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Sample records for repair genes predispose

  1. Double-strand break damage and associated DNA repair genes predispose smokers to gene methylation

    PubMed Central

    Leng, Shuguang; Stidley, Christine A.; Willink, Randy; Bernauer, Amanda; Do, Kieu; Picchi, Maria A.; Sheng, Xin; Frasco, Melissa, A.; Berg, David Van Den; Gilliland, Frank D.; Zima, Christopher; Crowell, Richard E.; Belinsky, Steven A.

    2008-01-01

    Gene promoter hypermethylation in sputum is a promising biomarker for predicting lung cancer. Identifying factors that predispose smokers to methylation of multiple gene promoters in the lung could impact strategies for early detection and chemoprevention. This study evaluated the hypothesis that double-strand break repair capacity and sequence variation in genes in this pathway are associated with a high methylation index in a cohort of current and former cancer-free smokers. A 50% reduction in the mean level of double-strand break repair capacity was seen in lymphocytes from smokers with a high methylation index, defined as ≥ 3 of 8 genes methylated in sputum, compared to smokers with no genes methylated. The classification accuracy for predicting risk for methylation was 88%. Single nucleotide polymorphisms within the MRE11A, CHEK2, XRCC3, DNA-Pkc, and NBN DNA repair genes were highly associated with the methylation index. A 14.5-fold increased odds for high methylation was seen for persons with ≥ 7 risk alleles of these genes. Promoter activity of the MRE11A gene that plays a critical role in recognition of DNA damage and activation of ATM was reduced in persons with the risk allele. Collectively, ours is the first population-based study to identify double-strand break DNA repair capacity and specific genes within this pathway as critical determinants for gene methylation in sputum, that is, in turn, associated with elevated risk for lung cancer. PMID:18413776

  2. Genomic scan for genes predisposing to schizophrenia

    SciTech Connect

    Coon, H.; Jensen. S.; Holik, J.

    1994-03-15

    We initiated a genome-wide search for genes predisposing to schizophrenia by ascertaining 9 families, each containing three to five cases of schizophrenia. The 9 pedigrees were initially genotyped with 329 polymorphic DNA loci distributed throughout the genome. Assuming either autosomal dominant or recessive inheritance, 254 DNA loci yielded lod scores less than -2.0 at {theta} = 0.0, 101 DNA markers gave lod scores less than -2.0 at {theta} = 0.05, while 5 DNA loci produced maximum lod scores greater than 1: D4S35, D14S17, D15S1, D22S84, and D22S55. Of the DNA markers yielding lod scores greater than 1, D4S35 and D22S55 also were suggestive of linkage when the Affected-Pedigree-Member method was used. The families were then genotyped with four highly polymorphic simple sequence repeat markers; possible linkage diminished with DNA markers mapping nearby D4S35, while suggestive evidence of linkage remained with loci in the region of D22S55. Although follow-up investigation of these chromosomal regions may be warranted, our linkage results should be viewed as preliminary observations, as 35 unaffected persons are not past the age of risk. 90 refs., 3 tabs.

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

    PubMed

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

    2014-04-01

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

  4. Human DNA repair genes.

    PubMed

    Wood, R D; Mitchell, M; Sgouros, J; Lindahl, T

    2001-02-16

    Cellular DNA is subjected to continual attack, both by reactive species inside cells and by environmental agents. Toxic and mutagenic consequences are minimized by distinct pathways of repair, and 130 known human DNA repair genes are described here. Notable features presently include four enzymes that can remove uracil from DNA, seven recombination genes related to RAD51, and many recently discovered DNA polymerases that bypass damage, but only one system to remove the main DNA lesions induced by ultraviolet light. More human DNA repair genes will be found by comparison with model organisms and as common folds in three-dimensional protein structures are determined. Modulation of DNA repair should lead to clinical applications including improvement of radiotherapy and treatment with anticancer drugs and an advanced understanding of the cellular aging process. PMID:11181991

  5. Gene Therapy for Cartilage Repair

    PubMed Central

    Madry, Henning; Orth, Patrick; Cucchiarini, Magali

    2011-01-01

    The concept of using gene transfer strategies for cartilage repair originates from the idea of transferring genes encoding therapeutic factors into the repair tissue, resulting in a temporarily and spatially defined delivery of therapeutic molecules to sites of cartilage damage. This review focuses on the potential benefits of using gene therapy approaches for the repair of articular cartilage and meniscal fibrocartilage, including articular cartilage defects resulting from acute trauma, osteochondritis dissecans, osteonecrosis, and osteoarthritis. Possible applications for meniscal repair comprise meniscal lesions, meniscal sutures, and meniscal transplantation. Recent studies in both small and large animal models have demonstrated the applicability of gene-based approaches for cartilage repair. Chondrogenic pathways were stimulated in the repair tissue and in osteoarthritic cartilage using genes for polypeptide growth factors and transcription factors. Although encouraging data have been generated, a successful translation of gene therapy for cartilage repair will require an ongoing combined effort of orthopedic surgeons and of basic scientists. PMID:26069580

  6. Human DNA repair and recombination genes

    SciTech Connect

    Thompson, L.H.; Weber, C.A.; Jones, N.J.

    1988-09-01

    Several genes involved in mammalian DNA repair pathways were identified by complementation analysis and chromosomal mapping based on hybrid cells. Eight complementation groups of rodent mutants defective in the repair of uv radiation damage are now identified. At least seven of these genes are probably essential for repair and at least six of them control the incision step. The many genes required for repair of DNA cross-linking damage show overlap with those involved in the repair of uv damage, but some of these genes appear to be unique for cross-link repair. Two genes residing on human chromosome 19 were cloned from genomic transformants using a cosmid vector, and near full-length cDNA clones of each gene were isolated and sequenced. Gene ERCC2 efficiently corrects the defect in CHO UV5, a nucleotide excision repair mutant. Gene XRCC1 normalizes repair of strand breaks and the excessive sister chromatid exchange in CHO mutant EM9. ERCC2 shows a remarkable /approximately/52% overall homology at both the amino acid and nucleotide levels with the yeast RAD3 gene. Evidence based on mutation induction frequencies suggests that ERCC2, like RAD3, might also be an essential gene for viability. 100 refs., 4 tabs.

  7. Variation in Telangiectasia Predisposing Genes Is Associated With Overall Radiation Toxicity

    SciTech Connect

    Tanteles, George A.; Murray, Robert J.S.; Mills, Jamie; Barwell, Julian; Chakraborti, Prabir; Chan, Steve; Cheung, Kwok-Leung; Ennis, Dawn; Khurshid, Nazish; Lambert, Kelly; Machhar, Rohan; Meisuria, Mitul; Osman, Ahmed; Peat, Irene; Sahota, Harjinder; Woodings, Pamela; Talbot, Christopher J.; and others

    2012-11-15

    Purpose: In patients receiving radiotherapy for breast cancer where the heart is within the radiation field, cutaneous telangiectasiae could be a marker of potential radiation-induced heart disease. We hypothesized that single nucleotide polymorphisms (SNPs) in genes known to cause heritable telangiectasia-associated disorders could predispose to such late, normal tissue vascular damage. Methods and Materials: The relationship between cutaneous telangiectasia as a late normal tissue radiation injury phenotype in 633 breast cancer patients treated with radiotherapy was examined. Patients were clinically assessed for the presence of cutaneous telangiectasia and genotyped at nine SNPs in three candidate genes. Candidate SNPs were within the endoglin (ENG) and activin A receptor, type II-like 1 (ACVRL1) genes, mutations in which cause hereditary hemorrhagic telangiectasia and the ataxia-telangiectasia mutated (ATM) gene associated with ataxia-telangiectasia. Results: A total of 121 (19.1%) patients exhibited a degree of cutaneous telangiectasiae on clinical examination. Regression was used to examine the associations between the presence of telangiectasiae in patients who underwent breast-conserving surgery, controlling for the effects of boost and known brassiere size (n=388), and individual geno- or haplotypes. Inheritance of ACVRL1 SNPs marginally contributed to the risk of cutaneous telangiectasiae. Haplotypic analysis revealed a stronger association between inheritance of a ATM haplotype and the presence of cutaneous telangiectasiae, fibrosis and overall toxicity. No significant association was observed between telangiectasiae and the coinheritance of the candidate ENG SNPs. Conclusions: Genetic variation in the ATM gene influences reaction to radiotherapy through both vascular damage and increased fibrosis. The predisposing variation in the ATM gene will need to be better defined to optimize it as a predictive marker for assessing radiotherapy late effects.

  8. A cancer-predisposing "hot spot" mutation of the fumarase gene creates a dominant negative protein.

    PubMed

    Lorenzato, Annalisa; Olivero, Martina; Perro, Mario; Brière, Jean Jacques; Rustin, Pierre; Di Renzo, Maria Flavia

    2008-02-15

    The Fumarase (Fumarate Hydratase, FH) is a tumor suppressor gene whose germline heterozygous mutations predispose to hereditary leiomyomatosis and renal cell cancer (HLRCC). The FH gene encodes an enzyme of the Krebs cycle, functioning as a homotetramer and catalyzing the hydration of fumarate to malate. Among the numerous FH mutations reported so far, the R190H missense mutation is the most frequent in HLRCC patients. Here we show the functional analyses of the R190H, in comparison to the better characterized E319Q mutation. We first expressed wild-type and mutated proteins in FH deficient human skin fibroblasts, using lentiviral vectors. The wild-type transgene was able to restore the FH enzymatic activity in cells, while the R190H- and E319Q-FH were not. More interestingly, when the same transgenes were expressed in normal, FH-proficient cells, only the R190H-FH reduced the endogenous FH enzymatic activity. By enforcing the expression of equal amount of wild-type and R190H-FH in the same cell, we showed that the mutated FH protein directly inhibited enzymatic activity by nearly abrogating the FH homotetramer formation. These data demonstrate the dominant negative effect of the R190H missense mutation in the FH gene and suggest that the FH tumor-suppressing activity might be impaired in cells carrying a heterozygous mutation.

  9. A cancer-predisposing "hot spot" mutation of the fumarase gene creates a dominant negative protein.

    PubMed

    Lorenzato, Annalisa; Olivero, Martina; Perro, Mario; Brière, Jean Jacques; Rustin, Pierre; Di Renzo, Maria Flavia

    2008-02-15

    The Fumarase (Fumarate Hydratase, FH) is a tumor suppressor gene whose germline heterozygous mutations predispose to hereditary leiomyomatosis and renal cell cancer (HLRCC). The FH gene encodes an enzyme of the Krebs cycle, functioning as a homotetramer and catalyzing the hydration of fumarate to malate. Among the numerous FH mutations reported so far, the R190H missense mutation is the most frequent in HLRCC patients. Here we show the functional analyses of the R190H, in comparison to the better characterized E319Q mutation. We first expressed wild-type and mutated proteins in FH deficient human skin fibroblasts, using lentiviral vectors. The wild-type transgene was able to restore the FH enzymatic activity in cells, while the R190H- and E319Q-FH were not. More interestingly, when the same transgenes were expressed in normal, FH-proficient cells, only the R190H-FH reduced the endogenous FH enzymatic activity. By enforcing the expression of equal amount of wild-type and R190H-FH in the same cell, we showed that the mutated FH protein directly inhibited enzymatic activity by nearly abrogating the FH homotetramer formation. These data demonstrate the dominant negative effect of the R190H missense mutation in the FH gene and suggest that the FH tumor-suppressing activity might be impaired in cells carrying a heterozygous mutation. PMID:17960613

  10. DNA repair genes in the Megavirales pangenome.

    PubMed

    Blanc-Mathieu, Romain; Ogata, Hiroyuki

    2016-06-01

    The order 'Megavirales' represents a group of eukaryotic viruses with a large genome encoding a few hundred up to two thousand five hundred genes. Several members of Megavirales possess genes involved in major DNA repair pathways. Some of these genes were likely inherited from an ancient virus world and some others were derived from the genomes of their hosts. Here we examine molecular phylogenies of key DNA repair enzymes in light of recent hypotheses on the origin of Megavirales, and propose that the last common ancestors of the individual families of the order Megavirales already possessed DNA repair functions to achieve and maintain a moderately large genome and that this repair capacity gradually increased, in a family-dependent manner, during their recent evolution.

  11. Gene Expression Profiling of Histiocytic Sarcomas in a Canine Model: The Predisposed Flatcoated Retriever Dog

    PubMed Central

    Boerkamp, Kim M.; van Wolferen, Monique E.; Groot Koerkamp, Marian J. A.; van Leenen, Dik; Grinwis, Guy C. M.; Penning, Louis C.; Wiemer, Erik A. C.; Rutteman, Gerard R.

    2013-01-01

    Background The determination of altered expression of genes in specific tumor types and their effect upon cellular processes may create insight in tumorigenesis and help to design better treatments. The Flatcoated retriever is a dog breed with an exceptionally high incidence of histiocytic sarcomas. The breed develops two distinct entities of histiocytic neoplasia, a soft tissue form and a visceral form. Gene expression studies of these tumors have value for comparable human diseases such as histiocytic/dendritic cell sarcoma for which knowledge is difficult to accrue due to their rare occurrence. In addition, such studies may help in the search for genetic aberrations underlying the genetic predisposition in this dog breed. Methods Microarray analysis and pathway analyses were performed on fresh-frozen tissues obtained from Flatcoated retrievers with localized, soft tissue histiocytic sarcomas (STHS) and disseminated, visceral histiocytic sarcomas (VHS) and on normal canine spleens from various breeds. Expression differences of nine genes were validated with quantitative real-time PCR (qPCR) analyses. Results QPCR analyses identified the significantly altered expression of nine genes; PPBP, SpiC, VCAM1, ENPEP, ITGAD (down-regulated), and GTSF1, Col3a1, CD90 and LUM (up-regulated) in the comparison of both the soft tissue and the visceral form with healthy spleen. DAVID pathway analyses revealed 24 pathways that were significantly involved in the development of HS in general, most of which were involved in the DNA repair and replication process. Conclusions This study identified altered expression of nine genes not yet implicated in histiocytic sarcoma manifestations in the dog nor in comparable human histiocytic/dendritic sarcomas. Exploration of the downside effect of canine inbreeding strategies for the study of similar sarcomas in humans might also lead to the identification of genes related to these rare malignancies in the human. PMID:23936488

  12. A Common Variant in the FTO Gene Is Associated with Body Mass Index and Predisposes to Childhood and Adult Obesity

    PubMed Central

    Frayling, Timothy M.; Timpson, Nicholas J.; Weedon, Michael N.; Zeggini, Eleftheria; Freathy, Rachel M.; Lindgren, Cecilia M.; Perry, John R. B.; Elliott, Katherine S.; Lango, Hana; Rayner, Nigel W.; Shields, Beverley; Harries, Lorna W.; Barrett, Jeffrey C.; Ellard, Sian; Groves, Christopher J.; Knight, Bridget; Patch, Ann-Marie; Ness, Andrew R.; Ebrahim, Shah; Lawlor, Debbie A.; Ring, Susan M.; Ben-Shlomo, Yoav; Jarvelin, Marjo-Riitta; Sovio, Ulla; Bennett, Amanda J.; Melzer, David; Ferrucci, Luigi; Loos, Ruth J. F.; Barroso, Inês; Wareham, Nicholas J.; Karpe, Fredrik; Owen, Katharine R.; Cardon, Lon R.; Walker, Mark; Hitman, Graham A.; Palmer, Colin N. A.; Doney, Alex S. F.; Morris, Andrew D.; Smith, George Davey; Hattersley, Andrew T.; McCarthy, Mark I.

    2009-01-01

    Obesity is a serious international health problem that increases the risk of several common diseases. The genetic factors predisposing to obesity are poorly understood. A genome-wide search for type 2 diabetes–susceptibility genes identified a common variant in the FTO (fat mass and obesity associated) gene that predisposes to diabetes through an effect on body mass index (BMI). An additive association of the variant with BMI was replicated in 13 cohorts with 38,759 participants. The 16% of adults who are homozygous for the risk allele weighed about 3 kilograms more and had 1.67-fold increased odds of obesity when compared with those not inheriting a risk allele. This association was observed from age 7 years upward and reflects a specific increase in fat mass. PMID:17434869

  13. SYN2 is an autism predisposing gene: loss-of-function mutations alter synaptic vesicle cycling and axon outgrowth.

    PubMed

    Corradi, Anna; Fadda, Manuela; Piton, Amélie; Patry, Lysanne; Marte, Antonella; Rossi, Pia; Cadieux-Dion, Maxime; Gauthier, Julie; Lapointe, Line; Mottron, Laurent; Valtorta, Flavia; Rouleau, Guy A; Fassio, Anna; Benfenati, Fabio; Cossette, Patrick

    2014-01-01

    An increasing number of genes predisposing to autism spectrum disorders (ASDs) has been identified, many of which are implicated in synaptic function. This 'synaptic autism pathway' notably includes disruption of SYN1 that is associated with epilepsy, autism and abnormal behavior in both human and mice models. Synapsins constitute a multigene family of neuron-specific phosphoproteins (SYN1-3) present in the majority of synapses where they are implicated in the regulation of neurotransmitter release and synaptogenesis. Synapsins I and II, the major Syn isoforms in the adult brain, display partially overlapping functions and defects in both isoforms are associated with epilepsy and autistic-like behavior in mice. In this study, we show that nonsense (A94fs199X) and missense (Y236S and G464R) mutations in SYN2 are associated with ASD in humans. The phenotype is apparent in males. Female carriers of SYN2 mutations are unaffected, suggesting that SYN2 is another example of autosomal sex-limited expression in ASD. When expressed in SYN2  knockout neurons, wild-type human Syn II fully rescues the SYN2 knockout phenotype, whereas the nonsense mutant is not expressed and the missense mutants are virtually unable to modify the SYN2 knockout phenotype. These results identify for the first time SYN2  as a novel predisposing gene for ASD and strengthen the hypothesis that a disturbance of synaptic homeostasis underlies ASD. PMID:23956174

  14. Germline variants in the SEMA4A gene predispose to familial colorectal cancer type X

    PubMed Central

    Schulz, Eduard; Klampfl, Petra; Holzapfel, Stefanie; Janecke, Andreas R.; Ulz, Peter; Renner, Wilfried; Kashofer, Karl; Nojima, Satoshi; Leitner, Anita; Zebisch, Armin; Wölfler, Albert; Hofer, Sybille; Gerger, Armin; Lax, Sigurd; Beham-Schmid, Christine; Steinke, Verena; Heitzer, Ellen; Geigl, Jochen B.; Windpassinger, Christian; Hoefler, Gerald; Speicher, Michael R.; Richard Boland, C.; Kumanogoh, Atsushi; Sill, Heinz

    2014-01-01

    Familial colorectal cancer type X (FCCTX) is characterized by clinical features of hereditary non-polyposis colorectal cancer with a yet undefined genetic background. Here we identify the SEMA4A p.Val78Met germline mutation in an Austrian kindred with FCCTX, using an integrative genomics strategy. Compared with wild-type protein, SEMA4AV78M demonstrates significantly increased MAPK/Erk and PI3K/Akt signalling as well as cell cycle progression of SEMA4A-deficient HCT-116 colorectal cancer cells. In a cohort of 53 patients with FCCTX, we depict two further SEMA4A mutations, p.Gly484Ala and p.Ser326Phe and the single-nucleotide polymorphism (SNP) p.Pro682Ser. This SNP is highly associated with the FCCTX phenotype exhibiting increased risk for colorectal cancer (OR 6.79, 95% CI 2.63 to 17.52). Our study shows previously unidentified germline variants in SEMA4A predisposing to FCCTX, which has implications for surveillance strategies of patients and their families. PMID:25307848

  15. Isolating human DNA repair genes using rodent-cell mutants

    SciTech Connect

    Thompson, L.H.; Weber, C.A.; Brookman, K.W.; Salazar, E.P.; Stewart, S.A.; Mitchell, D.L.

    1987-03-23

    The DNA repair systems of rodent and human cells appear to be at least as complex genetically as those in lower eukaryotes and bacteria. The use of mutant lines of rodent cells as a means of identifying human repair genes by functional complementation offers a new approach toward studying the role of repair in mutagenesis and carcinogenesis. In each of six cases examined using hybrid cells, specific human chromosomes have been identified that correct CHO cell mutations affecting repair of damage from uv or ionizing radiations. This finding suggests that both the repair genes and proteins may be virtually interchangeable between rodent and human cells. Using cosmid vectors, human repair genes that map to chromosome 19 have cloned as functional sequences: ERCC2 and XRCC1. ERCC1 was found to have homology with the yeast excision repair gene RAD10. Transformants of repair-deficient cell lines carrying the corresponding human gene show efficient correction of repair capacity by all criteria examined. 39 refs., 1 fig., 1 tab.

  16. Mutation of genes controlling mRNA metabolism and protein synthesis predisposes to neurodevelopmental disorders.

    PubMed

    Sartor, Francesca; Anderson, Jihan; McCaig, Colin; Miedzybrodzka, Zosia; Müller, Berndt

    2015-12-01

    Brain development is a tightly controlled process that depends upon differentiation and function of neurons to allow for the formation of functional neural networks. Mutation of genes encoding structural proteins is well recognized as causal for neurodevelopmental disorders (NDDs). Recent studies have shown that aberrant gene expression can also lead to disorders of neural development. Here we summarize recent evidence implicating in the aetiology of NDDs mutation of factors acting at the level of mRNA splicing, mRNA nuclear export, translation and mRNA degradation. This highlights the importance of these fundamental processes for human health and affords new strategies and targets for therapeutic intervention.

  17. Low copy number of the salivary amylase gene predisposes to obesity.

    PubMed

    Falchi, Mario; El-Sayed Moustafa, Julia Sarah; Takousis, Petros; Pesce, Francesco; Bonnefond, Amélie; Andersson-Assarsson, Johanna C; Sudmant, Peter H; Dorajoo, Rajkumar; Al-Shafai, Mashael Nedham; Bottolo, Leonardo; Ozdemir, Erdal; So, Hon-Cheong; Davies, Robert W; Patrice, Alexandre; Dent, Robert; Mangino, Massimo; Hysi, Pirro G; Dechaume, Aurélie; Huyvaert, Marlène; Skinner, Jane; Pigeyre, Marie; Caiazzo, Robert; Raverdy, Violeta; Vaillant, Emmanuel; Field, Sarah; Balkau, Beverley; Marre, Michel; Visvikis-Siest, Sophie; Weill, Jacques; Poulain-Godefroy, Odile; Jacobson, Peter; Sjostrom, Lars; Hammond, Christopher J; Deloukas, Panos; Sham, Pak Chung; McPherson, Ruth; Lee, Jeannette; Tai, E Shyong; Sladek, Robert; Carlsson, Lena M S; Walley, Andrew; Eichler, Evan E; Pattou, Francois; Spector, Timothy D; Froguel, Philippe

    2014-05-01

    Common multi-allelic copy number variants (CNVs) appear enriched for phenotypic associations compared to their biallelic counterparts. Here we investigated the influence of gene dosage effects on adiposity through a CNV association study of gene expression levels in adipose tissue. We identified significant association of a multi-allelic CNV encompassing the salivary amylase gene (AMY1) with body mass index (BMI) and obesity, and we replicated this finding in 6,200 subjects. Increased AMY1 copy number was positively associated with both amylase gene expression (P = 2.31 × 10(-14)) and serum enzyme levels (P < 2.20 × 10(-16)), whereas reduced AMY1 copy number was associated with increased BMI (change in BMI per estimated copy = -0.15 (0.02) kg/m(2); P = 6.93 × 10(-10)) and obesity risk (odds ratio (OR) per estimated copy = 1.19, 95% confidence interval (CI) = 1.13-1.26; P = 1.46 × 10(-10)). The OR value of 1.19 per copy of AMY1 translates into about an eightfold difference in risk of obesity between subjects in the top (copy number > 9) and bottom (copy number < 4) 10% of the copy number distribution. Our study provides a first genetic link between carbohydrate metabolism and BMI and demonstrates the power of integrated genomic approaches beyond genome-wide association studies. PMID:24686848

  18. Germline mutations in the PAF1 complex gene CTR9 predispose to Wilms tumour.

    PubMed

    Hanks, Sandra; Perdeaux, Elizabeth R; Seal, Sheila; Ruark, Elise; Mahamdallie, Shazia S; Murray, Anne; Ramsay, Emma; Del Vecchio Duarte, Silvana; Zachariou, Anna; de Souza, Bianca; Warren-Perry, Margaret; Elliott, Anna; Davidson, Alan; Price, Helen; Stiller, Charles; Pritchard-Jones, Kathy; Rahman, Nazneen

    2014-01-01

    Wilms tumour is a childhood kidney cancer. Here we identify inactivating CTR9 mutations in 3 of 35 Wilms tumour families, through exome and Sanger sequencing. By contrast, no similar mutations are present in 1,000 population controls (P<0.0001). Each mutation segregates with Wilms tumour in the family and a second mutational event is present in available tumours. CTR9 is a key component of the polymerase-associated factor 1 complex which has multiple roles in RNA polymerase II regulation and is implicated in embryonic organogenesis and maintenance of embryonic stem cell pluripotency. These data establish CTR9 as a Wilms tumour predisposition gene and suggest it acts as a tumour suppressor gene. PMID:25099282

  19. Nature and nurture predispose to violent behavior: serotonergic genes and adverse childhood environment.

    PubMed

    Reif, Andreas; Rösler, Michael; Freitag, Christine M; Schneider, Marc; Eujen, Andrea; Kissling, Christian; Wenzler, Denise; Jacob, Christian P; Retz-Junginger, Petra; Thome, Johannes; Lesch, Klaus-Peter; Retz, Wolfgang

    2007-11-01

    Aggressive behavior is influenced by variation in genes of the serotonergic circuitry and early-life experience alike. The present study aimed at investigating the contribution of polymorphisms shown to moderate transcription of two genes involved in serotonergic neurotransmission (serotonin transporter, 5HTT, and monoamine oxidase A, MAOA) to the development of violence and to test for gene-environment interactions relating to adverse childhood environment. A cohort of 184 adult male volunteers referred for forensic assessment participated in the study. Each individual was assigned to either a violent or a nonviolent group. Logistic regression was performed and the best-fitting model, with a predictive power of 74%, revealed independent effects of adverse childhood environment and MAOA genotype. High environmental adversity during childhood was associated significantly with violent behavior. Forty-five percent of violent, but only 30% of nonviolent individuals carried the low-activity, short MAOA allele. Most interestingly, an interaction effect between childhood environment and 5HTT genotype on violent behavior was found in that high adversity during childhood impacted only the later-life violence if the short promoter alleles were present. These findings indicate complex interactions between genetic variation of the serotonergic circuitry and environmental factors arguing against simplistic, mono-causal explanations of violent behavior.

  20. Disturbance of cardiac gene expression and cardiomyocyte structure predisposes Mecp2-null mice to arrhythmias

    PubMed Central

    Hara, Munetsugu; Takahashi, Tomoyuki; Mitsumasu, Chiaki; Igata, Sachiyo; Takano, Makoto; Minami, Tomoko; Yasukawa, Hideo; Okayama, Satoko; Nakamura, Keiichiro; Okabe, Yasunori; Tanaka, Eiichiro; Takemura, Genzou; Kosai, Ken-ichiro; Yamashita, Yushiro; Matsuishi, Toyojiro

    2015-01-01

    Methyl-CpG-binding protein 2 (MeCP2) is an epigenetic regulator of gene expression that is essential for normal brain development. Mutations in MeCP2 lead to disrupted neuronal function and can cause Rett syndrome (RTT), a neurodevelopmental disorder. Previous studies reported cardiac dysfunction, including arrhythmias in both RTT patients and animal models of RTT. In addition, recent studies indicate that MeCP2 may be involved in cardiac development and dysfunction, but its role in the developing and adult heart remains unknown. In this study, we found that Mecp2-null ESCs could differentiate into cardiomyocytes, but the development and further differentiation of cardiovascular progenitors were significantly affected in MeCP2 deficiency. In addition, we revealed that loss of MeCP2 led to dysregulation of endogenous cardiac genes and myocardial structural alterations, although Mecp2-null mice did not exhibit obvious cardiac functional abnormalities. Furthermore, we detected methylation of the CpG islands in the Tbx5 locus, and showed that MeCP2 could target these sequences. Taken together, these results suggest that MeCP2 is an important regulator of the gene-expression program responsible for maintaining normal cardiac development and cardiomyocyte structure. PMID:26073556

  1. Disturbance of cardiac gene expression and cardiomyocyte structure predisposes Mecp2-null mice to arrhythmias.

    PubMed

    Hara, Munetsugu; Takahashi, Tomoyuki; Mitsumasu, Chiaki; Igata, Sachiyo; Takano, Makoto; Minami, Tomoko; Yasukawa, Hideo; Okayama, Satoko; Nakamura, Keiichiro; Okabe, Yasunori; Tanaka, Eiichiro; Takemura, Genzou; Kosai, Ken-ichiro; Yamashita, Yushiro; Matsuishi, Toyojiro

    2015-06-15

    Methyl-CpG-binding protein 2 (MeCP2) is an epigenetic regulator of gene expression that is essential for normal brain development. Mutations in MeCP2 lead to disrupted neuronal function and can cause Rett syndrome (RTT), a neurodevelopmental disorder. Previous studies reported cardiac dysfunction, including arrhythmias in both RTT patients and animal models of RTT. In addition, recent studies indicate that MeCP2 may be involved in cardiac development and dysfunction, but its role in the developing and adult heart remains unknown. In this study, we found that Mecp2-null ESCs could differentiate into cardiomyocytes, but the development and further differentiation of cardiovascular progenitors were significantly affected in MeCP2 deficiency. In addition, we revealed that loss of MeCP2 led to dysregulation of endogenous cardiac genes and myocardial structural alterations, although Mecp2-null mice did not exhibit obvious cardiac functional abnormalities. Furthermore, we detected methylation of the CpG islands in the Tbx5 locus, and showed that MeCP2 could target these sequences. Taken together, these results suggest that MeCP2 is an important regulator of the gene-expression program responsible for maintaining normal cardiac development and cardiomyocyte structure.

  2. Knockout of the TauT Gene Predisposes C57BL/6 Mice to Streptozotocin-Induced Diabetic Nephropathy

    PubMed Central

    Han, Xiaobin; Patters, Andrea B.; Ito, Takashi; Schaffer, Stephen W.; Chesney, Russell W.

    2015-01-01

    Diabetic nephropathy is the leading cause of end stage renal disease in the world. Although tremendous efforts have been made, scientists have yet to identify an ideal animal model that can reproduce the characteristics of human diabetic nephropathy. In this study, we hypothesize that taurine insufficiency is a critical risk factor for development of diabetic nephropathy associated with diabetes mellitus. This hypothesis was tested in vivo in TauT heterozygous (TauT+/-) and homozygous (TauT-/-) knockout in C57BL/6 background mice. We have shown that alteration of the TauT gene (also known as SLC6A6) has a substantial effect on the susceptibility to development of extensive diabetic kidney disease in both TauT+/- and TauT-/-mouse models of diabetes. These animals developed histological changes characteristic of human diabetic nephropathy that included glomerulosclerosis, nodular lesions, arteriosclerosis, arteriolar dilation, and tubulointerstitial fibrosis. Immunohistochemical staining of molecular markers of smooth muscle actin, CD34, Ki67 and collagen IV further confirmed these observations. Our results demonstrated that both homozygous and heterozygous TauT gene deletion predispose C57BL/6 mice to develop end-stage diabetic kidney disease, which closely replicates the pathological features of diabetic nephropathy in human diabetic patients. PMID:25629817

  3. Detection of a Tumor Suppressor Gene Variant Predisposing to Colorectal Cancer in an 18th Century Hungarian Mummy

    PubMed Central

    Feldman, Michal; Hershkovitz, Israel; Sklan, Ella H.; Kahila Bar-Gal, Gila; Pap, Ildikó; Szikossy, Ildikó; Rosin-Arbesfeld, Rina

    2016-01-01

    Mutations of the Adenomatous polyposis coli (APC) gene are common and strongly associated with the development of colorectal adenomas and carcinomas. While extensively studied in modern populations, reports on visceral tumors in ancient populations are scarce. To the best of our knowledge, genetic characterization of mutations associated with colorectal cancer in ancient specimens has not yet been described. In this study we have sequenced hotspots for mutations in the APC gene isolated from 18th century naturally preserved human Hungarian mummies. While wild type APC sequences were found in two mummies, we discovered the E1317Q missense mutation, known to be a colorectal cancer predisposing mutation, in a large intestine tissue of an 18th century mummy. Our data suggests that this genetic predisposition to cancer already existed in the pre-industrialization era. This study calls for similar investigations of ancient specimens from different periods and geographical locations to be conducted and shared for the purpose of obtaining a larger scale analysis that will shed light on past cancer epidemiology and on cancer evolution. PMID:26863316

  4. Lipid Status and Predisposing Genes in Patients with Diabetes Mellitus Type 1 from Various Ethnic Groups.

    PubMed

    Kolesnikova, L I; Kolesnikov, S I; Darenskaya, M A; Grebenkina, L A; Semenova, N V; Osipova, E V; Gnusina, S V; Bardymova, T A

    2015-12-01

    The peculiarities of HLA class II profile and lipid metabolism were examined in Buryat and Russian ethnic groups of patients with diabetes mellitus type 1. The incidence of type 1 haplotypes in HLA class II gene family was lower in Buryats than that in Russians. In comparison with Russians, the course of diabetes mellitus type 1 in Buryat patients was characterized with a lower content of total lipids, triacylglycerols, total cholesterol, and LDL, which probably explains a more favorable course of the disease in Buryat population.

  5. Low-copy repeats at the human VIPR2 gene predispose to recurrent and nonrecurrent rearrangements

    PubMed Central

    Beri, Silvana; Bonaglia, Maria Clara; Giorda, Roberto

    2013-01-01

    Submicroscopic structural variations, including deletions, duplications, inversions and more complex rearrangements, are widespread in normal human genomes. Inverted segmental duplications or highly identical low-copy repeat (LCR) sequences can mediate the formation of inversions and more complex structural rearrangements through non-allelic homologous recombination. In a patient with 7q36 inverted duplication/terminal deletion, we demonstrated the central role of a pair of short inverted LCRs in the vasoactive intestinal peptide receptor gene (VIPR2)-LCRs in generating the rearrangement. We also revealed a relatively common VIPR2-LCR-associated inversion polymorphism disrupting the gene in almost 1% of healthy subjects, and a small number of complex duplications/triplications. In genome-wide studies of several thousand patients, a significant association of rare microduplications with variable size, all involving VIPR2, with schizophrenia was recently described, suggesting that altered vasoactive intestinal peptide signaling is likely implicated in the pathogenesis of schizophrenia. Genetic testing for VIPR2-LCR-associated inversions should be performed on available cohorts of psychiatric patients to evaluate their potential pathogenic role. PMID:23073313

  6. A genome-wide search for genes predisposing to manic-depression, assuming autosomal dominant inheritance

    SciTech Connect

    Coon, H.; Jensen, S.; Hoff, M.; Holik, J.; Plaetke, R.; Reimherr, F.; Wender, P.; Leppert, M.; Byerley, W. )

    1993-06-01

    Manic-depressive illness (MDI), also known as [open quotes]bipolar affective disorder[close quotes], is a common and devastating neuropsychiatric illness. Although pivotal biochemical alterations underlying the disease are unknown, results of family, twin, and adoption studies consistently implicate genetic transmission in the pathogenesis of MDI. In order to carry out linkage analysis, the authors ascertained eight moderately sized pedigrees containing multiple cases of the disease. For a four-allele marker mapping at 5 cM from the disease gene, the pedigree sample has >97% power to detect a dominant allele under genetic homogeneity and has >73% power under 20% heterogeneity. To date, the eight pedigrees have been genotyped with 328 polymorphic DNA loci throughout the genome. When autosomal dominant inheritance was assumed, 273 DNA markers gave lod scores <[minus]2.0 at [theta] = .05, and 4 DNA marker loci yielded lod scores >1 (chromosome 5 -- D5S39, D5S43, and D5S62; chromosome 11 -- D11S85). Of the markers giving lod scores >1, only D5S62 continued to show evidence for linkage when the affected-pedigree-member method was used. The D5S62 locus maps to distal 5q, a region containing neurotransmitter-receptor genes for dopamine, norepinephrine, glutamate, and gamma-aminobutyric acid. Although additional work in this region may be warranted, the linkage results should be interpreted as preliminary data, as 68 unaffected individuals are not past the age of risk. 72 refs., 2 tabs.

  7. Polymorphism in the Serotonin Receptor 2a (HTR2A) Gene as Possible Predisposal Factor for Aggressive Traits

    PubMed Central

    Banlaki, Zsofia; Elek, Zsuzsanna; Nanasi, Tibor; Szekely, Anna; Nemoda, Zsofia; Sasvari-Szekely, Maria; Ronai, Zsolt

    2015-01-01

    Aggressive manifestations and their consequences are a major issue of mankind, highlighting the need for understanding the contributory factors. Still, aggression-related genetic analyses have so far mainly been conducted on small population subsets such as individuals suffering from a certain psychiatric disorder or a narrow-range age cohort, but no data on the general population is yet available. In the present study, our aim was to identify polymorphisms in genes affecting neurobiological processes that might explain some of the inter-individual variation between aggression levels in the non-clinical Caucasian adult population. 55 single nucleotide polymorphisms (SNP) were simultaneously determined in 887 subjects who also filled out the self-report Buss-Perry Aggression Questionnaire (BPAQ). Single marker association analyses between genotypes and aggression scores indicated a significant role of rs7322347 located in the HTR2A gene encoding serotonin receptor 2a following Bonferroni correction for multiple testing (p = 0.0007) both for males and females. Taking the four BPAQ subscales individually, scores for Hostility, Anger and Physical Aggression showed significant association with rs7322347 T allele in themselves, while no association was found with Verbal Aggression. Of the subscales, relationship with rs7322347 was strongest in the case of Hostility, where statistical significance virtually equaled that observed with the whole BPAQ. In conclusion, this is the first study to our knowledge analyzing SNPs in a wide variety of genes in terms of aggression in a large sample-size non-clinical adult population, also describing a novel candidate polymorphism as predisposal to aggressive traits. PMID:25658328

  8. Genomic landscape of DNA repair genes in cancer.

    PubMed

    Chae, Young Kwang; Anker, Jonathan F; Carneiro, Benedito A; Chandra, Sunandana; Kaplan, Jason; Kalyan, Aparna; Santa-Maria, Cesar A; Platanias, Leonidas C; Giles, Francis J

    2016-04-26

    DNA repair genes are frequently mutated in cancer, yet limited data exist regarding the overall genomic landscape and functional implications of these alterations in their entirety. We created comprehensive lists of DNA repair genes and indirect caretakers. Mutation, copy number variation (CNV), and expression frequencies of these genes were analyzed in COSMIC. Mutation co-occurrence, clinical outcomes, and mutation burden were analyzed in TCGA. We report the 20 genes most frequently with mutations (n > 19,689 tumor samples for each gene), CNVs (n > 1,556), or up- or down-regulated (n = 7,998). Mutual exclusivity was observed as no genes displayed both high CNV gain and loss or high up- and down-regulation, and CNV gain and loss positively correlated with up- and down-regulation, respectively. Co-occurrence of mutations differed between cancers, and mutations in many DNA repair genes were associated with higher total mutation burden. Mutation and CNV frequencies offer insights into which genes may play tumor suppressive or oncogenic roles, such as NEIL2 and RRM2B, respectively. Mutual exclusivities within CNV and expression frequencies, and correlations between CNV and expression, support the functionality of these genomic alterations. This study provides comprehensive lists of candidate genes as potential biomarkers for genomic instability, novel therapeutic targets, or predictors of immunotherapy efficacy.

  9. Genomic landscape of DNA repair genes in cancer

    PubMed Central

    Carneiro, Benedito A.; Chandra, Sunandana; Kaplan, Jason; Kalyan, Aparna; Santa-Maria, Cesar A.; Platanias, Leonidas C.; Giles, Francis J.

    2016-01-01

    DNA repair genes are frequently mutated in cancer, yet limited data exist regarding the overall genomic landscape and functional implications of these alterations in their entirety.  We created comprehensive lists of DNA repair genes and indirect caretakers.  Mutation, copy number variation (CNV), and expression frequencies of these genes were analyzed in COSMIC. Mutation co-occurrence, clinical outcomes, and mutation burden were analyzed in TCGA. We report the 20 genes most frequently with mutations (n > 19,689 tumor samples for each gene), CNVs (n > 1,556), or up- or down-regulated (n = 7,998).  Mutual exclusivity was observed as no genes displayed both high CNV gain and loss or high up- and down-regulation, and CNV gain and loss positively correlated with up- and down-regulation, respectively. Co-occurrence of mutations differed between cancers, and mutations in many DNA repair genes were associated with higher total mutation burden. Mutation and CNV frequencies offer insights into which genes may play tumor suppressive or oncogenic roles, such as NEIL2 and RRM2B, respectively.  Mutual exclusivities within CNV and expression frequencies, and correlations between CNV and expression, support the functionality of these genomic alterations. This study provides comprehensive lists of candidate genes as potential biomarkers for genomic instability, novel therapeutic targets, or predictors of immunotherapy efficacy. PMID:27004405

  10. Non-functional genes repaired at the RNA level.

    PubMed

    Burger, Gertraud

    2016-01-01

    Genomes and genes continuously evolve. Gene sequences undergo substitutions, deletions or nucleotide insertions; mobile genetic elements invade genomes and interleave in genes; chromosomes break, even within genes, and pieces reseal in reshuffled order. To maintain functional gene products and assure an organism's survival, two principal strategies are used - either repair of the gene itself or of its product. I will introduce common types of gene aberrations and how gene function is restored secondarily, and then focus on systematically fragmented genes found in a poorly studied protist group, the diplonemids. Expression of their broken genes involves restitching of pieces at the RNA-level, and substantial RNA editing, to compensate for point mutations. I will conclude with thoughts on how such a grotesquely unorthodox system may have evolved, and why this group of organisms persists and thrives since tens of millions of years.

  11. Interaction of major genes predisposing to hepatocellular carcinoma with genes encoding signal transduction pathways influences tumor phenotype and prognosis

    PubMed Central

    Feo, Francesco; Frau, Maddalena; Pascale, Rosa Maria

    2008-01-01

    Studies on rodents and humans demonstrate an inherited predisposition to hepatocellular carcinoma (HCC). Analysis of the molecular alterations involved in the acquisition of a phenotype resistant or susceptible to hepatocarcinogenesis showed a deregulation of G1 and S phases in HCC of genetically susceptible F344 rats and a G1-S block in lesions of resistant Brown norway (BN) rats. Unrestrained extracellular signal-regulated kinase (ERK) activity linked to proteasomal degradation of dual-specificity phosphatase 1 (DUSP1), a specific ERK inhibitor, by the CKS1-SKP2 ubiquitin ligase complex occurs in more aggressive HCC of F344 rats and humans. This mechanism is less active in HCC of BN rats and human HCC with better prognosis. Upregulation of iNos cross-talk with IKK/NF-κB and RAS/ERK pathways occurs in rodent liver lesions at higher levels in the most aggressive models represented by HCC of F344 rats and c-Myc-TGF-α transgenic mice. iNOS, IKK/NF-κB, and RAS/ERK upregulation is highest in human HCC with a poorer prognosis and positively correlates with tumor proliferation, genomic instability and microvascularization, and negatively with apoptosis. Thus, cell cycle regulation and the activity of signal transduction pathways seem to be modulated by HCC modifier genes, and differences in their efficiency influence the susceptibility to hepatocarcinogenesis and probably the prognosis of human HCC. PMID:19034960

  12. Hodgkin Lymphoma Risk: Role of Genetic Polymorphisms and Gene-Gene Interactions in DNA repair pathways

    PubMed Central

    Monroy, Claudia M.; Cortes, Andrea C.; Lopez, Mirtha; Rourke, Elizabeth; Etzel, Carol J.; Younes, Anas; Strom, Sara S.; El-Zein, Randa

    2011-01-01

    DNA repair variants may play a potentially important role in an individual’s susceptibility to developing cancer. Numerous studies have reported the association between genetic single nucleotide polymorphisms (SNPs) in DNA repair genes and different types of hematologic cancers. However, to date, the effects of such SNPs on modulating Hodgkin Lymphoma (HL) risk have not yet been investigated. We hypothesized that gene-gene interaction between candidate genes in Direct Reversal, Nucleotide excision repair (NER), Base excision repair (BER) and Double strand break (DSB) pathways may contribute to susceptibility to HL. To test this hypothesis, we conducted a study on 200 HL cases and 220 controls to assess associations between HL risk and 21 functional SNPs in DNA repair genes. We evaluated potential gene-gene interactions and the association of multiple polymorphisms in a chromosome region using a multi-analytic strategy combining logistic regression, multi-factor dimensionality reduction and classification and regression tree approaches. We observed that, in combination, allelic variants in the XPC Ala499Val, NBN Glu185Gln, XRCC3 Thr241Me, XRCC1 Arg194Trp and XRCC1 399Gln polymorphisms modify the risk for developing HL. Moreover, the cumulative genetic risk score revealed a significant trend where the risk for developing HL increases as the number of adverse alleles in BER and DSB genes increase. These findings suggest that DNA repair variants in BER and DSB pathways may play an important role in the development of HL. PMID:21374732

  13. Regulation of targeted gene repair by intrinsic cellular processes.

    PubMed

    Engstrom, Julia U; Suzuki, Takayuki; Kmiec, Eric B

    2009-02-01

    Targeted gene alteration (TGA) is a strategy for correcting single base mutations in the DNA of human cells that cause inherited disorders. TGA aims to reverse a phenotype by repairing the mutant base within the chromosome itself, avoiding the introduction of exogenous genes. The process of how to accurately repair a genetic mutation is elucidated through the use of single-stranded DNA oligonucleotides (ODNs) that can enter the cell and migrate to the nucleus. These specifically designed ODNs hybridize to the target sequence and act as a beacon for nucleotide exchange. The key to this reaction is the frequency with which the base is corrected; this will determine whether the approach becomes clinically relevant or not. Over the course of the last five years, workers have been uncovering the role played by the cells in regulating the gene repair process. In this essay, we discuss how the impact of the cell on TGA has evolved through the years and illustrate ways that inherent cellular pathways could be used to enhance TGA activity. We also describe the cost to cell metabolism and survival when certain processes are altered to achieve a higher frequency of repair.

  14. Bone tissue engineering and repair by gene therapy.

    PubMed

    Betz, Volker M; Betz, Oliver B; Harris, Mitchel B; Vrahas, Mark S; Evans, Christopher H

    2008-01-01

    Many clinical conditions require the stimulation of bone growth. The use of recombinant bone morphogenetic proteins does not provide a satisfying solution to these conditions due to delivery problems and high cost. Gene therapy has emerged as a very promising approach for bone repair that overcomes limitations of protein-based therapy. Several preclinical studies have shown that gene transfer technology has the ability to deliver osteogenic molecules to precise anatomical locations at therapeutic levels for sustained periods of time. Both in-vivo and ex-vivo transduction of cells can induce bone formation at ectopic and orthotopic sites. Genetic engineering of adult stem cells from various sources with osteogenic genes has led to enhanced fracture repair, spinal fusion and rapid healing of bone defects in animal models. This review describes current viral and non-viral gene therapy strategies for bone tissue engineering and repair including recent work from the author's laboratory. In addition, the article discusses the potential of gene-enhanced tissue engineering to enter widespread clinical use.

  15. uv excision-repair gene transfer in Chinese hamster ovary (CHO) cells

    SciTech Connect

    MacInnes, M.A.; Bingham, J.M.; Strniste, G.F.; Thompson, L.H.

    1983-01-01

    uvc-sensitive mutants of CHO cells provide a model system for molecular studies of DNA repair. We present our recent results which show that these mutants are competent recipients for plasmid marker gene transfer and incorporation of a putative CHO repair gene. The applicability and advantages of this system for interspecies human repair gene identification are discussed.

  16. Mismatch-mediated error prone repair at the immunoglobulin genes.

    PubMed

    Chahwan, Richard; Edelmann, Winfried; Scharff, Matthew D; Roa, Sergio

    2011-12-01

    The generation of effective antibodies depends upon somatic hypermutation (SHM) and class-switch recombination (CSR) of antibody genes by activation induced cytidine deaminase (AID) and the subsequent recruitment of error prone base excision and mismatch repair. While AID initiates and is required for SHM, more than half of the base changes that accumulate in V regions are not due to the direct deamination of dC to dU by AID, but rather arise through the recruitment of the mismatch repair complex (MMR) to the U:G mismatch created by AID and the subsequent perversion of mismatch repair from a high fidelity process to one that is very error prone. In addition, the generation of double-strand breaks (DSBs) is essential during CSR, and the resolution of AID-generated mismatches by MMR to promote such DSBs is critical for the efficiency of the process. While a great deal has been learned about how AID and MMR cause hypermutations and DSBs, it is still unclear how the error prone aspect of these processes is largely restricted to antibody genes. The use of knockout models and mice expressing mismatch repair proteins with separation-of-function point mutations have been decisive in gaining a better understanding of the roles of each of the major MMR proteins and providing further insight into how mutation and repair are coordinated. Here, we review the cascade of MMR factors and repair signals that are diverted from their canonical error free role and hijacked by B cells to promote genetic diversification of the Ig locus. This error prone process involves AID as the inducer of enzymatically-mediated DNA mismatches, and a plethora of downstream MMR factors acting as sensors, adaptors and effectors of a complex and tightly regulated process from much of which is not yet well understood.

  17. N-acetyltransferase 2 (NAT2) gene polymorphism as a predisposing factor for phenytoin intoxication in tuberculous meningitis or tuberculoma patients having seizures - A pilot study

    PubMed Central

    Adole, Prashant S.; Kharbanda, Parampreet S.; Sharma, Sadhna

    2016-01-01

    Background & objectives: Simultaneous administration of phenytoin and isoniazid (INH) in tuberculous meningitis (TBM) or tuberculoma patients with seizures results in higher plasma phenytoin level and thus phenytoin intoxication. N-acetyltransferase 2 (NAT2) enzyme catalyses two acetylation reactions in INH metabolism and NAT2 gene polymorphism leads to slow and rapid acetylators. The present study was aimed to evaluate the effect of allelic variants of N-acetyltransferase 2 (NAT2) gene as a predisposing factor for phenytoin toxicity in patients with TBM or tuberculoma having seizures, and taking INH and phenytoin simultaneously. Methods: Sixty patients with TBM or tuberculoma with seizures and taking INH and phenytoin simultaneously for a minimum period of seven days were included in study. Plasma phenytoin was measured by high performance liquid chromatography. NAT2 gene polymorphism was studied using restriction fragment length polymorphism and allele specific PCR. Results: The patients were grouped into those having phenytoin intoxication and those with normal phenytoin level, and also classified as rapid or slow acetylators by NAT2 genotyping. Genotypic analysis showed that of the seven SNPs (single nucleotide polymorphisms) of NAT2 gene studied, six mutations were found to be associated with phenytoin intoxication. For rs1041983 (C282T), rs1799929 (C481T), rs1799931 (G857A), rs1799930 (G590A), rs1208 (A803G) and rs1801280 (T341C) allelic variants, the proportion of homozygous mutant was higher in phenytoin intoxicated group than in phenytoin non-intoxicated group. Interpretation & conclusions: Homozygous mutant allele of NAT2 gene at 481site may act as a predisposing factor for phenytoin intoxication among TBM or tuberculoma patients having seizures. PMID:27488001

  18. Expression of the excision repair gene, ERCC3 (excision repair cross-complementing), during mouse development.

    PubMed

    Hubank, M; Mayne, L

    1994-08-12

    Expression of the human ERCC3 (excision repair cross-complementing) gene in cells from patients with xeroderma pigmentosum (XP) group B (XP-B) corrects the defect in repair of UV light-induced DNA damage. XP-B is one of three groups of XP which exhibit the clinical symptoms of both XP and Cockayne's Syndrome (CS). CS and XP-B/CS patients develop severe neurological dysfunction during development. In order to explore the link between the defective gene and the neurological deficits in XP/CS, we have studied the expression of ERCC3 mRNA in developing mice by in situ hybridisation. ERCC3 was found to be ubiquitously expressed in cells from all regions and all developmental stages, from 9 day post-coitum embryo, to 15 day post-natal brain. In post-natal brain, regional differences in expression correlated with cell density and there was no evidence of cell specific or developmental alterations in levels of expression. These results indicate that the constitutively expressed gene does not perform a discrete developmental function. The neurological defects apparent in XP-B are likely to arise pleiotypically from the participation of ERCC3 in interactions with other elements involved in particular aspects of neurodevelopmental control. These results emphasise the developmental importance of genes whose primary functions are apparently unconnected with development. PMID:7805288

  19. Detection of a major gene predisposing to human T lymphotropic virus type I infection in children among an endemic population of African origin.

    PubMed

    Plancoulaine, S; Gessain, A; Joubert, M; Tortevoye, P; Jeanne, I; Talarmin, A; de Thé, G; Abel, L

    2000-08-01

    Human T lymphotropic virus type I (HTLV-I) is a human oncoretrovirus that causes an adult T cell leukemia/lymphoma and a chronic neuromyelopathy. To investigate whether familial aggregation of HTLV-I infection (as determined by specific seropositive status) could be explained in part by genetic factors, we conducted a large genetic epidemiological survey in an HTLV-I-endemic population of African origin from French Guiana. All of the families in 2 villages were included, representing 83 pedigrees with 1638 subjects, of whom 165 (10.1%) were HTLV-I seropositive. The results of segregation analysis are consistent with the presence of a dominant major gene predisposing to HTLV-I infection, in addition to the expected familial correlations (mother-offspring, spouse-spouse) due to the virus transmission routes. Under this genetic model, approximately 1. 5% of the population is predicted to be highly predisposed to HTLV-I infection, and almost all seropositive children <10 years of age are genetic cases, whereas most HTLV-I seropositive adults are sporadic cases.

  20. Non-DBS DNA Repair Genes Regulate Radiation-induced Cytogenetic Damage Repair and Cell Cycle Progression

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Rohde, Larry H.; Emami, Kamal; Casey, Rachael; Wu, Honglu

    2008-01-01

    Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in DSB repair, and its impact on cytogenetic responses has not been systematically studied. In the present study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by transfection with small interfering RNA in human fibroblast cells. The purpose of this study is to identify new roles of these selected genes on regulating DSB repair and cell cycle progression , as measured in the micronuclei formation and chromosome aberration. In response to IR, the formation of MN was significantly increased by suppressed expression of 5 genes: Ku70 in the DSB repair pathway, XPA in the NER pathway, RPA1 in the MMR pathway, and RAD17 and RBBP8 in cell cycle control. Knocked-down expression of 4 genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Furthermore, loss of XPA, P21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Most of the 11 genes that affected cytogenetic responses are not known to have clear roles influencing DBS repair. Nine of these 11 genes were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate the biological consequences after IR.

  1. DNA mismatch repair gene mutations in human cancer.

    PubMed Central

    Peltomäki, P

    1997-01-01

    A new pathogenetic mechanism leading to cancer has been delineated in the past 3 years when human homologues of DNA mismatch repair (MMR) genes have been identified and shown to be involved in various types of cancer. Germline mutations of MMR genes cause susceptibility to a hereditary form of colon cancer, hereditary nonpolyposis colon cancer (HNPCC), which represents one of the most common syndromes associated with cancer predisposition in man. Tumors from HNPCC patients are hypermutable and show length variation at short tandem repeat sequences, a phenomenon referred to as microsatellite instability or replication errors. A similar abnormality is found in a proportion of sporadic tumors of the colorectum as well as a variety of other organs; acquired mutations in MMR genes or other endogenous or exogenous causes may underlie these cases. Genetic and biochemical characterization of the functions of normal and mutated MMR genes elucidates mechanisms of cancer development and provides tools for diagnostic applications. PMID:9255561

  2. Sensitized phenotypic screening identifies gene dosage sensitive region on chromosome 11 that predisposes to disease in mice

    PubMed Central

    Ermakova, Olga; Piszczek, Lukasz; Luciani, Luisa; Cavalli, Florence M G; Ferreira, Tiago; Farley, Dominika; Rizzo, Stefania; Paolicelli, Rosa Chiara; Al-Banchaabouchi, Mumna; Nerlov, Claus; Moriggl, Richard; Luscombe, Nicholas M; Gross, Cornelius

    2011-01-01

    The identification of susceptibility genes for human disease is a major goal of current biomedical research. Both sequence and structural variation have emerged as major genetic sources of phenotypic variability and growing evidence points to copy number variation as a particularly important source of susceptibility for disease. Here we propose and validate a strategy to identify genes in which changes in dosage alter susceptibility to disease-relevant phenotypes in the mouse. Our approach relies on sensitized phenotypic screening of megabase-sized chromosomal deletion and deficiency lines carrying altered copy numbers of ∼30 linked genes. This approach offers several advantages as a method to systematically identify genes involved in disease susceptibility. To examine the feasibility of such a screen, we performed sensitized phenotyping in five therapeutic areas (metabolic syndrome, immune dysfunction, atherosclerosis, cancer and behaviour) of a 0.8 Mb reciprocal chromosomal duplication and deficiency on chromosome 11 containing 27 genes. Gene dosage in the region significantly affected risk for high-fat diet-induced metabolic syndrome, antigen-induced immune hypersensitivity, ApoE-induced atherosclerosis, and home cage activity. Follow up studies on individual gene knockouts for two candidates in the region showed that copy number variation in Stat5 was responsible for the phenotypic variation in antigen-induced immune hypersensitivity and metabolic syndrome. These data demonstrate the power of sensitized phenotypic screening of segmental aneuploidy lines to identify disease susceptibility genes. PMID:21204268

  3. Regulation of the Saccharomyces cerevisiae DNA repair gene RAD16.

    PubMed Central

    Bang, D D; Timmermans, V; Verhage, R; Zeeman, A M; van de Putte, P; Brouwer, J

    1995-01-01

    The RAD16 gene product has been shown to be essential for the repair of the silenced mating type loci [Bang et al. (1992) Nucleic Acids Res. 20, 3925-3931]. More recently we demonstrated that the RAD16 and RAD7 proteins are also required for repair of non-transcribed strands of active genes in Saccharomyces cerevisiae [Waters et al. (1993) Mol. Gen. Genet. 239, 28-32]. We have studied the regulation of the RAD16 gene and found that the RAD16 transcript levels increased up to 7-fold upon UV irradiation. Heat shock at 42 degrees C also results in elevated levels of RAD16 mRNA. In sporulating MAT alpha/MATa diploid cells RAD16 mRNA is also induced. The basal level of the RAD16 transcript is constant during the mitotic cell cycle. G1-arrested cells show normal induction of RAD16 mRNA upon UV irradiation demonstrating that the induction is not a secondary consequence of G2 cell cycle arrest following UV irradiation. However, in cells arrested in G1 the induction of RAD16 mRNA after UV irradiation is not followed by a rapid decline as occurs in normal growing cells suggesting that the down regulation of RAD16 transcription is dependent on progression into the cell cycle. Images PMID:7784171

  4. Regulation of the Saccharomyces cerevisiae DNA repair gene RAD16.

    PubMed

    Bang, D D; Timmermans, V; Verhage, R; Zeeman, A M; van de Putte, P; Brouwer, J

    1995-05-25

    The RAD16 gene product has been shown to be essential for the repair of the silenced mating type loci [Bang et al. (1992) Nucleic Acids Res. 20, 3925-3931]. More recently we demonstrated that the RAD16 and RAD7 proteins are also required for repair of non-transcribed strands of active genes in Saccharomyces cerevisiae [Waters et al. (1993) Mol. Gen. Genet. 239, 28-32]. We have studied the regulation of the RAD16 gene and found that the RAD16 transcript levels increased up to 7-fold upon UV irradiation. Heat shock at 42 degrees C also results in elevated levels of RAD16 mRNA. In sporulating MAT alpha/MATa diploid cells RAD16 mRNA is also induced. The basal level of the RAD16 transcript is constant during the mitotic cell cycle. G1-arrested cells show normal induction of RAD16 mRNA upon UV irradiation demonstrating that the induction is not a secondary consequence of G2 cell cycle arrest following UV irradiation. However, in cells arrested in G1 the induction of RAD16 mRNA after UV irradiation is not followed by a rapid decline as occurs in normal growing cells suggesting that the down regulation of RAD16 transcription is dependent on progression into the cell cycle.

  5. Review: Clinical aspects of hereditary DNA Mismatch repair gene mutations.

    PubMed

    Sijmons, Rolf H; Hofstra, Robert M W

    2016-02-01

    Inherited mutations of the DNA Mismatch repair genes MLH1, MSH2, MSH6 and PMS2 can result in two hereditary tumor syndromes: the adult-onset autosomal dominant Lynch syndrome, previously referred to as Hereditary Non-Polyposis Colorectal Cancer (HNPCC) and the childhood-onset autosomal recessive Constitutional Mismatch Repair Deficiency syndrome. Both conditions are important to recognize clinically as their identification has direct consequences for clinical management and allows targeted preventive actions in mutation carriers. Lynch syndrome is one of the more common adult-onset hereditary tumor syndromes, with thousands of patients reported to date. Its tumor spectrum is well established and includes colorectal cancer, endometrial cancer and a range of other cancer types. However, surveillance for cancers other than colorectal cancer is still of uncertain value. Prophylactic surgery, especially for the uterus and its adnexa is an option in female mutation carriers. Chemoprevention of colorectal cancer with aspirin is actively being investigated in this syndrome and shows promising results. In contrast, the Constitutional Mismatch Repair Deficiency syndrome is rare, features a wide spectrum of childhood onset cancers, many of which are brain tumors with high mortality rates. Future studies are very much needed to improve the care for patients with this severe disorder. PMID:26746812

  6. Simulated microgravity influenced the expression of DNA damage repair genes

    NASA Astrophysics Data System (ADS)

    Zhang, Meng; Sun, Yeqing; Jiawei, Liu; Wang, Ting

    2016-07-01

    Ionizing radiation and microgravity were considered to be the most important stress factors of space environmental the respective study of the biological effects of the radiation and microgravity carried out earlier, but the interaction of the effects of radiation with microgravity started later, and due to difference of the materials and methods the result of this experiment were not consistent. To further investigate the influence of microgravity on the expression of the radiation damage repair genes, the seed of Arabidopsis (Col) and its gravity-insensitive mutant (PIN2) were exposed to 0.1Gy of the dose of energetic carbon-ion beam radiation (LET = 30KeV / μm), and the germinated seed were than fixed in the 3D random positioning apparatus immediately for a 10-day simulated microgravity. By measuring the deflection angle of root tip and the changes of the expression of Ku70 and RAD51 protein, we investigated the impact of microgravity effect on radiation damage repair systems. The results shown that radiation, microgravity and microgravity with radiation could increase the angle of the root of the Col significantly, but no obvious effect on PIN2 type. The radiation could increase the expression of Ku70 significantly in both Col and PIN2, microgravity does not affect the expression, but the microgravity with radiation could decrease the expression of Ku70. This result shown that the microgravity could influence the radiation damage repair systems in molecular level. Moreover, our findings were important to understand the molecular mechanism of the impact of microgravity effect on radiation damage repair systems in vivo.

  7. POLE mutations in families predisposed to cutaneous melanoma.

    PubMed

    Aoude, Lauren G; Heitzer, Ellen; Johansson, Peter; Gartside, Michael; Wadt, Karin; Pritchard, Antonia L; Palmer, Jane M; Symmons, Judith; Gerdes, Anne-Marie; Montgomery, Grant W; Martin, Nicholas G; Tomlinson, Ian; Kearsey, Stephen; Hayward, Nicholas K

    2015-12-01

    Germline mutations in the exonuclease domain of POLE have been shown to predispose to colorectal cancers and adenomas. POLE is an enzyme involved in DNA repair and chromosomal DNA replication. In order to assess whether such mutations might also predispose to cutaneous melanoma, we interrogated whole-genome and exome data from probands of 34 melanoma families lacking pathogenic mutations in known high penetrance melanoma susceptibility genes: CDKN2A, CDK4, BAP1, TERT, POT1, ACD and TERF2IP. We found a novel germline mutation, POLE p.(Trp347Cys), in a 7-case cutaneous melanoma family. Functional assays in S. pombe showed that this mutation led to an increased DNA mutation rate comparable to that seen with a Pol ε mutant with no exonuclease activity. We then performed targeted sequencing of POLE in 1243 cutaneous melanoma cases and found that a further ten probands had novel or rare variants in the exonuclease domain of POLE. Although this frequency is not significantly higher than that in unselected Caucasian controls, we observed multiple cancer types in the melanoma families, suggesting that some germline POLE mutations may predispose to a broad spectrum of cancers, including melanoma. In addition, we found the first mutation outside the exonuclease domain, p.(Gln520Arg), in a family with an extensive history of colorectal cancer.

  8. POLE mutations in families predisposed to cutaneous melanoma.

    PubMed

    Aoude, Lauren G; Heitzer, Ellen; Johansson, Peter; Gartside, Michael; Wadt, Karin; Pritchard, Antonia L; Palmer, Jane M; Symmons, Judith; Gerdes, Anne-Marie; Montgomery, Grant W; Martin, Nicholas G; Tomlinson, Ian; Kearsey, Stephen; Hayward, Nicholas K

    2015-12-01

    Germline mutations in the exonuclease domain of POLE have been shown to predispose to colorectal cancers and adenomas. POLE is an enzyme involved in DNA repair and chromosomal DNA replication. In order to assess whether such mutations might also predispose to cutaneous melanoma, we interrogated whole-genome and exome data from probands of 34 melanoma families lacking pathogenic mutations in known high penetrance melanoma susceptibility genes: CDKN2A, CDK4, BAP1, TERT, POT1, ACD and TERF2IP. We found a novel germline mutation, POLE p.(Trp347Cys), in a 7-case cutaneous melanoma family. Functional assays in S. pombe showed that this mutation led to an increased DNA mutation rate comparable to that seen with a Pol ε mutant with no exonuclease activity. We then performed targeted sequencing of POLE in 1243 cutaneous melanoma cases and found that a further ten probands had novel or rare variants in the exonuclease domain of POLE. Although this frequency is not significantly higher than that in unselected Caucasian controls, we observed multiple cancer types in the melanoma families, suggesting that some germline POLE mutations may predispose to a broad spectrum of cancers, including melanoma. In addition, we found the first mutation outside the exonuclease domain, p.(Gln520Arg), in a family with an extensive history of colorectal cancer. PMID:26251183

  9. Polymorphism of the DNA Base Excision Repair Genes in Keratoconus

    PubMed Central

    Wojcik, Katarzyna A.; Synowiec, Ewelina; Sobierajczyk, Katarzyna; Izdebska, Justyna; Blasiak, Janusz; Szaflik, Jerzy; Szaflik, Jacek P.

