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Sample records for mismatch-repair mmr proteins

  1. Mismatch repair proteins: key regulators of genetic recombination.

    PubMed

    Surtees, J A; Argueso, J L; Alani, E

    2004-01-01

    Mismatch repair (MMR) systems are central to maintaining genome stability in prokaryotes and eukaryotes. MMR proteins play a fundamental role in avoiding mutations, primarily by removing misincorporation errors that occur during DNA replication. MMR proteins also act during genetic recombination in steps that include repairing mismatches in heteroduplex DNA, modulating meiotic crossover control, removing 3' non-homologous tails during double-strand break repair, and preventing recombination between divergent sequences. In this review we will, first, discuss roles for MMR proteins in repairing mismatches that occur during recombination, particularly during meiosis. We will also explore how studying this process has helped to refine models of double-strand break repair, and particularly to our understanding of gene conversion gradients. Second, we will examine the role of MMR proteins in repressing homeologous recombination, i.e. recombination between divergent sequences. We will also compare the requirements for MMR proteins in preventing homeologous recombination to the requirements for these proteins in mismatch repair.

  2. Mismatch repair system proteins in oral benign and malignant lesions.

    PubMed

    Amaral-Silva, Gleyson Kleber do; Martins, Manoela Domingues; Pontes, Hélder Antônio Rebelo; Fregnani, Eduardo Rodrigues; Lopes, Márcio Ajudarte; Fonseca, Felipe Paiva; Vargas, Pablo Agustin

    2017-04-01

    Different environmental agents may cause DNA mutations by disrupting its double-strand structure; however, even normal DNA polymerase function may synthesize mismatch nucleotide bases, occasionally demonstrating failure in its proofreading activity. To overcome this issue, mismatch repair (MMR) system, a group of proteins specialized in finding mispairing bases and small loops of insertion or deletion, works to avoid the occurrence of mutations that could ultimately lead to innumerous human diseases. In the last decades, the role of MMR proteins in oral carcinogenesis and in the development of other oral cavity neoplasms has grown, but their importance in the pathogenesis and their prognostic potential for patients affected by oral malignancies, especially oral squamous cell carcinoma (OSCC), remain unclear. Therefore, in this manuscript we aimed to review and critically discuss the currently available data on MMR proteins expression in oral potentially malignant lesions, in OSCC, and in other oral neoplasms to better understand their relevance in these lesions.

  3. Flanking sequence specificity determines coding microsatellite heteroduplex and mutation rates with defective DNA mismatch repair (MMR).

    PubMed

    Chung, H; Lopez, C G; Young, D J; Lai, J F; Holmstrom, J; Ream-Robinson, D; Cabrera, B L; Carethers, J M

    2010-04-15

    The activin type II receptor (ACVR2) contains two identical microsatellites in exons 3 and 10, but only the exon 10 microsatellite is frameshifted in mismatch repair (MMR)-defective colonic tumors. The reason for this selectivity is not known. We hypothesized that ACVR2 frameshifts were influenced by DNA sequences surrounding the microsatellite. We constructed plasmids in which exons 3 or 10 of ACVR2 were cloned +1 bp out of frame of enhanced green fluorescent protein (EGFP), allowing -1 bp frameshift to express EGFP. Plasmids were stably transfected into MMR-deficient cells, and subsequent non-fluorescent cells were sorted, cultured and harvested for mutation analysis. We swapped DNA sequences flanking the exon 3 and 10 microsatellites to test our hypothesis. Native ACVR2 exon 3 and 10 microsatellites underwent heteroduplex formation (A(7)/T(8)) in hMLH1(-/-) cells, but only exon 10 microsatellites fully mutated (A(7)/T(7)) in both hMLH1(-/-) and hMSH6(-/-) backgrounds, showing selectivity for exon 10 frameshifts and inability of exon 3 heteroduplexes to fully mutate. Substituting nucleotides flanking the exon 3 microsatellite for nucleotides flanking the exon 10 microsatellite significantly reduced heteroduplex and full mutation in hMLH1(-/-) cells. When the exon 3 microsatellite was flanked by nucleotides normally surrounding the exon 10 microsatellite, fully mutant exon 3 frameshifts appeared. Mutation selectivity for ACVR2 lies partly with flanking nucleotides surrounding each microsatellite.

  4. Mismatch repair.

    PubMed

    Fishel, Richard

    2015-10-30

    Highly conserved MutS homologs (MSH) and MutL homologs (MLH/PMS) are the fundamental components of mismatch repair (MMR). After decades of debate, it appears clear that the MSH proteins initiate MMR by recognizing a mismatch and forming multiple extremely stable ATP-bound sliding clamps that diffuse without hydrolysis along the adjacent DNA. The function(s) of MLH/PMS proteins is less clear, although they too bind ATP and are targeted to MMR by MSH sliding clamps. Structural analysis combined with recent real-time single molecule and cellular imaging technologies are providing new and detailed insight into the thermal-driven motions that animate the complete MMR mechanism.

  5. Mismatch Repair Proteins Are Activators of Toxic Responses to Chromium-DNA Damage

    PubMed Central

    Peterson-Roth, Elizabeth; Reynolds, Mindy; Quievryn, George; Zhitkovich, Anatoly

    2005-01-01

    Chromium(VI) is a toxic and carcinogenic metal that causes the formation of DNA phosphate-based adducts. Cr-DNA adducts are genotoxic in human cells, although they do not block replication in vitro. Here, we report that induction of cytotoxicity in Cr(VI)-treated human colon cells and mouse embryonic fibroblasts requires the presence of all major mismatch repair (MMR) proteins. Cr-DNA adducts lost their ability to block replication of Cr-modified plasmids in human colon cells lacking MLH1 protein. The presence of functional mismatch repair caused induction of p53-independent apoptosis associated with activation of caspases 2 and 7. Processing of Cr-DNA damage by mismatch repair resulted in the extensive formation of γ-H2AX foci in G2 phase, indicating generation of double-stranded breaks as secondary toxic lesions. Induction of γ-H2AX foci was observed at 6 to 12 h postexposure, which was followed by activation of apoptosis in the absence of significant G2 arrest. Our results demonstrate that mismatch repair system triggers toxic responses to Cr-DNA backbone modifications through stress mechanisms that are significantly different from those for other forms of DNA damage. Selection for Cr(VI) resistant, MMR-deficient cells may explain the very high frequency of lung cancers with microsatellite instability among chromate workers. PMID:15831465

  6. 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.

  7. Screening for Muir-Torre syndrome using mismatch repair protein immunohistochemistry of sebaceous neoplasms.

    PubMed

    Roberts, Maegan E; Riegert-Johnson, Douglas L; Thomas, Brittany C; Thomas, Colleen S; Heckman, Michael G; Krishna, Murli; DiCaudo, David J; Bridges, Alina G; Hunt, Katherine S; Rumilla, Kandelaria M; Cappel, Mark A

    2013-06-01

    Screening for the Muir-Torre variant of Lynch Syndrome (LS) using Mismatch Repair (MMR) gene immunohistochemistry (IHC) on sebaceous neoplasms (SNs) is technically feasible. To date, research into the clinical utility of MMR IHC for this indication is limited. We conducted a retrospective chart review of 90 patients with MMR IHC completed on at least one SN from January 2005 to May 2010. SNs included were adenomas, epitheliomas, carcinomas and basal and squamous cell carcinomas with sebaceous differentiation. Of the 90 patients, 13 (14 %) had genetically confirmed or fulfilled clinical criteria for a diagnosis of MTS and 51 patients (57 %) presented with an abnormal MMR IHC result (loss of one or more MMR proteins) on at least one SN. Abnormal IHC had a sensitivity of 85 %, specificity of 48 %, positive predictive value (PPV) of 22 % and negative predictive value (NPV) of 95 % when evaluating for MTS. When personal or family history of colorectal cancer (≥2 family members with a history of colorectal cancer) was taken into consideration, ignoring IHC results, sensitivity was 92 %, specificity was 99 %, PPV was 92 % and NPV was 99 %. MMR IHC on SNs when used to screen for MTS has poor diagnostic utility. We recommend that MMR IHC not be performed routinely on SNs when the patient does not have either personal or family history of colorectal cancer.

  8. Frequent Mismatch Repair Protein Deficiency in Mixed Endometrioid and Clear Cell Carcinoma of the Endometrium.

    PubMed

    Köbel, Martin; Tessier-Cloutier, Basile; Leo, Joyce; Hoang, Lien N; Gilks, C Blake; Soslow, Robert A; Delair, Deborah; Stewart, Colin J R; Lee, Cheng-Han

    2017-01-20

    Mixed endometrioid and clear cell carcinoma of the endometrium refers to a scenario in which the tumor exhibits histologic features of both endometrioid and clear cell carcinoma. We observed a tendency for these tumors to occur in a mismatch repair (MMR) protein-deficient molecular background in a prior study that examined a small cohort of mixed-type endometrial carcinomas. The aim of this study was to determine the rate of MMR protein deficiency in a larger series of endometrial mixed endometrioid and clear cell carcinomas, through a retrospective survey of MLH1, PMS2, MSH2, and MSH6 expression in such tumors at 5 tertiary centers. A total of 41 cases were identified and 27 (66%) tumors demonstrated MMR protein deficiency with a comparable frequency across the contributing centers (ranging from 56% to 83%). Among the MMR protein-deficient cases, 59% showed concurrent MLH1 and PMS2 loss, 33% showed concurrent MSH2 and MSH6 loss, and 4% showed isolated PMS2 or MSH6 loss. Compared with a previously published series of 15 pure endometrial clear cell carcinomas, mixed endometrioid and clear cell carcinomas are associated with significantly better disease-specific survival (P=0.02). In summary, endometrial carcinomas with mixed endometrioid and clear cell histology are frequently MMR protein deficient. This finding has implications both for our understanding of its tumor biology and for the identification of patients with potential Lynch syndrome.

  9. [Correlation between mismatch repair proteins status and clinicopathological characteristics in sporadic colorectal cancer patients].

    PubMed

    Xiao, Z T; Zhang, R X; Zhao, Y; Peng, J H; Lu, S X; Zhang, H Z; Ding, P R; Wu, X J; Lu, Z H; Li, L R; Wan, D S; Pan, Z Z; Chen, G

    2017-04-25

    Objective: To explore the expression of mismatch repair (MMR) proteins in sporadic colorectal cancer (SCRC) patients, and its association with clinicopathological characteristics of SCRC. Methods: Patients with histologically confirmed colorectal cancer were consecutively recruited between December 2011 and June 2015 at Sun Yat-sen University Cancer Center. The exclusion criteria included multiple primary colorectal tumors, hereditary colorectal cancer (including Lynch syndrome, familial adenomatous polyposis), and the patients without the MMR proteins status tested. A total of 2 684 patients were included. Correlations of MMR proteins status and patients' demographics (including gender, age), tumor characteristics (site and differentiation) and TNM staging (excluding 315 SCRC patients receiving neoadjuvant therapy) were investigated. Results: The percentage of deficient MMR (dMMR) in these SCRC patients was 10.2%, and that of proficient MMR (pMMR) was 89.8%. The dMMR was more likely to be detected in younger (≤59 old years) SCRC patients compared to the elderly (>59 years) [12.7%(179/1 406)vs 7.5%(96/1 278), P<0.001]. The dMMR rate in right colon cancer was significantly higher than that in left colon cancer and rectal cancer [22.7%(151/664)vs 7.2%(69/956)vs 5.2%(55/1 064), P<0.001]. Among the various pathological types of SCRC, mucinous adenocarcinoma showed the highest rate of dMMR (24.4%), and neuroendocrine carcinoma the lowest rate of dMMR (0) (P<0.001). In addition, the proportions of dMMR in stage Ⅰ, stage Ⅱ, stage Ⅲ and stage Ⅳ SCRC were 9.7%, 16.5%, 8.5%, and 3.9%, respectively (P<0.001). There is no significant difference in the proportion of dMMR between male and female (11.0% vs 9.1%, P=0.114). Conclusion: dMMR status may be most likely to exist in younger (≤59 years) patients with stage Ⅱ right colon mucinous adenocarcinoma among SCRC.

  10. Sebaceous neoplasms with mismatch repair protein expressions and the frequency of co-existing visceral tumors.

    PubMed

    Lee, Bonnie A; Yu, Limin; Ma, Linglei; Lind, Anne C; Lu, Dongsi

    2012-12-01

    Visceral malignancy has been associated with sebaceous neoplasms in patients with Muir-Torre syndrome. However, no large studies have been done to evaluate the frequency of visceral tumors in patients with sebaceous neoplasms and mismatch repair (MMR) protein expression of the sebaceous tumors. We sought to determine the frequency of visceral tumors in patients with sebaceous neoplasms, MMR protein expression of the sebaceous tumors, and the related surveillance practices of physicians. We identified 85 patients with sebaceous neoplasms. Relevant clinical information was obtained via chart review and database searches. MMR protein expression was examined by immunohistochemistry. Nineteen of the 85 patients had a total of 22 visceral malignancies, of which 41% were genitourinary in origin. Ten of the 17 patients (59%) with visceral malignancy had loss of MMR expression in their sebaceous neoplasms or somatic MMR mutation. Thirty patients had other findings such as colonic adenomas and polyps. Of the 23 patients who had a family history of visceral malignancy, 9 had a personal history of visceral malignancy. Only one sebaceous tumor from each patient (except one) was tested for MMR, which might reduce the sensitivity. Our findings demonstrate an increased frequency of internal malignancy in patients with sebaceous neoplasms compared with the general population, and highlight the heterogeneous nature of the visceral tumors. A majority of the sebaceous tumors show loss of MMR expression. The study reminds us to strive toward a consistent and comprehensive approach to screening for internal malignancy when a patient is given a diagnosis of a sebaceous neoplasm. Copyright © 2012 American Academy of Dermatology, Inc. Published by Mosby, Inc. All rights reserved.

  11. Detection of DNA mismatch repair (MMR) deficiencies by immunohistochemistry can effectively diagnose the microsatellite instability (MSI) phenotype in endometrial carcinomas.

    PubMed

    McConechy, M K; Talhouk, A; Li-Chang, H H; Leung, S; Huntsman, D G; Gilks, C B; McAlpine, J N

    2015-05-01

    A proportion of endometrial carcinomas (ECs) are associated with deficient DNA mismatch repair (MMR). These tumors are characterized by high levels of microsatellite instability (MSI). Identification of MSI is important in identifying women who should be tested for Lynch syndrome and identifying a phenotype that may have specific prognostic and predictive implications. Genomic characterization of ECs has shown that MSI tumors form a distinct subgroup. The two most common methodologies for MSI assessment have not been compared in EC. Pentaplex mono and di-nucleotide PCR for MSI testing was compared to MMR IHC (presence/absence of MLH1, MSH2, MSH6, PMS2) in a cohort of patients with EC. Concordance, Kappa statistic, sensitivity, specificity, positive and negative predictive values were obtained on the cross-tabulation of results. Comparison of both MSI and MMR status was complete for 89 cases. Overall agreement between methods (concordance) was 93.3% (95% CI[85.9%-97.5%]). A one-sided test to determine whether the accuracy is better than the "no information rate," which is taken to be the largest class percentage in the data, is significant (p<0.00001). Unweighted Kappa was 0.84, along with the sensitivity (88.5%), specificity (95.2%), PPV (88.5%), and NPV (95.2%). The balanced accuracy (i.e. the average between sensitivity and specificity) was 92%. We show the equivalence of MSI testing and MMR IHC. We advocate the implementation of MMR IHC in future EC classification schemes, enabling stratification of cases for future clinical trials as well as assisting identification of Lynch syndrome, so that screening and risk reducing interventions can be undertaken. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    PubMed

    Mohni, Kareem N; Mastrocola, Adam S; Bai, Ping; Weller, Sandra K; Heinen, Christopher D

    2011-12-01

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

  13. Expression of Mismatch Repair Proteins in Early and Advanced Gastric Cancer in Poland

    PubMed Central

    Karpińska-Kaczmarczyk, Katarzyna; Lewandowska, Magdalena; Ławniczak, Małgorzata; Białek, Andrzej; Urasińska, Elżbieta

    2016-01-01

    Background Mutations in DNA of mismatch repair (MMR) genes result in failure to repair errors that occur during DNA replication in microsatellites, resulting in accumulation of frameshift mutations in these genes and leading to DNA mismatch replication errors and microsatellite instability. Gastric cancers (GCs) with high MSI (MSI-H) are a well-defined subset of carcinomas showing distinctive clinicopathological features. In this study we investigated the rate of MSI and the correlation between MSI status and clinicopathological features of GC. Material/Methods The study included 107 patients with GCs: 61 with advanced gastric cancers (AGC) and 46 with early gastric cancer (EGC). MSI deficiency in GCs was assessed by the immunohistochemical analysis of expression of MMR proteins – MLH1, MSH2, MSH6, and PMS2 – using formalin-fixed and paraffin-embedded tissue. Results A total of 6 (5.6%) MSI-H were observed. The loss of MMR proteins expression was associated with the intestinal type of GC in Lauren classification, and tubular and papillary architecture in WHO classification. There was no statistically significant association between negative MMR expression and other selected clinical parameters: age, sex, tumor location, depth of invasion (EGC and AGC), lymph nodes status, presence of the ulceration, and lymphocytic infiltrate. Conclusions In the present era of personalized medicine, the histological type of GC and MMR proteins status in cancer cells are very important for the proper surveillance of patients with familial GC and sporadic GCs, as well as for selecting the proper follow-up and treatment. Larger collaborative studies are needed to verify the features of MSI-H GCs in Poland. PMID:27527654

  14. Interdependence of DNA mismatch repair proteins MLH1 and MSH2 in apoptosis in human colorectal carcinoma cell lines.

    PubMed

    Hassen, Samar; Ali, Akhtar A; Kilaparty, Surya P; Al-Anbaky, Qudes A; Majeed, Waqar; Boman, Bruce M; Fields, Jeremy Z; Ali, Nawab

    2016-01-01

    The mammalian DNA mismatch repair (MMR) system consists of a number of proteins that play important roles in repair of base pair mismatch mutations and in maintenance of genomic integrity. A defect in this system can cause genetic instability, which can lead to carcinogenesis. For instance, a germline mutation in one of the mismatch repair proteins, especially MLH1 or MSH2, is responsible for hereditary non-polyposis colorectal cancer. These MMR proteins also play an important role in the induction of apoptosis. Accordingly, altered expression of or a defect in MLH1 or MSH2 may confer resistance to anti-cancer drugs used in chemotherapy. We hypothesized that the ability of these two MMR proteins to regulate apoptosis are interdependent. Moreover, a defect in either one may confer resistance to chemotherapy by an inability to trigger apoptosis. To this end, we studied three cell lines-SW480, LoVo, and HTC116. These cell lines were selected based on their differential expression of MLH1 and MSH2 proteins. SW480 expresses both MLH1 and MSH2; LoVo expresses only MLH1 but not MSH2; HCT116 expresses only MSH2 but not MLH1 protein. MTT assays, a measure of cytotoxicity, showed that there were different cytotoxic effects of an anti-cancer drug, etoposide, on these cell lines, effects that were correlated with the MMR status of the cells. Cells that are deficient in MLH1 protein (HCT116 cells) were resistant to the drug. Cells that express both MLH1 and MSH2 proteins (SW480 cells) showed caspase-3 cleavage, an indicator of apoptosis. Cells that lack MLH1 (HCT116 cells) did not show any caspase-3 cleavage. Expression of full-length MLH1 protein was decreased in MMR proficient (SW480) cells during apoptosis; it remained unchanged in cells that lack MSH2 (LoVo cells). The expression of MSH2 protein remained unchanged during apoptosis both in MMR proficient (SW480) and deficient (HCT116) cells. Studies on translocation of MLH1 protein from nucleus to cytosolic fraction, an

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

    SciTech Connect

    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.

  16. Mismatch repair proteins collaborate with methyltransferases in the repair of O6-methylguanine

    PubMed Central

    Rye, Peter T.; Delaney, James C.; Netirojjanakul, Chawita; Sun, Dana X.; Liu, Jenny Z.; Essigmann, John M.

    2010-01-01

    DNA repair is essential for combatting the adverse effects of damage to the genome. One example of base damage is O6-methylguanine (O6mG), which stably pairs with thymine during replication and thereby creates a promutagenic O6mG:T mismatch. This mismatch has also been linked with cellular toxicity. Therefore, in the absence of repair, O6mG:T mismatches can lead to cell death or result in G:C→A:T transition mutations upon the next round of replication. Cysteine thiolate residues on the Ada and Ogt methyltransferase (MTase) proteins directly reverse the O6mG base damage to yield guanine. When a cytosine is opposite the lesion, MTase repair restores a normal G:C pairing. However, if replication past the lesion has produced an O6mG:T mismatch, MTase conversion to a G:T mispair must still undergo correction to avoid mutation. Two mismatch repair pathways in E. coli that convert G:T mispairs to native G:C pairings are methyl-directed mismatch repair (MMR) and very short patch repair (VSPR). This work examined the possible roles that proteins in these pathways play in coordination with the canonical MTase repair of O6mG:T mismatches. The possibility of this repair network was analyzed by probing the efficiency of MTase repair of a single O6mG residue in cells deficient in individual mismatch repair proteins (Dam, MutH, MutS, MutL, or Vsr). We found that MTase repair in cells deficient in Dam or MutH showed wild-type levels of MTase repair. In contrast, cells lacking any of the VSPR proteins MutS, MutL, or Vsr showed a decrease in repair of O6mG by the Ada and Ogt MTases. Evidence is presented that the VSPR pathway positively influences MTase repair of O6mG:T mismatches, and assists the efficiency of restoring these mismatches to native G:C base pairs. PMID:17951114

  17. Interpretation of immunohistochemistry for mismatch repair proteins is only reliable in a specialized setting.

    PubMed

    Overbeek, Lucia I H; Ligtenberg, Marjolijn J L; Willems, Riki W; Hermens, Rosella P M G; Blokx, Willeke A M; Dubois, Stefan V; van der Linden, Hans; Meijer, Jos W R; Mlynek-Kersjes, Maria L; Hoogerbrugge, Nicoline; Hebeda, Konnie M; van Krieken, Joannes H J M

    2008-08-01

    We examined the validity of immunohistochemistry for mismatch repair (MMR) proteins in colorectal cancer specimens to identify patients at risk for Lynch syndrome (hereditary nonpolyposis colorectal cancer) and patients with sporadic microsatellite instable colorectal cancer. This was assessed by observer agreement for and accuracy of interpretation of immunohistochemistry. Seven pathologists from 5 different pathology laboratories evaluated 100 molecularly defined colorectal cancers stained for MLH1, PMS2, MSH2, and MSH6. Two of the pathologists were experienced in interpretation of immunohistochemistry for MMR proteins. After evaluation of a subset of 20 cases, a discussion meeting was organized, after which pathologists evaluated all 100 cases. Staining patterns were interpreted as aberrant, normal, or indefinite. In 82% of tumors, 5 or more pathologists reached the same interpretation, which was considered the consensus diagnosis. Consensus was reached slightly less frequently in microsatellite instable than in stable tumors, and interobserver variation was moderate to substantial (kappa: 0.49-0.79). More microsatellite instable tumors showed an indefinite staining pattern compared with microsatellite stable tumors. Three out of 7 pathologists, including the 2 experienced pathologists, did not miss a microsatellite instable tumor. Each pathologist found at least 1 tumor with an aberrant staining pattern, whereas consensus was a normal staining pattern and the tumor was microsatellite stable. We conclude that, if restricted to experienced pathologists, immunohistochemistry is a valid tool to identify patients at risk for Lynch syndrome and patients with sporadic microsatellite instable colorectal cancer. An indefinite or aberrant staining result has to be followed by molecular microsatellite instability analysis to confirm the presence of a defective DNA MMR system.

  18. Roles for mismatch repair family proteins in promoting meiotic crossing over

    PubMed Central

    Manhart, Carol M.; Alani, Eric

    2015-01-01

    The mismatch repair (MMR) family complexes Msh4-Msh5 and Mlh1-Mlh3 act with Exo1 and Sgs1-Top3-Rmi1 in a meiotic double strand break repair pathway that results in the asymmetric cleavage of double Holliday junctions (dHJ) to form crossovers. This review discusses how meiotic roles for Msh4-Msh5 and Mlh1-Mlh3 do not fit paradigms established for post-replicative MMR. We also outline models used to explain how these factors promote the formation of meiotic crossovers required for the accurate segregation of chromosome homologs during the Meiosis I division. PMID:26686657

  19. Helicobacter pylori infection and expression of DNA mismatch repair proteins

    PubMed Central

    Mirzaee, Vahid; Molaei, Mahsa; Shalmani, Hamid Mohaghegh; Zali, Mohammad Reza

    2008-01-01

    AIM: To determine the expression of DNA (MMR) proteins, including hMLH1 and hMSH2, in gastric epithelial cells in the patients with or without Helicobacter pylori (H pylori)-infected gastritis. METHODS: Fifty H pylori-positive patients and 50 H pylori-negative patients were enrolled in the study. During endoscopy of patients with non-ulcer dyspepsia, two antral and two corpus biopsies were taken for histological examination (Giemsa stain) and for immunohistochemical staining of hMLH1 and hMSH2. RESULTS: The percentage of epithelial cell nuclei that demonstrated positivity for hMLH1 staining was 84.14 ± 7.32% in H pylori-negative patients, while it was 73.34 ± 10.10% in H pylori-positive patients (P < 0.0001). No significant difference was seen between the two groups regarding the percentage of epithelial cell nuclei that demonstrated positivity for hMSH2 staining (81.16 ± 8.32% in H pylori-negative versus 78.24 ± 8.71% in H pylori-positive patients; P = 0.09). CONCLUSION: This study indicates that H pylori might promote development of gastric carcinoma at least in part through its ability to affect the DNA MMR system. PMID:19034977

  20. [Incidence and Characteristics of Mismatch Repair Protein Deficiency in Elderly Gastric Cancer Patients].

    PubMed

    Chika, Noriyasu; Fukuchi, Minoru; Suzuki, Okihide; Ito, Tetsuya; Yamamoto, Azusa; Ishiguro, Toru; Kumagai, Youichi; Ishibashi, Keiichiro; Mochiki, Erito; Ishida, Hideyuki

    2016-10-01

    The loss of mismatch repair(MMR)function as a result of MLH1 promoter methylation is closely correlated with high frequency microsatellite instability, and tumors with such characteristics are resistant to anticancer drugs such as 5-FU. We examined the incidence and characteristics of deficient MMR(dMMR)in elderly gastric cancer patients by performing a comprehensive immunohistochemical screening. The study was conducted in 199 patients diagnosed with gastric cancer, aged 75 years or older, who underwent a gastrectomy between April 2005 and January 2014. dMMR was detected in 23 patients(12%). All the tumors with dMMR were deficient in MLH1 and PMS2. dMMR was significantly more common compared to proficient MMR(pMMR)in patients with a more advanced age(p=0.03), women(p<0.01), and a tumor location corresponding to the lower region(p<0.01). Considering the incidence of dMMR in elderly gastric cancer patients, only a limited proportion of patients are likely to be eligible for immune checkpoint inhibitor therapy, which is expected to become more popular in the near future.

  1. Minor Changes in Expression of the Mismatch Repair Protein MSH2 Exert a Major Impact on Glioblastoma Response to Temozolomide.

    PubMed

    McFaline-Figueroa, José L; Braun, Christian J; Stanciu, Monica; Nagel, Zachary D; Mazzucato, Patrizia; Sangaraju, Dewakar; Cerniauskas, Edvinas; Barford, Kelly; Vargas, Amanda; Chen, Yimin; Tretyakova, Natalia; Lees, Jacqueline A; Hemann, Michael T; White, Forest M; Samson, Leona D

    2015-08-01

    Glioblastoma (GBM) is often treated with the cytotoxic drug temozolomide, but the disease inevitably recurs in a drug-resistant form after initial treatment. Here, we report that in GBM cells, even a modest decrease in the mismatch repair (MMR) components MSH2 and MSH6 have profound effects on temozolomide sensitivity. RNAi-mediated attenuation of MSH2 and MSH6 showed that such modest decreases provided an unexpectedly strong mechanism of temozolomide resistance. In a mouse xenograft model of human GBM, small changes in MSH2 were sufficient to suppress temozolomide-induced tumor regression. Using The Cancer Genome Atlas to analyze mRNA expression patterns in tumors from temozolomide-treated GBM patients, we found that MSH2 transcripts in primary GBM could predict patient responses to initial temozolomide therapy. In recurrent disease, the absence of microsatellite instability (the standard marker for MMR deficiency) suggests a lack of involvement of MMR in the resistant phenotype of recurrent disease. However, more recent studies reveal that decreased MMR protein levels occur often in recurrent GBM. In accordance with our findings, these reported decreases may constitute a mechanism by which GBM evades temozolomide sensitivity while maintaining microsatellite stability. Overall, our results highlight the powerful effects of MSH2 attenuation as a potent mediator of temozolomide resistance and argue that MMR activity offers a predictive marker for initial therapeutic response to temozolomide treatment.

  2. Risk of secondary malignancy (including breast) in patients with mismatch-repair protein deficiency.

    PubMed

    Clay, Michael R; Allison, Kimberly H; Folkins, Ann K; Longacre, Teri A

    2014-11-01

    Lynch syndrome (LS) is an autosomal dominant inherited disease that is associated with an increased risk for colorectal and endometrial cancer due to germline mutations in mismatch-repair (MMR) genes. Whereas primary tumors in this syndrome are widely recognized, the relative risk(s) of secondary malignancies, particularly breast cancer, in LS patients are still poorly characterized. To provide an improved assessment of these risks, MMR status was evaluated in secondary tumors from a series of patients with index tumors of known MMR status (both proficient and deficient). A total of 1252 tumors (index tumors) and all secondary malignancies were tested for MMR by immunohistochemistry (MSH2, MSH6, MLH1, PMS2) between 1992 and 2013. Tumors with MLH1/PMS2 deficiency were tested for hypermethylation or BRAF mutation, when appropriate. Of the 1252 index tumors, 162 were MMR deficient (dMMR), and, of that subset, 32 secondary tumors were identified (19.7%). In contrast, 80 secondary tumors were identified in the proficient (intact) group (7.3%). Although secondary malignancies were more common in the dMMR group (P=0.0001), there was no trend in tumor type. Specifically, breast cancer was not overly represented in the dMMR group. When secondary tumors had dMMR, they were more likely to have deficiency in MSH2/MSH6 than in MLH1/PMS2 (P=0.01). Of the patients with tumors exhibiting dMMR, women were more likely to have a dMMR secondary tumor in this series (P=0.0001); however, breast cancer was not overly represented, and our study provides no evidence that it is more frequent in LS. MSH2/MSH6 deficiency is more commonly associated with a secondary tumor compared with MLH1/PMS2 deficiency, when methylation/BRAF status is taken into account.

  3. Clinical Impact of Mismatch Repair Protein Testing on Outcome of Early Staged Colorectal Carcinomas.

    PubMed

    Gandhi, Jatin Sundersham; Goswami, Malini; Sharma, Anila; Tanwar, Parul; Gupta, Gurudutt; Gupta, Nikhil; Pasricha, Sunil; Mehta, Anurag; Singh, Shivender; Agarwal, Mohit; Gupta, Nitin

    2017-06-05

    Colorectal cancer is the third most common cancer in men and second most common in women globally. In the present study, we aimed to analyse the proportion of patients with loss of immunostaining for mismatch repair (MMR) proteins in all newly diagnosed stage II cases of colorectal cancer for the purpose of prognostication, for determination of further chemotherapeutic strategy and for familial screening. From January 2014 to December 2015, 62 consecutive newly diagnosed cases of stage II colorectal cancer were included in the study. Details of each patient related to their demographic profile and tumour profile were recorded. All the cases were grossed and staged according to College of American Pathologist (CAP) guidelines. The expression of MMR proteins (which was earlier validated on normal as well as tumour tissue) in FFPE tumour tissue using IHC for mut L homologue 1 (MLH1), mut S homologue 2 (MSH2), mut S homologue 6 (MSH6) and post-meiotic segregation increased 2 (PMS2) was studied. Information regarding stage, treatment, clinical outcome and overall survival was retrieved when available. Out of a total of 371 cases, 62 (16.7%) cases were of stage II CRC, out of which 43 (12%) were treatment naive. Among the selected 62 cases, 26 (41.9%) demonstrated loss of MMR proteins and 36 (58.0%) cases had intact nuclear expression. Out of the cases with MMR loss, 38.4% showed loss of MLH1 and PMS2, 30.7% showed loss of MSH2 and MSH6, 26.9% showed isolated loss of PMS2 and 3.8% showed isolated loss of MSH6. Right-sided location (57.6%) was more common than left-sided (19.2%) and transverse colon (23.0%). Majority of the cases were moderately differentiated (65.3%) in morphology. There was no intratumoural infiltrate in most of the cases (53.8%), and only 3.8% cases showed marked intratumoural infiltrate. Also, peritumoural lymphocytic infiltrate was mild to moderate in most of the cases (26.9%) and marked Crohn's-like infiltrate was seen in only 7.6% cases. Our study

  4. Proteasome inhibition rescues clinically significant unstable variants of the mismatch repair protein Msh2

    PubMed Central

    Arlow, Tim; Scott, Kristan; Wagenseller, Aubrey; Gammie, Alison

    2013-01-01

    MSH2 is required for DNA mismatch repair recognition in eukaryotes. Deleterious mutations in human MSH2 account for approximately half of the alleles associated with a common hereditary cancer syndrome. Previously, we characterized clinically identified MSH2 missense mutations, using yeast as a model system, and found that the most common cause of defective DNA mismatch repair was low levels of the variant Msh2 proteins. Here, we show that increased protein turnover is responsible for the reduced cellular levels. Increasing gene dosage of more than half of the missense alleles fully restored function. A titration experiment revealed that raising the expression level of one variant to less than wild-type levels restored mismatch repair, suggesting that overexpression is not always required to regain function. We found that the ubiquitin-mediated proteasome degradation pathway is the major mechanism for increased turnover of the Msh2 variants and identified the primary ubiquitin ligase as San1. Deletion of San1 restored protein levels for all but one variant, but did not elevate wild-type Msh2 levels. The unstable variants interacted with San1, whereas wild-type Msh2 did not. Additionally, san1Δ suppressed the mismatch repair defect of unstable variants. Of medical significance, the clinically approved drug Bortezomib partially restored protein levels and mismatch repair function for low-level variants and reversed the resistance to cisplatin, a common chemotherapeutic. Our results provide the foundation for an innovative therapeutic regime for certain mismatch-repair-defective cancers that are refractory to conventional chemotherapies. PMID:23248292

  5. Non-canonical actions of mismatch repair

    PubMed Central

    Crouse, Gray F.

    2015-01-01

    At the heart of the mismatch repair (MMR) system are proteins that recognize mismatches in DNA. Such mismatches can be mispairs involving normal or damaged bases or insertion/deletion loops due to strand misalignment. When such mispairs are generated during replication or recombination, MMR will direct removal of an incorrectly paired base or block recombination between nonidentical sequences. However, when mispairs are recognized outside the context of replication, proper strand discrimination between old and new DNA is lost, and MMR can act randomly and mutagenically on mispaired DNA. Such non-canonical actions of MMR are important in somatic hypermutation and class switch recombination, expansion of triplet repeats, and potentially in mutations arising in nondividing cells. MMR involvement in damage recognition and signaling is complex, with the end result likely dependent on the amount of DNA damage in a cell. PMID:26698648

  6. Mismatch repair proteins MSH2, MLH1, and EXO1 are important for class-switch recombination events occurring in B cells that lack nonhomologous end joining.

    PubMed

    Eccleston, Jennifer; Yan, Catherine; Yuan, Karen; Alt, Frederick W; Selsing, Erik

    2011-02-15

    In the absence of core nonhomologous end-joining (NHEJ) factors, Ab gene class-switch recombination (CSR) uses an alternative end-joining (A-EJ) pathway to recombine switch (S) region DNA breaks. Previous reports showing decreased S-junction microhomologies in MSH2-deficient mice and an exonuclease 1 (EXO1) role in yeast microhomology-mediated end joining suggest that mismatch repair (MMR) proteins might influence A-EJ-mediated CSR. We have directly investigated whether MMR proteins collectively or differentially influence the A-EJ mechanism of CSR by analyzing CSR in mice deficient in both XRCC4 and individual MMR proteins. We find CSR is reduced and that Igh locus chromosome breaks are reduced in the MMR/XRCC4 double-deficient B cells compared with B cells deficient in XRCC4 alone, suggesting MMR proteins function upstream of double-strand break formation to influence CSR efficiency in these cells. Our results show that MLH1, EXO1, and MSH2 are all important for efficient A-EJ-mediated CSR, and we propose that MMR proteins convert DNA nicks and point mutations into dsDNA breaks for both C-NHEJ and A-EJ pathways of CSR. We also find Mlh1-XRCC4(-) B cells have an increased frequency of direct S junctions, suggesting that MLH1 proteins may have additional functions that influence A-EJ-mediated CSR.

  7. Evolutionary Covariance Combined with Molecular Dynamics Predicts a Framework for Allostery in the MutS DNA Mismatch Repair Protein

    PubMed Central

    2017-01-01

    Mismatch repair (MMR) is an essential, evolutionarily conserved pathway that maintains genome stability by correcting base-pairing errors in DNA. Here we examine the sequence and structure of MutS MMR protein to decipher the amino acid framework underlying its two key activities—recognizing mismatches in DNA and using ATP to initiate repair. Statistical coupling analysis (SCA) identified a network (sector) of coevolved amino acids in the MutS protein family. The potential functional significance of this SCA sector was assessed by performing molecular dynamics (MD) simulations for alanine mutants of the top 5% of 160 residues in the distribution, and control nonsector residues. The effects on three independent metrics were monitored: (i) MutS domain conformational dynamics, (ii) hydrogen bonding between MutS and DNA/ATP, and (iii) relative ATP binding free energy. Each measure revealed that sector residues contribute more substantively to MutS structure–function than nonsector residues. Notably, sector mutations disrupted MutS contacts with DNA and/or ATP from a distance via contiguous pathways and correlated motions, supporting the idea that SCA can identify amino acid networks underlying allosteric communication. The combined SCA/MD approach yielded novel, experimentally testable hypotheses for unknown roles of many residues distributed across MutS, including some implicated in Lynch cancer syndrome. PMID:28135092

  8. Mismatch repair protein deficient endometrioid adenocarcinomas, metastasizing to adrenal gland and lymph nodes: Unusual cases with diagnostic implications.

    PubMed

    Rekhi, Bharat

    2015-01-01

    Recently, certain endometrial carcinomas have been found to be associated with mismatch repair (MMR) protein defects/deficiency. A 39-year-old female presented with cough, decreased appetite and significant weight loss since 2 months. Earlier, she had undergone total abdominal hysterectomy with bilateral salpingo-oophorectomy (TAH-BSO) for endometrioid adenocarcinoma. Imaging disclosed an 8 cm-sized adrenal mass that was surgically excised. Histopathology of the adrenal tumor, endocervical tumor, and endometrial biopsy revealed Federation of Gynecology and Obstetrics (FIGO) Grade II to III endometrioid adenocarcinoma. By immunohistochemistry, tumor cells were positive for cytokeratin 7, epithelial membrane antigen, PAX8, MLH1 and PMS2 while negative for estrogen receptor (ER), progesterone receptor (PR), MSH2 and MSH6. She underwent adjuvant radiotherapy and chemotherapy. A 34-year-old lady presented with vaginal bleeding since 9 months. She underwent TAH-BSO, reported as FIGO Grade III endometrioid adenocarcinoma. By immunohistochemistry, tumor cells were negative for ER, PR, MLH1, and PMS2 while positive for MSH2 and MSH6. She underwent adjuvant radiotherapy and chemotherapy. However, she developed multiple nodal and pericardial metastases and succumbed to the disease within a year post-diagnosis. Certain high-grade endometrioid adenocarcinomas occurring in younger women are MMR protein deficient and display an aggressive clinical course. Adrenal metastasis in endometrial carcinomas is rare.

  9. 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.

  10. Correlation of tumour BRAF mutations and MLH1 methylation with germline mismatch repair (MMR) gene mutation status: a literature review assessing utility of tumour features for MMR variant classification.

    PubMed

    Parsons, Michael T; Buchanan, Daniel D; Thompson, Bryony; Young, Joanne P; Spurdle, Amanda B

    2012-03-01

    Colorectal cancer (CRC) that demonstrates microsatellite instability (MSI) is caused by either germline mismatch repair (MMR) gene mutations, or 'sporadic' somatic tumour MLH1 promoter methylation. MLH1 promoter methylation is reportedly correlated with tumour BRAF V600E mutation status. No systematic review has been undertaken to assess the value of BRAF V600E mutation and MLH1 promoter methylation tumour markers as negative predictors of germline MMR mutation status. A literature review of CRC cohorts tested for MMR mutations, and tumour BRAF V600E mutation and/or MLH1 promoter methylation was conducted using PubMed. Studies were assessed for tumour features, stratified by tumour MMR status based on immunohistochemistry or MSI where possible. Pooled frequencies and 95% CIs were calculated using a random effects model. BRAF V600E results for 4562 tumours from 35 studies, and MLH1 promoter methylation results for 2975 tumours from 43 studies, were assessed. In 550 MMR mutation carriers, the BRAF V600E mutation frequency was 1.40% (95% CI 0.06% to 3%). In MMR mutation-negative cases, the BRAF V600E mutation frequency was 5.00% (95% CI 4% to 7%) in 1623 microsatellite stable (MSS) cases and 63.50% (95% CI 47% to 79%) in 332 cases demonstrating MLH1 methylation or MLH1 expression loss. MLH1 promoter methylation of the 'A region' was reported more frequently than the 'C region' in MSS CRCs (17% vs 0.06%, p<0.0001) and in MLH1 mutation carriers (42% vs 6%, p<0.0001), but not in MMR mutation-negative MSI-H CRCs (40% vs 47%, p=0.12). Methylation of the 'C region' was a predictor of MMR mutation-negative status in MSI-H CRC cases (47% vs 6% in MLH1 mutation carriers, p<0.0001). This review demonstrates that tumour BRAF V600E mutation, and MLH1 promoter 'C region' methylation specifically, are strong predictors of negative MMR mutation status. It is important to incorporate these features in multifactorial models aimed at predicting MMR mutation status.

  11. Immunohistochemical analysis of DNA mismatch repair protein and O6-methylguanine-DNA methyltransferase in melanoma metastases in relation to clinical response to DTIC-based chemotherapy.

    PubMed

    Ma, Shuhua; Egyházi, Suzanne; Ringborg, Ulrik; Hansson, Johan

    2002-01-01

    DNA mismatch repair (MMR) deficiency and increased O6-methylguanine-DNA methyltransferase (MGMT) activity have been related to resistance to O6-guanine methylating agents in tumour cell lines. However, the clinical relevance of MMR and MGMT as drug resistance factors is still unclear. In a retrospective study, the expression levels of the MMR proteins, hMSH2, hMSH6 and hMLH1, were analysed by immunohistochemistry in melanoma metastases from 64 patients, who had received dacarbazine (DTIC) based chemotherapy. More than half of the melanoma patients had tumours with no nuclear staining for either hMSH2 or hMSH6 or both, while all tumours showed positive nuclear staining for hMLH1. The response rates were similar in patients with hMSH2 and/or hMSH6 positive tumours to these in patients with negative tumours. By combination of MMR with previously obtained MGMT data, only 2 of 12 responders had tumours with low MGMT and positive MMR expression. Still all except 3 of the non-responders were identified by having either high MGMT expression or absent staining for hMSH2 or hMSH6 or both in their tumours. However, there was no significant correlation of MMR expression alone or combined with MGMT levels with clinical response to DTIC-based chemotherapy in metastatic melanoma.

  12. 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.

  13. Reduction of DNA mismatch repair protein expression in airway epithelial cells of premenopausal women chronically exposed to biomass smoke.

    PubMed

    Mukherjee, Bidisha; Dutta, Anindita; Chowdhury, Saswati; Roychoudhury, Sanghita; Ray, Manas Ranjan

    2014-02-01

    Biomass burning is a major source of indoor air pollution in rural India. This study examined whether chronic inhalation of biomass smoke causes change in the DNA mismatch repair (MMR) pathway in the airway cells. For this, airway cells exfoliated in sputum were collected from 72 premenopausal nonsmoking rural women (median age 34 years) who cooked with biomass (wood, dung, crop residues) and 68 control women who cooked with cleaner fuel liquefied petroleum gas (LPG) for the past 5 years or more. The levels of particulate matters with diameters less than 10 and 2.5 μm (PM10 and PM2.5) in indoor air were measured by real-time aerosol monitor. Benzene exposure was monitored by measuring trans,trans-muconic acid (t,t-MA) in urine by high-performance liquid chromatography with ultraviolet detector. Generation of reactive oxygen species (ROS) and level of superoxide dismutase (SOD) in airway cells were measured by flow cytometry and spectrophotometry, respectively. Immunocytochemical assay revealed lower percentage of airway epithelial cells expressing MMR proteins mutL homolog 1 (MLH1) and mutS homolog 2 (MSH2) in biomass-using women compared to LPG-using controls. Women who cooked with biomass had 6.7 times higher level of urinary t,t-MA, twofold increase in ROS generation, and 31 % depletion of SOD. Indoor air of biomass-using households had three times more particulate matters than that of controls. ROS, urinary t,t-MA, and particulate pollution in biomass-using kitchen had negative correlation, while SOD showed positive correlation with MSH2 and MLH1 expression. It appears that chronic exposure to biomass smoke reduces MMR response in airway epithelial cells, and oxidative stress plays an important role in the process.

  14. 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 ...

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

    PubMed

    Qi, Yongmei; Schoene, Norberta W; Lartey, Frederick M; Cheng, Wen-Hsing

    2010-10-22

    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). Here, we have employed the MMR-deficient HCT 116 colorectal cancer cells and the MMR-proficient HCT 116 cells with hMLH1 complementation to investigate the role of hMLH1 in selenium-induced DNA damage response, a tumorigenesis barrier. The ATM (ataxia telangiectasia mutated) protein responds to clastogens and initiates DNA damage response. We show that hMLH1 complementation sensitizes HCT 116 cells to methylseleninic acid, methylselenocysteine, and sodium selenite via reactive oxygen species and facilitates the selenium-induced oxidative 8-oxoguanine damage, DNA breaks, G(2)/M checkpoint response, and ATM pathway activation. Pretreatment of the hMLH1-complemented HCT 116 cells with the antioxidant N-acetylcysteine or 2,2,6,6-tetramethylpiperidine-1-oxyl or the ATM kinase inhibitor KU55933 suppresses hMLH1-dependent DNA damage response to selenium exposure. Selenium treatment stimulates the association between hMLH1 and hPMS2 proteins, a heterodimer critical for functional MMR, in a manner dependent on ATM and reactive oxygen species. Taken together, the results suggest a new role of selenium in mitigating tumorigenesis by targeting the MMR pathway, whereby the lack of hMLH1 renders the HCT 116 colorectal cancer cells resistant to selenium-induced DNA damage response.

  16. Mismatch repair proteins recruit DNA methyltransferase 1 to sites of oxidative DNA damage.

    PubMed

    Ding, Ning; Bonham, Emily M; Hannon, Brooke E; Amick, Thomas R; Baylin, Stephen B; O'Hagan, Heather M

    2016-06-01

    At sites of chronic inflammation, epithelial cells are exposed to high levels of reactive oxygen species and undergo cancer-associated DNA methylation changes, suggesting that inflammation may initiate epigenetic alterations. Previously, we demonstrated that oxidative damage causes epigenetic silencing proteins to become part of a large complex that is localized to GC-rich regions of the genome, including promoter CpG islands that are epigenetically silenced in cancer. However, whether these proteins were recruited directly to damaged DNA or during the DNA repair process was unknown. Here we demonstrate that the mismatch repair protein heterodimer MSH2-MSH6 participates in the oxidative damage-induced recruitment of DNA methyltransferase 1 (DNMT1) to chromatin. Hydrogen peroxide treatment induces the interaction of MSH2-MSH6 with DNMT1, suggesting that the recruitment is through a protein-protein interaction. Importantly, the reduction in transcription for genes with CpG island-containing promoters caused by oxidative damage is abrogated by knockdown of MSH6 and/or DNMT1. Our findings provide evidence that the role of DNMT1 at sites of oxidative damage is to reduce transcription, potentially preventing transcription from interfering with the repair process. This study uniquely brings together several factors that are known to contribute to colon cancer, namely inflammation, mismatch repair proteins, and epigenetic changes. © The Author (2015). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

  17. Cytoskeletal scaffolding proteins interact with Lynch-Syndrome associated mismatch repair protein MLH1.

    PubMed

    Brieger, Angela; Adryan, Boris; Wolpert, Fabian; Passmann, Sandra; Zeuzem, Stefan; Trojan, Jörg

    2010-09-01

    The involvement of MLH1 in several mismatch repair-independent cellular processes has been reported. In an attempt to gain further insight into the protein's cellular functions, we screened for novel interacting partners of MLH1 utilizing a bacterial two-hybrid system. Numerous unknown interacting proteins were identified, suggesting novel biological roles of MLH1. The network of MLH1 and its partner proteins involves a multitude of cellular processes. Integration of our data with the "General Repository for Interaction Datasets" highlighted that MLH1 exhibits relationships to three interacting pairs of proteins involved in cytoskeletal and filament organization: Thymosin beta 4 and Actin gamma, Cathepsin B and Annexin A2 as well as Spectrin alpha and Desmin. Coimmunoprecipitation and colocalization experiments validated the interaction of MLH1 with these proteins. Differential mRNA levels of many of the identified proteins, detected by microarray analysis comparing MLH1-deficient and -proficient cell lines, support the assumed interplay of MLH1 and the identified candidate proteins. By siRNA knock down of MLH1, we demonstrated the functional impact of MLH1-Actin interaction on filament organization and propose that dysregulation of MLH1 plays an essential role in cytoskeleton dynamics. Our data suggest novel roles of MLH1 in cellular organization and colorectal cancerogenesis.

  18. 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.

  19. New insights into the mechanism of DNA mismatch repair

    PubMed Central

    Reyes, Gloria X.; Schmidt, Tobias T.; Kolodner, Richard D.; Hombauer, Hans

    2015-01-01

    The genome of all organisms is constantly being challenged by endogenous and exogenous sources of DNA damage. Errors like base:base mismatches or small insertions and deletions, primarily introduced by DNA polymerases during DNA replication are repaired by an evolutionary conserved DNA mismatch repair (MMR) system. The MMR system, together with the DNA replication machinery, promote repair by an excision and resynthesis mechanism during or after DNA replication, increasing replication fidelity by upto-three orders of magnitude. Consequently, inactivation of MMR genes results in elevated mutation rates that can lead to increased cancer susceptibility in humans. In this review, we summarize our current understanding of MMR with a focus on the different MMR protein complexes, their function and structure. We also discuss how recent findings have provided new insights in the spatio-temporal regulation and mechanism of MMR. PMID:25862369

  20. Mismatch Repair Protein Deficiency Is a Risk Factor for Aberrant Expression of HLA Class I Molecules: A Putative "Adaptive Immune Escape" Phenomenon.

    PubMed

    Kubo, Terufumi; Hirohashi, Yoshihiko; Matsuo, Kazuhiko; Sonoda, Tomoko; Sakamoto, Hiroki; Furumura, Kiyoshi; Tsukahara, Tomohide; Kanaseki, Takayuki; Nakatsugawa, Munehide; Hirano, Hiroshi; Furuhata, Tomohisa; Takemasa, Ichiro; Hasegawa, Tadashi; Torigoe, Toshihiko

    2017-03-01

    Accumulating evidence indicates that immune checkpoint inhibition-mediated cancer immunotherapies greatly improve the prognosis of certain types of cancer. This approach is now becoming a standard therapy, joining surgery, radiotherapy, and chemotherapy. Because the costs of antibody drugs are now a socioeconomic burden in many countries, an urgent need in cancer immunotherapy is the identification of relevant biomarkers that can predict therapy efficacy. Recent studies have reported that colorectal adenocarcinoma with hereditary or sporadic deficiency in mismatch repair (MMR) proteins has high antigenicity and that detection of these proteins could be a promising way to estimate clinical response. In this study of 135 patients with colorectal cancer, we used immunohistochemistry to investigate the correlation between deficiency in MMR proteins and expression of human leukocyte antigen (HLA) class I molecules, a prerequisite of cytotoxic T-cell-based immunotherapy. Interestingly, MMR protein deficiency was an independent risk factor for the impaired expression of HLA class I molecules (odds ratio (OR)=10.44, 95% confidence interval (CI)=3.15-34.62, p<0.001), suggesting the existence of a putative entity that we have named "adaptive immune escape". Moreover, our results might provide a potential novel biomarker for the selection of patients who would respond to cancer immunotherapies. At the same time, the results suggest that we have to overcome the impaired expression of HLA class I molecules to further improve the cure rate of cancer immunotherapies. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  1. 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

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

    PubMed

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

    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 the

  3. 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

  4. Small Bowel Adenocarcinoma Frequently Exhibits Lynch Syndrome-associated Mismatch Repair Protein Deficiency But Does Not Harbor Sporadic MLH1 Deficiency.

    PubMed

    Xia, Michelle; Singhi, Aatur D; Dudley, Beth; Brand, Randall; Nikiforova, Marina; Pai, Reetesh K

    2017-07-01

    Universal screening for Lynch syndrome has been advocated for colorectal carcinoma but its utility in small bowel adenocarcinoma has not been reported. We analyzed a consecutive series of 71 small bowel adenocarcinomas identified over an 8-year period for DNA mismatch repair (MMR) protein expression to (1) compare the clinicopathologic features of small bowel adenocarcinoma stratified into MMR-deficient (MMRD) and MMR-proficient (MMRP) groups and (2) examine the patterns of MMR protein expression in small bowel adenocarcinoma compared with colorectal carcinoma. Six of 71 (8.5%) small bowel adenocarcinomas and 149 of 1291 (11.5%) colorectal carcinomas demonstrated MMRD. The 6 MMRD small bowel adenocarcinomas had the following expression pattern: 3 with concurrent loss of MSH2 and MSH6, 1 with isolated loss of MSH6, and 2 with concurrent loss of MLH1 and PMS2 in patients with a family history suggestive of genetic cancer susceptibility. Histopathology suggestive of MMR protein deficiency as proposed by the revised Bethesda guidelines was commonly seen in both MMRP (63%) and MMRD (67%) small bowel adenocarcinomas (P>0.05). MMRD small bowel adenocarcinoma more frequently demonstrated abnormalities of MSH2 and/or MSH6 (4/6, 67%) compared with MMRD colorectal carcinoma (23/149, 15%) (P=0.01). None of the MMRD small bowel adenocarcinomas harbored the BRAF V600E mutation, whereas 60% of MMRD colorectal carcinomas were positive for BRAF V600E with concurrent loss of MLH1 and PMS2 expression. Small bowel adenocarcinoma more frequently harbored Lynch syndrome-associated MMRD compared with colorectal carcinoma, providing support for screening of small bowel adenocarcinoma to identify patients at risk for Lynch syndrome. In contrast to colorectal carcinoma, sporadic MLH1 deficiency is not seen in small bowel adenocarcinoma. Clinicopathologic and histologic features do not distinguish between MMRP and MMRD small bowel adenocarcinoma indicating that universal screening in small

  5. A Role for the MutL Mismatch Repair Mlh3 Protein in Immunoglobulin Class Switch DNA Recombination and Somatic Hypermutation1

    PubMed Central

    Wu, Xiaoping; Tsai, Connie Y.; Patam, Marienida B.; Zan, Hong; Chen, Jessica P.; Lipkin, Steve M.; Casali, Paolo

    2015-01-01

    Class switch DNA recombination (CSR) and somatic hypermutation (SHM) are central to the maturation of the Ab response. Both processes involve DNA mismatch repair (MMR). MMR proteins are recruited to dU:dG mispairs generated by activation-induced cytidine deaminase-mediated deamination of dC residues, thereby promoting S-S region synapses and introduction of mismatches (mutations). The MutL homolog Mlh3 is the last complement of the mammalian set of MMR proteins. It is highly conserved in evolution and is essential to meiosis and microsatellite stability. We used the recently generated knockout mlh3−/− mice to address the role of Mlh3 in CSR and SHM. We found that Mlh3 deficiency alters both CSR and SHM. mlh3−/− B cells switched in vitro to IgG and IgA but displayed preferential targeting of the RGYW/WRCY (R = A or G, Y = C or T, W = A or T) motif by Sγ1 and Sγ3 breakpoints and introduced more insertions and fewer donor/acceptor microhomologies in Sμ-Sγ1 and Sμ-Sγ3 DNA junctions, as compared with mlh3+/+ B cells. mlh3−/− mice showed only a slight decrease in the frequency of mutations in the intronic DNA downstream of the rearranged JH4 gene. However, the residual mutations were altered in spectrum. They comprised a decreased proportion of mutations at dA/dT and showed preferential RGYW/WRCY targeting by mutations at dC/dG. Thus, the MMR Mlh3 protein plays a role in both CSR and SHM. PMID:16622010

  6. Novel DNA mismatch-repair activity involving YB-1 in human mitochondria.

    PubMed

    de Souza-Pinto, Nadja C; Mason, Penelope A; Hashiguchi, Kazunari; Weissman, Lior; Tian, Jingyan; Guay, David; Lebel, Michel; Stevnsner, Tinna V; Rasmussen, Lene Juel; Bohr, Vilhelm A

    2009-06-04

    Maintenance of the mitochondrial genome (mtDNA) is essential for proper cellular function. The accumulation of damage and mutations in the mtDNA leads to diseases, cancer, and aging. Mammalian mitochondria have proficient base excision repair, but the existence of other DNA repair pathways is still unclear. Deficiencies in DNA mismatch repair (MMR), which corrects base mismatches and small loops, are associated with DNA microsatellite instability, accumulation of mutations, and cancer. MMR proteins have been identified in yeast and coral mitochondria; however, MMR proteins and function have not yet been detected in human mitochondria. Here we show that human mitochondria have a robust mismatch-repair activity, which is distinct from nuclear MMR. Key nuclear MMR factors were not detected in mitochondria, and similar mismatch-binding activity was observed in mitochondrial extracts from cells lacking MSH2, suggesting distinctive pathways for nuclear and mitochondrial MMR. We identified the repair factor YB-1 as a key candidate for a mitochondrial mismatch-binding protein. This protein localizes to mitochondria in human cells, and contributes significantly to the mismatch-binding and mismatch-repair activity detected in HeLa mitochondrial extracts, which are significantly decreased when the intracellular levels of YB-1 are diminished. Moreover, YB-1 depletion in cells increases mitochondrial DNA mutagenesis. Our results show that human mitochondria contain a functional MMR repair pathway in which YB-1 participates, likely in the mismatch-binding and recognition steps.

  7. DNA mismatch repair and its many roles in eukaryotic cells.

    PubMed

    Liu, Dekang; Keijzers, Guido; Rasmussen, Lene Juel

    2017-07-01

    DNA mismatch repair (MMR) is an important DNA repair pathway that plays critical roles in DNA replication fidelity, mutation avoidance and genome stability, all of which contribute significantly to the viability of cells and organisms. MMR is widely-used as a diagnostic biomarker for human cancers in the clinic, and as a biomarker of cancer susceptibility in animal model systems. Prokaryotic MMR is well-characterized at the molecular and mechanistic level; however, MMR is considerably more complex in eukaryotic cells than in prokaryotic cells, and in recent years, it has become evident that MMR plays novel roles in eukaryotic cells, several of which are not yet well-defined or understood. Many MMR-deficient human cancer cells lack mutations in known human MMR genes, which strongly suggests that essential eukaryotic MMR components/cofactors remain unidentified and uncharacterized. Furthermore, the mechanism by which the eukaryotic MMR machinery discriminates between the parental (template) and the daughter (nascent) DNA strand is incompletely understood and how cells choose between the EXO1-dependent and the EXO1-independent subpathways of MMR is not known. This review summarizes recent literature on eukaryotic MMR, with emphasis on the diverse cellular roles of eukaryotic MMR proteins, the mechanism of strand discrimination and cross-talk/interactions between and co-regulation of MMR and other DNA repair pathways in eukaryotic cells. The main conclusion of the review is that MMR proteins contribute to genome stability through their ability to recognize and promote an appropriate cellular response to aberrant DNA structures, especially when they arise during DNA replication. Although the molecular mechanism of MMR in the eukaryotic cell is still not completely understood, increased used of single-molecule analyses in the future may yield new insight into these unsolved questions. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Mismatch repair protein immunohistochemistry: a useful population screening strategy for Lynch syndrome.

    PubMed

    Musulén, Eva; Sanz, Carolina; Muñoz-Mármol, Ana María; Ariza, Aurelio

    2014-07-01

    Lynch syndrome (LS), the most frequent form of hereditary colorectal cancer, shows a highly penetrant, autosomal dominant pattern of inheritance. Distinction of LS colorectal carcinoma instances from the much more common sporadic colorectal carcinoma cases is of paramount importance. Revised Bethesda Guidelines were developed to diagnose LS by evaluating a combination of clinical and pathologic data. The aim of the present study was to evaluate the usefulness of the pathology items included in the Revised Bethesda Guidelines. We have prospectively studied a series of 1624 consecutive colorectal carcinomas with an algorithm including immunohistochemical analysis of mismatch repair proteins and molecular study of microsatellite instability and BRAF c.1799 T > A (p.V600E) gene mutations. Patients with tumors showing LS features were referred for germline mutation analysis. By applying our algorithmic approach, we were able to identify LS features in 89 colorectal cancer patients, of whom only 27 met Revised Bethesda Guidelines pathology criteria. Of the 89 patients, 47 were then studied at the Genetic Counseling Unit, and LS was confirmed in 18, of whom 7 had not been identified by the Revised Bethesda Guidelines. Our study shows that the Revised Bethesda Guidelines failed to detect 70% of patients at risk of LS. Our algorithmic approach is a realistic and effective tool for LS identification. We strongly recommend the implementation of universal population screening for LS among all patients with newly diagnosed colorectal carcinoma.

  9. How reliable is immunohistochemical staining for DNA mismatch repair proteins performed after neoadjuvant chemoradiation?

    PubMed

    Vilkin, Alex; Halpern, Marisa; Morgenstern, Sara; Brazovski, Eli; Gingold-Belfer, Rachel; Boltin, Doron; Purim, Ofer; Kundel, Yulia; Welinsky, Sara; Brenner, Baruch; Niv, Yaron; Levi, Zohar

    2014-10-01

    Immunohistochemistry (IHC) testing for mismatch repair proteins (MMRP) is currently being used primarily in colorectal cancer resection specimens. We aimed to compare the results of IHC staining performed on biopsy specimens obtained at endoscopy with that performed on surgical specimens after neoadjuvant therapy. Thirty-two rectal cancer subjects had paired preneoadjuvant and postneoadjuvant tissue available for IHC staining (MLH1, MSH2, MSH6, and PMS2), whereas 39 rectosigmoid cancer patients who did not receive neoadjuvant treatment served as controls. Each slide received a qualitative (absent, focal, and strong) and quantitative score (immunoreactivity [0-3] × percent positivity [0-4]). The quantitative scores of MMRP from the operative material were significantly lower in the neoadjuvant group than in the control (P < .05 for all).The scores of all MMRP from endoscopic biopsies were not significantly different between the neoadjuvant and the control groups. Disagreement between the endoscopic biopsy and the operative material was evident in 23 of 128 stains (18.5%) in the neoadjuvant group and in 12 of 156 stains (7.7%) in the control group (P = .009). In the neoadjuvant group, a disagreement pattern of "endoscopic strong operative focal" was observed in 28.1% for PMS2, 12.5% for MSH6, 12.5% for MLH1, and 6.3% for MSH2, and in the control group, this same disagreement pattern was found in 12.8% for PMS2, 7.7% for MSH6, 7.7% for MLH1, and 0% for MSH2. Based on our findings, we suggest that for rectal cancer, the endoscopic material rather than the operative material should serve as the primary material for IHC staining.

  10. Immunohistochemistry staining for mismatch repair proteins: the endoscopic biopsy material provides useful and coherent results.

    PubMed

    Vilkin, Alex; Leibovici-Weissman, Ya'ara; Halpern, Marisa; Morgenstern, Sara; Brazovski, Eli; Gingold-Belfer, Rachel; Wasserberg, Nir; Brenner, Baruch; Niv, Yaron; Sneh-Arbib, Orly; Levi, Zohar

    2015-11-01

    Immunohistochemistry (IHC) testing for mismatch repair proteins (MMRP) in patients with colorectal cancer can be performed on endoscopic biopsy material or the surgical resection material. Data are continuing to accumulate regarding the deleterious effect of neoadjuvant chemoradiation on MMRP expression. However, despite continuing rise in the use of endoscopic biopsies for IHC, most pathology departments still use mainly the surgical materials for IHC testing. In this study we compared the quality of stains among 96 colon cancer subjects with paired endoscopic and surgical material available for MLH1, MSH2, MSH6, and PMS2 stains (96 × 4, yielding 384 paired stains). Each slide received both a quantitative score (immunoreactivity [0-3] × percent positivity [0-4]) and a qualitative score (absent; weak and focal; strong). The quantitative scores of all MMRP were significantly higher among the endoscopic material (P<.001 for all). In 358 pairs (93.2%), both the endoscopic and operative material stained either strong (322, 83.9%) or absent (36, 9.4%). In 26 pairs (6.8%), the endoscopic material stained strong, whereas the operative material stained focal and weak. No endoscopic biopsy materials stained focal and weak. Our findings indicate that the biopsy material may provide more coherent results. Although these results may indicate that biopsy material provides coherent and useful results, it is yet to be determined if the demonstrated differences pose a real clinical problem in interpreting final results of IHC staining of such kind. Hence, we suggest that when available, the endoscopic material rather than the operative one should serve as the primary substrate for IHC staining.

  11. Human mismatch-repair protein MutL homologue 1 (MLH1) interacts with Escherichia coli MutL and MutS in vivo and in vitro: a simple genetic system to assay MLH1 function.

    PubMed Central

    Quaresima, Barbara; Alifano, Pietro; Tassone, Pierfrancesco; Avvedimento, Enrico V; Costanzo, Francesco S; Venuta, Salvatore

    2003-01-01

    A simple genetic system has been developed to test the effect of over-expression of wild-type or mutated human MutL homologue 1 (hMLH1) proteins on methyl-directed mismatch repair (MMR) in Escherichia coli. The system relies on detection of Lac(+) revertants using MMR-proficient or MMR-deficient E. coli strains carrying a lac +1 frameshift mutation expressing hMLH1 proteins. We report that expression of wild-type hMLH1 protein causes an approx. 19-fold increase in mutation rates. The mutator phenotype was due to the ability of hMLH1 protein to interact with bacterial MutL and MutS proteins, thereby interfering with the formation of complexes between MMR proteins and mismatched DNA. Conversely, expression of proteins encoded by alleles deriving from hereditary-non-polyposis-colon-cancer (HNPCC) families decreases mutation rates, depending on the specific amino acid substitutions. These effects parallel the MutL-and MutS-binding and ATP-binding/hydrolysis activities of the mutated proteins. PMID:12513688

  12. DNA Triplet Repeat Expansion and Mismatch Repair

    PubMed Central

    Iyer, Ravi R.; Pluciennik, Anna; Napierala, Marek; Wells, Robert D.

