protein was observed in 31.4% (66/210) of endometrial carcinomas, including 26 PLS cases, 15 of which had germline testing. Directed germline sequencing confirmed LS in 46.7% (7/15); the remaining cases were classified as LL. High-grade and/or biphasic morphology was seen in 42.9% (3/7) of LS and 62.5% (5/8) of LL cases; the remaining cases showed low-grade, conventional endometrioid morphology. High level microsatellite instability was observed in 71.4% (5/7) of LL cases. The majority of cases from both groups (LS: 85.7% [6/7]; LL: 87.5% [7/8]) were low-stage (T1a/T1b). Endometrial carcinoma was the presenting malignancy in 85.7% (6/7) of LS patients and 87.5% (7/8) of LL patients. Family history was suggestive of LS in 28.5% (2/7) of LS patients and 12.5% (1/8) of LL patients. Screening algorithms

Glycosyltransferase gene expression identifies a poor prognostic colorectal cancer subtype associated with mismatch repair deficiency and incomplete glycan synthesis.

PubMed

Noda, Masaru; Okayama, Hirokazu; Tachibana, Kazunoshin; Sakamoto, Wataru; Saito, Katsuharu; Thar Min, Aung Kyi; Ashizawa, Mai; Nakajima, Takahiro; Aoto, Keita; Momma, Tomoyuki; Katakura, Kyoko; Ohki, Shinji; Kono, Koji

2018-05-29

We aimed to discover glycosyltransferase gene (glycogene)-derived molecular subtypes of colorectal cancer (CRC) associated with patient outcomes. Transcriptomic and epigenomic datasets of non-tumor, pre-cancerous, cancerous tissues and cell lines with somatic mutations, mismatch repair status, clinicopathological and survival information, were assembled (n=4223) and glycogene profiles were analyzed. Immunohistochemistry for a glycogene, GALNT6, was conducted in adenoma and carcinoma specimens (n=403). The functional role and cell surface glycan profiles were further investigated by in vitro loss-of-function assays and lectin microarray analysis. We initially developed and validated a 15-glycogene signature that can identify a poor-prognostic subtype, which closely related to deficient mismatch repair (dMMR) and GALNT6 downregulation. The association of decreased GALNT6 with dMMR was confirmed in multiple datasets of tumors and cell lines, and was further recapitulated by immunohistochemistry, where approximately 15% tumors exhibited loss of GALNT6 protein. GALNT6 mRNA and protein was expressed in premalignant/preinvasive lesions but was subsequently downregulated in a subset of carcinomas, possibly through epigenetic silencing. Decreased GALNT6 was independently associated with poor prognosis in the immunohistochemistry cohort and an additional microarray meta-cohort, by multivariate analyses, and its discriminative power of survival was particularly remarkable in stage III patients. GALNT6 silencing in SW480 cells promoted invasion, migration, chemoresistance and increased cell surface expression of a cancer-associated truncated O-glycan, Tn-antigen. The 15-glycogene signature and the expression levels of GALNT6 mRNA and protein each serve as a novel prognostic biomarker, highlighting the role of dysregulated glycogenes in cancer-associated glycan synthesis and poor prognosis. Copyright ©2018, American Association for Cancer Research.

Distinct requirements within the Msh3 nucleotide binding pocket for mismatch and double-strand break repair.

PubMed

Kumar, Charanya; Williams, Gregory M; Havens, Brett; Dinicola, Michelle K; Surtees, Jennifer A

2013-06-12

In Saccharomyces cerevisiae, repair of insertion/deletion loops is carried out by Msh2-Msh3-mediated mismatch repair (MMR). Msh2-Msh3 is also required for 3' non-homologous tail removal (3' NHTR) in double-strand break repair. In both pathways, Msh2-Msh3 binds double-strand/single-strand junctions and initiates repair in an ATP-dependent manner. However, the kinetics of the two processes appear different; MMR is likely rapid in order to coordinate with the replication fork, whereas 3' NHTR has been shown to be a slower process. To understand the molecular requirements in both repair pathways, we performed an in vivo analysis of well-conserved residues in Msh3 that are hypothesized to be required for MMR and/or 3' NHTR. These residues are predicted to be involved in either communication between the DNA-binding and ATPase domains within the complex or nucleotide binding and/or exchange within Msh2-Msh3. We identified a set of aromatic residues within the FLY motif of the predicted Msh3 nucleotide binding pocket that are essential for Msh2-Msh3-mediated MMR but are largely dispensable for 3' NHTR. In contrast, mutations in other regions gave similar phenotypes in both assays. Based on these results, we suggest that the two pathways have distinct requirements with respect to the position of the bound ATP within Msh3. We propose that the differences are related, at least in part, to the kinetics of each pathway. Proper binding and positioning of ATP is required to induce rapid conformational changes at the replication fork, but is less important when more time is available for repair, as in 3' NHTR. Copyright © 2013 Elsevier Ltd. All rights reserved.

Distinct requirements within the Msh3 nucleotide binding pocket for mismatch and double-strand break repair

PubMed Central

Kumar, Charanya; Williams, Gregory M.; Havens, Brett; Dinicola, Michelle; Surtees, Jennifer A.

2013-01-01

In Saccharomyces cerevisiae, repair of insertion/deletion loops is carried out by Msh2-Msh3-mediated mismatch repair (MMR). Msh2-Msh3 is also required for 3’ non-homologous tail removal (3’NHTR) in double-strand break repair. In both pathways, Msh2-Msh3 binds double-strand/single-strand junctions and initiates repair in an ATP-dependent manner. However, the kinetics of the two processes appear different; MMR is likely rapid in order to coordinate with the replication fork, whereas 3’ NHTR has been shown to be a slower process. To understand the molecular requirements in both repair pathways, we performed an in vivo analysis of well conserved residues in Msh3 that are hypothesized to be required for MMR and/or 3’NHTR. These residues are predicted to be involved in either communication between the DNA-binding and ATPase domains within the complex or nucleotide binding and/or exchange within Msh2-Msh3. We identified a set of aromatic residues within the FLY motif of the predicted Msh3 nucleotide binding pocket that are essential for Msh2-Msh3-mediated MMR but are largely dispensable for 3’NHTR. In contrast, mutations in other regions gave similar phenotypes in both assays. Based on these results, we suggest the two pathways have distinct requirements with respect to the position of the bound ATP within Msh3. We propose that the differences are related, at least in part, to the kinetics of each pathway. Proper binding and positioning of ATP is required to induce rapid conformational changes at the replication fork, but is less important when more time is available for repair, as in 3’ NHTR. PMID:23458407

Inactivation of the Mismatch Repair System in Pseudomonas aeruginosa Attenuates Virulence but Favors Persistence of Oropharyngeal Colonization in Cystic Fibrosis Mice▿

PubMed Central

Mena, Ana; Maciá, María D.; Borrell, Nuria; Moya, Bartolomé; de Francisco, Teresa; Pérez, José L.; Oliver, Antonio

2007-01-01

The inactivation of the mismatch repair (MMR) system of Pseudomonas aeruginosa modestly reduced in vitro fitness, attenuated virulence in murine models of acute systemic and respiratory infections, and decreased the initial oropharyngeal colonization potential. In contrast, the inactivation of the MMR system favored long-term persistence of oropharyngeal colonization in cystic fibrosis mice. These results may help in understanding the reasons for the low and high prevalences, respectively, of hypermutable P. aeruginosa strains in acute and chronic infections. PMID:17307847

Mutation spectrum of MSH3-deficient HHUA/chr.2 cells reflects in vivo activity of the MSH3 gene product in mismatch repair.

PubMed

Tauchi, H; Komatsu, K; Ishizaki, K; Yatagai, F; Kato, T

2000-02-14

The endometrial tumor cell line HHUA carries mutations in two mismatch repair (MMR) genes MSH3 and MSH6. We have established an MSH3-deficient HHUA/chr.2 cell line by introducing human chromosome 2, which carries wild-type MSH6 and MSH2 genes, to HHUA cells. Introduction of chromosome 2 to HHUA cells partially restored G:G MMR activity to the cell extract and reduced the frequency of mutation at the hypoxanthine-guanine phosphoribosyltransferase (hprt*) locus to about 3% that of the parental HHUA cells, which is five-fold the frequency in MMR-proficient cells, indicating that the residual mutator activity in HHUA/chr.2 is due to an MSH3-deficiency in these cells. The spectrum of mutations occurring at the HPRT locus of HHUA/chr.2 was determined with 71 spontaneous 6TG(r) clones. Base substitutions and +/-1 bp frameshifts were the major mutational events constituting, respectively, 54% and 42% of the total mutations, and more than 70% of them occurred at A:T sites. A possible explanation for the apparent bias of mutations to A:T sites in HHUA/chr.2 is haploinsufficiency of the MSH6 gene on the transferred chromosome 2. Comparison of the mutation spectra of HHUA/chr.2 with that of the MSH6-deficient HCT-15 cell line [S. Ohzeki, A. Tachibana, K. Tatsumi, T. Kato, Carcinogenesis 18 (1997) 1127-1133.] suggests that in vivo the MutSalpha (MSH2:MSH6) efficiently repairs both mismatch and unpaired extrahelical bases, whereas MutSbeta (MSH2:MSH3) efficiently repairs extrahelical bases and repairs mismatch bases to a limited extent.

Clinical problems of colorectal cancer and endometrial cancer cases with unknown cause of tumor mismatch repair deficiency (suspected Lynch syndrome).

PubMed

Buchanan, Daniel D; Rosty, Christophe; Clendenning, Mark; Spurdle, Amanda B; Win, Aung Ko

2014-01-01

Carriers of a germline mutation in one of the DNA mismatch repair (MMR) genes have a high risk of developing numerous different cancers, predominantly colorectal cancer and endometrial cancer (known as Lynch syndrome). MMR gene mutation carriers develop tumors with MMR deficiency identified by tumor microsatellite instability or immunohistochemical loss of MMR protein expression. Tumor MMR deficiency is used to identify individuals most likely to carry an MMR gene mutation. However, MMR deficiency can also result from somatic inactivation, most commonly methylation of the MLH1 gene promoter. As tumor MMR testing of all incident colorectal and endometrial cancers (universal screening) is becoming increasingly adopted, a growing clinical problem is emerging for individuals who have tumors that show MMR deficiency who are subsequently found not to carry an MMR gene mutation after genetic testing using the current diagnostic approaches (Sanger sequencing and multiplex ligation-dependent probe amplification) and who also show no evidence of MLH1 methylation. The inability to determine the underlying cause of tumor MMR deficiency in these "Lynch-like" or "suspected Lynch syndrome" cases has significant implications on the clinical management of these individuals and their relatives. When the data from published studies are combined, 59% (95% confidence interval [CI]: 55% to 64%) of colorectal cancers and 52% (95% CI: 41% to 62%) of endometrial cancers with MMR deficiency were identified as suspected Lynch syndrome. Recent studies estimated that colorectal cancer risk for relatives of suspected Lynch syndrome cases is lower than for relatives of those with MMR gene mutations, but higher than for relatives of those with tumor MMR deficiency resulting from methylation of the MLH1 gene promoter. The cause of tumor MMR deficiency in suspected Lynch syndrome cases is likely due to either unidentified germline MMR gene mutations, somatic cell mosaicism, or biallelic somatic

Dominant Mutations in S. cerevisiae PMS1 Identify the Mlh1-Pms1 Endonuclease Active Site and an Exonuclease 1-Independent Mismatch Repair Pathway

PubMed Central

Smith, Catherine E.; Mendillo, Marc L.; Bowen, Nikki; Hombauer, Hans; Campbell, Christopher S.; Desai, Arshad; Putnam, Christopher D.; Kolodner, Richard D.

2013-01-01

Lynch syndrome (hereditary nonpolypsis colorectal cancer or HNPCC) is a common cancer predisposition syndrome. Predisposition to cancer in this syndrome results from increased accumulation of mutations due to defective mismatch repair (MMR) caused by a mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2/scPMS1. To better understand the function of Mlh1-Pms1 in MMR, we used Saccharomyces cerevisiae to identify six pms1 mutations (pms1-G683E, pms1-C817R, pms1-C848S, pms1-H850R, pms1-H703A and pms1-E707A) that were weakly dominant in wild-type cells, which surprisingly caused a strong MMR defect when present on low copy plasmids in an exo1Δ mutant. Molecular modeling showed these mutations caused amino acid substitutions in the metal coordination pocket of the Pms1 endonuclease active site and biochemical studies showed that they inactivated the endonuclease activity. This model of Mlh1-Pms1 suggested that the Mlh1-FERC motif contributes to the endonuclease active site. Consistent with this, the mlh1-E767stp mutation caused both MMR and endonuclease defects similar to those caused by the dominant pms1 mutations whereas mutations affecting the predicted metal coordinating residue Mlh1-C769 had no effect. These studies establish that the Mlh1-Pms1 endonuclease is required for MMR in a previously uncharacterized Exo1-independent MMR pathway. PMID:24204293

Dominant mutations in S. cerevisiae PMS1 identify the Mlh1-Pms1 endonuclease active site and an exonuclease 1-independent mismatch repair pathway.

PubMed

Smith, Catherine E; Mendillo, Marc L; Bowen, Nikki; Hombauer, Hans; Campbell, Christopher S; Desai, Arshad; Putnam, Christopher D; Kolodner, Richard D

2013-10-01

Lynch syndrome (hereditary nonpolypsis colorectal cancer or HNPCC) is a common cancer predisposition syndrome. Predisposition to cancer in this syndrome results from increased accumulation of mutations due to defective mismatch repair (MMR) caused by a mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2/scPMS1. To better understand the function of Mlh1-Pms1 in MMR, we used Saccharomyces cerevisiae to identify six pms1 mutations (pms1-G683E, pms1-C817R, pms1-C848S, pms1-H850R, pms1-H703A and pms1-E707A) that were weakly dominant in wild-type cells, which surprisingly caused a strong MMR defect when present on low copy plasmids in an exo1Δ mutant. Molecular modeling showed these mutations caused amino acid substitutions in the metal coordination pocket of the Pms1 endonuclease active site and biochemical studies showed that they inactivated the endonuclease activity. This model of Mlh1-Pms1 suggested that the Mlh1-FERC motif contributes to the endonuclease active site. Consistent with this, the mlh1-E767stp mutation caused both MMR and endonuclease defects similar to those caused by the dominant pms1 mutations whereas mutations affecting the predicted metal coordinating residue Mlh1-C769 had no effect. These studies establish that the Mlh1-Pms1 endonuclease is required for MMR in a previously uncharacterized Exo1-independent MMR pathway.

Epigenetic alteration of mismatch repair genes in the population chronically exposed to arsenic in West Bengal, India.

PubMed

Bhattacharjee, Pritha; Sanyal, Tamalika; Bhattacharjee, Sandip; Bhattacharjee, Pritha

2018-05-01

Arsenic exposure and its adverse health outcome, including the association with cancer risk are well established from several studies across the globe. The present study aims to analyze the epigenetic regulation of key mismatch repair (MMR) genes in the arsenic-exposed population. A case-control study was conducted involving two hundred twenty four (N=224) arsenic exposed [with skin lesion (WSL=110) and without skin lesion (WOSL=114)] and one hundred and two (N=102) unexposed individuals. The methylation status of key MMR genes i.e. MLH1, MSH2, and PMS2 were analyzed using methylation-specific PCR (MSP). The gene expression was studied by qRTPCR. The expression of H3K36me3, which was earlier reported to be an important regulator of MMR pathway, was assessed using ELISA. Arsenic-exposed individuals showed significant promoter hypermethylation (p < 0.0001) of MLH1 and MSH2 compared to those unexposed with consequent down-regulation in their gene expression [MLH1 (p=0.001) and MSH2 (p<0.05)]. However, no significant association was found in expression and methylation of PMS2 with arsenic exposure. We found significant down-regulation of H3K36me3 in the arsenic-exposed group, most significantly in the WSL group (p<0.0001). The expression of SETD2, the methyltransferase of an H3K36me3 moiety was found to be unaltered in arsenic exposure, suggesting the involvement of other regulatory factors yet to be identified. In summary, the epigenetic repression of DNA damage repair genes due to promoter hypermethylation of MLH1 and MSH2 and inefficient recruitment of MMR complex at the site of DNA damage owing to the reduced level of H3K36me3 impairs the mismatch repair pathway that might render the arsenic-exposed individuals more susceptible towards DNA damage and associated cancer risk. Copyright © 2018 Elsevier Inc. All rights reserved.

Loss of DNA mismatch repair imparts a selective advantage in planarian adult stem cells.

PubMed

Hollenbach, Jessica P; Resch, Alissa M; Palakodeti, Dasaradhi; Graveley, Brenton R; Heinen, Christopher D

2011-01-01

Lynch syndrome (LS) leads to an increased risk of early-onset colorectal and other types of cancer and is caused by germline mutations in DNA mismatch repair (MMR) genes. Loss of MMR function results in a mutator phenotype that likely underlies its role in tumorigenesis. However, loss of MMR also results in the elimination of a DNA damage-induced checkpoint/apoptosis activation barrier that may allow damaged cells to grow unchecked. A fundamental question is whether loss of MMR provides pre-cancerous stem cells an immediate selective advantage in addition to establishing a mutator phenotype. To test this hypothesis in an in vivo system, we utilized the planarian Schmidtea mediterranea which contains a significant population of identifiable adult stem cells. We identified a planarian homolog of human MSH2, a MMR gene which is mutated in 38% of LS cases. The planarian Smed-msh2 is expressed in stem cells and some progeny. We depleted Smed-msh2 mRNA levels by RNA-interference and found a striking survival advantage in these animals treated with a cytotoxic DNA alkylating agent compared to control animals. We demonstrated that this tolerance to DNA damage is due to the survival of mitotically active, MMR-deficient stem cells. Our results suggest that loss of MMR provides an in vivo survival advantage to the stem cell population in the presence of DNA damage that may have implications for tumorigenesis.

Loss of DNA Mismatch Repair Imparts a Selective Advantage in Planarian Adult Stem Cells

PubMed Central

Hollenbach, Jessica P.; Resch, Alissa M.; Palakodeti, Dasaradhi; Graveley, Brenton R.; Heinen, Christopher D.

2011-01-01

Lynch syndrome (LS) leads to an increased risk of early-onset colorectal and other types of cancer and is caused by germline mutations in DNA mismatch repair (MMR) genes. Loss of MMR function results in a mutator phenotype that likely underlies its role in tumorigenesis. However, loss of MMR also results in the elimination of a DNA damage-induced checkpoint/apoptosis activation barrier that may allow damaged cells to grow unchecked. A fundamental question is whether loss of MMR provides pre-cancerous stem cells an immediate selective advantage in addition to establishing a mutator phenotype. To test this hypothesis in an in vivo system, we utilized the planarian Schmidtea mediterranea which contains a significant population of identifiable adult stem cells. We identified a planarian homolog of human MSH2, a MMR gene which is mutated in 38% of LS cases. The planarian Smed-msh2 is expressed in stem cells and some progeny. We depleted Smed-msh2 mRNA levels by RNA-interference and found a striking survival advantage in these animals treated with a cytotoxic DNA alkylating agent compared to control animals. We demonstrated that this tolerance to DNA damage is due to the survival of mitotically active, MMR-deficient stem cells. Our results suggest that loss of MMR provides an in vivo survival advantage to the stem cell population in the presence of DNA damage that may have implications for tumorigenesis. PMID:21747960

Diffusion and Binding of Mismatch Repair Protein, MSH2, in Breast Cancer Cells at Different Stages of Neoplastic Transformation

PubMed Central

Sigley, Justin; Jarzen, John; Scarpinato, Karin; Guthold, Martin; Pu, Tracey; Nelli, Daniel; Low, Josiah

2017-01-01

The interior of cells is a highly complex medium, containing numerous organelles, a matrix of different fibers and a viscous, aqueous fluid of proteins and small molecules. The interior of cells is also a highly dynamic medium, in which many components move, either by active transport or passive diffusion. The mobility and localization of proteins inside cells can provide important insights into protein function and also general cellular properties, such as viscosity. Neoplastic transformation affects numerous cellular properties, and our goal was to investigate the diffusional and binding behavior of the important mismatch repair (MMR) protein MSH2 in live human cells at various stages of neoplastic transformation. Toward this end, noncancerous, immortal, tumorigenic, and metastatic mammary epithelial cells were transfected with EGFP and EGFP-tagged MSH2. MSH2 forms two MMR proteins (MutSα and MutSβ) and we assume MSH2 is in the complex MutSα, though our results are similar in either case. Unlike the MutS complexes that bind to nuclear DNA, EGFP diffuses freely. EGFP and MutSα-EGFP diffusion coefficients were determined in the cytoplasm and nucleus of each cell type using fluorescence recovery after photobleaching. Diffusion coefficients were 14–24 μm2/s for EGFP and 3–7 μm2/s for MutSα-EGFP. EGFP diffusion increased in going from noncancerous to immortal cells, indicating a decrease in viscosity, with smaller changes in subsequent stages. MutSα produces an effective diffusion coefficient that, coupled with the free EGFP diffusion measurements, can be used to extract a pure diffusion coefficient and a pseudo-equilibrium constant K*. The MutSα nuclear K* increased sixfold in the first stage of cancer and then decreased in the more advanced stages. The ratio of nuclear to cytoplasmic K*for MutSα increased almost two orders of magnitude in going from noncancerous to immortal cells, suggesting that this quantity may be a sensitive metric for recognizing

Expression of hMSH2 protein of the human DNA mismatch repair system in oral lichen planus

PubMed Central

2004-01-01

Lichen planus is a mucocutaneous disease of inflammatory nature and unknown etiology. It is characterized by a cell-mediated immunological response to induced antigenic change in skin and/or mucosa. The possible malignant transformation of lichen planus remains a subject of controversial discussions in the literature. hMSH2 is one of the human DNA mismatch repair (hMMR) genes and it plays an important role in reducing mutation and maintaining genomic stability. hMSH2 alterations have been reported in oral squamous cell carcinoma and there are evidences suggesting the association between oral lichen planus and squamous cell carcinoma. In this study, we aim to investigate the immunolocalization of hMSH2 protein in oral lichen planus compared to oral normal mucosa epithelium. We examined the expression of hMSH2 protein by immunohistochemistry in twenty-six cases of oral lichen planus. Clinically, 12 of them were categorized into reticular subtype and 14 were atrophic/erosive. Ten cases of normal mucosa were added to the control group. Results showed that the percentage of positive cells to hMSH2 was smaller in reticular (46.54%; p=0,006) and atrophic/erosive (48.79%; p=0,028) subtypes of oral lichen planus compared to normal mucosa (61.29%). The reduced expression of hMSH2 protein in oral lichen planus suggests that this lesion is more susceptible to mutation and therefore facilitate the development of oral squamous cell carcinoma. PMID:15912193

Base excision repair imbalance in colorectal cancer has prognostic value and modulates response to chemotherapy

PubMed Central

Leguisamo, Natalia M.; Gloria, Helena C.; Kalil, Antonio N.; Martins, Talita V.; Azambuja, Daniel B.

2017-01-01

Colorectal cancer (CRC) is prevalent worldwide, and treatment often involves surgery and genotoxic chemotherapy. DNA repair mechanisms, such as base excision repair (BER) and mismatch repair (MMR), may not only influence tumour characteristics and prognosis but also dictate chemotherapy response. Defective MMR contributes to chemoresistance in colorectal cancer. Moreover, BER affects cellular survival by repairing genotoxic base damage in a process that itself can disrupt metabolism. In this study, we characterized BER and MMR gene expression in colorectal tumours and the association between this repair profile with patients’ clinical and pathological features. In addition, we exploited the possible mechanisms underlying the association between altered DNA repair, metabolism and response to chemotherapy. Seventy pairs of sporadic colorectal tumour samples and adjacent non-tumour mucosal specimens were assessed for BER and MMR gene and protein expression and their association with pathological and clinical features. MMR-deficient colon cancer cells (HCT116) transiently overexpressing MPG or XRCC1 were treated with 5-FU or TMZ and evaluated for viability and metabolic intermediate levels. Increase in BER gene and protein expression is associated with more aggressive tumour features and poor pathological outcomes in CRC. However, tumours with reduced MMR gene expression also displayed low MPG, OGG1 and PARP1 expression. Imbalancing BER by overexpression of MPG, but not XRCC1, sensitises MMR-deficient colon cancer cells to 5-FU and TMZ and leads to ATP depletion and lactate accumulation. MPG overexpression alters DNA repair and metabolism and is a potential strategy to overcome 5-FU chemotherapeutic resistance in MMR-deficient CRC. PMID:28903334

Oligonucleotide-directed mutagenesis screen to identify pathogenic Lynch syndrome-associated MSH2 DNA mismatch repair gene variants

PubMed Central

Houlleberghs, Hellen; Dekker, Marleen; Lantermans, Hildo; Kleinendorst, Roos; Dubbink, Hendrikus Jan; Hofstra, Robert M. W.; Verhoef, Senno; te Riele, Hein

2016-01-01

Single-stranded DNA oligonucleotides can achieve targeted base-pair substitution with modest efficiency but high precision. We show that “oligo targeting” can be used effectively to study missense mutations in DNA mismatch repair (MMR) genes. Inherited inactivating mutations in DNA MMR genes are causative for the cancer predisposition Lynch syndrome (LS). Although overtly deleterious mutations in MMR genes can clearly be ascribed as the cause of LS, the functional implications of missense mutations are often unclear. We developed a genetic screen to determine the pathogenicity of these variants of uncertain significance (VUS), focusing on mutator S homolog 2 (MSH2). VUS were introduced into the endogenous Msh2 gene of mouse embryonic stem cells by oligo targeting. Subsequent selection for MMR-deficient cells using the guanine analog 6-thioguanine allowed the detection of MMR-abrogating VUS. The screen was able to distinguish weak and strong pathogenic variants from polymorphisms and was used to investigate 59 Msh2 VUS. Nineteen of the 59 VUS were identified as pathogenic. Functional assays revealed that 14 of the 19 detected variants fully abrogated MMR activity and that five of the detected variants attenuated MMR activity. Implementation of the screen in clinical practice allows proper counseling of mutation carriers and treatment of their tumors. PMID:26951660

Prognostic value of MACC1 and proficient mismatch repair status for recurrence risk prediction in stage II colon cancer patients: the BIOGRID studies.

PubMed

Rohr, U-P; Herrmann, P; Ilm, K; Zhang, H; Lohmann, S; Reiser, A; Muranyi, A; Smith, J; Burock, S; Osterland, M; Leith, K; Singh, S; Brunhoeber, P; Bowermaster, R; Tie, J; Christie, M; Wong, H-L; Waring, P; Shanmugam, K; Gibbs, P; Stein, U

2017-08-01

We assessed the novel MACC1 gene to further stratify stage II colon cancer patients with proficient mismatch repair (pMMR). Four cohorts with 596 patients were analyzed: Charité 1 discovery cohort was assayed for MACC1 mRNA expression and MMR in cryo-preserved tumors. Charité 2 comparison cohort was used to translate MACC1 qRT-PCR analyses to FFPE samples. In the BIOGRID 1 training cohort MACC1 mRNA levels were related to MACC1 protein levels from immunohistochemistry in FFPE sections; also analyzed for MMR. Chemotherapy-naïve pMMR patients were stratified by MACC1 mRNA and protein expression to establish risk groups based on recurrence-free survival (RFS). Risk stratification from BIOGRID 1 was confirmed in the BIOGRID 2 validation cohort. Pooled BIOGRID datasets produced a best effect-size estimate. In BIOGRID 1, using qRT-PCR and immunohistochemistry for MACC1 detection, pMMR/MACC1-low patients had a lower recurrence probability versus pMMR/MACC1-high patients (5-year RFS of 92% and 67% versus 100% and 68%, respectively). In BIOGRID 2, longer RFS was confirmed for pMMR/MACC1-low versus pMMR/MACC1-high patients (5-year RFS of 100% versus 90%, respectively). In the pooled dataset, 6.5% of patients were pMMR/MACC1-low with no disease recurrence, resulting in a 17% higher 5-year RFS [95% confidence interval (CI) (12.6%-21.3%)] versus pMMR/MACC1-high patients (P = 0.037). Outcomes were similar for pMMR/MACC1-low and deficient MMR (dMMR) patients (5-year RFS of 100% and 96%, respectively). MACC1 expression stratifies colon cancer patients with unfavorable pMMR status. Stage II colon cancer patients with pMMR/MACC1-low tumors have a similar favorable prognosis to those with dMMR with potential implications for the role of adjuvant therapy. © The Author 2017. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Construction and characterization of mismatch-containing circular DNA molecules competent for assessment of nick-directed human mismatch repair in vitro.

PubMed

Larson, Erik D; Nickens, David; Drummond, James T

2002-02-01

The ability of cell-free extracts to correct DNA mismatches has been demonstrated in both prokaryotes and eukaryotes. Such an assay requires a template containing both a mismatch and a strand discrimination signal, and the multi-step construction process can be technically difficult. We have developed a three-step procedure for preparing DNA heteroduplexes containing a site-specific nick. The mismatch composition, sequence context, distance to the strand signal, and the means for assessing repair in each strand are adjustable features built into a synthetic oligonucleotide. Controlled ligation events involving three of the four DNA strands incorporate the oligonucleotide into a circular template and generate the repair-directing nick. Mismatch correction in either strand of a prototype G.T mismatch was achieved by placing a nick 10-40 bp away from the targeted base. This proximity of nick and mismatch represents a setting where repair has not been well characterized, but the presence of a nick was shown to be essential, as was the MSH2/MSH6 heterodimer, although low levels of repair occurred in extract defective in each protein. All repair events were inhibited by a peptide that interacts with proliferating cell nuclear antigen and inhibits both mismatch repair and long-patch replication.

Determinants of Base-Pair Substitution Patterns Revealed by Whole-Genome Sequencing of DNA Mismatch Repair Defective Escherichia coli.

PubMed

Foster, Patricia L; Niccum, Brittany A; Popodi, Ellen; Townes, Jesse P; Lee, Heewook; MohammedIsmail, Wazim; Tang, Haixu

2018-06-15

Mismatch repair (MMR) is a major contributor to replication fidelity, but its impact varies with sequence context and the nature of the mismatch. Mutation accumulation experiments followed by whole-genome sequencing of MMR-defective E. coli strains yielded ≈30,000 base-pair substitutions, revealing mutational patterns across the entire chromosome. The base-pair substitution spectrum was dominated by A:T > G:C transitions, which occurred predominantly at the center base of 5'N A C3'+5'G T N3' triplets. Surprisingly, growth on minimal medium or at low temperature attenuated these mutations. Mononucleotide runs were also hotspots for base-pair substitutions, and the rate at which these occurred increased with run length. Comparison with ≈2000 base-pair substitutions accumulated in MMR-proficient strains revealed that both kinds of hotspots appeared in the wild-type spectrum and so are likely to be sites of frequent replication errors. In MMR-defective strains transitions were strand biased, occurring twice as often when A and C rather than T and G were on the lagging-strand template. Loss of nucleotide diphosphate kinase increases the cellular concentration of dCTP, which resulted in increased rates of mutations due to misinsertion of C opposite A and T. In an mmr ndk double mutant strain, these mutations were more frequent when the template A and T were on the leading strand, suggesting that lagging-strand synthesis was more error-prone or less well corrected by proofreading than was leading strand synthesis. Copyright © 2018, Genetics.

Destabilization of the MutSα's protein-protein interface due to binding to the DNA adduct induced by anticancer agent carboplatin via molecular dynamics simulations.

PubMed

Negureanu, Lacramioara; Salsbury, Freddie R

2013-11-01

DNA mismatch repair (MMR) proteins maintain genetic integrity in all organisms by recognizing and repairing DNA errors. Such alteration of hereditary information can lead to various diseases, including cancer. Besides their role in DNA repair, MMR proteins detect and initiate cellular responses to certain type of DNA damage. Its response to the damaged DNA has made the human MMR pathway a useful target for anticancer agents such as carboplatin. This study indicates that strong, specific interactions at the interface of MutSα in response to the mismatched DNA recognition are replaced by weak, non-specific interactions in response to the damaged DNA recognition. Data suggest a severe impairment of the dimerization of MutSα in response to the damaged DNA recognition. While the core of MutSα is preserved in response to the damaged DNA recognition, the loss of contact surface and the rearrangement of contacts at the protein interface suggest a different packing in response to the damaged DNA recognition. Coupled in response to the mismatched DNA recognition, interaction energies, hydrogen bonds, salt bridges, and solvent accessible surface areas at the interface of MutSα and within the subunits are uncoupled or asynchronously coupled in response to the damaged DNA recognition. These pieces of evidence suggest that the loss of a synchronous mode of response in the MutSα's surveillance for DNA errors would possibly be one of the mechanism(s) of signaling the MMR-dependent programed cell death much wanted in anticancer therapies. The analysis was drawn from dynamics simulations.

Constitutional mismatch repair deficiency in a healthy child: On the spot diagnosis?

PubMed

Suerink, M; Potjer, T P; Versluijs, A B; Ten Broeke, S W; Tops, C M; Wimmer, K; Nielsen, M

2018-01-01

Constitutional mismatch repair deficiency (CMMRD) is a rare, recessively inherited childhood cancer predisposition syndrome caused by biallelic germline mutations in one of the mismatch repair genes. The CMMRD phenotype overlaps with that of neurofibromatosis type 1 (NF1), since many patients have multiple café-au-lait macules (CALM) and other NF1 signs, but no germline NF1 mutations. We report of a case of a healthy 6-year-old girl who fulfilled the diagnostic criteria of NF1 with >6 CALM and freckling. Since molecular genetic testing was unable to confirm the diagnosis of NF1 or Legius syndrome and the patient was a child of consanguineous parents, we suspected CMMRD and found a homozygous PMS2 mutation that impairs MMR function. Current guidelines advise testing for CMMRD only in cancer patients. However, this case illustrates that including CMMRD in the differential diagnosis in suspected sporadic NF1 without causative NF1 or SPRED1 mutations may facilitate identification of CMMRD prior to cancer development. We discuss the advantages and potential risks of this CMMRD testing scenario. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Destabilization of the MutSα’s protein-protein interface due to binding to the DNA adduct induced by anticancer agent Carboplatin via molecular dynamics simulations

PubMed Central

Negureanu, Lacramioara; Salsbury, Freddie R

2013-01-01

DNA mismatch repair (MMR) proteins maintain genetic integrity in all organisms by recognizing and repairing DNA errors. Such alteration of hereditary information can lead to various diseases, including cancer. Besides their role in DNA repair, MMR proteins detect and initiate cellular responses to certain type of DNA damage. Its response to the damaged DNA has made the human MMR pathway a useful target for anticancer agents such as carboplatin. This study indicates that strong, specific interactions at the interface of MutSα in response to the mismatched DNA recognition are replaced by weak, non-specific interactions in response to the damaged DNA recognition. Data suggest a severe impairment of the dimerization of MutSα in response to the damaged DNA recognition. While the core of MutSα is preserved in response to the damaged DNA recognition, the loss of contact surface and the rearrangement of contacts at the protein interface suggest a different packing in response to the damaged DNA recognition. Coupled in response to the mismatched DNA recognition, interaction energies, hydrogen bonds, salt bridges, and solvent accessible surface areas at the interface of MutSα and within the subunits are uncoupled or asynchronously coupled in response to the damaged DNA recognition. These pieces of evidence suggest that the loss of a synchronous mode of response in the MutSα’s surveillance for DNA errors would possible be one of the mechanism(s) of signaling the MMR-dependent programed cell death much wanted in anticancer therapies. The analysis was drawn from dynamics simulations. PMID:24061854

Functional role of DNA mismatch repair gene PMS2 in prostate cancer cells.

PubMed

Fukuhara, Shinichiro; Chang, Inik; Mitsui, Yozo; Chiyomaru, Takeshi; Yamamura, Soichiro; Majid, Shahana; Saini, Sharanjot; Deng, Guoren; Gill, Ankurpreet; Wong, Darryn K; Shiina, Hiroaki; Nonomura, Norio; Lau, Yun-Fai C; Dahiya, Rajvir; Tanaka, Yuichiro

2015-06-30

DNA mismatch repair (MMR) enzymes act as proofreading complexes that maintains genomic integrity and MMR-deficient cells show an increased mutation rate. MMR has also been shown to influence cell signaling and the regulation of tumor development. MMR consists of various genes and includes post-meiotic segregation (PMS) 2 which is a vital component of mutL-alpha. In prostate, the functional role of this gene has never been reported and in this study, our aim was to investigate the effect of PMS2 on growth properties of prostate cancer (PCa) cells. Previous studies have shown PMS2 to be deficient in DU145 cells and this lack of expression was confirmed by Western blotting whereas normal prostatic PWR-1E and RWPE-1 cells expressed this gene. PMS2 effects on various growth properties of DU145 were then determined by creating stable gene transfectants. Interestingly, PMS2 caused decreased cell proliferation, migration, invasion, and in vivo growth; and increased apoptosis as compared to vector control. We further analyzed genes affected by PMS2 expression and observe the apoptosis-related TMS1 gene to be significantly upregulated whereas anti-apoptotic BCL2A1 was downregulated. These results demonstrate a functional role for PMS2 to protect against PCa progression by enhancing apoptosis of PCa cells.

Functional role of DNA mismatch repair gene PMS2 in prostate cancer cells

PubMed Central

Mitsui, Yozo; Chiyomaru, Takeshi; Yamamura, Soichiro; Majid, Shahana; Saini, Sharanjot; Deng, Guoren; Gill, Ankurpreet; Wong, Darryn K.; Shiina, Hiroaki; Nonomura, Norio; Lau, Yun-Fai C.; Dahiya, Rajvir; Tanaka, Yuichiro

2015-01-01

DNA mismatch repair (MMR) enzymes act as proofreading complexes that maintains genomic integrity and MMR-deficient cells show an increased mutation rate. MMR has also been shown to influence cell signaling and the regulation of tumor development. MMR consists of various genes and includes post-meiotic segregation (PMS) 2 which is a vital component of mutL-alpha. In prostate, the functional role of this gene has never been reported and in this study, our aim was to investigate the effect of PMS2 on growth properties of prostate cancer (PCa) cells. Previous studies have shown PMS2 to be deficient in DU145 cells and this lack of expression was confirmed by Western blotting whereas normal prostatic PWR-1E and RWPE-1 cells expressed this gene. PMS2 effects on various growth properties of DU145 were then determined by creating stable gene transfectants. Interestingly, PMS2 caused decreased cell proliferation, migration, invasion, and in vivo growth; and increased apoptosis as compared to vector control. We further analyzed genes affected by PMS2 expression and observe the apoptosis-related TMS1 gene to be significantly upregulated whereas anti-apoptotic BCL2A1 was downregulated. These results demonstrate a functional role for PMS2 to protect against PCa progression by enhancing apoptosis of PCa cells. PMID:26036629

Optic pathway glioma as part of a constitutional mismatch-repair deficiency syndrome in a patient meeting the criteria for neurofibromatosis type 1.

PubMed

Yeung, Jacky T; Pollack, Ian F; Shah, Sapana; Jaffe, Ronald; Nikiforova, Marina; Jakacki, Regina I

2013-01-01

Patients with constitutional mismatch repair-deficiency (CMMR-D) caused by the biallelic deletions of mismatch repair (MMR) genes have a high likelihood of developing malignancies of the bone marrow, bowel, and brain. Affected individuals often have phenotypic features of neurofibromatosis type 1 (NF-1), including café-au-lait spots. Optic pathway gliomas (OPGs), a common manifestation of NF-1, have not been reported. We report the case of a 3-year-old male with an extensive OPG who met the diagnostic criteria for NF-1. He was subsequently found to have multiple colonic polyps and bi-allelic loss of PMS2. Testing for NF-1 was negative. Copyright © 2012 Wiley Periodicals, Inc.

Mismatch repair factor MSH2-MSH3 binds and alters the conformation of branched DNA structures predicted to form during genetic recombination.

PubMed

Surtees, Jennifer A; Alani, Eric

2006-07-14

Genetic studies in Saccharomyces cerevisiae predict that the mismatch repair (MMR) factor MSH2-MSH3 binds and stabilizes branched recombination intermediates that form during single strand annealing and gene conversion. To test this model, we constructed a series of DNA substrates that are predicted to form during these recombination events. We show in an electrophoretic mobility shift assay that S. cerevisiae MSH2-MSH3 specifically binds branched DNA substrates containing 3' single-stranded DNA and that ATP stimulates its release from these substrates. Chemical footprinting analyses indicate that MSH2-MSH3 specifically binds at the double-strand/single-strand junction of branched substrates, alters its conformation and opens up the junction. Therefore, MSH2-MSH3 binding to its substrates creates a unique nucleoprotein structure that may signal downstream steps in repair that include interactions with MMR and nucleotide excision repair factors.

Café-au-lait macules and pediatric malignancy caused by biallelic mutations in the DNA mismatch repair (MMR) gene PMS2.

PubMed

Jackson, Carl-Christian; Holter, Spring; Pollett, Aaron; Clendenning, Mark; Chou, Shirley; Senter, Leigha; Ramphal, Raveena; Gallinger, Steven; Boycott, Kym

2008-06-01

A 14-year-old male presented with a T4 sigmoid adenocarcinoma, <10 colonic adenomas and multiple café-au-lait macules. Family history was not suggestive of a dominant hereditary form of colorectal cancer. Evaluation of the tumor revealed abnormal immunohistochemical staining of the PMS2 protein and high frequency microsatellite instability. Germline analysis identified biallelic PMS2 missense mutations. A new cancer syndrome caused by biallelic mutations in the mismatch repair genes, including PMS2, is now emerging and is characterized by café-au-lait macules, colonic polyps and a distinctive tumor spectrum. (c) 2007 Wiley-Liss, Inc.

Promoter methylation and expression of MGMT and the DNA mismatch repair genes MLH1, MSH2, MSH6 and PMS2 in paired primary and recurrent glioblastomas.

PubMed

Felsberg, Jörg; Thon, Niklas; Eigenbrod, Sabina; Hentschel, Bettina; Sabel, Michael C; Westphal, Manfred; Schackert, Gabriele; Kreth, Friedrich Wilhelm; Pietsch, Torsten; Löffler, Markus; Weller, Michael; Reifenberger, Guido; Tonn, Jörg C

2011-08-01

Epigenetic silencing of the O(6) -methylguanine-DNA methyltransferase (MGMT) gene promoter is associated with prolonged survival in glioblastoma patients treated with temozolomide (TMZ). We investigated whether glioblastoma recurrence is associated with changes in the promoter methylation status and the expression of MGMT and the DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2 in pairs of primary and recurrent glioblastomas of 80 patients, including 64 patients treated with radiotherapy and TMZ after the first operation. Among the primary tumors, the MGMT promoter was methylated in 31 patients and unmethylated in 49 patients. In 71 patients (89%), the MGMT promoter methylation status of the primary tumor was retained at recurrence. MGMT promoter methylation, but not MGMT protein expression, was associated with longer progression-free survival, overall survival and postrecurrence survival (PRS). Moreover, PRS was increased under salvage chemotherapy. Investigation of primary and recurrent glioblastomas of 43 patients did not identify promoter methylation in any of the four MMR genes. However, recurrent glioblastomas demonstrated significantly lower MSH2, MSH6 and PMS2 protein expression as detected by immunohistochemistry. In conclusion, reduced expression of MMR proteins, but not changes in MGMT promoter methylation, is characteristic of glioblastomas recurring after the current standards of care. Copyright © 2011 UICC.

Pms2 and uracil-DNA glycosylases act jointly in the mismatch repair pathway to generate Ig gene mutations at A-T base pairs.

PubMed

Girelli Zubani, Giulia; Zivojnovic, Marija; De Smet, Annie; Albagli-Curiel, Olivier; Huetz, François; Weill, Jean-Claude; Reynaud, Claude-Agnès; Storck, Sébastien

2017-04-03

During somatic hypermutation (SHM) of immunoglobulin genes, uracils introduced by activation-induced cytidine deaminase are processed by uracil-DNA glycosylase (UNG) and mismatch repair (MMR) pathways to generate mutations at G-C and A-T base pairs, respectively. Paradoxically, the MMR-nicking complex Pms2/Mlh1 is apparently dispensable for A-T mutagenesis. Thus, how detection of U:G mismatches is translated into the single-strand nick required for error-prone synthesis is an open question. One model proposed that UNG could cooperate with MMR by excising a second uracil in the vicinity of the U:G mismatch, but it failed to explain the low impact of UNG inactivation on A-T mutagenesis. In this study, we show that uracils generated in the G1 phase in B cells can generate equal proportions of A-T and G-C mutations, which suggests that UNG and MMR can operate within the same time frame during SHM. Furthermore, we show that Ung -/- Pms2 -/- mice display a 50% reduction in mutations at A-T base pairs and that most remaining mutations at A-T bases depend on two additional uracil glycosylases, thymine-DNA glycosylase and SMUG1. These results demonstrate that Pms2/Mlh1 and multiple uracil glycosylases act jointly, each one with a distinct strand bias, to enlarge the immunoglobulin gene mutation spectrum from G-C to A-T bases. © 2017 Girelli Zubani et al.

Pms2 and uracil-DNA glycosylases act jointly in the mismatch repair pathway to generate Ig gene mutations at A-T base pairs

PubMed Central

De Smet, Annie; Albagli-Curiel, Olivier; Huetz, François; Weill, Jean-Claude

2017-01-01

During somatic hypermutation (SHM) of immunoglobulin genes, uracils introduced by activation-induced cytidine deaminase are processed by uracil-DNA glycosylase (UNG) and mismatch repair (MMR) pathways to generate mutations at G-C and A-T base pairs, respectively. Paradoxically, the MMR-nicking complex Pms2/Mlh1 is apparently dispensable for A-T mutagenesis. Thus, how detection of U:G mismatches is translated into the single-strand nick required for error-prone synthesis is an open question. One model proposed that UNG could cooperate with MMR by excising a second uracil in the vicinity of the U:G mismatch, but it failed to explain the low impact of UNG inactivation on A-T mutagenesis. In this study, we show that uracils generated in the G1 phase in B cells can generate equal proportions of A-T and G-C mutations, which suggests that UNG and MMR can operate within the same time frame during SHM. Furthermore, we show that Ung−/−Pms2−/− mice display a 50% reduction in mutations at A-T base pairs and that most remaining mutations at A-T bases depend on two additional uracil glycosylases, thymine-DNA glycosylase and SMUG1. These results demonstrate that Pms2/Mlh1 and multiple uracil glycosylases act jointly, each one with a distinct strand bias, to enlarge the immunoglobulin gene mutation spectrum from G-C to A-T bases. PMID:28283534

The MutSβ complex is a modulator of p53-driven tumorigenesis through its functions in both DNA double-strand break repair and mismatch repair.

PubMed

van Oers, J M M; Edwards, Y; Chahwan, R; Zhang, W; Smith, C; Pechuan, X; Schaetzlein, S; Jin, B; Wang, Y; Bergman, A; Scharff, M D; Edelmann, W

2014-07-24

Loss of the DNA mismatch repair (MMR) protein MSH3 leads to the development of a variety of tumors in mice without significantly affecting survival rates, suggesting a modulating role for the MutSβ (MSH2-MSH3) complex in late-onset tumorigenesis. To better study the role of MSH3 in tumor progression, we crossed Msh3(-/-) mice onto a tumor predisposing p53-deficient background. Survival of Msh3/p53 mice was not reduced compared with p53 single mutant mice; however, the tumor spectrum changed significantly from lymphoma to sarcoma, indicating MSH3 as a potent modulator of p53-driven tumorigenesis. Interestingly, Msh3(-/-) mouse embryonic fibroblasts displayed increased chromatid breaks and persistence of γH2AX foci following ionizing radiation, indicating a defect in DNA double-strand break repair (DSBR). Msh3/p53 tumors showed increased loss of heterozygosity, elevated genome-wide copy-number variation and a moderate microsatellite instability phenotype compared with Msh2/p53 tumors, revealing that MSH2-MSH3 suppresses tumorigenesis by maintaining chromosomal stability. Our results show that the MSH2-MSH3 complex is important for the suppression of late-onset tumors due to its roles in DNA DSBR as well as in DNA MMR. Further, they demonstrate that MSH2-MSH3 suppresses chromosomal instability and modulates the tumor spectrum in p53-deficient tumorigenesis and possibly has a role in other chromosomally unstable tumors as well.

Proximity to AGCT sequences dictates MMR-independent versus MMR-dependent mechanisms for AID-induced mutation via UNG2

PubMed Central

Thientosapol, Eddy Sanchai; Sharbeen, George; Lau, K.K. Edwin; Bosnjak, Daniel; Durack, Timothy; Stevanovski, Igor; Weninger, Wolfgang

2017-01-01

Abstract AID deaminates C to U in either strand of Ig genes, exclusively producing C:G/G:C to T:A/A:T transition mutations if U is left unrepaired. Error-prone processing by UNG2 or mismatch repair diversifies mutation, predominantly at C:G or A:T base pairs, respectively. Here, we show that transversions at C:G base pairs occur by two distinct processing pathways that are dictated by sequence context. Within and near AGCT mutation hotspots, transversion mutation at C:G was driven by UNG2 without requirement for mismatch repair. Deaminations in AGCT were refractive both to processing by UNG2 and to high-fidelity base excision repair (BER) downstream of UNG2, regardless of mismatch repair activity. We propose that AGCT sequences resist faithful BER because they bind BER-inhibitory protein(s) and/or because hemi-deaminated AGCT motifs innately form a BER-resistant DNA structure. Distal to AGCT sequences, transversions at G were largely co-dependent on UNG2 and mismatch repair. We propose that AGCT-distal transversions are produced when apyrimidinic sites are exposed in mismatch excision patches, because completion of mismatch repair would require bypass of these sites. PMID:28039326

A massive parallel sequencing workflow for diagnostic genetic testing of mismatch repair genes

PubMed Central

Hansen, Maren F; Neckmann, Ulrike; Lavik, Liss A S; Vold, Trine; Gilde, Bodil; Toft, Ragnhild K; Sjursen, Wenche

2014-01-01

The purpose of this study was to develop a massive parallel sequencing (MPS) workflow for diagnostic analysis of mismatch repair (MMR) genes using the GS Junior system (Roche). A pathogenic variant in one of four MMR genes, (MLH1, PMS2, MSH6, and MSH2), is the cause of Lynch Syndrome (LS), which mainly predispose to colorectal cancer. We used an amplicon-based sequencing method allowing specific and preferential amplification of the MMR genes including PMS2, of which several pseudogenes exist. The amplicons were pooled at different ratios to obtain coverage uniformity and maximize the throughput of a single-GS Junior run. In total, 60 previously identified and distinct variants (substitutions and indels), were sequenced by MPS and successfully detected. The heterozygote detection range was from 19% to 63% and dependent on sequence context and coverage. We were able to distinguish between false-positive and true-positive calls in homopolymeric regions by cross-sample comparison and evaluation of flow signal distributions. In addition, we filtered variants according to a predefined status, which facilitated variant annotation. Our study shows that implementation of MPS in routine diagnostics of LS can accelerate sample throughput and reduce costs without compromising sensitivity, compared to Sanger sequencing. PMID:24689082

DNA mismatch repair gene polymorphisms affect survival in pancreatic cancer.