    2014-01-01

    Keratoconus (KC) is a degenerative corneal disorder for which the exact pathogenesis is not yet known. Oxidative stress is reported to be associated with this disease. The stress may damage corneal biomolecules, including DNA, and such damage is primarily removed by base excision repair (BER). Variation in genes encoding BER components may influence the effectiveness of corneal cells to cope with oxidative stress. In the present work we genotyped 5 polymorphisms of 4 BER genes in 284 patients and 353 controls. The A/A genotype of the c.–1370T>A polymorphism of the DNA polymerase γ (POLG) gene was associated with increased occurrence of KC, while the A/T genotype was associated with decreased occurrence of KC. The A/G genotype and the A allele of the c.1196A>G polymorphism of the X-ray repair cross-complementing group 1 (XRCC1) were associated with increased, and the G/G genotype and the G allele, with decreased KC occurrence. Also, the C/T and T as well as C/C genotypes and alleles of the c.580C>T polymorphism of the same gene displayed relationship with KC occurrence. Neither the g.46438521G>C polymorphism of the Nei endonuclease VIII-like 1 (NEIL1) nor the c.2285T>C polymorphism of the poly(ADP-ribose) polymerase-1 (PARP-1) was associated with KC. In conclusion, the variability of the XRCC1 and POLG genes may play a role in KC pathogenesis and determine the risk of this disease. PMID:25356504

  10. Effect of GDNF on depressive-like behavior, spatial learning and key genes of the brain dopamine system in genetically predisposed to behavioral disorders mouse strains.

    PubMed

    Naumenko, Vladimir S; Kondaurova, Elena M; Bazovkina, Daria V; Tsybko, Anton S; Ilchibaeva, Tatyana V; Khotskin, Nikita V; Semenova, Alina A; Popova, Nina K

    2014-11-01

    The effect of glial cell line-derived neurotrophic factor (GDNF) on behavior and brain dopamine system in predisposed to depressive-like behavior ASC (Antidepressant Sensitive Cataleptics) mice in comparison with the parental "nondepressive" CBA mice was studied. In 7days after administration (800ng, i.c.v.) GDNF decreased escape latency time and the path traveled to reach hidden platform in Morris water maze in ASC mice. GDNF enhanced depressive-like behavioral traits in both "nondepressive" CBA and "depressive" ASC mice. In CBA mice, GDNF decreased functional response to agonists of D1 (chloro-APB hydrobromide) and D2 (sumanirole maleate) receptors in tail suspension test, reduced D2 receptor gene expression in the substantia nigra and increased monoamine oxydase A (MAO A) gene expression in the striatum. GDNF increased D1 and D2 receptor genes expression in the nucleus accumbens of ASC mice but failed to alter expression of catechol-O-methyltransferase, dopamine transporter, MAO B and tyrosine hydroxylase genes in both investigated mouse strains. Thus, GDNF produced long-term genotype-dependent effect on behavior and the brain dopamine system. GDNF pretreatment (1) reduced D1 and D2 receptors functional responses and D2 receptor gene expression in s. nigra of CBA mice; (2) increased D1 and D2 receptor genes expression in n. accumbens of ASC mice and (3) improved spatial learning in ASC mice. GDNF enhanced depressive-like behavior both in CBA and ASC mice. The data suggest that genetically defined variance in the cross-talk between GDNF and brain dopamine system contributes to the variability of GDNF-induced responses and might be responsible for controversial GDNF effects.

  11. The Contribution of Whole Gene Deletions and Large Rearrangements to the Mutation Spectrum in Inherited Tumor Predisposing Syndromes.

    PubMed

    Smith, Miriam J; Urquhart, Jill E; Harkness, Elaine F; Miles, Emma K; Bowers, Naomi L; Byers, Helen J; Bulman, Michael; Gokhale, Carolyn; Wallace, Andrew J; Newman, William G; Evans, D Gareth

    2016-03-01

    Heterozygous whole gene deletions (WGDs), and intragenic microdeletions, account for a significant proportion of mutations underlying cancer predisposition syndromes. We analyzed the frequency and genotype-phenotype correlations of microdeletions in 12 genes (BRCA1, BRCA2, TP53, MSH2, MLH1, MSH6, PMS2, NF1, NF2, APC, PTCH1, and VHL) representing seven tumor predisposition syndromes in 5,897 individuals (2,611 families) from our center. Overall, microdeletions accounted for 14% of identified mutations. As expected, smaller deletions or duplications were more common (12%) than WGDs (2.2%). Where a WGD was identified in the germline in NF2, the mechanism of somatic second hit was not deletion, as previously described for NF1. For neurofibromatosis type 1 and 2, we compared the mechanism of germline deletion. Unlike NF1, where three specific deletion sizes account for most germline WGDs, NF2 deletion breakpoints were different across seven samples tested. One of these deletions was 3.93 Mb and conferred a severe phenotype, thus refining the region for a potential NF2 modifier gene to a 2.04-Mb region on chromosome 22. The milder phenotype of NF2 WGDs may be due to the apparent absence of chromosome 22 loss as the second hit. These observations of WGD phenotypes will be helpful for interpreting incidental findings from microarray analysis and next-generation sequencing. PMID:26615784

  12. A genetic association study of maternal and fetal candidate genes that predispose to preterm prelabor rupture of membranes (PROM)

    PubMed Central

    ROMERO, Roberto; FRIEL, Lara A.; EDWARDS, Digna R. VELEZ; KUSANOVIC, Juan Pedro; HASSAN, Sonia S.; MAZAKI-TOVI, Shali; VAISBUCH, Edi; KIM, Chong Jai; EREZ, Offer; CHAIWORAPONGSA, Tinnakorn; PEARCE, Brad D.; BARTLETT, Jacquelaine; SALISBURY, Benjamin A.; ANANT, Madan Kumar; VOVIS, Gerald F.; LEE, Min Seob; GOMEZ, Ricardo; BEHNKE, Ernesto; OYARZUN, Enrique; TROMP, Gerard; WILLIAMS, Scott M.; MENON, Ramkumar

    2010-01-01

    Objective To determine whether maternal/fetal SNPs in candidate genes are associated with preterm prelabor rupture of membranes (pPROM). Study Design A case-control study was conducted in patients with pPROM (225 mothers and 155 fetuses) and 599 mothers and 628 fetuses with a normal pregnancy; 190 candidate genes and 775 SNPs were studied. Single locus/haplotype association analyses were performed; FDR was used to correct for multiple testing (q*=0.15)]. Results 1) A SNP in TIMP2 in mothers was significantly associated with pPROM(OR=2.12 95% CI [1.47-3.07], p = 0.000068), and this association remained significant after correction for multiple comparisons; 2) Haplotypes for COL4A3 in the mother were associated with pPROM (global p = 0.003); 3) Multilocus analysis identified a three locus model, which included maternal SNPs in COL1A2, DEFA5, and EDN1. Conclusion DNA variants in a maternal gene involved in extracellular matrix metabolism doubled the risk of pPROM. PMID:20673868

  13. Identification of Fetal and Maternal Single Nucleotide Polymorphisms in Candidate Genes That Predispose to Spontaneous Preterm Labor with Intact Membranes

    PubMed Central

    Romero, Roberto; Velez, Digna R.; Kusanovic, Juan Pedro; Hassan, Sonia S.; Mazaki-Tovi, Shali; Vaisbuch, Edi; Kim, Chong Jai; Chaiworapongsa, Tinnakorn; Pearce, Brad; Friel, Lara A.; Bartlett, Jacquelaine; Anant, Madan Kumar; Salisbury, Benjamin A.; Vovis, Gerald F.; Lee, Min Seob; Gomez, Ricardo; Behnke, Ernesto; Oyarzun, Enrique; Tromp, Gerard; Williams, Scott M.; Menon, Ramkumar

    2013-01-01

    Objective To determine whether maternal/fetal SNPs in candidate genes are associated with spontaneous preterm labor/delivery. Study Design A genetic association study was conducted in 223 mothers and 179 fetuses [preterm labor with intact membranes who delivered <37 weeks (PTB)], and 599 mothers and 628 fetuses (normal pregnancy): 190 candidate genes and 775 SNPs were studied. Single locus/haplotype association analyses were performed; FDR was used to correct for multiple testing (q*=0.15)]. Results 1) The strongest single locus associations with PTB were IL6R (fetus: p=0.000148) and TIMP2 (mother: p=0.000197), remaining significant after correction for multiple comparisons; 2) Global haplotype analysis indicated an association between a fetal DNA variant in IGF2 and maternal COL4A3 (global p=0.004 and 0.007, respectively). Conclusion A SNP involved in controlling fetal inflammation (IL6R) and DNA variants in maternal genes encoding for proteins involved in extracellular matrix biology approximately doubled the risk of PTB. PMID:20452482

  14. Does 24bp Duplication of Human CHIT1 Gene (Chitotriosidase1) Predispose to Filarial Chyluria? A Case-Control Study

    PubMed Central

    Pant, Shriya; Agarwal, Jyotsna; Gangwar, Pravin K; Waseem, Mohammad; Gupta, Prashant; Sankhwar, Satya N; Purkait, Bimalesh

    2016-01-01

    Introduction Chyluria which is endemic in many parts of the world is mainly caused by Wuchereria bancrofti. CHIT1 (chitotriosidase) is produced by macrophages and plays an important role in the defense against chitin containing pathogen such as filarial parasite. Variation in the coding region with 24 bp duplication allele results in reduced CHIT1 activity that enhance the survival of parasite which may play a role in the occurrence of disease. Aim To examine the role of 24bp duplication of CHIT1 gene in patients of filarial chyluria (FC). Materials and Methods A case-control study was carried out where 155 confirmed FC patients and equal number of age-, sex- and residence-matched controls without any symptoms or signs of lymphatic filariasis, confirmed by negative immunochromatographic card test (ICT) and IgG/IgM combo rapid antibody test, from a hospital-based population were enrolled. Filarial aetiology was confirmed on the basis of DEC-provocative test (Giemsa staining), ICT and IgG/IgM- antifiarial antibody test. The patients positive by either of these tests were enrolled as FC cases. 24bp duplication in CHIT1 gene in FC was detected by the product size 99bp of amplified gene using polymerase chain reaction. Results The mean ages of patients and controls were 38.25±12.09 and 35.45±12.53 years, respectively while male: female ratio was 2.4:1. The mean duration of illness in chyluria patients was 62.81±60.83 months and mean number of episodes was 2.54±1.11. Homozygous wild type, heterozygous and homozygous mutant frequencies were 10.3%, 81.3% and 8.4% in FC patients and 18.7%, 75.5%, and 5.8% in controls, respectively. The 24bp duplication in CHIT1 gene showed a significant association in Heterozygous (HT) genotype with Odd Ratio (OR) of 1.95, 95% Confidence Interval (CI) (1.01-3.77); p=0.04. However, the homozygous mutant genotype (TT) was found to be non-significant with OR of 2.61, 95% CI (0.91-7.45); p=0.07. The combination of both HT+TT was also found

  15. Whole exome sequencing in families at high risk for Hodgkin lymphoma: identification of a predisposing mutation in the KDR gene

    PubMed Central

    Rotunno, Melissa; McMaster, Mary L.; Boland, Joseph; Bass, Sara; Zhang, Xijun; Burdett, Laurie; Hicks, Belynda; Ravichandran, Sarangan; Luke, Brian T.; Yeager, Meredith; Fontaine, Laura; Hyland, Paula L.; Goldstein, Alisa M.; Chanock, Stephen J.; Caporaso, Neil E.; Tucker, Margaret A.; Goldin, Lynn R.

    2016-01-01

    Hodgkin lymphoma shows strong familial aggregation but no major susceptibility genes have been identified to date. The goal of this study was to identify high-penetrance variants using whole exome sequencing in 17 Hodgkin lymphoma prone families with three or more affected cases or obligate carriers (69 individuals), followed by targeted sequencing in an additional 48 smaller HL families (80 individuals). Alignment and variant calling were performed using standard methods. Dominantly segregating, rare, coding or potentially functional variants were further prioritized based on predicted deleteriousness, conservation, and potential importance in lymphoid malignancy pathways. We selected 23 genes for targeted sequencing. Only the p.A1065T variant in KDR (kinase insert domain receptor) also known as VEGFR2 (vascular endothelial growth factor receptor 2) was replicated in two independent Hodgkin lymphoma families. KDR is a type III receptor tyrosine kinase, the main mediator of vascular endothelial growth factor induced proliferation, survival, and migration. Its activity is associated with several diseases including lymphoma. Functional experiments have shown that p.A1065T, located in the activation loop, can promote constitutive autophosphorylation on tyrosine in the absence of vascular endothelial growth factor and that the kinase activity was abrogated after exposure to kinase inhibitors. A few other promising mutations were identified but appear to be “private”. In conclusion, in the largest sequenced cohort of Hodgkin lymphoma families to date, we identified a causal mutation in the KDR gene. While independent validation is needed, this mutation may increase downstream tumor cell proliferation activity and might be a candidate for targeted therapy. PMID:27365461

  16. Whole exome sequencing in families at high risk for Hodgkin lymphoma: identification of a predisposing mutation in the KDR gene.

    PubMed

    Rotunno, Melissa; McMaster, Mary L; Boland, Joseph; Bass, Sara; Zhang, Xijun; Burdett, Laurie; Hicks, Belynda; Ravichandran, Sarangan; Luke, Brian T; Yeager, Meredith; Fontaine, Laura; Hyland, Paula L; Goldstein, Alisa M; Chanock, Stephen J; Caporaso, Neil E; Tucker, Margaret A; Goldin, Lynn R

    2016-07-01

    Hodgkin lymphoma shows strong familial aggregation but no major susceptibility genes have been identified to date. The goal of this study was to identify high-penetrance variants using whole exome sequencing in 17 Hodgkin lymphoma prone families with three or more affected cases or obligate carriers (69 individuals), followed by targeted sequencing in an additional 48 smaller HL families (80 individuals). Alignment and variant calling were performed using standard methods. Dominantly segregating, rare, coding or potentially functional variants were further prioritized based on predicted deleteriousness, conservation, and potential importance in lymphoid malignancy pathways. We selected 23 genes for targeted sequencing. Only the p.A1065T variant in KDR (kinase insert domain receptor) also known as VEGFR2 (vascular endothelial growth factor receptor 2) was replicated in two independent Hodgkin lymphoma families. KDR is a type III receptor tyrosine kinase, the main mediator of vascular endothelial growth factor induced proliferation, survival, and migration. Its activity is associated with several diseases including lymphoma. Functional experiments have shown that p.A1065T, located in the activation loop, can promote constitutive autophosphorylation on tyrosine in the absence of vascular endothelial growth factor and that the kinase activity was abrogated after exposure to kinase inhibitors. A few other promising mutations were identified but appear to be "private". In conclusion, in the largest sequenced cohort of Hodgkin lymphoma families to date, we identified a causal mutation in the KDR gene. While independent validation is needed, this mutation may increase downstream tumor cell proliferation activity and might be a candidate for targeted therapy. PMID:27365461

  17. ApcMin, A Mutation in the Murine Apc Gene, Predisposes to Mammary Carcinomas and Focal Alveolar Hyperplasias

    NASA Astrophysics Data System (ADS)

    Moser, Amy Rapaich; Mattes, Ellen M.; Dove, William F.; Lindstrom, Mary J.; Haag, Jill D.; Gould, Michael N.

    1993-10-01

    ApcMin (Min, multiple intestinal neoplasia) is a point mutation in the murine homolog of the APC gene. Min/+ mice develop multiple intestinal adenomas, as do humans carrying germ-line mutations in APC. Female mice carrying Min are also prone to develop mammary tumors. Min/+ mammary glands are more sensitive to chemical carcinogenesis than are +/+ mammary glands. Transplantation of mammary cells from Min/+ or +/+ donors into +/+ hosts demonstrates that the propensity to develop mammary tumors is intrinsic to the Min/+ mammary cells. Long-term grafts of Min/+ mammary glands also gave rise to focal alveolar hyperplasias, indicating that the presence of the Min mutation also has a role in the development of these lesions.

  18. The RETN gene rs1862513 polymorphism as a novel predisposing marker for familial Acne vulgaris in a Pakistani population.

    PubMed

    Hussain, Sabir; Faraz, Ahmad; Iqbal, Tahir

    2015-05-01

    Resistin (RETN), recently found to be relevant to inflammation and inflammatory disorders. We, therefore, aimed to investigate the potential role of RETN gene polymorphism in pathogenesis of acne vulgaris with familial history. We investigated the RETN-420C/G polymorphism in 180 patients with acne vulgaris and 180 healthy individuals in a case-control association analysis. In this study, we also investigated the heritability of the RETN susceptible allele from 140 trio families with acne affected offspring. The genotyping was performed by polymerase chain reaction and direct DNA sequencing. The RETN-420C/G polymorphism was significantly associated with acne in patients compared with healthy controls (P=0.014). The minor allele G at -420 was more prevalent in cases vs. controls (P=0.002). The RETN-420C/G polymorphism was significantly associated with severity of acne vulgaris in patients (P=0.0097). The results of a transmission disequilibrium test revealed a significant association between the RETN-420C/G polymorphism and acne vulgaris (P<0.001). For the first time in the literature, to our knowledge, we demonstrate a significant association of the RETN-420C/G functional polymorphism with familial acne vulgaris.

  19. Prevalent and rare mutations in the gene encoding filaggrin cause ichthyosis vulgaris and predispose individuals to atopic dermatitis.

    PubMed

    Sandilands, Aileen; O'Regan, Gráinne M; Liao, Haihui; Zhao, Yiwei; Terron-Kwiatkowski, Ana; Watson, Rosemarie M; Cassidy, Andrew J; Goudie, David R; Smith, Frances J D; McLean, W H Irwin; Irvine, Alan D

    2006-08-01

    Mutations in the filament aggregating protein (filaggrin) gene have recently been identified as the cause of the common genetic skin disorder ichthyosis vulgaris (IV), the most prevalent inherited disorder of keratinization. The main characteristics of IV are fine-scale on the arms and legs, palmar hyperlinearity, and keratosis pilaris. Here, we have studied six Irish families with IV for mutations in filaggrin. We have identified a new mutation, 3702delG, in addition to further instances of the reported mutations R501X and 2282del4, which are common in people of European origin. A case of a 2282del4 homozygote was also identified. Mutation 3702delG terminates protein translation in filaggrin repeat domain 3, whereas both recurrent mutations occur in repeat 1. These mutations are semidominant: heterozygotes have an intermediate phenotype most readily identified by palmar hyperlinearity and in some cases fine-scale and/or keratosis pilaris, whereas homozygotes or compound heterozygotes generally have more marked ichthyosis. Interestingly, the phenotypes of individuals homozygous for R501X, 2282del4, or compound heterozygous for R501X and 3702delG, were comparable, suggesting that mutations located centrally in the filaggrin repeats are also pathogenic.

  20. Immortalized neural progenitor cells for CNS gene transfer and repair.

    PubMed

    Martínez-Serrano, A; Björklund, A

    1997-11-01

    Immortalized multipotent neural stem and progenitor cells have emerged as a highly convenient source of tissue for genetic manipulation and ex vivo gene transfer to the CNS. Recent studies show that these cells, which can be maintained and genetically transduced as cell lines in culture, can survive, integrate and differentiate into both neurons and glia after transplantation to the intact or damaged brain. Progenitors engineered to secrete trophic factors, or to produce neurotransmitter-related or metabolic enzymes can be made to repopulate diseased or injured brain areas, thus providing a new potential therapeutic tool for the blockade of neurodegenerative processes and reversal of behavioural deficits in animal models of neurodegenerative diseases. With further technical improvements, the use of immortalized neural progenitors may bring us closer to the challenging goal of targeted and effective CNS repair.

  1. Energy and Technology Review: Unlocking the mysteries of DNA repair

    SciTech Connect

    Quirk, W.A.

    1993-04-01

    DNA, the genetic blueprint, has the remarkable property of encoding its own repair following diverse types of structural damage induced by external agents or normal metabolism. We are studying the interplay of DNA damaging agents, repair genes, and their protein products to decipher the complex biochemical pathways that mediate such repair. Our research focuses on repair processes that correct DNA damage produced by chemical mutagens and radiation, both ionizing and ultraviolet. The most important type of DNA repair in human cells is called excision repair. This multistep process removes damaged or inappropriate pieces of DNA -- often as a string of 29 nucleotides containing the damage -- and replaces them with intact ones. We have isolated, cloned, and mapped several human repair genes associated with the nucleotide excision repair pathway and involved in the repair of DNA damage after exposure to ultraviolet light or mutagens in cooked food. We have shown that a defect in one of these repair genes, ERCC2, is responsible for the repair deficiency in one of the groups of patients with the recessive genetic disorder xeroderma pigmentosum (XP group D). We are exploring ways to purify sufficient quantities (milligrams) of the protein products of these and other repair genes so that we can understand their functions. Our long-term goals are to link defective repair proteins to human DNA repair disorders that predispose to cancer, and to produce DNA-repair-deficient mice that can serve as models for the human disorders.

  2. Disruption of the Cockayne syndrome B gene impairs spontaneous tumorigenesis in cancer-predisposed Ink4a/ARF knockout mice.

    PubMed

    Lu, Y; Lian, H; Sharma, P; Schreiber-Agus, N; Russell, R G; Chin, L; van der Horst, G T; Bregman, D B

    2001-03-01

    Cells isolated from individuals with Cockayne syndrome (CS) have a defect in transcription-coupled DNA repair, which rapidly corrects certain DNA lesions located on the transcribed strand of active genes. Despite this DNA repair defect, individuals with CS group A (CSA) or group B (CSB) do not exhibit an increased spontaneous or UV-induced cancer rate. In order to investigate the effect of CSB deficiency on spontaneous carcinogenesis, we crossed CSB(-/-) mice with cancer-prone mice lacking the p16(Ink4a)/p19(ARF) tumor suppressor locus. CSB(-/-) mice are sensitive to UV-induced skin cancer but show no increased rate of spontaneous cancer. CSB(-/-) Ink4a/ARF(-/-) mice developed 60% fewer tumors than Ink4a/ARF(-/-) animals and demonstrated a longer tumor-free latency time (260 versus 150 days). Moreover, CSB(-/-) Ink4a/ARF(-/-) mouse embryo fibroblasts (MEFs) exhibited a lower colony formation rate after low-density seeding, a lower rate of H-Ras-induced transformation, slower proliferation, and a lower mRNA synthesis rate than Ink4a/ARF(-/-) MEFs. CSB(-/-) Ink4a/ARF(-/-) MEFs were also more sensitive to UV-induced p53 induction and UV-induced apoptosis than were Ink4a/ARF(-/-) MEFs. In order to investigate whether the apparent antineoplastic effect of CSB gene disruption was caused by sensitization to genotoxin-induced (p53-mediated) apoptosis or by p53-independent sequelae, we also generated p53(-/-) and CSB(-/-) p53(-/-) MEFs. The CSB(-/-) p53(-/-) MEFs demonstrated lower colony formation efficiency, a lower proliferation rate, a lower mRNA synthesis rate, and a higher rate of UV-induced cell death than p53(-/-) MEFs. Collectively, these results indicate that the antineoplastic effect of CSB gene disruption is at least partially p53 independent; it may result from impaired transcription or from apoptosis secondary to environmental or endogenous DNA damage. PMID:11238917

  3. Lifespan and Stress Resistance in Drosophila with Overexpressed DNA Repair Genes

    PubMed Central

    Shaposhnikov, Mikhail; Proshkina, Ekaterina; Shilova, Lyubov; Zhavoronkov, Alex; Moskalev, Alexey

    2015-01-01

    DNA repair declines with age and correlates with longevity in many animal species. In this study, we investigated the effects of GAL4-induced overexpression of genes implicated in DNA repair on lifespan and resistance to stress factors in Drosophila melanogaster. Stress factors included hyperthermia, oxidative stress, and starvation. Overexpression was either constitutive or conditional and either ubiquitous or tissue-specific (nervous system). Overexpressed genes included those involved in recognition of DNA damage (homologs of HUS1, CHK2), nucleotide and base excision repair (homologs of XPF, XPC and AP-endonuclease-1), and repair of double-stranded DNA breaks (homologs of BRCA2, XRCC3, KU80 and WRNexo). The overexpression of different DNA repair genes led to both positive and negative effects on lifespan and stress resistance. Effects were dependent on GAL4 driver, stage of induction, sex, and role of the gene in the DNA repair process. While the constitutive/neuron-specific and conditional/ubiquitous overexpression of DNA repair genes negatively impacted lifespan and stress resistance, the constitutive/ubiquitous and conditional/neuron-specific overexpression of Hus1, mnk, mei-9, mus210, and WRNexo had beneficial effects. This study demonstrates for the first time the effects of overexpression of these DNA repair genes on both lifespan and stress resistance in D. melanogaster. PMID:26477511

  4. Lifespan and Stress Resistance in Drosophila with Overexpressed DNA Repair Genes.

    PubMed

    Shaposhnikov, Mikhail; Proshkina, Ekaterina; Shilova, Lyubov; Zhavoronkov, Alex; Moskalev, Alexey

    2015-01-01

    DNA repair declines with age and correlates with longevity in many animal species. In this study, we investigated the effects of GAL4-induced overexpression of genes implicated in DNA repair on lifespan and resistance to stress factors in Drosophila melanogaster. Stress factors included hyperthermia, oxidative stress, and starvation. Overexpression was either constitutive or conditional and either ubiquitous or tissue-specific (nervous system). Overexpressed genes included those involved in recognition of DNA damage (homologs of HUS1, CHK2), nucleotide and base excision repair (homologs of XPF, XPC and AP-endonuclease-1), and repair of double-stranded DNA breaks (homologs of BRCA2, XRCC3, KU80 and WRNexo). The overexpression of different DNA repair genes led to both positive and negative effects on lifespan and stress resistance. Effects were dependent on GAL4 driver, stage of induction, sex, and role of the gene in the DNA repair process. While the constitutive/neuron-specific and conditional/ubiquitous overexpression of DNA repair genes negatively impacted lifespan and stress resistance, the constitutive/ubiquitous and conditional/neuron-specific overexpression of Hus1, mnk, mei-9, mus210, and WRNexo had beneficial effects. This study demonstrates for the first time the effects of overexpression of these DNA repair genes on both lifespan and stress resistance in D. melanogaster. PMID:26477511

  5. A new nucleotide-excision-repair gene associated with the disorder trichothiodystrophy

    SciTech Connect

    Stefanini, M.; Giliani, S. ); Vermuelen, W.; Weeda, G.; Hoeijmakers, H.J.; Mezzina, M.; Sarasin, A.; Harper, J.I.; Arlett, C.F.; Lehmann, A.R.

    1993-10-01

    The sun-sensitive, cancer-prone genetic disorder xeroderma pigmentosum (XP) is associated in most cases with a defect in the ability to carry out excision repair of UV damage. Seven genetically distinct complementation groups (i.e., A-G) have been identified. A large proportion of patients with the unrelated disorder trichothiodystrophy (TTD), which is characterized by hair-shaft abnormalities, as well as by physical and mental retardation, are also deficient in excision repair of UV damage. In most of these cases the repair deficiency is in the same complementation group as is XP group D. The authors report here on cells from a patient, TTD1BR, in which the repair defect complements all known XP groups (including XP-D). Furthermore, microinjection of various cloned human repair genes fails to correct the repair defect in this cell strain. The defect in TTD1BR cells is therefore in a new gene involved in excision repair in human cells. The finding of a second DNA repair gene that is associated with the clinical features of TTD argues strongly for an involvement of repair proteins in hair-shaft development. 20 refs., 2 figs., 1 tab.

  6. Triple Negative Breast Cancers Have a Reduced Expression of DNA Repair Genes

    PubMed Central

    Andreis, Daniele; Bertoni, Ramona; Giardini, Roberto; Fox, Stephen B.; Broggini, Massimo; Bottini, Alberto; Zanoni, Vanessa; Bazzola, Letizia; Foroni, Chiara; Generali, Daniele; Damia, Giovanna

    2013-01-01

    DNA repair is a key determinant in the cellular response to therapy and tumor repair status could play an important role in tailoring patient therapy. Our goal was to evaluate the mRNA of 13 genes involved in different DNA repair pathways (base excision, nucleotide excision, homologous recombination, and Fanconi anemia) in paraffin embedded samples of triple negative breast cancer (TNBC) compared to luminal A breast cancer (LABC). Most of the genes involved in nucleotide excision repair and Fanconi Anemia pathways, and CHK1 gene were significantly less expressed in TNBC than in LABC. PARP1 levels were higher in TNBC than in LABC. In univariate analysis high level of FANCA correlated with an increased overall survival and event free survival in TNBC; however multivariate analyses using Cox regression did not confirm FANCA as independent prognostic factor. These data support the evidence that TNBCs compared to LABCs harbour DNA repair defects. PMID:23825533

  7. Clinical features and mismatch repair gene mutation screening in Chinese patients with hereditary nonpolyposis colorectal carcinoma

    PubMed Central

    Liu, Shan-Run; Zhao, Bo; Wang, Zhen-Jun; Wan, Yuan-Lian; Huang, Yan-Ting

    2004-01-01

    AIM: Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominantly-inherited cancer-susceptibility syndrome that confers an increased risk for colorectal cancer and a variety of other tumors at a young age. It has been associated with germline mutations in five mismatch repair (MMR) genes (hMSH2, hMLH1, hPMS1, hPMS2, and hMSH6/GTBP). The great majority of germline mutations were found in hMSH2 and hMLH1. The purpose of this study was to analyze the clinical features of Chinese HNPCC patients and to screen hMSH2 and hMLH1 gene mutations. METHODS: Twenty-eight independent Chinese families were collected, of which 15 met Amsterdam criteria I and 13 met the Japanese clinical diagnosis criteria. The data were recorded including sex, site of colorectal cancer (CRC), age of diagnosis, history of synchronous and/or metachronous CRC, instance of extracolonic cancers, and histopathology of tumors. Peripheral blood samples were collected from all pedigrees after formal written consents were signed. PCR and denaturing high-performance liquid chromatography (DHPLC) were used to screen the coding regions of hMSH2 and hMLH1 genes. The samples showing abnormal DHPLC profiles were sequenced by a 377 DNA sequencer. RESULTS: One hundred and seventy malignant neoplasms were found in one hundred and twenty-six patients (multiple cancer in twenty-three), including one hundred and twenty-seven CRCs, fifteen gastric, seven endometrial, and five esophageal cancers. Seventy-seven point eight percent of the patients had CRCs, sharing the features of early occurrence (average age of onset, 45.9 years) and of the right-sided predominance reported in the literature. In Chinese HNPCC patients, gastric cancer occurred more frequently, accounting for 11.9% of all cancers patients and ranking second in the spectrum of HNPCC predisposing cancers. Synchronous CRCs occurred less frequently, only accounting for 3.1% of the total CRCs. Twenty percent of the colorectal patients had

  8. Expression profiles of DNA repair-related genes in rat target organs under subchronic cadmium exposure.

    PubMed

    Lei, Y X; Lu, Q; Shao, C; He, C C; Lei, Z N; Lian, Y Y

    2015-01-01

    We aimed to evaluate the toxicity of long-term exposure to different cadmium (Cd) doses in rats and expression profiles of DNA repair-related genes. The model rats were exposed to different concentrations of CdCl2 for 3 months, and 5 DNA repair-related genes - hMSH2, MLH1, XRCC1, hOGG1, ERCC1 - were cloned in different tissues, including the liver, kidney, heart, and lung. Accumulated amounts of Cd were detected in the tissues. Gene and protein detections were conducted via fluorescence quantitative real-time polymerase chain reaction and Western blotting, respectively. Methylated sequences of the 5 DNA repair-related gene promoters were used to investigate whether the low expression levels of the genes were related to methylation of the promoter. In the Cd-exposed group, 3 DNA repair genes (i.e., XRCC1, hOGG1, and ERCC1) significantly decreased in the rat liver, kidney, heart, and lung according to the β-actin internal standard (P < 0.01). Western blotting indicated the same trend for the different tissues. Each of the DNA repair genes had special characteristics; for example, hOGG1 gene expression decreased by 75% in the kidney, and XRCC1 gene expression decreased by 5% in the liver and heart when compared to the control group (P < 0.01). A negative correlation between the DNA repair gene expression levels and the cumulative levels of Cd was also suggested by malignancy pathology. The expression levels of 3 DNA repair genes (i.e., ERCC1, XRCC1, and hOGG1) played an important role in the rat response to Cd exposure but not DNA methylated protection. PMID:25729986

  9. Genes and Junk in Plant Mitochondria—Repair Mechanisms and Selection

    PubMed Central

    Christensen, Alan C.

    2014-01-01

    Plant mitochondrial genomes have very low mutation rates. In contrast, they also rearrange and expand frequently. This is easily understood if DNA repair in genes is accomplished by accurate mechanisms, whereas less accurate mechanisms including nonhomologous end joining or break-induced replication are used in nongenes. An important question is how different mechanisms of repair predominate in coding and noncoding DNA, although one possible mechanism is transcription-coupled repair (TCR). This work tests the predictions of TCR and finds no support for it. Examination of the mutation spectra and rates in genes and junk reveals what DNA repair mechanisms are available to plant mitochondria, and what selective forces act on the repair products. A model is proposed that mismatches and other DNA damages are repaired by converting them into double-strand breaks (DSBs). These can then be repaired by any of the DSB repair mechanisms, both accurate and inaccurate. Natural selection will eliminate coding regions repaired by inaccurate mechanisms, accounting for the low mutation rates in genes, whereas mutations, rearrangements, and expansions generated by inaccurate repair in noncoding regions will persist. Support for this model includes the structure of the mitochondrial mutS homolog in plants, which is fused to a double-strand endonuclease. The model proposes that plant mitochondria do not distinguish a damaged or mismatched DNA strand from the undamaged strand, they simply cut both strands and perform homology-based DSB repair. This plant-specific strategy for protecting future generations from mitochondrial DNA damage has the side effect of genome expansions and rearrangements. PMID:24904012

  10. Decreased DNA repair gene expression among individuals exposed to arsenic in United States drinking water.

    PubMed

    Andrew, Angeline S; Karagas, Margaret R; Hamilton, Joshua W

    2003-04-10

    Arsenic is well established as a human carcinogen, but its precise mechanism of action remains unknown. Arsenic does not directly damage DNA, but may act as a carcinogen through inhibition of DNA repair mechanisms, leading indirectly to increased mutations from other DNA damaging agents. The molecular mechanism underlying arsenic inhibition of nucleotide excision repair after UV irradiation (Hartwig et al., Carcinogenesis 1997;18:399-405) is unknown, but could be due to decreased expression of critical genes involved in nucleotide excision repair of damaged DNA. This hypothesis was tested by analyzing expression of repair genes and arsenic exposure in a subset of 16 individuals enrolled in a population based case-control study investigating arsenic exposure and cancer risk in New Hampshire. Toenail arsenic levels were inversely correlated with expression of critical members of the nucleotide excision repair complex, ERCC1 (r(2) = 0.82, p < 0.0001), XPF (r(2) = 0.56, p < 0.002), and XPB (r(2) = 0.75, p < 0.0001). The internal dose marker, toenail arsenic level, was more strongly associated with changes in expression of these genes than drinking water arsenic concentration. Our findings, based on human exposure to arsenic in a US population, show an association between biomarkers of arsenic exposure and expression of DNA repair genes. Although our findings need verification in a larger study group, they are consistent with the hypothesis that inhibition of DNA repair capacity is a potential mechanism for the co-carcinogenic activity of arsenic.

  11. Genomic survey and expression analysis of DNA repair genes in the genus Leptospira.

    PubMed

    Martins-Pinheiro, Marinalva; Schons-Fonseca, Luciane; da Silva, Josefa B; Domingos, Renan H; Momo, Leonardo Hiroyuki Santos; Simões, Ana Carolina Quirino; Ho, Paulo Lee; da Costa, Renata M A

    2016-04-01

    Leptospirosis is an emerging zoonosis with important economic and public health consequences and is caused by pathogenic leptospires. The genus Leptospira belongs to the order Spirochaetales and comprises saprophytic (L. biflexa), pathogenic (L. interrogans) and host-dependent (L. borgpetersenii) members. Here, we present an in silico search for DNA repair pathways in Leptospira spp. The relevance of such DNA repair pathways was assessed through the identification of mRNA levels of some genes during infection in animal model and after exposition to spleen cells. The search was performed by comparison of available Leptospira spp. genomes in public databases with known DNA repair-related genes. Leptospires exhibit some distinct and unexpected characteristics, for instance the existence of a redundant mechanism for repairing a chemically diverse spectrum of alkylated nucleobases, a new mutS-like gene and a new shorter version of uvrD. Leptospira spp. shares some characteristics from Gram-positive, as the presence of PcrA, two RecQ paralogs and two SSB proteins; the latter is considered a feature shared by naturally competent bacteria. We did not find a significant reduction in the number of DNA repair-related genes in both pathogenic and host-dependent species. Pathogenic leptospires were enriched for genes dedicated to base excision repair and non-homologous end joining. Their evolutionary history reveals a remarkable importance of lateral gene transfer events for the evolution of the genus. Up-regulation of specific DNA repair genes, including components of SOS regulon, during infection in animal model validates the critical role of DNA repair mechanisms for the complex interplay between host/pathogen.

  12. The effect of acute dose charge particle radiation on expression of DNA repair genes in mice.

    PubMed

    Tariq, Muhammad Akram; Soedipe, Ayodotun; Ramesh, Govindarajan; Wu, Honglu; Zhang, Ye; Shishodia, Shishir; Gridley, Daila S; Pourmand, Nader; Jejelowo, Olufisayo

    2011-03-01

    The space radiation environment consists of trapped particle radiation, solar particle radiation, and galactic cosmic radiation (GCR), in which protons are the most abundant particle type. During missions to the moon or to Mars, the constant exposure to GCR and occasional exposure to particles emitted from solar particle events (SPE) are major health concerns for astronauts. Therefore, in order to determine health risks during space missions, an understanding of cellular responses to proton exposure is of primary importance. The expression of DNA repair genes in response to ionizing radiation (X-rays and gamma rays) has been studied, but data on DNA repair in response to protons is lacking. Using qPCR analysis, we investigated changes in gene expression induced by positively charged particles (protons) in four categories (0, 0.1, 1.0, and 2.0 Gy) in nine different DNA repair genes isolated from the testes of irradiated mice. DNA repair genes were selected on the basis of their known functions. These genes include ERCC1 (5' incision subunit, DNA strand break repair), ERCC2/NER (opening DNA around the damage, Nucleotide Excision Repair), XRCC1 (5' incision subunit, DNA strand break repair), XRCC3 (DNA break and cross-link repair), XPA (binds damaged DNA in preincision complex), XPC (damage recognition), ATA or ATM (activates checkpoint signaling upon double strand breaks), MLH1 (post-replicative DNA mismatch repair), and PARP1 (base excision repair). Our results demonstrate that ERCC1, PARP1, and XPA genes showed no change at 0.1 Gy radiation, up-regulation at 1.0 Gy radiation (1.09 fold, 7.32 fold, 0.75 fold, respectively), and a remarkable increase in gene expression at 2.0 Gy radiation (4.83 fold, 57.58 fold and 87.58 fold, respectively). Expression of other genes, including ATM and XRCC3, was unchanged at 0.1 and 1.0 Gy radiation but showed up-regulation at 2.0 Gy radiation (2.64 fold and 2.86 fold, respectively). We were unable to detect gene expression for the

  13. Analysis of gene repair tracts from Cas9/gRNA double-stranded breaks in the human CFTR gene

    PubMed Central

    Hollywood, Jennifer A.; Lee, Ciaran M.; Scallan, Martina F.; Harrison, Patrick T.

    2016-01-01

    To maximise the efficiency of template-dependent gene editing, most studies describe programmable and/or RNA-guided endonucleases that make a double-stranded break at, or close to, the target sequence to be modified. The rationale for this design strategy is that most gene repair tracts will be very short. Here, we describe a CRISPR Cas9/gRNA selection-free strategy which uses deep sequencing to characterise repair tracts from a donor plasmid containing seven nucleotide differences across a 216 bp target region in the human CFTR gene. We found that 90% of the template-dependent repair tracts were >100 bp in length with equal numbers of uni-directional and bi-directional repair tracts. The occurrence of long repair tracts suggests that a single gRNA could be used with variants of the same template to create or correct specific mutations within a 200 bp range, the size of ~80% of human exons. The selection-free strategy used here also allowed detection of non-homologous end joining events in many of the homology-directed repair tracts. This indicates a need to modify the donor, possibly by silent changes in the PAM sequence, to prevent creation of a second double-stranded break in an allele that has already been correctly edited by homology-directed repair. PMID:27557525

  14. Analysis of gene repair tracts from Cas9/gRNA double-stranded breaks in the human CFTR gene.

    PubMed

    Hollywood, Jennifer A; Lee, Ciaran M; Scallan, Martina F; Harrison, Patrick T

    2016-01-01

    To maximise the efficiency of template-dependent gene editing, most studies describe programmable and/or RNA-guided endonucleases that make a double-stranded break at, or close to, the target sequence to be modified. The rationale for this design strategy is that most gene repair tracts will be very short. Here, we describe a CRISPR Cas9/gRNA selection-free strategy which uses deep sequencing to characterise repair tracts from a donor plasmid containing seven nucleotide differences across a 216 bp target region in the human CFTR gene. We found that 90% of the template-dependent repair tracts were >100 bp in length with equal numbers of uni-directional and bi-directional repair tracts. The occurrence of long repair tracts suggests that a single gRNA could be used with variants of the same template to create or correct specific mutations within a 200 bp range, the size of ~80% of human exons. The selection-free strategy used here also allowed detection of non-homologous end joining events in many of the homology-directed repair tracts. This indicates a need to modify the donor, possibly by silent changes in the PAM sequence, to prevent creation of a second double-stranded break in an allele that has already been correctly edited by homology-directed repair. PMID:27557525

  15. Preferential Repair of DNA Double-strand Break at the Active Gene in Vivo*

    PubMed Central

    Chaurasia, Priyasri; Sen, Rwik; Pandita, Tej K.; Bhaumik, Sukesh R.

    2012-01-01

    Previous studies have demonstrated transcription-coupled nucleotide/base excision repair. We report here for the first time that DNA double-strand break (DSB) repair is also coupled to transcription. We generated a yeast strain by introducing a homing (Ho) endonuclease cut site followed by a nucleotide sequence for multiple Myc epitopes at the 3′ end of the coding sequence of a highly active gene, ADH1. This yeast strain also contains the Ho cut site at the nearly silent or poorly active mating type α (MATα) locus and expresses Ho endonuclease under the galactose-inducible GAL1 promoter. Using this strain, DSBs were generated at the ADH1 and MATα loci in galactose-containing growth medium that induced HO expression. Subsequently, yeast cells were transferred to dextrose-containing growth medium to stop HO expression, and the DSB repair was monitored at the ADH1 and MATα loci by PCR, using the primer pairs flanking the Ho cut sites. Our results revealed a faster DSB repair at the highly active ADH1 than that at the nearly silent MATα locus, hence implicating a transcription-coupled DSB repair at the active gene in vivo. Subsequently, we extended this study to another gene, PHO5 (carrying the Ho cut site at its coding sequence), under transcriptionally active and inactive growth conditions. We found a fast DSB repair at the active PHO5 gene in comparison to its inactive state. Collectively, our results demonstrate a preferential DSB repair at the active gene, thus supporting transcription-coupled DSB repair in living cells. PMID:22910905

  16. Interaction of DNA repair gene polymorphisms and aflatoxin B1 in the risk of hepatocellular carcinoma

    PubMed Central

    Yao, Jin-Guang; Huang, Xiao-Ying; Long, Xi-Dai

    2014-01-01

    Aflatoxin B1 (AFB1) is an important environmental carcinogen and can induce DNA damage and involve in the carcinogenesis of hepatocellular carcinoma (HCC). The deficiency of DNA repair capacity related to the polymorphisms of DNA repair genes might play a central role in the process of HCC tumorigenesis. However, the interaction of DNA repair gene polymorphisms and AFB1 in the risk of hepatocellular carcinoma has not been elucidated. In this study, we investigated whether six polymorphisms (including rs25487, rs861539, rs7003908, rs28383151, rs13181, and rs2228001) in DNA repair genes (XPC, XRCC4, XRCC1, XRCC4, XPD, XRCC7, and XRCC3) interacted with AFB1, and the gene-environmental interactive role in the risk of HCC using hospital-based case-control study (including 1486 HCC cases and 1996 controls). Genotypes of DNA repair genes were tested using TaqMan-PCR technique. Higher AFB1 exposure was observed among HCC patients versus the control group [odds ratio (OR) = 2.08 for medium AFB1 exposure level and OR = 6.52 for high AFB1 exposure level]. Increasing risk of HCC was also observed in these with the mutants of DNA repair genes (risk values were from 1.57 to 5.86). Furthermore, these risk roles would be more noticeable under the conditions of two variables, and positive interactive effects were proved in the followed multiplicative interaction analysis. These results suggested that DNA repair risk genotypes might interact with AFB1 in the risk of HCC. PMID:25337275

  17. FISH comets show that the salvage enzyme TK1 contributes to gene-specific DNA repair

    PubMed Central

    McAllister, Katherine A.; Yasseen, Akeel A.; McKerr, George; Downes, C. S.; McKelvey-Martin, Valerie J.

    2014-01-01

    Thymidine kinase 1 (TK1) is a salvage enzyme that phosphorylates thymidine, imported from surrounding fluids, to create dTMP, which is further phosphorylated to the DNA precursor dTTP. TK1 deficiency has for a long time been known to cause increased cellular sensitivity to DNA damage. We have examined preferential strand break repair of DNA domains in TK1+ and TK1- clones of the Raji cell line, by the Comet-FISH technique, in bulk DNA and in the actively transcribed tumor suppressor (TP53) and human telomerase reverse transcriptase (hTERT) gene regions, over 1 h after 5Gy γ-irradiation. Results showed that repair of the TP53 and hTERT gene regions was more efficient in TK1+ compared to TK1- cells, a trend also reflected to a lesser degree in genomic DNA repair between the cell-lines. The targeted gene-specific repair in TK+ cells occurred rapidly, mainly over the first 15 min repair-period. Therefore, TK1 is needed for preferential repair of actively transcribed regions, through a previously unsuspected mechanism. In principle, TK1 could exert its protective effects through supply of a supplementary dTTP pool for accurate repair of damaged genes; but Raji TK1+ cells in thymidine free media still show preferential repair of transcribed regions. TK1 therefore does not exert its protective effects through dTTP pools, but through another unidentified mechanism, which affects sensitivity to and mutagenicity by DNA damaging agents. PMID:25152750

  18. FISH comets show that the salvage enzyme TK1 contributes to gene-specific DNA repair.

    PubMed

    McAllister, Katherine A; Yasseen, Akeel A; McKerr, George; Downes, C S; McKelvey-Martin, Valerie J

    2014-01-01

    Thymidine kinase 1 (TK1) is a salvage enzyme that phosphorylates thymidine, imported from surrounding fluids, to create dTMP, which is further phosphorylated to the DNA precursor dTTP. TK1 deficiency has for a long time been known to cause increased cellular sensitivity to DNA damage. We have examined preferential strand break repair of DNA domains in TK1(+) and TK1(-) clones of the Raji cell line, by the Comet-FISH technique, in bulk DNA and in the actively transcribed tumor suppressor (TP53) and human telomerase reverse transcriptase (hTERT) gene regions, over 1 h after 5Gy γ-irradiation. Results showed that repair of the TP53 and hTERT gene regions was more efficient in TK1(+) compared to TK1(-) cells, a trend also reflected to a lesser degree in genomic DNA repair between the cell-lines. The targeted gene-specific repair in TK(+) cells occurred rapidly, mainly over the first 15 min repair-period. Therefore, TK1 is needed for preferential repair of actively transcribed regions, through a previously unsuspected mechanism. In principle, TK1 could exert its protective effects through supply of a supplementary dTTP pool for accurate repair of damaged genes; but Raji TK1(+) cells in thymidine free media still show preferential repair of transcribed regions. TK1 therefore does not exert its protective effects through dTTP pools, but through another unidentified mechanism, which affects sensitivity to and mutagenicity by DNA damaging agents. PMID:25152750

  19. A new approach to tissue repair: gene therapy.

    PubMed

    Wei, Kuanhai; Pei, Guoxian; Hu, Basheng

    2000-11-15

    The process of tissue repair involves a complex tissue response to injury in which growth factors, playing a major role in this process, trigger, control and terminate soakage of inflammatory cells, cells proliferation, secretion of matrix and scars formation by autocrine, paracrine or both. Thus, growth factors can be used to alter the microenvironment of the wounded tissues and to promote their repair. But, there are notable disadvantages in using purified recombination growth factors, 1) the source is so limited that their prices are expensive; 2) the ir half-lives are short and easy to be destroyed by wound proteases; 3) there is no perfect carrier; 4) high initial doses are required but easy to bring toxicity; 5) it is difficult to apply growth factors in deep wounded tissues again and again, their function cannot be played enough accordingly; 6) most of growth factors are the products of recombination. All above-mentioned disadvantages result in a low activity.

  20. Polymorphisms in DNA repair genes, recreational physical activity and breast cancer risk.

    PubMed

    McCullough, Lauren E; Santella, Regina M; Cleveland, Rebecca J; Millikan, Robert C; Olshan, Andrew F; North, Kari E; Bradshaw, Patrick T; Eng, Sybil M; Terry, Mary Beth; Shen, Jing; Crew, Katherine D; Rossner, Pavel; Teitelbaum, Susan L; Neugut, Alfred I; Gammon, Marilie D

    2014-02-01

    The mechanisms driving the inverse association between recreational physical activity (RPA) and breast cancer risk are complex. While exercise is associated with increased reactive oxygen species production it may also improve damage repair systems, particularly those that operate on single-strand breaks including base excision repair (BER), nucleotide excision repair (NER) and mismatch repair (MMR). Of these repair pathways, the role of MMR in breast carcinogenesis is least investigated. Polymorphisms in MMR or other DNA repair gene variants may modify the association between RPA and breast cancer incidence. We investigated the individual and joint effects of variants in three MMR pathway genes (MSH3, MLH1 and MSH2) on breast cancer occurrence using resources from the Long Island Breast Cancer Study Project. We additionally characterized interactions between RPA and genetic polymorphisms in MMR, BER and NER pathways. We found statistically significant multiplicative interactions (p < 0.05) between MSH2 and MLH1, as well as between postmenopausal RPA and four variants in DNA repair (XPC-Ala499Val, XPF-Arg415Gln, XPG-Asp1104His and MLH1-lle219Val). Significant risk reductions were observed among highly active women with the common genotype for XPC (OR = 0.54; 95% CI, 0.36-0.81) and XPF (OR = 0.62; 95% CI, 0.44-0.87), as well as among active women who carried at least one variant allele in XPG (OR = 0.46; 95% CI, 0.29-0.77) and MLH1 (OR = 0.46; 95% CI, 0.30-0.71). Our data show that women with minor alleles in both MSH2 and MLH1 could be at increased breast cancer risk. RPA may be modified by genes in the DNA repair pathway, and merit further investigation.

  1. Polymorphisms in DNA Repair Genes, Recreational Physical Activity and Breast Cancer Risk

    PubMed Central

    McCullough, Lauren E.; Santella, Regina M.; Cleveland, Rebecca J.; Millikan, Robert C.; Olshan, Andrew F.; North, Kari E.; Bradshaw, Patrick T.; Eng, Sybil M.; Terry, Mary Beth; Shen, Jing; Crew, Katherine D.; Rossner, Pavel; Teitelbaum, Susan L.; Neugut, Alfred I.; Gammon, Marilie D.

    2013-01-01

    The mechanisms driving the inverse association between recreational physical activity (RPA) and breast cancer risk are complex. While exercise is associated with increased reactive oxygen species production it may also improve damage repair systems, particularly those that operate on single-strand breaks including base excision repair (BER), nucleotide excision repair (NER) and mismatch repair (MMR). Of these repair pathways, the role of MMR in breast carcinogenesis is least investigated. Polymorphisms in MMR or other DNA repair gene variants may modify the association between RPA and breast cancer incidence. We investigated the individual and joint effects of variants in three MMR pathway genes (MSH3, MLH1 and MSH2) on breast cancer occurrence using resources from the Long Island Breast Cancer Study Project. We additionally characterized interactions between RPA and genetic polymorphisms in MMR, BER and NER pathways. We found statistically significant multiplicative interactions (p<0.05) between MSH2 and MLH1, as well as between postmenopausal RPA and four variants in DNA repair (XPC-Ala499Val, XPF-Arg415Gln, XPG-Asp1104His and MLH1-lle219Val). Significant risk reductions were observed among highly active women with the common genotype for XPC (OR=0.54; 95% CI, 0.36–0.81) and XPF (OR=0.62; 95% CI, 0.44–0.87), as well as among active women who carried at least one variant allele in XPG (OR=0.46; 95% CI, 0.29–0.77) and MLH1 (OR=0.46; 95% CI, 0.30–0.71). Our data show that women with minor alleles in both MSH2 and MLH1 could be at increased breast cancer risk. RPA may be modified by genes in the DNA repair pathway, and merit further investigation. PMID:23852586

  2. Lack of association between polymorphisms of the DNA base excision repair genes MUTYH and hOGG1 and keratoconus in a Polish subpopulation

    PubMed Central

    Synowiec, Ewelina; Wójcik, Katarzyna A.; Czubatka, Anna; Polakowski, Piotr; Izdebska, Justyna; Szaflik, Jerzy; Błasiak, Janusz

    2015-01-01

    Introduction Keratoconus (KC) is a non-inflammatory thinning of the cornea and a leading indication for corneal transplantation. Oxidative stress plays a role in the pathogenesis of this disease. The products of the hOGG1 and MUTYH genes play an important role in the repair of oxidatively modified DNA in the base excision repair pathway. We hypothesized that variability in these genes may change susceptibility to oxidative stress and predispose individuals to the development of KC. We investigated the possible association between the c.977C>G polymorphism of the hOGG1 gene (rs1052133) and the c.972G>C polymorphism of the MUTYH gene (rs3219489) and KC occurrence as well as the modulation of this association by some KC risk factors. Material and methods A total of 205 patients with KC and 220 controls were included in this study. The polymorphisms were genotyped with polymerase chain reaction (PCR) restriction fragment length polymorphism and PCR-confronting two-pair primer techniques. Differences in genotype and allele frequency distributions were evaluated using the χ2 test, and KC risk was estimated with an unconditional multiple logistic regression with and without adjustment for co-occurrence of visual impairment, allergies, sex and family history for KC. Results We did not find any association between the genotypes and combined genotypes of the c.977C>G polymorphism of the hOGG1 gene and the c.972G>C polymorphism of the MUTYH gene and the occurrence of KC. Conclusions Our findings suggest that the c.977C>G-hOGG1 polymorphism and the c.972G>C-MUTYH polymorphism may not be linked with KC occurrence in this Polish subpopulation. PMID:26528356

  3. Gene Expression in Experimental Aortic Coarctation and Repair: Candidate Genes for Therapeutic Intervention?

    PubMed Central

    LaDisa, John F.; Bozdag, Serdar; Olson, Jessica; Ramchandran, Ramani; Kersten, Judy R.; Eddinger, Thomas J.

    2015-01-01

    Coarctation of the aorta (CoA) is a constriction of the proximal descending thoracic aorta and is one of the most common congenital cardiovascular defects. Treatments for CoA improve life expectancy, but morbidity persists, particularly due to the development of chronic hypertension (HTN). Identifying the mechanisms of morbidity is difficult in humans due to confounding variables such as age at repair, follow-up duration, coarctation severity and concurrent anomalies. We previously developed an experimental model that replicates aortic pathology in humans with CoA without these confounding variables, and mimics correction at various times using dissolvable suture. Here we present the most comprehensive description of differentially expressed genes (DEGs) to date from the pathology of CoA, which were obtained using this model. Aortic samples (n=4/group) from the ascending aorta that experiences elevated blood pressure (BP) from induction of CoA, and restoration of normal BP after its correction, were analyzed by gene expression microarray, and enriched genes were converted to human orthologues. 51 DEGs with >6 fold-change (FC) were used to determine enriched Gene Ontology terms, altered pathways, and association with National Library of Medicine Medical Subject Headers (MeSH) IDs for HTN, cardiovascular disease (CVD) and CoA. The results generated 18 pathways, 4 of which (cell cycle, immune system, hemostasis and metabolism) were shared with MeSH ID’s for HTN and CVD, and individual genes were associated with the CoA MeSH ID. A thorough literature search further uncovered association with contractile, cytoskeletal and regulatory proteins related to excitation-contraction coupling and metabolism that may explain the structural and functional changes observed in our experimental model, and ultimately help to unravel the mechanisms responsible for persistent morbidity after treatment for CoA. PMID:26207811

  4. The Convergence of Fracture Repair and Stem Cells: Interplay of Genes, Aging, Environmental Factors and Disease

    PubMed Central

    Hadjiargyrou, Michael; O’Keefe, Regis J

    2015-01-01

    The complexity of fracture repair makes it an ideal process for studying the interplay between the molecular, cellular, tissue, and organ level events involved in tissue regeneration. Additionally, as fracture repair recapitulates many of the processes that occur during embryonic development, investigations of fracture repair provide insights regarding skeletal embryogenesis. Specifically, inflammation, signaling, gene expression, cellular proliferation and differentiation, osteogenesis, chondrogenesis, angiogenesis, and remodeling represent the complex array of interdependent biological events that occur during fracture repair. Here we review studies of bone regeneration in genetically modified mouse models, during aging, following environmental exposure, and in the setting of disease that provide insights regarding the role of multipotent cells and their regulation during fracture repair. Complementary animal models and ongoing scientific discoveries define an increasing number of molecular and cellular targets to reduce the morbidity and complications associated with fracture repair. Last, some new and exciting areas of stem cell research such as the contribution of mitochondria function, limb regeneration signaling, and microRNA (miRNA) posttranscriptional regulation are all likely to further contribute to our understanding of fracture repair as an active branch of regenerative medicine. PMID:25264148

  5. Repair of UV damaged DNA, genes and proteins of yeast and human

    SciTech Connect

    Prakash, L.

    1991-04-01

    Our objectives are to determine the molecular mechanism of the incision step of excision repair of ultraviolet (UV) light damaged DNA in eukaryotic organisms, using the yeast Saccharomyces cerevisiae as a model system, as well as studying the human homologs of yeast excision repair and postreplication repair proteins. In addition to its single-stranded DNA-dependent A TPase and DNA helicase activities, we have found that RAD3 protein also possesses DNA-RNA helicase activity, and that like RAD3, the Schizosaccharomyces pombe RAD3 homolog, rhp3{sup +}, is an essential gene. We have overexpressed the human RAD3 homolog, ERCC2, in yeast to facilitate its purification. The RAD10 protein was purified to homogeneity and shown to bind DNA. ERCC3y, the yeast homolog of the human ERCC-3/XP-B gene, has been sequenced and shown to be essential for viability. The Drosophila and human homologs of RAD6, required for postreplication repair and UV induced mutagenesis, were shown to complement the rad6 {Delta} mutation of yeast. Since defective DNA repair and enhanced neoplasia characterize several human genetic diseases, and repair proteins are highly conserved between yeast and man, a thorough understanding of the molecular mechanisms of DNA repir in yeast should provide a better understanding of the causes of carcinogenesis.

  6. The Finland–United States Investigation of Non–Insulin-Dependent Diabetes Mellitus Genetics (FUSION) Study. I. An Autosomal Genome Scan for Genes That Predispose to Type 2 Diabetes

    PubMed Central

    Ghosh, Soumitra; Watanabe, Richard M.; Valle, Timo T.; Hauser, Elizabeth R.; Magnuson, Victoria L.; Langefeld, Carl D.; Ally, Delphine S.; Mohlke, Karen L.; Silander, Kaisa; Kohtamäki, Kimmo; Chines, Peter; Balow, Jr., James; Birznieks, Gunther; Chang, Jennie; Eldridge, William; Erdos, Michael R.; Karanjawala, Zarir E.; Knapp, Julie I.; Kudelko, Kristina; Martin, Colin; Morales-Mena, Anabelle; Musick, Anjene; Musick, Tiffany; Pfahl, Carrie; Porter, Rachel; Rayman, Joseph B.; Rha, David; Segal, Leonid; Shapiro, Shane; Sharaf, Ravi; Shurtleff, Ben; So, Alistair; Tannenbaum, Joyce; Te, Catherine; Tovar, Jason; Unni, Arun; Welch, Christian; Whiten, Ray; Witt, Alyson; Blaschak-Harvan, Jillian; Douglas, Julie A.; Duren, William L.; Epstein, Michael P.; Fingerlin, Tasha E.; Kaleta, Hong Shi; Lange, Ethan M.; Li, Chun; McEachin, Richard C.; Stringham, Heather M.; Trager, Edward; White, Peggy P.; Eriksson, Johan; Toivanen, Liisa; Vidgren, Gabriele; Nylund, Stella J.; Tuomilehto-Wolf, Eva; Ross, Edna H.; Demirchyan, Elza; Hagopian, William A.; Buchanan, Thomas A.; Tuomilehto, Jaakko; Bergman, Richard N.; Collins, Francis S.; Boehnke, Michael

    2000-01-01

    We performed a genome scan at an average resolution of 8 cM in 719 Finnish sib pairs with type 2 diabetes. Our strongest results are for chromosome 20, where we observe a weighted maximum LOD score (MLS) of 2.15 at map position 69.5 cM from pter and secondary weighted LOD-score peaks of 2.04 at 56.5 cM and 1.99 at 17.5 cM. Our next largest MLS is for chromosome 11 (MLS = 1.75 at 84.0 cM), followed by chromosomes 2 (MLS = 0.87 at 5.5 cM), 10 (MLS = 0.77 at 75.0 cM), and 6 (MLS = 0.61 at 112.5 cM), all under an additive model. When we condition on chromosome 2 at 8.5 cM, the MLS for chromosome 20 increases to 5.50 at 69.0 cM (P=.0014). An ordered-subsets analysis based on families with high or low diabetes-related quantitative traits yielded results that support the possible existence of disease-predisposing genes on chromosomes 6 and 10. Genomewide linkage-disequilibrium analysis using microsatellite marker data revealed strong evidence of association for D22S423 (P=.00007). Further analyses are being carried out to confirm and to refine the location of these putative diabetes-predisposing genes. PMID:11032783

  7. Coordination of cell cycle, DNA repair and muscle gene expression in myoblasts exposed to genotoxic stress

    PubMed Central

    Minetti, Giulia Claudia

    2011-01-01

    Upon exposure to genotoxic stress, skeletal muscle progenitors coordinate DNA repair and the activation of the differentiation program through the DNA damage-activated differentiation checkpoint, which holds the transcription of differentiation genes while the DNA is repaired. A conceptual hurdle intrinsic to this process relates to the coordination of DNA repair and muscle-specific gene transcription within specific cell cycle boundaries (cell cycle checkpoints) activated by different types of genotoxins. Here, we show that, in proliferating myoblasts, the inhibition of muscle gene transcription occurs by either a G1- or G2-specific differentiation checkpoint. In response to genotoxins that induce G1 arrest, MyoD binds target genes but is functionally inactivated by a c-Abl-dependent phosphorylation. In contrast, DNA damage-activated G2 checkpoint relies on the inability of MyoD to bind the chromatin at the G2 phase of the cell cycle. These results indicate an intimate relationship between DNA damage-activated cell cycle checkpoints and the control of tissue-specific gene expression to allow DNA repair in myoblasts prior to the activation of the differentiation program. PMID:21685725

  8. Repair of rDNA in Saccharomyces cerevisiae: RAD4-independent strand-specific nucleotide excision repair of RNA polymerase I transcribed genes.

    PubMed Central

    Verhage, R A; Van de Putte, P; Brouwer, J

    1996-01-01

    Removal of UV-induced pyrimidine dimers from the individual strands of the rDNA locus in Saccharomyces cerevisiae was studied. Yeast rDNA, that is transcribed by RNA polymerase I(RNA pol I), is repaired efficiently, slightly strand-specific and independently of RAD26, which has been implicated in transcription-coupled repair of the RNA pol II transcribed RPB2 gene. No repair of rDNA is observed in rad1,2,3 and 14 mutants, demonstrating that dimer removal from this highly repetitive DNA is accomplished by nucleotide excision repair (NER). In rad7 and rad16 mutants, which are specifically deficient in repair of non-transcribed DNA, there is a clear preferential repair of the transcribed strand of rDNA, indicating that strand-specific and therefore probably transcription-coupled repair of RNA pol I transcribed genes does exist in yeast. Unexpectedly, the transcribed but not the non-transcribed strand of rDNA can be repaired in rad4 mutants, which seem otherwise completely NER-deficient. PMID:8604332

  9. Gene expression profiling of archival tongue squamous cell carcinomas provides sub-classification based on DNA repair genes.

    PubMed

    Rentoft, Matilda; Laurell, Göran; Coates, Philip John; Sjöström, Björn; Nylander, Karin

    2009-12-01

    A subgroup of patients with squamous cell carcinoma of the head and neck (SCCHN) comprise young persons under the age of 40, who have not been heavily exposed to the classical risk factors, smoking and alcohol. The number of SCCHN in young adults, particularly tongue tumours, is increasing in several parts of the world. Here we employed a novel gene expression array methodology specifically developed for analysis of degraded RNA and investigated the expression of 502 cancer-related genes in archival paraffin-embedded SCCHN of the tongue from young (< or =40) and elderly patients (> or =50). Genes detected as de-regulated in tumours compared to non-malignant controls were in concordance with results from earlier studies of fresh frozen material. No genes were detected as significantly differentially expressed between young and old patients suggesting that the overall pathobiology of SCCHN is similar in young and old. Unsupervised clustering divided tumours into three groups, irrespective of age, where several differentially expressed DNA repair genes were a prominent separation factor. High levels of DNA repair genes associated with impaired therapeutic response to radiation, suggesting that DNA repair genes play a role in clinical outcome after radiotherapy.