    2016-01-01

    DNA mismatch repair is a conserved antimutagenic pathway that maintains genomic stability through rectification of DNA replication errors and attenuation of chromosomal rearrangements. Paradoxically, mutagenic action of mismatch repair has been implicated as a cause of triplet repeat expansions that cause neurological diseases such as Huntington disease and myotonic dystrophy. This mutagenic process requires the mismatch recognition factor MutSβ and the MutLα (and/or possibly MutLγ) endonuclease, and is thought to be triggered by the transient formation of unusual DNA structures within the expanded triplet repeat element. This review summarizes the current knowledge of DNA mismatch repair involvement in triplet repeat expansion, which encompasses in vitro biochemical findings, cellular studies, and various in vivo transgenic animal model experiments. We present current mechanistic hypotheses regarding mismatch repair protein function in mediating triplet repeat expansions and discuss potential therapeutic approaches targeting the mismatch repair pathway. PMID:25580529

  13. [Characteristics and Outcomes of Treatment in Patients with Stage IV Colorectal Cancer with Mismatch Repair Deficiency].

    PubMed

    Ishibashi, Keiichiro; Chika, Noriyasu; Suzuki, Okihide; Ito, Tetsuya; Amano, Kunihiko; Kumamoto, Kensuke; Fukuchi, Minoru; Kumagai, Youichi; Mochiki, Erito; Ishida, Hideyuki

    2016-11-01

    Mismatch repair(MMR)protein deficiency in colorectal cancer is well correlated with high-level microsatellite instability (MSI-H). There are little data on mismatch repair deficiency(dMMR)colorectal cancers in Japan. In addition, we have no available data on the therapeutic efficacy of oxaliplatin(oxa)-based chemotherapy, one of the standard treatment regimens for metastatic colorectal cancer, for patients with dMMR colorectal cancer. The subjects were 254 patients with Stage IV colorectal cancer whose tumors were immunohistochemically stained for MMR proteins, MLH1, MSH2, MSH6, and PMS2. Patients who underwent R0 resection were excluded. Clinicopathologic factors and the efficacy of oxa-based chemotherapy were compared between patients with dMMR colorectal cancer and those with mismatch repair proficient(pMMR)colorectal cancer. There were 7(2.8%)patients with dMMR. Four patients demonstrated both MLH1 and PMS2 loss, while 3 patients demonstrated both MSH2 and MSH6 loss. Though the dMMR had a higher frequency in female patients(p=0.02) and a lower frequency in those with liver metastasis(p<0.01), the other clinicopathologic factors evaluated did not significantly differ between the dMMR group and the pMMR group. One hundred and fifty patients with unresectable disease or R1/2 resection received first-line oxa-based chemotherapy. The median overall survival was 23.2 months and 16.2 months in patients with dMMR(n=4)and those with pMMR, respectively(n=146)(p=0.33). The frequency of dMMR amongStag e IV colorectal cancers was lower than those(4-11%)reported in Western countries. Therefore, the clinical significance of universal screeningfor dMMR in all colorectal cancer samples may not be valid. Concerningsurvival benefit, oxa-based chemotherapy seems to be an effective alternative in clinical practice for metastatic colorectal cancer patients with dMMR.

  14. A non-canonical mismatch repair pathway in prokaryotes

    PubMed Central

    Castañeda-García, A.; Prieto, A. I.; Rodríguez-Beltrán, J.; Alonso, N.; Cantillon, D.; Costas, C.; Pérez-Lago, L.; Zegeye, E. D.; Herranz, M.; Plociński, P.; Tonjum, T.; García de Viedma, D.; Paget, M.; Waddell, S. J.; Rojas, A. M.; Doherty, A. J.; Blázquez, J.

    2017-01-01

    Mismatch repair (MMR) is a near ubiquitous pathway, essential for the maintenance of genome stability. Members of the MutS and MutL protein families perform key steps in mismatch correction. Despite the major importance of this repair pathway, MutS–MutL are absent in almost all Actinobacteria and many Archaea. However, these organisms exhibit rates and spectra of spontaneous mutations similar to MMR-bearing species, suggesting the existence of an alternative to the canonical MutS–MutL-based MMR. Here we report that Mycobacterium smegmatis NucS/EndoMS, a putative endonuclease with no structural homology to known MMR factors, is required for mutation avoidance and anti-recombination, hallmarks of the canonical MMR. Furthermore, phenotypic analysis of naturally occurring polymorphic NucS in a M. smegmatis surrogate model, suggests the existence of M. tuberculosis mutator strains. The phylogenetic analysis of NucS indicates a complex evolutionary process leading to a disperse distribution pattern in prokaryotes. Together, these findings indicate that distinct pathways for MMR have evolved at least twice in nature. PMID:28128207

  15. A non-canonical mismatch repair pathway in prokaryotes.

    PubMed

    Castañeda-García, A; Prieto, A I; Rodríguez-Beltrán, J; Alonso, N; Cantillon, D; Costas, C; Pérez-Lago, L; Zegeye, E D; Herranz, M; Plociński, P; Tonjum, T; García de Viedma, D; Paget, M; Waddell, S J; Rojas, A M; Doherty, A J; Blázquez, J

    2017-01-27

    Mismatch repair (MMR) is a near ubiquitous pathway, essential for the maintenance of genome stability. Members of the MutS and MutL protein families perform key steps in mismatch correction. Despite the major importance of this repair pathway, MutS-MutL are absent in almost all Actinobacteria and many Archaea. However, these organisms exhibit rates and spectra of spontaneous mutations similar to MMR-bearing species, suggesting the existence of an alternative to the canonical MutS-MutL-based MMR. Here we report that Mycobacterium smegmatis NucS/EndoMS, a putative endonuclease with no structural homology to known MMR factors, is required for mutation avoidance and anti-recombination, hallmarks of the canonical MMR. Furthermore, phenotypic analysis of naturally occurring polymorphic NucS in a M. smegmatis surrogate model, suggests the existence of M. tuberculosis mutator strains. The phylogenetic analysis of NucS indicates a complex evolutionary process leading to a disperse distribution pattern in prokaryotes. Together, these findings indicate that distinct pathways for MMR have evolved at least twice in nature.

  16. Deficient mismatch repair: Read all about it (Review)

    PubMed Central

    RICHMAN, SUSAN

    2015-01-01

    Defects in the DNA mismatch repair (MMR) proteins, result in a phenotype called microsatellite instability (MSI), occurring in up to 15% of sporadic colorectal cancers. Approximately one quarter of colon cancers with deficient MMR (dMMR) develop as a result of an inherited predisposition syndrome, Lynch syndrome (formerly known as HNPCC). It is essential to identify patients who potentially have Lynch syndrome, as not only they, but also family members, may require screening and monitoring. Diagnostic criteria have been developed, based primarily on Western populations, and several methodologies are available to identify dMMR tumours, including immunohistochemistry and microsatellite testing. These criteria have provided evidence supporting the introduction of reflex testing. Yet, it is becoming increasingly clear that tests have a limited sensitivity and specificity and may yet be superseded by next generation sequencing. In this review, the limitations of diagnostic criteria are discussed, and current and emerging screening technologies explained. There is now useful evidence supporting the prognostic and predictive value of dMMR status in colorectal tumours, but much less is known about their value in extracolonic tumours, that may also feature in Lynch syndrome. This review assesses current literature relating to dMMR in endometrial, ovarian, gastric and melanoma cancers, which it would seem, may benefit from large-scale clinical trials in order to further close the gap in knowledge between colorectal and extracolonic tumours. PMID:26315971

  17. Identification of Polycomb Group Protein EZH2-Mediated DNA Mismatch Repair Gene MSH2 in Human Uterine Fibroids.

    PubMed

    Yang, Qiwei; Laknaur, Archana; Elam, Lelyand; Ismail, Nahed; Gavrilova-Jordan, Larisa; Lue, John; Diamond, Michael P; Al-Hendy, Ayman

    2016-10-01

    Uterine fibroids (UFs) are benign smooth muscle neoplasms affecting up to 70% of reproductive age women. Treatment of symptomatic UFs places a significant economic burden on the US health-care system. Several specific genetic abnormalities have been described as etiologic factors of UFs, suggesting that a low DNA damage repair capacity may be involved in the formation of UF. In this study, we used human fibroid and adjacent myometrial tissues, as well as an in vitro cell culture model, to evaluate the expression of MutS homolog 2 (MSH2), which encodes a protein belongs to the mismatch repair system. In addition, we deciphered the mechanism by which polycomb repressive complex 2 protein, EZH2, deregulates MSH2 in UFs. The RNA expression analysis demonstrated the deregulation of MSH2 expression in UF tissues in comparison to its adjacent myometrium. Notably, protein levels of MSH2 were upregulated in 90% of fibroid tissues (9 of 10) as compared to matched adjacent myometrial tissues. Human fibroid primary cells treated with 3-deazaneplanocin A (DZNep), chemical inhibitor of EZH2, exhibited a significant increase in MSH2 expression (P < .05). Overexpression of EZH2 using an adenoviral vector approach significantly downregulated the expression of MSH2 (P < .05). Chromatin immunoprecipitation assay demonstrated that enrichment of H3K27me3 in promoter regions of MSH2 was significantly decreased in DZNep-treated fibroid cells as compared to vehicle control. These data suggest that EZH2-H3K27me3 regulatory mechanism dynamically changes the expression levels of DNA mismatch repair gene MSH2, through epigenetic mark H3K27me3. MSH2 may be considered as a marker for early detection of UFs. © The Author(s) 2016.

  18. HEREDITARY, SPORADIC AND METASTATIC COLORECTAL CANCER ARE COMMONLY DRIVEN BY SPECIFIC SPECTRUMS OF DEFECTIVE DNA MISMATCH REPAIR COMPONENTS

    PubMed Central

    CARETHERS, JOHN M.

    2016-01-01

    DNA mismatch repair (MMR) is one of several human cell mechanisms utilized to repair mutable mistakes within DNA, particularly after DNA is replicated. MMR function is dependent upon heterodimerization of specific MMR proteins that can recognize base-base mispairs as well as frameshifts at microsatellite sequences, followed by the triggering of other complementary proteins that execute excision and repair or initiate cell demise if repair is futile. MMR function is compromised in specific disease states, all of which can be biochemically recognized by faulty repair of microsatellite sequences, causing microsatellite instability. Germline mutation of an MMR gene causes Lynch syndrome, the most common inherited form of colorectal cancer (CRC), and biallelic germline mutations cause the rare constitutional mismatch repair deficiency syndrome. Somatic inactivation of MMR through epigenetic mechanisms is observed in 15% of sporadic CRC, and a smaller portion of CRCs possess biallelic somatic mutations. A novel inflammation-driven nuclear-to-cytoplasmic shift of the specific MMR protein hMSH3 is seen in up to 60% of sporadic CRCs that associates with metastasis and poor patient prognosis, unlike improved outcome when MMR is genetically inactivated. The mechanism for MMR inactication as well as the component affected dictates the clinical spectrum and clinical response for patients. PMID:28066040

  19. Mismatch binding, ADP-ATP exchange and intramolecular signaling during mismatch repair

    PubMed Central

    Hingorani, Manju M.

    2015-01-01

    The focus of this article is on the DNA binding and ATPase activities of the mismatch repair (MMR) protein, MutS—our current understanding of how this protein uses ATP to fuel its actions on DNA and initiate repair via interactions with MutL, the next protein in the pathway. Structure-function and kinetic studies have yielded detailed views of the MutS mechanism of action in MMR. How MutS and MutL work together after mismatch recognition to enable strand-specific nicking, which leads to strand excision and synthesis, is less clear and remains an active area of investigation. PMID:26704427

  20. DNA mismatch repair and the DNA damage response to ionizing radiation: making sense of apparently conflicting data.

    PubMed

    Martin, Lynn M; Marples, Brian; Coffey, Mary; Lawler, Mark; Lynch, Thomas H; Hollywood, Donal; Marignol, Laure

    2010-11-01

    The DNA mismatch repair (MMR) pathway detects and repairs DNA replication errors. While DNA MMR-proficiency is known to play a key role in the sensitivity to a number of DNA damaging agents, its role in the cytotoxicity of ionizing radiation (IR) is less well characterized. Available literature to date is conflicting regarding the influence of MMR status on radiosensitivity, and this has arisen as a subject of controversy in the field. The aim of this paper is to provide the first comprehensive overview of the experimental data linking MMR proteins and the DNA damage response to IR. A PubMed search was conducted using the key words "DNA mismatch repair" and "ionizing radiation". Relevant articles and their references were reviewed for their association between DNA MMR and IR. Recent data suggest that radiation dose and the type of DNA damage induced may dictate the involvement of the MMR system in the cellular response to IR. In particular, the literature supports a role for the MMR system in DNA damage recognition, cell cycle arrest, DNA repair and apoptosis. In this review we discuss our current understanding of the impact of MMR status on the cellular response to radiation in mammalian cells gained from past and present studies and attempt to provide an explanation for how MMR may determine the response to radiation. Copyright © 2010 Elsevier Ltd. All rights reserved.

  1. High prevalence of mismatch repair deficiency in prostate cancers diagnosed in mismatch repair gene mutation carriers from the colon cancer family registry

    PubMed Central

    Rosty, Christophe; Walsh, Michael D.; Lindor, Noralane M.; Thibodeau, Stephen N.; Mundt, Erin; Gallinger, Steven; Aronson, Melyssa; Pollett, Aaron; Baron, John A.; Pearson, Sally; Clendenning, Mark; Walters, Rhiannon J.; Nagler, Belinda N.; Crawford, William J.; Young, Joanne P.; Winship, Ingrid; Win, Aung Ko; Hopper, John L.; Jenkins, Mark A.

    2015-01-01

    The question of whether prostate cancer is part of the Lynch syndrome spectrum of tumors is unresolved. We investigated the mismatch repair (MMR) status and pathologic features of prostate cancers diagnosed in MMR gene mutation carriers. Prostate cancers (mean age at diagnosis = 62 ± SD = 8 years) from 32 MMR mutation carriers (23 MSH2, 5 MLH1 and 4 MSH6) enrolled in the Australasian, Mayo Clinic and Ontario sites of the Colon Cancer Family Registry were examined for clinico-pathologic features and MMR-deficiency (immunohistochemical loss of MMR protein expression and high levels of microsatellite instability; MSI-H). Tumor MMR-deficiency was observed for 22 cases [69 %; 95 % confidence interval (CI) 50–83 %], with the highest prevalence of MMR-deficiency in tumors from MSH2 mutation carriers (19/23, 83 %) compared with MLH1 and MSH6 carriers combined (3/9, 33 %; p = 0.01). MMR-deficient tumors had increased levels of tumor infiltrating lymphocytes compared with tumors without MMR-deficiency (p = 0.04). Under the assumption that tumour MMR-deficiency occurred only because the cancer was caused by the germline mutation, mutation carriers are at 3.2-fold (95 % CI 2.0–6.3) increased risk of prostate cancer, and when assessed by gene, the relative risk was greatest for MSH2 carriers (5.8, 95 % CI 2.6–20.9). Prostate cancer was the first or only diagnosed tumor in 37 % of carriers. MMR gene mutation carriers have at least a twofold or greater increased risk of developing MMR-deficient prostate cancer where the risk is highest for MSH2 mutation carriers. MMR IHC screening of prostate cancers will aid in identifying MMR gene mutation carriers. PMID:25117503

  2. MUTYH and the mismatch repair system: partners in crime?

    PubMed

    Niessen, Renée C; Sijmons, Rolf H; Ou, J; Olthof, Sandra G M; Osinga, Jan; Ligtenberg, Marjolijn J; Hogervorst, Frans B L; Weiss, Marjan M; Tops, Carli M J; Hes, Frederik J; de Bock, Geertruida H; Buys, Charles H C M; Kleibeuker, Jan H; Hofstra, Robert M W

    2006-03-01

    Biallelic germline mutations of MUTYH-a gene encoding a base excision repair protein-are associated with an increased susceptibility of colorectal cancer. Whether monoallelic MUTYH mutations also increase cancer risk is not yet clear, although there is some evidence suggesting a slight increase of risk. As the MUTYH protein interacts with the mismatch repair (MMR) system, we hypothesised that the combination of a monoallelic MUTYH mutation with an MMR gene mutation increases cancer risk. We therefore investigated the prevalence of monoallelic MUTYH mutations in carriers of a germline MMR mutation: 40 carriers of a truncating mutation (group I) and 36 of a missense mutation (group II). These patients had been diagnosed with either colorectal or endometrial cancer. We compared their MUTYH mutation frequencies with those observed in a group of 134 Dutch colorectal and endometrial cancer patients without an MMR gene mutation (0.7%) and those reported for Caucasian controls (1.5%). In group I one monoallelic MUTYH mutation was found (2.5%). In group II five monoallelic germline MUTYH mutations were found (14%), four of them in MSH6 missense mutation carriers (20%). Of all patients with an MMR gene mutation, only those with a missense mutation showed a significantly higher frequency of (monoallelic) MUTYH mutations than the Dutch cancer patients without MMR gene mutations (P = 0.002) and the published controls (P = 0.001). These results warrant further study to test the hypothesis of mutations in MMR genes (in particular MSH6) and MUTYH acting together to increase cancer risk.

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

    SciTech Connect

    Tseng, Quincy; Orans, Jillian; Hast, Michael A.; Iyer, Ravi R.; Changela, Anita; Modrich, Paul L.; Beese, Lorena S.

    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 of 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.

  4. The DNA mismatch repair protein MutS forms a one-dimensional Tonks gas on DNA

    NASA Astrophysics Data System (ADS)

    Bundschuh, Ralf; Klajner, Piotr; Hanne, Jeungphill; Britton, Brooke M.; Liu, Jianquan; Park, Jonghyun; Lee, Jong-Bong; Fishel, Richard

    2014-03-01

    MutS is a protein involved in DNA mismatch repair. It recognizes the mismatch, forms a sliding clamp around the DNA, and displaces other proteins bound to the DNA prior to the actual repair process. Here, we present a quantitative model of an ensemble of MutS molecules on a short strand of DNA with one mismatch. We model the ensemble as a Tonks gas of passively diffusing one-dimensional particles of finite extension and include clamp formation at the mismatch and random detachment. The distributions of MutS number bound to the DNA for different mismatch positions and different MutS concentrations in solution fit very well with distributions determined by single molecule experiments, thereby establishing the Tonks gas as an excellent model of MutS action on DNA. This material is based upon work supported by the National Science Foundation under Grant No. 01105458 (RB), the National Institutes of Health under Grant No. CA67007 (RF), and the National Research Foundation of Korea under Grant No. 2011-0013901 (JBL).

  5. Molecular patterns in deficient mismatch repair colorectal tumours: results from a French prospective multicentric biological and genetic study

    PubMed Central

    Etienne-Grimaldi, M-C; Mahamat, A; Chazal, M; Laurent-Puig, P; Olschwang, S; Gaub, M-P; Formento, J-L; Formento, P; Sudaka, A; Boige, V; Abderrahim-Ferkoune, A; Benchimol, D; André, T; Houry, S; Faucheron, J-L; Letoublon, C; Gilly, F-N; Delpero, J-R; Lasser, P; Pradere, B; Pezet, D; Penault-Llorca, F; Milano, G

    2014-01-01

    Background: To test the prognostic value of tumour protein and genetic markers in colorectal cancer (CRC) and examine whether deficient mismatch repair (dMMR) tumours had a distinct profile relative to proficient mismatch repair (pMMR) tumours. Methods: This prospective multicentric study involved 251 stage I–III CRC patients. Analysed biomarkers were EGFR (binding assay), VEGFA, thymidylate synthase (TS), thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) expressions, MMR status, mutations of KRAS (codons 12–13), BRAF (V600E), PIK3CA (exons 9 and 20), APC (exon 15) and P53 (exons 4–9), CpG island methylation phenotype status, ploidy, S-phase, LOH. Results: The only significant predictor of relapse-free survival (RFS) was tumour staging. Analyses restricted to stage III showed a trend towards a shorter RFS in KRAS-mutated (P=0.005), BRAF wt (P=0.009) and pMMR tumours (P=0.036). Deficient mismatch repair tumours significantly demonstrated higher TS (median 3.1 vs 1.4) and TP (median 5.8 vs 3.5) expression relative to pMMR (P<0.001) and show higher DPD expression (median 14.9 vs 7.9, P=0.027) and EGFR content (median 69 vs 38, P=0.037) relative to pMMR. Conclusions: Present data suggesting that both TS and DPD are overexpressed in dMMR tumours as compared with pMMR tumours provide a strong rationale that may explain the resistance of dMMR tumours to 5FU-based therapy. PMID:24800948

  6. 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.

  7. Dynamics of DNA Mismatch Repair

    NASA Astrophysics Data System (ADS)

    Coats, Julie; Lin, Yuyen; Rasnik, Ivan

    2009-11-01

    DNA mismatch repair protects the genome from spontaneous mutations by recognizing errors, excising damage, and re-synthesizing DNA in a pathway that is highly conserved. Mismatch recognition is accomplished by the MutS family of proteins which are weak ATPases that bind specifically to damaged DNA, but the specific molecular mechanisms by which these proteins recognize damage and initiate excision are not known. Previous structural investigations have implied that protein-induced conformational changes are central to mismatch recognition. Because damage detection is a highly dynamic process in which conformational changes of the protein-DNA complexes occur on a time scale of a few seconds, it is difficult to obtain meaningful kinetic information with traditional ensemble techniques. In this work, we use single molecule fluorescence resonance energy transfer (smFRET) to study the conformational dynamics of fluorescently labeled DNA substrates in the presence of the mismatch repair protein MutS from E. coli and its human homolog MSH2/MSH6. Our studies allow us to obtain quantitative kinetic information about the rates of binding and dissociation and to determine the conformational states for each protein-DNA complex.

  8. Evidence that the DNA mismatch repair system removes 1-nucleotide Okazaki fragment flaps.

    PubMed

    Kadyrova, Lyudmila Y; Dahal, Basanta K; Kadyrov, Farid A

    2015-10-02

    The DNA mismatch repair (MMR) system plays a major role in promoting genome stability and suppressing carcinogenesis. In this work, we investigated whether the MMR system is involved in Okazaki fragment maturation. We found that in the yeast Saccharomyces cerevisiae, the MMR system and the flap endonuclease Rad27 act in overlapping pathways that protect the nuclear genome from 1-bp insertions. In addition, we determined that purified yeast and human MutSα proteins recognize 1-nucleotide DNA and RNA flaps. In reconstituted human systems, MutSα, proliferating cell nuclear antigen, and replication factor C activate MutLα endonuclease to remove the flaps. ATPase and endonuclease mutants of MutLα are defective in the flap removal. These results suggest that the MMR system contributes to the removal of 1-nucleotide Okazaki fragment flaps.

  9. New Therapeutic Opportunities Based on DNA Mismatch Repair and BRAF Status in Metastatic Colorectal Cancer.

    PubMed

    Cohen, Romain; Svrcek, Magali; Dreyer, Chantal; Cervera, Pascale; Duval, Alex; Pocard, Marc; Fléjou, Jean-François; de Gramont, Aimery; André, Thierry

    2016-03-01

    Recently, colorectal cancer (CRC) subtyping consortium identified four consensus molecular subtypes (CMS1-4). CMS1 is enriched for deficient mismatch repair (dMMR) and BRAF (V600E) tumors. Intriguingly, this subtype has better relapse-free survival but worse overall survival after relapse compared with the other subtypes. Growing evidence is accumulating on the benefit of specific therapeutic strategies such as immune checkpoint inhibition therapy in dMMR tumors and mitogen-activated protein kinase (MAPK) pathway targeted therapy in tumors harboring BRAF (V600E) mutation. After reviewing dMMR prognostic value, immune checkpoints as major targets for dMMR carcinomas will be highlighted. Following, BRAF (V600E) prognostic impact will be reviewed and therapeutic strategies with the combination of cytotoxic agents and especially the combinations of BRAF and MAPK inhibitors will be discussed.

  10. Oxidative mutagenesis, mismatch repair, and aging.

    PubMed

    Skinner, Amy M; Turker, Mitchell S

    2005-03-02

    A PubMed search for the term "oxidative stress" yields over 29,000 articles published on the subject over the past 10 years; more than 2000 of these articles also include the term "aging" in their title or abstract. Many theories of aging predict causal roles for oxidative stress in the myriad of pathological changes that occur as a function of age, including an increasing propensity to develop cancer. A possible link between aging and cancer is the induction and accumulation of somatic mutations caused by oxidative stress. This Review focuses on small mutational events that are induced by oxidative stress and the role of mismatch repair (MMR) in preventing their formation. It also discusses a possible inhibitory effect of oxidative stress on MMR. We speculate that a synergistic interaction between oxidative damage to DNA and reduced MMR levels will, in part, account for an accumulation of small mutational events, and hence cancer, with aging.

  11. Mismatch repair during homologous and homeologous recombination.

    PubMed

    Spies, Maria; Fishel, Richard

    2015-03-02

    Homologous recombination (HR) and mismatch repair (MMR) are inextricably linked. HR pairs homologous chromosomes before meiosis I and is ultimately responsible for generating genetic diversity during sexual reproduction. HR is initiated in meiosis by numerous programmed DNA double-strand breaks (DSBs; several hundred in mammals). A characteristic feature of HR is the exchange of DNA strands, which results in the formation of heteroduplex DNA. Mismatched nucleotides arise in heteroduplex DNA because the participating parental chromosomes contain nonidentical sequences. These mismatched nucleotides may be processed by MMR, resulting in nonreciprocal exchange of genetic information (gene conversion). MMR and HR also play prominent roles in mitotic cells during genome duplication; MMR rectifies polymerase misincorporation errors, whereas HR contributes to replication fork maintenance, as well as the repair of spontaneous DSBs and genotoxic lesions that affect both DNA strands. MMR suppresses HR when the heteroduplex DNA contains excessive mismatched nucleotides, termed homeologous recombination. The regulation of homeologous recombination by MMR ensures the accuracy of DSB repair and significantly contributes to species barriers during sexual reproduction. This review discusses the history, genetics, biochemistry, biophysics, and the current state of studies on the role of MMR in homologous and homeologous recombination from bacteria to humans.

  12. Mismatch Repair during Homologous and Homeologous Recombination

    PubMed Central

    Spies, Maria; Fishel, Richard

    2015-01-01

    Homologous recombination (HR) and mismatch repair (MMR) are inextricably linked. HR pairs homologous chromosomes before meiosis I and is ultimately responsible for generating genetic diversity during sexual reproduction. HR is initiated in meiosis by numerous programmed DNA double-strand breaks (DSBs; several hundred in mammals). A characteristic feature of HR is the exchange of DNA strands, which results in the formation of heteroduplex DNA. Mismatched nucleotides arise in heteroduplex DNA because the participating parental chromosomes contain nonidentical sequences. These mismatched nucleotides may be processed by MMR, resulting in nonreciprocal exchange of genetic information (gene conversion). MMR and HR also play prominent roles in mitotic cells during genome duplication; MMR rectifies polymerase misincorporation errors, whereas HR contributes to replication fork maintenance, as well as the repair of spontaneous DSBs and genotoxic lesions that affect both DNA strands. MMR suppresses HR when the heteroduplex DNA contains excessive mismatched nucleotides, termed homeologous recombination. The regulation of homeologous recombination by MMR ensures the accuracy of DSB repair and significantly contributes to species barriers during sexual reproduction. This review discusses the history, genetics, biochemistry, biophysics, and the current state of studies on the role of MMR in homologous and homeologous recombination from bacteria to humans. PMID:25731766

  13. Myc down-regulation sensitizes melanoma cells to radiotherapy by inhibiting MLH1 and MSH2 mismatch repair proteins.

    PubMed

    Bucci, Barbara; D'Agnano, Igea; Amendola, Donatella; Citti, Arianna; Raza, Giorgio H; Miceli, Roberto; De Paula, Ugo; Marchese, Rodolfo; Albini, Sonia; Felsani, Armando; Brunetti, Ercole; Vecchione, Aldo

    2005-04-01

    Melanoma patients have a very poor prognosis with a response rate of <1% due to advanced diagnosis. This type of tumor is particularly resistant to conventional chemotherapy and radiotherapy, and the surgery remains the principal treatment for patients with localized melanoma. For this reason, there is particular interest in the melanoma biological therapy. Using two p53 mutant melanoma models stably expressing an inducible c-myc antisense RNA, we have investigated whether Myc protein down-regulation could render melanoma cells more susceptible to radiotherapy, reestablishing apoptotic p53-independent pathway. In addition to address the role of p53 in the activation of apoptosis, we studied the effect of Myc down-regulation on radiotherapy sensitivity also in a p53 wild-type melanoma cell line. Myc down-regulation is able per se to induce apoptosis in a fraction of the cell population (approximately 40% at 72 hours) and in combination with gamma radiation efficiently enhances the death process. In fact, approximately 80% of apoptotic cells are evident in Myc down-regulated cells exposed to gamma radiation for 72 hours compared with approximately 13% observed after only gamma radiation treatment. Consistent with the enhanced apoptosis is the inhibition of the MLH1 and MSH2 mismatch repair proteins, which, preventing the correction of ionizing radiation mismatches occurring during DNA replication, renders the cells more prone to radiation-induced apoptosis. Data herein reported show that Myc down-regulation lowers the apoptotic threshold in melanoma cells by inhibiting MLH1 and MSH2 proteins, thus increasing cell sensitivity to gamma radiation in a p53-independent fashion. Our results indicate the basis for developing new antitumoral therapeutic strategy, improving the management of melanoma patients.

  14. The dual nature of mismatch repair as antimutator and mutator: for better or for worse

    PubMed Central

    Bak, Sara Thornby; Sakellariou, Despoina; Pena-Diaz, Javier

    2014-01-01

    DNA is constantly under attack by a number of both exogenous and endogenous agents that challenge its integrity. Among the mechanisms that have evolved to counteract this deleterious action, mismatch repair (MMR) has specialized in removing DNA biosynthetic errors that occur when replicating the genome. Malfunction or inactivation of this system results in an increase in spontaneous mutability and a strong predisposition to tumor development. Besides this key corrective role, MMR proteins are involved in other pathways of DNA metabolism such as mitotic and meiotic recombination and processing of oxidative damage. Surprisingly, MMR is also required for certain mutagenic processes. The mutagenic MMR has beneficial consequences contributing to the generation of a vast repertoire of antibodies through class switch recombination and somatic hypermutation processes. However, this non-canonical mutagenic MMR also has detrimental effects; it promotes repeat expansions associated with neuromuscular and neurodegenerative diseases and may contribute to cancer/disease-related aberrant mutations and translocations. The reaction responsible for replication error correction has been the most thoroughly studied and it is the subject to numerous reviews. This review describes briefly the biochemistry of MMR and focuses primarily on the non-canonical MMR activities described in mammals as well as emerging research implicating interplay of MMR and chromatin. PMID:25191341

  15. Phosphorylation of PCNA by EGFR inhibits mismatch repair and promotes misincorporation during DNA synthesis

    PubMed Central

    Ortega, Janice; Li, Jessie Y.; Lee, Sanghee; Tong, Dan; Gu, Liya; Li, Guo-Min

    2015-01-01

    Proliferating cell nuclear antigen (PCNA) plays essential roles in eukaryotic cells during DNA replication, DNA mismatch repair (MMR), and other events at the replication fork. Earlier studies show that PCNA is regulated by posttranslational modifications, including phosphorylation of tyrosine 211 (Y211) by the epidermal growth factor receptor (EGFR). However, the functional significance of Y211-phosphorylated PCNA remains unknown. Here, we show that PCNA phosphorylation by EGFR alters its interaction with mismatch-recognition proteins MutSα and MutSβ and interferes with PCNA-dependent activation of MutLα endonuclease, thereby inhibiting MMR at the initiation step. Evidence is also provided that Y211-phosphorylated PCNA induces nucleotide misincorporation during DNA synthesis. These findings reveal a novel mechanism by which Y211-phosphorylated PCNA promotes cancer development and progression via facilitating error-prone DNA replication and suppressing the MMR function. PMID:25825764

  16. Native mass spectrometry provides direct evidence for DNA mismatch-induced regulation of asymmetric nucleotide binding in mismatch repair protein MutS.

    PubMed

    Monti, Maria Chiara; Cohen, Serge X; Fish, Alexander; Winterwerp, Herrie H K; Barendregt, Arjan; Friedhoff, Peter; Perrakis, Anastassis; Heck, Albert J R; Sixma, Titia K; van den Heuvel, Robert H H; Lebbink, Joyce H G

    2011-10-01

    The DNA mismatch repair protein MutS recognizes mispaired bases in DNA and initiates repair in an ATP-dependent manner. Understanding of the allosteric coupling between DNA mismatch recognition and two asymmetric nucleotide binding sites at opposing sides of the MutS dimer requires identification of the relevant MutS.mmDNA.nucleotide species. Here, we use native mass spectrometry to detect simultaneous DNA mismatch binding and asymmetric nucleotide binding to Escherichia coli MutS. To resolve the small differences between macromolecular species bound to different nucleotides, we developed a likelihood based algorithm capable to deconvolute the observed spectra into individual peaks. The obtained mass resolution resolves simultaneous binding of ADP and AMP.PNP to this ABC ATPase in the absence of DNA. Mismatched DNA regulates the asymmetry in the ATPase sites; we observe a stable DNA-bound state containing a single AMP.PNP cofactor. This is the first direct evidence for such a postulated mismatch repair intermediate, and showcases the potential of native MS analysis in detecting mechanistically relevant reaction intermediates.

  17. Synergism of Dam, MutH, and MutS in methylation-directed mismatch repair in Escherichia coli.

    PubMed

    Hu, Changkun; Zhao, Yunqi; Sun, Huiyun; Yang, Yixin

    2017-01-01

    DNA mismatch repair (MMR) is a critical mutation surveillance system for recognizing and repairing erroneous insertion, deletion, and disincorporation of base. Major components of mismatch repair system consist of MutH, MutL, and MutS. Dam methylates adenine to distinguish newly synthesized daughter strands from the parent strands. Employing a tyrosine-auxotrophic E. coli FX-11 strain, the mutation frequency can be determined by the number of tyrosine revertants and the cell viability of FX-11 with deficiencies in dam and mismatch repair proteins. This study showed that mutS defect produced a higher mutation frequency than mutH did. Interestingly, double defects in dam and mutS synergistically produced a dramatically higher spontaneous mutation frequency than the summation of mutation frequencies of FX-11 strains with individual deficiency of dam or mutS, suggesting that Dam may work with MutHL to partially accomplish the task of recognizing the mismatch sites to retain partial mismatch repair capacity.

  18. Eukaryotic Mismatch Repair in Relation to DNA Replication.

    PubMed

    Kunkel, Thomas A; Erie, Dorothy A

    2015-01-01

    Three processes act in series to accurately replicate the eukaryotic nuclear genome. The major replicative DNA polymerases strongly prevent mismatch formation, occasional mismatches that do form are proofread during replication, and rare mismatches that escape proofreading are corrected by mismatch repair (MMR). This review focuses on MMR in light of increasing knowledge about nuclear DNA replication enzymology and the rate and specificity with which mismatches are generated during leading- and lagging-strand replication. We consider differences in MMR efficiency in relation to mismatch recognition, signaling to direct MMR to the nascent strand, mismatch removal, and the timing of MMR. These studies are refining our understanding of relationships between generating and repairing replication errors to achieve accurate replication of both DNA strands of the nuclear genome.

  19. Exonic deletions of mismatch repair genes MLH1 and MSH2 correlate with prognosis and protein expression levels in malignant melanomas.

    PubMed

    Korabiowska, Monika; Brinck, Ulrich; Stachura, Jerzy; Jawien, Jacek; Hasse, Frank Michael; Cordon-Cardos, Carlos; Fischer, Gösta

    2006-01-01

    The mutations of MLH1 and MSH2 have been reported to be responsible for malignant transformation and tumour progression in several sporadic tumours. Eighty-six primary malignant melanomas with known follow-up were investigated. Point mutations of DNA mismatch repair MLH1 and MSH2 in malignant melanomas were not found. Exon 12 (MSH2) was not present in 26 out of the 86 melanomas and exon 13 (MSH2) was lost in 25 of the tumours. The loss of exon 15 (MLH1) was observed in 22 out of the 86 tumours and the loss of exon 16 (MLH1) in 24 melanomas. The loss of exons correlated strongly with the loss of MLH1 and MSH2 protein expression. In multivariate analysis, including all 4 exons and expressions of MLH1 and MSH2, prognostic significance was found only for loss of exon 12 (MSH2) and loss of exon 15 (MLH1).

  20. Crosstalk between BRCA-Fanconi anemia and mismatch repair pathways prevents MSH2-dependent aberrant DNA damage responses.

    PubMed

    Peng, Min; Xie, Jenny; Ucher, Anna; Stavnezer, Janet; Cantor, Sharon B

    2014-08-01

    Several proteins in the BRCA-Fanconi anemia (FA) pathway, such as FANCJ, BRCA1, and FANCD2, interact with mismatch repair (MMR) pathway factors, but the significance of this link remains unknown. Unlike the BRCA-FA pathway, the MMR pathway is not essential for cells to survive toxic DNA interstrand crosslinks (ICLs), although MMR proteins bind ICLs and other DNA structures that form at stalled replication forks. We hypothesized that MMR proteins corrupt ICL repair in cells that lack crosstalk between BRCA-FA and MMR pathways. Here, we show that ICL sensitivity of cells lacking the interaction between FANCJ and the MMR protein MLH1 is suppressed by depletion of the upstream mismatch recognition factor MSH2. MSH2 depletion suppresses an aberrant DNA damage response, restores cell cycle progression, and promotes ICL resistance through a Rad18-dependent mechanism. MSH2 depletion also suppresses ICL sensitivity in cells deficient for BRCA1 or FANCD2, but not FANCA. Rescue by Msh2 loss was confirmed in Fancd2-null primary mouse cells. Thus, we propose that regulation of MSH2-dependent DNA damage response underlies the importance of interactions between BRCA-FA and MMR pathways.

  1. Crosstalk between BRCA-Fanconi anemia and mismatch repair pathways prevents MSH2-dependent aberrant DNA damage responses

    PubMed Central

    Peng, Min; Xie, Jenny; Ucher, Anna; Stavnezer, Janet; Cantor, Sharon B

    2014-01-01

    Several proteins in the BRCA-Fanconi anemia (FA) pathway, such as FANCJ, BRCA1, and FANCD2, interact with mismatch repair (MMR) pathway factors, but the significance of this link remains unknown. Unlike the BRCA-FA pathway, the MMR pathway is not essential for cells to survive toxic DNA interstrand crosslinks (ICLs), although MMR proteins bind ICLs and other DNA structures that form at stalled replication forks. We hypothesized that MMR proteins corrupt ICL repair in cells that lack crosstalk between BRCA-FA and MMR pathways. Here, we show that ICL sensitivity of cells lacking the interaction between FANCJ and the MMR protein MLH1 is suppressed by depletion of the upstream mismatch recognition factor MSH2. MSH2 depletion suppresses an aberrant DNA damage response, restores cell cycle progression, and promotes ICL resistance through a Rad18-dependent mechanism. MSH2 depletion also suppresses ICL sensitivity in cells deficient for BRCA1 or FANCD2, but not FANCA. Rescue by Msh2 loss was confirmed in Fancd2-null primary mouse cells. Thus, we propose that regulation of MSH2-dependent DNA damage response underlies the importance of interactions between BRCA-FA and MMR pathways. PMID:24966277

  2. Lynch Syndrome from a surgeon perspective: retrospective study of clinical impact of mismatch repair protein expression analysis in colorectal cancer patients less than 50 years old

    PubMed Central

    2014-01-01

    Background In clinical practice, unexpected diagnosis of colorectal cancer in young patients requires prompt surgery, thus genetic testing for Lynch Syndrome is frequently missed, and clinical management may result incorrect. Methods Patients younger than 50 years old undergoing colorectal resection for cancer in the period 1994-2007 were identified (Group A, 49 cases), and compared to a group of randomly selected patients more than 50 (Group B, 85 cases). In 31 group A patients, immunohistochemical expression analysis of MLH1, MSH2 and MSH6 was performed; personal and familial history of patients with defective MMR proteins expression was further investigated, searching for synchronous and metachronous tumors in probands and their families. Results Fifty-one percent of patients did not express one or more MMR proteins (MMR-) and should be considered Lynch Syndrome carriers (16 patients, group A1); while only 31.2% of them were positive for Amsterdam criteria, 50% had almost another tumor, 37.5% had another colorectal tumor and 68% had relatives with colorectal tumor. This group of patients, compared with A2 group (< 50 years old, MMR+) and B group, showed typical characteristics of HNPCC, such as proximal location, mucinous histotype, poor differentiation, high stage and shorter survival. Conclusions The present study confirms that preoperative knowledge of MMR proteins expression in colorectal cancer patients would allow correct staging, more extended colonic resection, specific follow-up and familial screening. PMID:24533633

  3. Mismatch repair genes expression defects & association with clinicopathological characteristics in colorectal carcinoma

    PubMed Central

    Kaur, Gurjeet; Masoud, Abdelhafid; Raihan, N.; Radzi, M.; Khamizar, W.; Kam, Lee Suk

    2011-01-01

    Background & objectives: DNA mismatch repair gene (MMR) abnormalities are seen in 95 per cent of hereditary nonpolyposis colorectal cancer (HNPCC) and 10-15 per cent of sporadic colorectal cancers. There are no data on MMR abnormalities in Malaysian colorectal cancer patients. This study was aimed to determine the frequency of abnormal MMR gene protein expression in colorectal carcinoma in Northern Peninsular Malaysia using immunohistochemistry. Methods: Clinicopathological information was obtained from 148 patients’ records who underwent bowel resection for colorectal cancer (CRC) at the three hospitals in Malaysia. Immunohistochemistry for MLH1, MSH2, MSH6 and PMS2 proteins were performed on paraffin embedded tissue containing carcinoma. Results: A total of 148 subjects and 150 colorectal carcinomas of sporadic and hereditary types were assessed. Three patients had synchronous tumours. Twenty eight cancers (18.6%) from 26 subjects (17.6%) had absent immunohistochemical expression of any one of the MMR gene proteins. This comprised absent MLH1 only – 3 cancers, absent MSH2 only – 3, absent MSH6 only – 2, absent PMS2 only – 3, absent MLH1 and PMS2 – 14, absent MSH2 and MSH6 – 2 and absent MLH1, MSH6 and PMS2 – 1. There was significant association between abnormal MMR gene protein expression and proximal colon cancers, mucinous, signet ring and poorly differentiated morphology. Interpretation & conclusions: Cancers with abnormal MMR gene expression were associated with microsatellite instability-high (MSI-H) phenotype. About 15 per cent demonstrated absent MSH2, MSH6 and PMS2 protein expression in isolation or in combination with other MMR genes, which often predicts a germline mutation, synonymous with a diagnosis of HNPCC. This appears to be high frequency compared to reported data. PMID:21911971

  4. LNA modification of single-stranded DNA oligonucleotides allows subtle gene modification in mismatch-repair-proficient cells.

    PubMed

    van Ravesteyn, Thomas W; Dekker, Marleen; Fish, Alexander; Sixma, Titia K; Wolters, Astrid; Dekker, Rob J; Te Riele, Hein P J

    2016-04-12

    Synthetic single-stranded DNA oligonucleotides (ssODNs) can be used to generate subtle genetic modifications in eukaryotic and prokaryotic cells without the requirement for prior generation of DNA double-stranded breaks. However, DNA mismatch repair (MMR) suppresses the efficiency of gene modification by >100-fold. Here we present a commercially available ssODN design that evades MMR and enables subtle gene modification in MMR-proficient cells. The presence of locked nucleic acids (LNAs) in the ssODNs at mismatching bases, or also at directly adjacent bases, allowed 1-, 2-, or 3-bp substitutions in MMR-proficient mouse embryonic stem cells as effectively as in MMR-deficient cells. Additionally, in MMR-proficient Escherichia coli, LNA modification of the ssODNs enabled effective single-base-pair substitution. In vitro, LNA modification of mismatches precluded binding of purified E. coli MMR protein MutS. These findings make ssODN-directed gene modification particularly well suited for applications that require the evaluation of a large number of sequence variants with an easy selectable phenotype.

  5. LNA modification of single-stranded DNA oligonucleotides allows subtle gene modification in mismatch-repair-proficient cells

    PubMed Central

    van Ravesteyn, Thomas W.; Dekker, Marleen; Fish, Alexander; Sixma, Titia K.; Wolters, Astrid; Dekker, Rob J.; te Riele, Hein P. J.

    2016-01-01

    Synthetic single-stranded DNA oligonucleotides (ssODNs) can be used to generate subtle genetic modifications in eukaryotic and prokaryotic cells without the requirement for prior generation of DNA double-stranded breaks. However, DNA mismatch repair (MMR) suppresses the efficiency of gene modification by >100-fold. Here we present a commercially available ssODN design that evades MMR and enables subtle gene modification in MMR-proficient cells. The presence of locked nucleic acids (LNAs) in the ssODNs at mismatching bases, or also at directly adjacent bases, allowed 1-, 2-, or 3-bp substitutions in MMR-proficient mouse embryonic stem cells as effectively as in MMR-deficient cells. Additionally, in MMR-proficient Escherichia coli, LNA modification of the ssODNs enabled effective single-base-pair substitution. In vitro, LNA modification of mismatches precluded binding of purified E. coli MMR protein MutS. These findings make ssODN-directed gene modification particularly well suited for applications that require the evaluation of a large number of sequence variants with an easy selectable phenotype. PMID:26951689

  6. Reliable Detection of Mismatch Repair Deficiency in Colorectal Cancers Using Mutational Load in Next-Generation Sequencing Panels

    PubMed Central

    Stadler, Zsofia K.; Battaglin, Francesca; Middha, Sumit; Hechtman, Jaclyn F.; Tran, Christina; Cercek, Andrea; Yaeger, Rona; Segal, Neil H.; Varghese, Anna M.; Reidy-Lagunes, Diane L.; Kemeny, Nancy E.; Salo-Mullen, Erin E.; Ashraf, Asad; Weiser, Martin R.; Garcia-Aguilar, Julio; Robson, Mark E.; Offit, Kenneth; Arcila, Maria E.; Berger, Michael F.; Shia, Jinru; Solit, David B.

    2016-01-01

    Purpose Tumor screening for Lynch syndrome is recommended in all or most patients with colorectal cancer (CRC). In metastatic CRC, sequencing of RAS/BRAF is necessary to guide clinical management. We hypothesized that a next-generation sequencing (NGS) panel that identifies RAS/BRAF and other actionable mutations could also reliably identify tumors with DNA mismatch repair protein deficiency (MMR-D) on the basis of increased mutational load. Methods We identified all CRCs that underwent genomic mutation profiling with a custom NGS assay (MSK-IMPACT) between March 2014 and July 2015. Tumor mutational load, with exclusion of copy number changes, was determined for each case and compared with MMR status as determined by routine immunohistochemistry. Results Tumors from 224 patients with unique CRC analyzed for MMR status also underwent MSK-IMPACT. Thirteen percent (n = 28) exhibited MMR-D by immunohistochemistry. Using the 341-gene assay, 100% of the 193 tumors with < 20 mutations were MMR-proficient. Of 31 tumors with ≥ 20 mutations, 28 (90%) were MMR-D. The three remaining tumors were easily identified as being distinct from the MMR-D tumors with > 150 mutations each. Each of these tumors harbored the P286R hotspot POLE mutation consistent with the ultramutator phenotype. Among MMR-D tumors, the median number of mutations was 50 (range, 20 to 90) compared with six (range, 0 to 17) in MMR-proficient/POLE wild-type tumors (P < .001). With a mutational load cutoff of ≥ 20 and < 150 for MMR-D detection, sensitivity and specificity were both 1.0 (95% CI, 0.93 to 1.0). Conclusion A cutoff for mutational load can be identified via multigene NGS tumor profiling, which provides a highly accurate means of screening for MMR-D in the same assay that is used for tumor genotyping. PMID:27022117

  7. Mismatch repair in heteroduplex DNA.

    PubMed Central

    Wildenberg, J; Meselson, M

    1975-01-01

    DNA with base pair mismatches was prepared by annealing mixtures of genetically marked DNA from bacteriophage lambda. This heteroduplex DNA was used to transfect bacteria under conditions minimizing recombination. Genetic analysis of the progeny phages indicates that: (i) Mismatch repair occurs, usually giving rise to a DNA molecule with one chain with the genotype arising from repair and one parental chain. (ii) The frequency of repair of a given mismatch to wild type depends on the marker, ranging from 3 to 20%. (iii) Excision tracts may extend several hundred nucleotides but are usually shorter than about 2000 nucleotides. (iv) In Rec-mediated bacteriophage crosses, recombination of markers closer than about 10-3 nucleotide pairs frequently occurs by mismatch repair within heteroduplex DNA. (V) The average amount of heteroduplex DNA formed in a Rec-mediated recombination event is a few thousand nucleotide pairs. PMID:1094458

  8. Approaches to diagnose DNA mismatch repair gene defects in cancer.

    PubMed

    Peña-Diaz, Javier; Rasmussen, Lene Juel

    2016-02-01

    The DNA repair pathway mismatch repair (MMR) is responsible for the recognition and correction of DNA biosynthetic errors caused by inaccurate nucleotide incorporation during replication. Faulty MMR leads to failure to address the mispairs or insertion deletion loops (IDLs) left behind by the replicative polymerases and results in increased mutation load at the genome. The realization that defective MMR leads to a hypermutation phenotype and increased risk of tumorigenesis highlights the relevance of this pathway for human disease. The association of MMR defects with increased risk of cancer development was first observed in colorectal cancer patients that carried inactivating germline mutations in MMR genes and the disease was named as hereditary non-polyposis colorectal cancer (HNPCC). Currently, a growing list of cancers is found to be MMR defective and HNPCC has been renamed Lynch syndrome (LS) partly to include the associated risk of developing extra-colonic cancers. In addition, a number of non-hereditary, mostly epigenetic, alterations of MMR genes have been described in sporadic tumors. Besides conferring a strong cancer predisposition, genetic or epigenetic inactivation of MMR genes also renders cells resistant to some chemotherapeutic agents. Therefore, diagnosis of MMR deficiency has important implications for the management of the patients, the surveillance of their relatives in the case of LS and for the choice of treatment. Some of the alterations found in MMR genes have already been well defined and their pathogenicity assessed. Despite this substantial wealth of knowledge, the effects of a large number of alterations remain uncharacterized (variants of uncertain significance, VUSs). The advent of personalized genomics is likely to increase the list of VUSs found in MMR genes and anticipates the need of diagnostic tools for rapid assessment of their pathogenicity. This review describes current tools and future strategies for addressing the relevance

  9. Involvement of p53 mutation and mismatch repair proteins dysregulation in NNK-induced malignant transformation of human bronchial epithelial cells.

    PubMed

    Shen, Ying; Zhang, Shuilian; Huang, Xiaobin; Chen, Kailin; Shen, Jing; Wang, Zhengyang

    2014-01-01

    Genome integrity is essential for normal cellular functions and cell survival. Its instability can cause genetic aberrations and is considered as a hallmark of most cancers. To investigate the carcinogenesis process induced by tobacco-specific carcinogen NNK, we studied the dynamic changes of two important protectors of genome integrity, p53 and MMR system, in malignant transformation of human bronchial epithelial cells after NNK exposure. Our results showed that the expression of MLH1, one of the important MMR proteins, was decreased early and maintained the downregulation during the transformation in a histone modification involved and DNA methylation-independent manner. Another MMR protein PMS2 also displayed a declined expression while being in a later stage of transformation. Moreover, we conducted p53 mutation analysis and revealed a mutation at codon 273 which led to the replacement of arginine by histidine. With the mutation, DNA damage-induced activation of p53 was significantly impaired. We further reintroduced the wild-type p53 into the transformed cells, and the malignant proliferation can be abrogated by inducing cell cycle arrest and apoptosis. These findings indicate that p53 and MMR system play an important role in the initiation and progression of NNK-induced transformation, and p53 could be a potential therapeutic target for tobacco-related cancers.

  10. Mismatch Repair Proficiency and In Vitro Response to 5-Fluorouracil

    PubMed Central

    CARETHERS, JOHN M.; CHAUHAN, DHARAM P.; FINK, DANIEL; NEBEL, SIBYLLE; BRESALIER, ROBERT S.; HOWELL, STEPHEN B.; BOLAND, C. RICHARD

    2015-01-01

    Background & Aims The DNA mismatch repair (MMR) system recognizes certain DNA adducts caused by alkylation damage in addition to its role in recognizing and directing repair of interstrand nucleotide mismatches and slippage mistakes at microsatellite sequences. Because defects in the MMR system can confer tolerance to acquired DNA damage and, by inference, the toxic effects of certain chemotherapeutic agents, we investigated the effect of 5-fluorouracil (5-FU) on colon cancer cell lines. Methods We determined growth selection by cell enrichment assay and cloning efficiency after treatment with 5 μmol/L 5-FU, assayed nucleic 3H–5-FU incorporation, and analyzed the cell cycle by flow cytometry. Results 5-FU treatment provided a growth advantage for MMR-deficient cell lines, indicating a relative degree of tolerance to 5-FU by the MMR-deficient cell lines. Enhanced survival was statistically significant after 5 days of growth, and a 28-fold reduction in survival was noted in the MMR-proficient cells by clonagenic assays after 10 days of growth. Differences in nucleotide uptake of 5-FU did not account for the observed growth differences, and specific cell cycle checkpoint arrest was not detected. Conclusions Intact DNA MMR seems to recognize 5-FU incorporated into DNA but may do so in a different manner than other types of alkylation damage. Defective DNA MMR might be one mechanism for tumor resistance to 5-FU. PMID:10381918

  11. Mouse models of DNA mismatch repair in cancer research

    PubMed Central

    Lee, Kyeryoung; Tosti, Elena; Edelmann, Winfried

    2016-01-01

    Germline mutations in DNA mismatch repair (MMR) genes are the cause of hereditary non-polyposis colorectal cancer/Lynch syndrome (HNPCC/LS) one of the most common cancer predisposition syndromes, and defects in MMR are also prevalent in sporadic colorectal cancers. In the past, the generation and analysis of mouse lines with knockout mutations in all of the known MMR genes has provided insight into how loss of individual MMR genes affects genome stability and contributes to cancer susceptibility. These studies also revealed essential functions for some of the MMR genes in B cell maturation and fertility. In this review, we will provide a brief overview of the cancer predisposition phenotypes of recently developed mouse models with targeted mutations in MutS and MutL homologs (Msh and Mlh, respectively) and their utility as preclinical models. The focus will be on mouse lines with conditional MMR mutations that have allowed more accurate modeling of human cancer syndromes in mice and that together with new technologies in gene targeting, hold great promise for the analysis of MMR-deficient intestinal tumors and other cancers which will drive the development of preventive and therapeutic treatment strategies. PMID:26708047

  12. Mouse models of DNA mismatch repair in cancer research.

    PubMed

    Lee, Kyeryoung; Tosti, Elena; Edelmann, Winfried

    2016-02-01

    Germline mutations in DNA mismatch repair (MMR) genes are the cause of hereditary non-polyposis colorectal cancer/Lynch syndrome (HNPCC/LS) one of the most common cancer predisposition syndromes, and defects in MMR are also prevalent in sporadic colorectal cancers. In the past, the generation and analysis of mouse lines with knockout mutations in all of the known MMR genes has provided insight into how loss of individual MMR genes affects genome stability and contributes to cancer susceptibility. These studies also revealed essential functions for some of the MMR genes in B cell maturation and fertility. In this review, we will provide a brief overview of the cancer predisposition phenotypes of recently developed mouse models with targeted mutations in MutS and MutL homologs (Msh and Mlh, respectively) and their utility as preclinical models. The focus will be on mouse lines with conditional MMR mutations that have allowed more accurate modeling of human cancer syndromes in mice and that together with new technologies in gene targeting, hold great promise for the analysis of MMR-deficient intestinal tumors and other cancers which will drive the development of preventive and therapeutic treatment strategies.

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

    PubMed Central

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

    2017-01-01

    BACKGROUND: 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. AIM: 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). MATERIAL AND METHODS: 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. RESULTS: 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. CONCLUSION: Testing for MSI should be done for all colorectal cancer patients, especially those younger than 50 years old, right sided and high-grade CRCs. PMID:28293308

  14. Mismatch repair status may predict response to adjuvant chemotherapy in resectable pancreatic ductal adenocarcinoma.

    PubMed

    Riazy, Maziar; Kalloger, Steve E; Sheffield, Brandon S; Peixoto, Renata D; Li-Chang, Hector H; Scudamore, Charles H; Renouf, Daniel J; Schaeffer, David F

    2015-10-01

    Deficiencies in DNA mismatch repair have been associated with inferior response to 5-FU in colorectal cancer. Pancreatic ductal adenocarcinoma is similarly treated with pyrimidine analogs, yet the predictive value of mismatch repair status for response to these agents has not been examined in this malignancy. A tissue microarray with associated clinical outcome, comprising 254 resected pancreatic ductal adenocarcinoma patients was stained for four mismatch repair proteins (MLH1, MSH2, MSH6 and PMS2). Mismatch repair deficiency and proficiency was determined by the absence or presence of uniform nuclear staining in tumor cells, respectively. Cases identified as mismatch repair deficient on the tissue microarray were confirmed by immunohistochemistry on whole slide sections. Of the 265 cases, 78 (29%) received adjuvant treatment with a pyrimidine analog and 41 (15%) showed a mismatch repair-deficient immunoprofile. Multivariable disease-specific survival in the mismatch repair-proficient cohort demonstrated that adjuvant chemotherapy, regional lymph-node status, gender, and the presence of tumor budding were significant independent prognostic variables (P≤0.04); however, none of the eight clinico-pathologic covariates examined in the mismatch repair-deficient cohort were of independent prognostic significance. Univariable assessment of disease-specific survival revealed an almost identical survival profile for both treated and untreated patients with a mismatch repair-deficient profile, while treatment in the mismatch repair-proficient cohort conferred a greater than 10-month median disease-specific survival advantage over their untreated counterparts (P=0.0018). In this cohort, adjuvant chemotherapy with a pyrimidine analog conferred no survival advantage to mismatch repair-deficient pancreatic ductal adenocarcinoma patients. Mismatch repair immunoprofiling is a feasible predictive marker in pancreatic ductal adenocarcinoma patients, and further prospective

  15. Genome Wide In silico Analysis of the Mismatch Repair Components of Plasmodium falciparum and Their Comparison with Human Host

    PubMed Central

    Tarique, Mohammed; Ahmad, Moaz; Chauhan, Manish; Tuteja, Renu

    2017-01-01

    Malaria a major parasitic infection globally particularly in tropical and sub-tropical regions of the world is responsible for about 198 million cases and estimated deaths due to this disease are about 0.6 million. The emergence of drug resistance in the malaria parasite is alarming and it is necessary to understand its underlying cause and molecular mechanisms. It has been established that drug resistant malaria parasites have defective mismatch repair (MMR) therefore it is essential to study this pathway and its components in detail. Recently a number of non-synonymous Single Nucleotide Polymorphisms have been reported in genes involved in MMR pathways. PfMLH is an endonuclease essential to restore the MMR in drug resistant strains of Plasmodium falciparum. Considering all these facts about the role of MMR in emergence of drug resistant parasite, in this manuscript we report a genome wide analysis of the components of the MMR pathway such as MLH, Pms1, MSH2-1, MSH2-2, MSH6, and UvrD using in silico bioinformatics based approaches. The phylogenetic analysis revealed evolutionary closeness with the MMR components of various organisms. It is noteworthy that P. falciparum contains two homologs of MSH2, which are located on different chromosomes. The structural modeling of these components showed their similarity with the human/yeast MMR components. The docking studies reveal that PfUvrD and PfMLH interact with each other. The in silico identification of interacting partners of the major MMR components identified numerous P. falciparum specific proteins. In line with our previous studies the present study will also contribute significantly to understand the MMR pathway of malaria parasite. PMID:28232818

  16. Screening for germline mismatch repair mutations following diagnosis of sebaceous neoplasm.

    PubMed

    Everett, Jessica N; Raymond, Victoria M; Dandapani, Monica; Marvin, Monica; Kohlmann, Wendy; Chittenden, Anu; Koeppe, Erika; Gustafson, Shanna L; Else, Tobias; Fullen, Douglas R; Johnson, Timothy M; Syngal, Sapna; Gruber, Stephen B; Stoffel, Elena M

    2014-12-01

    IMPORTANCE Sebaceous neoplasms (SNs) define the Muir-Torre syndrome variant of Lynch syndrome (LS), which is associated with increased risk for colon and other cancers necessitating earlier and more frequent screening to reduce morbidity and mortality.Immunohistochemical (IHC) staining for mismatch repair (MMR) proteins in SNs can be used to screen for LS, but data on subsequent germline genetic testing to confirm LS diagnosis are limited.OBJECTIVE To characterize the utility of IHC screening of SNs in identification of germline MMR mutations confirming LS.DESIGN, SETTING, AND PARTICIPANTS Retrospective study at 2 academic cancer centers of 86 adult patients referred for clinical genetics evaluation after diagnosis of SN.MAIN OUTCOMES AND MEASURES Results of tumor IHC testing and germline genetic testing were reviewed to determine positive predictive value and sensitivity of IHC testing in diagnosis of LS. Clinical variables, including age at diagnosis of SN, clinical diagnostic criteria for LS and Muir-Torre syndrome, and family history characteristics were compared between mutation carriers and noncarriers.RESULTS Of 86 patients with SNs, 25 (29%) had germline MMR mutations confirming LS.Among 77 patients with IHC testing on SNs, 38 (49%) had loss of staining of 1 or more MMR proteins and 14 had germline MMR mutations. Immunohistochemical analysis correctly identified 13 of 16 MMR mutation carriers, corresponding to 81% sensitivity. Ten of 12 patients(83%) with more than 1 SN had MMR mutations. Fifty-two percent of MMR mutation carriers did not meet clinical diagnostic criteria for LS, and 11 of 25 (44%) did not meet the clinical definition of Muir-Torre syndrome. CONCLUSIONS AND RELEVANCE Immunohistochemical screening of SNs is effective in identifying patients with germline MMR mutations and can be used as a first-line test when LSis suspected. Abnormal IHC results, including absence of MSH2, are not diagnostic of LS and should be interpreted cautiously in

  17. Biomarkers for immune therapy in colorectal cancer: mismatch-repair deficiency and others

    PubMed Central

    Bupathi, Manojkumar

    2016-01-01

    Colorectal cancer (CRC) is a heterogeneous disease for which the treatment backbone has primarily been cytotoxic chemotherapy. With better understanding of the involved molecular mechanisms, it is now known that there are a number of epigenetic and genetic events, which are involved in CRC pathogenesis. Specific biomarkers have been identified which can be used to determine the clinical outcome of patients beyond tumor staging and predict for treatment efficacy. Molecular testing is now routinely performed to select for patients that will benefit the most from targeted agents and immunotherapy. In addition to KRAS, NRAS, and BRAF mutation (MT), analysis of DNA mismatch repair (MMR) status, tumor infiltrating lymphocytes, and checkpoint protein expression may be helpful to determine whether patients are eligible for certain therapies. The focus of this article is to discuss present and upcoming biomarkers for immunotherapy in CRC. PMID:27747085

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

    PubMed

    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

    2013-10-22

    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.

  19. 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

  20. Mismatch repair genes identified using genetic screens in Blm-deficient embryonic stem cells.

    PubMed

    Guo, Ge; Wang, Wei; Bradley, Allan

    2004-06-24

    Phenotype-driven recessive genetic screens in diploid organisms require a strategy to render the mutation homozygous. Although homozygous mutant mice can be generated by breeding, a reliable method to make homozygous mutations in cultured cells has not been available, limiting recessive screens in culture. Cultured embryonic stem (ES) cells provide access to all of the genes required to elaborate the fundamental components and physiological systems of a mammalian cell. Here we have exploited the high rate of mitotic recombination in Bloom's syndrome protein (Blm)-deficient ES cells to generate a genome-wide library of homozygous mutant cells from heterozygous mutations induced with a revertible gene trap retrovirus. We have screened this library for cells with defects in DNA mismatch repair (MMR), a system that detects and repairs base-base mismatches. We demonstrate the recovery of cells with homozygous mutations in known and novel MMR genes. We identified Dnmt1(ref. 5) as a novel MMR gene and confirmed that Dnmt1-deficient ES cells exhibit micro-satellite instability, providing a mechanistic explanation for the role of Dnmt1 in cancer. The combination of insertional mutagenesis in Blm-deficient ES cells establishes a new approach for phenotype-based recessive genetic screens in ES cells.

  1. DNA Mismatch Repair System: Repercussions in Cellular Homeostasis and Relationship with Aging

    PubMed Central

    Conde-Pérezprina, Juan Cristóbal; León-Galván, Miguel Ángel; Konigsberg, Mina

    2012-01-01

    The mechanisms that concern DNA repair have been studied in the last years due to their consequences in cellular homeostasis. The diverse and damaging stimuli that affect DNA integrity, such as changes in the genetic sequence and modifications in gene expression, can disrupt the steady state of the cell and have serious repercussions to pathways that regulate apoptosis, senescence, and cancer. These altered pathways not only modify cellular and organism longevity, but quality of life (“health-span”). The DNA mismatch repair system (MMR) is highly conserved between species; its role is paramount in the preservation of DNA integrity, placing it as a necessary focal point in the study of pathways that prolong lifespan, aging, and disease. Here, we review different insights concerning the malfunction or absence of the DNA-MMR and its impact on cellular homeostasis. In particular, we will focus on DNA-MMR mechanisms regulated by known repair proteins MSH2, MSH6, PMS2, and MHL1, among others. PMID:23213348

  2. Molecular characterization of MSI-H colorectal cancer by MLHI promoter methylation, immunohistochemistry, and mismatch repair germline mutation screening.

    PubMed

    Poynter, Jenny N; Siegmund, Kimberly D; Weisenberger, Daniel J; Long, Tiffany I; Thibodeau, Stephen N; Lindor, Noralane; Young, Joanne; Jenkins, Mark A; Hopper, John L; Baron, John A; Buchanan, Dan; Casey, Graham; Levine, A Joan; Le Marchand, Loïc; Gallinger, Steven; Bapat, Bharati; Potter, John D; Newcomb, Polly A; Haile, Robert W; Laird, Peter W

    2008-11-01

    Microsatellite instability (MSI) occurs in 10% to 20% of colorectal cancers (CRC) and has been attributed to both MLH1 promoter hypermethylation and germline mutation in the mismatch repair (MMR) genes. We present results from a large population- and clinic-based study of MLH1 methylation, immunohistochemistry, and MMR germline mutations that enabled us to (a) estimate the prevalence of MMR germline mutations and MLH1 methylation among MSI-H cases and help us understand if all MSI-H CRC is explained by these mechanisms and (b) estimate the associations between MLH1 methylation and sex, age, and tumor location within the colon. MLH1 methylation was measured in 1,061 population-based and 172 clinic-based cases of CRC. Overall, we observed MLH1 methylation in 60% of population-based MSI-H cases and in 13% of clinic-based MSI-H cases. Within the population-based cases with MMR mutation screening and conclusive immunohistochemistry results, we identified a molecular event in MMR in 91% of MSI-H cases: 54% had MLH1 methylation, 14% had a germline mutation in a MMR gene, and 23% had immunohistochemistry evidence for loss of a MMR protein. We observed a striking age difference, with the prevalence of a MMR germline mutation more than 4-fold lower and the prevalence of MLH1 methylation more than 4-fold higher in cases diagnosed after the age of 50 years than in cases diagnosed before that age. We also determined that female sex is an independent predictor of MLH1 methylation within the MSI-H subgroup. These results reinforce the importance of distinguishing between the underlying causes of MSI in studies of etiology and prognosis.