PubMed

Dong, Xiaoqun; Li, Yanan; Hess, Kenneth R; Abbruzzese, James L; Li, Donghui

2011-01-01

DNA mismatch repair (MMR) maintains genomic stability and mediates cellular response to DNA damage. We aim to demonstrate whether MMR genetic variants affect overall survival (OS) in pancreatic cancer. Using the Sequenom method in genomic DNA, we retrospectively genotyped 102 single-nucleotide polymorphisms (SNPs) of 13 MMR genes from 706 patients with pancreatic adenocarcinoma seen at The University of Texas MD Anderson Cancer Center. Association between genotype and OS was evaluated using multivariable Cox proportional hazard regression models. At a false discovery rate of 1% (p ≤ .0015), 15 SNPs of EXO1, MLH1, MSH2, MSH3, MSH6, PMS2, PMS2L3, TP73, and TREX1 in patients with localized disease (n = 333) and 6 SNPs of MSH3, MSH6, and TP73 in patients with locally advanced or metastatic disease (n = 373) were significantly associated with OS. In multivariable Cox proportional hazard regression models, SNPs of EXO1, MSH2, MSH3, PMS2L3, and TP73 in patients with localized disease, MSH2, MSH3, MSH6, and TP73 in patients with locally advanced or metastatic disease, and EXO1, MGMT, MSH2, MSH3, MSH6, PMS2L3, and TP73 in all patients remained significant predictors for OS (p ≤ .0015) after adjusting for all clinical predictors and all SNPs with p ≤ .0015 in single-locus analysis. Sixteen haplotypes of EXO1, MLH1, MSH2, MSH3, MSH6, PMS2, PMS2L3, RECQL, TP73, and TREX1 significantly correlated with OS in all patients (p ≤ .001). MMR gene variants may have potential value as prognostic markers for OS in pancreatic cancer patients.

Topoisomerase-1 and -2A gene copy numbers are elevated in mismatch repair-proficient colorectal cancers.

PubMed

Sønderstrup, Ida Marie Heeholm; Nygård, Sune Boris; Poulsen, Tim Svenstrup; Linnemann, Dorte; Stenvang, Jan; Nielsen, Hans Jørgen; Bartek, Jiri; Brünner, Nils; Nørgaard, Peter; Riis, Lene

2015-06-01

Topoisomerase 1 (TOP1) and 2A (TOP2A) are potential predictive biomarkers for irinotecan and anthracycline treatment, respectively, in colorectal cancer (CRC), and we have recently reported a high frequency of gene gain of the TOP1 and TOP2A genes in CRC. Furthermore, Mismatch Repair (MMR) subtypes of CRC have been associated with benefit from adjuvant chemotherapy of primary CRC. Given the involvement of the topoisomerase enzymes in DNA replication and repair, we raised the hypothesis that an association may exist between TOP gene copy numbers and MMR proficiency/deficiency in CRC. Test cohort: FISH analysis with an in-house TOP1/CEN20 probe mix and a commercially available TOP2A/CEN17 (Dako, Glostrup, Denmark) probe mix was performed on archival formalin fixed paraffin embedded (FFPE) tissue samples from 18 patients with proficient MMR (pMMR) CRC and 18 patients with deficient MMR (dMMR) CRC. TOP1 and TOP2A gene copy numbers and their ratios per nucleus were correlated with MMR status using the Mann-Whitney test. Validation cohort: FFPE samples from 154 patients with primary stage III CRC (originally included in the RANX05 study) were classified according to MMR status by immunohistochemical analysis using validated antibodies for MLH1, MLH2, MSH6 and PMS2, and information on TOP1, CEN20, TOP2A and CEN17 status was previously published for this cohort. The observed TOP1 gene copy numbers in the 36 CRC test cohort were significantly greater (p < 0.01) in the pMMR subgroup (mean: 3.84, SD: 2.03) than in the dMMR subgroup (mean: 1.50, SD: 0.12). Similarly, the TOP2A copy numbers were significantly greater (p < 0.01) in the pMMR subgroup (mean: 1.99, SD: 0.52) than in the dMMR subgroup (mean: 1.52, SD: 0.10). These findings were confirmed in the validation cohort, where in the pMMR subgroup 51% had ≥2 extra TOP1 copies per cell, while all tumors classified as dMMR had diploid TOP1 status and mean TOP2A copy numbers were 2.30 (SD: 1.36) and 1.80 (SD: 0.31) (p = 0

Association between mismatch repair gene MSH3 codons 1036 and 222 polymorphisms and sporadic prostate cancer in the Iranian population.

PubMed

Jafary, Fariba; Salehi, Mansoor; Sedghi, Maryam; Nouri, Nayereh; Jafary, Farzaneh; Sadeghi, Farzaneh; Motamedi, Shima; Talebi, Maede

2012-01-01

The mismatch repair system (MMR) is a post-replicative DNA repair mechanism whose defects can lead to cancer. The MSH3 protein is an essential component of the system. We postulated that MSH3 gene polymorphisms might therefore be associated with prostate cancer (PC). We studied MSH3 codon 222 and MSH3 codon 1036 polymorphisms in a group of Iranian sporadic PC patients. A total of 60 controls and 18 patients were assessed using the polymerase chain reaction and single strand conformational polymorphism. For comparing the genotype frequencies of patients and controls the chi-square test was applied. The obtained result indicated that there was significantly association between G/A genotype of MSH3 codon 222 and G/G genotype of MSH3 codon 1036 with an increased PC risk (P=0.012 and P=0.02 respectively). Our results demonstrated that MSH3 codon 222 and MSH3 codon 1036 polymorphisms may be risk factors for sporadic prostate cancer in the Iranian population.

Four novel germline mutations in the MLH1 and PMS2 mismatch repair genes in patients with hereditary nonpolyposis colorectal cancer.

PubMed

Montazer Haghighi, Mahdi; Radpour, Ramin; Aghajani, Katayoun; Zali, Narges; Molaei, Mahsa; Zali, Mohammad Reza

2009-08-01

Hereditary nonpolyposis colorectal cancer (HNPCC) is the most common cause of early onset hereditary colorectal cancer. In the majority of HNPCC families, microsatellite instability (MSI) and germline mutation in one of the DNA mismatch repair (MMR) genes are found. The entire coding sequence of MMR genes (MLH1, MLH2, MLH6, and PMS2) was analyzed using direct sequencing. Also, tumor tests were done as MSI and immunohistochemistry testing. We were able to find three novel MLH1 and one novel PMS2 germline mutations in three Iranian HNPCC patients. The first was a transversion mutation c.346A>C (T116P) and happened in the highly conserved HATPase-c region of MLH1 protein. The second was a transversion mutation c.736A>T (I246L), which caused an amino acid change of isoleucine to leucine. The third mutation (c.2145,6 delTG) was frameshift and resulted in an immature stop codon in five codons downstream. All of these three mutations were detected in the MLH1 gene. The other mutation was a transition mutation, c.676G>A (G207E), which has been found in exon six of the PMS2 gene and caused an amino acid change of glycine to glutamic acid. MSI assay revealed high instability in microsatellite for two patients and microsatellite stable for one patient. In all patients, an abnormal expression of the MMR proteins in HNPCC was related to the above novel mutations.

The Heterogeneity Between Lynch-Associated and Sporadic MMR Deficiency in Colorectal Cancers.

PubMed

Liu, Guo-Chen; Liu, Ran-Yi; Yan, Jun-Ping; An, Xin; Jiang, Wu; Ling, Yi-Hong; Chen, Jie-Wei; Bei, Jin-Xin; Zuo, Xiao-Yu; Cai, Mu-Yan; Liu, Ze-Xian; Zuo, Zhi-Xiang; Liu, Ji-Hong; Pan, Zhi-Zhong; Ding, Pei-Rong

2018-02-20

Previous studies demonstrated that prognosis of germline deficiency in mismatch repair protein (dMMR) was different from that of sporadic dMMR. The underlying mechanism has not been studied. From a prospectively maintained database, we collected dMMR colorectal cancer (CRC) patients identified by postoperative immunohistochemistry screening. According to genetic test, patients were grouped as Lynch-associated or sporadic dMMR. We compared the clinical-pathological features, prognosis, and immunoreactive differences between the two groups. By whole-exome sequencing and neoantigen detection pipeline, mutational frequencies and neoantigen burdens were also compared. All statistical tests were two-sided. Sixty-seven sporadic dMMR and 85 Lynch-associated CRC patients were included in the study. Sporadic dMMR patients were older (P < .001) and their tumors were poorly differentiated (P = .03). The survival was better in the Lynch-associated group (P = .001). After adjustment, the difference still remained statistically significant (hazard ratio = 0.29, 95% confidence interval = 0.09 to 0.95, P = .04). The scores of Crohn's-like reaction (CRO; P < .001), immunoreactions in the invasive margin (IM; P = .01), tumor stroma (TS; P = .009), and cancer nest (CN; P = .02) of the Lynch-associated group were statistically significantly higher. The numbers of CD3+, CD8+, Foxp3+ tumor-infiltrating lymphocytes (TILs) in IM; CD3+, CD4+ TILs in TS; and CD3+, CD4+, CD8+ TILs in CN were statistically significantly higher in Lynch-associated dMMR patients. Based on the 16 patients who under went whole-exome sequencing, there were also more somatic mutations and neoantigen burdens in the Lynch-associated group compared with the sporadic dMMR group (439/pt vs 68/pt, P = .006; 628/pt vs 97/pt, P = .009). There are heterogeneities in dMMR CRCs. Lynch-associated dMMR patients present with more somatic mutations and neoantigens compared with sporadic dMMR, which probably results in stronger

Involvement of Mismatch Repair in the Reciprocal Control of Motility and Adherence of Uropathogenic Escherichia coli

PubMed Central

Cooper, Lauren A.; Simmons, Lyle A.

2012-01-01

Type 1 fimbriae and flagella, two surface organelles critical for colonization of the urinary tract by uropathogenic Escherichia coli (UPEC), mediate opposing virulence objectives. Type 1 fimbriae facilitate adhesion to mucosal cells and promote bacterial persistence in the urinary tract, while flagella propel bacteria through urine and along mucous layers during ascension to the upper urinary tract. Using a transposon screen of the E. coli CFT073 fim locked-ON (L-ON) mutant, a construct that constitutively expresses type 1 fimbriae and represses motility, we identified six mutants that exhibited a partial restoration of motility. Among these six mutated genes was mutS, which encodes a component of the methyl-directed mismatch repair (MMR) system. When complemented with mutS in trans, motility was again repressed. To determine whether the MMR system, in general, is involved in this reciprocal control, we characterized the effects of gene deletions of other MMR components on UPEC motility. Isogenic deletions of mutS, mutH, and mutL were constructed in both wild-type CFT073 and fim L-ON backgrounds. All MMR mutants showed an increase in motility in the wild-type background, and ΔmutH and ΔmutS mutations increased motility in the fim L-ON background. Cochallenge of the wild-type strain with an MMR-defective strain showed a subtle but significant competitive advantage in the bladder and spleen for the MMR mutant using the murine model of ascending urinary tract infection after 48 h. Our findings demonstrate that the MMR system generally affects the reciprocal regulation of motility and adherence and thus could contribute to UPEC pathogenesis during urinary tract infections. PMID:22473602

ATP binding and hydrolysis by Saccharomyces cerevisiae Msh2-Msh3 are differentially modulated by mismatch and double-strand break repair DNA substrates.

PubMed

Kumar, Charanya; Eichmiller, Robin; Wang, Bangchen; Williams, Gregory M; Bianco, Piero R; Surtees, Jennifer A

2014-06-01

In Saccharomyces cerevisiae, Msh2-Msh3-mediated mismatch repair (MMR) recognizes and targets insertion/deletion loops for repair. Msh2-Msh3 is also required for 3' non-homologous tail removal (3'NHTR) in double-strand break repair. In both pathways, Msh2-Msh3 binds double-strand/single-strand junctions and initiates repair in an ATP-dependent manner. However, we recently demonstrated that the two pathways have distinct requirements with respect to Msh2-Msh3 activities. We identified a set of aromatic residues in the nucleotide binding pocket (FLY motif) of Msh3 that, when mutated, disrupted MMR, but left 3'NHTR largely intact. One of these mutations, msh3Y942A, was predicted to disrupt the nucleotide sandwich and allow altered positioning of ATP within the pocket. To develop a mechanistic understanding of the differential requirements for ATP binding and/or hydrolysis in the two pathways, we characterized Msh2-Msh3 and Msh2-msh3Y942A ATP binding and hydrolysis activities in the presence of MMR and 3'NHTR DNA substrates. We observed distinct, substrate-dependent ATP hydrolysis and nucleotide turnover by Msh2-Msh3, indicating that the MMR and 3'NHTR DNA substrates differentially modify the ATP binding/hydrolysis activities of Msh2-Msh3. Msh2-msh3Y942A retained the ability to bind DNA and ATP but exhibited altered ATP hydrolysis and nucleotide turnover. We propose that both ATP and structure-specific repair substrates cooperate to direct Msh2-Msh3-mediated repair and suggest an explanation for the msh3Y942A separation-of-function phenotype. Copyright © 2014 Elsevier B.V. All rights reserved.

ATP binding and hydrolysis by Saccharomyces cerevisiae Msh2-Msh3 are differentially modulated by Mismatch and Double-strand Break Repair DNA substrates

PubMed Central

Kumar, Charanya; Eichmiller, Robin; Wang, Bangchen; Williams, Gregory M.; Bianco, Piero R.; Surtees, Jennifer A.

2014-01-01

In Saccharomyces cerevisiae, Msh2-Msh3-mediated mismatch repair (MMR) recognizes and targets insertion/deletion loops for repair. Msh2-Msh3 is also required for 3′ non-homologous tail removal (3′NHTR) in double-strand break repair. In both pathways, Msh2-Msh3 binds double-strand/single-strand junctions and initiates repair in an ATP-dependent manner. However, we recently demonstrated that the two pathways have distinct requirements with respect to Msh2-Msh3 activities. We identified a set of aromatic residues in the nucleotide binding pocket (FLY motif) of Msh3 that, when mutated, disrupted MMR, but left 3′ NHTR largely intact. One of these mutations, msh3Y942A, was predicted to disrupt the nucleotide sandwich and allow altered positioning of ATP within the pocket. To develop a mechanistic understanding of the differential requirements for ATP binding and/or hydrolysis in the two pathways, we characterized Msh2-Msh3 and Msh2-msh3Y942A ATP binding and hydrolysis activities in the presence of MMR and 3′ NHTR DNA substrates. We observed distinct, substrate-dependent ATP hydrolysis and nucleotide turnover by Msh2-Msh3, indicating that the MMR and 3′ NHTR DNA substrates differentially modify the ATP binding/hydrolysis activities of Msh2-Msh3. Msh2-msh3Y942A retained the ability to bind DNA and ATP but exhibited altered ATP hydrolysis and nucleotide turnover. We propose that both ATP and structure-specific repair substrates cooperate to direct Msh2-Msh3-mediated repair and suggest an explanation for the msh3Y942A separation-of-function phenotype. PMID:24746922

Immunohistochemical analysis of expression and allelotype of mismatch repair genes (hMLH1 and hMSH2) in bladder cancer

PubMed Central

Kassem, H Sh; Varley, J M; Hamam, S M; Margison, G P

2001-01-01

Mutation of human homologues of DNA mismatch repair (MMR) genes in tumours has been shown to be associated with the phenomenon of microsatellite instability (MSI). Several studies have reported the occurrence of MSI in bladder cancer, but evidence of involvement of MMR genes in the pathogenesis of this cancer is still unclear. We therefore utilized quantitative immunohistochemical (IHC) image analysis and PCR-based allelotype analysis to determine hMLH1 and hMSH2 genes alteration in a cohort of Egyptian bladder cancer samples. IHC analysis of 24 TCC and 12 SCC revealed marked- intra and intertumour heterogeneity in the levels of expression of the two MMR proteins. One TCC lost MLH1 expression and one lost MSH2, (1/24, 4%), and one SCC lost MSH2 (1/12, 8%). A large proportion of analysed tumours revealed a percentage positivity of less than 50% for MLH1 and MSH2 expression (44% and 69%, respectively). Complete loss of heterozygosity in three dinucleotide repeats lying within, or in close proximity to, hMLH1 and hMSH2 was rare (2/57, (4%) for MLH1; and 1/55, (2%) for MSH2), however allelic imbalance was detected in 11/57 (hMLH1) and 10/55 (hMSH2) at any of the informative microsatellite loci. These alterations in structure and expression of DNA MMR genes suggest their possible involvement in the tumorigenesis and/or progression of bladder cancer. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11161395

Analysis of in vivo correction of defined mismatches in the DNA mismatch repair mutants msh2, msh3 and msh6 of Saccharomyces cerevisiae.

PubMed

Lühr, B; Scheller, J; Meyer, P; Kramer, W

1998-02-01

We have analysed the correction of defined mismatches in wild-type and msh2, msh3, msh6 and msh3 msh6 mutants of Saccharomyces cerevisiae in two different yeast strain backgrounds by transformation with plasmid heteroduplex DNA constructs. Ten different base/base mismatches, two single-nucleotide loops and a 38-nucleotide loop were tested. Repair of all types of mismatches was severely impaired in msh2 and msh3 msh6 mutants. In msh6 mutants, repair efficiency of most base/base mismatches was reduced to a similar extent as in msh3 msh6 double mutants. G/T and A/C mismatches, however, displayed residual repair in msh6 mutants in one strain background, implying a role for Msh3p in recognition of base/base mismatches. Furthermore, the efficiency of repair of base/base mismatches was considerably reduced in msh3 mutants in one strain background, indicating a requirement for MSH3 for fully efficient mismatch correction. Also the efficiency of repair of the 38-nucleotide loop was reduced in msh3 mutants, and to a lesser extent in msh6 mutants. The single-nucleotide loop with an unpaired A was less efficiently repaired in msh3 mutants and that with an unpaired T was less efficiently corrected in msh6 mutants, indicating non-redundant functions for the two proteins in the recognition of single-nucleotide loops.

Overexpression of the DNA mismatch repair factor, PMS2, confers hypermutability and DNA damage tolerance.

PubMed

Gibson, Shannon L; Narayanan, Latha; Hegan, Denise Campisi; Buermeyer, Andrew B; Liskay, R Michael; Glazer, Peter M

2006-12-08

Inherited defects in genes associated with DNA mismatch repair (MMR) have been linked to familial colorectal cancer. Cells deficient in MMR are genetically unstable and demonstrate a tolerance phenotype in response to certain classes of DNA damage. Some sporadic human cancers also show abnormalities in MMR gene function, typically due to diminished expression of one of the MutL homologs, MLH1. Here, we report that overexpression of the MutL homolog, human PMS2, can also cause a disruption of the MMR pathway in mammalian cells, resulting in hypermutability and DNA damage tolerance. A mouse fibroblast cell line carrying a recoverable lambda phage shuttle vector for mutation detection was transfected with either a vector designed to express hPMS2 or with an empty vector control. Cells overexpressing hPMS2 were found to have elevated spontaneous mutation frequencies at the cII reporter gene locus. They also showed an increase in the level of mutations induced by the alkylating agent, methynitrosourea (MNU). Clonogenic survival assays demonstrated increased survival of the PMS2-overexpressing cells following exposure to MNU, consistent with the induction of a damage tolerance phenotype. Similar results were seen in cells expressing a mutant PMS2 gene, containing a premature stop codon at position 134 and representing a variant found in an individual with familial colon cancer. These results show that dysregulation of PMS2 gene expression can disrupt MMR function in mammalian cells and establish an additional carcinogenic mechanism by which cells can develop genetic instability and acquire resistance to cytotoxic cancer therapies.

Relationship between PTEN, DNA mismatch repair, and tumor histotype in endometrial carcinoma: retained positive expression of PTEN preferentially identifies sporadic non-endometrioid carcinomas.

PubMed

Djordjevic, Bojana; Barkoh, Bedia A; Luthra, Rajyalakshmi; Broaddus, Russell R

2013-10-01

Loss of PTEN (phosphatase and tensin homolog) expression and microsatellite instability are two of the more common molecular alterations in endometrial carcinoma. From the published literature, it is controversial as to whether there is a relationship between these different molecular mechanisms. Therefore, a cohort of 187 pure endometrioid and non-endometrioid endometrial carcinomas, carefully characterized as to clinical and pathological features, was examined for PTEN sequence abnormalities and the immunohistochemical expression of PTEN and the DNA mismatch repair proteins MLH1, MSH2, MSH6, and PMS2. MLH1 methylation analysis was performed when tumors had loss of MLH1 protein. Mismatch repair protein loss was more frequent in endometrioid carcinomas compared with non-endometrioid carcinomas, a difference primarily attributable to the presence of MLH1 methylation in a greater proportion of endometrioid tumors. Among the non-endometrioid group, mixed endometrioid/non-endometrioid carcinomas were the histotype that most commonly had loss of a mismatch repair protein. In endometrioid tumors, the frequency of PTEN loss measured by immunohistochemistry and mutation did not differ significantly between the mismatch repair protein intact or mismatch repair protein loss groups, suggesting that PTEN loss is independent of mismatch protein repair status in this group. However, in non-endometrioid carcinomas, both intact positive PTEN immunohistochemical expression and PTEN wild type were highly associated with retained positive expression of mismatch repair proteins in the tumor. Relevant to screening endometrial cancers for Lynch Syndrome, an initial PTEN immunohistochemistry determination may be able to replace the use of four mismatch repair immunohistochemical markers in 63% of patients with non-endometrioid endometrial carcinoma. Therefore, PTEN immunohistochemistry, in combination with tumor histotype, is a useful adjunct in the clinical evaluation of endometrial

Relationship between PTEN, DNA Mismatch Repair, and Tumor Histotype in Endometrial Carcinoma: Retained Positive Expression of PTEN Preferentially Identifies Sporadic Non-Endometrioid Carcinomas

PubMed Central

Djordjevic, Bojana; Barkoh, Bedia A.; Luthra, Rajyalakshmi; Broaddus, Russell R.

2013-01-01

Loss of PTEN (phosphatase and tensin homolog) expression and microsatellite instability are two of the more common molecular alterations in endometrial carcinoma. From the published literature, it is controversial as to whether there is a relationship between these different molecular mechanisms. Therefore, a cohort of 187 pure endometrioid and non-endometrioid endometrial carcinomas, carefully characterized as to clinical and pathological features, was examined for PTEN sequence abnormalities and the immunohistochemical expression of PTEN and the DNA mismatch repair proteins MLH1, MSH2, MSH6 and PMS2. MLH1 methylation analysis was performed when tumors had loss of MLH1 protein. Mismatch repair protein loss was more frequent in endometrioid carcinomas compared to non-endometrioid carcinomas, a difference primarily attributable to the presence of MLH1 methylation in a greater proportion of endometrioid tumors. Among the non-endometrioid group, mixed endometrioid/non-endometrioid carcinomas were the histotype that most commonly had loss of a mismatch repair protein. In endometrioid tumors, the frequency of PTEN loss measured by immunohistochemistry and mutation did not differ significantly between the mismatch repair protein intact or mismatch repair protein loss groups, suggesting that PTEN loss is independent of mismatch protein repair status in this group. However, in non-endometrioid carcinomas, both intact positive PTEN immunohistochemical expression and PTEN wild type were highly associated with retained positive expression of mismatch repair proteins in the tumor. Relevant to screening endometrial cancers for Lynch Syndrome, an initial PTEN immunohistochemistry determination may be able to replace the use of four mismatch repair immunohistochemical markers in 63% of patients with non-endometrioid endometrial carcinoma. Therefore, PTEN immunohistochemistry, in combination with tumor histotype, is a useful adjunct in the clinical evaluation of endometrial

Mismatch Repair Deficiency Testing in Patients With Colorectal Cancer and Nonadherence to Testing Guidelines in Young Adults.

PubMed

Shaikh, Talha; Handorf, Elizabeth A; Meyer, Joshua E; Hall, Michael J; Esnaola, Nestor F

2018-02-08

Mismatch repair (MMR) deficiency of DNA has been observed in up to 15% of sporadic colorectal cancers (CRCs) and is a characteristic feature of Lynch syndrome, which has a higher incidence in young adults (age, <50 years) with CRC. Mismatch repair deficiency can be due to germline mutations or epigenetic inactivation, affects prognosis and response to systemic therapy, and results in unrepaired repetitive DNA sequences, which increases the risk of multiple malignant tumors. To evaluate the utilization of MMR deficiency testing in adults with CRC and analyze nonadherence to long-standing testing guidelines in younger adults using a contemporary national data set to help identify potential risk factors for nonadherence to newly implemented universal testing guidelines. Adult (age, <30 to ≥70 years) and, of these, younger adult (<30 to 49 years) patients with invasive colorectal adenocarcinoma diagnosed between 2010 and 2012 and known MMR deficiency testing status were identified using the National Cancer Database. The study was conducted from March 16, 2016, to March 1, 2017. Patient sociodemographic, facility, tumor, and treatment characteristics. The primary outcome of interest was receipt of MMR deficiency testing. Multivariable logistic regression was used to identify independent predictors of testing in adult and/or young adult patients. A total of 152 993 adults with CRC were included in the study (78 579 [51.4%] men; mean [SD] age, 66.9 [13.9] years). Of these patients, only 43 143 (28.2%) underwent MMR deficiency testing; the proportion of patients tested increased between 2010 and 2012 (22.3% vs 33.1%; P<.001). Among 17 218 younger adult patients with CRC, only 7422 (43.1%) underwent MMR deficiency testing; the proportion tested increased between 2010 and 2012 (36.1% vs 48.0%; P < .001). Irrespective of age, higher educational level (OR, 1.38; 95% CI, 1.15-1.66), later diagnosis year (OR, 1.81; 95% CI, 1.65-1.98), early stage disease (OR, 1

BRCA2, EGFR, and NTRK mutations in mismatch repair-deficient colorectal cancers with MSH2 or MLH1 mutations.

PubMed

Deihimi, Safoora; Lev, Avital; Slifker, Michael; Shagisultanova, Elena; Xu, Qifang; Jung, Kyungsuk; Vijayvergia, Namrata; Ross, Eric A; Xiu, Joanne; Swensen, Jeffrey; Gatalica, Zoran; Andrake, Mark; Dunbrack, Roland L; El-Deiry, Wafik S

2017-06-20

Deficient mismatch repair (MMR) and microsatellite instability (MSI) contribute to ~15% of colorectal cancer (CRCs). We hypothesized MSI leads to mutations in DNA repair proteins including BRCA2 and cancer drivers including EGFR. We analyzed mutations among a discovery cohort of 26 MSI-High (MSI-H) and 558 non-MSI-H CRCs profiled at Caris Life Sciences. Caris-profiled MSI-H CRCs had high mutation rates (50% vs 14% in non-MSI-H, P < 0.0001) in BRCA2. Of 1104 profiled CRCs from a second cohort (COSMIC), MSH2/MLH1-mutant CRCs showed higher mutation rates in BRCA2 compared to non-MSH2/MLH1-mutant tumors (38% vs 6%, P < 0.0000001). BRCA2 mutations in MSH2/MLH1-mutant CRCs included 75 unique mutations not known to occur in breast or pancreatic cancer per COSMIC v73. Only 5 deleterious BRCA2 mutations in CRC were previously reported in the BIC database as germ-line mutations in breast cancer. Some BRCA2 mutations were predicted to disrupt interactions with partner proteins DSS1 and RAD51. Some CRCs harbored multiple BRCA2 mutations. EGFR was mutated in 45.5% of MSH2/MLH1-mutant and 6.5% of non-MSH2/MLH1-mutant tumors (P < 0.0000001). Approximately 15% of EGFR mutations found may be actionable through TKI therapy, including N700D, G719D, T725M, T790M, and E884K. NTRK gene mutations were identified in MSH2/MLH1-mutant CRC including NTRK1 I699V, NTRK2 P716S, and NTRK3 R745L. Our findings have clinical relevance regarding therapeutic targeting of BRCA2 vulnerabilities, EGFR mutations or other identified oncogenic drivers such as NTRK in MSH2/MLH1-mutant CRCs or other tumors with mismatch repair deficiency.

Removal of N-6-methyladenine by the nucleotide excision repair pathway triggers the repair of mismatches in yeast gap-repair intermediates.

PubMed

Guo, Xiaoge; Jinks-Robertson, Sue

2013-12-01

Gap-repair assays have been an important tool for studying the genetic control of homologous recombination in yeast. Sequence analysis of recombination products derived when a gapped plasmid is diverged relative to the chromosomal repair template additionally has been used to infer structures of strand-exchange intermediates. In the absence of the canonical mismatch repair pathway, mismatches present in these intermediates are expected to persist and segregate at the next round of DNA replication. In a mismatch repair defective (mlh1Δ) background, however, we have observed that recombination-generated mismatches are often corrected to generate gene conversion or restoration events. In the analyses reported here, the source of the aberrant mismatch removal during gap repair was examined. We find that most mismatch removal is linked to the methylation status of the plasmid used in the gap-repair assay. Whereas more than half of Dam-methylated plasmids had patches of gene conversion and/or restoration interspersed with unrepaired mismatches, mismatch removal was observed in less than 10% of products obtained when un-methylated plasmids were used in transformation experiments. The methylation-linked removal of mismatches in recombination intermediates was due specifically to the nucleotide excision repair pathway, with such mismatch removal being partially counteracted by glycosylases of the base excision repair pathway. These data demonstrate that nucleotide excision repair activity is not limited to bulky, helix-distorting DNA lesions, but also targets removal of very modest perturbations in DNA structure. In addition to its effects on mismatch removal, methylation reduced the overall gap-repair efficiency, but this reduction was not affected by the status of excision repair pathways. Finally, gel purification of DNA prior to transformation reduced gap-repair efficiency four-fold in a nucleotide excision repair-defective background, indicating that the collateral

Removal of N-6-methyladenine by the nucleotide excision repair pathway triggers the repair of mismatches in yeast gap-repair intermediates

PubMed Central

Guo, Xiaoge; Jinks-Robertson, Sue

2013-01-01

Gap-repair assays have been an important tool for studying the genetic control of homologous recombination in yeast. Sequence analysis of recombination products derived when a gapped plasmid is diverged relative to the chromosomal repair template additionally has been used to infer structures of strand-exchange intermediates. In the absence of the canonical mismatch repair pathway, mismatches present in these intermediates are expected to persist and segregate at the next round of DNA replication. In a mismatch repair defective (mlh1Δ) background, however, we have observed that recombination-generated mismatches are often corrected to generate gene conversion or restoration events. In the analyses reported here, the source of the aberrant mismatch removal during gap repair was examined. We find that most mismatch removal is linked to the methylation status of the plasmid used in the gap-repair assay. Whereas more than half of Dam-methylated plasmids had patches of gene conversion and/or restoration interspersed with unrepaired mismatches, mismatch removal was observed in less than 10% of products obtained when un-methylated plasmids were used in transformation experiments. The methylation-linked removal of mismatches in recombination intermediates was due specifically to the nucleotide excision repair pathway, with such mismatch removal being partially counteracted by glycosylases of the base excision repair pathway. These data demonstrate that nucleotide excision repair activity is not limited to bulky, helix-distorting DNA lesions, but also targets removal of very modest perturbations in DNA structure. In addition to its effects on mismatch removal, methylation reduced the overall gap-repair efficiency, but this reduction was not affected by the status of excision repair pathways. Finally, gel purification of DNA prior to transformation reduced gap-repair efficiency four-fold in a nucleotide excision repair-defective background, indicating that the cillateral

DNA Mismatch Repair Deficiency in Rectal Cancer: Benchmarking Its Impact on Prognosis, Neoadjuvant Response Prediction, and Clinical Cancer Genetics

PubMed Central

de Rosa, Nicole; Rodriguez-Bigas, Miguel A.; Chang, George J.; Veerapong, Jula; Borras, Ester; Krishnan, Sunil; Bednarski, Brian; Messick, Craig A.; Skibber, John M.; Feig, Barry W.; Lynch, Patrick M.; Vilar, Eduardo

2016-01-01

Purpose DNA mismatch repair deficiency (dMMR) hallmarks consensus molecular subtype 1 of colorectal cancer. It is being routinely tested, but little is known about dMMR rectal cancers. The efficacy of novel treatment strategies cannot be established without benchmarking the outcomes of dMMR rectal cancer with current therapy. We aimed to delineate the impact of dMMR on prognosis, the predicted response to fluoropyrimidine-based neoadjuvant therapy, and implications of germline alterations in the MMR genes in rectal cancer. Methods Between 1992 and 2012, 62 patients with dMMR rectal cancers underwent multimodality therapy. Oncologic treatment and outcomes as well as clinical genetics work-up were examined. Overall and rectal cancer–specific survival were calculated by the Kaplan-Meier method. Results The median age at diagnosis was 41 years. MMR deficiency was most commonly due to alterations in MSH2 (53%) or MSH6 (23%). After a median follow-up of 6.8 years, the 5-year rectal cancer–specific survival was 100% for stage I and II, 85.1% for stage III, and 60.0% for stage IV disease. Fluoropyrimidine-based neoadjuvant chemoradiation was associated with a complete pathologic response rate of 27.6%. The extent of surgical resection was influenced by synchronous colonic disease at presentation, tumor height, clinical stage, and pelvic radiation. An informed decision for a limited resection focusing on proctectomy did not compromise overall survival. Five of the 11 (45.5%) deaths during follow-up were due to extracolorectal malignancies. Conclusion dMMR rectal cancer had excellent prognosis and pathologic response with current multimodality therapy including an individualized surgical treatment plan. Identification of a dMMR rectal cancer should trigger germline testing, followed by lifelong surveillance for both colorectal and extracolorectal malignancies. We herein provide genotype-specific outcome benchmarks for comparison with novel interventions. PMID:27432916

DNA Mismatch Repair Deficiency in Rectal Cancer: Benchmarking Its Impact on Prognosis, Neoadjuvant Response Prediction, and Clinical Cancer Genetics.

PubMed

de Rosa, Nicole; Rodriguez-Bigas, Miguel A; Chang, George J; Veerapong, Jula; Borras, Ester; Krishnan, Sunil; Bednarski, Brian; Messick, Craig A; Skibber, John M; Feig, Barry W; Lynch, Patrick M; Vilar, Eduardo; You, Y Nancy

2016-09-01

DNA mismatch repair deficiency (dMMR) hallmarks consensus molecular subtype 1 of colorectal cancer. It is being routinely tested, but little is known about dMMR rectal cancers. The efficacy of novel treatment strategies cannot be established without benchmarking the outcomes of dMMR rectal cancer with current therapy. We aimed to delineate the impact of dMMR on prognosis, the predicted response to fluoropyrimidine-based neoadjuvant therapy, and implications of germline alterations in the MMR genes in rectal cancer. Between 1992 and 2012, 62 patients with dMMR rectal cancers underwent multimodality therapy. Oncologic treatment and outcomes as well as clinical genetics work-up were examined. Overall and rectal cancer-specific survival were calculated by the Kaplan-Meier method. The median age at diagnosis was 41 years. MMR deficiency was most commonly due to alterations in MSH2 (53%) or MSH6 (23%). After a median follow-up of 6.8 years, the 5-year rectal cancer-specific survival was 100% for stage I and II, 85.1% for stage III, and 60.0% for stage IV disease. Fluoropyrimidine-based neoadjuvant chemoradiation was associated with a complete pathologic response rate of 27.6%. The extent of surgical resection was influenced by synchronous colonic disease at presentation, tumor height, clinical stage, and pelvic radiation. An informed decision for a limited resection focusing on proctectomy did not compromise overall survival. Five of the 11 (45.5%) deaths during follow-up were due to extracolorectal malignancies. dMMR rectal cancer had excellent prognosis and pathologic response with current multimodality therapy including an individualized surgical treatment plan. Identification of a dMMR rectal cancer should trigger germline testing, followed by lifelong surveillance for both colorectal and extracolorectal malignancies. We herein provide genotype-specific outcome benchmarks for comparison with novel interventions. © 2016 by American Society of Clinical Oncology.

Connections between constitutional mismatch repair deficiency syndrome and neurofibromatosis type 1.

PubMed

Wimmer, K; Rosenbaum, T; Messiaen, L

2017-04-01

Constitutional mismatch repair (MMR) deficiency (CMMRD) is a rare childhood cancer susceptibility syndrome resulting from biallelic germline loss-of-function mutations in one of the MMR genes. Individuals with CMMRD have high risk to develop a broad spectrum of malignancies and frequently display features reminiscent of neurofibromatosis type 1 (NF1). Evaluation of the clinical findings of genetically proven CMMRD patients shows that not only multiple café-au-lait macules but also any of the diagnostic features of NF1 may be present in a CMMRD patient. This phenotypic overlap may lead to misdiagnosis of CMMRD patients as having NF1, which impedes adequate management of the patients and their families. The spectrum of CMMRD-associated childhood malignancies includes high-grade glioma, acute myeloid leukaemia or rhabdomyosarcoma, also reported as associated with NF1. Reported associations between NF1 and these malignancies are to a large extent based on studies that neither proved the presence of an NF1 germline mutation nor ruled-out CMMRD in the affected. Hence, these associations are challenged by our current knowledge of the phenotypic overlap between NF1 and CMMRD and should be re-evaluated in future studies. Recent advances in the diagnostics of CMMRD should render it possible to definitely state or refute this diagnosis in these individuals. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Two new subfamilies of DNA mismatch repair proteins (MutS) specifically abundant in the marine environment

PubMed Central

Ogata, Hiroyuki; Ray, Jessica; Toyoda, Kensuke; Sandaa, Ruth-Anne; Nagasaki, Keizo; Bratbak, Gunnar; Claverie, Jean-Michel

2011-01-01

MutS proteins are ubiquitous in cellular organisms and have important roles in DNA mismatch repair or recombination. In the virus world, the amoeba-infecting Mimivirus, as well as the recently sequenced Cafeteria roenbergensis virus are known to encode a MutS related to the homologs found in octocorals and ɛ-proteobacteria. To explore the presence of MutS proteins in other viral genomes, we performed a genomic survey of four giant viruses (‘giruses') (Pyramimonas orientalis virus (PoV), Phaeocystis pouchetii virus (PpV), Chrysochromulina ericina virus (CeV) and Heterocapsa circularisquama DNA virus (HcDNAV)) that infect unicellular marine algae. Our analysis revealed the presence of a close homolog of Mimivirus MutS in all the analyzed giruses. These viral homologs possess a specific domain structure, including a C-terminal HNH-endonuclease domain, defining the new MutS7 subfamily. We confirmed the presence of conserved mismatch recognition residues in all members of the MutS7 subfamily, suggesting their role in DNA mismatch repair rather than DNA recombination. PoV and PpV were found to contain an additional type of MutS, which we propose to call MutS8. The MutS8 proteins in PoV and PpV were found to be closely related to homologs from ‘Candidatus Amoebophilus asiaticus', an obligate intracellular amoeba-symbiont belonging to the Bacteroidetes. Furthermore, our analysis revealed that MutS7 and MutS8 are abundant in marine microbial metagenomes and that a vast majority of these environmental sequences are likely of girus origin. Giruses thus seem to represent a major source of the underexplored diversity of the MutS family in the microbial world. PMID:21248859

Clinical and molecular characterisation of hereditary and sporadic metastatic colorectal cancers harbouring microsatellite instability/DNA mismatch repair deficiency.

PubMed

Cohen, R; Buhard, O; Cervera, P; Hain, E; Dumont, S; Bardier, A; Bachet, J-B; Gornet, J-M; Lopez-Trabada, D; Dumont, S; Kaci, R; Bertheau, P; Renaud, F; Bibeau, F; Parc, Y; Vernerey, D; Duval, A; Svrcek, M; André, Thierry

2017-11-01

Patients treated with chemotherapy for microsatellite unstable (MSI) and/or mismatch repair deficient (dMMR) cancer metastatic colorectal cancer (mCRC) exhibit poor prognosis. We aimed to evaluate the relevance of distinguishing sporadic from Lynch syndrome (LS)-like mCRCs. MSI/dMMR mCRC patients were retrospectively identified in six French hospitals. Tumour samples were screened for MSI, dMMR, RAS/RAF mutations and MLH1 methylation. Sporadic cases were molecularly defined as those displaying MLH1/PMS2 loss of expression with BRAFV600E and/or MLH1 hypermethylation and no MMR germline mutation. Among 129 MSI/dMMR mCRC patients, 81 (63%) were LS-like and 48 (37%) had sporadic tumours; 22% of MLH1/PMS2-negative mCRCs would have been misclassified using an algorithm based on local medical records (age, Amsterdam II criteria, BRAF and MMR statuses when locally tested), compared to a systematical assessment of MMR, BRAF and MLH1 methylation statuses. In univariate analysis, parameters associated with better overall survival were age (P < 0.0001), metastatic resection (P = 0.001) and LS-like mCRC (P = 0.01), but not BRAFV600E. In multivariate analysis, age (hazard ratio (HR) = 3.19, P = 0.01) and metastatic resection (HR = 4.2, P = 0.001) were associated with overall survival, but not LS. LS-like patients were associated with more frequent liver involvement, metastatic resection and better disease-free survival after metastasectomy (HR = 0.28, P = 0.01). Median progression-free survival of first-line chemotherapy was similar between the two groups (4.2 and 4.2 months; P = 0.44). LS-like and sporadic MSI/dMMR mCRCs display distinct natural histories. MMR, BRAF mutation and MLH1 methylation testing should be mandatory to differentiate LS-like and sporadic MSI/dMMR mCRC, to determine in particular whether immune checkpoint inhibitors efficacy differs in these two populations. Copyright © 2017 Elsevier Ltd. All rights reserved.

Lymphocytic response to tumour and deficient DNA mismatch repair identify subtypes of stage II/III colorectal cancer associated with patient outcomes.

PubMed

Williams, David S; Mouradov, Dmitri; Jorissen, Robert N; Newman, Marsali R; Amini, Elham; Nickless, David K; Teague, Julie A; Fang, Catherine G; Palmieri, Michelle; Parsons, Marie J; Sakthianandeswaren, Anuratha; Li, Shan; Ward, Robyn L; Hawkins, Nicholas J; Faragher, Ian; Jones, Ian T; Gibbs, Peter; Sieber, Oliver M

2018-01-30

Tumour-infiltrating lymphocyte (TIL) response and deficient DNA mismatch repair (dMMR) are determinants of prognosis in colorectal cancer. Although highly correlated, evidence suggests that these are independent predictors of outcome. However, the prognostic significance of combined TIL/MMR classification and how this compares to the major genomic and transcriptomic subtypes remain unclear. A prospective cohort of 1265 patients with stage II/III cancer was examined for TIL/MMR status and BRAF / KRAS mutations. Consensus molecular subtype (CMS) status was determined for 142 cases. Associations with 5-year disease-free survival (DFS) were evaluated and validated in an independent cohort of 602 patients. Tumours were categorised into four subtypes based on TIL and MMR status: TIL-low/proficient-MMR (pMMR) (61.3% of cases), TIL-high/pMMR (14.8%), TIL-low/dMMR (8.6%) and TIL-high/dMMR (15.2%). Compared with TIL-high/dMMR tumours with the most favourable prognosis, both TIL-low/dMMR (HR=3.53; 95% CI=1.88 to 6.64; P multivariate <0.001) and TIL-low/pMMR tumours (HR=2.67; 95% CI=1.47 to 4.84; P multivariate =0.001) showed poor DFS. Outcomes of patients with TIL-low/dMMR and TIL-low/pMMR tumours were similar. TIL-high/pMMR tumours showed intermediate survival rates. These findings were validated in an independent cohort. TIL/MMR status was a more significant predictor of prognosis than National Comprehensive Cancer Network high-risk features and was a superior predictor of prognosis compared with genomic (dMMR, pMMR/ BRAF wt / KRAS wt , pMMR/ BRAF mut / KRAS wt , pMMR/ BRAF wt / KRAS mut ) and transcriptomic (CMS 1-4) subtypes. TIL/MMR classification identified subtypes of stage II/III colorectal cancer associated with different outcomes. Although dMMR status is generally considered a marker of good prognosis, we found this to be dependent on the presence of TILs. Prognostication based on TIL/MMR subtypes was superior compared with histopathological, genomic and

An Msh3 ATPase domain mutation has no effect on MMR function.

PubMed

Edwards, Yasmin

2017-11-25

To demonstrate that the Msh3 ATPase domain is required for DNA mismatch repair and tumor suppression in a murine model. The DNA mismatch repair proteins are members of the ABC family of ATPases. ATP binding and hydrolysis regulates their mismatch repair function. In the current study, a mouse model was generated harboring a glycine to aspartic acid residue change in the Walker A motif of the ATPase domain of Msh3. Impaired ATP mediated release of the Msh2-Msh3 GD/GD complex from it's DNA substrate in vitro confirmed the presence of an ATPase defect. However, the mismatch repair function of the protein was not significantly affected. Therefore, mutation of a critical residue within the ATPase domain of Msh3 did not preclude mismatch repair at the genomic sequences tested. Indicating that Msh3 mediated mismatch function is retained the absence of a functional ATPase domain.

Diagnosis of Constitutional Mismatch Repair-Deficiency Syndrome Based on Microsatellite Instability and Lymphocyte Tolerance to Methylating Agents.

PubMed

Bodo, Sahra; Colas, Chrystelle; Buhard, Olivier; Collura, Ada; Tinat, Julie; Lavoine, Noémie; Guilloux, Agathe; Chalastanis, Alexandra; Lafitte, Philippe; Coulet, Florence; Buisine, Marie-Pierre; Ilencikova, Denisa; Ruiz-Ponte, Clara; Kinzel, Miriam; Grandjouan, Sophie; Brems, Hilde; Lejeune, Sophie; Blanché, Hélène; Wang, Qing; Caron, Olivier; Cabaret, Odile; Svrcek, Magali; Vidaud, Dominique; Parfait, Béatrice; Verloes, Alain; Knappe, Ulrich J; Soubrier, Florent; Mortemousque, Isabelle; Leis, Alexander; Auclair-Perrossier, Jessie; Frébourg, Thierry; Fléjou, Jean-François; Entz-Werle, Natacha; Leclerc, Julie; Malka, David; Cohen-Haguenauer, Odile; Goldberg, Yael; Gerdes, Anne-Marie; Fedhila, Faten; Mathieu-Dramard, Michèle; Hamelin, Richard; Wafaa, Badre; Gauthier-Villars, Marion; Bourdeaut, Franck; Sheridan, Eamonn; Vasen, Hans; Brugières, Laurence; Wimmer, Katharina; Muleris, Martine; Duval, Alex

2015-10-01

Patients with bi-allelic germline mutations in mismatch repair (MMR) genes (MLH1, MSH2, MSH6, or PMS2) develop a rare but severe variant of Lynch syndrome called constitutional MMR deficiency (CMMRD). This syndrome is characterized by early-onset colorectal cancers, lymphomas or leukemias, and brain tumors. There is no satisfactory method for diagnosis of CMMRD because screens for mutations in MMR genes are noninformative for 30% of patients. MMR-deficient cancer cells are resistant to genotoxic agents and have microsatellite instability (MSI), due to accumulation of errors in repetitive DNA sequences. We investigated whether these features could be used to identify patients with CMMRD. We examined MSI by PCR analysis and tolerance to methylating or thiopurine agents (functional characteristics of MMR-deficient tumor cells) in lymphoblastoid cells (LCs) from 3 patients with CMMRD and 5 individuals with MMR-proficient LCs (controls). Using these assays, we defined experimental parameters that allowed discrimination of a series of 14 patients with CMMRD from 52 controls (training set). We then used the same parameters to assess 23 patients with clinical but not genetic features of CMMRD. In the training set, we identified parameters, based on MSI and LC tolerance to methylation, that detected patients with CMMRD vs controls with 100% sensitivity and 100% specificity. Among 23 patients suspected of having CMMRD, 6 had MSI and LC tolerance to methylation (CMMRD highly probable), 15 had neither MSI nor LC tolerance to methylation (unlikely to have CMMRD), and 2 were considered doubtful for CMMRD based on having only 1 of the 2 features. The presence of MSI and tolerance to methylation in LCs identified patients with CMMRD with 100% sensitivity and specificity. These features could be used in diagnosis of patients. Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.

Differing patterns of genetic instability in mice deficient in the mismatch repair genes Pms2, Mlh1, Msh2, Msh3 and Msh6.

PubMed

Hegan, Denise Campisi; Narayanan, Latha; Jirik, Frank R; Edelmann, Winfried; Liskay, R Michael; Glazer, Peter M

2006-12-01

Defects in genes associated with DNA mismatch repair (MMR) have been linked to hereditary colon cancer. Because the MMR pathway includes multiple factors with both overlapping and divergent functions, we sought to compare the impact of deficiencies in each of several MMR genes on genetic instability using a collection of knock-out mouse models. We investigated mutation frequencies and patterns in MMR-deficient mice using two transgenic reporter genes, supFG1 and cII, in the context of mice deficient for Pms2, Mlh1, Msh2, Msh3 or Msh6 or both Msh2 and Msh3 or both Msh3 and Msh6. We found that the mean mutation frequencies of all of the MMR-deficient mice were significantly higher than the mean mutation frequencies of wild-type mice. Mlh1-deficient mice and Msh2-deficient mice had the highest mutation frequencies in a comparison of the single nullizygous mice. Of all the mice studied, mice nullizygous for both Msh2 and Msh3 and those nullizygous for both Msh3 and Msh6 displayed the greatest overall increases in mutation frequencies compared with wild-type mice. Sequence analysis of the mutated reporter genes revealed significant differences between the individual groups of MMR-deficient mice. Taken together, our results further characterize the functions of the MMR factors in mutation avoidance and provide in vivo correlation to biochemical models of the MMR pathway.

The mouse mismatch repair protein, MSH3, is a nucleoplasmic protein that aggregates into denser nuclear bodies under conditions of stress.

PubMed

Holt, Ian; Thanh Lam, Le; Tomé, Stéphanie; Wansink, Derick G; Te Riele, Hein; Gourdon, Geneviève; Morris, Glenn E

2011-06-01

The mismatch repair protein, MSH3, together with MSH2, forms the MutSβ heterodimer which recognizes and repairs base pair mismatches and larger insertion/deletion loops in DNA. Lack of specific antibodies against mouse MSH3 has hampered studies of its expression and localization. Mouse MSH3 is not immunogenic in normal mice. This problem was overcome by immunizing msh3-knockout mice and generating a panel of ten monoclonal antibodies, two of which localize MSH3 specifically in cultured mouse cells and bind to an epitope containing amino-acids 33-37. The panel also includes two antibodies that recognise both mouse and human MSH3 and bind to a conserved epitope containing amino-acids 187-194. The mouse MSH3-specific antibodies show that MSH3 is a nuclear protein with a finely-granular nucleoplasmic distribution, largely absent from areas of condensed heterochromatin. Specificity of the localization was demonstrated by absence of immunostaining in a cell line from the msh3-knockout mouse. Furthermore, we show for the first time that stress treatment of mouse cells with ethanol or hydrogen peroxide caused the re-distribution of MSH3 into nuclear bodies containing the proliferating cell nuclear antigen (PCNA), a known binding partner of MutSβ. Copyright © 2011 Wiley-Liss, Inc.

The yeast MSH1 gene is not involved in DNA repair or recombination during meiosis.

PubMed

Sia, Elaine A; Kirkpatrick, David T

2005-02-03

Six strong homologs of the bacterial MutS DNA mismatch repair (MMR) gene have been identified in the yeast Saccharomyces cerevisiae. With the exception of the MSH1 gene, the involvement of each homolog in DNA repair and recombination during meiosis has been determined previously. Five of the homologs have been demonstrated to act in meiotic DNA repair (MSH2, MSH3, MSH6 and MSH4) and/or meiotic recombination (MSH4 and MSH5). Unfortunately the loss of mitochondrial function that results from deletion of MSH1 disrupts meiotic progression, precluding an analysis of MSH1 function in meiotic DNA repair and recombination. However, the recent identification of two separation-of-function alleles of MSH1 that interfere with protein function but still maintain functional mitochondria allow the meiotic activities of MSH1 to be determined. We show that the G776D and F105A alleles of MSH1 exhibit no defects in meiotic recombination, repair base-base mismatches and large loop mismatches efficiently during meiosis, and have high levels of spore viability. These data indicate that the MSH1 protein, unlike other MutS homologs in yeast, plays no role in DNA repair or recombination during meiosis.