  10. Higher expression of somatic repair genes in long-lived ant queens than workers

    PubMed Central

    Lucas, Eric R.; Privman, Eyal; Keller, Laurent

    2016-01-01

    Understanding why organisms senesce is a fundamental question in biology. One common explanation is that senescence results from an increase in macromolecular damage with age. The tremendous variation in lifespan between genetically identical queen and worker ants, ranging over an order of magnitude, provides a unique system to study how investment into processes of somatic maintenance and macromolecular repair influence lifespan. Here we use RNAseq to compare patterns of expression of genes involved in DNA and protein repair of age-matched queens and workers. There was no difference between queens and workers in 1-day-old individuals, but the level of expression of these genes increased with age and this up-regulation was greater in queens than in workers, resulting in significantly queen-biased expression in 2-month-old individuals in both legs and brains. Overall, these differences are consistent with the hypothesis that higher longevity is associated with increased investment into somatic repair. PMID:27617474

  11. Expression of DNA repair and metabolic genes in response to a flavonoid-rich diet.

    PubMed

    Guarrera, Simonetta; Sacerdote, Carlotta; Fiorini, Laura; Marsala, Rosa; Polidoro, Silvia; Gamberini, Sara; Saletta, Federica; Malaveille, Christian; Talaska, Glenn; Vineis, Paolo; Matullo, Giuseppe

    2007-09-01

    A diet rich in fruit and vegetables can be effective in the reduction of oxidative stress, through the antioxidant effects of phytochemicals and other mechanisms. Protection against the carcinogenic effects of chemicals may also be exerted by an enhancement of detoxification and DNA damage repair mechanisms. To investigate a putative effect of flavonoids, a class of polyphenols, on the regulation of the gene expression of DNA repair and metabolic genes, a 1-month flavonoid-rich diet was administered to thirty healthy male smokers, nine of whom underwent gene expression analysis. We postulated that tobacco smoke is a powerful source of reactive oxygen species. The expression level of twelve genes (APEX, ERCC1, ERCC2, ERCC4, MGMT, OGG1, XPA, XPC, XRCC1, XRCC3, AHR, CYP1A1) was investigated. We found a significant increase (P < 0.001) in flavonoid intake. Urinary phenolic content and anti-mutagenicity did not significantly change after diet, nor was a correlation found between flavonoid intake and urinary phenolic levels or anti-mutagenicity. Phenolic levels showed a significant positive correlation with urinary anti-mutagenicity. AHR levels were significantly reduced after the diet (P = 0.038), whereas the other genes showed a generalized up regulation, significant for XRCC3 gene (P = 0.038). Also in the context of a generalized up regulation of DNA repair genes, we found a non-significant negative correlation between flavonoid intake and the expression of all the DNA repair genes. Larger studies are needed to clarify the possible effects of flavonoids in vivo; our preliminary results could help to better plan new studies on gene expression and diet.

  12. Semiconservative replication, genetic repair, and many-gened genomes: Extending the quasispecies paradigm to living systems

    NASA Astrophysics Data System (ADS)

    Tannenbaum, Emmanuel; Shakhnovich, Eugene I.

    2005-12-01

    Quasispecies theory has emerged as an important tool for modeling the evolutionary dynamics of biological systems. We review recent advances in the field, with an emphasis on the quasispecies dynamics of semiconservatively replicating genomes. Applications to cancer and adult stem cell growth are discussed. Additional topics, such as genetic repair and many-gene genomes, are covered as well.

  13. Polymorphisms in DNA Repair Genes and MDR1 and the Risk for Non-Hodgkin Lymphoma

    PubMed Central

    Kim, Hee Nam; Kim, Nan Young; Yu, Li; Kim, Yeo-Kyeoung; Lee, Il-Kwon; Yang, Deok-Hwan; Lee, Je-Jung; Shin, Min-Ho; Park, Kyeong-Soo; Choi, Jin-Su; Kim, Hyeoung-Joon

    2014-01-01

    The damage caused by oxidative stress and exposure to cigarette smoke and alcohol necessitate DNA damage repair and transport by multidrug resistance-1 (MDR1). To explore the association between polymorphisms in these genes and non-Hodgkin lymphoma risk, we analyzed 15 polymorphisms of 12 genes in a population-based study in Korea (694 cases and 1700 controls). Four genotypes of DNA repair pathway genes (XRCC1 399 GA, OGG1 326 GG, BRCA1 871 TT, and WRN 787 TT) were associated with a decreased risk for NHL [odds ratio (OR)XRCC1 GA = 0.80, p = 0.02; OROGG1 GG = 0.70, p = 0.008; ORBRCA1 TT = 0.71, p = 0.048; ORWRN TT = 0.68, p = 0.01]. Conversely, the MGMT 115 CT genotype was associated with an increased risk for NHL (OR = 1.25, p = 0.04). In the MDR1 gene, the 1236 CC genotype was associated with a decreased risk for NHL (OR = 0.74, p = 0.04), and the 3435 CT and TT genotypes were associated with an increased risk (OR3435CT = 1.50, p < 0.0001; OR3435TT = 1.43, p = 0.02). These results suggest that polymorphisms in the DNA repair genes XRCC1, OGG1, BRCA1, WRN1, and MGMT and in the MDR1 gene may affect the risk for NHL in Korean patients. PMID:24756092

  14. Polymorphisms in DNA repair genes and MDR1 and the risk for non-Hodgkin lymphoma.

    PubMed

    Kim, Hee Nam; Kim, Nan Young; Yu, Li; Kim, Yeo-Kyeoung; Lee, Il-Kwon; Yang, Deok-Hwan; Lee, Je-Jung; Shin, Min-Ho; Park, Kyeong-Soo; Choi, Jin-Su; Kim, Hyeoung-Joon

    2014-04-21

    The damage caused by oxidative stress and exposure to cigarette smoke and alcohol necessitate DNA damage repair and transport by multidrug resistance-1 (MDR1). To explore the association between polymorphisms in these genes and non-Hodgkin lymphoma risk, we analyzed 15 polymorphisms of 12 genes in a population-based study in Korea (694 cases and 1700 controls). Four genotypes of DNA repair pathway genes (XRCC1 399 GA, OGG1 326 GG, BRCA1 871 TT, and WRN 787 TT) were associated with a decreased risk for NHL [odds ratio (OR)XRCC1 GA=0.80, p=0.02; OROGG1 GG=0.70, p=0.008; ORBRCA1 TT=0.71, p=0.048; ORWRN TT=0.68, p=0.01]. Conversely, the MGMT 115 CT genotype was associated with an increased risk for NHL (OR=1.25, p=0.04). In the MDR1 gene, the 1236 CC genotype was associated with a decreased risk for NHL (OR=0.74, p=0.04), and the 3435 CT and TT genotypes were associated with an increased risk (OR3435CT=1.50, p<0.0001; OR3435TT=1.43, p=0.02). These results suggest that polymorphisms in the DNA repair genes XRCC1, OGG1, BRCA1, WRN1, and MGMT and in the MDR1 gene may affect the risk for NHL in Korean patients.

  15. Cytogenetic Response to Ionizing Radiation Exposure in Human Fibroblasts with Suppressed Expression of Non-DSB Repair Genes

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Rohde, Larry H.; Emami, Kamal; Hammond, Dianne; Mehta, Satish K.; Jeevarajan, Antony S.; Pierson, Duane L.; Wu, Honglu

    2009-01-01

    Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in double-strand break (DSB) repair, and its impact on cytogenetic responses has not been well studied. The purpose of this study is to identify new roles of IR inducible genes in radiation-induced chromosome aberrations and micronuclei formation. In the study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by small interfering RNA in human fibroblast cells. Frequencies of micronuclei (MN) formation and chromosome aberrations were measured to determine the efficiency of cytogenetic repair, and the fraction of bi-nucleated cells in the MN analysis was used as a marker for cell cycle progression. In response to gamma radiation, the formation of MN was significantly increased by suppressed expression of five genes: Ku70 (DSB repair pathway), XPA (nucleotide excision repair pathway), RPA1 (mismatch repair pathway), RAD17 and RBBP8 (cell cycle control). Knocked-down expression of four genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Moreover, decreased XPA, p21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Nine of these eleven genes, whose knock-down expression affected cytogenetic repair, were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate IR

  16. Biochemical studies of DNA strand break repair and molecular characterization of mei-41, a gene involved in DNA break repair

    SciTech Connect

    Oliveri, D.R.

    1989-01-01

    The ability to repair X-irradiation induced single-strand DNA breaks was examined in mutagen-sensitive mutants of Drosophila melanogaster. This analysis demonstrated that examined stocks possess a normal capacity to repair X-ray induced single-strand breaks. One of the mutants in this study, mei-41, has been shown to be involved in a number of DNA metabolizing functions. A molecular characterization of this mutant is presented. A cDNA hybridizing to genomic DNA both proximal and distal to a P element inducing a mei-41 mutation was isolated from both embryonic and adult female recombinant lambda phage libraries. A 2.2 kilobase embryonic cDNA clone was sequenced; the sequence of an open reading frame was identified which would predict a protein of 384 amino acids with a molecular weight of 43,132 daltons. An examination of homologies to sequences in protein and nucleic acid data bases revealed no sequences with significant homology to mei-41, however, two potential Zinc-finger domains were identified. Analysis of RNA hybridizing to the embryonic cDNA demonstrated the existence of a major 2.2 kilobase transcript expressed primarily in embryos and adult flies. An examination of the transcription of this gene in mei-41 mutants revealed significant variation from wild-type, an indication that the embryonic cDNA does represent a mei-41 transcript. Expression in tissues from adult animals demonstrated that the 2.2 kilobase RNA is expressed primarily in reproductive tissues. A 3.8kb transcript is the major species of RNA in the adult head and thorax. Evidence is presented which implies that expression of the mei-41 gene is strongly induced by exposure of certain cells to mutagens.

  17. Transcription-coupled and global genome repair in the Saccharomyces cerevisiae RPB2 gene at nucleotide resolution.

    PubMed Central

    Tijsterman, M; Tasseron-de Jong, J G; van de Putte, P; Brouwer, J

    1996-01-01

    Repair of UV-induced cyclobutane pyrimidine dimers (CPDs) was examined at single nucleotide resolution in the yeast Saccharomyces cerevisiae, using an improved protocol for genomic end-labelling. To obtain the sensitivity required for adduct detection in yeast, an oligonucleotide-directed enrichment step was introduced into the current methodology developed for adduct detection in Escherichia coli. With this method, heterogeneous repair of CPDs within the RPB2 locus is observed. Individual CPDs positioned in the transcribed strand are removed very efficiently with identical kinetics. This fast repair starts within 23 bases downstream of the transcription initiation site. The non-transcribed strand of the active gene exhibits slow repair without detectable repair variations between individual lesions. In contrast, CPDs positioned in the promoter region show profound repair heterogeneity. Here, CPDs at specific sites are removed very quickly, with comparable rates to CPDs positioned in the transcribed strand, while at other positions lesions are not repaired at all during the period studied. Interestingly, the fast repair in the promoter region is dependent on the RAD7 and RAD16 genes, as are the slowly repaired CPDs in this region and in the non-transcribed strand. This indicates that the global genome repair pathway is not intrinsically slow and at specific positions can be as efficient as the transcription-coupled repair pathway. PMID:8836174

  18. Low-level infrared laser modulates muscle repair and chromosome stabilization genes in myoblasts.

    PubMed

    da Silva Neto Trajano, Larissa Alexsandra; Stumbo, Ana Carolina; da Silva, Camila Luna; Mencalha, Andre Luiz; Fonseca, Adenilson S

    2016-08-01

    Infrared laser therapy is used for skeletal muscle repair based on its biostimulative effect on satellite cells. However, shortening of telomere length limits regenerative potential in satellite cells, which occurs after each cell division cycle. Also, laser therapy could be more effective on non-physiologic tissues. This study evaluated low-level infrared laser exposure effects on mRNA expression from muscle injury repair and telomere stabilization genes in myoblasts in normal and stressful conditions. Laser fluences were those used in clinical protocols. C2C12 myoblast cultures were exposed to low-level infrared laser (10, 35, and 70 J/cm(2)) in standard or normal (10 %) and reduced (2 %) fetal bovine serum concentrations; total RNA was extracted for mRNA expression evaluation from muscle injury repair (MyoD and Pax7) and chromosome stabilization (TRF1 and TRF2) genes by real time quantitative polymerization chain reaction. Data show that low-level infrared laser increases the expression of MyoD and Pax7 in 10 J/cm(2) fluence, TRF1 expression in all fluences, and TRF2 expression in 70 J/cm(2) fluence in both 10 and 2 % fetal bovine serum. Low-level infrared laser increases mRNA expression from genes related to muscle repair and telomere stabilization in myoblasts in standard or normal and stressful conditions.

  19. Low-level infrared laser modulates muscle repair and chromosome stabilization genes in myoblasts.

    PubMed

    da Silva Neto Trajano, Larissa Alexsandra; Stumbo, Ana Carolina; da Silva, Camila Luna; Mencalha, Andre Luiz; Fonseca, Adenilson S

    2016-08-01

    Infrared laser therapy is used for skeletal muscle repair based on its biostimulative effect on satellite cells. However, shortening of telomere length limits regenerative potential in satellite cells, which occurs after each cell division cycle. Also, laser therapy could be more effective on non-physiologic tissues. This study evaluated low-level infrared laser exposure effects on mRNA expression from muscle injury repair and telomere stabilization genes in myoblasts in normal and stressful conditions. Laser fluences were those used in clinical protocols. C2C12 myoblast cultures were exposed to low-level infrared laser (10, 35, and 70 J/cm(2)) in standard or normal (10 %) and reduced (2 %) fetal bovine serum concentrations; total RNA was extracted for mRNA expression evaluation from muscle injury repair (MyoD and Pax7) and chromosome stabilization (TRF1 and TRF2) genes by real time quantitative polymerization chain reaction. Data show that low-level infrared laser increases the expression of MyoD and Pax7 in 10 J/cm(2) fluence, TRF1 expression in all fluences, and TRF2 expression in 70 J/cm(2) fluence in both 10 and 2 % fetal bovine serum. Low-level infrared laser increases mRNA expression from genes related to muscle repair and telomere stabilization in myoblasts in standard or normal and stressful conditions. PMID:27220530

  20. Irreversible UV inactivation of Cryptosporidium spp. despite the presence of UV repair genes.

    PubMed

    Rochelle, Paul A; Fallar, Daffodil; Marshall, Marilyn M; Montelone, Beth A; Upton, Steve J; Woods, Keith

    2004-01-01

    Ultraviolet light is being considered as a disinfectant by the water industry because it appears to be very effective for inactivating pathogens, including Cryptosporidium parvum. However, many organisms have mechanisms for repairing ultraviolet light-induced DNA damage, which may limit the utility of this disinfection technology. Inactivation of C. parvum was assessed by measuring infectivity in cells of the human ileocecal adenocarcinoma HCT-8 cell line, with an assay targeting a heat shock protein gene and using a reverse transcriptase polymerase chain reaction to detect infections. Oocysts of five different isolates displayed similar sensitivity to ultraviolet light. An average dosage of 7.6 mJ/cm2 resulted in 99.9% inactivation, providing the first evidence that multiple isolates of C. parvum are equally sensitive to ultraviolet disinfection. Irradiated oocysts were unable to regain pre-irradiation levels of infectivity, following exposure to a broad array of potential repair conditions, such as prolonged incubation, pre-infection excystation triggers, and post-ultraviolet holding periods. A combination of data-mining and sequencing was used to identify genes for all of the major components of a nucleotide excision repair complex in C. parvum and Cryptosporidium hominis. The average similarity between the two organisms for the various genes was 96.4% (range, 92-98%). Thus, while Cryptosporidum spp. may have the potential to repair ultraviolet light-induced damage, oocyst reactivation will not occur under the standard conditions used for storage and distribution of treated drinking water.

  1. Melatonin enhances DNA repair capacity possibly by affecting genes involved in DNA damage responsive pathways

    PubMed Central

    2013-01-01

    Background Melatonin, a hormone-like substance involved in the regulation of the circadian rhythm, has been demonstrated to protect cells against oxidative DNA damage and to inhibit tumorigenesis. Results In the current study, we investigated the effect of melatonin on DNA strand breaks using the alkaline DNA comet assay in breast cancer (MCF-7) and colon cancer (HCT-15) cell lines. Our results demonstrated that cells pretreated with melatonin had significantly shorter Olive tail moments compared to non-melatonin treated cells upon mutagen (methyl methanesulfonate, MMS) exposure, indicating an increased DNA repair capacity after melatonin treatment. We further examined the genome-wide gene expression in melatonin pretreated MCF-7 cells upon carcinogen exposure and detected altered expression of many genes involved in multiple DNA damage responsive pathways. Genes exhibiting altered expression were further analyzed for functional interrelatedness using network- and pathway-based bioinformatics analysis. The top functional network was defined as having relevance for “DNA Replication, Recombination, and Repair, Gene Expression, [and] Cancer”. Conclusions These findings suggest that melatonin may enhance DNA repair capacity by affecting several key genes involved in DNA damage responsive pathways. PMID:23294620

  2. Enhanced gene repair mediated by methyl-CpG-modified single-stranded oligonucleotides

    PubMed Central

    Bertoni, Carmen; Rustagi, Arjun; Rando, Thomas A.

    2009-01-01

    Gene editing mediated by oligonucleotides has been shown to induce stable single base alterations in genomic DNA in both prokaryotic and eukaryotic organisms. However, the low frequencies of gene repair have limited its applicability for both basic manipulation of genomic sequences and for the development of therapeutic approaches for genetic disorders. Here, we show that single-stranded oligodeoxynucleotides (ssODNs) containing a methyl-CpG modification and capable of binding to the methyl-CpG binding domain protein 4 (MBD4) are able to induce >10-fold higher levels of gene correction than ssODNs lacking the specific modification. Correction was stably inherited through cell division and was confirmed at the protein, transcript and genomic levels. Downregulation of MBD4 expression using RNAi prevented the enhancement of gene correction efficacy obtained using the methyl-CpG-modified ssODN, demonstrating the specificity of the repair mechanism being recruited. Our data demonstrate that efficient manipulation of genomic targets can be achieved and controlled by the type of ssODN used and by modulation of the repair mechanism involved in the correction process. This new generation of ssODNs represents an important technological advance that is likely to have an impact on multiple applications, especially for gene therapy where permanent correction of the genetic defect has clear advantages over viral and other nonviral approaches currently being tested. PMID:19854937

  3. Gene Delivery Strategies to Promote Spinal Cord Repair

    PubMed Central

    Walthers, Christopher M; Seidlits, Stephanie K

    2015-01-01

    Gene therapies hold great promise for the treatment of many neurodegenerative disorders and traumatic injuries in the central nervous system. However, development of effective methods to deliver such therapies in a controlled manner to the spinal cord is a necessity for their translation to the clinic. Although essential progress has been made to improve efficiency of transgene delivery and reduce the immunogenicity of genetic vectors, there is still much work to be done to achieve clinical strategies capable of reversing neurodegeneration and mediating tissue regeneration. In particular, strategies to achieve localized, robust expression of therapeutic transgenes by target cell types, at controlled levels over defined time periods, will be necessary to fully regenerate functional spinal cord tissues. This review summarizes the progress over the last decade toward the development of effective gene therapies in the spinal cord, including identification of appropriate target genes, improvements to design of genetic vectors, advances in delivery methods, and strategies for delivery of multiple transgenes with synergistic actions. The potential of biomaterials to mediate gene delivery while simultaneously providing inductive scaffolding to facilitate tissue regeneration is also discussed. PMID:25922572

  4. Transcriptional and Post-Transcriptional Regulation of Nucleotide Excision Repair Genes in Human Cells

    PubMed Central

    Lefkofsky, Hailey B.; Veloso, Artur; Ljungman, Mats

    2014-01-01

    Nucleotide excision repair (NER) removes DNA helix-distorting lesions induced by UV light and various chemotherapeutic agents such as cisplatin. These lesions efficiently block the elongation of transcription and need to be rapidly removed by transcription-coupled NER (TC-NER) to avoid the induction of apoptosis. Twenty-nine genes have been classified to code for proteins participating in nucleotide excision repair (NER) in human cells. Here we explored the transcriptional and post-transcriptional regulation of these NER genes across 13 human cell lines using Bru-seq and BruChase-seq, respectively. Many NER genes are relatively large in size and therefore will be easily inactivated by UV-induced transcription-blocking lesions. Furthermore, many of these genes produce transcripts that are rather unstable. Thus, these genes are expected to rapidly lose expression leading to a diminished function of NER. One such gene is ERCC6 that codes for the CSB protein critical for TC-NER. Due to its large gene size and high RNA turnover rate, the ERCC6 gene may act as dosimeter of DNA damage so that at high levels of damage, ERCC6 RNA levels would be diminished leading to the loss of CSB expression, inhibition of TC-NER and the promotion of cell death. PMID:26255935

  5. DNA damage and gene therapy of xeroderma pigmentosum, a human DNA repair-deficient disease.

    PubMed

    Dupuy, Aurélie; Sarasin, Alain

    2015-06-01

    Xeroderma pigmentosum (XP) is a genetic disease characterized by hypersensitivity to ultra-violet and a very high risk of skin cancer induction on exposed body sites. This syndrome is caused by germinal mutations on nucleotide excision repair genes. No cure is available for these patients except a complete protection from all types of UV radiations. We reviewed the various techniques to complement or to correct the genetic defect in XP cells. We, particularly, developed the correction of XP-C skin cells using the fidelity of the homologous recombination pathway during repair of double-strand break (DSB) in the presence of XPC wild type sequences. We used engineered nucleases (meganuclease or TALE nuclease) to induce a DSB located at 90 bp of the mutation to be corrected. Expression of specific TALE nuclease in the presence of a repair matrix containing a long stretch of homologous wild type XPC sequences allowed us a successful gene correction of the original TG deletion found in numerous North African XP patients. Some engineered nucleases are sensitive to epigenetic modifications, such as cytosine methylation. In case of methylated sequences to be corrected, modified nucleases or demethylation of the whole genome should be envisaged. Overall, we showed that specifically-designed TALE-nuclease allowed us to correct a 2 bp deletion in the XPC gene leading to patient's cells proficient for DNA repair and showing normal UV-sensitivity. The corrected gene is still in the same position in the human genome and under the regulation of its physiological promoter. This result is a first step toward gene therapy in XP patients. PMID:26255934

  6. DNA damage and gene therapy of xeroderma pigmentosum, a human DNA repair-deficient disease.

    PubMed

    Dupuy, Aurélie; Sarasin, Alain

    2015-06-01

    Xeroderma pigmentosum (XP) is a genetic disease characterized by hypersensitivity to ultra-violet and a very high risk of skin cancer induction on exposed body sites. This syndrome is caused by germinal mutations on nucleotide excision repair genes. No cure is available for these patients except a complete protection from all types of UV radiations. We reviewed the various techniques to complement or to correct the genetic defect in XP cells. We, particularly, developed the correction of XP-C skin cells using the fidelity of the homologous recombination pathway during repair of double-strand break (DSB) in the presence of XPC wild type sequences. We used engineered nucleases (meganuclease or TALE nuclease) to induce a DSB located at 90 bp of the mutation to be corrected. Expression of specific TALE nuclease in the presence of a repair matrix containing a long stretch of homologous wild type XPC sequences allowed us a successful gene correction of the original TG deletion found in numerous North African XP patients. Some engineered nucleases are sensitive to epigenetic modifications, such as cytosine methylation. In case of methylated sequences to be corrected, modified nucleases or demethylation of the whole genome should be envisaged. Overall, we showed that specifically-designed TALE-nuclease allowed us to correct a 2 bp deletion in the XPC gene leading to patient's cells proficient for DNA repair and showing normal UV-sensitivity. The corrected gene is still in the same position in the human genome and under the regulation of its physiological promoter. This result is a first step toward gene therapy in XP patients.

  7. The barley EST DNA Replication and Repair Database (bEST-DRRD) as a tool for the identification of the genes involved in DNA replication and repair

    PubMed Central

    2012-01-01

    Background The high level of conservation of genes that regulate DNA replication and repair indicates that they may serve as a source of information on the origin and evolution of the species and makes them a reliable system for the identification of cross-species homologs. Studies that had been conducted to date shed light on the processes of DNA replication and repair in bacteria, yeast and mammals. However, there is still much to be learned about the process of DNA damage repair in plants. Description These studies, which were conducted mainly using bioinformatics tools, enabled the list of genes that participate in various pathways of DNA repair in Arabidopsis thaliana (L.) Heynh to be outlined; however, information regarding these mechanisms in crop plants is still very limited. A similar, functional approach is particularly difficult for a species whose complete genomic sequences are still unavailable. One of the solutions is to apply ESTs (Expressed Sequence Tags) as the basis for gene identification. For the construction of the barley EST DNA Replication and Repair Database (bEST-DRRD), presented here, the Arabidopsis nucleotide and protein sequences involved in DNA replication and repair were used to browse for and retrieve the deposited sequences, derived from four barley (Hordeum vulgare L.) sequence databases, including the “Barley Genome version 0.05” database (encompassing ca. 90% of barley coding sequences) and from two databases covering the complete genomes of two monocot models: Oryza sativa L. and Brachypodium distachyon L. in order to identify homologous genes. Sequences of the categorised Arabidopsis queries are used for browsing the repositories, which are located on the ViroBLAST platform. The bEST-DRRD is currently used in our project during the identification and validation of the barley genes involved in DNA repair. Conclusions The presented database provides information about the Arabidopsis genes involved in DNA replication and

  8. Low intensity infrared laser affects expression of oxidative DNA repair genes in mitochondria and nucleus

    NASA Astrophysics Data System (ADS)

    Fonseca, A. S.; Magalhães, L. A. G.; Mencalha, A. L.; Geller, M.; Paoli, F.

    2014-11-01

    Practical properties and physical characteristics of low intensity lasers have made possible their application to treat soft tissue diseases. Excitation of intracellular chromophores by red and infrared radiation at low energy fluences with increase of mitochondrial metabolism is the basis of the biostimulation effect but free radicals can be produced. DNA lesions induced by free radicals are repaired by the base excision repair pathway. In this work, we evaluate the expression of POLγ and APEX2 genes related to repair of mitochondrial and nuclear DNA, respectively. Skin and muscle tissue of Wistar rats were exposed to low intensity infrared laser at different fluences. One hour and 24 hours after laser exposure, tissue samples were withdrawn for total RNA extraction, cDNA synthesis, and evaluation of POLγ and APEX2 mRNA expression by real time quantitative polymerase chain reaction. Skin and muscle tissue of Wistar rats exposed to laser radiation show different expression of POLγ and APEX2 mRNA depending of the fluence and time after exposure. Our study suggests that a low intensity infrared laser affects expression of genes involved in repair of oxidative lesions in mitochondrial and nuclear DNA.

  9. Coevolution between Nuclear-Encoded DNA Replication, Recombination, and Repair Genes and Plastid Genome Complexity

    PubMed Central

    Zhang, Jin; Ruhlman, Tracey A.; Sabir, Jamal S. M.; Blazier, John Chris; Weng, Mao-Lun; Park, Seongjun; Jansen, Robert K.

    2016-01-01

    Disruption of DNA replication, recombination, and repair (DNA-RRR) systems has been hypothesized to cause highly elevated nucleotide substitution rates and genome rearrangements in the plastids of angiosperms, but this theory remains untested. To investigate nuclear–plastid genome (plastome) coevolution in Geraniaceae, four different measures of plastome complexity (rearrangements, repeats, nucleotide insertions/deletions, and substitution rates) were evaluated along with substitution rates of 12 nuclear-encoded, plastid-targeted DNA-RRR genes from 27 Geraniales species. Significant correlations were detected for nonsynonymous (dN) but not synonymous (dS) substitution rates for three DNA-RRR genes (uvrB/C, why1, and gyrA) supporting a role for these genes in accelerated plastid genome evolution in Geraniaceae. Furthermore, correlation between dN of uvrB/C and plastome complexity suggests the presence of nucleotide excision repair system in plastids. Significant correlations were also detected between plastome complexity and 13 of the 90 nuclear-encoded organelle-targeted genes investigated. Comparisons revealed significant acceleration of dN in plastid-targeted genes of Geraniales relative to Brassicales suggesting this correlation may be an artifact of elevated rates in this gene set in Geraniaceae. Correlation between dN of plastid-targeted DNA-RRR genes and plastome complexity supports the hypothesis that the aberrant patterns in angiosperm plastome evolution could be caused by dysfunction in DNA-RRR systems. PMID:26893456

  10. Repair in ribosomal RNA genes is deficient in xeroderma pigmentosum group C and in Cockayne's syndrome cells.

    PubMed

    Christians, F C; Hanawalt, P C

    1994-04-01

    Previous studies have demonstrated transcription-coupled DNA repair in mammalian genes transcribed by RNA polymerase II but not in ribosomal RNA genes (rDNA), which are transcribed by RNA polymerase I. The removal of UV-induced cyclobutane pyrimidine dimers (CPD) from rDNA in repair-proficient human cells has been shown to be slow but detectable and apparently not coupled to transcription. We studied the induction and removal of CPD from rDNA in cultured cells from two repair-deficient human disorders. Primary xeroderma pigmentosum complementation group C (XP-C) cells, whether proliferating or nondividing, removed no CPD from either rDNA strand in 24 h post-UV, a result which supports earlier conclusions that XP-C cells lack the general, transcription-independent pathway of nucleotide excision repair. We also observed lower than normal repair of rDNA in Cockayne's syndrome (CS) cells from complementation groups A and B. In agreement with previous findings, the repair of both strands of the RNA polymerase II-transcribed dihydrofolate reductase gene was also deficient relative to that of normal cells. This strongly suggests that the defect in CS cells is not limited to a deficiency in a transcription-repair coupling factor. Rather, the defect may interfere with the ability of repair proteins to gain access to all expressed genes. PMID:7512688

  11. Capturing the biological impact of CDKN2A and MC1R genes as an early predisposing event in melanoma and non melanoma skin cancer

    PubMed Central

    Puig-Butille, Joan Anton; Escámez, María José; Garcia-Garcia, Francisco; Tell-Marti, Gemma; Fabra, Àngels; Martínez-Santamaría, Lucía; Badenas, Celia; Aguilera, Paula; Pevida, Marta; Dopazo, Joaquín; del Río, Marcela; Puig, Susana

    2014-01-01

    Germline mutations in CDKN2A and/or red hair color variants in MC1R genes are associated with an increased susceptibility to develop cutaneous melanoma or non melanoma skin cancer. We studied the impact of the CDKN2A germinal mutation p.G101W and MC1R variants on gene expression and transcription profiles associated with skin cancer. To this end we set-up primary skin cell co-cultures from siblings of melanoma prone-families that were later analyzed using the expression array approach. As a result, we found that 1535 transcripts were deregulated in CDKN2A mutated cells, with over-expression of immunity-related genes (HLA-DPB1, CLEC2B, IFI44, IFI44L, IFI27, IFIT1, IFIT2, SP110 and IFNK) and down-regulation of genes playing a role in the Notch signaling pathway. 3570 transcripts were deregulated in MC1R variant carriers. In particular, genes related to oxidative stress and DNA damage pathways were up-regulated as well as genes associated with neurodegenerative diseases such as Parkinson’s, Alzheimer and Huntington. Finally, we observed that the expression signatures indentified in phenotypically normal cells carrying CDKN2A mutations or MC1R variants are maintained in skin cancer tumors (melanoma and squamous cell carcinoma). These results indicate that transcriptome deregulation represents an early event critical for skin cancer development. PMID:24742402

  12. DNA Repair Gene Expression and Risk of Locoregional Relapse in Breast Cancer Patients

    SciTech Connect

    Le Scodan, Romuald; Cizeron-Clairac, Geraldine

    2010-10-01

    Purpose: Radiation therapy appears to kill cells mainly by inducing DNA double-strand breaks. We investigated whether the DNA repair gene expression status might influence the risk of locoregional recurrence (LRR) in breast cancer patients. Methods and Materials: We used a quantitative reverse transcriptase PCR-based approach to measure messenger RNA levels of 20 selected DNA repair genes in tumor samples from 97 breast cancer patients enrolled in a phase III trial (Centre Rene Huguenin cohort). Normalized mRNA levels were tested for an association with LRR-free survival (LRR-FS) and overall survival (OS). The findings were validated in comparison with those of an independent cohort (Netherlands Cancer Institute (NKI) cohort). Multivariate analysis encompassing known prognostic factors was used to assess the association between DNA repair gene expression and patient outcome. Results: RAD51 was the only gene associated with LRR in both cohorts. With a median follow-up of 126 months in the CRH cohort, the 5-year LRR-FS and OS rates were 100% and 95% in the 61 patients with low RAD51 expression, compared with 70% and 69% in the 36 patients with high RAD51 expression, respectively (p < 0.001). RAD51 overexpression was associated with a higher risk of LRR (hazard ratio [HR], 12.83; 95% confidence interval [CI], 3.6-45.6) and death (HR, 4.10; 95% CI, 1.7-9.7). RAD51 overexpression was also significantly associated with shorter LRR-FS and OS in the NKI cohort. Conclusions: Overexpression of RAD51, a key component of the homologous DNA repair pathway, is associated with poor breast cancer outcome. This finding warrants prospective studies of RAD51 as a prognosticator and therapeutic target.

  13. Rearrangement of Rag-1 recombinase gene in DNA-repair deficient/immunodeficient ``wasted`` mice

    SciTech Connect

    Woloschak, G.E.; Weaver, P.; Churchill, M.; Chang-Liu, C-M.; Libertin, C.R.

    1992-11-01

    Mice recessive for the autosomal gene ``wasted`` (wst) display a disease pattern which includes increased sensitivity to the killing effects of ionizing radiation, immunodeficiency, and neurologic dysfunction. The recent cloning and characterization of recombinase genes (Rag-l/Rag-2) expressed in lymphoid and possibly central nervous system tissues prompted us to examine expression of these genes in DNA repair-deficient/immunodeficient wasted mice. Our results revealed that in thymus tissue, a small Rag-I transcript (1.0 kb) was detected in wst/wst mice that was not evident in thymus from control mice. In wst/{sm_bullet} mice, a two-fold increase in Rag-1 mRNA was evident in thymus tissue. Rag-2 mRNA could only be detected in thymus tissue from wst/{sm_bullet} and not from wst/wst or parental control BCF, mice. Southern blots revealed a rearrangement or deletion within the Rag-1 gene of affected wasted mice that was not evident in known strain-specific parental or littermate controls. These results support the idea that the Rag-1 gene may map at or near the locus for the wasted mutation. In addition, they suggest the importance of recombinase function in normal immune and central nervous system development as well as the potential contribution of this gene family to the normal repair of radiation-induced DNA damage.

  14. Rearrangement of Rag-1 recombinase gene in DNA-repair deficient/immunodeficient wasted'' mice

    SciTech Connect

    Woloschak, G.E.; Weaver, P.; Churchill, M.; Chang-Liu, C-M. ); Libertin, C.R. )

    1992-01-01

    Mice recessive for the autosomal gene wasted'' (wst) display a disease pattern which includes increased sensitivity to the killing effects of ionizing radiation, immunodeficiency, and neurologic dysfunction. The recent cloning and characterization of recombinase genes (Rag-l/Rag-2) expressed in lymphoid and possibly central nervous system tissues prompted us to examine expression of these genes in DNA repair-deficient/immunodeficient wasted mice. Our results revealed that in thymus tissue, a small Rag-I transcript (1.0 kb) was detected in wst/wst mice that was not evident in thymus from control mice. In wst/[sm bullet] mice, a two-fold increase in Rag-1 mRNA was evident in thymus tissue. Rag-2 mRNA could only be detected in thymus tissue from wst/[sm bullet] and not from wst/wst or parental control BCF, mice. Southern blots revealed a rearrangement or deletion within the Rag-1 gene of affected wasted mice that was not evident in known strain-specific parental or littermate controls. These results support the idea that the Rag-1 gene may map at or near the locus for the wasted mutation. In addition, they suggest the importance of recombinase function in normal immune and central nervous system development as well as the potential contribution of this gene family to the normal repair of radiation-induced DNA damage.

  15. Evaluation of psychosocial effects of pre-symptomatic testing for breast/ovarian and colon cancer pre-disposing genes: a 12-month follow-up.

    PubMed

    Arver, Brita; Haegermark, Aina; Platten, Ulla; Lindblom, Annika; Brandberg, Yvonne

    2004-01-01

    A prospective study of psychosocial consequences following predictive testing for inherited mutations in breast/ovarian and colon cancer susceptibility genes BRCA1, BRCA2, MLH1, and MSH2 was performed. Eighty-seven healthy women were tested for known family mutations and self-assessment scales were used to evaluate anxiety, depression and quality of life. Extensive pre- and post-test information was given. Questionnaires were responded before testing and four times after during the following year. A statistically significant decrease in anxiety mean scores over time was observed among the studied participants. The levels of depression in cancer genes carriers decreased over time while, surprisingly the levels in non-carriers increased. Compared to a normative Swedish sample all women tested showed similar levels of anxiety but women tested for breast cancer genes showed statistically lower levels of depression. Vitality dropped initially after disclosure of the testing of colon cancer genes carriers, followed by increasing levels. No change in vitality or in other quality of life parameters was seen in the other groups and the levels were similar to Swedish norm data. Most tested individuals were satisfied with the testing procedure including genetic counselling and testing and all of them but one would redo the testing. Healthy self-referred women going through predictive breast/ovarian or colon cancer gene testing, including extensive pre- and post-test information and support, in general, will not experience adverse psychological consequences. PMID:15340261

  16. Incomplete complementation of the DNA repair defect in cockayne syndrome cells by the denV gene from bacteriophage T4 suggests a deficiency in base excision repair.

    PubMed

    Francis, M A; Bagga, P S; Athwal, R S; Rainbow, A J

    1997-10-01

    Endonuclease V (denV) from bacteriophage T4 has been examined for its ability to complement the repair defect in Cockayne syndrome (CS) cells of complementation groups A and B. CS is an autosomal recessive disorder characterized by hypersensitivity to UV light and a defect in the preferential repair of UV-induced lesions in transcriptionally active DNA by the nucleotide excision repair (NER) pathway. The denV gene was introduced into non-transformed normal and CS fibroblasts transiently via a recombinant adenovirus (Ad) vector and into SV40-transformed normal and CS cells via a retroviral vector. Expression of denV in CS-A cells resulted in partial correction of the UV-sensitive phenotype in assays of gene-specific repair and cell viability, while correction of CS-B cells by expression of denV in the same assays was minimal or non-existent. In contrast, denV expression led to enhanced host cell reactivation (HCR) of viral DNA synthesis in both CS complementation groups to near normal levels. DenV is a glycosylase which is specific for cyclobutane-pyrimidine dimers (CPDs) but does not recognize other UV-induced lesions. Previous work has indicated that CS cells can efficiently repair all non-CPD UV-induced transcription blocking lesions (S.F. Barrett et al.. Mutation Res. 255 (1991) 281-291 [1]) and that denV incised lesions are believed to be processed via the base excision repair (BER) pathway. The inability of denV to complement the NER defect in CS cells to normal levels implies an impaired ability to process denV incised lesions by the BER pathway, and suggests a role for the CS genes, particularly the CS-B gene, in BER. PMID:9372849

  17. The C677T mutation in the methylenetetrahydrofolate reductase gene predisposes to hyperhomocysteinemia in children with familial hypercholesterolemia treated with cholestyramine.

    PubMed

    Tonstad, S; Refsum, H; Ose, L; Ueland, P M

    1998-02-01

    In children with familial hypercholesterolemia, heterozygosity and homozygosity for the C677T mutation in the methylenetetrahydrofolate reductase gene was associated with low serum folate and increased susceptibility to elevation of plasma total homocysteine during cholestyramine treatment. Because of the independent relationship between elevated plasma total homocysteine and cardiovascular disease, folate supplementation may be prudent in these children. PMID:9506661

  18. 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.

  19. A comparison of synthetic oligodeoxynucleotides, DNA fragments and AAV-1 for targeted episomal and chromosomal gene repair

    PubMed Central

    Leclerc, Xavier; Danos, Olivier; Scherman, Daniel; Kichler, Antoine

    2009-01-01

    Background Current strategies for gene therapy of inherited diseases consist in adding functional copies of the gene that is defective. An attractive alternative to these approaches would be to correct the endogenous mutated gene in the affected individual. This study presents a quantitative comparison of the repair efficiency using different forms of donor nucleic acids, including synthetic DNA oligonucleotides, double stranded DNA fragments with sizes ranging from 200 to 2200 bp and sequences carried by a recombinant adeno-associated virus (rAAV-1). Evaluation of each gene repair strategy was carried out using two different reporter systems, a mutated eGFP gene or a dual construct with a functional eGFP and an inactive luciferase gene, in several different cell systems. Gene targeting events were scored either following transient co-transfection of reporter plasmids and donor DNAs, or in a system where a reporter construct was stably integrated into the chromosome. Results In both episomal and chromosomal assays, DNA fragments were more efficient at gene repair than oligonucleotides or rAAV-1. Furthermore, the gene targeting frequency could be significantly increased by using DNA repair stimulating drugs such as doxorubicin and phleomycin. Conclusion Our results show that it is possible to obtain repair frequencies of 1% of the transfected cell population under optimized transfection protocols when cells were pretreated with phleomycin using rAAV-1 and dsDNA fragments. PMID:19379497

  20. The rad18 gene of Schizosaccharomyces pombe defines a new subgroup of the SMC superfamily involved in DNA repair

    SciTech Connect

    Lehmann, A.R.; Walicka, M.; Griffiths, D.J.F.; Carr, A.M.

    1995-12-01

    This report describes the cloning and sequencing of the rad18 gene of Schizosaccharomyces pombe and its essential role in cell proliferation. It also describes the isolation and sequencing of its homolog from Saccharomyces cerevisiae, designated RHC18. Genetic radiation effects were explored and results indicate the gene product`s importance in a DNA repair pathway that is distinct from classical nucleotide excision repair. 57 refs., 20 figs., 1 tab.

  1. Microsatellite instability in yeast: dependence on repeat unit size and DNA mismatch repair genes.

    PubMed Central

    Sia, E A; Kokoska, R J; Dominska, M; Greenwell, P; Petes, T D

    1997-01-01

    We examined the stability of microsatellites of different repeat unit lengths in Saccharomyces cerevisiae strains deficient in DNA mismatch repair. The msh2 and msh3 mutations destabilized microsatellites with repeat units of 1, 2, 4, 5, and 8 bp; a poly(G) tract of 18 bp was destabilized several thousand-fold by the msh2 mutation and about 100-fold by msh3. The msh6 mutations destabilized microsatellites with repeat units of 1 and 2 bp but had no effect on microsatellites with larger repeats. These results argue that coding sequences containing repetitive DNA tracts will be preferred target sites for mutations in human tumors with mismatch repair defects. We find that the DNA mismatch repair genes destabilize microsatellites with repeat units from 1 to 13 bp but have no effect on the stability of minisatellites with repeat units of 16 or 20 bp. Our data also suggest that displaced loops on the nascent strand, resulting from DNA polymerase slippage, are repaired differently than loops on the template strand. PMID:9111357

  2. Nucleotide excision repair of the 5 S ribosomal RNA gene assembled into a nucleosome.

    PubMed

    Liu, X; Smerdon, M J

    2000-08-01

    A-175-base pair fragment containing the Xenopus borealis somatic 5 S ribosomal RNA gene was used as a model system to determine the effect of nucleosome assembly on nucleotide excision repair (NER) of the major UV photoproduct (cyclobutane pyrimidine dimer (CPD)) in DNA. Xenopus oocyte nuclear extracts were used to carry out repair in vitro on reconstituted, positioned 5 S rDNA nucleosomes. Nucleosome structure strongly inhibits NER at many CPD sites in the 5 S rDNA fragment while having little effect at a few sites. The time course of CPD removal at 35 different sites indicates that >85% of the CPDs in the naked DNA fragment have t(12) values <2 h, whereas <26% of the t(12) values in nucleosomes are <2 h, and 15% are >8 h. Moreover, removal of histone tails from these mononucleosomes has little effect on the repair rates. Finally, nucleosome inhibition of repair shows no correlation with the rotational setting of a 14-nucleotide-long pyrimidine tract located 30 base pairs from the nucleosome dyad. These results suggest that inhibition of NER by mononucleosomes is not significantly influenced by the rotational orientation of CPDs on the histone surface, and histone tails play little (or no) role in this inhibition. PMID:10821833

  3. Non-homologous DNA increases gene disruption efficiency by altering DNA repair outcomes

    PubMed Central

    Richardson, C. D.; Ray, G. J.; Bray, N. L.; Corn, J. E.

    2016-01-01

    The Cas9 endonuclease can be targeted to genomic sequences by programming the sequence of an associated single guide RNA (sgRNA). For unknown reasons, the activity of these Cas9–sgRNA combinations varies widely at different genomic loci and in different cell types. Thus, disrupting genes in polyploid cell lines or when using poorly performing sgRNAs can require extensive downstream screening to identify homozygous clones. Here we find that non-homologous single-stranded DNA greatly stimulates Cas9-mediated gene disruption in the absence of homology-directed repair. This stimulation increases the frequency of clones with homozygous gene disruptions and rescues otherwise ineffective sgRNAs. The molecular outcome of enhanced gene disruption depends upon cellular context, stimulating deletion of genomic sequence or insertion of non-homologous DNA at the edited locus in a cell line specific manner. Non-homologous DNA appears to divert cells towards error-prone instead of error-free repair pathways, dramatically increasing the frequency of gene disruption. PMID:27530320

  4. Non-homologous DNA increases gene disruption efficiency by altering DNA repair outcomes.

    PubMed

    Richardson, C D; Ray, G J; Bray, N L; Corn, J E

    2016-01-01

    The Cas9 endonuclease can be targeted to genomic sequences by programming the sequence of an associated single guide RNA (sgRNA). For unknown reasons, the activity of these Cas9-sgRNA combinations varies widely at different genomic loci and in different cell types. Thus, disrupting genes in polyploid cell lines or when using poorly performing sgRNAs can require extensive downstream screening to identify homozygous clones. Here we find that non-homologous single-stranded DNA greatly stimulates Cas9-mediated gene disruption in the absence of homology-directed repair. This stimulation increases the frequency of clones with homozygous gene disruptions and rescues otherwise ineffective sgRNAs. The molecular outcome of enhanced gene disruption depends upon cellular context, stimulating deletion of genomic sequence or insertion of non-homologous DNA at the edited locus in a cell line specific manner. Non-homologous DNA appears to divert cells towards error-prone instead of error-free repair pathways, dramatically increasing the frequency of gene disruption. PMID:27530320

  5. Assessment by Southern blot analysis of UV-induced damage and repair in human immunoglobulin genes.

    PubMed

    Bianchi, M S; Bianchi, N O; de la Chapelle, A

    1990-09-01

    Irradiation of DNA with UV light induces pyrimidine dimers and (6-4) photoproducts. The presence of one of these photolesions in the restriction site of a given endonuclease inhibits DNA cleavage and induces the formation of fragments by incomplete DNA digestion which appear as additional, facultative bands in Southern hybridization autoradiograms. The number and size of these fragments show a positive correlation with the UV dose. The response to UV light of immunoglobulin light-chain constant kappa and heavy-chain constant mu genes was analyzed with 2 specific probes. Constant kappa and mu genes when irradiated as part of the chromatin of living lymphocytes showed a UV sensitivity similar to that of naked DNA. The same genes from granulocytes had 50-60 times lower UV sensitivity. When cells were allowed to repair photolesions for 24 h the facultative bands from granulocytes disappeared indicating that these cells were able to remove photolesions from constant kappa and mu genes. Facultative bands from lymphocytes showed a smaller decrease of density after 24 h repair. This suggests that lymphocytes are less efficient than granulocytes in removing UV damage from constant kappa and mu genes.

  6. Existence and expression of photoreactivation repair genes in various yeast species.

    PubMed

    Yasui, A; Eker, A P; Koken, M

    1989-01-01

    Photoreactivation repair (Phr) activities in cell extracts of 13 different yeast species were measured by the Haemophilus influenzae transformation assay. Five species including Schizosaccharomyces pombe showed no or low enzymatic activity. In contrast to the other species, chromosomal DNAs of these 5 species did not show detectable hybridization using a DNA fragment of the photolyase PHR1 gene of Saccharomyces cervisiae as a probe even at a low stringency condition. When the PHR1 gene was attached to the 5'-flanking sequence of the iso-1-cytochrome c (CYC-1) gene of S. cerevisiae and introduced into S. pombe cells, the transformants acquired a high Phr activity, indicating that the PHR1 gene alone can provide a Phr-negative species with this repair activity and the light-absorbing cofactor(s) must be present in S. pombe. Our results also demonstrated that the 5'-flanking sequence of the S. cerevisiae CYC-1 gene works in S. pombe as a regulatory element. PMID:2911265

  7. The Polycomb Group Protein EZH2 Impairs DNA Damage Repair Gene Expression in Human Uterine Fibroids.

    PubMed

    Yang, Qiwei; Nair, Sangeeta; Laknaur, Archana; Ismail, Nahed; Diamond, Michael P; Al-Hendy, Ayman

    2016-03-01

    Uterine fibroids are benign, smooth muscle tumors that occur in approximately 70%-80% of women by age 50 yr. The cellular and molecular mechanism(s) by which uterine fibroids (UFs) develop are not fully understood. Accumulating evidence demonstrates that several genetic abnormalities, including deletions, rearrangements, translocations, as well as mutations, have been found in UFs. These genetic anomalies suggest that low DNA damage repair capacity may be involved in UF formation. The objective of this study was to determine whether expression levels of DNA damage repair-related genes were altered, and how they were regulated in the pathogenesis of UFs. Expression levels of DNA repair-related genes RAD51 and BRCA1 were deregulated in fibroid tissues as compared to adjacent myometrial tissues. Expression levels of chromatin protein enhancer of zeste homolog 2 (EZH2) were higher in a subset of fibroids as compared to adjacent myometrial tissues by both immunohistochemistry and Western blot analysis. Treatment with an inhibitor of EZH2 markedly increased expression levels of RAD51 and BRCA1 in fibroid cells and inhibited cell proliferation paired with cell cycle arrest. Restoring the expression of RAD51 and BRCA1 by treatment with EZH2 inhibitor was dependent on reducing the enrichment of trimethylation of histone 3 lysine 27 epigenetic mark in their promoter regions. This study reveals the important role of EZH2-regulated DNA damage-repair genes via histone methylation in fibroid biology, and may provide novel therapeutic targets for the medical treatment of women with symptomatic UFs. PMID:26888970

  8. Genetic Polymorphisms in DNA Repair Genes as Modulators of Hodgkin Disease Risk

    PubMed Central

    El-Zein, Randa; Monroy, Claudia M.; Etzel, Carol J.; Cortes, Andrea C.; Xing, Yun; Collier, Amanda L.; Strom, Sara S.

    2009-01-01

    BACKGROUND Although the pathogenesis of Hodgkin disease (HD) remains unknown, the results of epidemiologic studies suggest that heritable factors are important in terms of susceptibility. Polymorphisms in DNA repair genes may contribute to individual susceptibility for development of different cancers. However, to the authors’ knowledge, few studies to date have investigated the role of such polymorphisms as risk factors for development of HD. METHODS The authors evaluated the relation between polymorphisms in 3 nucleotide excision repair pathway genes (XPD [Lys751Gln], XPC [Lys939Gln], and XPG [Asp1104His]), the base excision repair XRCC1 (Arg399Gln), and double-strand break repair XRCC3 (Thr241Met) in a population of 200 HD cases and 220 matched controls. Variants were investigated independently and in combination; odd ratios (OR) were calculated. RESULTS A positive association was found for XRCC1 gene polymorphism Arg399Gln (OR, 1.77; 95% confidence interval [95% CI], 1.16−2.71) and risk of HD. The combined analysis demonstrated that XRCC1/XRCC3 and XRCC1/XPC polymorphisms were associated with a significant increase in HD risk. XRCC1 Arg/Arg and XRCC3 Thr/Met genotypes combined were associated with an OR of 2.38 (95% CI, 1.24−4.55). The XRCC1 Arg/Gln and XRCC3 Thr/Thr, Thr/Met, and Met/Met genotypes had ORs of 1.88 (95% CI, 1.02−4.10), 1.97 (95% CI, 1.05−3.73), and 4.13 (95% CI, 1.50−11.33), respectively. XRCC1 Gln/Gln and XRCC3 Thr/Thr variant led to a significant increase in risk, with ORs of 3.00 (95% CI, 1.15−7.80). Similarly, XRCC1 Arg/Gln together with XPC Lys/Lys was found to significantly increase the risk of HD (OR, 2.14; 95% CI, 1.09−4.23). CONCLUSIONS These data suggest that genetic polymorphisms in DNA repair genes may modify the risk of HD, especially when interactions between the pathways are considered. PMID:19280628

  9. Polymorphisms in DNA repair genes, traffic-related polycyclic aromatic hydrocarbon exposure and breast cancer incidence.

    PubMed

    Mordukhovich, Irina; Beyea, Jan; Herring, Amy H; Hatch, Maureen; Stellman, Steven D; Teitelbaum, Susan L; Richardson, David B; Millikan, Robert C; Engel, Lawrence S; Shantakumar, Sumitra; Steck, Susan E; Neugut, Alfred I; Rossner, Pavel; Santella, Regina M; Gammon, Marilie D

    2016-07-15

    Vehicular traffic polycyclic aromatic hydrocarbons (PAHs) have been associated with breast cancer incidence in epidemiologic studies, including our own. Because PAHs damage DNA by forming adducts and oxidative lesions, genetic polymorphisms that alter DNA repair capacity may modify associations between PAH-related exposures and breast cancer risk. Our goal was to examine the association between vehicular traffic exposure and breast cancer incidence within strata of a panel of nine biologically plausible nucleotide excision repair (NER) and base excision repair (BER) genotypes. Residential histories of 1,508 cases and 1,556 controls were assessed in the Long Island Breast Cancer Study Project between 1996 and 1997 and used to reconstruct residential traffic exposures to benzo[a]pyrene, as a proxy for traffic-related PAHs. Likelihood ratio tests from adjusted unconditional logistic regression models were used to assess multiplicative interactions. A gene-traffic interaction was evident (p = 0.04) for ERCC2 (Lys751); when comparing the upper and lower tertiles of 1995 traffic exposure estimates, the odds ratio (95% confidence interval) was 2.09 (1.13, 3.90) among women with homozygous variant alleles. Corresponding odds ratios for 1960-1990 traffic were also elevated nearly 2-3-fold for XRCC1(Arg194Trp), XRCC1(Arg399Gln) and OGG1(Ser326Cys), but formal multiplicative interaction was not evident. When DNA repair variants for ERCC2, XRCC1 and OGG1 were combined, among women with 4-6 variants, the odds ratios were 2.32 (1.22, 4.49) for 1995 traffic and 2.96 (1.06, 8.21) for 1960-1990 traffic. Our study is first to report positive associations between traffic-related PAH exposure and breast cancer incidence among women with select biologically plausible DNA repair genotypes.

  10. Cloning and molecular characterization of the Chinese hamster ERCC2 nucleotide excision repair gene

    SciTech Connect

    Kirchner, J.M.; Salazar, E.P.; Lamerdin, J.E.

    1994-10-01

    The Chinese hamster ERCC2 nucleotide excision repair gene, encoding a presumed ATP-dependent DNA helicase, was cloned from the V79 cell line, and its nucleotide sequence was determined. The {approximately}15-kb gene comprises 23 exons with a 2283-base open reading frame. The predicted 760-amino-acid protein is 98% identical to the human ERCC2/EXP (760 amino acids), 51% identical to the Saccharomyces cerevisiae RAD3 (778 amino acids), and 54% identical to the Schizosaccharomyces pombe rad15 (772 amino acids) proteins. The promoter region of the hamster ERCC2 gene contains a pyrimidine-rich stretch (42 nucleotides, 88% C+T) similar to sequences found in the promoter regions of two other nucleotide excision repair genes, a GC box, a putative {alpha}-Pal transcription factor binding site, and two CAAT boxes. There is no apparent TAATA box. No consensus polyadenylation sequence (AATAAA or its variants) was found with 663 bases 3{prime} of the translation termination codon. 54 refs., 2 figs., 2 tabs.

  11. Tissue repair genes: the TiRe database and its implication for skin wound healing.

    PubMed

    Yanai, Hagai; Budovsky, Arie; Tacutu, Robi; Barzilay, Thomer; Abramovich, Amir; Ziesche, Rolf; Fraifeld, Vadim E

    2016-04-19

    Wound healing is an inherent feature of any multicellular organism and recent years have brought about a huge amount of data regarding regular and abnormal tissue repair. Despite the accumulated knowledge, modulation of wound healing is still a major biomedical challenge, especially in advanced ages. In order to collect and systematically organize what we know about the key players in wound healing, we created the TiRe (Tissue Repair) database, an online collection of genes and proteins that were shown to directly affect skin wound healing. To date, TiRe contains 397 entries for four organisms: Mus musculus, Rattus norvegicus, Sus domesticus, and Homo sapiens. Analysis of the TiRe dataset of skin wound healing-associated genes showed that skin wound healing genes are (i) over-conserved among vertebrates, but are under-conserved in invertebrates; (ii) enriched in extracellular and immuno-inflammatory genes; and display (iii) high interconnectivity and connectivity to other proteins. The latter may provide potential therapeutic targets. In addition, a slower or faster skin wound healing is indicative of an aging or longevity phenotype only when assessed in advanced ages, but not in the young. In the long run, we aim for TiRe to be a one-station resource that provides researchers and clinicians with the essential data needed for a better understanding of the mechanisms of wound healing, designing new experiments, and the development of new therapeutic strategies. TiRe is freely available online at http://www.tiredb.org.

  12. Double-strand gap repair in a mammalian gene targeting reaction.

    PubMed Central

    Valancius, V; Smithies, O

    1991-01-01

    To better understand the mechanism of homologous recombination in mammalian cells that facilitates gene targeting, we have analyzed the recombination reaction that inserts a plasmid into a homologous chromosomal locus in mouse embryonic stem cells. A partially deleted HPRT gene was targeted with various plasmids capable of correcting the mutation at this locus, and HPRT+ recombinants were directly selected in HAT medium. The structures of the recombinant loci were then determined by genomic Southern blot hybridizations. We demonstrate that plasmid gaps of 200, 600, and 2,500 bp are efficiently repaired during the integrative recombination reaction. Targeting plasmids that carry a double-strand break or gap in the region of DNA homologous to the target locus produce 33- to 140-fold more hypoxanthine-aminopterin-thymidine-resistant recombinants than did these same plasmids introduced in their uncut (supercoiled) forms. Our data suggest that double-strand gaps and breaks may be enlarged prior to the repair reaction since sequence heterologies carried by the incoming plasmids located close to them are often lost. These results extend the known similarities between mammalian and yeast recombination mechanisms and suggest several features of the insertional (O-type) gene targeting reaction that should be considered when one is designing mammalian gene targeting experiments. Images PMID:1875928

  13. A mutation in the XPB/ERCC3 DNA repair transcription gene, associated with trichothiodystrophy.

    PubMed

    Weeda, G; Eveno, E; Donker, I; Vermeulen, W; Chevallier-Lagente, O; Taïeb, A; Stary, A; Hoeijmakers, J H; Mezzina, M; Sarasin, A

    1997-02-01

    Trichothiodystrophy (TTD) is a rare, autosomal recessive disorder characterized by sulfur-deficient brittle hair and nails, mental retardation, impaired sexual development, and ichthyosis. Photosensitivity has been reported in approximately 50% of the cases, but no skin cancer is associated with TTD. Virtually all photosensitive TTD patients have a deficiency in the nucleotide excision repair (NER) of UV-induced DNA damage that is indistinguishable from that of xeroderma pigmentosum (XP) complementation group D (XP-D) patients. DNA repair defects in XP-D are associated with two additional, quite different diseases; XP, a sun-sensitive and cancer-prone repair disorder, and Cockayne syndrome (CS), a photosensitive condition characterized by physical and mental retardation and wizened facial appearance. One photosensitive TTD case constitutes a new repair-deficient complementation group, TTD-A. Remarkably, both TTD-A and XP-D defects are associated with subunits of TFIIH, a basal transcription factor with a second function in DNA repair. Thus, mutations in TFIIH components may, on top of a repair defect, also cause transcriptional insufficiency, which may explain part of the non-XP clinical features of TTD. Besides XPD and TTDA, the XPB gene product is also part of TFIIH. To date, three patients with the remarkable conjunction of XP and CS but not TTD have been assigned to XP complementation group B (XP-B). Here we present the characterization of the NER defect in two mild TTD patients (TTD6VI and TTD4VI) and confirm the assignment to X-PB. The causative mutation was found to be a single base substitution resulting in a missense mutation (T119P) in a region of the XPB protein completely conserved in yeast, Drosophila, mouse, and man. These findings define a third TTD complementation group, extend the clinical heterogeneity associated with XP-B, stress the exclusive relationship between TTD and mutations in subunits of repair/transcription factor TFIIH, and strongly

  14. Molecular genetic and biochemical analyses of a DNA repair gene from Serratia marcescens

    SciTech Connect

    Murphy, K.E.

    1989-01-01

    In Escherichia coli, the SOS response and two 3-methyladenine DNA glycosylases (TagI and TagII) are required for repair of DNA damaged by alkylating agents such as methyl methanesulfonate (MMS). Mutations of the recA gene eliminate the SOS response. TagI and TagII are encoded by the tag and alkA genes, respectively. A gene (rpr) encoding 3-methyladenine DNA glycosylase activity was isolated from the Gram-negative bacterium Serratia marcescens. The gene, localized to a 1.5-kilobase pair SmaI-HindIII restriction fragment, was cloned into plasmid pUC18. The clone complemented E. coli tag alkA and recA mutations for MMS resistance. The rpr gene did not, however, complement recA mutations for resistance to ultraviolet light or the ability to perform homologous recombination reactions, nor did it complement E. coli ada or alkB mutations. Two proteins of molecular weights 42,000 and 16,000 were produced from the rpr locus. Analysis of deletion and insertion mutants of rpr suggested that the 42kD molecule is the active protein. The 16kD protein may either be a breakdown product of the 42kD species or may be encoded by another gene overlapping the reading frame of the rpr gene. Biochemical assays showed that the rpr gene product (Rpr) possesses 3-methyladenine DNA glycosylase activity.

  15. Reduced Activity of Double-Strand Break Repair Genes in Prostate Cancer Patients With Late Normal Tissue Radiation Toxicity

    SciTech Connect

    Oorschot, Bregje van; Hovingh, Suzanne E.; Moerland, Perry D.; Medema, Jan Paul; Stalpers, Lukas J.A.; Vrieling, Harry; Franken, Nicolaas A.P.

    2014-03-01

    Purpose: To investigate clinical parameters and DNA damage response as possible risk factors for radiation toxicity in the setting of prostate cancer. Methods and Materials: Clinical parameters of 61 prostate cancer patients, 34 with (overresponding, OR) and 27 without (non-responding, NR) severe late radiation toxicity were assembled. In addition, for a matched subset the DNA damage repair kinetics (γ-H2AX assay) and expression profiles of DNA repair genes were determined in ex vivo irradiated lymphocytes. Results: Examination of clinical data indicated none of the considered clinical parameters to be correlated with the susceptibility of patients to develop late radiation toxicity. Although frequencies of γ-H2AX foci induced immediately after irradiation were similar (P=.32), significantly higher numbers of γ-H2AX foci were found 24 hours after irradiation in OR compared with NR patients (P=.03). Patient-specific γ-H2AX foci decay ratios were significantly higher in NR patients than in OR patients (P<.0001). Consequently, NR patients seem to repair DNA double-strand breaks (DSBs) more efficiently than OR patients. Moreover, gene expression analysis indicated several genes of the homologous recombination pathway to be stronger induced in NR compared with OR patients (P<.05). A similar trend was observed in genes of the nonhomologous end-joining repair pathway (P=.09). This is congruent with more proficient repair of DNA DSBs in patients without late radiation toxicity. Conclusions: Both gene expression profiling and DNA DSB repair kinetics data imply that less-efficient repair of radiation-induced DSBs may contribute to the development of late normal tissue damage. Induction levels of DSB repair genes (eg, RAD51) may potentially be used to assess the risk for late radiation toxicity.

  16. Altered Gene Expressions and Cytogenetic Repair Efficiency in Cells with Suppressed Expression of XPA after Proton Exposure

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Rohde, Larry H.; Gridley, Daila S.; Mehta, Satish K.; Pierson, Duane L.; Wu, Honglu

    2009-01-01

    Cellular responses to damages from ionizing radiation (IR) exposure are influenced not only by the genes involved in DNA double strand break (DSB) repair, but also by non- DSB repair genes. We demonstrated previously that suppressed expression of several non-DSB repair genes, such as XPA, elevated IR-induced cytogenetic damages. In the present study, we exposed human fibroblasts that were treated with control or XPA targeting siRNA to 250 MeV protons (0 to 4 Gy), and analyzed chromosome aberrations and expressions of genes involved in DNA repair. As expected, after proton irradiation, cells with suppressed expression of XPA showed a significantly elevated frequency of chromosome aberrations compared with control siRNA treated (CS) cells. Protons caused more severe DNA damages in XPA knock-down cells, as 36% cells contained multiple aberrations compared to 25% in CS cells after 4Gy proton irradiation. Comparison of gene expressions using the real-time PCR array technique revealed that expressions of p53 and its regulated genes in irradiated XPA suppressed cells were altered similarly as in CS cells, suggesting that the impairment of IR induced DNA repair in XPA suppressed cells is p53-independent. Except for XPA, which was more than 2 fold down regulated in XPA suppressed cells, several other DNA damage sensing and repair genes (GTSE1, RBBP8, RAD51, UNG and XRCC2) were shown a more than 1.5 fold difference between XPA knock-down cells and CS cells after proton exposure. The possible involvement of these genes in the impairment of DNA repair in XPA suppressed cells will be further investigated.

  17. The human CSB (ERCC6) gene corrects the transcription-coupled repair defect in the CHO cell mutant UV61.

    PubMed

    Orren, D K; Dianov, G L; Bohr, V A

    1996-09-01

    The human CSB gene, mutated in Cockayne's syndrome group B (partially defective in both repair and transcription) was previously cloned by virtue of its ability to correct the moderate UV sensitivity of the CHO mutant UV61. To determine whether the defect in UV61 is the hamster equivalent of Cockayne's syndrome, the RNA polymerase II transcription and DNA repair characteristics of a repair-proficient CHO cell line (AA8), UV61 and a CSB transfectant of UV61 were compared. In each cell line, formation and removal of UV-induced cyclobutane pyrimidine dimers (CPDs) were measured in the individual strands of the actively transcribed DHFR gene and in a transcriptionally inactive region downstream of DHFR. AA8 cells efficiently remove CPDs from the transcribed strand, but not from either the non-transcribed strand or the inactive region. There was no detectable repair of CPDs in any region of the genome in UV61. Transfection of the human CSB gene into UV61 restores the normal repair pattern (CPD removal in only the transcribed strand), demonstrating that the DNA repair defect in UV61 is homologous to that in Cockayne's syndrome (complementation group B) cells. However, we observe no significant deficiency in RNA polymerase II-mediated transcription in UV61, suggesting that the CSB protein has independent roles in DNA repair and RNA transcription pathways. PMID:8811084

  18. Divergent genes in potential inoculant Sinorhizobium strains are related to DNA replication, recombination, and repair.

    PubMed

    Penttinen, Petri; Greco, Dario; Muntyan, Victoria; Terefework, Zewdu; De Lajudie, Philippe; Roumiantseva, Marina; Becker, Anke; Auvinen, Petri; Lindström, Kristina

    2016-06-01

    To serve as inoculants of legumes, nitrogen-fixing rhizobium strains should be competitive and tolerant of diverse environments. We hybridized the genomes of symbiotically efficient and salt tolerant Sinorhizobium inoculant strains onto the Sinorhizobium meliloti Rm1021 microarray. The number of variable genes, that is, divergent or putatively multiplied genes, ranged from 503 to 1556 for S. meliloti AK23, S. meliloti STM 1064 and S. arboris HAMBI 1552. The numbers of divergent genes affiliated with the symbiosis plasmid pSymA and related to DNA replication, recombination and repair were significantly higher than expected. The variation was mainly in the accessory genome, implying that it was important in shaping the adaptability of the strains.