  3. DNA mismatch repair and the DNA damage response

    PubMed Central

    Li, Zhongdao; Pearlman, Alexander H.; Hsieh, Peggy

    2015-01-01

    This review discusses the role of DNA mismatch repair (MMR) in the DNA damage response (DDR) that triggers cell cycle arrest and, in some cases, apoptosis. Although the focus is on findings from mammalian cells, much has been learned from studies in other organisms including bacteria and yeast [1,2]. MMR promotes a DDR mediated by a key signaling kinase, ATM and Rad3-related (ATR), in response to various types of DNA damage including some encountered in widely used chemotherapy regimes. An introduction to the DDR mediated by ATR reveals its immense complexity and highlights the many biological and mechanistic questions that remain. Recent findings and future directions are highlighted. PMID:26704428

  4. DNA mismatch repair and the DNA damage response.

    PubMed

    Li, Zhongdao; Pearlman, Alexander H; Hsieh, Peggy

    2016-02-01

    This review discusses the role of DNA mismatch repair (MMR) in the DNA damage response (DDR) that triggers cell cycle arrest and, in some cases, apoptosis. Although the focus is on findings from mammalian cells, much has been learned from studies in other organisms including bacteria and yeast [1,2]. MMR promotes a DDR mediated by a key signaling kinase, ATM and Rad3-related (ATR), in response to various types of DNA damage including some encountered in widely used chemotherapy regimes. An introduction to the DDR mediated by ATR reveals its immense complexity and highlights the many biological and mechanistic questions that remain. Recent findings and future directions are highlighted. Published by Elsevier B.V.

  5. Dynamic control of strand excision during human DNA mismatch repair.

    PubMed

    Jeon, Yongmoon; Kim, Daehyung; Martín-López, Juana V; Lee, Ryanggeun; Oh, Jungsic; Hanne, Jeungphill; Fishel, Richard; Lee, Jong-Bong

    2016-03-22

    Mismatch repair (MMR) is activated by evolutionarily conserved MutS homologs (MSH) and MutL homologs (MLH/PMS). MSH recognizes mismatched nucleotides and form extremely stable sliding clamps that may be bound by MLH/PMS to ultimately authorize strand-specific excision starting at a distant 3'- or 5'-DNA scission. The mechanical processes associated with a complete MMR reaction remain enigmatic. The purified human (Homo sapien or Hs) 5'-MMR excision reaction requires the HsMSH2-HsMSH6 heterodimer, the 5' → 3' exonuclease HsEXOI, and the single-stranded binding heterotrimer HsRPA. The HsMLH1-HsPMS2 heterodimer substantially influences 5'-MMR excision in cell extracts but is not required in the purified system. Using real-time single-molecule imaging, we show that HsRPA or Escherichia coli EcSSB restricts HsEXOI excision activity on nicked or gapped DNA. HsMSH2-HsMSH6 activates HsEXOI by overcoming HsRPA/EcSSB inhibition and exploits multiple dynamic sliding clamps to increase tract length. Conversely, HsMLH1-HsPMS2 regulates tract length by controlling the number of excision complexes, providing a link to 5' MMR.

  6. Truncation of the MSH2 C-terminal 60 amino acids disrupts effective DNA mismatch repair and is causative for Lynch syndrome.

    PubMed

    Wielders, Eva; Delzenne-Goette, Elly; Dekker, Rob; van der Valk, Martin; Te Riele, Hein

    2017-04-01

    Missense variants of DNA mismatch repair (MMR) genes pose a problem in clinical genetics as long as they cannot unambiguously be assigned as the cause of Lynch syndrome (LS). To study such variants of uncertain clinical significance, we have developed a functional assay based on direct measurement of MMR activity in mouse embryonic stem cells expressing mutant protein from the endogenous alleles. We have applied this protocol to a specific truncation mutant of MSH2 that removes 60 C-terminal amino acids and has been found in suspected LS families. We show that the stability of the MSH2/MSH6 heterodimer is severely perturbed, causing attenuated MMR in in vitro assays and cancer predisposition in mice. This mutation can therefore unambiguously be considered as deleterious and causative for LS.

  7. Prognostic Impact of Deficient DNA Mismatch Repair in Patients With Stage III Colon Cancer From a Randomized Trial of FOLFOX-Based Adjuvant Chemotherapy

    PubMed Central

    Sinicrope, Frank A.; Mahoney, Michelle R.; Smyrk, Thomas C.; Thibodeau, Stephen N.; Warren, Robert S.; Bertagnolli, Monica M.; Nelson, Garth D.; Goldberg, Richard M.; Sargent, Daniel J.; Alberts, Steven R.

    2013-01-01

    Purpose The association of deficient DNA mismatch repair (dMMR) with prognosis in patients with colon cancer treated with adjuvant fluorouracil, leucovorin, and oxaliplatin (FOLFOX) chemotherapy remains unknown. Patients and Methods Resected, stage III colon carcinomas from patients (N = 2,686) randomly assigned to FOLFOX ± cetuximab (North Central Cancer Treatment Group N0147 trial) were analyzed for mismatch repair (MMR) protein expression and mutations in BRAFV600E (exon 15) and KRAS (codons 12 and 13). Association of biomarkers with disease-free survival (DFS) was determined using Cox models. A validation cohort (Cancer and Leukemia Group B 88903 trial) was used. Results dMMR was detected in 314 (12%) of 2,580 tumors, of which 49.3% and 10.6% had BRAFV600E or KRAS mutations, respectively. MMR status was not prognostic overall (adjusted hazard ratio [HR], 0.82; 95% CI, 0.64 to 1.07; P = .14), yet significant interactions were found between MMR and primary tumor site (Pinteraction = .009) and lymph node category (N1 v N2; Pinteraction = .014). Favorable DFS was observed for dMMR versus proficient MMR proximal tumors (HR, 0.71; 95% CI, 0.53 to 0.94; P = .018) but not dMMR distal tumors (HR, 1.71; 95% CI, 0.99 to 2.95; P = .056), adjusting for mutations and covariates. Any survival benefit of dMMR was lost in N2 tumors. Mutations in BRAFV600E (HR, 1.37; 95% CI, 1.08 to 1.70; P = .009) or KRAS (HR, 1.44; 95% CI, 1.21 to 1.70; P < .001) were independently associated with worse DFS. The observed MMR by tumor site interaction was validated in an independent cohort of stage III colon cancers (Pinteraction = .037). Conclusion The prognostic impact of MMR depended on tumor site, and this interaction was validated in an independent cohort. Among dMMR cancers, proximal tumors had favorable outcome, whereas distal or N2 tumors had poor outcome. BRAF or KRAS mutations were independently associated with adverse outcome. PMID:24019539

  8. Mismatch repair balances leading and lagging strand DNA replication fidelity.

    PubMed

    Lujan, Scott A; Williams, Jessica S; Pursell, Zachary F; Abdulovic-Cui, Amy A; Clark, Alan B; Nick McElhinny, Stephanie A; Kunkel, Thomas A

    2012-01-01

    The two DNA strands of the nuclear genome are replicated asymmetrically using three DNA polymerases, α, δ, and ε. Current evidence suggests that DNA polymerase ε (Pol ε) is the primary leading strand replicase, whereas Pols α and δ primarily perform lagging strand replication. The fact that these polymerases differ in fidelity and error specificity is interesting in light of the fact that the stability of the nuclear genome depends in part on the ability of mismatch repair (MMR) to correct different mismatches generated in different contexts during replication. Here we provide the first comparison, to our knowledge, of the efficiency of MMR of leading and lagging strand replication errors. We first use the strand-biased ribonucleotide incorporation propensity of a Pol ε mutator variant to confirm that Pol ε is the primary leading strand replicase in Saccharomyces cerevisiae. We then use polymerase-specific error signatures to show that MMR efficiency in vivo strongly depends on the polymerase, the mismatch composition, and the location of the mismatch. An extreme case of variation by location is a T-T mismatch that is refractory to MMR. This mismatch is flanked by an AT-rich triplet repeat sequence that, when interrupted, restores MMR to > 95% efficiency. Thus this natural DNA sequence suppresses MMR, placing a nearby base pair at high risk of mutation due to leading strand replication infidelity. We find that, overall, MMR most efficiently corrects the most potentially deleterious errors (indels) and then the most common substitution mismatches. In combination with earlier studies, the results suggest that significant differences exist in the generation and repair of Pol α, δ, and ε replication errors, but in a generally complementary manner that results in high-fidelity replication of both DNA strands of the yeast nuclear genome.

  9. 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

  10. 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.

  11. 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

  12. Comparison of the Mismatch Repair System between Primary and Metastatic Colorectal Cancers Using Immunohistochemistry

    PubMed Central

    Jung, Jiyoon; Kang, Youngjin; Lee, Yoo Jin; Kim, Eojin; Ahn, Bokyung; Lee, Eunjung; Kim, Joo Young; Lee, Jeong Hyeon; Lee, Youngseok; Kim, Chul Hwan; Chae, Yang-Seok

    2017-01-01

    Background Colorectal cancer (CRC) is one of the most common malignancies worldwide. Approximately 10%–15% of the CRC cases have defective DNA mismatch repair (MMR) genes. Although the high level of microsatellite instability status is a predictor of favorable outcome in primary CRC, little is known about its frequency and importance in secondary CRC. Immunohistochemical staining (IHC) for MMR proteins (e.g., MLH1, MSH2, MSH6, and PMS2) has emerged as a useful technique to complement polymerase chain reaction (PCR) analyses. Methods In this study, comparison between the MMR system of primary CRCs and paired liver and lung metastatic lesions was done using IHC and the correlation with clinical outcomes was also examined. Results Based on IHC, 7/61 primary tumors (11.4%) showed deficient MMR systems, while 13/61 secondary tumors (21.3%) showed deficiencies. In total, 44 cases showed proficient expression in both the primary and metastatic lesions. Three cases showed deficiencies in both the primary and paired metastatic lesions. In 10 cases, proficient expression was found only in the primary lesions, and not in the corresponding metastatic lesions. In four cases, proficient expression was detected in the secondary tumor, but not in the primary tumor. Conclusions Although each IHC result and the likely defective genes were not exactly matched between the primary and the metastatic tumors, identical results for primary and metastatic lesions were obtained in 77% of the cases (47/61). These data are in agreement with the previous microsatellite detection studies that used PCR and IHC. PMID:28192899

  13. Association between IHC and MSI testing to identify mismatch repair-deficient patients with ovarian cancer.

    PubMed

    Lee, Ji-Hyun; Cragun, Deborah; Thompson, Zachary; Coppola, Domenico; Nicosia, Santo V; Akbari, Mohammad; Zhang, Shiyu; McLaughlin, John; Narod, Steven; Schildkraut, Joellen; Sellers, Thomas A; Pal, Tuya

    2014-04-01

    In epithelial ovarian cancer, concordance between results of microsatellite instability (MSI) and immunohistochemical (IHC) testing has not been demonstrated. This study evaluated the association of MSI-high (MSI-H) status with loss of expression (LoE) of mismatch repair (MMR) proteins on IHC and assessed for potential factors affecting the strength of the association. Tumor specimens from three population-based studies of epithelial ovarian cancer were stained for MMR proteins through manual or automated methods, and results were interpreted by one of two pathologists. Tumor and germline DNA was extracted and MSI testing performed. Multivariable logistic regression models were fitted to predict loss of IHC expression based on MSI status after adjusting for staining method and reading pathologist. Of 834 cases, 564 (67.6%) were concordant; 41 were classified as MSI-H with LoE and 523 as microsatellite stable (MSS) with no LoE. Of the 270 discordant cases, 83 were MSI-H with no LoE and 187 were MSS with LoE. Both IHC staining method and reading pathologist were strongly associated with discordant results. Lack of concordance in the current study may be related to inconsistencies in IHC testing methods and interpretation. Results support the need for validation studies before routine screening of ovarian tumors is implemented in clinical practice for the purpose of identifying Lynch syndrome.

  14. Cyclin E and histone H3 levels are regulated by 5-fluorouracil in a DNA mismatch repair-dependent manner

    PubMed Central

    Chung, Heekyung; Chaudhry, Joy; Lopez, Claudia G

    2010-01-01

    Several studies indicate that the DNA mismatch repair (MMR) system may trigger cytotoxicity upon 5-fluorouracil (5-FU) recognition, but signaling pathways regulated by MMR in response to 5-FU are unknown. We hypothesize that recognition of 5-FU in DNA by MMR proteins trigger specific signaling cascades that results in slowing of the cell cycle and cell death. Whole human genome cDNA microarrays were used to examine relative signaling responses induced in MMR-proficient cells after 5-FU (5 µM) treatment for 24 hours. Analysis revealed 43 pathways differentially affected by 5-FU compared to control (p < 0.05), including cyclin and cell cycle regulation involving G1-S cell cycle transition, activation of Src, MAP K, p53 and base excision repair. In particular, 5-FU upregulated cyclins E1 and E2 (≥1.4-fold) and downregulated cdc25C, cyclins B1 and B2, histone H2A, H2B and H3 (≤-1.4-fold) over control. Cell cycle analysis revealed a G1/S arrest by 5-FU that was congruent with increased cyclin E and decreased cdc25C protein expression. Importantly, with knockdown of hMLH1 and hMSH2, we observed that decreased histone H3 expression by 5-FU was dependent on hMLH1. Additionally, 5-FU treatment dramatically decreased levels of several histone H3 modifications. Our data suggest that 5-FU induces a G1/S arrest by regulating cyclin E and cdc25C expression and MMR recognition of 5-FU in DNA may modulate cyclin E to affect the cell cycle. Furthermore, MMR recognition of 5-FU reduces histone H3 levels that could be related to DNA access by proteins and/or cell death during the G1/S phase of the cell cycle. PMID:20930505

  15. DNA Mismatch Repair-Induced Double-Strand Breaks

    PubMed Central

    Nowosielska, Anetta; Marinus, M. G.

    2007-01-01

    Escherichia coli dam mutants are sensitized to the cytotoxic action of base analogs, cisplatin and N-methyl-N’-nitro-N-nitrosoguanidine (MNNG), while their mismatch repair (MMR)-deficient derivatives are tolerant to these agents. We showed previously, using pulse field gel electrophoresis, that MMR-mediated double-strand breaks (DSBs) are produced by cisplatin in dam recB (Ts) cells at the non-permissive temperature. We demonstrate here that the majority of these DSBs require DNA replication for their formation, consistent with a model in which replication forks collapse at nicks or gaps formed during MMR. DSBs were also detected in dam recB(Ts) ada ogt cells exposed to MNNG in a dose- and MMR-dependent manner. In contrast to cisplatin, the formation of these DSBs was not affected by DNA replication and it is proposed that two separate mechanisms result in DSB formation. Replication-independent DSBs arise from overlapping base excision and MMR repair tracts on complementary strands and constitute the majority of detectable DSBs in dam recB(Ts) ada ogt cells exposed to MNNG. Replication-dependent DSBs result from replication fork collapse at O6-meG base pairs undergoing MMR futile cycling and are more likely to contribute to cytotoxicity. This model is consistent with the observation that fast-growing dam recB (Ts) ada ogt cells, which have more chromosome replication origins, are more sensitive to the cytotoxic effect of MNNG than the same cells growing slowly. PMID:17827074

  16. The Histone Mark H3K36me3 Regulates Human DNA Mismatch Repair through its Interaction with MutSα

    PubMed Central

    Li, Feng; Mao, Guogen; Tong, Dan; Huang, Jian; Gu, Liya; Yang, Wei; Li, Guo-Min

    2013-01-01

    Summary DNA mismatch repair (MMR) ensures replication fidelity by correcting mismatches generated during DNA replication. Although human MMR has been reconstituted in vitro, how MMR occurs in vivo is unknown. Here, we show that an epigenetic histone mark, H3K36me3, is required in vivo to recruit the mismatch recognition protein hMutSα (hMSH2-hMSH6) onto chromatin through direct interactions with the hMSH6 PWWP domain. The abundance of H3K36me3 in G1 and early S phases ensures that hMutSα is enriched on chromatin before mispairs are introduced during DNA replication. Cells lacking the H3K36 tri-methyltransferase SETD2 display microsatellite instability (MSI) and an elevated spontaneous mutation frequency, characteristic of MMR-deficient cells. This work reveals that a histone mark regulates MMR in human cells and explains the long-standing puzzle of MSI-positive cancer cells that lack detectable mutations in known MMR genes. PMID:23622243

  17. Is thymidine glycol containing DNA a substrate of E. coli DNA mismatch repair system?

    PubMed

    Perevozchikova, Svetlana A; Trikin, Roman M; Heinze, Roger J; Romanova, Elena A; Oretskaya, Tatiana S; Friedhoff, Peter; Kubareva, Elena A

    2014-01-01

    The DNA mismatch repair (MMR) system plays a crucial role in the prevention of replication errors and in the correction of some oxidative damages of DNA bases. In the present work the most abundant oxidized pyrimidine lesion, 5,6-dihydro-5,6-dihydroxythymidine (thymidine glycol, Tg) was tested for being recognized and processed by the E. coli MMR system, namely complex of MutS, MutL and MutH proteins. In a partially reconstituted MMR system with MutS-MutL-MutH proteins, G/Tg and A/Tg containing plasmids failed to provoke the incision of DNA. Tg residue in the 30-mer DNA duplex destabilized double helix due to stacking disruption with neighboring bases. However, such local structural changes are not important for E. coli MMR system to recognize this lesion. A lack of repair of Tg containing DNA could be due to a failure of MutS (a first acting protein of MMR system) to interact with modified DNA in a proper way. It was shown that Tg in DNA does not affect on ATPase activity of MutS. On the other hand, MutS binding affinities to DNA containing Tg in G/Tg and A/Tg pairs are lower than to DNA with a G/T mismatch and similar to canonical DNA. Peculiarities of MutS interaction with DNA was monitored by Förster resonance energy transfer (FRET) and fluorescence anisotropy. Binding of MutS to Tg containing DNAs did not result in the formation of characteristic DNA kink. Nevertheless, MutS homodimer orientation on Tg-DNA is similar to that in the case of G/T-DNA. In contrast to G/T-DNA, neither G/Tg- nor A/Tg-DNA was able to stimulate ADP release from MutS better than canonical DNA. Thus, Tg residue in DNA is unlikely to be recognized or processed by the E. coli MMR system. Probably, the MutS transformation to active "sliding clamp" conformation on Tg-DNA is problematic.

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

    PubMed

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

    2012-09-14

    DNA mismatch repair (MMR) models have proposed that MSH (MutS homolog) proteins identify DNA polymerase errors while interacting with the DNA replication fork. MLH (MutL homolog) 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 nm) unstructured arms that connect two terminal globular domains. These arms can vary between 100 and 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.

  19. Mismatch Repair Gene Expression as a Predictor of Tumor Responses in Patients With Rectal Cancer Treated With Preoperative Chemoradiation

    PubMed Central

    Huh, Jung Wook; Kim, Hee Cheol; Kim, Seok Hyung; Park, Yoon Ah; Cho, Yong Beom; Yun, Seong Hyeon; Lee, Woo Yong; Park, Hee Chul; Choi, Doo Ho; Park, Joon Oh; Park, Young Suk; Chun, Ho-Kyung

    2016-01-01

    Abstract This study evaluated the predictive and prognostic value of expression of mismatch repair (MMR) protein, including MLH1, MSH2, and MSH6 in rectal cancer patients with preoperative chemoradiotherapy. MMR protein expression was measured by immunohistochemistry in both pretreatment biopsies (pre-) and pathologic specimens (post-) from 209 patients with locally advanced rectal cancer who underwent preoperative chemoradiotherapy and radical surgery. The patients were followed for a median period of 44 months. A pathologic complete response (pCR) was observed in 30 patients (14.4%). The expression levels of MLH1, MSH2, and MSH6 were not significantly different between the pCR and non-pCR groups. A multivariate analysis revealed that tumor differentiation, postoperative chemotherapy, and pre-MSH6 expression were independent predictors of overall survival; ypN category and perineural invasion were independent predictors of disease-free survival. The pre-MSH6 expression was significantly associated with tumor budding and expression of all MMR proteins. On multivariate analysis, ypN category and post-MSH6 expression were independent predictors for local recurrence. In our study, we observed the independent prognostic value of MSH6 expression in pretreatment tissue on overall survival and MSH6 expression after chemoradiation on local recurrence. Constitutive MSH6 expression before and after preoperative therapy may be a useful tool for prediction of oncologic outcome in locally advanced rectal cancer. PMID:26817916

  20. Interaction between Mismatch Repair and Genetic Recombination in Saccharomyces Cerevisiae

    PubMed Central

    Alani, E.; Reenan, RAG.; Kolodner, R. D.

    1994-01-01

    The yeast Saccharomyces cerevisiae encodes a set of genes that show strong amino acid sequence similarity to MutS and MutL, proteins required for mismatch repair in Escherichia coli. We examined the role of MSH2 and PMS1, yeast homologs of mutS and mutL, respectively, in the repair of base pair mismatches formed during meiotic recombination. By using specifically marked HIS4 and ARG4 alleles, we showed that msh2 mutants displayed a severe defect in the repair of all base pair mismatches as well as 1-, 2- and 4-bp insertion/deletion mispairs. The msh2 and pms1 phenotypes were indistinguishable, suggesting that the wild-type gene products act in the same repair pathway. A comparison of gene conversion events in wild-type and msh2 mutants indicated that mismatch repair plays an important role in genetic recombination. (1) Tetrad analysis at five different loci revealed that, in msh2 mutants, the majority of aberrant segregants displayed a sectored phenotype, consistent with a failure to repair mismatches created during heteroduplex formation. In wild type, base pair mismatches were almost exclusively repaired toward conversion rather than restoration. (2) In msh2 strains 10-19% of the aberrant tetrads were Ab4:4. (3) Polarity gradients at HIS4 and ARG4 were nearly abolished in msh2 mutants. The frequency of gene conversion at the 3' end of these genes was increased and was nearly the frequency observed at the 5' end. (4) Co-conversion studies were consistent with mismatch repair acting to regulate heteroduplex DNA tract length. We favor a model proposing that recombination events occur through the formation and resolution of heteroduplex intermediates and that mismatch repair proteins specifically interact with recombination enzymes to regulate the length of symmetric heteroduplex DNA. PMID:8056309

  1. Loss of DNA mismatch repair facilitates reactivation of a reporter plasmid damaged by cisplatin

    PubMed Central

    Cenni, B; Kim, H-K; Bubley, G J; Aebi, S; Fink, D; Teicher, B A; Howell, S B; Christen, R D

    1999-01-01

    In addition to recognizing and repairing mismatched bases in DNA, the mismatch repair (MMR) system also detects cisplatin DNA adducts and loss of MMR results in resistance to cisplatin. A comparison was made of the ability of MMR-proficient and -deficient cells to remove cisplatin adducts from their genome and to reactivate a transiently transfected plasmid that had previously been inactivated by cisplatin to express the firefly luciferase enzyme. MMR deficiency due to loss of hMLH1 function did not change the extent of platinum (Pt) accumulation or kinetics of removal from total cellular DNA. However, MMR-deficient cells, lacking either hMLH1 or hMSH2, generated twofold more luciferase activity from a cisplatin-damaged reporter plasmid than their MMR-proficient counterparts. Thus, detection of the cisplatin adducts by the MMR system reduced the efficiency of reactivation of the damaged luciferase gene compared to cells lacking this detector. The twofold reduction in reactivation efficiency was of the same order of magnitude as the difference in cisplatin sensitivity between the MMR-proficient and -deficient cells. We conclude that although MMR-proficient and -deficient cells remove Pt from their genome at equal rates, the loss of a functional MMR system facilitates the reactivation of a cisplatin-damaged reporter gene. © 1999 Cancer Research Campaign PMID:10360646

  2. Effects of suppressing the DNA mismatch repair system on homeologous recombination in tomato.

    PubMed

    Tam, Sheh May; Hays, John B; Chetelat, Roger T

    2011-12-01

    In plant breeding, the ability to manipulate genetic (meiotic) recombination would be beneficial for facilitating gene transfer from wild relatives of crop plants. The DNA mismatch repair (MMR) system helps maintain genetic integrity by correcting base mismatches that arise via DNA synthesis or damage, and antagonizes recombination between homeologous (divergent) DNA sequences. Previous studies have established that the genomes of cultivated tomato (Solanum lycopersicum) and the wild relative S. lycopersicoides are substantially diverged (homeologous) such that recombination between their chromosomes is strongly reduced. Here, we report the effects on homeologous recombination of suppressing endogenous MMR genes in S. lycopersicum via RNAi-induced silencing of SlMSH2 and SlMSH7 or overexpressing dominant negatives of Arabidopsis MSH2 (AtMSH2-DN) in an alien substitution line (SL-8) of S. lycopersicoides in tomato. We show that certain inhibitions of MMR (RNAi of SlMSH7, AtMSH2-DN) are associated with modest increases in homeologous recombination, ranging from 3.8 to 29.2% (average rate of 17.8%) compared to controls. Unexpectedly, only the AtMSH2-DN proteins but not RNAi-induced silencing of MSH2 was found to increase homeologous recombination. The ratio of single to double crossovers (SCO:DCO ratio) decreased by approximately 50% in progeny of the AtMSH2-DN parents. An increase in the frequency of heterozygous SL-8 plants was also observed in the progeny of the SlMSH7-RNAi parents. Our findings may contribute to acceleration of introgression in cultivated tomato.

  3. Role of Deficient Mismatch Repair in the Personalized Management of Colorectal Cancer

    PubMed Central

    Zhang, Cong-Min; Lv, Jin-Feng; Gong, Liang; Yu, Lin-Yu; Chen, Xiao-Ping; Zhou, Hong-Hao; Fan, Lan

    2016-01-01

    Colorectal cancer (CRC) represents the third most common type of cancer in developed countries and one of the leading causes of cancer deaths worldwide. Personalized management of CRC has gained increasing attention since there are large inter-individual variations in the prognosis and response to drugs used to treat CRC owing to molecular heterogeneity. Approximately 15% of CRCs are caused by deficient mismatch repair (dMMR) characterized by microsatellite instability (MSI) phenotype. The present review is aimed at highlighting the role of MMR status in informing prognosis and personalized treatment of CRC including adjuvant chemotherapy, targeted therapy, and immune checkpoint inhibitor therapy to guide the individualized therapy of CRC. PMID:27618077

  4. 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.

  5. Alleles of the yeast Pms1 mismatch-repair gene that differentially affect recombination- and replication-related processes.

    PubMed Central

    Welz-Voegele, Caroline; Stone, Jana E; Tran, Phuoc T; Kearney, Hutton M; Liskay, R Michael; Petes, Thomas D; Jinks-Robertson, Sue

    2002-01-01

    Mismatch-repair (MMR) systems promote eukaryotic genome stability by removing errors introduced during DNA replication and by inhibiting recombination between nonidentical sequences (spellchecker and antirecombination activities, respectively). Following a common mismatch-recognition step effected by MutS-homologous Msh proteins, homologs of the bacterial MutL ATPase (predominantly the Mlh1p-Pms1p heterodimer in yeast) couple mismatch recognition to the appropriate downstream processing steps. To examine whether the processing steps in the spellchecker and antirecombination pathways might differ, we mutagenized the yeast PMS1 gene and screened for mitotic separation-of-function alleles. Two alleles affecting only the antirecombination function of Pms1p were identified, one of which changed an amino acid within the highly conserved ATPase domain. To more specifically address the role of ATP binding/hydrolysis in MMR-related processes, we examined mutations known to compromise the ATPase activity of Pms1p or Mlh1p with respect to the mitotic spellchecker and antirecombination activities and with respect to the repair of mismatches present in meiotic recombination intermediates. The results of these analyses confirm a differential requirement for the Pms1p ATPase activity in replication vs. recombination processes, while demonstrating that the Mlh1p ATPase activity is important for all examined MMR-related functions. PMID:12454061

  6. Abnormal mismatch repair and other clinicopathologic predictors of poor response to progestin treatment in young women with endometrial complex atypical hyperplasia and well-differentiated endometrial adenocarcinoma: a consecutive case series.

    PubMed

    Zakhour, M; Cohen, J G; Gibson, A; Walts, A E; Karimian, B; Baltayan, A; Aoyama, C; Garcia, L; Dhaliwal, S K; Elashoff, D; Amneus, M; Walsh, C

    2017-09-01

    To report the response to progestin therapy in young women with endometrial complex atypical hyperplasia (CAH) or FIGO grade 1 endometrial adenocarcinoma (FIGO 1 EAC) based on clinicopathologic features, including abnormal DNA mismatch repair (MMR) by immunohistochemistry (IHC). Consecutive case series. Olive View-UCLA Medical Center in Sylmar, CA, USA, and Cedars-Sinai Medical Center in Los Angeles, CA, USA. Women ≤55 years old with CAH or FIGO 1 EAC. Response to progestin therapy in 84 consecutive patients was assessed based on clinicopathologic factors, including age, body mass index (BMI), initial histology, and IHC staining for MMR proteins. Rates of abnormal MMR protein expression and response to progestin therapy were determined. Six (7%) patients had abnormal IHC staining, of whom five (83%) had FIGO 1 EAC at initial diagnosis. Following progestin treatment, none of the endometrial lesions in patients with abnormal IHC for MMR proteins had resolution of hyperplasia or malignancy, in contrast to 41 (53%) with normal staining (P = 0.028). Age ≤40 years and initial lesion (CAH versus FIGO 1 EAC) were predictors of response to progestin; BMI was not. In this cohort, 7% of women ≤55 years of age with CAH or FIGO 1 EAC had loss of MMR proteins by IHC. These patients had a higher incidence of invasive cancer and a lower incidence of resolution with progestin therapy. Abnormal MMR protein expression predicts poor response to progestins in young women with CAH or FIGO 1 EAC. © 2017 Royal College of Obstetricians and Gynaecologists.

  7. 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.

  8. Evidence for presence of mismatch repair gene expression positive Lynch syndrome cases in India.

    PubMed

    Bashyam, Murali D; Kotapalli, Viswakalyan; Raman, Ratheesh; Chaudhary, Ajay K; Yadav, Brijesh K; Gowrishankar, Swarnalata; Uppin, Shantveer G; Kongara, Ravikanth; Sastry, Regulagadda A; Vamsy, Mohana; Patnaik, Sujit; Rao, Satish; Dsouza, Shoba; Desai, Devendra; Tester, Ashavaid

    2015-12-01

    Lynch syndrome (LS), the most common form of familial CRC predisposition that causes tumor onset at a young age, is characterized by the presence of microsatellite instability (MSI) in tumors due to germline inactivation of mismatch repair (MMR) system. Two MMR genes namely MLH1 and MSH2 account for majority of LS cases while MSH6 and PMS2 may account for a minor proportion. In order to identify MMR genes causing LS in India, we analyzed MSI and determined expression status of the four MMR genes in forty eight suspected LS patient colorectal tumor samples. Though a majority exhibited MSI, only 58% exhibited loss of MMR expression, a significantly low proportion compared to reports from other populations. PCR-DNA sequencing and MLPA-based mutation and exonic deletion/duplication screening respectively, revealed genetic lesions in samples with and without MMR gene expression. Interestingly, tumor samples with and without MMR expression exhibited significant differences with respect to histological (mucin content) and molecular (instability exhibited by mononucleotide microsatellites) features. The study has revealed for the first time a significant proportion of LS tumors not exhibiting loss of MMR expression. © 2014 Wiley Periodicals, Inc.

  9. MutSα maintains the mismatch repair capability by inhibiting PCNA unloading

    PubMed Central

    Kawasoe, Yoshitaka; Tsurimoto, Toshiki; Nakagawa, Takuro; Masukata, Hisao; Takahashi, Tatsuro S

    2016-01-01

    Eukaryotic mismatch repair (MMR) utilizes single-strand breaks as signals to target the strand to be repaired. DNA-bound PCNA is also presumed to direct MMR. The MMR capability must be limited to a post-replicative temporal window during which the signals are available. However, both identity of the signal(s) involved in the retention of this temporal window and the mechanism that maintains the MMR capability after DNA synthesis remain unclear. Using Xenopus egg extracts, we discovered a mechanism that ensures long-term retention of the MMR capability. We show that DNA-bound PCNA induces strand-specific MMR in the absence of strand discontinuities. Strikingly, MutSα inhibited PCNA unloading through its PCNA-interacting motif, thereby extending significantly the temporal window permissive to strand-specific MMR. Our data identify DNA-bound PCNA as the signal that enables strand discrimination after the disappearance of strand discontinuities, and uncover a novel role of MutSα in the retention of the post-replicative MMR capability. DOI: http://dx.doi.org/10.7554/eLife.15155.001 PMID:27402201

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

    PubMed Central

    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. PMID:26739522

  11. 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.

  12. 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

  13. Identification of mismatch repair gene mutations in young patients with colorectal cancer and in patients with multiple tumours associated with hereditary non-polyposis colorectal cancer.

    PubMed

    Niessen, R C; Berends, M J W; Wu, Y; Sijmons, R H; Hollema, H; Ligtenberg, M J L; de Walle, H E K; de Vries, E G E; Karrenbeld, A; Buys, C H C M; van der Zee, A G J; Hofstra, R M W; Kleibeuker, J H

    2006-12-01

    Patients with early-onset colorectal cancer (CRC) or those with multiple tumours associated with hereditary non-polyposis colorectal cancer (HNPCC) raise suspicion of the presence of germline DNA mismatch repair (MMR) gene mutations. To analyse the value of family history, microsatellite instability (MSI) analysis and MMR protein staining in the tumour to predict the presence of an MMR gene mutation in such patients. In 281 patients diagnosed with CRC before the age of 50 years or with CRC and at least one additional HNPCC-associated cancer, germline mutation analysis in MLH1, MSH2 and MSH6 was carried out with denaturing gradient gel electrophoresis and multiplex ligation-dependent probe amplification. MSI analysis with five consensus markers and MMR protein staining for MLH1, MSH2 and MSH6 were carried out in the tumours. 25 pathogenic mutations (8 in MLH1, 9 in MSH2 and 8 in MSH6) were found. MSI analysis missed three and immunohistochemistry (IHC) missed two mutation carriers. Sensitivities of family history, MSI analysis and IHC for the presence of a mutation were 76%, 82% and 88%, specificities were 64%, 70% and 84%, and positive predictive values were 19%, 23% and 38%, respectively. Multivariate analysis showed the highest odds ratio for IHC (38.3, 95% confidence interval 9.0 to 184). Prevalence of pathogenic germline MMR gene mutations in patients with CRC before the age of 50 years was 6% and in those with > or =2 HNPCC-associated tumours was 22%. In the second group, no mutation carriers were found among the 29 patients who were diagnosed with their first tumour after the age of 60 years. Family history, MSI analysis and IHC are indicative parameters to select patients with CRC for MMR gene mutation analysis. The data show that IHC is the best single selection criterion.

  14. Identification of novel leads applying in silico studies for Mycobacterium multidrug resistant (MMR) protein.

    PubMed

    Malkhed, Vasavi; Mustyala, Kiran Kumar; Potlapally, Sarita Rajender; Vuruputuri, Uma

    2014-12-01

    Multidrug efflux mechanism is the main cause of intrinsic drug resistance in bacteria. Mycobacterium multidrug resistant (MMR) protein belongs to small multidrug resistant family proteins (SMR), causing multidrug resistance to proton (H(+))-linked lipophilic cationic drug efflux across the cell membrane. In the present work, MMR is treated as a novel target to identify new molecular entities as inhibitors for drug resistance in Mycobacterium tuberculosis. In silico techniques are applied to evaluate the 3D structure of MMR protein. The putative amino acid residues present in the active site of MMR protein are predicted. Protein-ligand interactions are studied by docking cationic ligands transported by MMR protein. Virtual screening is carried out with an in-house library of small molecules against the grid created at the predicted active site residues in the MMR protein. Absorption distribution metabolism and elimination (ADME) properties of the molecules with best docking scores are predicted. The studies with cationic ligands and those of virtual screening are analysed for identification of new lead molecules as inhibitors for drug resistance caused by the MMR protein.

  15. Helicobacter pylori infection modulates the expression of miRNAs associated with DNA mismatch repair pathway.

    PubMed

    Santos, Juliana C; Brianti, Mitsue T; Almeida, Victor R; Ortega, Manoela M; Fischer, Wolfgang; Haas, Rainer; Matheu, Ander; Ribeiro, Marcelo L

    2017-04-01

    Genetic and epigenetic inactivation of DNA mismatch repair (MMR) genes might lead to modifications in cancer-related gene expression and cancer development. Recently, it has been shown that the infection by Helicobacter pylori, the major causative agent of gastric cancer, induces DNA damage and inhibits MMR DNA repair. Also, it has been reported that microRNAs (miRs) have an important role in regulating genomic stability and MMR DNA repair. Thus, the aim of this study was to identify miRs regulating MMR pathway in H. pylori-associated gastric carcinogenesis. To address this question, a gastric epithelial cell line and AGS cancer gastric cells were infected with several H. pylori strains. MMR gene expression and miRs correlating with H. pylori strain infection were evaluated. The results showed that H. pylori infection significantly down-regulated the expression of all selected MMR genes. Also, H. pylori infection modulated the expression of several miRs (including miR-150-5p, miR-155-5p, and miR-3163), after 4, 8, and 12 h of infection. Computational prediction of candidate miRs and their predicted MMR targeting sites were obtained from TargetScan, mirDB, and MetaCore. The generated data indicated that the selected miRs (miR-150-5p, miR-155-5p, and miR-3163) could possibly target and modulate MMR genes (POLD3, MSH2, and MSH3, respectively). The target validation was performed using mimics and luciferase gene reporter assays. Briefly, this study shows that H. pylori impairs MMR DNA repair pathway and identifies miRs that regulate MMR gene expression in gastric cancer. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

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

    PubMed Central

    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.

    2017-01-01

    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. PMID:28591715

  17. Aberrant DNA Methylation in Hereditary Non-Polyposis Colorectal Cancer without Mismatch Repair Deficiency

    PubMed Central

    Goel, Ajay; Xicola, Rosa M.; Nguyen, Thuy-Phuong; Doyle, Brian J; Sohn, Vanessa R.; Bandipalliam, Prathap; Reyes, Josep; Cordero, Carmen; Balaguer, Francesc; Castells, Antoni; Jover, Rodrigo; Andreu, Montserrat; Syngal, Sapna; Boland, C. Richard; Llor, Xavier

    2010-01-01

    Background & Aims Approximately half of the families that fulfill Amsterdam criteria for Lynch syndrome or hereditary non-polyposis colorectal cancer (HNPCC) do not have evidence of the germline mismatch repair (MMR) gene mutations that define this syndrome and result in microsatellite instability. The carcinogenic pathways and the best diagnostic approaches to detect microsatellite stable (MSS) HNPCC tumors are unclear. We investigated the contribution of epigenetic alterations to development of MSS HNPCC tumors. Methods Colorectal cancers were divided in four groups: 1. Microsatellite stable, Amsterdam positive (MSS HNPCC) (N=22); 2. Lynch syndrome cancers (identified mismatch repair mutations) (N=21); 3. Sporadic MSS (N=92); 4. Sporadic MSI (N=46). Methylation status was evaluated for CACNAG1, SOCS1, RUNX3, NEUROG1, MLH1, and LINE-1. KRAS and BRAF mutations status was analyzed. Results MSS HNPCC tumors displayed a significantly lower degree of LINE-1 methylation, marker for global methylation, than any other group. Whereas most MSS HNPCC tumors had some degree of CpG island methylation, none presented a high index of methylation. MSS HNPCC tumors had KRAS mutations exclusively in codon 12, but none harbored V600E BRAF mutations. Conclusions Tumors from Amsterdam-positive patients without mismatch repair deficiency (MSS HNPCC) have certain molecular features, including global hypomethylation that distinguish them from all other colorectal cancers. These characteristics could have an important impact on tumor behavior or treatment response. Studies are underway to further assess the cause and effects of these features. PMID:20102720

  18. Competency in mismatch repair prohibits clonal expansion of cancer cells treated with N-methyl-N'-nitro-N-nitrosoguanidine.

    PubMed Central

    Carethers, J M; Hawn, M T; Chauhan, D P; Luce, M C; Marra, G; Koi, M; Boland, C R

    1996-01-01

    The phenomenon of alkylation tolerance has been observed in cells that are deficient in some component of the DNA mismatch repair (MMR) system. An alkylation-induced cell cycle arrest had been reported previously in one MMR-proficient cell line, whereas a MMR-defective clone derived from this line escapes from this arrest. We examined human cancer cell lines to determine if the cell cycle arrest were dependent upon the MMR system. Growth characteristics and cell cycle analysis after MNNG treatment were ascertained in seven MMR-deficient and proficient cell lines, with and without confirmed mutations in hMLH1 or hMSH2 by an in vitro transcription/translation assay. MMR-proficient cells underwent growth arrest in the G2 phase of the cell cycle after the first S phase, whereas MMR-deficient cells escaped an initial G2 delay and resumed a normal growth pattern. In the HCT116 line corrected for defective MMR by chromosome 3 transfer, the G2 phase arrest lasted more than five days. In another MMR-proficient colon cancer cell line, SW480, cell death occurred five days after MNNG treatment. A competent MMR system appears to be necessary for G2 arrest or cell death after alkylation damage, and this cell cycle checkpoint may allow the cell to repair damaged DNA, or prevent the replication of mutated DNA by prohibiting clonal expansion. PMID:8690794

  19. [Prevalence of altered mismatch repair protein nuclear expression detected by immunohistochemistry on adenomas with high-grade dysplasia and features associated with this risk in a population-based study].

    PubMed

    Basterra, Marta; Gomez, Marta; Mercado, María Del Rosario; Irisarri, Rebeca; Amorena, Edurne; Arrospide, Arantzazu; Montes, Marta; Aisa, Gregorio; Cambra, Koldo Iñaki; Urman, Jesús

    2016-10-01

    Alteration of mismatch repair system protein expression detected by immunohistochemistry (IHQ) in tumoural tissue is a useful technique for Lynch Syndrome (LS) screening. A recent review proposes LS screening through immunohistochemical study not only in all diagnosed cases of colorectal cancer (CRC) but also in advanced adenomas, especially in young patients. To assess the prevalence of altered IHQ carried out in all adenomas with high-grade dysplasia (HGD) diagnosed in our community in 2011, as well as the variables associated with this alteration. We included all the cases of adenomatous polyps with HGD diagnosed in the three public pathology laboratories of Navarre during 2011 and performed a statistical study to assess the association between different patient and lesion characteristics and altered IHQ results. A total of 213 colonic adenomas with HGD were diagnosed, and 26 (12.2%) cases were excluded from the final analysis (2 known LS, 22 without IHQ study and 2 with inconclusive IHQ studies). The final number of adenomas included was 187. Pathologic results were found in 10 cases (5.35%)-6 cases in MLH1 and PMS2, 2 cases in PMS2, 1 case in MSH6 and 1 case in MSH2 and MSH6. The factors showing a statistically significant association with the presence of abnormal proteins were the synchronous presence of CRC, the presence of only one advanced adenoma, proximal location of HGD and age <50 years. The percentage of pathologic nuclear expression found in IHQ is high. Consequently, screening of all diagnosed HGD could be indicated, especially in young patients, with a single AA and proximal HGD. Copyright © 2015 Elsevier España, S.L.U. y AEEH y AEG. All rights reserved.

  20. Differential DNA mismatch repair underlies mutation rate variation across the human genome.

    PubMed

    Supek, Fran; Lehner, Ben

    2015-05-07

    Cancer genome sequencing has revealed considerable variation in somatic mutation rates across the human genome, with mutation rates elevated in heterochromatic late replicating regions and reduced in early replicating euchromatin. Multiple mechanisms have been suggested to underlie this, but the actual cause is unknown. Here we identify variable DNA mismatch repair (MMR) as the basis of this variation. Analysing ∼17 million single-nucleotide variants from the genomes of 652 tumours, we show that regional autosomal mutation rates at megabase resolution are largely stable across cancer types, with differences related to changes in replication timing and gene expression. However, mutations arising after the inactivation of MMR are no longer enriched in late replicating heterochromatin relative to early replicating euchromatin. Thus, differential DNA repair and not differential mutation supply is the primary cause of the large-scale regional mutation rate variation across the human genome.

  1. An interplay of the base excision repair and mismatch repair pathways in active DNA demethylation

    PubMed Central

    Grin, Inga; Ishchenko, Alexander A.

    2016-01-01

    Active DNA demethylation (ADDM) in mammals occurs via hydroxylation of 5-methylcytosine (5mC) by TET and/or deamination by AID/APOBEC family enzymes. The resulting 5mC derivatives are removed through the base excision repair (BER) pathway. At present, it is unclear how the cell manages to eliminate closely spaced 5mC residues whilst avoiding generation of toxic BER intermediates and whether alternative DNA repair pathways participate in ADDM. It has been shown that non-canonical DNA mismatch repair (ncMMR) can remove both alkylated and oxidized nucleotides from DNA. Here, a phagemid DNA containing oxidative base lesions and methylated sites are used to examine the involvement of various DNA repair pathways in ADDM in murine and human cell-free extracts. We demonstrate that, in addition to short-patch BER, 5-hydroxymethyluracil and uracil mispaired with guanine can be processed by ncMMR and long-patch BER with concomitant removal of distant 5mC residues. Furthermore, the presence of multiple mispairs in the same MMR nick/mismatch recognition region together with BER-mediated nick formation promotes proficient ncMMR resulting in the reactivation of an epigenetically silenced reporter gene in murine cells. These findings suggest cooperation between BER and ncMMR in the removal of multiple mismatches that might occur in mammalian cells during ADDM. PMID:26843430

  2. 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

  3. Immunoscore in mismatch repair-proficient and -deficient colon cancer.

    PubMed

    Wirta, Erkki-Ville; Seppälä, Toni; Friman, Marjukka; Väyrynen, Juha; Ahtiainen, Maarit; Kautiainen, Hannu; Kuopio, Teijo; Kellokumpu, Ilmo; Mecklin, Jukka-Pekka; Böhm, Jan

    2017-07-01

    The aim of this study was to investigate immune response and its prognostic significance in colon carcinomas using the previously described Immunoscore (IS). A population-based series of 779 colorectal cancers, operated on between 2000 and 2010, were classified according to tumour, node, metastasis (TNM) status, mismatch repair (MMR), and BRAF mutation status. Rectal cancer cases (n = 203) were excluded as a high proportion of these patients received preoperative neoadjuvant chemoradiotherapy. Tissue microarray (TMA) samples collected from the tumour centre and invasive front were immunostained for CD3 and CD8. Lymphocytes were then digitally calculated to categorize IS from grade 0 to 4. Samples adequate for IS were available from 510 tumours. IS was significantly associated with AJCC/UICC stage, T stage, lymph node and distant metastases, perineural and lymphovascular invasion, MMR status, and BRAF mutation status. For IS0, IS1, IS2, IS3 and IS4, respectively, the 5-year disease-free survival (DFS) rates were 59, 68, 78, 83 and 94% (p < 0.001); 5-year disease-specific survival (DSS) rates were 47, 55, 75, 80, and 89% (p < 0.001); and 5-year overall survival (OS) rates were 40, 44, 66, 61, and 76% (p < 0.001). IS was also prognostic for DFS, DSS, and OS within subsets of microsatellite-stable (MSS) and microsatellite-instable (MSI) disease. Multivariable analysis showed that IS, AJCC/UICC stage, lymphovascular invasion, and lymph node ratio in AJCC/UICC stage III disease were independent prognostic factors for DFS, DSS, and OS. Age was an independent prognostic factor for DSS and OS. Gender and BRAF mutation were independent prognostic factors for OS. In conclusion, IS differentiated patients with poor versus improved prognosis in MSS and MSI disease and across AJCC/UICC stages. IS, AJCC/UICC stage, lymphovascular invasion, and lymph node ratio in AJCC/UICC stage III disease were independent prognostic factors for DFS, DSS, and OS.

  4. Evolving approach and clinical significance of detecting DNA mismatch repair deficiency in colorectal carcinoma

    PubMed Central

    Shia, Jinru

    2016-01-01

    The last two decades have seen significant advancement in our understanding of colorectal tumors with DNA mismatch repair (MMR) deficiency. The ever-emerging revelations of new molecular and genetic alterations in various clinical conditions have necessitated constant refinement of disease terminology and classification. Thus, a case with the clinical condition of hereditary non-polyposis colorectal cancer as defined by the Amsterdam criteria may be one of Lynch syndrome characterized by a germline defect in one of the several MMR genes, one of the yet-to-be-defined “Lynch-like syndrome” if there is evidence of MMR deficiency in the tumor but no detectable germline MMR defect or tumor MLH1 promoter methylation, or “familial colorectal cancer type X” if there is no evidence of MMR deficiency. The detection of these conditions carries significant clinical implications. The detection tools and strategies are constantly evolving. The Bethesda guidelines symbolize a selective approach that uses clinical information and tumor histology as the basis to select high-risk individuals. Such a selective approach has subsequently been found to have limited sensitivity, and is thus gradually giving way to the alternative universal approach that tests all newly diagnosed colorectal cancers. Notably, the universal approach also has its own limitations; its cost-effectiveness in real practice, in particular, remains to be determined. Meanwhile, technological advances such as the next-generation sequencing are offering the promise of direct genetic testing for MMR deficiency at an affordable cost probably in the near future. This article reviews the up-to-date molecular definitions of the various conditions related to MMR deficiency, and discusses the tools and strategies that have been used in detecting these conditions. Special emphasis will be placed on the evolving nature and the clinical importance of the disease definitions and the detection strategies. PMID:25716099

  5. MonoSeq Variant Caller Reveals Novel Mononucleotide Run Indel Mutations in Tumors with Defective DNA Mismatch Repair

    PubMed Central

    Walker, Christopher J.; Miranda, Mario A.; O’Hern, Matthew J.; Blachly, James S.; Moyer, Cassandra L.; Ivanovich, Jennifer; Kroll, Karl W.; Eisfeld, Ann-Kathrin; Sapp, Caroline E.; Mutch, David G.; Cohn, David E.; Bundschuh, Ralf; Goodfellow, Paul J

    2016-01-01

    Next-generation sequencing has revolutionized cancer genetics, but accurately detecting mutations in repetitive DNA sequences, especially mononucleotide runs, remains a challenge. This is a particular concern for tumors with defective mismatch repair (MMR) that accumulate strand-slippage mutations. We developed MonoSeq to improve indel mutation detection in mononucleotide runs, and used MonoSeq to investigate strand-slippage mutations in endometrial cancers, a tumor type that has frequent loss of MMR. We performed extensive Sanger sequencing to validate both clonal and sub-clonal MonoSeq mutation calls. Eighty-one regions containing mononucleotide runs were sequenced in 542 primary endometrial cancers (223 with defective MMR). Our analyses revealed that the overall mutation rate in MMR-deficient tumors was 20–30-fold higher than in MMR normal tumors. MonoSeq analysis identified several previously unreported mutations, including a novel hotspot in an A7 run in the terminal exon of ARID5B.The ARID5B indel mutations were seen in both MMR-deficient and MMR normal tumors, suggesting biologic selection. Analysis of tumor mRNAs revealed the presence of mutant transcripts that could result in translation of neopeptides. Improved detection of mononucleotide run strand-slippage mutations has clear implications for comprehensive mutation detection in tumors with defective MMR. Indel frameshift mutations and the resultant antigenic peptides could help guide immunotherapy strategies. PMID:27346418

  6. MonoSeq Variant Caller Reveals Novel Mononucleotide Run Indel Mutations in Tumors with Defective DNA Mismatch Repair.

    PubMed

    Walker, Christopher J; Miranda, Mario A; O'Hern, Matthew J; Blachly, James S; Moyer, Cassandra L; Ivanovich, Jennifer; Kroll, Karl W; Eisfeld, Ann-Kathrin; Sapp, Caroline E; Mutch, David G; Cohn, David E; Bundschuh, Ralf; Goodfellow, Paul J

    2016-10-01

    Next-generation sequencing has revolutionized cancer genetics, but accurately detecting mutations in repetitive DNA sequences, especially mononucleotide runs, remains a challenge. This is a particular concern for tumors with defective mismatch repair (MMR) that accumulate strand-slippage mutations. We developed MonoSeq to improve indel mutation detection in mononucleotide runs, and used MonoSeq to investigate strand-slippage mutations in endometrial cancers, a tumor type that has frequent loss of MMR. We performed extensive Sanger sequencing to validate both clonal and subclonal MonoSeq mutation calls. Eighty-one regions containing mononucleotide runs were sequenced in 540 primary endometrial cancers (223 with defective MMR). Our analyses revealed that the overall mutation rate in MMR-deficient tumors was 20-30-fold higher than in MMR-normal tumors. MonoSeq analysis identified several previously unreported mutations, including a novel hotspot in an A7 run in the terminal exon of ARID5B.The ARID5B indel mutations were seen in both MMR-deficient and MMR-normal tumors, suggesting biologic selection. The analysis of tumor mRNAs revealed the presence of mutant transcripts that could result in translation of neopeptides. Improved detection of mononucleotide run strand-slippage mutations has clear implications for comprehensive mutation detection in tumors with defective MMR. Indel frameshift mutations and the resultant antigenic peptides could help guide immunotherapy strategies.

  7. DNA Helicases in NER, BER, and MMR.

    PubMed

    Kuper, Jochen; Kisker, Caroline

    2013-01-01

    Different DNA repair mechanisms have evolved to protect our genome from modifications caused by endogenous and exogenous agents, thus maintaining the integrity of the DNA. Helicases often play a central role in these repair pathways and have shown to be essential for diverse tasks within these mechanisms. In prokaryotic nucleotide excision repair (NER) for example the two helicases UvrB and UvrD assume vastly different functions. While UvrB is intimately involved in damage verification and acts as an anchor for the other prokaryotic NER proteins UvrA and UvrC, UvrD is required to resolve the post-incision complex leading to the release of UvrC and the incised ssDNA fragment. For the XPD helicase in eukaryotic NER a similar function in analogy to UvrB has been proposed, whereas XPB the second helicase uses only its ATPase activity during eukaryotic NER. In prokaryotic mismatch repair (MMR) UvrD again plays a central role. The different tasks of this protein in the different repair pathways highlight the importance of regulative protein-protein interactions to fine-tune its helicase activity. In other DNA repair pathways the role of the helicases involved is sometimes not as well characterized, and no helicase has so far been described to assume the function of UvrD in eukaryotic MMR. RecQ helicases and FancJ interact with eukaryotic MMR proteins but their involvement in this repair pathway is unclear. Lastly, long-patch base excision repair is linked to the WRN helicase and many proteins within this pathway interact with the helicase leading to increased activity of the interacting proteins as observed for pol β and FEN-1 or the helicase itself is negatively regulated through the interaction with APE-1. However, compared to the precise functions described for the helicases in the other DNA repair mechanisms the role of WRN in BER remains speculative and requires further analysis.

  8. 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.

  9. The impact of sequence divergence and DNA mismatch repair on homeologous recombination in Arabidopsis.

    PubMed

    Li, Liangliang; Jean, Martine; Belzile, François

    2006-03-01

    We examined the effects of substrate divergence and DNA mismatch repair (MMR) on recombination in Arabidopsis thaliana. Relative to the frequency observed in plants with a homologous construct (0% divergence), recombination was decreased 4.1-, 9.6-, 11.7- or 20.3-fold, respectively, in lines with constructs containing 0.5%, 2%, 4% or 9% divergence between the recombination substrates. To evaluate the contribution of the MMR system in this decrease, 12 independent reporter lines (two or three lines per reporter construct) were crossed to an AtMSH2 T-DNA insertional mutant. We examined the recombination frequency in progeny homozygous for a reporter T-DNA and homozygous either for the wild type or the mutant allele of AtMSH2. The loss of MMR activity led to a two- to ninefold increase in homeologous recombination and the size of the increase did not seem to correlate with the amount of divergence. Inversely, complementation of the insertional mutant with a wild-type cDNA of AtMSH2 reduced recombination. Our results demonstrate clearly that sequence divergence can dramatically reduce the recombination frequency in plants and that the MMR system plays a part in this decrease.

  10. Mismatch repair genes founder mutations and cancer susceptibility in Lynch syndrome.

    PubMed

    Ponti, G; Castellsagué, E; Ruini, C; Percesepe, A; Tomasi, A

    2015-06-01

    Founder mutations in specific populations are common in several Mendelian disorders. They are shared by apparently unrelated families that inherited them from a common ancestor that existed hundreds to thousands of years ago. They have been proven to impact in molecular diagnostics strategies in specific populations, where they can be assessed as the first screening step and, if positive, avoid further expensive gene scanning. In Lynch syndrome (LS), a dominantly inherited colorectal cancer disease, more than 50 founder pathogenic mutations have been described so far in the mismatch repair (MMR) genes (MLH1, MSH2, MSH6 and PMS2). We here provide a comprehensive summary of the founder mutations found in the MMR genes and an overview of their main characteristics. At a time when high-throughput strategies are being introduced in the molecular diagnostics of cancer, genetic testing for founder mutations can complement next generation sequencing (NGS) technologies to most efficiently identify MMR gene mutations in any given population. Additionally, special attention is paid to MMR founder mutations with interesting anthropological significance. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  11. Mutations affecting a putative MutLα endonuclease motif impact multiple mismatch repair functions

    PubMed Central

    Erdeniz, Naz; Nguyen, Megan; Deschênes, Suzanne M.; Liskay, R. Michael

    2008-01-01

    Mutations in DNA mismatch repair (MMR) lead to increased mutation rates and higher recombination between similar, but not identical sequences, as well as resistance to certain DNA methylating agents. Recently, a component of human MMR machinery, MutLα, has been shown to display a latent endonuclease activity. The endonuclease active site appears to include a conserved motif, DQHA(X)2E(X)4E, within the COOH-terminus of human PMS2. Substitution of the glutamic acid residue (E705) abolished the endonuclease activity and mismatch-dependent excision in vitro. Previously, we showed that the PMS2-E705K mutation and the corresponding mutation in Saccharomyces cerevisiae were both recessive loss of function alleles for mutation avoidance in vivo. Here, we show that mutations impacting this endonuclease motif also significantly affect MMR-dependent suppression of homeologous recombination in yeast and responses to Sn1-type methylating agents in both yeast and mammalian cells. Thus, our in vivo results suggest that the endonuclease activity of MutLα is important not only in MMR-dependent mutation avoidance but also for recombination and damage response functions. PMID:17567544

  12. Evolution of the methyl directed mismatch repair system in Escherichia coli.

    PubMed

    Putnam, Christopher D

    2016-02-01

    DNA mismatch repair (MMR) repairs mispaired bases in DNA generated by replication errors. MutS or MutS homologs recognize mispairs and coordinate with MutL or MutL homologs to direct excision of the newly synthesized DNA strand. In most organisms, the signal that discriminates between the newly synthesized and template DNA strands has not been definitively identified. In contrast, Escherichia coli and some related gammaproteobacteria use a highly elaborated methyl-directed MMR system that recognizes Dam methyltransferase modification sites that are transiently unmethylated on the newly synthesized strand after DNA replication. Evolution of methyl-directed MMR is characterized by the acquisition of Dam and the MutH nuclease and by the loss of the MutL endonuclease activity. Methyl-directed MMR is present in a subset of Gammaproteobacteria belonging to the orders Enterobacteriales, Pasteurellales, Vibrionales, Aeromonadales, and a subset of the Alteromonadales (the EPVAA group) as well as in gammaproteobacteria that have obtained these genes by horizontal gene transfer, including the medically relevant bacteria Fluoribacter, Legionella, and Tatlockia and the marine bacteria Methylophaga and Nitrosococcus. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Arsenic Inhibits DNA Mismatch Repair by Promoting EGFR Expression and PCNA Phosphorylation*

    PubMed Central

    Tong, Dan; Ortega, Janice; Kim, Christine; Huang, Jian; Gu, Liya; Li, Guo-Min

    2015-01-01

    Both genotoxic and non-genotoxic chemicals can act as carcinogens. However, while genotoxic compounds lead directly to mutations that promote unregulated cell growth, the mechanism by which non-genotoxic carcinogens lead to cellular transformation is poorly understood. Using a model non-genotoxic carcinogen, arsenic, we show here that exposure to arsenic inhibits mismatch repair (MMR) in human cells, possibly through its ability to stimulate epidermal growth factor receptor (EGFR)-dependent tyrosine phosphorylation of proliferating cellular nuclear antigen (PCNA). HeLa cells exposed to exogenous arsenic demonstrate a dose- and time-dependent increase in the levels of EGFR and tyrosine 211-phosphorylated PCNA. Cell extracts derived from arsenic-treated HeLa cells are defective in MMR, and unphosphorylated recombinant PCNA restores normal MMR activity to these extracts. These results suggest a model in which arsenic induces expression of EGFR, which in turn phosphorylates PCNA, and phosphorylated PCNA then inhibits MMR, leading to increased susceptibility to carcinogenesis. This study suggests a putative novel mechanism of action for arsenic and other non-genotoxic carcinogens. PMID:25907674

  14. Mismatch repair genes in renal cortical neoplasms.

    PubMed

    Baiyee, Daniel; Banner, Barbara

    2006-02-01

    Mutation of human mutL homolog 1 (MLH-1) and human mutS homolog 2 (MSH-2) has been linked with the pathogenesis of colorectal carcinoma in hereditary nonpolyposis colorectal cancer syndrome and other carcinomas. Mutations of these genes in renal cell carcinomas were recently described. The aim of this study was to examine the expression of MLH-1 and MSH-2 in renal cortical neoplasms of various histological types by immunohistochemistry. Thirty-eight (n = 38) resected renal tumors were obtained from the surgical pathology files of the UMass Memorial Healthcare, including clear cell carcinomas (CLEARs, n = 20), papillary carcinomas (PAPs, n = 8), chromophobe carcinomas (CHRs, n = 4), and oncocytomas (ONCs, n = 6). Positive immunostaining for MLH-1 and MSH-2 was graded by the number of positive tumor cell nuclei, as follows: 0, negative; 1, up to one third of positive nuclei; 2, one to two thirds positive; and 3, greater than two thirds positive. Loss of MLH-1 or MSH-2 was defined as a tumor with grade 0 or 1, compared with the normal tubules. Normal tubules and intercalated ducts contained cells positive for MLH-1 and MSH-2 in all cases. For both antibodies, positive staining in tumors ranged from grade 1 to 3 in the CLEAR and PAP but was only grade 2 to 3 in the CHR and ONC. Loss of MLH-1 and/or MSH-2 occurred in malignant tumors but not in ONC. Loss of MLH-1 was present in 8 (40%) of 20 CLEARs and 4 (50%) of 8 PAPs, compared with loss of MSH-2 in 4 (20%) of 20 CLEARs and 1 (25%) of 4 CHRs. Our results suggest that loss of mismatch repair genes is involved in the malignant transformation in some renal carcinomas, particularly those derived from the proximal tubules.

  15. E. coli mismatch repair acts downstream of replication fork stalling to stabilize the expanded (GAA·TTC)n sequence

    PubMed Central

    Bourn, Rebecka L.; Rindler, Paul M.; Pollard, Laura M.; Bidichandani, Sanjay I.

    2008-01-01

    Expanded triplet repeat sequences are known to cause at least 16 inherited neuromuscular diseases. In addition to short length changes, expanded triplet repeat tracts frequently undergo large changes, often amounting to hundreds of base-pairs. Such changes might occur when template or primer slipping creates insertion/deletion loops (IDLs), which are normally repaired by the mismatch repair system (MMR). However, in prokaryotes and eukaryotes, MMR promotes large changes in the length of (CTG·CAG)n sequences, the motif most commonly associated with human disease. We tested the effect of MMR on instability of the expanded (GAA·TTC)n sequence, which causes Friedreich ataxia, by comparing repeat instability in wild-type and MMR-deficient strains of E. coli. As expected, the prevalence of small mutations increased in the MMR-deficient strains. However, the prevalence of large contractions increased in the MMR mutants specifically when GAA was the lagging strand template, the orientation in which replication fork stalling is known to occur. After hydroxyurea-induced stalling, both orientations of replication showed significantly more large contractions in MMR mutants than in the wild-type, suggesting that fork stalling may be responsible for the large contractions. Deficiency of MMR promoted large contractions independently of RecA status, a known determinant of (GAA·TTC)n instability. These data suggest that two independent mechanisms act in response to replication stalling to prevent instability of the (GAA·TTC)n sequence in E. coli, when GAA serves as the lagging strand template: one that is dependent on RecA-mediated restart of stalled forks, and another that is dependent on MMR-mediated repair of IDLs. While MMR destabilizes the (CTG·CAG)n sequence, it is involved in stabilization of the (GAA·TTC)n sequence. The role of MMR in triplet repeat instability therefore depends on the repeat sequence and the orientation of replication. PMID:19046977

  16. Quantifying the contributions of base selectivity, proofreading and mismatch repair to nuclear DNA replication in Saccharomyces cerevisiae.

    PubMed

    St Charles, Jordan A; Liberti, Sascha E; Williams, Jessica S; Lujan, Scott A; Kunkel, Thomas A

    2015-07-01

    Mismatches generated during eukaryotic nuclear DNA replication are removed by two evolutionarily conserved error correction mechanisms acting in series, proofreading and mismatch repair (MMR). Defects in both processes are associated with increased susceptibility to cancer. To better understand these processes, we have quantified base selectivity, proofreading and MMR during nuclear DNA replication in Saccharomyces cerevisiae. In the absence of proofreading and MMR, the primary leading and lagging strand replicases, polymerase ɛ and polymerase δ respectively, synthesize DNA in vivo with somewhat different error rates and specificity, and with apparent base selectivity that is more than 100 times higher than measured in vitro. Moreover, leading and lagging strand replication fidelity rely on a different balance between proofreading and MMR. On average, proofreading contributes more to replication fidelity than does MMR, but their relative contributions vary from nearly all proofreading of some mismatches to mostly MMR of other mismatches. Thus accurate replication of the two DNA strands results from a non-uniform and variable balance between error prevention, proofreading and MMR.

  17. Absence of chicken myelin basic protein residues in commercial formulations of MMR vaccine.

    PubMed

    Afzal, M A; Pipkin, P A; Minor, P D

    2000-10-15

    Several preparations of MMR vaccines and their progenitor monovalent vaccine bulks produced by two different manufacturers were examined serologically for the presence of chicken myelin basic protein (MBP) residues. The products were challenged against several commercial preparations of anti-hMBP antisera that reacted positively with the control MBP preparations of human and chicken origins. There was no evidence of the presence of MBP components in MMR vaccines or their progenitor vaccine bulks as shown by the reactivity profiles of the antibody preparations against control and test antigens.

  18. Comprehensive characterization of HNPCC-related colorectal cancers reveals striking molecular features in families with no germline mismatch repair gene mutations.