[Constitutional mismatch repair deficiency syndrome].

PubMed

Jongmans, Marjolijn C; Gidding, Corrie E; Loeffen, Jan; Wesseling, Pieter; Mensenkamp, Arjen; Hoogerbrugge, Nicoline

2015-01-01

Constitutional mismatch repair deficiency (CMMR-D) syndrome is characterised by a significantly increased risk for developing cancer in childhood. It arises when both parents have a mutation in the same mismatch repair gene and pass it on to their child. An 8-year-old girl was diagnosed with CMMR-D syndrome after she developed a brain tumour at the age of 4 and a T-cell non-Hodgkin lymphoma at the age of 6. She had multiple hyperpigmented skin lesions and died of myelodysplastic syndrome at the age of 11. In children with cancer CMMR-D syndrome can be recognized particularly if there are multiple primary malignancies and skin hyperpigmentations and hypopigmentations. The parents of these children are at high risk for colorectal and endometrial cancer (Lynch syndrome), amongst others.

Alcohol Consumption and the Risk of Colorectal Cancer for Mismatch Repair Gene Mutation Carriers

PubMed Central

Dashti, S. Ghazaleh; Buchanan, Daniel D.; Jayasekara, Harindra; Ouakrim, Driss Ait; Clendenning, Mark; Rosty, Christophe; Winship, Ingrid M.; Macrae, Finlay A.; Giles, Graham G.; Parry, Susan; Casey, Graham; Haile, Robert W.; Gallinger, Steven; Le Marchand, Loïc; Thibodeau, Stephen N.; Lindor, Noralane M.; Newcomb, Polly A.; Potter, John D.; Baron, John A.; Hopper, John L.; Jenkins, Mark A.; Win, Aung Ko

2016-01-01

Background People with germline mutation in one of the DNA mismatch repair (MMR) genes have increased colorectal cancer risk. For these high-risk people, study findings of the relationship between alcohol consumption and colorectal cancer risk have been inconclusive. Methods 1,925 MMR gene mutations carriers recruited into the Colon Cancer Family Registry who had completed a questionnaire on lifestyle factors were included. Weighted Cox proportional hazard regression models were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for the association between alcohol consumption and colorectal cancer. Results Colorectal cancer was diagnosed in 769 carriers (40%) at a mean (standard deviation) age of 42.6 (10.3) years. Compared with abstention, ethanol consumption from any alcoholic beverage up to 14 grams/day and >28 grams/day were associated with increased colorectal cancer risk (HR, 1.50; 95%CI, 1.09–2.07 and 1.69; 95%CI, 1.07–2.65 respectively; P-trend=0.05), and colon cancer risk (HR, 1.78; 95%CI, 1.27–2.49 and 1.94; 95%CI, 1.19–3.18 respectively; P-trend=0.02). However, there was no clear evidence for an association with rectal cancer risk. Also, there was no evidence for associations between consumption of individual alcoholic beverage types (beer, wine, spirits) and colorectal, colon, or rectal cancer risk. Conclusion Our data suggests that alcohol consumption, particularly more than 28 grams/day of ethanol (~2 standard drinks of alcohol in the US), is associated with increased colorectal cancer risk for MMR gene mutation carriers. Impact Although these data suggested that alcohol consumption in MMR carriers was associated with increased colorectal cancer risk, there was no evidence of a dose-response, and not all types of alcohol consumption were associated with increased risk. PMID:27811119

The multifaceted influence of histone deacetylases on DNA damage signalling and DNA repair

PubMed Central

Roos, Wynand Paul; Krumm, Andrea

2016-01-01

Histone/protein deacetylases play multiple roles in regulating gene expression and protein activation and stability. Their deregulation during cancer initiation and progression cause resistance to therapy. Here, we review the role of histone deacetylases (HDACs) and the NAD+ dependent sirtuins (SIRTs) in the DNA damage response (DDR). These lysine deacetylases contribute to DNA repair by base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), non-homologous end joining (NHEJ), homologous recombination (HR) and interstrand crosslink (ICL) repair. Furthermore, we discuss possible mechanisms whereby these histone/protein deacetylases facilitate the switch between DNA double-strand break (DSB) repair pathways, how SIRTs play a central role in the crosstalk between DNA repair and cell death pathways due to their dependence on NAD+, and the influence of small molecule HDAC inhibitors (HDACi) on cancer cell resistance to genotoxin based therapies. Throughout the review, we endeavor to identify the specific HDAC targeted by HDACi leading to therapy sensitization. PMID:27738139

Preoperative diagnosis of Lynch syndrome with DNA mismatch repair immunohistochemistry on a diagnostic biopsy.

PubMed

Warrier, S K; Trainer, A H; Lynch, A C; Mitchell, C; Hiscock, R; Sawyer, S; Boussioutas, A; Heriot, A G

2011-12-01

DNA mismatch repair immunohistochemistry on tumor tissue is a simple, readily available, and cost-effective method of identifying patients with Lynch syndrome in the postoperative setting. The aim of the study was to assess whether the mismatch repair status of a colorectal cancer can be confirmed by mismatch repair immunohistochemistry on preoperative biopsy. Germline positive patients with Lynch syndrome were identified from a prospectively collected Familial Cancer Clinic database. Preoperative colorectal cancer biopsy specimens were obtained from the source pathology provider to generate a cohort of matched preoperative and postoperative specimens. The specimens were sectioned and stained for 4 mismatch repair proteins (MLH1, MSH2, MSH6, PMS2). An age-matched cohort to compare specimens was selected from Bethesda positive but mismatch repair immunohistochemistry negative patients. All slides were reviewed by a single blinded pathologist. The Wilson method was used to calculate a true underlying proportion of patients for whom the preoperative result matched the postoperative test result with a 95% confidence interval. Of 128 germline positive mutation carriers, 40 patients (mean age 41, SD 11.3) had colorectal resections. Thirty-three preoperative specimens were retrievable and were matched with biopsies from 33 controls. The germline mutations included in the study were 8 MLH1, 19 MSH2, 3 MSH6, and 2 PMS2. In patients where germline positive status was known, sensitivity was 100% (95% CI 89.2-100) and specificity was 100% (95% CI 89.2-100). Identical sensitivity and specificity were observed in 33 age-matched patients. The sensitivity of the endoscopic biopsy in predicting germline status was 94.9% (95% CI 80.4-98.3). The mismatch repair disease status of a colorectal cancer can be reliably confirmed by mismatch repair immunohistochemistry on a diagnostic colorectal cancer biopsy sample before definitive surgery. Ascertaining a diagnosis of Lynch syndrome

MSH3 mismatch repair protein regulates sensitivity to cytotoxic drugs and a histone deacetylase inhibitor in human colon carcinoma cells.

PubMed

Park, Jae Myung; Huang, Shengbing; Tougeron, David; Sinicrope, Frank A

2013-01-01

MSH3 is a DNA mismatch repair (MMR) gene that undergoes frequent somatic mutation in colorectal cancers (CRCs) with MMR deficiency. MSH3, together with MSH2, forms the MutSβ heteroduplex that interacts with interstrand cross-links induced by drugs such as cisplatin. To date, the impact of MSH3 on chemosensitivity is unknown. We utilized isogenic HCT116 (MLH1-/MSH3-) cells where MLH1 is restored by transfer of chromosome 3 (HCT116+ch3) and also MSH3 by chromosome 5 (HCT116+3+5). We generated HCT116+3+5, SW480 (MLH1+/MSH3+) and SW48 (MLH1-/MSH3+) cells with shRNA knockdown of MSH3. Cells were treated with 5-fluorouracil (5-FU), SN-38, oxaliplatin, or the histone deacetylase (HDAC) inhibitor PCI-24781 and cell viability, clonogenic survival, DNA damage and apoptosis were analyzed. MSH3-deficient vs proficient CRC cells showed increased sensitivity to the irinotecan metabolite SN-38 and to oxaliplatin, but not 5-FU, as shown in assays for apoptosis and clonogenic survival. In contrast, suppression of MLH1 attenuated the cytotoxic effect of 5-FU, but did not alter sensitivity to SN-38 or oxaliplatin. The impact of MSH3 knockdown on chemosensitivity to SN-38 and oxaliplatin was maintained independent of MLH1 status. In MSH3-deficient vs proficient cells, SN-38 and oxaliplatin induced higher levels of phosphorylated histone H2AX and Chk2, and similar results were found in MLH1-proficient SW480 cells. MSH3-deficient vs proficient cells showed increased 53BP1 nuclear foci after irradiation, suggesting that MSH3 can regulate DNA double strand break (DSB) repair. We then utilized PCI-24781 that interferes with homologous recombination (HR) indicated by a reduction in Rad51 expression. The addition of PCI-24781 to oxaliplatin enhanced cytotoxicity to a greater extent compared to either drug alone. MSH3 status can regulate the DNA damage response and extent of apoptosis induced by chemotherapy. The ability of MSH3 to regulate chemosensitivity was independent of MLH1 status

Homozygous germ-line mutation of the PMS2 mismatch repair gene: a unique case report of constitutional mismatch repair deficiency (CMMRD).

PubMed

Ramchander, N C; Ryan, N A J; Crosbie, E J; Evans, D G

2017-04-05

Constitutional mismatch repair deficiency syndrome results from bi-allelic inheritance of mutations affecting the key DNA mismatch repair genes: MLH1, MSH2, MSH6 or PMS2. Individuals with bi-allelic mutations have a dysfunctional mismatch repair system from birth; as a result, constitutional mismatch repair deficiency syndrome is characterised by early onset malignancies. Fewer than 150 cases have been reported in the literature over the past 20 years. This is the first report of the founder PMS2 mutation - NM_000535.5:c.1500del (p.Val501TrpfsTer94) in exon 11 and its associated cancers in this family. The proband is 30 years old and is alive today. She is of Pakistani ethnic origin and a product of consanguinity. She initially presented aged 24 with painless bleeding per-rectum from colorectal polyps and was referred to clinical genetics. Clinical examination revealed two café-au-lait lesions, lichen planus, and a dermoid cyst. Her sister had been diagnosed in childhood with an aggressive brain tumour followed by colorectal cancer. During follow up, the proband developed 37 colorectal adenomatous polyps, synchronous ovarian and endometrial adenocarcinomas, and ultimately a metachronous gastric adenocarcinoma. DNA sequencing of peripheral lymphocytes revealed a bi-allelic inheritance of the PMS2 mutation NM_000535.5:c.1500del (p.Val501TrpfsTer94) in exon 11. Ovarian tumour tissue demonstrated low microsatellite instability. To date, she has had a total abdominal hysterectomy, bilateral salpingo-oophorectomy, and a total gastrectomy. Aspirin and oestrogen-only hormone replacement therapy provide some chemoprophylaxis and manage postmenopausal symptoms, respectively. An 18-monthly colonoscopy surveillance programme has led to the excision of three high-grade dysplastic colorectal tubular adenomatous polyps. The proband's family pedigree displays multiple relatives with cancers including a likely case of 'true' Turcot syndrome. Constitutional mismatch repair

Purification, crystallization and preliminary X-ray diffraction analysis of the human mismatch repair protein MutS[beta

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tseng, Quincy; Orans, Jillian; Hast, Michael A.

2012-03-16

MutS{beta} is a eukaryotic mismatch repair protein that preferentially targets extrahelical unpaired nucleotides and shares partial functional redundancy with MutS{alpha} (MSH2-MSH6). Although mismatch recognition by MutS{alpha} has been shown to involve a conserved Phe-X-Glu motif, little is known about the lesion-binding mechanism of MutS{beta}. Combined MSH3/MSH6 deficiency triggers a strong predisposition to cancer in mice and defects in msh2 and msh6 account for roughly half of hereditary nonpolyposis colorectal cancer mutations. These three MutS homologs are also believed to play a role in trinucleotide repeat instability, which is a hallmark of many neurodegenerative disorders. The baculovirus overexpression and purification ofmore » recombinant human MutS{beta} and three truncation mutants are presented here. Binding assays with heteroduplex DNA were carried out for biochemical characterization. Crystallization and preliminary X-ray diffraction analysis of the protein bound to a heteroduplex DNA substrate are also reported.« less

MMR Deficiency Does Not Sensitize or Compromise the Function of Hematopoietic Stem Cells to Low and High LET Radiation.

PubMed

Patel, Rutulkumar; Qing, Yulan; Kennedy, Lucy; Yan, Yan; Pink, John; Aguila, Brittany; Desai, Amar; Gerson, Stanton L; Welford, Scott M

2018-04-14

One of the major health concerns on long-duration space missions will be radiation exposure to the astronauts. Outside the earth's magnetosphere, astronauts will be exposed to galactic cosmic rays (GCR) and solar particle events that are principally composed of protons and He, Ca, O, Ne, Si, Ca, and Fe nuclei. Protons are by far the most common species, but the higher atomic number particles are thought to be more damaging to biological systems. Evaluation and amelioration of risks from GCR exposure will be important for deep space travel. The hematopoietic system is one of the most radiation-sensitive organ systems, and is highly dependent on functional DNA repair pathways for survival. Recent results from our group have demonstrated an acquired deficiency in mismatch repair (MMR) in human hematopoietic stem cells (HSCs) with age due to functional loss of the MLH1 protein, suggesting an additional risk to astronauts who may have significant numbers of MMR deficient HSCs at the time of space travel. In the present study, we investigated the effects gamma radiation, proton radiation, and 56 Fe radiation on HSC function in Mlh1 +/+ and Mlh1 -/- marrow from mice in a variety of assays and have determined that while cosmic radiation is a major risk to the hematopoietic system, there is no dependence on MMR capacity. Stem Cells Translational Medicine 2018. © 2018 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Mismatch repair deficiency associated with overexpression of the MSH3 gene.

PubMed

Marra, G; Iaccarino, I; Lettieri, T; Roscilli, G; Delmastro, P; Jiricny, J

1998-07-21

We tested the ability of recombinant hMutSalpha (hMSH2/hMSH6) and hMutSbeta (hMSH2/hMSH3) heterodimers to complement the mismatch repair defect of HEC59, a human cancer cell line whose extracts lack all three MutS homologues. Although repair of both base/base mispairs and insertion-deletion loops was restored by hMutSalpha, only the latter substrates were addressed in extracts supplemented with hMutSbeta. hMutSalpha was also able to complement a defect in the repair of base/base mispairs in CHO R and HL60R cell extracts. In these cells, methotrexate-induced amplification of the dihydrofolate reductase (DHFR) locus, which also contains the MSH3 gene, led to an overexpression of MSH3 and thus to a dramatic change in the relative levels of MutSalpha and MutSbeta. As a rule, MSH2 is primarily complexed with MSH6. MutSalpha is thus relatively abundant in mammalian cell extracts, whereas MutSbeta levels are generally low. In contrast, in cells that overexpress MSH3, the available MSH2 protein is sequestered predominantly into MutSbeta. This leads to degradation of the partnerless MSH6 and depletion of MutSalpha. CHO R and HL60R cells therefore lack correction of base/base mispairs, whereas loop repair is maintained by MutSbeta. Consequently, frameshift mutations in CHO R are rare, whereas transitions and transversions are acquired at a rate two orders of magnitude above background. Our data thus support and extend the findings of Drummond et al. [Drummond, J. T., Genschel, J., Wolf, E. & Modrich, P. (1997) Proc. Natl. Acad. Sci. USA 94, 10144-10149] and demonstrate that mismatch repair deficiency can arise not only through mutation or transcriptional silencing of a mismatch repair gene, but also as a result of imbalance in the relative amounts of the MSH3 and MSH6 proteins.

Endometrial tumour BRAF mutations and MLH1 promoter methylation as predictors of germline mismatch repair gene mutation status: a literature review.

PubMed

Metcalf, Alexander M; Spurdle, Amanda B

2014-03-01

Colorectal cancer (CRC) that displays high microsatellite instability (MSI-H) can be caused by either germline mutations in mismatch repair (MMR) genes, or non-inherited transcriptional silencing of the MLH1 promoter. A correlation between MLH1 promoter methylation, specifically the 'C' region, and BRAF V600E status has been reported in CRC studies. Germline MMR mutations also greatly increase risk of endometrial cancer (EC), but no systematic review has been undertaken to determine if these tumour markers may be useful predictors of MMR mutation status in EC patients. Endometrial cancer cohorts meeting review inclusion criteria encompassed 2675 tumours from 20 studies for BRAF V600E, and 447 tumours from 11 studies for MLH1 methylation testing. BRAF V600E mutations were reported in 4/2675 (0.1%) endometrial tumours of unknown MMR mutation status, and there were 7/823 (0.9%) total sequence variants in exon 11 and 27/1012 (2.7%) in exon 15. Promoter MLH1 methylation was not observed in tumours from 32 MLH1 mutation carriers, or for 13 MSH2 or MSH6 mutation carriers. MMR mutation-negative individuals with tumour MLH1 and PMS2 IHC loss displayed MLH1 methylation in 48/51 (94%) of tumours. We have also detailed specific examples that show the importance of MLH1 promoter region, assay design, and quantification of methylation. This review shows that BRAF mutations occurs so infrequently in endometrial tumours they can be discounted as a useful marker for predicting MMR-negative mutation status, and further studies of endometrial cohorts with known MMR mutation status are necessary to quantify the utility of tumour MLH1 promoter methylation as a marker of negative germline MMR mutation status in EC patients.

The DNA mismatch repair genes Msh3 and Msh6 cooperate in intestinal tumor suppression.

PubMed

Edelmann, W; Umar, A; Yang, K; Heyer, J; Kucherlapati, M; Lia, M; Kneitz, B; Avdievich, E; Fan, K; Wong, E; Crouse, G; Kunkel, T; Lipkin, M; Kolodner, R D; Kucherlapati, R

2000-02-15

Repair of mismatches in DNA in mammalian cells is mediated by a complex of proteins that are members of two highly conserved families of genes referred to as MutS and MutL homologues. Germline mutations in several members of these families, MSH2, MSH6, MLH1, and PMS2, but not MSH3, are responsible for hereditary non-polyposis colorectal cancer. To examine the role of MSH3, we generated a mouse with a null mutation in this gene. Cells from Msh3-/- mice are defective in repair of insertion/ deletion mismatches but can repair base-base mismatches. Msh3-/- mice develop tumors at a late age. When the Msh3-/- and Msh6-/- mutations are combined, the tumor predisposition phenotype is indistinguishable from Msh2-/- or Mlh1-/- mice. These results suggest that MSH3 cooperates with MSH6 in tumor suppression.

Measuring strand discontinuity-directed mismatch repair in yeast Saccharomyces cerevisiae by cell-free nuclear extracts.

PubMed

Yuan, Fenghua; Lai, Fangfang; Gu, Liya; Zhou, Wen; El Hokayem, Jimmy; Zhang, Yanbin

2009-05-01

Mismatch repair corrects biosynthetic errors generated during DNA replication, whose deficiency causes a mutator phenotype and directly underlies hereditary non-polyposis colorectal cancer and sporadic cancers. Because of remarkably high conservation of the mismatch repair machinery between the budding yeast (Saccharomyces cerevisiae) and humans, the study of mismatch repair in yeast has provided tremendous insights into the mechanisms of this repair pathway in humans. In addition, yeast cells possess an unbeatable advantage over human cells in terms of the easy genetic manipulation, the availability of whole genome deletion strains, and the relatively low cost for setting up the system. Although many components of eukaryotic mismatch repair have been identified, it remains unclear if additional factors, such as DNA helicase(s) and redundant nuclease(s) besides EXO1, participate in eukaryotic mismatch repair. To facilitate the discovery of novel mismatch repair factors, we developed a straightforward in vitro cell-free repair system. Here, we describe the practical protocols for preparation of yeast cell-free nuclear extracts and DNA mismatch substrates, and the in vitro mismatch repair assay. The validity of the cell-free system was confirmed by the mismatch repair deficient yeast strain (Deltamsh2) and the complementation assay with purified yeast MSH2-MSH6.

Tissue expression of MLH1, PMS2, MSH2, and MSH6 proteins and prognostic value of microsatellite instability in Wilms tumor: experience of 45 cases.

PubMed

Diniz, Gulden; Aktas, Safiye; Cubuk, Cankut; Ortac, Ragip; Vergin, Canan; Olgun, Nur

2013-05-01

Although the importance of microsatellite instability (MSI) and mismatch repair genes (MMR) is strongly established in colorectal cancer seen in the Lynch syndrome, its significance has not been fully established in Wilms tumor (WT). The aim of this study was to determine the prognostic value of MSI and MMR proteins in WT. This study included 45 pediatric cases with nephroblastoma. Protein expression was analyzed by immunohistochemistry of archival tissue sections. Real-time PCR melting analysis and fluorescence capillary electrophoresis (FCE) were performed to evaluate the MSI markers BAT25, BAT26, NR21, NR24, MONO27, penta D, and penta C in DNA extracted from tumor and normal tissues. Lower levels of MSI were observed in six cases (13.3%). There were no statistically significant correlations between MSI and some clinical prognostic factors such as stage of the tumors, and survival rates. Nineteen tumors (42.2%) showed loss of protein expression of MLH1, PMS2, MSH2, or MSH6. MMR protein defects were correlated with size (P = .021), and stage (P = .019) of the tumor, and survival rates (P < .01).Similarly MSI was also correlated with the size of the tumor (P = .046). This study showed that a small proportion of WT might be associated with the presence of MSI, as is the case with defects of DNA mismatch repair genes in the pathogenesis of WT. However, there was no concordance with the frequency of tissue expression of MMR proteins and MSI. These findings suggest that MMR genes may play an important role in the development of WT via different pathways.

GLI1 interferes with the DNA mismatch repair system in pancreatic cancer through BHLHE41-mediated suppression of MLH1.

PubMed

Inaguma, Shingo; Riku, Miho; Hashimoto, Mitsuyoshi; Murakami, Hideki; Saga, Shinsuke; Ikeda, Hiroshi; Kasai, Kenji

2013-12-15

The mismatch repair (MMR) system is indispensable for the fidelity of DNA replication, the impairment of which predisposes to the development and progression of many types of cancers. To date, GLI1 transcription factor, a key molecule of the Hedgehog signaling pathway, has been shown to regulate the expression of several genes crucial for a variety of cancer cell properties in many types of cancers, including pancreatic ductal adenocarcinoma (PDAC), but whether GLI1 could control the MMR system was not known. Here, we showed that GLI1 and GLI2 indirectly suppressed the expression of MLH1 in PDAC cells. Through GLI1 target gene screening, we found that GLI1 and GLI2 activated the expression of a basic helix-loop-helix type suppressor BHLHE41/DEC2/SHARP1 through a GLI-binding site in the promoter. Consistent with a previous report that BHLHE41 suppresses the MLH1 promoter activity, we found that the activation of GLI1 led to the BHLHE41-dependent suppression of MLH1, and a double knockdown of GLI1 and GLI2 conversely increased the MLH1 protein in PDAC cells. Using TALEN-based modification of the MLH1 gene, we further showed that GLI1 expression was indeed associated with an increased tolerance to a methylating agent, methylnitrosourea cooperatively with a lower copy number status of MLH1. Finally, GLI1 expression was immunohistochemically related positively with BHLHE41 and inversely with MLH1 in PDAC cells and precancerous lesions of the pancreas. On the basis of these results, we propose that GLI1 depresses the MMR activity and might contribute to the development and progression of PDAC. ©2013 AACR.

MSH3 Mismatch Repair Protein Regulates Sensitivity to Cytotoxic Drugs and a Histone Deacetylase Inhibitor in Human Colon Carcinoma Cells

PubMed Central

Park, Jae Myung; Huang, Shengbing; Tougeron, David; Sinicrope, Frank A.

2013-01-01

Background MSH3 is a DNA mismatch repair (MMR) gene that undergoes frequent somatic mutation in colorectal cancers (CRCs) with MMR deficiency. MSH3, together with MSH2, forms the MutSβ heteroduplex that interacts with interstrand cross-links induced by drugs such as cisplatin. To date, the impact of MSH3 on chemosensitivity is unknown. Methods We utilized isogenic HCT116 (MLH1−/MSH3−) cells where MLH1 is restored by transfer of chromosome 3 (HCT116+ch3) and also MSH3 by chromosome 5 (HCT116+3+5). We generated HCT116+3+5, SW480 (MLH1+/MSH3+) and SW48 (MLH1−/MSH3+) cells with shRNA knockdown of MSH3. Cells were treated with 5-fluorouracil (5-FU), SN-38, oxaliplatin, or the histone deacetylase (HDAC) inhibitor PCI-24781 and cell viability, clonogenic survival, DNA damage and apoptosis were analyzed. Results MSH3-deficient vs proficient CRC cells showed increased sensitivity to the irinotecan metabolite SN-38 and to oxaliplatin, but not 5-FU, as shown in assays for apoptosis and clonogenic survival. In contrast, suppression of MLH1 attenuated the cytotoxic effect of 5-FU, but did not alter sensitivity to SN-38 or oxaliplatin. The impact of MSH3 knockdown on chemosensitivity to SN-38 and oxaliplatin was maintained independent of MLH1 status. In MSH3-deficient vs proficient cells, SN-38 and oxaliplatin induced higher levels of phosphorylated histone H2AX and Chk2, and similar results were found in MLH1-proficient SW480 cells. MSH3-deficient vs proficient cells showed increased 53BP1 nuclear foci after irradiation, suggesting that MSH3 can regulate DNA double strand break (DSB) repair. We then utilized PCI-24781 that interferes with homologous recombination (HR) indicated by a reduction in Rad51 expression. The addition of PCI-24781 to oxaliplatin enhanced cytotoxicity to a greater extent compared to either drug alone. Conclusion MSH3 status can regulate the DNA damage response and extent of apoptosis induced by chemotherapy. The ability of MSH3 to regulate

Alcohol Consumption and the Risk of Colorectal Cancer for Mismatch Repair Gene Mutation Carriers.

PubMed

Dashti, S Ghazaleh; Buchanan, Daniel D; Jayasekara, Harindra; Ait Ouakrim, Driss; Clendenning, Mark; Rosty, Christophe; Winship, Ingrid M; Macrae, Finlay A; Giles, Graham G; Parry, Susan; Casey, Graham; Haile, Robert W; Gallinger, Steven; Le Marchand, Loïc; Thibodeau, Stephen N; Lindor, Noralane M; Newcomb, Polly A; Potter, John D; Baron, John A; Hopper, John L; Jenkins, Mark A; Win, Aung Ko

2017-03-01

Background: People with germline mutation in one of the DNA mismatch repair (MMR) genes have increased colorectal cancer risk. For these high-risk people, study findings of the relationship between alcohol consumption and colorectal cancer risk have been inconclusive. Methods: 1,925 MMR gene mutations carriers recruited into the Colon Cancer Family Registry who had completed a questionnaire on lifestyle factors were included. Weighted Cox proportional hazard regression models were used to estimate hazard ratios (HR) and 95% confidence intervals (CI) for the association between alcohol consumption and colorectal cancer. Results: Colorectal cancer was diagnosed in 769 carriers (40%) at a mean (SD) age of 42.6 (10.3) years. Compared with abstention, ethanol consumption from any alcoholic beverage up to 14 g/day and >28 g/day was associated with increased colorectal cancer risk (HR, 1.50; 95% CI, 1.09-2.07 and 1.69; 95% CI, 1.07-2.65, respectively; P trend = 0.05), and colon cancer risk (HR, 1.78; 95% CI, 1.27-2.49 and 1.94; 95% CI, 1.19-3.18, respectively; P trend = 0.02). However, there was no clear evidence for an association with rectal cancer risk. Also, there was no evidence for associations between consumption of individual alcoholic beverage types (beer, wine, spirits) and colorectal, colon, or rectal cancer risk. Conclusions: Our data suggest that alcohol consumption, particularly more than 28 g/day of ethanol (∼2 standard drinks of alcohol in the United States), is associated with increased colorectal cancer risk for MMR gene mutation carriers. Impact: Although these data suggested that alcohol consumption in MMR carriers was associated with increased colorectal cancer risk, there was no evidence of a dose-response, and not all types of alcohol consumption were associated with increased risk. Cancer Epidemiol Biomarkers Prev; 26(3); 366-75. ©2016 AACR . ©2016 American Association for Cancer Research.

Hijacking of the mismatch repair system to cause CAG expansion and cell death in neurodegenerative disease.

PubMed

McMurray, Cynthia T

2008-07-01

Mammalian cells have evolved sophisticated DNA repair systems to correct mispaired or damaged bases and extrahelical loops. Emerging evidence suggests that, in some cases, the normal DNA repair machinery is "hijacked" to become a causative factor in mutation and disease, rather than act as a safeguard of genomic integrity. In this review, we consider two cases in which active MMR leads to mutation or to cell death. There may be similar mechanisms by which uncoupling of normal MMR recognition from downstream repair allows triplet expansions underlying human neurodegenerative disease, or cell death in response to chemical lesion.

Comparison of the anti-proliferation and apoptosis-induction activities of sulindac, celecoxib, curcumin, and nifedipine in mismatch repair-deficient cell lines.

PubMed

Wei, Shu-Chen; Lin, Young-Sun; Tsao, Po-Nien; Wu-Tsai, Jyy-Ji; Wu, C H Herbert; Wong, Jau-Min

2004-08-01

The adenomatous polyposis coli (APC) and mismatch repair (MMR) pathways are both involved in the tumorigenesis of hereditary colorectal cancers. Chemoprevention focuses on the APC pathway in the absence of information concerning MMR targets. This study compared the anticancer effects of sulindac, celecoxib, curcumin, and nifedipine in MMR-deficient cell lines, in order to determine the most appropriate chemopreventive agent for long-term use in patients with hereditary colorectal cancer. Five human colorectal cell lines (SW480, HCT116, LoVo, SW48, and HCT15) and an endometrial cancer cell line (HEC-1-A) were used for susceptibility testing. Tests included assays for growth inhibition, cell-cycle arrest, and apoptosis. Sulindac, celecoxib, curcumin, and nifedipine all displayed dose- and time-dependent anti-proliferation activities. Celecoxib was the most effective anti-proliferative agent, and increased the G0/G1 phase proportion in the cell cycle after treatment more significantly than the other agents in all cell lines. Curcumin displayed a more potent apoptosis-inducing activity than the other agents in treated cells. The tested drugs were effective against colorectal and endometrial cancer cell lines. Celecoxib is more potent with fewer side effects than sulindac. Nifedipine's observed chemopreventive efficacy may complement its known therapeutic application in patients with hypertension.

DNA Excision Repair at Telomeres

PubMed Central

Jia, Pingping; Her, Chengtao; Chai, Weihang

2015-01-01

DNA damage is caused by either endogenous cellular metabolic processes such as hydrolysis, oxidation, alkylation, and DNA base mismatches, or exogenous sources including ultraviolet (UV) light, ionizing radiation, and chemical agents. Damaged DNA that is not properly repaired can lead to genomic instability, driving tumorigenesis. To protect genomic stability, mammalian cells have evolved highly conserved DNA repair mechanisms to remove and repair DNA lesions. Telomeres are composed of long tandem TTAGGG repeats located at the ends of chromosomes. Maintenance of functional telomeres is critical for preventing genome instability. The telomeric sequence possesses unique features that predispose telomeres to a variety of DNA damage induced by environmental genotoxins. This review briefly describes the relevance of excision repair pathways in telomere maintenance, with the focus on base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR). By summarizing current knowledge on excision repair of telomere damage and outlining many unanswered questions, it is our hope to stimulate further interest in a better understanding of excision repair processes at telomeres and in how these processes contribute to telomere maintenance. PMID:26422132

Constitutional mismatch repair deficiency and Lynch syndrome among consecutive Arab Bedouins with colorectal cancer in Israel.

PubMed

Abu Freha, Naim; Leibovici Weissman, Yaara; Fich, Alexander; Barnes Kedar, Inbal; Halpern, Marisa; Sztarkier, Ignacio; Behar, Doron M; Arbib Sneh, Orly; Vilkin, Alex; Baris, Hagit N; Gingold, Rachel; Lejbkowicz, Flavio; Niv, Yaron; Goldberg, Yael; Levi, Zohar

2018-01-01

We assessed the molecular characteristics and the frequency of mutations in mismatch-repair genes among Bedouin patients with colorectal cancer (CRC) in Israel. Bedouin patients with a diagnosis of CRC at a major hospital in the southern part of Israel were deemed eligible for this study. The primary screening method was immunohistochemical staining for mismatch-repair proteins (MLH1, MSH2, MSH6, and PMS2). For subjects with abnormal immunohistochemical staining, we performed microsatellite instability (MSI) analyses, and for tumors with a loss of MLH1 expression we also performed BRAF testing. In MSI high cases we searched further for germline mutations. Of the 24 patients enrolled, four subjects (16.7%) had MSI high tumors: one subject was found to harbor a biallelic PMS2 mutation, one subject had Lynch syndrome (LS) with MSH6 mutation and two subjects had a loss of MLH1/PMS2 proteins/BRAF wild type /normal MLH1 sequence. Ten patients (41.7%) were younger than 50 at the time of diagnosis and none had first degree relatives with CRC. In conclusion, in this cohort of 24 consecutive Arab Bedouins with CRC, one patient was found to harbor a constitutional mismatch repair deficiency, one patient had LS with MSH6 mutation, and two patients had unresolved loss of MLH1/PMS2 proteins/BRAF wild type phenotype.

DNA Damage Induced by Alkylating Agents and Repair Pathways

PubMed Central

Kondo, Natsuko; Takahashi, Akihisa; Ono, Koji; Ohnishi, Takeo

2010-01-01

The cytotoxic effects of alkylating agents are strongly attenuated by cellular DNA repair processes, necessitating a clear understanding of the repair mechanisms. Simple methylating agents form adducts at N- and O-atoms. N-methylations are removed by base excision repair, AlkB homologues, or nucleotide excision repair (NER). O6-methylguanine (MeG), which can eventually become cytotoxic and mutagenic, is repaired by O6-methylguanine-DNA methyltransferase, and O6MeG:T mispairs are recognized by the mismatch repair system (MMR). MMR cannot repair the O6MeG/T mispairs, which eventually lead to double-strand breaks. Bifunctional alkylating agents form interstrand cross-links (ICLs) which are more complex and highly cytotoxic. ICLs are repaired by complex of NER factors (e.g., endnuclease xeroderma pigmentosum complementation group F-excision repair cross-complementing rodent repair deficiency complementation group 1), Fanconi anemia repair, and homologous recombination. A detailed understanding of how cells cope with DNA damage caused by alkylating agents is therefore potentially useful in clinical medicine. PMID:21113301

Homologous and Homeologous Intermolecular Gene Conversion Are Not Differentially Affected by Mutations in the DNA Damage or the Mismatch Repair Genes Rad1, Rad50, Rad51, Rad52, Rad54, Pms1 and Msh2

PubMed Central

Porter, G.; Westmoreland, J.; Priebe, S.; Resnick, M. A.

1996-01-01

Mismatch repair (MMR) genes or genes involved in both DNA damage repair and homologous recombination might affect homeologous vs. homologous recombination differentially. Spontaneous mitotic gene conversion between a chromosome and a homologous or homeologous donor sequence (14% diverged) on a single copy plasmid was examined in wild-type Saccharomyces cerevisiae strains and in MMR or DNA damage repair mutants. Homologous recombination in rad51, rad52 and rad54 mutants was considerably reduced, while there was little effect of rad1, rad50, pms1 and msh2 null mutations. DNA divergence resulted in no differential effect on recombination rates in the wild type or the mutants; there was only a five- to 10-fold reduction in homeologous relative to homologous recombination regardless of background. Since DNA divergence is known to affect recombination in some systems, we propose that differences in the role of MMR depends on the mode of recombination and/or the level of divergence. Based on analysis of the recombination breakpoints, there is a minimum of three homologous bases required at a recombination junction. A comparison of Rad(+) vs. rad52 strains revealed that while all conversion tracts are continuous, elimination of RAD52 leads to the appearance of a novel class of very short conversion tracts. PMID:8725224

Redundancy of Saccharomyces cerevisiae MSH3 and MSH6 in MSH2-dependent mismatch repair.

PubMed

Marsischky, G T; Filosi, N; Kane, M F; Kolodner, R

1996-02-15

Saccharomyces cerevisiae encodes six genes, MSH1-6, which encode proteins related to the bacterial MutS protein. In this study the role of MSH2, MSH3, and MSH6 in mismatch repair has been examined by measuring the rate of accumulating mutations and mutation spectrum in strains containing different combinations of msh2, msh3, and msh6 mutations and by studying the physical interaction between the MSH2 protein and the MSH3 and MSH6 proteins. The results indicate that S. cerevisiae has two pathways of MSH2-dependent mismatch repair: one that recognized single-base mispairs and requires MSH2 and MSH6, and a second that recognizes insertion/deletion mispairs and requires a combination of either MSH2 and MSH6 or MSH2 and MSH3. The redundancy of MSH3 and MSH6 explains the greater prevalence of hmsh2 mutations in HNPCC families and suggests how the role of hmsh3 and hmsh6 mutations in cancer susceptibility could be analyzed.

Constitutional mismatch repair deficiency syndrome: Do we know it?

PubMed

Ramachandra, C; Challa, Vasu Reddy; Shetty, Rachan

2014-04-01

Constitutional mismatch repair deficiency syndrome is a rare autosomal recessive syndrome caused by homozygous mutations in mismatch repair genes. This is characterized by the childhood onset of brain tumors, colorectal cancers, cutaneous manifestations of neurofibromatosis-1 like café au lait spots, hematological malignancies, and occasionally other rare malignancies. Here, we would like to present a family in which the sibling had glioblastoma, and the present case had acute lymphoblastic lymphoma and colorectal cancer. We would like to present this case because of its rarity and would add to literature.

Validation of predictive models for germline mutations in DNA mismatch repair genes in colorectal cancer.

PubMed

Monzon, Jose G; Cremin, Carol; Armstrong, Linlea; Nuk, Jennifer; Young, Sean; Horsman, Doug E; Garbutt, Kristy; Bajdik, Chris D; Gill, Sharlene

2010-02-15

Lynch syndrome is defined by the presence of germline mutations in mismatch repair (MMR) genes. Several models have been recently devised that predict mutation carrier status (Myriad Genetics, Wijnen, Barnetson, PREMM and MMRpro models). Families at moderate-high risk for harboring a Lynch-associated mutation, referred to the BC Cancer Agency (BCCA) Hereditary Cancer Program (HCP), underwent mutation analysis, immunohistochemistry and/or microsatellite testing. Seventy-two tested cases were included. Twenty-five patients were mutation positive (34.7%) and 47 were mutation negative (65.3%). Nineteen of 43 patients who were both microsatellite stable and normal on immunohistochemistry for MLH1 and MSH2 were also genotyped for mutations in these genes; all 19 were negative for MMR gene mutations. Model-derived probabilities of harboring a MMR gene mutation in the proband were calculated and compared to observed results. The area under the ROC curves were 0.75 (95%CI; 0.63-0.87), 0.86 (0.7-0.96), 0.89 (0.82-0.97), 0.89 (0.81-0.98) and 0.93 (0.86-0.99) for the Myriad, Barnetson, Wijnen, MMRpro and PREMM models, respectively. The Amsterdam II criteria had a sensitivity and specificity of 0.76 and 0.74, respectively, in this cohort. The PREMM model demonstrated the best performance for predicting carrier status based on the positive likelihood ratios at the >10%, >20% and >30% probability thresholds. In this referred cohort, the PREMM model had the most favorable concordance index and predictive performance for carrier status based on the positive LR. These prediction models (PREMM, MMRPro and Wijnen) may soon replace the Amsterdam II and revised Bethesda criteria as a prescreening tool for Lynch mutations.

Analysis of the functional domains of the mismatch repair homologue Msh1p and its role in mitochondrial genome maintenance.

PubMed

Mookerjee, Shona A; Lyon, Hiram D; Sia, Elaine A

2005-02-01

Mitochondrial DNA (mtDNA) repair occurs in all eukaryotic organisms and is essential for the maintenance of mitochondrial function. Evidence from both humans and yeast suggests that mismatch repair is one of the pathways that functions in overall mtDNA stability. In the mitochondria of the yeast Saccharomyces cerevisiae, the presence of a homologue to the bacterial MutS mismatch repair protein, MSH1, has long been known to be essential for mitochondrial function. The mechanisms for which it is essential are unclear, however. Here, we analyze the effects of two point mutations, msh1-F105A and msh1-G776D, both predicted to be defective in mismatch repair; and we show that they are both able to maintain partial mitochondrial function. Moreover, there are significant differences in the severity of mitochondrial disruption between the two mutants that suggest multiple roles for Msh1p in addition to mismatch repair. Our overall findings suggest that these additional predicted functions of Msh1p, including recombination surveillance and heteroduplex rejection, may be primarily responsible for its essential role in mtDNA stability.

Colon and Endometrial Cancers with Mismatch Repair Deficiency can Arise from Somatic, Rather Than Germline, Mutations

PubMed Central

Haraldsdottir, Sigurdis; Hampel, Heather; Tomsic, Jerneja; Frankel, Wendy L.; Pearlman, Rachel; de la Chapelle, Albert; Pritchard, Colin C.

2014-01-01

Background & Aims Patients with Lynch syndrome carry germline mutations in single alleles of genes encoding the MMR proteins MLH1, MSH2, MSH6 and PMS2; when the second allele becomes mutated, cancer can develop. Increased screening for Lynch syndrome has identified patients with tumors that have deficiency in MMR, but no germline mutations in genes encoding MMR proteins. We investigated whether tumors with deficient MMR had acquired somatic mutations in patients without germline mutations in MMR genes using next-generation sequencing. Methods We analyzed blood and tumor samples from 32 patients with colorectal or endometrial cancer who participated in Lynch syndrome screening studies in Ohio and were found to have tumors with MMR deficiency (based on microsatellite instability and/or absence of MMR proteins in immunohistochemical analysis, without hypermethylation of MLH1), but no germline mutations in MMR genes. Tumor DNA was sequenced for MLH1, MSH2, MSH6, PMS2, EPCAM, POLE and POLD1 with ColoSeq and mutation frequencies were established. Results Twenty-two of 32 patients (69%) were found to have two somatic (tumor) mutations in MMR genes encoding proteins that were lost from tumor samples, based on immunohistochemistry. Of the 10 tumors without somatic mutations in MMR genes, 3 had somatic mutations with possible loss of heterozygosity that could lead to MMR deficiency, 6 were found to be false-positive results (19%), and 1 had no mutations known to be associated with MMR deficiency. All of the tumors found to have somatic MMR mutations were of the hypermutated phenotype (>12 mutations/Mb); 6 had mutation frequencies >200 per Mb, and 5 of these had somatic mutations in POLE, which encodes a DNA polymerase. Conclusions Some patients are found to have tumors with MMR deficiency during screening for Lynch syndrome, yet have no identifiable germline mutations in MMR genes. We found that almost 70% of these patients acquire somatic mutations in MMR genes, leading to

Agenesis of the corpus callosum and gray matter heterotopia in three patients with constitutional mismatch repair deficiency syndrome

PubMed Central

Baas, Annette F; Gabbett, Michael; Rimac, Milan; Kansikas, Minttu; Raphael, Martine; Nievelstein, Rutger AJ; Nicholls, Wayne; Offerhaus, Johan; Bodmer, Danielle; Wernstedt, Annekatrin; Krabichler, Birgit; Strasser, Ulrich; Nyström, Minna; Zschocke, Johannes; Robertson, Stephen P; van Haelst, Mieke M; Wimmer, Katharina

2013-01-01

Constitutional mismatch repair deficiency (CMMR-D) syndrome is a rare inherited childhood cancer predisposition caused by biallelic germline mutations in one of the four mismatch repair (MMR)-genes, MLH1, MSH2, MSH6 or PMS2. Owing to a wide tumor spectrum, the lack of specific clinical features and the overlap with other cancer predisposing syndromes, diagnosis of CMMR-D is often delayed in pediatric cancer patients. Here, we report of three new CMMR-D patients all of whom developed more than one malignancy. The common finding in these three patients is agenesis of the corpus callosum (ACC). Gray matter heterotopia is present in two patients. One of the 57 previously reported CMMR-D patients with brain tumors (therefore all likely had cerebral imaging) also had ACC. With the present report the prevalence of cerebral malformations is at least 4/60 (6.6%). This number is well above the population birth prevalence of 0.09–0.36 live births with these cerebral malformations, suggesting that ACC and heterotopia are features of CMMR-D. Therefore, the presence of cerebral malformations in pediatric cancer patients should alert to the possible diagnosis of CMMR-D. ACC and gray matter heterotopia are the first congenital malformations described to occur at higher frequency in CMMR-D patients than in the general population. Further systematic evaluations of CMMR-D patients are needed to identify possible other malformations associated with this syndrome. PMID:22692065

Agenesis of the corpus callosum and gray matter heterotopia in three patients with constitutional mismatch repair deficiency syndrome.

PubMed

Baas, Annette F; Gabbett, Michael; Rimac, Milan; Kansikas, Minttu; Raphael, Martine; Nievelstein, Rutger Aj; Nicholls, Wayne; Offerhaus, Johan; Bodmer, Danielle; Wernstedt, Annekatrin; Krabichler, Birgit; Strasser, Ulrich; Nyström, Minna; Zschocke, Johannes; Robertson, Stephen P; van Haelst, Mieke M; Wimmer, Katharina

2013-01-01

Constitutional mismatch repair deficiency (CMMR-D) syndrome is a rare inherited childhood cancer predisposition caused by biallelic germline mutations in one of the four mismatch repair (MMR)-genes, MLH1, MSH2, MSH6 or PMS2. Owing to a wide tumor spectrum, the lack of specific clinical features and the overlap with other cancer predisposing syndromes, diagnosis of CMMR-D is often delayed in pediatric cancer patients. Here, we report of three new CMMR-D patients all of whom developed more than one malignancy. The common finding in these three patients is agenesis of the corpus callosum (ACC). Gray matter heterotopia is present in two patients. One of the 57 previously reported CMMR-D patients with brain tumors (therefore all likely had cerebral imaging) also had ACC. With the present report the prevalence of cerebral malformations is at least 4/60 (6.6%). This number is well above the population birth prevalence of 0.09-0.36 live births with these cerebral malformations, suggesting that ACC and heterotopia are features of CMMR-D. Therefore, the presence of cerebral malformations in pediatric cancer patients should alert to the possible diagnosis of CMMR-D. ACC and gray matter heterotopia are the first congenital malformations described to occur at higher frequency in CMMR-D patients than in the general population. Further systematic evaluations of CMMR-D patients are needed to identify possible other malformations associated with this syndrome.

Polymorphisms in mismatch repair genes are associated with risk and microsatellite instability of gastric cancer, and interact with life exposures.

PubMed

Zhu, Haifeng; Li, Xiaoqin; Zhang, Xiaomei; Chen, Deyu; Li, Dan; Ren, Jin; Gu, Hangang; Shu, Yongqian; Wang, Deqiang

2016-03-15

Epigenetic alterations of DNA mismatch repair (MMR) genes are associated with risk of gastric cancer (GC) by causing microsatellite instability (MSI). Less understood is the association of common polymorphisms in MMR genes with the risk and MSI phenotype of GC. A hospital-based study was conducted in China with 423 cases and 454 matched controls. Four potentially functional polymorphisms were selected and analyzed: rs1800734 in MLH1, rs2303428 in MSH2, rs735943 in EXO1, and rs11797 in TREX1. The rs1800734 G-allele was associated with decreased risk of GC (GA or GG vs AA, OR=0.72; 95% CI: 0.50-1.05; Ptrend=0.029). For combined effects, a dose-response manner was observed in which GC risk was increased with increasing number of at-risk genotypes (Ptrend=0.039); this manner mainly existed in MSI GC (Ptrend=0.047) rather than in microsatellite stability GC, though neither single polymorphism was linked with MSI. For exposures, modified effects were observed from green tea drinking and soy foods intake on rs11797 (P for interaction=0.007 and 0.016, respectively). The MLH1 rs1800734 polymorphism is associated with GC risk. Those at-risk genotypes have a joint effect on GC risk, which contributes to the MSI phenotype of GC. Life exposures modify GC risk, stratified by MMR genotypes. Copyright © 2015 Elsevier B.V. All rights reserved.

Clinical Management and Tumor Surveillance Recommendations of Inherited Mismatch Repair Deficiency in Childhood.

PubMed

Tabori, Uri; Hansford, Jordan R; Achatz, Maria Isabel; Kratz, Christian P; Plon, Sharon E; Frebourg, Thierry; Brugières, Laurence

2017-06-01

Replication proofreading is crucial to avoid mutation accumulation in dividing cells. In humans, proofreading and replication repair is maintained by the exonuclease domains of DNA polymerases and the mismatch repair system. Individuals harboring germline mutations in genes involved in this process are at increased risk of early cancers from multiple organs. Biallelic mutations in any of the four mismatch repair genes MSH2, MSH6, MLH1 , and PMS2 result in one of the most aggressive childhood cancer predisposition syndromes, termed constitutional mismatch repair deficiency or constitutional mismatch repair deficiency syndrome (CMMRD). Data gathered in the last decade allow us to better define the clinical manifestations, tumor spectrum, and diagnostic algorithms for CMMRD. In this article, we summarize this information and present a comprehensive consensus surveillance protocol for these individuals. Ongoing research will allow for further definition of replication repair-deficient cancer syndromes, assessing the cost-effectiveness of such surveillance protocols and potential therapeutic interventions for these children and families. Clin Cancer Res; 23(11); e32-e37. ©2017 AACR See all articles in the online-only CCR Pediatric Oncology Series. ©2017 American Association for Cancer Research.

Repair of naturally occurring mismatches can induce mutations in flanking DNA

PubMed Central

Chen, Jia; Miller, Brendan F; Furano, Anthony V

2014-01-01

‘Normal’ genomic DNA contains hundreds of mismatches that are generated daily by the spontaneous deamination of C (U/G) and methyl-C (T/G). Thus, a mutagenic effect of their repair could constitute a serious genetic burden. We show here that while mismatches introduced into human cells on an SV40-based episome were invariably repaired, this process induced mutations in flanking DNA at a significantly higher rate than no mismatch controls. Most mutations involved the C of TpC, the substrate of some single strand-specific APOBEC cytidine deaminases, similar to the mutations that can typify the ‘mutator phenotype’ of numerous tumors. siRNA knockdowns and chromatin immunoprecipitation showed that TpC preferring APOBECs mediate the mutagenesis, and siRNA knockdowns showed that both the base excision and mismatch repair pathways are involved. That naturally occurring mispairs can be converted to mutators, represents an heretofore unsuspected source of genetic changes that could underlie disease, aging, and evolutionary change. DOI: http://dx.doi.org/10.7554/eLife.02001.001 PMID:24843013

Frequent PIK3CA Mutations in Colorectal and Endometrial Cancer with Double Somatic Mismatch Repair Mutations

PubMed Central

Cohen, Stacey A.; Turner, Emily H.; Beightol, Mallory B.; Jacobson, Angela; Gooley, Ted A.; Salipante, Stephen J.; Haraldsdottir, Sigurdis; Smith, Christina; Scroggins, Sheena; Tait, Jonathan F.; Grady, William M.; Lin, Edward H.; Cohn, David E.; Goodfellow, Paul J.; Arnold, Mark W.; de la Chapelle, Albert; Pearlman, Rachel; Hampel, Heather; Pritchard, Colin C.