  19. The swi4+ gene of Schizosaccharomyces pombe encodes a homologue of mismatch repair enzymes.

    PubMed Central

    Fleck, O; Michael, H; Heim, L

    1992-01-01

    The swi4+ gene of Schizosaccharomyces pombe is involved in termination of copy-synthesis during mating-type switching. The gene was cloned by functional complementation of a swi4 mutant transformed with a genomic library. Determination of the nucleotide sequence revealed an open reading frame of 2979 nucleotides which is interrupted by a 68 bp long intron. The putative Swi4 protein shows homology to Duc-1 (human), Rep-3 (mouse), HexA (Streptococcus pneumoniae) and MutS (Salmonella typhimurium). The prokaryotic proteins are known as essential components involved in mismatch repair. A strain with a disrupted swi4+ gene was constructed and analysed with respect to the switching process. As in swi4 mutants duplications occur in the mating-type region of the swi4 (null) strain, reducing the efficiency of switching. Images PMID:1317550

  20. Rearrangement of RAG-1 recombinase gene in DNA-repair deficient ``wasted`` mice

    SciTech Connect

    Woloschak, G.E.; Libertin, C.R.; Weaver, P.; Churchill, M.; Chang-Liu, C.M.

    1993-11-01

    Mice recessive for the autosomal gene ``wasted`` wst display a disease pattern which includes increased sensitivity to the killing effects of ionizing radiation, immunodeficiency, and neurologic dysfunction. The recent cloning and characterization of recombinase genes (RAG-l/RAG-2) expressed in lymphoid and possibly central nervous system tissues prompted us to examine expression of these genes in DNA repair-deficient/immunodeficient wasted mice. Our results revealed expression of RAG-1 mRNA in spinal cord (but not brain) of control mice; no expression of RAG-1 mRNA was detected in spinal cord or brain from wst/wst mice or their normal littermates (wst/{center_dot}mice). In thymus tissue, a small RAG-1 transcript (1.0 kb) was detected in wst/wst mice that was not evident in thymus from control mice. In wst/{center_dot}mice, a two-fold increase in RAG-1 mRNA was evident in thymus tissue. RAG-2 mRNA could only be detected in thymus tissue from wst/{center_dot} and not from wst/wst or parental control BCF{sub 1} mice. Southern blots revealed a rearrangement/deletion within the RAG-1 gene of affected wasted mice, not evident in known strain-specific parental or littermate controls. These results support the idea that the RAG-1 gene may map at or near the locus for the wasted mutation. In addition, they suggest the importance of recombinase function in normal immune and central nervous system development as well as the potential contribution of this gene family to the normal repair of radiation-induced DNA damage.

  1. Widespread transcriptional gene inactivation initiated by a repair intermediate of 8-oxoguanine

    PubMed Central

    Allgayer, Julia; Kitsera, Nataliya; Bartelt, Solveig; Epe, Bernd; Khobta, Andriy

    2016-01-01

    DNA damage can significantly modulate expression of the affected genes either by direct structural interference with transcription components or as a collateral outcome of cellular repair attempts. Thus, DNA glycosylases of the base excision repair (BER) pathway have been implicated in negative transcriptional response to several spontaneously generated DNA base modifications, including a common oxidative DNA base modification 8-oxoguanine (8-oxoG). Here, we report that single 8-oxoG situated in the non-transcribed DNA strand of a reporter gene has a pronounced negative effect on transcription, driven by promoters of various strength and with different structural properties, including viral, human, and artificial promoters. We further show that the magnitude of the negative effect on the gene expression correlates with excision of the modified base by OGG1 in all promoter constructs tested. Moreover, by using expression vectors with nuclease resistant backbone modifications, we demonstrate that OGG1 does not catalyse DNA strand cleavage in vivo. Rather, cleavage of the phosphate bond 5′ to 8-oxodG (catalysed by APE1) is essential and universally required for the onset of transcriptional silencing, regardless of the promoter structure. Hence, induction of transcriptional silencing emerges as a ubiquitous mode of biological response to 8-oxoG in DNA. PMID:27220469

  2. Are SNP-Smoking Association Studies Needed in Controls? DNA Repair Gene Polymorphisms and Smoking Intensity

    PubMed Central

    Verde, Zoraida; Reinoso, Luis; Chicharro, Luis Miguel; Resano, Pilar; Sánchez-Hernández, Ignacio; Rodríguez González-Moro, Jose Miguel; Bandrés, Fernando

    2015-01-01

    Variations in tobacco-related cancers, incidence and prevalence reflect differences in tobacco consumption in addition to genetic factors. Besides, genes related to lung cancer risk could be related to smoking behavior. Polymorphisms altering DNA repair capacity may lead to synergistic effects with tobacco carcinogen-induced lung cancer risk. Common problems in genetic association studies, such as presence of gene-by-environment (G x E) correlation in the population, may reduce the validity of these designs. The main purpose of this study was to evaluate the independence assumption for selected SNPs and smoking behaviour in a cohort of 320 healthy Spanish smokers. We found an association between the wild type alleles of XRCC3 Thr241Met or KLC3 Lys751Gln and greater smoking intensity (OR = 12.98, 95% CI = 2.86–58.82 and OR=16.90, 95% CI=2.09-142.8; respectively). Although preliminary, the results of our study provide evidence that genetic variations in DNA-repair genes may influence both smoking habits and the development of lung cancer. Population-specific G x E studies should be carried out when genetic and environmental factors interact to cause the disease. PMID:26017978

  3. Are SNP-Smoking Association Studies Needed in Controls? DNA Repair Gene Polymorphisms and Smoking Intensity.

    PubMed

    Verde, Zoraida; Reinoso, Luis; Chicharro, Luis Miguel; Resano, Pilar; Sánchez-Hernández, Ignacio; Rodríguez González-Moro, Jose Miguel; Bandrés, Fernando; Gómez-Gallego, Félix; Santiago, Catalina

    2015-01-01

    Variations in tobacco-related cancers, incidence and prevalence reflect differences in tobacco consumption in addition to genetic factors. Besides, genes related to lung cancer risk could be related to smoking behavior. Polymorphisms altering DNA repair capacity may lead to synergistic effects with tobacco carcinogen-induced lung cancer risk. Common problems in genetic association studies, such as presence of gene-by-environment (G x E) correlation in the population, may reduce the validity of these designs. The main purpose of this study was to evaluate the independence assumption for selected SNPs and smoking behaviour in a cohort of 320 healthy Spanish smokers. We found an association between the wild type alleles of XRCC3 Thr241Met or KLC3 Lys751Gln and greater smoking intensity (OR = 12.98, 95% CI = 2.86-58.82 and OR=16.90, 95% CI=2.09-142.8; respectively). Although preliminary, the results of our study provide evidence that genetic variations in DNA-repair genes may influence both smoking habits and the development of lung cancer. Population-specific G x E studies should be carried out when genetic and environmental factors interact to cause the disease.

  4. Expression of DNA repair genes in burned skin exposed to low-level red laser.

    PubMed

    Trajano, Eduardo Tavares Lima; Mencalha, Andre Luiz; Monte-Alto-Costa, Andréa; Pôrto, Luís Cristóvão; de Souza da Fonseca, Adenilson

    2014-11-01

    Although red laser lights lie in the region of non-ionizing radiations in the electromagnetic spectrum, there are doubts whether absorption of these radiations causes lesions in the DNA molecule. Our aim was to investigate the expression of the genes involved with base excision and nucleotide excision repair pathways in skin tissue submitted to burn injury and exposed to low-level red laser. Wistar rats were divided as follows: control group-rats burned and not irradiated, laser group-rats burned and irradiated 1 day after injury for five consecutive days, and later laser group-rats injured and treated 4 days after injury for five consecutive days. Irradiation was performed according to a clinical protocol (20 J/cm(2), 100 mW, continuous wave emission mode). The animals were sacrificed on day 10, and scarred tissue samples were withdrawn for total RNA extraction, complementary DNA (cDNA) synthesis, and evaluation of gene expression by quantitative polymerase chain reaction. Low-level red laser exposure (1) reduces the expression of APE1 messenger (mRNA), (2) increases the expression of OGG1 mRNA, (3) reduces the expression of XPC mRNA, and (4) increases the expression of XPA mRNA both in laser and later laser groups. Red laser exposure at therapeutic fluences alters the expression of genes related to base excision and nucleotide excision pathways of DNA repair during wound healing of burned skin.

  5. Increased expression of p53 enhances transcription-coupled repair and global genomic repair of a UVC-damaged reporter gene in human cells.

    PubMed

    Dregoesc, Diana; Rybak, Adrian P; Rainbow, Andrew J

    2007-05-01

    Ultraviolet (UV) light-induced DNA damage is repaired by nucleotide excision repair, which is divided into two sub-pathways: global genome repair (GGR) and transcription-coupled repair (TCR). While it is well established that the GGR pathway is dependent on the p53 tumour suppressor protein in human cells, both p53-dependent and p53-independent pathways have been reported for TCR. In the present work, we investigated the role of p53 in both GGR and TCR of a UVC-damaged reporter gene in human fibroblasts. We employed a non-replicating recombinant human adenovirus, AdCA17lacZ, that can efficiently infect human fibroblasts and express the beta-galactosidase (beta-gal) reporter gene under the control of the human cytomegalovirus promoter. We examined host cell reactivation (HCR) of beta-gal expression for the UVC-treated reporter construct in normal fibroblasts and in xeroderma pigmentosum (XP) and Cockayne syndrome (CS) fibroblasts deficient in GGR, TCR, or both. HCR was examined in fibroblasts that had been pre-infected with Ad5p53wt, which expresses wild-type p53, or a control adenovirus, AdCA18luc, which expresses the luciferase gene. We show that increased expression of p53 results in enhanced HCR of the UVC-damaged reporter gene in both untreated and UVC-treated cells for normal, CS-B (TCR-deficient), and XP-C (GGR-deficient), but not XP-A (TCR- and GGR-deficient) fibroblasts. These results indicate an involvement of p53 in both TCR and GGR of the UV-damaged reporter gene in human cells. PMID:17196445

  6. A systematic gene-gene and gene-environment interaction analysis of DNA repair genes XRCC1, XRCC2, XRCC3, XRCC4, and oral cancer risk.

    PubMed

    Yang, Cheng-Hong; Lin, Yu-Da; Yen, Ching-Yui; Chuang, Li-Yeh; Chang, Hsueh-Wei

    2015-04-01

    Oral cancer is the sixth most common cancer worldwide with a high mortality rate. Biomarkers that anticipate susceptibility, prognosis, or response to treatments are much needed. Oral cancer is a polygenic disease involving complex interactions among genetic and environmental factors, which require multifaceted analyses. Here, we examined in a dataset of 103 oral cancer cases and 98 controls from Taiwan the association between oral cancer risk and the DNA repair genes X-ray repair cross-complementing group (XRCCs) 1-4, and the environmental factors of smoking, alcohol drinking, and betel quid (BQ) chewing. We employed logistic regression, multifactor dimensionality reduction (MDR), and hierarchical interaction graphs for analyzing gene-gene (G×G) and gene-environment (G×E) interactions. We identified a significantly elevated risk of the XRCC2 rs2040639 heterozygous variant among smokers [adjusted odds ratio (OR) 3.7, 95% confidence interval (CI)=1.1-12.1] and alcohol drinkers [adjusted OR=5.7, 95% CI=1.4-23.2]. The best two-factor based G×G interaction of oral cancer included the XRCC1 rs1799782 and XRCC2 rs2040639 [OR=3.13, 95% CI=1.66-6.13]. For the G×E interaction, the estimated OR of oral cancer for two (drinking-BQ chewing), three (XRCC1-XRCC2-BQ chewing), four (XRCC1-XRCC2-age-BQ chewing), and five factors (XRCC1-XRCC2-age-drinking-BQ chewing) were 32.9 [95% CI=14.1-76.9], 31.0 [95% CI=14.0-64.7], 49.8 [95% CI=21.0-117.7] and 82.9 [95% CI=31.0-221.5], respectively. Taken together, the genotypes of XRCC1 rs1799782 and XRCC2 rs2040639 DNA repair genes appear to be significantly associated with oral cancer. These were enhanced by exposure to certain environmental factors. The observations presented here warrant further research in larger study samples to examine their relevance for routine clinical care in oncology.

  7. Transplantation of human telomerase reverse transcriptase gene-transfected Schwann cells for repairing spinal cord injury

    PubMed Central

    Zhang, Shu-quan; Wu, Min-fei; Liu, Jia-bei; Li, Ye; Zhu, Qing-san; Gu, Rui

    2015-01-01

    Transfection of the human telomerase reverse transcriptase (hTERT) gene has been shown to increase cell proliferation and enhance tissue repair. In the present study, hTERT was transfected into rat Schwann cells. A rat model of acute spinal cord injury was established by the modified free-falling method. Retrovirus PLXSN was injected at the site of spinal cord injury as a vector to mediate hTERT gene-transfected Schwann cells (1 × 1010/L; 10 μL) or Schwann cells (1 × 1010/L; 10 μL) without hTERT gene transfection. Between 1 and 4 weeks after model establishment, motor function of the lower limb improved in the hTERT-transfected group compared with the group with non-transfected Schwann cells. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and reverse transcription-polymerase chain reaction results revealed that the number of apoptotic cells, and gene expression of aquaporin 4/9 and matrix metalloproteinase 9/2 decreased at the site of injury in both groups; however, the effect improved in the hTERT-transfected group compared with the Schwann cells without hTERT transfection group. Hematoxylin and eosin staining, PKH26 fluorescent labeling, and electrophysiological testing demonstrated that compared with the non-transfected group, spinal cord cavity and motor and sensory evoked potential latencies were reduced, while the number of PKH26-positive cells and the motor and sensory evoked potential amplitude increased at the site of injury in the hTERT-transfected group. These findings suggest that transplantation of hTERT gene-transfected Schwann cells repairs the structure and function of the injured spinal cord. PMID:26889196

  8. DNA repair mechanisms in cancer development and therapy

    PubMed Central

    Torgovnick, Alessandro; Schumacher, Björn

    2015-01-01

    DNA damage has been long recognized as causal factor for cancer development. When erroneous DNA repair leads to mutations or chromosomal aberrations affecting oncogenes and tumor suppressor genes, cells undergo malignant transformation resulting in cancerous growth. Genetic defects can predispose to cancer: mutations in distinct DNA repair systems elevate the susceptibility to various cancer types. However, DNA damage not only comprises a root cause for cancer development but also continues to provide an important avenue for chemo- and radiotherapy. Since the beginning of cancer therapy, genotoxic agents that trigger DNA damage checkpoints have been applied to halt the growth and trigger the apoptotic demise of cancer cells. We provide an overview about the involvement of DNA repair systems in cancer prevention and the classes of genotoxins that are commonly used for the treatment of cancer. A better understanding of the roles and interactions of the highly complex DNA repair machineries will lead to important improvements in cancer therapy. PMID:25954303

  9. Global genome repair of 8-oxoG in hamster cells requires a functional CSB gene product.

    PubMed

    Sunesen, Morten; Stevnsner, Tinna; Brosh, Robert M; Dianov, Grigory L; Bohr, Vilhelm A

    2002-05-16

    Cockayne syndrome (CS) is an autosomal recessive human disease characterized by UV-sensitivity as well as neurological and developmental abnormalities. Two complementation groups have been established, designated CS-A and CS-B. Traditionally, CSA and CSB have been ascribed a function in the transcription-coupled repair (TCR) pathway of nucleotide excision repair (NER) that efficiently removes bulky lesions from the transcribed strand of RNA polymerase II transcribed genes. To assess the role of the CSB protein in the repair of the highly mutagenic base lesion 7,8-dihydro-8-oxoguanine (8-oxoG), we have investigated the removal of this lesion using an in vitro incision approach with cell extracts as well as an in vivo approach with a modified protocol of the gene-specific repair assay, which allows the measurement of base lesion repair in intragenomic sequences. Our results demonstrate that the integrity of the CSB protein is pivotal for processes leading to incision at the site of 8-oxoG and that the global genome repair (GGR) of this lesion requires a functional CSB gene product in vivo. PMID:12032859

  10. The Schizosaccharomyces pombe rhp3+ gene required for DNA repair and cell viability is functionally interchangeable with the RAD3 gene of Saccharomyces cerevisiae.

    PubMed Central

    Reynolds, P R; Biggar, S; Prakash, L; Prakash, S

    1992-01-01

    The RAD3 gene of Saccharomyces cerevisiae is required for excision repair and is essential for cell viability. RAD3 encoded protein possesses a single stranded DNA-dependent ATPase and DNA and DNA.RNA helicase activities. Mutational studies have indicated a requirement for the RAD3 helicase activities in excision repair. To examine the extent of conservation of structure and function of RAD3 during eukaryotic evolution, we have cloned the RAD3 homolog, rhp3+, from the distantly related yeast Schizosaccharomyces pombe. RAD3 and rhp3+ encoded proteins are highly similar, sharing 67% identical amino acids. We show that like RAD3, rhp3+ is indispensable for excision repair and cell viability, and our studies indicate a requirement of the putative rhp3+ DNA helicase activity in DNA repair. We find that the RAD3 and rhp3+ genes can functionally substitute for one another. The level of complementation provided by the rhp3+ gene in S.cerevisiae rad3 mutants or by the RAD3 gene in S.pombe rhp3 mutants is remarkable in that both the excision repair and viability defects in both yeasts are restored to wild type levels. These observations suggest a parallel evolutionary conservation of other protein components with which RAD3 interacts in mediating its DNA repair and viability functions. Images PMID:1534406

  11. Repairing DNA damage in xeroderma pigmentosum: T4N5 lotion and gene therapy.

    PubMed

    Zahid, Sarwar; Brownell, Isaac

    2008-04-01

    Patients with xeroderma pigmentosum (XP) have defective DNA repair and are at a high risk for cutaneous malignancies. Standard treatments for XP are limited in scope and effectiveness. Understanding the molecular etiology of XP has led to the development of novel therapeutic approaches, including enzyme and gene therapies. One new topical treatment utilizing bacteriophage T4 endonuclease 5 (T4N5) in a liposomal lotion is currently in clinical trials and has received a Fast Track designation from the FDA. Gene therapy for XP, while making leaps in preclinical studies, has been slower to develop due to tactical hurdles, but seems to have much potential for future treatment. If these treatments prove effective in lowering the risk of cancer in patients with XP, they may also be found useful in reducing skin cancers in other at-risk patient populations.

  12. p63 and p73 Transcriptionally Regulate Genes Involved in DNA Repair

    PubMed Central

    Gurdziel, Katherine; Bell, George W.; Jacks, Tyler; Flores, Elsa R.

    2009-01-01

    The p53 family activates many of the same genes in response to DNA damage. Because p63 and p73 have structural differences from p53 and play distinct biological functions in development and metastasis, it is likely that they activate a unique transcriptional network. Therefore, we performed a genome-wide analysis using cells lacking the p53 family members after treatment with DNA damage. We identified over 100 genes involved in multiple pathways that were uniquely regulated by p63 or p73, and not p53. Further validation indicated that BRCA2, Rad51, and mre11 are direct transcriptional targets of p63 and p73. Additionally, cells deficient for p63 and p73 are impaired in DNA repair and p63+/−;p73+/− mice develop mammary tumors suggesting a novel mechanism whereby p63 and p73 suppress tumorigenesis. PMID:19816568

  13. RAD6/sup +/ gene of Saccharomyces cerevisiae codes for two mutationally separable deoxyribonucleic acid repair functions

    SciTech Connect

    Tuite, M.F.; Cox, B.S.

    1981-02-01

    The response of two mutant alleles of the RAD6/sup +/ gene of Saccharomyces cerevisiae to the ochre translational suppressor SUQ5 was determined. Both the ultraviolet sensitivity phenotype and the deficiency in ultraviolet-induced mutagenesis phenotype of the rad6-1 allelle were suppressed in a (psi/sup +/) background. For the rad6-3 allelle, only the ultraviolet-sensitivity phenotype was suppressible in a (psi/sup +/) background. An SUQ5 rad6-3 (psi/sup +/) strain that was examined showed the normal rad6-3 deficiency in ultraviolet-induced mutagenesis. The authors propose that the RAD6/sup +/ gene is divided into two cistrons, RAD6A and RAD6B. RAD6A codes for an activity responsible for the error-prone repair of ultraviolet-induced lesions in deoxyribonucleic acid but is not involved in a cell's resistance to the lethal effects of ultraviolet light. RAD6B codes for an activity essential for error-free repair of potentially lethal mutagenic damage.

  14. Tissue repair genes: the TiRe database and its implication for skin wound healing

    PubMed Central

    Yanai, Hagai; Budovsky, Arie; Tacutu, Robi; Barzilay, Thomer; Abramovich, Amir; Ziesche, Rolf; Fraifeld, Vadim E.

    2016-01-01

    Wound healing is an inherent feature of any multicellular organism and recent years have brought about a huge amount of data regarding regular and abnormal tissue repair. Despite the accumulated knowledge, modulation of wound healing is still a major biomedical challenge, especially in advanced ages. In order to collect and systematically organize what we know about the key players in wound healing, we created the TiRe (Tissue Repair) database, an online collection of genes and proteins that were shown to directly affect skin wound healing. To date, TiRe contains 397 entries for four organisms: Mus musculus, Rattus norvegicus, Sus domesticus, and Homo sapiens. Analysis of the TiRe dataset of skin wound healing-associated genes showed that skin wound healing genes are (i) over-conserved among vertebrates, but are under-conserved in invertebrates; (ii) enriched in extracellular and immuno-inflammatory genes; and display (iii) high interconnectivity and connectivity to other proteins. The latter may provide potential therapeutic targets. In addition, a slower or faster skin wound healing is indicative of an aging or longevity phenotype only when assessed in advanced ages, but not in the young. In the long run, we aim for TiRe to be a one-station resource that provides researchers and clinicians with the essential data needed for a better understanding of the mechanisms of wound healing, designing new experiments, and the development of new therapeutic strategies. TiRe is freely available online at http://www.tiredb.org. PMID:27049721

  15. Tissue-engineering strategies to repair joint tissue in osteoarthritis: nonviral gene-transfer approaches.

    PubMed

    Madry, Henning; Cucchiarini, Magali

    2014-10-01

    Loss of articular cartilage is a common clinical consequence of osteoarthritis (OA). In the past decade, substantial progress in tissue engineering, nonviral gene transfer, and cell transplantation have provided the scientific foundation for generating cartilaginous constructs from genetically modified cells. Combining tissue engineering with overexpression of therapeutic genes enables immediate filling of a cartilage defect with an engineered construct that actively supports chondrogenesis. Several pioneering studies have proved that spatially defined nonviral overexpression of growth-factor genes in constructs of solid biomaterials or hydrogels is advantageous compared with gene transfer or scaffold alone, both in vitro and in vivo. Notably, these investigations were performed in models of focal cartilage defects, because advanced cartilage-repair strategies based on the principles of tissue engineering have not advanced sufficiently to enable resurfacing of extensively degraded cartilage as therapy for OA. These studies serve as prototypes for future technological developments, because they raise the possibility that cartilage constructs engineered from genetically modified chondrocytes providing autocrine and paracrine stimuli could similarly compensate for the loss of articular cartilage in OA. Because cartilage-tissue-engineering strategies are already used in the clinic, combining tissue engineering and nonviral gene transfer could prove a powerful approach to treat OA.

  16. The study of the relation of DNA repair pathway genes SNPs and the sensitivity to radiotherapy and chemotherapy of NSCLC

    PubMed Central

    Wang, Chunbo; Nie, Huan; Li, Yiqun; Liu, Guiyou; Wang, Xu; Xing, Shijie; Zhang, Liping; Chen, Xin; Chen, Yue; Li, Yu

    2016-01-01

    To analyze the relation between SNPs in DNA repair pathway-related genes and sensitivity of tumor radio-chemotherapy, 26 SNPs in 20 DNA repair genes were genotyped on 176 patients of NSCLC undertaking radio-chemotherapy treatment. In squamous cell carcinoma (SCC), as the rs2228000, rs2228001 (XPC), rs2273953 (TP73), rs2279744 (MDM2), rs2299939 (PTEN) and rs8178085, rs12334811 (DNA-PKcs) affected the sensitivity to chemotherapy, so did the rs8178085, rs12334811 to radiotherapy. Moreover rs344781, rs2273953 and rs12334811 were related with the survival time of SCC. In general, the “good” genotype GG (rs12334811) showed greater efficacy of radio-chemotherapy and MSF (24 months) on SCC. In adenocarcinoma, as the rs2699887 (PIK3), rs12334811 (DNA-PKcs) influenced the sensitivity to chemotherapy, so did the rs2299939, rs2735343 (PTEN) to radiotherapy. And rs402710, rs80270, rs2279744 and rs2909430 impacted the survival time of the adenocarcinoma patients. Both GG (rs2279744) and AG (rs2909430) showed a shorter survival time (MFS = 6). Additionally, some SNPs such as rs2228000, rs2228001 and rs344781 were found to regulate the expression of DNA repair pathway genes through eQTLs dataset analysis. These results indicate that SNPs in DNA repair pathway genes might regulate the expression and affect the DNA damage repair, and thereby impact the efficacy of radio-chemotherapy and the survival time of NSCLC. PMID:27246533

  17. Study of gene-specific DNA repair in the comet assay with padlock probes and rolling circle amplification.

    PubMed

    Henriksson, Sara; Shaposhnikov, Sergey; Nilsson, Mats; Collins, Andrew

    2011-04-25

    We used padlock probes to study the rate of gene specific repair of three genes, OGG1 (8-oxoguanine-DNA glycosylase-1), XPD (xeroderma pigmentosum group D), and HPRT (hypoxanthine-guanine phosphoribosyltransferase) in human lymphocytes, in relation to the repair rate of Alu repeats and total genomic DNA. Padlock probes offer highly specific detection of short target sequences by combining detection by ligation and signal amplification. In this approach only genes in sequences containing strand breaks, which become single-stranded in the tail, are available for hybridisation. Thus the total number of signals from the padlock probes per comet gives a direct measure of the amount of damage (strand-breaks) present and allows the repair process to be monitored. This method could provide insights on the organisation of genomic DNA in the comet tail. Alu repeat containing DNA was repaired rapidly in comparison with total genomic DNA, and the studied genes were generally repaired more rapidly than the Alu repeats.

  18. Cockayne syndrome exhibits dysregulation of p21 and other gene products that may be independent of transcription-coupled repair.

    PubMed

    Cleaver, J E; Hefner, E; Laposa, R R; Karentz, D; Marti, T

    2007-04-14

    Cockayne syndrome (CS) is a progressive childhood neurodegenerative disorder associated with a DNA repair defect caused by mutations in either of two genes, CSA and CSB. These genes are involved in nucleotide excision repair (NER) of DNA damage from ultraviolet (UV) light, other bulky chemical adducts and reactive oxygen in transcriptionally active genes (transcription-coupled repair, TCR). For a long period it has been assumed that the symptoms of CS patients are all due to reduced TCR of endogenous DNA damage in the brain, together with unexplained unique sensitivity of specific neural cells in the cerebellum. Not all the symptoms of CS patients are however easily related to repair deficiencies, so we hypothesize that there are additional pathways relevant to the disease, particularly those that are downstream consequences of a common defect in the E3 ubiquitin ligase associated with the CSA and CSB gene products. We have found that the CSB defect results in altered expression of anti-angiogenic and cell cycle genes and proteins at the level of both gene expression and protein lifetime. We find an over-abundance of p21 due to reduced protein turnover, possibly due to the loss of activity of the CSA/CSB E3 ubiquitylation pathway. Increased levels of p21 can result in growth inhibition, reduced repair from the p21-PCNA interaction, and increased generation of reactive oxygen. Consistent with increased reactive oxygen levels we find that CS-A and -B cells grown under ambient oxygen show increased DNA breakage, as compared with xeroderma pigmentosum cells. Thus the complex symptoms of CS may be due to multiple, independent downstream targets of the E3 ubiquitylation system that results in increased DNA damage, reduced transcription coupled repair, and inhibition of cell cycle progression and growth. PMID:17055654

  19. DNA repair gene XPD and susceptibility to arsenic-induced hyperkeratosis.

    PubMed

    Ahsan, Habibul; Chen, Yu; Wang, Qiao; Slavkovich, Vesna; Graziano, Joseph H; Santella, Regina M

    2003-07-20

    Chronic exposure to inorganic arsenic is known to cause non-melanocytic skin and internal cancers in humans. An estimated 50-70 million people in Bangladesh have been chronically exposed to arsenic from drinking water and are at risk of skin and other cancers. We undertook the first study to examine whether genetic susceptibility, as determined by the codon 751 SNP (A-->C) of the DNA repair gene XPD, influences the risk of arsenic-induced hyperkeratotic skin lesions, precursors of skin cancer, in a case-control study of 29 hyperkeratosis cases and 105 healthy controls from the same community in an area of Bangladesh. As expected, there was a monotonic increase in risk of hyperkeratosis in relation to urinary arsenic measures but the XPD genotype was not independently associated with the risk. However, the increase in hyperkeratosis risk in relation to urinary arsenic measures genotype was borderline significant for urinary total arsenic (P for trend=0.06) and statistically significant for urinary creatinine adjusted arsenic (P for trend=0.01) among subjects with the XPD A allele (AA) but not among subjects with the other XPD genotypes. Among AA carriers, the risk for the highest arsenic exposed group compared with the lowest was more than 7-fold for urinary total arsenic and about 11-fold for urinary creatinine adjusted arsenic. In conclusion, our findings suggest that the DNA repair gene XPD may influence the risk of arsenic-induced premalignant hyperkeratotic skin lesions. Future larger studies are needed to confirm this novel finding and investigate how combinations of different candidate genes and/or other host and environmental factors may influence the risk of arsenic induced skin and other cancers.

  20. Dimethylarsinic acid in drinking water changed the morphology of urinary bladder but not the expression of DNA repair genes of bladder transitional epithelium in F344 rats.

    PubMed

    Wang, Amy; Wolf, Douglas C; Sen, Banalata; Knapp, Geremy W; Holladay, Steven D; Huckle, William R; Caceci, Thomas; Robertson, John L

    2009-06-01

    Inorganic arsenic increases urinary bladder transitional cell carcinoma in humans. In F344 rats, dimethylarsinic acid (DMA[V]) increases transitional cell carcinoma. Arsenic-induced inhibition of DNA repair has been reported in cultured cell lines and in lymphocytes of arsenic-exposed humans, but it has not been studied in urinary bladder. Should inhibition of DNA damage repair in transitional epithelium occur, it may contribute to carcinogenesis or cocarcinogenesis. We investigated morphology and expression of DNA repair genes in F344 rat transitional cells following up to 100 ppm DMA(V) in drinking water for four weeks. Mitochondria were very sensitive to DMA(V), and swollen mitochondria appeared to be the main source of vacuoles in the transitional epithelium. Real-time reverse transcriptase polymerase chain reaction (Real-Time RT PCR) showed the mRNA levels of tested DNA repair genes, ataxia telangectasia mutant (ATM), X-ray repair cross-complementing group 1 (XRCC1), excision repair cross-complementing group 3/xeroderma pigmentosum B (ERCC3/XPB), and DNA polymerase beta (Polbeta), were not altered by DMA(V). These data suggested that either DMA(V) does not affect DNA repair in the bladder or DMA(V) affects DNA repair without affecting baseline mRNA levels of repair genes. The possibility remains that DMA(V) may lower damage-induced increases in repair gene expression or cause post-translational modification of repair enzymes.

  1. Isolation of a human DNA repair gene by selection in Chinese hamster ovary cells

    SciTech Connect

    Ding, R.C.; Eastman, A.; Bresnick, E.

    1987-05-01

    Alkylation of DNA at the O/sup 6/-position of guanine represents a potent mutagenic and carcinogenic lesion. O/sup 6/-Methylguanine DNA methyltransferase is the repair system responsible for catalyzing the transfer of the methyl group to a cysteine of the protein in a suicide reaction. The gene controlling its expression in mammalian systems is designated mex. Resistance to chloroethylnitrosourea (CNU) is also mediated by this protein; this was used to select cells into which the max gene has been introduced. DNA purified from human liver has been transfected into mex/sup -/ CHO cells by the CaPO/sub 4/ method. pSV2gpt, containing a marker gene, gpt, was cotransfected. The transformed cells were initially selected for the expression of gpt (mycophenolic acid resistance) and reselected in CNU for mex/sup +/. Several clones were resistant to both demonstrating the linkage of these genes. A cosmid library was made from a mex/sup +/gpt/sup +/ clone and grown in a gpt/sup -/ strain of E. coli. gpt/sup +/ colonies were selected and the cosmid DNA rescued. One of the tested cosmid DNA's produced CNU resistance upon introduction into CHO cells. This cosmid was subcloned, restriction endonuclease-treated and a 5.3 kb fragment showed mex activity. This fragment is being further characterized and the DNA sequenced.

  2. 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.

  3. Resveratrol improves bone repair by modulation of bone morphogenetic proteins and osteopontin gene expression in rats.

    PubMed

    Casarin, R C; Casati, M Z; Pimentel, S P; Cirano, F R; Algayer, M; Pires, P R; Ghiraldini, B; Duarte, P M; Ribeiro, F V

    2014-07-01

    This study investigated the effect of resveratrol on bone healing and its influence on the gene expression of osteogenic markers. Two calvarial defects were created and one screw-shaped titanium implant was inserted in the tibia of rats that were assigned to daily administration of placebo (control group, n=15) or 10mg/kg of resveratrol (RESV group, n=15) for 30 days. The animals were then sacrificed. One of the calvarial defects was processed for histomorphometric analysis and the tissue relative to the other was collected for mRNA quantification of bone morphogenetic protein (BMP)-2, BMP-7, osteopontin (OPN), bone sialoprotein (BSP), osteoprotegrin (OPG), and receptor activator of NF-κB ligand (RANKL). Implants were removed by applying a counter-torque force. Histomorphometric analysis revealed higher remaining defect in the calvarial defects of the control group than the RESV group (P=0.026). Resveratrol increased the counter-torque values of implant removal when compared to control therapy (P=0.031). Gene expression analysis showed a higher expression of BMP-2 (P=0.011), BMP-7 (P=0.049), and OPN (P=0.002) genes in the RESV group than in the control group. In conclusion, resveratrol improved the repair of critical-sized bone defects and the biomechanical retention of implants. Indeed, this natural agent may up-regulate the gene expression of important osteogenic markers. PMID:24530035

  4. Fibrin patch-based insulin-like growth factor-1 gene-modified stem cell transplantation repairs ischemic myocardium

    PubMed Central

    Li, Jun; Zhu, Kai; Yang, Shan; Wang, Yulin; Guo, Changfa; Yin, Kanhua; Wang, Chunsheng

    2015-01-01

    Bone marrow mesenchymal stem cells (BMSCs), tissue-engineered cardiac patch, and therapeutic gene have all been proposed as promising therapy strategies for cardiac repair after myocardial infarction. In our study, BMSCs were modified with insulin-like growth factor-1 (IGF-1) gene, loaded into a fibrin patch, and then transplanted into a porcine model of ischemia/reperfusion (I/R) myocardium injury. The results demonstrated that IGF-1 gene overexpression could promote proliferation of endothelial cells and cardiomyocyte-like differentiation of BMSCs in vitro. Four weeks after transplantation of fibrin patch loaded with gene-modified BMSCs, IGF-1 overexpression could successfully promote angiogenesis, inhibit remodeling, increase grafted cell survival and reduce apoptosis. In conclusion, the integrated strategy, which combined fibrin patch with IGF-1 gene modified BMSCs, could promote the histological cardiac repair for a clinically relevant porcine model of I/R myocardium injury. PMID:25767192

  5. Changes in the expression of DNA double strand break repair genes in primordial follicles from immature and aged rats.

    PubMed

    Govindaraj, Vijayakumar; Keralapura Basavaraju, Rajani; Rao, Addicam Jagannadha

    2015-03-01

    Oocytes present at birth undergo a progressive process of apoptosis in humans and other mammals as they age. Accepted opinion is that no fresh oocytes are produced other than those present at the time of birth. Studies have shown that DNA repair genes in oocytes of mice and women decline with age, and lack of these genes show higher DNA breaks and increased oocyte death rates. In contrast to the ethical problems associated with monitoring the changes in DNA double-strand breaks in oocytes from young and old humans, it is relatively easy to carry out such a study using a rodent model. In this study, the mRNA levels of DNA repair genes are compared with protein products of some of the genes in the primordial follicles isolated from immature (18-20 days) and aged (400-450 days) female rats. Results revealed a significant decline in mRNA levels of BRAC1 (P < 0.01), RAD51 (P < 0.05), ERCC2 (P < 0.05), and H2AX (P < 0.01) of DNA repair genes and phospho-protein levels of BRAC1 (P < 0.01) and H2AX (P < 0.05) in primordial follicles of aged rats. Impaired DNA repair is confirmed as a mechanism of oocyte ageing.

  6. Enzymatic repair of selected cross-linked homoduplex molecules enhances nuclear gene rescue from Pompeii and Herculaneum remains.

    PubMed

    Di Bernardo, Giovanni; Del Gaudio, Stefania; Cammarota, Marcella; Galderisi, Umberto; Cascino, Antonino; Cipollaro, Marilena

    2002-02-15

    Ancient DNA (aDNA) samples extracted from the bone remains of six equids buried by the Vesuvius eruption in 79 AD were investigated to test pre-amplification and enzymatic repair procedures designed to enhance the rescue of nuclear genes. The extracts, which proved all positive for Equidae mtDNA amplification, proved positive only four times out of 18 when tested for single-copy Equidae nuclear genes (epsilon globin, p53 and gamma interferon). Pre-amplification did not change the number of retrieved aDNA sequences but 10 times out of 14 enzymatic repair restored the amplifiability of the genes analysed, proving that repair increases the rate of successful rescue from 22 to alpha(lambda)mu(omicron)sigma(tau) 80%. These findings support the hypothesis that some of these cross-linked aDNA molecules, which are not completely separated when DNA is extracted under denaturing conditions, become homoduplex substrates for Pol I and/or T4 ligase action upon renaturation. aDNA authenticity is proved by the homology of the nucleotide sequences of loci tested to the corresponding modern Equidae sequences. Data also indicate that cross-linked homoduplex molecules selected by denaturation of the extract are repaired without any chimera formation. The general features of aDNA amplification with and without denaturation and enzymatic repair are discussed.

  7. Benzo(a)pyrene induces similar gene expression changes in testis of DNA repair proficient and deficient mice

    PubMed Central

    2010-01-01

    Background Benzo [a]pyrene (B[a]P) exposure induces DNA adducts at all stages of spermatogenesis and in testis, and removal of these lesions is less efficient in nucleotide excision repair deficient Xpc-/- mice than in wild type mice. In this study, we investigated by using microarray technology whether compromised DNA repair in Xpc-/- mice may lead to a transcriptional reaction of the testis to cope with increased levels of B[a]P induced DNA damage. Results Two-Way ANOVA revealed only 4 genes differentially expressed between wild type and Xpc-/- mice, and 984 genes between testes of B[a]P treated and untreated mice irrespective of the mouse genotype. However, the level in which these B[a]P regulated genes are expressed differs between Wt and Xpc-/- mice (p = 0.000000141), and were predominantly involved in the regulation of cell cycle, translation, chromatin structure and spermatogenesis, indicating a general stress response. In addition, analysis of cell cycle phase dependent gene expression revealed that expression of genes involved in G1-S and G2-M phase arrest was increased after B[a]P exposure in both genotypes. A slightly higher induction of average gene expression was observed at the G2-M checkpoint in Xpc-/- mice, but this did not reach statistical significance (P = 0.086). Other processes that were expected to have changed by exposure, like apoptosis and DNA repair, were not found to be modulated at the level of gene expression. Conclusion Gene expression in testis of untreated Xpc-/- and wild type mice were very similar, with only 4 genes differentially expressed. Exposure to benzo(a)pyrene affected the expression of genes that are involved in cell cycle regulation in both genotypes, indicating that the presence of unrepaired DNA damage in testis blocks cell proliferation to protect DNA integrity in both DNA repair proficient and deficient animals. PMID:20504355

  8. 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

  9. The Gene Targeting Approach of Small Fragment Homologous Replacement (SFHR) Alters the Expression Patterns of DNA Repair and Cell Cycle Control Genes

    PubMed Central

    Pierandrei, Silvia; Luchetti, Andrea; Sanchez, Massimo; Novelli, Giuseppe; Sangiuolo, Federica; Lucarelli, Marco

    2016-01-01

    Cellular responses and molecular mechanisms activated by exogenous DNA that invades cells are only partially understood. This limits the practical use of gene targeting strategies. Small fragment homologous replacement (SFHR) uses a small exogenous wild-type DNA fragment to restore the endogenous wild-type sequence; unfortunately, this mechanism has a low frequency of correction. In this study, we used a mouse embryonic fibroblast cell line with a stably integrated mutated gene for enhanced green fluorescence protein. The restoration of a wild-type sequence can be detected by flow cytometry analysis. We quantitatively analyzed the expression of 84 DNA repair genes and 84 cell cycle control genes. Peculiar temporal gene expression patterns were observed for both pathways. Different DNA repair pathways, not only homologous recombination, as well as the three main cell cycle checkpoints appeared to mediate the cellular response. Eighteen genes were selected as highly significant target/effectors of SFHR. We identified a wide interconnection between SFHR, DNA repair, and cell cycle control. Our results increase the knowledge of the molecular mechanisms involved in cell invasion by exogenous DNA and SFHR. Specific molecular targets of both the cell cycle and DNA repair machineries were selected for manipulation to enhance the practical application of SFHR. PMID:27045208

  10. A critical re-assessment of DNA repair gene promoter methylation in non-small cell lung carcinoma

    PubMed Central

    Do, Hongdo; Wong, Nicholas C.; Murone, Carmel; John, Thomas; Solomon, Benjamin; Mitchell, Paul L.; Dobrovic, Alexander

    2014-01-01

    DNA repair genes that have been inactivated by promoter methylation offer potential therapeutic targets either by targeting the specific repair deficiency, or by synthetic lethal approaches. This study evaluated promoter methylation status for eight selected DNA repair genes (ATM, BRCA1, ERCC1, MGMT, MLH1, NEIL1, RAD23B and XPC) in 56 non-small cell lung cancer (NSCLC) tumours and 11 lung cell lines using the methylation-sensitive high resolution melting (MS-HRM) methodology. Frequent methylation in NEIL1 (42%) and infrequent methylation in ERCC1 (2%) and RAD23B (2%) are reported for the first time in NSCLC. MGMT methylation was detected in 13% of the NSCLCs. Contrary to previous studies, methylation was not detected in ATM, BRCA1, MLH1 and XPC. Data from The Cancer Genome Atlas (TCGA) was consistent with these findings. The study emphasises the importance of using appropriate methodology for accurate assessment of promoter methylation. PMID:24569633

  11. Detoxification and repair process of ozone injury: from O3 uptake to gene expression adjustment.

    PubMed

    Castagna, A; Ranieri, A

    2009-05-01

    Plants react to O(3) threat by setting up a variety of defensive strategies involving the co-ordinated modulation of stress perception, signalling and metabolic responses. Although stomata largely controls O(3) uptake, differences in O(3) tolerance cannot always be ascribed to changes in stomatal conductance but cell protective and repair processes should be taken into account. O(3)-driven ROS production in the apoplast induces a secondary, active, self-propagating generation of ROS, whose levels must be finely tuned, by many enzymatic and non-enzymatic antioxidant systems, to induce gene activation without determining uncontrolled cell death. Additional signalling molecules, as ethylene, jasmonic and salicylic acid are also crucial to determine the spreading and the containment of leaf lesions. The main recent results obtained on O(3) sensing, signal transduction, ROS formation and detoxification mechanisms are here discussed.

  12. ABCB5 is a limbal stem cell gene required for corneal development and repair

    PubMed Central

    Ksander, Bruce R.; Kolovou, Paraskevi E.; Wilson, Brian J.; Saab, Karim R.; Guo, Qin; Ma, Jie; McGuire, Sean P.; Gregory, Meredith S.; Vincent, William J. B.; Perez, Victor L.; Cruz-Guilloty, Fernando; Kao, Winston W. Y.; Call, Mindy K.; Tucker, Budd A.; Zhan, Qian; Murphy, George F.; Lathrop, Kira L.; Alt, Clemens; Mortensen, Luke J.; Lin, Charles P.; Zieske, James D.; Frank, Markus H.; Frank, Natasha Y.

    2014-01-01

    Corneal epithelial homeostasis and regeneration are sustained by limbal stem cells (LSCs)1–3, and LSC deficiency is a major cause of blindness worldwide4. Transplantation is often the only therapeutic option available to patients with LSC deficiency. However, while transplant success depends foremost on LSC frequency within grafts5, a gene allowing for prospective LSC enrichment has not been identified so far5. Here we show that ATP-binding cassette, sub-family B, member 5 (ABCB5)6,7 marks LSCs and is required for LSC maintenance, corneal development and repair. Furthermore, we demonstrate that prospectively isolated human or murine ABCB5-positive LSCs possess the exclusive capacity to fully restore the cornea upon grafting to LSC-deficient mice in xenogeneic or syngeneic transplantation models. ABCB5 is preferentially expressed on label-retaining LSCs2 in mice and p63α-positive LSCs8 in humans. Consistent with these findings, ABCB5-positive LSC frequency is reduced in LSC-deficient patients. Abcb5 loss of function in Abcb5 knockout mice causes depletion of quiescent LSCs due to enhanced proliferation and apoptosis, and results in defective corneal differentiation and wound healing. Our results from gene knockout studies, LSC tracing and transplantation models, as well as phenotypic and functional analyses of human biopsy specimens, provide converging lines of evidence that ABCB5 identifies mammalian LSCs. Identification and prospective isolation of molecularly defined LSCs with essential functions in corneal development and repair has important implications for the treatment of corneal disease, particularly corneal blindness due to LSC deficiency. PMID:25030174

  13. Characterization of Chondrocyte Scaffold Carriers for Cell-based Gene Therapy in Articular Cartilage Repair

    PubMed Central

    Shui, Wei; Yin, Liangjun; Luo, Jeffrey; Li, Ruidong; Zhang, Wenwen; Zhang, Jiye; Huang, Wei; Hu, Ning; Liang, Xi; Deng, Zhong-Liang; Hu, Zhenming; Shi, Lewis; Luu, Hue H.; Haydon, Rex C.; He, Tong-Chuan; Ho, Sherwin

    2014-01-01

    Articular cartilage lesions in the knee are common injuries. Chondrocyte transplant represents a promising therapeutic modality for articular cartilage injuries. Here, we characterize the viability and transgene expression of articular chondrocytes cultured in 3-D scaffolds provided by four types of carriers. Articular chondrocytes are isolated from rabbit knees and cultured in four types of scaffolds: type I collagen sponge, fibrin glue, hyaluronan, and Open-cell PolyLactic Acid (OPLA). The cultured cells are transduced with adenovirus expressing green fluorescence protein (AdGFP) and luciferase (AdGL3-Luc). The viability and gene expression in the chondrocytes are determined with fluorescence microscopy and luciferase assay. Cartilage matrix production is assessed by Alcian blue staining. Rabbit articular chondrocytes are effectively infected by AdGFP and exhibited sustained GFP expression. All tested scaffolds support the survival and gene expression of the infected chondrocytes. However, the highest transgene expression is observed in the OPLA carrier. At four weeks, Alcian blue-positive matrix materials are readily detected in OPLA cultures. Thus, our results indicate that, while all tested carriers can support the survival of chondrocytes, OPLA supports the highest transgene expression and is the most conductive scaffold for matrix production, suggesting that OPLA may be a suitable scaffold for cell-based gene therapy of articular cartilage repairs. PMID:23629940

  14. The ATPase domain but not the acidic region of Cockayne syndrome group B gene product is essential for DNA repair.

    PubMed

    Brosh, R M; Balajee, A S; Selzer, R R; Sunesen, M; Proietti De Santis, L; Bohr, V A

    1999-11-01

    Cockayne syndrome (CS) is a human genetic disorder characterized by UV sensitivity, developmental abnormalities, and premature aging. Two of the genes involved, CSA and CSB, are required for transcription-coupled repair (TCR), a subpathway of nucleotide excision repair that removes certain lesions rapidly and efficiently from the transcribed strand of active genes. CS proteins have also been implicated in the recovery of transcription after certain types of DNA damage such as those lesions induced by UV light. In this study, site-directed mutations have been introduced to the human CSB gene to investigate the functional significance of the conserved ATPase domain and of a highly acidic region of the protein. The CSB mutant alleles were tested for genetic complementation of UV-sensitive phenotypes in the human CS-B homologue of hamster UV61. In addition, the CSB mutant alleles were tested for their ability to complement the sensitivity of UV61 cells to the carcinogen 4-nitroquinoline-1-oxide (4-NQO), which introduces bulky DNA adducts repaired by global genome repair. Point mutation of a highly conserved glutamic acid residue in ATPase motif II abolished the ability of CSB protein to complement the UV-sensitive phenotypes of survival, RNA synthesis recovery, and gene-specific repair. These data indicate that the integrity of the ATPase domain is critical for CSB function in vivo. Likewise, the CSB ATPase point mutant failed to confer cellular resistance to 4-NQO, suggesting that ATP hydrolysis is required for CSB function in a TCR-independent pathway. On the contrary, a large deletion of the acidic region of CSB protein did not impair the genetic function in the processing of either UV- or 4-NQO-induced DNA damage. Thus the acidic region of CSB is likely to be dispensable for DNA repair, whereas the ATPase domain is essential for CSB function in both TCR-dependent and -independent pathways. PMID:10564257

  15. DNA repair of oxidative DNA damage in human carcinogenesis

    PubMed Central

    Paz-Elizur, Tamar; Sevilya, Ziv; Leitner-Dagan, Yael; Elinger, Dalia; Roisman, Laila; Livneh, Zvi

    2008-01-01

    Efficient DNA repair mechanisms comprise a critical component in the protection against human cancer, as indicated by the high predisposition to cancer of individuals with germ-line mutations in DNA repair genes. This includes biallelic germ-line mutations in the MUYH gene, encoding a DNA glycosylase that is involved in the repair of oxidative DNA damage, which strongly predispose humans to a rare hereditary form of colorectal cancer. Extensive research efforts including biochemical, enzymological and genetic studies in model organisms established that the oxidative DNA lesion 8-oxoguanine is mutagenic, and that several DNA repair mechanisms operate to prevent its potentially mutagenic and carcinogenic outcome. Epidemiological studies on the association with sporadic cancers of single nucleotide polymorphisms in genes such as OGG1, involved in the repair of 8-oxoguanine yielded conflicting results, and suggest a minor effect at best. A new approach based on the functional analysis of DNA repair enzymatic activity showed that reduced activity of 8-oxoguanine DNA glycosylase (OGG) is a risk factor in lung and head and neck cancer. Moreover, the combination of smoking and low OGG activity was associated with a higher risk, suggesting a potential strategy for risk assessment and prevention of lung cancer, as well as other types of cancer. PMID:18374480

  16. ERCC6, a member of a subfamily of putative helicases, is involved in Cockayne's syndrome and preferential repair of active genes.

    PubMed

    Troelstra, C; van Gool, A; de Wit, J; Vermeulen, W; Bootsma, D; Hoeijmakers, J H

    1992-12-11

    Cells from patients with the UV-sensitive nucleotide excision repair disorder Cockayne's syndrome (CS) have a specific defect in preferential repair of lesions from the transcribed strand of active genes. This system permits quick resumption of transcription after UV exposure. Here we report the characterization of ERCC6, a gene involved in preferential repair in eukaryotes. ERCC6 corrects the repair defect of CS complementation group B (CS-B). It encodes a protein of 1493 amino acids, containing seven consecutive domains conserved between DNA and RNA helicases. The entire helicase region bears striking homology to segments in recently discovered proteins involved in transcription regulation, chromosome stability, and DNA repair. Mutation analysis of a CS-B patient indicates that the gene is not essential for cell viability and is specific for preferential repair of transcribed sequences. PMID:1339317

  17. Repair of uv damaged DNA: Genes and proteins of yeast and human

    SciTech Connect

    Prakash, L.

    1992-04-01

    Our objectives are to determine the molecular mechanism of the incision step of excision repair of ultraviolet (UV) light damaged DNA in eukaryotic organisms, using the yeast Saccharomyces cerevisiae as a model system, and to study the human homologs of yeast excision repair and postreplication repair proteins progress is described.

  18. Necrotic enteritis predisposing factors in broiler chickens.

    PubMed

    Moore, Robert J

    2016-06-01

    Necrotic enteritis in chickens develops as a result of infection with pathogenic strains of Clostridium perfringens and the presence of predisposing factors. Predisposing factors include elements that directly change the physical properties of the gut, either damaging the epithelial surface, inducing mucus production, or changing gut transit times; factors that disrupt the gut microbiota; and factors that alter the immune status of birds. In the past research into necrotic enteritis predisposing factors was directed by the simple hypothesis that low-level colonization of C. perfringens commonly occurred within the gut of healthy chickens and the predisposing factors lead to a proliferation of those bacteria to produce disease. More recently, with an increasing understanding of the major virulence factors of C. perfringens and the application of molecular techniques to define different clades of C. perfringens strains, it has become clear that the C. perfringens isolates commonly found in healthy chickens are generally not strains that have the potential to cause disease. Therefore, we need to re-evaluate hypotheses regarding the development of disease, the origin of disease causing isolates of C. perfringens, and the importance of interactions with other C. perfringens strains and with predisposing factors. Many predisposing factors that affect the physical and immunological characteristics of the gastrointestinal tract may also change the resident microbiota. Research directed towards defining the relative importance of each of these different actions of predisposing factors will improve the understanding of disease pathogenesis and may allow refinement of experiment disease models. PMID:26926926

  19. Necrotic enteritis predisposing factors in broiler chickens.

    PubMed

    Moore, Robert J

    2016-06-01

    Necrotic enteritis in chickens develops as a result of infection with pathogenic strains of Clostridium perfringens and the presence of predisposing factors. Predisposing factors include elements that directly change the physical properties of the gut, either damaging the epithelial surface, inducing mucus production, or changing gut transit times; factors that disrupt the gut microbiota; and factors that alter the immune status of birds. In the past research into necrotic enteritis predisposing factors was directed by the simple hypothesis that low-level colonization of C. perfringens commonly occurred within the gut of healthy chickens and the predisposing factors lead to a proliferation of those bacteria to produce disease. More recently, with an increasing understanding of the major virulence factors of C. perfringens and the application of molecular techniques to define different clades of C. perfringens strains, it has become clear that the C. perfringens isolates commonly found in healthy chickens are generally not strains that have the potential to cause disease. Therefore, we need to re-evaluate hypotheses regarding the development of disease, the origin of disease causing isolates of C. perfringens, and the importance of interactions with other C. perfringens strains and with predisposing factors. Many predisposing factors that affect the physical and immunological characteristics of the gastrointestinal tract may also change the resident microbiota. Research directed towards defining the relative importance of each of these different actions of predisposing factors will improve the understanding of disease pathogenesis and may allow refinement of experiment disease models.

  20. Mutational analysis of the Drosophila DNA repair and recombination gene mei-9.

    PubMed Central

    Yildiz, Ozlem; Kearney, Hutton; Kramer, Benjamin C; Sekelsky, Jeff J

    2004-01-01

    Drosophila mei-9 is essential for several DNA repair and recombination pathways, including nucleotide excision repair (NER), interstrand crosslink repair, and meiotic recombination. To better understand the role of MEI-9 in these processes, we characterized 10 unique mutant alleles of mei-9. These include a P-element insertion that disrupts repair functions but not the meiotic function; three nonsense mutations, one of which has nearly wild-type levels of protein; three missense mutations, one of which disrupts the meiotic function but not repair functions; two small in-frame deletions; and one frameshift. PMID:15166153

  1. Cell and gene therapy for arrhythmias: Repair of cardiac conduction damage

    PubMed Central

    Xiao, Yong-Fu

    2011-01-01

    Action potentials generated in the sinoatrial node (SAN) dominate the rhythm and rate of a healthy human heart. Subsequently, these action potentials propagate to the whole heart via its conduction system. Abnormalities of impulse generation and/or propagation in a heart can cause arrhythmias. For example, SAN dysfunction or conduction block of the atrioventricular node can lead to serious bradycardia which is currently treated with an implanted electronic pacemaker. On the other hand, conduction damage may cause reentrant tachyarrhythmias which are primarily treated pharmacologically or by medical device-based therapies, including defibrillation and tissue ablation. However, drug therapies sometimes may not be effective or are associated with serious side effects. Device-based therapies for cardiac arrhythmias, even with well developed technology, still face inadequacies, limitations, hardware complications, and other challenges. Therefore, scientists are actively seeking other alternatives for antiarrhythmic therapy. In particular, cells and genes used for repairing cardiac conduction damage/defect have been investigated in various studies both in vitro and in vivo. Despite the complexities of the excitation and conduction systems of the heart, cell and gene-based strategies provide novel alternatives for treatment or cure of cardiac arrhythmias. This review summarizes some highlights of recent research progress in this field. PMID:22783301

  2. Influence of functional polymorphisms in DNA repair genes of myelodysplastic syndrome.

    PubMed

    Ribeiro, Howard Lopes; Soares Maia, Allan Rodrigo; Costa, Marília Braga; Farias, Izabelle Rocha; de Paula Borges, Daniela; de Oliveira, Roberta Taiane Germano; de Sousa, Juliana Cordeiro; Magalhães, Silvia Maria Meira; Pinheiro, Ronald Feitosa

    2016-09-01

    Myelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic stem cell (HSC) malignances characterized by peripheral cytopenias and predisposition to acute myeloid leukemia transformation. Several studies show that the MDS pathogenesis is a complex and heterogeneous process that involves multiple steps through a sequence of genetic lesions in the DNA which lead to functional changes in the cell and the emergence and subsequent evolution of pre-malignant clone. Double strand breaks (DSB) lesions are the most severe type of DNA damage in HSCs, which, if not properly repaired, might contribute to the development of chromosomal abnormalities, which in turn may lead to leukemia development. We assessed the mRNA expression levels of ATM, BRCA1, BRCA2, RAD51, XRCC5, XRCC6 and LIG4 genes in bone marrow samples of 47 MDS patients in order to evaluate the association with functional polymorphisms rs228593, rs4793191, rs9567623, rs1801320, rs3835, rs2267437 and rs1805388, respectively, and try to detect clinical associations. We found that the rs228593, rs2267437 and rs1805388 functional polymorphisms probably alter the level of expression of the ATM, XRCC6 and LIG4 genes, respectively, being important in the maintenance of genomic instability in MDS. PMID:27497341

  3. Variable continental distribution of polymorphisms in the coding regions of DNA-repair genes.

    PubMed

    Mathonnet, Géraldine; Labuda, Damian; Meloche, Caroline; Wambach, Tina; Krajinovic, Maja; Sinnett, Daniel

    2003-01-01

    DNA-repair pathways are critical for maintaining the integrity of the genetic material by protecting against mutations due to exposure-induced damages or replication errors. Polymorphisms in the corresponding genes may be relevant in genetic epidemiology by modifying individual cancer susceptibility or therapeutic response. We report data on the population distribution of potentially functional variants in XRCC1, APEX1, ERCC2, ERCC4, hMLH1, and hMSH3 genes among groups representing individuals of European, Middle Eastern, African, Southeast Asian and North American descent. The data indicate little interpopulation differentiation in some of these polymorphisms and typical FST values ranging from 10 to 17% at others. Low FST was observed in APEX1 and hMSH3 exon 23 in spite of their relatively high minor allele frequencies, which could suggest the effect of balancing selection. In XRCC1, hMSH3 exon 21 and hMLH1 Africa clusters either with Middle East and Europe or with Southeast Asia, which could be related to the demographic history of human populations, whereby human migrations and genetic drift rather than selection would account for the observed differences.

  4. DNA repair gene XRCC4 codon 247 polymorphism modified diffusely infiltrating astrocytoma risk and prognosis.

    PubMed

    Lin, Zhong-Hui; Chen, Jin-Chun; Wang, Yun-Sun; Huang, Teng-Jiao; Wang, Jin; Long, Xi-Dai

    2013-12-27

    The DNA repair gene X-ray cross-complementary group 4 (XRCC4), an important caretaker of the overall genome stability, is thought to play a major role in human tumorigenesis. We investigated the association between an important polymorphic variant of this gene at codon 247 (rs373409) and diffusely infiltrating astrocytoma (DIA) risk and prognosis. This hospital-based case-control study investigated this association in the Guangxi population. In total, 242 cases with DIA and 358 age-, sex-, and race-matched healthy controls were genotyped using TaqMan-PCR technique. We found a significant difference in the frequency of XRCC4 genotypes between cases and controls. Compared with the homozygote of XRCC4 codon 247 Ala alleles (XRCC4-AA), the genotypes of XRCC4 codon 247 Ser alleles (namely XRCC4-AS or -SS) increased DIA risk (odds ratios [OR], 1.82 and 2.89, respectively). Furthermore, XRCC4 polymorphism was correlated with tumor dedifferentiation of DIA (r = 0.261, p < 0.01). Additionally, this polymorphism modified the overall survival of DIA patients (the median survival times were 26, 14, and 8 months for patients with XRCC4-AA, -AS, and -SS, respectively). Like tumor grade, XRCC4 codon 247 polymorphism was an independent prognostic factor influencing the survival of DIA. These results suggest that XRCC4 codon 247 polymorphism may be associated with DIA risk and prognosis among the Guangxi population.

  5. Retrovirus-mediated gene transfer corrects DNA repair defect of xeroderma pigmentosum cells of complementation groups A, B and C.

    PubMed

    Zeng, L; Quilliet, X; Chevallier-Lagente, O; Eveno, E; Sarasin, A; Mezzina, M

    1997-10-01

    With the aim to devise a long-term gene therapy protocol for skin cancers in individuals affected by the inherited autosomal recessive xeroderma pigmentosum we transferred the human DNA repair XPA, XPB/ERCC3 and XPC cDNAs, by using the recombinant retroviral vector LXSN, into primary and immortalized fibroblasts obtained from two XP-A, one XP-B (associated with Cockayne's syndrome) and two XP-C patients. After transduction, the complete correction of DNA repair deficiency and functional expression of the transgenes were monitored by UV survival, unscheduled DNA synthesis and recovery of RNA synthesis, and Western blots. The results show that the recombinant retroviruses are highly efficient vectors to transfer and stably express the human DNA repair genes in XP cells and correct the defect of DNA repair of group A, B and C. With our previous results with XPD/ERCC2, the present work extends further promising issues for the gene therapy strategy for most patients suffering from this cancer-prone syndrome. PMID:9415314

  6. Genome-wide Transcriptome Profiling of Homologous Recombination DNA Repair

    PubMed Central

    Peng, Guang; Lin, Curtis Chun-Jen; Mo, Wei; Dai, Hui; Park, Yun-Yong; Kim, Soo-Mi; Peng, Yang; Mo, Qianxing; Siwko, Stefan; Hu, Ruozhen; Lee, Ju-Seog; Hennessy, Bryan; Hanash, Samir; Mills, Gordon B.; Lin, Shiaw-Yih

    2014-01-01

    Homologous recombination (HR) repair deficiency predisposes to cancer development, but also sensitizes cancer cells to DNA-damage-inducing therapeutics. Here we identify an HR-defect (HRD) gene signature, which can be used to functionally assess HR repair status without interrogating individual genetic alterations in cells. By using this HRD gene signature as a functional network analysis tool, we discover that simultaneous loss of two major tumor suppressors BRCA1 and PTEN extensively rewire the HR repair-deficient phenotype, which is found in cells with defects in either BRCA1 or PTEN alone. Moreover, the HRD gene signature serves as an effective drug discovery platform to identify agents targeting HR repair as potential chemo/radio-sensitizers. More importantly, this HRD gene signature is able to predict clinical outcomes across multiple cancer lineages. Our findings, therefore, provide a molecular profile of HR repair to assess its status at a functional network level, which can provide both biological insights and have clinical implications in cancer. PMID:24553445

  7. DNA repair gene ERCC1 polymorphisms may contribute to the risk of glioma.

    PubMed

    Yuan, Guanqian; Gao, Dandan; Ding, Shaofeng; Tan, Jun

    2014-05-01

    Polymorphisms in excision repair cross-complementing rodent repair deficiency complementation group 1 (ERCC1) gene have been shown to affect individual susceptibility to glioma, though studies have yielded conflicting results. This meta-analysis aims to derive a more precise estimation of the association between ERCC1 C8092A and C118T polymorphisms and glioma risk. A literature search of PubMed, Embase, Web of Science, Cochrane Library, and CBM databases was conducted to identify all eligible studies published before August 5, 2013. Crude odds ratios (ORs) with their corresponding confidence intervals (95% CIs) were used to assess the strength of this association. A meta-analysis was performed by reviewing seven studies on the C8092A polymorphism (2,978 cases and 4,051 controls) and four studies on the C118T polymorphism (1,390 Asian cases and 1,546 Asian controls). Pooled analysis yielded a significant association between the C8092A variant genotype and increased risk of glioma. As for ethnicity, the A allele was associated with increased risk of glioma in Asians, while no similar finding was observed in Caucasians. Stratified analyses by histological subtype indicated that the C8092A polymorphism showed a significant association with the risk of non-glioblastoma multiforme. For the C118T polymorphism, increased glioma susceptibility was also observed among Asians. Taken together, results from our meta-analysis support the view that common variants in ERCC1 may contribute to susceptibility to glioma, especially in Asians. However, further studies investigating the significance of these two polymorphisms as markers of susceptibility to and disease progression of glioma are still needed. PMID:24453030

  8. Increased susceptibility to ultraviolet-B and carcinogens of mice lacking the DNA excision repair gene XPA.

    PubMed

    de Vries, A; van Oostrom, C T; Hofhuis, F M; Dortant, P M; Berg, R J; de Gruijl, F R; Wester, P W; van Kreijl, C F; Capel, P J; van Steeg, H; Verbeek, S J

    1995-09-14

    Xeroderma pigmentosum patients with a defect in the nucleotide-excision repair gene XPA are characterized by, for example, a > 1,000-fold higher risk of developing sunlight-induced skin cancer. Nucleotide-excision repair (NER) is involved in the removal of a wide spectrum of DNA lesions. The XPA protein functions in a pre-incision step, the recognition of DNA damage. To permit the functional analysis of the XPA gene in vivo, we have generated XPA-deficient mice by gene targeting in embryonic stem cells. The XPA-/-mice appear normal, at least until the age of 13 months. XPA-/-mice are highly susceptible to ultraviolet (UV)-B-induced skin and eye tumours and to 7,12-dimethylbenz[a]anthracene (DMBA)-induced skin tumours. We conclude that the XPA-deficient mice strongly mimic the phenotype of humans with xeroderma pigmentosum. PMID:7675086

  9. DNA repair and recovery of RNA synthesis following exposure to ultraviolet light are delayed in long genes.

    PubMed

    Andrade-Lima, Leonardo C; Veloso, Artur; Paulsen, Michelle T; Menck, Carlos F M; Ljungman, Mats

    2015-03-11

    The kinetics of DNA repair and RNA synthesis recovery in human cells following UV-irradiation were assessed using nascent RNA Bru-seq and quantitative long PCR. It was found that UV light inhibited transcription elongation and that recovery of RNA synthesis occurred as a wave in the 5'-3' direction with slow recovery and TC-NER at the 3' end of long genes. RNA synthesis resumed fully at the 3'-end of genes after a 24 h recovery in wild-type fibroblasts, but not in cells deficient in transcription-coupled nucleotide excision repair (TC-NER) or global genomic NER (GG-NER). Different transcription recovery profiles were found for individual genes but these differences did not fully correlate to differences in DNA repair of these genes. Our study gives the first genome-wide view of how UV-induced lesions affect transcription and how the recovery of RNA synthesis of large genes are particularly delayed by the apparent lack of resumption of transcription by arrested polymerases.