    PubMed

    Abdel-Rahman, Wael M; Ollikainen, Miina; Kariola, Reetta; Järvinen, Heikki J; Mecklin, Jukka-Pekka; Nyström-Lahti, Minna; Knuutila, Sakari; Peltomäki, Päivi

    2005-02-24

    A considerable fraction of families with HNPCC shows no germline mismatch repair (MMR) gene mutations. We previously detected 'hidden' MMR gene defects in 42% of such families, leaving the remaining 58% 'truly' mutation negative. Here, we characterized 50 colorectal carcinomas and five adenomas arising in HNPCC families; 24 truly MMR gene mutation negative and 31 MMR gene mutation positive. Among 31 tumors from MMR gene mutation positive families, 25 (81%) had active Wnt signaling as indicated by aberrant beta-catenin localization with or without CTNNB1 mutations, compared to only 7/18 tumors from MMR gene mutation negative families (39%; P=0.005). CGH studies revealed stable profiles in 9/16 (56%) of MMR gene mutation negative tumors, which was significantly associated with membranous beta-catenin (P=0.005). Tumors with membranous beta-catenin from the MMR gene mutation negative group also showed low frequency of TP53 mutations compared to those with nuclear beta-catenin. Thus, a majority of the MMR gene mutation negative cases exhibited a novel molecular pattern characterized by the paucity of changes in common pathways to colorectal carcinogenesis. This feature distinguishes the MMR gene mutation negative families from both HNPCC families linked to MMR defects and sporadic cases, suggesting the involvement of novel predisposition genes and pathways in such families.

  19. Mismatch repair at stop codons is directed independent of GATC methylation on the Escherichia coli chromosome.

    PubMed

    Sneppen, Kim; Semsey, Szabolcs

    2014-12-05

    The mismatch repair system (MMR) corrects replication errors that escape proofreading. Previous studies on extrachromosomal DNA in Escherichia coli suggested that MMR uses hemimethylated GATC sites to identify the newly synthesized strand. In this work we asked how the distance of GATC sites and their methylation status affect the occurrence of single base substitutions on the E. coli chromosome. As a reporter system we used a lacZ gene containing an early TAA stop codon. We found that occurrence of point mutations at this stop codon is unaffected by GATC sites located more than 115 base pairs away. However, a GATC site located about 50 base pairs away resulted in a decreased mutation rate. This effect was independent of Dam methylation. The reversion rate of the stop codon increased only slightly in dam mutants compared to mutL and mutS mutants. We suggest that unlike on extrachromosomal DNA, GATC methylation is not the only strand discrimination signal for MMR on the E. coli chromosome.

  20. Mismatch repair at stop codons is directed independent of GATC methylation on the Escherichia coli chromosome

    PubMed Central

    Sneppen, Kim; Semsey, Szabolcs

    2014-01-01

    The mismatch repair system (MMR) corrects replication errors that escape proofreading. Previous studies on extrachromosomal DNA in Escherichia coli suggested that MMR uses hemimethylated GATC sites to identify the newly synthesized strand. In this work we asked how the distance of GATC sites and their methylation status affect the occurrence of single base substitutions on the E. coli chromosome. As a reporter system we used a lacZ gene containing an early TAA stop codon. We found that occurrence of point mutations at this stop codon is unaffected by GATC sites located more than 115 base pairs away. However, a GATC site located about 50 base pairs away resulted in a decreased mutation rate. This effect was independent of Dam methylation. The reversion rate of the stop codon increased only slightly in dam mutants compared to mutL and mutS mutants. We suggest that unlike on extrachromosomal DNA, GATC methylation is not the only strand discrimination signal for MMR on the E. coli chromosome. PMID:25475788

  1. Mutation mismatch repair gene deletions in diffuse large B-cell lymphoma.

    PubMed

    Couronné, Lucile; Ruminy, Philippe; Waultier-Rascalou, Agathe; Rainville, Vinciane; Cornic, Marie; Picquenot, Jean-Michel; Figeac, Martin; Bastard, Christian; Tilly, Hervé; Jardin, Fabrice

    2013-05-01

    To further unravel the molecular pathogenesis of diffuse large B-cell lymphoma (DLBCL), we performed high-resolution comparative genomic hybridization on lymph node biopsies from 70 patients. With this strategy, we identified microdeletions of genes involved in the mutation mismatch repair (MMR) pathway in two samples. The first patient presented with a homozygous deletion of MSH2-MSH6 due to duplication of an unbalanced pericentric inversion of chromosome 2. The other case showed a PMS2 heterozygous deletion. PMS2 and MSH2-MSH6 abnormalities, respectively, resulted in a decrease and complete loss of gene expression. However, unlike tumors associated with the hereditary non-polyposis colorectal cancer syndrome or immunodeficiency-related lymphomas, no microsatellite instability was detected. Mutational profiles revealed especially in one patient an aberrant hypermutation without a clear activation-induced cytidine deaminase signature, indicating a breakdown of the high-fidelity repair in favor of the error-prone repair pathway. Our findings suggest that in a rare subset of patients, inactivation of the genes of the MMR pathway is likely an important step in the molecular pathogenesis of DLBCL and does not involve the same molecular mechanisms as other common neoplasms with MMR deficiency.

  2. Concurrent genetic alterations in DNA polymerase proofreading and mismatch repair in human colorectal cancer

    PubMed Central

    Yoshida, Rintaro; Miyashita, Kaname; Inoue, Mayuko; Shimamoto, Akiyoshi; Yan, Zhao; Egashira, Akinori; Oki, Eiji; Kakeji, Yoshishiro; Oda, Shinya; Maehara, Yoshihiko

    2011-01-01

    Genomic sequences encoding the 3′ exonuclease (proofreading) domains of both replicative DNA polymerases, pol delta and pol epsilon, were explored simultaneously in human colorectal carcinomas including six established cell lines. Three unequivocal sequence alterations, including one previously reported, were found, and all these were considered as dysfunctional mutations in light of the local amino-acid sequences. In particular, the F367S mutation found in the POLE gene encoding the pol epsilon catalytic subunit, which includes the proofreading domain, is the first found in human diseases. Surprisingly, the tumours carrying these proofreading domain mutations were all defective in DNA mismatch repair (MMR). In addition to the two cell lines with acknowledged MMR gene mutations, the third tumour was also demonstrated to harbour a distinct mutation in MLH1, and indeed exhibited a microsatellite-unstable phenotype. These findings suggest that, in concert with MMR deficiency, defective polymerase proofreading may also contribute to the mutator phenotype observed in human colorectal cancer. Our observations may suggest previously unrecognised complexities in the molecular abnormalities underlying the mutator phenotype in human neoplasms. PMID:21157497

  3. 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

  4. Mismatch repair at stop codons is directed independent of GATC methylation on the Escherichia coli chromosome

    NASA Astrophysics Data System (ADS)

    Sneppen, Kim; Semsey, Szabolcs

    2014-12-01

    The mismatch repair system (MMR) corrects replication errors that escape proofreading. Previous studies on extrachromosomal DNA in Escherichia coli suggested that MMR uses hemimethylated GATC sites to identify the newly synthesized strand. In this work we asked how the distance of GATC sites and their methylation status affect the occurrence of single base substitutions on the E. coli chromosome. As a reporter system we used a lacZ gene containing an early TAA stop codon. We found that occurrence of point mutations at this stop codon is unaffected by GATC sites located more than 115 base pairs away. However, a GATC site located about 50 base pairs away resulted in a decreased mutation rate. This effect was independent of Dam methylation. The reversion rate of the stop codon increased only slightly in dam mutants compared to mutL and mutS mutants. We suggest that unlike on extrachromosomal DNA, GATC methylation is not the only strand discrimination signal for MMR on the E. coli chromosome.

  5. Detection of coding microsatellite frameshift mutations in DNA mismatch repair-deficient mouse intestinal tumors.

    PubMed

    Woerner, Stefan M; Tosti, Elena; Yuan, Yan P; Kloor, Matthias; Bork, Peer; Edelmann, Winfried; Gebert, Johannes

    2015-11-01

    Different DNA mismatch repair (MMR)-deficient mouse strains have been developed as models for the inherited cancer predisposing Lynch syndrome. It is completely unresolved, whether coding mononucleotide repeat (cMNR) gene mutations in these mice can contribute to intestinal tumorigenesis and whether MMR-deficient mice are a suitable molecular model of human microsatellite instability (MSI)-associated intestinal tumorigenesis. A proof-of-principle study was performed to identify mouse cMNR-harboring genes affected by insertion/deletion mutations in MSI murine intestinal tumors. Bioinformatic algorithms were developed to establish a database of mouse cMNR-harboring genes. A panel of five mouse noncoding mononucleotide markers was used for MSI classification of intestinal matched normal/tumor tissues from MMR-deficient (Mlh1(-/-) , Msh2(-/-) , Msh2(LoxP/LoxP) ) mice. cMNR frameshift mutations of candidate genes were determined by DNA fragment analysis. Murine MSI intestinal tumors but not normal tissues from MMR-deficient mice showed cMNR frameshift mutations in six candidate genes (Elavl3, Tmem107, Glis2, Sdccag1, Senp6, Rfc3). cMNRs of mouse Rfc3 and Elavl3 are conserved in type and length in their human orthologs that are known to be mutated in human MSI colorectal, endometrial and gastric cancer. We provide evidence for the utility of a mononucleotide marker panel for detection of MSI in murine tumors, the existence of cMNR instability in MSI murine tumors, the utility of mouse subspecies DNA for identification of polymorphic repeats, and repeat conservation among some orthologous human/mouse genes, two of them showing instability in human and mouse MSI intestinal tumors. MMR-deficient mice hence are a useful molecular model system for analyzing MSI intestinal carcinogenesis.

  6. MLH1 promoter methylation, diet, and lifestyle factors in mismatch repair deficient colorectal cancer patients from EPIC-Norfolk.

    PubMed

    Gay, Laura J; Arends, Mark J; Mitrou, Panagiota N; Bowman, Richard; Ibrahim, Ashraf E; Happerfield, Lisa; Luben, Robert; McTaggart, Alison; Ball, Richard Y; Rodwell, Sheila A

    2011-01-01

    There is conflicting evidence for the role diet and lifestyle play in the development of mismatch repair (MMR)-deficient colorectal cancers (CRC). In this study, associations between MMR deficiency, clinicopathological characteristics, and dietary and lifestyle factors in sporadic CRC were investigated. Tumor samples from 185 individuals in the EPIC-Norfolk study were analyzed for MLH1 gene promoter methylation and microsatellite instability (MSI). Dietary and lifestyle data were collected prospectively using 7-day food diaries (7dd) and questionnaires. MMR-deficient tumor cases (MLH1 promoter methylation positive, MSI-H) were more likely to be female, older at diagnosis, early Dukes' stage (A/B), and proximal in location (MSI-H P = 0.03, 0.03, 0.02, and 0.001, respectively). Tumors with positive MLH1 promoter methylation (>20%) were associated with poor differentiation (P = 0.03). Low physical activity was associated with cases without MSI (P = 0.05). MMR deficiency was not significantly associated with cigarette smoking or alcohol, folate, fruit, vegetable, or meat consumption. We conclude that MMR-deficient tumors represent a distinct subset of sporadic CRC that are proximal in location, early Dukes' stage, and poorly differentiated, in cases that are female and older at diagnosis. There is no overall role for diet and lifestyle in MMR status in CRC, consistent with age-related susceptibility to MLH1 promoter methylation.

  7. A mononucleotide repeat in PRRT2 is an important, frequent target of mismatch repair deficiency in cancer

    PubMed Central

    Alves, Inês Teles; Cano, David; Böttcher, René; van der Korput, Hetty; Dinjens, Winand; Jenster, Guido; Trapman, Jan

    2017-01-01

    The DNA mismatch repair (MMR) system corrects DNA replication mismatches thereby contributing to the maintenance of genomic stability. MMR deficiency has been observed in prostate cancer but its impact on the genomic landscape of these tumours is not known. In order to identify MMR associated mutations in prostate cancer we have performed whole genome sequencing of the MMR deficient PC346C prostate cancer cell line. We detected a total of 1196 mutations in PC346C which was 1.5-fold higher compared to a MMR proficient prostate cancer sample (G089). Of all different mutation classes, frameshifts in mononucleotide repeat (MNR) sequences were significantly enriched in the PC346C sample. As a result, a selection of genes with frameshift mutations in MNR was further assessed regarding its mutational status in a comprehensive panel of prostate, ovarian, endometrial and colorectal cancer cell lines. We identified PRRT2 and DAB2IP to be frequently mutated in MMR deficient cell lines, colorectal and endometrial cancer patient samples. Further characterization of PRRT2 revealed an important role of this gene in cancer biology. Both normal prostate cell lines and a colorectal cancer cell line showed increased proliferation, migration and invasion when expressing the mutated form of PRRT2 (ΔPRRT2). The wild-type PRRT2 (PRRT2wt) had an inhibitory effect in proliferation, consistent with the low expression level of PRRT2 in cancer versus normal prostate samples. PMID:27907910

  8. GADD45α modulates curcumin sensitivity through c-Abl- and JNK-dependent signaling pathways in a mismatch repair-dependent manner.

    PubMed

    Naick, Hemanth; Jin, Shunqian; Baskaran, R

    2016-03-01

    Colorectal cancer is a critical health concern because of its incidence as the third most prevalent cancer in the world. Currently, 5-fluorouracil (5-FU), 6-thioguanine, and certain other genotoxic agents are mainstays of treatment; however, patients often die due to emergence of resistant population. Curcumin, a bioactive compound derived from the dietary turmeric (Curcuma longa) is an effective anticancer, anti-inflammatory, and antioxidant agent. Previously, we reported that human colorectal cancer cell lines compromised for mismatch repair (MMR) function exhibit heightened sensitivity to curcumin due to sustained curcumin-induced unrepaired DNA damage compared to proficient population counterparts. In this report, we show that the protein levels of gadd45α, whose transcript levels are increased during DNA damage and stress signals, are upregulated following curcumin treatment in a dose- and time-dependent manner. We further observed that cells compromised for Mlh1 function (HCT116 + Ch2) displayed ~twofold increased GADD45α upregulation compared to similarly treated proficient counterparts (HCT116 + Ch3). Similarly, suppression of Mlh1 using ShRNA increased GADD45α upregulation upon curcumin treatment. On the other hand, suppression of GADD45α using SiRNA-blocked curcumin-induced cell death induction in Mlh1-deficient cells. Moreover, inhibition of Abl through ST571 treatment and its downstream effector JNK through SP600125 treatment blocked GADD45α upregulation and cell death triggered by curcumin. Collective results lead us to conclude that GADD45α modulates curcumin sensitivity through activation of c-Abl > JNK signaling in a mismatch repair-dependent manner.

  9. A novel DNA damage response mediated by DNA mismatch repair in Caenorhabditis elegans: induction of programmed autophagic cell death in non-dividing cells

    PubMed Central

    Moriwaki, Takahito; Kato, Yuichi; Nakamura, Chihiro; Ishikawa, Satoru; Zhang-Akiyama, Qiu-Mei

    2015-01-01

    DNA mismatch repair (MMR) contributes to genome integrity by correcting errors of DNA polymerase and inducing cell death in response to DNA damage. Dysfunction of MMR results in increased mutation frequency and cancer risk. Clinical researches revealed that MMR abnormalities induce cancers of non-dividing tissues, such as kidney and liver. However, how MMR suppresses cancer in non-dividing tissues is not understood. To address that mechanism, we analyzed the roles of MMR in non-dividing cells using Caenorhabditis elegans (C. elegans), in which all somatic cells are non-dividing in the adult stage. In this study, we used stable MMR-mutant lines with a balancer chromosome. First, we confirmed that deficiency of MMR leads to resistance to various mutagens in C. elegans dividing cells. Next, we performed drug resistance assays, and found that MMR-deficient adult worms were resistant to SN1-type alkylating and oxidizing agents. In addition, dead cell staining and reporter assays of an autophagy-related gene demonstrated that the cell death was autophagic cell death. Interestingly, this autophagic cell death was not suppressed by caffeine, implying that MMR induces death of non-dividing cells in an atl-1-independent manner. Hence, we propose the hypothesis that MMR prevents cancers in non-dividing tissues by directly inducing cell death. PMID:26413217

  10. Lower Cancer Incidence in Amsterdam-I Criteria Families Without Mismatch Repair Deficiency

    PubMed Central

    Lindor, Noralane M.; Rabe, Kari; Petersen, Gloria M.; Haile, Robert; Casey, Graham; Baron, John; Gallinger, Steve; Bapat, Bharati; Aronson, Melyssa; Hopper, John; Jass, Jeremy; LeMarchand, Loic; Grove, John; Potter, John; Newcomb, Polly; Terdiman, Jonathan P.; Conrad, Peggy; Moslein, Gabriella; Goldberg, Richard; Ziogas, Argyrios; Anton-Culver, Hoda; de Andrade, Mariza; Siegmund, Kim; Thibodeau, Stephen N.; Boardman, Lisa A.; Seminara, Daniela

    2010-01-01

    Context Approximately 60% of families that meet the Amsterdam-I criteria (AC-I) for hereditary nonpolyposis colorectal cancer (HNPCC) have a hereditary abnormality in a DNA mismatch repair (MMR) gene. Cancer incidence in AC-I families with MMR gene mutations is reported to be very high, but cancer incidence for individuals in AC-I families with no evidence of an MMR defect is unknown. Objective To determine if cancer risks in AC-I families with no apparent deficiency in DNA MMR are different from cancer risks in AC-I families with DNA MMR abnormalities. Design, Setting, and Participants Identification (1997–2001) of 161 AC-I pedigrees from multiple population- and clinic-based sources in North America and Germany, with families grouped into those with (group A) or without (group B) MMR deficiency by tumor testing. A total of 3422 relatives were included in the analyses. Main Outcome Measures Cancer incidence in groups A and B (excluding the 3 affected members used to define each pedigree as AC-I) and computed age- and sex-adjusted standardized incidence ratios (SIRs) using Surveillance, Epidemiology, and End Results data. Results Group A families from both population- and clinic-based series showed increased incidence of the HNPCC-related cancers. Group B families showed increased incidence only for colorectal cancer (SIR, 2.3; 95% confidence interval, 1.7–3.0) and to a lesser extent than group A (SIR, 6.1; 95% confidence interval, 5.2–7.2) (P<.001). Conclusions Families who fulfill AC-I criteria but who have no evidence of a DNA MMR defect do not share the same cancer incidence as families with HNPCC-Lynch syndrome (ie, hereditary MMR deficiency). Relatives in such families have a lower incidence of colorectal cancer than those in families with HNPCC-Lynch syndrome, and incidence may not be increased for other cancers. These families should not be described or counseled as having HNPCC-Lynch syndrome. To facilitate distinguishing these entities, the

  11. The mismatch repair system reduces meiotic homeologous recombination and stimulates recombination-dependent chromosome loss.

    PubMed Central

    Chambers, S R; Hunter, N; Louis, E J; Borts, R H

    1996-01-01

    Efficient genetic recombination requires near-perfect homology between participating molecules. Sequence divergence reduces the frequency of recombination, a process that is dependent on the activity of the mismatch repair system. The effects of chromosomal divergence in diploids of Saccharomyces cerevisiae in which one copy of chromosome III is derived from a closely related species, Saccharomyces paradoxus, have been examined. Meiotic recombination between the diverged chromosomes is decreased by 25-fold. Spore viability is reduced with an observable increase in the number of tetrads with only two or three viable spores. Asci with only two viable spores are disomic for chromosome III, consistent with meiosis I nondisjunction of the homeologs. Asci with three viable spores are highly enriched for recombinants relative to tetrads with four viable spores. In 96% of the class with three viable spores, only one spore possesses a recombinant chromosome III, suggesting that the recombination process itself contributes to meiotic death. This phenomenon is dependent on the activities of the mismatch repair genes PMS1 and MSH2. A model of mismatch-stimulated chromosome loss is proposed to account for this observation. As expected, crossing over is increased in pms1 and msh2 mutants. Furthermore, genetic exchange in pms1 msh2 double mutants is affected to a greater extent than in either mutant alone, suggesting that the two proteins act independently to inhibit homeologous recombination. All mismatch repair-deficient strains exhibited reductions in the rate of chromosome III nondisjunction. PMID:8887641

  12. Prediction of biological behavior and prognosis of colorectal cancer patients by tumor MSI/MMR in the Chinese population

    PubMed Central

    Yan, Wen-Yue; Hu, Jing; Xie, Li; Cheng, Lei; Yang, Mi; Li, Li; Shi, Jiong; Liu, Bao-Rui; Qian, Xiao-Ping

    2016-01-01

    Colorectal cancers (CRCs) exhibiting microsatellite instability (MSI) have special biological behavior. The clinical predictors for MSI and its survival relevance for the Chinese population were still unclear. Seven hundred ninety-five CRC patients were retrospectively assessed. Mismatch repair (MMR) proteins (MSH2, MSH6, PMS1, and MLH1) expression was detected by immunohistochemistry using tumor tissues of all patients. DNA MSI status was analyzed by polymerase chain reaction in 182 samples randomly selected from the 795 cases. Among all CRC tumor tissues, 97 cases (12.2%) were with an MMR protein-deficient (MMR-D) phenotype, whereas 698 cases (87.8%) were with an MMR proteins intact (MMR-I) phenotype. A total of 21 (11.5%) CRCs were identified as having high microsatellite instability, 156 (85.7%) tumors were having microsatellite stability (MSS), and five (2.7%) were having low microsatellite instability. Importantly, MMR status was demonstrated to be moderately consistent with MSI status (κ=0.845, 95% confidence interval [CI] 0.721, 0.969). Unconditional logistic regression analysis revealed age, number of lymph node, tumor diameter, and tumor site as predictors for MSI with a substantial ability to discriminate different MSI status by area under curve of 80.62% using receiver operation curve. Compared with MMR-I, MMR-D was an independent prognostic factor for longer overall survival (hazard ratio =0.340, 95% CI 0.126, 0.919; P=0.034). MMR-D is an independent prognostic factor for better outcome. Our results may provide evidence for individualized diagnosis and treatment of CRC, but this will require further validation in larger sample studies. PMID:27994472

  13. Prediction of biological behavior and prognosis of colorectal cancer patients by tumor MSI/MMR in the Chinese population.

    PubMed

    Yan, Wen-Yue; Hu, Jing; Xie, Li; Cheng, Lei; Yang, Mi; Li, Li; Shi, Jiong; Liu, Bao-Rui; Qian, Xiao-Ping

    2016-01-01

    Colorectal cancers (CRCs) exhibiting microsatellite instability (MSI) have special biological behavior. The clinical predictors for MSI and its survival relevance for the Chinese population were still unclear. Seven hundred ninety-five CRC patients were retrospectively assessed. Mismatch repair (MMR) proteins (MSH2, MSH6, PMS1, and MLH1) expression was detected by immunohistochemistry using tumor tissues of all patients. DNA MSI status was analyzed by polymerase chain reaction in 182 samples randomly selected from the 795 cases. Among all CRC tumor tissues, 97 cases (12.2%) were with an MMR protein-deficient (MMR-D) phenotype, whereas 698 cases (87.8%) were with an MMR proteins intact (MMR-I) phenotype. A total of 21 (11.5%) CRCs were identified as having high microsatellite instability, 156 (85.7%) tumors were having microsatellite stability (MSS), and five (2.7%) were having low microsatellite instability. Importantly, MMR status was demonstrated to be moderately consistent with MSI status (κ=0.845, 95% confidence interval [CI] 0.721, 0.969). Unconditional logistic regression analysis revealed age, number of lymph node, tumor diameter, and tumor site as predictors for MSI with a substantial ability to discriminate different MSI status by area under curve of 80.62% using receiver operation curve. Compared with MMR-I, MMR-D was an independent prognostic factor for longer overall survival (hazard ratio =0.340, 95% CI 0.126, 0.919; P=0.034). MMR-D is an independent prognostic factor for better outcome. Our results may provide evidence for individualized diagnosis and treatment of CRC, but this will require further validation in larger sample studies.

  14. The Mre11/Rad50/Nbs1 complex interacts with the mismatch repair system and contributes to temozolomide-induced G2 arrest and cytotoxicity.

    PubMed

    Mirzoeva, Olga K; Kawaguchi, Tomohiro; Pieper, Russell O

    2006-11-01

    The chemotherapeutic agent temozolomide produces O(6)-methylguanine (O6MG) in DNA, which triggers futile DNA mismatch repair, DNA double-strand breaks (DSB), G(2) arrest, and ultimately cell death. Because the protein complex consisting of Mre11/Rad50/Nbs1 (MRN complex) plays a key role in DNA damage detection and signaling, we asked if this complex also played a role in the cellular response to temozolomide. Temozolomide exposure triggered the assembly of MRN complex into chromatin-associated nuclear foci. MRN foci formed significantly earlier than gamma-H2AX and 53BP1 foci that assembled in response to temozolomide-induced DNA DSBs. MRN foci formation was suppressed in cells that incurred lower levels of temozolomide-induced O6MG lesions and/or had decreased mismatch repair capabilities, suggesting that the MRN foci formed not in response to temozolomide-induced DSB but rather in response to mismatch repair processing of mispaired temozolomide-induced O6MG lesions. Consistent with this idea, the MRN foci colocalized with those of proliferating cell nuclear antigen (a component of the mismatch repair complex), and the MRN complex component Nbs1 coimmunoprecipitated with the mismatch repair protein Mlh1 specifically in response to temozolomide treatment. Furthermore, small inhibitory RNA-mediated suppression of Mre11 levels decreased temozolomide-induced G(2) arrest and cytotoxicity in a manner comparable to that achieved by suppression of mismatch repair. These data show that temozolomide-induced O6MG lesions, acted upon by the mismatch repair system, drive formation of the MRN complex foci and the interaction of this complex with the mismatch repair machinery. The MRN complex in turn contributes to the control of temozolomide-induced G(2) arrest and cytotoxicity, and as such is an additional determining factor in glioma sensitivity to DNA methylating chemotherapeutic drugs such as temozolomide.

  15. Interaction of MutS and Vsr: Some Dominant-Negative mutS Mutations That Disable Methyladenine-Directed Mismatch Repair Are Active in Very-Short-Patch Repair

    PubMed Central

    Lieb, Margaret; Rehmat, Shehnaz; Bhagwat, Ashok S.

    2001-01-01

    In Escherichia coli and related bacteria, the very-short-patch (VSP) repair pathway uses an endonuclease, Vsr, to correct T · G mismatches that result from the deamination of 5-methylcytosines in DNA to C · G. The products of mutS and mutL, which are required for adenine methylation-directed mismatch repair (MMR), enhance VSP repair. Multicopy plasmids carrying mutS alleles that are dominant negative for MMR were tested for their effects on VSP repair. Some mutS mutations (class I) did not lower VSP repair in a mutS+ background, and most class I mutations increased VSP repair in mutS cells more than plasmids containing mutS+. Other plasmid-borne mutS mutations (class II) and mutS+ decreased VSP repair in the mutS+ background. Thus, MutS protein lacking functions required for MMR can still participate in VSP repair, and our results are consistent with a model in which MutS binds transiently to the mispair and then translocates away from the mispair to create a specialized structure that enhances the binding of Vsr. PMID:11591694

  16. Interaction of MutS and Vsr: some dominant-negative mutS mutations that disable methyladenine-directed mismatch repair are active in very-short-patch repair.

    PubMed

    Lieb, M; Rehmat, S; Bhagwat, A S

    2001-11-01

    In Escherichia coli and related bacteria, the very-short-patch (VSP) repair pathway uses an endonuclease, Vsr, to correct T-G mismatches that result from the deamination of 5-methylcytosines in DNA to C-G. The products of mutS and mutL, which are required for adenine methylation-directed mismatch repair (MMR), enhance VSP repair. Multicopy plasmids carrying mutS alleles that are dominant negative for MMR were tested for their effects on VSP repair. Some mutS mutations (class I) did not lower VSP repair in a mutS(+) background, and most class I mutations increased VSP repair in mutS cells more than plasmids containing mutS(+). Other plasmid-borne mutS mutations (class II) and mutS(+) decreased VSP repair in the mutS(+) background. Thus, MutS protein lacking functions required for MMR can still participate in VSP repair, and our results are consistent with a model in which MutS binds transiently to the mispair and then translocates away from the mispair to create a specialized structure that enhances the binding of Vsr.

  17. Mechanisms in E. coli and Human Mismatch Repair (Nobel Lecture).

    PubMed

    Modrich, Paul

    2016-07-18

    DNA molecules are not completely stable, they are subject to chemical or photochemical damage and errors that occur during DNA replication resulting in mismatched base pairs. Through mechanistic studies Paul Modrich showed how replication errors are corrected by strand-directed mismatch repair in Escherichia coli and human cells.

  18. Avalanching mutations in biallelic mismatch repair deficiency syndrome.

    PubMed

    Waterfall, Joshua J; Meltzer, Paul S

    2015-03-01

    Tumors from pediatric patients generally contain relatively few somatic mutations. A new study reports a striking exception in individuals in whom biallelic germline deficiency for mismatch repair is compounded by somatic loss of function in DNA proofreading polymerases, resulting in 'ultra-hypermutated' malignant brain tumors.

  19. DNA mismatch repair: Dr. Jekyll and Mr. Hyde?

    PubMed

    Hsieh, Peggy

    2012-09-14

    In this issue, Peña-Diaz et al. (2012) describe a pathway for somatic mutation in nonlymphoid cells termed noncanonical DNA mismatch repair, whereby the error-prone translesion polymerase Pol-η substitutes for high-fidelity replicative polymerases to resynthesize excised regions opposite DNA damage. Copyright © 2012 Elsevier Inc. All rights reserved.

  20. High prevalence of deficient mismatch repair phenotype and the V600E BRAF mutation in elderly patients with colorectal cancer.

    PubMed

    Aparicio, Thomas; Schischmanoff, Olivier; Poupardin, Cecile; Mary, Florence; Soufir, Nadem; Barrat, Christophe; Bellaiche, Guy; Boubaya, Marouane; Choudat, Laurence; Cucherousset, Joel; DesGuetz, Gaetan; Wind, Philippe; Benamouzig, Robert

    2014-10-01

    Colorectal cancer (CRC) occurs mostly in the elderly. However, the biology of CRC in elderly has been poorly studied. This study examined the prevalence of deficient mismatch repair phenotype (dMMR) and BRAF mutations according to age. MMR phenotype was prospectively determined by molecular analysis in patients of all ages undergoing surgery for CRC. BRAF V600E mutation status was analysed in a subset of dMMR tumours. A total of 754 patients who underwent surgery between 2005 and 2008 were included in the study. Amongst them, 272 (36%) were ≥75years old. The proportion of women <75 was 38% and that ≥75 was 53% (p<0.0001). The prevalence of dMMR was 19.4% in patients ≥75 and 10.7% in patients <75 (p=0.0017). For patients ≥75, the prevalence of dMMR was significantly higher in women than in men (27% vs 10.2%, respectively; p=0.003) but was similar in women and men <75 (12.5% vs 9.7%, respectively; p=0.4). We examined BRAF mutation status in 80 patients with dMMR tumours. The V600E BRAF mutation was significantly more frequent in patients ≥75 than in patients <75 (72.2% vs 11.4%, respectively; p<0.001). In patients ≥75, there was no difference in the prevalence of the BRAF V600E mutation according to sex (78% in women and 70% in men, p=0.9). The prevalence of dMMR in CRC is high in patients over 75. In elderly patients, dMMR tumours are significantly more frequent in women than in men. The BRAF mutation is frequent in elderly patients with CRC. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. DREMECELS: A Curated Database for Base Excision and Mismatch Repair Mechanisms Associated Human Malignancies

    PubMed Central

    Shukla, Ankita; Singh, Tiratha Raj

    2016-01-01

    DNA repair mechanisms act as a warrior combating various damaging processes that ensue critical malignancies. DREMECELS was designed considering the malignancies with frequent alterations in DNA repair pathways, that is, colorectal and endometrial cancers, associated with Lynch syndrome (also known as HNPCC). Since lynch syndrome carries high risk (~40–60%) for both cancers, therefore we decided to cover all three diseases in this portal. Although a large population is presently affected by these malignancies, many resources are available for various cancer types but no database archives information on the genes specifically for only these cancers and disorders. The database contains 156 genes and two repair mechanisms, base excision repair (BER) and mismatch repair (MMR). Other parameters include some of the regulatory processes that have roles in these disease progressions due to incompetent repair mechanisms, specifically BER and MMR. However, our unique database mainly provides qualitative and quantitative information on these cancer types along with methylation, drug sensitivity, miRNAs, copy number variation (CNV) and somatic mutations data. This database would serve the scientific community by providing integrated information on these disease types, thus sustaining diagnostic and therapeutic processes. This repository would serve as an excellent accompaniment for researchers and biomedical professionals and facilitate in understanding such critical diseases. DREMECELS is publicly available at http://www.bioinfoindia.org/dremecels. PMID:27276067

  2. 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

  3. 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

  4. Evidence that hMLH3 functions primarily in meiosis and in hMSH2-hMSH3 mismatch repair.

    PubMed

    Charbonneau, Nicole; Amunugama, Ravindra; Schmutte, Christoph; Yoder, Kristine; Fishel, Richard

    2009-07-01

    The MutS (MSH) and MutL (MLH) homologs are conserved proteins that function in mismatch repair (MMR) and meiosis. We examined mRNA and protein expression of hMLH3 compared to other human MSH and MLH in a panel of human tissues and the HeLa cell line. Quantitative PCR suggests that MSH and MLH transcripts are expressed ubiquitously. hMLH3 mRNA is present at low levels in numerous tissues. Protein expression appears to correlate with a threshold of mRNA expression with hMLH3 present at high levels in testis. In addition, we have found and mapped interactions between hMLH1 and hMLH3 with hMSH3. These data are consistent with yeast studies and suggest a role for hMLH3 in meiosis as well as hMSH2-hMSH3 repair processes and little if any role in Hereditary Non-Polyposis Colorectal Cancer (HNPCC).

  5. Emerging importance of mismatch repair components including UvrD helicase and their cross-talk with the development of drug resistance in malaria parasite.

    PubMed

    Ahmad, Moaz; Tuteja, Renu

    2014-12-01

    Human malaria is an important parasitic infection responsible for a significant number of deaths worldwide, particularly in tropical and subtropical regions. The recent scenario has worsened mainly because of the emergence of drug-resistant malaria parasites having the potential to spread across the world. Drug-resistant parasites possess a defective mismatch repair (MMR); therefore, it is essential to explore its mechanism in detail to determine the underlying cause. Recently, artemisinin-resistant parasites have been reported to exhibit nonsynonymous single nucleotide polymorphisms in genes involved in MMR pathways such as MutL homolog (MLH) and UvrD. Plasmodium falciparum MLH is an endonuclease required to restore the defective MMR in drug-resistant W2 strain of P. falciparum. Although the role of helicases in eukaryotic MMR has been questioned, the identification and characterization of the UvrD helicase and their cross-talk with MLH in P. falciparum suggests the possible involvement of UvrD in MMR. A comparative genome-wide analysis revealed the presence of the UvrD helicase in Plasmodium species, while it is absent in human host. Therefore, PfUvrD may emerge as a suitable drug target to control malaria. This review study is focused on recent developments in MMR biochemistry, emerging importance of the UvrD helicase, possibility of its involvement in MMR and the emerging cross-talk between MMR components and drug resistance in malaria parasite. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Role of mismatch repair in the Escherichia coli UVM response.

    PubMed

    Murphy, H S; Palejwala, V A; Rahman, M S; Dunman, P M; Wang, G; Humayun, M Z

    1996-12-01

    Mutagenesis at 3,N4-ethenocytosine (epsilonC), a nonpairing mutagenic lesion, is significantly enhanced in Escherichia coli cells pretreated with UV, alkylating agents, or H2O2. This effect, termed UVM (for UV modulation of mutagenesis), is distinct from known DNA damage-inducible responses, such as the SOS response, the adaptive response to alkylating agents, or the oxyR-mediated response to oxidative agents. Here, we have addressed the hypothesis that UVM results from transient depletion of a mismatch repair activity that normally acts to reduce mutagenesis. To test whether the loss of mismatch repair activities results in the predicted constitutive UVM phenotype, E. coli cells defective for methyl-directed mismatch repair, for very-short-patch repair, or for the N-glycosylase activities MutY and MutM were treated with the UVM-inducing agent 1-methyl-3-nitro-1-nitrosoguanidine, with subsequent transfection of M13 viral single-stranded DNA bearing a site-specific epsilonC lesion. Survival of the M13 DNA was measured as transfection efficiency, and mutation fixation at the lesion was characterized by multiplex sequencing technology. The results showed normal UVM induction patterns in all the repair-defective strains tested. In addition, normal UVM induction was observed in cells overexpressing MutH, MutL, or MutS. All strains displayed UVM reactivation, the term used to describe the increased survival of epsilonC-containing DNA in UVM-induced cells. Taken together, these results indicate that the UVM response is independent of known mismatch repair systems in E. coli and may thus represent a previously unrecognized misrepair or misreplication pathway.

  7. Clinical and pathologic features of young endometrial cancer patients with loss of mismatch repair expression.

    PubMed

    Grzankowski, Kassondra S; Shimizu, David M; Kimata, Chieko; Black, Michael; Terada, Keith Y

    2012-09-01

    This study examines premenopausal and early menopause patients in a unique population with endometrial cancer and loss of mismatch repair (MMR) gene expression. The purpose is to compare clinical and pathologic differences in patients with loss of expression (LOE) to those with normal expression (NE). Endometrial cancer patients under age 60 in-between 1998 and 2008 were identified from a single tumor registry. Clinical and pathologic data were abstracted from records. Staining for expression of MSH6, MSH2, MLH1, and PMS2 were performed on archived tissue blocks. Statistical analysis was performed. 158 patients were analyzed; 58% Asian, 34% Pacific Islander, and 8% Caucasian. 31 demonstrated LOE of at least one MMR gene; 127 retained NE. 50% Caucasian, 21.9% Asian, and 12.5% Pacific Island populations had LOE of one or more MMR genes. LOE was found to have a higher incidence of Grade III (p=0.0013) and stage 3-4 tumors (p=0.0079), mean depth of myometrial invasion (p=0.0019), lymphovascular space invasion (p=0.0020), nodal metastases (p=0.0157), and a lower incidence of Grade I (p=0.0020) and stage 1A tumors (p=0.0085). LOE had a significantly lower mean BMI (p=0.0001). 35% of patients in the NE vs zero in the LOE group had a BMI greater than 40. Younger patients with LOE endometrial cancer appear to represent a clinically significant subgroup of patients without features characteristically found in classic type 1 endometrial cancer generally demonstrating lower BMI and tumors associated with poor prognostic characteristics. It is unclear if the distinctive ethnicity found in Hawaii has a significant impact on outcome. Further investigation is necessary to identify appropriate treatment strategies. Copyright © 2012 Elsevier Inc. All rights reserved.

  8. 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

  9. An Optimized Pentaplex PCR for Detecting DNA Mismatch Repair-Deficient Colorectal Cancers

    PubMed Central

    Hamelin, Richard; Boland, C. Richard

    2010-01-01

    Purpose Microsatellite instability (MSI) is used to screen colorectal cancers (CRC) for Lynch Syndrome, and to predict outcome and response to treatment. The current technique for measuring MSI requires DNA from normal and neoplastic tissues, and fails to identify tumors with specific DNA mismatch repair (MMR) defects. We tested a panel of five quasi-monomorphic mononucleotide repeat markers amplified in a single multiplex PCR reaction (pentaplex PCR) to detect MSI. Experimental Design We investigated a cohort of 213 CRC patients, comprised of 114 MMR-deficient and 99 MMR-proficient tumors. Immunohistochemical (IHC) analysis evaluated the expression of MLH1, MSH2, PMS2 and MSH6. MSI status was defined by differences in the quasi-monomorphic variation range (QMVR) from a pool of normal DNA samples, and measuring differences in allele lengths in tumor DNA. Results Amplification of 426 normal alleles allowed optimization of the QMVR at each marker, and eliminated the requirement for matched reference DNA to define MSI in each sample. Using ≥2/5 unstable markers as the criteria for MSI resulted in a sensitivity of 95.6% (95% CI = 90.1–98.1%) and a positive predictive value of 100% (95% CI = 96.6%–100%). Detection of MSH6-deficiency was limited using all techniques. Data analysis with a three-marker panel (BAT26, NR21 and NR27) was comparable in sensitivity (97.4%) and positive predictive value (96.5%) to the five marker panel. Both approaches were superior to the standard approach to measuring MSI. Conclusions An optimized pentaplex (or triplex) PCR offers a facile, robust, very inexpensive, highly sensitive, and specific assay for the identification of MSI in CRC. PMID:20195377

  10. Human mismatch repair system balances mutation rates between strands by removing more mismatches from the lagging strand.

    PubMed

    Andrianova, Maria A; Bazykin, Georgii A; Nikolaev, Sergey I; Seplyarskiy, Vladimir B

    2017-08-01

    Mismatch repair (MMR) is one of the main systems maintaining fidelity of replication. Differences in correction of errors produced during replication of the leading and the lagging DNA strands were reported in yeast and in human cancers, but the causes of these differences remain unclear. Here, we analyze data on human cancers with somatic mutations in two of the major DNA polymerases, delta and epsilon, that replicate the genome. We show that these cancers demonstrate a substantial asymmetry of the mutations between the leading and the lagging strands. The direction of this asymmetry is the opposite between cancers with mutated polymerases delta and epsilon, consistent with the role of these polymerases in replication of the lagging and the leading strands in human cells, respectively. Moreover, the direction of strand asymmetry observed in cancers with mutated polymerase delta is similar to that observed in MMR-deficient cancers. Together, these data indicate that polymerase delta (possibly together with polymerase alpha) contributes more mismatches during replication than its leading-strand counterpart, polymerase epsilon; that most of these mismatches are repaired by the MMR system; and that MMR repairs about three times more mismatches produced in cells during lagging strand replication compared with the leading strand. © 2017 Andrianova et al.; Published by Cold Spring Harbor Laboratory Press.

  11. Loss of ARID1A Expression in Gastric Cancer: Correlation with Mismatch Repair Deficiency and Clinicopathologic Features.

    PubMed

    Kim, Kyung-Ju; Jung, Hae Yoen; Oh, Mee-Hye; Cho, Hyundeuk; Lee, Ji-Hye; Lee, Hyun Ju; Jang, Si-Hyong; Lee, Moon Soo

    2015-09-01

    The AT-rich interactive domain 1A (ARID1A) gene encodes BRG1-associated factor 250a, a component of the SWItch/Sucrose NonFermentable chromatin remodeling complex, which is considered a tumor suppressor in many tumors. We aimed to investigate the prognostic significance of ARID1A expression in gastric cancers and explore its relationship with clinicopathologic parameters such as mismatch repair protein expression. Four tissue microarrays were constructed from 191 resected specimens obtained at Soonchunhyang University Cheonan Hospital from 2006 to 2008. Nuclear expression of ARID1A was semiquantitatively assessed and binarized into retained and lost expression. Loss of ARID1A expression was observed in 62 cases (32.5%). This was associated with more frequent vascular invasion (P=0.019) and location in the upper third of the stomach (P=0.001), and trended toward more poorly differentiated subtypes (P=0.054). ARID1A loss was significantly associated with the mismatch repair-deficient phenotype (P=0.003). ARID1A loss showed a statistically significant correlation with loss of MLH1 (P=0.001) but not MSH2 expression (P=1.000). Kaplan-Meier survival analysis showed no statistically significant difference in overall survival; however, patients with retained ARID1A expression tended to have better overall survival than those with loss of ARID1A expression (P=0.053). In both mismatch repair-deficient and mismatch repair-proficient groups, survival analysis showed no differences related to ARID1A expression status. Our results demonstrated that loss of ARID1A expression is closely associated with the mismatch repair-deficient phenotype, especially in sporadic microsatellite instability-high gastric cancers.

  12. Cascading MutS and MutL sliding clamps control DNA diffusion to activate mismatch repair.

    PubMed

    Liu, Jiaquan; Hanne, Jeungphill; Britton, Brooke M; Bennett, Jared; Kim, Daehyung; Lee, Jong-Bong; Fishel, Richard

    2016-11-24

    Mismatched nucleotides arise from polymerase misincorporation errors, recombination between heteroallelic parents and chemical or physical DNA damage. Highly conserved MutS (MSH) and MutL (MLH/PMS) homologues initiate mismatch repair and, in higher eukaryotes, act as DNA damage sensors that can trigger apoptosis. Defects in human mismatch repair genes cause Lynch syndrome or hereditary non-polyposis colorectal cancer and 10-40% of related sporadic tumours. However, the collaborative mechanics of MSH and MLH/PMS proteins have not been resolved in any organism. We visualized Escherichia coli (Ec) ensemble mismatch repair and confirmed that EcMutS mismatch recognition results in the formation of stable ATP-bound sliding clamps that randomly diffuse along the DNA with intermittent backbone contact. The EcMutS sliding clamps act as a platform to recruit EcMutL onto the mismatched DNA, forming an EcMutS-EcMutL search complex that then closely follows the DNA backbone. ATP binding by EcMutL establishes a second long-lived DNA clamp that oscillates between the principal EcMutS-EcMutL search complex and unrestricted EcMutS and EcMutL sliding clamps. The EcMutH endonuclease that targets mismatch repair excision only binds clamped EcMutL, increasing its DNA association kinetics by more than 1,000-fold. The assembly of an EcMutS-EcMutL-EcMutH search complex illustrates how sequential stable sliding clamps can modulate one-dimensional diffusion mechanics along the DNA to direct mismatch repair.

  13. Antagonism of ultraviolet-light mutagenesis by the methyl-directed mismatch-repair system of Escherichia coli.

    PubMed Central

    Liu, H; Hewitt, S R; Hays, J B

    2000-01-01

    Previous studies have demonstrated that the Escherichia coli MutHLS mismatch-repair system can process UV-irradiated DNA in vivo and that the human MSH2.MSH6 mismatch-repair protein binds more strongly in vitro to photoproduct/base mismatches than to "matched" photoproducts in DNA. We tested the hypothesis that mismatch repair directed against incorrect bases opposite photoproducts might reduce UV mutagenesis, using two alleles at E. coli lacZ codon 461, which revert, respectively, via CCC --> CTC and CTT --> CTC transitions. F' lacZ targets were mated from mut(+) donors into mutH, mutL, or mutS recipients, once cells were at substantial densities, to minimize spontaneous mutation prior to irradiation. In umu(+) mut(+) recipients, a range of UV fluences induced lac(+) revertant frequencies of 4-25 x 10(-8); these frequencies were consistently 2-fold higher in mutH, mutL, or mutS recipients. Since this effect on mutation frequency was unaltered by an Mfd(-) defect, it appears not to involve transcription-coupled excision repair. In mut(+) umuC122::Tn5 bacteria, UV mutagenesis (at 60 J/m(2)) was very low, but mutH or mutL or mutS mutations increased reversion of both lacZ alleles roughly 25-fold, to 5-10 x 10(-8). Thus, at UV doses too low to induce SOS functions, such as Umu(2)'D, most incorrect bases opposite occasional photoproducts may be removed by mismatch repair, whereas in heavily irradiated (SOS-induced) cells, mismatch repair may only correct some photoproduct/base mismatches, so UV mutagenesis remains substantial. PMID:10655206

  14. Improved generation of rat gene knockouts by target-selected mutagenesis in mismatch repair-deficient animals

    PubMed Central

    van Boxtel, Ruben; Toonen, Pim W; Verheul, Mark; van Roekel, Henk S; Nijman, Isaac J; Guryev, Victor; Cuppen, Edwin

    2008-01-01

    Background The laboratory rat (Rattus norvegicus) is one of the preferred model organisms in physiological and pharmacological research, although the availability of specific genetic models, especially gene knockouts, is limited. N-ethyl-N-nitrosourea (ENU)-driven target-selected mutagenesis is currently the most successful method in rats, although it is still very laborious and expensive. Results As ENU-induced DNA damage is normally recognized by the mismatch repair (MMR) system, we hypothesized that the effectiveness of the target-selected mutagenesis approach could be improved by using a MMR-deficient genetic background. Indeed, Msh6 knockout rats were found to be more sensitive to ENU treatment and the germ line mutation rate was boosted more than two-fold to 1 mutation per 585 kb. In addition, the molecular mutation spectrum was found to be changed in favor of generating knockout-type alleles by ~20%, resulting in an overall increase in efficiency of ~2.5 fold. The improved effectiveness was demonstrated by high throughput mutation discovery in 70 Mb of sequence in a set of only 310 mutant F1 rats. This resulted in the identification of 89 mutations of which four introduced a premature stopcodon and 64 resulted in amino acid changes. Conclusion Taken together, we show that the use of a MMR-deficient background considerably improves ENU-driven target-selected mutagenesis in the rat, thereby reducing animal use as well as screening costs. The use of a mismatch repair-deficient genetic background for improving mutagenesis and target-selected knockout efficiency is in principle applicable to any organism of interest. PMID:18840264

  15. Improved generation of rat gene knockouts by target-selected mutagenesis in mismatch repair-deficient animals.

    PubMed

    van Boxtel, Ruben; Toonen, Pim W; Verheul, Mark; van Roekel, Henk S; Nijman, Isaac J; Guryev, Victor; Cuppen, Edwin

    2008-10-07

    The laboratory rat (Rattus norvegicus) is one of the preferred model organisms in physiological and pharmacological research, although the availability of specific genetic models, especially gene knockouts, is limited. N-ethyl-N-nitrosourea (ENU)-driven target-selected mutagenesis is currently the most successful method in rats, although it is still very laborious and expensive. As ENU-induced DNA damage is normally recognized by the mismatch repair (MMR) system, we hypothesized that the effectiveness of the target-selected mutagenesis approach could be improved by using a MMR-deficient genetic background. Indeed, Msh6 knockout rats were found to be more sensitive to ENU treatment and the germ line mutation rate was boosted more than two-fold to 1 mutation per 585 kb. In addition, the molecular mutation spectrum was found to be changed in favor of generating knockout-type alleles by approximately 20%, resulting in an overall increase in efficiency of approximately 2.5 fold. The improved effectiveness was demonstrated by high throughput mutation discovery in 70 Mb of sequence in a set of only 310 mutant F1 rats. This resulted in the identification of 89 mutations of which four introduced a premature stopcodon and 64 resulted in amino acid changes. Taken together, we show that the use of a MMR-deficient background considerably improves ENU-driven target-selected mutagenesis in the rat, thereby reducing animal use as well as screening costs. The use of a mismatch repair-deficient genetic background for improving mutagenesis and target-selected knockout efficiency is in principle applicable to any organism of interest.

  16. Survey of unaffected BRCA and mismatch repair (MMR) mutation positive individuals

    PubMed Central

    Banks, Kimberly C.; Skelly, Joan; Kohlmann, Wendy; Bennett, Robin; Shannon, Kristen; Larson-Haidle, Joy; Ashakaga, Taka; Weitzel, Jeffrey N.; Wood, Marie

    2012-01-01

    Many individuals do not proceed with cancer predisposition testing due to fears of genetic discrimination (GD). We report the results of a survey of 47 unaffected, mutation positive individuals regarding insurance outcomes. Participants recruited from six different Cancer Risk Programs across the country were queried about their experiences with health, life, and disability insurance, as well as employment issues. Eighty-seven percent of participants carried a BRCA mutation and 87% were part of a group insurance plan at the time of testing. Forty-seven percent of participants self-paid for testing. Less than 10% of participants reported that their results were placed in the general medical record, while 43% did not know where their results were placed. Due to concerns about GD, 13% of participants stated they avoided changing jobs. Thirteen percent stated that their at-risk relatives had not undergone testing for the familial mutation due to fears about GD. Adverse events following genetic testing included two denials from private health insurers, one denial for average life insurance coverage and one denial for additional disability insurance. There were no reports of job discrimination. Results suggest fear of GD is prevalent, yet data do not support evidence that GD exists. PMID:19466581

  17. The cumulative effects of polymorphisms in the DNA mismatch repair genes and tobacco smoking in oesophageal cancer risk.

    PubMed

    Vogelsang, Matjaz; Wang, Yabing; Veber, Nika; Mwapagha, Lamech M; Parker, M Iqbal

    2012-01-01

    The DNA mismatch repair (MMR) enzymes repair errors in DNA that occur during normal DNA metabolism or are induced by certain cancer-contributing exposures. We assessed the association between 10 single-nucleotide polymorphisms (SNPs) in 5 MMR genes and oesophageal cancer risk in South Africans. Prior to genotyping, SNPs were selected from the HapMap database, based on their significantly different genotypic distributions between European ancestry populations and four HapMap populations of African origin. In the Mixed Ancestry group, the MSH3 rs26279 G/G versus A/A or A/G genotype was positively associated with cancer (OR = 2.71; 95% CI: 1.34-5.50). Similar associations were observed for PMS1 rs5742938 (GG versus AA or AG: OR = 1.73; 95% CI: 1.07-2.79) and MLH3 rs28756991 (AA or GA versus GG: OR = 2.07; 95% IC: 1.04-4.12). In Black individuals, however, no association between MMR polymorhisms and cancer risk was observed in individual SNP analysis. The interactions between MMR genes were evaluated using the model-based multifactor-dimensionality reduction approach, which showed a significant genetic interaction between SNPs in MSH2, MSH3 and PMS1 genes in Black and Mixed Ancestry subjects, respectively. The data also implies that pathogenesis of common polymorphisms in MMR genes is influenced by exposure to tobacco smoke. In conclusion, our findings suggest that common polymorphisms in MMR genes and/or their combined effects might be involved in the aetiology of oesophageal cancer.

  18. Association of obesity with DNA mismatch repair status and clinical outcome in patients with stage II or III colon carcinoma participating in NCCTG and NSABP adjuvant chemotherapy trials.

    PubMed

    Sinicrope, Frank A; Foster, Nathan R; Yoon, Harry H; Smyrk, Thomas C; Kim, George P; Allegra, Carmen J; Yothers, Greg; Nikcevich, Daniel A; Sargent, Daniel J

    2012-02-01

    Although the importance of obesity in colon cancer risk and outcome is recognized, the association of body mass index (BMI) with DNA mismatch repair (MMR) status is unknown. BMI (kg/m(2)) was determined in patients with TNM stage II or III colon carcinomas (n = 2,693) who participated in randomized trials of adjuvant chemotherapy. The association of BMI with MMR status and survival was analyzed by logistic regression and Cox models, respectively. Overall, 427 (16%) tumors showed deficient MMR (dMMR), and 630 patients (23%) were obese (BMI ≥ 30 kg/m(2)). Obesity was significantly associated with younger age (P = .021), distal tumor site (P = .012), and a lower rate of dMMR tumors (10% v 17%; P < .001) compared with normal weight. Obesity remained associated with lower rates of dMMR (odds ratio, 0.57; 95% CI, 0.41 to 0.79; P < .001) after adjusting for tumor site, stage, sex, and age. Among obese patients, rates of dMMR were lower in men compared with women (8% v 13%; P = .041). Obesity was associated with higher recurrence rates (P = .0034) and independently predicted worse disease-free survival (DFS; hazard ratio [HR], 1.37; 95% CI, 1.14 to 1.64; P = .0010) and overall survival (OS), whereas dMMR predicted better DFS (HR, 0.59; 95% CI, 0.47 to 0.74; P < .001) and OS. The favorable prognosis of dMMR was maintained in obese patients. Colon cancers from obese patients are less likely to show dMMR, suggesting obesity-related differences in the pathogenesis of colon cancer. Although obesity was independently associated with adverse outcome, the favorable prognostic impact of dMMR was maintained among obese patients.

  19. 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.

  20. Production of extrachromosomal microDNAs is linked to mismatch repair pathways and transcriptional activity

    PubMed Central

    Dillon, Laura W.; Kumar, Pankaj; Shibata, Yoshiyuki; Wang, Yuh-Hwa; Willcox, Smaranda; Griffith, Jack D.; Pommier, Yves; Takeda, Shunichi; Dutta, Anindya

    2015-01-01

    SUMMARY MicroDNAs are <400-base extrachromosomal circles found in mammalian cells. Tens of thousands of microDNAs have been found in all tissue types, including sperm. MicroDNAs arise preferentially from areas with high gene density, GC content, and exon density, from promoters with activating chromatin modifications and in sperm from the 5'-UTR of full-length LINE-1 elements, but are depleted from lamin-associated heterochromatin. Analysis of microDNAs from a set of human cancer cell lines revealed lineage-specific patterns of microDNA origins. A survey of microDNAs from chicken cells defective in various DNA repair proteins reveal that homologous recombination and nonhomologous end joining repair pathways are not required for microDNA production. Deletion of the MSH3 DNA mismatch repair protein results in a significant decrease in microDNA abundance, specifically from non-CpG genomic regions. Thus, microDNAs arise as part of normal cellular physiology; either from DNA breaks associated with RNA metabolism or from replication slippage followed by mismatch repair. PMID:26051933

  1. Production of Extrachromosomal MicroDNAs Is Linked to Mismatch Repair Pathways and Transcriptional Activity.

    PubMed

    Dillon, Laura W; Kumar, Pankaj; Shibata, Yoshiyuki; Wang, Yuh-Hwa; Willcox, Smaranda; Griffith, Jack D; Pommier, Yves; Takeda, Shunichi; Dutta, Anindya

    2015-06-23

    MicroDNAs are <400-base extrachromosomal circles found in mammalian cells. Tens of thousands of microDNAs have been found in all tissue types, including sperm. MicroDNAs arise preferentially from areas with high gene density, GC content, and exon density from promoters with activating chromatin modifications and in sperm from the 5'-UTR of full-length LINE-1 elements, but are depleted from lamin-associated heterochromatin. Analysis of microDNAs from a set of human cancer cell lines revealed lineage-specific patterns of microDNA origins. A survey of microDNAs from chicken cells defective in various DNA repair proteins reveals that homologous recombination and non-homologous end joining repair pathways are not required for microDNA production. Deletion of the MSH3 DNA mismatch repair protein results in a significant decrease in microDNA abundance, specifically from non-CpG genomic regions. Thus, microDNAs arise as part of normal cellular physiology—either from DNA breaks associated with RNA metabolism or from replication slippage followed by mismatch repair.

  2. 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; Ptrend = 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; Ptrend = 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.

  3. Association between expression of DNA mismatch repair genes and clinical features and prognosis of patients with radical resection of colon cancer.

    PubMed

    Wang, J B; Ma, D L; Li, J Y; Sun, Q D; Liu, Y E

    2016-08-19

    The aim of this study was to investigate the clinical significance of the expression of DNA mismatch repair (MMR) genes in patients subjected to radical surgical removal of colon cancer, as well as their correlation with disease prognosis. Ninety stage II and III colon cancer patients who received laparoscopic radical resection of colon cancer at our hospital were recruited in this study. The expression of hMLH1, hMSH2, hMSH6, and hPMS2 in the resected tumor tissues was examined by SP immunohistochemistry, in order to analyze the relationship between defective DNA MMR (dMMR) and the clinico-pathological features and prognosis of colon cancer. Patients were followed up over a period of 5-35 months, and the Kaplan-Meier survival curve was plotted. dMMR was confirmed in 27 subjects (30.0%), among whom recurrence with metastasis and death was reported in 5 (18.5%) and 2 (7.4%) patients, respectively. The remaining 63 subjects displayed proficient DNA MMR (pMMR); among these, 19 (30.2%) and 7 (11.1%) recurrences with metastasis and death were reported, respectively. dMMR showed no significant correlation with gender, age, or therapeutic modality (P > 0.05), but was significantly correlated with the degree of differentiation, tumor location, number of resected lymph nodes, presence of ileus, and TNM stage (P < 0.05). The prognosis of patients with dMMR was better than that of patients with pMMR. dMMR serves as a biomarker for the prognosis of stage II/III colon cancers.

  4. The Arabidopsis DNA mismatch repair gene PMS1 restricts somatic recombination between homeologous sequences.

    PubMed

    Li, Liangliang; Dion, Eric; Richard, Gabriel; Domingue, Olivier; Jean, Martine; Belzile, François J

    2009-04-01

    The eukaryotic DNA mismatch repair (MMR) system contributes to maintaining the fidelity of genetic information by correcting replication errors and preventing illegitimate recombination events. This study aimed to examine the function(s) of the Arabidopsis thaliana PMS1 gene (AtPMS1), one of three homologs of the bacterial MutL gene in plants. Two independent mutant alleles (Atpms1-1 and Atpms1-2) were obtained and one of these (Atpms1-1) was studied in detail. The mutant exhibited a reduction in seed set and a bias against the transmission of the mutant allele. Somatic recombination, both homologous and homeologous, was examined using a set of reporter constructs. Homologous recombination remained unchanged in the mutant while homeologous recombination was between 1.7- and 4.8-fold higher than in the wild type. This increase in homeologous recombination frequency was not correlated with the degree of sequence divergence. In RNAi lines, a range of increases in homeologous recombination were observed with two lines showing a 3.3-fold and a 3.6-fold increase. These results indicate that the AtPMS1 gene contributes to an antirecombination activity aimed at restricting recombination between diverged sequences.

  5. Associations of defect mismatch repair genes with prognosis and heredity in sporadic colorectal cancer.

    PubMed

    Ghanipour, L; Jirström, K; Sundström, M; Glimelius, B; Birgisson, H

    2017-02-01

    Microsatellite instability arises due to defect mismatch repair (MMR) and occurs in 10-20% of sporadic colorectal cancer. The purpose was to investigate correlations between defect MMR, prognosis and heredity for colorectal cancer in first-degree relatives. Tumour tissues from 318 patients consecutively operated for colorectal cancer were analysed for immunohistochemical expression of MLH1, MSH2 and MSH6 on tissue microarrays. Information on KRAS and BRAF mutation status was available for selected cases. Forty-seven (15%) tumours displayed MSI. No correlation was seen between patients exhibiting MSI in the tumour and heredity (p = 0.789). Patients with proximal colon cancer and MSI had an improved cancer-specific survival (p = 0.006) and prolonged time to recurrence (p = 0.037). In a multivariate analysis including MSI status, gender, CEA, vascular and neural invasion, patients with MSS and proximal colon cancer had an impaired cancer-specific survival compared with patients with MSI (HR, 4.32; CI, 1.46-12.78). The same prognostic information was also seen in distal colon cancer; no recurrences seen in the eight patients with stages II and III distal colon cancer and MSI, but the difference was not statistically significant. No correlation between MSI and heredity for colorectal cancer in first-degree relatives was seen. Patients with MSI tumours had improved survival. Copyright © 2016 Elsevier Ltd, BASO ~ The Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved.

  6. Crosstalk between mismatch repair and base excision repair in human gastric cancer.

    PubMed

    Simonelli, Valeria; Leuzzi, Giuseppe; Basile, Giorgia; D'Errico, Mariarosaria; Fortini, Paola; Franchitto, Annapaola; Viti, Valentina; Brown, Ashley R; Parlanti, Eleonora; Pascucci, Barbara; Palli, Domenico; Giuliani, Alessandro; Palombo, Fabio; Sobol, Robert W; Dogliotti, Eugenia

    2016-06-20

    DNA repair gene expression in a set of gastric cancers suggested an inverse association between the expression of the mismatch repair (MMR) gene MLH1 and that of the base excision repair (BER) gene DNA polymerase β (Polβ). To gain insight into possible crosstalk of these two repair pathways in cancer, we analysed human gastric adenocarcinoma AGS cells over-expressing Polβ or Polβ active site mutants, alone or in combination with MLH1 silencing. Next, we investigated the cellular response to the alkylating agent methyl methanesulfonate (MMS) and the purine analogue 6-thioguanine (6-TG), agents that induce lesions that are substrates for BER and/or MMR. AGS cells over-expressing Polβ were resistant to 6-TG to a similar extent as when MLH1 was inactivated while inhibition of O6-methylguanine-DNA methyltransferase (MGMT) was required to detect resistance to MMS. Upon either treatment, the association with MLH1 down-regulation further amplified the resistant phenotype. Moreover, AGS cells mutated in Polβ were hypersensitive to both 6-TG and MMS killing and their sensitivity was partially rescued by MLH1 silencing. We provide evidence that the critical lethal lesions in this new pathway are double strand breaks that are exacerbated when Polβ is defective and relieved when MLH1 is silenced. In conclusion, we provide evidence of crosstalk between MLH1 and Polβ that modulates the response to alkylation damage. These studies suggest that the Polβ/MLH1 status should be taken into consideration when designing chemotherapeutic approaches for gastric cancer.

  7. The MutSα-Proliferating Cell Nuclear Antigen Interaction in Human DNA Mismatch Repair*S⃞♦

    PubMed Central

    Iyer, Ravi R.; Pohlhaus, Timothy J.; Chen, Sihong; Hura, Gregory L.; Dzantiev, Leonid; Beese, Lorena S.; Modrich, Paul

    2008-01-01

    We have examined the interaction parameters, conformation, and functional significance of the human MutSα· proliferating cell nuclear antigen (PCNA) complex in mismatch repair. The two proteins associate with a 1:1 stoichiometry and a KD of 0.7 μm in the absence or presence of heteroduplex DNA. PCNA does not influence the affinity of MutSα for a mismatch, and mismatch-bound MutSα binds PCNA. Small angle x-ray scattering studies have established the molecular parameters of the complex, which are consistent with an elongated conformation in which the two proteins associate in an end-to-end fashion in a manner that does not involve an extended unstructured tether, as has been proposed for yeast MutSα and PCNA (Shell, S. S., Putnam, C. D., and Kolodner, R. D. (2007) Mol. Cell26 ,565 -57817531814). MutSα variants lacking the PCNA interaction motif are functional in 3′- or 5′-directed mismatch-provoked excision, but display a partial defect in 5′-directed mismatch repair. This finding is consistent with the modest mutability conferred by inactivation of the MutSα PCNA interaction motif and suggests that interaction of the replication clamp with other repair protein(s) accounts for the essential role of PCNA in MutSα-dependent mismatch repair. PMID:18326858

  8. Cell cycle and mismatch repair genes as potential biomarkers in Arabidopsis thaliana seedlings exposed to silver nanoparticles.

    PubMed

    Gopalakrishnan Nair, Prakash M; Chung, Ill-Min

    2014-06-01

    The expression of cell cycle genes and DNA mismatch repair (MMR) genes were analyzed in Arabidopsis thaliana seedlings exposed to 0, 0.2, 0.5 and 1 mg/L of silver nanoparticles for 24, 48 and 72 h using real-time PCR. Significant up-regulation of AtPCNA1 was observed after 24 h exposure to 0.2 and 0.5 mg/L of silver nanoparticles. AtPCNA2 gene was up-regulated after 24, 48 and 72 h exposure to 0.5 and 1 mg/L of silver nanoparticles. AtMLH1 gene was up-regulated after 48 h exposure to 0.5 and 1 mg/L of silver nanoparticles and down-regulated after 72 h. Down-regulation of AtMSH2, AtMSH3, AtMSH6 and AtMSH7 mRNA was observed after exposure to all concentrations of silver nanoparticles for different time periods. Exposure to silver ions showed no significant change in the expression levels of AtPCNA and MMR genes. The results show that AtPCNA and MMR genes could be used as potential molecular biomarkers.