2016-01-01

Background & Aims Double somatic mutations in mismatch repair (MMR) genes have recently been described in colorectal and endometrial cancers with microsatellite instability (MSI) not attributable to MLH1 hypermethylation or germline mutation. We sought to define the molecular phenotype of this newly recognized tumor subtype. Methods From two prospective Lynch syndrome screening studies, we identified patients with colorectal and endometrial tumors harboring ≥2 somatic MMR mutations, but normal germline MMR testing (“double somatic”). We determined the frequencies of tumor PIK3CA, BRAF, KRAS, NRAS, and PTEN mutations by targeted next-generation sequencing and used logistic-regression models to compare them to: Lynch syndrome, MLH1 hypermethylated, and microsatellite stable (MSS) tumors. We validated our findings using independent datasets from The Cancer Genome Atlas (TCGA). Results Among colorectal cancer cases, we found that 14/21 (67%) of double somatic cases had PIK3CA mutations vs. 4/18 (22%) Lynch syndrome, 2/10 (20%) MLH1 hypermethylated, and 12/78 (15%) MSS tumors; p<0.0001. PIK3CA mutations were detected in 100% of 13 double somatic endometrial cancers (p=0.04). BRAF mutations were absent in double somatic and Lynch syndrome colorectal tumors. We found highly similar results in a validation cohort from TCGA (113 colorectal, 178 endometrial cancer), with 100% of double somatic cases harboring a PIK3CA mutation (p<0.0001). Conclusions PIK3CA mutations are present in double somatic mutated colorectal and endometrial cancers at substantially higher frequencies than other MSI subgroups. PIK3CA mutation status may better define an emerging molecular entity in colorectal and endometrial cancers, with the potential to inform screening and therapeutic decision making. PMID:27302833

Up-regulation of mismatch repair genes MSH6, PMS2 and MLH1 parallels development of genetic instability and is linked to tumor aggressiveness and early PSA recurrence in prostate cancer.

PubMed

Wilczak, Waldemar; Rashed, Semin; Hube-Magg, Claudia; Kluth, Martina; Simon, Ronald; Büscheck, Franziska; Clauditz, Till Sebastian; Grupp, Katharina; Minner, Sarah; Tsourlakis, Maria Christina; Möller-Koop, Christina; Graefen, Markus; Adam, Meike; Haese, Alexander; Wittmer, Corinna; Sauter, Guido; Izbicki, Jakob Robert; Huland, Hartwig; Schlomm, Thorsten; Steurer, Stefan; Krech, Till; Lebok, Patrick

2017-01-01

DNA mismatch repair (MMR) is integral to the maintenance of genetic stability. We aimed to evaluate the clinical impact of MMR gene expression in prostate cancer. The MMR genes MSH6, MLH1 and PMS2 were analyzed by immunohistochemistry on a tissue microarray containing 11152 prostate cancer specimens. Results were compared with ETS-related gene status and deletions of PTEN, 3p13, 5q21 and 6q15. MSH6, MLH1 and PMS2 expression was detectable in 89.5%, 85.4% and 85.0% of cancers and was particularly strong in cancers with advanced pathological tumor stage (P < 0.0001 each), high Gleason grade (P < 0.0001 each), nodal metastasis (P ≤ 0.0083) and early biochemical recurrence (P < 0.0001). High levels of MMR gene expression paralleled features of genetic instability, such as the number of genomic deletions per cancer; strong expression of all three MMR genes was found in 24%, 29%, 30%, 33% and 42% of cancers with no, one, two, three or four to five deletions (P < 0.0001). The prognostic value of the analyzed MMR genes was largely driven by the subset of cancers lacking ERG fusion (P < 0.0001), while the prognostic impact of MMR gene overexpression was only marginal in ERG-positive cancers. Multivariate analyses suggested an independent prognostic relevance of MMR genes in ERG-negative prostate cancers when compared with prognostic parameters available at the time of initial biopsy. In conclusion, MMR overexpression is common in prostate cancer and is linked to poor outcome as well as features indicating genetic instability. ERG fusion should be analyzed along with MMR gene expression in potential clinical tests. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Expression of DNA mismatch repair proteins MLH1, MSH2, and MSH6 in recurrent glioblastoma.

PubMed

Stark, Andreas M; Doukas, Alexander; Hugo, Heinz-Herrmann; Hedderich, Jürgen; Hattermann, Kirsten; Maximilian Mehdorn, H; Held-Feindt, Janka

2015-02-01

Methylated O6-methylguanin-DNA-methytransferase (MGMT) promoter methylation is associated with survival in patients with glioblastoma. Current evidence suggests that further mismatch repair genes play a pivotal role in the tumor response to treatment. Candidate genes are MLH1, MSH2, and MSH6. Formerly, we found evidence of prognostic impact of MLH1 and MSH6 immunohistochemical expression in a small series of patients with initial glioblastoma. Two hundred and eleven patients were included who underwent macroscopically total removal of primary glioblastoma and at least one re-craniotomy for recurrence. Immunohistochemical staining was performed on paraffin-embedded specimens of initial tumors with specific antibodies against MLH1, MSH2, and MSH6. RESULTS were compared to the Ki67 proliferation index and patient survival. Additionally, fresh frozen samples from 16 paired initial and recurrent specimens were examined using real-time reverse transcription polymerase chain reaction (RT-PCR) with specific primers against MLH1, MSH2, and MSH6. RESULTS were compared to MGMT status and survival. (1) Immunohistochemical expression of MSH6 was significantly associated with the Ki67 proliferation index (P<0.001) but not with survival. (2) PCR revealed two patients with increasing expression of MLH1, MLH2, and MSH6 over treatment combined with lacking MGMT methylation. In another two patients, decreased MLH1, MSH2, and MSH6 expression was observed in combination with MGMT promoter methylation. Our data indicate that there may be glioblastoma patient subgroups characterized by MMR-expression changes beyond MGMT promoter methylation. The immunohistochemical expression of MLH1, MSH2, and MSH6 in initial glioblastoma is not associated with patient survival.

Methylation Analysis of DNA Mismatch Repair Genes Using DNA Derived from the Peripheral Blood of Patients with Endometrial Cancer: Epimutation in Endometrial Carcinogenesis.

PubMed

Takeda, Takashi; Banno, Kouji; Yanokura, Megumi; Adachi, Masataka; Iijima, Moito; Kunitomi, Haruko; Nakamura, Kanako; Iida, Miho; Nogami, Yuya; Umene, Kiyoko; Masuda, Kenta; Kobayashi, Yusuke; Yamagami, Wataru; Hirasawa, Akira; Tominaga, Eiichiro; Susumu, Nobuyuki; Aoki, Daisuke

2016-10-14

Germline mutation of DNA mismatch repair (MMR) genes is a cause of Lynch syndrome. Methylation of MutL homolog 1 ( MLH1 ) and MutS homolog 2 ( MSH2 ) has been detected in peripheral blood cells of patients with colorectal cancer. This methylation is referred to as epimutation. Methylation of these genes has not been studied in an unselected series of endometrial cancer cases. Therefore, we examined methylation of MLH1 , MSH2 , and MSH6 promoter regions of peripheral blood cells in 206 patients with endometrial cancer using a methylation-specific polymerase chain reaction (MSP). Germline mutation of MMR genes, microsatellite instability (MSI), and immunohistochemistry (IHC) were also analyzed in each case with epimutation. MLH1 epimutation was detected in a single patient out of a total of 206 (0.49%)-1 out of 58 (1.72%) with an onset age of less than 50 years. The patient with MLH1 epimutation showed high level MSI (MSI-H), loss of MLH1 expression and had developed endometrial cancer at 46 years old, complicated with colorectal cancer. No case had epimutation of MSH2 or MSH6 . The MLH1 epimutation detected in a patient with endometrial cancer may be a cause of endometrial carcinogenesis. This result indicates that it is important to check epimutation in patients with endometrial cancer without a germline mutation of MMR genes.

Mismatch repair polymorphisms and the risk of colorectal cancer.

PubMed

Berndt, Sonja I; Platz, Elizabeth A; Fallin, M Daniele; Thuita, Lucy W; Hoffman, Sandra C; Helzlsouer, Kathy J

2007-04-01

Rare germline variants in mismatch repair genes have been linked to hereditary nonpolyposis colorectal cancer; however, it is unknown whether common polymorphisms in these genes alter the risk of colorectal cancer. To examine the association between common variants in mismatch repair genes and colorectal cancer, we conducted a case-cohort study within the CLUE II cohort. Four single nucleotide polymorphisms in 3 mismatch repair genes (MSH3 R940Q, MSH3 T1036A, MSH6 G39E and MLH1 I219V) were genotyped in 237 colorectal cancer cases and a subcohort of 2,189 participants. Incidence rate ratios (RRs) and 95% confidence intervals (95% CIs) for each polymorphism were estimated. The MSH3 1036A variant was found to be associated with an increased risk of colorectal cancer (RR=1.28, 95% CI: 0.94-1.74 and RR=1.65, 95% CI: 1.01-2.70 for the AT and TT genotypes, respectively, with p(trend)=0.02), particularly proximal colon cancer. Although the MSH3 940Q variant was only weakly associated with colorectal cancer overall (p(trend)=0.07), it was associated with a significant increased risk of proximal colon cancer (RR=1.69, 95% CI: 1.10-2.61 and RR=2.68, 95% CI: 0.96-7.47 for the RQ and QQ genotypes, respectively with p(trend)=0.005). Processed meat intake appeared to modify the association between the MSH3 polymorphisms and colorectal cancer (p(interaction) < 0.10 for both). No association was observed with the MSH6 and MLH1 polymorphisms overall. This study suggests that common polymorphisms in the mismatch repair gene, MSH3, may increase the risk of colorectal cancer, especially proximal colon cancer. (c) 2006 Wiley-Liss, Inc.

Bacterial genes mutL, mutS, and dcm participate in repair of mismatches at 5-methylcytosine sites.

PubMed Central

Lieb, M

1987-01-01

Certain amber mutations in the cI gene of bacteriophage lambda appear to recombine very frequently with nearby mutations. The aberrant mutations included C-to-T transitions at the second cytosine in 5'CC(A/T)GG sequences (which are subject to methylation by bacterial cytosine methylase) and in 5'CCAG and 5'CAGG sequences. Excess cI+ recombinants arising in crosses that utilize these mutations are attributable to the correction of mismatches by a bacterial very-short-patch (VSP) mismatch repair system. In the present study I found that two genes required for methyladenine-directed (long-patch) mismatch repair, mutL and mutS, also functioned in VSP mismatch repair; mutH and mutU (uvrD) were dispensable. VSP mismatch repair was greatly reduced in a dcm Escherichia coli mutant, in which 5-methylcytosine was not methylated. However, mismatches in heteroduplexes prepared from lambda DNA lacking 5-methylcytosine were repaired in dcm+ bacteria. These results indicate that the product of gene dcm has a repair function in addition to its methylase activity. PMID:2959653

MSH-2 and MLH-1 Protein Expression in Muir Torre Syndrome-Related and Sporadic Sebaceous Neoplasms

PubMed Central

Morales-Burgos, Adisbeth; Sánchez, Jorge L.; Figueroa, Luz D.; De Jesús-Monge, Wilfredo E.; Cruz-Correa, Marcia R.; González-Keelan, Carmen; Nazario, Cruz María

2009-01-01

Background Muir-Torre Syndrome (MTS) is a rare autosomal-dominant disorder characterized by the predisposition to both sebaceous neoplasm and internal malignancies. MTS-associated sebaceous neoplasms reveal mutations in DNA mismatch repair (MMR) genes and microsatellite instability. A significant part of MTS patients represents a phenotypic variant, the hereditary nonpolyposis colorectal cancer (HNPCC). A strong correlation between microsatellite instability and immunostaining has been demonstrated. The early recognition of sebaceous neoplasm as part of MTS, and their differentiation from sporadic sebaceous neoplasm may have an important application in a clinical setting. The absence of MLH-1 or MSH-2 expression by immunostaining identifies tumors with mismatch repair deficiency. Objectives Our aim is to determine whether an immunohistochemical approach, targeting DNA repair proteins MSH-2 and MLH-1 in MTS-related sebaceous neoplasm and their sporadic counterparts, can be used for their identification. Methods We examined 15 sebaceous neoplasms (including 6 internal malignancy- associated sebaceous neoplasms and 8 sporadic sebaceous neoplasms) from 11 patients for the expression of MSH-2 and MLH-1 by immunohistochemistry. Results Four of 5 internal malignancy-associated sebaceous neoplasms showed loss of expression of MSH-2 or MLH-1. Correlation of the immunostaining pattern of the sebaceous neoplasms and the patients’ positive history of colon carcinoma was 80%. Seven of 8 sporadic sebaceous neoplasms showed a positive expression of MSH-2 and MLH-1. The prevalence for loss of expression of MMR proteins in sebaceous neoplasms was 38.5%. MMR immunostaining had 87.5% specificity and 80% sensitivity. Limitations This study is limited by a small sample size, and by bias selection due to the use of non nationwide data-base as the resource of cases. Conclusions Our findings demonstrate that immunohistochemical testing for internal malignancy-associated sebaceous

DNA repair and tumorigenesis: lessons from hereditary cancer syndromes.

PubMed

Heinen, Christopher D; Schmutte, Christoph; Fishel, Richard

2002-01-01

The discovery that alterations of the DNA mismatch repair system (MMR) were linked to the common human cancer susceptibility syndrome hereditary nonpolyposis colon cancer (HNPCC) resulted in the declaration of a third class of genes involved in tumor development. In addition to oncogenes and tumor suppressors, alterations of DNA repair genes involved in maintaining genomic stability were found to be a clear cause of tum the level of the single nucleotides or chromosomes. This observation suggested that the establishment of genomic instability, termed the Mutator Phenotype, was an important aspect of tumor development.(1,2) Since the initial identification of the human MutS homolog hMSH2 nearly a decade ago,(3,4) more links have been described between human cancers and genes involved in maintaining genomic stability. Work in recent years has revealed that DNA repair proteins may also function in signaling pathways that provoke cell cycle arrest and apoptosis. This review will focus on the genetic and biochemical functions of DNA repair genes linked to hereditary cancer predisposition characterized by genomic instability (Table 1). Interestingly, the protein products of these genes have been directly or indirectly linked to the DNA damage-induce cell cycle arrest and apoptosis. We conclude that a robust connection between DNA repair proteins and damage-induced apoptosis may be as important for tumorigenesis as their role in maintaining genome stability.

Characterization of the immunological microenvironment of tumour buds and its impact on prognosis in mismatch repair-proficient and -deficient colorectal cancers.

PubMed

Zlobec, Inti; Minoo, Parham; Terracciano, Luigi; Baker, Kristi; Lugli, Alessandro

2011-09-01

Tumour budding in colorectal cancer is established as a poor prognostic factor. The inverse correlation of tumour buds with peritumoural lymphocytic inflammation suggests an interaction with specific immune responses. The aims of this study were to characterize the immunological microenvironment of tumour buds and its impact on prognosis in mismatch repair (MMR)-proficient and -deficient colorectal cancers. A total of 297 colorectal cancers were double-immunostained for CK22 plus one of the following: CD138, CD16, CD20, CD21, CD56, CD68, CD8, forkhead box P2 (FoxP3), granzyme B, mast cell tryptase, CD3 or T cell intracellular antigen-1 (TIA)-1. Tumour buds and immune cells within the region of densest budding were evaluated [×40 high-power field (HPF)] simultaneously. In both MMR-proficient and -deficient cancers, CD8(+), FoxP3(+) and CD68(+) cells were observed most frequently (>40 cells/HPF) and were independent prognostic factors. A combined prognostic score of tumour budding and CD8(+), FoxP3(+) and CD68(+) distinctly identified patients with low-, moderate- or high-risk colorectal cancers with 5-year survival rates of 75.2% [confidence interval 95% (CI): 66-83], 56.3% (95% CI: 43-68) and 25.2% (95% CI: 14-38), respectively, in MMR-proficient and -deficient cancers. The combined assessment of tumour budding with CD8, FoxP3 and CD68 lymphocytes could represent a basis for a prognostic score similar to the Bloom Richardson grade (BRE) and Gleason scores for breast and prostatic cancers. © 2011 Blackwell Publishing Limited.

Population-based study of the association of variants in mismatch repair genes with prostate cancer risk and outcomes

PubMed Central

Langeberg, Wendy J.; Kwon, Erika M.; Koopmeiners, Joseph S.; Ostrander, Elaine A.; Stanford, Janet L.

2009-01-01

Background Mismatch repair (MMR) gene activity may be associated with prostate cancer (PC) risk and outcomes. This study evaluated whether single nucleotide polymorphisms (SNPs) in key MMR genes are related to PC outcomes. Methods Data from two population-based case-control studies of PC among Caucasian and African-American men residing in King County, Washington were combined for this analysis. Cases (n=1,458) were diagnosed with PC in 1993–96 or 2002–05 and identified via the Seattle-Puget Sound SEER cancer registry. Controls (n=1,351) were age-matched to cases and identified via random digit dialing. Logistic regression was used to assess the relationship between haplotype-tagging SNPs and PC risk and disease aggressiveness. Cox proportional hazards regression was used to assess the relationship between SNPs and PC recurrence and PC-specific death. Results Nineteen SNPs were evaluated in the key MMR genes: five in MLH1, 10 in MSH2, and 4 in PMS2. Among Caucasian men, one SNP in MLH1 (rs9852810) was associated with: overall PC risk (OR=1.21, 95% CI=1.02, 1.44; p=0.03), more aggressive PC (OR=1.49, 95% CI=1.15–1.91; p<0.01), and PC recurrence (HR=1.83, 95% CI=1.18, 2.86; p<0.01), but not PC-specific mortality. A non-synonymous coding SNP in MLH1, rs1799977 (I219V), was also found to be associated with more aggressive disease. These results did not remain significant after adjusting for multiple comparisons. Conclusion This population-based case-control study provides evidence for a possible association with a gene variant in MLH1 in relation to risk of overall PC, more aggressive disease, and PC recurrence, which warrants replication. PMID:20056646

An Efficient Site-Specific Method for Irreversible Covalent Labeling of Proteins with a Fluorophore.

PubMed

Liu, Jiaquan; Hanne, Jeungphill; Britton, Brooke M; Shoffner, Matthew; Albers, Aaron E; Bennett, Jared; Zatezalo, Rachel; Barfield, Robyn; Rabuka, David; Lee, Jong-Bong; Fishel, Richard

2015-11-19

Fluorophore labeling of proteins while preserving native functions is essential for bulk Förster resonance energy transfer (FRET) interaction and single molecule imaging analysis. Here we describe a versatile, efficient, specific, irreversible, gentle and low-cost method for labeling proteins with fluorophores that appears substantially more robust than a similar but chemically distinct procedure. The method employs the controlled enzymatic conversion of a central Cys to a reactive formylglycine (fGly) aldehyde within a six amino acid Formylglycine Generating Enzyme (FGE) recognition sequence in vitro. The fluorophore is then irreversibly linked to the fGly residue using a Hydrazinyl-Iso-Pictet-Spengler (HIPS) ligation reaction. We demonstrate the robust large-scale fluorophore labeling and purification of E.coli (Ec) mismatch repair (MMR) components. Fluorophore labeling did not alter the native functions of these MMR proteins in vitro or in singulo. Because the FGE recognition sequence is easily portable, FGE-HIPS fluorophore-labeling may be easily extended to other proteins.

Expression levels of DNA replication and repair genes predict regional somatic repeat instability in the brain but are not altered by polyglutamine disease protein expression or age.

PubMed

Mason, Amanda G; Tomé, Stephanie; Simard, Jodie P; Libby, Randell T; Bammler, Theodor K; Beyer, Richard P; Morton, A Jennifer; Pearson, Christopher E; La Spada, Albert R

2014-03-15

Expansion of CAG/CTG trinucleotide repeats causes numerous inherited neurological disorders, including Huntington's disease (HD), several spinocerebellar ataxias and myotonic dystrophy type 1. Expanded repeats are genetically unstable with a propensity to further expand when transmitted from parents to offspring. For many alleles with expanded repeats, extensive somatic mosaicism has been documented. For CAG repeat diseases, dramatic instability has been documented in the striatum, with larger expansions noted with advancing age. In contrast, only modest instability occurs in the cerebellum. Using microarray expression analysis, we sought to identify the genetic basis of these regional instability differences by comparing gene expression in the striatum and cerebellum of aged wild-type C57BL/6J mice. We identified eight candidate genes enriched in cerebellum, and validated four--Pcna, Rpa1, Msh6 and Fen1--along with a highly associated interactor, Lig1. We also explored whether expression levels of mismatch repair (MMR) proteins are altered in a line of HD transgenic mice, R6/2, that is known to show pronounced regional repeat instability. Compared with wild-type littermates, MMR expression levels were not significantly altered in R6/2 mice regardless of age. Interestingly, expression levels of these candidates were significantly increased in the cerebellum of control and HD human samples in comparison to striatum. Together, our data suggest that elevated expression levels of DNA replication and repair proteins in cerebellum may act as a safeguard against repeat instability, and may account for the dramatically reduced somatic instability present in this brain region, compared with the marked instability observed in the striatum.

MSH6- or PMS2-deficiency causes re-replication in DT40 B cells, but it has little effect on immunoglobulin gene conversion or on repair of AID-generated uracils

PubMed Central

Campo, Vanina A.; Patenaude, Anne-Marie; Kaden, Svenja; Horb, Lori; Firka, Daniel; Jiricny, Josef; Di Noia, Javier M.

2013-01-01

The mammalian antibody repertoire is shaped by somatic hypermutation (SHM) and class switch recombination (CSR) of the immunoglobulin (Ig) loci of B lymphocytes. SHM and CSR are triggered by non-canonical, error-prone processing of G/U mismatches generated by activation-induced deaminase (AID). In birds, AID does not trigger SHM, but it triggers Ig gene conversion (GC), a ‘homeologous’ recombination process involving the Ig variable region and proximal pseudogenes. Because recombination fidelity is controlled by the mismatch repair (MMR) system, we investigated whether MMR affects GC in the chicken B cell line DT40. We show here that Msh6−/− and Pms2−/− DT40 cells display cell cycle defects, including genomic re-replication. However, although IgVλ GC tracts in MMR-deficient cells were slightly longer than in normal cells, Ig GC frequency, donor choice or the number of mutations per sequence remained unaltered. The finding that the avian MMR system, unlike that of mammals, does not seem to contribute towards the processing of G/U mismatches in vitro could explain why MMR is unable to initiate Ig GC in this species, despite initiating SHM and CSR in mammalian cells. Moreover, as MMR does not counteract or govern Ig GC, we report a rare example of ‘homeologous’ recombination insensitive to MMR. PMID:23314153

Explosive mutation accumulation triggered by heterozygous human Pol ε proofreading-deficiency is driven by suppression of mismatch repair

PubMed Central

Campbell, Brittany B; Ungerleider, Nathan; Light, Nicholas; Wu, Tong; LeCompte, Kimberly G; Goksenin, A Yasemin; Bunnell, Bruce A; Tabori, Uri; Shlien, Adam

2018-01-01

Tumors defective for DNA polymerase (Pol) ε proofreading have the highest tumor mutation burden identified. A major unanswered question is whether loss of Pol ε proofreading by itself is sufficient to drive this mutagenesis, or whether additional factors are necessary. To address this, we used a combination of next generation sequencing and in vitro biochemistry on human cell lines engineered to have defects in Pol ε proofreading and mismatch repair. Absent mismatch repair, monoallelic Pol ε proofreading deficiency caused a rapid increase in a unique mutation signature, similar to that observed in tumors from patients with biallelic mismatch repair deficiency and heterozygous Pol ε mutations. Restoring mismatch repair was sufficient to suppress the explosive mutation accumulation. These results strongly suggest that concomitant suppression of mismatch repair, a hallmark of colorectal and other aggressive cancers, is a critical force for driving the explosive mutagenesis seen in tumors expressing exonuclease-deficient Pol ε. PMID:29488881

Explosive mutation accumulation triggered by heterozygous human Pol ε proofreading-deficiency is driven by suppression of mismatch repair.

PubMed

Hodel, Karl P; de Borja, Richard; Henninger, Erin E; Campbell, Brittany B; Ungerleider, Nathan; Light, Nicholas; Wu, Tong; LeCompte, Kimberly G; Goksenin, A Yasemin; Bunnell, Bruce A; Tabori, Uri; Shlien, Adam; Pursell, Zachary F

2018-02-28

Tumors defective for DNA polymerase (Pol) ε proofreading have the highest tumor mutation burden identified. A major unanswered question is whether loss of Pol ε proofreading by itself is sufficient to drive this mutagenesis, or whether additional factors are necessary. To address this, we used a combination of next generation sequencing and in vitro biochemistry on human cell lines engineered to have defects in Pol ε proofreading and mismatch repair. Absent mismatch repair, monoallelic Pol ε proofreading deficiency caused a rapid increase in a unique mutation signature, similar to that observed in tumors from patients with biallelic mismatch repair deficiency and heterozygous Pol ε mutations. Restoring mismatch repair was sufficient to suppress the explosive mutation accumulation. These results strongly suggest that concomitant suppression of mismatch repair, a hallmark of colorectal and other aggressive cancers, is a critical force for driving the explosive mutagenesis seen in tumors expressing exonuclease-deficient Pol ε. © 2018, Hodel et al.

Unnatural substrates reveal the importance of 8-oxoguanine for in vivo mismatch repair by MutY

PubMed Central

Livingston, Alison L.; O’Shea, Valerie L.; Kim, Taewoo; Kool, Eric T.; David, Sheila S.

2009-01-01

Escherchia coli MutY plays an important role in preventing mutations associated with the oxidative lesion 7,8-dihydro-8-oxo-2′-deoxyguanosine (OG) in DNA by excising adenines from OG:A mismatches as the first step of base excision repair. To determine the importance of specific steps in the base pair recognition and base removal process of MutY, we have evaluated the effects of modifications of the OG:A substrate on the kinetics of base removal, mismatch affinity and repair to G:C in an Escherchia coli-based assay. Surprisingly, adenine modification was tolerated in the cellular assay, while modification of OG results in minimal cellular repair. High affinity for the mismatch and efficient base removal require the presence of OG. Taken together, these results suggest that the presence of OG is a critical feature for MutY to locate OG:A mismatches and select the appropriate adenines for excision to initiate repair in vivo prior to replication. PMID:18026095

Overcoming temozolomide resistance in glioblastoma via dual inhibition of NAD+ biosynthesis and base excision repair

PubMed Central

Goellner, Eva M.; Grimme, Bradford; Brown, Ashley R.; Lin, Ying-Chih; Wang, Xiao-Hong; Sugrue, Kelsey F.; Mitchell, Leah; Trivedi, Ram N.; Tang, Jiang-bo; Sobol, Robert W.

2011-01-01

Glioblastoma multiforme (GBM) is a devastating brain tumor with poor prognosis and low median survival time. Standard treatment includes radiation and chemotherapy with the DNA alkylating agent temozolomide (TMZ). However, a large percentage of tumors are resistant to the cytotoxic effects of the TMZ-induced DNA lesion O6-methylguanine (O6-MeG) due to elevated expression of the repair protein O6-methylguanine-DNA methyltransferase (MGMT) or a defect in the mismatch repair (MMR) pathway. Although a majority of the TMZ induced lesions (N7-methylguanine and N3-methyladenine) are base excision repair (BER) substrates, these DNA lesions are also readily repaired. However, blocking BER can enhance response to TMZ and therefore the BER pathway has emerged as an attractive target for reversing TMZ resistance. Our lab has recently reported that inhibition of BER leads to the accumulation of repair intermediates that induce energy depletion-mediated cell death via hyperactivation of poly(ADP-ribose) polymerase. Based on our observation that TMZ-induced cell death via BER inhibition is dependent on the availability of NAD+, we have hypothesized that combined BER and NAD+ biosynthesis inhibition will increase TMZ efficacy in glioblastoma cell lines greater than BER inhibition alone. Importantly, we find that the combination of BER and NAD+ biosynthesis inhibition significantly sensitizes glioma cells with elevated expression of MGMT and those deficient in MMR, two genotypes normally associated with TMZ resistance. Dual targeting of these two interacting pathways (DNA repair and NAD+ biosynthesis) may prove to be an effective treatment combination for patients with resistant and recurrent GBM. PMID:21406402

Rhabdomyosarcoma in patients with constitutional mismatch-repair-deficiency syndrome.

PubMed

Kratz, C P; Holter, S; Etzler, J; Lauten, M; Pollett, A; Niemeyer, C M; Gallinger, S; Wimmer, K

2009-06-01

Biallelic germline mutations in the mismatch repair genes MLH1, MSH2, MSH6 or PMS2 cause a recessive childhood cancer syndrome characterised by early-onset malignancies and signs reminiscent of neurofibromatosis type 1 (NF1). Alluding to the underlying genetic defect, we refer to this syndrome as constitutional mismatch repair-deficiency (CMMR-D) syndrome. The tumour spectrum of CMMR-D syndrome includes haematological neoplasias, brain tumours and Lynch syndrome-associated tumours. Other tumours, such as neuroblastoma, Wilm tumour, ovarian neuroectodermal tumour or infantile myofibromatosis, have so far been found only in individual cases. We analysed two consanguineous families that had members with suspected CMMR-D syndrome who developed rhabdomyosarcoma among other neoplasias. In the first family, we identified a pathogenic PMS2 mutation for which the affected patient was homozygous. In family 2, immunohistochemistry analysis showed isolated loss of PMS2 expression in all tumours in the affected patients, including rhabdomyosarcoma itself and the surrounding normal tissue. Together with the family history and microsatellite instability observed in one tumour this strongly suggests an underlying PMS2 alteration in family 2 also. Together, these two new cases show that rhabdomyosarcoma and possibly other embryonic tumours, such as neuroblastoma and Wilm tumour, belong to the tumour spectrum of CMMR-D syndrome. Given the clinical overlap of CMMR-D syndrome with NF1, we suggest careful examination of the family history in patients with embryonic tumours and signs of NF1 as well as analysis of the tumours for loss of one of the mismatch repair genes and microsatellite instability. Subsequent mutation analysis will lead to a definitive diagnosis of the underlying disorder.

Clinicopathological features of 22C3 PD-L1 expression with mismatch repair, Epstein-Barr virus status, and cancer genome alterations in metastatic gastric cancer.

PubMed

Kawazoe, Akihito; Shitara, Kohei; Kuboki, Yasutoshi; Bando, Hideaki; Kojima, Takashi; Yoshino, Takayuki; Ohtsu, Atsushi; Ochiai, Atsushi; Togashi, Yosuke; Nishikawa, Hiroyoshi; Doi, Toshihiko; Kuwata, Takeshi

2018-06-01

Recently, the U.S. Food and Drug Administration approved pembrolizumab for patients (pts) with PD-L1-positive metastatic gastric cancer (MGC) based on 22C3 immunohistochemistry (IHC) assay. However, little is known about detailed clinicopathological features of 22C3 PD-L1 expression in MGC. Pts with histologically confirmed MGC were eligible for this prospective observational study. PD-L1 expression (22C3) on tumor cell (TC) or immune cell (IC) and mismatch repair (MMR) were analyzed by IHC. Epstein-Barr virus (EBV) was detected by in situ hybridization. The expressions of tyrosine kinase receptors (RTKs) and cancer genome alterations were evaluated by IHC or next-generation sequencing. A total of 225 pts were analyzed in this study. PD-L1 expression on TC, PD-L1 on IC, MMR-deficient (D-MMR), and EBV positivity were identified in 8.4, 65.3, 6.2, and 6.2% cases, respectively. PD-L1 expression in TC was more frequently observed in pts with D-MMR (P < 0.001), PIK3CA mutation (P = 0.020), and KRAS mutation (P = 0.002), and PD-L1 on IC was associated with EBV positivity (P = 0.034), and lymph-node metastasis (P < 0.001). PD-L1 expression on either IC or TC was less frequently observed in pts with peritoneal metastasis and Borrmann Type 4. A significant association was not observed between PD-L1 expression and RTKs expression or presence of other gene alterations. PD-L1 expression on either TC or IC was not prognostic factor. 22C3 PD-L1 expression in MGC was associated with distinct clinicopathological features, but was not a prognostic factor.

Constitutional mismatch repair deficiency syndrome: clinical description in a French cohort.

PubMed

Lavoine, N; Colas, C; Muleris, M; Bodo, S; Duval, A; Entz-Werle, N; Coulet, F; Cabaret, O; Andreiuolo, F; Charpy, C; Sebille, G; Wang, Q; Lejeune, S; Buisine, M P; Leroux, D; Couillault, G; Leverger, G; Fricker, J P; Guimbaud, R; Mathieu-Dramard, M; Jedraszak, G; Cohen-Hagenauer, O; Guerrini-Rousseau, L; Bourdeaut, F; Grill, J; Caron, O; Baert-Dusermont, S; Tinat, J; Bougeard, G; Frébourg, T; Brugières, L

2015-11-01

Constitutional mismatch repair deficiency (CMMRD) syndrome is a childhood cancer predisposition syndrome involving biallelic germline mutations of MMR genes, poorly recognised by clinicians so far. Retrospective review of all 31 patients with CMMRD diagnosed in French genetics laboratories in order to describe the characteristics, treatment and outcome of the malignancies and biological diagnostic data. 67 tumours were diagnosed in 31 patients, 25 (37%) Lynch syndrome-associated malignancies, 22 (33%) brain tumours, 17 (25%) haematological malignancies and 3 (5%) sarcomas. The median age of onset of the first tumour was 6.9 years (1.2-33.5). Overall, 22 patients died, 9 (41%) due to the primary tumour. Median survival after the diagnosis of the primary tumour was 27 months (0.26-213.2). Failure rate seemed to be higher than expected especially for T-cell non-Hodgkin's lymphoma (progression/relapse in 6/12 patients). A familial history of Lynch syndrome was identified in 6/23 families, and consanguinity in 9/23 families. PMS2 mutations (n=18) were more frequent than other mutations (MSH6 (n=6), MLH1 (n=4) and MSH2 (n=3)). In conclusion, this unselected series of patients confirms the extreme severity of this syndrome with a high mortality rate mostly related to multiple childhood cancers, and highlights the need for its early detection in order to adapt treatment and surveillance. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Blockade of LAG3 enhances responses of tumor-infiltrating T cells in mismatch repair-proficient liver metastases of colorectal cancer

PubMed Central

Noordam, Lisanne; Sprengers, Dave; Boor, Patrick P. C.; Mancham, Shanta; Menon, Anand G.; Lange, Johan F.; Burger, Pim J. W. A.; Brandt, Alexandra; Galjart, Boris; Kwekkeboom, Jaap; Bruno, Marco J.

2018-01-01

ABSTRACT Purpose: Liver metastasis develops in >50% of patients with colorectal cancer (CRC), and is a leading cause of CRC-related mortality. We aimed to identify which inhibitory immune checkpoint pathways can be targeted to enhance functionality of intra-tumoral T-cells in mismatch repair-proficient liver metastases of colorectal cancer (LM-CRC). Methodology: Intra-tumoral expression of multiple inhibitory molecules was compared among mismatch repair-proficient LM-CRC, peritoneal metastases of colorectal cancer (PM-CRC) and primary CRC. Expression of inhibitory molecules was also analyzed on leukocytes isolated from paired resected metastatic liver tumors, tumor-free liver tissues, and blood of patients with mismatch repair-proficient LM-CRC. The effects of blocking inhibitory pathways on tumor-infiltrating T-cell responses were studied in ex vivo functional assays. Results: Mismatch repair-proficient LM-CRC showed higher expression of inhibitory receptors on intra-tumoral T-cells and contained higher proportions of CD8+ T-cells, dendritic cells and monocytes than mismatch repair-proficient primary CRC and/or PM-CRC. Inhibitory receptors LAG3, PD-1, TIM3 and CTLA4 were higher expressed on CD8+ T-cells, CD4+ T-helper and/or regulatory T-cells in LM-CRC tumors compared with tumor-free liver and blood. Antibody blockade of LAG3 or PD-L1 increased proliferation and effector cytokine production of intra-tumoral T-cells isolated from LM-CRC in response to both polyclonal and autologous tumor-specific stimulations. Higher LAG3 expression on intra-tumoral CD8+ T-cells associated with longer progression-free survival of LM-CRC patients. Conclusion: Mismatch repair-proficient LM-CRC may be more sensitive to immune checkpoint inhibitors than mismatch repair-proficient primary CRC. Blocking LAG3 enhances tumor-infiltrating T-cell responses of mismatch repair-proficient LM-CRC, and therefore may be a new promising immunotherapeutic target for LM-CRC.

Indoleamine 2,3-dioxygenase in endometrial cancer: a targetable mechanism of immune resistance in mismatch repair-deficient and intact endometrial carcinomas.

PubMed

Mills, Anne; Zadeh, Sara; Sloan, Emily; Chinn, Zachary; Modesitt, Susan C; Ring, Kari L

2018-03-20

Mismatch repair-deficient endometrial carcinomas are optimal candidates for immunotherapy given their high neoantigen loads, robust lymphoid infiltrates, and frequent PD-L1 expression. However, co-opting the PD-1/PD-L1 pathway is just one mechanism that tumors can utilize to evade host immunity. Another immune modulatory molecule that has been demonstrated in endometrial carcinoma is indoleamine 2,3-dioxygenase (IDO). We herein evaluate IDO expression in 60 endometrial carcinomas and assess results in relation to PD-L1 and mismatch repair status. IDO immunohistochemistry was performed on 60 endometrial carcinomas (20 Lynch syndrome (LS)-associated, 20 MLH1 promoter hypermethylated, and 20 mismatch repair-intact). Eight-five percent of endometrial carcinomas showed IDO tumor staining in >1% of cells. Twenty-five percent were positive in >25% of tumor cells and only 7% exceeded 50% staining. Mismatch repair-deficient cancers were more likely than mismatch repair-intact cancers to be >25% IDO-positive (35% vs. 5% p = 0.024). Differences were amplified when Lynch syndrome-associated cases were evaluated in isolation (50% Lynch syndrome-associated vs. 10% mismatch repair-intact and MLH1-hypermethylated, p = 0.001). Of the four cases showing >50% staining, three were Lynch syndrome-associated and one was MLH1-hypermethylated; no mismatch repair-intact cases had >50% staining. Forty-three percent of IDO-positive tumors were also positive for PD-L1, whereas only two cases showed tumoral PD-L1 in the absence of IDO. In summary, IDO expression is prevalent in endometrial carcinomas and diffuse staining is significantly more common in mismatch repair-deficient cancers, particularly Lynch syndrome-associated cases. Given that the majority of PD-L1 positive cancers also express IDO, synergistic combination therapy with anti-IDO and anti-PD1/PD-L1 may be relevant in this tumor type. Furthermore, anti-IDO therapy may be an option for a small subset of mismatch repair

Immunohistochemistry for PMS2 and MSH6 alone can replace a four antibody panel for mismatch repair deficiency screening in colorectal adenocarcinoma.

PubMed

Hall, Geoffrey; Clarkson, Adele; Shi, Amanda; Langford, Eileen; Leung, Helen; Eckstein, Robert P; Gill, Anthony J

2010-01-01

Currently, testing for mismatch repair deficiency in colorectal cancers is initiated by performing immunohistochemistry with four antibodies (MLH1, PMS2, MSH2 and MSH6). If any one of these stains is negative the tumour is considered microsatellite unstable and, if clinical circumstances warrant it, the patient is offered genetic testing for Lynch's syndrome. Due to the binding properties of the mismatch repair heterodimer complexes, gene mutation and loss of MLH1 and MSH2 invariably result in the degradation of PMS2 and MSH6, respectively, but the converse is not true. We propose that staining for PMS2 and MSH6 alone will be sufficient to detect all cases of mismatch repair deficiency and should replace routine screening with all four antibodies. The electronic database of the department of Anatomical Pathology, Royal North Shore Hospital, Sydney, Australia, was searched for all colorectal carcinomas on which a four panel immunohistochemical microsatellite instability screen was performed. An audit of the slides for concordant loss of MLH1-PMS2 and MSH2-MSH6 was then undertaken. Unusual or discordant cases were reviewed and, in some cases, re-stained to confirm the staining pattern. Of 344 cases of colorectal cancer which underwent four antibody immunohistochemistry, 104 displayed loss of at least one mismatch repair protein. Of these, 100 showed concordant mismatch repair loss (i.e., loss of MLH1 and PMS2 or loss of MSH2 and MSH6). The four discordant cases comprised two single negative cases (1 MSH6 negative/MSH2 positive case, 1 PMS2 negative/MLH1 positive) and two triple negative (both MLH1/PMS2/MSH6 negative). The microsatellite instability (MSI) group showed a relatively high median age (69.3 years) due to the departmental policy of testing all cases with possible MSI morphology regardless of age. The sensitivity and specificity of a two panel test comprised of PMS2 and MSH6, compared to a four panel test, is 100%. No false negatives or positives were

Loss of DNA Mismatch Repair Protein hMSH6 in Ovarian Cancer is Histotype-Specific

PubMed Central

Zhai, Qihui “Jim”; Rosen, Daniel Gustavo; Lu, Karen; Liu, Jinsong

2008-01-01

Microsatellite instability (MSI) due to defects in DNA mismatch repair genes may be involved in the development of a subset of human ovarian carcinomas. The role of one such gene, hMSH6, in ovarian cancer is not well documented. We investigated the expression of hMSH6 protein in different histotypes of ovarian carcinoma and the associations between loss of hMSH6 protein and tumor grade, disease stage, familial history of cancer and patient survival. We stained an ovarian carcinoma tissue microarray consisting of formalin-fixed, paraffin-embedded tissue samples from 322 patients with an anti-hMSH6 antibody and scored the results semiquantitatively as negative or positive. Twelve cases were excluded owing to loss of cores during staining. Absence of hMSH6 protein was noted in 20 of 230 serous carcinomas (8.7%), in 7 of 16 clear cell carcinomas (43.7%), in 4 of 34 endometrioid carcinomas (11.7%), in 1 of 14 malignant mixed Müllerian tumors, 2 of 6 mucinous carcinomas, 0 of 2 transitional cell carcinomas and in 0 of 8 undifferentiated carcinomas. Loss of hMSH6 protein was not associated with survival, patient age, tumor grade, or disease stage but was associated with clear cell, mucinous and endometrioid carcinoma histology (P<0.007). These findings indicate that loss of hMSH6 expression in ovarian carcinoma is more common in certain histologic subtypes, particularly in clear cell, endometrioid, and mucinous carcinoma, suggesting that loss of hMSH6 function may participate in the pathogenesis of these subtypes of cancer. Loss of hMSH6 expression did not predict survival and was not associated with disease stage, tumor grade, patient age or family history of cancer. PMID:18787632

Curcumin Chemosensitizes 5-Fluorouracil Resistant MMR-Deficient Human Colon Cancer Cells in High Density Cultures

PubMed Central

Shakibaei, Mehdi; Buhrmann, Constanze; Kraehe, Patricia; Shayan, Parviz; Lueders, Cora; Goel, Ajay

2014-01-01

Objective Treatment of colorectal cancer (CRC) remains a clinical challenge, as more than 15% of patients are resistant to 5-Fluorouracil (5-FU)-based chemotherapeutic regimens, and tumor recurrence rates can be as high as 50–60%. Cancer stem cells (CSC) are capable of surviving conventional chemotherapies that permits regeneration of original tumors. Therefore, we investigated the effectiveness of 5-FU and plant polyphenol (curcumin) in context of DNA mismatch repair (MMR) status and CSC activity in 3D cultures of CRC cells. Methods High density 3D cultures of CRC cell lines HCT116, HCT116+ch3 (complemented with chromosome 3) and their corresponding isogenic 5-FU-chemo-resistant derivative clones (HCT116R, HCT116+ch3R) were treated with 5-FU either without or with curcumin in time- and dose-dependent assays. Results Pre-treatment with curcumin significantly enhanced the effect of 5-FU on HCT116R and HCR116+ch3R cells, in contrast to 5-FU alone as evidenced by increased disintegration of colonospheres, enhanced apoptosis and by inhibiting their growth. Curcumin and/or 5-FU strongly affected MMR-deficient CRC cells in high density cultures, however MMR-proficient CRC cells were more sensitive. These effects of curcumin in enhancing chemosensitivity to 5-FU were further supported by its ability to effectively suppress CSC pools as evidenced by decreased number of CSC marker positive cells, highlighting the suitability of this 3D culture model for evaluating CSC marker expression in a close to vivo setting. Conclusion Our results illustrate novel and previously unrecognized effects of curcumin in enhancing chemosensitization to 5-FU-based chemotherapy on DNA MMR-deficient and their chemo-resistant counterparts by targeting the CSC sub-population. (246 words in abstract). PMID:24404205

Curcumin chemosensitizes 5-fluorouracil resistant MMR-deficient human colon cancer cells in high density cultures.

PubMed

Shakibaei, Mehdi; Buhrmann, Constanze; Kraehe, Patricia; Shayan, Parviz; Lueders, Cora; Goel, Ajay

2014-01-01

Treatment of colorectal cancer (CRC) remains a clinical challenge, as more than 15% of patients are resistant to 5-Fluorouracil (5-FU)-based chemotherapeutic regimens, and tumor recurrence rates can be as high as 50-60%. Cancer stem cells (CSC) are capable of surviving conventional chemotherapies that permits regeneration of original tumors. Therefore, we investigated the effectiveness of 5-FU and plant polyphenol (curcumin) in context of DNA mismatch repair (MMR) status and CSC activity in 3D cultures of CRC cells. High density 3D cultures of CRC cell lines HCT116, HCT116+ch3 (complemented with chromosome 3) and their corresponding isogenic 5-FU-chemo-resistant derivative clones (HCT116R, HCT116+ch3R) were treated with 5-FU either without or with curcumin in time- and dose-dependent assays. Pre-treatment with curcumin significantly enhanced the effect of 5-FU on HCT116R and HCR116+ch3R cells, in contrast to 5-FU alone as evidenced by increased disintegration of colonospheres, enhanced apoptosis and by inhibiting their growth. Curcumin and/or 5-FU strongly affected MMR-deficient CRC cells in high density cultures, however MMR-proficient CRC cells were more sensitive. These effects of curcumin in enhancing chemosensitivity to 5-FU were further supported by its ability to effectively suppress CSC pools as evidenced by decreased number of CSC marker positive cells, highlighting the suitability of this 3D culture model for evaluating CSC marker expression in a close to vivo setting. Our results illustrate novel and previously unrecognized effects of curcumin in enhancing chemosensitization to 5-FU-based chemotherapy on DNA MMR-deficient and their chemo-resistant counterparts by targeting the CSC sub-population. (246 words in abstract).

DNA repair pathways and mitochondrial DNA mutations in gastrointestinal carcinogenesis.

PubMed

Basso, Daniela; Navaglia, Filippo; Fogar, Paola; Zambon, Carlo-Federico; Greco, Eliana; Schiavon, Stefania; Fasolo, Michela; Stranges, Alessia; Falda, Alessandra; Padoan, Andrea; Fadi, Elisa; Pedrazzoli, Sergio; Plebani, Mario

2007-05-01

This work focuses on the main DNA repair pathways, highlighting their role in gastrointestinal carcinogenesis and the role of mitochondrial DNA (mtDNA), mutations being described in several tumor types, including those of the gastrointestinal tract. The mismatch repair (MMR) system is inherently altered in patients with hereditary non-polyposis colorectal cancer, and plays a role in carcinogenesis in a subset of sporadic colorectal, gastric and esophageal cancers. Alterations in homologous recombination (HR) and non-homologous end-joining (NHEJ) also contribute to the development of pancreatic cancer. Gene polymorphisms of some X-ray cross-complementing (XRCCs), cofactor proteins involved in the base excision repair pathway, have been investigated in relation to gastric, colorectal and pancreatic cancer. Yet only one polymorphism, XRCC1 Arg194Trp, appears to be involved in smoking-related cancers and in early onset pancreatic cancer. Although evidence in the literature indicates that mtDNA somatic mutations play a role in gastric and colorectal carcinogenesis, no sound conclusions have yet been drawn regarding this issue in pancreatic cancer, although an mtDNA variant at 16519 is believed to worsen the outcome of pancreatic cancer patients, possibly because it is involved in altering cellular metabolism.

Pembrolizumab, Capecitabine, and Radiation Therapy in Treating Patients With Mismatch-Repair Deficient and Epstein-Barr Virus Positive Gastric Cancer

ClinicalTrials.gov

2017-11-15

Epstein-Barr Virus Positive; Gastric Adenocarcinoma; Mismatch Repair Protein Deficiency; Stage IB Gastric Cancer AJCC v7; Stage II Gastric Cancer AJCC v7; Stage IIA Gastric Cancer AJCC v7; Stage IIB Gastric Cancer AJCC v7; Stage III Gastric Cancer AJCC v7; Stage IIIA Gastric Cancer AJCC v7; Stage IIIB Gastric Cancer AJCC v7; Stage IIIC Gastric Cancer AJCC v7

A relationship to survival is seen by combining the factors of mismatch repair status, tumor location and age of onset in colorectal cancer patients.

PubMed

Li, Pan; Xiao, Zhitao; Braciak, Todd A; Ou, Qingjian; Chen, Gong; Oduncu, Fuat S

2017-01-01

The progression of colorectal cancer (CRC) may differ depending on the location of the tumor and the age of onset of the disease. Previous studies also suggested that the molecular basis of CRC varies with tumor location, which could affect the clinical management of patients. Therefore, we performed survival analysis looking at different age groups and mismatch repair status (MMR) of CRC patients according to primary tumor location in an attempt to identify subgroups of CRC that might help in the prognosis of disease. A group of 2233 patients operated on to remove their CRC tumors were analyzed (521 with right colon cancer, 740 with left colon cancer and 972 with rectal cancer). The expression of four MMR genes was assessed by immunohistochemistry (IHC), independent of clinical criteria. From the data collected, a predictive model for overall survival (OS) could be constructed for some associations of tumor location and age of onset using Kaplan-Meier, logistic and Cox regression analysis. When tumor location was considered as the lone factor, we found no statistical difference in overall survival (OS) between right cancer (68%), left cancer (67%) or rectal cancer tumor locations (71%) (HR: 1.17, 95%CI (confidence interval): 0.97-1.43, P = 0.057). When age of onset was considered, middle age (40-59 years) and older (60-85 years) patients were found to have higher OS than younger onset cancer (20-39 years) patients (69% vs 71% vs 59%, HR: 1.07, 95% confidence interval (CI): 0.91-1.25, P = 0.008). When both age of onset and tumor location were considered in combination as disease factors, we found that the subgroup of patients with left colon cancer from middle age (69%) and older (67%) aged patients had higher OS than younger (54%) patients (HR: 0.89, 95%CI: 0.68-1.16, P = 0.048). However in patients with right colon cancers, we found no statistical difference is OS between younger, middle age or older grouped patients (60% vs 71% vs 67%, HR: 0.84, 95% CI: 0

Schizosaccharomyces pombe MutSα and MutLα Maintain Stability of Tetra-Nucleotide Repeats and Msh3 of Hepta-Nucleotide Repeats

PubMed Central

Villahermosa, Desirée; Christensen, Olaf; Knapp, Karen; Fleck, Oliver

2017-01-01

Defective mismatch repair (MMR) in humans is associated with colon cancer and instability of microsatellites, that is, DNA sequences with one or several nucleotides repeated. Key factors of eukaryotic MMR are the heterodimers MutSα (Msh2-Msh6), which recognizes base-base mismatches and unpaired nucleotides in DNA, and MutLα (Mlh1-Pms1), which facilitates downstream steps. In addition, MutSβ (Msh2-Msh3) recognizes DNA loops of various sizes, although our previous data and the data presented here suggest that Msh3 of Schizosaccharomyces pombe does not play a role in MMR. To test microsatellite stability in S. pombe and hence DNA loop repair, we have inserted tetra-, penta-, and hepta-nucleotide repeats in the ade6 gene and determined their Ade+ reversion rates and spectra in wild type and various mutants. Our data indicate that loops with four unpaired nucleotides in the nascent and the template strand are the upper limit of MutSα- and MutLα-mediated MMR in S. pombe. Stability of hepta-nucleotide repeats requires Msh3 and Exo1 in MMR-independent processes as well as the DNA repair proteins Rad50, Rad51, and Rad2FEN1. Most strikingly, mutation rates in the double mutants msh3 exo1 and msh3 rad51 were decreased when compared to respective single mutants, indicating that Msh3 prevents error prone processes carried out by Exo1 and Rad51. We conclude that Msh3 has no obvious function in MMR in S. pombe, but contributes to DNA repeat stability in MMR-independent processes. PMID:28341698

Schizosaccharomyces pombe MutSα and MutLα Maintain Stability of Tetra-Nucleotide Repeats and Msh3 of Hepta-Nucleotide Repeats.