  10. NKX3.1 Suppresses TMPRSS2-ERG Gene Rearrangement and Mediates Repair of Androgen Receptor-Induced DNA Damage

    PubMed Central

    Bowen, Cai; Zheng, Tian; Gelmann, Edward P.

    2015-01-01

    TMPRSS2 gene rearrangements occur at DNA breaks formed during androgen receptor-mediated transcription and activate expression of ETS transcription factors at the early stages of more than half of prostate cancers. NKX3.1, a prostate tumor suppressor that accelerates the DNA repair response, binds to androgen receptor at the ERG gene breakpoint and inhibits both the juxtaposition of the TMPRSS2 and ERG gene loci and also their recombination. NKX3.1 acts by accelerating DNA repair after androgen-induced transcriptional activation. NKX3.1 influences the recruitment of proteins that promote homology-directed DNA repair. Loss of NKX3.1 favors recruitment to the ERG gene breakpoint of proteins that promote error-prone nonhomologous end-joining. Analysis of prostate cancer tissues showed that the presence of a TMPRSS2-ERG rearrangement was highly correlated with lower levels of NKX3.1 expression consistent with the role of NKX3.1 as a suppressor of the pathogenic gene rearrangement. PMID:25977336

  11. Association of genomic instability, and the methylation status of imprinted genes and mismatch-repair genes, with neural tube defects.

    PubMed

    Liu, Zhuo; Wang, Zhigang; Li, Yuanyuan; Ouyang, Shengrong; Chang, Huibo; Zhang, Ting; Zheng, Xiaoying; Wu, Jianxin

    2012-05-01

    We studied the genomic instability and methylation status of the mismatch-repair (MMR) genes hMLH1 and hMSH2, and the imprinted genes H19/IGF2, in fetuses with neural tube defects (NTDs) to explore the pathogenesis of the disease. Microsatellite instability (MSI) was observed in 23 of 50 NTD patients. Five NTD patients showed high-degree MSI (MSI-H) and 18 showed low-degree MSI (MSI-L). The frequencies of mutated microsatellite loci were 3/50 (6%) for BatT-25, 10/50 (20%) for Bat-26, 3/50 (6%) for Bat34C4, 6/50 (12%) for D2S123, 4/50 (8%) for D2S119, and 3/50 (6%) for D3S1611. The promoter regions of the hMLH1 and hMSH2 genes were unmethylated in NTD patients, as determined by methylation-specific PCR. The hMLH1 and hMSH2 promoter methylation patterns, the methylation levels of H19 DMR1, and IGF2 DMR0 were detected by bisulfite sequencing PCR, sub-cloning, and sequencing. The hMSH2 promoter sequence was unmethylated, and the hMLH1 promoter showed a specific methylation pattern at two CpG sites. The methylation levels of H19 DMR1 in the NTD and control groups are 73.3% ± 15.9 and 58.3% ± 11.2, respectively. The methylation level of the NTD group was higher than that of the control group (Student's t-test, P<0.05). There is no significant difference in IGF2 DMR0 methylation level between the two groups. All of the results presented here suggest that genomic instability, the MMR system, and hyper-methylation of the H19 DMR1 may be correlated with the occurrence of NTDs.

  12. Preferential repair of ionizing radiation-induced damage in the transcribed strand of an active human gene is defective in Cockayne syndrome.

    PubMed Central

    Leadon, S. A.; Cooper, P. K.

    1993-01-01

    Cells from patients with Cockayne syndrome (CS), which are sensitive to killing by UV although overall damage removal appears normal, are specifically defective in repair of UV damage in actively transcribed genes. Because several CS strains display cross-sensitivity to killing by ionizing radiation, we examined whether ionizing radiation-induced damage in active genes is preferentially repaired by normal cells and whether the radiosensitivity of CS cells can be explained by a defect in this process. We found that ionizing radiation-induced damage was repaired more rapidly in the transcriptionally active metallothionein IIA (MTIIA) gene than in the inactive MTIIB gene or in the genome overall in normal cells as a result of faster repair on the transcribed strand of MTIIA. Cells of the radiosensitive CS strain CS1AN are completely defective in this strand-selective repair of ionizing radiation-induced damage, although their overall repair rate appears normal. CS3BE cells, which are intermediate in radiosensitivity, do exhibit more rapid repair of the transcribed strand but at a reduced rate compared to normal cells. Xeroderma pigmentosum complementation group A cells, which are hypersensitive to UV light because of a defect in the nucleotide excision repair pathway but do not show increased sensitivity to ionizing radiation, preferentially repair ionizing radiation-induced damage on the transcribed strand of MTIIA. Thus, the ability to rapidly repair ionizing radiation-induced damage in actively transcribing genes correlates with cell survival. Our results extend the generality of preferential repair in active genes to include damage other than bulky lesions. Images Fig. 2 Fig. 3 PMID:8248136

  13. Role of the silkworm argonaute2 homolog gene in double-strand break repair of extrachromosomal DNA.

    PubMed

    Tsukioka, Haruna; Takahashi, Masateru; Mon, Hiroaki; Okano, Kazuhiro; Mita, Kazuei; Shimada, Toru; Lee, Jae Man; Kawaguchi, Yutaka; Koga, Katsumi; Kusakabe, Takahiro

    2006-01-01

    The argonaute protein family provides central components for RNA interference (RNAi) and related phenomena in a wide variety of organisms. Here, we isolated, from a Bombyx mori cell, a cDNA clone named BmAGO2, which is homologous to Drosophila ARGONAUTE2, the gene encoding a repressive factor for the recombination repair of extrachromosomal double-strand breaks (DSBs). RNAi-mediated silencing of the BmAGO2 sequence markedly increased homologous recombination (HR) repair of DSBs in episomal DNA, but had no effect on that in chromosomes. Moreover, we found that RNAi for BmAGO2 enhanced the integration of linearized DNA into a silkworm chromosome via HR. These results suggested that BmAgo2 protein plays an indispensable role in the repression of extrachromosomal DSB repair.

  14. DNA Double-Strand Break Repair Genes and Oxidative Damage in Brain Metastasis of Breast Cancer

    PubMed Central

    Evans, Lynda; Duchnowska, Renata; Reed, L. Tiffany; Palmieri, Diane; Qian, Yongzhen; Badve, Sunil; Sledge, George; Gril, Brunilde; Aladjem, Mirit I.; Fu, Haiqing; Flores, Natasha M.; Gökmen-Polar, Yesim; Biernat, Wojciech; Szutowicz-Zielińska, Ewa; Mandat, Tomasz; Trojanowski, Tomasz; Och, Waldemar; Czartoryska-Arlukowicz, Bogumiła; Jassem, Jacek; Mitchell, James B.

    2014-01-01

    Background Breast cancer frequently metastasizes to the brain, colonizing a neuro-inflammatory microenvironment. The molecular pathways facilitating this colonization remain poorly understood. Methods Expression profiling of 23 matched sets of human resected brain metastases and primary breast tumors by two-sided paired t test was performed to identify brain metastasis–specific genes. The implicated DNA repair genes BARD1 and RAD51 were modulated in human (MDA-MB-231-BR) and murine (4T1-BR) brain-tropic breast cancer cell lines by lentiviral transduction of cDNA or short hairpin RNA (shRNA) coding sequences. Their functional contribution to brain metastasis development was evaluated in mouse xenograft models (n = 10 mice per group). Results Human brain metastases overexpressed BARD1 and RAD51 compared with either matched primary tumors (1.74-fold, P < .001; 1.46-fold, P < .001, respectively) or unlinked systemic metastases (1.49-fold, P = .01; 1.44-fold, P = .008, respectively). Overexpression of either gene in MDA-MB-231-BR cells increased brain metastases by threefold to fourfold after intracardiac injections, but not lung metastases upon tail-vein injections. In 4T1-BR cells, shRNA-mediated RAD51 knockdown reduced brain metastases by 2.5-fold without affecting lung metastasis development. In vitro, BARD1- and RAD51-overexpressing cells showed reduced genomic instability but only exhibited growth and colonization phenotypes upon DNA damage induction. Reactive oxygen species were present in tumor cells and elevated in the metastatic neuro-inflammatory microenvironment and could provide an endogenous source of genotoxic stress. Tempol, a brain-permeable oxygen radical scavenger suppressed brain metastasis promotion induced by BARD1 and RAD51 overexpression. Conclusions BARD1 and RAD51 are frequently overexpressed in brain metastases from breast cancer and may constitute a mechanism to overcome reactive oxygen species–mediated genotoxic stress in the metastatic

  15. Single nucleotide polymorphisms in nucleotide excision repair genes, cancer treatment, and head and neck cancer survival

    PubMed Central

    Wyss, Annah B.; Weissler, Mark C.; Avery, Christy L.; Herring, Amy H.; Bensen, Jeannette T.; Barnholtz-Sloan, Jill S.; Funkhouser, William K.

    2014-01-01

    Purpose Head and neck cancers (HNC) are commonly treated with radiation and platinum-based chemotherapy, which produce bulky DNA adducts to eradicate cancerous cells. Because nucleotide excision repair (NER) enzymes remove adducts, variants in NER genes may be associated with survival among HNC cases both independently and jointly with treatment. Methods Cox proportional hazards models were used to estimate race-stratified (White, African American) hazard ratios (HRs) and 95 % confidence intervals for overall (OS) and disease-specific (DS) survival based on treatment (combinations of surgery, radiation, and chemotherapy) and 84 single nucleotide polymorphisms (SNPs) in 15 NER genes among 1,227 HNC cases from the Carolina Head and Neck Cancer Epidemiology Study. Results None of the NER variants evaluated were associated with survival at a Bonferroni-corrected alpha of 0.0006. However, rs3136038 [OS HR = 0.79 (0.65, 0.97), DS HR = 0.69 (0.51, 0.93)] and rs3136130 [OS HR = 0.78 (0.64, 0.96), DS HR = 0.68 (0.50, 0.92)] of ERCC4 and rs50871 [OS HR = 0.80 (0.64, 1.00), DS HR = 0.67 (0.48, 0.92)] of ERCC2 among Whites, and rs2607755 [OS HR = 0.62 (0.45, 0.86), DS HR = 0.51 (0.30, 0.86)] of XPC among African Americans were suggestively associated with survival at an uncorrected alpha of 0.05. Three SNP-treatment joint effects showed possible departures from additivity among Whites. Conclusions Our study, a large and extensive evaluation of SNPs in NER genes and HNC survival, identified mostly null associations, though a few variants were suggestively associated with survival and potentially interacted additively with treatment. PMID:24487794

  16. Nucleotide fluctuation of RecA repair gene in Siberian permafrost Psychrobacter cryohalolentis K5

    NASA Astrophysics Data System (ADS)

    Tremberger, George, Jr.; Holden, T.; Cheung, E.; Subramaniam, R.; Sullivan, R.; Schneider, P.; Flamholz, A.; Marchese, P.; Lieberman, D.; Cheung, T.

    2008-08-01

    A nucleotide sequence can be expressed as a numerical sequence when each nucleotide is assigned its proton number. A resulting gene numerical sequence can be investigated for its fractal dimension in terms of evolution and chemical properties for comparative studies. We have investigated such nucleotide fluctuation in the RecA repair gene of Psychrobacter cryohalolentis K5, Psychrobacter arcticus 273-4, and Psychrobacter sp. PRwf-1. The fractal dimension was found to correlate with the gene's operating temperature with the highest fractal dimension associated with P. cryohalolentis K5 living at the low temperatures found in Siberian permafrost. The CpG dinucleotide content was found to be about 5% for the three species of Psychrobacters, which is substantially lower than that of Deinococcus radiodurans at about 12%. The average nucleotide pair-wise free energy was found to be lowest for Psychrobacter sp. PRwf-1, the species with the lowest fractal dimension of the three, consistent with the recent finding that Psychrobacter sp. PRw-f1 has a temperature growth maximum of 15-20°C higher than P. arcticus 273-4 and P. cryohaloentis K5. The results suggest that microbial vitality in extreme environments is associated with fractal dimension as well as high CpG dinucleotide content, while the average nucleotide pair-wise free energy is related to the operating environment. Evidence that extreme temperature operation would impose constraints measurable by Shannon entropy is also discussed. A quantitative estimate of an entropy-based measure having the characteristics of a mechanical pressure shows that the Psychrobacter RecA sequence experiences lower pressure than that of the human HAR1 sequence.

  17. NOD2 prevents emergence of disease-predisposing microbiota

    PubMed Central

    Secher, Thomas; Normand, Sylvain; Chamaillard, Mathias

    2013-01-01

    The gut flora is composed of a huge number of diverse, well-adapted symbionts that interact with epithelial lining throughout the host's entire life. Not all commensals have the same ability to maintain quiescent, protective inflammation. Importantly, instability in the composition of gut microbial communities (referred to as dysbiosis) has been linked to loss of gut barrier in the context of common human illnesses with increasing socio-economic impacts, such as Crohn disease and colorectal cancer. Our recent findings suggest that disease-predisposing dysbiosis can now be intentionally manipulated by targeting the major Crohn disease-predisposing NOD2 gene. That knowledge will not only add a new dimension to the often overlooked microbiology of Crohn disease and colorectal cancer, but will also have a broad impact on biomedical sciences worldwide. PMID:23778641

  18. Gypenosides causes DNA damage and inhibits expression of DNA repair genes of human oral cancer SAS cells.

    PubMed

    Lu, Kung-Wen; Chen, Jung-Chou; Lai, Tung-Yuan; Yang, Jai-Sing; Weng, Shu-Wen; Ma, Yi-Shih; Tang, Nou-Ying; Lu, Pei-Jung; Weng, Jing-Ru; Chung, Jing-Gung

    2010-01-01

    Gypenosides (Gyp) are the major components of Gynostemma pentaphyllum Makino, a Chinese medical plant. Recently, Gyp has been shown to induce cell cycle arrest and apoptosis in many human cancer cell lines. However, there is no available information to address the effects of Gyp on DNA damage and DNA repair-associated gene expression in human oral cancer cells. Therefore, we investigated whether Gyp induced DNA damage and DNA repair gene expression in human oral cancer SAS cells. The results from flow cytometric assay indicated that Gyp-induced cytotoxic effects led to a decrease in the percentage of viable SAS cells. The results from comet assay revealed that the incubation of SAS cells with Gyp led to a longer DNA migration smear (comet tail) when compared with control and this effect was dose-dependent. The results from real-time PCR analysis indicated that treatment of SAS cells with 180 mug/ml of Gyp for 24 h led to a decrease in 14-3-3sigma, DNA-dependent serine/threonine protein kinase (DNAPK), p53, ataxia telangiectasia mutated (ATM), ataxia-telangiectasia and Rad3-related (ATR) and breast cancer gene 1 (BRCA1) mRNA expression. These observations may explain the cell death caused by Gyp in SAS cells. Taken together, Gyp induced DNA damage and inhibited DNA repair-associated gene expressions in human oral cancer SAS cells in vitro.

  19. Frequency distribution of DNA repair genes ERCC1 and ERCC2 polymorphisms in South Indian healthy population.

    PubMed

    Rao, Katiboina Srinivasa; SureshKumar, Srinivasamurthy; Umamaheswaran, Gurusamy; Paul, Abialbon; Dubashi, Biswajit; Gunaseelan, Karunanithi; Dkhar, Steven Aibor

    2014-09-01

    DNA repair genes are crucial in maintaining the integrity of the whole genome. Single nucleotide polymorphisms (SNPs) in DNA repair genes have been attributed to the development of various cancers. SNPs of DNA repair genes (ERCC1 and ERCC2) have been implicated in the causation of various cancers as well as inter-individual variability in the therapeutic outcomes of platinum based therapy. Thus establishing the frequency of these functional SNPs in the healthy population is of significance. The present study was aimed to establish the allele and genotype frequencies of ERCC1 (19007C>T, rs11615; 8092C>A, rs3212986) and ERCC2 (Asp312Asn, rs1799793) genes in South Indian healthy population and to compare the data from HapMap populations. The study population consisted of 128 healthy South Indian unrelated individuals of either sex aged between 18 and 60 years. Standard phenol-chloroform method was used to extract DNA from peripheral leukocytes. The genotype of DNA repair gene polymorphisms was determined by quantitative real-time polymerase chain reaction using TaqMan genotyping assay. The observed frequency of the studied polymorphisms followed Hardy-Weinberg equilibrium (p>0.05). The frequencies of the minor alleles of the SNPs rs11615 (T), rs3212986 (A) and rs1799793 (A) were 43.8%, 29.3% and 35.6%, respectively. Gender-based analysis showed no significant difference in the frequency pattern. The observed allele and genotype frequencies showed significant ethnic difference between South Indians and other HapMap populations. This is the first study to provide the normative frequency data of allele and genotype distribution of three SNPs of ERCC1 and ERCC2 in South Indian healthy population. It might be useful in future genotype-phenotype association studies, especially for predicting the efficacy and adverse events of platinum based drugs. PMID:25155628

  20. Contribution of DNA double-strand break repair gene XRCC3 genotypes to oral cancer susceptibility in Taiwan.

    PubMed

    Tsai, Chia-Wen; Chang, Wen-Shin; Liu, Juhn-Cherng; Tsai, Ming-Hsui; Lin, Cheng-Chieh; Bau, Da-Tian

    2014-06-01

    The DNA repair gene X-ray repair cross complementing protein 3 (XRCC3) is thought to play a major role in double-strand break repair and in maintaining genomic stability. Very possibly, defective double-strand break repair of cells can lead to carcinogenesis. Therefore, a case-control study was performed to reveal the contribution of XRCC3 genotypes to individual oral cancer susceptibility. In this hospital-based research, the association of XRCC3 rs1799794, rs45603942, rs861530, rs3212057, rs1799796, rs861539, rs28903081 genotypes with oral cancer risk in a Taiwanese population was investigated. In total, 788 patients with oral cancer and 956 age- and gender-matched healthy controls were genotyped. The results showed that there was significant differential distribution among oral cancer and controls in the genotypic (p=0.001428) and allelic (p=0.0013) frequencies of XRCC3 rs861539. As for the other polymorphisms, there was no difference between case and control groups. In gene-lifestyle interaction analysis, we have provided the first evidence showing that there is an obvious joint effect of XRCC3 rs861539 genotype with individual areca chewing habits on oral cancer risk. In conclusion, the T allele of XRCC3 rs861539, which has an interaction with areca chewing habit in oral carcinogenesis, may be an early marker for oral cancer in Taiwanese.

  1. Mating-type suppression of the DNA-repair defect of the yeast rad6 delta mutation requires the activity of genes in the RAD52 epistasis group.

    PubMed

    Yan, Y X; Schiestl, R H; Prakash, L

    1995-06-01

    The RAD6 gene of Saccharomyces cerevisiae is required for post-replication repair of UV-damaged DNA, UV mutagenesis, and sporulation. Here, we show that the radiation sensitivity of a MATa rad6 delta strain can be suppressed by the MAT alpha 2 gene carried on a multicopy plasmid. The a1-alpha 2 suppression is specific to the RAD6 pathway, as mutations in genes required for nucleotide excision repair or for recombinational repair do not show such mating-type suppression. The a1-alpha 2 suppression of the rad6 delta mutation requires the activity of the RAD52 group of genes, suggesting that suppression occurs by channelling of post-replication gaps present in the rad6 delta mutant into the RAD52 recombinational repair pathway. The a1-alpha 2 repressor could mediate this suppression via an enhancement in the expression, or the activity, of recombination genes.

  2. Molecular cloning of Drosophila mus308, a gene involved in DNA cross-link repair with homology to prokaryotic DNA polymerase I genes.

    PubMed Central

    Harris, P V; Mazina, O M; Leonhardt, E A; Case, R B; Boyd, J B; Burtis, K C

    1996-01-01

    Mutations in the Drosophila mus308 gene confer specific hypersensitivity to DNA-cross-linking agents as a consequence of defects in DNA repair. The mus308 gene is shown here to encode a 229-kDa protein in which the amino-terminal domain contains the seven conserved motifs characteristic of DNA and RNA helicases and the carboxy-terminal domain shares over 55% sequence similarity with the polymerase domains of prokaryotic DNA polymerase I-like enzymes. This is the first reported member of this family of DNA polymerases in a eukaryotic organism, as well as the first example of a single polypeptide with homology to both DNA polymerase and helicase motifs. Identification of a closely related gene in the genome of Caenorhabditis elegans suggests that this novel polypeptide may play an evolutionarily conserved role in the repair of DNA damage in eukaryotic organisms. PMID:8816490

  3. Functional repair of human donor lungs by IL-10 gene therapy.

    PubMed

    Cypel, Marcelo; Liu, Mingyao; Rubacha, Matt; Yeung, Jonathan C; Hirayama, Shin; Anraku, Masaki; Sato, Masaaki; Medin, Jeffrey; Davidson, Beverly L; de Perrot, Marc; Waddell, Thomas K; Slutsky, Arthur S; Keshavjee, Shaf

    2009-10-28

    More than 80% of potential donor lungs are injured during brain death of the donor and from complications experienced in the intensive care unit, and therefore cannot be used for transplantation. These lungs show inflammation and disruption of the alveolar-capillary barrier, leading to poor gas exchange. Although the number of patients in need of lung transplantation is increasing, the number of donors is static. We investigated the potential to use gene therapy with an adenoviral vector encoding human interleukin-10 (AdhIL-10) to repair injured donor lungs ex vivo before transplantation. IL-10 is an anti-inflammatory cytokine that mainly exerts its suppressive functions by the inactivation of antigen-presenting cells with consequent inhibition of proinflammatory cytokine secretion. In pigs, AdhIL-10-treated lungs exhibited attenuated inflammation and improved function after transplantation. Lungs from 10 human multiorgan donors that had suffered brain death were determined to be clinically unsuitable for transplantation. They were then maintained for 12 hours at body temperature in an ex vivo lung perfusion system with or without intra-airway delivery of AdhIL-10 gene therapy. AdhIL-10-treated lungs showed significant improvement in function (arterial oxygen pressure and pulmonary vascular resistance) when compared to controls, a favorable shift from proinflammatory to anti-inflammatory cytokine expression, and recovery of alveolar-blood barrier integrity. Thus, treatment of injured human donor lungs with the cytokine IL-10 can improve lung function, potentially rendering injured lungs suitable for transplantation into patients. PMID:20368171

  4. 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.

  5. Nucleotide Excision Repair Gene Polymorphisms, Meat Intake and Colon Cancer Risk

    PubMed Central

    Steck, Susan E.; Butler, Lesley M.; Keku, Temitope; Antwi, Samuel; Galanko, Joseph; Sandler, Robert S.; Hu, Jennifer J.

    2014-01-01

    Purpose Much of the DNA damage from colon cancer-related carcinogens, including heterocyclic amines (HCA) and polycyclic aromatic hydrocarbons (PAH) from red meat cooked at high temperature, are repaired by the nucleotide excision repair (NER) pathway. Thus, we examined whether NER non-synonymous single nucleotide polymorphisms (nsSNPs) modified the association between red meat intake and colon cancer risk. Methods The study consists of 244 African-American and 311 white colon cancer cases and population-based controls (331 African Americans and 544 whites) recruited from 33 counties in North Carolina from 1996 to 2000. Information collected by food frequency questionnaire on meat intake and preparation methods were used to estimate HCA and benzo(a)pyrene (BaP, a PAH) intake. We tested 7 nsSNPs in 5 NER genes: XPC A499V and K939Q, XPD D312N and K751Q, XPF R415Q, XPG D1104H, and RAD23B A249V. Adjusted odds ratios (OR) and 95% confidence intervals (CI) were calculated using unconditional logistic regression. Results Among African Americans, we observed a statistically significant positive association between colon cancer risk and XPC 499 AV+VV genotype (OR=1.7, 95% CI: 1.1, 2.7, AA as referent), and an inverse association with XPC 939 QQ (OR=0.3, 95%CI: 0.2, 0.8, KK as referent). These associations were not observed among whites. For both races combined, there was interaction between the XPC 939 genotype, well-done red meat intake and colon cancer risk (OR=1.5, 95% CI=1.0, 2.2 for high well-done red meat and KK genotype as compared to low well-done red meat and KK genotype, pinteraction =0.05). Conclusions Our data suggest that NER nsSNPs are associated with colon cancer risk and may modify the association between well-done red meat intake and colon cancer risk. PMID:24607854

  6. Expression of genes responsible for the repair of mispaired bases of the DNA (MLH1) in invasive ductal breast carcinoma.

    PubMed

    Milanović, Rudolf; Stanec, Sanda; Stanec, Mladen; Korusić, Andelko; Husedzinović, Ino; Razumović, Jasminka Jakić

    2013-09-01

    Breast cancer is a heterogeneous group of diseases determined and distinguished by cellular type, gene expression and clinical signs and symptoms. Identification of histological and biological markers is of great value in predicting the progression of tumor growth and anticipating the expected response to various treatment options. Due to a high degree of cell proliferation in breast tumors and high genetic instability of these tumors, as a consequence of defective DNA repair mechanisms, chemotherapy as a treatment option often renders very successful results. During our scientific research we wanted to determine the involvement of the genetic polymorphisms of DNA mismatch repair system (MLH1 gene) and the subsequent development of breast carcinoma. This study included 108 patients who were surgically treated for invasive breast cancer at the Department of Plastic, Reconstructive and Aesthetic Surgery, University Hospital "Dubrava". The expression of the MLH1 gene was determined by immunohistochemical methods. The results showed that 82.9% of tumor cells expressed the MLH1 gene. Analysis of survival rate for patients with invasive ductal breast cancer showed a statistically significant (p = 0.043) correlation with the expression of MLH1 genes. The overall five year survival rate of our patients was 78.7%. These results indicate that there is a possible involvement of MLH1 gene in the progression and development of breast cancer.

  7. Occupational solvent exposure, genetic variation of DNA repair genes, and the risk of non-Hodgkin's lymphoma.

    PubMed

    Jiao, Jie; Zheng, Tongzhang; Lan, Qing; Chen, Yingtai; Deng, Qian; Bi, Xiaofeng; Kim, Christopher; Holford, Theodore; Leaderer, Brian; Boyle, Peter; Ba, Yue; Xia, Zhaolin; Chanock, Stephen J; Rothman, Nathaniel; Zhang, Yawei

    2012-11-01

    The main objective of this study was to test the hypothesis that genetic variations in DNA repair genes may modify the association between occupational exposure to solvents and the risk of non-Hodgkin's lymphoma (NHL). A population-based case-control study was conducted on Connecticut women including 518 histologically confirmed incident NHL cases and 597 controls. Unconditional logistic regression models were used to estimate the odds ratios and effect modification from the 30 single nucleotide polymorphisms in 16 DNA repair genes of the association between solvent exposure and the risk of NHL overall and subtypes. Single nucleotide polymorphisms in MGMT (rs12917) and NBS1 (rs1805794) significantly modified the association between exposure to chlorinated solvents and the risk of NHL (Pfor interaction=0.0003 and 0.0048, respectively). After stratification by major NHL histological subtypes, MGMT (rs12917) modified the association between chlorinated solvents and the risk of diffuse large B-cell lymphoma (Pfor interaction=0.0027) and follicular lymphoma (Pfor interaction=0.0024). A significant interaction was also observed between occupational exposure to benzene and BRCA2 (rs144848) for NHL overall (Pfor interaction=0.0042). Our study results suggest that genetic variations in DNA repair genes modify the association between occupational exposure to solvents and the risk of NHL. PMID:22430443

  8. Polymorphisms in DNA repair genes, smoking, and bladder cancer risk: findings from the International Consortium of Bladder Cancer

    PubMed Central

    Stern, Mariana C.; Lin, Jie; Figueroa, Jonine D.; Kelsey, Karl T.; Kiltie, Anne E.; Yuan, Jian-Min; Matullo, Giuseppe; Fletcher, Tony; Benhamou, Simone; Taylor, Jack A.; Placidi, Donatella; Zhang, Zuo-Feng; Steineck, Gunnar; Rothman, Nathaniel; Kogevinas, Manolis; Silverman, Debra; Malats, Nuria; Chanock, Stephen; Wu, Xifeng; Karagas, Margaret R.; Andrew, Angeline S.; Nelson, Heather H.; Bishop, D. Timothy; Sak, Sei Chung; Choudhury, Ananya; Barrett, Jennifer H; Elliot, Faye; Corral, Román; Joshi, Amit D.; Gago-Dominguez, Manuela; Cortessis, Victoria K.; Xiang, Yong-Bing; Vineis, Paolo; Sacerdote, Carlotta; Guarrera, Simonetta; Polidoro, Silvia; Allione, Alessandra; Gurzau, Eugen; Koppova, Kvetoslava; Kumar, Rajiv; Rudnai, Peter; Porru, Stefano; Carta, Angela; Campagna, Marcello; Arici, Cecilia; Park, SungShim Lani; Garcia-Closas, Montserrat

    2009-01-01

    Tobacco smoking is the most important and well-established bladder cancer risk factor, and a rich source of chemical carcinogens and reactive oxygen species that can induce damage to DNA in urothelial cells. Therefore, common variation in DNA repair genes might modify bladder cancer risk. In this study we present results from meta- and pooled analyses conducted as part of the International Consortium of Bladder Cancer. We included data on 10 single nucleotide polymorphisms corresponding to 7 DNA repair genes from 13 studies. Pooled- and meta-analyses included 5,282 cases and 5,954 controls of non-Latino white origin. We found evidence for weak but consistent associations with ERCC2 D312N (rs1799793) (per allele OR = 1.10; 95% CI = 1.01–1.19; p = 0.021), NBN E185Q (rs1805794) (per allele OR = 1.09; 95% CI = 1.01–1.18; p = 0.028), and XPC A499V (rs2228000) (per allele OR = 1.10; 95% CI = 1.00–1.21, p = 0.044). The association with NBN E185Q was limited to ever smokers (interaction p = 0.002), and was strongest for the highest levels of smoking dose and smoking duration. Overall, our study provides the strongest evidence to date for a role of common variants in DNA repair genes in bladder carcinogenesis. PMID:19706757

  9. A unique horizontal gene transfer event has provided the octocoral mitochondrial genome with an active mismatch repair gene that has potential for an unusual self-contained function

    PubMed Central

    2011-01-01

    Background The mitochondrial genome of the Octocorallia has several characteristics atypical for metazoans, including a novel gene suggested to function in DNA repair. This mtMutS gene is favored for octocoral molecular systematics, due to its high information content. Several hypotheses concerning the origins of mtMutS have been proposed, and remain equivocal, although current weight of support is for a horizontal gene transfer from either an epsilonproteobacterium or a large DNA virus. Here we present new and compelling evidence on the evolutionary origin of mtMutS, and provide the very first data on its activity, functional capacity and stability within the octocoral mitochondrial genome. Results The mtMutS gene has the expected conserved amino acids, protein domains and predicted tertiary protein structure. Phylogenetic analysis indicates that mtMutS is not a member of the MSH family and therefore not of eukaryotic origin. MtMutS clusters closely with representatives of the MutS7 lineage; further support for this relationship derives from the sharing of a C-terminal endonuclease domain that confers a self-contained mismatch repair function. Gene expression analyses confirm that mtMutS is actively transcribed in octocorals. Rates of mitochondrial gene evolution in mtMutS-containing octocorals are lower than in their hexacoral sister-group, which lacks the gene, although paradoxically the mtMutS gene itself has higher rates of mutation than other octocoral mitochondrial genes. Conclusions The octocoral mtMutS gene is active and codes for a protein with all the necessary components for DNA mismatch repair. A lower rate of mitochondrial evolution, and the presence of a nicking endonuclease domain, both indirectly support a theory of self-sufficient DNA mismatch repair within the octocoral mitochondrion. The ancestral affinity of mtMutS to non-eukaryotic MutS7 provides compelling support for an origin by horizontal gene transfer. The immediate vector of transmission

  10. Transplantation of erythropoietin gene-modified neural stem cells improves the repair of injured spinal cord

    PubMed Central

    Wu, Min-fei; Zhang, Shu-quan; Gu, Rui; Liu, Jia-bei; Li, Ye; Zhu, Qing-san

    2015-01-01

    The protective effects of erythropoietin on spinal cord injury have not been well described. Here, the eukaryotic expression plasmid pcDNA3.1 human erythropoietin was transfected into rat neural stem cells cultured in vitro. A rat model of spinal cord injury was established using a free falling object. In the human erythropoietin-neural stem cells group, transfected neural stem cells were injected into the rat subarachnoid cavity, while the neural stem cells group was injected with non-transfected neural stem cells. Dulbecco's modified Eagle's medium/F12 medium was injected into the rats in the spinal cord injury group as a control. At 1–4 weeks post injury, the motor function in the rat lower limbs was best in the human erythropoietin-neural stem cells group, followed by the neural stem cells group, and lastly the spinal cord injury group. At 72 hours, compared with the spinal cord injury group, the apoptotic index and Caspase-3 gene and protein expressions were apparently decreased, and the bcl-2 gene and protein expressions were noticeably increased, in the tissues surrounding the injured region in the human erythropoietin-neural stem cells group. At 4 weeks, the cavities were clearly smaller and the motor and somatosensory evoked potential latencies were remarkably shorter in the human erythropoietin-neural stem cells group and neural stem cells group than those in the spinal cord injury group. These differences were particularly obvious in the human erythropoietin-neural stem cells group. More CM-Dil-positive cells and horseradish peroxidase-positive nerve fibers and larger amplitude motor and somatosensory evoked potentials were found in the human erythropoietin-neural stem cells group and neural stem cells group than in the spinal cord injury group. Again, these differences were particularly obvious in the human erythropoietin-neural stem cells group. These data indicate that transplantation of erythropoietin gene-modified neural stem cells into the

  11. Transplantation of erythropoietin gene-modified neural stem cells improves the repair of injured spinal cord.

    PubMed

    Wu, Min-Fei; Zhang, Shu-Quan; Gu, Rui; Liu, Jia-Bei; Li, Ye; Zhu, Qing-San

    2015-09-01

    The protective effects of erythropoietin on spinal cord injury have not been well described. Here, the eukaryotic expression plasmid pcDNA3.1 human erythropoietin was transfected into rat neural stem cells cultured in vitro. A rat model of spinal cord injury was established using a free falling object. In the human erythropoietin-neural stem cells group, transfected neural stem cells were injected into the rat subarachnoid cavity, while the neural stem cells group was injected with non-transfected neural stem cells. Dulbecco's modified Eagle's medium/F12 medium was injected into the rats in the spinal cord injury group as a control. At 1-4 weeks post injury, the motor function in the rat lower limbs was best in the human erythropoietin-neural stem cells group, followed by the neural stem cells group, and lastly the spinal cord injury group. At 72 hours, compared with the spinal cord injury group, the apoptotic index and Caspase-3 gene and protein expressions were apparently decreased, and the bcl-2 gene and protein expressions were noticeably increased, in the tissues surrounding the injured region in the human erythropoietin-neural stem cells group. At 4 weeks, the cavities were clearly smaller and the motor and somatosensory evoked potential latencies were remarkably shorter in the human erythropoietin-neural stem cells group and neural stem cells group than those in the spinal cord injury group. These differences were particularly obvious in the human erythropoietin-neural stem cells group. More CM-Dil-positive cells and horseradish peroxidase-positive nerve fibers and larger amplitude motor and somatosensory evoked potentials were found in the human erythropoietin-neural stem cells group and neural stem cells group than in the spinal cord injury group. Again, these differences were particularly obvious in the human erythropoietin-neural stem cells group. These data indicate that transplantation of erythropoietin gene-modified neural stem cells into the

  12. Two DNA repair and recombination genes in Saccharomyces cerevisiae, RAD52 and RAD54, are induced during meiosis

    SciTech Connect

    Cole, G.M.; Mortimer, R.K. ); Schild, D. )

    1989-07-01

    The DNA repair and recombination genes of Saccharomyces cerevisiae, RAD52 and RAD54, were transcriptionally induced approximately 10- to 15-fold in sporulating MATa/{alpha} cells. Congenic MATa/a cells, which did not sporulate, did not show similar increases. Assays of {beta}-galactosidase activity in strains harboring either a RAD52- or RAD54-lacZ gene fusion indicated that this induction occurred at a time concomitant with a commitment to meiotic recombination, as measured by prototroph formation from his1 heteroalleles.

  13. RAD1 and RAD10, but not other excision repair genes, are required for double-strand break-induced recombination in Saccharomyces cerevisiae.

    PubMed

    Ivanov, E L; Haber, J E

    1995-04-01

    HO endonuclease-induced double-strand breaks (DSBs) in the yeast Saccharomyces cerevisiae can be repaired by the process of gap repair or, alternatively, by single-strand annealing if the site of the break is flanked by directly repeated homologous sequences. We have shown previously (J. Fishman-Lobell and J. E. Haber, Science 258:480-484, 1992) that during the repair of an HO-induced DSB, the excision repair gene RAD1 is needed to remove regions of nonhomology from the DSB ends. In this report, we present evidence that among nine genes involved in nucleotide excision repair, only RAD1 and RAD10 are required for removal of nonhomologous sequences from the DSB ends. rad1 delta and rad10 delta mutants displayed a 20-fold reduction in the ability to execute both gap repair and single-strand annealing pathways of HO-induced recombination. Mutations in RAD2, RAD3, and RAD14 reduced HO-induced recombination by about twofold. We also show that RAD7 and RAD16, which are required to remove UV photodamage from the silent HML, locus, are not required for MAT switching with HML or HMR as a donor. Our results provide a molecular basis for understanding the role of yeast nucleotide excision repair gene and their human homologs in DSB-induced recombination and repair.

  14. Association of base excision repair gene polymorphisms with ESRD risk in a Chinese population.

    PubMed

    Cai, Zhenming; Chen, Huimei; Tao, Jing; Guo, Wenwen; Liu, Xiufang; Zheng, Bixia; Sun, Wei; Wang, Yaping

    2012-01-01

    The base excision repair (BER) pathway, containing OGG1, MTH1 and MUTYH, is a major protector from oxidative DNA damage in humans, while 8-oxoguanine (8-OHdG), an index of DNA oxidation, is increased in maintenance hemodialysis (HD) patients. Four polymorphisms of BER genes, OGG1 c.977C > G (rs1052133), MTH1 c.247G > A (rs4866), MUTYH c.972G > C (rs3219489), and AluYb8MUTYH (rs10527342), were examined in 337 HD patients and 404 healthy controls. And the 8-OHdG levels in leukocyte DNA were examined in 116 HD patients. The distribution of MUTYH c.972 GG or AluYb8MUTYH differed between the two groups and was associated with a moderately increased risk for end-stage renal disease (ESRD) (P = 0.013 and 0.034, resp.). The average 8-OHdG/10(6) dG value was significantly higher in patients with the OGG1 c.977G, MUTYH c.972G or AluYb8MUTYH alleles (P < 0.001 via ANOVA). Further analysis showed that combination of MUTYH c.972GG with OGG1 c.977GG or AluYb8MUTYH increased both the risk for ESRD and leukocyte DNA 8-OHdG levels in HD patients. Our study showed that MUTYH c.972GG, AluYb8MUTYH, and combination of OGG1 c.977GG increased the risk for ESRD development in China and suggested that DNA oxidative damage might be involved in such process.

  15. Ovarian steroids regulate gene expression related to DNA repair and neurodegenerative diseases in serotonin neurons of macaques

    PubMed Central

    Bethea, Cynthia L.; Reddy, Arubala P.

    2015-01-01

    Depression often accompanies the peri-menopausal transition and it often precedes overt symptomology in common neurodegenerative diseases (NDD; Alzheimer's, Parkinson's, Huntington, ALS). Serotonin dysfunction is frequently found in the different etiologies of depression. We have shown that ovariectomized (Ovx) monkeys treated with estradiol (E) for 28 days supplement with placebo or progesterone (P) on days 14-28 had reduced DNA fragmentation in serotonin neurons of the dorsal raphe nucleus; and long-term Ovx monkeys had fewer serotonin neurons than intact controls. We questioned the effect of E alone or E+P on gene expression related to DNA repair, protein folding (chaperones), the ubiquitin proteosome, axon transport, and NDD specific genes in serotonin neurons. Ovx macaques were treated with placebo, E or E+P (n=3/group) for 1 month. Serotonin neurons were laser captured and subjected to microarray analysis and qRT-PCR. Increases were confirmed with qRT-PCR in 5 genes that code for proteins involved in repair of strand breaks and nucleotide excision. NBN1, PCNA, GADD45A, RAD23A and GTF2H5 significantly increased with E or E+P treatment (all ANOVA p< 0.01). Chaperone genes HSP70, HSP60 and HSP27 significantly increased with E or E+P treatment (all ANOVA p<0.05). HSP90 showed a similar trend. Ubiquinase coding genes UBEA5, UBE2D3 and UBE3A (Parkin) increased with E or E+P (all ANOVA p<0.003). Transport related genes coding kinesin, dynein, and dynactin increased with E or E+P (all ANOVA p<0.03). SCNA (α synuclein) and ADAM10 (α secretase) increased (both ANOVA p<0.02), but PSEN1 (presenilin1) decreased (ANOVA p<0.02) with treatment. APP decreased 10-fold with E or E+P administration. Newman-Keuls posthoc comparisons indicated variation in the response to E alone versus E+P across the different genes. In summary, E or E+P increased gene expression for DNA repair mechanisms in serotonin neurons, thereby rendering them less vulnerable to stress-induced DNA

  16. Preferential repair of cyclobutane pyrimidine dimers in the transcribed strand of a gene in yeast chromosomes and plasmids is dependent on transcription.

    PubMed Central

    Sweder, K S; Hanawalt, P C

    1992-01-01

    While preferential repair of the transcribed strands within active genes has been demonstrated in organisms as diverse as humans and Escherichia coli, it has not previously been shown to occur in chromosomal genes in the yeast Saccharomyces cerevisiae. We found that repair of cyclobutane pyrimidine dimers in the transcribed strand of the expressed RPB2 gene in the chromosome of a repair-proficient strain is much more rapid than that in the nontranscribed strand. Furthermore, a copy of the RPB2 gene borne on a centromeric ARS1 plasmid showed the same strand bias in repair. To investigate the relation of this strand bias to transcription, we studied repair in a yeast strain with the temperature-sensitive mutation, rpb1-1, in the largest subunit of RNA polymerase II. When exponentially growing rpb1-1 cells are shifted to the nonpermissive temperature, they rapidly cease mRNA synthesis. At the permissive temperature, both rpb1-1 and the wild-type, parental cells exhibited rapid, proficient repair in the transcribed strand of chromosomal and plasmid-borne copies of the RPB2 gene. At the nonpermissive temperature, the rate of repair in the transcribed strand in rpb1-1 cells was reduced to that in the nontranscribed strand. These findings establish the dependence of strand bias in repair on transcription by RNA polymerase II in the chromosomes and in plasmids, and they validate the use of plasmids for analysis of the relation of repair to transcription in yeast. Images PMID:1438266

  17. Histone modifications predispose genome regions to breakage and translocation

    PubMed Central

    Burman, Bharat; Zhang, Zhuzhu Z.; Pegoraro, Gianluca; Lieb, Jason D.; Misteli, Tom

    2015-01-01

    Chromosome translocations are well-established hallmarks of cancer cells and often occur at nonrandom sites in the genome. The molecular features that define recurrent chromosome breakpoints are largely unknown. Using a combination of bioinformatics, biochemical analysis, and cell-based assays, we identify here specific histone modifications as facilitators of chromosome breakage and translocations. We show enrichment of several histone modifications over clinically relevant translocation-prone genome regions. Experimental modulation of histone marks sensitizes genome regions to breakage by endonuclease challenge or irradiation and promotes formation of chromosome translocations of endogenous gene loci. Our results demonstrate that histone modifications predispose genome regions to chromosome breakage and translocations. PMID:26104467

  18. Deficiency in Nucleotide Excision Repair Family Gene Activity, Especially ERCC3, Is Associated with Non-Pigmented Hair Fiber Growth

    PubMed Central

    Yu, Mei; Bell, Robert H.; Ho, Maggie M.; Leung, Gigi; Haegert, Anne; Carr, Nicholas; Shapiro, Jerry; McElwee, Kevin J.

    2012-01-01

    We conducted a microarray study to discover gene expression patterns associated with a lack of melanogenesis in non-pigmented hair follicles (HF) by microarray. Pigmented and non-pigmented HFs were collected and micro-dissected into the hair bulb (HB) and the upper hair sheaths (HS) including the bulge region. In comparison to pigmented HS and HBs, nucleotide excision repair (NER) family genes ERCC1, ERCC2, ERCC3, ERCC4, ERCC5, ERCC6, XPA, NTPBP, HCNP, DDB2 and POLH exhibited statistically significantly lower expression in non- pigmented HS and HBs. Quantitative PCR verified microarray data and identified ERCC3 as highly differentially expressed. Immunohistochemistry confirmed ERCC3 expression in HF melanocytes. A reduction in ERCC3 by siRNA interference in human melanocytes in vitro reduced their tyrosinase production ability. Our results suggest that loss of NER gene function is associated with a loss of melanin production capacity. This may be due to reduced gene transcription and/or reduced DNA repair in melanocytes which may eventually lead to cell death. These results provide novel information with regard to melanogenesis and its regulation. PMID:22615732

  19. Restoration of Chinese hamster cell radiation resistance by the human repair gene ERCC-5 and progress in molecular cloning of this gene

    SciTech Connect

    Strniste, G.F.; Chen, D.J.; deBruin, D.; McCoy, L.S.; Luke, J.A.; Mudgett, J.S.; Nickols, J.W.; Okinaka, R.T.; Tesmer, J.G.; MacInnes, M.A.

    1988-01-01

    The uv-sensitive Chinese hamster cell uv-135 is being used to identify and isolate the human gene, ERCC-5, which corrects nucleotide excision repair in this incision-defective mutant. A cosmid library, constructed from a 3/sup 0/ transformant of uv-135, has been screened for transfected gpt and human Alu family sequences. An ordered physical map of overlapping positives cosmids has been determined. Molecular evidence suggests a region of this map of <40 Kbp contains the ERCC-5 gene. 10 refs., 2 figs.

  20. TaMSH7: A cereal mismatch repair gene that affects fertility in transgenic barley (Hordeum vulgare L.)

    PubMed Central

    Lloyd, Andrew H; Milligan, Andrew S; Langridge, Peter; Able, Jason A

    2007-01-01

    Background Chromosome pairing, recombination and DNA repair are essential processes during meiosis in sexually reproducing organisms. Investigating the bread wheat (Triticum aestivum L.) Ph2 (Pairing homoeologous) locus has identified numerous candidate genes that may have a role in controlling such processes, including TaMSH7, a plant specific member of the DNA mismatch repair family. Results Sequencing of the three MSH7 genes, located on the short arms of wheat chromosomes 3A, 3B and 3D, has revealed no significant sequence divergence at the amino acid level suggesting conservation of function across the homoeogroups. Functional analysis of MSH7 through the use of RNAi loss-of-function transgenics was undertaken in diploid barley (Hordeum vulgare L.). Quantitative real-time PCR revealed several T0 lines with reduced MSH7 expression. Positive segregants from two T1 lines studied in detail showed reduced MSH7 expression when compared to transformed controls and null segregants. Expression of MSH6, another member of the mismatch repair family which is most closely related to the MSH7 gene, was not significantly reduced in these lines. In both T1 lines, reduced seed set in positive segregants was observed. Conclusion Results presented here indicate, for the first time, a distinct functional role for MSH7 in vivo and show that expression of this gene is necessary for wild-type levels of fertility. These observations suggest that MSH7 has an important function during meiosis and as such remains a candidate for Ph2. PMID:18096080

  1. Analysis of gene expression dynamics revealed delayed and abnormal epidermal repair process in aged compared to young skin.

    PubMed

    Sextius, Peggy; Marionnet, Claire; Tacheau, Charlotte; Bon, François-Xavier; Bastien, Philippe; Mauviel, Alain; Bernard, Bruno A; Bernerd, Françoise; Dubertret, Louis

    2015-05-01

    With aging, epidermal homeostasis and barrier function are disrupted. In a previous study, we analyzed the transcriptomic response of young skin epidermis after stratum corneum removal, and obtained a global kinetic view of the molecular processes involved in barrier function recovery. In the present study, the same analysis was performed in aged skin in order to better understand the defects which occur with aging. Thirty healthy male volunteers (67 ± 4 years old) were involved. Tape-strippings were carried out on the inner face of one forearm, the other unstripped forearm serving as control. At 2, 6, 18, 30 and 72 h after stripping, TEWL measurements were taken, and epidermis samples were collected. Total RNA was extracted and analyzed using DermArray(®) cDNA microarrays. The results highlighted that barrier function recovery and overall kinetics of gene expression were delayed following stripping in aged skin. Indeed, the TEWL measurements showed that barrier recovery in the young group appeared to be dramatically significant during the overall kinetics, while there were no significant evolution in the aged group until 30 h. Moreover, gene expression analysis revealed that the number of modulated genes following tape stripping increased as a function of time and reached a peak at 6 h after tape stripping in young skin, while it was at 30 h in aged skin, showing that cellular activity linked to the repair process may be engaged earlier in young epidermis than in aged epidermis. A total of 370 genes were modulated in the young group. In the aged group, 382 genes were modulated, whose 184 were also modulated in the young group. Only eight genes that were modulated in both groups were significantly differently modulated. The characterization of these genes into 15 functional families helped to draw a scenario for the aging process affecting epidermal repair capacity. PMID:25740152

  2. A mutation in the XPB/ERCC3 DNA repair transcription gene, associated with trichothiodystrophy

    SciTech Connect

    Weeda, G.; Donker, I.; Vermeulen, W.

    1997-02-01

    Trichothiodystrophy (TTD) is a rare, autosomal recessive disorder characterized by sulfur-deficient brittle hair and nails, mental retardation, impaired sexual development, and ichthyosis. Photosensitivity has been reported in {approximately}50% of the cases, but no skin cancer is associated with TTD. Virtually all photosensitive TTD patients have a deficiency in the nucleotide excision repair (NER) of UV-induced DNA damage that is indistinguishable from that of xeroderma pigmentosum (XP) complementation group D (XP-D) patients. DNA repair defects in XP-D are associated with two additional, quite different diseases; XP, a sun-sensitive and cancer-prone repair disorder, and Cockayne syndrome (CS), a photosensitive condition characterized by physical and mental retardation and wizened facial appearance. One photosensitive TTD case constitutes a new repair-deficient complementation group, TTD-A. Remarkably, both TTD-A and XP-D defects are associated with subunits of TFIIH, a basal transcription factor with a second function in DNA repair. Thus, mutations in TFIIH components may, on top of a repair defect, also cause transcriptional insufficiency, which may explain part of the non-XP clinical features of TTD. To date, three patients with the remarkable conjunction of XP and CS but not TM have been assigned to XP complementation group B (XP-B). Here we present the characterization of the NER defect in two mild TTD patients (TTD6VI and TTD4VI) and confirm the assignment to X-PB. The causative mutation was found to be a single base substitution resulting in a missense mutation (T119P) in a region of the XPB protein. These findings define a third TTD complementation group, extend the clinical heterogeneity associated with XP-B, stress the exclusive relationship between TTD and mutations in subunits of repair/transcription factor TFIIH, and strongly support the concept of {open_quotes}transcription syndromes.{close_quotes} 46 refs., 6 figs., 2 tabs.

  3. Smoking and selected DNA repair gene polymorphisms in controls: Systematic review and meta-analysis

    PubMed Central

    Hodgson, M. Elizabeth; Poole, Charles; Olshan, Andrew F.; North, Kari E.; Zeng, Donglin; Millikan, Robert C.

    2010-01-01

    Background When the case-only study design is used to estimate statistical interaction between genetic (G) and environmental (E) exposures, G and E must be independent in the underlying population, or the case-only estimate of interaction (COR) will be biased. Few studies have examined the occurrence of G-E association in published control group data. Methods To examine the assumption of G-E independence in empirical data, we conducted a systematic review and meta-analysis of G-E associations in controls for frequently investigated DNA repair genes (XRCC1 Arg399Gln, Arg194Trp, or Arg280His, XPD Lys751Gln, and Asp312Asn, and XRCC3 Thr241Met) and smoking (ever/never smoking, current/not current smoker, smoking duration, smoking intensity and pack-years). Results Across the 55 included studies, SNP-smoking associations in controls (ORz) were not reliably at the null value of 1.0 for any SNP-smoking combinations. Two G-E combinations were too heterogeneous for summary estimates: XRCC1 399 and ever-never smoking (N=21), and XPD 751 and pack-years (N=12). ORz ranges for these combinations were: [ORz (95% confidence interval (CI)] 0.7 (0.4, 1.2) – 1.9 (1.2, 2.8) and 0.8 (0.5, 1.3) – 2.3 (0.8, 6.1), respectively). Estimates for studies considered homogeneous (Cochran’s Q p-value <0.10) varied 2- to 5-fold. No study characteristics were identified that could explain heterogeneity. Conclusions We recommend the independence assumption be evaluated in the population underlying any potential case-only study, rather than in a proxy control group(s) or pooled controls. Impact These results suggest that G-E association in controls may be population-specific. Increased access to control data would improve evaluation of the independence assumption. PMID:20935063

  4. Mismatch repair gene defects in sporadic colorectal cancer enhance immune surveillance

    PubMed Central

    Canal, Fabio; Scarpa, Melania; Basato, Silvia; Erroi, Francesca; Fiorot, Alain; Dall'Agnese, Lucia; Pozza, Anna; Porzionato, Andrea; Castagliuolo, Ignazio; Dei Tos, Angelo P.; Bassi, Nicolò; Castoro, Carlo

    2015-01-01

    Background There is evidence that colorectal cancers (CRC) with DNA mismatch repair deficiency (MMR-D) are associated with a better prognosis than the generality of large bowel malignancies. Since an active immune surveillance process has been demonstrated to influence CRC outcome, we investigated whether MMR-D can enhance the immune response in CRC. Patients and Methods A group of 113 consecutive patients operated for CRC (42 stage I or II and 71 with stage III or IV) was retrospectively analyzed. The expression of MMR genes (MSH2, MLH1, MSH6 and PSM2) and co-stimulatory molecule CD80 was assessed by tissue microarray immunohistochemistry. In addition, tumor infiltrating mononuclear cells (TIMC) and T cell subpopulations (CD4, CD8, T-bet and FoxP-3) were quantified. The effect of specific siRNA (siMSH2, siMLH1, siMSH6 and siPSM2) transfection in HT29 on CD80 expression was quantified by flow cytometry. Non parametric statistics and survival analysis were used. Results Patients with MMR-D showed a higher T-bet/CD4 ratio (p = 0.02), a higher rate of CD80 expression and CD8 lymphocyte infiltration compared to those with no MMR-D. Moreover, in the MMR-D group, the Treg marker FoxP-3 was not expressed (p = 0.05). MMR-D patients with stage I or II and T-bet expression had a significant better survival (p = 0.009). Silencing of MSH2, MLH1 and MSH6, but not PSM2, significantly increased the rate of CD80+ HT29 cells (p = 0.007, p = 0.023 and p = 0.015, respectively). Conclusions CRC with MMR-D showed a higher CD80 expression, and CD8+ and Th1 T-cell infiltration. In vitro silencing of MSH2, MLH1 and MSH6 significantly increased CD80+ cell rate. These results suggest an enhanced immune surveillance mechanism in presence of MMR-D. PMID:26496037

  5. Purkinje Cell Degeneration in pcd Mice Reveals Large Scale Chromatin Reorganization and Gene Silencing Linked to Defective DNA Repair*

    PubMed Central

    Baltanás, Fernando C.; Casafont, Iñigo; Lafarga, Vanesa; Weruaga, Eduardo; Alonso, José R.; Berciano, María T.; Lafarga, Miguel

    2011-01-01

    DNA repair protects neurons against spontaneous or disease-associated DNA damage. Dysfunctions of this mechanism underlie a growing list of neurodegenerative disorders. The Purkinje cell (PC) degeneration mutation causes the loss of nna1 expression and is associated with the postnatal degeneration of PCs. This PC degeneration dramatically affects nuclear architecture and provides an excellent model to elucidate the nuclear mechanisms involved in a whole array of neurodegenerative disorders. We used immunocytochemistry for histone variants and components of the DNA damage response, an in situ transcription assay, and in situ hybridization for telomeres to analyze changes in chromatin architecture and function. We demonstrate that the phosphorylation of H2AX, a DNA damage signal, and the trimethylation of the histone H4K20, a repressive mark, in extensive domains of genome are epigenetic hallmarks of chromatin in degenerating PCs. These histone modifications are associated with a large scale reorganization of chromatin, telomere clustering, and heterochromatin-induced gene silencing, all of them key factors in PC degeneration. Furthermore, ataxia telangiectasia mutated and 53BP1, two components of the DNA repair pathway, fail to be concentrated in the damaged chromatin compartments, even though the expression levels of their coding genes were slightly up-regulated. Although the mechanism by which Nna1 loss of function leads to PC neurodegeneration is undefined, the progressive accumulation of DNA damage in chromosome territories irreversibly compromises global gene transcription and seems to trigger PC degeneration and death. PMID:21700704

  6. Beryllium chloride-induced oxidative DNA damage and alteration in the expression patterns of DNA repair-related genes.

    PubMed

    Attia, Sabry M; Harisa, Gamaleldin I; Hassan, Memy H; Bakheet, Saleh A

    2013-09-01

    Beryllium metal has physical properties that make its use essential for very specific applications, such as medical diagnostics, nuclear/fusion reactors and aerospace applications. Because of the widespread human exposure to beryllium metals and the discrepancy of the genotoxic results in the reported literature, detail assessments of the genetic damage of beryllium are warranted. Mice exposed to beryllium chloride at an oral dose of 23mg/kg for seven consecutive days exhibited a significant increase in the level of DNA-strand breaking and micronuclei formation as detected by a bone marrow standard comet assay and micronucleus test. Whereas slight beryllium chloride-induced oxidative DNA damage was detected following formamidopyrimidine DNA glycosylase digestion, digestion with endonuclease III resulted in considerable increases in oxidative DNA damage after the 11.5 and 23mg/kg/day treatment as detected by enzyme-modified comet assays. Increased 8-hydroxydeoxyguanosine was also directly correlated with increased bone marrow micronuclei formation and DNA strand breaks, which further confirm the involvement of oxidative stress in the induction of bone marrow genetic damage after exposure to beryllium chloride. Gene expression analysis on the bone marrow cells from beryllium chloride-exposed mice showed significant alterations in genes associated with DNA damage repair. Therefore, beryllium chloride may cause genetic damage to bone marrow cells due to the oxidative stress and the induced unrepaired DNA damage is probably due to the down-regulation in the expression of DNA repair genes, which may lead to genotoxicity and eventually cause carcinogenicity.

  7. Isolation and Characterization of Two Saccharomyces Cerevisiae Genes Encoding Homologs of the Bacterial Hexa and Muts Mismatch Repair Proteins

    PubMed Central

    Reenan, R. A.; Kolodner, R. D.

    1992-01-01

    Homologs of the Escherichia coli (mutL, S and uvrD) and Streptococcus pneumoniae (hexA, B) genes involved in mismatch repair are known in several distantly related organisms. Degenerate oligonucleotide primers based on conserved regions of E. coli MutS protein and its homologs from Salmonella typhimurium, S. pneumoniae and human were used in the polymerase chain reaction (PCR) to amplify and clone mutS/hexA homologs from Saccharomyces cerevisiae. Two DNA sequences were amplified whose deduced amino acid sequences both shared a high degree of homology with MutS. These sequences were then used to clone the full-length genes from a yeast genomic library. Sequence analysis of the two MSH genes (MSH = mutS homolog), MSH1 and MSH2, revealed open reading frames of 2877 bp and 2898 bp. The deduced amino acid sequences predict polypeptides of 109.3 kD and 109.1 kD, respectively. The overall amino acid sequence identity with the E. coli MutS protein is 28.6% for MSH1 and 25.2% for MSH2. Features previously found to be shared by MutS homologs, such as the nucleotide binding site and the helix-turn-helix DNA binding motif as well as other highly conserved regions whose function remain unknown, were also found in the two yeast homologs. Evidence presented in this and a companion study suggest that MSH1 is involved in repair of mitochondrial DNA and that MSH2 is involved in nuclear DNA repair. PMID:1459447

  8. Radiation-Induced Upregulation of Gene Expression From Adenoviral Vectors Mediated by DNA Damage Repair and Regulation

    SciTech Connect

    Nokisalmi, Petri; Rajecki, Maria; Pesonen, Sari; Escutenaire, Sophie; Soliymani, Rabah; Tenhunen, Mikko; Ahtiainen, Laura; Hemminki, Akseli

    2012-05-01

    Purpose: In the present study, we evaluated the combination of replication-deficient adenoviruses and radiotherapy in vitro. The purpose of the present study was to analyze the mechanism of radiation-mediated upregulation of adenoviral transgene expression. Methods and Materials: Adenoviral transgene expression (luciferase or green fluorescent protein) was studied with and without radiation in three cell lines: breast cancer M4A4-LM3, prostate cancer PC-3MM2, and lung cancer LNM35/enhanced green fluorescent protein. The effect of the radiation dose, modification of the viral capsid, and five different transgene promoters were studied. The cellular responses were studied using mass spectrometry and immunofluorescence analysis. Double strand break repair was modulated by inhibitors of heat shock protein 90, topoisomerase-I, and DNA protein kinase, and transgene expression was measured. Results: We found that a wide range of radiation doses increased adenoviral transgene expression regardless of the cell line, transgene, promoter, or viral capsid modification. Treatment with adenovirus, radiation, and double strand break repair inhibitors resulted in persistence of double strand breaks and subsequent increases in adenovirus transgene expression. Conclusions: Radiation-induced enhancement of adenoviral transgene expression is linked to DNA damage recognition and repair. Radiation induces a global cellular response that results in increased production of RNA and proteins, including adenoviral transgene products. This study provides a mechanistic rationale for combining radiation with adenoviral gene delivery.

  9. The sensitivity of Cockayne's syndrome cells to DNA-damaging agents is not due to defective transcription-coupled repair of active genes.

    PubMed

    van Oosterwijk, M F; Versteeg, A; Filon, R; van Zeeland, A A; Mullenders, L H

    1996-08-01

    Two of the hallmarks of Cockayne's syndrome (CS) are the hypersensitivity of cells to UV light and the lack of recovery of the ability to synthesize RNA following exposure of cells to UV light, in spite of the normal repair capacity at the overall genome level. The prolonged repressed RNA synthesis has been attributed to a defect in transcription-coupled repair, resulting in slow removal of DNA lesions from the transcribed strand of active genes. This model predicts that the sensitivity of CS cells to another DNA-damaging agent, i.e., the UV-mimetic agent N-acetoxy-2-acetylaminofluorene (NA-AAF), should also be associated with a lack of resumption of RNA synthesis and defective transcription-coupled repair of NA-AAF-induced DNA adducts. We tested this by measuring the rate of excision of DNA adducts in the adenosine deaminase gene of primary normal human fibroblasts and two CS (complementation group A and B) fibroblast strains. High-performance liquid chromatography analysis of DNA adducts revealed that N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) was the main adduct induced by NA-AAF in both normal and CS cells. No differences were found between normal and CS cells with respect to induction of this lesion either at the level of the genome overall or at the gene level. Moreover, repair of dG-C8-AF in the active adenosine deaminase gene occurred at similar rates and without strand specificity in normal and CS cells, indicating that transcription-coupled repair does not contribute significantly to repair of dG-C8-AF in active genes. Yet CS cells are threefold more sensitive to NA-AAF than are normal cells and are unable to recover the ability to synthesize RNA. Our data rule out defective transcription-coupled repair as the cause of the increased sensitivity of CS cells to DNA-damaging agents and suggest that the cellular sensitivity and the prolonged repressed RNA synthesis are primarily due to a transcription defect. We hypothesize that upon treatment of cells

  10. Impaired Cytogenetic Damage Repair and Cell Cycle Regulation in Response to Ionizing Radiation in Human Fibroblast Cells with Individual Knock-down of 25 Genes

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Rohde, Larry; Emami, Kamal; Hammond, Dianne; Casey, Rachael; Mehta, Satish; Jeevarajan, Antony; Pierson, Duane; Wu, Honglu

    2008-01-01

    Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have demonstrated that genes with upregulated expression induced by IR may play important roles in DNA damage sensing, cell cycle checkpoint and chromosomal repair, the relationship between the regulation of gene expression by IR and its impact on cytogenetic responses to ionizing radiation has not been systematically studied. In our present study, the expression of 25 genes selected based on their transcriptional changes in response to IR or from their known DNA repair roles were individually knocked down by siRNA transfection in human fibroblast cells. Chromosome aberrations (CA) and micronuclei (MN) formation were measured as the cytogenetic endpoints. Our results showed that the yield of MN and/or CA formation were significantly increased by suppressed expression of 5 genes that included Ku70 in the DSB repair pathway; XPA in the NER pathway; RPA1 in the MMR pathway; RAD17 and RBBP8 in cell cycle control. Knocked-down expression of 4 genes including MRE11A, RAD51 in the DSB pathway, and SESN1 and SUMO1 showed significant inhibition of cell cycle progression, possibly because of severe impairment of DNA damage repair. Furthermore, loss of XPA, p21 and MLH1 expression resulted in both enhanced cell cycle progression and significantly higher yield of cytogenetic damage, indicating the involvement of these gene products in both cell cycle control and DNA damage repair. Of these 11 genes that affected the cytogenetic response, 9 were up-regulated in the cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulating the biological consequences after IR. Failure to express these IR-responsive genes, such as by gene mutation, could seriously change the outcome of the post IR scenario and lead to carcinogenesis.

  11. Functional nucleotide excision repair is required for the preferential removal of N-ethylpurines from the transcribed strand of the dihydrofolate reductase gene of Chinese hamster ovary cells.