  9. Relevance of GC content to the conservation of DNA polymerase III/mismatch repair system in Gram-positive bacteria

    PubMed Central

    Akashi, Motohiro; Yoshikawa, Hirofumi

    2013-01-01

    The mechanism of DNA replication is one of the driving forces of genome evolution. Bacterial DNA polymerase III, the primary complex of DNA replication, consists of PolC and DnaE. PolC is conserved in Gram-positive bacteria, especially in the Firmicutes with low GC content, whereas DnaE is widely conserved in most Gram-negative and Gram-positive bacteria. PolC contains two domains, the 3′-5′exonuclease domain and the polymerase domain, while DnaE only possesses the polymerase domain. Accordingly, DnaE does not have the proofreading function; in Escherichia coli, another enzyme DnaQ performs this function. In most bacteria, the fidelity of DNA replication is maintained by 3′-5′ exonuclease and a mismatch repair (MMR) system. However, we found that most Actinobacteria (a group of Gram-positive bacteria with high GC content) appear to have lost the MMR system and chromosomes may be replicated by DnaE-type DNA polymerase III with DnaQ-like 3′-5′ exonuclease. We tested the mutation bias of Bacillus subtilis, which belongs to the Firmicutes and found that the wild type strain is AT-biased while the mutS-deletant strain is remarkably GC-biased. If we presume that DnaE tends to make mistakes that increase GC content, these results can be explained by the mutS deletion (i.e., deletion of the MMR system). Thus, we propose that GC content is regulated by DNA polymerase and MMR system, and the absence of polC genes, which participate in the MMR system, may be the reason for the increase of GC content in Gram-positive bacteria such as Actinobacteria. PMID:24062730

  10. PD-1 Blockade in Tumors with Mismatch-Repair Deficiency

    PubMed Central

    Le, D.T.; Uram, J.N.; Wang, H.; Bartlett, B.R.; Kemberling, H.; Eyring, A.D.; Skora, A.D.; Luber, B.S.; Azad, N.S.; Laheru, D.; Biedrzycki, B.; Donehower, R.C.; Zaheer, A.; Fisher, G.A.; Crocenzi, T.S.; Lee, J.J.; Duffy, S.M.; Goldberg, R.M.; de la Chapelle, A.; Koshiji, M.; Bhaijee, F.; Huebner, T.; Hruban, R.H.; Wood, L.D.; Cuka, N.; Pardoll, D.M.; Papadopoulos, N.; Kinzler, K.W.; Zhou, S.; Cornish, T.C.; Taube, J.M.; Anders, R.A.; Eshleman, J.R.; Vogelstein, B.; Diaz, L.A.

    2015-01-01

    BACKGROUND Somatic mutations have the potential to encode “non-self” immunogenic antigens. We hypothesized that tumors with a large number of somatic mutations due to mismatch-repair defects may be susceptible to immune checkpoint blockade. METHODS We conducted a phase 2 study to evaluate the clinical activity of pembrolizumab, an anti–programmed death 1 immune checkpoint inhibitor, in 41 patients with progressive metastatic carcinoma with or without mismatch-repair deficiency. Pembrolizumab was administered intravenously at a dose of 10 mg per kilogram of body weight every 14 days in patients with mismatch repair–deficient colorectal cancers, patients with mismatch repair–proficient colorectal cancers, and patients with mismatch repair–deficient cancers that were not colorectal. The coprimary end points were the immune-related objective response rate and the 20-week immune-related progression-free survival rate. RESULTS The immune-related objective response rate and immune-related progression-free survival rate were 40% (4 of 10 patients) and 78% (7 of 9 patients), respectively, for mismatch repair–deficient colorectal cancers and 0% (0 of 18 patients) and 11% (2 of 18 patients) for mismatch repair–proficient colorectal cancers. The median progression-free survival and overall survival were not reached in the cohort with mismatch repair–deficient colorectal cancer but were 2.2 and 5.0 months, respectively, in the cohort with mismatch repair–proficient colorectal cancer (hazard ratio for disease progression or death, 0.10 [P<0.001], and hazard ratio for death, 0.22 [P = 0.05]). Patients with mismatch repair–deficient noncolorectal cancer had responses similar to those of patients with mismatch repair–deficient colorectal cancer (immune-related objective response rate, 71% [5 of 7 patients]; immune-related progression-free survival rate, 67% [4 of 6 patients]). Whole-exome sequencing revealed a mean of 1782 somatic mutations per tumor in

  11. 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.

  12. Human MutL-complexes monitor homologous recombination independently of mismatch repair.

    PubMed

    Siehler, Simone Yasmin; Schrauder, Michael; Gerischer, Ulrike; Cantor, Sharon; Marra, Giancarlo; Wiesmüller, Lisa

    2009-02-01

    The role of mismatch repair proteins has been well studied in the context of DNA repair following DNA polymerase errors. Particularly in yeast, MSH2 and MSH6 have also been implicated in the regulation of genetic recombination, whereas MutL homologs appeared to be less important. So far, little is known about the role of the human MutL homolog hMLH1 in recombination, but recently described molecular interactions suggest an involvement. To identify activities of hMLH1 in this process, we applied an EGFP-based assay for the analysis of different mechanisms of DNA repair, initiated by a targeted double-stranded DNA break. We analysed 12 human cellular systems, differing in the hMLH1 and concomitantly in the hPMS1 and hPMS2 status via inducible protein expression, genetic reconstitution, or RNA interference. We demonstrate that hMLH1 and its complex partners hPMS1 and hPMS2 downregulate conservative homologous recombination (HR), particularly when involving DNA sequences with only short stretches of uninterrupted homology. Unexpectedly, hMSH2 is dispensable for this effect. Moreover, the damage-signaling kinase ATM and its substrates BLM and BACH1 are not strictly required, but the combined effect of ATM/ATR-signaling components may mediate the anti-recombinogenic effect. Our data indicate a protective role of hMutL-complexes in a process which may lead to detrimental genome rearrangements, in a manner which does not depend on mismatch repair.

  13. 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

  14. 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

  15. Germline variants in POLE are associated with early onset mismatch repair deficient colorectal cancer

    PubMed Central

    Elsayed, Fadwa A; Kets, C Marleen; Ruano, Dina; van den Akker, Brendy; Mensenkamp, Arjen R; Schrumpf, Melanie; Nielsen, Maartje; Wijnen, Juul T; Tops, Carli M; Ligtenberg, Marjolijn J; Vasen, Hans FA; Hes, Frederik J; Morreau, Hans; van Wezel, Tom

    2015-01-01

    Germline variants affecting the exonuclease domains of POLE and POLD1 predispose to multiple colorectal adenomas and/or colorectal cancer (CRC). The aim of this study was to estimate the prevalence of previously described heterozygous germline variants POLE c.1270C>G, p.(Leu424Val) and POLD1 c.1433G>A, p.(Ser478Asn) in a Dutch series of unexplained familial, early onset CRC and polyposis index cases. We examined 1188 familial CRC and polyposis index patients for POLE p.(Leu424Val) and POLD1 p.(Ser478Asn) variants using competitive allele-specific PCR. In addition, protein expression of the POLE and DNA mismatch repair genes was studied by immunohistochemistry in tumours from POLE carriers. Somatic mutations were screened using semiconductor sequencing. We detected three index patients (0.25%) with a POLE p.(Leu424Val) variant. In one patient, the variant was found to be de-novo. Tumours from three patients from two families were microsatellite instable, and immunohistochemistry showed MSH6/MSH2 deficiency suggestive of Lynch syndrome. Somatic mutations but no germline MSH6 and MSH2 variants were subsequently found, and one tumour displayed a hypermutator phenotype. None of the 1188 patients carried the POLD1 p.(Ser478Asn) variant. POLE germline variant carriers are also associated with a microsatellite instable CRC. POLE DNA analysis now seems warranted in microsatellite instable CRC, especially in the absence of a causative DNA mismatch repair gene germline variant. PMID:25370038

  16. Cloning of the hexA mismatch-repair gene of Streptococcus pneumoniae and identification of the product.

    PubMed

    Martin, B; Prats, H; Claverys, J P

    1985-01-01

    The hexA mismatch repair gene of Streptococcus pneumoniae has been cloned into multicopy plasmid vectors. The cloned hexA gene is expressed as judged from its ability to complement various chromosomal hexA- alleles. Its direction of transcription was defined and the functional limits were localized by original methods relying on homology-dependent integration of nonautonomous chimeric plasmids carrying chromosomal inserts into the chromosome. Comparison of the proteins encoded by recombinant plasmids and by restriction fragments allowed us to identify an Mr 94 000 protein as the probable product of the hexA gene.

  17. Maternal effect for DNA mismatch repair in the mouse.

    PubMed Central

    Gurtu, Vanessa E; Verma, Shelly; Grossmann, Allie H; Liskay, R Michael; Skarnes, William C; Baker, Sean M

    2002-01-01

    DNA mismatch repair (DMR) functions to maintain genome stability. Prokaryotic and eukaryotic cells deficient in DMR show a microsatellite instability (MSI) phenotype characterized by repeat length alterations at microsatellite sequences. Mice deficient in Pms2, a mammalian homolog of bacterial mutL, develop cancer and display MSI in all tissues examined, including the male germ line where a frequency of approximately 10% was observed. To determine the consequences of maternal DMR deficiency on genetic stability, we analyzed F(1) progeny from Pms2(-/-) female mice mated with wild-type males. Our analysis indicates that MSI in the female germ line was approximately 9%. MSI was also observed in paternal alleles, a surprising result since the alleles were obtained from wild-type males and the embryos were therefore DMR proficient. We propose that mosaicism for paternal alleles is a maternal effect that results from Pms2 deficiency during the early cleavage divisions. The absence of DMR in one-cell embryos leads to the formation of unrepaired replication errors in early cell divisions of the zygote. The occurrence of postzygotic mutation in the early mouse embryo suggests that Pms2 deficiency is a maternal effect, one of a limited number identified in the mouse and the first to involve a DNA repair gene. PMID:11805062

  18. Eliminating both canonical and short-patch mismatch repair in Drosophila melanogaster suggests a new meiotic recombination model.

    PubMed

    Crown, K Nicole; McMahan, Susan; Sekelsky, Jeff

    2014-09-01

    In most meiotic systems, recombination is essential to form connections between homologs that ensure their accurate segregation from one another. Meiotic recombination is initiated by DNA double-strand breaks that are repaired using the homologous chromosome as a template. Studies of recombination in budding yeast have led to a model in which most early repair intermediates are disassembled to produce noncrossovers. Selected repair events are stabilized so they can proceed to form double-Holliday junction (dHJ) intermediates, which are subsequently resolved into crossovers. This model is supported in yeast by physical isolation of recombination intermediates, but the extent to which it pertains to animals is unknown. We sought to test this model in Drosophila melanogaster by analyzing patterns of heteroduplex DNA (hDNA) in recombination products. Previous attempts to do this have relied on knocking out the canonical mismatch repair (MMR) pathway, but in both yeast and Drosophila the resulting recombination products are complex and difficult to interpret. We show that, in Drosophila, this complexity results from a secondary, short-patch MMR pathway that requires nucleotide excision repair. Knocking out both canonical and short-patch MMR reveals hDNA patterns that reveal that many noncrossovers arise after both ends of the break have engaged with the homolog. Patterns of hDNA in crossovers could be explained by biased resolution of a dHJ; however, considering the noncrossover and crossover results together suggests a model in which a two-end engagement intermediate with unligated HJs can be disassembled by a helicase to a produce noncrossover or nicked by a nuclease to produce a crossover. While some aspects of this model are similar to the model from budding yeast, production of both noncrossovers and crossovers from a single, late intermediate is a fundamental difference that has important implications for crossover control.

  19. Epigenetic Enhancement of the Post-replicative DNA Mismatch Repair of Mammalian Genomes by a Hemi-mCpG-Np95-Dnmt1 Axis

    PubMed Central

    Wang, Keh-Yang; Chen, Chun-Chang; Tsai, Shih-Feng; Shen, Che-Kun James

    2016-01-01

    DNA methylation at C of CpG dyads (mCpG) in vertebrate genomes is essential for gene regulation, genome stability and development. We show in this study that proper functioning of post-replicative DNA mismatch repair (MMR) in mammalian cells relies on the presence of genomic mCpG, as well as on the maintenance DNA methyltransferase Dnmt1 independently of its catalytic activity. More importantly, high efficiency of mammalian MMR surveillance is achieved through a hemi-mCpG-Np95(Uhrf1)-Dnmt1 axis, in which the MMR surveillance complex(es) is recruited to post-replicative DNA by Dnmt1, requiring its interactions with MutSα, as well as with Np95 bound at the hemi-methylated CpG sites. Thus, efficiency of MMR surveillance over the mammalian genome in vivo is enhanced at the epigenetic level. This synergy endows vertebrate CpG methylation with a new biological significance and, consequently, an additional mechanism for the maintenance of vertebrate genome stability. PMID:27886214

  20. A study of molecular signals deregulating mismatch repair genes in prostate cancer compared to benign prostatic hyperplasia.

    PubMed

    Basu, Sanmitra; Majumder, Subhadipa; Bhowal, Ankur; Ghosh, Alip; Naskar, Sukla; Nandy, Sumit; Mukherjee, Subhabrata; Sinha, Rajan Kumar; Basu, Keya; Karmakar, Dilip; Banerjee, Soma; Sengupta, Sanghamitra

    2015-01-01

    Prostate cancer is one of the leading causes of mortality among aging males. There is an unmet requirement of clinically useful biomarkers for early detection of prostate cancer to reduce the liabilities of overtreatment and accompanying morbidity. The present population-based study investigates the factors disrupting expression of multiple functionally related genes of DNA mismatch repair pathway in prostate cancer patients to identify molecular attributes distinguishing adenocarcinoma from benign hyperplasia of prostate. Gene expression was compared between tissue samples from prostate cancer and benign prostatic hyperplasia using real-time-PCR, western blot and immunohistochemistry. Assessment of genotypes of seven single-nucleotide-polymorphisms of three MMR genes was conducted using PCR-coupled RFLP and sequencing. Promoter methylation was interrogated by methylation-specific-PCR and bisulfite-sequencing. Interaction between microRNAs and MMR genes was verified by 3'UTR-based dual luciferase assays. Concurrent reduction of three MMR genes namely hMLH1, hMSH6 and hMSH2 (34-85%, P<0.05) was observed in prostate cancer tissues. hMSH6 polymorphism rs1800932(Pro92Pro) conferred a borderline protection in cancer patients (OR = 0.33, 95% CI = 0.15-0.75). Relative transcript level of hMLH1 was inversely related (r = -0.59, P<0.05) with methylation quotient of its promoter which showed a significantly higher methylation density (P = 0.008, Z = -2.649) in cancer patients. hsa-miR-155, hsa-miR-141 and hsa-miR-21 gene expressions were significantly elevated (66-85%, P<0.05) in tumor specimens and negatively correlated (r = -0.602 to -0.527, P<0.05) with that of MMR genes. hsa-miR-155 & hsa-miR-141 and hsa-miR-155 & hsa-miR-21 were demonstrated to bind to their putative seed sequences in hMLH1 and hMSH6 3'UTRs respectively. Relatively higher expression of DNA methyl-transferases (DNMT1 and DNMT3b) and HIF-1α genes (34-50%, P<0.05) were also detected in tumor tissues. This

  1. A Study of Molecular Signals Deregulating Mismatch Repair Genes in Prostate Cancer Compared to Benign Prostatic Hyperplasia

    PubMed Central

    Basu, Sanmitra; Majumder, Subhadipa; Bhowal, Ankur; Ghosh, Alip; Naskar, Sukla; Nandy, Sumit; Mukherjee, Subhabrata; Sinha, Rajan Kumar; Basu, Keya; Karmakar, Dilip; Banerjee, Soma; Sengupta, Sanghamitra

    2015-01-01

    Prostate cancer is one of the leading causes of mortality among aging males. There is an unmet requirement of clinically useful biomarkers for early detection of prostate cancer to reduce the liabilities of overtreatment and accompanying morbidity. The present population-based study investigates the factors disrupting expression of multiple functionally related genes of DNA mismatch repair pathway in prostate cancer patients to identify molecular attributes distinguishing adenocarcinoma from benign hyperplasia of prostate. Gene expression was compared between tissue samples from prostate cancer and benign prostatic hyperplasia using real-time-PCR, western blot and immunohistochemistry. Assessment of genotypes of seven single-nucleotide-polymorphisms of three MMR genes was conducted using PCR-coupled RFLP and sequencing. Promoter methylation was interrogated by methylation-specific-PCR and bisulfite-sequencing. Interaction between microRNAs and MMR genes was verified by 3'UTR-based dual luciferase assays. Concurrent reduction of three MMR genes namely hMLH1, hMSH6 and hMSH2 (34-85%, P<0.05) was observed in prostate cancer tissues. hMSH6 polymorphism rs1800932(Pro92Pro) conferred a borderline protection in cancer patients (OR = 0.33, 95% CI = 0.15-0.75). Relative transcript level of hMLH1 was inversely related (r = -0.59, P<0.05) with methylation quotient of its promoter which showed a significantly higher methylation density (P = 0.008, Z = -2.649) in cancer patients. hsa-miR-155, hsa-miR-141 and hsa-miR-21 gene expressions were significantly elevated (66-85%, P<0.05) in tumor specimens and negatively correlated (r = -0.602 to -0.527, P<0.05) with that of MMR genes. hsa-miR-155 & hsa-miR-141 and hsa-miR-155 & hsa-miR-21 were demonstrated to bind to their putative seed sequences in hMLH1 and hMSH6 3’UTRs respectively. Relatively higher expression of DNA methyl-transferases (DNMT1 and DNMT3b) and HIF-1α genes (34-50%, P<0.05) were also detected in tumor tissues

  2. Evaluating the performance of clinical criteria for predicting mismatch repair gene mutations in Lynch syndrome: a comprehensive analysis of 3,671 families.

    PubMed

    Steinke, Verena; Holzapfel, Stefanie; Loeffler, Markus; Holinski-Feder, Elke; Morak, Monika; Schackert, Hans K; Görgens, Heike; Pox, Christian; Royer-Pokora, Brigitte; von Knebel-Doeberitz, Magnus; Büttner, Reinhard; Propping, Peter; Engel, Christoph

    2014-07-01

    Carriers of mismatch repair (MMR) gene mutations have a high lifetime risk for colorectal and endometrial cancers, as well as other malignancies. As mutation analysis to detect these patients is expensive and time-consuming, clinical criteria and tumor-tissue analysis are widely used as pre-screening methods. The aim of our study was to evaluate the performance of commonly applied clinical criteria (the Amsterdam I and II Criteria, and the original and revised Bethesda Guidelines) and the results of tumor-tissue analysis in predicting MMR gene mutations. We analyzed 3,671 families from the German HNPCC Registry and divided them into nine mutually exclusive groups with different clinical criteria. A total of 680 families (18.5%) were found to have a pathogenic MMR gene mutation. Among all 1,284 families with microsatellite instability-high (MSI-H) colorectal cancer, the overall mutation detection rate was 53.0%. Mutation frequencies and their distribution between the four MMR genes differed significantly between clinical groups (p < 0.001). The highest frequencies were found in families fulfilling the Amsterdam Criteria (46.4%). Families with loss of MSH2 expression had higher mutation detection rates (69.5%) than families with loss of MLH1 expression (43.1%). MMR mutations were found significantly more often in families with at least one MSI-H small-bowel cancer (p < 0.001). No MMR mutations were found among patients under 40-years-old with only colorectal adenoma. Familial clustering of Lynch syndrome-related tumors, early age of onset, and familial occurrence of small-bowel cancer were clinically relevant predictors for Lynch syndrome. © 2013 UICC.

  3. Acquired temozolomide resistance in human glioblastoma cell line U251 is caused by mismatch repair deficiency and can be overcome by lomustine.

    PubMed

    Stritzelberger, J; Distel, L; Buslei, R; Fietkau, R; Putz, F

    2017-08-20

    Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor in adults. While the alkylating agent temozolomide (TMZ) has prolonged overall survival, resistance evolution represents an important clinical problem. Therefore, we studied the effectiveness of radiotherapy and CCNU in an in vitro model of acquired TMZ resistance. We studied the MGMT-methylated GBM cell line U251 and its in vitro derived TMZ-resistant subline, U251/TMZ-R. Cytotoxicity of TMZ, CCNU, and radiation was tested. Both cell lines were analyzed for MGMT promotor status and expression of mismatch repair genes (MMR). The influence of MMR inhibition by cadmium chloride (CdCl2) on the effects of both drugs was evaluated. During the resistance evolution process in vitro, U251/TMZ-R developed MMR deficiency, but MGMT status did not change. U251/TMZ-R cells were more resistant to TMZ than parental U251 cells (cell viability: 92.0% in U251/TMZ-R/69.2% in U251; p = 0.032) yet more sensitive to CCNU (56.4%/80.8%; p = 0.023). The effectiveness of radiotherapy was not reduced in the TMZ-resistant cell line. Combination of CCNU and TMZ showed promising results for both cell lines and overcame resistance. CdCl2-induced MMR deficiency increased cytotoxicity of CCNU. Our results confirm MMR deficiency as a crucial process for resistance evolution to TMZ. MMR-deficient TMZ-resistant GBM cells were particularly sensitive to CCNU and to combined CCNU/TMZ. Effectiveness of radiotherapy was preserved in TMZ-resistant cells. Consequently, CCNU might be preferentially considered as a treatment option for recurrent MGMT-methylated GBM and may even be suitable for prevention of resistance evolution in primary treatment.

  4. Dynamic basis for one-dimensional DNA scanning by the mismatch repair complex Msh2-Msh6.

    PubMed

    Gorman, Jason; Chowdhury, Arindam; Surtees, Jennifer A; Shimada, Jun; Reichman, David R; Alani, Eric; Greene, Eric C

    2007-11-09

    The ability of proteins to locate specific sites or structures among a vast excess of nonspecific DNA is a fundamental theme in biology. Yet the basic principles that govern these mechanisms remain poorly understood. For example, mismatch repair proteins must scan millions of base pairs to find rare biosynthetic errors, and they then must probe the surrounding region to identify the strand discrimination signals necessary to distinguish the parental and daughter strands. To determine how these proteins might function we used single-molecule optical microscopy to answer the following question: how does the mismatch repair complex Msh2-Msh6 interrogate undamaged DNA? Here we show that Msh2-Msh6 slides along DNA via one-dimensional diffusion. These findings indicate that interactions between Msh2-Msh6 and DNA are dominated by lateral movement of the protein along the helical axis and have implications for how MutS family members travel along DNA at different stages of the repair reaction.

  5. 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

  6. Honokiol radiosensitizes colorectal cancer cells: enhanced activity in cells with mismatch repair defects

    PubMed Central

    He, Zhiyun; Subramaniam, Dharmalingam; Ramalingam, Satish; Dhar, Animesh; Postier, Russell G.; Umar, Shahid; Zhang, Youcheng

    2011-01-01

    DNA mismatch repair is required for correcting any mismatches that are created during replication and recombination, and a defective mismatch repair system contributes to DNA damage-induced growth arrest. The colorectal cancer cell line HCT116 is known to have a mutation in the hMLH1 mismatch repair gene resulting in microsatellite instability and defective mismatch repair. Honokiol is a biphenolic compound that has been used in traditional Chinese medicine for treating various ailments including cancer. This study was designed to test the hypothesis that honokiol enhances the radiosensitivity of cancer cells with mismatch repair defect (HCT116) compared with those that are mismatch repair proficient (HCT116-CH3). We first determined that the combination of honokiol and γ-irradiation treatment resulted in dose-dependent inhibition of proliferation and colony formation in both cell lines. However, the effects were more pronounced in HCT116 cells. Similarly, the combination induced higher levels of apoptosis (caspase 3 activation, Bax to Bcl2 ratio) in the HCT116 cells compared with HCT116-CH3 cells. Cell cycle analyses revealed higher levels of dead cells in HCT116 cells. The combination treatment reduced expression of cyclin A1 and D1 and increased phosphorylated p53 in both cell lines, although there were significantly lower amounts of phosphorylated p53 in the HCT116-CH3 cells, suggesting that high levels of hMLH1 reduce radiosensitivity. These data demonstrate that honokiol is highly effective in radiosensitizing colorectal cancer cells, especially those with a mismatch repair defect. PMID:21836060

  7. Honokiol radiosensitizes colorectal cancer cells: enhanced activity in cells with mismatch repair defects.

    PubMed

    He, Zhiyun; Subramaniam, Dharmalingam; Ramalingam, Satish; Dhar, Animesh; Postier, Russell G; Umar, Shahid; Zhang, Youcheng; Anant, Shrikant

    2011-11-01

    DNA mismatch repair is required for correcting any mismatches that are created during replication and recombination, and a defective mismatch repair system contributes to DNA damage-induced growth arrest. The colorectal cancer cell line HCT116 is known to have a mutation in the hMLH1 mismatch repair gene resulting in microsatellite instability and defective mismatch repair. Honokiol is a biphenolic compound that has been used in traditional Chinese medicine for treating various ailments including cancer. This study was designed to test the hypothesis that honokiol enhances the radiosensitivity of cancer cells with mismatch repair defect (HCT116) compared with those that are mismatch repair proficient (HCT116-CH3). We first determined that the combination of honokiol and γ-irradiation treatment resulted in dose-dependent inhibition of proliferation and colony formation in both cell lines. However, the effects were more pronounced in HCT116 cells. Similarly, the combination induced higher levels of apoptosis (caspase 3 activation, Bax to Bcl2 ratio) in the HCT116 cells compared with HCT116-CH3 cells. Cell cycle analyses revealed higher levels of dead cells in HCT116 cells. The combination treatment reduced expression of cyclin A1 and D1 and increased phosphorylated p53 in both cell lines, although there were significantly lower amounts of phosphorylated p53 in the HCT116-CH3 cells, suggesting that high levels of hMLH1 reduce radiosensitivity. These data demonstrate that honokiol is highly effective in radiosensitizing colorectal cancer cells, especially those with a mismatch repair defect.

  8. Screening for germline mutations in mismatch repair genes in patients with Lynch syndrome by next generation sequencing.

    PubMed

    Soares, Barbara Luísa; Brant, Ayslan Castro; Gomes, Renan; Pastor, Tatiane; Schneider, Naye Balzan; Ribeiro-Dos-Santos, Ândrea; de Assumpção, Paulo Pimentel; Achatz, Maria Isabel W; Ashton-Prolla, Patrícia; Moreira, Miguel Angelo Martins

    2017-09-20

    Lynch syndrome (LS) is an autosomal dominant disorder, with high penetrance that affects approximately 3% of the cases of colorectal cancer. Affected individuals inherit germline mutations in genes responsible for DNA mismatch repair, mainly at MSH2, MLH1, MSH6 and PMS2. The molecular screening of these individuals is frequently costly and time consuming due to the large size of these genes. In addition, PMS2 mutation detection is often a challenge because there are 16 different pseudogenes identified until now. In the present work we evaluate a molecular screening strategy based in next generation sequencing (NGS) in order to optimize the mutation detection in LS patients. We established 16 multiplex PCRs for MSH2, MSH6 and MLH1 and 5 Long-Range PCRs for PMS2, coupled with NGS. The strategy was validated by screening 66 patients who filled Bethesda and Amsterdam criteria for LS from health institutions of Brazil. The mean depth of coverage for MSH2, MSH6, MLH1 and PMS2 genes was 7.988, 36.313, 11.899 and 4.772 times, respectively. Ninety-four variants were found in exons and flanking intron/exon regions for the four MMR genes. Twenty-five were pathogenic or VUS and found in 32 patients (7 in MSH2, 5 in MSH6, 12 in MLH1 e 1 in PMS2). All variants were confirmed by Sanger sequencing. The strategy was efficient to reduce time consuming and costs to identify genetic changes at these MMR genes, reducing in three times the number of PCR reactions performed per patient and was efficient in identifying variants at PMS2 gene.

  9. Mismatch repair regulates homologous recombination, but has little influence on antigenic variation, in Trypanosoma brucei.

    PubMed

    Bell, Joanna S; McCulloch, Richard

    2003-11-14

    Antigenic variation is critical in the life of the African trypanosome, as it allows the parasite to survive in the face of host immunity and enhance its transmission to other hosts. Much of trypanosome antigenic variation uses homologous recombination of variant surface glycoprotein (VSG)-encoding genes into specialized transcription sites, but little is known about the processes that regulate it. Here we describe the effects on VSG switching when two central mismatch repair genes, MSH2 and MLH1, are mutated. We show that disruption of the parasite mismatch repair system causes an increased frequency of homologous recombination, both between perfectly matched DNA molecules and between DNA molecules with divergent sequences. Mismatch repair therefore provides an important regulatory role in homologous recombination in this ancient eukaryote. Despite this, the mismatch repair system has no detectable role in regulating antigenic variation, meaning that VSG switching is either immune to mismatch selection or that mismatch repair acts in a subtle manner, undetectable by current assays.

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

    ClinicalTrials.gov

    2017-09-14

    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

  11. 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.

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

    PubMed Central

    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

    2016-01-01

    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. PMID:27754426

  13. DNA tandem repeat instability in the Escherichia coli chromosome is stimulated by mismatch repair at an adjacent CAG·CTG trinucleotide repeat

    PubMed Central

    Blackwood, John K.; Okely, Ewa A.; Zahra, Rabaab; Eykelenboom, John K.; Leach, David R. F.

    2010-01-01

    Approximately half the human genome is composed of repetitive DNA sequences classified into microsatellites, minisatellites, tandem repeats, and dispersed repeats. These repetitive sequences have coevolved within the genome but little is known about their potential interactions. Trinucleotide repeats (TNRs) are a subclass of microsatellites that are implicated in human disease. Expansion of CAG·CTG TNRs is responsible for Huntington disease, myotonic dystrophy, and a number of spinocerebellar ataxias. In yeast DNA double-strand break (DSB) formation has been proposed to be associated with instability and chromosome fragility at these sites and replication fork reversal (RFR) to be involved either in promoting or in preventing instability. However, the molecular basis for chromosome fragility of repetitive DNA remains poorly understood. Here we show that a CAG·CTG TNR array stimulates instability at a 275-bp tandem repeat located 6.3 kb away on the Escherichia coli chromosome. Remarkably, this stimulation is independent of both DNA double-strand break repair (DSBR) and RFR but is dependent on a functional mismatch repair (MMR) system. Our results provide a demonstration, in a simple model system, that MMR at one type of repetitive DNA has the potential to influence the stability of another. Furthermore, the mechanism of this stimulation places a limit on the universality of DSBR or RFR models of instability and chromosome fragility at CAG·CTG TNR sequences. Instead, our data suggest that explanations of chromosome fragility should encompass the possibility of chromosome gaps formed during MMR. PMID:21149728

  14. The role of the bacterial mismatch repair system in SOS-induced mutagenesis: a theoretical background.

    PubMed

    Belov, Oleg V; Chuluunbaatar, Ochbadrakh; Kapralov, Mikhail I; Sweilam, Nasser H

    2013-09-07

    A theoretical study is performed of the possible role of the methyl-directed mismatch repair system in the ultraviolet-induced mutagenesis of Escherichia coli bacterial cells. For this purpose, mathematical models of the SOS network, translesion synthesis and mismatch repair are developed. Within the proposed models, the key pathways of these repair systems were simulated on the basis of modern experimental data related to their mechanisms. Our model approach shows a possible mechanistic explanation of the hypothesis that the bacterial mismatch repair system is responsible for attenuation of mutation frequency during ultraviolet-induced SOS response via removal of the nucleotides misincorporated by DNA polymerase V (the UmuD'2C complex). Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. Saturation of DNA mismatch repair and error catastrophe by a base analogue in Escherichia coli.

    PubMed Central

    Negishi, Kazuo; Loakes, David; Schaaper, Roel M

    2002-01-01

    Deoxyribosyl-dihydropyrimido[4,5-c][1,2]oxazin-7-one (dP) is a potent mutagenic deoxycytidine-derived base analogue capable of pairing with both A and G, thereby causing G. C --> A. T and A. T --> G. C transition mutations. We have found that the Escherichia coli DNA mismatch-repair system can protect cells against this mutagenic action. At a low dose, dP is much more mutagenic in mismatch-repair-defective mutH, mutL, and mutS strains than in a wild-type strain. At higher doses, the difference between the wild-type and the mutator strains becomes small, indicative of saturation of mismatch repair. Introduction of a plasmid containing the E. coli mutL(+) gene significantly reduces dP-induced mutagenesis. Together, the results indicate that the mismatch-repair system can remove dP-induced replication errors, but that its capacity to remove dP-containing mismatches can readily be saturated. When cells are cultured at high dP concentration, mutant frequencies reach exceptionally high levels and viable cell counts are reduced. The observations are consistent with a hypothesis in which dP-induced cell killing and growth impairment result from excess mutations (error catastrophe), as previously observed spontaneously in proofreading-deficient mutD (dnaQ) strains. PMID:12196386

  16. A quantitative model of bacterial mismatch repair as applied to studying induced mutagenesis

    NASA Astrophysics Data System (ADS)

    Belov, O. V.; Chuluunbaatar, O.; Kapralov, M. I.; Sweilam, N. H.

    2013-11-01

    The paper presents a mathematical model of the DNA mismatch repair system in Escherichia coli bacterial cells. The key pathways of this repair mechanism were simulated on the basis of modern experimental data. We have modelled in detail five main pathways of DNA misincorporation removal with different DNA exonucleases. Here we demonstrate an application of the model to problems of radiation-induced mutagenesis.

  17. Aspirin-induced nuclear translocation of NFκB and apoptosis in colorectal cancer is independent of p53 status and DNA mismatch repair proficiency

    PubMed Central

    Din, F V N; Stark, L A; Dunlop, M G

    2005-01-01

    Substantial evidence indicates nonsteroidal anti-inflammatory drugs (NSAIDs) protect against colorectal cancer (CRC). However, the molecular basis for this anti-tumour activity has not been fully elucidated. We previously reported that aspirin induces signal-specific IκBα degradation followed by NFκB nuclear translocation in CRC cells, and that this mechanism contributes substantially to aspirin-induced apoptosis. We have also reported the relative specificity of this aspirin-induced NFκB-dependent apoptotic effect for CRC cells, in comparison to other cancer cell types. It is now important to establish whether there is heterogeneity within CRC, with respect to the effects of aspirin on the NFκB pathway and apoptosis. p53 signalling and DNA mismatch repair (MMR) are known to be deranged in CRC and have been reported as potential molecular targets for the anti-tumour activity of NSAIDs. Furthermore, both p53 and MMR dysfunction have been shown to confer resistance to chemotherapeutic agents. Here, we set out to determine the p53 and hMLH1 dependency of the effects of aspirin on NFκB signalling and apoptosis in CRC. We specifically compared the effects of aspirin treatment on cell viability, apoptosis and NFκB signalling in an HCT-116 CRC cell line with the p53 gene homozygously disrupted (HCT-116p53−/−) and an HCT-116 cell line rendered MMR proficient by chromosomal transfer (HCT-116+ch3), to the parental HCT-116 CRC cell line. We found that aspirin treatment induced apoptosis following IκBα degradation, NFκB nuclear translocation and repression of NFκB-driven transcription, irrespective of p53 and DNA MMR status. These findings are relevant for design of both novel chemopreventative agents and chemoprevention trials in CRC. PMID:15770215

  18. 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

  19. 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 Central

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

    2017-01-01

    Background 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. Methods 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. Results 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

  20. The effects of mismatch repair and RAD1 genes on interchromosomal crossover recombination in Saccharomyces cerevisiae.

    PubMed

    Nicholson, Ainsley; Fabbri, Rebecca M; Reeves, Jason W; Crouse, Gray F

    2006-06-01

    We have previously shown that recombination between 400-bp substrates containing only 4-bp differences, when present in an inverted repeat orientation, is suppressed by >20-fold in wild-type strains of S. cerevisiae. Among the genes involved in this suppression were three genes involved in mismatch repair--MSH2, MSH3, and MSH6--and one in nucleotide excision repair, RAD1. We now report the involvement of these genes in interchromosomal recombination occurring via crossovers using these same short substrates. In these experiments, recombination was stimulated by a double-strand break generated by the HO endonuclease and can occur between completely identical (homologous) substrates or between nonidentical (homeologous) substrates. In addition, a unique feature of this system is that recombining DNA strands can be given a choice of either type of substrate. We find that interchromosomal crossover recombination with these short substrates is severely inhibited in the absence of MSH2, MSH3, or RAD1 and is relatively insensitive to the presence of mismatches. We propose that crossover recombination with these short substrates requires the products of MSH2, MSH3, and RAD1 and that these proteins have functions in recombination in addition to the removal of terminal nonhomology. We further propose that the observed insensitivity to homeology is a result of the difference in recombinational mechanism and/or the timing of the observed recombination events. These results are in contrast with those obtained using longer substrates and may be particularly relevant to recombination events between the abundant short repeated sequences that characterize the genomes of higher eukaryotes.

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

    PubMed

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

    2016-12-29

    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.

  2. Screening of the DNA mismatch repair genes MLH1, MSH2 and MSH6 in a Greek cohort of Lynch syndrome suspected families

    PubMed Central

    2010-01-01

    Background Germline mutations in the DNA mismatch repair genes predispose to Lynch syndrome, thus conferring a high relative risk of colorectal and endometrial cancer. The MLH1, MSH2 and MSH6 mutational spectrum reported so far involves minor alterations scattered throughout their coding regions as well as large genomic rearrangements. Therefore, a combination of complete sequencing and a specialized technique for the detection of genomic rearrangements should be conducted during a proper DNA-testing procedure. Our main goal was to successfully identify Lynch syndrome families and determine the spectrum of MLH1, MSH2 and MSH6 mutations in Greek Lynch families in order to develop an efficient screening protocol for the Greek colorectal cancer patients' cohort. Methods Forty-two samples from twenty-four families, out of which twenty two of Greek, one of Cypriot and one of Serbian origin, were screened for the presence of germline mutations in the major mismatch repair genes through direct sequencing and MLPA. Families were selected upon Amsterdam criteria or revised Bethesda guidelines. Results Ten deleterious alterations were detected in twelve out of the twenty-four families subjected to genetic testing, thus our detection rate is 50%. Four of the pathogenic point mutations, namely two nonsense, one missense and one splice site change, are novel, whereas the detected genomic deletion encompassing exon 6 of the MLH1 gene has been described repeatedly in the LOVD database. The average age of onset for the development of both colorectal and endometrial cancer among mutation positive families is 43.2 years. Conclusion The mutational spectrum of the MMR genes investigated as it has been shaped by our analysis is quite heterogeneous without any strong indication for the presence of a founder effect. PMID:20937110

  3. Loss of DNA mismatch repair function and cancer predisposition in the mouse: animal models for human hereditary nonpolyposis colorectal cancer.

    PubMed

    Edelmann, Lisa; Edelmann, Winfried

    2004-08-15

    Germline mutations in DNA mismatch repair genes underlie one of the most common hereditary cancer predisposition syndromes known in humans, hereditary nonpolyposis colorectal cancer (HNPCC). Defects of the DNA mismatch repair system are also prevalent in sporadic colorectal cancers. The generation of mice with targeted inactivating mutations in the mismatch repair genes has facilitated the in vivo study of how these genes function and how their individual loss contributes to tumorigenesis. Although there are notable limitations when using murine models to study the molecular basis of human cancer, there is remarkable similarity between the two species with respect to the contribution of individual members of the mismatch repair system to cancer susceptibility, and mouse mutants have greatly enhanced our understanding of the normal role of these genes in mutation avoidance and suppression of tumorigenesis.

  4. Colorectal cancer intrinsic subtypes predict chemotherapy benefit, deficient mismatch repair and epithelial-to-mesenchymal transition

    PubMed Central

    Roepman, Paul; Schlicker, Andreas; Tabernero, Josep; Majewski, Ian; Tian, Sun; Moreno, Victor; Snel, Mireille H; Chresta, Christine M; Rosenberg, Robert; Nitsche, Ulrich; Macarulla, Teresa; Capella, Gabriel; Salazar, Ramon; Orphanides, George; Wessels, Lodewyk FA; Bernards, Rene; Simon, Iris M

    2014-01-01

    In most colorectal cancer (CRC) patients, outcome cannot be predicted because tumors with similar clinicopathological features can have differences in disease progression and treatment response. Therefore, a better understanding of the CRC biology is required to identify those patients who will benefit from chemotherapy and to find a more tailored therapy plan for other patients. Based on unsupervised classification of whole genome data from 188 stages I–IV CRC patients, a molecular classification was developed that consist of at least three major intrinsic subtypes (A-, B- and C-type). The subtypes were validated in 543 stages II and III patients and were associated with prognosis and benefit from chemotherapy. The heterogeneity of the intrinsic subtypes is largely based on three biological hallmarks of the tumor: epithelial-to-mesenchymal transition, deficiency in mismatch repair genes that result in high mutation frequency associated with microsatellite instability and cellular proliferation. A-type tumors, observed in 22% of the patients, have the best prognosis, have frequent BRAF mutations and a deficient DNA mismatch repair system. C-type patients (16%) have the worst outcome, a mesenchymal gene expression phenotype and show no benefit from adjuvant chemotherapy treatment. Both A-type and B-type tumors have a more proliferative and epithelial phenotype and B-types benefit from adjuvant chemotherapy. B-type tumors (62%) show a low overall mutation frequency consistent with the absence of DNA mismatch repair deficiency. Classification based on molecular subtypes made it possible to expand and improve CRC classification beyond standard molecular and immunohistochemical assessment and might help in the future to guide treatment in CRC patients. PMID:23852808

  5. Characterisation of the oxysterol metabolising enzyme pathway in mismatch repair proficient and deficient colorectal cancer

    PubMed Central

    Swan, Rebecca; Alnabulsi, Abdo; Cash, Beatriz; Alnabulsi, Ayham; Murray, Graeme I.

    2016-01-01

    Oxysterols are oxidised derivatives of cholesterol, formed by the enzymatic activity of several cytochrome P450 enzymes and tumour-derived oxysterols have been implicated in tumour growth and survival. The aim of this study was to profile the expression of oxysterol metabolising enzymes in primary colorectal cancer and assess the association between expression and prognosis. Immunohistochemistry was performed on a colorectal cancer tissue microarray containing 650 primary colorectal cancers using monoclonal antibodies to CYP2R1, CYP7B1, CYP8B1, CYP27A1, CYP39A1, CYP46A1 and CYP51A1, which we have developed. Unsupervised hierarchical cluster analysis was used to examine the overall relationship of oxysterol metabolising enzyme expression with outcome and based on this identify an oxysterol metabolising enzyme signature associated with prognosis. Cluster analysis of the whole patient cohort identified a good prognosis group (mean survival=146 months 95% CI 127-165 months) that had a significantly better survival (δ2=12.984, p<0.001, HR=1.983, 95% CI 1.341-2.799) than the poor prognosis group (mean survival=107 months, 95% CI 98-123 months). For the mismatch repair proficient cohort, the good prognosis group had a significantly better survival (δ2=8.985, p=0.003, HR=1.845, 95% CI 1.227-2.774) than the poor prognosis group. Multi-variate analysis showed that cluster group was independently prognostically significant in both the whole patient cohort (p=0.02, HR=1.554, 95% CI 1.072-2.252) and the mismatch repair proficient group (p=0.04, HR=1.530, 95% CI 1.014-2.310). Individual oxysterol metabolising enzymes are overexpressed in colorectal cancer and an oxysterol metabolising enzyme expression profile associated with prognosis has been identified in the whole patient cohort and in mismatch repair proficient colorectal cancers. PMID:27341022

  6. 'Escherichia Coli' MutS Tetramerization Domain Structure Reveals That Stable Dimers But Not Tetramers are Essential for DNA Mismatch Repair in Vivo

    SciTech Connect

    Mendillo, M.L.; Putnam, C.D.; Kolodner, R.D.; /UC, San Diego

    2007-07-10

    The E. coli mispair binding protein MutS forms dimers and tetramers in vitro, although the functional form in vivo is under debate. Here we demonstrate that the MutS tetramer is extended in solution using small angle x-ray scattering (SAXS) and the crystal structure of the C-terminal 34 amino acids of MutS containing the tetramer-forming domain fused to maltose binding protein (MBP). Wild-type C-terminal MBP fusions formed tetramers and could bind MutS and MutS-MutL-DNA complexes. In contrast, Asp835Arg and Arg840Glu mutations predicted to disrupt tetrameric interactions only allowed dimerization of MBP. A chromosomal MutS truncation mutation eliminating the dimerization/tetramerization domain eliminated mismatch repair, whereas the tetramer-disrupting MutS Asp835Arg and Arg840Glu mutations only modestly affected MutS function. These results demonstrate that dimerization but not tetramerization of the MutS C- terminus is essential for mismatch repair.

  7. Nucleotide sequence of the hexA gene for DNA mismatch repair in Streptococcus pneumoniae and homology of hexA to mutS of Escherichia coli and Salmonella typhimurium

    SciTech Connect

    Priebe, S.D.; Hadi, S.M.; Greenberg, B.; Lacks, S.A.

    1988-01-01

    The Hex system of heteroduplex DNA base mismatch repair operates in Streptococcus pneumoniae after transformation and replication to correct donor and nascent DNA strands, respectively. A functionally similar system, called Mut, operates in Escherichia coli and Salmonella typhimurium. The nucleotide sequence of a 3.8-kilobase segment from the S. pneumoniae chromosome that includes the 2.7-kilobase hexA gene was determined. Chromosomal DNA used as donor to measure Hex phenotype was irradiated with UV light. An open reading frame that could encode a 17-kilodalton polypeptide (OrfC) was located just upstream of the gene encoding a polypeptide of 95 kilodaltons corresponding to HexA. Shine-Dalgarno sequences and putative promoters were identified upstream of each protein start site. Insertion mutations showed that only HexA functioned in mismatch repair and that the promoter for hexA transcription was located within the OrfC-coding region. The HexA polypeptide contains a consensus sequence for ATP- or GTP-binding sites in proteins. Comparison of the entire HexA protein sequence to that of MutS of S. typhimurium, showed the proteins to be homologous, inasmuch as 36% of their amino acid residues were identical. This homology indicates that the Hex and Mut systems of mismatch repair evolved from an ancestor common to the gram-positive streptococci and the gram-negative enterobacteria. It is the first direct evidence linking the two systems.

  8. 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

    SciTech Connect

    Porter, G.; Westmoreland, J.; Priebe, S.

    1996-06-01

    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{sup +} 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. 67 refs., 5 figs., 4 tabs.

  9. 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

  10. Single nucleotide variations in cultured cancer cells: Effect of mismatch repair.

    PubMed

    Panyutin, Igor G; Panyutin, Irina V; Powell-Castilla, Ian; Felix, Laura; Neumann, Ronald D

    2017-10-01

    We assessed single nucleotide variations (SNVs) between individual cells in two cancer cell lines; DU145, from brain metastasis of prostate tumor with deficient mismatch repair; and HT1080, a fibrosarcoma cell line. Clones of individual cells were isolated, and sequenced using Ion Ampliseq comprehensive cancer panel that covered the exomes of 409 oncogenes and tumor suppressor genes. Five clones of DU145 and four clones of HT1080 cells were analyzed. We found from 7 to 12 unique SNVs between DU145 clones, while HT1080 clones showed no more than one unique SNV. We then sub-cloned individual cells from some of these isolated clones of DU145 and HT1080 cells. The sub-clones were expanded from a single cell to approximately one million cells after about 20 cell divisions. The sub-clones of DU145 cells had from one to four new unique SNVs within the sequenced regions. No unique SNVs were found between sub-clones of HT1080 cells. Our data demonstrate that the extent of genetic variation at the single nucleotide level in cultured cancer cells is significantly affected by the status of the DNA mismatch repair system. Published by Elsevier B.V.

  11. Sequence and stress-response analyses of the DNA mismatch repair gene hexA in Lactococcus lactis.

    PubMed

    Ren, J; Park, J H; Dunn, N W; Kim, W S

    2001-10-01

    The DNA mismatch repair gene hexA was identified in Lactococcus lactis by PCR amplification by using a pair of primers homologous to the DNA-binding Dps protein. The gene in its entirety, including the regulatory regions, was sequenced, by using a strategy of chromosomal walking based on two PCR protocols. The open reading frame of 2526 bp was preceded by a strong ribosome-binding site (AGGAAG) and was followed by a potential transcription terminator (hairpin loop structure). The 5' terminus of the hexA mRNA was located 135 bp upstream of the start codon, and putative -10 and -35 regions were identified. The deduced amino acid sequence revealed two motifs, the ATP/GTP-binding site (P-loop) and the "MutS family signature". The hexA promoter was cloned into pMU1327, which contained a promoter-less CAT reporter gene, and the promoter activity was examined under oxidative-stress conditions. It appears that the promoter activity is down-shifted by H2O2 at 4 mM.

  12. Mismatch Repair in Schizosaccharomyces Pombe Requires the Mutl Homologous Gene Pms1: Molecular Cloning and Functional Analysis

    PubMed Central

    Schar, P.; Baur, M.; Schneider, C.; Kohli, J.

    1997-01-01

    Homologues of the bacterial mutS and mutL genes involved in DNA mismatch repair have been found in organisms from bacteria to humans. Here, we describe the structure and function of a newly identified Schizosaccharomyces pombe gene that encodes a predicted amino acid sequence of 794 residues with a high degree of homology to MutL related proteins. On the basis of its closer relationship to the eukaryotic ``PMS'' genes than to the ``MLH'' genes, we have designated the S. pombe homologue pms1. Disruption of the pms1 gene causes a significant increase of spontaneous mutagenesis as documented by reversion rate measurements. Tetrad analyses of crosses homozygous for the pms1 mutation reveal a reduction of spore viability from >92% to 80% associated with a low proportion (~50%) of meioses producing four viable spores and a significant, allele-dependent increase of the level of post-meiotic segregation of genetic marker allele pairs. The mutant phenotypes are consistent with a general function of pms1 in correction of mismatched base pairs arising as a consequence of DNA polymerase errors during DNA synthesis, or of hybrid DNA formation between homologous but not perfectly complementary DNA strands during meiotic recombination. PMID:9258673

  13. Management of Acute Myeloblastic Leukemia in a Child With Biallelic Mismatch Repair Deficiency.

    PubMed

    Elhasid, Ronit; Dvir, Rina; Rosenfeld Keidar, Hila; Ben Shachar, Shay; Bitan, Menachem; Solar, Irit; Durno, Carol; Aronson, Melyssa; Malkin, David; Hawkins, Cynthia; Bouffet, Eric; Tabori, Uri

    2015-11-01

    Germline biallelic mismatch repair deficiency (bMMRD) results in a unique cancer predisposition syndrome in which the affected children are susceptible to the development of malignancies, especially brain, gastrointestinal, and lymphoid cancers. Acute myeloblastic leukemia is rarely reported in this syndrome. Here we report the decision-making challenges in a bMMRD child with acute myeloblastic leukemia. Our experience should alert physicians to include bMMRD in the differential diagnosis of a child with hyper/hypopigmented spots and leukemia. Furthermore, the presence of the above and consanguinity emphasizes the need to rule out bMMRD when an allogeneic bone marrow transplant is considered and to enable the surveillance of other family members for earlier detection of cancers in these children.

  14. Phenotypic Heterogeneity by Germline Mismatch Repair Gene Defect in Lynch Syndrome Patients.

    PubMed

    Hernâni-Eusébio, Jorge; Barbosa, Elisabete

    2016-10-01

    Introdução: A síndrome de Lynch é a forma hereditária mais comum de cancro colo-rectal, sendo também responsável por cancro do endométrio e de outros tipos. Associa-se a mutações germinativas nos genes de mismatch repair do ADN e a instabilidade de microssatélites. As mutações MLH1 e MSH2 têm um fenótipo de síndrome de Lynch ‘clássico’, sendo o MSH2 mais associado a cancro extra-cólico. Mutações do MSH6 e PMS2 têm um fenótipo atípico. A expressão clínica é heterogénea, existindo uma correlação entre o gene mismatch repair mutado e o padrão fenotípico. Material e Métodos: Análise retrospetiva dos dados clínicos de doentes que cumpriam os critérios de Amesterdão ou que tinha mutações nos genes mismatch repair, entre setembro de 2012 e outubro de 2015. Resultados: Identificámos 28 doentes. Dezassete tinham cancro colo-rectal sendo a localização no cólon direito predominante. Cinco tiveram cancro do endométrio (mediana da idade de diagnóstico – 53), sem qualquer mutação no MSH6. Cinco desenvolveram outros cancros. Todos os casos com mutações mismatch repair estudados tinham instabilidade de microssatélites. Discussão: Na maioria dos casos foi encontrada mutação no MSH2 apesar de o MLH1 ser descrito na literatura como o gene mais frequentemente mutado. Interessa dizer que os doentes com cancro colo-rectal não evidenciam uma tendência para ter muito infiltrado inflamatório. Na maioria dos casos foi realizada colectomia parcial apesar da incidência elevada de lesões síncronas e metácronas associadas. Histerectomia e anexectomia profilática foi realizada em doentes em menopausa/perimenopausa. Conclusão: O registo standardizado dos dados dos doentes poderá levar a um melhor acompanhamento e conhecimento desta síndrome. O uso das Guidelines de Bethesda poderá identificar novos casos que escapam aos critérios de Amesterdão. A pesquisa de instabilidade de microssatélites deve ser feita em muito maior n

  15. Mismatch repair of heteroduplex DNA intermediates of extrachromosomal recombination in mammalian cells.

    PubMed Central

    Deng, W P; Nickoloff, J A

    1994-01-01

    Previous work indicated that extrachromosomal recombination in mammalian cells could be explained by the single-strand annealing (SSA) model. This model predicts that extrachromosomal recombination leads to nonconservative crossover products and that heteroduplex DNA (hDNA) is formed by annealing of complementary single strands. Mismatched bases in hDNA may subsequently be repaired to wild-type or mutant sequences, or they may remain unrepaired and segregate following DNA replication. We describe a system to examine the formation and mismatch repair of hDNA in recombination intermediates. Our results are consistent with extrachromosomal recombination occurring via SSA and producing crossover recombinant products. As predicted by the SSA model, hDNA was present in double-strand break-induced recombination intermediates. By placing either silent or frameshift mutations in the predicted hDNA region, we have shown that mismatches are efficiently repaired prior to DNA replication. Images PMID:8264607

  16. Working through a diagnostic challenge: colonic polyposis, Amsterdam criteria, and a mismatch repair mutation.

    PubMed

    Jasperson, Kory W; Blazer, Kathleen R; Lowstuter, Katrina; Weitzel, Jeffrey N

    2008-01-01

    The two most common causes of hereditary colorectal cancer are Lynch syndrome and familial adenomatous polyposis (FAP). The phenotype of Lynch syndrome, also known as hereditary nonpolyposis colorectal cancer (HNPCC), is differentiated in part from FAP by the lack of profuse colonic polyposis. Here we describe a proband who presented with greater than 50 adenomatous colonic polyps prior to developing cancer of the colon and urinary bladder, and a family history that fulfills the Amsterdam criteria. Germline analyses of APC and MYH in the proband did not reveal any mutations. Comprehensive analysis of the mismatch repair genes associated with Lynch syndrome revealed a germline hMSH6 missense mutation 2314C>T (arg772trp) and normal sequencing for hMSH2 and hMLH1. We outline evidence supporting the pathogenicity of the identified hMSH6 mutation (arg772trp) and suggest possible etiologies for the unexplained colonic adenomatous polyposis.

  17. MISMATCH REPAIR-DEPENDENT ITERATIVE EXCISION AT IRREPARABLE O6-METHYLGUANINE LESIONS IN HUMAN NUCLEAR EXTRACTS*

    PubMed Central

    York, Sally J.; Modrich, Paul

    2008-01-01

    The response of mammalian cells to SN1 DNA methylators depends on functional MutSα and MutLα. Cells deficient in either of these activities are resistant to the cytotoxic effects of this class of chemotherapeutic drug. Because killing by SN1 methylators has been attributed to O6-methylguanine (MeG), we have constructed nicked circular heteroduplexes that contain a single MeG-T mispair and have examined processing of these molecules by mismatch repair in nuclear extracts of human cells. Excision provoked by MeG-T is restricted to the incised heteroduplex strand, leading to removal of the MeG when it resides on this strand. However, when the MeG is located on the continuous strand, the heteroduplex is irreparable. MeG-T-dependent repair DNA synthesis is observed on both reparable and irreparable, 3’ and 5’ heteroduplexes as judged by [32P]dAMP incorporation. Labeling with [α-32P]dATP followed by a cold dATP chase has demonstrated that newly synthesized DNA on irreparable molecules is subject to re-excision in a reaction that is MutLα-dependent, an effect attributable to presence of MeG on the template strand. Processing of the irreparable 3’ heteroduplex is also associated with incision of the discontinuous strand of a few percent of molecules near the thymidylate of the MeG-T base pair. These results provide the first direct evidence for mismatch repair-mediated iterative processing of DNA methylator damage, an effect that may be relevant to damage signaling events triggered by this class of chemotherapeutic agent. PMID:16772289

  18. Correlation between germline mutations in MMR genes and microsatellite instability in ovarian cancer specimens.

    PubMed

    Akbari, Mohammad R; Zhang, Shiyu; Cragun, Deborah; Lee, Ji-Hyun; Coppola, Domenico; McLaughlin, John; Risch, Harvey A; Rosen, Barry; Shaw, Patricia; Sellers, Thomas A; Schildkraut, Joellen; Narod, Steven A; Pal, Tuya

    2017-02-07

    A high proportion of ovarian cancers from women who carry germline mutations in mismatch repair (MMR) genes demonstrate microsatellite instability (MSI). The utility of pre-screening ovarian cancer specimens for MSI to identify potential patients for germline screening for MMR mutations is uncertain. 656 women with malignant ovarian cancer underwent both MSI testing and germline mutation testing for large rearrangements in three MMR genes, MLH1, MSH2 and MSH6. Germline DNA sequencing data for the same genes was available. Among the 656 women, only four (0.6%) carried a clearly pathogenic MMR mutation. All four cancers from patients with mutations had loss of two or more microsatellite markers (MSI-high). Eighty-four of 652 (13.0%) women without a mutation had MSI-high ovarian cancers. Using MSI-high as a prescreening criterion, the sensitivity of MSI testing to identify germline MMR gene mutations was 100% and the positive predictive value was 4.5%. Germline mutations in MLH1, MSH2 and MSH6 are rare among unselected cases of ovarian cancer. Patients with germline mutations often will have MSI-positive cancers and pre-screening of ovarian cancer specimens may be an efficient way of identifying patients with Lynch syndrome.

  19. Diversity of the clinical presentation of the MMR gene biallelic mutations.

    PubMed

    Bougeard, Gaëlle; Olivier-Faivre, Laurence; Baert-Desurmont, Stéphanie; Tinat, Julie; Martin, Cosette; Bouvignies, Emilie; Vasseur, Stéphanie; Huet, Frédéric; Couillault, Gérard; Vabres, Pierre; Le Pessot, Florence; Chapusot, Caroline; Malka, David; Bressac-de Paillerets, Brigitte; Tosi, Mario; Frebourg, Thierry

    2014-03-01

    Constitutional mismatch repair-deficiency, due to biallelic mutations of MMR genes, results in a tumour spectrum characterized by leukaemias, lymphomas, brain tumours and adenocarcinomas of the gastro-intestinal tract, occurring mostly in childhood. We report here two families illustrating the phenotypic diversity associated with biallelic MMR mutations. In the first family, two siblings developed six malignancies including glioblastoma, lymphoblastic T cell lymphoma, rectal and small bowel adenocarcinoma with onset as early as 6 years of age. We showed that this dramatic clinical presentation was due to the presence of two complex genomic PMS2 deletions in each patient predicted to result into complete PMS2 inactivation. In the second family, the index case presented with an early form of Lynch syndrome with colorectal adenocarcinomas at ages 17 and 20 years, and urinary tract tumours at the age of 25 years. We identified in this patient two MSH6 mutations corresponding to a frameshift deletion and an in frame deletion. The latter was not predicted to result into complete inactivation of MSH6. These reports show that the clinical expression of biallelic MMR mutations depends on the biological impact of the second MMR mutation and that, in clinical practice, the presence of a second MMR mutation located in trans should also be considered in patients suspected to present a Lynch syndrome with an unusual early-onset of tumours.

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

    PubMed Central

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

    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. PMID:28286799

  1. Spontaneous frameshift mutations in Saccharomyces cerevisiae: accumulation during DNA replication and removal by proofreading and mismatch repair activities.

    PubMed Central

    Greene, C N; Jinks-Robertson, S

    2001-01-01

    The accumulation of frameshift mutations during DNA synthesis is determined by the rate at which frameshift intermediates are generated during DNA polymerization and the efficiency with which frameshift intermediates are removed by DNA polymerase-associated exonucleolytic proofreading activity and/or the postreplicative mismatch repair machinery. To examine the relative contributions of these factors to replication fidelity in Saccharomyces cerevisiae, we determined the reversion rates and spectra of the lys2 Delta Bgl +1 frameshift allele. Wild-type and homozygous mutant diploid strains with all possible combinations of defects in the exonuclease activities of DNA polymerases delta and epsilon (conferred by the pol3-01 and pol2-4 alleles, respectively) and in mismatch repair (deletion of MSH2) were analyzed. Although there was no direct correlation between homopolymer run length and frameshift accumulation in the wild-type strain, such a correlation was evident in the triple mutant strain lacking all repair capacity. Furthermore, examination of strains defective in one or two repair activities revealed distinct biases in the removal of the corresponding frameshift intermediates by exonucleolytic proofreading and/or mismatch repair. Finally, these analyses suggest that the mismatch repair machinery may be important for generating some classes of frameshift mutations in yeast. PMID:11560887

  2. 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.

  3. Somatic MMR gene mutations as a cause for MSI-H sebaceous neoplasms in Muir-Torre syndrome-like patients.

    PubMed

    Joly, Marie-Odile; Attignon, Valéry; Saurin, Jean-Christophe; Desseigne, Françoise; Leroux, Dominique; Martin-Denavit, Tanguy; Giraud, Sophie; Bonnet-Dupeyron, Marie-Noëlle; Faivre, Laurence; Auclair, Jessie; Grand-Masson, Chloé; Audoynaud, Carole; Wang, Qing

    2015-03-01

    Sebaceous neoplasms are a major clinical feature of Muir-Torre syndrome (MTS) associated with visceral malignancies, especially colorectal and endometrial tumors. The diagnosis of MTS relies largely on the microsatellite instability (MSI) phenotype in tumors, suggesting germline mutations in DNA mismatch repair (MMR) genes responsible for the inherited disease. We hypothesized that in some MSI-H sebaceous tumors, acquired rather than inherited mutations in MMR genes could be involved. Using next-generation sequencing, we screened MMR gene mutations in 18 MSI-H sebaceous tumors. We found mutations in 17 samples (94%). Indeed, 12/17 (71%) were shown to carry acquired somatic mutations and among 12 samples, seven were shown to be associated with additional somatic alterations like loss of heterozygosity or multiple mutations, suggesting somatic second hits. Our findings strongly suggest that somatic MMR deficiency is responsible for a proportion of MSI-H sebaceous tumors. © 2014 WILEY PERIODICALS, INC.

  4. Insights into protein -- DNA interactions, stability and allosteric communications: A computational study of MutS-DNA recognition complexes

    NASA Astrophysics Data System (ADS)

    Negureanu, Lacramioara; Salsbury, Freddie

    2012-02-01

    DNA mismatch repair proteins (MMR) maintain genetic stability by recognizing and repairing mismatched bases and insertion/deletion loops mistakenly incorporated during DNA replication, and initiate cellular response to certain types of DNA damage. The most abundant MMR mismatch-binding factor in eukaryotes, MutS, recognizes and initiates the repair of base-base mismatches and small insertion/deletions. We performed molecular dynamics simulations on mismatched and damaged MutS-DNA complexes. A comprehensive DNA binding site analysis of relevant conformations shows that MutS proteins recognize the mismatched and platinum cross-linked DNA substrates in significantly different modes. Distinctive conformational changes associated with MutS binding to mismatched and damaged DNA have been identified and they provide insight into the involvement of MMR proteins in DNA-repair and DNA-damage pathways. Stability and allosteric interactions at the heterodimer interface associated with the mismatch and damage recognition step allow for prediction of key residues in MMR cancer-causing mutations. A rigorous hydrogen bonding analysis for ADP molecules at the ATPase binding sites is also presented. A large number of known MMR cancer causing mutations among the residues were found.

  5. Mismatch repair genes of Streptococcus pneumoniae: HexA confers a mutator phenotype in Escherichia coli by negative complementation.

    PubMed

    Prudhomme, M; Méjean, V; Martin, B; Claverys, J P

    1991-11-01

    DNA repair systems able to correct base pair mismatches within newly replicated DNA or within heteroduplex molecules produced during recombination are widespread among living organisms. Evidence that such generalized mismatch repair systems evolved from a common ancestor is particularly strong for two of them, the Hex system of the gram-positive Streptococcus pneumoniae and the Mut system of the gram-negative Escherichia coli and Salmonella typhimurium. The homology existing between HexA and MutS and between HexB and MutL prompted us to investigate the effect of expressing hex genes in E. coli. Complementation of mutS or mutL mutations, which confer a mutator phenotype, was assayed by introducing on a multicopy plasmid the hexA and hexB genes, under the control of an inducible promoter, either individually or together in E. coli strains. No decrease in mutation rate was conferred by either hexA or hexB gene expression. However, a negative complementation effect was observed in wild-type E. coli cells: expression of hexA resulted in a typical Mut- mutator phenotype. hexB gene expression did not increase the mutation rate either individually or in conjunction with hexA. Since expression of hexA did not affect the mutation rate in mutS mutant cells and the hexA-induced mutator effect was recA independent, it is concluded that this effect results from inhibition of the Mut system. We suggest that HexA, like its homolog MutS, binds to mismatches resulting from replication errors, but in doing so it protects them from repair by the Mut system. In agreement with this hypothesis, an increase in mutS gene copy number abolished the hexA-induced mutator phenotype. HexA protein could prevent repair either by being unable to interact with Mut proteins or by producing nonfunctional repair complexes.

  6. 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

  7. Mitotane sensitizes adrenocortical cancer cells to ionizing radiations by involvement of the cyclin B1/CDK complex in G2 arrest and mismatch repair enzymes modulation.

    PubMed

    Cerquetti, Lidia; Sampaoli, Camilla; Amendola, Donatella; Bucci, Barbara; Misiti, Silvia; Raza, Giorgio; De Paula, Ugo; Marchese, Rodolfo; Brunetti, Ercole; Toscano, Vincenzo; Stigliano, Antonio

    2010-08-01

    Mitotane inhibits steroid synthesis by an action on steroidogenic enzymes, as 11beta-hydroxylase and cholesterol side chain cleavage. It also has a cytotoxic effect on the adrenocortical cells and represents a primary drug used in the adrenocortical carcinoma (ACC). H295R and SW13 cell lines were treated with mitotane 10(-5) M and ionizing radiations (IR) in combination therapy, inducing an irreversible inhibition of cell growth in both adrenocortical cancer cells. As shown in a previous report, mitotane/IR combination treatment induced a cell accumulation in the G2 phase. Here, we report the radiosensitizing properties of mitotane in two different ACC cell lines. The drug reveals the effectiveness to enhance the cytotoxic effects of IR by attenuating DNA repair and interfering on the activation of mitosis promoting factor (MPF), mainly regulated by the degradation of cyclin B1 in the mitotic process. These events may explain the inappropriate activation of cdc2, implicated in G2/M phase arrest and probably induced by the mitotane and IR in the combined treatment. Indeed, treatment with purvalanol, a cdc2-inhibitor prevents cell cycle arrest, triggering the G2/M transition. The observation that mitotane and IR in combination treatment amplifies the activation level of cyclin B/cdc2 complexes contributing to cell cycle arrest, suggests that the MPF could function as a master signal for controlling the temporal order of different mitotic events. Moreover, we report that mitotane interferes in modulation of mismatch repair (MMR) enzymes, revealing radiosensitizing drug ability.