PubMed

Villahermosa, Desirée; Christensen, Olaf; Knapp, Karen; Fleck, Oliver

2017-05-05

Defective mismatch repair (MMR) in humans is associated with colon cancer and instability of microsatellites, that is, DNA sequences with one or several nucleotides repeated. Key factors of eukaryotic MMR are the heterodimers MutSα (Msh2-Msh6), which recognizes base-base mismatches and unpaired nucleotides in DNA, and MutLα (Mlh1-Pms1), which facilitates downstream steps. In addition, MutSβ (Msh2-Msh3) recognizes DNA loops of various sizes, although our previous data and the data presented here suggest that Msh3 of Schizosaccharomyces pombe does not play a role in MMR. To test microsatellite stability in S. pombe and hence DNA loop repair, we have inserted tetra-, penta-, and hepta-nucleotide repeats in the ade6 gene and determined their Ade + reversion rates and spectra in wild type and various mutants. Our data indicate that loops with four unpaired nucleotides in the nascent and the template strand are the upper limit of MutSα- and MutLα-mediated MMR in S. pombe Stability of hepta-nucleotide repeats requires Msh3 and Exo1 in MMR-independent processes as well as the DNA repair proteins Rad50, Rad51, and Rad2 FEN1 Most strikingly, mutation rates in the double mutants msh3 exo1 and msh3 rad51 were decreased when compared to respective single mutants, indicating that Msh3 prevents error prone processes carried out by Exo1 and Rad51. We conclude that Msh3 has no obvious function in MMR in S. pombe , but contributes to DNA repeat stability in MMR-independent processes. Copyright © 2017 Villahermosa et al.

Phosphorylation-dependent signaling controls degradation of DNA mismatch repair protein PMS2.

PubMed

Hinrichsen, Inga; Weßbecher, Isabel M; Huhn, Meik; Passmann, Sandra; Zeuzem, Stefan; Plotz, Guido; Biondi, Ricardo M; Brieger, Angela

2017-12-01

MutLα, a heterodimer consisting of MLH1 and PMS2, plays an important role in DNA mismatch repair and has been shown to be additionally involved in several other important cellular mechanisms. Previous work indicated that AKT could modulate PMS2 stability by phosphorylation. Still, the mechanisms of regulation of MutLα remain unclear. The stability of MutLα subunits was investigated by transiently overexpression of wild type and mutant forms of MLH1 and PMS2 using immunoblotting for measuring the protein levels after treatment. We found that treatment with the cell-permeable serine/threonine phosphatase inhibitor, Calyculin, leads to degradation of PMS2 when MLH1 or its C-terminal domain is missing or if amino acids of MLH1 essential for PMS2 interaction are mutated. In addition, we discovered that the C-terminal tail of PMS2 is relevant for this Calyculin-dependent degradation. A direct involvement of AKT, which was previously described to be responsible for PMS2 degradation, could not be detected. The multi-kinase inhibitor Sorafenib, in contrast, was able to avoid the degradation of PMS2 which postulates that cellular phosphorylation is involved in this process. Together, we show that pharmacologically induced phosphorylation by Calyculin can induce the selective proteasome-dependent degradation of PMS2 but not of MLH1 and that the PMS2 degradation could be blocked by Sorafenib treatment. Curiously, the C-terminal Lynch Syndrome-variants MLH1 L749P and MLH1 Y750X make PMS2 prone to Calyculin induced degradation. Therefore, we conclude that the specific degradation of PMS2 may represent a new mechanism to regulate MutLα. © 2017 Wiley Periodicals, Inc.

Mismatch Responses to Lexical Tone, Initial Consonant, and Vowel in Mandarin-Speaking Preschoolers

ERIC Educational Resources Information Center

Lee, Chia-Ying; Yen, Huei-ling; Yeh, Pei-wen; Lin, Wan-Hsuan; Cheng, Ying-Ying; Tzeng, Yu-Lin; Wu, Hsin-Chi

2012-01-01

The present study investigates how age, phonological saliency, and deviance size affect the presence of mismatch negativity (MMN) and positive mismatch response (P-MMR). This work measured the auditory mismatch responses to Mandarin lexical tones, initial consonants, and vowels in 4- to 6-year-old preschoolers using the multiple-deviant oddball…

Role of Saccharomyces cerevisiae Msh2 and Msh3 repair proteins in double-strand break-induced recombination

PubMed Central

Sugawara, Neal; Pâques, Frédéric; Colaiácovo, Mónica; Haber, James E.

1997-01-01

When gene conversion is initiated by a double-strand break (DSB), any nonhomologous DNA that may be present at the ends must be removed before new DNA synthesis can be initiated. In Saccharomyces cerevisiae, removal of nonhomologous ends depends not only on the nucleotide excision repair endonuclease Rad1/Rad10 but also on Msh2 and Msh3, two proteins that are required to correct mismatched bp. These proteins have no effect when DSB ends are homologous to the donor, either in the kinetics of recombination or in the proportion of gene conversions associated with crossing-over. A second DSB repair pathway, single-strand annealing also requires Rad1/Rad10 and Msh2/Msh3, but reveals a difference in their roles. When the flanking homologous regions that anneal are 205 bp, the requirement for Msh2/Msh3 is as great as for Rad1/Rad10; but when the annealing partners are 1,170 bp, Msh2/Msh3 have little effect, while Rad1/Rad10 are still required. Mismatch repair proteins Msh6, Pms1, and Mlh1 are not required. We suggest Msh2 and Msh3 recognize not only heteroduplex loops and mismatched bp, but also branched DNA structures with a free 3′ tail. PMID:9256462

Role of Saccharomyces cerevisiae Msh2 and Msh3 repair proteins in double-strand break-induced recombination.

PubMed

Sugawara, N; Pâques, F; Colaiácovo, M; Haber, J E

1997-08-19

When gene conversion is initiated by a double-strand break (DSB), any nonhomologous DNA that may be present at the ends must be removed before new DNA synthesis can be initiated. In Saccharomyces cerevisiae, removal of nonhomologous ends depends not only on the nucleotide excision repair endonuclease Rad1/Rad10 but also on Msh2 and Msh3, two proteins that are required to correct mismatched bp. These proteins have no effect when DSB ends are homologous to the donor, either in the kinetics of recombination or in the proportion of gene conversions associated with crossing-over. A second DSB repair pathway, single-strand annealing also requires Rad1/Rad10 and Msh2/Msh3, but reveals a difference in their roles. When the flanking homologous regions that anneal are 205 bp, the requirement for Msh2/Msh3 is as great as for Rad1/Rad10; but when the annealing partners are 1,170 bp, Msh2/Msh3 have little effect, while Rad1/Rad10 are still required. Mismatch repair proteins Msh6, Pms1, and Mlh1 are not required. We suggest Msh2 and Msh3 recognize not only heteroduplex loops and mismatched bp, but also branched DNA structures with a free 3' tail.

Interaction of proliferating cell nuclear antigen with PMS2 is required for MutLα activation and function in mismatch repair

PubMed Central

Genschel, Jochen; Kadyrova, Lyudmila Y.; Iyer, Ravi R.; Dahal, Basanta K.; Kadyrov, Farid A.; Modrich, Paul

2017-01-01

Eukaryotic MutLα (mammalian MLH1–PMS2 heterodimer; MLH1–PMS1 in yeast) functions in early steps of mismatch repair as a latent endonuclease that requires a mismatch, MutSα/β, and DNA-loaded proliferating cell nuclear antigen (PCNA) for activation. We show here that human PCNA and MutLα interact specifically but weakly in solution to form a complex of approximately 1:1 stoichiometry that depends on PCNA interaction with the C-terminal endonuclease domain of the MutLα PMS2 subunit. Amino acid substitution mutations within a PMS2 C-terminal 721QRLIAP motif attenuate or abolish human MutLα interaction with PCNA, as well as PCNA-dependent activation of MutLα endonuclease, PCNA- and DNA-dependent activation of MutLα ATPase, and MutLα function in in vitro mismatch repair. Amino acid substitution mutations within the corresponding yeast PMS1 motif (723QKLIIP) reduce or abolish mismatch repair in vivo. Coupling of a weak allele within this motif (723AKLIIP) with an exo1Δ null mutation, which individually confer only weak mutator phenotypes, inactivates mismatch repair in the yeast cell. PMID:28439008

Interaction of proliferating cell nuclear antigen with PMS2 is required for MutLα activation and function in mismatch repair.

PubMed

Genschel, Jochen; Kadyrova, Lyudmila Y; Iyer, Ravi R; Dahal, Basanta K; Kadyrov, Farid A; Modrich, Paul

2017-05-09

Eukaryotic MutLα (mammalian MLH1-PMS2 heterodimer; MLH1-PMS1 in yeast) functions in early steps of mismatch repair as a latent endonuclease that requires a mismatch, MutSα/β, and DNA-loaded proliferating cell nuclear antigen (PCNA) for activation. We show here that human PCNA and MutLα interact specifically but weakly in solution to form a complex of approximately 1:1 stoichiometry that depends on PCNA interaction with the C-terminal endonuclease domain of the MutLα PMS2 subunit. Amino acid substitution mutations within a PMS2 C-terminal 721 QRLIAP motif attenuate or abolish human MutLα interaction with PCNA, as well as PCNA-dependent activation of MutLα endonuclease, PCNA- and DNA-dependent activation of MutLα ATPase, and MutLα function in in vitro mismatch repair. Amino acid substitution mutations within the corresponding yeast PMS1 motif ( 723 QKLIIP) reduce or abolish mismatch repair in vivo. Coupling of a weak allele within this motif ( 723 AKLIIP) with an exo1 Δ null mutation, which individually confer only weak mutator phenotypes, inactivates mismatch repair in the yeast cell.

Reduced mutation rate in exons due to differential mismatch repair

PubMed Central

Mularoni, Loris; Muiños, Ferran; Gonzalez-Perez, Abel; López-Bigas, Núria

2017-01-01

While recent studies have revealed higher than anticipated heterogeneity of mutation rate across genomic regions, mutations in exons and introns are assumed to be generated at the same rate. Here we find fewer somatic mutations in exons than expected based on their sequence content, and demonstrate that this is not due to purifying selection. Moreover, we show that it is caused by higher mismatch repair activity in exonic than in intronic regions. Our findings have important implications for our understanding of mutational and DNA repair processes, our knowledge of the evolution of eukaryotic genes, and practical ramifications for the study of the evolution of both tumors and species. PMID:29106418

Reflex test reminders in required cancer synoptic templates decrease order entry error: An analysis of mismatch repair immunohistochemical orders to screen for Lynch syndrome.

PubMed

Kilgore, Mark R; McIlwain, Carrie A; Schmidt, Rodney A; Norquist, Barbara M; Swisher, Elizabeth M; Garcia, Rochelle L; Rendi, Mara H

2016-01-01

Endometrial carcinoma (EC) is the most common extracolonic malignant neoplasm associated with Lynch syndrome (LS). LS is caused by autosomal dominant germline mutations in DNA mismatch repair (MMR) genes. Screening for LS in EC is often evaluated by loss of immunohistochemical (IHC) expression of DNA MMR enzymes MLH1, MSH2, MSH6, and PMS2 (MMR IHC). In July 2013, our clinicians asked that we screen all EC in patients ≤60 for loss of MMR IHC expression. Despite this policy, several cases were not screened or screening was delayed. We implemented an informatics-based approach to ensure that all women who met criteria would have timely screening. Reports are created in PowerPath (Sunquest Information Systems, Tucson, AZ) with custom synoptic templates. We implemented an algorithm on March 6, 2014 requiring pathologists to address MMR IHC in patients ≤60 with EC before sign out (S/O). Pathologists must answer these questions: is patient ≤60 (yes/no), if yes, follow-up questions (IHC done previously, ordered with addendum to follow, results included in report, N/A, or not ordered), if not ordered, one must explain. We analyzed cases from July 18, 2013 to August 31, 2016 preimplementation (PreImp) and postimplementation (PostImp) that met criteria. Data analysis was performed using the standard data package included with GraphPad Prism ® 7.00 (GraphPad Software, Inc., La Jolla, CA, USA). There were 147 patients who met criteria (29 PreImp and 118 PostImp). IHC was ordered in a more complete and timely fashion PostImp than PreImp. PreImp, 4/29 (13.8%) cases did not get any IHC, but PostImp, only 4/118 (3.39%) were missed ( P = 0.0448). Of cases with IHC ordered, 60.0% (15/25) were ordered before or at S/O PreImp versus 91.2% (104/114) PostImp ( P = 0.0004). Relative to day of S/O, the mean days of order delay were longer and more variable PreImp versus PostImp (12.9 ± 40.7 vs. -0.660 ± 1.15; P = 0.0227), with the average being before S/O PostImp. This algorithm

Balancing repair and tolerance of DNA damage caused by alkylating agents.

PubMed

Fu, Dragony; Calvo, Jennifer A; Samson, Leona D

2012-01-12

Alkylating agents constitute a major class of frontline chemotherapeutic drugs that inflict cytotoxic DNA damage as their main mode of action, in addition to collateral mutagenic damage. Numerous cellular pathways, including direct DNA damage reversal, base excision repair (BER) and mismatch repair (MMR), respond to alkylation damage to defend against alkylation-induced cell death or mutation. However, maintaining a proper balance of activity both within and between these pathways is crucial for a favourable response of an organism to alkylating agents. Furthermore, the response of an individual to alkylating agents can vary considerably from tissue to tissue and from person to person, pointing to genetic and epigenetic mechanisms that modulate alkylating agent toxicity.

The MutSβ complex is a modulator of p53-driven tumorigenesis through its functions in both DNA double strand break repair and mismatch repair

PubMed Central

van Oers, Johanna M. M.; Edwards, Yasmin; Chahwan, Richard; Zhang, Weijia; Smith, Cameron; Pechuan, Joaquín; Schaetzlein, Sonja; Jin, Bo; Wang, Yuxun; Bergman, Aviv; Scharff, Matthew D.; Edelmann, Winfried

2014-01-01

Loss of the DNA mismatch repair protein MSH3 leads to the development of a variety of tumors in mice without significantly affecting survival rates, suggesting a modulating role for the MutSβ (MSH2-MSH3) complex in late onset tumorigenesis. To better study the role of MSH3 in tumor progression, we crossed Msh3−/− mice onto a tumor predisposing p53-deficient background. Survival of Msh3/p53 mice was not reduced compared to single p53 mutant mice; however, the tumor spectrum changed significantly from lymphoma to sarcoma, indicating MSH3 as a potent modulator of p53-driven tumorigenesis. Interestingly, Msh3−/− mouse embryonic fibroblasts displayed increased chromatid breaks and persistence of γH2AX foci following ionizing radiation, indicating a defect in DNA double strand break repair. Msh3/p53 tumors showed increased loss of heterozygosity, elevated genome-wide copy number variation, and a moderate microsatellite instability phenotype compared to Msh2/p53 tumors, revealing that MSH2-MSH3 suppresses tumorigenesis by maintaining chromosomal stability. Our results show that the MSH2-MSH3 complex is important for the suppression of late onset tumors due to its role in DNA double strand break repair as well as in DNA mismatch repair. Furthermore, they demonstrate that MSH2-MSH3 suppresses chromosomal instability and modulates the tumor spectrum in p53-deficient tumorigenesis, and possibly plays a role in other chromosomally unstable tumors as well. PMID:24013230

Cancer screening behaviors and risk perceptions among family members of colorectal cancer patients with unexplained mismatch repair deficiency.

PubMed

Katz, Lior H; Advani, Shailesh; Burton-Chase, Allison M; Fellman, Bryan; Polivka, Katrina M; Yuan, Ying; Lynch, Patrick M; Peterson, Susan K

2017-04-01

Communication gaps in families with unexplained mismatch repair (MMR) deficiency (UMMRD) could negatively impact the screening behaviors of relatives of individual with UMMRD. We evaluated cancer risk perception, screening behaviors, and family communication among relatives of colorectal cancer (CRC) patients with UMMRD. Fifty-one family members of 17 probands with UMMRD completed a questionnaire about cancer risk perception, adherence to Lynch syndrome (LS) screening recommendations, and communication with relatives. Clinical data about the probands were obtained from medical records. Thirty-eight participants (78%) were worried from having cancer and twenty-one participants (42%) had undergone colonoscopy in the past 2 years, as recommended for LS families. In terms of screening for extracolonic cancers, only two eligible participants (3.9%) were screened for gastric, endometrial (10.0%), and ovarian (9.5%) cancers. Additionally, 5 participants (10%) underwent genetic counseling. Most participants were not told by anyone to be screened for extracolonic cancers (84, 85, and 95% for gastric, ovarian, and endometrial cancers, respectively). A minority of family members of CRC patients with UMMRD follow cancer screening as recommended for LS families. Health care providers should encourage patients with UMMRD to share information on LS-related cancers screening, especially extracolonic cancers, with their relatives.

Cancer screening behaviors and risk perceptions among family members of colorectal cancer patients with unexplained mismatch repair deficiency

PubMed Central

Advani, Shailesh; Burton-Chase, Allison M.; Fellman, Bryan; Polivka, Katrina M.; Yuan, Ying; Lynch, Patrick M.; Peterson, Susan K.

2018-01-01

Communication gaps in families with unexplained mismatch repair (MMR) deficiency (UMMRD) could negatively impact the screening behaviors of relatives of individual with UMMRD. We evaluated cancer risk perception, screening behaviors, and family communication among relatives of colorectal cancer (CRC) patients with UMMRD. Fifty-one family members of 17 probands with UMMRD completed a questionnaire about cancer risk perception, adherence to Lynch syndrome (LS) screening recommendations, and communication with relatives. Clinical data about the probands were obtained from medical records. Thirty-eight participants (78%) were worried from having cancer and twenty-one participants (42%) had undergone colonoscopy in the past 2 years, as recommended for LS families. In terms of screening for extracolonic cancers, only two eligible participants (3.9%) were screened for gastric, endometrial (10.0%), and ovarian (9.5%) cancers. Additionally, 5 participants (10%) underwent genetic counseling. Most participants were not told by anyone to be screened for extracolonic cancers (84, 85, and 95% for gastric, ovarian, and endometrial cancers, respectively). A minority of family members of CRC patients with UMMRD follow cancer screening as recommended for LS families. Health care providers should encourage patients with UMMRD to share information on LS-related cancers screening, especially extracolonic cancers, with their relatives. PMID:27832499

Cytosine-based nucleoside analogs are selectively lethal to DNA mismatch repair-deficient tumour cells by enhancing levels of intracellular oxidative stress

PubMed Central

Hewish, M; Martin, S A; Elliott, R; Cunningham, D; Lord, C J; Ashworth, A

2013-01-01

Background: DNA mismatch repair deficiency is present in a significant proportion of a number of solid tumours and is associated with distinct clinical behaviour. Methods: To identify the therapeutic agents that might show selectivity for mismatch repair-deficient tumour cells, we screened a pair of isogenic MLH1-deficient and MLH1-proficient tumour cell lines with a library of clinically used drugs. To test the generality of hits in the screen, selective agents were retested in cells deficient in the MSH2 mismatch repair gene. Results: We identified cytarabine and other related cytosine-based nucleoside analogues as being selectively toxic to MLH1 and MSH2-deficient tumour cells. The selective cytotoxicity we observed was likely caused by increased levels of cellular oxidative stress, as it could be abrogated by antioxidants. Conclusion: We propose that cytarabine-based chemotherapy regimens may represent a tumour-selective treatment strategy for mismatch repair-deficient cancers. PMID:23361057

The biochemical basis of microsatellite instability and abnormal immunohistochemistry and clinical behavior in Lynch Syndrome: from bench to bedside

PubMed Central

Koi, Minoru; Chang, Dong K.; Carethers, John M.

2010-01-01

Lynch syndrome is an inherited disease caused by a germline mutation in one of four DNA mismatch repair (MMR) genes. The clinical manifestations can be somewhat variable depending upon which gene is involved, and where the mutation occurs. Moreover, the approach to the diagnosis of Lynch syndrome is becoming more complex as more is learned about the disease, and one needs to understand how the DNA MMR proteins function, and what makes them malfunction, to have an optimal appreciation of how to interpret diagnostic studies such as microsatellite instability and immunohistochemistry of the DNA MMR proteins. Finally, an understanding of the role of the DNA MMR system in regulation of the cell cycle and the response to DNA damage helps illuminate the differences in natural history and response to chemotherapeutic agents seen in Lynch syndrome. PMID:17636426

Hyaluronan Tumor Cell Interactions in Prostate Cancer Growth and Survival

DTIC Science & Technology

2008-12-01

different outcomes. For example, colo- rectal cancers can be grouped into DNA mismatch repair-proficient, MLH1 negative and presumed Lynch syndrome. Although...a prognostic factor in DNA-mismatch repair-proficient (MMR-proficient) and presumed Lynch syndrome forms of colorectal cancer but not in MLH1 negative

The structural impact of DNA mismatches

PubMed Central

Rossetti, Giulia; Dans, Pablo D.; Gomez-Pinto, Irene; Ivani, Ivan; Gonzalez, Carlos; Orozco, Modesto

2015-01-01

The structure and dynamics of all the transversion and transition mismatches in three different DNA environments have been characterized by molecular dynamics simulations and NMR spectroscopy. We found that the presence of mismatches produced significant local structural alterations, especially in the case of purine transversions. Mismatched pairs often show promiscuous hydrogen bonding patterns, which interchange among each other in the nanosecond time scale. This therefore defines flexible base pairs, where breathing is frequent, and where distortions in helical parameters are strong, resulting in significant alterations in groove dimension. Even if the DNA structure is plastic enough to absorb the structural impact of the mismatch, local structural changes can be propagated far from the mismatch site, following the expected through-backbone and a previously unknown through-space mechanism. The structural changes related to the presence of mismatches help to understand the different susceptibility of mismatches to the action of repairing proteins. PMID:25820425

Splicing analysis for exonic and intronic mismatch repair gene variants associated with Lynch syndrome confirms high concordance between minigene assays and patient RNA analyses

PubMed Central

van der Klift, Heleen M; Jansen, Anne M L; van der Steenstraten, Niki; Bik, Elsa C; Tops, Carli M J; Devilee, Peter; Wijnen, Juul T

2015-01-01

A subset of DNA variants causes genetic disease through aberrant splicing. Experimental splicing assays, either RT-PCR analyses of patient RNA or functional splicing reporter minigene assays, are required to evaluate the molecular nature of the splice defect. Here, we present minigene assays performed for 17 variants in the consensus splice site regions, 14 exonic variants outside these regions, and two deep intronic variants, all in the DNA mismatch-repair (MMR) genes MLH1, MSH2, MSH6, and PMS2, associated with Lynch syndrome. We also included two deep intronic variants in APC and PKD2. For one variant (MLH1 c.122A>G), our minigene assay and patient RNA analysis could not confirm the previously reported aberrant splicing. The aim of our study was to further investigate the concordance between minigene splicing assays and patient RNA analyses. For 30 variants results from patient RNA analyses were available, either performed by our laboratory or presented in literature. Some variants were deliberately included in this study because they resulted in multiple aberrant transcripts in patient RNA analysis, or caused a splice effect other than the prevalent exon skip. While both methods were completely concordant in the assessment of splice effects, four variants exhibited major differences in aberrant splice patterns. Based on the present and earlier studies, together showing an almost 100% concordance of minigene assays with patient RNA analyses, we discuss the weight given to minigene splicing assays in the current criteria proposed by InSiGHT for clinical classification of MMR variants. PMID:26247049

Clustered Mutation Signatures Reveal that Error-Prone DNA Repair Targets Mutations to Active Genes.

PubMed

Supek, Fran; Lehner, Ben

2017-07-27

Many processes can cause the same nucleotide change in a genome, making the identification of the mechanisms causing mutations a difficult challenge. Here, we show that clustered mutations provide a more precise fingerprint of mutagenic processes. Of nine clustered mutation signatures identified from >1,000 tumor genomes, three relate to variable APOBEC activity and three are associated with tobacco smoking. An additional signature matches the spectrum of translesion DNA polymerase eta (POLH). In lymphoid cells, these mutations target promoters, consistent with AID-initiated somatic hypermutation. In solid tumors, however, they are associated with UV exposure and alcohol consumption and target the H3K36me3 chromatin of active genes in a mismatch repair (MMR)-dependent manner. These regions normally have a low mutation rate because error-free MMR also targets H3K36me3 chromatin. Carcinogens and error-prone repair therefore redistribute mutations to the more important regions of the genome, contributing a substantial mutation load in many tumors, including driver mutations. Copyright © 2017 Elsevier Inc. All rights reserved.

SERIES: Genomic instability in cancer Balancing repair and tolerance of DNA damage caused by alkylating agents

PubMed Central

Fu, Dragony; Calvo, Jennifer A.; Samson, Leona D

2013-01-01

Alkylating agents comprise a major class of frontline chemotherapeutic drugs that inflict cytotoxic DNA damage as their main mode of action, in addition to collateral mutagenic damage. Numerous cellular pathways, including direct DNA damage reversal, base excision repair (BER), and mismatch repair (MMR) respond to alkylation damage to defend against alkylation-induced cell death or mutation. However, maintaining a proper balance of activity both within and between these pathways is crucial for an organism's favorable response to alkylating agents. Furthermore, an individual's response to alkylating agents can vary considerably from tissue to tissue and from person to person, pointing to genetic and epigenetic mechanisms that modulate alkylating agent toxicity. PMID:22237395

Hydrophobic mismatch sorts SNARE proteins into distinct membrane domains

PubMed Central

Milovanovic, Dragomir; Honigmann, Alf; Koike, Seiichi; Göttfert, Fabian; Pähler, Gesa; Junius, Meike; Müllar, Stefan; Diederichsen, Ulf; Janshoff, Andreas; Grubmüller, Helmut; Risselada, Herre J.; Eggeling, Christian; Hell, Stefan W.; van den Bogaart, Geert; Jahn, Reinhard

2015-01-01

The clustering of proteins and lipids in distinct microdomains is emerging as an important principle for the spatial patterning of biological membranes. Such domain formation can be the result of hydrophobic and ionic interactions with membrane lipids as well as of specific protein–protein interactions. Here using plasma membrane-resident SNARE proteins as model, we show that hydrophobic mismatch between the length of transmembrane domains (TMDs) and the thickness of the lipid membrane suffices to induce clustering of proteins. Even when the TMDs differ in length by only a single residue, hydrophobic mismatch can segregate structurally closely homologous membrane proteins in distinct membrane domains. Domain formation is further fine-tuned by interactions with polyanionic phosphoinositides and homo and heterotypic protein interactions. Our findings demonstrate that hydrophobic mismatch contributes to the structural organization of membranes. PMID:25635869

Molecular Mechanisms of Ultraviolet Radiation-Induced DNA Damage and Repair

PubMed Central

Rastogi, Rajesh P.; Richa; Kumar, Ashok; Tyagi, Madhu B.; Sinha, Rajeshwar P.

2010-01-01

DNA is one of the prime molecules, and its stability is of utmost importance for proper functioning and existence of all living systems. Genotoxic chemicals and radiations exert adverse effects on genome stability. Ultraviolet radiation (UVR) (mainly UV-B: 280–315 nm) is one of the powerful agents that can alter the normal state of life by inducing a variety of mutagenic and cytotoxic DNA lesions such as cyclobutane-pyrimidine dimers (CPDs), 6-4 photoproducts (6-4PPs), and their Dewar valence isomers as well as DNA strand breaks by interfering the genome integrity. To counteract these lesions, organisms have developed a number of highly conserved repair mechanisms such as photoreactivation, base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR). Additionally, double-strand break repair (by homologous recombination and nonhomologous end joining), SOS response, cell-cycle checkpoints, and programmed cell death (apoptosis) are also operative in various organisms with the expense of specific gene products. This review deals with UV-induced alterations in DNA and its maintenance by various repair mechanisms. PMID:21209706

Estrogen enhances mismatch repair by induction of MLH1 expression via estrogen receptor-β

PubMed Central

Lu, Jun-Yu; Jin, Peng; Gao, Wei; Wang, De-Zhi; Sheng, Jian-Qiu

2017-01-01

Epidemiological data demonstrated that hormone replace treatment has protective effect against colorectal cancer (CRC). Our previous studies showed that this effect may be associated with DNA mismatch repair. This study aims to investigate the mechanism of estrogen induction of MLH1, and whether colorectal tumor proliferation can be inhibited through induction of MLH1 by estrogen signal pathway. Human CRC cell lines were used to examine the regulation of MLH1 expression by over-expression and depletion of estrogen receptor-α (ERα) and estrogen receptor-β (ERβ), under the treatment with 17β-estradiol or β-Estradiol 6-(O-carboxy-methyl)oxime:BSA, followed by a real-time Q-PCR and Western blotting analysis. Luciferase reporter and chromatin immunoprecipitation assays were used to identify the estrogen response elements in the proximal promoter of MLH1 gene. Then, the influence of estrogen-induced MLH1 on CRC tumor growth were determined in vitro and in vivo. We found that mismatch repair ability and microsatellite stability of cells were enhanced by estrogen via induction of MLH1 expression, which was mediated by ERβ, through a transcriptional activation process. Furthermore, we identified that ERβ exerted an inhibitory effect on CRC tumor proliferation in vitro and in vivo, combined with 5-FU, through up-regulation of MLH1 expression. Finally, we concluded that estrogen enhances mismatch repair ability and tumor inhibition effect in vitro and in vivo, via induction of MLH1 expression mediated by ERβ. PMID:28404976

Estrogen enhances mismatch repair by induction of MLH1 expression via estrogen receptor-β.

PubMed

Lu, Jun-Yu; Jin, Peng; Gao, Wei; Wang, De-Zhi; Sheng, Jian-Qiu

2017-06-13

Epidemiological data demonstrated that hormone replace treatment has protective effect against colorectal cancer (CRC). Our previous studies showed that this effect may be associated with DNA mismatch repair. This study aims to investigate the mechanism of estrogen induction of MLH1, and whether colorectal tumor proliferation can be inhibited through induction of MLH1 by estrogen signal pathway. Human CRC cell lines were used to examine the regulation of MLH1 expression by over-expression and depletion of estrogen receptor-α (ERα) and estrogen receptor-β (ERβ), under the treatment with 17β-estradiol or β-Estradiol 6-(O-carboxy-methyl)oxime:BSA, followed by a real-time Q-PCR and Western blotting analysis. Luciferase reporter and chromatin immunoprecipitation assays were used to identify the estrogen response elements in the proximal promoter of MLH1 gene. Then, the influence of estrogen-induced MLH1 on CRC tumor growth were determined in vitro and in vivo. We found that mismatch repair ability and microsatellite stability of cells were enhanced by estrogen via induction of MLH1 expression, which was mediated by ERβ, through a transcriptional activation process. Furthermore, we identified that ERβ exerted an inhibitory effect on CRC tumor proliferation in vitro and in vivo, combined with 5-FU, through up-regulation of MLH1 expression. Finally, we concluded that estrogen enhances mismatch repair ability and tumor inhibition effect in vitro and in vivo, via induction of MLH1 expression mediated by ERβ.

Genomic amplification of the human DHFR/MSH3 locus remodels mismatch recognition and repair activities.

PubMed

Drummond, J T

1999-01-01

Mismatch recognition in human cells is mediated by two heterodimers, MutS alpha and MutS beta. MutS alpha appears to shoulder primary responsibility for mismatch correction during replication, based on its relative abundance and ability to recognize a broad spectrum of base-base and base-insertion mismatches. Because MutS alpha and MutS beta share a common component, MSH2, conditions that influence the expression or degradation of MSH3 or MSH6 can redistribute the profile of mismatch recognition and repair. MSH3 is linked by a shared promoter with DHFR, connecting two pathways with key roles in DNA metabolism. In a classic example of gene amplification, the DHFR (and MSH3) locus can become amplified to several hundred copies in the presence of methotrexate. Under these conditions, MutS beta forms at the expense of MutS alpha, and the mutation rate in these tumor cells rises more than 100-fold. The implications for cancer chemotherapy include a potential increase in mutability when tumors are treated with methotrexate, which could increase the frequency of subsequent mutations that influence the tumor's drug sensitivity or aggressiveness. Because processing certain types of DNA damage by the mismatch repair pathway has also been implicated in tumor sensitivity to agents such as cisplatin, changes in expression at the DHFR/MSH3 locus may have further relevance to the outcome of multi-drug treatment regimens.

Requirement of mismatch repair genes MSH2 and MSH3 in the RAD1-RAD10 pathway of mitotic recombination in Saccharomyces cerevisiae.

PubMed

Saparbaev, M; Prakash, L; Prakash, S

1996-03-01

The RAD1 and RAD10 genes of Saccharomyces cerevisiae are required for nucleotide excision repair and they also act in mitotic recombination. The Rad1-Rad10 complex has a single-stranded DNA endonuclease activity. Here, we show that the mismatch repair genes MSH2 and MSH3 function in mitotic recombination. For both his3 and his4 duplications, and for homologous integration of a linear DNA fragment into the genome, the msh3 delta mutation has an effect on recombination similar to that of the rad1 delta and rad10 delta mutations. The msh2 delta mutation also reduces the rate of recombination of the his3 duplication and lowers the incidence of homologous integration of a linear DNA fragment. Epistasis analyses indicate that MSH2 and MSH3 function in the RAD1-RAD10 recombination pathway, and studies presented here suggest an involvement of the RAD1-RAD10 pathway in reciprocal recombination. The possible roles of Msh2, Msh3, Rad1, and Rad10 proteins in genetic recombination are discussed. Coupling of mismatch binding proteins with the recombinational machinery could be important for ensuring genetic fidelity in the recombination process.

Association between genetic polymorphisms in DNA mismatch repair-related genes with risk and prognosis of head and neck squamous cell carcinoma.

PubMed

Nogueira, Guilherme Augusto Silva; Lourenço, Gustavo Jacob; Oliveira, Camila Borges Martins; Marson, Fernando Augusto Lima; Lopes-Aguiar, Leisa; Costa, Ericka Francislaine Dias; Lima, Tathiane Regine Penna; Liutti, Vitor Teixeira; Leal, Frederico; Santos, Vivian Castro Antunes; Rinck-Junior, José Augusto; Lima, Carmen Silvia Passos

2015-08-15

We examined the influence of MLH1 c.-93G>A, MSH2 c.211 + 9C>G, MSH3 c.3133G>A and EXO1 c.1765G>A polymorphisms, involved in DNA mismatch repair (MMR), on head and neck (HN) squamous cell carcinoma (SCC) risk and prognosis. Aiming to identify genotypes, DNA from 450 HNSCC patients and 450 controls was analyzed by PCR-RFLP or real time PCR. MSH2 GG plus MSH3 GG (31.7% vs. 18.7%, p = 0.003) genotypes were higher in laryngeal SCC (LSCC) patients than in controls. Carriers of the respective combined genotype were under a 3.69 (95% CI: 1.54-8.81)-fold increased risk of LSCC. Interactions of tobacco and tobacco plus all the above-mentioned polymorphisms on HNSCC and LSCC risk were also evident in study (p = 0.001). At 60 months of follow-up, relapse-free survival (RFS) was shorter in patients with EXO1 GG genotype (54.8% vs. 61.1%, p = 0.03) and overall survival (OS) was shorter in patients with MSH3 GG genotype (42.8% vs. 52.5%, p = 0.02) compared to those with other genotypes, respectively. After multivariate Cox analysis, patients with EXO1 GG and MSH3 GG genotypes had worst RFS (HR: 1.50, 95% CI: 1.03-2.20, p = 0.03) and OS (HR: 1.59, 95% CI: 1.19-2.13, P = 0.002) than those with the remaining genotypes, respectively. Our data present, for the first time, evidence that inherited MLH1 c.-93G>A, MSH2 c.211 + 9C>G, MSH3 c.3133G>A, and EXO1 c.1765G>A abnormalities of DNA MMR pathway are important determinants of HNSCC, particularly among smokers, and predictors of patient outcomes. © 2015 UICC.

DNA Mismatch Repair Status Predicts Need for Future Colorectal Surgery for Metachronous Neoplasms in Young Individuals Undergoing Colorectal Cancer Resection.

PubMed

Aronson, Melyssa; Holter, Spring; Semotiuk, Kara; Winter, Laura; Pollett, Aaron; Gallinger, Steven; Cohen, Zane; Gryfe, Robert

2015-07-01

The treatment of colorectal cancer in young patients involves both management of the incident cancer and consideration of the possibility of Lynch syndrome and the development of metachronous colorectal cancers. This study aims to assess the prognostic role of DNA mismatch repair deficiency and extended colorectal resection for metachronous colorectal neoplasia risk in young patients with colorectal cancer. This is a retrospective review of 285 patients identified in our GI cancer registry with colorectal cancer diagnosed at 35 years or younger in the absence of polyposis. Using univariate and multivariate analysis, we assessed the prognostic role of mismatch repair deficiency and standard clinicopathologic characteristics, including the extent of resection, on the rate of developing metachronous colorectal neoplasia requiring resection. Mismatch repair deficiency was identified in biospecimens from 44% of patients and was significantly associated with an increased risk for metachronous colorectal neoplasia requiring resection (10-year cumulative risk, 13.5% ± 4.2%) compared with 56% of patients with mismatch repair-intact colorectal cancer (10-year cumulative risk, 5.8% ± 3.3%; p = 0.011). In multivariate analysis, mismatch repair deficiency was associated with a HR of 3.65 (95% CI, 1.44-9.21; p = 0.006) for metachronous colorectal neoplasia, whereas extended resection with ileorectal or ileosigmoid anastomosis significantly decreased the risk of metachronous colorectal neoplasia (HR, 0.21; 95% CI, 0.05-0.90; p = 0.036). This study had a retrospective design, and, therefore, recommendations for colorectal cancer surgery and screening were not fully standardized. Quality of life after colorectal cancer surgery was not assessed. Young patients with colorectal cancer with molecular hallmarks of Lynch syndrome were at significantly higher risk for the development of subsequent colorectal neoplasia. This risk was significantly reduced in those who underwent extended

The changing landscape of Lynch syndrome due to PMS2 mutations.

PubMed

Blount, J; Prakash, A

2018-07-01

DNA repair pathways are essential for cellular survival as our DNA is constantly under assault from both exogenous and endogenous DNA damaging agents. Five major mammalian DNA repair pathways exist within a cell to maintain genomic integrity. Of these, the DNA mismatch repair (MMR) pathway is highly conserved among species and is well documented in bacteria. In humans, the importance of MMR is underscored by the discovery that a single mutation in any 1 of 4 genes within the MMR pathway (MLH1, MSH2, MSH6 and PMS2) results in Lynch syndrome (LS). LS is a autosomal dominant condition that predisposes individuals to a higher incidence of many malignancies including colorectal, endometrial, ovarian, and gastric cancers. In this review, we discuss the role of PMS2 in the MMR pathway, the evolving testing criteria used to identify variants in the PMS2 gene, the LS phenotype as well as the autosomal recessive condition called constitutional mismatch repair deficiency syndrome, and current methods used to elucidate the clinical impact of PMS2 mutations. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

PMS2 expression in epithelial ovarian cancer is posttranslationally regulated by Akt and essential for platinum-induced apoptosis.

PubMed

Jia, Jinghui; Wang, Zehua; Cai, Jing; Zhang, Yuan

2016-03-01

Epithelial ovarian cancer (EOC) is the most lethal of the gynecologic malignancies, mainly due to the advanced stage at diagnosis and development of cisplatin resistance. The sensitivity of tumor cells to cisplatin is frequently affected by defect in DNA mismatch repair (MMR), which repairs mispaired DNA sequences and regulates DNA-damage-induced apoptosis. However, the role of postmeiotic segregation increased 2 (PMS2), a member of MMR protein family, in cisplatin resistance remains elusive. In the present study, we demonstrated the frequent deficiency of PMS2 and phosphorylation of Akt in EOC cell lines and tissues. Results of complex immunoprecipitation (co-IP) and protein stability assay indicated that activated Akt could directly bind to PMS2 and cause degradation of PMS2 in EOC cells. In addition, functional experiments revealed that PMS2 was required for cisplatin-induced apoptosis and cell cycle arrest in G2/M phase. These findings provide a novel insight into molecular mechanisms linking MMR with chemoresistance and suggest that stabilization of PMS2 expression may be useful in overcoming the cisplatin resistance in EOC.

Conformational trapping of mismatch recognition complex MSH2/MSH3 on repair-resistant DNA loops.

PubMed

Lang, Walter H; Coats, Julie E; Majka, Jerzy; Hura, Greg L; Lin, Yuyen; Rasnik, Ivan; McMurray, Cynthia T

2011-10-18

Insertion and deletion of small heteroduplex loops are common mutations in DNA, but why some loops are prone to mutation and others are efficiently repaired is unknown. Here we report that the mismatch recognition complex, MSH2/MSH3, discriminates between a repair-competent and a repair-resistant loop by sensing the conformational dynamics of their junctions. MSH2/MSH3 binds, bends, and dissociates from repair-competent loops to signal downstream repair. Repair-resistant Cytosine-Adenine-Guanine (CAG) loops adopt a unique DNA junction that traps nucleotide-bound MSH2/MSH3, and inhibits its dissociation from the DNA. We envision that junction dynamics is an active participant and a conformational regulator of repair signaling, and governs whether a loop is removed by MSH2/MSH3 or escapes to become a precursor for mutation.

Requirement for Msh6, but not for Swi4 (Msh3), in Msh2-dependent repair of base-base mismatches and mononucleotide loops in Schizosaccharomyces pombe.

PubMed

Tornier, C; Bessone, S; Varlet, I; Rudolph, C; Darmon, M; Fleck, O

2001-05-01

The msh6 mismatch repair gene of Schizosaccharomyces pombe was cloned, sequenced, and inactivated. Strains bearing all combinations of inactivated msh6, msh2, and swi4 (the S. pombe MSH3 ortholog) alleles were tested for their defects in mitotic and meiotic mismatch repair. Mitotic mutation rates were similarly increased in msh6 and msh2 mutants, both for reversion of a base-base substitution as well as of an insertion of one nucleotide in a mononucleotide run. Tetrad analysis and intragenic two-factor crosses revealed that meiotic mismatch repair was affected in msh6 to the same extent as in msh2 background. In contrast, loss of Swi4 likely did not cause a defect in mismatch repair, but rather resulted in reduced recombination frequency. Consistently, a mutated swi4 caused a two- to threefold reduction of recombinants in intergenic crosses, while msh2 and msh6 mutants were not significantly different from wild type. In summary, our study showed that Msh6 plays the same important role as Msh2 in the major mismatch repair pathway of S. pombe, while Swi4 rather functions in recombination.

Solution structure and intramolecular exchange of methyl-cytosine binding domain protein 4 (MBD4) on DNA suggests a mechanism to scan for mCpG/TpG mismatches

PubMed Central

Walavalkar, Ninad M.; Cramer, Jason M.; Buchwald, William A.; Scarsdale, J. Neel; Williams, David C.

2014-01-01

Unlike other members of the methyl-cytosine binding domain (MBD) family, MBD4 serves as a potent DNA glycosylase in DNA mismatch repair specifically targeting mCpG/TpG mismatches arising from spontaneous deamination of methyl-cytosine. The protein contains an N-terminal MBD (MBD4MBD) and a C-terminal glycosylase domain (MBD4GD) separated by a long linker. This arrangement suggests that the MBD4MBD either directly augments enzymatic catalysis by the MBD4GD or targets the protein to regions enriched for mCpG/TpG mismatches. Here we present structural and dynamic studies of MBD4MBD bound to dsDNA. We show that MBD4MBD binds with a modest preference formCpG as compared to mismatch, unmethylated and hydroxymethylated DNA. We find that while MBD4MBD exhibits slow exchange between molecules of DNA (intermolecular exchange), the domain exhibits fast exchange between two sites in the same molecule of dsDNA (intramolecular exchange). Introducing a single-strand defect between binding sites does not greatly reduce the intramolecular exchange rate, consistent with a local hopping mechanism for moving along the DNA. These results support a model in which the MBD4MBD4 targets the intact protein to mCpG islands and promotes scanning by rapidly exchanging between successive mCpG sites which facilitates repair of nearby mCpG/TpG mismatches by the glycosylase domain. PMID:25183517

Requirement of mismatch repair genes MSH2 and MSH3 in the RAD1-RAD10 pathway of mitotic recombination in Saccharomyces cerevisiae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saparbaev, M.; Prakash, L.; Prakash, S.

1996-03-01

The RAD1 and RAD10 genes of Saccharomyces cerevisiae are required for nucleotide excision repair and they also act in mitotic recombination. The Rad1-Rad10 complex has a single-stranded DNA endonuclease activity. Here, we show that the mismatch repair genes MSH2 and MSH3 function in mitotic recombination. For both his3 and his4 duplications, and for homologous integration of a linear DNA fragment into the genome, the msh3-A mutation has an effect on recombination similar to that of the rad1{Delta} and rad10{Delta} mutations. The msh2{Delta} mutation also reduces the rate of recombination of the his3 duplication and lowers the incidence of homologous integrationmore » of a linear DNA fragment. Epistasis analyses indicate that MSH2 and MSH3 function in the RAD1-RAD10 recombination pathway, and studies presented here suggest an involvement of the RAM-RAD10 pathway in reciprocal recombination. The possible roles of Msh2, Msh3, Rad1, and Rad10 proteins in genetic recombination are discussed. Coupling of mismatch binding proteins with the recombinational machinery could be important for ensuring genetic fidelity in the recombination process. 59 refs., 2 figs., 7 tabs.« less

Constitutional mismatch repair deficiency presenting in childhood as three simultaneous malignancies.

PubMed

Walter, Andrew W; Ennis, Sara; Best, Hunter; Vaughn, Cecily P; Swensen, Jeffrey J; Openshaw, Amanda; Gripp, Karen W

2013-11-01

A 13-year-old child presented with three simultaneous malignancies: glioblastoma multiforme, Burkitt lymphoma, and colonic adenocarcinoma. She was treated for her diseases without success and died 8 months after presentation. Genetic analysis revealed a homozygous mutation in the PMS2 gene, consistent with constitutional mismatch repair deficiency. Her siblings and parents were screened: three of four siblings and both parents were heterozygous for this mutation; the fourth sibling did not have the mutation. Copyright © 2013 Wiley Periodicals, Inc.

Interaction of the E. coli DNA G:T-mismatch endonuclease (vsr protein) with oligonucleotides containing its target sequence.

PubMed

Turner, D P; Connolly, B A

2000-12-15

The Escherichia coli vsr endonuclease recognises G:T base-pair mismatches in double-stranded DNA and initiates a repair pathway by hydrolysing the phosphate group 5' to the incorrectly paired T. The enzyme shows a preference for G:T mismatches within a particular sequence context, derived from the recognition site of the E. coli dcm DNA-methyltransferase (CC[A/T]GG). Thus, the preferred substrate for the vsr protein is (CT[A/T]GG), where the underlined T is opposed by a dG base. This paper provides quantitative data for the interaction of the vsr protein with a number of oligonucleotides containing G:T mismatches. Evaluation of specificity constant (k(st)/K(D); k(st)=rate constant for single turnover, K(D)=equilibrium dissociation constant) confirms vsr's preference for a G:T mismatch within a hemi-methylated dcm sequence, i.e. the best substrate is a duplex (both strands written in the 5'-3' orientation) composed of CT[A/T]GG and C(5Me)C[T/A]GG. Conversion of the mispaired T (underlined) to dU or the d(5Me)C to dC gave poorer substrates. No interaction was observed with oligonucleotides that lacked a G:T mismatch or did not possess a dcm sequence. An analysis of the fraction of active protein, by "reverse-titration" (i.e. adding increasing amounts of DNA to a fixed amount of protein followed by gel-mobility shift analysis) showed that less than 1% of the vsr endonuclease was able to bind to the substrate. This was confirmed using "competitive titrations" (where competitor oligonucleotides are used to displace a (32)P-labelled nucleic acid from the vsr protein) and burst kinetic analysis. This result is discussed in the light of previous in vitro and in vivo data which indicate that the MutL protein may be needed for full vsr activity. Copyright 2000 Academic Press.

Biological effects of simple changes in functionality on rhodium metalloinsertors

PubMed Central

Weidmann, Alyson G.; Komor, Alexis C.; Barton, Jacqueline K.

2013-01-01

DNA mismatch repair (MMR) is crucial to ensuring the fidelity of the genome. The inability to correct single base mismatches leads to elevated mutation rates and carcinogenesis. Using metalloinsertors–bulky metal complexes that bind with high specificity to mismatched sites in the DNA duplex–our laboratory has adopted a new chemotherapeutic strategy through the selective targeting of MMR-deficient cells, that is, those that have a propensity for cancerous transformation. Rhodium metalloinsertors display inhibitory effects selectively in cells that are deficient in the MMR machinery, consistent with this strategy. However, a highly sensitive structure–function relationship is emerging with the development of new complexes that highlights the importance of subcellular localization. We have found that small structural modifications, for example a hydroxyl versus a methyl functional group, can yield profound differences in biological function. Despite similar binding affinities and selectivities for DNA mismatches, only one metalloinsertor shows selective inhibition of cellular proliferation in MMR-deficient versus -proficient cells. Studies of whole-cell, nuclear and mitochondrial uptake reveal that this selectivity depends upon targeting DNA mismatches in the cell nucleus. PMID:23776288

PMS2 gene mutation results in DNA mismatch repair system failure in a case of adult granulosa cell tumor.

PubMed

Wang, Wen-Chung; Lee, Ya-Ting; Lai, Yen-Chein

2017-03-27

Granulosa cell tumors are rare ovarian malignancies. Their characteristics include unpredictable indolent growth with malignant potential and late recurrence. Approximately 95% are of adult type. Recent molecular studies have characterized the FOXL2 402C > G mutation in adult granulosa cell tumor. Our previous case report showed that unique FOXL2 402C > G mutation and defective DNA mismatch repair system are associated with the development of adult granulosa cell tumor. In this study, the DNA sequences of four genes, MSH2, MLH1, MSH6, and PMS2, in the DNA mismatch repair system were determined via direct sequencing to elucidate the exact mechanism for the development of this granulosa cell tumor. The results showed that two missense germline mutations, T485K and N775L, inactivate the PMS2 gene. The results of this case study indicated that although FOXL2 402C > G mutation determines the development of granulosa cell tumor, PMS2 mutation may be the initial driver of carcinogenesis. Immunohistochemistry-based tumor testing for mismatch repair gene expression may be necessary for granulosa cell tumors to determine their malignant potential or if they are part of Lynch syndrome.