    PubMed Central

    Sitaram, A; Plitas, G; Wang, W; Scicchitano, D A

    1997-01-01

    Transcription-coupled repair of DNA adducts is an essential factor that must be considered when one is elucidating biological endpoints resulting from exposure to genotoxic agents. Alkylating agents comprise one group of chemical compounds which modify DNA by reacting with oxygen and nitrogen atoms in the bases of the double helix. To discern the role of transcription-coupled DNA repair of N-ethylpurines present in discrete genetic domains, Chinese hamster ovary cells were exposed to N-ethyl-N-nitrosourea, and the clearance of the damage from the dihydrofolate reductase gene was investigated. The results indicate that N-ethylpurines were removed from the dihydrofolate reductase gene of nucleotide excision repair-proficient Chinese hamster ovary cells; furthermore, when repair rates in the individual strands were determined, a statistically significant bias in the removal of ethyl-induced, alkali-labile sites was observed, with clearance occurring 30% faster from the transcribed strand than from its nontranscribed counterpart at early times after exposure. In contrast, removal of N-ethylpurines was observed in the dihydrofolate reductase locus in cells that lacked nucleotide excision repair, but both strands were repaired at the same rate, indicating that transcription-coupled clearance of these lesions requires the presence of active nucleotide excision repair. PMID:9001209

  12. POLYMORPHISMS IN THE DNA NUCLEOTIDE EXCISION REPAIR GENES AND LUNG CANCER RISK IN XUAN WEI, CHINA

    EPA Science Inventory

    The lung cancer mortality rate in Xuan Wei County, China is among the highest in the country and has been etiologically attributed to exposure to indoor smoky coal emissions that contain very high levels of polycyclic aromatic hydrocarbons (PAHs). Nucleotide excision repair (NE...

  13. Repair of benzo[a]pyrene diol epoxide-DNA adducts in the DHFR gene of a human embryonic kidney cell line

    SciTech Connect

    Schild, L.J.; Baird, W.M.; Smith, C.A.

    1997-10-01

    Benzo[a]pyrene (BaP) can be metabolized in cells to (+) anti-BaP-7,8-diol-9,10-epoxide (BPDE), an ultimate carcinogen which binds extensively to dG in DNA. Chen et al. found that BPDE adducts were removed by transcription coupled repair in the HPRT gene of human fibroblast cultures. We examined repair of BPDE adducts in the amplified DHFR gene of a human fibroblast cultures. We examined repair of BPDE adducts in the amplified DHFR gene of a human embryonic kidney cell line, 293c18 mtx-r, using a laser cleavage-Southern analysis technique. Attempts to determine whether BPDE adducts were repaired by transcription coupled repair were very toxic to the cells and little total repair was observed. Cells treated with 1.5 {mu}M BPDE contained 26 pmol BPDE/mg DNA after 1 hr and 19 pmol BPDE/mg DNA after 24 hr. To determine if the toxicity of the BPDE affected repair in these cells, cultures were treated with 0.5 {mu}M and 1 {mu}M BPDE. Postlabeling and HPLC analysis of total adducts demonstrated that the 1{mu}M treatment resulted in 18 pmol BPDE/mg DNA at 1 hr and this was reduced to 3 pmol BPDE/mg DNA by 48 hr. Laser induced cleavage of KpnI-restricted DNA demonstrated breakdown of a 20 kb DHFR fragment. Analysis of the repair of BPDE-DNA adducts in the transcribed and non-transcribed strands of the DHFR gene are in progress.

  14. Predisposing factors and prevention of frostbite.

    PubMed

    Rintamäki, H

    2000-04-01

    This review focuses on the physiological, behavioural and environmental factors which predispose to frostbite. Also prevention of frostbite is summarised. Predisposing factors may increase heat loss, decrease heat production, decrease the insulation of the clothing, make people especially susceptible to cold or make them to behave inadequately. Marked increase in convective or conductive heat loss is often the immediate reason for frostbite. Wind (as described by wind chill index) increases convective heat loss and touching of metal objects increases conductive cooling. Poor insulation of the clothing is also a common reason of frostbite. The insulation can be insufficient when clothing is wet, tight, permeable to wind or does not cover the cold sensitive body parts. Individual factors predisposing to frostbite are inadequate behaviour, low physical fitness, fatigue, dehydration, earlier cold injuries, sickness or poor circulation in peripheral parts of the body. Frostbite is often associated with the use of alcohol. To prevent frostbite, it is necessary to recognise cold risks, practise tasks in the cold, eat and drink well, have physical exercise, have sufficient clothing (also spare clothing), change into dry clothing if necessary and take care of companions. In the cold it is not advisable to get fatigued until exhaustion, sweat excessively, use tight and/or wet clothing, drink alcohol, smoke and expose oneself unnecessarily to wind, metals or fluids. PMID:10998828

  15. Predisposing factors and prevention of frostbite.

    PubMed

    Rintamäki, H

    2000-04-01

    This review focuses on the physiological, behavioural and environmental factors which predispose to frostbite. Also prevention of frostbite is summarised. Predisposing factors may increase heat loss, decrease heat production, decrease the insulation of the clothing, make people especially susceptible to cold or make them to behave inadequately. Marked increase in convective or conductive heat loss is often the immediate reason for frostbite. Wind (as described by wind chill index) increases convective heat loss and touching of metal objects increases conductive cooling. Poor insulation of the clothing is also a common reason of frostbite. The insulation can be insufficient when clothing is wet, tight, permeable to wind or does not cover the cold sensitive body parts. Individual factors predisposing to frostbite are inadequate behaviour, low physical fitness, fatigue, dehydration, earlier cold injuries, sickness or poor circulation in peripheral parts of the body. Frostbite is often associated with the use of alcohol. To prevent frostbite, it is necessary to recognise cold risks, practise tasks in the cold, eat and drink well, have physical exercise, have sufficient clothing (also spare clothing), change into dry clothing if necessary and take care of companions. In the cold it is not advisable to get fatigued until exhaustion, sweat excessively, use tight and/or wet clothing, drink alcohol, smoke and expose oneself unnecessarily to wind, metals or fluids.

  16. Antioxidative Dietary Compounds Modulate Gene Expression Associated with Apoptosis, DNA Repair, Inhibition of Cell Proliferation and Migration

    PubMed Central

    Wang, Likui; Gao, Shijuan; Jiang, Wei; Luo, Cheng; Xu, Maonian; Bohlin, Lars; Rosendahl, Markus; Huang, Wenlin

    2014-01-01

    Many dietary compounds are known to have health benefits owing to their antioxidative and anti-inflammatory properties. To determine the molecular mechanism of these food-derived compounds, we analyzed their effect on various genes related to cell apoptosis, DNA damage and repair, oxidation and inflammation using in vitro cell culture assays. This review further tests the hypothesis proposed previously that downstream products of COX-2 (cyclooxygenase-2) called electrophilic oxo-derivatives induce antioxidant responsive elements (ARE), which leads to cell proliferation under antioxidative conditions. Our findings support this hypothesis and show that cell proliferation was inhibited when COX-2 was down-regulated by polyphenols and polysaccharides. Flattened macrophage morphology was also observed following the induction of cytokine production by polysaccharides extracted from viili, a traditional Nordic fermented dairy product. Coix lacryma-jobi (coix) polysaccharides were found to reduce mitochondrial membrane potential and induce caspase-3- and 9-mediated apoptosis. In contrast, polyphenols from blueberries were involved in the ultraviolet-activated p53/Gadd45/MDM2 DNA repair system by restoring the cell membrane potential. Inhibition of hypoxia-inducible factor-1 by saponin extracts of ginsenoside (Ginsen) and Gynostemma and inhibition of S100A4 by coix polysaccharides inhibited cancer cell migration and invasion. These observations suggest that antioxidants and changes in cell membrane potential are the major driving forces that transfer signals through the cell membrane into the cytosol and nucleus, triggering gene expression, changes in cell proliferation and the induction of apoptosis or DNA repair. PMID:25226533

  17. Decreased DNA repair gene XRCC1 expression is associated with radiotherapy-induced acute side effects in breast cancer patients.

    PubMed

    Batar, Bahadir; Guven, Gulgun; Eroz, Seda; Bese, Nuran Senel; Guven, Mehmet

    2016-05-10

    DNA repair plays a critical role in response to ionizing radiation (IR) and developing of radiotherapy induced normal tissue reactions. In our study, we investigated the association of radiotherapy related acute side effects, with X-ray repair cross complementing group 1 (XRCC1) and Poly (ADP-ribose) polymerase 1 (PARP1) DNA repair gene expression levels, their changes in protein expression and DNA damage levels in breast cancer patients. The study included 40 women with newly diagnosed breast cancer; an experimental case group (n=20) with acute side effects and the control group (n=20) without side effects. For gene and protein expression analysis, lymphocytes were cultured for 72 h and followed by in vitro 2 Gray (Gy) gamma-irradiation. For detection of DNA damage levels, lymphocytes were irradiated with in vitro 2 Gy gamma-rays and followed by incubation for 72 h. XRCC1 mRNA and protein expression levels were significantly higher in controls than in experimental cases (P=0.020). In terms of DNA damage levels, an increased frequency of micronucleus (MN) was observed in experimental cases versus controls, but this association was not significant (P=0.206). We also observed a significant negative correlation between MN frequency and XRCC1 protein levels in experimental (r=-0.469, P=0.037) vs control (r=-0.734, P<0.001). Our results suggested that decreased XRCC1 expression levels might be associated with the increased risk of therapeutic IR-related acute side effects in patients with breast cancer.

  18. Decreased DNA repair gene XRCC1 expression is associated with radiotherapy-induced acute side effects in breast cancer patients.

    PubMed

    Batar, Bahadir; Guven, Gulgun; Eroz, Seda; Bese, Nuran Senel; Guven, Mehmet

    2016-05-10

    DNA repair plays a critical role in response to ionizing radiation (IR) and developing of radiotherapy induced normal tissue reactions. In our study, we investigated the association of radiotherapy related acute side effects, with X-ray repair cross complementing group 1 (XRCC1) and Poly (ADP-ribose) polymerase 1 (PARP1) DNA repair gene expression levels, their changes in protein expression and DNA damage levels in breast cancer patients. The study included 40 women with newly diagnosed breast cancer; an experimental case group (n=20) with acute side effects and the control group (n=20) without side effects. For gene and protein expression analysis, lymphocytes were cultured for 72 h and followed by in vitro 2 Gray (Gy) gamma-irradiation. For detection of DNA damage levels, lymphocytes were irradiated with in vitro 2 Gy gamma-rays and followed by incubation for 72 h. XRCC1 mRNA and protein expression levels were significantly higher in controls than in experimental cases (P=0.020). In terms of DNA damage levels, an increased frequency of micronucleus (MN) was observed in experimental cases versus controls, but this association was not significant (P=0.206). We also observed a significant negative correlation between MN frequency and XRCC1 protein levels in experimental (r=-0.469, P=0.037) vs control (r=-0.734, P<0.001). Our results suggested that decreased XRCC1 expression levels might be associated with the increased risk of therapeutic IR-related acute side effects in patients with breast cancer. PMID:26826460

  19. Regulated expression of the Saccharomyces cerevisiae DNA repair gene RAD7 in response to DNA damage and during sporulation.

    PubMed

    Jones, J S; Prakash, L; Prakash, S

    1990-06-11

    The RAD7 gene of Saccharomyces cerevisiae affects the proficiency of excision repair of DNA damaged by UV light. Here, we report our studies on the regulation of the RAD7 gene in response to UV irradiation and during sporulation. RAD7 transcript levels increased 6-fold within 40 min of exposure of cells to 37 J/m2 of UV light. Higher UV doses also elicited rapid increases in the level of RAD7 mRNA. RAD7 mRNA levels increased in sporulating MATa/MAT alpha diploid cells, but not in the asporogenous MATa/MATa strain exposed to sporulation conditions. The increase in RAD7 mRNA level in MATa/MAT alpha cells was 15-fold after 6 h and 9-fold after 7 h in sporulation medium; thereafter, RAD7 mRNA levels declined. Periodic transcription of RAD7 during sporulation suggests a role for RAD7 in this process.

  20. Analysis of aberrant methylation in DNA repair genes during malignant transformation of human bronchial epithelial cells induced by cadmium.

    PubMed

    Zhou, Zhi-heng; Lei, Yi-xiong; Wang, Cai-xia

    2012-02-01

    Cadmium (Cd) and its compounds are well-known human carcinogens, but the mechanisms underlying the carcinogenesis are not entirely understood yet. Aberrant methylation was investigated in order to obtain insight into the DNA repair-related epigenetic mechanisms underlying CdCl(2)-induced malignant transformation of human bronchial epithelial cells (16HBE). Gene expression and DNA methylation were assessed in untreated control cells; 5th, 15th, and 35th passage of CdCl2-treated cells and tumorigenic cells (TCs) from nude mice by using high-performance liquid chromatography, real-time PCR, Western blot analysis, and methylation-specific PCR assay. During Cd-induced malignant transformation, global DNA methylation progressively increased and was associated with the overexpression of the DNA methyltransferase genes DNMT1 and DNMT3a but not DNMT3b. Expression of both the messenger RNA and proteins of the DNA repair genes (hMSH2, ERCC1, XRCC1, and hOGG1) progressively reduced and DNA damage increased with Cd-induced transformation. The promoter regions of hMSH2, ERCC1, XRCC1, and hOGG1 were heavily methylated in the 35th passage transformed cells and the TCs. The DNA demethylating agent 5-aza-2'-deoxycytidine could reverse the Cd-induced global DNA hypermethylation, DNMT hyperactivity, and the silencing of hMSH2, ERCC1, XRCC1, and hOGG1 in a time-dependent manner. The results indicate that DNMT1 and DNMT3a overexpression can result in global DNA hypermethylation and silencing of the hMSH2, ERCC1, XRCC1, and hOGG1 genes. They may partly explain the epigenetic mechanisms underlying the carcinogenesis due to Cd.

  1. Primary sequence and biological functions of a Saccharomyces cerevisiae O6-methylguanine/O4-methylthymine DNA repair methyltransferase gene.

    PubMed Central

    Xiao, W; Derfler, B; Chen, J; Samson, L

    1991-01-01

    We previously identified and characterized biochemically an O6-methylguanine (O6MeG) DNA repair methyltransferase (MTase) in the yeast Saccharomyces cerevisiae and showed that it recognizes both O6MeG and O4-methylthymine (O4MeT) in vitro. Here we characterize the cloned S. cerevisiae O6MeG DNA MTase gene (MGT1) and determine its in vivo role in protecting yeast from DNA alkylation damage. We isolated a yeast DNA fragment that suppressed alkylation-induced killing and mutation in Escherichia coli ada ogt MTase deficient mutants and produced in these cells a protein similar to the yeast MTase. The cloned yeast fragment was mapped to chromosome IV and DNA sequencing identified an open reading frame, designated MGT1, which encodes a 188 amino acid protein with a molecular weight of 21,500 daltons. An 88 amino acid stretch of the MGT1 protein displays remarkable homology with four bacterial MTases and the human DNA MTase. S.cerevisiae mutants bearing an insertion in the MGT1 gene lacked DNA MTase activity and were very sensitive to alkylation induced killing and mutation. MGT1 transcript levels are not increased in response to DNA alkylation damage, nor is the MGT1 MTase involved in the regulation of the yeast 3-methyladenine DNA glycosylase gene (MAG). Expression of the MGT1 gene in E.coli prevented the induction by alkylating agents of both G:C to A:T and A:T to G:C transition mutations indicating that this eukaryotic MTase repairs both O6MeG and O4MeT in vivo. Images PMID:2065659

  2. Frequency of mutations in mismatch repair genes in a population-based study of women with ovarian cancer

    PubMed Central

    Pal, T; Akbari, M R; Sun, P; Lee, J-H; Fulp, J; Thompson, Z; Coppola, D; Nicosia, S; Sellers, T A; McLaughlin, J; Risch, H A; Rosen, B; Shaw, P; Schildkraut, J; Narod, S A

    2012-01-01

    Background: Mutations in genes for hereditary non-polyposis colorectal cancer (HNPCC) in ovarian cancer patients remains poorly defined. We sought to estimate the frequency and characteristics of HNPCC gene mutations in a population-based sample of women with epithelial ovarian cancer. Methods: The analysis included 1893 women with epithelial ovarian cancer ascertained from three population-based studies. Full-germline DNA sequencing of the coding regions was performed on three HNPCC genes, MLH1, MSH2 and MSH6. Collection of demographic, clinical and family history information was attempted in all women. Results: Nine clearly pathogenic mutations were identified, including five in MSH6, two each in MLH1 and MSH2. In addition, 28 unique predicted pathogenic missense variants were identified in 55 patients. Pathogenic mutation carriers had an earlier mean age at diagnosis of ovarian cancer, overrepresentation of cancers with non-serous histologies and a higher number of relatives with HNPCC-related cancers. Conclusions: Our findings suggest that fewer than 1% of women with ovarian cancer harbour a germline mutation in the HNPCC genes, with overrepresentation of MSH6 mutations. This represents a lower-range estimate due to the large number of predicted pathogenic variants in which pathogenicity could not definitively be determined. Identification of mismatch repair gene mutations has the potential to impact screening and treatment decisions in these women. PMID:23047549

  3. 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

  4. Heterogeneity of excision repair cross-complementation group 1 gene expression in non-small-cell lung cancer patients

    PubMed Central

    SMIRNOV, SERHEY; PASHKEVICH, ANASTASIYA; LIUNDYSHEVA, VALERIYA; BABENKO, ANDREY; SMOLYAKOVA, RAISA

    2015-01-01

    Excision repair cross-complementation group 1 (ERCC1) gene expression analysis is currently used widely in the molecular diagnosis of cancer. According to numerous studies, ERCC1 gene expression correlates with overall survival and effectiveness of chemotherapy with platinum agents. However, the degree of this correlation differs among various studies, with certain authors reporting a complete lack of such a correlation. These contradictions may be attributed to a number of factors, including the heterogeneity of the tumor tissue. In this study, we attempted to assess the degree of genetic heterogeneity exhibited by tissue samples obtained from non-small-cell lung cancer (NSCLC) through the expression of the ERCC1 gene. This study included 25 samples of tumor tissue from patients with a morphologically confirmed NSCLC diagnosis. A total of three randomized sections of each specimen were used. The ERCC1 gene expression was assessed by quantitative polymerase chain reaction (qPCR) in the TaqMan format. When planning the experiment and analysis of qPCR data, the MIQE guidelines were taken into consideration. We established that the coefficient of variation of the relative level of ERCC1 gene expression in the majority of the samples exceeded 33% (P<0.05), indicating the significant heterogeneity of the sample. We also demonstrated that the degree of heterogeneity of the tumor tissue is largely dependent on disease stage. PMID:25469300

  5. MicroRNA regulation of DNA repair gene expression in hypoxic stress.

    PubMed

    Crosby, Meredith E; Kulshreshtha, Ritu; Ivan, Mircea; Glazer, Peter M

    2009-02-01

    Genetic instability is a hallmark of cancer; the hypoxic tumor microenvironment has been implicated as a cause of this phenomenon. MicroRNAs (miR) are small nonprotein coding RNAs that can regulate various cellular pathways. We report here that two miRs, miR-210 and miR-373, are up-regulated in a hypoxia-inducible factor-1alpha-dependent manner in hypoxic cells. Bioinformatics analyses suggested that these miRs could regulate factors implicated in DNA repair pathways. Forced expression of miR-210 was found to suppress the levels of RAD52, which is a key factor in homology-dependent repair (HDR); the forced expression of miR-373 led to a reduction in the nucleotide excision repair (NER) protein, RAD23B, as well as in RAD52. Consistent with these results, both RAD52 and RAD23B were found to be down-regulated in hypoxia, but in both cases, the hypoxia-induced down-regulation could be partially reversed by antisense inhibition of miR-210 and miR-373. Importantly, luciferase reporter assays indicated that miR-210 is capable of interacting with the 3' untranslated region (UTR) of RAD52 and that miR-373 can act on the 3' UTR of RAD23B. These results indicate that hypoxia-inducible miR-210 and miR-373 play roles in modulating the expression levels of key proteins involved in the HDR and NER pathways, providing new mechanistic insight into the effect of hypoxia on DNA repair and genetic instability in cancer.

  6. Genetic variants in DNA repair genes as potential predictive markers for oxaliplatin chemotherapy in colorectal cancer.

    PubMed

    Kap, E J; Seibold, P; Richter, S; Scherer, D; Habermann, N; Balavarca, Y; Jansen, L; Becker, N; Pfütze, K; Popanda, O; Hoffmeister, M; Ulrich, A; Benner, A; Ulrich, C M; Burwinkel, B; Brenner, H; Chang-Claude, J

    2015-12-01

    Oxaliplatin-based chemotherapy exerts its effects through generating DNA damage. Hence, genetic variants in DNA repair pathways could modulate treatment response. We used a prospective cohort of 623 colorectal cancer patients with stage II-IV disease treated with adjuvant/palliative chemotherapy to comprehensively investigate 1727 genetic variants in the DNA repair pathways as potential predictive markers for oxaliplatin treatment. Single nucleotide polymorphisms (SNP) associations with overall survival and recurrence-free survival were assessed using a Cox regression model. Pathway analysis was performed using the gamma method. Patients carrying variant alleles of rs3783819 (MNAT1) and rs1043953 (XPC) experienced a longer overall survival after treatment with oxaliplatin than patients who did not carry the variant allele, while the opposite association was found in patients who were not treated with oxaliplatin (false discovery rate-adjusted P-values for heterogeneity 0.0047 and 0.0237, respectively). The nucleotide excision repair (NER) pathway was found to be most likely associated with overall survival in patients who received oxaliplatin (P-value=0.002). Our data show that genetic variants in the NER pathway are potentially predictive of treatment response to oxaliplatin.

  7. Polymorphisms in DNA repair genes as risk factors for asbestos-related malignant mesothelioma in a general population study.

    PubMed

    Dianzani, I; Gibello, L; Biava, A; Giordano, M; Bertolotti, M; Betti, M; Ferrante, D; Guarrera, S; Betta, G P; Mirabelli, D; Matullo, G; Magnani, C

    2006-07-25

    Differences in response to carcinogenic agents are due to the allelic variants of the genes that control it. Key genes are those involved in the repair of the DNA damage caused by such agents. This paper describes the results of a case-control epidemiological study designed to determine the genotypes of four of these genes in persons exposed to a single genotoxic factor, i.e. asbestos, who had or had not developed malignant mesothelioma (MM). Our working hypothesis was that an imperfect DNA repair, as revealed by subtle polymorphic variants, could reduce protection against the chronic DNA insult provoked by asbestos and eventually result in mutagenesis and cancer. Seven variants (i.e. XRCC1-R399Q-NCBI SNP, XRCC1-R194W, XRCC3-T241M, XRCC3-IVS6-14, XPD-K751Q, XPD-D312N, OGG1-S326C) were investigated in 81 patients and 110 age and sex-matched controls, all residents at Casale Monferrato, a Piedmontese town highly exposed to asbestos pollution. Unconditional multivariable logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs). When considered as a categorical variable, XRCC1-399Q showed an increased OR both in heterozygotes (OR=2.08; 95% CI=1.00-4.33) and homozygotes (2.38; 95% CI=0.82-6.94), although individual ORs were not significant. When it was considered as a continuous variable OR was significant (OR=1.68; 95% CI: 1.02-2.75). When genotypes were divided into "non-risk" and "risk" genotypes, i.e. those thought to be associated with increased risk in the light of the functional significance of the variants, XRCC1-399Q (Q homozygotes+Q/R heterozygotes versus R homozygotes) had an OR=2.147 (95% CI: 1.08-4.28), whereas that of XRCC3-241T (T homozygotes+M/T heterozygotes versus M homozygotes) was 4.09 (95% CI: 1.26-13.21) and that of OGG1-326C was increased, though not significantly. None of the haplotypes showed a significantly different frequency between patients and controls. This is the first report of an association between

  8. UV-enhanced reactivation of a UV-damaged reporter gene suggests transcription-coupled repair is UV-inducible in human cells.

    PubMed

    Francis, M A; Rainbow, A J

    1999-01-01

    The genetic disorders xeroderma pigmentosum (XP) and Cockayne syndrome (CS) exhibit deficiencies in the repair of UV-induced DNA damage. CS fibroblasts retain proficient nucleotide excision repair (NER) of inactive (or bulk) DNA, but are deficient in the transcription-coupled repair (TCR) of active genes. In contrast, XP complementation group C (XP-C) fibroblasts retain proficient TCR, but are deficient in bulk DNA repair. The remaining NER-deficient XP groups exhibit deficiencies in both repair pathways. Ad5HCMVsp1lacZ is a recombinant adenovirus vector that is unable to replicate in human fibroblasts, but can efficiently infect and express the beta-galactosidase reporter gene in these cells. We have examined the host cell reactivation (HCR) of beta-galactosidase activity for UV-irradiated Ad5HCMVsp1lacZ in non-irradiated and UV-irradiated normal, XP-B, XP-C, XP-D, XP-F, XP-G, CS-A and CS-B fibroblasts. HCR of beta-galactosidase activity for UV-irradiated Ad5HCMVsp1lacZ was reduced in non-irradiated cells from each of the repair-deficient groups examined (including XP-C) relative to that in non-irradiated normal cells. Prior irradiation of cells with low UV fluences resulted in an enhancement of HCR for normal and XP-C strains, but not for the remaining XP and CS strains. HCR of the UV-damaged reporter gene in UV-irradiated XP and CS strains was similar to measurements of TCR reported previously for these cells. These results suggest that UV treatment results in an induced repair of UV-damaged DNA in the transcribed strand of an active gene in XP-C and normal cells through an enhancement of TCR or a mechanism which involves the TCR pathway. PMID:9934845

  9. Repair of endonuclease-induced double-strand breaks in Saccharomyces cerevisiae: essential role for genes associated with nonhomologous end-joining.

    PubMed Central

    Lewis, L K; Westmoreland, J W; Resnick, M A

    1999-01-01

    Repair of double-strand breaks (DSBs) in chromosomal DNA by nonhomologous end-joining (NHEJ) is not well characterized in the yeast Saccharomyces cerevisiae. Here we demonstrate that several genes associated with NHEJ perform essential functions in the repair of endonuclease-induced DSBs in vivo. Galactose-induced expression of EcoRI endonuclease in rad50, mre11, or xrs2 mutants, which are deficient in plasmid DSB end-joining and some forms of recombination, resulted in G2 arrest and rapid cell killing. Endonuclease synthesis also produced moderate cell killing in sir4 strains. In contrast, EcoRI caused prolonged cell-cycle arrest of recombination-defective rad51, rad52, rad54, rad55, and rad57 mutants, but cells remained viable. Cell-cycle progression was inhibited in excision repair-defective rad1 mutants, but not in rad2 cells, indicating a role for Rad1 processing of the DSB ends. Phenotypic responses of additional mutants, including exo1, srs2, rad5, and rdh54 strains, suggest roles in recombinational repair, but not in NHEJ. Interestingly, the rapid cell killing in haploid rad50 and mre11 strains was largely eliminated in diploids, suggesting that the cohesive-ended DSBs could be efficiently repaired by homologous recombination throughout the cell cycle in the diploid mutants. These results demonstrate essential but separable roles for NHEJ pathway genes in the repair of chromosomal DSBs that are structurally similar to those occurring during cellular development. PMID:10430580

  10. DNA repair genes implicated in triple negative familial non-BRCA1/2 breast cancer predisposition

    PubMed Central

    Ollier, Marie; Radosevic-Robin, Nina; Kwiatkowski, Fabrice; Ponelle, Flora; Viala, Sandrine; Privat, Maud; Uhrhammer, Nancy; Bernard-Gallon, Dominique; Penault-Llorca, Frédérique; Bignon, Yves-Jean; Bidet, Yannick

    2015-01-01

    Among breast cancers, 10 to 15% of cases would be due to hereditary risk. In these familial cases, mutations in BRCA1 and BRCA2 are found in only 15% to 20%, meaning that new susceptibility genes remain to be found. Triple-negative breast cancers represent 15% of all breast cancers, and are generally aggressive tumours without targeted therapies available. Our hypothesis is that some patients with triple negative breast cancer could share a genetic susceptibility different from other types of breast cancers. We screened 36 candidate genes, using pyrosequencing, in all the 50 triple negative breast cancer patients with familial history of cancer but no BRCA1 or BRCA2 mutation of a population of 3000 families who had consulted for a familial breast cancer between 2005 and 2013. Any mutations were also sequenced in available relatives of cases. Protein expression and loss of heterozygosity were explored in tumours. Seven deleterious mutations in 6 different genes (RAD51D, MRE11A, CHEK2, MLH1, MSH6, PALB2) were observed in one patient each, except the RAD51D mutation found in two cases. Loss of heterozygosity in the tumour was found for 2 of the 7 mutations. Protein expression was absent in tumour tissue for 5 mutations. Taking into consideration a specific subtype of tumour has revealed susceptibility genes, most of them in the homologous recombination DNA repair pathway. This may provide new possibilities for targeted therapies, along with better screening and care of patients. PMID:26328243

  11. Polysaccharides of Aloe vera induce MMP-3 and TIMP-2 gene expression during the skin wound repair of rat.

    PubMed

    Tabandeh, Mohammad Reza; Oryan, Ahmad; Mohammadalipour, Adel

    2014-04-01

    Polysaccharides are the main macromolecules of Aloe vera gel but no data about their effect on extracellular matrix (ECM) elements are available. Here, mannose rich Aloe vera polysaccharides (AVP) with molecular weight between 50 and 250 kDa were isolated and characterized. Open cutaneous wounds on the back of 45 rats (control and treated) were daily treated with 25mg (n=15) and 50 mg (n=15) AVP for 30 days. The levels of MMP-3 and TIMP-2 gene expression were analyzed using real time PCR. The levels of n-acetyl glucosamine (NAGA), n-acetyl galactosamine (NAGLA) and collagen contents were also measured using standard biochemical methods. Faster wound closure was observed at day 15 post wounding in AVP treated animals in comparison with untreated group. At day 10 post wounding, AVP inhibited MMP-3 gene expression, while afterwards MMP-3 gene expression was upregulated. AVP enhanced TIMP-2 gene expression, collagen, NAGLA and NAGA synthesis in relation to untreated wounds. Our results suggest that AVP has positive effects on the regulation of ECM factor synthesis, which open up new perspectives for the wound repair activity of Aloe vera polysaccharide at molecular level.

  12. DNA repair genes implicated in triple negative familial non-BRCA1/2 breast cancer predisposition.

    PubMed

    Ollier, Marie; Radosevic-Robin, Nina; Kwiatkowski, Fabrice; Ponelle, Flora; Viala, Sandrine; Privat, Maud; Uhrhammer, Nancy; Bernard-Gallon, Dominique; Penault-Llorca, Frédérique; Bignon, Yves-Jean; Bidet, Yannick

    2015-01-01

    Among breast cancers, 10 to 15% of cases would be due to hereditary risk. In these familial cases, mutations in BRCA1 and BRCA2 are found in only 15% to 20%, meaning that new susceptibility genes remain to be found. Triple-negative breast cancers represent 15% of all breast cancers, and are generally aggressive tumours without targeted therapies available. Our hypothesis is that some patients with triple negative breast cancer could share a genetic susceptibility different from other types of breast cancers. We screened 36 candidate genes, using pyrosequencing, in all the 50 triple negative breast cancer patients with familial history of cancer but no BRCA1 or BRCA2 mutation of a population of 3000 families who had consulted for a familial breast cancer between 2005 and 2013. Any mutations were also sequenced in available relatives of cases. Protein expression and loss of heterozygosity were explored in tumours. Seven deleterious mutations in 6 different genes (RAD51D, MRE11A, CHEK2, MLH1, MSH6, PALB2) were observed in one patient each, except the RAD51D mutation found in two cases. Loss of heterozygosity in the tumour was found for 2 of the 7 mutations. Protein expression was absent in tumour tissue for 5 mutations. Taking into consideration a specific subtype of tumour has revealed susceptibility genes, most of them in the homologous recombination DNA repair pathway. This may provide new possibilities for targeted therapies, along with better screening and care of patients. PMID:26328243

  13. Base excision repair genes and risk of lung cancer among San Francisco Bay Area Latinos and African-Americans

    PubMed Central

    Chang, Jeffrey S.; Wrensch, Margaret R.; Hansen, Helen M.; Sison, Jennette D.; Aldrich, Melinda C.; Quesenberry, Charles P.; Seldin, Michael F.; Kelsey, Karl T.; Wiencke, John K.

    2009-01-01

    Base excision repair (BER) is the primary DNA damage repair mechanism for repairing small base lesions resulting from oxidation and alkylation damage. This study examines the association between 24 single-nucleotide polymorphisms (SNPs) belonging to five BER genes (XRCC1, APEX1, PARP1, MUTYH and OGG1) and lung cancer among Latinos (113 cases and 299 controls) and African-Americans (255 cases and 280 controls). The goal was to evaluate the differences in genetic contribution to lung cancer risk by ethnic groups. Analyses of individual SNPs and haplotypes were performed using unconditional logistic regressions adjusted for age, sex and genetic ancestry. Four SNPs among Latinos and one SNP among African-Americans were significantly (P < 0.05) associated with either risk of all lung cancer or non-small cell lung cancer (NSCLC). However, only the association between XRCC1 Arg399Gln (rs25487) and NSCLC among Latinos (odds ratio associated with every copy of Gln = 1.52; 95% confidence interval: 1.01–2.28) had a false-positive report probability of <0.5. Arg399Gln is a SNP with some functional evidence and has been shown previously to be an important SNP associated with lung cancer, mostly for Asians. Since the analyses were adjusted for genetic ancestry, the observed association between Arg399Gln and NSCLC among Latinos is unlikely to be confounded by population stratification; however, this result needs to be confirmed by additional studies among the Latino population. This study suggests that there are genetic differences in the association between BER pathway and lung cancer between Latinos and African-Americans. PMID:19029194

  14. The RAD7 and RAD16 genes, which are essential for pyrimidine dimer removal from the silent mating type loci, are also required for repair of the nontranscribed strand of an active gene in Saccharomyces cerevisiae.

    PubMed Central

    Verhage, R; Zeeman, A M; de Groot, N; Gleig, F; Bang, D D; van de Putte, P; Brouwer, J

    1994-01-01

    The rad16 mutant of Saccharomyces cerevisiae was previously shown to be impaired in removal of UV-induced pyrimidine dimers from the silent mating-type loci (D. D. Bang, R. A. Verhage, N. Goosen, J. Brouwer, and P. van de Putte, Nucleic Acids Res. 20:3925-3931, 1992). Here we show that rad7 as well as rad7 rad16 double mutants have the same repair phenotype, indicating that the RAD7 and RAD16 gene products might operate in the same nucleotide excision repair subpathway. Dimer removal from the genome overall is essentially incomplete in these mutants, leaving about 20 to 30% of the DNA unrepaired. Repair analysis of the transcribed RPB2 gene shows that the nontranscribed strand is not repaired at all in rad7 and rad16 mutants, whereas the transcribed strand is repaired in these mutants at a fast rate similar to that in RAD+ cells. When the results obtained with the RPB2 gene can be generalized, the RAD7 and RAD16 proteins not only are essential for repair of silenced regions but also function in repair of nontranscribed strands of active genes in S. cerevisiae. The phenotype of rad7 and rad16 mutants closely resembles that of human xeroderma pigmentosum complementation group C (XP-C) cells, suggesting that RAD7 and RAD16 in S. cerevisiae function in the same pathway as the XPC gene in human cells. RAD4, which on the basis of sequence homology has been proposed to be the yeast XPC counterpart, seems to be involved in repair of both inactive and active yeast DNA, challenging the hypothesis that RAD4 and XPC are functional homologs. Images PMID:8065346

  15. RAD25 (SSL2), the yeast homolog of the human xeroderma pigmentosum group B DNA repair gene, is essential for viability.

    PubMed

    Park, E; Guzder, S N; Koken, M H; Jaspers-Dekker, I; Weeda, G; Hoeijmakers, J H; Prakash, S; Prakash, L

    1992-12-01

    Xeroderma pigmentosum (XP) patients are extremely sensitive to ultraviolet (UV) light and suffer from a high incidence of skin cancers, due to a defect in nucleotide excision repair. The disease is genetically heterogeneous, and seven complementation groups, A-G, have been identified. Homologs of human excision repair genes ERCC1, XPDC/ERCC2, and XPAC have been identified in the yeast Saccharomyces cerevisiae. Since no homolog of human XPBC/ERCC3 existed among the known yeast genes, we cloned the yeast homolog by using XPBC cDNA as a hybridization probe. The yeast homolog, RAD25 (SSL2), encodes a protein of 843 amino acids (M(r) 95,356). The RAD25 (SSL2)- and XPBC-encoded proteins share 55% identical and 72% conserved amino acid residues, and the two proteins resemble one another in containing the conserved DNA helicase sequence motifs. A nonsense mutation at codon 799 that deletes the 45 C-terminal amino acid residues in RAD25 (SSL2) confers UV sensitivity. This mutation shows epistasis with genes in the excision repair group, whereas a synergistic increase in UV sensitivity occurs when it is combined with mutations in genes in other DNA repair pathways, indicating that RAD25 (SSL2) functions in excision repair but not in other repair pathways. We also show that RAD25 (SSL2) is an essential gene. A mutation of the Lys392 residue to arginine in the conserved Walker type A nucleotide-binding motif is lethal, suggesting an essential role of the putative RAD25 (SSL2) ATPase/DNA helicase activity in viability. PMID:1333609

  16. Low-level laser irradiation alters mRNA expression from genes involved in DNA repair and genomic stabilization in myoblasts

    NASA Astrophysics Data System (ADS)

    Trajano, L. A. S. N.; Sergio, L. P. S.; Silva, C. L.; Carvalho, L.; Mencalha, A. L.; Stumbo, A. C.; Fonseca, A. S.

    2016-07-01

    Low-level lasers are used for the treatment of diseases in soft and bone tissues, but few data are available regarding their effects on genomic stability. In this study, we investigated mRNA expression from genes involved in DNA repair and genomic stabilization in myoblasts exposed to low-level infrared laser. C2C12 myoblast cultures in different fetal bovine serum concentrations were exposed to low-level infrared laser (10, 35 and 70 J cm‑2), and collected for the evaluation of DNA repair gene expression. Laser exposure increased gene expression related to base excision repair (8-oxoguanine DNA glycosylase and apurinic/apyrimidinic endonuclease 1), nucleotide excision repair (excision repair cross-complementation group 1 and xeroderma pigmentosum C protein) and genomic stabilization (ATM serine/threonine kinase and tumor protein p53) in normal and low fetal bovine serum concentrations. Results suggest that genomic stability could be part of a biostimulation effect of low-level laser therapy in injured muscles.

  17. Low-level laser irradiation alters mRNA expression from genes involved in DNA repair and genomic stabilization in myoblasts

    NASA Astrophysics Data System (ADS)

    Trajano, L. A. S. N.; Sergio, L. P. S.; Silva, C. L.; Carvalho, L.; Mencalha, A. L.; Stumbo, A. C.; Fonseca, A. S.

    2016-07-01

    Low-level lasers are used for the treatment of diseases in soft and bone tissues, but few data are available regarding their effects on genomic stability. In this study, we investigated mRNA expression from genes involved in DNA repair and genomic stabilization in myoblasts exposed to low-level infrared laser. C2C12 myoblast cultures in different fetal bovine serum concentrations were exposed to low-level infrared laser (10, 35 and 70 J cm-2), and collected for the evaluation of DNA repair gene expression. Laser exposure increased gene expression related to base excision repair (8-oxoguanine DNA glycosylase and apurinic/apyrimidinic endonuclease 1), nucleotide excision repair (excision repair cross-complementation group 1 and xeroderma pigmentosum C protein) and genomic stabilization (ATM serine/threonine kinase and tumor protein p53) in normal and low fetal bovine serum concentrations. Results suggest that genomic stability could be part of a biostimulation effect of low-level laser therapy in injured muscles.

  18. Many amino acid substitution variants identified in DNA repair genes during human population screenings are predicted to impact protein function

    SciTech Connect

    Xi, T; Jones, I M; Mohrenweiser, H W

    2003-11-03

    Over 520 different amino acid substitution variants have been previously identified in the systematic screening of 91 human DNA repair genes for sequence variation. Two algorithms were employed to predict the impact of these amino acid substitutions on protein activity. Sorting Intolerant From Tolerant (SIFT) classified 226 of 508 variants (44%) as ''Intolerant''. Polymorphism Phenotyping (PolyPhen) classed 165 of 489 amino acid substitutions (34%) as ''Probably or Possibly Damaging''. Another 9-15% of the variants were classed as ''Potentially Intolerant or Damaging''. The results from the two algorithms are highly associated, with concordance in predicted impact observed for {approx}62% of the variants. Twenty one to thirty one percent of the variant proteins are predicted to exhibit reduced activity by both algorithms. These variants occur at slightly lower individual allele frequency than do the variants classified as ''Tolerant'' or ''Benign''. Both algorithms correctly predicted the impact of 26 functionally characterized amino acid substitutions in the APE1 protein on biochemical activity, with one exception. It is concluded that a substantial fraction of the missense variants observed in the general human population are functionally relevant. These variants are expected to be the molecular genetic and biochemical basis for the associations of reduced DNA repair capacity phenotypes with elevated cancer risk.

  19. What aspects of autism predispose to talent?

    PubMed Central

    Happé, Francesca; Vital, Pedro

    2009-01-01

    In this paper, we explore the question, why are striking special skills so much more common in autism spectrum conditions (ASC) than in other groups? Current cognitive accounts of ASC are briefly reviewed in relation to special skills. Difficulties in ‘theory of mind’ may contribute to originality in ASC, since individuals who do not automatically ‘read other minds’ may be better able to think outside prevailing fashions and popular theories. However, originality alone does not confer talent. Executive dysfunction has been suggested as the ‘releasing’ mechanism for special skills in ASC, but other groups with executive difficulties do not show raised incidence of talents. Detail-focused processing bias (‘weak coherence’, ‘enhanced perceptual functioning’) appears to be the most promising predisposing characteristic, or ‘starting engine’, for talent development. In support of this notion, we summarize data from a population-based twin study in which parents reported on their 8-year-olds' talents and their ASC-like traits. Across the whole sample, ASC-like traits, and specifically ‘restricted and repetitive behaviours and interests’ related to detail focus, were more pronounced in children reported to have talents outstripping older children. We suggest that detail-focused cognitive style predisposes to talent in savant domains in, and beyond, autism spectrum disorders. PMID:19528019

  20. Enrichment of G2/M cell cycle phase in human pluripotent stem cells enhances HDR-mediated gene repair with customizable endonucleases

    PubMed Central

    Yang, Diane; Scavuzzo, Marissa A; Chmielowiec, Jolanta; Sharp, Robert; Bajic, Aleksandar; Borowiak, Malgorzata

    2016-01-01

    Efficient gene editing is essential to fully utilize human pluripotent stem cells (hPSCs) in regenerative medicine. Custom endonuclease-based gene targeting involves two mechanisms of DNA repair: homology directed repair (HDR) and non-homologous end joining (NHEJ). HDR is the preferred mechanism for common applications such knock-in, knock-out or precise mutagenesis, but remains inefficient in hPSCs. Here, we demonstrate that synchronizing synchronizing hPSCs in G2/M with ABT phase increases on-target gene editing, defined as correct targeting cassette integration, 3 to 6 fold. We observed improved efficiency using ZFNs, TALENs, two CRISPR/Cas9, and CRISPR/Cas9 nickase to target five genes in three hPSC lines: three human embryonic stem cell lines, neural progenitors and diabetic iPSCs. neural progenitors and diabetic iPSCs. Reversible synchronization has no effect on pluripotency or differentiation. The increase in on-target gene editing is locus-independent and specific to the cell cycle phase as G2/M phase enriched cells show a 6-fold increase in targeting efficiency compared to cells in G1 phase. Concurrently inhibiting NHEJ with SCR7 does not increase HDR or improve gene targeting efficiency further, indicating that HR is the major DNA repair mechanism after G2/M phase arrest. The approach outlined here makes gene editing in hPSCs a more viable tool for disease modeling, regenerative medicine and cell-based therapies. PMID:26887909

  1. Evolutionary conservation of excision repair in Schizosaccharomyces pombe: evidence for a family of sequences related to the Saccharomyces cerevisiae RAD2 gene.

    PubMed Central

    Carr, A M; Sheldrick, K S; Murray, J M; al-Harithy, R; Watts, F Z; Lehmann, A R

    1993-01-01

    Cells mutated at the rad13 locus in the fission yeast, Schizosaccharomyces pombe are deficient in excision-repair of UV damage. We have cloned the S.pombe rad13 gene by its ability to complement the UV sensitivity of a rad13 mutant. The gene is not essential for cell proliferation. Sequence analysis of the cloned gene revealed an open reading-frame of 1113 amino acids with structural homology to the RAD2 gene of the distantly related Saccharomyces cerevisiae. The sequence similarity is confined to three domains, two close to the N-terminus of the encoded protein, the third being close to the C-terminus. The central region of about 500 amino acids shows little similarity between the two organisms. The first and third domains are also found in a related yet distinct pair of homologous S.pombe/S.cerevisiae DNA repair genes (rad2/YKL510), which have only a very short region between these two conserved domains. Using the polymerase chain reaction with degenerate primers, we have isolated fragments from a gene homologous to rad13/RAD2 from Aspergillus nidulans. These findings define new functional domains involved in excision-repair, as well as identifying a conserved family of genes related to RAD2. Images PMID:8464724

  2. Male infertility, genetic analysis of the DAZ genes on the human Y chromosome and genetic analysis of DNA repair.

    PubMed

    Fox, M S; Reijo Pera, R A

    2001-11-26

    Many genes that are required for fertility have been identified in model organisms (). Mutations in these genes cause infertility due to defects in development of the germ cell lineage, but the organism is otherwise healthy. Although human reproduction is undoubtedly as complex as that of other organisms, very few fertility loci have been mapped (). This is in spite of the prevalence of human infertility, the lack of effective treatments to remedy germ cell defects, and the cost to couples and society of assisted reproductive techniques. Fifteen percent of couples are infertile and half of all cases can be traced to the male partner. Aside from defects in sperm production, most infertile men are otherwise healthy. This review is divided into two distinct parts to discuss work that: (i) led to the identification of several genes on the Y chromosome that likely function in sperm production; and (ii) implicates DNA repair in male infertility via increased frequency of mutations in DNA from men with meiotic arrest. PMID:11694340

  3. Novel hyperbranched polyamidoamine nanoparticles for transfecting skeletal myoblasts with vascular endothelial growth factor gene for cardiac repair.

    PubMed

    Zhu, Kai; Guo, Changfa; Lai, Hao; Yang, Wuli; Xia, Yu; Zhao, Dong; Wang, Chunsheng

    2011-11-01

    We investigated the feasibility and efficacy of hyperbranched polyamidoamine (hPAMAM) mediated human vascular endothelial growth factor-165 (hVEGF(165)) gene transfer into skeletal myoblasts for cardiac repair. The hPAMAM was synthesized using a modified one-pot method. Encapsulated DNA was protected by hPAMAM from degradation for over 120 min. The transfection efficiency of hPAMAM in myoblasts was 82.6 ± 7.0% with cell viability of 94.6 ± 1.4% under optimal conditions. The hPAMAM showed much higher transfection efficiency (P < 0.05) than polyetherimide and Lipofectamine 2000 with low cytotoxicity. The transfected skeletal myoblasts gave stable hVEGF(165) expression for 18 days. After transplantation of hPAMAM-hVEGF(165) transfected cells, apoptotic myocardial cells decreased at day 1 and heart function improved at day 28, with increased neovascularization (P < 0.05). These results indicate that hPAMAM-based gene delivery into myoblasts is feasible and effective and may serve as a novel and promising non-viral DNA vehicle for gene therapy in myocardial infarction.

  4. 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

  5. Challenges and complexities in estimating both the functional impact and the disease risk associated with the extensive genetic variation in human DNA repair genes.

    PubMed

    Mohrenweiser, Harvey W; Wilson, David M; Jones, Irene M

    2003-05-15

    Individual risk and the population incidence of disease result from the interaction of genetic susceptibility and exposure. DNA repair is an example of a cellular process where genetic variation in families with extreme predisposition is documented to be associated with high disease likelihood, including syndromes of premature aging and cancer. Although the identification and characterization of new genes or variants in cancer families continues to be important, the focus of this paper is the current status of efforts to define the impact of polymorphic amino acid substitutions in DNA repair genes on individual and population cancer risk. There is increasing evidence that mild reductions in DNA repair capacity, assumed to be the consequence of common genetic variation, affect cancer predisposition. The extensive variation being found in the coding regions of DNA repair genes and the large number of genes in each of the major repair pathways results in complex genotypes with potential to impact cancer risk in the general population. The implications of this complexity for molecular epidemiology studies, as well as concepts that may make these challenges more manageable, are discussed. The concepts include both experimental and computational approaches that could be employed to develop predictors of disease susceptibility based on DNA repair genotype, focusing initially on studies to assess functional impact on individual proteins and pathways and then on molecular epidemiology studies to assess exposure-dependent health risk. In closing, we raise some of the non-technical challenges to the utilization of the full richness of the genetic variation to reduce disease occurrence and ultimately improve health care. PMID:12714187

  6. Alterations of DNA repair genes in the NCI-60 cell lines and their predictive value for anticancer drug activity

    PubMed Central

    Sousa, Fabricio G.; Matuo, Renata; Tang, Sai-Wen; Rajapakse, Vinodh N.; Luna, Augustin; Sander, Chris; Varma, Sudhir; Simon, Paul H.G.; Doroshow, James H.; Reinhold, William C.; Pommier, Yves

    2015-01-01

    Loss of function of DNA repair (DNAR) genes is associated with genomic instability and cancer predisposition; it also makes cancer cells reliant on a reduced set of DNAR pathways to resist DNA-targeted therapy, which remains the core of the anticancer armamentarium. Because the landscape of DNAR defects across numerous types of cancers and its relation with drug activity have not been systematically examined, we took advantage of the unique drug and genomic databases of the US National Cancer Institute cancer cell lines (the NCI-60) to characterize 260 DNAR genes with respect to deleterious mutations and expression down-regulation; 169 genes exhibited a total of 549 function-affecting alterations, with 39 of them scoring as putative knockouts across 31 cell lines. Those mutations were compared to tumor samples from 12 studies of The Cancer Genome Atlas (TCGA) and The Cancer Cell Line Encyclopedia (CCLE). Based on this compendium of alterations, we determined which DNAR genomic alterations predicted drug response for 20,195 compounds present in the NCI-60 drug database. Among 242 DNA damaging agents, 202 showed associations with at least one DNAR genomic signature. In addition to SLFN11, the Fanconi anemia-scaffolding gene SLX4 (FANCP/BTBD12) stood out among the genes most significantly related with DNA synthesis and topoisomerase inhibitors. Depletion and complementation experiments validated the causal relationship between SLX4 defects and sensitivity to raltitrexed and cytarabine in addition to camptothecin. Therefore, we propose new rational uses for existing anticancer drugs based on a comprehensive analysis of DNAR genomic parameters. PMID:25758781

  7. Alterations of DNA repair genes in the NCI-60 cell lines and their predictive value for anticancer drug activity.

    PubMed

    Sousa, Fabricio G; Matuo, Renata; Tang, Sai-Wen; Rajapakse, Vinodh N; Luna, Augustin; Sander, Chris; Varma, Sudhir; Simon, Paul H G; Doroshow, James H; Reinhold, William C; Pommier, Yves

    2015-04-01

    Loss of function of DNA repair (DNAR) genes is associated with genomic instability and cancer predisposition; it also makes cancer cells reliant on a reduced set of DNAR pathways to resist DNA-targeted therapy, which remains the core of the anticancer armamentarium. Because the landscape of DNAR defects across numerous types of cancers and its relation with drug activity have not been systematically examined, we took advantage of the unique drug and genomic databases of the US National Cancer Institute cancer cell lines (the NCI-60) to characterize 260 DNAR genes with respect to deleterious mutations and expression down-regulation; 169 genes exhibited a total of 549 function-affecting alterations, with 39 of them scoring as putative knockouts across 31 cell lines. Those mutations were compared to tumor samples from 12 studies of The Cancer Genome Atlas (TCGA) and The Cancer Cell Line Encyclopedia (CCLE). Based on this compendium of alterations, we determined which DNAR genomic alterations predicted drug response for 20,195 compounds present in the NCI-60 drug database. Among 242 DNA damaging agents, 202 showed associations with at least one DNAR genomic signature. In addition to SLFN11, the Fanconi anemia-scaffolding gene SLX4 (FANCP/BTBD12) stood out among the genes most significantly related with DNA synthesis and topoisomerase inhibitors. Depletion and complementation experiments validated the causal relationship between SLX4 defects and sensitivity to raltitrexed and cytarabine in addition to camptothecin. Therefore, we propose new rational uses for existing anticancer drugs based on a comprehensive analysis of DNAR genomic parameters.

  8. Selection-free gene repair after adenoviral vector transduction of designer nucleases: rescue of dystrophin synthesis in DMD muscle cell populations

    PubMed Central

    Maggio, Ignazio; Stefanucci, Luca; Janssen, Josephine M.; Liu, Jin; Chen, Xiaoyu; Mouly, Vincent; Gonçalves, Manuel A.F.V.

    2016-01-01

    Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle-wasting disorder caused by mutations in the 2.4 Mb dystrophin-encoding DMD gene. The integration of gene delivery and gene editing technologies based on viral vectors and sequence-specific designer nucleases, respectively, constitutes a potential therapeutic modality for permanently repairing defective DMD alleles in patient-derived myogenic cells. Therefore, we sought to investigate the feasibility of combining adenoviral vectors (AdVs) with CRISPR/Cas9 RNA-guided nucleases (RGNs) alone or together with transcriptional activator-like effector nucleases (TALENs), for endogenous DMD repair through non-homologous end-joining (NHEJ). The strategies tested involved; incorporating small insertions or deletions at out-of-frame sequences for reading frame resetting, splice acceptor knockout for DNA-level exon skipping, and RGN-RGN or RGN-TALEN multiplexing for targeted exon(s) removal. We demonstrate that genome editing based on the activation and recruitment of the NHEJ DNA repair pathway after AdV delivery of designer nuclease genes, is a versatile and robust approach for repairing DMD mutations in bulk populations of patient-derived muscle progenitor cells (up to 37% of corrected DMD templates). These results open up a DNA-level genetic medicine strategy in which viral vector-mediated transient designer nuclease expression leads to permanent and regulated dystrophin synthesis from corrected native DMD alleles. PMID:26762977

  9. Selection-free gene repair after adenoviral vector transduction of designer nucleases: rescue of dystrophin synthesis in DMD muscle cell populations.

    PubMed

    Maggio, Ignazio; Stefanucci, Luca; Janssen, Josephine M; Liu, Jin; Chen, Xiaoyu; Mouly, Vincent; Gonçalves, Manuel A F V

    2016-02-18

    Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle-wasting disorder caused by mutations in the 2.4 Mb dystrophin-encoding DMD gene. The integration of gene delivery and gene editing technologies based on viral vectors and sequence-specific designer nucleases, respectively, constitutes a potential therapeutic modality for permanently repairing defective DMD alleles in patient-derived myogenic cells. Therefore, we sought to investigate the feasibility of combining adenoviral vectors (AdVs) with CRISPR/Cas9 RNA-guided nucleases (RGNs) alone or together with transcriptional activator-like effector nucleases (TALENs), for endogenous DMD repair through non-homologous end-joining (NHEJ). The strategies tested involved; incorporating small insertions or deletions at out-of-frame sequences for reading frame resetting, splice acceptor knockout for DNA-level exon skipping, and RGN-RGN or RGN-TALEN multiplexing for targeted exon(s) removal. We demonstrate that genome editing based on the activation and recruitment of the NHEJ DNA repair pathway after AdV delivery of designer nuclease genes, is a versatile and robust approach for repairing DMD mutations in bulk populations of patient-derived muscle progenitor cells (up to 37% of corrected DMD templates). These results open up a DNA-level genetic medicine strategy in which viral vector-mediated transient designer nuclease expression leads to permanent and regulated dystrophin synthesis from corrected native DMD alleles. PMID:26762977

  10. The Cellular Response to Oxidatively Induced DNA Damage and Polymorphism of Some DNA Repair Genes Associated with Clinicopathological Features of Bladder Cancer.

    PubMed

    Savina, Nataliya V; Nikitchenko, Nataliya V; Kuzhir, Tatyana D; Rolevich, Alexander I; Krasny, Sergei A; Goncharova, Roza I

    2016-01-01

    Genome instability and impaired DNA repair are hallmarks of carcinogenesis. The study was aimed at evaluating the DNA damage response in H2O2-treated lymphocytes using the alkaline comet assay in bladder cancer (BC) patients as compared to clinically healthy controls, elderly persons, and individuals with chronic inflammations. Polymorphism in DNA repair genes involved in nucleotide excision repair (NER) and base excision repair (BER) was studied using the PCR-RFLP method in the Belarusian population to elucidate the possible association of their variations with both bladder cancer risk and clinicopathological features of tumors. The increased level of H2O2-induced DNA damage and a higher proportion of individuals sensitive to oxidative stress were found among BC patients as compared to other groups under study. Heterozygosity in the XPD gene (codon 751) increased cancer risk: OR (95% CI) = 1.36 (1.03-1.81), p = 0.031. The frequency of the XPD 312Asn allele was significantly higher in T ≥ 2 high grade than in T ≥ 2 low grade tumors (p = 0.036); the ERCC6 1097Val/Val genotype was strongly associated with muscle-invasive tumors. Combinations of homozygous wild type alleles occurred with the increased frequency in patients with non-muscle-invasive tumors suggesting that the maintenance of normal DNA repair activity may prevent cancer progression.

  11. The Cellular Response to Oxidatively Induced DNA Damage and Polymorphism of Some DNA Repair Genes Associated with Clinicopathological Features of Bladder Cancer

    PubMed Central

    Savina, Nataliya V.; Nikitchenko, Nataliya V.; Kuzhir, Tatyana D.; Rolevich, Alexander I.; Krasny, Sergei A.; Goncharova, Roza I.

    2016-01-01

    Genome instability and impaired DNA repair are hallmarks of carcinogenesis. The study was aimed at evaluating the DNA damage response in H2O2-treated lymphocytes using the alkaline comet assay in bladder cancer (BC) patients as compared to clinically healthy controls, elderly persons, and individuals with chronic inflammations. Polymorphism in DNA repair genes involved in nucleotide excision repair (NER) and base excision repair (BER) was studied using the PCR-RFLP method in the Belarusian population to elucidate the possible association of their variations with both bladder cancer risk and clinicopathological features of tumors. The increased level of H2O2-induced DNA damage and a higher proportion of individuals sensitive to oxidative stress were found among BC patients as compared to other groups under study. Heterozygosity in the XPD gene (codon 751) increased cancer risk: OR (95% CI) = 1.36 (1.03–1.81), p = 0.031. The frequency of the XPD 312Asn allele was significantly higher in T ≥ 2 high grade than in T ≥ 2 low grade tumors (p = 0.036); the ERCC6 1097Val/Val genotype was strongly associated with muscle-invasive tumors. Combinations of homozygous wild type alleles occurred with the increased frequency in patients with non-muscle-invasive tumors suggesting that the maintenance of normal DNA repair activity may prevent cancer progression. PMID:26649138

  12. Suppression of a DNA base excision repair gene, hOGG1, increases bleomycin sensitivity of human lung cancer cell line

    SciTech Connect

    Wu Mei; Zhang Zunzhen Che Wangjun

    2008-05-01

    Bleomycin (BLM) has been found to induce 8-oxoguanine and DNA strand breaks through producing oxidative free radicals, thereby leading to cell cycle arrest, apoptosis and cell death. Cellular DNA damage repair mechanisms such as single strand DNA break repair/base excision repair (BER) are responsible for removing bleomycin-induced DNA damage, therefore confer chemotherapeutic resistance to bleomycin. In this study, we have investigated if down-regulation of human 8-oxoguanine DNA glycosylase (hOGG1), an important BER enzyme, could alter cellular sensitivity to bleomycin, thereby reducing chemotherapeutic resistance in human tumor cell. A human lung cancer cell line with hOGG1 deficiency (A549-R) was created by ribozyme gene knockdown technique. Bleomycin cellular sensitivity and DNA/chromosomal damages were examined using MTT, colony forming assay, comet assay as well as micronucleus assay. We demonstrated that hOGG1 gene knockdown enhanced bleomycin cytotoxicity and reduced the ability of colony formation of the lung cancer cell lines. We further demonstrated that bleomycin-induced DNA strand breaks resulted in an increase of micronucleus rate. hOGG1 deficiency significantly reduced DNA damage repair capacity of the lung cancer cell lines. Our results indicated that hOGG1 deficiency allowed the accumulation of bleomycin-induced DNA damage and chromosomal breaks by compromising DNA damage repair capacity, thereby increasing cellular sensitivity to bleomycin.

  13. 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

  14. Repair of uv damaged DNA: Genes and proteins of yeast and human. Progress report, November 1, 1991--April 15, 1992

    SciTech Connect

    Prakash, L.

    1992-04-01

    Our objectives are to determine the molecular mechanism of the incision step of excision repair of ultraviolet (UV) light damaged DNA in eukaryotic organisms, using the yeast Saccharomyces cerevisiae as a model system, and to study the human homologs of yeast excision repair and postreplication repair proteins progress is described.

  15. The Saccharomyces cerevisiae RAD7 and RAD16 genes are required for inducible excision of endonuclease III sensitive-sites, yet are not needed for the repair of these lesions following a single UV dose.

    PubMed

    Scott, A D; Waters, R

    1997-01-31

    The RAD7 and RAD16 genes of Saccharomyces cerevisiae have roles in the repair of UV induced CPDs in nontranscribed genes [1], and in the repair of CPDs in the nontranscribed strand of transcribed genes [2]. Previously, we identified an inducible component to nucleotide excision repair (NER), which is absent in a rad16 delta strain [3]. We have examined the repair of UV induced endonuclease III sensitive-sites (EIIISS), and have shown repair of these lesions to proceed by NER but their removal from nontranscribed regions is independent of RAD7 and RAD16. Furthermore, EIIISS are repaired with equal efficiency from both transcribed and nontranscribed genes [4]. In order to dissect the roles of RAD7 and RAD16 in the above processes we examined the repair of EIIISS in the MAT alpha and HML alpha loci, which are, respectively, transcriptionally active and inactive in alpha haploid cells. These loci have elevated levels of these lesions after UV (in genomic DNA EIIISS constitute about 10% of total lesions, whereas CPDs are about 70% of total lesions). We have shown that excision of UV induced EIIISS is enhanced following a prior UV irradiation. No enhancement of repair was detected in either the rad7 delta or the rad16 delta mutant. The fact that RAD7 and RAD16 are not required for the repair of EIIISS per se yet are required for the enhanced excision of these lesions from MAT alpha and HML alpha suggests two possibilities. These genes have two roles in NER, namely in the repair of CPDs from nontranscribed sequences, and in enhancing NER itself regardless of whether these genes' products are required for the excision of the specific lesion being repaired. In the latter case, the induction of RAD7 and RAD16 may increase the turnover of complexes stalled in nontranscribed DNA so as to increase the availability of NER proteins for the repair of CPDs and EIIISS in all regions of the genome.

  16. Immunoglobulin genes undergo legitimate repair in human B cells not only after cis- but also frequent trans-class switch recombination.

    PubMed

    Laffleur, B; Bardet, S M; Garot, A; Brousse, M; Baylet, A; Cogné, M

    2014-01-01

    Immunoglobulin (Ig) genes specifically recruit activation-induced deaminase (AID) for 'on-target' DNA deamination, initiating either variable (V) region somatic hypermutation, or double-strand break intermediates of class switch recombination (CSR). Such breaks overwhelmingly undergo legitimate intra-Ig repair rather than rare illegitimate and potentially oncogenic junctions outside of Ig loci. We show that in human B cells, legitimate synapsis and repair efficiently join Ig genes whether physically linked on one chromosome or located apart on both alleles. This indicates mechanisms faithfully recognizing and/or pairing loci with homology in structure and accessibility, thus licensing interchromosomal trans-CSR junctions while usually preventing illegitimate interchromosomal recombination with AID off-target genes. Physical linkage of IgH genes in cis on the same allele just increases the likelihood of legitimate repair by another fourfold. The strongest force driving CSR might thus be recognition of legitimate target genes. Formation of IgH intra-allelic loops along this process would then constitute a consequence rather than a pre-requisite of this gene-pairing process.

  17. Cox4i2, Ifit2, and Prdm11 Mutant Mice: Effective Selection of Genes Predisposing to an Altered Airway Inflammatory Response from a Large Compendium of Mutant Mouse Lines.

    PubMed

    Horsch, Marion; Aguilar-Pimentel, Juan Antonio; Bönisch, Clemens; Côme, Christophe; Kolster-Fog, Cathrine; Jensen, Klaus T; Lund, Anders H; Lee, Icksoo; Grossman, Lawrence I; Sinkler, Christopher; Hüttemann, Maik; Bohn, Erwin; Fuchs, Helmut; Ollert, Markus; Gailus-Durner, Valérie; de Angelis, Martin Hrabĕ; Beckers, Johannes

    2015-01-01

    We established a selection strategy to identify new models for an altered airway inflammatory response from a large compendium of mutant mouse lines that were systemically phenotyped in the German Mouse Clinic (GMC). As selection criteria we included published gene functional data, as well as immunological and transcriptome data from GMC phenotyping screens under standard conditions. Applying these criteria we identified a few from several hundred mutant mouse lines and further characterized the Cox4i2tm1Hutt, Ifit2tm1.1Ebsb, and Prdm11tm1.1ahl lines following ovalbumin (OVA) sensitization and repeated OVA airway challenge. Challenged Prdm11tm1.1ahl mice exhibited changes in B cell counts, CD4+ T cell counts, and in the number of neutrophils in bronchoalveolar lavages, whereas challenged Ifit2tm1.1Ebsb mice displayed alterations in plasma IgE, IgG1, IgG3, and IgM levels compared to the challenged wild type littermates. In contrast, challenged Cox4i2tm1Hutt mutant mice did not show alterations in the humoral or cellular immune response compared to challenged wild type mice. Transcriptome analyses from lungs of the challenged mutant mouse lines showed extensive changes in gene expression in Prdm11tm1.1ahl mice. Functional annotations of regulated genes of all three mutant mouse lines were primarily related to inflammation and airway smooth muscle (ASM) remodeling. We were thus able to define an effective selection strategy to identify new candidate genes for the predisposition to an altered airway inflammatory response under OVA challenge conditions. Similar selection strategies may be used for the analysis of additional genotype-envirotype interactions for other diseases.