  8. Longer telomeres are associated with cancer risk in MMR-proficient hereditary non-polyposis colorectal cancer.

    PubMed

    Seguí, Nuria; Guinó, Elisabet; Pineda, Marta; Navarro, Matilde; Bellido, Fernando; Lázaro, Conxi; Blanco, Ignacio; Moreno, Victor; Capellá, Gabriel; Valle, Laura

    2014-01-01

    Aberrant telomere length measured in blood has been associated with increased risk of several cancer types. In the field of hereditary non-polyposis colorectal cancer (CRC), and more particularly in Lynch syndrome, caused by germline mutations in the mismatch repair (MMR) genes, we recently found that cancer-affected MMR gene mutation carriers had shorter telomeres and more pronounced shortening of telomere length with age than controls and unaffected MMR gene mutation carriers. Here we evaluate blood telomere length in MMR-proficient hereditary non-polyposis CRC, i.e. familial CRC type X (fCRC-X). A total of 57 cancer-affected and 57 cancer-free individuals from 34 Amsterdam-positive fCRC-X families were analyzed and compared to the data previously published on 144 cancer-affected and 100 cancer-free MMR gene mutation carriers, and 234 controls. Relative telomere length was measured using a monochrome multiplex quantitative PCR method, following strict measures to avoid sources of bias and adjusting by age. Despite the retrospective nature of our study, the results show that longer telomeres associate with cancer risk in fCRC-X, thus identifying different patterns of telomere length according to the status of the MMR system.

  9. Longer Telomeres Are Associated with Cancer Risk in MMR-Proficient Hereditary Non-Polyposis Colorectal Cancer

    PubMed Central

    Seguí, Nuria; Guinó, Elisabet; Pineda, Marta; Navarro, Matilde; Bellido, Fernando; Lázaro, Conxi; Blanco, Ignacio; Moreno, Victor; Capellá, Gabriel; Valle, Laura

    2014-01-01

    Aberrant telomere length measured in blood has been associated with increased risk of several cancer types. In the field of hereditary non-polyposis colorectal cancer (CRC), and more particularly in Lynch syndrome, caused by germline mutations in the mismatch repair (MMR) genes, we recently found that cancer-affected MMR gene mutation carriers had shorter telomeres and more pronounced shortening of telomere length with age than controls and unaffected MMR gene mutation carriers. Here we evaluate blood telomere length in MMR-proficient hereditary non-polyposis CRC, i.e. familial CRC type X (fCRC-X). A total of 57 cancer-affected and 57 cancer-free individuals from 34 Amsterdam-positive fCRC-X families were analyzed and compared to the data previously published on 144 cancer-affected and 100 cancer-free MMR gene mutation carriers, and 234 controls. Relative telomere length was measured using a monochrome multiplex quantitative PCR method, following strict measures to avoid sources of bias and adjusting by age. Despite the retrospective nature of our study, the results show that longer telomeres associate with cancer risk in fCRC-X, thus identifying different patterns of telomere length according to the status of the MMR system. PMID:24498269

  10. 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

  11. The mismatch repair system modulates curcumin sensitivity through induction of DNA strand breaks and activation of G2-M checkpoint.

    PubMed

    Jiang, Zhihua; Jin, ShunQian; Yalowich, Jack C; Brown, Kevin D; Rajasekaran, Baskaran

    2010-03-01

    The highly conserved mismatch (MMR) repair system corrects postreplicative errors and modulates cellular responses to genotoxic agents. Here, we show that the MMR system strongly influences cellular sensitivity to curcumin. Compared with MMR-proficient cells, isogenically matched MMR-deficient cells displayed enhanced sensitivity to curcumin. Similarly, cells suppressed for MLH1 or MSH2 expression by RNA interference displayed increased curcumin sensitivity. Curcumin treatment generated comparable levels of reactive oxygen species and the mutagenic adduct 8-oxo-guanine in MMR-proficient and MMR-deficient cells; however, accumulation of gammaH2AX foci, a marker for DNA double-strand breaks (DSB), occurred only in MMR-positive cells in response to curcumin treatment. Additionally, MMR-positive cells showed activation of Chk1 and induction of G(2)-M cell cycle checkpoint following curcumin treatment and inhibition of Chk1 by UCN-01 abrogated Chk1 activation and heightened apoptosis in MMR-proficient cells. These results indicate that curcumin triggers the accumulation of DNA DSB and induction of a checkpoint response through a MMR-dependent mechanism. Conversely, in MMR-compromised cells, curcumin-induced DSB is significantly blunted, and as a result, cells fail to undergo cell cycle arrest, enter mitosis, and die through mitotic catastrophe. The results have potential therapeutic value, especially in the treatment of tumors with compromised MMR function.

  12. Recombination between divergent sequences leads to cell death in a mismatch-repair-independent manner.

    PubMed

    Inbar, O; Kupiec, M

    2000-07-01

    Homologous recombination is an important DNA repair mechanism in vegetative cells. During the repair of double-strand breaks, genetic information is transferred between the interacting DNA sequences, thus creating a gene-conversion event. Gene conversion of a functional member of a gene family, which uses an inactive member (such as a pseudogene) as a template, might have deleterious consequences. It is therefore important for the cell to prevent recombination between divergent sequences. We have studied the repair of a double-strand break by recombination in a haploid yeast strain carrying 99% identical alleles located on different chromosomes. The fate of the broken chromosome was followed in the whole cell population without imposing selective constraints. Our results show that all the cells were able to repair the broken chromosome by gene conversion. During the repair, the cells arrest in the cell cycle with a "dumbbell" configuration characteristic of G2/M-arrested cells. Surprisingly, although all the cells repaired the broken chromosome, 60% of them were unable to resume growth and to form colonies after the repair was completed. The low level of cell recovery was due to the 1% divergence between the alleles, but was not dependent on the function of the mismatch-repair system. Cell death, however, could be prevented by the presence of an alternative source of perfect homology located on a different chromosome.

  13. Evolution and Adaptation in Pseudomonas aeruginosa Biofilms Driven by Mismatch Repair System-Deficient Mutators

    PubMed Central

    Yang, Liang; Molin, Søren; Oliver, Antonio; Smania, Andrea M.

    2011-01-01

    Pseudomonas aeruginosa is an important opportunistic pathogen causing chronic airway infections, especially in cystic fibrosis (CF) patients. The majority of the CF patients acquire P. aeruginosa during early childhood, and most of them develop chronic infections resulting in severe lung disease, which are rarely eradicated despite intensive antibiotic therapy. Current knowledge indicates that three major adaptive strategies, biofilm development, phenotypic diversification, and mutator phenotypes [driven by a defective mismatch repair system (MRS)], play important roles in P. aeruginosa chronic infections, but the relationship between these strategies is still poorly understood. We have used the flow-cell biofilm model system to investigate the impact of the mutS associated mutator phenotype on development, dynamics, diversification and adaptation of P. aeruginosa biofilms. Through competition experiments we demonstrate for the first time that P. aeruginosa MRS-deficient mutators had enhanced adaptability over wild-type strains when grown in structured biofilms but not as planktonic cells. This advantage was associated with enhanced micro-colony development and increased rates of phenotypic diversification, evidenced by biofilm architecture features and by a wider range and proportion of morphotypic colony variants, respectively. Additionally, morphotypic variants generated in mutator biofilms showed increased competitiveness, providing further evidence for mutator-driven adaptive evolution in the biofilm mode of growth. This work helps to understand the basis for the specific high proportion and role of mutators in chronic infections, where P. aeruginosa develops in biofilm communities. PMID:22114708

  14. 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

  15. Mitochondrial dysfunction and increased sensitivity to excitotoxicity in mice deficient in DNA mismatch repair.

    PubMed

    Francisconi, Simona; Codenotti, Mara; Ferrari Toninelli, Giulia; Uberti, Daniela; Memo, Maurizio

    2006-07-01

    The expression profile in the hippocampus of mice lacking one allele of the MutS homologue (Msh2), gene, which is one of the most representative components of the DNA mismatch repair system, was analysed to understand whether defects in the repair or in response to DNA damage could impact significantly on brain function. The overall results suggested a reduction in mitochondrial function as indicated by gene expression analysis, biochemical and behavioural studies. In the hippocampus of Msh2+/- mice, array data, validated by RT-PCR and western blot analysis, showed reduced expression levels of genes for cytochrome c oxidase subunit 2 (CoxII), ATP synthase subunit beta and superoxide dismutase 1. Biochemically, mitochondria from the hippocampus and cortex of these mice show reduced CoxII and increased aconitase activity. Behaviourally, these alterations resulted in mice with increased vulnerability to kainic acid-induced epileptic seizures and hippocampal neuronal loss. These data suggest that lack of an efficient system involved in recognizing and repairing DNA damage may generate a brain mitochondriopathy.

  16. Altered expression and new mutations in DNA mismatch repair genes MLH1 and MSH2 in melanoma brain metastases.

    PubMed

    Korabiowska, Monika; König, Fatima; Verheggen, Raphaela; Schlott, Thilo; Cordon-Cardo, Carlos; Romeike, Bernd; Brinck, Ulrich

    2004-01-01

    Brain metastases, including those of malignant melanoma (known for its high genomic instability), are the most common intracranial tumors. The main objective of this study was to investigate expression and mutation in the DNA mismatch repair system in melanoma brain metastases. Expression of MLH1, MSH2, PMS1 and PMS2 was investigated immunohistochemically in 31 melanoma metastatic tumors. Mutational analysis of MLH1 and MSH2 was performed in 17 melanoma brain metastases. Loss of MLH1 and MSH2 expression was found in 10/31 and 12/31 tumors. PMS1 (27/31) and PMS2 (28/31) expression was preserved in the majority of lesions. Potential missense mutation was found in MSH2 (exon 13) in 2/17 melanomas. Mutation in the intron sequence between exon 14 and 15 of MLH1 (exon 15) was observed in 4/17 cases. Our results indicate that the two major DNA mismatch repair genes, MLH1 and MSH2, are more frequently affected by alterations in the DNA mismatch repair system than the helper genes PMS1 and PMS2. The presence of mutations of MSH2 and MLH1 in melanoma brain metastases, which has not been found in primary melanomas, indicates the high genomic instability of melanoma brain metastases.

  17. Relationship between DNA mismatch repair genes expression, Ku-genes expression and ploidy-related parameters in the progression of pigmented lesions of the skin.

    PubMed

    Korabiowska, Monika; Tscherny, Michael; Stachura, Jerzy; Ruschenburg, Ilka; Cordon-Cardo, Carlos; Brinck, Ulrich

    2002-01-01

    Defects of DNA repair systems in cutaneous tumours are related to DNA mismatch repair genes (MLH1, MSH2, PMS1, PMS2) and Ku70/80 genes involved in double- strand repair. In this study we investigated the statistical relationship between these systems and DNA-ploidy-related parameters in 19 naevus cell naevi, 23 lentigos maligna, 76 primary melanomas and 31 melanoma metastases, applying the correlation coefficient according to Spearman. In naevi significant correlations were found between Ku70/80 gene expression and some ploidy-related parameters. In lentigos, additionally, some significant correlations between the expression of DNA mismatch repair genes were found. Similar results were demonstrated for primary melanomas. In metastases no one significant correlation between DNA mismatch repair genes and Ku-genes was present. We postulate that DNA mismatch repair genes and Ku70/80 genes are functionally independent and that some of them are able to influence ploidy-related parameters.

  18. Muir-Torre Syndrome and founder mismatch repair gene mutations: A long gone historical genetic challenge.

    PubMed

    Ponti, G; Manfredini, M; Tomasi, A; Pellacani, G

    2016-09-10

    A "cancer predisposing syndrome" later labeled as Hereditary Non-Polyposis Colorectal Cancer (HNPCC) or Lynch Syndrome, was firstly described by Warthin, about one century ago. An increased predisposition to the development of multiple familial tumors is described as characteristic of this syndrome where visceral and cutaneous malignancies may appear at an early age namely endometrial, gastric, small bowel, ureteral and renal pelvis, ovarian, hepatobiliary tract, pancreatic, brain (Turcot Syndrome) and sebaceous glands (Muir-Torre Syndrome). The latter, a variant of Lynch Syndrome, is characterized by the presence of sebaceous skin adenomas, carcinomas and/or keratoacanthomas associated with visceral malignancies. Both Lynch Syndrome and Muir-Torre Syndrome have been recognized due to germline mutations in mismatch repair genes MLH1, MSH2 and MSH6. To date, 56 Lynch Syndrome founder mutations dependent on MLH1, MSH2 and, although less frequently found, MSH6 and PMS2 are described. Some of these founder mutations, principally of MSH2 gene, have been described to cause Muir-Torre phenotype and have been traced in large and outbreed Muir-Torre Syndrome families living in different US and European territories. Due to the evidences of highly specific Muir-Torre phenotypes related to the presence of widespread MSH2 founder mutations, preliminary search for these MSH2 common mutations in individuals carrying sebaceous tumors and/or keratoacanthomas, at early age or in association to visceral and familial tumors, permits cost-effective and time-saving diagnostic strategies for Lynch/Muir-Torre Syndromes. Copyright © 2015. Published by Elsevier B.V.

  19. 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

  20. Immune Checkpoint Inhibition for Hypermutant Glioblastoma Multiforme Resulting From Germline Biallelic Mismatch Repair Deficiency.

    PubMed

    Bouffet, Eric; Larouche, Valérie; Campbell, Brittany B; Merico, Daniele; de Borja, Richard; Aronson, Melyssa; Durno, Carol; Krueger, Joerg; Cabric, Vanja; Ramaswamy, Vijay; Zhukova, Nataliya; Mason, Gary; Farah, Roula; Afzal, Samina; Yalon, Michal; Rechavi, Gideon; Magimairajan, Vanan; Walsh, Michael F; Constantini, Shlomi; Dvir, Rina; Elhasid, Ronit; Reddy, Alyssa; Osborn, Michael; Sullivan, Michael; Hansford, Jordan; Dodgshun, Andrew; Klauber-Demore, Nancy; Peterson, Lindsay; Patel, Sunil; Lindhorst, Scott; Atkinson, Jeffrey; Cohen, Zane; Laframboise, Rachel; Dirks, Peter; Taylor, Michael; Malkin, David; Albrecht, Steffen; Dudley, Roy W R; Jabado, Nada; Hawkins, Cynthia E; Shlien, Adam; Tabori, Uri

    2016-07-01

    Recurrent glioblastoma multiforme (GBM) is incurable with current therapies. Biallelic mismatch repair deficiency (bMMRD) is a highly penetrant childhood cancer syndrome often resulting in GBM characterized by a high mutational burden. Evidence suggests that high mutation and neoantigen loads are associated with response to immune checkpoint inhibition. We performed exome sequencing and neoantigen prediction on 37 bMMRD cancers and compared them with childhood and adult brain neoplasms. Neoantigen prediction bMMRD GBM was compared with responsive adult cancers from multiple tissues. Two siblings with recurrent multifocal bMMRD GBM were treated with the immune checkpoint inhibitor nivolumab. All malignant tumors (n = 32) were hypermutant. Although bMMRD brain tumors had the highest mutational load because of secondary polymerase mutations (mean, 17,740 ± standard deviation, 7,703), all other high-grade tumors were hypermutant (mean, 1,589 ± standard deviation, 1,043), similar to other cancers that responded favorably to immune checkpoint inhibitors. bMMRD GBM had a significantly higher mutational load than sporadic pediatric and adult gliomas and all other brain tumors (P < .001). bMMRD GBM harbored mean neoantigen loads seven to 16 times higher than those in immunoresponsive melanomas, lung cancers, or microsatellite-unstable GI cancers (P < .001). On the basis of these preclinical data, we treated two bMMRD siblings with recurrent multifocal GBM with the anti-programmed death-1 inhibitor nivolumab, which resulted in clinically significant responses and a profound radiologic response. This report of initial and durable responses of recurrent GBM to immune checkpoint inhibition may have implications for GBM in general and other hypermutant cancers arising from primary (genetic predisposition) or secondary MMRD. © 2016 by American Society of Clinical Oncology.

  1. MMR: risk, choice, chance.

    PubMed

    Fitzpatrick, Michael

    2004-01-01

    The unfolding of the measles, mumps and rubella (MMR) controversy reveals some of the key features of the cultural climate affecting matters of health and illness in contemporary society. A high level of anxiety around issues of health is reflected in a heightened sense of individual vulnerability to environmental dangers (such as atmospheric pollution, electromagnetic fields, bioterrorism) and in a general aversion to risk, particularly in relation to children. This mood has proved responsive to views sceptical, if not hostile, towards science and medicine and associated professionals, particularly in the sphere of immunization. The result is that uptake of MMR vaccination in the UK has fallen, from a peak of 92% in the mid-1990s to a national level of 82% in 2003 (at the age of two); in London uptake is now less than 75%-much less in some areas-causing a significant risk of outbreaks of measles. In the USA too, the proportion of parents opting out of regulations requiring immunization as a condition of school entry has increased significantly in some areas, though these controversies appear to have had little impact so far in continental Europe.

  2. Recall bias, MMR, and autism.

    PubMed

    Andrews, N; Miller, E; Taylor, B; Lingam, R; Simmons, A; Stowe, J; Waight, P

    2002-12-01

    Parents of autistic children with regressive symptoms who were diagnosed after the publicity alleging a link with measles, mumps, and rubella (MMR) vaccine tended to recall the onset as shortly after MMR more often than parents of similar children who were diagnosed prior to the publicity. This is consistent with the recall bias expected under such circumstances.

  3. Case report: impressive response to pembrolizumab in a patient with mismatch-repair deficient metastasized colorectal cancer and bulky disease

    PubMed Central

    Kieler, Markus; Scheithauer, Werner; Zielinski, Christoph C; Chott, Andreas; Al-Mukhtar, Ali; Prager, Gerald W

    2016-01-01

    Here, we report the history of a 42-year-old female patient with sporadic mismatch-repair-deficient metastatic colorectal cancer and abdominal bulky disease, who received pembrolizumab (200 mg every 3 weeks) after the failure of third-line treatment. Restaging 3 months after initiation of treatment revealed a striking response with shrinkage of the bulky peritoneal tumour mass (baseline size 11×11×14 cm) to nearly 25% of the original tumour volume (6.2×7.1×10.4 cm). Restaging 8 months after initiation showed further downsizing of the tumour mass (5.5×7.0×8.0 cm). Tumour markers CEA and CA 19-9 decreased to normal levels, haemoglobin level increased from 8 to 13 mg/dL and her overall clinical performance status increased from ECOG 3 to 1 within 3 months. Therapy with pembrolizumab was continued and is still ongoing. We emphasise the importance of testing for mismatch-repair status in metastatic disease. PMID:28255450

  4. Introduction of specific point mutations into RNA polymerase II by gene targeting in mouse embryonic stem cells: evidence for a DNA mismatch repair mechanism.

    PubMed Central

    Steeg, C M; Ellis, J; Bernstein, A

    1990-01-01

    We have introduced two specific point mutations, located 20 base pairs apart, into the endogenous murine gene that encodes the largest subunit of RNA polymerase II (RPII215). The first mutation conferred resistance to the mushroom toxin alpha-amanitin (amar), and the second mutation generated a restriction fragment length polymorphism without altering the protein sequence. Targeted amar clones were generated at a frequency of 1 in 30 totipotent embryonic stem cells that expressed stably integrated DNA vectors after electroporation. Thirty to 40% of these clones had acquired both mutations, whereas, surprisingly, the remaining clones had acquired the specific amar point mutation but lacked the restriction fragment length polymorphism. We suggest that the latter clones were generated by independent DNA mismatch repair rather than by double crossover or gene conversion. These results demonstrate that it is possible to introduce specific point mutations into an endogenous gene in embryonic stem cells. Thus it should be possible to introduce single base substitutions into other cellular genes, including nonselectable genes, by optimizing the efficiency of gene transfer and/or the sensitivity of screening for targeted clones. Images PMID:1972278

  5. Evolutionary Rate Covariation in Meiotic Proteins Results from Fluctuating Evolutionary Pressure in Yeasts and Mammals

    PubMed Central

    Clark, Nathan L.; Alani, Eric; Aquadro, Charles F.

    2013-01-01

    Evolutionary rates of functionally related proteins tend to change in parallel over evolutionary time. Such evolutionary rate covariation (ERC) is a sequence-based signature of coevolution and a potentially useful signature to infer functional relationships between proteins. One major hypothesis to explain ERC is that fluctuations in evolutionary pressure acting on entire pathways cause parallel rate changes for functionally related proteins. To explore this hypothesis we analyzed ERC within DNA mismatch repair (MMR) and meiosis proteins over phylogenies of 18 yeast species and 22 mammalian species. We identified a strong signature of ERC between eight yeast proteins involved in meiotic crossing over, which seems to have resulted from relaxation of constraint specifically in Candida glabrata. These and other meiotic proteins in C. glabrata showed marked rate acceleration, likely due to its apparently clonal reproductive strategy and the resulting infrequent use of meiotic proteins. This correlation between change of reproductive mode and change in constraint supports an evolutionary pressure origin for ERC. Moreover, we present evidence for similar relaxations of constraint in additional pathogenic yeast species. Mammalian MMR and meiosis proteins also showed statistically significant ERC; however, there was not strong ERC between crossover proteins, as observed in yeasts. Rather, mammals exhibited ERC in different pathways, such as piRNA-mediated defense against transposable elements. Overall, if fluctuation in evolutionary pressure is responsible for ERC, it could reveal functional relationships within entire protein pathways, regardless of whether they physically interact or not, so long as there was variation in constraint on that pathway. PMID:23183665

  6. mmr, a Mycobacterium tuberculosis Gene Conferring Resistance to Small Cationic Dyes and Inhibitors

    PubMed Central

    De Rossi, Edda; Branzoni, Manuela; Cantoni, Rita; Milano, Anna; Riccardi, Giovanna; Ciferri, Orio

    1998-01-01

    The mmr gene, cloned from Mycobacterium tuberculosis, was shown to confer to Mycobacterium smegmatis resistance to tetraphenylphosphonium (TPP), erythromycin, ethidium bromide, acriflavine, safranin O, and pyronin Y. The gene appears to code for a protein containing four transmembrane domains. Studies of [3H]TPP intracellular accumulation strongly suggest that the resistance mediated by the Mmr protein involves active extrusion of TPP. PMID:9811672

  7. Development of DNA mismatch repair gene, MutS, as a diagnostic marker for detection and phylogenetic analysis of algal Megaviruses.

    PubMed

    Wilson, William H; Gilg, Ilana C; Duarte, Amy; Ogata, Hiroyuki

    2014-10-01

    Megaviruses are generically defined as giant viruses with genomes up to 1.26Mb that infect eukaryotic unicellular protists; they are clearly delineated in DNA polymerase B phylogenetic trees; in addition, common features often include an associated virophage observed during infection; the presence of an amino acyl tRNA synthetase gene; and a nucleic acid mismatch repair protein, MutS gene. The archetypal representative of this evolving putative family is Mimivirus, an opportunistic pathogen of Acanthamoeba spp. originally thought to be a bacterium until its genome sequence was published in 2004. Subsequent analysis of marine metagenomic data revealed Megaviruses are likely ubiquitous on the surface ocean. Analysis of genome sequences of giant viruses isolated from naturally occurring marine protists such as microalgae and a microflagellate grazer, started the expansion of the Megaviridae. Here, we explored the possibility of developing Megavirus specific markers for mutS that could be used in virus molecular ecology studies. MutS is split into 15 different clades representing a wide range of cellular life, and two that contain Megaviruses, clade MutS7 and clade MutS8. We developed specific PCR primers that recognized Megavirus clade MutS8, a clade that we propose discriminates most of the algal Megaviruses. Analysis of seawater off the coast of Maine, US, revealed novel groups of algal Megaviruses that were present in all samples tested. The Megavirus clade MutS8 marker should be considered as a tool to reveal new diversity and distribution of this enigmatic group of viruses.

  8. Heteroduplex formation and mismatch repair of the "stuck" mutation during mating-type switching in Saccharomyces cerevisiae.

    PubMed Central

    Ray, B L; White, C I; Haber, J E

    1991-01-01

    We sequenced two alleles of the MATa locus of Saccharomyces cerevisiae that reduce homothallic switching and confer viability to HO rad52 strains. Both the MATa-stk (J. E. Haber, W. T. Savage, S. M. Raposa, B. Weiffenbach, and L. B. Rowe, Proc. Natl. Acad. Sci. USA 77:2824-2828, 1980) and MATa-survivor (R. E. Malone and D. Hyman, Curr. Genet. 7:439-447, 1983) alleles result from a T----A base change at position Z11 of the MAT locus. These strains also contain identical base substitutions at HMRa, so that the mutation is reintroduced when MAT alpha switches to MATa. Mating-type switching in a MATa-stk strain relative to a MATa Z11T strain is reduced at least 50-fold but can be increased by expression of HO from a galactose-inducible promoter. We confirmed by Southern analysis that the Z11A mutation reduced the efficiency of double-strand break formation compared with the Z11T variant; the reduction was more severe in MAT alpha than in MATa. In MAT alpha, the Z11A mutation also creates a mat alpha 1 (sterile) mutation that distinguishes switches of MATa-stk to either MAT alpha or mat alpha 1-stk. Pedigree analysis of cells induced to switch in G1 showed that MATa-stk switched frequently (23% of the time) to produce one mat alpha 1-stk and one MAT alpha progeny. This postswitching segregation suggests that Z11 was often present in heteroduplex DNA that was not mismatch repaired. When mismatch repair was prevented by deletion of the PMS1 gene, there was an increase in the proportion of mat alpha 1-stk/MAT alpha sectors (59%) and in pairs of switched cells that both retained the stk mutation (27%). We conclude that at least one strand of DNA only 4 bp from the HO cut site is not degraded in most of the gene conversion events that accompany MAT switching. Images PMID:1922052

  9. Heteroduplex formation and mismatch repair of the "stuck" mutation during mating-type switching in Saccharomyces cerevisiae.

    PubMed

    Ray, B L; White, C I; Haber, J E

    1991-10-01

    We sequenced two alleles of the MATa locus of Saccharomyces cerevisiae that reduce homothallic switching and confer viability to HO rad52 strains. Both the MATa-stk (J. E. Haber, W. T. Savage, S. M. Raposa, B. Weiffenbach, and L. B. Rowe, Proc. Natl. Acad. Sci. USA 77:2824-2828, 1980) and MATa-survivor (R. E. Malone and D. Hyman, Curr. Genet. 7:439-447, 1983) alleles result from a T----A base change at position Z11 of the MAT locus. These strains also contain identical base substitutions at HMRa, so that the mutation is reintroduced when MAT alpha switches to MATa. Mating-type switching in a MATa-stk strain relative to a MATa Z11T strain is reduced at least 50-fold but can be increased by expression of HO from a galactose-inducible promoter. We confirmed by Southern analysis that the Z11A mutation reduced the efficiency of double-strand break formation compared with the Z11T variant; the reduction was more severe in MAT alpha than in MATa. In MAT alpha, the Z11A mutation also creates a mat alpha 1 (sterile) mutation that distinguishes switches of MATa-stk to either MAT alpha or mat alpha 1-stk. Pedigree analysis of cells induced to switch in G1 showed that MATa-stk switched frequently (23% of the time) to produce one mat alpha 1-stk and one MAT alpha progeny. This postswitching segregation suggests that Z11 was often present in heteroduplex DNA that was not mismatch repaired. When mismatch repair was prevented by deletion of the PMS1 gene, there was an increase in the proportion of mat alpha 1-stk/MAT alpha sectors (59%) and in pairs of switched cells that both retained the stk mutation (27%). We conclude that at least one strand of DNA only 4 bp from the HO cut site is not degraded in most of the gene conversion events that accompany MAT switching.

  10. Measles, mumps, rubella (MMR) vaccine.

    PubMed

    Dubey, A P; Banerjee, S

    2003-07-01

    MMR is a live attenuated vaccine. Indian children show almost 90% seroconversion against measles and rubella and 90% against mumps. Several adverse effects have been reported. Epidemiological studies do not support a causative link between MMR and autism, IBD or GBS. There is an association between the Urabe strain of mumps vaccine and viral meningitis. Vaccine associated thrombocytopenia has been reported. Severe hypersensitivity reactions occur, mainly due to the gelatin component. Outbreaks of measles occur in areas of high measles vaccine coverage, when susceptible individuals accumulate. A second dose is given mainly to vaccinate those who missed the first dose or had primary vaccine failure, rather than to boost waning antibody levels. The possibility or eradication of mumps with a second dose of mumps vaccine is being considered.

  11. Reduced Mismatch Repair of Heteroduplexes Reveals “Non”-interfering Crossing Over in Wild-Type Saccharomyces cerevisiae

    PubMed Central

    Getz, Tony J.; Banse, Stephen A.; Young, Lisa S.; Banse, Allison V.; Swanson, Johanna; Wang, Grace M.; Browne, Barclay L.; Foss, Henriette M.; Stahl, Franklin W.

    2008-01-01

    Using small palindromes to monitor meiotic double-strand-break-repair (DSBr) events, we demonstrate that two distinct classes of crossovers occur during meiosis in wild-type yeast. We found that crossovers accompanying 5:3 segregation of a palindrome show no conventional (i.e., positive) interference, while crossovers with 6:2 or normal 4:4 segregation for the same palindrome, in the same cross, do manifest interference. Our observations support the concept of a “non”-interference class and an interference class of meiotic double-strand-break-repair events, each with its own rules for mismatch repair of heteroduplexes. We further show that deletion of MSH4 reduces crossover tetrads with 6:2 or normal 4:4 segregation more than it does those with 5:3 segregation, consistent with Msh4p specifically promoting formation of crossovers in the interference class. Additionally, we present evidence that an ndj1 mutation causes a shift of noncrossovers to crossovers specifically within the “non”-interference class of DSBr events. We use these and other data in support of a model in which meiotic recombination occurs in two phases—one specializing in homolog pairing, the other in disjunction—and each producing both noncrossovers and crossovers. PMID:18385111

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

    PubMed

    Heinen, Christopher D

    2016-02-01

    We have currently entered a genomic era of cancer research which may soon lead to a genomic era of cancer treatment. Patient DNA sequencing information may lead to a personalized approach to managing an individual's cancer as well as future cancer risk. The success of this approach, however, begins not necessarily in the clinician's office, but rather at the laboratory bench of the basic scientist. The basic scientist plays a critical role since the DNA sequencing information is of limited use unless one knows the function of the gene that is altered and the manner by which a sequence alteration affects that function. The role of basic science research in aiding the clinical management of a disease is perhaps best exemplified by considering the case of Lynch syndrome, a hereditary disease that predisposes patients to colorectal and other cancers. This review will examine how the diagnosis, treatment and even prevention of Lynch syndrome-associated cancers has benefitted from extensive basic science research on the DNA mismatch repair genes whose alteration underlies this condition. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Mechanism of cadmium-mediated inhibition of Msh2-Msh6 function in DNA mismatch repair.

    PubMed

    Wieland, Markus; Levin, Mikhail K; Hingorani, Karan S; Biro, F Noah; Hingorani, Manju M

    2009-10-13

    The observation that Cadmium (Cd(2+)) inhibits Msh2-Msh6, which is responsible for identifying base pair mismatches and other discrepancies in DNA, has led to the proposal that selective targeting of this protein and consequent suppression of DNA repair or apoptosis promote the carcinogenic effects of the heavy metal toxin. It has been suggested that Cd(2+) binding to specific sites on Msh2-Msh6 blocks its DNA binding and ATPase activities. To investigate the mechanism of inhibition, we measured Cd(2+) binding to Msh2-Msh6, directly and by monitoring changes in protein structure and enzymatic activity. Global fitting of the data to a multiligand binding model revealed that binding of about 100 Cd(2+) ions per Msh2-Msh6 results in its inactivation. This finding indicates that the inhibitory effect of Cd(2+) occurs via a nonspecific mechanism. Cd(2+) and Msh2-Msh6 interactions involve cysteine sulfhydryl groups, and the high Cd(2+):Msh2-Msh6 ratio implicates other ligands such as histidine, aspartate, glutamate, and the peptide backbone as well. Our study also shows that cadmium inactivates several unrelated enzymes similarly, consistent with a nonspecific mechanism of inhibition. Targeting of a variety of proteins, including Msh2-Msh6, in this generic manner would explain the marked broad-spectrum impact of Cd(2+) on biological processes. We propose that the presence of multiple nonspecific Cd(2+) binding sites on proteins and their propensity to change conformation on interaction with Cd(2+) are critical determinants of the susceptibility of corresponding biological systems to cadmium toxicity.

  14. 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.

  15. 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

  16. The E705K mutation in hPMS2 exerts recessive, not dominant, effects on mismatch repair

    PubMed Central

    Deschênes, Suzanne M.; Tomer, Guy; Nguyen, Megan; Erdeniz, Naz; Juba, Nicole C.; Sepúlveda, Natalia; Pisani, Jenna E.; Liskay, R. Michael

    2008-01-01

    The hPMS2 mutation E705K is associated with Turcot syndrome. To elucidate the pathogenesis of hPMS2-E705K, we modeled this mutation in yeast and characterized its expression and effects on mutation avoidance in mammalian cells. We found that while hPMS2-E705K (pms1-E738K in yeast) did not significantly affect hPMS2 (Pms1p in yeast) stability or interaction with MLH1, it could not complement the mutator phenotype in MMR-deficient mouse or yeast cells. Further-more, hPMS2-E705K/pms1-E738K inhibited MMR in wild-type (WT) mammalian cell extracts or yeast cells only when present in excess amounts relative to WT PMS2. Our results strongly suggest that hPMS2-E705K is a recessive loss-of-function allele. PMID:17029773

  17. [Cloning of mMR-1 gene and expression in Pichia pastoris systems].

    PubMed

    Li, Tian-Bo; Hu, Yang; Wang, Yi-Guang; Xia, Huan-Zhang

    2005-01-01

    hMR-1 (Homo Myofibrillogenesis Regulator 1, AF417001) is a novel homo gene, which was firstly cloned in our laboratory. The former studies revealed that hMR-1 is a transmembrane protein which shows protein interaction with sarcomeric proteins like myomesin I, myosin regulatory light chain, alpha-enolase and some cell regulator proteins such as eukaryotic translation initiation factor3 subunit 5 (eIF3S5) and etc. In this work, we focused on cloning the homologous gene of hMR-1 from mouse C57BL/6J and exploring its expression using Pichia pastoris yeast system. Two pairs of primers were synthesized according to the hMR-1 gene homologous sequence on mouse genome chromosome 1. The mouse MR-1 gene (mMR-1) was cloned by PCR following the first round RT-PCR from mouse C57BL/6J spleen total RNA. Sequence analysis verified that mMR-1 gene and amino acids sequence showed 90.4% and 90.1% identity with hMR-1, respectively. The prediction of hydrophobic transmembrane structure of mMR-1 suggested it is also a transmembrane protein. The mMR-1 Pichia pastoris expression vector pPIC9-mMR-1 was constructed by fusion of the flanking mMR-1 ORF in the pPIC9 plasmid. After linearization of pPIC9-mMR-1 with Sal I, the 8.5kb DNA fragment was transformed into Pichia pastoris GS115 strain by electroporation. GS115/Mut+ pPIC9-mMR-1 transformants were selected on minimal methanol medium. Integration of mMR-1 gene into the yeast genome in the recombinants was verified by PCR from the transformants total DNA. The mMR-1 protein was expressed by induction under the concentration of 0.5 % methanol. The specific induced protein of 25 kD molecular mass in SDS-PAGE was confirmed to be the mMR-1 protein by Western blot rsing hMR-1 polyclonal antibody. The expression level of this recombinant mMR-1 protein was about 50 mg/L. The successful expression of mMR-1 in the Pichia pastoris GS115 will facilitate the further functional analysis of the novel gene MR-1 in animal model.

  18. Temozolomide Resistance in Glioblastoma Cell Lines: Implication of MGMT, MMR, P-Glycoprotein and CD133 Expression.

    PubMed

    Perazzoli, Gloria; Prados, Jose; Ortiz, Raul; Caba, Octavio; Cabeza, Laura; Berdasco, Maria; Gónzalez, Beatriz; Melguizo, Consolación

    2015-01-01

    The use of temozolomide (TMZ) has improved the prognosis for glioblastoma multiforme patients. However, TMZ resistance may be one of the main reasons why treatment fails. Although this resistance has frequently been linked to the expression of O6-methylguanine-DNA methyltransferase (MGMT) it seems that this enzyme is not the only molecular mechanism that may account for the appearance of drug resistance in glioblastoma multiforme patients as the mismatch repair (MMR) complex, P-glycoprotein, and/or the presence of cancer stem cells may also be implicated. Four nervous system tumor cell lines were used to analyze the modulation of MGMT expression and MGMT promoter methylation by TMZ treatment. Furthermore, 5-aza-2'-deoxycytidine was used to demethylate the MGMT promoter and O(6)-benzylguanine to block GMT activity. In addition, MMR complex and P-glycoprotein expression were studied before and after TMZ exposure and correlated with MGMT expression. Finally, the effect of TMZ exposure on CD133 expression was analyzed. Our results showed two clearly differentiated groups of tumor cells characterized by low (A172 and LN229) and high (SF268 and SK-N-SH) basal MGMT expression. Interestingly, cell lines with no MGMT expression and low TMZ IC50 showed a high MMR complex expression, whereas cell lines with high MGMT expression and high TMZ IC50 did not express the MMR complex. In addition, modulation of MGMT expression in A172 and LN229 cell lines was accompanied by a significant increase in the TMZ IC50, whereas no differences were observed in SF268 and SK-N-SH cell lines. In contrast, P-glycoprotein and CD133 was found to be unrelated to TMZ resistance in these cell lines. These results may be relevant in understanding the phenomenon of TMZ resistance, especially in glioblastoma multiforme patients laking MGMT expression, and may also aid in the design of new therapeutic strategies to improve the efficacy of TMZ in glioblastoma multiforme patients.

  19. Relationship between MMR vaccine and autism.

    PubMed

    Klein, Kristin C; Diehl, Emily B

    2004-01-01

    To evaluate the proposed link between the administration of the measles, mumps, and rubella (MMR) vaccine and the development of autism. A literature search utilizing MEDLINE (1966-November 2003), with the key terms measles, mumps, rubella, and autism, was conducted. Review of the references listed in the articles identified was also performed. Ten articles that specifically evaluated the possible relationship between the MMR vaccine and autism were identified. Review articles, commentaries, and evaluations of a link between gastrointestinal symptoms in autistic children and MMR immunization were excluded. Based upon the current literature, it appears that there is no relationship between MMR vaccination and the development of autism.

  20. 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.

  1. 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.)

  2. 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.)

  3. Case-case study of factors associated to hMLH1, hMSH2, and hMSH6 protein expression among endometrial cancer patients of the University District Hospital of San Juan, Puerto Rico

    PubMed Central

    González, Lorena; Ortiz, Ana P.; Suárez, Erick; Umpierre, Sharee; Marcos, Maria J.; Billoch, Jorge; Joy, Leilani; Charneco, Eileen; Lacourt, Mercedes Y.; Bernabe-Dones, Raúl D.; Cruz-Correa, Marcia

    2012-01-01

    Lynch syndrome (LS) is an autosomal dominant disorder caused by DNA mismatch repair (MMR) system deficiencies. Women affected by LS present a 40 to 60% lifetime risk of endometrial cancer (EC). Objective This case-case study aims to determine the frequency of the hMLH1, hMSH2, and hMSH6 MMR proteins and the factors (age, family history of cancer [FHC] related to LS, and body mass index [BMI]) associated to their absence in EC patients attending the University District Hospital of San Juan, Puerto Rico. Method/Materials Twenty cases were preliminary evaluated for the MMR protein expression by immunohistochemistry testing and classified as positive-cases (presence of protein) or negative-cases (absence of protein). The statistical analysis was based on the logistic regression model using the Maximum Likelihood estimation (MLE). The Bayesian approach was used to determine the posterior probability {posterior Pr[OR>1]}. Results Results showed absence for at least one MMR protein in 25% of the cases; 15% for hMLH1 and 10% for hMSH2. None of the cases showed an absence for hMSH6. The MLE demonstrated that women diagnosed with EC before the age of 50 (OR: 12.4; 95%CI = 0.5–322.7), having FHC related to LS (OR: 17.7; 95%CI = 0.6–534.6), and having lower BMI (OR: 2.38; 95%CI = 0.39–14.28) present higher odds than their counterparts of lacking an MMR protein, once adjusting for potential predictors (p > .05). The posterior probability that an excess risk of lacking an MMR protein occurs was ≥ 95% for each predictor. Conclusion Our study in this Hispanic population supports previous studies in that younger age, FHC, and lower BMI are associated with increased odds of having an absence of MMR protein expression. Further studies with larger sample sizes should be performed. PMID:22635031

  4. Red meat and poultry intake, polymorphisms in the nucleotide excision repair and mismatch repair pathways and colorectal cancer risk.

    PubMed

    Joshi, Amit D; Corral, Román; Siegmund, Kimberly D; Haile, Robert W; Le Marchand, Loïc; Martínez, Maria Elena; Ahnen, Dennis J; Sandler, Robert S; Lance, Peter; Stern, Mariana C

    2009-03-01

    Diets high in red meat have been consistently associated with colorectal cancer (CRC) risk and may result in exposure to carcinogens that cause DNA damage [i.e polycyclic aromatic hydrocarbons, heterocyclic amines (HCAs) and N-nitroso compounds]. Using a family-based study, we investigated whether polymorphisms in the nucleotide excision repair (NER) (ERCC1 3' untranslated region (UTR) G/T, XPD Asp312Asn and Lys751Gln, XPC intron 11 C/A, XPA 5' UTR C/T, XPF Arg415Gln and XPG Asp1104His) and mismatch repair (MLH1 Ile219Val and MSH2 Gly322Asp) pathways modified the association with red meat and poultry intake. We tested for gene-environment interactions using case-only analyses (n = 577) and compared the results using case-unaffected sibling comparisons (n = 307 sibships). Increased risk of CRC was observed for intake of more than or equal to three servings per week of red meat [odds ratio (OR) = 1.8, 95% confidence interval (CI) = 1.3-2.5)] or high-temperature cooked red meat (OR = 1.6, 95% CI = 1.1-2.2). Intake of red meat heavily brown on the outside or inside increased CRC risk only among subjects who carried the XPD codon 751 Lys/Lys genotype (case-only interaction P = 0.006 and P = 0.001, respectively, for doneness outside or inside) or the XPD codon 312 Asp/Asp genotype (case-only interaction P = 0.090 and P < 0.001, respectively). These interactions were stronger for rectal cancer cases (heterogeneity test P = 0.002 for XPD Asp312Asn and P = 0.03 for XPD Lys751Gln) and remained statistically significant after accounting for multiple testing. Case-unaffected sibling analyses were generally supportive of the case-only results. These findings highlight the possible contribution of diets high in red meat to the formation of lesions that elicit the NER pathway, such as carcinogen-induced bulky adducts.

  5. Red meat and poultry intake, polymorphisms in the nucleotide excision repair and mismatch repair pathways and colorectal cancer risk

    PubMed Central

    Joshi, Amit D.; Corral, Román; Siegmund, Kimberly D.; Haile, Robert W.; Le Marchand, Loïc; Martínez, Maria Elena; Ahnen, Dennis J.; Sandler, Robert S.; Lance, Peter; Stern, Mariana C.

    2009-01-01

    Diets high in red meat have been consistently associated with colorectal cancer (CRC) risk and may result in exposure to carcinogens that cause DNA damage [i.e polycyclic aromatic hydrocarbons, heterocyclic amines (HCAs) and N-nitroso compounds]. Using a family-based study, we investigated whether polymorphisms in the nucleotide excision repair (NER) (ERCC1 3′ untranslated region (UTR) G/T, XPD Asp312Asn and Lys751Gln, XPC intron 11 C/A, XPA 5′ UTR C/T, XPF Arg415Gln and XPG Asp1104His) and mismatch repair (MLH1 Ile219Val and MSH2 Gly322Asp) pathways modified the association with red meat and poultry intake. We tested for gene–environment interactions using case-only analyses (n = 577) and compared the results using case-unaffected sibling comparisons (n = 307 sibships). Increased risk of CRC was observed for intake of more than or equal to three servings per week of red meat [odds ratio (OR) = 1.8, 95% confidence interval (CI) = 1.3–2.5)] or high-temperature cooked red meat (OR = 1.6, 95% CI = 1.1–2.2). Intake of red meat heavily brown on the outside or inside increased CRC risk only among subjects who carried the XPD codon 751 Lys/Lys genotype (case-only interaction P = 0.006 and P = 0.001, respectively, for doneness outside or inside) or the XPD codon 312 Asp/Asp genotype (case-only interaction P = 0.090 and P < 0.001, respectively). These interactions were stronger for rectal cancer cases (heterogeneity test P = 0.002 for XPD Asp312Asn and P = 0.03 for XPD Lys751Gln) and remained statistically significant after accounting for multiple testing. Case-unaffected sibling analyses were generally supportive of the case-only results. These findings highlight the possible contribution of diets high in red meat to the formation of lesions that elicit the NER pathway, such as carcinogen-induced bulky adducts. PMID:19029193

  6. Simple Sequence Repeats Together with Mismatch Repair Deficiency Can Bias Mutagenic Pathways in Pseudomonas aeruginosa during Chronic Lung Infection

    PubMed Central

    Moyano, Alejandro J.; Feliziani, Sofía; Di Rienzo, Julio A.; Smania, Andrea M.

    2013-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen that chronically infects the airways of cystic fibrosis (CF) patients and undergoes a process of genetic adaptation based on mutagenesis. We evaluated the role of mononucleotide G:C and A:T simple sequence repeats (SSRs) in this adaptive process. An in silico survey of the genome sequences of 7 P. aeruginosa strains showed that mononucleotide G:C SSRs but not A:T SSRs were greatly under-represented in coding regions, suggesting a strong counterselection process for G:C SSRs with lengths >5 bp but not for A:T SSRs. A meta-analysis of published whole genome sequence data for a P. aeruginosa strain from a CF patient with chronic airway infection showed that G:C SSRs but not A:T SSRs were frequently mutated during the infection process through the insertion or deletion of one or more SSR subunits. The mutation tendency of G:C SSRs was length-dependent and increased exponentially as a function of SSR length. When this strain naturally became a stable Mismatch Repair System (MRS)-deficient mutator, the degree of increase of G:C SSRs mutations (5-fold) was much higher than that of other types of mutation (2.2-fold or less). Sequence analysis of several mutated genes reported for two different collections, both containing mutator and non-mutator strains of P. aeruginosa from CF chronic infections, showed that the proportion of G:C SSR mutations was significantly higher in mutators than in non-mutators, whereas no such difference was observed for A:T SSR mutations. Our findings, taken together, provide genome-scale evidences that under a MRS-deficient background, long G:C SSRs are able to stochastically bias mutagenic pathways by making the genes in which they are harbored more prone to mutation. The combination of MRS deficiency and virulence-related genes that contain long G:C SSRs is therefore a matter of concern in P. aeruginosa CF chronic infection. PMID:24278287

  7. Mucoidy, Quorum Sensing, Mismatch Repair and Antibiotic Resistance in Pseudomonas aeruginosa from Cystic Fibrosis Chronic Airways Infections

    PubMed Central

    Feliziani, Sofía; Sola, Claudia; Bocco, José L.; Montanaro, Patricia; Canigia, Liliana Fernández; Argaraña, Carlos E.; Smania, Andrea M.

    2010-01-01

    Survival of Pseudomonas aeruginosa in cystic fibrosis (CF) chronic infections is based on a genetic adaptation process consisting of mutations in specific genes, which can produce advantageous phenotypic switches and ensure its persistence in the lung. Among these, mutations inactivating the regulators MucA (alginate biosynthesis), LasR (quorum sensing) and MexZ (multidrug-efflux pump MexXY) are the most frequently observed, with those inactivating the DNA mismatch repair system (MRS) being also highly prevalent in P. aeruginosa CF isolates, leading to hypermutator phenotypes that could contribute to this adaptive mutagenesis by virtue of an increased mutation rate. Here, we characterized the mutations found in the mucA, lasR, mexZ and MRS genes in P. aeruginosa isolates obtained from Argentinean CF patients, and analyzed the potential association of mucA, lasR and mexZ mutagenesis with MRS-deficiency and antibiotic resistance. Thus, 38 isolates from 26 chronically infected CF patients were characterized for their phenotypic traits, PFGE genotypic patterns, mutations in the mucA, lasR, mexZ, mutS and mutL gene coding sequences and antibiotic resistance profiles. The most frequently mutated gene was mexZ (79%), followed by mucA (63%) and lasR (39%) as well as a high prevalence (42%) of hypermutators being observed due to loss-of-function mutations in mutL (60%) followed by mutS (40%). Interestingly, mutational spectra were particular to each gene, suggesting that several mechanisms are responsible for mutations during chronic infection. However, no link could be established between hypermutability and mutagenesis in mucA, lasR and mexZ, indicating that MRS-deficiency was not involved in the acquisition of these mutations. Finally, although inactivation of mucA, lasR and mexZ has been previously shown to confer resistance/tolerance to antibiotics, only mutations in MRS genes could be related to an antibiotic resistance increase. These results help to unravel the

  8. hMYH and hMTH1 cooperate for survival in mismatch repair defective T-cell acute lymphoblastic leukemia

    PubMed Central

    Eshtad, S; Mavajian, Z; Rudd, S G; Visnes, T; Boström, J; Altun, M; Helleday, T

    2016-01-01

    hMTH1 is an 8-oxodGTPase that prevents mis-incorporation of free oxidized nucleotides into genomic DNA. Base excision and mismatch repair pathways also restrict the accumulation of oxidized lesions in DNA by removing the mis-inserted 8-oxo-7,8-dihydro-2'-deoxyguanosines (8-oxodGs). In this study, we aimed to investigate the interplay between hMYH DNA glycosylase and hMTH1 for cancer cell survival by using mismatch repair defective T-cell acute lymphoblastic leukemia (T-ALL) cells. To this end, MYH and MTH1 were silenced individually or simultaneously using small hairpin RNAs. Increased sub-G1 population and apoptotic cells were observed upon concurrent depletion of both enzymes. Elevated cell death was consistent with cleaved caspase 3 accumulation in double knockdown cells. Importantly, overexpression of the nuclear isoform of hMYH could remove the G1 arrest and partially rescue the toxicity observed in hMTH1-depleted cells. In addition, expression profiles of human DNA glycosylases were generated using quantitative reverse transcriptase–PCR in MTH1 and/or MYH knockdown cells. NEIL1 DNA glycosylase, involved in repair of oxidized nucleosides, was found to be significantly downregulated as a cellular response to MTH1–MYH co-suppression. Overall, the results suggest that hMYH and hMTH1 functionally cooperate for effective repair and survival in mismatch repair defective T-ALL Jurkat A3 cells. PMID:27918552

  9. A PLAC8-containing protein from an endomycorrhizal fungus confers cadmium resistance to yeast cells by interacting with Mlh3p

    PubMed Central

    Abbà, S.; Vallino, M.; Daghino, S.; Di Vietro, L.; Borriello, R.; Perotto, S.

    2011-01-01

    Cadmium is a genotoxic pollutant known to target proteins that are involved in DNA repair and in antioxidant defence, altering their functions and ultimately causing mutagenic and carcinogenic effects. We have identified a PLAC8 domain-containing protein, named OmFCR, by a yeast functional screen aimed at identifying genes involved in cadmium resistance in the endomycorrhizal fungus Oidiodendron maius. OmFCR shows a remarkable specificity in mediating cadmium resistance. Both its function and its nuclear localization in yeast strictly depend on the interaction with Mlh3p, a subunit of the mismatch repair (MMR) system. Although proteins belonging to the PLAC8 family are widespread in eukaryotes, they are poorly characterized and their biological role still remains elusive. Our work represents the first report about the potential role of a PLAC8 protein in physically coupling DNA lesion recognition by the MMR system to appropriate effectors that affect cell cycle checkpoint pathways. On the basis of cell survival assays and yeast growth curves, we hypothesize that, upon cadmium exposure, OmFCR might promote a higher rate of cell division as compared to control cells. PMID:21672957

  10. Escherichia coli strains (ndk) lacking nucleoside diphosphate kinase are powerful mutators for base substitutions and frameshifts in mismatch-repair-deficient strains.

    PubMed

    Miller, Jeffrey H; Funchain, Pauline; Clendenin, Wendy; Huang, Tiffany; Nguyen, Anh; Wolff, Erika; Yeung, Annie; Chiang, Ju-Huei; Garibyan, Lilit; Slupska, Malgorzata M; Yang, Hanjing

    2002-09-01

    Nucleoside diphosphate (NDP) kinase is one of the enzymes that maintains triphosphate pools. Escherichia coli strains (ndk) lacking this enzyme have been shown to be modest base substitution mutators, and two members of the human family of NDP kinases act as tumor suppressors. We show here that in E. coli strains lacking NDP kinase high levels of mispairs are generated, but most of these are corrected by the mismatch-repair system. Double mutants that are ndk mutS, lacking both the NDP kinase and mismatch repair, have levels of base substitutions 15-fold higher and levels of certain frameshifts up to 10-fold higher than those of the respective mutations in mutS strains that are NDP kinase proficient. A sequence analysis of the specificity of base substitution mutations generated in ndk and ndk mutS backgrounds as well as other experiments suggests that NDP kinase deficiency stimulates polymerase errors that lead to A:T --> G:C transitions and that the editing capacity of cells may be affected, leading to additional uncorrected mispairs and to A:T --> T:A transversions.

  11. Development of an Immunosensor Based on Layered Double Hydroxides for MMR Cancer Biomarker Detection.

    PubMed

    Hammami, M; Soussou, A; Idoudi, F; Cohen-Bouhacina, T; Bouhaouala-Zahar, B; Baccar, Z M

    2015-10-01

    As a potential biomarker for the investigation of cancer inflammatory profiles, macrophage mannose receptor (MMR, CD206) is herein selected to develop an immunosensor based on layered double hydroxide (LDH). Like an endocyte C-type lectin receptor, MMR plays an important role in immune homeostasis by scavenging unwanted mannose glycoproteins. It attracts a progressive attention thanks to its particularly high expression within the tumor microenvironment. There is a great of interest to develop an immunosensor based on an antibody specific to MMR for detection of stroma versus tumor cells. In this work, we studied the feasibility of high sensitive MMR cancer Screen Printed Electrode (SPE) immunosensor. Working electrode of commercialized SPE was modified by immobilization of specific antibody (anti-MMR) into thin layer of LDH nanomaterials. Structural, morphological, and surface properties of LDHs were studied by X-Ray diffraction, atomic force microscopy and Infrared spectroscopy in ATR. Cyclic Voltammetry technique was used to study interaction between the human recombinant MMR protein (rHu-MMR, NSO derived) and an immobilized antibody into developed immunosensor. High specific response of -11.72 μA/ng.mL(-1) (with a correlation coefficient of R(2)=0.994 ) were obtained in linear range of 0.05 ng/mL to 10.0 ng/mL of specific recombinant antigen. The limit of detection (LOD) was less than 15.0 pg/mL. From these attractive results, the feasibility of an electrochemical immunosensor for cancer was proved. Additional experiments to study stability and reproducibility the immunosensor should be completed in perspective to use these anti-MMR based immunosensors for sensing human MMR in patient biopsies and sera.

  12. 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.

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

    PubMed Central

    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

    2016-01-01

    Abstract 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. PMID:27258499

  14. Frequent loss of heterozygosity at the DNA mismatch-repair loci hMLH1 and hMSH3 in sporadic breast cancer

    PubMed Central

    Benachenhou, N; Guiral, S; Gorska-Flipot, I; Labuda, D; Sinnett, D

    1999-01-01

    To study the involvement of DNA mismatch-repair genes in sporadic breast cancer, matched normal and tumoral DNA samples of 22 patients were analysed for genetic instability and loss of heterozygosity (LOH) with 42 microsatellites at or linked to hMLH1 (3p21), hMSH2 (2p16), hMSH3 (5q11–q13), hMSH6 (2p16), hPMS1 (2q32) and hPMS2 (7p22) loci. Chromosomal regions 3p21 and 5q11–q13 were found hemizygously deleted in 46% and 23% of patients respectively. Half of the patients deleted at hMLH1 were also deleted at hMSH3. The shortest regions of overlapping (SRO) deletions were delimited by markers D3S1298 and D3S1266 at 3p21 and by D5S647 and D5S418 at 5q11–q13. Currently, the genes hMLH1 (3p21) and hMSH3 (5q11–q13) are the only known candidates located within these regions. The consequence of these allelic losses is still unclear because none of the breast cancers examined displayed microsatellite instability, a hallmark of mismatch-repair defect during replication error correction. We suggest that hMLH1 and hMSH3 could be involved in breast tumorigenesis through cellular functions other than replication error correction. © 1999 Cancer Research Campaign PMID:10098729

  15. Role of the Mmr Efflux Pump in Drug Resistance in Mycobacterium tuberculosis

    PubMed Central

    Rodrigues, Liliana; Villellas, Cristina; Bailo, Rebeca; Viveiros, Miguel

    2013-01-01

    Efflux pumps are membrane proteins capable of actively transporting a broad range of substrates from the cytoplasm to the exterior of the cell. Increased efflux activity in response to drug treatment may be the first step in the development of bacterial drug resistance. Previous studies showed that the efflux pump Mmr was significantly overexpressed in strains exposed to isoniazid. In the work to be described, we constructed mutants lacking or overexpressing Mmr in order to clarify the role of this efflux pump in the development of resistance to isoniazid and other drugs in M. tuberculosis. The mmr knockout mutant showed an increased susceptibility to ethidium bromide, tetraphenylphosphonium, and cetyltrimethylammonium bromide (CTAB). Overexpression of mmr caused a decreased susceptibility to ethidium bromide, acriflavine, and safranin O that was obliterated in the presence of the efflux inhibitors verapamil and carbonyl cyanide m-chlorophenylhydrazone. Isoniazid susceptibility was not affected by the absence or overexpression of mmr. The fluorometric method allowed the detection of a decreased efflux of ethidium bromide in the knockout mutant, whereas the overexpressed strain showed increased efflux of this dye. This increased efflux activity was inhibited in the presence of efflux inhibitors. Under our experimental conditions, we have found that efflux pump Mmr is mainly involved in the susceptibility to quaternary compounds such as ethidium bromide and disinfectants such as CTAB. The contribution of this efflux pump to isoniazid resistance in Mycobacterium tuberculosis still needs to be further elucidated. PMID:23165464

  16. Influencing factors in MMR immunisation decision making.

    PubMed

    Hill, Marie C; Cox, Carol L

    Immunisation decision making is not a straightforward process for parents. Many factors influence parental decision making on whether they immunise their child with the measles, mumps and rubella (MMR) vaccine. The feasibility study described in this article provides insight into influencing factors associated with decisions regarding the immunisation of children by parents. The study findings suggest that the practice nurse is a credible source of information for parents seeking informed decision making. At a time when the incidence of measles and mumps is rising in the UK, the provision of appropriate information by the practice nurse has the potential to increase uptake of the MMR vaccine.

  17. Temozolomide Resistance in Glioblastoma Cell Lines: Implication of MGMT, MMR, P-Glycoprotein and CD133 Expression

    PubMed Central

    Prados, Jose; Caba, Octavio; Cabeza, Laura; Berdasco, Maria; Gónzalez, Beatriz; Melguizo, Consolación

    2015-01-01

    Background The use of temozolomide (TMZ) has improved the prognosis for glioblastoma multiforme patients. However, TMZ resistance may be one of the main reasons why treatment fails. Although this resistance has frequently been linked to the expression of O6-methylguanine-DNA methyltransferase (MGMT) it seems that this enzyme is not the only molecular mechanism that may account for the appearance of drug resistance in glioblastoma multiforme patients as the mismatch repair (MMR) complex, P-glycoprotein, and/or the presence of cancer stem cells may also be implicated. Methods Four nervous system tumor cell lines were used to analyze the modulation of MGMT expression and MGMT promoter methylation by TMZ treatment. Furthermore, 5-aza-2’-deoxycytidine was used to demethylate the MGMT promoter and O(6)-benzylguanine to block GMT activity. In addition, MMR complex and P-glycoprotein expression were studied before and after TMZ exposure and correlated with MGMT expression. Finally, the effect of TMZ exposure on CD133 expression was analyzed. Results Our results showed two clearly differentiated groups of tumor cells characterized by low (A172 and LN229) and high (SF268 and SK-N-SH) basal MGMT expression. Interestingly, cell lines with no MGMT expression and low TMZ IC50 showed a high MMR complex expression, whereas cell lines with high MGMT expression and high TMZ IC50 did not express the MMR complex. In addition, modulation of MGMT expression in A172 and LN229 cell lines was accompanied by a significant increase in the TMZ IC50, whereas no differences were observed in SF268 and SK-N-SH cell lines. In contrast, P-glycoprotein and CD133 was found to be unrelated to TMZ resistance in these cell lines. Conclusions These results may be relevant in understanding the phenomenon of TMZ resistance, especially in glioblastoma multiforme patients laking MGMT expression, and may also aid in the design of new therapeutic strategies to improve the efficacy of TMZ in glioblastoma

  18. Identification of the mismatch repair genes PMS2 and MLH1 as p53 target genes by using serial analysis of binding elements

    PubMed Central

    Chen, Jiguo; Sadowski, Ivan

    2005-01-01

    The ability to determine the global location of transcription factor binding sites in vivo is important for a comprehensive understanding of gene regulation in human cells. We have developed a technology, called serial analysis of binding elements (SABE), involving subtractive hybridization of chromatin immunoprecipitation-enriched DNA fragments followed by the generation and analysis of concatamerized sequence tags. We applied the SABE technology to search for p53 target genes in the human genome, and have identified several previously described p53 targets in addition to numerous potentially novel targets, including the DNA mismatch repair genes MLH1 and PMS2. Both of these genes were determined to be responsive to DNA damage and p53 activation in normal human fibroblasts, and have p53-response elements within their first intron. These two genes may serve as a sensor in DNA repair mechanisms and a critical determinant for the decision between cell-cycle arrest and apoptosis. These results also demonstrate the potential for use of SABE as a broadly applicable means to globally identify regulatory elements for human transcription factors in vivo. PMID:15781865

  19. Identification of a deletion in the mismatch repair gene, MSH2, using mouse-human cell hybrids monosomal for chromosome 2.

    PubMed

    Pyatt, R E; Nakagawa, H; Hampel, H; Sedra, M; Fuchik, M B; Comeras, I; de la Chapelle, A; Prior, T W

    2003-03-01

    Hereditary non-polyposis colorectal cancer is characterized by mutations in one of the DNA mismatch repair genes, primarily MLH1, MSH2, or MSH6. We report here the identification of a genomic deletion of approximately 11.4 kb encompassing the first two exons of the MSH2 gene in two generations of an Ohio family. By Southern blot analysis, using a cDNA probe spanning the first seven exons of MSH2, an alteration in each of three different enzyme digests (including a unique 13-kb band on HindIII digests) was observed, which suggested the presence of a large alteration in the 5' region of this gene. Mouse-human cell hybrids from a mutation carrier were then generated which contained a single copy each of human chromosome 2 on which the MSH2 gene resides. Southern blots on DNA from the cell hybrids demonstrated the same, unique 13-kb band from one MSH2 allele, as seen in the diploid DNA. DNA from this same monosomal cell hybrid failed to amplify in polymerase chain reactions (PCRs) using primers to exons 1 and 2, demonstrating the deletion of these sequences in one MSH2 allele, and the breakpoints involving Alu repeats were identified by PCR amplification and sequence analysis.

  20. The gastrointestinal manifestation of constitutional mismatch repair deficiency syndrome: from a single adenoma to polyposis-like phenotype and early onset cancer.

    PubMed

    Levi, Z; Kariv, R; Barnes-Kedar, I; Goldberg, Y; Half, E; Morgentern, S; Eli, B; Baris, H N; Vilkin, A; Belfer, R G; Niv, Y; Elhasid, R; Dvir, R; Abu-Freha, N; Cohen, S

    2015-11-01

    Data on the clinical presentation of constitutional mismatch repair deficiency syndrome (CMMRD) is accumulating. However, as the extraintestinal manifestations are often fatal and occur at early age, data on the systematic evaluation of the gastrointestinal tract is scarce. Here we describe 11 subjects with verified biallelic carriage and who underwent colonoscopy, upper endoscopy and small bowel evaluation. Five subjects were symptomatic and in six subjects the findings were screen detected. Two subjects had colorectal cancer and few adenomatous polyps (19, 20 years), three subjects had polyposis-like phenotype (13, 14, 16 years), four subjects had few adenomatous polyps (8, 12-14 years) and two subjects had no polyps (both at age 6). Of the three subjects in the polyposis-like group, two subjects had already developed high-grade dysplasia or cancer and one subject had atypical juvenile polyps suggesting juvenile polyposis. Three out of the five subjects that underwent repeated exams had significant findings during short interval. The gastrointestinal manifestations of CMMRD are highly dependent upon age of examination and highly variable. The polyps may also resemble juvenile polyposis. Intensive surveillance according to current guidelines is mandatory. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  1. Germline PMS2 and somatic POLEexo mutations cause hypermutability of the leading DNA strand in Biallelic Mismatch Repair Deficiency syndrome brain tumors.

    PubMed

    Andrianova, Maria A; Chetan, Ghati Kasturirangan; Sibin, Madathan Kandi; Mckee, Thomas; Merkler, Doron; Narasinga, Rao Kvl; Ribaux, Pascale; Blouin, Jean-Louis; Makrythanasis, Periklis; Seplyarskiy, Vladimir B; Antonarakis, Stylianos E; Nikolaev, Sergey I

    2017-08-14

    Biallelic Mismatch Repair Deficiency (bMMRD) in tumors is frequently associated with somatic mutations in the exonuclease domains of DNA polymerases POLE or POLD1 and results to a characteristic mutational profile. In this study we describe the genetic basis of ultramutated high grade brain tumors in the context of bMMRD. We performed exome sequencing of two second-cousin patients from a large consanguineous family of Indian origin with early onset of high grade glioblastoma and astrocytoma. We identified a germline homozygous nonsense variant R802X in the PMS2 gene. Additionally, by genome sequencing of these tumors we have observed extremely high somatic mutation rates (237 and 123 mut/Mb) as well as somatic mutations in the proofreading domain of POLE polymerase (P436H and L424V), that replicates the leading DNA strand. Most interestingly, we have observed in both cancers that the vast majority of mutations were consistent with the signature of PolE exo-, i.e. the abundance of C > A and C > T mutations, particularly in special contexts, on the leading strand. We showed that the fraction of mutations under positive selection among mutations in tumor suppressor genes is more than 2-fold lower in ultramutated tumors compared to other glioblastomas. Genetic analyses enabled the diagnosis of the two consanguineous childhood brain tumors due to a combination of PMS2 germline and POLE somatic variants and confirmed them as a bMMRD/POLEexo- disorder. This article is protected by copyright. All rights reserved.

  2. The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans

    PubMed Central

    Manhart, Carol M.; Ni, Xiaodan; White, Martin A.; Ortega, Joaquin; Surtees, Jennifer A.