Biallelic PMS2 Mutation and Heterozygous DICER1 Mutation Presenting as Constitutional Mismatch Repair Deficiency With Corpus Callosum Agenesis: Case Report and Review of Literature.

PubMed

Cheyuo, Cletus; Radwan, Walid; Ahn, Janice; Gyure, Kymberly; Qaiser, Rabia; Tomboc, Patrick

2017-10-01

Constitutional mismatch repair deficiency syndrome is a cancer predisposition syndrome caused by autosomal recessive biallelic (homozygous) germline mutations in the mismatch repair genes (MLH1, MSH2, MSH6, and PMS2). The clinical spectrum includes neoplastic and non-neoplastic manifestations. We present the case of a 7-year-old boy who presented with T-lymphoblastic lymphoma and glioblastoma, together with non-neoplastic manifestations including corpus callosum agenesis, arachnoid cyst, developmental venous anomaly, and hydrocephalus. Gene mutation analysis revealed pathogenic biallelic mutations of PMS2 and heterozygous DICER1 variant predicted to be pathogenic. This report is the first to allude to a possible interaction of the mismatch repair system with DICER1 to cause corpus callosum agenesis.

Mismatch Repair Proteins and Microsatellite Instability in Colorectal Carcinoma (MLH1, MSH2, MSH6 and PMS2): Histopathological and Immunohistochemical Study.

PubMed

Ismael, Nour El Hoda S; El Sheikh, Samar A; Talaat, Suzan M; Salem, Eman M

2017-03-15

Colorectal cancer (CRC) is one of the most common cancers worldwide. Microsatellite instability (MSI) is detected in about 15% of all colorectal cancers. CRC with MSI has particular characteristics such as improved survival rates and better prognosis. They also have a distinct sensitivity to the action of chemotherapy. The aim of the study was to detect microsatellite instability in a cohort of colorectal cancer Egyptian patients using the immunohistochemical expression of mismatch repair proteins (MLH1, MSH2, MSH6 and PMS2). Cases were divided into Microsatellite stable (MSS), Microsatellite unstable low (MSI-L) and Microsatellite unstable high (MSI-H). This Microsatellite stability status was correlated with different clinicopathological parameters. There was a statistically significant correlation between the age of cases, tumor site & grade and the microsatellite stability status. There was no statistically significant correlation between the gender of patients, tumor subtype, stage, mucoid change, necrosis, tumor borders, lymphocytic response, lymphovascular emboli and the microsatellite stability status. Testing for MSI should be done for all colorectal cancer patients, especially those younger than 50 years old, right sided and high-grade CRCs.

TumorNext-Lynch-MMR: a comprehensive next generation sequencing assay for the detection of germline and somatic mutations in genes associated with mismatch repair deficiency and Lynch syndrome.

PubMed

Gray, Phillip N; Tsai, Pei; Chen, Daniel; Wu, Sitao; Hoo, Jayne; Mu, Wenbo; Li, Bing; Vuong, Huy; Lu, Hsiao-Mei; Batth, Navanjot; Willett, Sara; Uyeda, Lisa; Shah, Swati; Gau, Chia-Ling; Umali, Monalyn; Espenschied, Carin; Janicek, Mike; Brown, Sandra; Margileth, David; Dobrea, Lavinia; Wagman, Lawrence; Rana, Huma; Hall, Michael J; Ross, Theodora; Terdiman, Jonathan; Cullinane, Carey; Ries, Savita; Totten, Ellen; Elliott, Aaron M

2018-04-17

The current algorithm for Lynch syndrome diagnosis is highly complex with multiple steps which can result in an extended time to diagnosis while depleting precious tumor specimens. Here we describe the analytical validation of a custom probe-based NGS tumor panel, TumorNext-Lynch-MMR, which generates a comprehensive genetic profile of both germline and somatic mutations that can accelerate and streamline the time to diagnosis and preserve specimen. TumorNext-Lynch-MMR can detect single nucleotide variants, small insertions and deletions in 39 genes that are frequently mutated in Lynch syndrome and colorectal cancer. Moreover, the panel provides microsatellite instability status and detects loss of heterozygosity in the five Lynch genes; MSH2 , MSH6 , MLH1 , PMS2 and EPCAM . Clinical cases are described that highlight the assays ability to differentiate between somatic and germline mutations, precisely classify variants and resolve discordant cases.

TumorNext-Lynch-MMR: a comprehensive next generation sequencing assay for the detection of germline and somatic mutations in genes associated with mismatch repair deficiency and Lynch syndrome

PubMed Central

Gray, Phillip N.; Tsai, Pei; Chen, Daniel; Wu, Sitao; Hoo, Jayne; Mu, Wenbo; Li, Bing; Vuong, Huy; Lu, Hsiao-Mei; Batth, Navanjot; Willett, Sara; Uyeda, Lisa; Shah, Swati; Gau, Chia-Ling; Umali, Monalyn; Espenschied, Carin; Janicek, Mike; Brown, Sandra; Margileth, David; Dobrea, Lavinia; Wagman, Lawrence; Rana, Huma; Hall, Michael J.; Ross, Theodora; Terdiman, Jonathan; Cullinane, Carey; Ries, Savita; Totten, Ellen; Elliott, Aaron M.

2018-01-01

The current algorithm for Lynch syndrome diagnosis is highly complex with multiple steps which can result in an extended time to diagnosis while depleting precious tumor specimens. Here we describe the analytical validation of a custom probe-based NGS tumor panel, TumorNext-Lynch-MMR, which generates a comprehensive genetic profile of both germline and somatic mutations that can accelerate and streamline the time to diagnosis and preserve specimen. TumorNext-Lynch-MMR can detect single nucleotide variants, small insertions and deletions in 39 genes that are frequently mutated in Lynch syndrome and colorectal cancer. Moreover, the panel provides microsatellite instability status and detects loss of heterozygosity in the five Lynch genes; MSH2, MSH6, MLH1, PMS2 and EPCAM. Clinical cases are described that highlight the assays ability to differentiate between somatic and germline mutations, precisely classify variants and resolve discordant cases. PMID:29755653

PCNA function in the activation and strand direction of MutLα endonuclease in mismatch repair

PubMed Central

Pluciennik, Anna; Dzantiev, Leonid; Iyer, Ravi R.; Constantin, Nicoleta; Kadyrov, Farid A.; Modrich, Paul

2010-01-01

MutLα (MLH1–PMS2) is a latent endonuclease that is activated in a mismatch-, MutSα-, proliferating cell nuclear antigen (PCNA)-, replication factor C (RFC)-, and ATP-dependent manner, with nuclease action directed to the heteroduplex strand that contains a preexisting break. RFC depletion experiments and use of linear DNAs indicate that RFC function in endonuclease activation is limited to PCNA loading. Whereas nicked circular heteroduplex DNA is a good substrate for PCNA loading and for endonuclease activation on the incised strand, covalently closed, relaxed circular DNA is a poor substrate for both reactions. However, covalently closed supercoiled or bubble-containing relaxed heteroduplexes, which do support PCNA loading, also support MutLα activation, but in this case cleavage strand bias is largely abolished. Based on these findings we suggest that PCNA has two roles in MutLα function: The clamp is required for endonuclease activation, an effect that apparently involves interaction of the two proteins, and by virtue of its loading orientation, PCNA determines the strand direction of MutLα incision. These results also provide a potential mechanism for activation of mismatch repair on nonreplicating DNA, an effect that may have implications for the somatic phase of triplet repeat expansion. PMID:20713735

Predictive genetic testing in children: constitutional mismatch repair deficiency cancer predisposing syndrome.

PubMed

Bruwer, Zandrè; Algar, Ursula; Vorster, Alvera; Fieggen, Karen; Davidson, Alan; Goldberg, Paul; Wainwright, Helen; Ramesar, Rajkumar

2014-04-01

Biallelic germline mutations in mismatch repair genes predispose to constitutional mismatch repair deficiency syndrome (CMMR-D). The condition is characterized by a broad spectrum of early-onset tumors, including hematological, brain and bowel and is frequently associated with features of Neurofibromatosis type 1. Few definitive screening recommendations have been suggested and no published reports have described predictive testing. We report on the first case of predictive testing for CMMR-D following the identification of two non-consanguineous parents, with the same heterozygous mutation in MLH1: c.1528C > T. The genetic counseling offered to the family, for their two at-risk daughters, is discussed with a focus on the ethical considerations of testing children for known cancer-causing variants. The challenges that are encountered when reporting on heterozygosity in a child younger than 18 years (disclosure of carrier status and risk for Lynch syndrome), when discovered during testing for homozygosity, are addressed. In addition, the identification of CMMR-D in a three year old, and the recommended clinical surveillance that was proposed for this individual is discussed. Despite predictive testing and presymptomatic screening, the sudden death of the child with CMMR-D syndrome occurred 6 months after her last surveillance MRI. This report further highlights the difficulty of developing guidelines, as a result of the rarity of cases and diversity of presentation.

Cell-Selective Biological Activity of Rhodium Metalloinsertors Correlates with Subcellular Localization

PubMed Central

Komor, Alexis C.; Schneider, Curtis J.; Weidmann, Alyson G.; Barton, Jacqueline K.

2013-01-01

Deficiencies in the mismatch repair (MMR) pathway are associated with several types of cancers, as well as resistance to commonly used chemotherapeutics. Rhodium metalloinsertors have been found to bind DNA mismatches with high affinity and specificity in vitro, and also exhibit cell-selective cytotoxicity, targeting MMR-deficient cells over MMR-proficient cells. Ten distinct metalloinsertors with varying lipophilicities have been synthesized and their mismatch binding affinities and biological activities determined. Although DNA photocleavage experiments demonstrate that their binding affinities are quite similar, their cell-selective antiproliferative and cytotoxic activities vary significantly. Inductively coupled plasma mass spectrometry (ICP-MS) experiments have uncovered a relationship between the subcellular distribution of these metalloinsertors and their biological activities. Specifically, we find that all of our metalloinsertors localize in the nucleus at sufficient concentrations for binding to DNA mismatches. However, the metalloinsertors with high rhodium localization in the mitochondria show toxicity that is not selective for MMR-deficient cells, whereas metalloinsertors with less mitochondrial rhodium show activity that is highly selective for MMR-deficient versus proficient cells. This work supports the notion that specific targeting of the metalloinsertors to nuclear DNA gives rise to their cell-selective cytotoxic and antiproliferative activities. The selectivity in cellular targeting depends upon binding to mismatches in genomic DNA. PMID:23137296

Overexpression of MutSα Complex Proteins Predicts Poor Prognosis in Oral Squamous Cell Carcinoma.

PubMed

Wagner, Vivian Petersen; Webber, Liana Preto; Salvadori, Gabriela; Meurer, Luise; Fonseca, Felipe Paiva; Castilho, Rogério Moraes; Squarize, Cristiane Helena; Vargas, Pablo Agustin; Martins, Manoela Domingues

2016-05-01

The DNA mismatch repair (MMR) system is responsible for the detection and correction of errors created during DNA replication, thereby avoiding the incorporation of mutations in dividing cells. The prognostic value of alterations in MMR system has not previously been analyzed in oral squamous cell carcinoma (OSCC).The study comprised 115 cases of OSCC diagnosed between 1996 and 2010. The specimens collected were constructed into tissue microarray blocks. Immunohistochemical staining for MutSα complex proteins hMSH2 and hMSH6 was performed. The slides were subsequently scanned into high-resolution images, and nuclear staining of hMSH2 and hMSH6 was analyzed using the Nuclear V9 algorithm. Univariable and multivariable Cox proportional hazard regression models were performed to evaluate the prognostic value of hMSH2 and hMSH6 in OSCC.All cases in the present cohort were positive for hMSH2 and hMSH6 and a direct correlation was found between the expression of the proteins (P < 0.05). The mean number of positive cells for hMSH2 and hMSH6 was 64.44 ± 15.21 and 31.46 ± 22.38, respectively. These values were used as cutoff points to determine high protein expression. Cases with high expression of both proteins simultaneously were classified as having high MutSα complex expression. In the multivariable analysis, high expression of the MutSα complex was an independent prognostic factor for poor overall survival (hazard ratio: 2.75, P = 0.02).This study provides a first insight of the prognostic value of alterations in MMR system in OSCC. We found that MutSα complex may constitute a molecular marker for the poor prognosis of OSCC.

Repair of 8-oxo-7,8-dihydroguanine in prokaryotic and eukaryotic cells: Properties and biological roles of the Fpg and OGG1 DNA N-glycosylases.

PubMed

Boiteux, Serge; Coste, Franck; Castaing, Bertrand

2017-06-01

Oxidatively damaged DNA results from the attack of sugar and base moieties by reactive oxygen species (ROS), which are formed as byproducts of normal cell metabolism and during exposure to endogenous or exogenous chemical or physical agents. Guanine, having the lowest redox potential, is the DNA base the most susceptible to oxidation, yielding products such as 8-oxo-7,8-dihydroguanine (8-oxoG) and 2-6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG). In DNA, 8-oxoG was shown to be mutagenic yielding GC to TA transversions upon incorporation of dAMP opposite this lesion by replicative DNA polymerases. In prokaryotic and eukaryotic cells, 8-oxoG is primarily repaired by the base excision repair pathway (BER) initiated by a DNA N-glycosylase, Fpg and OGG1, respectively. In Escherichia coli, Fpg cooperates with MutY and MutT to prevent 8-oxoG-induced mutations, the "GO-repair system". In Saccharomyces cerevisiae, OGG1 cooperates with nucleotide excision repair (NER), mismatch repair (MMR), post-replication repair (PRR) and DNA polymerase η to prevent mutagenesis. Human and mouse cells mobilize all these pathways using OGG1, MUTYH (MutY-homolog also known as MYH), MTH1 (MutT-homolog also known as NUDT1), NER, MMR, NEILs and DNA polymerases η and λ, to prevent 8-oxoG-induced mutations. In fact, mice deficient in both OGG1 and MUTYH develop cancer in different organs at adult age, which points to the critical impact of 8-oxoG repair on genetic stability in mammals. In this review, we will focus on Fpg and OGG1 proteins, their biochemical and structural properties as well as their biological roles. Other DNA N-glycosylases able to release 8-oxoG from damaged DNA in various organisms will be discussed. Finally, we will report on the role of OGG1 in human disease and the possible use of 8-oxoG DNA N-glycosylases as therapeutic targets. Copyright © 2017 Elsevier Inc. All rights reserved.

Expression of ERCC1, RRM1, TUBB3 in correlation with apoptosis repressor ARC, DNA mismatch repair proteins and p53 in liver metastasis of colorectal cancer.

PubMed

Tóth, Csaba; Sükösd, Farkas; Valicsek, Erzsébet; Herpel, Esther; Schirmacher, Peter; Renner, Marcus; Mader, Christoph; Tiszlavicz, László; Kriegsmann, Jörg

2017-11-01

Liver metastasis in colorectal cancer is common and the primary treatment is chemotherapy. To date, there is no routinely used test in clinical practice to predict the effectiveness of conventional chemotherapy. Therefore, biomarkers with predictive value for conventional chemotherapy would be of considerable benefit in treatment planning. We analysed three proteins [excision repair cross-complementing 1 (ERCC1), ribonucleoside-diphosphate reductase 1 (RRM1) and class III β-tubulin (TUBB3)] in colorectal cancer liver metastasis. We used tissue microarray slides with 101 liver metastasis samples, stained for ERCC1, RRM1 and TUBB3 and established scoring systems (fitted for tissue microarray) for each protein. In statistical analysis, we compared the expression of ERCC1, RRM1 and TUBB3 to mismatch proteins (MLH1, MSH2, MSH6 and PMS2), p53 and to apoptosis repressor protein (ARC). Statistically significant correlations were found between ERCC1, TUBB3 and MLH1, MSH2 and RRM1 and MSH2, MSH6. Noteworthy, our analysis revealed a strong significant correlation between cytoplasmic ARC expression and RRM1, TUBB3 (p=0.000 and p=0.001, respectively), implying an additional role of TUBB3 and RRM1 not only in therapy resistance, but also in the apoptotic machinery. Our data strengthens the importance of ERCC1, TUBB3 and RRM1 in the prediction of chemotherapy effectiveness and suggest new functional connections in DNA repair, microtubule network and apoptotic signaling (i.e. ARC protein). In conclusion, we showed the importance and need of predictive biomarkers in metastasized colorectal cancer and pointed out the relevance not only of single predictive markers but also of their interactions with other known and newly explored relations between different signaling pathways.

Mlh1 is required for female fertility in Drosophila melanogaster: An outcome of effects on meiotic crossing over, ovarian follicles and egg activation.

PubMed

Vimal, Divya; Kumar, Saurabh; Pandey, Ashutosh; Sharma, Divya; Saini, Sanjay; Gupta, Snigdha; Ravi Ram, Kristipati; Chowdhuri, Debapratim Kar

2018-03-01

Mismatch repair (MMR) system, a conserved DNA repair pathway, plays crucial role in DNA recombination and is involved in gametogenesis. The impact of alterations in MMR family of proteins (bacterial MutS and MutL homologues) on mammalian fertility is well documented. However, an insight to the role of MMR in reproduction of non-mammalian organisms is limited. Hence, in the present study, we analysed the impact of mlh1 (a MutL homologue) on meiotic crossing over/recombination and fertility in a genetically tractable model, Drosophila melanogaster. Using mlh1 e00130 hypomorphic allele, we report female specific adverse reproductive outcome for reduced mlh1 in Drosophila: mlh1 e00130 homozygous females had severely reduced fertility while males were fertile. Further, mlh1 e00130 females contained small ovaries with large number of early stages as well as significantly reduced mature oocytes, and laid fewer eggs, indicating discrepancies in egg production and ovulation. These observations contrast the sex independent and/or male specific sterility and normal follicular development as well as ovulation reported so far for MMR family proteins in mammals. However, analogous to the role(s) of mlh1 in meiotic crossing over and DNA repair processes underlying mammalian fertility, ovarian follicles from mlh1 e00130 females contained significantly increased DNA double strand breaks (DSBs) and reduced synaptonemal complex foci. In addition, large proportion of fertilized eggs display discrepancies in egg activation and fail to proceed beyond stage 5 of embryogenesis. Hence, reduction of the Mlh1 protein level leads to defective oocytes that fail to complete embryogenesis after fertilization thereby reducing female fertility. Copyright © 2017 Elsevier GmbH. All rights reserved.

The distinct spectra of tumor-associated Apc mutations in mismatch repair-deficient Apc1638N mice define the roles of MSH3 and MSH6 in DNA repair and intestinal tumorigenesis.

PubMed

Kuraguchi, M; Yang, K; Wong, E; Avdievich, E; Fan, K; Kolodner, R D; Lipkin, M; Brown, A M; Kucherlapati, R; Edelmann, W

2001-11-01

In mammalian cells, mismatch recognition has been attributed to two partially redundant heterodimeric protein complexes of MutS homologues, MSH2-MSH3 and MSH2-MSH6. We have conducted a comparative analysis of Msh3 and Msh6 deficiency in mouse intestinal tumorigenesis by generating Apc1638N mice deficient in Msh3, Msh6 or both. We have found that Apc1638N mice defective in Msh6 show reduced survival and a 6-7-fold increase in intestinal tumor multiplicity. In contrast, Msh3-deficient Apc1638N mice showed no difference in survival and intestinal tumor multiplicity as compared with Apc1638N mice. However, when Msh3 deficiency is combined with Msh6 deficiency (Msh3(-/-)Msh6(-/-)Apc1638N), the survival rate of the mice was further reduced compared to Msh6(-/-)Apc(1638N) mice because of a high multiplicity of intestinal tumors at a younger age. Almost 90% of the intestinal tumors from both Msh6(-/-)Apc1638N and Msh3(-/-)Msh6(-/-)Apc1638N mice contained truncation mutations in the wild-type Apc allele. Apc mutations in Msh6(-/-)Apc1638N mice consisted predominantly of base substitutions (93%) creating stop codons, consistent with a major role for Msh6 in the repair of base-base mismatches. However, in Msh3(-/-)Msh6(-/-)Apc1638N tumors, we observed a mixture of base substitutions (46%) and frameshifts (54%), indicating that in Msh6(-/-)Apc1638N mice frameshift mutations in the Apc gene were suppressed by Msh3. Interestingly, all except one of the Apc mutations detected in mismatch repair-deficient intestinal tumors were located upstream of the third 20-amino acid beta-catenin binding repeat and before all of the Ser-Ala-Met-Pro repeats, suggesting that there is selection for loss of multiple domains involved in beta-catenin regulation. Our analysis therefore has revealed distinct mutational spectra and clarified the roles of Msh3 and Msh6 in DNA repair and intestinal tumorigenesis.

Differential cellular responses to prolonged LDR-IR in MLH1-proficient and MLH1-deficient colorectal cancer HCT116 cells.

PubMed

Yan, Tao; Seo, Yuji; Kinsella, Timothy J

2009-11-15

MLH1 is a key DNA mismatch repair (MMR) protein involved in maintaining genomic stability by participating in the repair of endogenous and exogenous mispairs in the daughter strands during S phase. Exogenous mispairs can result following treatment with several classes of chemotherapeutic drugs, as well as with ionizing radiation. In this study, we investigated the role of the MLH1 protein in determining the cellular and molecular responses to prolonged low-dose rate ionizing radiation (LDR-IR), which is similar to the clinical use of cancer brachytherapy. An isogenic pair of MMR(+) (MLH1(+)) and MMR(-) (MLH1(-)) human colorectal cancer HCT116 cells was exposed to prolonged LDR-IR (1.3-17 cGy/h x 24-96 h). The clonogenic survival and gene mutation rates were examined. Cell cycle distribution was analyzed with flow cytometry. Changes in selected DNA damage repair proteins, DNA damage response proteins, and cell death marker proteins were examined with Western blotting. MLH1(+) HCT116 cells showed greater radiosensitivity with enhanced expression of apoptotic and autophagic markers, a reduced HPRT gene mutation rate, and more pronounced cell cycle alterations (increased late-S population and a G(2)/M arrest) following LDR-IR compared with MLH1(-) HCT116 cells. Importantly, a progressive increase in MLH1 protein levels was found in MLH1(+) cells during prolonged LDR-IR, which was temporally correlated with a progressive decrease in Rad51 protein (involved in homologous recombination) levels. MLH1 status significantly affects cellular responses to prolonged LDR-IR. MLH1 may enhance cell radiosensitivity to prolonged LDR-IR through inhibition of homologous recombination (through inhibition of Rad51).

Acute lymphoblastic leukemia and lymphoma in the context of constitutional mismatch repair deficiency syndrome.

PubMed

Ripperger, Tim; Schlegelberger, Brigitte

2016-03-01

Constitutional mismatch repair deficiency (CMMRD) syndrome is one of the rare diseases associated with a high risk of cancer. Causative mutations are found in DNA mismatch repair genes PMS2, MSH6, MSH2 or MLH1 that are well known in the context of Lynch syndrome. CMMRD follows an autosomal recessive inheritance trait and is characterized by childhood brain tumors and hematological malignancies as well as gastrointestinal cancer in the second and third decades of life. There is a high risk of multiple cancers, occurring synchronously and metachronously. In general, the prognosis is poor. About one third of CMMRD patients develop hematological malignancies as primary (sometimes the only) malignancy or as secondary neoplasm. T-cell non-Hodgkin lymphomas, mainly of mediastinal origin, are the most frequent hematological malignancies. Besides malignant diseases, non-neoplastic features are frequently observed, e.g. café-au-lait spots sometimes resembling neurofibromatosis type I, hypopigmented skin lesions, numerous adenomatous polyps, multiple pilomatricomas, or impaired immunoglobulin class switch recombination. Within the present review, we summarize previously published CMMRD patients with at least one hematological malignancy, provide an overview of steps necessary to substantiate the diagnosis of CMMRD, and refer to the recent most relevant literature. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

The interaction between cytosine methylation and processes of DNA replication and repair shape the mutational landscape of cancer genomes

PubMed Central

Poulos, Rebecca C.

2017-01-01

Abstract Methylated cytosines (5mCs) are frequently mutated in the genome. However, no studies have yet comprehensively analysed mutation–methylation associations across cancer types. Here we analyse 916 cancer genomes, together with tissue type-specific methylation and replication timing data. We describe a strong mutation–methylation association across colorectal cancer subtypes, most interestingly in samples with microsatellite instability (MSI) or Polymerase epsilon (POLE) exonuclease domain mutations. By analysing genomic regions with differential mismatch repair (MMR) efficiency, we suggest a possible role for MMR in the correction of 5mC deamination events, potentially accounting for the high rate of 5mC mutation accumulation in MSI tumours. Additionally, we propose that mutant POLE asserts a mutator phenotype specifically at 5mCs, and we find coding mutation hotspots in POLE-mutant cancers at highly-methylated CpGs in the tumour-suppressor genes APC and TP53. Finally, using multivariable regression models, we demonstrate that different cancers exhibit distinct mutation–methylation associations, with DNA repair influencing such associations in certain cancer genomes. Taken together, we find differential associations with methylation that are vital for accurately predicting expected mutation loads across cancer types. Our findings reveal links between methylation and common mutation and repair processes, with these mechanisms defining a key part of the mutational landscape of cancer genomes. PMID:28531315

A 30-Year-Old Man with Three Primary Malignancies: A Case of Constitutional Mismatch Repair Deficiency.

PubMed

Rengifo-Cam, William; Jasperson, Kory; Garrido-Laguna, Ignacio; Colman, Howard; Scaife, Courtney; Samowitz, Wade; Samadder, N Jewel

2017-01-01

Constitutional mismatch repair deficiency (CMMRD) is a devastating cancer predisposition syndrome for which clinical manifestations, genetic screening, and cancer prevention strategies are limited. We report a case of CMMRD presenting with metachronous colorectal cancer and brain cancer. Oncologists and gastroenterologists should be aware of the CMMRD syndrome as a rare cause of very early-onset colorectal cancer.

Chromosomal location and genetic mapping of the mismatch repair gene homologs MSH2, MSH3, and MSH6 in rye and wheat

PubMed

Korzun; Borner; Siebert; Malyshev; Hilpert; Kunze; Puchta

1999-12-01

The efficiency of homeologous recombination is influenced by mismatch repair genes in bacteria, yeast, and mammals. To elucidate a possible role of these genes in homeologous pairing and cross-compatibility in plants, gene probes of wheat (Triticum aestivum) specific for the mismatch repair gene homologues MSH2, MSH3, and MSH6 were used to map them to their genomic positions in rye (Secale cereale). Whereas MSH2 was mapped to the short arm of chromosome 1R, MSH3 was mapped to the long arm of chromosome 2R and MSH6 to the long arm of chromosome 5R. Southern blots with nullisomic-tetrasomic (NT) lines of wheat indicated the presence of the sequences on the respective homeologous group of wheat chromosomes. Additionally, an MSH6-specific homologue could also be detected on homoeologous group 3 of wheat. However, in the well-known, highly homoeologous pairing wheat mutant ph1b the MSH6-specific sequence is not within the deleted part of chromosome 5BL, indicating that the pairing phenotype is not due to a loss of one of the mismatch repair genes tested.

Apoptotic function of human PMS2 compromised by the nonsynonymous single-nucleotide polymorphic variant R20Q

PubMed Central

Marinovic-Terzic, Ivana; Yoshioka-Yamashita, Atsuko; Shimodaira, Hideki; Avdievich, Elena; Hunton, Irina C.; Kolodner, Richard D.; Edelmann, Winfried; Wang, Jean Y. J.

2008-01-01

Mismatch repair (MMR) corrects replication errors during DNA synthesis. The mammalian MMR proteins also activate cell cycle checkpoints and apoptosis in response to persistent DNA damage. MMR-deficient cells are resistant to cisplatin, a DNA cross-linking agent used in chemotherapy, because of impaired activation of apoptotic pathways. It is shown that postmeiotic segregation 2 (PMS2), an MMR protein, is required for cisplatin-induced activation of p73, a member of the p53 family of transcription factors with proapoptotic activity. The human PMS2 is highly polymorphic, with at least 12 known nonsynonymous codon changes identified. We show here that the PMS2(R20Q) variant is defective in activating p73-dependent apoptotic response to cisplatin. When expressed in Pms2-deficient mouse fibroblasts, human PMS2(R20Q) but not PMS2 interfered with the apoptotic response to cisplatin. Correspondingly, PMS2 but not PMS2(R20Q) enhanced the cytotoxic effect of cisplatin measured by clonogenic survival. Because PMS2(R20Q) lacks proapoptotic activity, this polymorphic allele may modulate tumor responses to cisplatin among cancer patients. PMID:18768816

Apoptotic function of human PMS2 compromised by the nonsynonymous single-nucleotide polymorphic variant R20Q.

PubMed

Marinovic-Terzic, Ivana; Yoshioka-Yamashita, Atsuko; Shimodaira, Hideki; Avdievich, Elena; Hunton, Irina C; Kolodner, Richard D; Edelmann, Winfried; Wang, Jean Y J

2008-09-16

Mismatch repair (MMR) corrects replication errors during DNA synthesis. The mammalian MMR proteins also activate cell cycle checkpoints and apoptosis in response to persistent DNA damage. MMR-deficient cells are resistant to cisplatin, a DNA cross-linking agent used in chemotherapy, because of impaired activation of apoptotic pathways. It is shown that postmeiotic segregation 2 (PMS2), an MMR protein, is required for cisplatin-induced activation of p73, a member of the p53 family of transcription factors with proapoptotic activity. The human PMS2 is highly polymorphic, with at least 12 known nonsynonymous codon changes identified. We show here that the PMS2(R20Q) variant is defective in activating p73-dependent apoptotic response to cisplatin. When expressed in Pms2-deficient mouse fibroblasts, human PMS2(R20Q) but not PMS2 interfered with the apoptotic response to cisplatin. Correspondingly, PMS2 but not PMS2(R20Q) enhanced the cytotoxic effect of cisplatin measured by clonogenic survival. Because PMS2(R20Q) lacks proapoptotic activity, this polymorphic allele may modulate tumor responses to cisplatin among cancer patients.

Aberrant protein expression and frequent allelic loss of MSH3 in colorectal cancer with low-level microsatellite instability.

PubMed

Plaschke, Jens; Preußler, Mark; Ziegler, Andreas; Schackert, Hans K

2012-07-01

High level of microsatellite instability (MSI-H) in colorectal cancer (CRC) is caused by the inactivation of mismatch repair (MMR) genes; however, it is unknown for tumors with low level MSI (MSI-L). The protein complex involving MSH3 preferentially recognizes insertion/deletion loops (IDLs) of two to eight bases and di- and tetranucleotide repeats are affected in the majority of MSI-L CRC. We selected 10 and eight MSI-L CRCs from 228 and 204 patients with sporadic and hereditary disease, respectively. The tumors were analyzed for protein expression of MSH3, MSH2, MSH6, MLH1, and PMS2, and for mutations and loss of heterozygosity (LOH) in MSH3. Four tumors showed a markedly reduced MSH3 expression, whereas all 18 tumors had normal expression of the remaining MMR proteins. Twenty-five different sequence variants were identified. None of these results in a truncated protein, though L902W represents the first constitutional missense mutation in MSH3 predicted to be functional based on conservation among mutS homologues. All variants have also been found in normal DNA of the patients and in controls. LOH intragenic to MSH3 was evident for 12 of 16 (75%) informative tumors. Occurrence of sequence variants in normal DNA of the patients and in controls excludes somatic mutations and mutations specific to the CRC patient population, respectively. In contrast, the high frequency of LOH as well as the aberrant protein expression in some tumors indicates an involvement of MSH3 impairment in MSI-L CRC.

Enhancement of MSH2-MSH3-mediated mismatch recognition by the yeast MLH1-PMS1 complex.

PubMed

Habraken, Y; Sung, P; Prakash, L; Prakash, S

1997-10-01

DNA mismatch repair has a key role in maintaining genomic stability. Defects in mismatch repair cause elevated spontaneous mutation rates and increased instability of simple repetitive sequences, while mutations in human mismatch repair genes result in hereditary nonpolyposis colorectal cancers. Mismatch recognition represents the first critical step of mismatch repair. Genetic and biochemical studies in yeast and humans have indicated a requirement for MSH2-MSH3 and MSH2-MSH6 heterodimers in mismatch recognition. These complexes have, to some extent, overlapping mismatch binding specificities. MLH1 and PMS1 are the other essential components of mismatch repair, but how they function in this process is not known. We have purified the yeast MLH1-PMS1 heterodimer to near homogeneity, and examined its effect on MSH2-MSH3 binding to DNA mismatches. By itself, the MLH1-PMS1 complex shows no affinity for mismatched DNA, but it greatly enhances the mismatch binding ability of MSH2-MSH3.

Targeting the FANCJ–BRCA1 interaction promotes a switch from recombination to polη-dependent bypass

PubMed Central

Xie, J; Litman, R; Wang, S; Peng, M; Guillemette, S; Rooney, T; Cantor, SB

2010-01-01

BRCA1 and the DNA helicase FANCJ (also known as BACH1 or BRIP1) have common functions in breast cancer suppression and DNA repair. However, the functional significance of the direct interaction between BRCA1 and FANCJ remains unclear. Here, we have discovered that BRCA1 binding to FANCJ regulates DNA damage repair choice. Thus, when FANCJ binding to BRCA1 is ablated, the molecular mechanism chosen for the repair of damaged DNA is dramatically altered. Specifically, a FANCJ protein that cannot be phosphorylated at serine 990 or bind BRCA1 inhibits DNA repair via homologous recombination and promotes polη-dependent bypass. Furthermore, the polη-dependent bypass promoted by FANCJ requires the direct binding to the mismatch repair (MMR) protein, MLH1. Together, our findings implicate that in human cells BRCA1 binding to FANCJ is critical to regulate DNA repair choice and promote genomic stability. Moreover, unregulated FANCJ function could be associated with cancer and/or chemoresistance. PMID:20173781

Elucidating the clinical significance of two PMS2 missense variants coexisting in a family fulfilling hereditary cancer criteria.

PubMed

González-Acosta, Maribel; Del Valle, Jesús; Navarro, Matilde; Thompson, Bryony A; Iglesias, Sílvia; Sanjuan, Xavier; Paúles, María José; Padilla, Natàlia; Fernández, Anna; Cuesta, Raquel; Teulé, Àlex; Plotz, Guido; Cadiñanos, Juan; de la Cruz, Xavier; Balaguer, Francesc; Lázaro, Conxi; Pineda, Marta; Capellá, Gabriel

2017-10-01

The clinical spectrum of germline mismatch repair (MMR) gene variants continues increasing, encompassing Lynch syndrome, Constitutional MMR Deficiency (CMMRD), and the recently reported MSH3-associated polyposis. Genetic diagnosis of these hereditary cancer syndromes is often hampered by the presence of variants of unknown significance (VUS) and overlapping phenotypes. Two PMS2 VUS, c.2149G>A (p.V717M) and c.2444C>T (p.S815L), were identified in trans in one individual diagnosed with early-onset colorectal cancer (CRC) who belonged to a family fulfilling clinical criteria for hereditary cancer. Clinico-pathological data, multifactorial likelihood calculations and functional analyses were used to refine their clinical significance. Likelihood analysis based on cosegregation and tumor data classified the c.2444C>T variant as pathogenic, which was supported by impaired MMR activity associated with diminished protein expression in functional assays. Conversely, the c.2149G>A variant displayed MMR proficiency and protein stability. These results, in addition to the conserved PMS2 expression in normal tissues and the absence of germline microsatellite instability (gMSI) in the biallelic carrier ruled out a CMMRD diagnosis. The use of comprehensive strategies, including functional and clinico-pathological information, is mandatory to improve the clinical interpretation of naturally occurring MMR variants. This is critical for appropriate clinical management of cancer syndromes associated to MMR gene mutations.

Characterization of pathogenic human MSH2 missense mutations using yeast as a model system: a laboratory course in molecular biology.

PubMed

Gammie, Alison E; Erdeniz, Naz

2004-01-01

This work describes the project for an advanced undergraduate laboratory course in cell and molecular biology. One objective of the course is to teach students a variety of cellular and molecular techniques while conducting original research. A second objective is to provide instruction in science writing and data presentation by requiring comprehensive laboratory reports modeled on the primary literature. The project for the course focuses on a gene, MSH2, implicated in the most common form of inherited colorectal cancer. Msh2 is important for maintaining the fidelity of genetic material where it functions as an important component of the DNA mismatch repair machinery. The goal of the project has two parts. The first part is to create mapped missense mutation listed in the human databases in the cognate yeast MSH2 gene and to assay for defects in DNA mismatch repair. The second part of the course is directed towards understanding in what way are the variant proteins defective for mismatch repair. Protein levels are analyzed to determine if the missense alleles display decreased expression. Furthermore, the students establish whether the Msh2p variants are properly localized to the nucleus using indirect immunofluorescence and whether the altered proteins have lost their ability to interact with other subunits of the MMR complex by creating recombinant DNA molecules and employing the yeast 2-hybrid assay.

Enhanced spontaneous DNA twisting/bending fluctuations unveiled by fluorescence lifetime distributions promote mismatch recognition by the Rad4 nucleotide excision repair complex

PubMed Central

Chakraborty, Sagnik; Steinbach, Peter J; Paul, Debamita; Mu, Hong; Broyde, Suse

2018-01-01

Abstract Rad4/XPC recognizes diverse DNA lesions including ultraviolet-photolesions and carcinogen-DNA adducts, initiating nucleotide excision repair. Studies have suggested that Rad4/XPC senses lesion-induced helix-destabilization to flip out nucleotides from damaged DNA sites. However, characterizing how DNA deformability and/or distortions impact recognition has been challenging. Here, using fluorescence lifetime measurements empowered by a maximum entropy algorithm, we mapped the conformational heterogeneities of artificially destabilized mismatched DNA substrates of varying Rad4-binding specificities. The conformational distributions, as probed by FRET between a cytosine-analog pair exquisitely sensitive to DNA twisting/bending, reveal a direct connection between intrinsic DNA deformability and Rad4 recognition. High-specificity CCC/CCC mismatch, free in solution, sampled a strikingly broad range of conformations from B-DNA-like to highly distorted conformations that resembled those observed with Rad4 bound; the extent of these distortions increased with bound Rad4 and with temperature. Conversely, the non-specific TAT/TAT mismatch had a homogeneous, B-DNA-like conformation. Molecular dynamics simulations also revealed a wide distribution of conformations for CCC/CCC, complementing experimental findings. We propose that intrinsic deformability promotes Rad4 damage recognition, perhaps by stalling a diffusing protein and/or facilitating ‘conformational capture’ of pre-distorted damaged sites. Surprisingly, even mismatched DNA specifically bound to Rad4 remains highly dynamic, a feature that may reflect the versatility of Rad4/XPC to recognize many structurally dissimilar lesions. PMID:29267981

DNA repair targeted therapy: the past or future of cancer treatment?

PubMed Central

Gavande, Navnath S.; VanderVere-Carozza, Pamela S.; Hinshaw, Hilary D.; Jalal, Shadia I.; Sears, Catherine R.; Pawelczak, Katherine S.; Turchi, John J.

2016-01-01

The repair of DNA damage is a complex process that relies on particular pathways to remedy specific types of damage to DNA. The range of insults to DNA includes small, modest changes in structure including mismatched bases and simple methylation events to oxidized bases, intra- and interstrand DNA crosslinks, DNA double strand breaks and protein-DNA adducts. Pathways required for the repair of these lesions include mismatch repair, base excision repair, nucleotide excision repair, and the homology directed repair/Fanconi anemia pathway. Each of these pathways contributes to genetic stability, and mutations in genes encoding proteins involved in these pathways have been demonstrated to promote genetic instability and cancer. In fact, it has been suggested all cancers display defects in DNA repair. It has also been demonstrated that the ability of cancer cells to repair therapeutically induced DNA damage impacts therapeutic efficacy. This has led to targeting DNA repair pathways and proteins to develop anti-cancer agents that will increase sensitivity to traditional chemotherapeutics. While initial studies languished and were plagued by a lack of specificity and a defined mechanism of action, more recent approaches to exploit synthetic lethal interaction and develop high affinity chemical inhibitors have proven considerably more effective. In this review we will highlight recent advances and discuss previous failures in targeting DNA repair to pave the way for future DNA repair targeted agents and their use in cancer therapy. PMID:26896565

Identification of Lynch syndrome mutations in the MLH1-PMS2 interface that disturb dimerization and mismatch repair

PubMed Central

Kosinski, Jan; Hinrichsen, Inga; Bujnicki, Janusz M.; Friedhoff, Peter; Plotz, Guido

2010-01-01

Missense alterations of the mismatch repair gene MLH1 have been identified in a significant proportion of individuals suspected of having Lynch syndrome, a hereditary syndrome which predisposes for cancer of colon and endometrium. The pathogenicity of many of these alterations, however, is unclear. A number of MLH1 alterations are located in the C-terminal domain (CTD) of MLH1, which is responsible for constitutive dimerization with PMS2. We analyzed which alterations may result in pathogenic effects due to interference with dimerization. We used a structural model of CTD of MLH1-PMS2 heterodimer to select 19 MLH1 alterations located inside and outside two candidate dimerization interfaces in the MLH1-CTD. Three alterations (p.Gln542Leu, p.Leu749Pro, p.Tyr750X) caused decreased co-expression of PMS2, which is unstable in the absence of interaction with MLH1, suggesting that these alterations interfere with dimerization. All three alterations are located within the dimerization interface suggested by our model. They also compromised mismatch repair, suggesting that defects in dimerization abrogate repair and confirming that all three alterations are pathogenic. Additionally, we provided biochemical evidence that four alterations with uncertain pathogenicity (p.Ala586Pro, p.Leu636Pro, p.Thr662Pro, and p.Arg755Trp) are deleterious because of poor expression or poor repair efficiency, and confirm the deleterious effect of eight further alterations. PMID:20533529

Identification of Lynch syndrome mutations in the MLH1-PMS2 interface that disturb dimerization and mismatch repair.

PubMed

Kosinski, Jan; Hinrichsen, Inga; Bujnicki, Janusz M; Friedhoff, Peter; Plotz, Guido

2010-08-01

Missense alterations of the mismatch repair gene MLH1 have been identified in a significant proportion of individuals suspected of having Lynch syndrome, a hereditary syndrome that predisposes for cancer of colon and endometrium. The pathogenicity of many of these alterations, however, is unclear. A number of MLH1 alterations are located in the C-terminal domain (CTD) of MLH1, which is responsible for constitutive dimerization with PMS2. We analyzed which alterations may result in pathogenic effects due to interference with dimerization. We used a structural model of CTD of MLH1-PMS2 heterodimer to select 19 MLH1 alterations located inside and outside two candidate dimerization interfaces in the MLH1-CTD. Three alterations (p.Gln542Leu, p.Leu749Pro, p.Tyr750X) caused decreased coexpression of PMS2, which is unstable in the absence of interaction with MLH1, suggesting that these alterations interfere with dimerization. All three alterations are located within the dimerization interface suggested by our model. They also compromised mismatch repair, suggesting that defects in dimerization abrogate repair and confirming that all three alterations are pathogenic. Additionally, we provided biochemical evidence that four alterations with uncertain pathogenicity (p.Ala586Pro, p.Leu636Pro, p.Thr662Pro, and p.Arg755Trp) are deleterious because of poor expression or poor repair efficiency, and confirm the deleterious effect of eight further alterations.

EMAST is a Form of Microsatellite Instability That is Initiated by Inflammation and Modulates Colorectal Cancer Progression.

PubMed

Carethers, John M; Koi, Minoru; Tseng-Rogenski, Stephanie S

2015-03-31

DNA mismatch repair (MMR) function is critical for correcting errors coincident with polymerase-driven DNA replication, and its proteins are frequent targets for inactivation (germline or somatic), generating a hypermutable tumor that drives cancer progression. The biomarker for defective DNA MMR is microsatellite instability-high (MSI-H), observed in ~15% of colorectal cancers, and defined by mono- and dinucleotide microsatellite frameshift mutations. MSI-H is highly correlated with loss of MMR protein expression, is commonly diploid, is often located in the right side of the colon, prognosticates good patient outcome, and predicts poor efficacy with 5-fluorouracil treatment. Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) is another form of MSI at tetranucleotide repeats that has been observed in multiple cancers, but its etiology and clinical relevance to patient care has only been recently illuminated. Specifically, EMAST is an acquired somatic defect observed in up to 60% of colorectal cancers and caused by unique dysfunction of the DNA MMR protein MSH3 (and its DNA MMR complex MutSβ, a heterodimer of MSH2-MSH3), and in particular a loss-of-function phenotype due to a reversible shift from its normal nuclear location into the cytosol in response to oxidative stress and the pro-inflammatory cytokine interleukin-6. Tumor hypoxia may also be a contributor. Patients with EMAST colorectal cancers show diminished prognosis compared to patients without the presence of EMAST in their cancer. In addition to defective DNA MMR recognized by tetranucleotide (and di- and tri-nucleotide) frameshifts, loss of MSH3 also contributes to homologous recombination-mediated repair of DNA double stranded breaks, indicating the MSH3 dysfunction is a complex defect for cancer cells that generates not only EMAST but also may contribute to chromosomal instability and aneuploidy. Areas for future investigation for this most common DNA MMR defect among

EMAST is a Form of Microsatellite Instability That is Initiated by Inflammation and Modulates Colorectal Cancer Progression

PubMed Central

Carethers, John M.; Koi, Minoru; Tseng-Rogenski, Stephanie S.

2015-01-01

DNA mismatch repair (MMR) function is critical for correcting errors coincident with polymerase-driven DNA replication, and its proteins are frequent targets for inactivation (germline or somatic), generating a hypermutable tumor that drives cancer progression. The biomarker for defective DNA MMR is microsatellite instability-high (MSI-H), observed in ~15% of colorectal cancers, and defined by mono- and dinucleotide microsatellite frameshift mutations. MSI-H is highly correlated with loss of MMR protein expression, is commonly diploid, is often located in the right side of the colon, prognosticates good patient outcome, and predicts poor efficacy with 5-fluorouracil treatment. Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) is another form of MSI at tetranucleotide repeats that has been observed in multiple cancers, but its etiology and clinical relevance to patient care has only been recently illuminated. Specifically, EMAST is an acquired somatic defect observed in up to 60% of colorectal cancers and caused by unique dysfunction of the DNA MMR protein MSH3 (and its DNA MMR complex MutSβ, a heterodimer of MSH2-MSH3), and in particular a loss-of-function phenotype due to a reversible shift from its normal nuclear location into the cytosol in response to oxidative stress and the pro-inflammatory cytokine interleukin-6. Tumor hypoxia may also be a contributor. Patients with EMAST colorectal cancers show diminished prognosis compared to patients without the presence of EMAST in their cancer. In addition to defective DNA MMR recognized by tetranucleotide (and di- and tri-nucleotide) frameshifts, loss of MSH3 also contributes to homologous recombination-mediated repair of DNA double stranded breaks, indicating the MSH3 dysfunction is a complex defect for cancer cells that generates not only EMAST but also may contribute to chromosomal instability and aneuploidy. Areas for future investigation for this most common DNA MMR defect among

The interaction between cytosine methylation and processes of DNA replication and repair shape the mutational landscape of cancer genomes.

PubMed

Poulos, Rebecca C; Olivier, Jake; Wong, Jason W H

2017-07-27

Methylated cytosines (5mCs) are frequently mutated in the genome. However, no studies have yet comprehensively analysed mutation-methylation associations across cancer types. Here we analyse 916 cancer genomes, together with tissue type-specific methylation and replication timing data. We describe a strong mutation-methylation association across colorectal cancer subtypes, most interestingly in samples with microsatellite instability (MSI) or Polymerase epsilon (POLE) exonuclease domain mutations. By analysing genomic regions with differential mismatch repair (MMR) efficiency, we suggest a possible role for MMR in the correction of 5mC deamination events, potentially accounting for the high rate of 5mC mutation accumulation in MSI tumours. Additionally, we propose that mutant POLE asserts a mutator phenotype specifically at 5mCs, and we find coding mutation hotspots in POLE-mutant cancers at highly-methylated CpGs in the tumour-suppressor genes APC and TP53. Finally, using multivariable regression models, we demonstrate that different cancers exhibit distinct mutation-methylation associations, with DNA repair influencing such associations in certain cancer genomes. Taken together, we find differential associations with methylation that are vital for accurately predicting expected mutation loads across cancer types. Our findings reveal links between methylation and common mutation and repair processes, with these mechanisms defining a key part of the mutational landscape of cancer genomes. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

The relationship between mismatch response and the acoustic change complex in normal hearing infants.

PubMed

Uhler, Kristin M; Hunter, Sharon K; Tierney, Elyse; Gilley, Phillip M

2018-06-01

To examine the utility of the mismatch response (MMR) and acoustic change complex (ACC) for assessing speech discrimination in infants. Continuous EEG was recorded during sleep from 48 (24 male, 20 female) normally hearing aged 1.77 to -4.57 months in response to two auditory discrimination tasks. ACC was recorded in response to a three-vowel sequence (/i/-/a/-/i/). MMR was recorded in response to a standard vowel, /a/, (probability 85%), and to a deviant vowel, /i/, (probability of 15%). A priori comparisons included: age, sex, and sleep state. These were conducted separately for each of the three bandpass filter settings were compared (1-18, 1-30, and 1-40 Hz). A priori tests revealed no differences in MMR or ACC for age, sex, or sleep state for any of the three filter settings. ACC and MMR responses were prominently observed in all 44 sleeping infants (data from four infants were excluded). Significant differences observed for ACC were to the onset and offset of stimuli. However, neither group nor individual differences were observed to changes in speech stimuli in the ACC. MMR revealed two prominent peaks occurring at the stimulus onset and at the stimulus offset. Permutation t-tests revealed significant differences between the standard and deviant stimuli for both the onset and offset MMR peaks (p < 0.01). The 1-18 Hz filter setting revealed significant differences for all participants in the MMR paradigm. Both ACC and MMR responses were observed to auditory stimulation suggesting that infants perceive and process speech information even during sleep. Significant differences between the standard and deviant responses were observed in the MMR, but not ACC paradigm. These findings suggest that the MMR is sensitive to detecting auditory/speech discrimination processing. This paper identified that MMR can be used to identify discrimination in normal hearing infants. This suggests that MMR has potential for use in infants with hearing loss to validate

Novel MSH2 splice-site mutation in a young patient with Lynch syndrome

PubMed Central

Liccardo, Raffaella; De Rosa, Marina; Izzo, Paola; Duraturo, Francesca

2018-01-01

Lynch Syndrome (LS) is associated with germline mutations in one of the mismatch repair (MMR) genes, including MutL homolog 1 (MLH1), MutS homolog 2 (MSH2), MSH6, PMS1 homolog 2, mismatch repair system component (PMS2), MLH3 and MSH3. The mutations identified in MMR genes are point mutations or large rearrangements. The point mutations are certainly pathogenetic whether they determine formation of truncated protein. The mutations that arise in splice sites are classified as ‘likely pathogenic’ variants. In the present study, a novel splicing mutation was identified, (named c.212-1g>a), in the MSH2 gene. This novel mutation in the consensus splice site of MSH2 exon 2 leads to the loss of the canonical splice site, without skipping in-frame of exon 2; also with the formation of 2 aberrant transcripts, due to the activation of novel splice sites in exon 2. This mutation was identified in a young patient who developed colon cancer at the age of 26 years and their belongs to family that met the ‘Revised Amsterdam Criteria’. The present study provided insight into the molecular mechanism determining the pathogenicity of this novel MSH2 mutation and it reaffirms the importance of genetic testing in LS. PMID:29568967

A rare case of Crohn's ileitis in a patient with constitutional mismatch repair deficiency.