  18. Cox4i2, Ifit2, and Prdm11 Mutant Mice: Effective Selection of Genes Predisposing to an Altered Airway Inflammatory Response from a Large Compendium of Mutant Mouse Lines

    PubMed Central

    Bönisch, Clemens; Côme, Christophe; Kolster-Fog, Cathrine; Jensen, Klaus T.; Lund, Anders H.; Lee, Icksoo; Grossman, Lawrence I.; Sinkler, Christopher; Hüttemann, Maik; Bohn, Erwin; Fuchs, Helmut; Ollert, Markus; Gailus-Durner, Valérie; Hrabĕ de Angelis, Martin; Beckers, Johannes

    2015-01-01

    We established a selection strategy to identify new models for an altered airway inflammatory response from a large compendium of mutant mouse lines that were systemically phenotyped in the German Mouse Clinic (GMC). As selection criteria we included published gene functional data, as well as immunological and transcriptome data from GMC phenotyping screens under standard conditions. Applying these criteria we identified a few from several hundred mutant mouse lines and further characterized the Cox4i2tm1Hutt, Ifit2tm1.1Ebsb, and Prdm11tm1.1ahl lines following ovalbumin (OVA) sensitization and repeated OVA airway challenge. Challenged Prdm11tm1.1ahl mice exhibited changes in B cell counts, CD4+ T cell counts, and in the number of neutrophils in bronchoalveolar lavages, whereas challenged Ifit2tm1.1Ebsb mice displayed alterations in plasma IgE, IgG1, IgG3, and IgM levels compared to the challenged wild type littermates. In contrast, challenged Cox4i2tm1Hutt mutant mice did not show alterations in the humoral or cellular immune response compared to challenged wild type mice. Transcriptome analyses from lungs of the challenged mutant mouse lines showed extensive changes in gene expression in Prdm11tm1.1ahl mice. Functional annotations of regulated genes of all three mutant mouse lines were primarily related to inflammation and airway smooth muscle (ASM) remodeling. We were thus able to define an effective selection strategy to identify new candidate genes for the predisposition to an altered airway inflammatory response under OVA challenge conditions. Similar selection strategies may be used for the analysis of additional genotype – envirotype interactions for other diseases. PMID:26263558

  19. Double-strand break repair and colorectal cancer: gene variants within 3′ UTRs and microRNAs binding as modulators of cancer risk and clinical outcome

    PubMed Central

    Naccarati, Alessio; Rosa, Fabio; Vymetalkova, Veronika; Barone, Elisa; Jiraskova, Katerina; Di Gaetano, Cornelia; Novotny, Jan; Levy, Miroslav; Vodickova, Ludmila; Gemignani, Federica; Buchler, Tomas; Landi, Stefano

    2016-01-01

    Genetic variations in 3′ untranslated regions of target genes may affect microRNA binding, resulting in differential protein expression. microRNAs regulate DNA repair, and single-nucleotide polymorphisms in miRNA binding sites (miRSNPs) may account for interindividual differences in the DNA repair capacity. Our hypothesis is that miRSNPs in relevant DNA repair genes may ultimately affect cancer susceptibility and impact prognosis. In the present study, we analysed the association of polymorphisms in predicted microRNA target sites of double-strand breaks (DSBs) repair genes with colorectal cancer (CRC) risk and clinical outcome. Twenty-one miRSNPs in non-homologous end-joining and homologous recombination pathways were assessed in 1111 cases and 1469 controls. The variant CC genotype of rs2155209 in MRE11A was strongly associated with decreased cancer risk when compared with the other genotypes (OR 0.54, 95% CI 0.38–0.76, p = 0.0004). A reduced expression of the reporter gene was observed for the C allele of this polymorphism by in vitro assay, suggesting a more efficient interaction with potentially binding miRNAs. In colon cancer patients, the rs2155209 CC genotype was associated with shorter survival while the TT genotype of RAD52 rs11226 with longer survival when both compared with their respective more frequent genotypes (HR 1.63, 95% CI 1.06-2.51, p = 0.03 HR 0.60, 95% CI 0.41–0.89, p = 0.01, respectively). miRSNPs in DSB repair genes involved in the maintenance of genomic stability may have a role on CRC susceptibility and clinical outcome. PMID:26735576

  20. Genetic variants in DNA double-strand break repair genes and risk of salivary gland carcinoma: a case-control study.

    PubMed

    Xu, Li; Tang, Hongwei; El-Naggar, Adel K; Wei, Peng; Sturgis, Erich M

    2015-01-01

    DNA double strand break (DSB) repair is the primary defense mechanism against ionizing radiation-induced DNA damage. Ionizing radiation is the only established risk factor for salivary gland carcinoma (SGC). We hypothesized that genetic variants in DSB repair genes contribute to individual variation in susceptibility to SGC. To test this hypothesis, we conducted a case-control study in which we analyzed 415 single nucleotide polymorphisms (SNPs) in 45 DSB repair genes in 352 SGC cases and 598 controls. Multivariate logistic regression analysis was performed to calculate odds ratios (ORs) and 95% confidence intervals (CIs). Rs3748522 in RAD52 and rs13180356 in XRCC4 were significantly associated with SGC after Bonferroni adjustment; ORs (95% CIs) for the variant alleles of these SNPs were 1.71 (1.40-2.09, P = 1.70 × 10(-7)) and 0.58 (0.45-0.74, P = 2.00 × 10(-5)) respectively. The genetic effects were modulated by histological subtype. The association of RAD52-rs3748522 with SGC was strongest for mucoepidermoid carcinoma (OR = 2.21, 95% CI: 1.55-3.15, P = 1.25 × 10(-5), n = 74), and the association of XRCC4-rs13180356 with SGC was strongest for adenoid cystic carcinoma (OR = 0.60, 95% CI: 0.42-0.87, P = 6.91 × 10(-3), n = 123). Gene-level association analysis revealed one gene, PRKDC, with a marginally significant association with SGC risk in non-Hispanic whites. To our knowledge, this study is the first to comprehensively evaluate the genetic effect of DSB repair genes on SGC risk. Our results indicate that genetic variants in the DSB repair pathways contribute to inter-individual differences in susceptibility to SGC and show that the impact of genetic variants differs by histological subtype. Independent studies are warranted to confirm these findings. PMID:26035306

  1. Mismatch repair gene defects contribute to the genetic basis of double primary cancers of the colorectum and endometrium.

    PubMed

    Millar, A L; Pal, T; Madlensky, L; Sherman, C; Temple, L; Mitri, A; Cheng, H; Marcus, V; Gallinger, S; Redston, M; Bapat, B; Narod, S

    1999-05-01

    Hereditary non-polyposis colorectal cancer (HNPCC) is a dominantly inherited cancer syndrome caused by germline defects of mismatch repair (MMR) genes. Endometrial cancer is the most common extracolonic neoplasm in HNPCC and is the primary clinical manifestation of the syndrome in some families. The cumulative incidence of endometrial cancer among HNPCC mutation carriers is high, estimated to be from 22 to 43%. We hypothesized that women with double primary cancers of the colorectum and endometrium are likely to be members of HNPCC families. In order to determine how frequently HNPCC manifests in the context of double primary cancers, we examined alterations of two MMR genes, hMSH2 and hMLH1, in 40 unrelated women affected with double primary cancers. These cases were identified using hospital-based and population-based cancer registries in Ontario, Canada. MMR gene mutations were screened by single-strand conformation polymorphism analysis and confirmed by direct sequencing. Eighteen percent (seven of 40) were found to harbor mutations of one of the two MMR genes. Analysis of colorectal and/or endometrial tumors of mutation-negative probands found microsatellite instability in seven of 20 cases. Six of seven mutation-positive probands had strong family histories suggestive of HNPCC. First degree relatives of mutation-positive probands had a very high relative risk (RR) of colorectal cancer (RR = 8.1, CI 3. 5-15.9) and endometrial cancer (RR = 23.8, CI 6.4-61.0). The relative risk of mutation-negative cases was 2.8 (CI 1.7-4.5) for colorectal cancer and 5.4 (CI 2.0-11.7) for endometrial cancer. We recommend that all double primary patients with cancers at these sites should have a genetic evaluation, including molecular analysis for HNPCC where appropriate. PMID:10196371

  2. Mutations in the transcriptional repressor REST predispose to Wilms tumor.

    PubMed

    Mahamdallie, Shazia S; Hanks, Sandra; Karlin, Kristen L; Zachariou, Anna; Perdeaux, Elizabeth R; Ruark, Elise; Shaw, Chad A; Renwick, Alexander; Ramsay, Emma; Yost, Shawn; Elliott, Anna; Birch, Jillian; Capra, Michael; Gray, Juliet; Hale, Juliet; Kingston, Judith; Levitt, Gill; McLean, Thomas; Sheridan, Eamonn; Renwick, Anthony; Seal, Sheila; Stiller, Charles; Sebire, Neil; Westbrook, Thomas F; Rahman, Nazneen

    2015-12-01

    Wilms tumor is the most common childhood renal cancer. To identify mutations that predispose to Wilms tumor, we are conducting exome sequencing studies. Here we describe 11 different inactivating mutations in the REST gene (encoding RE1-silencing transcription factor) in four familial Wilms tumor pedigrees and nine non-familial cases. Notably, no similar mutations were identified in the ICR1000 control series (13/558 versus 0/993; P < 0.0001) or in the ExAC series (13/558 versus 0/61,312; P < 0.0001). We identified a second mutational event in two tumors, suggesting that REST may act as a tumor-suppressor gene in Wilms tumor pathogenesis. REST is a zinc-finger transcription factor that functions in cellular differentiation and embryonic development. Notably, ten of 11 mutations clustered within the portion of REST encoding the DNA-binding domain, and functional analyses showed that these mutations compromise REST transcriptional repression. These data establish REST as a Wilms tumor predisposition gene accounting for ∼2% of Wilms tumor. PMID:26551668

  3. Recent developments with the human repair genes ERCC2, ERCC4, and XRCC1

    SciTech Connect

    Thompson, L.H.; Caldecott, K.W.; Brookman, K.W.; Weber, C.A.; Salazar, E.S.; Takayama, K.; Fornace, A.J.

    1992-11-06

    ERCC2 was first identified as a gene on human chromosome 19 that complemented the UV sensitivity of CHO UV5 cells in somatic cell hybrids. Subsequent studies localized ERCC2 to the same chromosomal region (19q13.2--13.3) as the ERCC1 gene and showed that the two genes were less than 250 kb apart. Cloning of ERCC2 was accomplished by transfection of genomic DNA into UV5 cells and rescue of the gene from a secondary transformant. Recovery of the gene was aided by the presence of repetitive sequences that were detected on Southern blots with a probe for Alu-family repeats. ERCC2, which is 19 kb in size, quantitatively corrected the UV sensitivity and incision defect in UV5 cells upon transfection. An ERCC2 CDNA clone was recovered from the pcD2 expression library. Although this clone was truncated at the 5 in. end, it conferred transient, but not stable, correction to UV5 cells upon transfection. Based on genomic sequence, this clone was extended by oligonucleotide addition to obtain minigene constructs in which the complete open reading frame (ORF) was present. Translation of the ERCC2 ORF gives an amino acid sequence that has 72% similarity with the S. cerevisiae RAD3 protein, which encodes a DNA helicase.

  4. Hereditary truncating mutations of DNA repair and other genes in BRCA1/BRCA2/PALB2-negatively tested breast cancer patients.

    PubMed

    Lhota, F; Zemankova, P; Kleiblova, P; Soukupova, J; Vocka, M; Stranecky, V; Janatova, M; Hartmannova, H; Hodanova, K; Kmoch, S; Kleibl, Z

    2016-10-01

    Hereditary breast cancer comprises a minor but clinically meaningful breast cancer (BC) subgroup. Mutations in the major BC-susceptibility genes are important prognostic and predictive markers; however, their carriers represent only 25% of high-risk BC patients. To further characterize variants influencing BC risk, we performed SOLiD sequencing of 581 genes in 325 BC patients (negatively tested in previous BRCA1/BRCA2/PALB2 analyses). In 105 (32%) patients, we identified and confirmed 127 truncating variants (89 unique; nonsense, frameshift indels, and splice site), 19 patients harbored more than one truncation. Forty-six (36 unique) truncating variants in 25 DNA repair genes were found in 41 (12%) patients, including 16 variants in the Fanconi anemia (FA) genes. The most frequent variant in FA genes was c.1096_1099dupATTA in FANCL that also show a borderline association with increased BC risk in subsequent analysis of enlarged groups of BC patients and controls. Another 81 (53 unique) truncating variants were identified in 48 non-DNA repair genes in 74 patients (23%) including 16 patients carrying variants in genes coding proteins of estrogen metabolism/signaling. Our results highlight the importance of mutations in the FA genes' family, and indicate that estrogen metabolism genes may reveal a novel candidate genetic component for BC susceptibility. PMID:26822949

  5. Regulated expression of the Saccharomyces cerevisiae DNA repair gene RAD7 in response to DNA damage and during sporulation.

    PubMed Central

    Jones, J S; Prakash, L; Prakash, S

    1990-01-01

    The RAD7 gene of Saccharomyces cerevisiae affects the proficiency of excision repair of DNA damaged by UV light. Here, we report our studies on the regulation of the RAD7 gene in response to UV irradiation and during sporulation. RAD7 transcript levels increased 6-fold within 40 min of exposure of cells to 37 J/m2 of UV light. Higher UV doses also elicited rapid increases in the level of RAD7 mRNA. RAD7 mRNA levels increased in sporulating MATa/MAT alpha diploid cells, but not in the asporogenous MATa/MATa strain exposed to sporulation conditions. The increase in RAD7 mRNA level in MATa/MAT alpha cells was 15-fold after 6 h and 9-fold after 7 h in sporulation medium; thereafter, RAD7 mRNA levels declined. Periodic transcription of RAD7 during sporulation suggests a role for RAD7 in this process. Images PMID:2192359

  6. CYS3, a hotspot of meiotic recombination in Saccharomyces cerevisiae. Effects of heterozygosity and mismatch repair functions on gene conversion and recombination intermediates.

    PubMed Central

    Vedel, M; Nicolas, A

    1999-01-01

    We have examined meiotic recombination at the CYS3 locus. Genetic analysis indicates that CYS3 is a hotspot of meiotic gene conversion, with a putative 5'-3' polarity gradient of conversion frequencies. This gradient is relieved in the presence of msh2 and pms1 mutations, indicating an involvement of mismatch repair functions in meiotic recombination. To investigate the role of mismatch repair proteins in meiotic recombination, we performed a physical analysis of meiotic DNA in wild-type and msh2 pms1 strains in the presence or absence of allelic differences at CYS3. Neither the mutations in CYS3 nor the absence of mismatch repair functions affects the frequency and distribution of nearby recombination-initiating DNA double-strand breaks (DSBs). Processing of DSBs is also similar in msh2 pms1 and wild-type strains. We conclude that mismatch repair functions do not control the distribution of meiotic gene conversion events at the initiating steps. In the MSH2 PMS1 background, strains heteroallelic for frameshift mutations in CYS3 exhibit a frequency of gene conversion greater than that observed for either marker alone. Physical analysis revealed no modification in the formation of DSBs, suggesting that this marker effect results from subsequent processing events that are not yet understood. PMID:10101154

  7. Candidate driver genes involved in genome maintenance and DNA repair in Sézary syndrome.

    PubMed

    Woollard, Wesley J; Pullabhatla, Venu; Lorenc, Anna; Patel, Varsha M; Butler, Rosie M; Bayega, Anthony; Begum, Nelema; Bakr, Farrah; Dedhia, Kiran; Fisher, Joshua; Aguilar-Duran, Silvia; Flanagan, Charlotte; Ghasemi, Aria A; Hoffmann, Ricarda M; Castillo-Mosquera, Nubia; Nuttall, Elisabeth A; Paul, Arisa; Roberts, Ceri A; Solomonidis, Emmanouil G; Tarrant, Rebecca; Yoxall, Antoinette; Beyers, Carl Z; Ferreira, Silvia; Tosi, Isabella; Simpson, Michael A; de Rinaldis, Emanuele; Mitchell, Tracey J; Whittaker, Sean J

    2016-06-30

    Sézary syndrome (SS) is a leukemic variant of cutaneous T-cell lymphoma (CTCL) and represents an ideal model for study of T-cell transformation. We describe whole-exome and single-nucleotide polymorphism array-based copy number analyses of CD4(+) tumor cells from untreated patients at diagnosis and targeted resequencing of 101 SS cases. A total of 824 somatic nonsynonymous gene variants were identified including indels, stop-gain/loss, splice variants, and recurrent gene variants indicative of considerable molecular heterogeneity. Driver genes identified using MutSigCV include POT1, which has not been previously reported in CTCL; and TP53 and DNMT3A, which were also identified consistent with previous reports. Mutations in PLCG1 were detected in 11% of tumors including novel variants not previously described in SS. This study is also the first to show BRCA2 defects in a significant proportion (14%) of SS tumors. Aberrations in PRKCQ were found to occur in 20% of tumors highlighting selection for activation of T-cell receptor/NF-κB signaling. A complex but consistent pattern of copy number variants (CNVs) was detected and many CNVs involved genes identified as putative drivers. Frequent defects involving the POT1 and ATM genes responsible for telomere maintenance were detected and may contribute to genomic instability in SS. Genomic aberrations identified were enriched for genes implicated in cell survival and fate, specifically PDGFR, ERK, JAK STAT, MAPK, and TCR/NF-κB signaling; epigenetic regulation (DNMT3A, ASLX3, TET1-3); and homologous recombination (RAD51C, BRCA2, POLD1). This study now provides the basis for a detailed functional analysis of malignant transformation of mature T cells and improved patient stratification and treatment. PMID:27121473

  8. Defects in the DNA repair and transcription gene ERCC2(XPD) in trichothiodystrophy

    SciTech Connect

    Takayama, K.; Salazar, E.P.; Thompson, L.H.

    1996-02-01

    Trichothiodystrophy (TTD) is a rare autosomal recessive disorder characterized by brittle hair with reduced sulfur content, ichthyosis, peculiar face, and mental and growth retardation. Clinical photosensitivity is present in {approximately}50% of TTD patients but is not associated with an elevated frequency of cancers. Previous complementation studies show that the photosensitivity in nearly all of the studied patients is due to a defect in the same genetic locus that underlies the cancer-prone genetic disorder xeroderma pigmentosum group D (XP-D). Nucleotide-sequence analysis of the ERCC2 cDNA from three TTD cell strains (TTD1VI, TTD3VI, and TTD1RO) revealed mutations within the region from amino acid 713-730 and within previously identified helicase functional domains. The various clinical presentations and DNA repair characteristics of the cell strains can be correlated with the particular mutations found in the ERCC2 locus. Mutations of Arg658 to either His or Cys correlate with TTD cell strains with intermediate UV-sensitivity, mutation of Arg722 to Trp correlates with highly UV-sensitive TTD cell strains, and mutation of Arg683 to Trp correlates with XP-D. Alleles with mutation of Arg616 to Pro or with the combined mutation of Leu461 to Val and deletion of 716-730 are found in both XP-D and TTD cell strains. 39 refs., 2 figs., 3 tabs.

  9. Defects in the DNA repair and transcription gene ERCC2(XPD) in trichothiodystrophy.

    PubMed Central

    Takayama, K.; Salazar, E. P.; Broughton, B. C.; Lehmann, A. R.; Sarasin, A.; Thompson, L. H.; Weber, C. A.

    1996-01-01

    Trichothiodystrophy (TTD) is a rare autosomal recessive disorder characterized by brittle hair with reduced sulfur content, ichthyosis, peculiar face, and mental and growth retardation. Clinical photosensitivity is present in approximately 50% of TTD patients but is not associated with an elevated frequency of cancers. Previous complementation studies show that the photosensitivity in nearly all of the studied patients is due to a defect in the same genetic locus that underlies the cancer-prone genetic disorder xeroderma pigmentosum group D (XP-D). Nucleotide-sequence analysis of the ERCC2 cDNA from three TTD cell strains (TTD1V1, TTD3VI, and TTD1RO) revealed mutations within the region from amino acid 713-730 and within previously identified helicase functional domains. The various clinical presentations and DNA repair characteristics of the cell strains can be correlated with the particular mutations found in the ERCC2 locus. Mutations of Arg658 to either His or Cys correlate with TTD cell strains with intermediate UV-sensitivity, mutation of Arg722 to Trp correlates with highly UV-sensitive TTD cell strains, and mutation of Arg683 to Trp correlates with XP-D. Alleles with mutation of Arg616 to Pro or with the combined mutation of Leu461 to Val and deletion of 716-730 are found in both XP-D and TTD cell strains. PMID:8571952

  10. Induction of resistance to alkylating agents in E. coli: the ada+ gene product serves both as a regulatory protein and as an enzyme for repair of mutagenic damage.

    PubMed Central

    Teo, I; Sedgwick, B; Demple, B; Li, B; Lindahl, T

    1984-01-01

    The expression of several inducible enzymes for repair of alkylated DNA in Escherichia coli is controlled by the ada+ gene. This regulatory gene has been cloned into a multicopy plasmid and shown to code for a 37-kd protein. Antibodies raised against homogeneous O6-methylguanine-DNA methyltransferase (the main repair activity for mutagenic damage in alkylated DNA) were found to cross-react with this 37-kd protein. Cell extracts from several independently derived ada mutants contain variable amounts of an altered 37-kd protein after an inducing alkylation treatment. In addition, an 18-kd protein identical with the previously isolated O6-methyl-guanine-DNA methyltransferase has been identified as a product of the ada+ gene. The smaller polypeptide is derived from the 37-kd protein by proteolytic processing. Images Fig. 1. Fig. 2. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. PMID:6092060

  11. Endovascular repair of a Kommerell diverticulum anomaly.

    PubMed

    Gao, Peixian; Wang, Mo; Dong, Dianning; Kong, Xiangqian; Jin, Xing; Zhang, Shiyi

    2015-05-01

    A Kommerell diverticulum (KD) may predispose toward aortic aneurysm, dissection, or rupture, although they are primarily asymptomatic. We report a case of an aberrant left subclavian artery arising from a KD in a right-side aortic arch. The lesions were successfully treated by an endovascular approach involving Amplatzer vascular plug embolization of the aberrant left subclavian artery and endovascular repair of the KD.

  12. Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases.

    PubMed

    Remy, Séverine; Tesson, Laurent; Menoret, Séverine; Usal, Claire; De Cian, Anne; Thepenier, Virginie; Thinard, Reynald; Baron, Daniel; Charpentier, Marine; Renaud, Jean-Baptiste; Buelow, Roland; Cost, Gregory J; Giovannangeli, Carine; Fraichard, Alexandre; Concordet, Jean-Paul; Anegon, Ignacio

    2014-08-01

    The generation of genetically modified animals is important for both research and commercial purposes. The rat is an important model organism that until recently lacked efficient genetic engineering tools. Sequence-specific nucleases, such as ZFNs, TALE nucleases, and CRISPR/Cas9 have allowed the creation of rat knockout models. Genetic engineering by homology-directed repair (HDR) is utilized to create animals expressing transgenes in a controlled way and to introduce precise genetic modifications. We applied TALE nucleases and donor DNA microinjection into zygotes to generate HDR-modified rats with large new sequences introduced into three different loci with high efficiency (0.62%-5.13% of microinjected zygotes). Two of these loci (Rosa26 and Hprt1) are known to allow robust and reproducible transgene expression and were targeted for integration of a GFP expression cassette driven by the CAG promoter. GFP-expressing embryos and four Rosa26 GFP rat lines analyzed showed strong and widespread GFP expression in most cells of all analyzed tissues. The third targeted locus was Ighm, where we performed successful exon exchange of rat exon 2 for the human one. At all three loci we observed HDR only when using linear and not circular donor DNA. Mild hypothermic (30°C) culture of zygotes after microinjection increased HDR efficiency for some loci. Our study demonstrates that TALE nuclease and donor DNA microinjection into rat zygotes results in efficient and reproducible targeted donor integration by HDR. This allowed creation of genetically modified rats in a work-, cost-, and time-effective manner.

  13. DNA repair gene ERCC1 polymorphisms and glioma susceptibility among Chinese population: a meta-analysis

    PubMed Central

    Jiang, Chunming; Shen, Fang; Du, Jianmin; Wang, Xiaohua; Su, Jin; Liu, Zhanli; Huang, Xianmei

    2015-01-01

    Background: Excision repair cross complementation group 1 (ERCC1) has been shown to be involved in the progression of glioma susceptibility. However, the results remain conflict. The aim of this study was to systematically review and evaluate the role of ERCC1 C118T and C8092A polymorphisms in glioma risk among Chinese population. Methods: Related case-control studies were searched in online electronic databases. Odds ratio (OR) with its 95% confidence interval (CI) were employed to calculate the extracted data. Results: Total seven articles were retrieved, including 4426 subjects (1926 were glioma patients and 2500 were matched controls). No significant heterogeneity was found between studies (I2=0%, P>0.01). Our results demonstrated that A allele and AA genotype of ERCC1 C8092A polymorphism have a positive association with increasing the risk of glioma in the fixed-effect model (A vs. C: OR=1.13, 95% CI=1.02-1.25, P=0.02; AA vs. CC: OR=1.29, 95% CI=1.04-1.61, P=0.02; AA vs. CA+CC: OR=1.25, 95% CI=1.01-1.55, P=0.04). However, no significant relationship was found between C118T variant and glioma susceptibility. Conclusions: Our results indicated that ERCC1 C8092A, not C118T polymorphism might be a biomarker for patients with glioma among Chinese population. Future studies with more ethnicities are needed to explore the precise association. PMID:26379816

  14. Single Nucleotide Polymorphisms in Nucleotide Excision Repair Genes, Cigarette Smoking, and the Risk of Head and Neck Cancer

    PubMed Central

    Wyss, Annah B.; Herring, Amy H.; Avery, Christy L.; Weissler, Mark C.; Bensen, Jeannette T.; Barnholtz-Sloan, Jill S.; Funkhouser, William K.; Olshan, Andrew F.

    2013-01-01

    Background Cigarette smoking is associated with increased head and neck cancer (HNC) risk. Tobacco-related carcinogens are known to cause bulky DNA adducts. Nucleotide excision repair (NER) genes encode enzymes that remove adducts and may be independently associated with HNC, as well as modifiers of the association between smoking and HNC. Methods Using population-based case-control data from the Carolina Head and Neck Cancer Epidemiology Study (1,227 cases, 1,325 controls), race-stratified (white, African American) conventional and hierarchical logistic regression models were utilized to estimate odds ratios (OR) with 95% intervals (I) for the independent and joint effects of cigarette smoking and 84 single nucleotide polymorphisms (SNPs) from 15 NER genes on HNC risk. Results The odds of HNC were elevated among ever cigarette smokers, and increased with smoking duration and frequency. Among whites, rs4150403 on ERCC3 was associated with increased HNC odds (AA+AG vs. GG, OR=1.28, 95% I=1.01,1.61). Among African Americans, rs4253132 on ERCC6 was associated with decreased HNC odds (CC+CT vs. TT, OR=0.62, 95% I=0.45,0.86). Interactions between ever cigarette smoking and three SNPs (rs4253132 on ERCC6, rs2291120 on DDB2, and rs744154 on ERCC4) suggested possible departures from additivity among whites. Conclusions We did not find associations between some previously studied NER variants and HNC. We did identify new associations between two SNPs and HNC and three suggestive cigarette-SNP interactions to consider in future studies. Impact We conducted one of the most comprehensive evaluations of NER variants, identifying a few SNPs from biologically plausible candidate genes associated with HNC and possibly interacting with cigarette smoking. PMID:23720401

  15. Common variants in mismatch repair genes associated with increased risk of sperm DNA damage and male infertility

    PubMed Central

    2012-01-01

    Background The mismatch repair (MMR) pathway plays an important role in the maintenance of the genome integrity, meiotic recombination and gametogenesis. This study investigated whether genetic variations in MMR genes are associated with an increased risk of sperm DNA damage and male infertility. Methods We selected and genotyped 21 tagging single nucleotide polymorphisms (SNPs) in five MMR genes (MLH1, MLH3, PMS2, MSH4 and MSH5) using the SNPstream 12-plex platform in a case-control study of 1,292 idiopathic infertility patients and 480 fertile controls in a Chinese population. Sperm DNA damage levels were detected with the Tdt-mediated dUTP nick end labelling (TUNEL) assay in 450 cases. Fluorescence resonance energy transfer (FRET) and co-immunoprecipitation techniques were employed to determine the effects of functional variants. Results One intronic SNP in MLH1 (rs4647269) and two non-synonymous SNPs in PMS2 (rs1059060, Ser775Asn) and MSH5 (rs2075789, Pro29Ser) seem to be risk factors for the development of azoospermia or oligozoospermia. Meanwhile, we also identified a possible contribution of PMS2 rs1059060 to the risk of male infertility with normal sperm count. Among patients with normal sperm count, MLH1 rs4647269 and PMS2 rs1059060 were associated with increased sperm DNA damage. Functional analysis revealed that the PMS2 rs1059060 can affect the interactions between MLH1 and PMS2. Conclusions Our results provide evidence supporting the involvement of genetic polymorphisms in MMR genes in the aetiology of male infertility. PMID:22594646

  16. Reduced XPC DNA repair gene mRNA levels in clinically normal parents of xeroderma pigmentosum patients.

    PubMed

    Khan, Sikandar G; Oh, Kyu-Seon; Shahlavi, Tala; Ueda, Takahiro; Busch, David B; Inui, Hiroki; Emmert, Steffen; Imoto, Kyoko; Muniz-Medina, Vanessa; Baker, Carl C; DiGiovanna, John J; Schmidt, Deborah; Khadavi, Arash; Metin, Ahmet; Gozukara, Engin; Slor, Hanoch; Sarasin, Alain; Kraemer, Kenneth H

    2006-01-01

    Xeroderma pigmentosum group C (XP-C) is a rare autosomal recessive disorder. Patients with two mutant alleles of the XPC DNA repair gene have sun sensitivity and a 1000-fold increase in skin cancers. Clinically normal parents of XP-C patients have one mutant allele and one normal allele. As a step toward evaluating cancer risk in these XPC heterozygotes we characterized cells from 16 XP families. We identified 15 causative mutations (5 frameshift, 6 nonsense and 4 splicing) in the XPC gene in cells from 16 XP probands. All had premature termination codons (PTC) and absence of normal XPC protein on western blotting. The cell lines from 26 parents were heterozygous for the same mutations. We employed a real-time quantitative reverse transcriptase-PCR assay as a rapid and sensitive method to measure XPC mRNA levels. The mean XPC mRNA levels in the cell lines from the XP-C probands were 24% (P<10(-7)) of that in 10 normal controls. This reduced XPC mRNA level in cells from XP-C patients was caused by the PTC that induces nonsense-mediated mRNA decay. The mean XPC mRNA levels in cell lines from the heterozygous XP-C carriers were intermediate (59%, P=10(-4)) between the values for the XP patients and the normal controls. This study demonstrates reduced XPC mRNA levels in XP-C patients and heterozygotes. Thus, XPC mRNA levels may be evaluated as a marker of cancer susceptibility in carriers of mutations in the XPC gene. PMID:16081512

  17. Might there be a link between intron 3 VNTR polymorphism in the XRCC4 DNA repair gene and the etiopathogenesis of rheumatoid arthritis?

    PubMed

    Pehlivan, Sacide; Balci, Sibel Oguzkan; Aydeniz, Ali; Pehlivan, Mustafa; Sever, Tugce; Gursoy, Savas

    2015-01-01

    DNA repair genes are involved in several diseases such as cancers and autoimmune diseases. Previous studies indicated that a DNA repair system was involved in the development of rheumatoid arthritis (RA). In this study, we aimed to examine whether four polymorphisms in the DNA repair genes (xeroderma pigmentosum complementation group D [XPD], X-ray repair cross-complementing group 1 [XRCC1], and X-ray repair cross-complementing group 4 [XRCC4]) were associated with RA. Sixty-five patients with RA and 70 healthy controls (HCs) were examined for XPD (A-751G), XRCC1 (A399G), and XRCC4 (intron 3 VNTR and G-1394T) polymorphisms. All polymorphisms were genotyped by PCR and/or PCR-RFLP. The association between the polymorphisms and RA was analyzed using the chi-square test and de Finetti program. The intron 3 VNTR polymorphism in the XRCC4 gene showed an association with RA patients. The DI genotype was found lower in RA patients (χ(2)=8.227; p=0.0021), while the II genotype was higher in RA patients (χ(2)=5.285; p=0.010). There were deviations from the Hardy-Weinberg Equilibrium (HWE) in both intron 3 VNTR and G-1394T polymorphisms in the XRCC4 gene and in the polymorphism in the XRCC1 gene, and the observed genotype counts deviated from those expected according to the HWE (p=0.027, 0.004, and 0.002, respectively); however, there was no deviation in the other gene polymorphisms. There is no statistical difference between the RA patients and HCs for XPD (A-751G), XRCC1 (A399G), and XRCC4 (G-1394T) gene polymorphisms (p>0.05). Although XPD (A-751G), XRCC1 (A399G), and XRCC4 (G-1394T) gene polymorphisms have been extensively investigated in different clinical pictures, this is the first study to evaluate the role of these polymorphisms in the genetic etiopathogenesis of RA in Turkish patients. In conclusion, we suggested that the intron 3 VNTR polymorphism in the XRCC4 gene may be associated with the etiopathogenesis of RA as a marker of immune aging.

  18. Might there be a link between intron 3 VNTR polymorphism in the XRCC4 DNA repair gene and the etiopathogenesis of rheumatoid arthritis?

    PubMed

    Pehlivan, Sacide; Balci, Sibel Oguzkan; Aydeniz, Ali; Pehlivan, Mustafa; Sever, Tugce; Gursoy, Savas

    2015-01-01

    DNA repair genes are involved in several diseases such as cancers and autoimmune diseases. Previous studies indicated that a DNA repair system was involved in the development of rheumatoid arthritis (RA). In this study, we aimed to examine whether four polymorphisms in the DNA repair genes (xeroderma pigmentosum complementation group D [XPD], X-ray repair cross-complementing group 1 [XRCC1], and X-ray repair cross-complementing group 4 [XRCC4]) were associated with RA. Sixty-five patients with RA and 70 healthy controls (HCs) were examined for XPD (A-751G), XRCC1 (A399G), and XRCC4 (intron 3 VNTR and G-1394T) polymorphisms. All polymorphisms were genotyped by PCR and/or PCR-RFLP. The association between the polymorphisms and RA was analyzed using the chi-square test and de Finetti program. The intron 3 VNTR polymorphism in the XRCC4 gene showed an association with RA patients. The DI genotype was found lower in RA patients (χ(2)=8.227; p=0.0021), while the II genotype was higher in RA patients (χ(2)=5.285; p=0.010). There were deviations from the Hardy-Weinberg Equilibrium (HWE) in both intron 3 VNTR and G-1394T polymorphisms in the XRCC4 gene and in the polymorphism in the XRCC1 gene, and the observed genotype counts deviated from those expected according to the HWE (p=0.027, 0.004, and 0.002, respectively); however, there was no deviation in the other gene polymorphisms. There is no statistical difference between the RA patients and HCs for XPD (A-751G), XRCC1 (A399G), and XRCC4 (G-1394T) gene polymorphisms (p>0.05). Although XPD (A-751G), XRCC1 (A399G), and XRCC4 (G-1394T) gene polymorphisms have been extensively investigated in different clinical pictures, this is the first study to evaluate the role of these polymorphisms in the genetic etiopathogenesis of RA in Turkish patients. In conclusion, we suggested that the intron 3 VNTR polymorphism in the XRCC4 gene may be associated with the etiopathogenesis of RA as a marker of immune aging. PMID:25494482

  19. Gene- and stem cell-based therapeutics for cartilage regeneration and repair.

    PubMed

    Tang, Ying; Wang, Bing

    2015-04-15

    Cell-based regeneration of damaged or diseased articular cartilage still faces significant clinical challenge due to inadequate environmental regulation of stem cell proliferation and chondrogenic differentiation. The role of insulin-like growth factor in critical steps of human bone marrow-derived mesenchymal stem cell chondrogenesis has potential in optimizing the therapeutic use of mesenchymal stem cells in cartilage disorders. In addition to the previously described benefits of recombinant adeno-associated viral vector for in vivo gene therapy, demonstrated by Frisch and colleagues, such vector is also a safe and efficient delivery system for the genetic modification of human bone marrow-derived mesenchymal stem cells via ex vivo insulin-like growth factor 1 gene transfer, so that implanted mesenchymal stem cells continuously release a therapeutic level of insulin-like growth factor 1 to achieve sustained mesenchymal stem cell chondrogenesis for cartilage regeneration.

  20. Injectable graphene oxide/hydrogel-based angiogenic gene delivery system for vasculogenesis and cardiac repair.

    PubMed

    Paul, Arghya; Hasan, Anwarul; Kindi, Hamood Al; Gaharwar, Akhilesh K; Rao, Vijayaraghava T S; Nikkhah, Mehdi; Shin, Su Ryon; Krafft, Dorothee; Dokmeci, Mehmet R; Shum-Tim, Dominique; Khademhosseini, Ali

    2014-08-26

    The objective of this study was to develop an injectable and biocompatible hydrogel which can efficiently deliver a nanocomplex of graphene oxide (GO) and vascular endothelial growth factor-165 (VEGF) pro-angiogenic gene for myocardial therapy. For the study, an efficient nonviral gene delivery system using polyethylenimine (PEI) functionalized GO nanosheets (fGO) complexed with DNAVEGF was formulated and incorporated in the low-modulus methacrylated gelatin (GelMA) hydrogel to promote controlled and localized gene therapy. It was hypothesized that the fGOVEGF/GelMA nanocomposite hydrogels can efficiently transfect myocardial tissues and induce favorable therapeutic effects without invoking cytotoxic effects. To evaluate this hypothesis, a rat model with acute myocardial infarction was used, and the therapeutic hydrogels were injected intramyocardially in the peri-infarct regions. The secreted VEGF from in vitro transfected cardiomyocytes demonstrated profound mitotic activities on endothelial cells. A significant increase in myocardial capillary density at the injected peri-infarct region and reduction in scar area were noted in the infarcted hearts with fGOVEGF/GelMA treatment compared to infarcted hearts treated with untreated sham, GelMA and DNAVEGF/GelMA groups. Furthermore, the fGOVEGF/GelMA group showed significantly higher (p < 0.05, n = 7) cardiac performance in echocardiography compared to other groups, 14 days postinjection. In addition, no significant differences were noticed between GO/GelMA and non-GO groups in the serum cytokine levels and quantitative PCR based inflammatory microRNA (miRNA) marker expressions at the injected sites. Collectively, the current findings suggest the feasibility of a combined hydrogel-based gene therapy system for ischemic heart diseases using nonviral hybrid complex of fGO and DNA.

  1. Injectable Graphene Oxide/Hydrogel-Based Angiogenic Gene Delivery System for Vasculogenesis and Cardiac Repair

    PubMed Central

    2015-01-01

    The objective of this study was to develop an injectable and biocompatible hydrogel which can efficiently deliver a nanocomplex of graphene oxide (GO) and vascular endothelial growth factor-165 (VEGF) pro-angiogenic gene for myocardial therapy. For the study, an efficient nonviral gene delivery system using polyethylenimine (PEI) functionalized GO nanosheets (fGO) complexed with DNAVEGF was formulated and incorporated in the low-modulus methacrylated gelatin (GelMA) hydrogel to promote controlled and localized gene therapy. It was hypothesized that the fGOVEGF/GelMA nanocomposite hydrogels can efficiently transfect myocardial tissues and induce favorable therapeutic effects without invoking cytotoxic effects. To evaluate this hypothesis, a rat model with acute myocardial infarction was used, and the therapeutic hydrogels were injected intramyocardially in the peri-infarct regions. The secreted VEGF from in vitro transfected cardiomyocytes demonstrated profound mitotic activities on endothelial cells. A significant increase in myocardial capillary density at the injected peri-infarct region and reduction in scar area were noted in the infarcted hearts with fGOVEGF/GelMA treatment compared to infarcted hearts treated with untreated sham, GelMA and DNAVEGF/GelMA groups. Furthermore, the fGOVEGF/GelMA group showed significantly higher (p < 0.05, n = 7) cardiac performance in echocardiography compared to other groups, 14 days postinjection. In addition, no significant differences were noticed between GO/GelMA and non-GO groups in the serum cytokine levels and quantitative PCR based inflammatory microRNA (miRNA) marker expressions at the injected sites. Collectively, the current findings suggest the feasibility of a combined hydrogel-based gene therapy system for ischemic heart diseases using nonviral hybrid complex of fGO and DNA. PMID:24988275

  2. Environmental arsenic exposure and DNA methylation of the tumor suppressor gene p16 and the DNA repair gene MLH1: effect of arsenic metabolism and genotype.

    PubMed

    Hossain, Mohammad Bakhtiar; Vahter, Marie; Concha, Gabriela; Broberg, Karin

    2012-11-01

    Arsenic is carcinogenic, possibly partly through epigenetic mechanisms. We evaluated the effects of arsenic exposure and metabolism on DNA methylation. Arsenic exposure and methylation efficiency in 202 women in the Argentinean Andes were assessed from concentrations of arsenic metabolites in urine (inorganic arsenic, methylarsonic acid [MMA], and dimethylarsinic acid [DMA]), measured by HPLC-ICPMS. Methylation of CpGs of the tumor suppressor gene p16, the DNA repair gene MLH1, and the repetitive elements LINE1 was measured by PCR pyrosequencing of blood DNA. Genotyping (N = 172) for AS3MT was performed using Sequenom™, and gene expression (N = 90) using Illumina DirectHyb HumanHT-12 v3.0. Median arsenic concentration in urine was 230 μg L(-1) (range 10.1-1251). In linear regression analysis, log(2)-transformed urinary arsenic concentrations were positively associated with methylation of p16 (β = 0.14, P = 0.0028) and MLH1 (β = 0.28, P = 0.0011), but not with LINE1. Arsenic concentrations were of borderline significance negatively correlated with expression of p16 (r(s) = -0.20; P = 0.066)), but not with MLH1. The fraction of inorganic arsenic was positively (β = 0.026; P = 0.010) and DMA was negatively (β = -0.017, P = 0.043) associated with p16 methylation with no effect of MMA. Carriers of the slow-metabolizing AS3MT haplotype were associated with more p16 methylation (P = 0.022). Arsenic exposure was correlated with increased methylation, in blood, of genes encoding enzymes that suppress carcinogenesis, and the arsenic metabolism efficiency modified the degree of epigenetic alterations.

  3. Xeroderma pigmentosum and the role of DNA repair in oncogenesis.

    PubMed

    Giannelli, F

    1978-01-01

    Biochemical and genetic information on xeroderma pigmentosum (XP) has been briefly reviewed. This indicates that 80 to 90 percent of all XP patients are defective in the excision repair of pyrimidine dimers and are unable to perform the first step of this process as shown, for example, by their inability to undergo the DNA superhelical changes which accompany the initiation of excision repair in normal cells. However, in spite of its apparent biochemical homogeneity, XP is genetically heterogeneous and many genes appear to be responsible for the function of the factor defective in XP. Ten to 20 percent of all XP patients (called XP-variants) are capable of "dimer excision repair" but have difficulties in replicating UV-damaged DNA. The defects of XP and XP-variant affect also the repair of DNA damage caused by a number of chemical mutagens and carcinogens. This has important theoretical and practical implications since it indicates, for example, that the repair systems defective in XP must have broad specificity and that even XP cells not exposed to the harmful effect of light may suffer from poor repair of DNA damage. With regard to cancer, two questions have been considered. Namely, does XP provide a valid general model for UV-carcinogenesis in man and does it show how DNA damage leads to malignant transformation? The first question was answered in the affirmative in view of some clinical but, mainly, of cell biological data indicating that normal and excision defective XP cells differ, more quantitatively than qualitatively, in their response to UV-light. With regard to the second question XP seems to provide some support for various theories on carcinogenesis and, DNA repair defects may favour actinic carcinogenesis in a complex, non-univocous manner. Possibly the most important lesson imparted by XP is that, in man, the stability of the genetic material is dependent on the function of repair systems whose failure may predispose to cancer. In addition, the

  4. Inverse Association between Obesity Predisposing FTO Genotype and Completed Suicide

    PubMed Central

    Chojnicka, Izabela; Fudalej, Sylwia; Walczak, Anna; Wasilewska, Krystyna; Fudalej, Marcin; Stawiński, Piotr; Strawa, Katarzyna; Pawlak, Aleksandra; Wojnar, Marcin; Krajewski, Paweł; Płoski, Rafał

    2014-01-01

    The A allele of rs9939609 in the FTO gene predisposes to increased body mass index (BMI) and obesity. Recently we showed an inverse association between the obesity related A allele of rs9939609 and alcohol dependence which was replicated by others. Since this finding raises a possibility that FTO may be associated with other psychiatric phenotypes, we aimed to examine association of rs9939609 with completed suicide. We genotyped rs9939609 in 912 suicide victims and 733 controls using TaqMan approach. We observed an inverse association between suicide and the rs9939609 A allele (OR = 0.80, P = 0.002, Pcor = 0.006) with genotype distribution suggesting a co-dominant effect. Given the link between alcoholism and suicide under influence of alcohol reported in Polish population, confounding by alcohol addiction was unlikely due to apparently similar effect size among cases who were under influence of ethanol at the time of death (OR = 0.76, P = 0.003, N = 361) and those who were not (OR = 0.80, P = 0.007, N = 469). The search for genotype-phenotype correlations did not show significant results. In conclusion, our study proves that there is an inverse association between rs9939609 polymorphism in FTO gene and completed suicide which is independent from association between FTO and alcohol addiction. PMID:25265168

  5. Inverse association between obesity predisposing FTO genotype and completed suicide.

    PubMed

    Chojnicka, Izabela; Fudalej, Sylwia; Walczak, Anna; Wasilewska, Krystyna; Fudalej, Marcin; Stawiński, Piotr; Strawa, Katarzyna; Pawlak, Aleksandra; Wojnar, Marcin; Krajewski, Paweł; Płoski, Rafał

    2014-01-01

    The A allele of rs9939609 in the FTO gene predisposes to increased body mass index (BMI) and obesity. Recently we showed an inverse association between the obesity related A allele of rs9939609 and alcohol dependence which was replicated by others. Since this finding raises a possibility that FTO may be associated with other psychiatric phenotypes, we aimed to examine association of rs9939609 with completed suicide. We genotyped rs9939609 in 912 suicide victims and 733 controls using TaqMan approach. We observed an inverse association between suicide and the rs9939609 A allele (OR = 0.80, P = 0.002, Pcor = 0.006) with genotype distribution suggesting a co-dominant effect. Given the link between alcoholism and suicide under influence of alcohol reported in Polish population, confounding by alcohol addiction was unlikely due to apparently similar effect size among cases who were under influence of ethanol at the time of death (OR = 0.76, P = 0.003, N = 361) and those who were not (OR = 0.80, P = 0.007, N = 469). The search for genotype-phenotype correlations did not show significant results. In conclusion, our study proves that there is an inverse association between rs9939609 polymorphism in FTO gene and completed suicide which is independent from association between FTO and alcohol addiction.

  6. Interactive effects of ultraviolet-B radiation and pesticide exposure on DNA photo-adduct accumulation and expression of DNA damage and repair genes in Xenopus laevis embryos.

    PubMed

    Yu, Shuangying; Tang, Song; Mayer, Gregory D; Cobb, George P; Maul, Jonathan D

    2015-02-01

    Pesticide use and ultraviolet-B (UVB) radiation have both been suggested to adversely affect amphibians; however, little is known about their interactive effects. One potential adverse interaction could involve pesticide-induced dysregulation of DNA repair pathways, resulting in greater numbers of DNA photo-adducts from UVB exposure. In the present study, we investigated the interactive effects of UVB radiation and two common pesticides (endosulfan and α-cypermethrin) on induction of DNA photo-adducts and expression of DNA damage and repair related genes in African clawed frog (Xenopus laevis) embryos. We examined 13 genes that are, collectively, involved in stress defense, cell cycle arrest, nucleotide excision repair (NER), base excision repair, mismatch repair, DNA repair regulation, and apoptosis. We exposed X. laevis embryos to 0, 25, and 50 μg/L endosulfan or 0, 2.5, and 5.0 μg/L α-cypermethrin for 96 h, with environmentally relevant exposures of UVB radiation during the last 7 h of the 96 h exposure. We measured the amount of cyclobutane pyrimidine dimers (CPDs) and mRNA abundance of the 13 genes among treatments including control, pesticide only, UVB only, and UVB and pesticide co-exposures. Each of the co-exposure scenarios resulted in elevated CPD levels compared to UVB exposure alone, suggesting an inhibitory effect of endosulfan and α-cypermethrin on CPD repair. This is attributed to results indicating that α-cypermethrin and endosulfan reduced mRNA abundance of XPA and HR23B, respectively, to levels that may affect the initial recognition of DNA lesions. In contrast, both pesticides increased transcript abundance of CSA and MUTL. In addition, mRNA abundance of HSP70 and GADD45α were increased by endosulfan and mRNA abundance of XPG was increased by α-cypermethrin. XPC, HR23B, XPG, and GADD45α exhibited elevated mRNA concentrations whereas there was a reduction in MUTL transcript concentrations in UVB-alone treatments. It appeared that even

  7. DNA repair gene polymorphisms and micronucleus frequencies in Chinese workers exposed to vinyl chloride monomer.

    PubMed

    Qiu, Yu-lan; Wang, Wei; Wang, Tong; Sun, Pin; Wu, Fen; Zhu, Shou-min; Qian, Ji; Jin, Li; Au, William; Xia, Zhao-lin

    2011-06-01

    Vinyl chloride monomer (VCM) is genotoxic and cancerogen agent. Individual variations in response to the exposure have been noticed and the variations may be due to genetic differences in the removal of VCM-DNA adducts, such as polymorphism in genes NER pathway and BER pathway. This study explores the relationship between genetic polymorphism of seven genes within the NER pathway (XPA, XPD, XPC, XPG, XPF, and ERCC1) and BER pathway (APE1), and susceptibility to chromosomal damage after exposure to VCM. One hundred and eighty workers occupationally exposed to VCM and 43 unexposed controls were investigated. Chromosome damage in peripheral lymphocytes was measured by the cytokinesis-blocked micronucleus assay. PCR-RFLP technique was applied to detect polymorphisms of the seven genes. The influence of genotype, age, gender, cumulative exposure dose, alcohol consumption, and smoking status on the frequencies of MN was determined using univariate and multiple Poisson regression analyses. We found XPA A23G variant workers had significantly higher MN frequencies than those from the wild-type homozygous counterparts (P = 0.01). Those with the variant XPD Lys751Gln genotype had marginally higher MN frequencies (P = 0.07). On the other hand, XPC PAT and XPF 5'-UTR T2063A variant workers had significantly lower MN frequencies compared with those from their wild-type homozygous counterparts (P = 0.04 and P = 0.004, respectively). Our findings suggest that XPC PAT, XPD Lys751Gln, XPA A23G and XPF 5'-UTR T2063A contribute to the level of chromosome damage in occupational exposure to VCM in Chinese population.

  8. Ultrasound-induced hyperthermia for the spatio-temporal control of gene expression in bone repair

    NASA Astrophysics Data System (ADS)

    Wilson, Christopher; Padilla, Frédéric; Zhang, Man; Vilaboa, Nuria; Kripfgans, Oliver; Fowlkes, Brian; Franceschi, Renny

    2012-10-01

    Spatial and temporal control over the expression of growth/differentiation factors is of great interest for regeneration of bone, but technologies capable of providing tight and active control over gene expression remain elusive. We propose the use of focused ultrasound for the targeted activation of heat shock-sensitive expression systems in engineered bone. We report in vitro results with cells that express firefly luciferase (fLuc) under the control of a heat shock protein promoter. Cells were embedded in fibrin scaffolds and exposed to focused ultrasound, using a custom 3.3MHz transducer (focal length 4", f-number 1.33", focal dimension 1.2mm lateral FWHM) in CW mode for 2-20 minutes at intensities ISPTA=120-440 W/cm2. The kinetics of ultrasound-mediated activation of the cells was compared with that of strictly thermal activation. Bioluminescence imaging revealed fLuc expression in an area ≥2.5mm in diameter at the position of the ultrasound focus, and the diameter and intensity of the signal increased with the amplitude of the acoustic energy. We also found that ultrasound activated fLuc expression with substantially shorter exposures than thermal activation. Our results demonstrate the potential for focused ultrasound to selectively activate the expression of a gene of interest in an engineered tissue and suggest that focused ultrasound activates the heat shock pathway by a combination of thermal and non-thermal mechanisms.

  9. Distinct expression profiles of stress defense and DNA repair genes in Daphnia pulex exposed to cadmium, zinc, and quantum dots.

    PubMed

    Tang, Song; Wu, Yonggan; Ryan, Caitlin N; Yu, Shuangying; Qin, Guangqiu; Edwards, Donn S; Mayer, Gregory D

    2015-02-01

    The ever-increasing production and use of nanocrystaline semiconductors (Quantum dots; QDs) will inevitably result in increased appearance of these nanomaterials in the aquatic environment. However, the behavior and potential toxicity of heavy metal constituted nanoparticulates in aquatic invertebrates is largely unknown, especially with regard to molecular responses. The freshwater crustacean Daphnia pulex is a well-suited toxicological and ecological model to study molecular responses to environmental stressors. In this study, D. pulex were exposed for 48 h to sublethal doses of QDs (25% and 50% of LC50) with differing spectral properties (CdTe and CdSe/ZnS QDs) and Cd and Zn salts. Our data suggest that acute exposure to both CdSO4 and Cd-based QDs leads to Cd uptake in vivo, which was biologically supported by the observation of increased expression of metallothionein (MT-1). Furthermore, Cd, Zn, and CdSe/ZnS QDs induced different patterns of gene expression regarding stress defense and DNA repair, which furthers our knowledge regarding which response pathways are affected by nanoparticulate forms of metals versus ionic forms in aquatic crustaceans. PMID:25014899

  10. Polymorphisms in the DNA repair gene XPD: correlations with risk and age at onset of basal cell carcinoma.

    PubMed

    Dybdahl, M; Vogel, U; Frentz, G; Wallin, H; Nexø, B A

    1999-01-01

    The XPD protein has a dual function, both in nucleotide excision repair and in basal transcription. We have studied the role of two nucleotide substitutions in the XPD gene, one in exon 23 leading to an amino acid substitution (Lys751Gln) and one silent in exon 6 in relation to basal cell carcinoma (BCC). Both are two-allele polymorphisms, with the nucleobases A and C at the given positions. We genotyped psoriasis patients with and without BCC and nonpsoriatic persons with and without BCC (4 x 20 persons). The choice to study psoriasis patients was motivated by their high genotoxic exposure via treatment and their high relative rate of early BCC. Subjects carrying two A alleles (AA genotype) in exon 23 were at 4.3-fold higher risk of BCC than subjects with two C alleles (95% CI, 0.79-23.57). In addition, the mean age at first skin tumor for BCC cases with the AA genotype was significantly lower than the mean age for BCC cases with the AC or CC genotype (P = 0.012). Thus, the variant C-allele of exon 23 may be protective. The exon 6 genotype was associated with the risk of BCC among the psoriasis patients; psoriatics carrying two A alleles in exon 6 were at 5.3-fold higher risk of BCC than psoriatics with two C alleles (95% CI, 0.78-36.31). For the psoriatics, the mean age at onset of BCC for cases with the AA genotype was marginally lower than the mean age for cases with genotype AC or CC (P = 0.060). Our results raise the possibility that the polymorphisms in the XPD gene may be contributing factors in the risk of BCC development. They are, therefore, important candidates for future studies in susceptibility to cancer.

  11. Nucleotide excision repair genes and risk of lung cancer among San Francisco bay area Latinos and African Americans

    PubMed Central

    Chang, Jeffrey S.; Wrensch, Margaret R.; Hansen, Helen M.; Sison, Jennette D.; Aldrich, Melinda C.; Quesenberry, Charles P.; Seldin, Michael F.; Kelsey, Karl T.; Kittles, Rick A.; Silva, Gabriel; Wiencke, John K.

    2009-01-01

    Few studies on the association between nucleotide excision repair (NER) variants and lung cancer risk have included Latinos and African Americans. We examine variants in six NER genes (ERCC2, ERCC4, ERCC5, LIG1, RAD23B and XPC) in association with primary lung cancer risk among 113 Latino and 255 African American subjects newly diagnosed with primary lung cancer from 1998 to 2003 in the San Francisco Bay Area, and 579 healthy controls (299 Latinos and 280 African Americans). Individual single nucleotide polymorphism and haplotype analyses, multifactor dimensionality reduction, and principal components analysis were performed to assess the association between six genes in the NER pathway and lung cancer risk. Among Latinos, ERCC2 haplotype CGA (rs238406, rs11878644, rs6966) was associated with reduced lung cancer risk [odds ratio (OR) of 0.65 and 95% confidence interval (CI): 0.44-0.97], especially among non-smokers (OR=0.29; 95% CI: 0.12-0.67). From multifactor dimensionality reduction analysis, in Latinos, smoking and three SNPs (ERCC2 rs171140, ERCC5 rs17655, and LIG1 rs20581) together had a prediction accuracy of 67.4% (p=0.001) for lung cancer. Among African Americans, His/His genotype of ERCC5 His1104Asp (rs17655) was associated with increased lung cancer risk (OR=1.78; 95% CI: 1.09-2.91), and LIG1 haplotype GGGAA (rs20581, rs156641, rs3730931, rs20579, and rs439132) was associated with reduced lung cancer risk (OR=0.61; 95% CI: 0.42-0.88). Our study suggests different elements of the NER pathway may be important in the different ethnic groups resulting either from different linkage relationship, genetic backgrounds, and/or exposure histories. PMID:18709642

  12. SGLT2 Inhibitors May Predispose to Ketoacidosis

    PubMed Central

    Blau, Jenny E.; Rother, Kristina I.

    2015-01-01

    Context: Sodium glucose cotransporter 2 (SGLT2) inhibitors are antidiabetic drugs that increase urinary excretion of glucose, thereby improving glycemic control and promoting weight loss. Since approval of the first-in-class drug in 2013, data have emerged suggesting that these drugs increase the risk of diabetic ketoacidosis. In May 2015, the Food and Drug Administration issued a warning that SGLT2 inhibitors may lead to ketoacidosis. Evidence Acquisition: Using PubMed and Google, we conducted Boolean searches including terms related to ketone bodies or ketoacidosis with terms for SGLT2 inhibitors or phlorizin. Priority was assigned to publications that shed light on molecular mechanisms whereby SGLT2 inhibitors could affect ketone body metabolism. Evidence Synthesis: SGLT2 inhibitors trigger multiple mechanisms that could predispose to diabetic ketoacidosis. When SGLT2 inhibitors are combined with insulin, it is often necessary to decrease the insulin dose to avoid hypoglycemia. The lower dose of insulin may be insufficient to suppress lipolysis and ketogenesis. Furthermore, SGLT2 is expressed in pancreatic α-cells, and SGLT2 inhibitors promote glucagon secretion. Finally, phlorizin, a nonselective inhibitor of SGLT family transporters decreases urinary excretion of ketone bodies. A decrease in the renal clearance of ketone bodies could also increase the plasma ketone body levels. Conclusions: Based on the physiology of SGLT2 and the pharmacology of SGLT2 inhibitors, there are several biologically plausible mechanisms whereby this class of drugs has the potential to increase the risk of developing diabetic ketoacidosis. Future research should be directed toward identifying which patients are at greatest risk for this side effect and also to optimizing pharmacotherapy to minimize the risk to patients. PMID:26086329

  13. Use of tissue engineering strategies to repair joint tissues in osteoarthritis: viral gene transfer approaches.

    PubMed

    Cucchiarini, Magali; Madry, Henning

    2014-10-01

    Osteoarthritis (OA) is a major chronic disease of the joints, affecting mostly the articular cartilage but also all the surrounding tissues including the subchondral bone, synovium, meniscus, tendons, and ligaments. Despite the availability in the clinic of a variety of therapeutic approaches, there is crucial need for improved treatment to protect and regenerate the cartilage with full integrity and function. In this regard, combining gene, cell, and tissue engineering-based procedures is an attractive concept for novel, effective therapy against AO, a slow, progressive, and irreversible disease. Here, we provide an overview of the treatment available for management of the progression of the OA phenotype and discuss current progress and remaining challenges for potential future treatment of patients.

  14. Inhibition of DNA-repair genes Ercc1 and Mgmt enhances temozolomide efficacy in gliomas treatment: a pre-clinical study.

    PubMed

    Boccard, Sandra G; Marand, Sandie V; Geraci, Sandra; Pycroft, Laurie; Berger, François R; Pelletier, Laurent A

    2015-10-01

    Gliomas are the most common primary brain tumors. To date, therapies do not allow curing patients, and glioblastomas (GBMs) are associated with remarkably poor prognosis. This situation is at least partly due to intrinsic or acquired resistance to treatment, especially to chemotherapy. In 2005, temozolomide (TMZ) has become the first chemotherapeutic drug validated for GBM. Nevertheless TMZ efficacy depends on Mgmt status. While the methylation of Mgmt promoter was considered so far as a prognostic marker, its targeting is becoming an effective therapeutic opportunity. Thus, arrival of both TMZ and Mgmt illustrated that considerable progress can still be realized by optimizing adjuvant chemotherapy. A part of this progress could be accomplished in the future by overcoming residual resistance. The aim of the present study was to investigate the involvement of a set of other DNA-repair genes in glioma resistance to temozolomide. We focused on DNA-repair genes located in the commonly deleted chromosomal region in oligodendroglioma (1p/19q) highly correlated with patient response to chemotherapy. We measured effects of inhibition of ten DNA-repair genes expression using siRNAs on astrocytoma cell response to cisplatin (CDDP) and TMZ. SiRNAs targeting ercc1, ercc2, mutyh, and pnkp significantly sensitized cells to chemotherapy, increasing cell death by up to 25%. In vivo we observed a decrease of subcutaneous glioma tumor growth after injection of siRNA in conjunction with absorption of TMZ. We demonstrated in this pre-clinical study that targeting of DNA-repair genes such as Ercc1 could be used as an adjuvant chemosensitization treatment, similarly to Mgmt inhibition. PMID:26336131

  15. Inhibition of DNA-repair genes Ercc1 and Mgmt enhances temozolomide efficacy in gliomas treatment: a pre-clinical study

    PubMed Central

    Boccard, Sandra G.; Marand, Sandie V.; Geraci, Sandra; Pycroft, Laurie; Berger, François R.; Pelletier, Laurent A.

    2015-01-01

    Gliomas are the most common primary brain tumors. To date, therapies do not allow curing patients, and glioblastomas (GBMs) are associated with remarkably poor prognosis. This situation is at least partly due to intrinsic or acquired resistance to treatment, especially to chemotherapy. In 2005, temozolomide (TMZ) has become the first chemotherapeutic drug validated for GBM. Nevertheless TMZ efficacy depends on Mgmt status. While the methylation of Mgmt promoter was considered so far as a prognostic marker, its targeting is becoming an effective therapeutic opportunity. Thus, arrival of both TMZ and Mgmt illustrated that considerable progress can still be realized by optimizing adjuvant chemotherapy. A part of this progress could be accomplished in the future by overcoming residual resistance. The aim of the present study was to investigate the involvement of a set of other DNA-repair genes in glioma resistance to temozolomide. We focused on DNA-repair genes located in the commonly deleted chromosomal region in oligodendroglioma (1p/19q) highly correlated with patient response to chemotherapy. We measured effects of inhibition of ten DNA-repair genes expression using siRNAs on astrocytoma cell response to cisplatin (CDDP) and TMZ. SiRNAs targeting ercc1, ercc2, mutyh, and pnkp significantly sensitized cells to chemotherapy, increasing cell death by up to 25%. In vivo we observed a decrease of subcutaneous glioma tumor growth after injection of siRNA in conjunction with absorption of TMZ. We demonstrated in this pre-clinical study that targeting of DNA-repair genes such as Ercc1 could be used as an adjuvant chemosensitization treatment, similarly to Mgmt inhibition. PMID:26336131

  16. Association of nucleotide excision repair pathway gene polymorphisms with gastric cancer and atrophic gastritis risks.

    PubMed

    Liu, Jingwei; Sun, Liping; Xu, Qian; Tu, Huakang; He, Caiyun; Xing, Chengzhong; Yuan, Yuan

    2016-02-01

    Polymorphisms of NER genes could change NER ability, thereby altering individual susceptibility to GC. We systematically analyzed 39 SNPs of 8 key genes of NER pathway in 2686 subjects including 898 gastric cancer (GC), 851 atrophic gastritis (AG) and 937 controls (CON) in northern Chinese. SNP genotyping were performed using Sequenom MassARRAY platform. The results demonstrated that DDB2 rs830083 GG genotype was significantly associated with increased GC risk compared with wild-type CC (OR=2.32, P= 6.62 × 10-9); XPC rs2607775 CG genotype conferred a 1.73 increased odds of GC risk than non-cancer subjects compared with wild-type CC (OR=1.73, P= 3.04 × 10-4). The combined detection of these two polymorphisms demonstrated even higher GC risk (OR=3.05). Haplotype analysis suggested that DDB2 rs2029298-rs326222-rs3781619-rs830083 GTAG haplotype was significantly associated with disease risk in each step of CON→AG→GC development (AG vs. CON: OR=2.88, P= 7.51 × 10-7; GC vs. AG: OR=2.90, P=5.68 × 10-15; GC vs. CON: OR=8.42, P=2.22 × 10-15); DDB2 GTAC haplotype was associated with reduced risk of GC compared with CON (OR=0.63, P= 8.31 × 10-12). XPC rs1870134-rs2228000-rs2228001-rs2470352-rs2607775 GCAAG haplotype conferred increased risk of GC compared with AG (OR=1.88, P= 6.98 × 10-4). XPA rs2808668 and drinking, DDB2 rs326222, rs3781619, rs830083 and smoking demonstrated significant interactions in AG; XPC rs2607775 had significant interaction with smoking in GC. In conclusion, NER pathway polymorphisms especially in "damage incision" step were significantly associated with GC risk and had interactions with environment factors. The detection of NER pathway polymorphisms such as DDB2 and XPC might be applied in the prediction of GC risk and personalized prevention in the future. NER pathway polymorphisms especially in "damage incision" step were significantly associated with GC risk and had interactions with environment factors, which might be applied in the

  17. Normal repair of ultraviolet radiation-induced DNA damage in familial melanoma without CDKN2A or CDK4 gene mutation.

    PubMed

    Shannon, J A; Matias, C; Luxford, C; Kefford, R F; Mann, G J

    1999-04-01

    Excessive sun exposure and family history are strong risk factors for the development of cutaneous melanoma. Inherited susceptibility to this type of skin cancer could therefore result from constitutively impaired capacity to repair ultraviolet (UV)-induced DNA lesions. While a proportion of familial melanoma kindreds exhibit germline mutations in the cell cycle regulatory gene CDKN2A (p16INK4a) or its protein target, cyclin-dependent kinase 4 (CDK4), the biochemical basis of most familial melanoma is unknown. We have examined lymphoblastoid cell lines from melanoma-affected and unaffected individuals from large hereditary melanoma kindreds which are not attributable to CDKN2A or CDK4 gene mutation. These lines were tested for sensitivity of clonogenic growth to UV radiation and for their ability to repair transfected UV-damaged plasmid templates (host cell reactivation). Two of seven affected-unaffected pairs differed in colony survival after exposure to UVB radiation; however, no significant differences were observed in the host-cell reactivation assays. These results indicate that melanoma susceptibility genes other than CDKN2A and CDK4 do not impair net capacity to repair UV-induced DNA damage.