    2017-01-01

    Crossing over between homologs is initiated in meiotic prophase by the formation of DNA double-strand breaks that occur throughout the genome. In the major interference-responsive crossover pathway in baker’s yeast, these breaks are resected to form 3' single-strand tails that participate in a homology search, ultimately forming double Holliday junctions (dHJs) that primarily include both homologs. These dHJs are resolved by endonuclease activity to form exclusively crossovers, which are critical for proper homolog segregation in Meiosis I. Recent genetic, biochemical, and molecular studies in yeast are consistent with the hypothesis of Mlh1-Mlh3 DNA mismatch repair complex acting as the major endonuclease activity that resolves dHJs into crossovers. However, the mechanism by which the Mlh1-Mlh3 endonuclease is activated is unknown. Here, we provide evidence that Mlh1-Mlh3 does not behave like a structure-specific endonuclease but forms polymers required to generate nicks in DNA. This conclusion is supported by DNA binding studies performed with different-sized substrates that contain or lack polymerization barriers and endonuclease assays performed with varying ratios of endonuclease-deficient and endonuclease-proficient Mlh1-Mlh3. In addition, Mlh1-Mlh3 can generate religatable double-strand breaks and form an active nucleoprotein complex that can nick DNA substrates in trans. Together these observations argue that Mlh1-Mlh3 may not act like a canonical, RuvC-like Holliday junction resolvase and support a novel model in which Mlh1-Mlh3 is loaded onto DNA to form an activated polymer that cleaves DNA. PMID:28453523

  3. Hypermutation in Burkholderia cepacia complex is mediated by DNA mismatch repair inactivation and is highly prevalent in cystic fibrosis chronic respiratory infection.

    PubMed

    Martina, Pablo; Feliziani, Sofía; Juan, Carlos; Bettiol, Marisa; Gatti, Blanca; Yantorno, Osvaldo; Smania, Andrea M; Oliver, Antonio; Bosch, Alejandra

    2014-11-01

    The Burkholderia cepacia complex (Bcc) represents an important group of pathogens involved in long-term lung infection in cystic fibrosis (CF) patients. A positive selection of hypermutators, linked to antimicrobial resistance development, has been previously reported for Pseudomonas aeruginosa in this chronic infection setting. Hypermutability, however, has not yet been systematically evaluated in Bcc species. A total of 125 well characterized Bcc isolates recovered from 48 CF patients, 10 non-CF patients and 15 environmental samples were analyzed. In order to determine the prevalence of mutators their spontaneous mutation rates to rifampicin resistance were determined. In addition, the genetic basis of the mutator phenotypes was investigated by sequencing the mutS and mutL genes, the main components of the mismatch repair system (MRS). The overall prevalence of hypermutators in the collection analyzed was 13.6%, with highest occurrence (40.7%) among the chronically infected CF patients, belonging mainly to B. cenocepacia, B. multivorans, B. cepacia, and B. contaminans -the most frequently recovered Bcc species from CF patients worldwide. Thirteen (76.5%) of the hypermutators were defective in mutS and/or mutL. Finally, searching for a possible association between antimicrobial resistance and hypermutability, the resistance-profiles to 17 antimicrobial agents was evaluated. High antimicrobial resistance rates were documented for all the Bcc species recovered from CF patients, but, except for ciprofloxacin, a significant association with hypermutation was not detected. In conclusion, in the present study we demonstrate for the first time that, MRS-deficient Bcc species mutators are highly prevalent and positively selected in CF chronic lung infections. Hypermutation therefore, might be playing a key role in increasing bacterial adaptability to the CF-airway environment, facilitating the persistence of chronic lung infections.

  4. Distinct patterns of KRAS mutations in colorectal carcinomas according to germline mismatch repair defects and hMLH1 methylation status.

    PubMed

    Oliveira, Carla; Westra, Jantine L; Arango, Diego; Ollikainen, Miina; Domingo, Enric; Ferreira, Ana; Velho, Sérgia; Niessen, Renee; Lagerstedt, Kristina; Alhopuro, Pia; Laiho, Paivi; Veiga, Isabel; Teixeira, Manuel R; Ligtenberg, Marjolijn; Kleibeuker, Jan H; Sijmons, Rolf H; Plukker, John T; Imai, Kohzoh; Lage, Pedro; Hamelin, Richard; Albuquerque, Cristina; Schwartz, Simo; Lindblom, Annika; Peltomaki, Päivi; Yamamoto, Hiroyuki; Aaltonen, Lauri A; Seruca, Raquel; Hofstra, Robert M W

    2004-10-01

    In sporadic colorectal tumours the BRAFV600E is associated with microsatellite instability (MSI-H) and inversely associated to KRAS mutations. Tumours from hereditary non-polyposis colorectal cancer (HNPCC) patients carrying germline mutations in hMSH2 or hMLH1 do not show BRAFV600E, however no consistent data exist regarding KRAS mutation frequency and spectrum in HNPCC tumours. We investigated KRAS in 158 HNPCC tumours from patients with germline hMLH1, hMSH2 or hMSH6 mutations, 166 MSI-H and 688 microsatellite stable (MSS) sporadic carcinomas. All tumours were characterized for MSI and 81 of 166 sporadic MSI-H colorectal cancer (CRCs) were analysed for hMLH1 promoter hypermethylation. KRAS mutations were observed in 40% of HNPCC tumours, and the mutation frequency varied upon the mismatch repair gene affected: 48% (29/61) in hMSH2, 32% (29/91) in hMLH1 and 83% (5/6) in hMSH6 (P = 0.01). KRAS mutation frequency was different between HNPCC, MSS and MSI-H CRCs (P = 0.002), and MSI-H with hMLH1 hypermethylation (P = 0.005). Furthermore, HNPCC CRCs had more G13D mutations than MSS (P < 0.0001), MSI-H (P = 0.02) or MSI-H tumours with hMLH1 hypermethylation (P = 0.03). HNPCC colorectal and sporadic MSI-H tumours without hMLH1 hypermethylation shared similar KRAS mutation frequency, in particular G13D. In conclusion, we show that depending on the genetic/epigenetic mechanism leading to MSI-H, the outcome in terms of oncogenic activation may be different, reinforcing the idea that HNPCC, sporadic MSI-H (depending on the hMLH1 status) and MSS CRCs, may target distinct kinases within the RAS/RAF/MAPK pathway.

  5. 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.

  6. Tracking mothers' attitudes to MMR immunisation 1996-2006.

    PubMed

    Smith, Alan; Yarwood, Joanne; Salisbury, David M

    2007-05-16

    This paper presents the findings of surveys that have tracked mothers' attitudes towards MMR over the period 1996-2006. The main aim was to demonstrate how attitudes in relation to MMR have evolved over the last 10 years incorporating the periods of time before, during and after the height of the MMR controversy within the UK. MMR vaccine remains the number one 'top of mind' vaccination issue for parents. The proportion of parents believing MMR to be a greater risk than the diseases it protects against has fallen from 24% in 2002 to 14% in 2006. The proportion of 'hard-core rejectors' of MMR vaccine remains stable at 6%. There has been a gradual and sustained increase in the proportion of parents across all social groups saying MMR was completely safe/slight risk rising from 60% in 2002 to a current level of 74%. There now appears to be a sustained move away from fears over MMR safety and belief in the unfounded link to autism towards a more positive perception of the vaccine.

  7. Unintended events following immunization with MMR: a systematic review.

    PubMed

    Jefferson, Tom; Price, Deirdre; Demicheli, Vittorio; Bianco, Elvira

    2003-09-08

    Public debate over the safety of the trivalent measles, mumps and rubella (MMR) vaccine and the drop in vaccination rates in several countries persists despite its almost universal use and accepted effectiveness. We carried out a systematic review to assess the evidence of unintended effects (beneficial or harmful) associated with MMR and the applicability of systematic reviewing methods to the field of safety evaluation. Eligible studies were comparative prospective or retrospective on healthy individuals up to 15 years of age, carried out or published by 2003. We identified 120 articles satisfying our inclusion criteria and included 22. MMR is associated with a lower incidence of upper respiratory tract infections, a higher incidence of irritability, similar incidence of other adverse effects compared to placebo and is likely to be associated with benign thrombocytopenic purpura (TP), parotitis, joint and limb complaints and aseptic meningitis (mumps Urabe strain-containing MMR). Exposure to MMR is unlikely to be associated with Crohn's disease, ulcerative colitis, autism or aseptic meningitis (mumps Jeryl-Lynn strain-containing MMR). The design and reporting of safety outcomes in MMR vaccine studies, both pre- and post-marketing, are largely inadequate. The evidence of adverse events following immunization with MMR cannot be separated from its role in preventing the target diseases.

  8. Addressing MMR Vaccine Resistance in Minnesota's Somali Community.

    PubMed

    Bahta, Lynn; Ashkir, Asli

    2015-10-01

    Over the past 10 years, Minnesota clinicians have noticed increased resistance to MMR vaccination among Somali Minnesotans. Misinformation about a discredited study asserting a link between the MMR vaccine and autism has permeated this community as parents have increasingly become concerned about the prevalence of autism spectrum disorder among their children. As a result, MMR vaccination rates among U.S.-born children of Somali descent are declining. This article reports findings from an investigation by the Minnesota Department of Health, which was undertaken to better understand vaccine hesitancy among Somali Minnesotans. Based on these and other findings, we propose a multi-pronged approach for increasing vaccination rates in this population.

  9. Parents' perspectives on the MMR immunisation: a focus group study.

    PubMed

    Evans, M; Stoddart, H; Condon, L; Freeman, E; Grizzell, M; Mullen, R

    2001-11-01

    The uptake of the combined measles, mumps and rubella immunisation (MMR) in Britain has fallen since 1998, when a link was hypothesised with the development of bowel disorders and childhood autism. Despite reassurances about the safety of MMR, uptake levels remain lower than optimal. We need to understand what influences parents' decisions on whether to accept MMR or not so that health professionals can provide a service responsive to their needs. To investigate what influences parents' decisions on whether to accept or refuse the primary MMR immunisation and the impact of the recent controversy over its safety. Qualitative study using focus group discussions. Forty-eight parents, whose youngest child was between 14 months and three years old, attended groups at community halls in six localities in Avon and Gloucestershire. Purposive sampling strategy was used to include parents from a variety of socioeconomic backgrounds. Three groups comprised parents who had accepted MMR and three groups comprised parents who had refused MMR. Data analysis used modified grounded theory techniques incorporating the constant comparative method. All parents felt that the decision about MMR was difficult and stressful, and experienced unwelcome pressure from health professionals to comply. Parents were not convinced by Department of Health reassurances that MMR was the safest and best option for their children and many had accepted MMR unwillingly. Four key factors influenced parents' decisions: (a) beliefs about the risks and benefits of MMR compared with contracting the diseases, (b) information from the media and other sources about the safety of MMR, (c) confidence and trust in the advice of health professionals and attitudes towards compliance with this advice, and (d) views on the importance of individual choice within Government policy on immunisation. Parents wanted up-to-date information about the risks and benefits of MMR to be available in advance of their immunisation

  10. Dispelling vaccine myths: MMR and considerations for practicing pharmacists.

    PubMed

    Mormann, Megan; Gilbertson, Carolyn; Milavetz, Gary; Vos, Susan

    2012-01-01

    To discuss information surrounding the erroneous association between the measles-mumps-rubella (MMR) vaccine and autism spectrum disorders (ASDs) and to provide pharmacists with information to dispel vaccine myths. Pharmaceutical and medical literature and public media (e.g., newspapers). The diagnosis of ASDs is on the rise, and many speculations have been made as to the cause, including the MMR vaccine. A small case series article published in The Lancet in 1998 and later retracted has been the center of the controversy over whether the MMR vaccine causes ASDs. New definitive research demonstrates no link, and medical organizations state that evidence does not support a link between the MMR vaccine and ASDs. Pharmacists can play a role in providing up-do-date information to patients to dispel myths concerning vaccine safety. Accurate peer review remains an important step to ensure correct information is given to health care providers and the public.

  11. Measles, Mumps, Rubella and the MMR Vaccine during Pregnancy

    MedlinePlus

    ... women of childbearing age who do not have immunity to MMR receive the vaccine before pregnancy. In ... that have health conditions that severely lower their immunity (such as HIV/AIDS or steroid treatments) or ...

  12. 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.

  13. Active segregation of yeast mitochondria by Myo2 is essential and mediated by Mmr1 and Ypt11.

    PubMed

    Chernyakov, Irina; Santiago-Tirado, Felipe; Bretscher, Anthony

    2013-09-23

    Active segregation of essential organelles is required for successful cell division. The essential budding yeast myosin V Myo2 actively segregates most organelles along polarized actin cables. The mechanism of mitochondrial segregation remains controversial, with movement driven by actin polymerization, movement driven by association with transported cortical endoplasmic reticulum (ER), and direct transport by Myo2 proposed as models. Two nonessential proteins, Mmr1 and the Rab GTPase Ypt11, bind Myo2 and have been implicated in mitochondrial inheritance, although their specific roles are also contended. We generated myo2(sens) mutations that exhibit no overt phenotype but render MMR1 essential and have compromised Ypt11 binding. We then isolated myo2(sens)mmr1(ts) conditional mutants and determined that they have a specific and severe defect in active mitochondrial inheritance, revealing mitochondrial transport by Myo2 as an essential function. ypt11Δ mmr1(ts) cells also have conditional defects in growth and active transport of mitochondria into the bud, both of which are suppressed by artificially forcing mitochondrial inheritance. At the restrictive temperature, cells defective in mitochondrial inheritance give rise to dead buds that go through cytokinesis normally, showing no evidence of a proposed cell-cycle mitochondrial inheritance checkpoint. Thus, active mitochondrial inheritance is an essential process and a function of Myo2 that requires either Mmr1 or Ypt11.

  14. 'MMR talk' and vaccination choices: an ethnographic study in Brighton.

    PubMed

    Poltorak, Mike; Leach, Melissa; Fairhead, James; Cassell, Jackie

    2005-08-01

    In the context of the high-profile controversy that has unfolded in the UK around the measles, mumps and rubella (MMR) vaccine and its possible adverse effects, this paper explores how parents in Brighton, southern England, are thinking about MMR for their own children. Research focusing on parents' engagement with MMR has been dominated by analysis of the proximate influences on their choices, and in particular scientific and media information, which have led health policy to focus on information and education campaigns. This paper reports ethnographic work including narratives by mothers in Brighton. Our work questions such reasoning in showing how wider personal and social issues shape parents' immunisation actions. The narratives by mothers show how practices around MMR are shaped by personal histories, by birth experiences and related feelings of control, by family health histories, by their readings of their child's health and particular strengths and vulnerabilities, by particular engagements with health services, by processes building or undermining confidence, and by friendships and conversations with others, which are themselves shaped by wider social differences and transformations. Although many see vaccination as a personal decision which must respond to the particularities of a child's immune system, 'MMR talk', which affirms these conceptualisations, has become a social phenomenon in itself. These perspectives suggest ways in which people's engagements with MMR reflect wider changes in their relations with science and the state.

  15. The Escherichia coli mismatch repair protein MutL recruits the Vsr and MutH endonucleases in response to DNA damage.

    PubMed

    Polosina, Yaroslava Y; Mui, Justin; Pitsikas, Photini; Cupples, Claire G

    2009-06-01

    The activities of the Vsr and MutH endonucleases of Escherichia coli are stimulated by MutL. The interaction of MutL with each enzyme is enhanced in vivo by 2-aminopurine treatment and by inactivation of the mutY gene. We hypothesize that MutL recruits the endonucleases to sites of DNA damage.

  16. The Escherichia coli Mismatch Repair Protein MutL Recruits the Vsr and MutH Endonucleases in Response to DNA Damage▿

    PubMed Central

    Polosina, Yaroslava Y.; Mui, Justin; Pitsikas, Photini; Cupples, Claire G.

    2009-01-01

    The activities of the Vsr and MutH endonucleases of Escherichia coli are stimulated by MutL. The interaction of MutL with each enzyme is enhanced in vivo by 2-aminopurine treatment and by inactivation of the mutY gene. We hypothesize that MutL recruits the endonucleases to sites of DNA damage. PMID:19376855

  17. Reporting of MMR evidence in professional publications: 1988-2007.

    PubMed

    Hilton, S; Hunt, K; Langan, M; Hamilton, V; Petticrew, M

    2009-11-01

    To examine how journals and magazines disseminate research evidence and guidance on best practice to health professionals by aligning commentaries on measles, mumps, and rubella vaccine (MMR) evidence in journals with key events in the MMR controversy. Content analysis. Comment articles in six commonly read UK publications. Number of comment pieces by publication, year and article type; trends in the focus, tone and inclusion of recommendations on MMR. 860 articles met the inclusion criteria (BMJ n = 104, Community Practitioner n = 45, Health Visitor n = 24, Practice Nurse n = 61, Nursing Standard n = 61 and Pulse n = 565). Of these 860 comment pieces, 264 made some reference to evidence endorsing the safety of MMR. Around one in 10 were rated as negative (10.9%, n = 29) or neutral (11.3%, n = 30) in relation to MMR safety, and nearly a quarter (22.7%, n = 60) were rated as mixed. Following the publication of Wakefield et al's 1998 paper there was a period of neutrality. In 2000, despite growing public concerns and widespread media coverage, fewer than 20 comment pieces were published. Less than a quarter of comment pieces (n = 196, 22.7%) included recommendations. While a period of neutrality may reflect a professional response to uncertainty by holding back until consensus emerges, it may also represent a missed opportunity to promote evidence-based practice.

  18. Parents' reported reasons for avoiding MMR vaccination. A telephone survey.

    PubMed

    Dannetun, Eva; Tegnell, Anders; Hermansson, Göran; Giesecke, Johan

    2005-09-01

    During the second half of the 1990s and the first years of the 2000s a declining coverage for MMR vaccination in two-year-olds was observed in Sweden. The aim was to assess reasons for postponement or non-vaccination. A telephone survey using a structured questionnaire on parents' attitudes regarding their choice to postpone or abstain from vaccinating their child. The County of Ostergötland in Sweden. A total of 203 parents of children who had no registered date for MMR vaccination at a Child Health Centre. Parental reasons for non-vaccination. In all, 26 of the 203 children had received MMR vaccination but this had not been registered. Of those not vaccinated, 40% of the parents had decided to abstain and 60% to postpone vaccination. Fear of side effects was the most common reason for non-vaccination in both groups. The main source of information was the media followed by the Child Health Centre. Parents with a single child more often postponed vaccination and those who abstained were more likely to have had a discussion with a doctor or nurse about MMR vaccine. Postponers and abstainers may have different reasons for their decision. The role of well-trained healthcare staff in giving advice and an opportunity to discuss MMR vaccination with concerned parents is very important.

  19. Exploring the evidence surrounding the debate on MMR and autism.

    PubMed

    Purssell, Edward

    Autism is a poorly understood condition that would appear to be on the increase. There is currently much concern about a possible link between the measles, mumps and rubella (MMR) vaccination and autism which has resulted in a substantial reduction in the uptake of MMR, putting children at risk of three significant childhood diseases. This article looks at the evidence for a link between MMR and autism, finding that while a plausible hypothesis has been constructed, there is not substantive evidence for such a link and that the quality of this evidence is, in many cases, poor. This is due, in part, to the difficulties inherent in pre- and post-licensure vaccination research. These difficulties and the importance of vaccination and parental understanding of risk are discussed.

  20. MMR vaccine--how effective and how safe?

    PubMed

    2003-04-01

    The introduction of combined measles, mumps and rubella (MMR) vaccine into the UK childhood immunisation schedule in 1988 has markedly reduced the incidence of these diseases and their complications, with, for example, no deaths from acute measles occurring since 1992. However, uptake of MMR vaccine has fallen since the publication, in 1998, of a study that suggested a link between exposure to the vaccine and the development of intestinal inflammation and autism. The study, and the subsequent debate, have attracted considerable media coverage and left many parents and some healthcare professionals uncertain about what to do. While in 1996, 92% of children in England and Wales had received their first dose of the vaccine by the age of 2 years, by 2001-2 this figure had fallen to 84%, with much lower uptake in some regions, threatening a resurgence of all three diseases. Here, we review the evidence for the effectiveness and safety of MMR vaccine.

  1. 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

  2. MMR vaccination and autism: is there a link?

    PubMed

    DeStefano, Frank; Thompson, William W

    2002-07-01

    In 1998, a report was published describing 12 patients with inflammatory bowel conditions and regressive developmental disorders consisting primarily of autism. The authors hypothesised that MMR vaccine may have been responsible for the bowel dysfunction which subsequently resulted in the neurodevelopmental disorders. The suggestion that measles vaccine may cause autism through a persistent bowel infection generated much interest since it provided a possible biological mechanism for a causal association. Epidemiological studies, however, have not found an association between MMR vaccination and autism. Autism has a strong genetic component and its associated neurological defects probably occur during embryonic development. It seems unlikely that a vaccination that is given after birth could cause autism. In a minority of cases, autism may have onset after 1 year of age (regressive autism) but the one epidemiological study that included such cases did not find an association with MMR vaccination. Currently, the weight of the available epidemiological and related evidence does not support a causal link between MMR vaccine and autism.

  3. Danish MMR vaccination coverage is considerably higher than reported.

    PubMed

    Holt, Nanna; Mygind, Anna; Bro, Flemming

    2017-02-01

    The Danish childhood vaccination programme offers protection against measles, mumps, and rubella (MMR). Nevertheless, many children appear to be unvaccinated according to the national registers. The aim of this study was to estimate the MMR1 vaccination coverage based on a medical record review of children whose vaccination status is negative according to the register-based data. We conducted a cross-sectional study of 19 randomly selected general practices in the Central Denmark Region including 1,712 children aged 18-42 months. The practices received a registration form listing children with a negative MMR1 vaccination status in the register-based data. The general practices then validated the children's vaccination status by medical record review. In total, 94% of the children had been vaccinated according to the medical records in general practice compared with 86% according to the register-based data. Of the 246 children who were unvaccinated according to the register-based data, 135 (55%) had been vaccinated according to the medical records. This discrepancy was due mainly to administrative reimbursement errors. The MMR1 vaccination coverage in Denmark seems to be considerably higher than reflected in national registers. Using medical record review to re-assess the vaccination status revealed that most of the supposedly unvaccinated children had, in fact, been vaccinated. The Danish Research Foundation for General Practice and the General Practitioners' Foundation for Education and Development. not relevant.

  4. Reporting of MMR evidence in professional publications: 1988–2007

    PubMed Central

    Hilton, S; Hunt, K; Langan, M; Hamilton, V; Petticrew, M

    2009-01-01

    Objective: To examine how journals and magazines disseminate research evidence and guidance on best practice to health professionals by aligning commentaries on measles, mumps, and rubella vaccine (MMR) evidence in journals with key events in the MMR controversy. Design: Content analysis. Data sources: Comment articles in six commonly read UK publications. Main outcome measures: Number of comment pieces by publication, year and article type; trends in the focus, tone and inclusion of recommendations on MMR. Results: 860 articles met the inclusion criteria (BMJ n = 104, Community Practitioner n = 45, Health Visitor n = 24, Practice Nurse n = 61, Nursing Standard n = 61 and Pulse n = 565). Of these 860 comment pieces, 264 made some reference to evidence endorsing the safety of MMR. Around one in 10 were rated as negative (10.9%, n = 29) or neutral (11.3%, n = 30) in relation to MMR safety, and nearly a quarter (22.7%, n = 60) were rated as mixed. Following the publication of Wakefield et al’s 1998 paper there was a period of neutrality. In 2000, despite growing public concerns and widespread media coverage, fewer than 20 comment pieces were published. Less than a quarter of comment pieces (n = 196, 22.7%) included recommendations. Conclusion: While a period of neutrality may reflect a professional response to uncertainty by holding back until consensus emerges, it may also represent a missed opportunity to promote evidence-based practice. PMID:19414434

  5. MMR-Vaccine and Regression in Autism Spectrum Disorders: Negative Results Presented from Japan

    ERIC Educational Resources Information Center

    Uchiyama, Tokio; Kurosawa, Michiko; Inaba, Yutaka

    2007-01-01

    It has been suggested that the measles, mumps, and rubella vaccine (MMR) is a cause of regressive autism. As MMR was used in Japan only between 1989 and 1993, this time period affords a natural experiment to examine this hypothesis. Data on 904 patients with autism spectrum disorders (ASD) were analyzed. During the period of MMR usage no…

  6. MMR-Vaccine and Regression in Autism Spectrum Disorders: Negative Results Presented from Japan

    ERIC Educational Resources Information Center

    Uchiyama, Tokio; Kurosawa, Michiko; Inaba, Yutaka

    2007-01-01

    It has been suggested that the measles, mumps, and rubella vaccine (MMR) is a cause of regressive autism. As MMR was used in Japan only between 1989 and 1993, this time period affords a natural experiment to examine this hypothesis. Data on 904 patients with autism spectrum disorders (ASD) were analyzed. During the period of MMR usage no…

  7. U.K. parents' decision-making about measles-mumps-rubella (MMR) vaccine 10 years after the MMR-autism controversy: a qualitative analysis.

    PubMed

    Brown, Katrina F; Long, Susannah J; Ramsay, Mary; Hudson, Michael J; Green, John; Vincent, Charles A; Kroll, J Simon; Fraser, Graham; Sevdalis, Nick

    2012-02-27

    Public concern about an unsubstantiated link between MMR vaccine and autism stemmed from a 1998 paper by Dr Andrew Wakefield and colleagues, and the substantial media coverage which that work attracted. Though the Wakefield paper is now discredited and an MMR-autism link has never been demonstrated empirically, this concern has manifested in over a decade of suboptimal MMR uptake. Few qualitative studies have explored parents' MMR decision-making since uptake began to improve in 2004. This study updates and adds methodological rigour to the evidence base. 24 mothers planning to accept, postpone or decline the first MMR dose (MMR1) for their 11-36 month-old children, described their decision-making in semi-structured interviews. Mothers were recruited via General Practice, parents' groups/online forums, and chain referral. MMR1 status was obtained from General Practice records 6 months post-interview. Interview transcripts were coded and interpreted using a modified Grounded Theory approach. Five themes were identified: MMR vaccine and controversy; Social and personal consequences of MMR decision; Health professionals and policy; Severity and prevalence of measles, mumps and rubella infections; Information about MMR and alternatives. Results indicated that MMR1 acceptors were sympathetic toward Wakefield as a person, but universally rejected his study which sparked the controversy; parents opting for single vaccines expressed the sense that immune overload is not a consideration but that not all three components of MMR are warranted by disease severity; and MMR1 rejectors openly criticised other parents' MMR decisions and decision-making. This study corroborated some previous qualitative work but indicated that the shrinking group of parents now rejecting MMR comprises mainly those with more extreme and complex anti-immunisation views, whilst parents opting for single vaccines may use second-hand information about the controversy. In response, policymakers and

  8. Misled and confused? Telling the public about MMR vaccine safety

    PubMed Central

    Clements, C; Ratzan, S

    2003-01-01

    The extraordinary events surrounding the measles, mumps, and rubella (MMR) vaccine in the United Kingdom have not only placed in jeopardy the use of this triple vaccine but have also spread concern to other parts of the world. Examination of the public's worry about MMR vaccine reveals they have been exposed to a range of conflicting views resulting in the feeling of having been misled about the safety of the vaccine. There are various groups and individuals who have legitimate roles in informing the public about such subjects. But is each one behaving in an ethically responsible way? And if confidence falters, vaccine coverage dips, and an outbreak of measles, mumps, or rubella ensues, who, if anyone, will stand and say "I misled them, I confused them, this is my responsibility"? We examine the ethical issues of each group with a voice in the debate about vaccine safety. PMID:12569190

  9. Preservation of DNA integrity and neuronal degeneration.

    PubMed

    Francisconi, Simona; Codenotti, Mara; Ferrari-Toninelli, Giulia; Uberti, Daniela; Memo, Maurizio

    2005-04-01

    The mismatch repair system (MMR) is an important member of the DNA checkpoint, that includes a number of protein deputed to control genomic stability through cell cycle arrest, DNA repair, and apoptosis. Here we summarize some recent data from our and other groups underlining the contribution to neurodegeneration of MSH2, perhaps the most relevant component of the MMR system. These data suggest that this protein participates not only in the cancer prevention machinery for the body but also in neurodegenerative processes.

  10. Expression analysis of heat shock protein 90 (HSP90) and Her2 in colon carcinoma.

    PubMed

    Drecoll, Enken; Nitsche, Ulrich; Bauer, Karina; Berezowska, Sabina; Slotta-Huspenina, Julia; Rosenberg, Robert; Langer, Rupert

    2014-06-01

    The molecular chaperone heat shock protein 90 (HSP90) plays an important role in several types of tumors also participating in the modulation of the activity of receptor tyrosine kinases activity such as members of the Her family. We evaluated the significance of HSP90 and Her2 expression in colon cancer. HSP90 and Her2 expression was determined by immunohistochemistry and by fluorescence in situ hybridization (FISH) on 355 primary resected colon carcinomas. Results were correlated with pathologic features (Union for International Cancer Control (UICC) pTNM category, tumor localisation, tumor differentiation), additional molecular genetic characteristics (BRAF, KRAS mutational status, mismatch repair genes (MMR)), and survival. HSP90 immunoreactivity was observed in various degrees. Fifty-one cases (14 %) were positive for Her2 (score 2+ and 3+) with 16/43 cases with Her2 2+ staining pattern showing amplification of Her2 determined by FISH. There was a significant correlation between high HSP90 expression and Her2 overexpression (p = 0.011). High HSP90 expression was associated with earlier tumor stages (p = 0.019), absence of lymph node (p = 0.006), and absence of distant metastases (p = 0.001). Patients with high tumoral HSP90 levels had a better survival (p = 0.032), but this was not independent from other prognostic relevant pathologic parameters. Her2 expression was not associated with any of the investigated histopathological, molecular, or clinical parameters. High HSP90 levels are reflecting lower malignant potential in colon cancer. Her2 positivity can be observed in a small number of cases. Targeting HSP90 and/or Her2 may be an alternative therapeutic approach in colon cancer in a subset of patients.

  11. A polymorphism in the MSH3 mismatch repair gene is associated with the levels of somatic instability of the expanded CTG repeat in the blood DNA of myotonic dystrophy type 1 patients.

    PubMed

    Morales, Fernando; Vásquez, Melissa; Santamaría, Carolina; Cuenca, Patricia; Corrales, Eyleen; Monckton, Darren G

    2016-04-01

    Somatic mosaicism of the expanded CTG repeat in myotonic dystrophy type 1 is age-dependent, tissue-specific and expansion-biased, contributing toward the tissue-specificity and progressive nature of the symptoms. Previously, using regression modelling of repeat instability we showed that variation in the rate of somatic expansion in blood DNA contributes toward variation in age of onset, directly implicating somatic expansion in the disease pathway. Here, we confirm these results using a larger more genetically homogenous Costa Rican DM1 cohort (p<0.001). Interestingly, we also provide evidence that supports subtle sex-dependent differences in repeat length-dependent age at onset and somatic mutational dynamics. Previously, we demonstrated that variation in the rate of somatic expansion was a heritable quantitative trait. Given the important role that DNA mismatch repair genes play in mediating expansions in mouse models, we tested for modifier gene effects with 13 DNA mismatch gene polymorphisms (one each in MSH2, PMS2, MSH6 and MLH1; and nine in MSH3). After correcting for allele length and age effects, we identified three polymorphisms in MSH3 that were associated with variation in somatic instability: Rs26279 (p=0.003); Rs1677658 (p=0.009); and Rs10168 (p=0.031). However, only the association with Rs26279 remained significant after multiple testing correction. Although we revealed a statistically significant association between Rs26279 and somatic instability, we did not detect an association with the age at onset. Individuals with the A/A genotype for Rs26279 tended to show a greater propensity to expand the CTG repeat than other genotypes. Interestingly, this SNP results in an amino acid change in the critical ATPase domain of MSH3 and is potentially functionally dimorphic. These data suggest that MSH3 is a key player in generating somatic variation in DM1 patients and further highlight MSH3 as a potential therapeutic target.

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

    PubMed

    Thompson, Bryony A; Spurdle, Amanda B; Plazzer, John-Paul; Greenblatt, Marc S; Akagi, Kiwamu; Al-Mulla, Fahd; Bapat, Bharati; Bernstein, Inge; Capellá, Gabriel; den Dunnen, Johan T; du Sart, Desiree; Fabre, Aurelie; Farrell, Michael P; Farrington, Susan M; Frayling, Ian M; Frebourg, Thierry; Goldgar, David E; Heinen, Christopher D; Holinski-Feder, Elke; Kohonen-Corish, Maija; Robinson, Kristina Lagerstedt; Leung, Suet Yi; Martins, Alexandra; Moller, Pal; Morak, Monika; Nystrom, Minna; Peltomaki, Paivi; Pineda, Marta; Qi, Ming; Ramesar, Rajkumar; Rasmussen, Lene Juel; Royer-Pokora, Brigitte; Scott, Rodney J; Sijmons, Rolf; Tavtigian, Sean V; Tops, Carli M; Weber, Thomas; Wijnen, Juul; Woods, Michael O; Macrae, Finlay; Genuardi, Maurizio

    2014-02-01

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

  13. Risk, its perception and the media: the MMR controversy.

    PubMed

    Hackett, Alison Jane

    2008-07-01

    This article aims to explore how the media contributes to and generates 'risk' and 'risk perception.' The example of parents refusing to have their children immunised with the measles, mumps and rubella (MMR) vaccine following negative media reporting will be discussed. The media appears to have an important influence on the perception of risk. We are living in a society that is increasingly aware of risk, and in which risk is socially constructed. It is important that healthcare professionals provide clear, consistent, evidence-based information to clients, ensuring that any areas of uncertainty are acknowledged. Otherwise, the public's trust in the healthcare professional will be undermined.

  14. 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.

  15. MMR and autism: an overview of the debate to date.

    PubMed

    Phelan, Anastasia T

    The ongoing controversial debate about the measles mumps rubella (MMR) vaccine and its links to regressional autism and specific bowel disorders appears to experience 'peaks and table troughs' of public interest correlated with specific media attention at any one time. It is not the intention of this article to either refute or substantiate the ongoing controversy, as this is obviously a scientific debate, but rather to offer an overview of the studies to date in the interest of helping practitioners in the frontline to engage in informed debate with concerned parents. The conclusions from this review stem from two major studies conducted by the Institute of Medicine (IoM, 2001) in the USA, and Medical Research Council (MRC, 2001) in the UK. Both conclude that although epidemiological studies so far do not support a link between MMR and autism, nonetheless the studies have been too imprecise to rule out the prospect of the vaccination being involved in a small number of cases, and the need for further research has been pointed out.

  16. Distinctive patterns of p53 protein expression and microsatellite instability in human colorectal cancer.

    PubMed

    Nyiraneza, Christine; Jouret-Mourin, Anne; Kartheuser, Alex; Camby, Philippe; Plomteux, Olivier; Detry, Roger; Dahan, Karin; Sempoux, Christine

    2011-12-01

    Although evidence suggests an inverse relationship between microsatellite instability and p53 alterations in colorectal cancer, no study has thoroughly examined the use of p53 immunohistochemistry in phenotyping colorectal cancers. We investigated the value of p53 immunohistochemistry in microsatellite instability-positive colorectal cancers prescreening and attempted to clarify the relationship between DNA mismatch repair system and p53 pathway. In a series of 104 consecutive colorectal cancers, we performed p53 immunohistochemistry, TP53 mutational analysis, DNA mismatch repair system efficiency evaluation (DNA mismatch repair system immunohistochemistry, microsatellite instability status, MLH1/MSH2 germ line, and BRAF, murine double minute 2, and p21 immunohistochemistry. Microsatellite instability high was observed in 25 of 104 colorectal cancers, with DNA mismatch repair system protein loss (24/25) and germ line (8/25) or BRAF mutations (8/25). p53 immunohistochemistry revealed 3 distinct patterns of expression: complete negative immunostaining associated with truncating TP53 mutations (P < .0001), diffuse overexpression associated with missense TP53 mutations (P < .0001), and restricted overexpression characterized by a limited number of homogenously scattered strongly positive tumor cells in 36.5% of colorectal cancers. This latest pattern was associated with wild-type TP53 and microsatellite instability high colorectal cancers (P < .0001) including all Lynch tumors (8/8), but its presence among 22% of DNA mismatch repair system-competent colorectal cancers decreased its positive predictive value (55.2% [95% confidence interval, 45%-65%]). It was also correlated with murine double minute 2 overexpression (P < .0001) and inversely with p21 loss (P = .0002), independently of microsatellite instability status. In conclusion, a restricted pattern of p53 overexpression is preferentially associated with microsatellite instability high phenotype and could

  17. Media Reports of Links between MMR and Autism: A Discourse Analysis

    ERIC Educational Resources Information Center

    O'Dell, Lindsay; Brownlow, Charlotte

    2005-01-01

    This paper details an analysis of BBC reporting of the proposed links between MMR and autism. The study aimed to identify main issues arising from the media reports into the link between MMR and the development of autism, and how these contribute to common understandings about people with autism. The study employed a form of discourse analysis to…

  18. No Effect of MMR Withdrawal on the Incidence of Autism: A Total Population Study

    ERIC Educational Resources Information Center

    Honda, Hideo; Shimizu, Yasuo; Rutter, Michael

    2005-01-01

    Background: A causal relationship between the measles, mumps, and rubella (MMR) vaccine and occurrence of autism spectrum disorders (ASD) has been claimed, based on an increase in ASD in the USA and the UK after introduction of the MMR vaccine. However, the possibility that this increase is coincidental has not been eliminated. The unique…

  19. 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

  20. Media Reports of Links between MMR and Autism: A Discourse Analysis

    ERIC Educational Resources Information Center

    O'Dell, Lindsay; Brownlow, Charlotte

    2005-01-01

    This paper details an analysis of BBC reporting of the proposed links between MMR and autism. The study aimed to identify main issues arising from the media reports into the link between MMR and the development of autism, and how these contribute to common understandings about people with autism. The study employed a form of discourse analysis to…

  1. No Effect of MMR Withdrawal on the Incidence of Autism: A Total Population Study

    ERIC Educational Resources Information Center

    Honda, Hideo; Shimizu, Yasuo; Rutter, Michael

    2005-01-01

    Background: A causal relationship between the measles, mumps, and rubella (MMR) vaccine and occurrence of autism spectrum disorders (ASD) has been claimed, based on an increase in ASD in the USA and the UK after introduction of the MMR vaccine. However, the possibility that this increase is coincidental has not been eliminated. The unique…

  2. MSI phenotype and MMR alterations in familial and sporadic gastric cancer.

    PubMed

    Leite, Marina; Corso, Giovanni; Sousa, Sónia; Milanezi, Fernanda; Afonso, Luís P; Henrique, Rui; Soares, José Manuel; Castedo, Sérgio; Carneiro, Fátima; Roviello, Franco; Oliveira, Carla; Seruca, Raquel

    2011-04-01

    Microsatellite instability (MSI) is a major pathway involved in gastric carcinogenesis occurring in 20% of gastric cancer (GC). However, it is not clear whether MSI phenotype preferentially occurs in the sporadic or familial GC, when stringent inclusion criteria are used. The aim of this study was to compare the frequency of MSI and hypermethylation of MLH1 promoter in a large series of familial GC patients (non-HNPCC and non-CDH1-related) and sporadic cases. Additionally, we analysed the immunoexpression of MMR proteins in a fraction of cases. Overall, the frequency of familial GC was 7.1%, and the frequency of hereditary tumours was 4.6%. MSI phenotype and MLH1 hypermethylation frequencies were not statistical different between familial and sporadic GC settings. Further, the MSI phenotype was not associated with any clinico-pathological features studied in the familial GC setting, whereas in the sporadic setting, it was associated with older age, female gender and intestinal histotype. Using our stringent Amsterdam-based clinical criteria to select familial GC (number of cases, age of onset), we verified that sporadic and familial cases differed in gender but shared histopathological features. We verified that the frequency of MSI was similar in familial and sporadic GC settings, demonstrating that this molecular phenotype is not a hallmark of familial GC in contrast to what is verified in HNPCC. Moreover, we observed that the frequency of MLH1 hypermethylation is similar in sporadic and familial cases suggesting that in both settings MSI is not associated to MMR genetic alterations but in contrast to epigenetic deregulation. Copyright © 2010 UICC.

  3. An MLH1 Mutation Links BACH1/FANCJ to Colon Cancer, Signaling, and Insight toward Directed Therapy

    PubMed Central

    Xie, Jenny; Guillemette, Shawna; Peng, Min; Gilbert, Candace; Buermeyer, Andrew; Cantor, Sharon B.

    2017-01-01

    Defects in MLH1, as with other mismatch repair (MMR) proteins, are the primary cause of hereditary nonpolyposis colon cancer (HNPCC). Mutations in MMR genes often disrupt mismatch repair and MMR signaling functions. However, some HNPCC-associated mutations have unknown pathogenicity. Here, we uncover an MLH1 clinical mutation with a leucine (L)-to-histidine (H) amino acid change at position 607 that ablates MLH1 binding to FANCJ. Given that a DNA helicase is not essential for mammalian MMR in vitro, we considered that loss of MLH1 binding to FANCJ could alter MMR signaling. Consistent with this hypothesis, FANCJ-deficient cells exhibit delayed MMR signaling and apoptotic responses that generate resistance to agents that induce O6-methylguanine lesions. Our data indicate that the delay in MMR signaling provides time for the methylguanine methyltransferase (MGMT) enzyme to reverse DNA methylation. In essence, FANCJ deficiency alters the competition between two pathways: MGMT-prosurvival versus MMR-prodeath. This outcome could explain the HNPCC familial cancers that present as microsatellite stable and with intact MMR, such as MLHL607H. Importantly, the link between FANCJ and HNPCC provides insight toward directed therapies because loss of the FANCJ/MLH1 interaction also uniquely sensitizes cells to DNA cross-linking agents. PMID:20978114

  4. BOREAS RSS-3 Reflectance Measured from a Helicopter-Mounted Barnes MMR

    NASA Technical Reports Server (NTRS)

    Hall, Forrest G. (Editor); Nickeson, Jaime (Editor); Walthall, Charles L.; Loechel, Sara; deColstoun, Eric Brown

    2000-01-01

    The BOREAS RSS-3 team acquired helicopter-based radiometric measurements of forested sites with a Barnes MMR. The data were collected in 1994 during the three BOREAS IFCs at numerous tower and auxiliary sites in both the NSA and SSA. The 15-degree FOV of the MMR yielded approximately 79-m ground resolution from an altitude of 300 m. The MMR has seven spectral bands that are similar to the Landsat TM bands, ranging from the blue region to the thermal. The data are stored in tabular ASCII files. The data are stored in tabular ASCII files.

  5. A survey of UK parental attitudes to the MMR vaccine and trust in medical authority.

    PubMed

    Casiday, Rachel; Cresswell, Tricia; Wilson, Deb; Panter-Brick, Catherine

    2006-01-12

    Contested reports associating the MMR vaccine with autism have resulted in diminished confidence and uptake of the vaccine in the UK. This postal survey of parent's decisions, attitudes and use of information about MMR immunisation was constructed from questions derived from in-depth qualitative work. The setting was a Primary Care Trust in northeast England (N=996). Both MMR-accepting and refusing parents were supportive of immunisation, yet the high level of concern about the safety of the vaccine expressed even by parents who had immunised their children is worrying in its implications for public confidence and trust in health care. The findings suggest that the ability of practitioners to provide effective professional advice about MMR vaccine could be undermined if a government were to directly promote the vaccine to parents. Practitioners should continue to provide parents with accurate information, while communicating respect for parents' intentions to protect their children's health.

  6. MMR vaccination and autism : what is the evidence for a causal association?

    PubMed

    Madsen, Kreesten M; Vestergaard, Mogens

    2004-01-01

    It has been suggested that vaccination with the measles-mumps-rubella (MMR) vaccine causes autism. The wide-scale use of the MMR vaccine has been reported to coincide with the apparent increase in the incidence of autism. Case reports have described children who developed signs of both developmental regression and gastrointestinal symptoms shortly after MMR vaccination.A review of the literature revealed no convincing scientific evidence to support a causal relationship between the use of MMR vaccines and autism. No primate models exist to support the hypothesis. The biological plausibility remains questionable and there is a sound body of epidemiological evidence to refute the hypothesis. The hypothesis has been subjected to critical evaluation in many different ways, using techniques from molecular biology to population-based epidemiology, and with a vast number of independent researchers involved, none of which has been able to corroborate the hypothesis.

  7. 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

  8. [The relationship between MMR vaccination level and the number of new cases of autism in children].

    PubMed

    Mrozek-Budzyn, Dorota; Kiełtyka, Agnieszka

    2008-01-01

    The MMR vaccination coverage in Malopolskie voivodeship improved rapidly and finally reached a high level during last years. The number of new cases of autism spectrum disorders in children during that time revealed a slightly rising but not significant trend, while the number of childhood autism were stable. Ecological study showed no correlation between MMR vaccination and an increased risk of childhood autism and autism spectrum disorders in children.

  9. No effect of MMR withdrawal on the incidence of autism: a total population study.

    PubMed

    Honda, Hideo; Shimizu, Yasuo; Rutter, Michael

    2005-06-01

    A causal relationship between the measles, mumps, and rubella (MMR) vaccine and occurrence of autism spectrum disorders (ASD) has been claimed, based on an increase in ASD in the USA and the UK after introduction of the MMR vaccine. However, the possibility that this increase is coincidental has not been eliminated. The unique circumstances of a Japanese MMR vaccination program provide an opportunity for comparison of ASD incidence before and after termination of the program. This study examined cumulative incidence of ASD up to age seven for children born from 1988 to 1996 in Kohoku Ward (population approximately 300,000), Yokohama, Japan. ASD cases included all cases of pervasive developmental disorders according to ICD-10 guidelines. The MMR vaccination rate in the city of Yokohama declined significantly in the birth cohorts of years 1988 through 1992, and not a single vaccination was administered in 1993 or thereafter. In contrast, cumulative incidence of ASD up to age seven increased significantly in the birth cohorts of years 1988 through 1996 and most notably rose dramatically beginning with the birth cohort of 1993. The significance of this finding is that MMR vaccination is most unlikely to be a main cause of ASD, that it cannot explain the rise over time in the incidence of ASD, and that withdrawal of MMR in countries where it is still being used cannot be expected to lead to a reduction in the incidence of ASD.

  10. Text message reminders for timely routine MMR vaccination: A randomized controlled trial.

    PubMed

    Hofstetter, Annika M; DuRivage, Nathalie; Vargas, Celibell Y; Camargo, Stewin; Vawdrey, David K; Fisher, Allison; Stockwell, Melissa S

    2015-10-26

    Measles-mumps-rubella (MMR) vaccination is important for preventing disease outbreaks, yet pockets of under-vaccination persist. Text message reminders have been employed successfully for other pediatric vaccines, but studies examining their use for MMR vaccination are limited. This study assessed the impact of text message reminders on timely MMR vaccination. Parents (n=2054) of 9.5-10.5-month-old children from four urban academically-affiliated pediatric clinics were randomized to scheduling plus appointment text message reminders, appointment text message reminder-only, or usual care. The former included up to three text reminders to schedule the one-year preventive care visit. Both text messaging arms included a text reminder sent 2 days before that visit. Outcomes included appointment scheduling, appointment attendance, and MMR vaccination by age 13 months, the standard of care at study sites. Children of parents in the scheduling plus appointment text message reminders arm were more likely to have a scheduled one-year visit than those in the other arms (71.9% vs. 67.4%, relative risk ratio (RRR) 1.07 [95% CI 1.005-1.13]), particularly if no appointment was scheduled before randomization (i.e., no baseline appointment) (62.1% vs. 54.7%, RRR 1.14 [95% CI 1.04-1.24]). One-year visit attendance and timely MMR vaccination were similar between arms. However, among children without a baseline appointment, those with parents in the scheduling plus appointment text message reminders arm were more likely to undergo timely MMR vaccination (61.1% vs. 55.1%, RRR 1.11 [95% CI 1.01-1.21]). Text message reminders improved timely MMR vaccination of high-risk children without a baseline one-year visit. Copyright © 2015. Published by Elsevier Ltd.

  11. Vaccine Message Framing and Parents’ Intent to Immunize Their Infants for MMR

    PubMed Central

    Finnell, S. Maria E.; Zimet, Gregory D.; Sturm, Lynne A.; Lane, Kathleen A.; Downs, Stephen M.

    2014-01-01

    BACKGROUND AND OBJECTIVE: Emphasizing societal benefits of vaccines has been linked to increased vaccination intentions in adults. It is unclear if this pattern holds for parents deciding whether to vaccinate their children. The objective was to determine whether emphasizing the benefits of measles-mumps-rubella (MMR) vaccination directly to the vaccine recipient or to society differentially impacts parents' vaccine intentions for their infants. METHODS: In a national online survey, parents (N = 802) of infants <12 months old were randomly assigned to receive 1 of 4 MMR vaccine messages: (1) the Centers for Disease Control and Prevention Vaccine Information Statement (VIS), (2) VIS and information emphasizing the MMR vaccine's benefits to the child, (3) VIS and information emphasizing societal benefits, or (4) VIS and information emphasizing benefits both to the child and society. Parents reported their likelihood of vaccinating their infants for MMR on a response scale of 0 (extremely unlikely) to 100 (extremely likely). RESULTS: Compared with the VIS-only group (mean intention = 86.3), parents reported increased vaccine intentions for their infants when receiving additional information emphasizing the MMR vaccine’s benefits either directly to the child (mean intention = 91.6, P = .01) or to both the child and society (mean intention = 90.8, P = .03). Emphasizing the MMR vaccine’s benefits only to society did not increase intentions (mean intention = 86.4, P = .97). CONCLUSIONS: We did not see increases in parents’ MMR vaccine intentions for their infants when societal benefits were emphasized without mention of benefits directly to the child. This finding suggests that providers should emphasize benefits directly to the child. Mentioning societal benefits seems to neither add value to, nor interfere with, information highlighting benefits directly to the child. PMID:25136038

  12. Opportunistic MMR vaccination for unimmunized children at the time of routine teenage booster vaccination in secondary schools: implications for policy.

    PubMed

    Paranthaman, K; Bunce, A

    2012-09-01

    With the aim of minimizing adverse health outcomes and reducing the risk of outbreaks, we offered one dose of MMR vaccine to children known to be incompletely immunized at the time of teenage booster vaccination in secondary schools in Swindon in 2011. The Child Health Department database was queried to identify Year 10 children who had had zero or one dose of MMR vaccine previously. Of the 316 children offered vaccination, 60 received a first dose and 87 received a second dose of MMR vaccine. Fourteen children had two documented doses in the past and two had contraindications to the vaccine. Overall uptake of two doses of MMR vaccine increased from 86·3% to 90·6%. The valuable uptake achieved demonstrates that an opportunistic offer of MMR vaccine for unimmunized children at schools is feasible and beneficial. MMR vaccine should be offered routinely to unimmunized children at the time of school vaccination programmes, especially in areas with sub-optimal coverage.

  13. Safety and Immunogenicity of Human Serum Albumin-Free MMR Vaccine in US Children Aged 12–15 Months

    PubMed Central

    Mufson, Maurice A.; Diaz, Clemente; Leonardi, Michael; Harrison, Christopher J.; Grogg, Stanley; Carbayo, Antonio; Carlo-Torres, Simon; JeanFreau, Robert; Quintero-Del-Rio, Ana; Bautista, Gisele; Povey, Michael; Da Costa, Christopher; Nicholson, Ouzama; Innis, Bruce L.

    2015-01-01

    Background M-M-RTMII (MMRII; Merck & Co) is currently the only measles-mumps-rubella (MMR) vaccine licensed in the United States. Another licensed vaccine would reinforce MMR supply. This study assessed the immunogenicity of a candidate vaccine (PriorixTM, GlaxoSmithKline Vaccines [MMR-RIT]) when used as a first dose among eligible children in the United States. Methods In this exploratory Phase-2, multicenter, observer-blind study, 1220 healthy subjects aged 12–15 months were randomized (3:3:3:3) and received 1 dose of 1 of 3 MMR-RIT lots with differing mumps virus titers (MMR-RIT-1 [4.8 log10]; MMR-RIT-2 [4.1 log10]; MMR-RIT-3 [3.7 log10] CCID50) or MMRII co-administered with hepatitis A vaccine (HAV), varicella vaccine (VAR) and 7-valent pneumococcal conjugate vaccine (PCV7). Immune response to measles, mumps, and rubella viruses was evaluated at Day 42 post-vaccination. Incidence of solicited injection site, general, and serious adverse events was assessed. Results Seroresponse rates for MMR vaccine viral components in MMR-RIT lots were 98.3–99.2% (measles), 89.7–90.7% (mumps), and 97.5–98.8% (rubella), and for MMRII were 99.6%, 91.1%, and 100%, respectively. Immune responses to HAV, VAR, and PCV7 were similar when co-administered with any of the 3 MMR-RIT lots or MMRII. There were no apparent differences in solicited or serious adverse events among the 4 groups. Conclusions Immune responses were above threshold levels for projected protection against the 3 viruses from MMR-RIT lots with differing mumps virus titers. MMR-RIT had an acceptable safety profile when co-administered with HAV, VAR, and PCV7. Clinical Trials Registration NCT00861744; etrack; 111870 PMID:26582873

  14. Erybraedin C and bitucarpin A, two structurally related pterocarpans purified from Bituminaria bituminosa, induced apoptosis in human colon adenocarcinoma cell lines MMR- and p53-proficient and -deficient in a dose-, time-, and structure-dependent fashion.

    PubMed

    Maurich, Tiziana; Iorio, Mariacarla; Chimenti, Daniele; Turchi, Gino

    2006-02-01

    Pterocarpans, the second group of natural isoflavonoids, have received considerable interest on account of their medicinal properties. These drugs are employed as antitoxins, but display antifungal, antiviral and antibacterial properties as well. Erybraedin C and bitucarpin A are two new structurally related pterocarpans recently purified and characterized. Bitucarpin A differs from erybraedin C for the absence of a prenyl group in 5' position and the presence of a methoxylate hydroxyl group in 7, 4' positions. These compounds proved not to be clastogens in human lymphocytes per se but displayed anticlastogenic activity against mytomicin C and bleomycin C. Here we extended the study of their antiproliferative and apoptosis-inducing mechanism on human cell lines. Two human adenocarcinoma cell lines, LoVo and HT29, as examples of slow-growing solid tumors, proficient and deficient in mismatch repair system (MMR), p53 and Bcl-2, were used to evaluate the cytotoxicity of the drugs and their effects on the cell cycle, measured by flow cytometry. Erybraedin C similarly affects the survival of HT29 (MMR +/+, p53 -/- and Bcl-2 +/+) and LoVo (MMR -/-, p53 +/+ and Bcl-2 -/-) cells (LD(50): 1.94 and 1.73 microg/ml, respectively). By contrast, bitucarpin A exhibits a differential cytotoxicity in the cell lines (LD(50): 6.00 microg/ml, HT29, and 1.84 microg/ml, LoVo). The cell cycle distributions of the LoVo and HT29 cells treated with erybraedin C lacked a specific checkpoint arrest, whereas they underwent a characteristic sub-G(1) peak, time- and drug-concentration dependent. So that apoptotic process induced by erybraedin C in both adenocarcinoma cell lines is independent of cell cycle arrest and of phenotypic status of the cells as well. By contrast, bitucarpin A affects cell cycle progression on both cell lines, inducing a transient block in G(0)/G(1) along 24-96 h, and induces apoptosis with a cell density and treatment time dependency. Similar results were obtained with

  15. Common variants associated with general and MMR vaccine-related febrile seizures

    PubMed Central

    Feenstra, Bjarke; Pasternak, Björn; Geller, Frank; Carstensen, Lisbeth; Wang, Tongfei; Huang, Fen; Eitson, Jennifer L.; Hollegaard, Mads V.; Svanström, Henrik; Vestergaard, Mogens; Hougaard, David M.; Schoggins, John W.; Jan, Lily Yeh; Melbye, Mads; Hviid, Anders

    2014-01-01

    Febrile seizures represent a recognized serious adverse event following measles, mumps, and rubella (MMR) vaccination. We conducted a series of genome-wide association scans comparing children with MMR-related febrile seizures, children with febrile seizures unrelated to vaccination, and controls with no history of febrile seizures. Two loci were distinctly associated with MMR-related febrile seizures, harboring the interferon-stimulated gene IFI44L (rs273259; P = 5.9×10−12 vs. controls; P =1.2×10−9 vs. MMR-unrelated febrile seizures) and the measles virus receptor CD46 (rs1318653; P = 9.6×10−11 vs. controls; P = 1.6×10−9 vs. MMR-unrelated febrile seizures). Furthermore, four loci were associated with febrile seizures in general implicating the sodium channel genes SCN1A (rs6432860; P = 2.2×10−16) and SCN2A (rs3769955; P = 3.1×10−10), a TMEM16 family gene (TMEM16C; rs114444506; P = 3.7×10−20), and a region associated with magnesium levels (12q21.33; rs11105468; P = 3.4×10−11). Finally, functional relevance of TMEM16C was demonstrated with electrophysiological experiments in wild-type and knockout rats. PMID:25344690

  16. Coexistence of MSI with KRAS mutation is associated with worse prognosis in colorectal cancer

    PubMed Central

    Hu, Jing; Yan, Wen-Yue; Xie, Li; Cheng, Lei; Yang, Mi; Li, Li; Shi, Jiong; Liu, Bao-Rui; Qian, Xiao-Ping

    2016-01-01

    Abstract Kristen rat sarcoma viral oncogene homolog (KRAS) and microsatellite instability (MSI) are prognostic markers of colorectal cancer (CRC). However, the clinical value is still not fully understood, when giving the consideration to both the molecular makers. Five hundred fifty-one patients with CRC were retrospectively assessed by determining their clinicopathological features. KRAS mutations were detected by polymerase chain reaction. MSI, a defect in the mismatch repair (MMR) system, was detected by immunohistochemistry. The prognostic value of KRAS in combination with MSI was studied. Among 551 CRC patients, mutations in KRAS codon 12 and KRAS codon 13 were detected in 34.5% and 10.5% of patients, respectively. Four hundred one tumors were randomly selected to detect for MMR proteins expression. In this analysis, 30 (7.5%) tumors that had at least 1 MMR protein loss were defined as MMR protein-deficient (MMR-D), and the remaining tumors were classed as MMR protein-intact (MMR-I). According to KRAS mutation and MSI status, CRC was classified into 4 groups: Group 1, KRAS-mutated and MMR-I; Group 2, KRAS-mutated and MMR-D; Group 3, KRAS wild and MMR-I; and Group 4, KRAS wild and MMR-D. We found that patients in Group4 had the best prognosis. In conclusion, combination status of KRAS and MSI status may be used as a prognostic biomarker for CRC patient, if validated by larger studies. PMID:27977612

  17. Coexistence of MSI with KRAS mutation is associated with worse prognosis in colorectal cancer.

    PubMed

    Hu, Jing; Yan, Wen-Yue; Xie, Li; Cheng, Lei; Yang, Mi; Li, Li; Shi, Jiong; Liu, Bao-Rui; Qian, Xiao-Ping

    2016-12-01

    Kristen rat sarcoma viral oncogene homolog (KRAS) and microsatellite instability (MSI) are prognostic markers of colorectal cancer (CRC). However, the clinical value is still not fully understood, when giving the consideration to both the molecular makers. Five hundred fifty-one patients with CRC were retrospectively assessed by determining their clinicopathological features. KRAS mutations were detected by polymerase chain reaction. MSI, a defect in the mismatch repair (MMR) system, was detected by immunohistochemistry. The prognostic value of KRAS in combination with MSI was studied. Among 551 CRC patients, mutations in KRAS codon 12 and KRAS codon 13 were detected in 34.5% and 10.5% of patients, respectively. Four hundred one tumors were randomly selected to detect for MMR proteins expression. In this analysis, 30 (7.5%) tumors that had at least 1 MMR protein loss were defined as MMR protein-deficient (MMR-D), and the remaining tumors were classed as MMR protein-intact (MMR-I). According to KRAS mutation and MSI status, CRC was classified into 4 groups: Group 1, KRAS-mutated and MMR-I; Group 2, KRAS-mutated and MMR-D; Group 3, KRAS wild and MMR-I; and Group 4, KRAS wild and MMR-D. We found that patients in Group4 had the best prognosis. In conclusion, combination status of KRAS and MSI status may be used as a prognostic biomarker for CRC patient, if validated by larger studies.

  18. Age-stratified seroprevalence of measles, mumps and rubella (MMR) virus infections in Switzerland after the introduction of MMR mass vaccination.

    PubMed

    Matter, L; Germann, D; Bally, F; Schopfer, K

    1997-01-01

    We have performed age-stratified seroprevalence studies for MMR to evaluate these vaccinations. Serum samples submitted for diagnostic testing were randomly selected for unlinked anonymous panels. IgG antibodies were tested by ELISA and indirect immunofluorescence. In the vaccination cohort (age 1.5 to 6.5 years), seroprevalence attained 80%. For measles and mumps it continued to increase to 95%, while for rubella it declined transiently to 60% between 7 and 12 years of age. We observed no differences according to gender in any age group in 1991-1992. (Semi)quantitative values of the IgG antibodies against all three viruses increased during adolescence, suggesting wild virus circulation. In 1992, MMR vaccination has reached < 80% of the children during their second year of age. Due to previous monovalent measles and mumps vaccinations in pre-school children and due to endemic and epidemic activity, particularly of mumps virus, a trough of the seroprevalence in adolescents was evident only for rubella. MMR vaccination campaigns performed at school since 1987 have increase seroprevalence in this population segment and have probably over-compensated for the expected shift to the right of the seroprevalence curves. A more compulsive implementation of the recommended childhood vaccination schedule and continued efforts at catchup vaccinations during school age especially for rubella are necessary to avoid the accumulation of susceptible young adults during the forthcoming decades.

  19. Media coverage of the measles-mumps-rubella vaccine and autism controversy and its relationship to MMR immunization rates in the United States.

    PubMed

    Smith, Michael J; Ellenberg, Susan S; Bell, Louis M; Rubin, David M

    2008-04-01

    The purpose of this work was to assess the association between media coverage of the MMR-autism controversy and MMR immunization in the United States. The public-use files of the National Immunization Survey were used to estimate annual MMR coverage from 1995 to 2004. The primary outcome was selective measles-mumps-rubella nonreceipt, that is, those children who received all childhood immunizations except MMR. Media coverage was measured by using LexisNexis, a comprehensive database of national and local news media. Factors associated with MMR nonreceipt were identified by using a logistic regression model. Selective MMR nonreceipt, occurring in as few as 0.77% of children in the 1995 cohort, rose to 2.1% in the 2000 National Immunization Survey. Children included in the 2000 National Immunization Survey were born when the putative link between MMR and autism surfaced in the medical literature but before any significant media attention occurred. Selective nonreceipt was more prevalent in private practices and unrelated to family characteristics. MMR nonreceipt returned to baseline before sustained media coverage of the MMR-autism story began. There was a significant increase in selective MMR nonreceipt that was temporally associated with the publication of the original scientific literature, suggesting a link between MMR and autism, which preceded media coverage of the MMR-autism controversy. This finding suggests a limited influence of mainstream media on MMR immunization in the United States.

  20. Mmr1p is a mitochondrial factor for Myo2p-dependent inheritance of mitochondria in the budding yeast.

    PubMed

    Itoh, Takashi; Toh-E, Akio; Matsui, Yasushi

    2004-07-07

    Class V myosins play a pivotal role in organelle distribution. In the budding yeast, Myo2p, a class V myosin, is essential for mitochondrial distribution. We identified MMR1 as a high-dose suppressor of the myo2 mitochondrial defect and that Mmr1p resides restrictively on the bud-localizing mitochondria and forms a complex with Myo2p tail. Mmr1p loss delayed mitochondrial transfer to buds and completely abolished mitochondrial distribution in the absence of Ypt11p, which promotes mitochondrial distribution by complex formation with Myo2p tail. The myo2-573 mutation, which causes a mitochondrial distribution defect and inactivates the Mmr1p function, reduced association between Myo2p and Mmr1p and depolarized Mmr1p localization on mitochondria. These strongly suggest that Mmr1p is a key mitochondrial component of the link between Myo2p and mitochondria for Myo2p-dependent mitochondrial distribution. Genetical analysis revealed that the Mmr1p-Myo2p pathway is independent of the Ypt11p-Myo2p pathway, suggesting that an essential system for mitochondrial distribution is composed of two independent Myo2p pathways.

  1. Anatomy of a health scare: education, income and the MMR controversy in the UK.

    PubMed

    Anderberg, Dan; Chevalier, Arnaud; Wadsworth, Jonathan

    2011-05-01

    The measles, mumps and rubella (MMR) controversy provides an interesting case where, for a short period of time, research publicized in the media, suggested a potential risk of serious side-effects associated with the vaccine, where there was also a sharp behavioral response from the public, and where the initial information was subsequently overturned. We consider the controversy from the perspective of health inequalities and the assimilation of information, focusing on whether and how vaccine uptake behavior in the wake of the controversy differed among groups of parents by education and income. Using panel data on the variation in the uptake of the MMR, and other childhood immunizations, across local Health Authority areas we find that the uptake rate of the MMR declined faster in areas where a larger fraction of parents had stayed in education past the age of 18 than in areas with less educated parents. We also find that the same areas reduced their relative uptake of other uncontroversial childhood immunizations, suggesting a "spillover" effect. Using a supplementary data source we find evidence of a corresponding positive income effect, indicating that wealthier parents avoided the MMR dilemma by purchasing single vaccines. Copyright © 2011 Elsevier B.V. All rights reserved.

  2. Similar immunogenicity of measles-mumps-rubella (MMR) vaccine administrated at 8 months versus 12 months age in children.

    PubMed

    He, Hanqing; Chen, Enfu; Chen, Haiping; Wang, Zhifang; Li, Qian; Yan, Rui; Guo, Jing; Zhou, Yang; Pan, Jinren; Xie, Shuyun

    2014-06-30

    Two doses of measles-mumps-rubella (MMR) strategy has been recommended by World Health Organization and is also widely adopted in many countries. In order to provide the evidence for perfecting the immunization strategy of MMR, this study evaluated the safety and immunogenicity of MMR with different two-dose schedule in infants. 280 participants were enrolled and randomly allocated to Group 1 (first dose at 8 months) or Group 2 (first dose at 12 months), and both groups administered the second dose at 10 months later. Solicited local and general symptoms after each vaccination with MMR were mild and infrequent in all participants of two groups. After administration of the first dose of MMR, seropositive rates were 100% in both groups for measles, 89.3% in Group 1 and 87.1% in Group 2 for mumps (P=0.578), 92.0% in Group 1 and 92.9% in Group 2 (P=0.393). The seropositive rates of mumps decreased significantly (from >86% to <65%) both in two groups (P<0.001) 10 months after the first dose of MMR, but no significant change was found in measles and rubella. All children get the positive titer for three vaccines in two groups after given the second dose MMR, higher seroconversion rate was found for mumps both in two groups (71.7% vs 77.2%, P=0.370). In conclusion, this study indicated that the MMR was well tolerated and immunogenic against measles, mumps and rubella with schedule of first dose both at 8 months and 12 months age. Our findings strongly supported that two doses of MMR can be introduced by replacing the first dose of MR in current EPI with MMR at 8 months age and the second dose at 18 months in China.