PubMed

Kaimakliotis, Pavlos; Giardiello, Francis; Eze, Ogechukwu; Truta, Brindusa

2017-01-01

Constitutional mismatch repair deficiency (CMMRD), a variant of Lynch syndrome, is a rare disease characterized by café-au-lait spots, oligopolyposis, glioblastoma and lymphoma. A 24-year-old male, under surveillance for CMMRD, developed Crohn's ileitis after total colectomy with end ileostomy for colorectal cancer and failed to respond to oral corticosteroids. The patient underwent induction and maintenance of remission with vedolizumab infusions. We report the first patient with CMMRD developing Crohn's disease. The choice of immunosuppressive therapy in these patients is challenging and needs to be made according to their risk for malignancy.

Early onset of colorectal cancer in a 13-year-old girl with Lynch syndrome.

PubMed

Ahn, Do Hee; Rho, Jung Hee; Tchah, Hann; Jeon, In-Sang

2016-01-01

Lynch syndrome is the most common inherited colon cancer syndrome. Patients with Lynch syndrome develop a range of cancers including colorectal cancer (CRC) and carry a mutation on one of the mismatched repair (MMR) genes. Although CRC usually occurs after the fourth decade in patients with Lynch syndrome harboring a heterozygous MMR gene mutation, it can occur in children with Lynch syndrome who have a compound heterozygous or homozygous MMR gene mutation. We report a case of CRC in a 13-year-old patient with Lynch syndrome and congenital heart disease. This patient had a heterozygous mutation in MLH1 (an MMR gene), but no compound MMR gene defects, and a K-RAS somatic mutation in the cancer cells.

mlh3 mutations in baker’s yeast alter meiotic recombination outcomes by increasing noncrossover events genome-wide

PubMed Central

Al-Sweel, Najla; Raghavan, Vandana; Khondakar, Nabila; Manhart, Carol M.; Surtees, Jennifer A.

2017-01-01

Mlh1-Mlh3 is an endonuclease hypothesized to act in meiosis to resolve double Holliday junctions into crossovers. It also plays a minor role in eukaryotic DNA mismatch repair (MMR). To understand how Mlh1-Mlh3 functions in both meiosis and MMR, we analyzed in baker’s yeast 60 new mlh3 alleles. Five alleles specifically disrupted MMR, whereas one (mlh3-32) specifically disrupted meiotic crossing over. Mlh1-mlh3 representatives for each class were purified and characterized. Both Mlh1-mlh3-32 (MMR+, crossover-) and Mlh1-mlh3-45 (MMR-, crossover+) displayed wild-type endonuclease activities in vitro. Msh2-Msh3, an MSH complex that acts with Mlh1-Mlh3 in MMR, stimulated the endonuclease activity of Mlh1-mlh3-32 but not Mlh1-mlh3-45, suggesting that Mlh1-mlh3-45 is defective in MSH interactions. Whole genome recombination maps were constructed for wild-type and MMR+ crossover-, MMR- crossover+, endonuclease defective and null mlh3 mutants in an S288c/YJM789 hybrid background. Compared to wild-type, all of the mlh3 mutants showed increases in the number of noncrossover events, consistent with recombination intermediates being resolved through alternative recombination pathways. Our observations provide a structure-function map for Mlh3 that reveals the importance of protein-protein interactions in regulating Mlh1-Mlh3’s enzymatic activity. They also illustrate how defective meiotic components can alter the fate of meiotic recombination intermediates, providing new insights for how meiotic recombination pathways are regulated. PMID:28827832

Population Modelling with M&M's[R

ERIC Educational Resources Information Center

Winkel, Brian

2009-01-01

Several activities in which population dynamics can be modelled by tossing M&M's[R] candy are presented. Physical activities involving M&M's[R] can be modelled by difference equations and several population phenomena, including death and immigration, are studied. (Contains 1 note.)

(CAG)(n)-hairpin DNA binds to Msh2-Msh3 and changes properties of mismatch recognition.

PubMed

Owen, Barbara A L; Yang, Zungyoon; Lai, Maoyi; Gajec, Maciej; Gajek, Maciez; Badger, John D; Hayes, Jeffrey J; Edelmann, Winfried; Kucherlapati, Raju; Wilson, Teresa M; McMurray, Cynthia T

2005-08-01

Cells have evolved sophisticated DNA repair systems to correct damaged DNA. However, the human DNA mismatch repair protein Msh2-Msh3 is involved in the process of trinucleotide (CNG) DNA expansion rather than repair. Using purified protein and synthetic DNA substrates, we show that Msh2-Msh3 binds to CAG-hairpin DNA, a prime candidate for an expansion intermediate. CAG-hairpin binding inhibits the ATPase activity of Msh2-Msh3 and alters both nucleotide (ADP and ATP) affinity and binding interfaces between protein and DNA. These changes in Msh2-Msh3 function depend on the presence of A.A mispaired bases in the stem of the hairpin and on the hairpin DNA structure per se. These studies identify critical functional defects in the Msh2-Msh3-CAG hairpin complex that could misdirect the DNA repair process.

Diagnostic criteria for constitutional mismatch repair deficiency syndrome: suggestions of the European consortium 'care for CMMRD' (C4CMMRD).

PubMed

Wimmer, Katharina; Kratz, Christian P; Vasen, Hans F A; Caron, Olivier; Colas, Chrystelle; Entz-Werle, Natacha; Gerdes, Anne-Marie; Goldberg, Yael; Ilencikova, Denisa; Muleris, Martine; Duval, Alex; Lavoine, Noémie; Ruiz-Ponte, Clara; Slavc, Irene; Burkhardt, Brigit; Brugieres, Laurence

2014-06-01

Constitutional mismatch repair deficiency (CMMRD) syndrome is a distinct childhood cancer predisposition syndrome that results from biallelic germline mutations in one of the four MMR genes, MLH1, MSH2, MSH6 or PMS2. The tumour spectrum is very broad, including mainly haematological, brain and intestinal tract tumours. Patients show a variety of non-malignant features that are indicative of CMMRD. However, currently no criteria that should entail diagnostic evaluation of CMMRD exist. We present a three-point scoring system for the suspected diagnosis CMMRD in a paediatric/young adult cancer patient. Tumours highly specific for CMMRD syndrome are assigned three points, malignancies overrepresented in CMMRD two points and all other malignancies one point. According to their specificity for CMMRD and their frequency in the general population, additional features are weighted with 1-2 points. They include multiple hyperpigmented and hypopigmented skin areas, brain malformations, pilomatricomas, a second childhood malignancy, a Lynch syndrome (LS)-associated tumour in a relative and parental consanguinity. According to the scoring system, CMMRD should be suspected in any cancer patient who reaches a minimum of three points by adding the points of the malignancy and the additional features. The diagnostic steps to confirm or refute the suspected diagnosis are outlined. We expect that application of the suggested strategy for CMMRD diagnosis will increase the number of patients being identified at the time when they develop their first tumour. This will allow adjustment of the treatment modalities, offering surveillance strategies for second malignancies and appropriate counselling of the entire family. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Mitochondrial DNA repair and damage tolerance.

PubMed

Stein, Alexis; Sia, Elaine A

2017-01-01

The accurate maintenance of mitochondrial DNA (mtDNA) is required in order for eukaryotic cells to assemble a functional electron transport chain. This independently-maintained genome relies on nuclear-encoded proteins that are imported into the mitochondria to carry out replication and repair processes. Decades of research has made clear that mitochondria employ robust and varied mtDNA repair and damage tolerance mechanisms in order to ensure the proper maintenance of the mitochondrial genome. This review focuses on our current understanding of mtDNA repair and damage tolerance pathways including base excision repair, mismatch repair, homologous recombination, non-homologous end joining, translesion synthesis and mtDNA degradation in both yeast and mammalian systems.

Expression of DNA repair proteins MSH2, MLH1 and MGMT in human benign and malignant thyroid lesions: An immunohistochemical study

PubMed Central

Giaginis, Constantinos; Michailidi, Christina; Stolakis, Vasileios; Alexandrou, Paraskevi; Tsourouflis, Gerasimos; Klijanienko, Jerzy; Delladetsima, Ioanna; Theocharis, Stamatios

2011-01-01

Summary Background DNA repair is a major defense mechanism, which contributes to the maintenance of genetic sequence, and minimizes cell death, mutation rates, replication errors, DNA damage persistence and genomic instability. Alterations in the expression levels of proteins participating in DNA repair mechanisms have been associated with several aspects of cancer biology. The present study aimed to evaluate the clinical significance of DNA repair proteins MSH2, MLH1 and MGMT in benign and malignant thyroid lesions. Material/Methods MSH2, MLH1 and MGMT protein expression was assessed immunohistochemically on paraffin-embedded thyroid tissues from 90 patients with benign and malignant lesions. Results The expression levels of MLH1 was significantly upregulated in cases with malignant compared to those with benign thyroid lesions (p=0.038). The expression levels of MGMT was significantly downregulated in malignant compared to benign thyroid lesions (p=0.001). Similar associations for both MLH1 and MGMT between cases with papillary carcinoma and hyperplastic nodules were also noted (p=0.014 and p=0.026, respectively). In the subgroup of malignant thyroid lesions, MSH2 downregulation was significantly associated with larger tumor size (p=0.031), while MLH1 upregulation was significantly associated with the presence of lymphatic and vascular invasion (p=0.006 and p=0.002, respectively). Conclusions Alterations in the mismatch repair proteins MSH2 and MLH1 and the direct repair protein MGMT may result from tumor development and/or progression. Further studies are recommended to draw definite conclusions on the clinical significance of DNA repair proteins in thyroid neoplasia. PMID:21358597

The effect of S-substitution at the O6-guanine site on the structure and dynamics of a DNA oligomer containing a G:T mismatch

PubMed Central

2017-01-01

The effect of S-substitution on the O6 guanine site of a 13-mer DNA duplex containing a G:T mismatch is studied using molecular dynamics. The structure, dynamic evolution and hydration of the S-substituted duplex are compared with those of a normal duplex, a duplex with S-substitution on guanine, but no mismatch and a duplex with just a G:T mismatch. The S-substituted mismatch leads to cell death rather than repair. One suggestion is that the G:T mismatch recognition protein recognises the S-substituted mismatch (GS:T) as G:T. This leads to a cycle of futile repair ending in DNA breakage and cell death. We find that some structural features of the helix are similar for the duplex with the G:T mismatch and that with the S-substituted mismatch, but differ from the normal duplex, notably the helical twist. These differences arise from the change in the hydrogen-bonding pattern of the base pair. However a marked feature of the S-substituted G:T mismatch duplex is a very large opening. This showed considerable variability. It is suggested that this enlarged opening would lend support to an alternative model of cell death in which the mismatch protein attaches to thioguanine and activates downstream damage-response pathways. Attack on the sulphur by reactive oxygen species, also leading to cell death, would also be aided by the large, variable opening. PMID:28910418

A novel germline POLE mutation causes an early onset cancer prone syndrome mimicking constitutional mismatch repair deficiency.

PubMed

Wimmer, Katharina; Beilken, Andreas; Nustede, Rainer; Ripperger, Tim; Lamottke, Britta; Ure, Benno; Steinmann, Diana; Reineke-Plaass, Tanja; Lehmann, Ulrich; Zschocke, Johannes; Valle, Laura; Fauth, Christine; Kratz, Christian P

2017-01-01

In a 14-year-old boy with polyposis and rectosigmoid carcinoma, we identified a novel POLE germline mutation, p.(Val411Leu), previously found as recurrent somatic mutation in 'ultramutated' sporadic cancers. This is the youngest reported cancer patient with polymerase proofreading-associated polyposis indicating that POLE mutation p.(Val411Leu) may confer a more severe phenotype than previously reported POLE and POLD1 germline mutations. The patient had multiple café-au-lait macules and a pilomatricoma mimicking the clinical phenotype of constitutional mismatch repair deficiency. We hypothesize that these skin features may be common to different types of constitutional DNA repair defects associated with polyposis and early-onset cancer.

Tumour testing to identify Lynch syndrome in two Australian colorectal cancer cohorts

PubMed Central

Eriksen, Stine V.; Walsh, Michael D.; Walters, Rhiannon J.; Thibodeau, Stephen N.; Stewart, Jenna; Preston, Susan; Win, Aung Ko; Flander, Louisa; Ouakrim, Driss Ait; Macrae, Finlay A.; Boussioutas, Alex; Winship, Ingrid M.; Giles, Graham G.; Hopper, John L.; Southey, Melissa C.

2016-01-01

Background and Aim Tumour testing of colorectal cancers (CRC) for mismatch repair (MMR) deficiency is an effective approach to identify carriers of germline MMR gene mutation (Lynch syndrome). The aim of this study was to identify MMR gene mutation carriers in two cohorts of population-based CRC utilising a combination of tumour and germline testing approaches. Methods CRCs from 813 patients diagnosed with CRC <60 years of age from the Australasian Colorectal Cancer Family Registry (ACCFR) and from 826 patients from the Melbourne Collaborative Cohort Study (MCCS) were tested for MMR protein expression using immunohistochemistry (IHC), microsatellite instability (MSI), BRAFV600E somatic mutation and for MLH1 methylation. MMR gene mutation testing (Sanger sequencing and MLPA) was performed on germline DNA of patients with MMR-deficient tumours and a subset of MMR-proficient CRCs. Results Of the 813 ACCFR probands, 90 probands demonstrated tumour MMR-deficiency (11.1%) and 42 had a MMR gene germline mutation (5.2%). For the MCCS, MMR-deficiency was identified in the tumours of 103 probands (12.5%) and 7 had a germline mutation (0.8%). All the mutation carriers were diagnosed prior to 70 years of age. Probands with a MMR-deficient CRC without MLH1 methylation and a gene mutation were considered Lynch-like and comprised 41.1% and 22.3% of the MMR-deficient CRCs for the ACCFR and MCCS, respectively. Conclusions Identification of MMR gene mutation carriers in Australian CRC-affected patients is optimised by IHC screening of CRC diagnosed before 70 years. A significant proportion of MMR-deficient CRCs will have unknown aetiology (Lynch-like) proving problematic for clinical management. PMID:27273229

Pms2 Suppresses Large Expansions of the (GAA·TTC)n Sequence in Neuronal Tissues

PubMed Central

Bourn, Rebecka L.; De Biase, Irene; Pinto, Ricardo Mouro; Sandi, Chiranjeevi; Al-Mahdawi, Sahar; Pook, Mark A.; Bidichandani, Sanjay I.

2012-01-01

Expanded trinucleotide repeat sequences are the cause of several inherited neurodegenerative diseases. Disease pathogenesis is correlated with several features of somatic instability of these sequences, including further large expansions in postmitotic tissues. The presence of somatic expansions in postmitotic tissues is consistent with DNA repair being a major determinant of somatic instability. Indeed, proteins in the mismatch repair (MMR) pathway are required for instability of the expanded (CAG·CTG)n sequence, likely via recognition of intrastrand hairpins by MutSβ. It is not clear if or how MMR would affect instability of disease-causing expanded trinucleotide repeat sequences that adopt secondary structures other than hairpins, such as the triplex/R-loop forming (GAA·TTC)n sequence that causes Friedreich ataxia. We analyzed somatic instability in transgenic mice that carry an expanded (GAA·TTC)n sequence in the context of the human FXN locus and lack the individual MMR proteins Msh2, Msh6 or Pms2. The absence of Msh2 or Msh6 resulted in a dramatic reduction in somatic mutations, indicating that mammalian MMR promotes instability of the (GAA·TTC)n sequence via MutSα. The absence of Pms2 resulted in increased accumulation of large expansions in the nervous system (cerebellum, cerebrum, and dorsal root ganglia) but not in non-neuronal tissues (heart and kidney), without affecting the prevalence of contractions. Pms2 suppressed large expansions specifically in tissues showing MutSα-dependent somatic instability, suggesting that they may act on the same lesion or structure associated with the expanded (GAA·TTC)n sequence. We conclude that Pms2 specifically suppresses large expansions of a pathogenic trinucleotide repeat sequence in neuronal tissues, possibly acting independently of the canonical MMR pathway. PMID:23071719

Pms2 suppresses large expansions of the (GAA·TTC)n sequence in neuronal tissues.

PubMed

Bourn, Rebecka L; De Biase, Irene; Pinto, Ricardo Mouro; Sandi, Chiranjeevi; Al-Mahdawi, Sahar; Pook, Mark A; Bidichandani, Sanjay I

2012-01-01

Expanded trinucleotide repeat sequences are the cause of several inherited neurodegenerative diseases. Disease pathogenesis is correlated with several features of somatic instability of these sequences, including further large expansions in postmitotic tissues. The presence of somatic expansions in postmitotic tissues is consistent with DNA repair being a major determinant of somatic instability. Indeed, proteins in the mismatch repair (MMR) pathway are required for instability of the expanded (CAG·CTG)(n) sequence, likely via recognition of intrastrand hairpins by MutSβ. It is not clear if or how MMR would affect instability of disease-causing expanded trinucleotide repeat sequences that adopt secondary structures other than hairpins, such as the triplex/R-loop forming (GAA·TTC)(n) sequence that causes Friedreich ataxia. We analyzed somatic instability in transgenic mice that carry an expanded (GAA·TTC)(n) sequence in the context of the human FXN locus and lack the individual MMR proteins Msh2, Msh6 or Pms2. The absence of Msh2 or Msh6 resulted in a dramatic reduction in somatic mutations, indicating that mammalian MMR promotes instability of the (GAA·TTC)(n) sequence via MutSα. The absence of Pms2 resulted in increased accumulation of large expansions in the nervous system (cerebellum, cerebrum, and dorsal root ganglia) but not in non-neuronal tissues (heart and kidney), without affecting the prevalence of contractions. Pms2 suppressed large expansions specifically in tissues showing MutSα-dependent somatic instability, suggesting that they may act on the same lesion or structure associated with the expanded (GAA·TTC)(n) sequence. We conclude that Pms2 specifically suppresses large expansions of a pathogenic trinucleotide repeat sequence in neuronal tissues, possibly acting independently of the canonical MMR pathway.

Distinct DNA-binding surfaces in the ATPase and linker domains of MutLγ determine its substrate specificities and exert separable functions in meiotic recombination and mismatch repair

PubMed Central

2017-01-01

Mlh1-Mlh3 (MutLγ) is a mismatch repair factor with a central role in formation of meiotic crossovers, presumably through resolution of double Holliday junctions. MutLγ has DNA-binding, nuclease, and ATPase activities, but how these relate to one another and to in vivo functions are unclear. Here, we combine biochemical and genetic analyses to characterize Saccharomyces cerevisiae MutLγ. Limited proteolysis and atomic force microscopy showed that purified recombinant MutLγ undergoes ATP-driven conformational changes. In vitro, MutLγ displayed separable DNA-binding activities toward Holliday junctions (HJ) and, surprisingly, single-stranded DNA (ssDNA), which was not predicted from current models. MutLγ bound DNA cooperatively, could bind multiple substrates simultaneously, and formed higher-order complexes. FeBABE hydroxyl radical footprinting indicated that the DNA-binding interfaces of MutLγ for ssDNA and HJ substrates only partially overlap. Most contacts with HJ substrates were located in the linker regions of MutLγ, whereas ssDNA contacts mapped within linker regions as well as the N-terminal ATPase domains. Using yeast genetic assays for mismatch repair and meiotic recombination, we found that mutations within different DNA-binding surfaces exert separable effects in vivo. For example, mutations within the Mlh1 linker conferred little or no meiotic phenotype but led to mismatch repair deficiency. Interestingly, mutations in the N-terminal domain of Mlh1 caused a stronger meiotic defect than mlh1Δ, suggesting that the mutant proteins retain an activity that interferes with alternative recombination pathways. Furthermore, mlh3Δ caused more chromosome missegregation than mlh1Δ, whereas mlh1Δ but not mlh3Δ partially alleviated meiotic defects of msh5Δ mutants. These findings illustrate functional differences between Mlh1 and Mlh3 during meiosis and suggest that their absence impinges on chromosome segregation not only via reduced formation of

Mlh1-Mlh3, a Meiotic Crossover and DNA Mismatch Repair Factor, Is a Msh2-Msh3-stimulated Endonuclease*

PubMed Central

Rogacheva, Maria V.; Manhart, Carol M.; Chen, Cheng; Guarne, Alba; Surtees, Jennifer; Alani, Eric

2014-01-01

Crossing over between homologous chromosomes is initiated in meiotic prophase in most sexually reproducing organisms by the appearance of programmed double strand breaks throughout the genome. In Saccharomyces cerevisiae the double-strand breaks are resected to form three prime single-strand tails that primarily invade complementary sequences in unbroken homologs. These invasion intermediates are converted into double Holliday junctions and then resolved into crossovers that facilitate homolog segregation during Meiosis I. Work in yeast suggests that Msh4-Msh5 stabilizes invasion intermediates and double Holliday junctions, which are resolved into crossovers in steps requiring Sgs1 helicase, Exo1, and a putative endonuclease activity encoded by the DNA mismatch repair factor Mlh1-Mlh3. We purified Mlh1-Mlh3 and showed that it is a metal-dependent and Msh2-Msh3-stimulated endonuclease that makes single-strand breaks in supercoiled DNA. These observations support a direct role for an Mlh1-Mlh3 endonuclease activity in resolving recombination intermediates and in DNA mismatch repair. PMID:24403070

Mlh1-Mlh3, a meiotic crossover and DNA mismatch repair factor, is a Msh2-Msh3-stimulated endonuclease.

PubMed

Rogacheva, Maria V; Manhart, Carol M; Chen, Cheng; Guarne, Alba; Surtees, Jennifer; Alani, Eric

2014-02-28

Crossing over between homologous chromosomes is initiated in meiotic prophase in most sexually reproducing organisms by the appearance of programmed double strand breaks throughout the genome. In Saccharomyces cerevisiae the double-strand breaks are resected to form three prime single-strand tails that primarily invade complementary sequences in unbroken homologs. These invasion intermediates are converted into double Holliday junctions and then resolved into crossovers that facilitate homolog segregation during Meiosis I. Work in yeast suggests that Msh4-Msh5 stabilizes invasion intermediates and double Holliday junctions, which are resolved into crossovers in steps requiring Sgs1 helicase, Exo1, and a putative endonuclease activity encoded by the DNA mismatch repair factor Mlh1-Mlh3. We purified Mlh1-Mlh3 and showed that it is a metal-dependent and Msh2-Msh3-stimulated endonuclease that makes single-strand breaks in supercoiled DNA. These observations support a direct role for an Mlh1-Mlh3 endonuclease activity in resolving recombination intermediates and in DNA mismatch repair.

A rhodium(III) complex for high-affinity DNA base-pair mismatch recognition

PubMed Central

Junicke, Henrik; Hart, Jonathan R.; Kisko, Jennifer; Glebov, Oleg; Kirsch, Ilan R.; Barton, Jacqueline K.

2003-01-01

A rhodium(III) complex, rac-[Rh(bpy)2phzi]3+ (bpy, 2,2′-bipyridine; phzi, benzo[a]phenazine-5,6-quinone diimine) has been designed as a sterically demanding intercalator targeted to destabilized mismatched sites in double-helical DNA. The complex is readily synthesized by condensation of the phenazine quinone with the corresponding diammine complex. Upon photoactivation, the complex promotes direct strand scission at single-base mismatch sites within the DNA duplex. As with the parent mismatch-specific reagent, [Rh(bpy)2(chrysi)]3+ [chrysene-5,6-quinone diimine (chrysi)], mismatch selectivity depends on the helix destabilization associated with mispairing. Unlike the parent chrysi complex, the phzi analogue binds and cleaves with high affinity and efficiency. The specific binding constants for CA, CC, and CT mismatches within a 31-mer oligonucleotide duplex are 0.3, 1, and 6 × 107 M−1, respectively; site-specific photocleavage is evident at nanomolar concentrations. Moreover, the specificity, defined as the ratio in binding affinities for mispaired vs. well paired sites, is maintained. The increase in affinity is attributed to greater stability in the mismatched site associated with stacking by the heterocyclic aromatic ligand. The high-affinity complex is also applied in the differential cleavage of DNA obtained from cell lines deficient in mismatch repair vs. those proficient in mismatch repair. Agreement is found between photocleavage by the mismatch-specific probes and deficiency in mismatch repair. This mismatch-specific targeting, therefore, offers a potential strategy for new chemotherapeutic design. PMID:12610209

DNA mismatch repair gene MSH6 implicated in determining age at natural menopause

PubMed Central

Perry, John R.B.; Hsu, Yi-Hsiang; Chasman, Daniel I.; Johnson, Andrew D.; Elks, Cathy; Albrecht, Eva; Andrulis, Irene L.; Beesley, Jonathan; Berenson, Gerald S.; Bergmann, Sven; Bojesen, Stig E.; Bolla, Manjeet K.; Brown, Judith; Buring, Julie E.; Campbell, Harry; Chang-Claude, Jenny; Chenevix-Trench, Georgia; Corre, Tanguy; Couch, Fergus J.; Cox, Angela; Czene, Kamila; D'adamo, Adamo Pio; Davies, Gail; Deary, Ian J.; Dennis, Joe; Easton, Douglas F.; Engelhardt, Ellen G.; Eriksson, Johan G.; Esko, Tõnu; Fasching, Peter A.; Figueroa, Jonine D.; Flyger, Henrik; Fraser, Abigail; Garcia-Closas, Montse; Gasparini, Paolo; Gieger, Christian; Giles, Graham; Guenel, Pascal; Hägg, Sara; Hall, Per; Hayward, Caroline; Hopper, John; Ingelsson, Erik; Kardia, Sharon L.R.; Kasiman, Katherine; Knight, Julia A.; Lahti, Jari; Lawlor, Debbie A.; Magnusson, Patrik K.E.; Margolin, Sara; Marsh, Julie A.; Metspalu, Andres; Olson, Janet E.; Pennell, Craig E.; Polasek, Ozren; Rahman, Iffat; Ridker, Paul M.; Robino, Antonietta; Rudan, Igor; Rudolph, Anja; Salumets, Andres; Schmidt, Marjanka K.; Schoemaker, Minouk J.; Smith, Erin N.; Smith, Jennifer A.; Southey, Melissa; Stöckl, Doris; Swerdlow, Anthony J.; Thompson, Deborah J.; Truong, Therese; Ulivi, Sheila; Waldenberger, Melanie; Wang, Qin; Wild, Sarah; Wilson, James F; Wright, Alan F.; Zgaga, Lina; Ong, Ken K.; Murabito, Joanne M.; Karasik, David; Murray, Anna

2014-01-01

The length of female reproductive lifespan is associated with multiple adverse outcomes, including breast cancer, cardiovascular disease and infertility. The biological processes that govern the timing of the beginning and end of reproductive life are not well understood. Genetic variants are known to contribute to ∼50% of the variation in both age at menarche and menopause, but to date the known genes explain <15% of the genetic component. We have used genome-wide association in a bivariate meta-analysis of both traits to identify genes involved in determining reproductive lifespan. We observed significant genetic correlation between the two traits using genome-wide complex trait analysis. However, we found no robust statistical evidence for individual variants with an effect on both traits. A novel association with age at menopause was detected for a variant rs1800932 in the mismatch repair gene MSH6 (P = 1.9 × 10−9), which was also associated with altered expression levels of MSH6 mRNA in multiple tissues. This study contributes to the growing evidence that DNA repair processes play a key role in ovarian ageing and could be an important therapeutic target for infertility. PMID:24357391

Lynch syndrome-related small intestinal adenocarcinomas.

PubMed

Jun, Sun-Young; Lee, Eui-Jin; Kim, Mi-Ju; Chun, Sung Min; Bae, Young Kyung; Hong, Soon Uk; Choi, Jene; Kim, Joon Mee; Jang, Kee-Taek; Kim, Jung Yeon; Kim, Gwang Il; Jung, Soo Jin; Yoon, Ghilsuk; Hong, Seung-Mo

2017-03-28

Lynch syndrome is an autosomal-dominant disorder caused by defective DNA mismatch repair (MMR) genes and is associated with increased risk of malignancies in multiple organs. Small-intestinal adenocarcinomas are common initial manifestations of Lynch syndrome. To define the incidence and characteristics of Lynch syndrome-related small-intestinal adenocarcinomas, meticulous familial and clinical histories were obtained from 195 patients with small-intestinal adenocarcinoma, and MMR protein immunohistochemistry, microsatellite instability, MLH1 methylation, and germline mutational analyses were performed. Lynch syndrome was confirmed in eight patients (4%), all of whom had synchronous/metachronous malignancies without noticeable familial histories. Small-intestinal adenocarcinomas were the first clinical manifestation in 37% (3/8) of Lynch syndrome patients, and second malignancies developed within 5 years in 63% (5/8). The patients with accompanying Lynch syndrome were younger (≤50 years; P=0.04) and more likely to have mucinous adenocarcinomas (P=0.003), and tended to survive longer (P=0.11) than those with sporadic cases. A meticulous patient history taking, MMR protein immunolabeling, and germline MMR gene mutational analysis are important for the diagnosis of Lynch syndrome-related small-intestinal adenocarcinomas. Identifying Lynch syndrome in patients with small-intestinal adenocarcinoma can be beneficial for the early detection and treatment of additional Lynch syndrome-related cancers, especially in patients who are young or have mucinous adenocarcinomas.

Lynch syndrome-related small intestinal adenocarcinomas

PubMed Central

Jun, Sun-Young; Lee, Eui-Jin; Kim, Mi-Ju; Chun, Sung Min; Bae, Young Kyung; Hong, Soon Uk; Choi, Jene; Kim, Joon Mee; Jang, Kee-Taek; Kim, Jung Yeon; Kim, Gwang Il; Jung, Soo Jin; Yoon, Ghilsuk; Hong, Seung-Mo

2017-01-01

Lynch syndrome is an autosomal-dominant disorder caused by defective DNA mismatch repair (MMR) genes and is associated with increased risk of malignancies in multiple organs. Small-intestinal adenocarcinomas are common initial manifestations of Lynch syndrome. To define the incidence and characteristics of Lynch syndrome-related small-intestinal adenocarcinomas, meticulous familial and clinical histories were obtained from 195 patients with small-intestinal adenocarcinoma, and MMR protein immunohistochemistry, microsatellite instability, MLH1 methylation, and germline mutational analyses were performed. Lynch syndrome was confirmed in eight patients (4%), all of whom had synchronous/metachronous malignancies without noticeable familial histories. Small-intestinal adenocarcinomas were the first clinical manifestation in 37% (3/8) of Lynch syndrome patients, and second malignancies developed within 5 years in 63% (5/8). The patients with accompanying Lynch syndrome were younger (≤50 years; P=0.04) and more likely to have mucinous adenocarcinomas (P=0.003), and tended to survive longer (P=0.11) than those with sporadic cases. A meticulous patient history taking, MMR protein immunolabeling, and germline MMR gene mutational analysis are important for the diagnosis of Lynch syndrome-related small-intestinal adenocarcinomas. Identifying Lynch syndrome in patients with small-intestinal adenocarcinoma can be beneficial for the early detection and treatment of additional Lynch syndrome-related cancers, especially in patients who are young or have mucinous adenocarcinomas. PMID:28206961

Tumor testing to identify lynch syndrome in two Australian colorectal cancer cohorts.

PubMed

Buchanan, Daniel D; Clendenning, Mark; Rosty, Christophe; Eriksen, Stine V; Walsh, Michael D; Walters, Rhiannon J; Thibodeau, Stephen N; Stewart, Jenna; Preston, Susan; Win, Aung Ko; Flander, Louisa; Ouakrim, Driss Ait; Macrae, Finlay A; Boussioutas, Alex; Winship, Ingrid M; Giles, Graham G; Hopper, John L; Southey, Melissa C; English, Dallas; Jenkins, Mark A

2017-02-01

Tumor testing of colorectal cancers (CRC) for mismatch repair (MMR) deficiency is an effective approach to identify carriers of germline MMR gene mutation (Lynch syndrome). The aim of this study was to identify MMR gene mutation carriers in two cohorts of population-based CRC utilizing a combination of tumor and germline testing approaches. Colorectal cancers from 813 patients diagnosed with CRC < 60 years of age from the Australasian Colorectal Cancer Family Registry (ACCFR) and from 826 patients from the Melbourne Collaborative Cohort Study (MCCS) were tested for MMR protein expression using immunohistochemistry, microsatellite instability (MSI), BRAF V600E somatic mutation, and for MLH1 methylation. MMR gene mutation testing (Sanger sequencing and Multiplex Ligation Dependent Probe Amplification) was performed on germline DNA of patients with MMR-deficient tumors and a subset of MMR-proficient CRCs. Of the 813 ACCFR probands, 90 probands demonstrated tumor MMR deficiency (11.1%), and 42 had a MMR gene germline mutation (5.2%). For the MCCS, MMR deficiency was identified in the tumors of 103 probands (12.5%) and seven had a germline mutation (0.8%). All the mutation carriers were diagnosed prior to 70 years of age. Probands with a MMR-deficient CRC without MLH1 methylation and a gene mutation were considered Lynch-like and comprised 41.1% and 25.2% of the MMR-deficient CRCs for the ACCFR and MCCS, respectively. Identification of MMR gene mutation carriers in Australian CRC-affected patients is optimized by immunohistochemistry screening of CRC diagnosed before 70 years of age. A significant proportion of MMR-deficient CRCs will have unknown etiology (Lynch-like) proving problematic for clinical management. © 2016 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.

Systematic immunohistochemical screening for Lynch syndrome in colorectal cancer: a single centre experience of 486 patients.

PubMed

Zumstein, Valentin; Vinzens, Fabrizio; Zettl, Andreas; Heinimann, Karl; Koeberle, Dieter; von Flüe, Markus; Bolli, Martin

2016-01-01

Germline mutations in DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2 cause autosomal dominantly inherited Lynch syndrome. Lynch syndrome patients and their families benefit from life-saving intensive cancer surveillance. Approximately one in 30 colorectal cancers arises in the setting of Lynch syndrome. The aim of this study was to assess the detection rate of Lynch syndrome at our institution after introduction of systematic immunohistochemical screening for MMR deficiency in colorectal cancers from 2011 to 2015. Following the recommendations by the Evaluation of Genomic Applications in Practice and Prevention working group all colorectal cancers were immunohistochemically stained for the presence of MMR proteins MLH1, PMS2, MSH2 and MSH6, independent of clinical criteria. In the case of loss of MLH1, the somatic BRAF mutation V600E was assessed with molecular testing and/or immunohistochemistry. Clinical follow-up of potential Lynch syndrome carriers (patients with tumours showing loss of MLH1 expression with absence of BRAFV600E, loss of PMS2, MSH2 or MSH6) was evaluated. Of all patients (n = 486), loss of MMR protein expression was found in 73 (15.0%) tumours. Twenty-eight (6.0%) were classified as potential Lynch syndrome carriers. Of the genetically tested potential Lynch syndrome carriers (10 out of 28 patients), 40% were first diagnosed with Lynch syndrome. Implementation of systematic immunohistochemistry screening for Lynch syndrome showed that 6% of colorectal cancers were potentially Lynch-syndrome related. Tumour board protocols should systematically contain information on MMR status of all colorectal cancers and, in MMR deficient cases, include clear recommendations for genetic counselling for all potential Lynch syndrome patients.

Promoter methylation of MLH1, PMS2, MSH2 and p16 is a phenomenon of advanced-stage HCCs.

PubMed

Hinrichsen, Inga; Kemp, Matthias; Peveling-Oberhag, Jan; Passmann, Sandra; Plotz, Guido; Zeuzem, Stefan; Brieger, Angela

2014-01-01

Epigenetic silencing of tumour suppressor genes has been observed in various cancers. Looking at hepatocellular carcinoma (HCC) specific protein silencing was previously demonstrated to be associated with the Hepatitis C virus (HCV). However, the proposed HCV dependent promoter methylation of DNA mismatch repair (MMR) genes and thereby enhanced progression of hepatocarcinogenesis has been the subject of controversial discussion. We investigated promoter methylation pattern of the MMR genes MLH1, MSH2 and PMS2 as well as the cyclin-dependent kinase inhibitor 2A gene (p16) in 61 well characterized patients with HCCs associated with HCV, Hepatitis B virus infection or alcoholic liver disease. DNA was isolated from formalin-fixed, paraffin-embedded tumour and non-tumour adjacent tissue and analysed by methylation-specific PCR. Moreover, microsatellite analysis was performed in tissues showing methylation in MMR gene promoters. Our data demonstrated that promoter methylation of MLH1, MSH2, PMS2 and p16 is present among all considered HCCs. Hereby, promoter silencing was detectable more frequently in advanced-stage HCCs than in low-stage ones. However, there was no significant correlation between aberrant DNA methylation of MMR genes or p16 and HCV infection in related HCC specimens. In summary, we show that promoter methylation of essential MMR genes and p16 is detectable in HCCs most dominantly in pT3 stage tumour cases. Since loss of MMR proteins was previously described to be not only responsible for tumour development but also for chemotherapy resistance, the knowledge of mechanisms jointly responsible for HCC progression might enable significant improvement of individual HCC therapy in the future.

Stabilization of perfect and imperfect tandem repeats by single-strand DNA exonucleases

PubMed Central

Feschenko, Vladimir V.; Rajman, Luis A.; Lovett, Susan T.

2003-01-01

Rearrangements between tandemly repeated DNA sequences are a common source of genetic instability. Such rearrangements underlie several human genetic diseases. In many organisms, the mismatch-repair (MMR) system functions to stabilize repeats when the repeat unit is short or when sequence imperfections are present between the repeats. We show here that the action of single-stranded DNA (ssDNA) exonucleases plays an additional, important role in stabilizing tandem repeats, independent of their role in MMR. For perfect repeats of ≈100 bp in Escherichia coli that are not susceptible to MMR, exonuclease (Exo)-I, ExoX, and RecJ exonuclease redundantly inhibit deletion. Our data suggest that >90% of potential deletion events are avoided by the combined action of these three exonucleases. Imperfect tandem repeats, less prone to rearrangements, are stabilized by both the MMR-pathway and ssDNA-specific exonucleases. For 100-bp repeats containing four mispairs, ExoI alone aborts most deletion events, even in the presence of a functional MMR system. By genetic analysis, we show that the inhibitory effect of ssDNA exonucleases on deletion formation is independent of the MutS and UvrD proteins. Exonuclease degradation of DNA displaced during the deletion process may abort slipped misalignment. Exonuclease action is therefore a significant force in genetic stabilization of many forms of repetitive DNA. PMID:12538867

Clinical impact of endometrial cancer stratified by genetic mutational profiles, POLE mutation, and microsatellite instability.

PubMed

Haruma, Tomoko; Nagasaka, Takeshi; Nakamura, Keiichiro; Haraga, Junko; Nyuya, Akihiro; Nishida, Takeshi; Goel, Ajay; Masuyama, Hisashi; Hiramatsu, Yuji

2018-01-01

The molecular characterization of endometrial cancer (EC) can facilitate identification of various tumor subtypes. Although EC patients with POLE mutations reproducibly demonstrate better prognosis, the outcome of patients with microsatellite instability (MSI) remains controversial. This study attempted to interrogate whether genetic stratification of EC can identify distinct subsets with prognostic significance. A cohort of 138 EC patients who underwent surgical resection with curative intent was enrolled. Sanger sequencing was used to evaluate mutations in the POLE and KRAS genes. MSI analysis was performed using four mononucleotide repeat markers and methylation status of the MLH1 promoter was measured by a fluorescent bisulfite polymerase chain reaction (PCR). Protein expression for mismatch repair (MMR) proteins was evaluated by immunohistochemistry (IHC). Extensive hypermethylation of the MLH1 promoter was observed in 69.6% ECs with MLH1 deficiency and 3.5% with MMR proficiency, but in none of the ECs with loss of other MMR genes (P < .0001). MSI-positive and POLE mutations were found in 29.0% and 8.7% EC patients, respectively. Our MSI analysis showed a sensitivity of 92.7% for EC patients with MMR deficiency, and a specificity of 97.9% for EC patients with MMR proficiency. In univariate and multivariate analyses, POLE mutations and MSI status was significantly associated with progression-free survival (P = 0.0129 and 0.0064, respectively) but not with endometrial cancer-specific survival. This study provides significant evidence that analyses of proofreading POLE mutations and MSI status based on mononucleotide repeat markers are potentially useful biomarkers to identify EC patients with better prognosis.

Metachronous T-Lymphoblastic Lymphoma and Burkitt Lymphoma in a Child With Constitutional Mismatch Repair Deficiency Syndrome.

PubMed

Alexander, Thomas B; McGee, Rose B; Kaye, Erica C; McCarville, Mary Beth; Choi, John K; Cavender, Cary P; Nichols, Kim E; Sandlund, John T

2016-08-01

Constitutional mismatch repair deficiency (CMMRD) is a cancer predisposition syndrome associated with a high risk of developing early-onset malignancies of the blood, brain, and intestinal tract. We present the case of a patient with T-lymphoblastic lymphoma at the age of 3 years, followed by Burkitt lymphoma 10 years later. This patient also exhibited numerous nonmalignant findings including café au lait spots, lipomas, bilateral renal nodules, a nonossifying fibroma, multiple colonic adenomas, and a rapidly enlarging pilomatrixoma. The spectrum of malignant and nonmalignant neoplasms in this patient highlights the remarkable diversity, and early onset, of lesions seen in children with CMMRD. © 2016 Wiley Periodicals, Inc.

Mismatch Repair Balances Leading and Lagging Strand DNA Replication Fidelity

DTIC Science & Technology

2012-10-11

mismatched base stacks with a conserved phenylalanine in Msh6, and/or (iii) DNA flexibility, since MutSa-bound mismatched DNA is kinked, and a...AB, Watt DL , Watts BE, et al. (2010) Genome instability due to ribonucleotide incorporation into DNA. Nat Chem Biol 6: 774–781. 24. Poloumienko A

Abrogation of Microsatellite-instable Tumors Using a Highly Selective Suicide Gene/Prodrug Combination

PubMed Central

Ferrás, Cristina; Oude Vrielink, Joachim AF; Verspuy, Johan WA; te Riele, Hein; Tsaalbi-Shtylik, Anastasia; de Wind, Niels

2009-01-01

A substantial fraction of sporadic and inherited colorectal and endometrial cancers in humans is deficient in DNA mismatch repair (MMR). These cancers are characterized by length alterations in ubiquitous simple sequence repeats, a phenotype called microsatellite instability. Here we have exploited this phenotype by developing a novel approach for the highly selective gene therapy of MMR-deficient tumors. To achieve this selectivity, we mutated the VP22FCU1 suicide gene by inserting an out-of-frame microsatellite within its coding region. We show that in a significant fraction of microsatellite-instable (MSI) cells carrying the mutated suicide gene, full-length protein becomes expressed within a few cell doublings, presumably resulting from a reverting frameshift within the inserted microsatellite. Treatment of these cells with the innocuous prodrug 5-fluorocytosine (5-FC) induces strong cytotoxicity and we demonstrate that this owes to multiple bystander effects conferred by the suicide gene/prodrug combination. In a mouse model, MMR-deficient tumors that contained the out-of-frame VP22FCU1 gene displayed strong remission after treatment with 5-FC, without any obvious adverse systemic effects to the mouse. By virtue of its high selectivity and potency, this conditional enzyme/prodrug combination may hold promise for the treatment or prevention of MMR-deficient cancer in humans. PMID:19471249

Single nucleotide polymorphisms of DNA mismatch repair genes MSH2 and MLH1 confer susceptibility to esophageal cancer.

PubMed

Sun, Ming-Zhong; Ju, Hui-Xiang; Zhou, Zhong-Wei; Jin, Hao; Zhu, Rong

2014-01-01

Defects in DNA mismatch repair genes like MSH2 and MLH1 confer increased risk of cancers. Here, single nucleotide polymorphisms (SNPs) in MSH2 and MLH1 were investigated for their potential contribution to the risk of esophageal cancer. This study recruited 614 participants from Affiliated Yancheng Hospital, School of Medicine, Southeast University, of which 289 were patients with esophageal cancer, and the remainder was healthy individuals who served as a control group. Two SNPs, MSH2 c.2063T>G and MLH1 IVS14-19A>G, were genotyped using PCR-RFLP. Statistical analysis was performed using chi-square test and logistic regression analysis. Carriers of the MSH2 c.2063G allele were at significantly higher risk for esophageal cancer compared to individuals with the TT genotype [OR = 3.36, 95% confidence interval (CI): 1.18-11.03]. The MLH1 IVS14-19A>G allele also conferred significantly increased (1.70-fold) for esophageal cancer compared to the AA genotype (OR = 1.70, 95% CI: 1.13-5.06). Further, the variant alleles interacted such that individuals with the susceptible genotypes at both MSH2 and MLH1 had a significantly exacerbated risk for esophageal cancer (OR = 12.38, 95% CI: 3.09-63.11). In brief, SNPs in the DNA mismatch repair genes MSH2 and MLH1 increase the risk of esophageal cancer. Molecular investigations are needed to uncover the mechanism behind their interaction effect.

Reduction of MLH1 and PMS2 confers temozolomide resistance and is associated with recurrence of glioblastoma.

PubMed

Shinsato, Yoshinari; Furukawa, Tatsuhiko; Yunoue, Shunji; Yonezawa, Hajime; Minami, Kentarou; Nishizawa, Yukihiko; Ikeda, Ryuji; Kawahara, Kohichi; Yamamoto, Masatatsu; Hirano, Hirofumi; Tokimura, Hiroshi; Arita, Kazunori

2013-12-01

Although there is a relationship between DNA repair deficiency and temozolomide (TMZ) resistance in glioblastoma (GBM), it remains unclear which molecule is associated with GBM recurrence. We isolated three TMZ-resistant human GBM cell lines and examined the expression of O6-methylguanine-DNA methyltransferase (MGMT) and mismatch repair (MMR) components. We used immunohistochemical analysis to compare MutL homolog 1 (MLH1), postmeiotic segregation increased 2 (PMS2) and MGMT expression in primary and recurrent GBM specimens obtained from GBM patients during TMZ treatment. We found a reduction in MLH1 expression and a subsequent reduction in PMS2 protein levels in TMZ-resistant cells. Furthermore, MLH1 or PMS2 knockdown confered TMZ resistance. In recurrent GBM tumours, the expression of MLH1 and PMS2 was reduced when compared to primary tumours.

Guidelines for surveillance of individuals with constitutional mismatch repair-deficiency proposed by the European Consortium "Care for CMMR-D" (C4CMMR-D).

PubMed

Vasen, H F A; Ghorbanoghli, Z; Bourdeaut, F; Cabaret, O; Caron, O; Duval, A; Entz-Werle, N; Goldberg, Y; Ilencikova, D; Kratz, C P; Lavoine, N; Loeffen, J; Menko, F H; Muleris, M; Sebille, G; Colas, C; Burkhardt, B; Brugieres, L; Wimmer, K

2014-05-01

Lynch syndrome (LS) is an autosomal dominant disorder caused by a defect in one of the DNA mismatch repair genes: MLH1, MSH2, MSH6 and PMS2. In the last 15 years, an increasing number of patients have been described with biallelic mismatch repair gene mutations causing a syndrome referred to as 'constitutional mismatch repair-deficiency' (CMMR-D). The spectrum of cancers observed in this syndrome differs from that found in LS, as about half develop brain tumours, around half develop digestive tract cancers and a third develop haematological malignancies. Brain tumours and haematological malignancies are mainly diagnosed in the first decade of life, and colorectal cancer (CRC) and small bowel cancer in the second and third decades of life. Surveillance for CRC in patients with LS is very effective. Therefore, an important question is whether surveillance for the most common CMMR-D-associated cancers will also be effective. Recently, a new European consortium was established with the aim of improving care for patients with CMMR-D. At a workshop of this group held in Paris in June 2013, one of the issues addressed was the development of surveillance guidelines. In 1968, criteria were proposed by WHO that should be met prior to the implementation of screening programmes. These criteria were used to assess surveillance in CMMR-D. The evaluation showed that surveillance for CRC is the only part of the programme that largely complies with the WHO criteria. The values of all other suggested screening protocols are unknown. In particular, it is questionable whether surveillance for haematological malignancies improves the already favourable outcome for patients with these tumours. Based on the available knowledge and the discussions at the workshop, the European consortium proposed a surveillance protocol. Prospective collection of all results of the surveillance is needed to evaluate the effectiveness of the programme.

MMR vaccination status of children exempted from school-entry immunization mandates.

PubMed

Buttenheim, Alison M; Sethuraman, Karthik; Omer, Saad B; Hanlon, Alexandra L; Levy, Michael Z; Salmon, Daniel

2015-11-17

Child immunizations are one of the most successful public health interventions of the past century. Still, parental vaccine hesitancy is widespread and increasing. One manifestation of this are rising rates of nonmedical or "personal beliefs" exemptions (PBEs) from school-entry immunization mandates. Exemptions have been shown to be associated with increased risk of disease outbreak, but the strength of this association depends critically on the true vaccination status of exempted children, which has not been assessed. To estimate the true measles-mumps-rubella (MMR) vaccination status of children with PBEs. We use administrative data collected by the California Department of Public Health in 2009 and imputation to estimate the MMR vaccination status of children with PBEs under varying scenarios. Results from 2009 surveillance data indicate MMR1/MMR2 coverage of 18-47% among children with PBEs at typical schools and 11-34% among children with PBEs at schools with high PBE rates. Imputation scenarios point to much higher coverage (64-92% for MMR1 and 25-58% for MMR2 at typical schools; 49-90% for MMR1 and 16-63% for MMR2 at high PBE schools) but still below levels needed to maintain herd immunity against measles. These coverage estimates suggest that prior analyses of the relative risk of measles associated with vaccine refusal underestimate that risk by an order of magnitude of 2-10 times. Copyright © 2015 Elsevier Ltd. All rights reserved.

MMR vaccination status of children exempted from school-entry immunization mandates

PubMed Central

Sethuraman, Karthik; Omer, Saad B.; Hanlon, Alexandra L.; Levy, Michael Z.; Salmon, Daniel

2015-01-01

BACKGROUND Child immunizations are one of the most successful public health interventions of the past century. Still, parental vaccine hesitancy is widespread and increasing. One manifestation of this are rising rates of nonmedical or “personal beliefs” exemptions (PBEs) from school-entry immunization mandates. Exemptions have been shown to be associated with increased risk of disease outbreak, but the strength of this association depends critically on the true vaccination status of exempted children, which has not been assessed. OBJECTIVE To estimate the true measles-mumps-rubella (MMR) vaccination status of children with PBEs. METHODS We use administrative data collected by the California Department of Public Health in 2009 and imputation to estimate the MMR vaccination status of children with PBEs under varying scenarios. RESULTS Results from 2009 surveillance data indicate MMR1/MMR2 coverage of 18–47% among children with PBEs at typical schools and 11–34% among children with PBEs at schools with high PBE rates. Imputation scenarios point to much higher coverage (64–92% for MMR1 and 25–58% for MMR2 at typical schools; 49–90% for MMR1 and 16–63% for MMR2 at high PBE schools) but still below levels needed to maintain herd immunity against measles. CONCLUSIONS These coverage estimates suggest that prior analyses of the relative risk of measles associated with vaccine refusal underestimate that risk by an order of magnitude of 2–10 times. PMID:26431991

Tolerance of DNA Mismatches in Dmc1 Recombinase-mediated DNA Strand Exchange*

PubMed Central

Borgogno, María V.; Monti, Mariela R.; Zhao, Weixing; Sung, Patrick; Argaraña, Carlos E.; Pezza, Roberto J.