  3. Quantitative comparison of PET performance-Siemens Biograph mCT and mMR.

    PubMed

    Karlberg, Anna M; Sæther, Oddbjørn; Eikenes, Live; Goa, Pål Erik

    2016-12-01

    Integrated clinical whole-body PET/MR systems were introduced in 2010. In order to bring this technology into clinical usage, it is of great importance to compare the performance with the well-established PET/CT. The aim of this study was to evaluate PET performance, with focus on image quality, on Siemens Biograph mMR (PET/MR) and Siemens Biograph mCT (PET/CT). A direct quantitative comparison of the performance characteristics between the mMR and mCT system was performed according to National Electrical Manufacturers Association (NEMA) NU 2-2007 protocol. Spatial resolution, sensitivity, count rate and image quality were evaluated. The evaluation was supplemented with additional standardized uptake value (SUV) measurements. The spatial resolution was similar for the two systems. Average sensitivity was higher for the mMR (13.3 kcps/MBq) compared to the mCT system (10.0 kcps/MBq). Peak noise equivalent count rate (NECR) was slightly higher for the mMR (196 kcps @ 24.4 kBq/mL) compared to the mCT (186 kcps @ 30.1 kBq/mL). Scatter fractions in the clinical activity concentration range yielded lower values for the mCT (34.9 %) compared to those for the mMR (37.0 %). Best image quality of the systems resulted in approximately the same mean hot sphere contrast and a difference of 19 percentage points (pp) in mean cold contrast, in favour of the mCT. In general, point spread function (PSF) increased hot contrast and time of flight (TOF) increased both hot and cold contrast. Highest hot contrast for the smallest sphere (10 mm) was achieved with the combination of TOF and PSF on the mCT. Lung residual error was higher for the mMR (22 %) than that for the mCT (17 %), with no effect of PSF. With TOF, lung residual error was reduced to 8 % (mCT). SUV was accurate for both systems, but PSF caused overestimations for the 13-, 17- and 22-mm spheres. Both systems proved good performance characteristics, and the PET image quality of the mMR was close to that of the m

  4. Delayed adaptive immunity is related to higher MMR vaccine-induced antibody titers in children.

    PubMed

    Strömbeck, Anna; Lundell, Anna-Carin; Nordström, Inger; Andersson, Kerstin; Adlerberth, Ingegerd; Wold, Agnes E; Rudin, Anna

    2016-04-01

    There are notable inter-individual variations in vaccine-specific antibody responses in vaccinated children. The aim of our study was to investigate whether early-life environmental factors and adaptive immune maturation prior and close to measles-mumps-rubella (MMR) immunization relate to magnitudes of vaccine-specific antibody titers. In the FARMFLORA birth cohort, including both farming and non-farming families, children were immunized with the MMR vaccine at 18 months of age. MMR vaccine-induced antibody titers were measured in plasma samples obtained at 36 months of age. Infants' blood samples obtained at birth, 3-5 days and at 4 and 18 months of age were analyzed for T- and B-cell numbers, proportions of naive and memory T and B cells, and fractions of putative regulatory T cells. Multivariate factor analyses show that higher anti-MMR antibody titers were associated with a lower degree of adaptive immune maturation, that is, lower proportions of memory T cells and a lower capacity of mononuclear cells to produce cytokines, but with higher proportions of putative regulatory T cells. Further, children born by cesarean section (CS) had significantly higher anti-measles titers than vaginally-born children; and CS was found to be associated with delayed adaptive immunity. Also, girls presented with significantly higher anti-mumps and anti-rubella antibody levels than boys at 36 months of age. These results indicate that delayed adaptive immune maturation before and in close proximity to immunization seems to be advantageous for the ability of children to respond with higher anti-MMR antibody levels after vaccination.

  5. Delayed adaptive immunity is related to higher MMR vaccine-induced antibody titers in children

    PubMed Central

    Strömbeck, Anna; Lundell, Anna-Carin; Nordström, Inger; Andersson, Kerstin; Adlerberth, Ingegerd; Wold, Agnes E; Rudin, Anna

    2016-01-01

    There are notable inter-individual variations in vaccine-specific antibody responses in vaccinated children. The aim of our study was to investigate whether early-life environmental factors and adaptive immune maturation prior and close to measles–mumps–rubella (MMR) immunization relate to magnitudes of vaccine-specific antibody titers. In the FARMFLORA birth cohort, including both farming and non-farming families, children were immunized with the MMR vaccine at 18 months of age. MMR vaccine-induced antibody titers were measured in plasma samples obtained at 36 months of age. Infants' blood samples obtained at birth, 3–5 days and at 4 and 18 months of age were analyzed for T- and B-cell numbers, proportions of naive and memory T and B cells, and fractions of putative regulatory T cells. Multivariate factor analyses show that higher anti-MMR antibody titers were associated with a lower degree of adaptive immune maturation, that is, lower proportions of memory T cells and a lower capacity of mononuclear cells to produce cytokines, but with higher proportions of putative regulatory T cells. Further, children born by cesarean section (CS) had significantly higher anti-measles titers than vaginally-born children; and CS was found to be associated with delayed adaptive immunity. Also, girls presented with significantly higher anti-mumps and anti-rubella antibody levels than boys at 36 months of age. These results indicate that delayed adaptive immune maturation before and in close proximity to immunization seems to be advantageous for the ability of children to respond with higher anti-MMR antibody levels after vaccination. PMID:27195118

  6. Evaluating a Web-Based MMR Decision Aid to Support Informed Decision-Making by UK Parents: A Before-and-After Feasibility Study

    ERIC Educational Resources Information Center

    Jackson, Cath; Cheater, Francine M.; Peacock, Rose; Leask, Julie; Trevena, Lyndal

    2010-01-01

    Objective: The objective of this feasibility study was to evaluate the acceptability and potential effectiveness of a web-based MMR decision aid in supporting informed decision-making for the MMR vaccine. Design: This was a prospective before-and-after evaluation. Setting: Thirty parents of children eligible for MMR vaccination were recruited from…

  7. Evaluating a Web-Based MMR Decision Aid to Support Informed Decision-Making by UK Parents: A Before-and-After Feasibility Study

    ERIC Educational Resources Information Center

    Jackson, Cath; Cheater, Francine M.; Peacock, Rose; Leask, Julie; Trevena, Lyndal

    2010-01-01

    Objective: The objective of this feasibility study was to evaluate the acceptability and potential effectiveness of a web-based MMR decision aid in supporting informed decision-making for the MMR vaccine. Design: This was a prospective before-and-after evaluation. Setting: Thirty parents of children eligible for MMR vaccination were recruited from…

  8. Association between perfluoroalkyl substance exposure and asthma and allergic disease in children as modified by MMR vaccination.

    PubMed

    Timmermann, Clara Amalie Gade; Budtz-Jørgensen, Esben; Jensen, Tina Kold; Osuna, Christa Elyse; Petersen, Maria Skaalum; Steuerwald, Ulrike; Nielsen, Flemming; Poulsen, Lars K; Weihe, Pál; Grandjean, Philippe

    2017-12-01

    Perfluoroalkyl substances (PFASs) are highly persistent chemicals that might be associated with asthma and allergy, but the associations remain unclear. Therefore, this study examined whether pre- and postnatal PFAS exposure was associated with childhood asthma and allergy. Measles, mumps, and rubella (MMR) vaccination in early life may have a protective effect against asthma and allergy, and MMR vaccination is therefore taken into account when evaluating these associations. In a cohort of Faroese children whose mothers were recruited during pregnancy, serum concentrations of five PFASs - Perfluorohexane sulfonic acid (PFHxS), perfluorooctane sulfonic acid (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and perfluorodecanoic acid (PFDA) - were measured at three timepoints (maternal serum in pregnancy week 34-36 and child serum at ages 5 and 13 years) and their association with immunoglobulin E (IgE) (cord blood and at age 7 years) and asthma/allergic diseases (questionnaires at ages 5 and 13 years and skin prick test at age 13 years) was determined. A total of 559 children were included in the analyses. Interactions with MMR vaccination were evaluated. Among 22 MMR-unvaccinated children, higher levels of the five PFASs at age 5 years were associated with increased odds of asthma at ages 5 and 13. The associations were reversed among MMR-vaccinated children. Prenatal PFAS exposure was not associated with childhood asthma or allergic diseases regardless of MMR vaccination status. In conclusion, PFAS exposure at age 5 was associated with increased risk of asthma among a small subgroup of MMR-unvaccinated children but not among MMR-vaccinated children. While PFAS exposure may impact immune system functions, this study suggests that MMR vaccination might be a potential effect-modifier.

  9. Acetylation regulates DNA repair mechanisms in human cells.

    PubMed

    Piekna-Przybylska, Dorota; Bambara, Robert A; Balakrishnan, Lata

    2016-06-02

    The p300-mediated acetylation of enzymes involved in DNA repair and replication has been previously shown to stimulate or inhibit their activities in reconstituted systems. To explore the role of acetylation on DNA repair in cells we constructed plasmid substrates carrying inactivating damages in the EGFP reporter gene, which should be repaired in cells through DNA mismatch repair (MMR) or base excision repair (BER) mechanisms. We analyzed efficiency of repair within these plasmid substrates in cells exposed to deacetylase and acetyltransferase inhibitors, and also in cells deficient in p300 acetyltransferase. Our results indicate that protein acetylation improves DNA mismatch repair in MMR-proficient HeLa cells and also in MMR-deficient HCT116 cells. Moreover, results suggest that stimulated repair of mismatches in MMR-deficient HCT116 cells is done though a strand-displacement synthesis mechanism described previously for Okazaki fragments maturation and also for the EXOI-independent pathway of MMR. Loss of p300 reduced repair of mismatches in MMR-deficient cells, but did not have evident effects on BER mechanisms, including the long patch BER pathway. Hypoacetylation of the cells in the presence of acetyltransferase inhibitor, garcinol generally reduced efficiency of BER of 8-oxoG damage, indicating that some steps in the pathway are stimulated by acetylation.

  10. Autism occurrence by MMR vaccine status among US children with older siblings with and without autism.

    PubMed

    Jain, Anjali; Marshall, Jaclyn; Buikema, Ami; Bancroft, Tim; Kelly, Jonathan P; Newschaffer, Craig J

    2015-04-21

    Despite research showing no link between the measles-mumps-rubella (MMR) vaccine and autism spectrum disorders (ASD), beliefs that the vaccine causes autism persist, leading to lower vaccination levels. Parents who already have a child with ASD may be especially wary of vaccinations. To report ASD occurrence by MMR vaccine status in a large sample of US children who have older siblings with and without ASD. A retrospective cohort study using an administrative claims database associated with a large commercial health plan. Participants included children continuously enrolled in the health plan from birth to at least 5 years of age during 2001-2012 who also had an older sibling continuously enrolled for at least 6 months between 1997 and 2012. MMR vaccine receipt (0, 1, 2 doses) between birth and 5 years of age. ASD status defined as 2 claims with a diagnosis code in any position for autistic disorder or other specified pervasive developmental disorder (PDD) including Asperger syndrome, or unspecified PDD (International Classification of Diseases, Ninth Revision, Clinical Modification 299.0x, 299.8x, 299.9x). Of 95,727 children with older siblings, 994 (1.04%) were diagnosed with ASD and 1929 (2.01%) had an older sibling with ASD. Of those with older siblings with ASD, 134 (6.9%) had ASD, vs 860 (0.9%) children with unaffected siblings (P < .001). MMR vaccination rates (≥1 dose) were 84% (n = 78,564) at age 2 years and 92% (n = 86,063) at age 5 years for children with unaffected older siblings, vs 73% (n = 1409) at age 2 years and 86% (n = 1660) at age 5 years for children with affected siblings. MMR vaccine receipt was not associated with an increased risk of ASD at any age. For children with older siblings with ASD, at age 2, the adjusted relative risk (RR) of ASD for 1 dose of MMR vaccine vs no vaccine was 0.76 (95% CI, 0.49-1.18; P = .22), and at age 5, the RR of ASD for 2 doses compared with no vaccine was 0.56 (95% CI, 0.31-1.01; P

  11. What Every Behavior Analyst Should Know About the “MMR Causes Autism” Hypothesis

    PubMed Central

    Ahearn, William H

    2010-01-01

    In 1998, the English physician Andrew Wakefield suggested that the MMR vaccine insults the guts of children who then regress developmentally and become autistic. Although his research did not provide firm evidence for this hypothesis, many believe that (a) the MMR vaccine can cause autism; (b) children with autism typically have gastrointestinal problems; and, (c) a necessary component of treating autism is “treating the gut” through dietary restrictions. Research has subsequently shown that Wakefield's hypothesis is unquestionably false, children with autism are not more likely to have gastrointestinal problems, and there is no sound evidence that diets are a valid treatment for autism. This paper will critically review these topics. PMID:22479671

  12. The blame frame: media attribution of culpability about the MMR-autism vaccination scare.

    PubMed

    Holton, Avery; Weberling, Brooke; Clarke, Christopher E; Smith, Michael J

    2012-01-01

    Scholars have examined how news media frame events, including responsibility for causing and fixing problems, and how these frames inform public judgment. This study analyzed 281 newspaper articles about a controversial medical study linking the measles, mumps, and rubella (MMR) vaccination with autism. Given criticism of the study and its potential negative impact on vaccination rates across multiple countries, the current study examined actors to whom news media attributed blame for the MMR-vaccine association, sources used to support those attributions, and what solutions (e.g., mobilizing information), if any, were offered. This study provides unique insight by examining the evolution of these attributions over the lifetime of the controversy. Findings emphasize how news media may attribute blame in health risk communication and how that ascription plays a potentially vital role in shaping public behavior. Theoretical and practical implications are discussed.

  13. Evidence and policymaking: The introduction of MMR vaccine in the Netherlands

    PubMed Central

    Blume, Stuart; Tump, Janneke

    2010-01-01

    Based on a case-study of the introduction of measles-mumps-rubella (MMR) vaccine in the Netherlands two decades ago, using documentary and archival sources, this paper examines the way evidence is used in policymaking. Starting from the question of ‘what counts as evidence’, two central claims are developed. First, the decision to introduce MMR was not one but a series of decisions going back at least seven years, over the course of which the significance attached to various forms of evidence changed. Second, results of international studies were coming gradually to be of greater significance than evidence gathered from within the Netherlands itself. These developments had, and continue to have, major consequences for national scientific competences. PMID:20667640

  14. Measles, the media, and MMR: Impact of the 2014-15 measles outbreak.

    PubMed

    Cataldi, Jessica R; Dempsey, Amanda F; O'Leary, Sean T

    2016-12-07

    In late 2014, a measles outbreak beginning in California received significant media attention. To better understand the impact of this outbreak, we conducted a survey to assess and compare among vaccine hesitant and non-hesitant new mothers how this outbreak affected vaccine knowledge, attitudes, vaccination plans, and media use. A cross-sectional email survey of English-speaking women with a child ⩽1year old using a convenience sample of women from nine obstetrics and gynecology (OB/GYN) practices in Colorado assessed vaccine hesitancy, knowledge and attitudes about MMR vaccines and the outbreak, MMR vaccination plans before and after the outbreak, and use of and trust for media sources related to the outbreak. The response rate was 50% (351/701). Knowledge about the outbreak was high and vaccination attitudes were mostly favorable. Forty-eight percent of respondents thought MMR vaccine was more important after the outbreak. Online news (76%), television news (75%), and social media (68%) were the most frequently used media sources, yet were highly trusted by only 18%, 22%, and 1% of respondents respectively. Government websites (34%) and information from a doctor's office (34%) were infrequently used, but were highly trusted by 62% and 60% of respondents. Knowledge of the outbreak was lower among vaccine-hesitant respondents. Few mothers changed MMR vaccination plans after the outbreak. New mothers had high levels of knowledge and favorable attitudes about vaccination after the 2014-15 measles outbreak. Media sources used the most are not the most trusted. Communication about outbreaks of vaccine-preventable diseases should include spread of accurate information to new media sources and strengthening of existing trust in traditional media. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. A New Approach to Nuclear Power The Multi-Module Reactor (MMR) Concept

    SciTech Connect

    Vernon, Milton E.

    2002-07-01

    While fuel cost for nuclear power is incredibly low relative to fossil fuel, the capital investment needed to build today's nuclear power plant is substantial. Utilities are reluctant to build new nuclear power plants because of the long construction time and the associated uncertainty of investment recovery. This paper introduces a new modular reactor concept, the Multi-Module Reactor (MMR), that reduces both the construction cost and time in an attempt to renew commercial interest in nuclear power. (authors)

  16. Factors associated with poor adherence to MMR vaccination in parents who follow vaccination schedule.

    PubMed

    Restivo, Vincenzo; Napoli, Giuseppe; Marsala, Maria Grazia Laura; Bonanno, Valentina; Sciuto, Valentina; Amodio, Emanuele; Calamusa, Giuseppe; Vitale, Francesco; Firenze, Alberto

    2015-01-01

    Due to median vaccination coverage far from elimination level, Italy is still an European country with high number of measles cases per million of people. In this study we explored potential socioeconomic, medical and demographic factors which could influence the propensity of family members for measles vaccination schedule. A cross-sectional study was performed through a questionnaire administered to the parents of children who received the first dose of MMR vaccine in two different vaccination centers in the Palermo area from November 2012 to May 2013. Overall, the role played by internet (OR 19.8 P = 0.001) and the large number of children in a family (OR 7.3 P ≤ 0.001) were the factors more associated to be unvaccinated, whereas the birth order of the child (OR 0.3 P = < 0.05 for the oldest children vs. the closer young one) and reporting a lack of MMR vaccination as a "personal decision" (OR 0.19 P ≤ 0.01) inversely correlated with the risk of quitting vaccination. These findings can be useful for a better knowledge of disaffection to vaccination practice in local settings and could contribute to improve and maintain timely uptake, suggesting approaches to optimize the uptake of MMR tailored to the needs of local populations.

  17. [Lack of association between MMR vaccination and the incidence of autism in children: a case-control study].

    PubMed

    Mrozek-Budzyn, Dorota; Kiełtyka, Agnieszka; Majewska, Renata

    2009-01-01

    The matched case-control study has been undertook to investigate whether measles, mumps, and rubella (MMR) vaccine may be casually associated with autism in children. Cases were children to 14-year old with diagnosis of core autism or atypical autism. Controls were matched on age, sex and general practice. The 96 cases and 192 controls were included. The study provides strong evidence against association of autism with both MMR and a single measles individual vaccine. Additionally children vaccinated with MMR, regardless of age of vaccination (to 18th, 24th and 36th month of life), had risk equal half of that of single measles vaccinated (for vaccinated to 18th month OR=0.41 95%PU: 0.20-0.85). Our findings confirm that MMR vaccination is not associated with an increased risk of autism in children.

  18. Healthcare workers and measles-mumps-rubella (MMR) status: how worried should we be about further outbreaks?

    PubMed

    Basu, S; Giri, P; Adisesh, A; McNAUGHT, R

    2014-08-01

    Recently, a number of outbreaks of measles and mumps have occurred within the UK and Europe. Healthcare workers (HCWs) are at risk of contracting and transmitting disease to patients and staff. To examine this risk at the point of entry to healthcare, we assessed the serological results of new HCWs presenting for pre-placement clearance without evidence of measles-mumps-rubella (MMR) immunity between 1 April 2010 and 31 March 2012. Overall rates of serological positivity to MMR across all age groups were 88·2%, 68·8% and 93·9%, respectively. With regard to measles and mumps, there were statistically significant decreases in the percentage of HCWs born after 1980 that had positive serology (P < 0·05). No such differences were seen between healthcare groups. Most seronegative HCWs accepted MMR vaccination. Despite our entry-level findings, the ongoing risk of a MMR outbreak within this cohort of HCWs appears low.

  19. Mismatch repair gene MLH3 Pro844Leu and Thr942Ile polymorphisms and the susceptibility to cervical carcinoma and HPV infection: a case-control study in a Chinese population.

    PubMed

    Ye, Feng; Cheng, Qi; Shen, Jiajie; Zhou, Caiyun; Chen, Huaizeng

    2014-01-01

    To investigate the association between MLH3 Pro844Leu, Thr942Ile polymorphisms and potential linkage with the risk of cervical carcinoma and potential effect on protein function, we carried out a case-control study with 400 cervical squamous cell carcinoma, 400 CIN3 and 1200 normal controls in a Chinese population. The results showed that there was an increased risk of cervical carcinoma and CIN3 associated with the genotype 844CT [OR 2.17 (1.61-2.94); P<0.001; OR 1.49 (1.08-2.07), P 0.017, respectively] and a decreased risk with the 942CT genotype [OR 0.56 (0.38-0.82); P<0.001; OR 0.37 (0.24-0.58), P<0.001, respectively]. Most 844CT genotypes were linkage CT(844)-CC(942), which increased the risk of cervical carcinoma and CIN3 [77/83, OR 2.04 (1.48-2.80), P<0.001; 55/61, OR 1.46 (1.03-2.06), P 0.035, respectively]. Most 942CT were linkage CC(844)-CT(942), which decreased the risk of cervical carcinoma [29/35, OR 0.60 (0.40-0.91); P 0.017; 18/24, OR 0.33 (0.20-0.55), P<0.001, respectively]. In some grouping, the 844CT and 942CT were further enriched; especially HR-HPV-positive subjects both in the CIN3 and the cervical carcinoma, the 844CT had greater enrichment. These results included that CT(844)-CC(942) was associated with a high risk of cervical carcinoma and CIN3, and the CC(844)-CT(942) decreased the risk. The 844CT had a higher level of enrichment in HR-HPV positive individuals, which is probably related to HR-HPV susceptibility. There was no significant difference of the MLH3 mRNA expression and these two amino acid substitutions did not impact on the protein function.

  20. Mismatch Repair Gene MLH3 Pro844Leu and Thr942Ile Polymorphisms and the Susceptibility to Cervical Carcinoma and HPV Infection: A Case-Control Study in a Chinese Population

    PubMed Central

    Ye, Feng; Cheng, Qi; Shen, Jiajie; Zhou, Caiyun; Chen, Huaizeng

    2014-01-01

    To investigate the association between MLH3 Pro844Leu, Thr942Ile polymorphisms and potential linkage with the risk of cervical carcinoma and potential effect on protein function, we carried out a case-control study with 400 cervical squamous cell carcinoma, 400 CIN3 and 1200 normal controls in a Chinese population. The results showed that there was an increased risk of cervical carcinoma and CIN3 associated with the genotype 844CT [OR 2.17 (1.61–2.94); P<0.001; OR 1.49 (1.08–2.07), P 0.017, respectively] and a decreased risk with the 942CT genotype [OR 0.56 (0.38–0.82); P<0.001; OR 0.37 (0.24–0.58), P<0.001, respectively]. Most 844CT genotypes were linkage CT(844)-CC(942), which increased the risk of cervical carcinoma and CIN3 [77/83, OR 2.04 (1.48–2.80), P<0.001; 55/61, OR 1.46 (1.03–2.06), P 0.035, respectively]. Most 942CT were linkage CC(844)-CT(942), which decreased the risk of cervical carcinoma [29/35, OR 0.60 (0.40–0.91); P 0.017; 18/24, OR 0.33 (0.20–0.55), P<0.001, respectively]. In some grouping, the 844CT and 942CT were further enriched; especially HR-HPV-positive subjects both in the CIN3 and the cervical carcinoma, the 844CT had greater enrichment. These results included that CT(844)-CC(942) was associated with a high risk of cervical carcinoma and CIN3, and the CC(844)-CT(942) decreased the risk. The 844CT had a higher level of enrichment in HR-HPV positive individuals, which is probably related to HR-HPV susceptibility. There was no significant difference of the MLH3 mRNA expression and these two amino acid substitutions did not impact on the protein function. PMID:24759751

  1. Application of a five-tiered scheme for standardized classification of 2,360 unique mismatch repair gene variants lodged on the InSiGHT locus-specific database

    PubMed Central

    Plazzer, John-Paul; Greenblatt, Marc S.; Akagi, Kiwamu; Al-Mulla, Fahd; Bapat, Bharati; Bernstein, Inge; Capellá, Gabriel; den Dunnen, Johan T.; du Sart, Desiree; Fabre, Aurelie; Farrell, Michael P.; Farrington, Susan M.; Frayling, Ian M.; Frebourg, Thierry; Goldgar, David E.; Heinen, Christopher D.; Holinski-Feder, Elke; Kohonen-Corish, Maija; Robinson, Kristina Lagerstedt; Leung, Suet Yi; Martins, Alexandra; Moller, Pal; Morak, Monika; Nystrom, Minna; Peltomaki, Paivi; Pineda, Marta; Qi, Ming; Ramesar, Rajkumar; Rasmussen, Lene Juel; Royer-Pokora, Brigitte; Scott, Rodney J.; Sijmons, Rolf; Tavtigian, Sean V.; Tops, Carli M.; Weber, Thomas; Wijnen, Juul; Woods, Michael O.; Macrae, Finlay; Genuardi, Maurizio

    2015-01-01

    Clinical classification of sequence variants identified in hereditary disease genes directly affects clinical management of patients and their relatives. The International Society for Gastrointestinal Hereditary Tumours (InSiGHT) undertook a collaborative effort to develop, test and apply a standardized classification scheme to constitutional variants in the Lynch Syndrome genes MLH1, MSH2, MSH6 and PMS2. Unpublished data submission was encouraged to assist variant classification, and recognized by microattribution. The scheme was refined by multidisciplinary expert committee review of clinical and functional data available for variants, applied to 2,360 sequence alterations, and disseminated online. Assessment using validated criteria altered classifications for 66% of 12,006 database entries. Clinical recommendations based on transparent evaluation are now possible for 1,370 variants not obviously protein-truncating from nomenclature. This large-scale endeavor will facilitate consistent management of suspected Lynch Syndrome families, and demonstrates the value of multidisciplinary collaboration for curation and classification of variants in public locus-specific databases. PMID:24362816

  2. Does the relative importance of MMR vaccine concerns differ by degree of parental vaccine hesitancy?: An exploratory study.

    PubMed

    Gowda, Charitha; Schaffer, Sarah E; Kopec, Kristin; Markel, Arielle; Dempsey, Amanda F

    2013-02-01

    There has been a rise in the number of vaccine-hesitant parents (VHPs) in the US, many of whom express reservations about administering the MMR vaccine to their children. We studied the relative importance of attitudinal barriers to MMR vaccination among VHPs with differing levels of MMR vaccine-hesitancy. We performed a cross-sectional exploratory analysis of a parental survey that assessed common vaccination barriers among MMR vaccine-hesitant parents in Michigan. The outcome of interest was parental MMR vaccination intention, measured on an 11-point scale, with higher numbers corresponding to greater intent. The relative importance of identified barriers to MMR vaccination was assessed across levels of vaccine hesitancy. Exploratory factor analysis was performed to identify underlying attitudinal constructs and assess if these constructs' importance varied depending on the degree of parental vaccine hesitancy. Our study population included 79 Michigan parents who initially screened positive for MMR vaccine-hesitancy. Within this sample, 47% of parents were unsure about their vaccination intentions and 20% expressed negative intentions, while a third (33%) of parents had positive vaccination intentions when further questioned. After grouping the barriers in our study into four underlying factors, parents with negative vaccination intentions had statistically significant higher factor score for the factor "risks versus benefits" and a statistically significant lower mean score for "vaccine importance," compared with parents with unsure or positive intentions. In this exploratory study we found that vaccine-specific concerns have varying salience for parents based on their vaccination intention. Thus, future educational programs likely should tailor messages based on the degree of vaccine hesitancy expressed in their target populations in order to improve their overall effectiveness.

  3. What are parents' perspectives on psychological empowerment in the MMR vaccination decision? A focus group study

    PubMed Central

    Fadda, Marta; Galimberti, Elisa; Carraro, Valter; Schulz, Peter J

    2016-01-01

    Objectives Most developed countries do not have compulsory immunisation requirements, but instead issue recommendations. Although parents are expected to make an informed, autonomous (ie, empowered) decision regarding their children's vaccinations, there is no evidence about how parents' interpret this demand nor on the latitude of their decision-making. The goal of this study is to gain insights from parents residing in a low measles-mumps-rubella (MMR) uptake area on what constitutes feelings of empowerment in the decision they have to make on their child's MMR vaccination. Design A qualitative study employing focus group interviews. Setting 11 vaccination centres and hospitals in the Province of Trento, Italy. Participants 24 mothers and 4 fathers of children for whom the MMR vaccination decision was still pending participated in 6 focus groups. Results Autonomy and competence were salient themes in relation to empowerment, and were further connected with beliefs regarding legal responsibility and ethics of freedom concerning the decision, parents' relationship with the paediatrician (trust), feelings of relevance of the decision and related stress, and seeking, avoidance, or fear of vaccination-related information. Competence was interpreted as medical knowledge and information-seeking skills, but it was also related to the extent parents perceived the paediatrician to be competent. Conclusions Since parents' interpretation of empowerment goes beyond mere perceptions of being informed and autonomous and differs across individuals, it is important that this construct be correctly interpreted and implemented by best practice, for instance by explicitly adopting a relational conception of autonomy. Knowing whether parents want to make an empowered decision and what their information and autonomy needs are might help health professionals adapt their communication about immunisation, and promote parental perception of making an informed, autonomous decision. PMID

  4. What are parents' perspectives on psychological empowerment in the MMR vaccination decision? A focus group study.

    PubMed

    Fadda, Marta; Galimberti, Elisa; Carraro, Valter; Schulz, Peter J

    2016-04-15

    Most developed countries do not have compulsory immunisation requirements, but instead issue recommendations. Although parents are expected to make an informed, autonomous (ie, empowered) decision regarding their children's vaccinations, there is no evidence about how parents' interpret this demand nor on the latitude of their decision-making. The goal of this study is to gain insights from parents residing in a low measles-mumps-rubella (MMR) uptake area on what constitutes feelings of empowerment in the decision they have to make on their child's MMR vaccination. A qualitative study employing focus group interviews. 11 vaccination centres and hospitals in the Province of Trento, Italy. 24 mothers and 4 fathers of children for whom the MMR vaccination decision was still pending participated in 6 focus groups. Autonomy and competence were salient themes in relation to empowerment, and were further connected with beliefs regarding legal responsibility and ethics of freedom concerning the decision, parents' relationship with the paediatrician (trust), feelings of relevance of the decision and related stress, and seeking, avoidance, or fear of vaccination-related information. Competence was interpreted as medical knowledge and information-seeking skills, but it was also related to the extent parents perceived the paediatrician to be competent. Since parents' interpretation of empowerment goes beyond mere perceptions of being informed and autonomous and differs across individuals, it is important that this construct be correctly interpreted and implemented by best practice, for instance by explicitly adopting a relational conception of autonomy. Knowing whether parents want to make an empowered decision and what their information and autonomy needs are might help health professionals adapt their communication about immunisation, and promote parental perception of making an informed, autonomous decision. Published by the BMJ Publishing Group Limited. For permission to

  5. Booster immune response in children 6-7 years of age, randomly assigned to four groups with two MMR vaccines applied by aerosol or by injection.

    PubMed

    Díaz-Ortega, José-Luis; Bennett, John V; Castañeda-Desales, Deyanira; Quintanilla, Doris-Ma Arellano; Martínez, David; de Castro, Jorge Fernández

    2014-06-17

    Aerosol immunization may be a useful tool to reach and sustain the elimination of measles, rubella, and congenital rubella syndrome. We compared booster seroresponses to aerosolized or injected MMR vaccines containing different strains of measles (Attenuvax or Edmonston-Zagreb) and mumps (Jeryl-Lynn or Leningrad-Zagreb). To assess the safety and immunogenicity of two MMR: Vaccines administered by aerosol. A randomized and controlled clinical trial was conducted to evaluate the safety and booster responses to the MMR SII (Serum Institute of India) and MMR II (Merck Sharp & Dhome) vaccines, both of which were administered by aerosol (ae) or injection (inj) to Mexican children aged 6-7 years in elementary schools. The seroresponses were evaluated by PRN (measles) and ELISA (rubella and mumps). Adverse events were followed-up for 28 days after the immunization. Two hundred and fifty-three of 260 children completed the one-month follow-up. All participants reached protective seropositivity for measles and rubella after immunization, and 98.3 to 100% reached protective seropositivity for mumps (p=0.552). The proportions of the seroresponses (a 2-fold rise from the baseline antibody titers) to measles were 38.3% for MMR SII (ae), 31.3% for MMR II (ae), 37.5% for MMR SII (inj), and 44.6% for MMR II (inj) (p=0.483). The seroresponses for rubella were 26.7% for MMR SII (ae), 31.3% for MMR II (ae), 46.9% for MMR SII (inj), and 40.0% for MMR II (inj) (p=0.086). The seroresponse to mumps were 31.7% for MMR SII (ae), 25.0% for MMR II (ae), 48.4% for MMR SII (inj), and 53.9% for MMR II (inj) (p=0.002). The difference in the seroresponse of a 4-fold rise from the baseline antibody titers was not statistically significant. Only mild adverse events were noted. Aerosolized vaccines were as safe and as immunogenic as injected vaccines. CMN 2010-005 (National Regulatory Authority). Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Attitudinal and Demographic Predictors of Measles-Mumps-Rubella Vaccine (MMR) Uptake during the UK Catch-Up Campaign 2008–09: Cross-Sectional Survey

    PubMed Central

    Brown, Katrina; Fraser, Graham; Ramsay, Mary; Shanley, Ruth; Cowley, Noel; van Wijgerden, Johan; Toff, Penelope; Falconer, Michelle; Hudson, Michael; Green, John; Kroll, J. Simon; Vincent, Charles; Sevdalis, Nick

    2011-01-01

    Background and Objective Continued suboptimal measles-mumps-rubella (MMR) vaccine uptake has re-established measles epidemic risk, prompting a UK catch-up campaign in 2008–09 for children who missed MMR doses at scheduled age. Predictors of vaccine uptake during catch-ups are poorly understood, however evidence from routine schedule uptake suggests demographics and attitudes may be central. This work explored this hypothesis using a robust evidence-based measure. Design Cross-sectional self-administered questionnaire with objective behavioural outcome. Setting and Participants 365 UK parents, whose children were aged 5–18 years and had received <2 MMR doses before the 2008–09 UK catch-up started. Main Outcome Measures Parents' attitudes and demographics, parent-reported receipt of invitation to receive catch-up MMR dose(s), and catch-up MMR uptake according to child's medical record (receipt of MMR doses during year 1 of the catch-up). Results Perceived social desirability/benefit of MMR uptake (OR = 1.76, 95% CI = 1.09–2.87) and younger child age (OR = 0.78, 95% CI = 0.68–0.89) were the only independent predictors of catch-up MMR uptake in the sample overall. Uptake predictors differed by whether the child had received 0 MMR doses or 1 MMR dose before the catch-up. Receipt of catch-up invitation predicted uptake only in the 0 dose group (OR = 3.45, 95% CI = 1.18–10.05), whilst perceived social desirability/benefit of MMR uptake predicted uptake only in the 1 dose group (OR = 9.61, 95% CI = 2.57–35.97). Attitudes and demographics explained only 28% of MMR uptake in the 0 dose group compared with 61% in the 1 dose group. Conclusions Catch-up MMR invitations may effectively move children from 0 to 1 MMR doses (unimmunised to partially immunised), whilst attitudinal interventions highlighting social benefits of MMR may effectively move children from 1 to 2 MMR doses (partially to fully immunised). Older children may be

  7. Mismatch Repair Balances Leading and Lagging Strand DNA Replication Fidelity

    DTIC Science & Technology

    2012-10-11

    Pol e mutator variant to confirm that Pol e is the primary leading strand replicase in Saccharomyces cerevisiae . We then use polymerase-specific error...variables using Saccharomyces cerevisiae strains containing mutant alleles of the POL1 (Pol a), POL2 (Pol e) and POL3 (Pol d) genes. These mutant...AL, Johnston LH, Sugino A (1993) Pathway correcting DNA replication errors in Saccharomyces cerevisiae . Embo J 12: 1467–1473. 17. Morrison A, Sugino A

  8. Risks and benefits of the single versus the triple MMR vaccine: how can health professionals reassure parents?

    PubMed

    McGreevy, Deborah

    2005-03-01

    Measles, mumps and rubella (MMR) are all preventable but infectious diseases caused by viruses. A particular study by Wakefield et al suggests that there are potentially adverse effects of having the triple MMR vaccine. This has been reported widely by the media and has caused alarm to parents of young children, probably contributing to the decline in its uptake. In order to provide the context for the debate regarding the single versus the triple vaccine, this paper briefly appraises firstly, the Wakefield et al research paper that has led to public health concerns and secondly, a more rigorous research study (Madsen et al) that contradicts the findings; the paper then explores the risks and benefits of the single and the triple MMR vaccine programmes, finally providing a short discussion on factors that might influence the decision-making process by parents when faced with the dilemma of not having their child vaccinated, or opting for either the single or triple vaccination programme.

  9. [The putative link between the MMR vaccine and autism and refusal to vaccinate].

    PubMed

    Segura Benedicto, Andreu

    2012-01-01

    The paper of Wakefield et al. in The Lancet, triggered a negative reaction to the MMR vaccine, even though it was just a series of cases and the association between vaccination and autism could well be anecdotal. However, it was found that this association was spurious, not only because of hidden biases but also to alterations of the data and other improper behavior of the two authors that they were expelled from medical council. Finally, the article was removed from the magazine. This episode invites to think about the credibility and trust in the authorities and professionals to the population, as well as the suspicions that may arise when there are potential conflicts of interest among professionals, industry magazines and the population. A special area of interest is on the distorted expectations of health interventions, including vaccination, particularly with regard to both individual and collective prevention. Copyright © 2011 SESPAS. Published by Elsevier España, S.L. All rights reserved.

  10. Reasons for measles cases not being vaccinated with MMR: investigation into parents' and carers' views following a large measles outbreak.

    PubMed

    McHale, P; Keenan, A; Ghebrehewet, S

    2016-03-01

    Uptake rates for the combined measles, mumps and rubella (MMR) vaccine have been below the required 95% in the UK since a retracted and discredited article linking the MMR vaccine with autism and inflammatory bowel disease was released in 1998. This study undertook semi-structured telephone interviews among parents or carers of 47 unvaccinated measles cases who were aged between 13 months and 9 years, during a large measles outbreak in Merseyside. Results showed that concerns over the specific links with autism remain an important cause of refusal to vaccinate, with over half of respondents stating this as a reason. A quarter stated child illness during scheduled vaccination time, while other reasons included general safety concerns and access issues. Over half of respondents felt that more information or a discussion with a health professional would help the decision-making process, while a third stated improved access. There was clear support for vaccination among respondents when asked about current opinions regarding MMR vaccine. The findings support the hypothesis that safety concerns remain a major barrier to MMR vaccination, and also support previous evidence that experience of measles is an important determinant in the decision to vaccinate.

  11. Persistence of measles antibodies, following changes in the recommended age for the second dose of MMR-vaccine in Portugal.

    PubMed

    Gonçalves, Guilherme; Frade, João; Nunes, Carla; Mesquita, João Rodrigo; Nascimento, Maria São José

    2015-09-22

    In populations vaccinated with two doses of combined measles-mumps-rubella vaccine (MMR), the serum levels of antibodies against measles depend on the vaccination schedule, time elapsed from the last dose and the area-specific epidemiological situation. Variables measuring "schedule" are age at first and second doses of MMR and intervals derived from that. Changes in vaccination schedules have been made in Portugal. The specific objectives of this study were to measure the association between those potential determinants and the concentration of measles-specific IgG antibodies, after the second dose of MMR. Convenience samples of three Portuguese birth cohorts were selected for this study (41, 66 and 60 born, respectively, in 2001-2003, 1990-1993 and 1994-1995). Geometric mean concentrations (GMC) for measles IgG were, respectively, 934, 251 and 144mIU/ml; p<0.001). Anti-measles-IgG serum concentration decreased with time since last vaccination (waning immunity) and was not influenced by any other component of vaccination schedule, namely age at vaccination with the second dose of MMR. Waning levels of measles antibodies have been observed elsewhere but not as fast as it was observed in Portuguese birth cohorts in this study. Changes in the vaccination schedules might have to be considered in the future.

  12. iPE-MMR: An integrated approach to accurately assign monoisotopic precursor masses to tandem mass spectrometric data

    PubMed Central

    Jung, Hee-Jung; Purvine, Samuel O.; Kim, Hokeun; Petyuk, Vladislav A.; Hyung, Seok-Won; Monroe, Matthew E.; Mun, Dong-Gi; Kim, Kyong-Chul; Park, Jong-Moon; Kim, Su-Jin; Tolic, Nikola; Slysz, Gordon W.; Moore, Ronald J.; Zhao, Rui; Adkins, Joshua N.; Anderson, Gordon A.; Lee, Hookeun; Camp, David G.; Yu, Myeong-Hee; Smith, Richard D.; Lee, Sang-Won

    2010-01-01

    Accurate assignment of monoisotopic precursor masses to tandem mass spectrometric (MS/MS) data is a fundamental and critically important step for successful peptide identifications in mass spectrometry based proteomics. Here we describe an integrated approach that combines three previously reported methods of treating MS/MS data for precursor mass refinement. This combined method, “integrated Post-Experiment Monoisotopic Mass Refinement” (iPE-MMR), integrates steps: 1) generation of refined MS/MS data by DeconMSn; 2) additional refinement of the resultant MS/MS data by a modified version of PE-MMR; 3) elimination of systematic errors of precursor masses using DtaRefinery. iPE-MMR is the first method that utilizes all MS information from multiple MS scans of a precursor ion including multiple charge states, in an MS scan, to determine precursor mass. By combining these methods, iPE-MMR increases sensitivity in peptide identification and provides increased accuracy when applied to complex high-throughput proteomics data. PMID:20863060

  13. Cross-calibration of the Siemens mMR: easily acquired accurate PET phantom measurements, long-term stability and reproducibility.

    PubMed

    Keller, Sune H; Jakoby, Björn; Svalling, Susanne; Kjaer, Andreas; Højgaard, Liselotte; Klausen, Thomas L

    2016-12-01

    We present a quick and easy method to perform quantitatively accurate PET scans of typical water-filled PET plastic shell phantoms on the Siemens Biograph mMR PET/MR system. We perform regular cross-calibrations (Xcal) of our PET systems, including the PET/MR, using a Siemens mCT water phantom. The mMR calibration stability was evaluated over a 3-year period where 54 cross-calibrations were acquired, showing that the mMR on average underestimated the concentration by 16 %, consistently due to the use of MR-based μ-maps. The mMR produced the narrowest calibration ratio range with the lowest standard deviation, implying it is the most stable of the six systems in the study over a 3-year period. MMR ACCURACY WITH PREDEFINED μ-MAPS: With the latest mMR software version, VB20P, it is possible to utilize predefined phantom μ-maps. We evaluated both the system-integrated, predefined μ-map of the long mMR water phantom and our own user-defined CT-based μ-map of the mCT water phantom, which is used for cross-calibration. For seven scans, which were reconstructed with correctly segmented μ-maps, the mMR produced cross-calibration ratios of 1.00-1.02, well within the acceptance range [0.95-1.05], showing high accuracy. The mMR is the most stable PET system in this study, and the mean underestimation is no longer an issue with the easily accessible μ-map, which resulted in correct cross-calibration ratios in all seven tests. We will share the user-defined μ-map of the mCT phantom and the protocol with interested mMR users.

  14. Reproductive Decision-Making in MMR Mutation Carriers After Results Disclosure: Impact of Psychological Status in Childbearing Options.

    PubMed

    Duffour, Jacqueline; Combes, Audrey; Crapez, Evelyne; Boissière-Michot, Florence; Bibeau, Frédéric; Senesse, Pierre; Ychou, Marc; Courraud, Julie; de Forges, Hélène; Roca, Lise

    2016-06-01

    Reproductive techniques such as prenatal diagnosis (PND) or preimplantation genetic diagnosis (PGD), although debated, are legally forbidden in France in case of Lynch syndrome. The preference of mutation carriers about their reproductive options is not systematically considered in France. We aimed to prospectively assess the reproductive preferences of mismatch repair mutation carriers consulting in our institution (2003-2010, n = 100). We also considered the short- and long-term post-disclosure psychological impact using the Impact of Events Scale-Revised questionnaire to measure the prevalence of posttraumatic stress disorder (PTSD) in those patients. Complete data were obtained for 34 respondents (17 males, 17 females, median age of 33.5 years [22-59]). Seventeen respondents (57 %) preferred spontaneous natural conception versus 28 % and 35 % choosing PND and PGD, respectively. At results disclosure, respondents mainly explained their distress by fear of premature death (43 %) and transmitting mutated genes (42 %). One year later, this last fear remained predominant in 55 % of subjects. None of the main socio-demographical, psychological or medical variables (including fear of transmitting mutations) was significantly associated with the reproductive preferences. Results disclosure had a real and time-decreasing psychological impact on mutation carriers. Reproductive techniques, expected to decrease the hereditary risk, were not significantly preferred to natural conception.

  15. Guanine- 5-carboxylcytosine base pairs mimic mismatches during DNA replication.

    PubMed

    Shibutani, Toshihiro; Ito, Shinsuke; Toda, Mariko; Kanao, Rie; Collins, Leonard B; Shibata, Marika; Urabe, Miho; Koseki, Haruhiko; Masuda, Yuji; Swenberg, James A; Masutani, Chikahide; Hanaoka, Fumio; Iwai, Shigenori; Kuraoka, Isao

    2014-06-09

    The genetic information encoded in genomes must be faithfully replicated and transmitted to daughter cells. The recent discovery of consecutive DNA conversions by TET family proteins of 5-methylcytosine into 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine (5caC) suggests these modified cytosines act as DNA lesions, which could threaten genome integrity. Here, we have shown that although 5caC pairs with guanine during DNA replication in vitro, G·5caC pairs stimulated DNA polymerase exonuclease activity and were recognized by the mismatch repair (MMR) proteins. Knockdown of thymine DNA glycosylase increased 5caC in genome, affected cell proliferation via MMR, indicating MMR is a novel reader for 5caC. These results suggest the epigenetic modification products of 5caC behave as DNA lesions.

  16. Dilemmas of a vitalizing vaccine market: lessons from the MMR vaccine/autism debate.

    PubMed

    Bragesjö, Fredrik; Hallberg, Margareta

    2011-03-01

    A number of issues related to vaccines and vaccinations in society are discussed in this paper. Our purpose is to merge an analysis of some recent changes in the vaccine market with social science research on the relationship between citizens and authorities. The article has two empirical parts. The first shows how the vaccine market, which for many years has had immense financial problems, nowadays seems to becoming economically vitalized, mostly due to the production of new and profitable vaccines. However prosperous the future may appear, certain reactions from the public regarding vaccination initiatives offer insight into inherent problems of vaccine policies in many Western countries. In the second part of the article, these problems are exemplified with the recent controversy over the MMR (measles, mumps, and rubella) vaccine. We conclude that in spite of the improving profit-margins, the vaccine market remains vulnerable and insecure. Vaccines are permeated by society, even more so than pharmaceutics that are used to cure or alleviate illnesses. Radical changes in financial conditions with promises of a more profitable market will not, we argue, solve other even more fundamental problems.

  17. Using electronic mail to improve MMR uptake amongst third level students.

    PubMed

    Cooney, F; Ryan, A; Schinaia, N; Breslin, A

    2010-03-01

    This study assessed the usefulness of email in informing third level students about special MMR clinics being provided on campus during a mumps outbreak in the North West of Ireland. Email messages were sent directly to students, informing them of the clinics, inviting them to make a clinic appointment by email and providing details of walk-in clinics. At the clinics, all 177 attendees were asked to fill out a questionnaire and the response rate was 89% (n=158). Regarding the main sources of information about the vaccination clinics, email was selected by 117 (74%) students, word-of-mouth by 27 (17%), posters/leaflets by 8 (5%), and other sources by 6 (4%). Use of email as a source of information was rated as very good/excellent by 115 (73%), as good by 35 (22%) and poor/fair by 8 (5%). This study demonstrates that email is a useful and acceptable way of informing third level students about immunisation clinics in an outbreak situation.

  18. 'That's just what's expected of you … so you do it': mothers discussions around choice and the MMR vaccination.

    PubMed

    Johnson, Sally; Capdevila, Rose

    2014-01-01

    One of the major shifts in the form and experience of contemporary family life has been the increasing insertion of the 'expert' voice into the relationship between parents and children. This paper focuses on an exploration of mothers' engagement with advice around the combined measles, mumps and rubella (MMR) vaccine. Much of the previous literature utilises a 'decision-making' framework, based on 'risk assessment' whereby mothers' decisions are conceptualised as rooted in complex belief systems, and supposes that that by gaining an understanding of these systems, beliefs and behaviour can be modified and uptake improved. However, less attention has been paid to the ways in which mothers negotiate such advice or the ways in which advice is mediated by positionings, practices and relationships. Analysis of data from a focus group with five mothers identified three themes: (i) Sourcing advice and information, (ii) Constructing 'Mother knows best' and (iii) Negotiating agency. Despite the trustworthiness of advice and information being questioned, an awareness of concerns about the MMR, and health professionals being constructed as remote, ultimate conformity to, and compliance with, the 'system' and 'society' were described as determining MMR 'decisions'.

  19. Antibody persistence in children aged 6-7years one year following booster immunization with two MMR vaccines applied by aerosol or by injection.

    PubMed

    Díaz Ortega, José Luis; Castaneda, Deyanira; Arellano Quintanilla, Doris Ma; Martínez, David; Trumbo, Silas P; Fernández de Castro, Jorge

    2017-05-25

    In a previous study on booster vaccination, we reported that two aerosolized MMR vaccines were as safe and immunogenic as injectable vaccines containing the same antigens. We now present results of antibody persistence one year after immunization. To assess the antibody persistence for measles, mumps, and rubella one year following booster immunization. We performed clinical and serological follow-up of participants in a previous study of Mexican children aged 6-7years, in which participants were randomized to four groups receiving, by aerosolized or by injection, the MMR SII vaccine (Serum Institute of India), or the MMR II (Merck Sharp & Dhome). We evaluated the antibody persistence by PRN test for measles and by ELISA for rubella and mumps. The occurrence of clinical events was evaluated via periodic visits of a nurse team to children's schools and homes. Of the 260 initial participants, 241 completed one-year follow-up. There were only statistically significant differences in baseline seropositivity for mumps. One year after immunization, seropositivity in all groups was 100% for measles and rubella. The seropositivity rank for mumps was from 90.3% for the injected vaccine MMR II to 96.6% for vaccine MMR SII applied by aerosol; these differences were not statistically significant. With exception of the aerosolized vaccine MMR SII for the geometric mean titer (GMT) for measles, all study groups presented declination of GMT for the three viruses. The difference between the aerosolized vaccines MMR SII and MMR RII was statistically significant for mumps antibodies. Only mild clinical events were identified. Under conditions of no endemic transmission for measles and rubella, and of low circulation of mumps virus, school-aged children remained seropositive to the three viruses one year following booster immunization. The study was registered under CMN 2010-005 number at COFEPRIS (National Regulatory Authority). Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. MutS Homologues hMSH4 and hMSH5: Genetic Variations, Functions, and Implications in Human Diseases.

    PubMed

    Clark, Nicole; Wu, Xiling; Her, Chengtao

    2013-04-01

    The prominence of the human mismatch repair (MMR) pathway is clearly reflected by the causal link between MMR gene mutations and the occurrence of Lynch syndrome (or HNPCC). The MMR family of proteins also carries out a plethora of diverse cellular functions beyond its primary role in MMR and homologous recombination. In fact, members of the MMR family of proteins are being increasingly recognized as critical mediators between DNA damage repair and cell survival. Thus, a better functional understanding of MMR proteins will undoubtedly aid the development of strategies to effectively enhance apoptotic signaling in response to DNA damage induced by anti-cancer therapeutics. Among the five known human MutS homologs, hMSH4 and hMSH5 form a unique heterocomplex. However, the expression profiles of the two genes are not correlated in a number of cell types, suggesting that they may function independently as well. Consistent with this, these two proteins are promiscuous and thought to play distinct roles through interacting with different binding partners. Here, we describe the gene and protein structures of eukaryotic MSH4 and MSH5 with a particular emphasis on their human homologues, and we discuss recent findings of the roles of these two genes in DNA damage response and repair. Finally, we delineate the potential links of single nucleotide polymorphism (SNP) loci of these two genes with several human diseases.

  1. MutS Homologues hMSH4 and hMSH5: Genetic Variations, Functions, and Implications in Human Diseases

    PubMed Central

    Clark, Nicole; Wu, Xiling; Her, Chengtao

    2013-01-01

    The prominence of the human mismatch repair (MMR) pathway is clearly reflected by the causal link between MMR gene mutations and the occurrence of Lynch syndrome (or HNPCC). The MMR family of proteins also carries out a plethora of diverse cellular functions beyond its primary role in MMR and homologous recombination. In fact, members of the MMR family of proteins are being increasingly recognized as critical mediators between DNA damage repair and cell survival. Thus, a better functional understanding of MMR proteins will undoubtedly aid the development of strategies to effectively enhance apoptotic signaling in response to DNA damage induced by anti-cancer therapeutics. Among the five known human MutS homologs, hMSH4 and hMSH5 form a unique heterocomplex. However, the expression profiles of the two genes are not correlated in a number of cell types, suggesting that they may function independently as well. Consistent with this, these two proteins are promiscuous and thought to play distinct roles through interacting with different binding partners. Here, we describe the gene and protein structures of eukaryotic MSH4 and MSH5 with a particular emphasis on their human homologues, and we discuss recent findings of the roles of these two genes in DNA damage response and repair. Finally, we delineate the potential links of single nucleotide polymorphism (SNP) loci of these two genes with several human diseases. PMID:24082819

  2. Missed Opportunities for Measles, Mumps, and Rubella (MMR) Immunization in Mesoamerica: Potential Impact on Coverage and Days at Risk.

    PubMed

    Mokdad, Ali H; Gagnier, Marielle C; Colson, K Ellicott; Dansereau, Emily; Zúñiga-Brenes, Paola; Ríos-Zertuche, Diego; Haakenstad, Annie; Johanns, Casey K; Palmisano, Erin B; Hernandez, Bernardo; Iriarte, Emma

    2015-01-01

    Recent outbreaks of measles in the Americas have received news and popular attention, noting the importance of vaccination to population health. To estimate the potential increase in immunization coverage and reduction in days at risk if every opportunity to vaccinate a child was used, we analyzed vaccination histories of children 11-59 months of age from large household surveys in Mesoamerica. Our study included 22,234 children aged less than 59 months in El Salvador, Guatemala, Honduras, Mexico, Nicaragua, and Panama. Child vaccination cards were used to calculate coverage of measles, mumps, and rubella (MMR) and to compute the number of days lived at risk. A child had a missed opportunity for vaccination if their card indicated a visit for vaccinations at which the child was not caught up to schedule for MMR. A Cox proportional hazards model was used to compute the hazard ratio associated with the reduction in days at risk, accounting for missed opportunities. El Salvador had the highest proportion of children with a vaccine card (91.2%) while Nicaragua had the lowest (76.5%). Card MMR coverage ranged from 44.6% in Mexico to 79.6% in Honduras while potential coverage accounting for missed opportunities ranged from 70.8% in Nicaragua to 96.4% in El Salvador. Younger children were less likely to have a missed opportunity. In Panama, children from households with higher expenditure were more likely to have a missed opportunity for MMR vaccination compared to the poorest (OR 1.62, 95% CI: 1.06-2.47). In Nicaragua, compared to children of mothers with no education, children of mothers with primary education and secondary education were less likely to have a missed opportunity (OR 0.46, 95% CI: 0.24-0.88 and OR 0.25, 95% CI: 0.096-0.65, respectively). Mean days at risk for MMR ranged from 158 in Panama to 483 in Mexico while potential days at risk ranged from 92 in Panama to 239 in El Salvador. Our study found high levels of missed opportunities for immunizing

  3. Missed Opportunities for Measles, Mumps, and Rubella (MMR) Immunization in Mesoamerica: Potential Impact on Coverage and Days at Risk

    PubMed Central

    Mokdad, Ali H.; Gagnier, Marielle C.; Colson, K. Ellicott; Dansereau, Emily; Zúñiga-Brenes, Paola; Ríos-Zertuche, Diego; Haakenstad, Annie; Johanns, Casey K.; Palmisano, Erin B.; Hernandez, Bernardo; Iriarte, Emma

    2015-01-01

    Background Recent outbreaks of measles in the Americas have received news and popular attention, noting the importance of vaccination to population health. To estimate the potential increase in immunization coverage and reduction in days at risk if every opportunity to vaccinate a child was used, we analyzed vaccination histories of children 11–59 months of age from large household surveys in Mesoamerica. Methods Our study included 22,234 children aged less than 59 months in El Salvador, Guatemala, Honduras, Mexico, Nicaragua, and Panama. Child vaccination cards were used to calculate coverage of measles, mumps, and rubella (MMR) and to compute the number of days lived at risk. A child had a missed opportunity for vaccination if their card indicated a visit for vaccinations at which the child was not caught up to schedule for MMR. A Cox proportional hazards model was used to compute the hazard ratio associated with the reduction in days at risk, accounting for missed opportunities. Results El Salvador had the highest proportion of children with a vaccine card (91.2%) while Nicaragua had the lowest (76.5%). Card MMR coverage ranged from 44.6% in Mexico to 79.6% in Honduras while potential coverage accounting for missed opportunities ranged from 70.8% in Nicaragua to 96.4% in El Salvador. Younger children were less likely to have a missed opportunity. In Panama, children from households with higher expenditure were more likely to have a missed opportunity for MMR vaccination compared to the poorest (OR 1.62, 95% CI: 1.06–2.47). In Nicaragua, compared to children of mothers with no education, children of mothers with primary education and secondary education were less likely to have a missed opportunity (OR 0.46, 95% CI: 0.24–0.88 and OR 0.25, 95% CI: 0.096–0.65, respectively). Mean days at risk for MMR ranged from 158 in Panama to 483 in Mexico while potential days at risk ranged from 92 in Panama to 239 in El Salvador. Conclusions Our study found high levels

  4. Persistence of rubella and mumps antibodies, following changes in the recommended age for the second dose of MMR vaccine in Portugal.

    PubMed

    Gonçalves, G; Frade, J; Nascimento, M S J; Mesquita, J R; Nunes, C

    2016-11-01

    In Portugal, the recommended age for the second dose of MMR (MMR2) was changed from 10-13 years to 5-6 years for those born in 1994 and afterwards. This study aimed to assess if MMR schedule and time elapsed from the last dose are associated with the concentration of rubella and mumps IgG antibodies. Three Portuguese birth cohorts (convenience samples) were selected for this study (66, 59 and 41 participants born respectively in 1990-1993, 1994-1995 and 2001-2003). Geometric mean concentrations (GMC) for mumps IgG were respectively 36, 30 and 38 RU/ml (P = 0·236) and for rubella IgG were 18, 20 and 17 IU/ml (P = 0·641). For both specific antibodies, no differences were observed with time since MMR2. Receiving MMR2 at 5-6 or 10-13 years was not associated with concentration of both antibodies. The GMC of rubella IgG was lower in males (P = 0·029). Taking into account previous evidence and the logistics needed to change vaccination schedules, it seems reasonable that sustaining very high coverage with two doses of MMR is currently the most pragmatic way to control mumps and rubella rather than any changes to the schedule.

  5. 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.

  6. Characterization of Pathogenic Human MSH2 Missense Mutations Using Yeast as a Model System: A Laboratory Course in Molecular Biology

    PubMed Central

    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. PMID:22039344

  7. Radiometric calibration of the reflective bands of NS001-Thematic Mapper Simulator (TMS) and modular multispectral radiometers (MMR)

    NASA Technical Reports Server (NTRS)

    Markham, Brian L.; Wood, Frank M., Jr.; Ahmad, Suraiya P.

    1988-01-01

    The NS001 Thematic Mapper Simulator scanner (TMS) and several modular multispectral radiometers (MMRs) are among the primary instruments used in the First ISLSCP (International Satellite Land Surface Climatology Project) Field Experiment (FIFE). The NS001 has a continuously variable gain setting. Calibration of the NS001 data is influenced by drift in the dark current level of up to six counts during a mirror scan at typical gain settings. The MMR instruments are being used in their 1 deg FOV configuration on the helicopter and 15 deg FOV on the ground.

  8. Transient suppression of MLH1 allows effective single-nucleotide substitution by single-stranded DNA oligonucleotides.

    PubMed

    Dekker, Marleen; de Vries, Sandra; Aarts, Marieke; Dekker, Robert; Brouwers, Conny; Wiebenga, Oliver; de Wind, Niels; Cantelli, Erika; Tonelli, Roberto; Te Riele, Hein

    2011-10-01

    Short synthetic single-stranded oligodeoxyribonucleotides (ssODNs) can be used to introduce subtle modifications into the genome of mouse embryonic stem cells (ESCs). We have previously shown that effective application of ssODN-mediated gene targeting in ESC requires (transient) suppression of DNA mismatch repair (MMR). However, whereas transient down-regulation of the mismatch recognition protein MSH2 allowed substitution of 3 or 4 nucleotides, 1 or 2 nucleotide substitutions were still suppressed. We now demonstrate that single- or dinucleotide substitution can effectively be achieved by transient down-regulation of the downstream MMR protein MLH1. By exploiting highly specific real-time PCR, we demonstrate the feasibility of substituting a single basepair in a non-selectable gene. However, disabling the MMR machinery may lead to inadvertent mutations. To obtain insight into the mutation rate associated with transient MMR suppression, we have compared the impact of transient and constitutive MMR deficiency on the repair of frameshift intermediates at mono- and dinucleotide repeats. Repair at these repeats relied on the substrate specificity and functional redundancy of the MSH2/MSH6 and MSH2/MSH3 MMR complexes. MLH1 knockdown increased the level of spontaneous mutagenesis, but modified ESCs remained germ line competent. Thus, transient MLH1 suppression provides a valuable extension of the MSH2 knockdown strategy, allowing rapid generation of mice carrying single basepair alterations in their genome.

  9. 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

    2017-04-01

    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.

  10. Imaging of DNA and Protein-DNA Complexes with Atomic Force Microscopy.

    PubMed

    Lyubchenko, Yuri L; Shlyakhtenko, Luda S

    2016-01-01

    This article reviews atomic force microscopy (AFM) studies of DNA structure and dynamics and protein-DNA complexes, including recent advances in the visualization of protein-DNA complexes with the use of cutting-edge, high-speed AFM. Special emphasis is given to direct nanoscale visualization of dynamics of protein-DNA complexes. In the area of DNA structure and dynamics, structural studies of local non-B conformations of DNA and the interplay of local and global DNA conformations are reviewed. The application of time-lapse AFM nanoscale imaging of DNA dynamics is illustrated by studies of Holliday junction branch migration. Structure and dynamics of protein-DNA interactions include problems related to site-specific DNA recombination, DNA replication, and DNA mismatch repair. Studies involving the structure and dynamics of chromatin are also described.

  11. Functional interactions between the MutL and Vsr proteins of Escherichia coli are dependent on the N-terminus of Vsr.

    PubMed

    Monastiriakos, Stavroula K; Doiron, Kathy M J; Siponen, Marina I; Cupples, Claire G

    2004-06-03

    The crystal structure of the Escherichia coli Vsr endonuclease bound to a C(T/G)AGG substrate revealed that the DNA is held by a pincer composed of a trio of aromatic residues which intercalate into the major groove, and an N-terminus alpha helix which lies across the minor groove. We have constructed an N-terminus truncation (Delta14) which removes most of the alpha helix. The mutant is still fairly proficient in mediating very short patch repair. However, its endonuclease activity is considerably reduced and, in contrast to that of the wild type protein, cannot be stimulated by MutL. We had shown previously that excess Vsr in vivo causes mutagenesis, probably by inhibiting the participation of MutL in mismatch repair. The Delta14 mutant has diminished mutagenicity. In contrast, four enzymatically inactive mutants, with intact N-termini, are as mutagenic as the wild type protein. On the basis of these results we suggest that MutL causes a conformational change in the N-terminus of Vsr which enhances Vsr activity, and that this functional interaction between Vsr and MutL decreases the ability of MutL to carry out mismatch repair.

  12. Chromatographic isolation of the functionally active MutS protein covalently linked to deoxyribonucleic acid.

    PubMed

    Monakhova, Mayya; Ryazanova, Alexandra; Hentschel, Andreas; Viryasov, Mikhail; Oretskaya, Tatiana; Friedhoff, Peter; Kubareva, Elena

    2015-04-10

    DNA metabolism is based on formation of different DNA-protein complexes which can adopt various conformations. To characterize functioning of such complexes, one needs a solution-based technique which allows fixing a complex in a certain transient conformation. The crosslinking approach is a popular tool for such studies. However, it is under debate if the protein components retain their natural activities in the resulting crosslinked complexes. In the present work we demonstrate the possibility of obtaining pure DNA conjugate with functionally active protein using as example MutS protein from Escherichia coli mismatch repair system. A conjugate of a chemically modified mismatch-containing DNA duplex with MutS is fixed by thiol-disulfide exchange reaction. To perform a reliable test of the protein activity in the conjugate, such conjugate must be thoroughly separated from the uncrosslinked protein and DNA prior to the test. In the present work, we employ anion exchange chromatography for this purpose for the first time and demonstrate this technique to be optimal for the conjugate purification. The activity test is a FRET-based detection of DNA unbending. We show experimentally that MutS in the conjugate retains its ability to unbend DNA in response to ATP addition and find out for the first time that the DNA unbending rate increases with increasing ATP concentration. Since the crosslinked complexes contain active MutS protein, they can be used in further experiments to investigate MutS interactions with other proteins of the mismatch repair system.

  13. [Wakefield's affair: 12 years of uncertainty whereas no link between autism and MMR vaccine has been proved].

    PubMed

    Maisonneuve, Hervé; Floret, Daniel

    2012-09-01

    In 1998, a Lancet paper described 12 cases of children with autism, and having been vaccinated (MMR) in the United Kingdom; medias presented the information to the lay public, stating that a link was possible. In 2004, The Lancet published letters responding to allegations against the paper. Later, it was established that no link existed between MMR and autism; few years and many publications were necessary to conclude to the absence of evidence. In 2010, the General Medical Council published a report against Dr Wakefield, first author of the 1998 paper, and showing that the children hospital records did not contain the evidence; hospital records differed from the published paper; the Lancet retracted the 1998 paper. In 2011, Brian Deer, a journalist, published the complete story in theBMJ: in 1996, Wakefield was approached by lawyers representing an anti-vaccine lobby, and they supported the Wakefield research. Dr Wakefield left England; in 2012 he works in Texas, USA, for anti-vaccine lobbies. Copyright © 2012. Published by Elsevier Masson SAS.

  14. Applications of geographic information systems in public health: A geospatial approach to analyzing MMR immunization uptake in Alberta.

    PubMed

    Eccles, Kristin M; Bertazzon, Stefania

    2015-06-18

    This study evaluates the temporal, spatial, and spatio-temporal variation of immunization rates for measles, mumps and rubella (MMR) immunization in the province of Alberta. The study uses yearly immunization rate data for Health Zones and Local Geographic Areas (2004-2012), which were obtained from Alberta Health's Interactive Health Data Application (IHDA). Spatial analyses include a global spatial analysis, Moran's I, and local indicators of spatial association (LISA) analysis - Getis and Ord's G* - to identify clusters of high or low immunization rates. Spatial methods are then applied to a time series analysis to examine how the immunization rates change over time in conjunction with space. Mapped results indicate decreasing immunization rates over time for the majority of the province where most local geographic areas (LGAs) fall short of the 95% herd immunity threshold. Clusters of high immunization rates in the metropolitan centres, and clusters of low immunization rates in the southern and northern region of the province exist spatially and spatio-temporally. Over time, the high rate clusters are decreasing in size and the low rate clust