2016-01-01

Recombination between homologous chromosomes is required for the faithful meiotic segregation of chromosomes and leads to the generation of genetic diversity. The conserved meiosis-specific Dmc1 recombinase catalyzes homologous recombination triggered by DNA double strand breaks through the exchange of parental DNA sequences. Although providing an efficient rate of DNA strand exchange between polymorphic alleles, Dmc1 must also guard against recombination between divergent sequences. How DNA mismatches affect Dmc1-mediated DNA strand exchange is not understood. We have used fluorescence resonance energy transfer to study the mechanism of Dmc1-mediated strand exchange between DNA oligonucleotides with different degrees of heterology. The efficiency of strand exchange is highly sensitive to the location, type, and distribution of mismatches. Mismatches near the 3′ end of the initiating DNA strand have a small effect, whereas most mismatches near the 5′ end impede strand exchange dramatically. The Hop2-Mnd1 protein complex stimulates Dmc1-catalyzed strand exchange on homologous DNA or containing a single mismatch. We observed that Dmc1 can reject divergent DNA sequences while bypassing a few mismatches in the DNA sequence. Our findings have important implications in understanding meiotic recombination. First, Dmc1 acts as an initial barrier for heterologous recombination, with the mismatch repair system providing a second level of proofreading, to ensure that ectopic sequences are not recombined. Second, Dmc1 stepping over infrequent mismatches is likely critical for allowing recombination between the polymorphic sequences of homologous chromosomes, thus contributing to gene conversion and genetic diversity. PMID:26709229

Dynamics of spontaneous flipping of a mismatched base in DNA duplex.

PubMed

Yin, Yandong; Yang, Lijiang; Zheng, Guanqun; Gu, Chan; Yi, Chengqi; He, Chuan; Gao, Yi Qin; Zhao, Xin Sheng

2014-06-03

DNA base flipping is a fundamental theme in DNA biophysics. The dynamics for a B-DNA base to spontaneously flip out of the double helix has significant implications in various DNA-protein interactions but are still poorly understood. The spontaneous base-flipping rate obtained previously via the imino proton exchange assay is most likely the rate of base wobbling instead of flipping. Using the diffusion-decelerated fluorescence correlation spectroscopy together with molecular dynamics simulations, we show that a base of a single mismatched base pair (T-G, T-T, or T-C) in a double-stranded DNA can spontaneously flip out of the DNA duplex. The extrahelical lifetimes are on the order of 10 ms, whereas the intrahelical lifetimes range from 0.3 to 20 s depending on the stability of the base pairs. These findings provide detailed understanding on the dynamics of DNA base flipping and lay down foundation to fully understand how exactly the repair proteins search and locate the target mismatched base among a vast excess of matched DNA bases.

Severity of mumps disease is related to MMR vaccination status and viral shedding.

PubMed

Gouma, Sigrid; Hahné, Susan J M; Gijselaar, Daphne B; Koopmans, Marion P G; van Binnendijk, Rob S

2016-04-07

During recent years, various mumps outbreaks have occurred among measles, mumps, and rubella (MMR) vaccinated persons in various countries worldwide, including the Netherlands. We studied mumps virus shedding in MMR vaccinated and unvaccinated mumps patients and related these findings to clinical data. In this study, we included 1112 mumps patients of whom diagnostic samples were tested positive in our laboratory between 1 January 2007 and 31 December 2014. We compared mumps virus shedding and severity of disease between patients who had received 2 doses of MMR (n=592) and unvaccinated mumps patients (n=195). Mumps virus shedding in saliva and urine specimens was measured by qPCR. Severity of disease was studied in a subset of patients with clinical data available. Mumps patients who had received 2 MMR doses shed less often mumps virus in their urine than unvaccinated patients. Salivary viral loads were higher at day of onset of disease in twice MMR vaccinated patients with viruria than in twice MMR vaccinated patients without viruria. However, salivary viral loads did not significantly differ between patients who had received 2 MMR doses and unvaccinated patients. Bilateral parotitis and orchitis were less often reported in patients who had received 2 MMR doses than in unvaccinated patients. Furthermore, the prevalence of bilateral parotitis and orchitis was higher among twice MMR vaccinated patients with viruria than among twice MMR vaccinated patients without viruria. MMR vaccination was associated with less severe disease among mumps patients. Systemic spread of virus was associated with more severe disease. The elevated salivary viral loads in patients with systemic mumps disease suggest that these patients pose a higher risk for mumps virus transmission. Our study contributes to the understanding of mumps virus pathogenesis and shows the protective effect of MMR vaccination on severity of disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

An Unusual Case of Constitutional Mismatch Repair Deficiency Syndrome With Anaplastic Ganglioglioma, Colonic Adenocarcinoma, Osteosarcoma, Acute Myeloid Leukemia, and Signs of Neurofibromatosis Type 1: Case Report.

PubMed

Daou, Badih; Zanello, Marc; Varlet, Pascale; Brugieres, Laurence; Jabbour, Pascal; Caron, Olivier; Lavoine, Noémie; Dhermain, Frederic; Willekens, Christophe; Beuvon, Frederic; Malka, David; Lechapt-Zalcmann, Emmanuèle; Abi Lahoud, Georges

2015-07-01

Constitutional mismatch repair deficiency (CMMRD) syndrome is a disorder with recessive inheritance caused by biallelic mismatch repair gene mutations, in which mismatch repair defects are inherited from both parents. This syndrome is associated with multiple cancers occurring in childhood. The most common tumors observed with CMMRD include brain tumors, digestive tract tumors, and hematological malignancies. The aim of this study was to report new phenotypic expressions of CMMRD syndrome and add new insight to the existing knowledge about this disease. A review of the literature was conducted and recommendation for surveillance and follow-up in patients with CMMRD are proposed. We report for the first time in the literature, the case of a 22-year-old female patient who was diagnosed with CMMRD syndrome, with the development of 2 unusual tumors: an anaplastic ganglioglioma and an osteosarcoma. She presented initially with an anaplastic ganglioglioma and later developed several malignancies including colonic adenocarcinoma, osteosarcoma, and acute myeloid leukemia. The patient had an atypical course of her disease with development of the initial malignancy at an older age and a remarkably long survival period despite developing aggressive tumors. Many aspects of this disease are still unknown. We identified a case of CMMRD in a patient presenting with an anaplastic ganglioglioma, who underwent successful surgical resection, chemotherapy, and radiotherapy and has had one of the longest survival periods known with this disease. This case broadens the tumor spectrum observed with CMMRD syndrome with anaplastic ganglioglioma and osteosarcoma as new phenotypic expressions of this genetic defect.

Lynch syndrome and Lynch syndrome mimics: The growing complex landscape of hereditary colon cancer

PubMed Central

Carethers, John M; Stoffel, Elena M

2015-01-01

Hereditary non-polyposis colorectal cancer (HNPCC) was previously synonymous with Lynch syndrome; however, identification of the role of germline mutations in the DNA mismatch repair (MMR) genes has made it possible to differentiate Lynch syndrome from other conditions associated with familial colorectal cancer (CRC). Broadly, HNPCC may be dichotomized into conditions that demonstrate defective DNA MMR and microsatellite instability (MSI) vs those conditions that demonstrate intact DNA MMR. Conditions characterized by MMR deficient CRCs include Lynch syndrome (germline MMR mutation), Lynch-like syndrome (biallelic somatic MMR mutations), constitutional MMR deficiency syndrome (biallelic germline MMR mutations), and sporadic MSI CRC (somatic biallelic methylation of MLH1). HNPCC conditions with intact DNA MMR associated with familial CRC include polymerase proofreading associated polyposis and familial colorectal cancer type X. Although next generation sequencing technologies have elucidated the genetic cause for some HNPCC conditions, others remain genetically undefined. Differentiating between Lynch syndrome and the other HNPCC disorders has profound implications for cancer risk assessment and surveillance of affected patients and their at-risk relatives. Clinical suspicion coupled with molecular tumor analysis and testing for germline mutations can help differentiate the clinical mimicry within HNPCC and facilitate diagnosis and management. PMID:26309352

Lynch syndrome and Lynch syndrome mimics: The growing complex landscape of hereditary colon cancer.

PubMed

Carethers, John M; Stoffel, Elena M

2015-08-21

Hereditary non-polyposis colorectal cancer (HNPCC) was previously synonymous with Lynch syndrome; however, identification of the role of germline mutations in the DNA mismatch repair (MMR) genes has made it possible to differentiate Lynch syndrome from other conditions associated with familial colorectal cancer (CRC). Broadly, HNPCC may be dichotomized into conditions that demonstrate defective DNA MMR and microsatellite instability (MSI) vs those conditions that demonstrate intact DNA MMR. Conditions characterized by MMR deficient CRCs include Lynch syndrome (germline MMR mutation), Lynch-like syndrome (biallelic somatic MMR mutations), constitutional MMR deficiency syndrome (biallelic germline MMR mutations), and sporadic MSI CRC (somatic biallelic methylation of MLH1). HNPCC conditions with intact DNA MMR associated with familial CRC include polymerase proofreading associated polyposis and familial colorectal cancer type X. Although next generation sequencing technologies have elucidated the genetic cause for some HNPCC conditions, others remain genetically undefined. Differentiating between Lynch syndrome and the other HNPCC disorders has profound implications for cancer risk assessment and surveillance of affected patients and their at-risk relatives. Clinical suspicion coupled with molecular tumor analysis and testing for germline mutations can help differentiate the clinical mimicry within HNPCC and facilitate diagnosis and management.

In Silico Systems Biology Analysis of Variants of Uncertain Significance in Lynch Syndrome Supports the Prioritization of Functional Molecular Validation.

PubMed

Borras, Ester; Chang, Kyle; Pande, Mala; Cuddy, Amanda; Bosch, Jennifer L; Bannon, Sarah A; Mork, Maureen E; Rodriguez-Bigas, Miguel A; Taggart, Melissa W; Lynch, Patrick M; You, Y Nancy; Vilar, Eduardo

2017-10-01

Lynch syndrome (LS) is a genetic condition secondary to germline alterations in the DNA mismatch repair (MMR) genes with 30% of changes being variants of uncertain significance (VUS). Our aim was to perform an in silico reclassification of VUS from a large single institutional cohort that will help prioritizing functional validation. A total of 54 VUS were detected with 33 (61%) novel variants. We integrated family history, pathology, and genetic information along with supporting evidence from eight different in silico tools at the RNA and protein level. Our assessment allowed us to reclassify 54% (29/54) of the VUS as probably damaging, 13% (7/54) as possibly damaging, and 28% (15/54) as probably neutral. There are more than 1,000 VUS reported in MMR genes and our approach facilitates the prioritization of further functional efforts to assess the pathogenicity to those classified as probably damaging. Cancer Prev Res; 10(10); 580-7. ©2017 AACR . ©2017 American Association for Cancer Research.

Uncertainties in the Management of a Lynch Syndrome Patient: A Case Report

PubMed Central

Campos, Sara; Amaro, Pedro; Cunha, Inês; Fraga, João; Cipriano, Maria Augusta; Tomé, Luís

2017-01-01

Introduction Lynch syndrome (LS), the most common hereditary colorectal cancer syndrome, is characterized by mutations in mismatch repair (MMR) genes leading to an increased cancer risk, namely colorectal cancer. Case In the context of surveillance colonoscopy, a 40-mm flat lesion (0-IIa+b, Paris classification) was identified and submitted to piecemeal mucosal endoscopic resection in a 64-year-old LS patient with an MLH1 germline mutation (262delATC) and two previous segmental resections due to metachronous colorectal cancer. Pathology raised the suspicion of superficial submucosal invasive carcinoma with poor differentiation. Immunochemistry showed heterogeneous MLH1 expression and PMS2 loss. In a short-term follow-up colonoscopy, another 30-mm advanced carcinoma was identified. The patient was referred to surgery. Conclusion This case raises several issues: (1) the potentially fast tumorigenesis and progression to carcinoma in LS and implications for endoscopic screening and surveillance; (2) pitfalls in the interpretation of MMR proteins immunochemistry; (3) the role of endoscopic resection in LS. PMID:29255760

Role for cER and Mmr1p in anchorage of mitochondria at sites of polarized surface growth in budding yeast.

PubMed

Swayne, Theresa C; Zhou, Chun; Boldogh, Istvan R; Charalel, Joseph K; McFaline-Figueroa, José Ricardo; Thoms, Sven; Yang, Christine; Leung, Galen; McInnes, Joseph; Erdmann, Ralf; Pon, Liza A

2011-12-06

Mitochondria accumulate at neuronal and immunological synapses and yeast bud tips and associate with the ER during phospholipid biosynthesis, calcium homeostasis, and mitochondrial fission. Here we show that mitochondria are associated with cortical ER (cER) sheets underlying the plasma membrane in the bud tip and confirm that a deletion in YPT11, which inhibits cER accumulation in the bud tip, also inhibits bud tip anchorage of mitochondria. Time-lapse imaging reveals that mitochondria are anchored at specific sites in the bud tip. Mmr1p, a member of the DSL1 family of tethering proteins, localizes to punctate structures on opposing surfaces of mitochondria and cER sheets underlying the bud tip and is recovered with isolated mitochondria and ER. Deletion of MMR1 impairs bud tip anchorage of mitochondria without affecting mitochondrial velocity or cER distribution. Deletion of the phosphatase PTC1 results in increased Mmr1p phosphorylation, mislocalization of Mmr1p, defects in association of Mmr1p with mitochondria and ER, and defects in bud tip anchorage of mitochondria. These findings indicate that Mmr1p contributes to mitochondrial inheritance as a mediator of anchorage of mitochondria to cER sheets in the yeast bud tip and that Ptc1p regulates Mmr1p phosphorylation, localization, and function. Copyright © 2011 Elsevier Ltd. All rights reserved.

Natural mismatch repair mutations mediate phenotypic diversity and drug resistance in Cryptococcus deuterogattii.

PubMed

Billmyre, R Blake; Clancey, Shelly Applen; Heitman, Joseph

2017-09-26

Pathogenic microbes confront an evolutionary conflict between the pressure to maintain genome stability and the need to adapt to mounting external stresses. Bacteria often respond with elevated mutation rates, but little evidence exists of stable eukaryotic hypermutators in nature. Whole genome resequencing of the human fungal pathogen Cryptococcus deuterogattii identified an outbreak lineage characterized by a nonsense mutation in the mismatch repair component MSH2. This defect results in a moderate mutation rate increase in typical genes, and a larger increase in genes containing homopolymer runs. This allows facile inactivation of genes with coding homopolymer runs including FRR1 , which encodes the target of the immunosuppresive antifungal drugs FK506 and rapamycin. Our study identifies a eukaryotic hypermutator lineage spread over two continents and suggests that pathogenic eukaryotic microbes may experience similar selection pressures on mutation rate as bacterial pathogens, particularly during long periods of clonal growth or while expanding into new environments.

Complexes of mismatched and complementary DNA with minor groove binders. Structures at nucleotide resolution via an improved hydroxyl radical cleavage methodology

PubMed Central

Bialonska, Dobroslawa; Song, Kenneth; Bolton, Philip H.

2011-01-01

Tumor cell lines can replicate faster than normal cells and many also have defective DNA repair pathways. This has lead to the investigation of the inhibition of DNA repair proteins as a means of therapeutic intervention. An alternative approach is to hide or mask damaged DNA from the repair systems. We have developed a protocol to investigate the structures of the complexes of damaged DNA with drug like molecules. Nucleotide resolution structural information can be obtained using an improved hydroxyl radical cleavage protocol. The use of a dTn tail increases the length of the smallest fragments of interest and allows efficient co-precipitation of the fragments with poly(A). The use of a fluorescent label, on the 5′ end of the dTn tail, in conjunction with modified cleavage reaction conditions, avoids the lifetime and other problems with 32P labeling. The structures of duplex DNAs containing AC and CC mismatches in the presence and absence of minor groove binders have been investigated as have those of the fully complementary DNA. The results indicate that the structural perturbations of the mismatches are localized, are sequence dependent and that the presence of a mismatch can alter the binding of drug like molecules. PMID:21893212

Complexes of mismatched and complementary DNA with minor groove binders. Structures at nucleotide resolution via an improved hydroxyl radical cleavage methodology.

PubMed

Bialonska, Dobroslawa; Song, Kenneth; Bolton, Philip H

2011-11-27

Tumor cell lines can replicate faster than normal cells and many also have defective DNA repair pathways. This has lead to the investigation of the inhibition of DNA repair proteins as a means of therapeutic intervention. An alternative approach is to hide or mask damaged DNA from the repair systems. We have developed a protocol to investigate the structures of the complexes of damaged DNA with drug like molecules. Nucleotide resolution structural information can be obtained using an improved hydroxyl radical cleavage protocol. The use of a dT(n) tail increases the length of the smallest fragments of interest and allows efficient co-precipitation of the fragments with poly(A). The use of a fluorescent label, on the 5' end of the dT(n) tail, in conjunction with modified cleavage reaction conditions, avoids the lifetime and other problems with (32)P labeling. The structures of duplex DNAs containing AC and CC mismatches in the presence and absence of minor groove binders have been investigated as have those of the fully complementary DNA. The results indicate that the structural perturbations of the mismatches are localized, are sequence dependent and that the presence of a mismatch can alter the binding of drug like molecules. Copyright © 2011 Elsevier B.V. All rights reserved.

A homozygous PMS2 founder mutation with an attenuated constitutional mismatch repair deficiency phenotype.

PubMed

Li, Lili; Hamel, Nancy; Baker, Kristi; McGuffin, Michael J; Couillard, Martin; Gologan, Adrian; Marcus, Victoria A; Chodirker, Bernard; Chudley, Albert; Stefanovici, Camelia; Durandy, Anne; Hegele, Robert A; Feng, Bing-Jian; Goldgar, David E; Zhu, Jun; De Rosa, Marina; Gruber, Stephen B; Wimmer, Katharina; Young, Barbara; Chong, George; Tischkowitz, Marc D; Foulkes, William D

2015-05-01

Inherited mutations in DNA mismatch repair genes predispose to different cancer syndromes depending on whether they are mono-allelic or bi-allelic. This supports a causal relationship between expression level in the germline and phenotype variation. As a model to study this relationship, our study aimed to define the pathogenic characteristics of a recurrent homozygous coding variant in PMS2 displaying an attenuated phenotype identified by clinical genetic testing in seven Inuit families from Northern Quebec. Pathogenic characteristics of the PMS2 mutation NM_000535.5:c.2002A>G were studied using genotype-phenotype correlation, single-molecule expression detection and single genome microsatellite instability analysis. This PMS2 mutation generates a de novo splice site that competes with the authentic site. In homozygotes, expression of the full-length protein is reduced to a level barely detectable by conventional diagnostics. Median age at primary cancer diagnosis is 22 years among 13 NM_000535.5:c.2002A>G homozygotes, versus 8 years in individuals carrying bi-allelic truncating mutations. Residual expression of full-length PMS2 transcript was detected in normal tissues from homozygotes with cancers in their 20s. Our genotype-phenotype study of c.2002A>G illustrates that an extremely low level of PMS2 expression likely delays cancer onset, a feature that could be exploited in cancer preventive intervention. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Tolerance of DNA Mismatches in Dmc1 Recombinase-mediated DNA Strand Exchange.

PubMed

Borgogno, María V; Monti, Mariela R; Zhao, Weixing; Sung, Patrick; Argaraña, Carlos E; Pezza, Roberto J

2016-03-04

Recombination between homologous chromosomes is required for the faithful meiotic segregation of chromosomes and leads to the generation of genetic diversity. The conserved meiosis-specific Dmc1 recombinase catalyzes homologous recombination triggered by DNA double strand breaks through the exchange of parental DNA sequences. Although providing an efficient rate of DNA strand exchange between polymorphic alleles, Dmc1 must also guard against recombination between divergent sequences. How DNA mismatches affect Dmc1-mediated DNA strand exchange is not understood. We have used fluorescence resonance energy transfer to study the mechanism of Dmc1-mediated strand exchange between DNA oligonucleotides with different degrees of heterology. The efficiency of strand exchange is highly sensitive to the location, type, and distribution of mismatches. Mismatches near the 3' end of the initiating DNA strand have a small effect, whereas most mismatches near the 5' end impede strand exchange dramatically. The Hop2-Mnd1 protein complex stimulates Dmc1-catalyzed strand exchange on homologous DNA or containing a single mismatch. We observed that Dmc1 can reject divergent DNA sequences while bypassing a few mismatches in the DNA sequence. Our findings have important implications in understanding meiotic recombination. First, Dmc1 acts as an initial barrier for heterologous recombination, with the mismatch repair system providing a second level of proofreading, to ensure that ectopic sequences are not recombined. Second, Dmc1 stepping over infrequent mismatches is likely critical for allowing recombination between the polymorphic sequences of homologous chromosomes, thus contributing to gene conversion and genetic diversity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

DNA mismatch repair complex MutSβ promotes GAA·TTC repeat expansion in human cells.

PubMed

Halabi, Anasheh; Ditch, Scott; Wang, Jeffrey; Grabczyk, Ed

2012-08-24

While DNA repair has been implicated in CAG·CTG repeat expansion, its role in the GAA·TTC expansion of Friedreich ataxia (FRDA) is less clear. We have developed a human cellular model that recapitulates the DNA repeat expansion found in FRDA patient tissues. In this model, GAA·TTC repeats expand incrementally and continuously. We have previously shown that the expansion rate is linked to transcription within the repeats. Our working hypothesis is that structures formed within the GAA·TTC repeat during transcription attract DNA repair enzymes that then facilitate the expansion process. MutSβ, a heterodimer of MSH2 and MSH3, is known to have a role in CAG·CTG repeat expansion. We now show that shRNA knockdown of either MSH2 or MSH3 slowed GAA·TTC expansion in our system. We further characterized the role of MutSβ in GAA·TTC expansion using a functional assay in primary FRDA patient-derived fibroblasts. These fibroblasts have no known propensity for instability in their native state. Ectopic expression of MSH2 and MSH3 induced GAA·TTC repeat expansion in the native FXN gene. MSH2 is central to mismatch repair and its absence or reduction causes a predisposition to cancer. Thus, despite its essential role in GAA·TTC expansion, MSH2 is not an attractive therapeutic target. The absence or reduction of MSH3 is not strongly associated with cancer predisposition. Accordingly, MSH3 has been suggested as a therapeutic target for CAG·CTG repeat expansion disorders. Our results suggest that MSH3 may also serve as a therapeutic target to slow the expansion of GAA·TTC repeats in the future.

DNA Mismatch Repair Complex MutSβ Promotes GAA·TTC Repeat Expansion in Human Cells*

PubMed Central

Halabi, Anasheh; Ditch, Scott; Wang, Jeffrey; Grabczyk, Ed

2012-01-01

While DNA repair has been implicated in CAG·CTG repeat expansion, its role in the GAA·TTC expansion of Friedreich ataxia (FRDA) is less clear. We have developed a human cellular model that recapitulates the DNA repeat expansion found in FRDA patient tissues. In this model, GAA·TTC repeats expand incrementally and continuously. We have previously shown that the expansion rate is linked to transcription within the repeats. Our working hypothesis is that structures formed within the GAA·TTC repeat during transcription attract DNA repair enzymes that then facilitate the expansion process. MutSβ, a heterodimer of MSH2 and MSH3, is known to have a role in CAG·CTG repeat expansion. We now show that shRNA knockdown of either MSH2 or MSH3 slowed GAA·TTC expansion in our system. We further characterized the role of MutSβ in GAA·TTC expansion using a functional assay in primary FRDA patient-derived fibroblasts. These fibroblasts have no known propensity for instability in their native state. Ectopic expression of MSH2 and MSH3 induced GAA·TTC repeat expansion in the native FXN gene. MSH2 is central to mismatch repair and its absence or reduction causes a predisposition to cancer. Thus, despite its essential role in GAA·TTC expansion, MSH2 is not an attractive therapeutic target. The absence or reduction of MSH3 is not strongly associated with cancer predisposition. Accordingly, MSH3 has been suggested as a therapeutic target for CAG·CTG repeat expansion disorders. Our results suggest that MSH3 may also serve as a therapeutic target to slow the expansion of GAA·TTC repeats in the future. PMID:22787155

Intralesional tuberculin (PPD) versus measles, mumps, rubella (MMR) vaccine in treatment of multiple warts: a comparative clinical and immunological study.

PubMed

Shaheen, Maha Adel; Salem, Samar Abdallah M; Fouad, Dina Adel; El-Fatah, Abeer Aly Abd

2015-01-01

Intralesional purified protein derivative (PPD) or mumps, measles, rubella (MMR) were not previously compared regarding their efficacy or mechanism of action in treatment of warts. We aimed to compare their efficacy in treatment of multiple warts and investigate their effect on serum interleukin (IL)-4 and IL-12. Thirty patients with multiple warts were included (10 treated with PPD, 10 with MMR, and 10 with normal saline (control)). Injection was done every 3 weeks until clearance or maximum of three treatments. Clinical response of target and distant warts was evaluated. Serum ILs-4 and -12 were assessed before and after treatment. A significantly higher rate of complete response was found in target and distant warts with PPD (60% each) and MMR (80%, 40%, respectively) compared with controls (0%), with no significant difference between both treatments. After treatment, the control group showed the lowest serum IL-12 and IL-4 levels compared with the MMR- and PPD-treated groups with statistically significant difference in between. MMR resulted in a significantly higher serum IL-12 than PPD. With PPD, IL-4 was increased with statistically significant change compared with pretreat-ment level. Intralesional PPD and MMR show comparable efficacy and safety in treatment of multiple warts. Serum ILs-4 and-12 increase following antigen injection. © 2015 Wiley Periodicals, Inc.

Dissociating Long and Short-term Memory in Three-Month-Old Infants Using the Mismatch Response to Voice Stimuli.

PubMed

Zinke, Katharina; Thöne, Leonie; Bolinger, Elaina M; Born, Jan

2018-01-01

Auditory event-related potentials (ERPs) have been successfully used in adults as well as in newborns to discriminate recall of longer-term and shorter-term memories. Specifically the Mismatch Response (MMR) to deviant stimuli of an oddball paradigm is larger if the deviant stimuli are highly familiar (i.e., retrieved from long-term memory) than if they are unfamiliar, representing an immediate change to the standard stimuli kept in short-term memory. Here, we aimed to extend previous findings indicating a differential MMR to familiar and unfamiliar deviants in newborns (Beauchemin et al., 2011), to 3-month-old infants who are starting to interact more with their social surroundings supposedly based on forming more (social) long-term representations. Using a voice discrimination paradigm, each infant was repeatedly presented with the word "baby" (400 ms, interstimulus interval: 600 ms, 10 min overall duration) pronounced by three different female speakers. One voice that was unfamiliar to the infants served as the frequently presented "standard" stimulus, whereas another unfamiliar voice served as the "unfamiliar deviant" stimulus, and the voice of the infant's mother served as the "familiar deviant." Data collection was successful for 31 infants (mean age = 100 days). The MMR was determined by the difference between the ERP to standard stimuli and the ERP to the unfamiliar and familiar deviant, respectively. The MMR to the familiar deviant (mother's voice) was larger, i.e., more positive, than that to the unfamiliar deviant between 100 and 400 ms post-stimulus over the frontal and central cortex. However, a genuine MMR differentiating, as a positive deflection, between ERPs to familiar deviants and standard stimuli was only found in the 300-400 ms interval. On the other hand, a genuine MMR differentiating, as a negative deflection, between ERPs to unfamiliar deviants from ERPs to standard stimuli was revealed for the 200-300 ms post-stimulus interval. Overall

Dissociating Long and Short-term Memory in Three-Month-Old Infants Using the Mismatch Response to Voice Stimuli

PubMed Central

Zinke, Katharina; Thöne, Leonie; Bolinger, Elaina M.; Born, Jan

2018-01-01

Auditory event-related potentials (ERPs) have been successfully used in adults as well as in newborns to discriminate recall of longer-term and shorter-term memories. Specifically the Mismatch Response (MMR) to deviant stimuli of an oddball paradigm is larger if the deviant stimuli are highly familiar (i.e., retrieved from long-term memory) than if they are unfamiliar, representing an immediate change to the standard stimuli kept in short-term memory. Here, we aimed to extend previous findings indicating a differential MMR to familiar and unfamiliar deviants in newborns (Beauchemin et al., 2011), to 3-month-old infants who are starting to interact more with their social surroundings supposedly based on forming more (social) long-term representations. Using a voice discrimination paradigm, each infant was repeatedly presented with the word “baby” (400 ms, interstimulus interval: 600 ms, 10 min overall duration) pronounced by three different female speakers. One voice that was unfamiliar to the infants served as the frequently presented “standard” stimulus, whereas another unfamiliar voice served as the “unfamiliar deviant” stimulus, and the voice of the infant’s mother served as the “familiar deviant.” Data collection was successful for 31 infants (mean age = 100 days). The MMR was determined by the difference between the ERP to standard stimuli and the ERP to the unfamiliar and familiar deviant, respectively. The MMR to the familiar deviant (mother’s voice) was larger, i.e., more positive, than that to the unfamiliar deviant between 100 and 400 ms post-stimulus over the frontal and central cortex. However, a genuine MMR differentiating, as a positive deflection, between ERPs to familiar deviants and standard stimuli was only found in the 300–400 ms interval. On the other hand, a genuine MMR differentiating, as a negative deflection, between ERPs to unfamiliar deviants from ERPs to standard stimuli was revealed for the 200–300 ms post

Reduction of MLH1 and PMS2 confers temozolomide resistance and is associated with recurrence of glioblastoma

PubMed Central

Shinsato, Yoshinari; Furukawa, Tatsuhiko; Yunoue, Shunji; Yonezawa, Hajime; Minami, Kentarou; Nishizawa, Yukihiko; Ikeda, Ryuji; Kawahara, Kohichi; Yamamoto, Masatatsu; Hirano, Hirofumi; Tokimura, Hiroshi; Arita, Kazunori

2013-01-01

Although there is a relationship between DNA repair deficiency and temozolomide (TMZ) resistance in glioblastoma (GBM), it remains unclear which molecule is associated with GBM recurrence. We isolated three TMZ-resistant human GBM cell lines and examined the expression of O6-methylguanine-DNA methyltransferase (MGMT) and mismatch repair (MMR) components. We used immunohistochemical analysis to compare MutL homolog 1 (MLH1), postmeiotic segregation increased 2 (PMS2) and MGMT expression in primary and recurrent GBM specimens obtained from GBM patients during TMZ treatment. We found a reduction in MLH1 expression and a subsequent reduction in PMS2 protein levels in TMZ-resistant cells. Furthermore, MLH1 or PMS2 knockdown confered TMZ resistance. In recurrent GBM tumours, the expression of MLH1 and PMS2 was reduced when compared to primary tumours. PMID:24259277

Detailed characterization of MLH1 p.D41H and p.N710D variants coexisting in a Lynch syndrome family with conserved MLH1 expression tumors.

PubMed

Pineda, M; González-Acosta, M; Thompson, B A; Sánchez, R; Gómez, C; Martínez-López, J; Perea, J; Caldés, T; Rodríguez, Y; Landolfi, S; Balmaña, J; Lázaro, C; Robles, L; Capellá, G; Rueda, D

2015-06-01

Lynch syndrome (LS) is an autosomal dominant cancer-susceptibility disease caused by inactivating germline mutations in mismatch repair (MMR) genes. Variants of unknown significance (VUS) are often detected in mutational analysis of MMR genes. Here we describe a large family fulfilling Amsterdam I criteria carrying two rare VUS in the MLH1 gene: c.121G > C (p.D41H) and c.2128A > G (p.N710D). Collection of clinico-pathological data, multifactorial analysis, in silico predictions, and functional analyses were used to elucidate the clinical significance of the identified MLH1 VUS. Only the c.121G > C variant cosegregated with LS-associated tumors in the family. Diagnosed colorectal tumors were microsatellite unstable although immunohistochemical staining revealed no loss of MMR proteins expression. Multifactorial likelihood analysis classified c.2128A > G as a non-pathogenic variant and c.121G > C as pathogenic. In vitro functional tests revealed impaired MMR activity and diminished expression of c.121G > C. Accordingly, the N710 residue is located in the unconserved MLH1 C-terminal domain, whereas D41 is highly conserved and located in the ATPase domain. The obtained results will enable adequate genetic counseling of c.121G > C and c.2128A > G variant carriers and their families. Furthermore, they exemplify how cumulative data and comprehensive analyses are mandatory to refine the classification of MMR variants. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The key role of CYC2 during meiosis in Tetrahymena thermophila.

PubMed

Xu, Qianlan; Wang, Ruoyu; Ghanam, A R; Yan, Guanxiong; Miao, Wei; Song, Xiaoyuan

2016-04-01

Meiotic recombination is carried out through a specialized pathway for the formation and repair of DNA double-strand breaks (DSBs) made by the Spo11 protein. The present study shed light on the functional role of cyclin, CYC2, in Tetrahymena thermophila which has transcriptionally high expression level during meiosis process. Knocking out the CYC2 gene results in arrest of meiotic conjugation process at 2.5-3.5 h after conjugation initiation, before the meiosis division starts, and in company with the absence of DSBs. To investigate the underlying mechanism of this phenomenon, a complete transcriptome profile was performed between wild-type strain and CYC2 knock-out strain. Functional analysis of RNA-Seq results identifies related differentially expressed genes (DEGs) including SPO11 and these DEGs are enriched in DNA repair/mismatch repair (MMR) terms in homologous recombination (HR), which indicates that CYC2 could play a crucial role in meiosis by regulating SPO11 and participating in HR.

MMR: marginalised, misrepresented and rejected? Autism: a focus group study

PubMed Central

Hilton, Shona; Hunt, Kate; Petticrew, Mark

2007-01-01

Objective To explore how the measles, mumps, and rubella (MMR) vaccine controversy impacted on the lives of parents caring for children with autism. Design Qualitative focus group study. Setting United Kingdom. Patients A purposively selected sample of 38 parents took part in 10 focus group discussions between March 2003 and May 2005. Results Many parents felt that the MMR vaccine could be too potent for children who are susceptible to developing autism. Of the parents whose children received the MMR vaccine, many felt guilty that they may have caused or contributed to their child's autism. Some parents felt frustrated by health professionals' lack of understanding of the negative impact the MMR controversy has had on them. Some parents were anxious about subsequent MMR decision‐making for their children. Conclusions The controversy has had a negative impact on some parents of children with autism. This has implications for health professionals, who need to be particularly aware of the issues these parents face in future MMR decision‐making for their affected child and younger siblings. It is anticipated that these findings will raise awareness among health professionals of the difficulties faced by such parents. More generally, there is a need to promote a greater awareness of the important role health visitors can play in parental decision‐making and for research examining whether health professionals feel they receive sufficient training in communication skills. It is also essential that the latest scientific research findings are disseminated quickly to these parents and to those health professionals advising parents on matters of vaccine safety. PMID:17376937

Mutsβ generates both expansions and contractions in a mouse model of the Fragile X-associated disorders

PubMed Central

Zhao, Xiao-Nan; Kumari, Daman; Gupta, Shikha; Wu, Di; Evanitsky, Maya; Yang, Wei; Usdin, Karen

2015-01-01

Fragile X-associated disorders are Repeat Expansion Diseases that result from expansion of a CGG/CCG-repeat in the FMR1 gene. Contractions of the repeat tract also occur, albeit at lower frequency. However, these contractions can potentially modulate disease symptoms or generate an allele with repeat numbers in the normal range. Little is known about the expansion mechanism and even less about contractions. We have previously demonstrated that the mismatch repair (MMR) protein MSH2 is required for expansions in a mouse model of these disorders. Here, we show that MSH3, the MSH2-binding partner in the MutSβ complex, is required for 98% of germ line expansions and all somatic expansions in this model. In addition, we provide evidence for two different contraction mechanisms that operate in the mouse model, a MutSβ-independent one that generates small contractions and a MutSβ-dependent one that generates larger ones. We also show that MutSβ complexes formed with the repeats have altered kinetics of ATP hydrolysis relative to complexes with bona fide MMR substrates and that MutSβ increases the stability of the CCG-hairpins at physiological temperatures. These data may have important implications for our understanding of the mechanism(s) of repeat instability and for the role of MMR proteins in this process. PMID:26420841

MMR (measles, mumps, and rubella) vaccine - what you need to know

MedlinePlus

Centers for Disease Control and Prevention website. MMR (measles, mumps, and rubella) vaccine. www.cdc.gov/vaccines/hcp/vis/vis-statements/mmr.html . Updated February 12, 2018. Accessed February 14, 2018.

Genetic Analysis of mlh3 Mutations Reveals Interactions Between Crossover Promoting Factors During Meiosis in Baker’s Yeast

PubMed Central

Brown, Megan Sonntag; Lim, Elisha; Chen, Cheng; Nishant, K. T.; Alani, Eric

2013-01-01

Crossing over between homologous chromosomes occurs during the prophase of meiosis I and is critical for chromosome segregation. In baker’s yeast, two heterodimeric complexes, Msh4-Msh5 and Mlh1-Mlh3, act in meiosis to promote interference-dependent crossing over. Mlh1-Mlh3 also plays a role in DNA mismatch repair (MMR) by interacting with Msh2-Msh3 to repair insertion and deletion mutations. Mlh3 contains an ATP-binding domain that is highly conserved among MLH proteins. To explore roles for Mlh3 in meiosis and MMR, we performed a structure−function analysis of eight mlh3 ATPase mutants. In contrast to previous work, our data suggest that ATP hydrolysis by both Mlh1 and Mlh3 is important for both meiotic and MMR functions. In meiotic assays, these mutants showed a roughly linear relationship between spore viability and genetic map distance. To further understand the relationship between crossing over and meiotic viability, we analyzed crossing over on four chromosomes of varying lengths in mlh3Δ mms4Δ strains and observed strong decreases (6- to 17-fold) in crossing over in all intervals. Curiously, mlh3Δ mms4Δ double mutants displayed spore viability levels that were greater than observed in mms4Δ strains that show modest defects in crossing over. The viability in double mutants also appeared greater than would be expected for strains that show such severe defects in crossing over. Together, these observations provide insights for how Mlh1-Mlh3 acts in crossover resolution and MMR and for how chromosome segregation in Meiosis I can occur in the absence of crossing over. PMID:23316435

The web and public confidence in MMR vaccination in Italy.

PubMed

Aquino, Francesco; Donzelli, Gabriele; De Franco, Emanuela; Privitera, Gaetano; Lopalco, Pier Luigi; Carducci, Annalaura

2017-08-16

Measles, mumps and rubella (MMR) vaccination coverage in Italy has been decreasing starting from 2012 and, at the present, none of the Italian regions has achieved the goal of 95% coverage target. A decision of the Court of Justice of Rimini in March 2012 that awarded vaccine-injury compensation for a case of autism has been indicated as a probable trigger event leading to a reduction of vaccine confidence in Italy. The aim of the study was to explore the relationship between MMR vaccination coverage to online search trends and social network activity on the topic "autism and MMR vaccine", during the period 2010-2015. A significant inverse correlation was found between MMR vaccination coverage and Internet search activity, tweets and Facebook posts. New media might have played a role in spreading misinformation. Media monitoring could be useful to assess the level of vaccine hesitancy and to plan and target effective information campaigns. Copyright © 2017 Elsevier Ltd. All rights reserved.

MSH3 protein expression and nodal status in MLH1-deficient colorectal cancers.

PubMed

Laghi, Luigi; Bianchi, Paolo; Delconte, Gabriele; Celesti, Giuseppe; Di Caro, Giuseppe; Pedroni, Monica; Chiaravalli, Anna Maria; Jung, Barbara; Capella, Carlo; de Leon, Maurizio Ponz; Malesci, Alberto

2012-06-01

Patients with colorectal cancers (CRC) and high microsatellite instability (MSI) have a better outcome than their chromosome-unstable counterpart. Given the heterogeneity of microsatellite-unstable CRCs, we wanted to see whether any MSI-associated molecular features are specifically associated with prognosis. One hundred and nine MSI-high CRCs were typed for primary mismatch repair (MMR) defect and for secondary loss of MMR proteins. Frameshifts at seven target genes, mutations in the RAS pathway, and methylation at MLH1/CDKN2A promoters were also searched. The interplay of molecular findings with clinicopathologic features and patient survival was analyzed. Of 84 MLH1-deficient CRCs, 31 (36.9%) had MSH3 and 11 (13.1%) had MSH6 loss (P < 0.001), biallelic frameshift mutations at mononucleotide repeats accounting for most (78%) MSH3 losses. As compared with MSH3-retaining cancers, MLH1-deficient tumors with MSH3 loss showed a higher number of mutated target genes (3.94 ± 1.56 vs. 2.79 ± 1.75; P = 0.001), absence of nodal involvement at pathology [N0; OR, 0.11; 95% confidence interval (CI), 0.04-0.43, P < 0.001], and better disease-free survival (P = 0.06). No prognostic value was observed for KRAS status and for MLH1/CDKN2A promoter methylation. The association between MSH3 loss and N0 was confirmed in an independent cohort of 71 MLH1-deficient CRCs (OR, 0.23; 95% CI, 0.06-0.83, P = 0.02). MLH1-deficient CRCs not expressing MSH3 have a more severe MSI, a lower rate of nodal involvement, and a better postsurgical outcome.

Inflammation-associated microsatellite alterations: Mechanisms and significance in the prognosis of patients with colorectal cancer.

PubMed

Koi, Minoru; Tseng-Rogenski, Stephanie S; Carethers, John M

2018-01-15

Microsatellite alterations within genomic DNA frameshift as a result of defective DNA mismatch repair (MMR). About 15% of sporadic colorectal cancers (CRCs) manifest hypermethylation of the DNA MMR gene MLH1 , resulting in mono- and di-nucleotide frameshifts to classify it as microsatellite instability-high (MSI-H) and hypermutated, and due to frameshifts at coding microsatellites generating neo-antigens, produce a robust protective immune response that can be enhanced with immune checkpoint blockade. More commonly, approximately 50% of sporadic non-MSI-H CRCs demonstrate frameshifts at di- and tetra-nucleotide microsatellites to classify it as MSI-low/elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) as a result of functional somatic inactivation of the DNA MMR protein MSH3 via a nuclear-to-cytosolic displacement. The trigger for MSH3 displacement appears to be inflammation and/or oxidative stress, and unlike MSI-H CRC patients, patients with MSI-L/EMAST CRCs show poor prognosis. These inflammatory-associated microsatellite alterations are a consequence of the local tumor microenvironment, and in theory, if the microenvironment is manipulated to lower inflammation, the microsatellite alterations and MSH3 dysfunction should be corrected. Here we describe the mechanisms and significance of inflammatory-associated microsatellite alterations, and propose three areas to deeply explore the consequences and prevention of inflammation's effect upon the DNA MMR system.

Inflammation-associated microsatellite alterations: Mechanisms and significance in the prognosis of patients with colorectal cancer

PubMed Central

Koi, Minoru; Tseng-Rogenski, Stephanie S; Carethers, John M

2018-01-01

Microsatellite alterations within genomic DNA frameshift as a result of defective DNA mismatch repair (MMR). About 15% of sporadic colorectal cancers (CRCs) manifest hypermethylation of the DNA MMR gene MLH1, resulting in mono- and di-nucleotide frameshifts to classify it as microsatellite instability-high (MSI-H) and hypermutated, and due to frameshifts at coding microsatellites generating neo-antigens, produce a robust protective immune response that can be enhanced with immune checkpoint blockade. More commonly, approximately 50% of sporadic non-MSI-H CRCs demonstrate frameshifts at di- and tetra-nucleotide microsatellites to classify it as MSI-low/elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) as a result of functional somatic inactivation of the DNA MMR protein MSH3 via a nuclear-to-cytosolic displacement. The trigger for MSH3 displacement appears to be inflammation and/or oxidative stress, and unlike MSI-H CRC patients, patients with MSI-L/EMAST CRCs show poor prognosis. These inflammatory-associated microsatellite alterations are a consequence of the local tumor microenvironment, and in theory, if the microenvironment is manipulated to lower inflammation, the microsatellite alterations and MSH3 dysfunction should be corrected. Here we describe the mechanisms and significance of inflammatory-associated microsatellite alterations, and propose three areas to deeply explore the consequences and prevention of inflammation’s effect upon the DNA MMR system. PMID:29375743

Lynch syndrome in the 21st century: clinical perspectives.

PubMed

Tiwari, A K; Roy, H K; Lynch, H T

2016-03-01

Lynch syndrome (LS) is the most common of all inherited cancer syndromes, associated with substantially elevated risks for colonic and extracolonic malignancies, earlier onset and high rates of multiple primary cancers. At the genetic level, it is caused by a defective mismatch repair (MMR) system due to presence of germline defects in at least one of the MMR genes- MLH1, MSH2, MSH6, PMS2 or EPCAM. An impaired MMR function during replication introduces infidelity in DNA sequence and leads to ubiquitous mutations at simple repetitive sequences (microsatellites), causing microsatellite instability (MSI). Although previously, clinicopathological criteria such as Amsterdam I/II and Revised Bethesda Guidelines were commonly used to identify suspected LS mutation carriers, there has been a recent push towards universally testing, especially in case of colorectal cancers (CRCs), through immunohistochemistry for expression of MMR proteins or through molecular tests (polymerase chain reaction, PCR) for MSI, in order to identify LS mutation carriers and subject them to genetic testing to ascertain the specific gene implicated. In this review, we have discussed the latest diagnostic strategies and the current screening and treatment guidelines for colonic and extracolonic cancers in clinically affected and at-risk individuals for LS. © The Author 2015. Published by Oxford University Press on behalf of the Association of Physicians. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Using a multi-feature paradigm to measure mismatch responses to minimal sound contrasts in children with cochlear implants and hearing aids.

PubMed

Uhlén, Inger; Engström, Elisabet; Kallioinen, Petter; Nakeva von Mentzer, Cecilia; Lyxell, Björn; Sahlén, Birgitta; Lindgren, Magnus; Ors, Marianne

2017-10-01

Our aim was to explore whether a multi-feature paradigm (Optimum-1) for eliciting mismatch negativity (MMN) would objectively capture difficulties in perceiving small sound contrasts in children with hearing impairment (HI) listening through their hearing aids (HAs) and/or cochlear implants (CIs). Children aged 5-7 years with HAs, CIs and children with normal hearing (NH) were tested in a free-field setting using a multi-feature paradigm with deviations in pitch, intensity, gap, duration, and location. There were significant mismatch responses across all subjects that were positive (p-MMR) for the gap and pitch deviants (F(1,43) = 5.17, p = 0.028 and F(1,43) = 6.56, p = 0.014, respectively) and negative (MMN) for the duration deviant (F(1,43) = 4.74, p = 0.035). Only the intensity deviant showed a significant group interaction with MMN in the HA group and p-MMR in the CI group (F(2,43) = 3.40, p = 0.043). The p-MMR correlated negatively with age, with the strongest correlation in the NH subjects. In the CI group, the late discriminative negativity (LDN) was replaced by a late positivity with a significant group interaction for the location deviant. Children with severe HI can be assessed through their hearing device with a fast multi-feature paradigm. For further studies a multi-feature paradigm including more complex speech sounds may better capture variation in auditory processing in these children. © 2017 Scandinavian Psychological Associations and John Wiley & Sons Ltd.

Randomised cluster trial to support informed parental decision-making for the MMR vaccine

PubMed Central

2011-01-01

Background In the UK public concern about the safety of the combined measles, mumps and rubella [MMR] vaccine continues to impact on MMR coverage. Whilst the sharp decline in uptake has begun to level out, first and second dose uptake rates remain short of that required for population immunity. Furthermore, international research consistently shows that some parents lack confidence in making a decision about MMR vaccination for their children. Together, this work suggests that effective interventions are required to support parents to make informed decisions about MMR. This trial assessed the impact of a parent-centred, multi-component intervention (balanced information, group discussion, coaching exercise) on informed parental decision-making for MMR. Methods This was a two arm, cluster randomised trial. One hundred and forty two UK parents of children eligible for MMR vaccination were recruited from six primary healthcare centres and six childcare organisations. The intervention arm received an MMR information leaflet and participated in the intervention (parent meeting). The control arm received the leaflet only. The primary outcome was decisional conflict. Secondary outcomes were actual and intended MMR choice, knowledge, attitude, concern and necessity beliefs about MMR and anxiety. Results Decisional conflict decreased for both arms to a level where an 'effective' MMR decision could be made one-week (effect estimate = -0.54, p < 0.001) and three-months (effect estimate = -0.60, p < 0.001) post-intervention. There was no significant difference between arms (effect estimate = 0.07, p = 0.215). Heightened decisional conflict was evident for parents making the MMR decision for their first child (effect estimate = -0.25, p = 0.003), who were concerned (effect estimate = 0.07, p < 0.001), had less positive attitudes (effect estimate = -0.20, p < 0.001) yet stronger intentions (effect estimate = 0.09, p = 0.006). Significantly more parents in the intervention arm

Constitutional mismatch repair-deficiency syndrome presenting as colonic adenomatous polyposis: clues from the skin.

PubMed

Jasperson, K W; Samowitz, W S; Burt, R W

2011-10-01

Constitutional mismatch repair-deficiency (CMMR-D) syndrome is an autosomal recessive condition characterized by hematologic malignancies, brain tumors, Lynch syndrome-associated cancers and skin manifestations reminiscent of neurofibromatosis type 1 (NF1). In contrast to Lynch syndrome, CMMR-D syndrome is exceptionally rare, onset typically occurs in infancy or early childhood and, as described in this report, may also present with colonic polyposis suggestive of attenuated familial adenomatous polyposis (AFAP) or MUTYH associated polyposis (MAP). Here we describe two sisters with CMMR-D syndrome due to germline bi-allelic MSH6 mutations. Both sisters are without cancer, are older than typical for this condition, have NF1 associated features and a colonic phenotype suspicious for an attenuated polyposis syndrome. This report highlights the role of skin examinations in leading to an underlying genetic diagnosis in individuals with colonic adenomatous polyposis, but without mutations associated with AFAP or MAP. © 2010 John Wiley & Sons A/S.

Pancreatic non-functioning neuroendocrine tumor: a new entity genetically related to Lynch syndrome

PubMed Central

Serracant Barrera, Anna; Serra Pla, Sheila; Blázquez Maña, Carmen María; Salas, Rubén Carrera; García Monforte, Neus; Bejarano González, Natalia; Romaguera Monzonis, Andreu; Andreu Navarro, Francisco Javier; Bella Cueto, Maria Rosa

2017-01-01

Some pancreatic neuroendocrine tumors (P-NETs) are associated with hereditary syndromes. An association between Lynch syndrome (LS) and P-NETs has been suggested, however it has not been confirmed to date. We describe the first case associating LS and P-NETs. Here we report a 65-year-old woman who in the past 20 years presented two colorectal carcinomas (CRC) endometrial carcinoma (EC), infiltrating ductal breast carcinoma, small intestine adenocarcinoma, two non-functioning P-NETs and sebomatricoma. With the exception of one P-NET, all these conditions were associated with LS, as confirmed by immunohistochemistry (IHC) and polymerase chain reaction (PCR). LS is caused by a mutation of a mismatch repair (MMR) gene which leads to a loss of expression of its protein. CRC is the most common tumor, followed by EC. Pancreatic tumors have also been associated with LS. Diagnosis of LS is based on clinical criteria (Amsterdam II and Bethesda) and genetic study (MMR gene mutation). The association between LS and our patient’s tumors was confirmed by IHC (loss of expression of proteins MLH1 and its dimer PMS2) and the detection of microsatellite instability (MSI) using PCR. PMID:29184699