  18. The function of the Periaxin gene during nerve repair in a model of CMT4F.

    PubMed

    Williams, Anna C; Brophy, Peter J

    2002-04-01

    Mutations in the Periaxin (PRX) gene are known to cause autosomal recessive demyelinating Charcot-Marie-Tooth (CMT4F) and Dejerine-Sottas disease. The pathogenesis of these diseases is not fully understood. However, progress is being made by studying both the periaxin-null mouse, a mouse model of the disease, and the protein-protein interactions of periaxin. L-periaxin is a constituent of the dystroglycan-dystrophin-related protein-2 complex linking the Schwann cell cytoskeleton to the extracellular matrix. Although periaxin-null mice myelinate normally, they develop a demyelinating peripheral neuropathy later in life. This suggests that periaxin is required for the stable maintenance of a normal myelin sheath. We carried out sciatic nerve crushes in 6-week-old periaxin-null mice, and, 6 weeks later, found that although the number of myelinated axons had returned to normal, the axon diameters remained smaller than in the contralateral uncrushed nerve. Not only do periaxin-null mice have more hyper-myelinated axons than their wild-type counterparts but they also recapitulate this hypermyelination during regeneration. Therefore, periaxin-null mice can undergo peripheral nerve remyelination, but the regulation of peripheral myelin thickness is disrupted. PMID:12090399

  19. Polymorphisms in metabolism and repair genes affects DNA damage caused by open-cast coal mining exposure.

    PubMed

    Espitia-Pérez, Lyda; Sosa, Milton Quintana; Salcedo-Arteaga, Shirley; León-Mejía, Grethel; Hoyos-Giraldo, Luz Stella; Brango, Hugo; Kvitko, Katia; da Silva, Juliana; Henriques, João A P

    2016-09-15

    Increasing evidence suggest that occupational exposure to open-cast coal mining residues like dust particles, heavy metals and Polycyclic Aromatic Hydrocarbons (PAHs) may cause a wide range of DNA damage and genomic instability that could be associated to initial steps in cancer development and other work-related diseases. The aim of our study was to evaluate if key polymorphisms in metabolism genes CYP1A1Msp1, GSTM1Null, GSTT1Null and DNA repair genes XRCC1Arg194Trp and hOGG1Ser326Cys could modify individual susceptibility to adverse coal exposure effects, considering the DNA damage (Comet assay) and micronucleus formation in lymphocytes (CBMN) and buccal mucosa cells (BMNCyt) as endpoints for genotoxicity. The study population is comprised of 200 healthy male subjects, 100 open-cast coal-mining workers from "El Cerrejón" (world's largest open-cast coal mine located in Guajira - Colombia) and 100 non-exposed referents from general population. The data revealed a significant increase of CBMN frequency in peripheral lymphocytes of occupationally exposed workers carrying the wild-type variant of GSTT1 (+) gene. Exposed subjects carrying GSTT1null polymorphism showed a lower micronucleus frequency compared with their positive counterparts (FR: 0.83; P=0.04), while BMNCyt, frequency and Comet assay parameters in lymphocytes: Damage Index (DI) and percentage of DNA in the tail (Tail % DNA) were significantly higher in exposed workers with the GSTM1Null polymorphism. Other exfoliated buccal mucosa abnormalities related to cell death (Karyorrhexis and Karyolysis) were increased in GSTT/M1Null carriers. Nuclear buds were significantly higher in workers carrying the CYP1A1Msp1 (m1/m2, m2/m2) allele. Moreover, BMNCyt frequency and Comet assay parameters were significantly lower in exposed carriers of XRCC1Arg194Trp (Arg/Trp, Trp/Trp) and hOGG1Ser326Cys (Ser/Cys, Cys/Cys), thereby providing new data to the increasing evidence about the protective role of these polymorphisms

  20. Polymorphisms in metabolism and repair genes affects DNA damage caused by open-cast coal mining exposure.

    PubMed

    Espitia-Pérez, Lyda; Sosa, Milton Quintana; Salcedo-Arteaga, Shirley; León-Mejía, Grethel; Hoyos-Giraldo, Luz Stella; Brango, Hugo; Kvitko, Katia; da Silva, Juliana; Henriques, João A P

    2016-09-15

    Increasing evidence suggest that occupational exposure to open-cast coal mining residues like dust particles, heavy metals and Polycyclic Aromatic Hydrocarbons (PAHs) may cause a wide range of DNA damage and genomic instability that could be associated to initial steps in cancer development and other work-related diseases. The aim of our study was to evaluate if key polymorphisms in metabolism genes CYP1A1Msp1, GSTM1Null, GSTT1Null and DNA repair genes XRCC1Arg194Trp and hOGG1Ser326Cys could modify individual susceptibility to adverse coal exposure effects, considering the DNA damage (Comet assay) and micronucleus formation in lymphocytes (CBMN) and buccal mucosa cells (BMNCyt) as endpoints for genotoxicity. The study population is comprised of 200 healthy male subjects, 100 open-cast coal-mining workers from "El Cerrejón" (world's largest open-cast coal mine located in Guajira - Colombia) and 100 non-exposed referents from general population. The data revealed a significant increase of CBMN frequency in peripheral lymphocytes of occupationally exposed workers carrying the wild-type variant of GSTT1 (+) gene. Exposed subjects carrying GSTT1null polymorphism showed a lower micronucleus frequency compared with their positive counterparts (FR: 0.83; P=0.04), while BMNCyt, frequency and Comet assay parameters in lymphocytes: Damage Index (DI) and percentage of DNA in the tail (Tail % DNA) were significantly higher in exposed workers with the GSTM1Null polymorphism. Other exfoliated buccal mucosa abnormalities related to cell death (Karyorrhexis and Karyolysis) were increased in GSTT/M1Null carriers. Nuclear buds were significantly higher in workers carrying the CYP1A1Msp1 (m1/m2, m2/m2) allele. Moreover, BMNCyt frequency and Comet assay parameters were significantly lower in exposed carriers of XRCC1Arg194Trp (Arg/Trp, Trp/Trp) and hOGG1Ser326Cys (Ser/Cys, Cys/Cys), thereby providing new data to the increasing evidence about the protective role of these polymorphisms

  1. Single-nucleotide polymorphisms in base excision repair, nucleotide excision repair, and double strand break genes as markers for response to radiotherapy in patients with Stage I to II head-and-neck cancer

    SciTech Connect

    Carles, Joan . E-mail: jcarles@imas.imim.es; Monzo, Mariano; Amat, Marta; Jansa, Sonia; Artells, Rosa; Navarro, Alfons; Foro, Palmira; Alameda, Francesc; Gayete, Angel; Gel, Bernat; Miguel, Maribel; Albanell, Joan; Fabregat, Xavier

    2006-11-15

    Purpose: Polymorphisms in DNA repair genes can influence response to radiotherapy. We analyzed single-nucleotide polymorphisms (SNP) in nine DNA repair genes in 108 patients with head-and-neck cancer (HNSCC) who had received radiotherapy only. Methods and Materials: From May 1993 to December 2004, patients with Stage I and II histopathologically confirmed HNSCC underwent radiotherapy. DNA was obtained from paraffin-embedded tissue, and SNP analysis was performed using a real-time polymerase chain reaction allelic discrimination TaqMan assay with minor modifications. Results: Patients were 101 men (93.5%) and 7 (6.5%) women, with a median age of 64 years (range, 40 to 89 years). Of the patients, 76 (70.4%) patients were Stage I and 32 (29.6%) were Stage II. The XPF/ERCC1 SNP at codon 259 and XPG/ERCC5 at codon 46 emerged as significant predictors of progression (p 0.00005 and 0.049, respectively) and survival (p = 0.0089 and 0.0066, respectively). Similarly, when variant alleles of XPF/ERCC1, XPG/ERCC5 and XPA were examined in combination, a greater number of variant alleles was associated with shorter time to progression (p = 0.0003) and survival (p 0.0002). Conclusions: Genetic polymorphisms in XPF/ERCC1, XPG/ERCC5, and XPA may significantly influence response to radiotherapy; large studies are warranted to confirm their role in HNSCC.

  2. Influence of DNA repair gene polymorphisms of hOGG1, XRCC1, XRCC3, ERCC2 and the folate metabolism gene MTHFR on chromosomal aberration frequencies.

    PubMed

    Skjelbred, Camilla Furu; Svendsen, Marit; Haugan, Vera; Eek, Anette Kildal; Clausen, Kjell Oskar; Svendsen, Martin Veel; Hansteen, Inger-Lise

    2006-12-01

    We have studied the effect of genetic polymorphisms in the DNA repair genes hOGG1, XRCC1, XRCC3, ERCC2 and the MTHFR gene in the folate metabolism on the frequencies of cells with chromosomal aberrations (CA), chromosome-type aberrations (CSA), chromatid-type aberrations (CTA), chromatid breaks (CTB) and chromatid gaps (CTG) scored in peripheral blood lymphocytes from 651 Norwegian subjects of Caucasian descendant. DNA was extracted from fixed cell suspensions. The log-linear Poisson regression model was used for the combined data which included age, smoking, occupational exposure and genotype for 449 subjects. Our results suggest that individuals carrying the hOGG1 326Cys or the XRCC1 399Gln allele have an increased risk of chromosomal damage, while individuals carrying the XRCC1 194Trp or the ERCC2 751Gln allele have a reduced risk regardless of smoking habits and age. Individuals carrying the XRCC1 280His allele had an increased risk of CSA which was only apparent in non-smokers. This was independent of age. A protective effect of the XRCC3 241Met allele was only found in the older age group in non-smokers for CA, CSA and CTA, and in smokers for CSA. In the youngest age group, the opposite effect was found, with an increased risk for CA, CTA and CTG in smokers. Carrying the MTHFR 222Val allele gave an increased risk for chromosome and chromatid-type aberrations for both non-smokers and smokers, especially for individuals in the older age group, and with variable results in the youngest age group. The variables included in the different regression models accounted, however, for only 4-10% of the variation. The frequency ratio for CTG was significantly higher than for CTA and CTB for only 7 of the 43 comparisons performed. Some of the gap frequencies diverge from the trend in the CA, CSA, CTA and CTB results.

  3. Post-transcriptional Repair of a Split Heat Shock Protein 90 Gene by mRNA trans-Splicing*♦

    PubMed Central

    Nageshan, Rishi Kumar; Roy, Nainita; Hehl, Adrian B.; Tatu, Utpal

    2011-01-01

    Heat shock protein 90 participates in diverse biological processes ranging from protein folding, cell cycle, signal transduction and development to evolution in all eukaryotes. It is also critically involved in regulating growth of protozoa such as Dictyostelium discoideum, Leishmania donovani, Plasmodium falciparum, Trypanosoma cruzi, and Trypanosoma evansi. Selective inhibition of Hsp90 has also been explored as an intervention strategy against important human diseases such as cancer, malaria, or trypanosomiasis. Giardia lamblia, a simple protozoan parasite of humans and animals, is an important cause of diarrheal disease with significant morbidity and some mortality in tropical countries. Here we show that the G. lamblia cytosolic hsp90 (glhsp90) is split in two similar sized fragments located 777 kb apart on the same scaffold. Intrigued by this unique arrangement, which appears to be specific for the Giardiinae, we have investigated the biosynthesis of GlHsp90. We used genome sequencing to confirm the split nature of the giardial hsp90. However, a specific antibody raised against the peptide detected a product with a mass of about 80 kDa, suggesting a post-transcriptional rescue of the genomic defect. We show evidence for the joining of the two independent Hsp90 transcripts in-trans to one long mature mRNA presumably by RNA splicing. The splicing junction carries hallmarks of classical cis-spliced introns, suggesting that the regular cis-splicing machinery may be sufficient for repair of the open reading frame. A complementary 26-nt sequence in the “intron” regions adjacent to the splice sites may assist in positioning the two pre-mRNAs for processing. This is the first example of post-transcriptional rescue of a split gene by trans-splicing. PMID:21209094

  4. Post-transcriptional repair of a split heat shock protein 90 gene by mRNA trans-splicing.

    PubMed

    Nageshan, Rishi Kumar; Roy, Nainita; Hehl, Adrian B; Tatu, Utpal

    2011-03-01

    Heat shock protein 90 participates in diverse biological processes ranging from protein folding, cell cycle, signal transduction and development to evolution in all eukaryotes. It is also critically involved in regulating growth of protozoa such as Dictyostelium discoideum, Leishmania donovani, Plasmodium falciparum, Trypanosoma cruzi, and Trypanosoma evansi. Selective inhibition of Hsp90 has also been explored as an intervention strategy against important human diseases such as cancer, malaria, or trypanosomiasis. Giardia lamblia, a simple protozoan parasite of humans and animals, is an important cause of diarrheal disease with significant morbidity and some mortality in tropical countries. Here we show that the G. lamblia cytosolic hsp90 (glhsp90) is split in two similar sized fragments located 777 kb apart on the same scaffold. Intrigued by this unique arrangement, which appears to be specific for the Giardiinae, we have investigated the biosynthesis of GlHsp90. We used genome sequencing to confirm the split nature of the giardial hsp90. However, a specific antibody raised against the peptide detected a product with a mass of about 80 kDa, suggesting a post-transcriptional rescue of the genomic defect. We show evidence for the joining of the two independent Hsp90 transcripts in-trans to one long mature mRNA presumably by RNA splicing. The splicing junction carries hallmarks of classical cis-spliced introns, suggesting that the regular cis-splicing machinery may be sufficient for repair of the open reading frame. A complementary 26-nt sequence in the "intron" regions adjacent to the splice sites may assist in positioning the two pre-mRNAs for processing. This is the first example of post-transcriptional rescue of a split gene by trans-splicing. PMID:21209094

  5. DNA Repair Genes ERCC1 and BRCA1 Expression in Non-Small Cell Lung Cancer Chemotherapy Drug Resistance.

    PubMed

    Wang, Shuai; Liu, Feng; Zhu, Jingyan; Chen, Peng; Liu, Hongxing; Liu, Qi; Han, Junqing

    2016-06-12

    BACKGROUND Surgery combined with chemotherapy is an important therapy for non-small cell lung cancer (NSCLC). However, chemotherapy drug resistance seriously hinders the curative effect. Studies show that DNA repair genes ERCC1 and BRCA1 are associated with NSCLC chemotherapy, but their expression and mechanism in NSCLC chemotherapy drug-resistant cells has not been elucidated. MATERIAL AND METHODS NSCLC cell line A549 and drug resistance cell line A549/DDP were cultured. Real-time PCR and Western blot analyses were used to detect ERCC1 and BRCA1 mRNA expression. A549/DDP cells were randomly divided into 3 groups: the control group; the siRNA-negative control group (scramble group); and the siRNA ERCC1 and BRCA1siRNA transfection group. Real-time PCR and Western blot analyses were used to determine ERCC1 and BRCA1 mRNA and protein expression. MTT was used to detect cell proliferation activity. Caspase 3 activity was tested by use of a kit. Western blot analysis was performed to detect PI3K, AKT, phosphorylated PI3K, and phosphorylated AKT protein expression. RESULTS ERCC1 and BRCA1 were overexpressed in A549/DDP compared with A549 (P<0.05). ERCC1 and BRCA1siRNA transfection can significantly reduce ERCC1 and BRCA1 mRNA and protein expression (P<0.05). Downregulating ERCC1 and BRCA1 expression obviously inhibited cell proliferation and increased caspase 3 activity (P<0.05). Downregulating ERCC1 and BRCA1 significantly decreased PI3K and AKT phosphorylation levels (P<0.05). CONCLUSIONS ERCC1 and BRCA1 were overexpressed in NSCLC drug-resistant cells, and they regulated lung cancer occurrence and development through the phosphorylating PI3K/AKT signaling pathway.

  6. DNA Repair Genes ERCC1 and BRCA1 Expression in Non-Small Cell Lung Cancer Chemotherapy Drug Resistance

    PubMed Central

    Wang, Shuai; Liu, Feng; Zhu, Jingyan; Chen, Peng; Liu, Hongxing; Liu, Qi; Han, Junqing

    2016-01-01

    Background Surgery combined with chemotherapy is an important therapy for non-small cell lung cancer (NSCLC). However, chemotherapy drug resistance seriously hinders the curative effect. Studies show that DNA repair genes ERCC1 and BRCA1 are associated with NSCLC chemotherapy, but their expression and mechanism in NSCLC chemotherapy drug-resistant cells has not been elucidated. Material/Methods NSCLC cell line A549 and drug resistance cell line A549/DDP were cultured. Real-time PCR and Western blot analyses were used to detect ERCC1 and BRCA1 mRNA expression. A549/DDP cells were randomly divided into 3 groups: the control group; the siRNA-negative control group (scramble group); and the siRNA ERCC1 and BRCA1siRNA transfection group. Real-time PCR and Western blot analyses were used to determine ERCC1 and BRCA1 mRNA and protein expression. MTT was used to detect cell proliferation activity. Caspase 3 activity was tested by use of a kit. Western blot analysis was performed to detect PI3K, AKT, phosphorylated PI3K, and phosphorylated AKT protein expression. Results ERCC1 and BRCA1 were overexpressed in A549/DDP compared with A549 (P<0.05). ERCC1 and BRCA1siRNA transfection can significantly reduce ERCC1 and BRCA1 mRNA and protein expression (P<0.05). Downregulating ERCC1 and BRCA1 expression obviously inhibited cell proliferation and increased caspase 3 activity (P<0.05). Downregulating ERCC1 and BRCA1 significantly decreased PI3K and AKT phosphorylation levels (P<0.05). Conclusions ERCC1 and BRCA1 were overexpressed in NSCLC drug-resistant cells, and they regulated lung cancer occurrence and development through the phosphorylating PI3K/AKT signaling pathway. PMID:27289442

  7. Evaluating for suspected child abuse: conditions that predispose to bleeding.

    PubMed

    Carpenter, Shannon L; Abshire, Thomas C; Anderst, James D

    2013-04-01

    Child abuse might be suspected when children present with cutaneous bruising, intracranial hemorrhage, or other manifestations of bleeding. In these cases, it is necessary to consider medical conditions that predispose to easy bleeding/bruising. When evaluating for the possibility of bleeding disorders and other conditions that predispose to hemorrhage, the pediatrician must consider the child's presenting history, medical history, and physical examination findings before initiating a laboratory investigation. Many medical conditions can predispose to easy bleeding. Before ordering laboratory tests for a disease, it is useful to understand the biochemical basis and clinical presentation of the disorder, condition prevalence, and test characteristics. This technical report reviews the major medical conditions that predispose to bruising/bleeding and should be considered when evaluating for abusive injury.

  8. Evaluating for suspected child abuse: conditions that predispose to bleeding.

    PubMed

    Carpenter, Shannon L; Abshire, Thomas C; Anderst, James D

    2013-04-01

    Child abuse might be suspected when children present with cutaneous bruising, intracranial hemorrhage, or other manifestations of bleeding. In these cases, it is necessary to consider medical conditions that predispose to easy bleeding/bruising. When evaluating for the possibility of bleeding disorders and other conditions that predispose to hemorrhage, the pediatrician must consider the child's presenting history, medical history, and physical examination findings before initiating a laboratory investigation. Many medical conditions can predispose to easy bleeding. Before ordering laboratory tests for a disease, it is useful to understand the biochemical basis and clinical presentation of the disorder, condition prevalence, and test characteristics. This technical report reviews the major medical conditions that predispose to bruising/bleeding and should be considered when evaluating for abusive injury. PMID:23530171

  9. Sequence conservation of the rad21 Schizosaccharomyces pombe DNA double-strand break repair gene in human and mouse

    SciTech Connect

    McKay, M.J.; Troelstra, C.; Kanaar, R.

    1996-09-01

    The rad21 gene of Schizosaccharomyces pombe is involved in the repair of ionizing radiation-induced DNA double-strand breaks. The isolation of mouse and human putative homologs of rad21 is reported here. Alignment of the predicted amino acid sequence of Rad21 with the mammalian proteins showed that the similarity was distributed across the length of the proteins, with more highly conserved regions at both termini. The mHR21{sup sp} (mouse homolog of Rad21, S. pombe) and hHR21{sup sp} (human homolog of Rad21, S. pombe) predicted proteins were 96% identical, whereas the human and S. pombe proteins were 25% identical and 47% similar. RNA blot analysis showed that mHR21{sup sp} mRNA was abundant in all adult mouse tissues examined, with highest expression in testis and thymus. In addition to a 3.1-kb constitutive mRNA transcript, a 2.2-kb transcript was present at a high level in postmeiotic spermatids, while expression of the 3.1-kb mRNA in testis was confined to the meiotic compartment. hHR21{sup sp} mRNA was cell-cycle regulated in human cells, increasing in late S phase to a peak in G2 phase. The level of hHR21{sup sp} transcripts was not altered by exposure of normal diploid fibroblasts to 10 Gy ionizing radiation. In situ hybridization showed that mHR21{sup sp} resided on chromosome 15D3, whereas hHR21{sup sp} localized to the syntenic 8q24 region. Elevated expression of mHR21{sup sp} in testis and thymus supports a possible role for the rad21 mammalian homologs in V(D)J and meiotic recombination, respectively. Cell cycle regulation of rad21, retained from S. pombe to human, is consistent with a conservation of function between S. pombe and human rad21 genes. 62 refs., 8 figs., 1 tab.

  10. β-Cyclodextrin-Linked Polyethylenimine Nanoparticles Facilitate Gene Transfer and Enhance the Angiogenic Capacity of Mesenchymal Stem Cells for Wound Repair and Regeneration.

    PubMed

    Peng, Li-Hua; Wei, Wei; Shan, Ying-Hui; Zhang, Tian-Yuan; Zhang, Chen-Zhen; Wu, Jia-He; Yu, Lian; Lin, Jun; Liang, Wen-Quan; Khang, Gilson; Gao, Jian-Qing

    2015-04-01

    Repair of deep wounds by cell transplantation strongly depends on angiogenesis and on the regeneration of skin and appendages. In this study, plasmid DNA encoding vascular endothelial growth factor-165 (VEGF-165) was transduced into bone-marrow mesenchymal stem cells (MSCs) using a nonviral vector, β-cyclodextrin-linked polyethylenimine, to enhance angiogenic capacity. The effects of MSCs administered by intradermal injection or transplantation on wound closure were compared in a full-thickness excision wound model. The results showed that the MSC-seeded sponge had significantly stronger acceleration in wound closure than the MSC injection. The effects on wound repair and regeneration of transplanted MSCs and pDNA-VEGF1 65-transfected MSCs (TMSCs) with gelatin/β-tricalcium phosphate scaffold were also investigated. Compared with MSC transplantation, TMSC transplantation showed higher efficacy in stimulating wound closure, promoting dermal collagen synthesis and regulating the deposition of newly formed collagen. In addition, the angiogenic capacity of the TMSCs was higher than that of the MSCs. The results indicate that the nonviral genetic engineering of the MSCs is a promising strategy to enhance the angiogenic capacity of MSCs for wound repair and angiogenesis. Functional gene-activated MSCs may be used as cost-effective and accessible seed cells for skin tissue engineering and as novel carriers for wound gene therapy. PMID:26310074

  11. [Photoreactivating Activity of Bioluminescence: Repair of UV-damaged DNA of Escherichia coli Occurs with Assistance of lux-Genes of Marine Bacteria].

    PubMed

    Zavilgelsky, G B; Melkina, O E; Kotova, V Yu; Konopleva, M N; Manukhov, I V; Pustovoit, K Ss

    2015-01-01

    The UV resistance of luminescent bacteria Escherichia coli AB1886 uvrA6 (pLeo1) containing the plasmid with luxCDABE genes of marine bacteria Photobacterium leiognathi is approximately two times higher than the UV resistance of non-luminous bacteria E. coli AB1886 uvrA6. Introduction of phr::kan(r) mutations (a defect in the functional activity of photolyase) into the genome of E. coli AB1886 uvrA6 (pLeo1) completely removes the high UV resistance of the cells. Therefore, photoreactivation that involves bacterial photolyase contributes mainly to the bioluminescence-induced DNA repair. It is shown that photoreactivating activity of bioluminescence of P. leiognathi is about 2.5 times lower compared with that one induced by a light source with λ > 385 nm. It is also shown that an increase in the bioluminescence intensity, induced by UV radiation in E. coli bacterial cells with a plasmid containing the luxCD ABE genes under RecA-LexA-regulated promoters, occurs only 25-30 min later after UV irradiation of cells and does not contribute to DNA repair. A quorum sensing regulatory system is not involved in the DNA repair by photolyase.

  12. Differences in Expression of Key DNA Damage Repair Genes after Epigenetic-Induced BRCAness Dictate Synthetic Lethality with PARP1 Inhibition.

    PubMed

    Wiegmans, Adrian P; Yap, Pei-Yi; Ward, Ambber; Lim, Yi Chieh; Khanna, Kum Kum

    2015-10-01

    The triple-negative breast cancer (TNBC) subtype represents a cancer that is highly aggressive with poor patient outcome. Current preclinical success has been gained through synthetic lethality, targeting genome instability with PARP inhibition in breast cancer cells that harbor silencing of the homologous recombination (HR) pathway. Histone deacetylase inhibitors (HDACi) are a class of drugs that mediate epigenetic changes in expression of HR pathway genes. Here, we compare the activity of the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA), the class I/IIa HDAC inhibitor valproic acid (VPA), and the HDAC1/2-specific inhibitor romidepsin (ROMI) for their capability to regulate DNA damage repair gene expression and in sensitizing TNBC to PARPi. We found that two of the HDACis tested, SAHA and ROMI, but not VPA, indeed inhibit HR repair and that RAD51, BARD1, and FANCD2 represent key proteins whose inhibition is required for HDACi-mediated therapy with PARP inhibition in TNBC. We also observed that restoration of BRCA1 function stabilizes the genome compared with mutant BRCA1 that results in enhanced polyploid population after combination treatment with HDACi and PARPi. Furthermore, we found that overexpression of the key HR protein RAD51 represents a mechanism for this resistance, promoting aberrant repair and the enhanced polyploidy observed. These findings highlight the key components of HR in guiding synthetic lethality with PARP inhibition and support the rationale for utilizing the novel combination of HDACi and PARPi against TNBC in the clinical setting. PMID:26294743

  13. Modulation of DNA repair capacity and mRNA expression levels of XRCC1, hOGG1 and XPC genes in styrene-exposed workers

    SciTech Connect

    Hanova, Monika; Stetina, Rudolf; Vodickova, Ludmila; Vaclavikova, Radka; Hlavac, Pavel; Smerhovsky, Zdenek; Naccarati, Alessio; Polakova, Veronika; Soucek, Pavel; Kuricova, Miroslava; Manini, Paola; Kumar, Rajiv; Hemminki, Kari; Vodicka, Pavel

    2010-11-01

    Decreased levels of single-strand breaks in DNA (SSBs), reflecting DNA damage, have previously been observed with increased styrene exposure in contrast to a dose-dependent increase in the base-excision repair capacity. To clarify further the above aspects, we have investigated the associations between SSBs, micronuclei, DNA repair capacity and mRNA expression in XRCC1, hOGG1 and XPC genes on 71 styrene-exposed and 51 control individuals. Styrene concentrations at workplace and in blood characterized occupational exposure. The workers were divided into low (below 50 mg/m{sup 3}) and high (above 50 mg/m{sup 3}) styrene exposure groups. DNA damage and DNA repair capacity were analyzed in peripheral blood lymphocytes by Comet assay. The mRNA expression levels were determined by qPCR. A significant negative correlation was observed between SSBs and styrene concentration at workplace (R = - 0.38, p = 0.001); SSBs were also significantly higher in men (p = 0.001). The capacity to repair irradiation-induced DNA damage was the highest in the low exposure group (1.34 {+-} 1.00 SSB/10{sup 9} Da), followed by high exposure group (0.72 {+-} 0.81 SSB/10{sup 9} Da) and controls (0.65 {+-} 0.82 SSB/10{sup 9} Da). The mRNA expression levels of XRCC1, hOGG1 and XPC negatively correlated with styrene concentrations in blood and at workplace (p < 0.001) and positively with SSBs (p < 0.001). Micronuclei were not affected by styrene exposure, but were higher in older persons and in women (p < 0.001). In this study, we did not confirm previous findings on an increased DNA repair response to styrene-induced genotoxicity. However, negative correlations of SSBs and mRNA expression levels of XRCC1, hOGG1 and XPC with styrene exposure warrant further highly-targeted study.

  14. Mismatch repair genes on chromosomes 2p and 3p account for a major share of hereditary nonpolyposis colorectal cancer families evaluable by linkage

    SciTech Connect

    Nystroem-Lahti, M.; Pylkkaenen, L.; Aaltonen, L.A.; Parsons, R.; Leach, F.S.; Hamilton, S.R.; Sistonen, P. |; Watson, P.; Bronson, E.; Fusaro, R.

    1994-10-01

    Two susceptibility loci for hereditary nonpolyposis colorectal cancer (HNPCC) have been identified, and each contains a mismatch repair gene: MSH2 on chromosome 2p and MLH1 on chromosome 3p. We studied the involvement of these loci in 13 large HNPCC kindreds originating from three different continents. Six families showed close linkage to the 2p locus, and a heritable mutation of the MSH2 gene was subsequently found in four. The 2p-linked kindreds included a family characterized by the lack of extracolonic manifestations (Lynch I syndrome), as well as two families with cutaneous manifestations typical of the Muir-Torre syndrome. Four families showed evidence for linkage to the 3p locus, and a heritable mutation of the MLH1 gene was later detected in three. One 3p-linked kindred was of Amerindian origin. Of the remaining three families studied for linkage, one showed lod scores compatible with exclusion of both MSH2 and MLH1, while lod scores obtained in the other two families suggested exclusion of one HNPCC locus (MSH2 or MLH1) but were uninformative for markers flanking the other locus. Our results suggest that mismatch repair genes on 2p and 3p account for a major share of HNPCC in kindreds that can be evaluated by linkage analysis. 36 refs., 2 figs., 3 tabs.

  15. UV-induced skin carcinogenesis in xeroderma pigmentosum group A (XPA) gene-knockout mice with nucleotide excision repair-deficiency.

    PubMed

    Tanaka, K; Kamiuchi, S; Ren, Y; Yonemasu, R; Ichikawa, M; Murai, H; Yoshino, M; Takeuchi, S; Saijo, M; Nakatsu, Y; Miyauchi-Hashimoto, H; Horio, T

    2001-06-01

    Nucleotide excision repair (NER) removes a wide variety of lesions from the genome and is deficient in the genetic disorder, xeroderma pigmentosum (XP). In this paper, an in vitro analysis of the XP group A gene product (XPA protein) is reported. Results of an analysis on the pathogenesis of ultraviolet (UV)-B-induced skin cancer in the XPA gene-knockout mouse are also described: (1) contrary to wild type mice, significant bias of p53 mutations to the transcribed strand and no evident p53 mutational hot spots were detected in the skin tumors of XPA-knockout mice. (2) Skin cancer cell lines from UVB-irradiated XPA-knockout mice had a decreased mismatch repair activity and an abnormal cell cycle checkpoint, suggesting that the downregulation of mismatch repair helps cells escape killing by UVB and that mismatch repair-deficient clones are selected for during the tumorigenic transformation of XPA (-/-) cells. (3) The XPA-knockout mice showed a higher frequency of UVB-induced mutation in the rpsL transgene at a low dose of UVB-irradiation than the wild type mice. CC-->TT tandem transition, a hallmark of UV-induced mutation, was detected at higher frequency in the rpsL transgene in the XPA-knockout mice than the wild type mice. This rpsL/XPA mouse system will be useful for further analysing the role of NER in the mutagenesis induced by various carcinogens. (4) The UVB-induced immunosuppression was greatly enhanced in the XPA-knockout mice. It is possible that an enhanced impairment of the immune system by UVB irradiation is involved in the high incidence of skin cancer in XP. PMID:11376684

  16. Clubfoot repair

    MedlinePlus

    ... release; Talipes equinovarus - repair; Tibialis anterior tendon transfer Images Clubfoot repair - series References Kelly DM. Congenital Anomalies ... provided herein should not be used during any medical emergency or for the diagnosis or treatment of ...

  17. Tendon repair

    MedlinePlus

    Repair of tendon ... Tendon repair can be performed using: Local anesthesia (the immediate area of the surgery is pain-free) ... a cut on the skin over the injured tendon. The damaged or torn ends of the tendon ...

  18. International congress on DNA damage and repair: Book of abstracts

    SciTech Connect

    Not Available

    1987-01-01

    This document contains the abstracts of 105 papers presented at the Congress. Topics covered include the Escherichia coli nucleotide excision repair system, DNA repair in malignant transformations, defective DNA repair, and gene regulation. (TEM)

  19. Programmed genetic instability: a tumor-permissive mechanism for maintaining the evolvability of higher species through methylation-dependent mutation of DNA repair genes in the male germ line.

    PubMed

    Zhao, Yongzhong; Epstein, Richard J

    2008-08-01

    Tumor suppressor genes are classified by their somatic behavior either as caretakers (CTs) that maintain DNA integrity or as gatekeepers (GKs) that regulate cell survival, but the germ line role of these disease-related gene subgroups may differ. To test this hypothesis, we have used genomic data mining to compare the features of human CTs (n = 38), GKs (n = 36), DNA repair genes (n = 165), apoptosis genes (n = 622), and their orthologs. This analysis reveals that repair genes are numerically less common than apoptosis genes in the genomes of multicellular organisms (P < 0.01), whereas CT orthologs are commoner than GK orthologs in unicellular organisms (P < 0.05). Gene targeting data show that CTs are less essential than GKs for survival of multicellular organisms (P < 0.0005) and that CT knockouts often permit offspring viability at the cost of male sterility. Patterns of human familial oncogenic mutations confirm that isolated CT loss is commoner than is isolated GK loss (P < 0.00001). In sexually reproducing species, CTs appear subject to less efficient purifying selection (i.e., higher Ka/Ks) than GKs (P = 0.000003); the faster evolution of CTs seems likely to be mediated by gene methylation and reduced transcription-coupled repair, based on differences in dinucleotide patterns (P = 0.001). These data suggest that germ line CT/repair gene function is relatively dispensable for survival, and imply that milder (e.g., epimutational) male prezygotic repair defects could enhance sperm variation-and hence environmental adaptation and speciation-while sparing fertility. We submit that CTs and repair genes are general targets for epigenetically initiated adaptive evolution, and propose a model in which human cancers arise in part as an evolutionarily programmed side effect of age- and damage-inducible genetic instability affecting both somatic and germ line lineages. PMID:18535014

  20. 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

  1. Combined hereditary and somatic mutations of replication error repair genes result in rapid onset of ultra-hypermutated cancers.

    PubMed

    Shlien, Adam; Campbell, Brittany B; de Borja, Richard; Alexandrov, Ludmil B; Merico, Daniele; Wedge, David; Van Loo, Peter; Tarpey, Patrick S; Coupland, Paul; Behjati, Sam; Pollett, Aaron; Lipman, Tatiana; Heidari, Abolfazl; Deshmukh, Shriya; Avitzur, Na'ama; Meier, Bettina; Gerstung, Moritz; Hong, Ye; Merino, Diana M; Ramakrishna, Manasa; Remke, Marc; Arnold, Roland; Panigrahi, Gagan B; Thakkar, Neha P; Hodel, Karl P; Henninger, Erin E; Göksenin, A Yasemin; Bakry, Doua; Charames, George S; Druker, Harriet; Lerner-Ellis, Jordan; Mistry, Matthew; Dvir, Rina; Grant, Ronald; Elhasid, Ronit; Farah, Roula; Taylor, Glenn P; Nathan, Paul C; Alexander, Sarah; Ben-Shachar, Shay; Ling, Simon C; Gallinger, Steven; Constantini, Shlomi; Dirks, Peter; Huang, Annie; Scherer, Stephen W; Grundy, Richard G; Durno, Carol; Aronson, Melyssa; Gartner, Anton; Meyn, M Stephen; Taylor, Michael D; Pursell, Zachary F; Pearson, Christopher E; Malkin, David; Futreal, P Andrew; Stratton, Michael R; Bouffet, Eric; Hawkins, Cynthia; Campbell, Peter J; Tabori, Uri

    2015-03-01

    DNA replication-associated mutations are repaired by two components: polymerase proofreading and mismatch repair. The mutation consequences of disruption to both repair components in humans are not well studied. We sequenced cancer genomes from children with inherited biallelic mismatch repair deficiency (bMMRD). High-grade bMMRD brain tumors exhibited massive numbers of substitution mutations (>250/Mb), which was greater than all childhood and most cancers (>7,000 analyzed). All ultra-hypermutated bMMRD cancers acquired early somatic driver mutations in DNA polymerase ɛ or δ. The ensuing mutation signatures and numbers are unique and diagnostic of childhood germ-line bMMRD (P < 10(-13)). Sequential tumor biopsy analysis revealed that bMMRD/polymerase-mutant cancers rapidly amass an excess of simultaneous mutations (∼600 mutations/cell division), reaching but not exceeding ∼20,000 exonic mutations in <6 months. This implies a threshold compatible with cancer-cell survival. We suggest a new mechanism of cancer progression in which mutations develop in a rapid burst after ablation of replication repair.

  2. A Novel Germline Mutation in BAP1 Predisposes to Familial Clear-Cell Renal Cell Carcinoma

    PubMed Central

    Mester, Jessica L.; Pena-Llopis, Samuel; Pavia-Jimenez, Andrea; Christie, Alana; Vocke, Cathy D.; Ricketts, Christopher J.; Peterson, James; Middelton, Lindsay; Kinch, Lisa; Grishin, Nick; Merino, Maria J.; Metwalli, Adam R.; Xing, Chao; Xie, Xian-Jin; Dahia, Patricia L.M.; Eng, Charis

    2013-01-01

    Renal cell carcinoma (RCC) clusters in some families. Familial RCC arises from mutations in several genes, including VHL, which is also mutated in sporadic RCC. However, a significant percentage of familial RCC remains unexplained. Recently, we discovered that the BAP1 gene is mutated in sporadic RCC. BAP1, which encodes a nuclear deubiquitinase, is a two-hit tumor suppressor gene. Somatic BAP1 mutations are associated with high-grade ccRCC and poor patient outcomes. To determine whether BAP1 predisposes to familial RCC, we sequenced the BAP1 gene in 83 unrelated probands with unexplained familial RCC. We identified a novel variant (c.41T>A; p.L14H), which cosegregated with the RCC phenotype. The p.L14H variant disrupts a highly conserved residue in the catalytic domain, a domain frequently targeted by missense mutations. The family with the BAP1 variant was characterized by early-onset clear cell RCC, occasionally of high Fuhrman grade, and lacked other features that characterize von Hippel-Lindau syndrome. These findings suggest that BAP1 is a familial RCC predisposing gene. PMID:23709298

  3. Host genetic factors predisposing to HIV-associated neurocognitive disorder.

    PubMed

    Kallianpur, Asha R; Levine, Andrew J

    2014-09-01

    The success of combination antiretroviral therapy (cART) in transforming the lives of HIV-infected individuals with access to these drugs is tempered by the increasing threat of HIV-associated neurocognitive disorders (HAND) to their overall health and quality of life. Intensive investigations over the past two decades have underscored the role of host immune responses, inflammation, and monocyte-derived macrophages in HAND, but the precise pathogenic mechanisms underlying HAND remain only partially delineated. Complicating research efforts and therapeutic drug development are the sheer complexity of HAND phenotypes, diagnostic imprecision, and the growing intersection of chronic immune activation with aging-related comorbidities. Yet, genetic studies still offer a powerful means of advancing individualized care for HIV-infected individuals at risk. There is an urgent need for 1) longitudinal studies using consistent phenotypic definitions of HAND in HIV-infected subpopulations at very high risk of being adversely impacted, such as children, 2) tissue studies that correlate neuropathological changes in multiple brain regions with genomic markers in affected individuals and with changes at the RNA, epigenomic, and/or protein levels, and 3) genetic association studies using more sensitive subphenotypes of HAND. The NIH Brain Initiative and Human Connectome Project, coupled with rapidly evolving systems biology and machine learning approaches for analyzing high-throughput genetic, transcriptomic and epigenetic data, hold promise for identifying actionable biological processes and gene networks that underlie HAND. This review summarizes the current state of understanding of host genetic factors predisposing to HAND in light of past challenges and suggests some priorities for future research to advance the understanding and clinical management of HAND in the cART era. PMID:24996618

  4. Classical non-homologous end-joining pathway utilizes nascent RNA for error-free double-strand break repair of transcribed genes

    PubMed Central

    Chakraborty, Anirban; Tapryal, Nisha; Venkova, Tatiana; Horikoshi, Nobuo; Pandita, Raj K.; Sarker, Altaf H.; Sarkar, Partha S.; Pandita, Tej K.; Hazra, Tapas K.

    2016-01-01

    DNA double-strand breaks (DSBs) leading to loss of nucleotides in the transcribed region can be lethal. Classical non-homologous end-joining (C-NHEJ) is the dominant pathway for DSB repair (DSBR) in adult mammalian cells. Here we report that during such DSBR, mammalian C-NHEJ proteins form a multiprotein complex with RNA polymerase II and preferentially associate with the transcribed genes after DSB induction. Depletion of C-NHEJ factors significantly abrogates DSBR in transcribed but not in non-transcribed genes. We hypothesized that nascent RNA can serve as a template for restoring the missing sequences, thus allowing error-free DSBR. We indeed found pre-mRNA in the C-NHEJ complex. Finally, when a DSB-containing plasmid with several nucleotides deleted within the E. coli lacZ gene was allowed time to repair in lacZ-expressing mammalian cells, a functional lacZ plasmid could be recovered from control but not C-NHEJ factor-depleted cells, providing important mechanistic insights into C-NHEJ-mediated error-free DSBR of the transcribed genome. PMID:27703167

  5. Zinc finger transcription factor CASZ1 interacts with histones, DNA repair proteins and recruits NuRD complex to regulate gene transcription

    PubMed Central

    Liu, Zhihui; Lam, Norris; Thiele, Carol J.

    2015-01-01

    The zinc finger transcription factor CASZ1 has been found to control neural fate-determination in flies, regulate murine and frog cardiac development, control murine retinal cell progenitor expansion and function as a tumor suppressor gene in humans. However, the molecular mechanism by which CASZ1 regulates gene transcription to exert these diverse biological functions has not been described. Here we identify co-factors that are recruited by CASZ1b to regulate gene transcription using co-immunoprecipitation (co-IP) and mass spectrometry assays. We find that CASZ1b binds to the nucleosome remodeling and histone deacetylase (NuRD) complex, histones and DNA repair proteins. Mutagenesis of the CASZ1b protein assay demonstrates that the N-terminus of CASZ1b is required for NuRD binding, and a poly(ADP-ribose) binding motif in the CASZ1b protein is required for histone H3 and DNA repair proteins binding. The N-terminus of CASZ1b fused to an artificial DNA-binding domain (GAL4DBD) causes a significant repression of transcription (5xUAS-luciferase assay), which could be blocked by treatment with an HDAC inhibitor. Realtime PCR results show that the transcriptional activity of CASZ1b mutants that abrogate NuRD or histone H3/DNA binding is significantly decreased. This indicates a model in which CASZ1b binds to chromatin and recruits NuRD complexes to orchestrate epigenetic-mediated transcriptional programs. PMID:26296975

  6. Increasing the efficiency of homologous recombination vector-mediated end joining repair by inhibition of Lig4 gene using siRNA in sheep embryo fibroblasts.

    PubMed

    Wei, Wang; Yushuang, Wang; Lanlan, Huang; Zijian, Jian; Xinhua, Wang; Shouren, Liu; Wenhui, Pi

    2016-09-01

    In animal cells, inhibition of non-homologous end joining (NHEJ) pathway improves the efficiency of homologous recombination (HR)-mediated double-strand brakes (DSBs) repair. To improve the efficiency of HR in sheep embryo fibroblasts, the NHEJ key molecule DNA ligase 4 (Lig4) was suppressed by siRNA interference. Four pairs of siRNA targeting Lig4 were designed and chemically synthesized. These siRNA were electro-transferred into sheep embryo fibroblasts respectively. Compared with the control groups, two pairs of siRNA were identified to effectively inhibit the expression of sheep Lig4 gene by qRT-PCR and Western blotting. The plasmid rejoining assay was adopted for examining the efficiency of HR-mediated DSB repair. I-SceⅠ endonuclease linearized vector and siRNA were co-transfected into sheep embryo fibroblasts. Flow cytometry analysis of cells after transfection for 72 h showed that suppression of Lig4 using siRNAs increased the rejoining efficiency of HR vector by 3-4 times compared with the control groups. Therefore, enhanced HR vector rejoining frequency by instant inhabition of Lig4 gene provides theoretical basis for improving gene targeting efficiency of sheep embryo fibroblasts. PMID:27644744

  7. 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.

  8. Radio-adaptive response of base excision repair genes and proteins in human peripheral blood mononuclear cells exposed to gamma radiation.

    PubMed

    Toprani, Sneh M; Das, Birajalaxmi

    2015-09-01

    Radio-adaptive response is a mechanism whereby a low-dose exposure (priming dose) induces resistance to a higher dose (challenging dose) thus significantly reducing its detrimental effects. Radiation-induced DNA damage gets repaired through various DNA repair pathways in human cells depending upon the type of lesion. The base excision repair (BER) pathway repairs radiation-induced base damage, abasic sites and single-strand breaks in cellular DNA. In the present study, an attempt has been made to investigate the involvement of BER genes and proteins in the radio-adaptive response in human resting peripheral blood mononuclear cells (PBMC). Venous blood samples were collected from 20 randomly selected healthy male individuals with written informed consent. PBMC were isolated and irradiated at a priming dose of 0.1 Gy followed 4h later with a challenging dose of 2.0 Gy (primed cells). Quantitation of DNA damage was done using the alkaline comet assay immediately and expression profile of BER genes and proteins were studied 30 min after the challenging dose using real-time quantitative polymerase chain reaction and western blot, respectively. The overall result showed significant (P ≤ 0.05) reduction of DNA damage in terms of percentage of DNA in tail (%T) with a priming dose of 0.1 Gy followed by a challenging dose of 2.0 Gy after 4 h. Twelve individuals showed significant (P ≤ 0.05) reduction in %T whereas eight individuals showed marginal reduction in DNA damage that was not statistically significant. However, at the transcriptional level, BER genes such as APE1, FEN1 and LIGASE1 showed significant (P ≤ 0.05) up-regulation in both groups. Significant (P ≤ 0.05) up-regulation was also observed at the protein level for OGG1, APE1, MBD4, FEN1 and LIGASE1 in primed cells. Up-regulation of some BER genes and proteins such as APE1, FEN1 and LIGASE1 in primed cells of resting PBMC is suggestive of active involvement of the BER pathway in radio-adaptive response

  9. Oxidative stress and DNA repair and detoxification gene expression in adolescents exposed to heavy metals living in the Milazzo-Valle del Mela area (Sicily, Italy)

    PubMed Central

    Pizzino, Gabriele; Bitto, Alessandra; Interdonato, Monica; Galfo, Federica; Irrera, Natasha; Mecchio, Anna; Pallio, Giovanni; Ramistella, Vincenzo; Luca, Filippo De; Minutoli, Letteria; Squadrito, Francesco; Altavilla, Domenica

    2014-01-01

    Background The area of Milazzo-Valle del Mela (Sicily, Italy) is considered at high risk of environmental crisis by regional authorities. Objective To measure oxidative-stress, DNA repair and detoxification genes in school children living near the industrial area and in age-matched controls. Methods The parent study was a biomonitoring investigation evaluating heavy metal urine levels in 226 children aged 12–14 years, living in the high risk area, and in 29 age-matched controls living 45 km far from the industrial site. In the present study 67 exposed adolescents and 29 controls were included. Samples were analyzed for urinary 8-hydroxydeoxyguanosine (8OHdG) levels, and gene expression of OGG1 (DNA repair gene), NQO1, ST13, and MT1A (detoxifying genes). Results Urinary cadmium was higher (p = 0.0004) in exposed [geometric mean, 0.46 µg/L; 25th–75th percentile: 0.3–0.56] than in control adolescents [geometric mean, 0.26 µg/L; 25th–75th percentile: 0.2–0.3]. Chromium was also significantly elevated in exposed [geometric mean, 1.52 µg/L; 25th–75th percentile: 1.19–1.93] compared with controls [geometric mean, 1.25 µg/L; 25th–75th percentile: 1.05–1.48; p = 0.02]. Urinary 8-OHdG concentration was greater in exposed than in controls (71.49 vs 61.87 µg/L, p = 0.02), and it was correlated with cadmium levels (r = 0.46, p < 0.0001), and with the combined exposure index (r = 0.43, p < 0.0001). Moreover, cadmium levels showed a robust correlation with OGG1 and MT1A gene expression levels (r = 0.44, p < 0.0001; r = 0.39, p < 0.0001, respectively). Finally, OGG1 and MT1A were over-expressed in adolescents from Milazzo-Valle del Mela area compared with controls (p = 0.0004; p < 0.0001, respectively). Conclusions Continuous exposure at relatively low concentrations of heavy metals is associated with increased oxidative DNA damage and impaired expression of DNA repair and detoxification genes in adolescents. PMID:24936443

  10. Detecting disease-predisposing variants: The haplotype method

    SciTech Connect

    Valdes, A.M.; Thomson, G.

    1997-03-01

    For many HLA-associated diseases, multiple alleles - and, in some cases, multiple loci - have been suggested as the causative agents. The haplotype method for identifying disease-predisposing amino acids in a genetic region is a stratification analysis. We show that, for each haplotype combination containing all the amino acid sites involved in the disease process, the relative frequencies of amino acid variants at sites not involved in disease but in linkage disequilibrium with the disease-predisposing sites are expected to be the same in patients and controls. The haplotype method is robust to mode of inheritance and penetrance of the disease and can be used to determine unequivocally whether all amino acid sites involved in the disease have not been identified. Using a resampling technique, we developed a statistical test that takes account of the nonindependence of the sites sampled. Further, when multiple sites in the genetic region are involved in disease, the test statistic gives a closer fit to the null expectation when some - compared with none - of the true predisposing factors are included in the haplotype analysis. Although the haplotype method cannot distinguish between very highly correlated sites in one population, ethnic comparisons may help identify the true predisposing factors. The haplotype method was applied to insulin-dependent diabetes mellitus (IDDM) HLA class II DQA1-DQB1 data from Caucasian, African, and Japanese populations. Our results indicate that the combination DQA1 No. 52 (Arg predisposing) DQB1 No. 57 (Asp protective), which has been proposed as an important IDDM agent, does not include all the predisposing elements. With rheumatoid arthritis HLA class H DRB1 data, the results were consistent with the shared-epitope hypothesis. 35 refs., 2 figs., 6 tabs.

  11. Targeting DNA repair pathways for cancer treatment: what's new?

    PubMed Central

    Kelley, Mark R; Logsdon, Derek; Fishel, Melissa L

    2014-01-01

    Disruptions in DNA repair pathways predispose cells to accumulating DNA damage. A growing body of evidence indicates that tumors accumulate progressively more mutations in DNA repair proteins as cancers progress. DNA repair mechanisms greatly affect the response to cytotoxic treatments, so understanding those mechanisms and finding ways to turn dysregulated repair processes against themselves to induce tumor death is the goal of all DNA repair inhibition efforts. Inhibition may be direct or indirect. This burgeoning field of research is replete with promise and challenge, as more intricacies of each repair pathway are discovered. In an era of increasing concern about healthcare costs, use of DNA repair inhibitors can prove to be highly effective stewardship of R&D resources and patient expenses. PMID:24947262

  12. Evidence that the product of the xrs gene is predominantly involved in the repair of a subset of radiation-induced interphase chromosome breaks rejoining with fast kinetics

    SciTech Connect

    Okayasu, R.; Iliakis, G. )

    1994-04-01

    We classified interphase chromosome breaks into [alpha] and [beta] forms to study the requirement for the xrs gene product in the repair of each of these forms of damage. The [alpha] form of damage comprises radiation-induced interphase chromosome breaks whose rejoining is slow and sensitive to treatment with [beta]-arabinofuranosyladenine ([beta]-araA), whereas the [beta] form of damage comprises interphase chromosome breaks whose rejoining is fast and sensitive to treatment in hypertonic medium. Interphase chromosome breaks of the [alpha] form are visualized in plateau-phase cells by premature chromosome condensation (PCC) carried out in the absence of any treatment during the condensation period. More interphase chromosome breaks of the [beta] form are not visualized in experiments using standard PCC protocols but can be uncovered by treatment in hypertonic growth medium during the period allowed for PCC. In the present report, we show that the yield of interphase chromosome breaks of the [alpha] form is similar in CHO and xrs-5 cells and demonstrate that xrs-5 cells rejoin this type of interphase chromosome breaks with an efficiency similar to that observed in repair-proficient CHO cells. Furthermore, we provide evidence supporting the notion that xrs-5 cells are deficient in the rejoining of the [beta] form of interphase chromosome breaks. These results strongly suggest that the product of the xrs gene is required predominantly in the repair of the [beta] form of interphase chromosome damage and emphasize the need for discrimination between different forms of interphase chromosome breaks in irradiated cells. 41 refs., 8 figs., 1 tab.

  13. Early postnatal ataxia and abnormal cerebellar development in mice lacking Xeroderma pigmentosum Group A and Cockayne syndrome Group B DNA repair genes.

    PubMed

    Murai, M; Enokido, Y; Inamura, N; Yoshino, M; Nakatsu, Y; van der Horst, G T; Hoeijmakers, J H; Tanaka, K; Hatanaka, H

    2001-11-01

    Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are rare autosomal recessive disorders associated with a defect in the nucleotide excision repair (NER) pathway required for the removal of DNA damage induced by UV light and distorting chemical adducts. Although progressive neurological dysfunction is one of the hallmarks of CS and of some groups of XP patients, the causative mechanisms are largely unknown. Here we show that mice lacking both the XPA (XP-group A) and CSB (CS-group B) genes in contrast to the single mutants display severe growth retardation, ataxia, and motor dysfunction during early postnatal development. Their cerebella are hypoplastic and showed impaired foliation and stunted Purkinje cell dendrites. Reduced neurogenesis and increased apoptotic cell death occur in the cerebellar external granular layer. These findings suggest that XPA and CSB have additive roles in the mouse nervous system and support a crucial role for these genes in normal brain development. PMID:11687625

  14. Differential expression of genes involved in immunity and biomineralization during Brown Ring Disease development and shell repair in the Manila clam, Ruditapes philippinarum.

    PubMed

    Jeffroy, Fanny; Brulle, Franck; Paillard, Christine

    2013-06-01

    Severe drop in Manila clams production in French aquacultured fields since the end of the 1980's is associated to Brown Ring Disease (BRD). This disease, caused by the bacteria Vibrio tapetis, is characterized by specific symptoms on the inner face of the shell. Diseased animals develop conchiolin deposit to enrobe bacteria and form new calcified layers on the shell. Suppression subtractive hybridization was performed to identify genes differentially expressed during the early interaction of V. tapetis and Ruditapes philippinarum. Results revealed 301 unique genes differentially expressed during V. tapetis challenge. Several candidates involved in immune and biomineralization processes were selected from libraries. Transcriptional expression of selected candidates was determined in hemolymph and mantle tissues and revealed spatial and temporal variations. At 56 days after infection, when clams were in phase of shell repair, transcripts of galectin and ferritin in hemocytes showed higher expression. Ca-like and serpin transcripts were specifically expressed in mantle and could contribute to defense against BRD.

  15. Transcript levels of the Saccharomyes cerevisiae DNA repair gene RAD23 increase in response to UV light and in meiosis but remain constant in the mitotic cell cycle.

    PubMed

    Madura, K; Prakash, S

    1990-08-25

    The RAD23 gene of Saccharomyces cerevisiae is required for excision-repair of UV damaged DNA. In this paper, we determine the location of the RAD23 gene in a cloned DNA fragment, identify the 1.6 kb RAD23 transcript, and examine RAD23 transcript levels in UV damaged cells, during the mitotic cell cycle, and in meiosis. The RAD23 mRNA levels are elevated 5-fold between 30 to 60 min after 37 J/m2 of UV light. RAD23 mRNA levels rise over 6-fold during meiosis at a stage coincident with high levels of genetic recombination. This response is specific to sporulation competent MATa/MAT alpha diploid cells, and is not observed in asporogenous MATa/MATa diploids. RAD23 mRNA levels, however, remain constant during the mitotic cell cycle.

  16. The Cytosolic Iron-Sulfur Cluster Assembly Protein MMS19 Regulates Transcriptional Gene Silencing, DNA Repair, and Flowering Time in Arabidopsis.

    PubMed

    Han, Yong-Feng; Huang, Huan-Wei; Li, Lin; Cai, Tao; Chen, She; He, Xin-Jian

    2015-01-01

    MMS19 is an essential component of the cytoplasmic iron-sulfur (Fe-S) cluster assembly complex in fungi and mammals; the mms19 null mutant alleles are lethal. Our study demonstrates that MMS19/MET18 in Arabidopsis thaliana interacts with the cytoplasmic Fe-S cluster assembly complex but is not an essential component of the complex. We find that MMS19 also interacts with the catalytic subunits of DNA polymerases, which have been demonstrated to be involved in transcriptional gene silencing (TGS), DNA repair, and flowering time regulation. Our results indicate that MMS19 has a similar biological function, suggesting a functional link between MMS19 and DNA polymerases. In the mms19 null mutant, the assembly of Fe-S clusters on the catalytic subunit of DNA polymerase α is reduced but not blocked, which is consistent with the viability of the mutant. Our study suggests that MMS19 assists the assembly of Fe-S clusters on DNA polymerases in the cytosol, thereby facilitating transcriptional gene silencing, DNA repair, and flowering time control.

  17. The CREB Coactivator CRTC2 is a Lymphoma Tumor Suppressor that Preserves Genome Integrity Through Transcription of DNA Mismatch Repair Genes

    PubMed Central

    Fang, Minggang; Pak, Magnolia L.; Chamberlain, Lynn; Xing, Wei; Yu, Hongbo; Green, Michael R.

    2015-01-01

    SUMMARY The CREB-regulated transcription coactivator CRTC2 stimulates CREB target gene expression and has a well-established role in modulating glucose and lipid metabolism. Here we find, unexpectedly, that loss of CRTC2, as well as CREB1 and its coactivator CREB-binding protein (CBP), results in a deficiency in DNA mismatch repair (MMR) and a resultant increased mutation frequency. We show that CRTC2, CREB1 and CBP are transcriptional activators of well-established MMR genes, including EXO1, MSH6, PMS1 and POLD2. Mining of expression profiling databases and analysis of patient samples reveal that CRTC2 and its target MMR genes are down-regulated in specific T-cell lymphoma subtypes, which are microsatellite unstable. The levels of acetylated histone H3 on the CRTC2 promoter are significantly reduced in lymphoma compared to normal tissue, explaining the decreased CRTC2 expression. Our results establish a role for CRTC2 as a lymphoma tumor suppressor gene that preserves genome integrity by stimulating transcription of MMR genes. PMID:26004186

  18. Maternal folate depletion and high-fat feeding from weaning affects DNA methylation and DNA repair in brain of adult offspring.

    PubMed

    Langie, Sabine A S; Achterfeldt, Sebastian; Gorniak, Joanna P; Halley-Hogg, Kirstin J A; Oxley, David; van Schooten, Frederik J; Godschalk, Roger W L; McKay, Jill A; Mathers, John C

    2013-08-01

    The mechanisms through which environmental and dietary factors modulate DNA repair are still unclear but may include dysregulation of gene expression due to altered epigenetic markings. In a mouse model, we investigated the effect of maternal folate depletion during pregnancy and lactation, and high-fat feeding from weaning, on base excision repair (BER) and DNA methylation and expression of selected BER-related genes in the brain of adult offspring. While folate depletion did not affect BER activity of the mothers, BER increased in the offspring at weaning (P=0.052). In the long term, as observed in 6-mo-old offspring, the double insult, i.e., maternal low-folate supply and high-fat feeding from weaning, decreased BER activity significantly in the cortex, cerebellum, hippocampus, and subcortical regions (P≤0.017). This fall in BER activity was associated with small changes in methylation or expression of BER-related genes. Maternal folate depletion led to slightly increased oxidative DNA damage levels in subcortical regions of adult offspring, which may increase sensitivity to oxidative stress and predispose to neurological disorders. In summary, our data suggest that low-folate supply during early life may leave an epigenetic mark that can predispose the offspring to further dietary insults, causing adverse effects during adult life. PMID:23603834

  19. Maternal folate depletion and high-fat feeding from weaning affects DNA methylation and DNA repair in brain of adult offspring.

    PubMed

    Langie, Sabine A S; Achterfeldt, Sebastian; Gorniak, Joanna P; Halley-Hogg, Kirstin J A; Oxley, David; van Schooten, Frederik J; Godschalk, Roger W L; McKay, Jill A; Mathers, John C

    2013-08-01

    The mechanisms through which environmental and dietary factors modulate DNA repair are still unclear but may include dysregulation of gene expression due to altered epigenetic markings. In a mouse model, we investigated the effect of maternal folate depletion during pregnancy and lactation, and high-fat feeding from weaning, on base excision repair (BER) and DNA methylation and expression of selected BER-related genes in the brain of adult offspring. While folate depletion did not affect BER activity of the mothers, BER increased in the offspring at weaning (P=0.052). In the long term, as observed in 6-mo-old offspring, the double insult, i.e., maternal low-folate supply and high-fat feeding from weaning, decreased BER activity significantly in the cortex, cerebellum, hippocampus, and subcortical regions (P≤0.017). This fall in BER activity was associated with small changes in methylation or expression of BER-related genes. Maternal folate depletion led to slightly increased oxidative DNA damage levels in subcortical regions of adult offspring, which may increase sensitivity to oxidative stress and predispose to neurological disorders. In summary, our data suggest that low-folate supply during early life may leave an epigenetic mark that can predispose the offspring to further dietary insults, causing adverse effects during adult life.

  20. Mechanisms predisposing penile fracture and long-term outcomes on erectile and voiding functions.

    PubMed

    Reis, Leonardo O; Cartapatti, Marcelo; Marmiroli, Rafael; de Oliveira Júnior, Eduardo Jeronimo; Saade, Ricardo Destro; Fregonesi, Adriano

    2014-01-01

    Purpose. To determine the mechanisms predisposing penile fracture as well as the rate of long-term penile deformity and erectile and voiding functions. Methods. All fractures were repaired on an emergency basis via subcoronal incision and absorbable suture with simultaneous repair of eventual urethral lesion. Patients' status before fracture and voiding and erectile functions at long term were assessed by periodic follow-up and phone call. Detailed history included cause, symptoms, and single-question self-report of erectile and voiding functions. Results. Among the 44 suspicious cases, 42 (95.4%) were confirmed, mean age was 34.5 years (range: 18-60), mean follow-up 59.3 months (range 9-155). Half presented the classical triad of audible crack, detumescence, and pain. Heterosexual intercourse was the most common cause (28 patients, 66.7%), followed by penile manipulation (6 patients, 14.3%), and homosexual intercourse (4 patients, 9.5%). "Woman on top" was the most common heterosexual position (n = 14, 50%), followed by "doggy style" (n = 8, 28.6%). Four patients (9.5%) maintained the cause unclear. Six (14.3%) patients had urethral injury and two (4.8%) had erectile dysfunction, treated by penile prosthesis and PDE-5i. No patient showed urethral fistula, voiding deterioration, penile nodule/curve or pain. Conclusions. "Woman on top" was the potentially riskiest sexual position (50%). Immediate surgical treatment warrants long-term very low morbidity. PMID:24822062

  1. Incisional Hernia in Women: Predisposing Factors and Management Where Mesh is not Readily Available

    PubMed Central

    Agbakwuru, EA; Olabanji, JK; Alatise, OI; Okwerekwu, RO; Esimai, OA

    2009-01-01

    Background / Aim: Incisional hernia is still relatively common in our practice. The aim of the study was to identify risk factors associated with incisional hernia in our region. The setting is the Obafemi Awolowo University Teaching Hospitals Complex, Ile-Ife, Nigeria during a period when prosthetic mesh was not readily available. Patients and Methods: All the women who presented with incisional hernia between 1996 and 2005 were prospectively studied using a standard form to obtain information on pre-hernia (index) operations and possible predisposing factors. They all had open surgical repair and were followed up for 18–60 months. Results: Forty-four women were treated during study period. The index surgeries leading to the hernias were emergency caesarian section 26/44 (59.1%), emergency exploratory laparotomy 6/44 (13.6%), and elective surgeries 12/44 (27.3%). Major associated risk factors were the use of wrong suture materials for fascia repair, midline incisions, wound sepsis, and overweight. Conclusion: For elective surgeries, reduction of weight should be encouraged when appropriate, and transverse incisions are preferred. Absorbable sutures, especially chromic catgut, should be avoided in fascia closure. Antibiotics should be used for complicated obstetric cases. PMID:21483511

  2. Microarray analysis of DNA damage repair gene expression profiles in cervical cancer cells radioresistant to 252Cf neutron and X-rays

    PubMed Central

    2010-01-01

    Background The aim of the study was to obtain stable radioresistant sub-lines from the human cervical cancer cell line HeLa by prolonged exposure to 252Cf neutron and X-rays. Radioresistance mechanisms were investigated in the resulting cells using microarray analysis of DNA damage repair genes. Methods HeLa cells were treated with fractionated 252Cf neutron and X-rays, with a cumulative dose of 75 Gy each, over 8 months, yielding the sub-lines HeLaNR and HeLaXR. Radioresistant characteristics were detected by clone formation assay, ultrastructural observations, cell doubling time, cell cycle distribution, and apoptosis assay. Gene expression patterns of the radioresistant sub-lines were studied through microarray analysis and verified by Western blotting and real-time PCR. Results The radioresistant sub-lines HeLaNR and HeLaXR were more radioresisitant to 252Cf neutron and X-rays than parental HeLa cells by detecting their radioresistant characteristics, respectively. Compared to HeLa cells, the expression of 24 genes was significantly altered by at least 2-fold in HeLaNR cells. Of these, 19 genes were up-regulated and 5 down-regulated. In HeLaXR cells, 41 genes were significantly altered by at least 2-fold; 38 genes were up-regulated and 3 down-regulated. Conclusions Chronic exposure of cells to ionizing radiation induces adaptive responses that enhance tolerance of ionizing radiation and allow investigations of cellular radioresistance mechanisms. The insights gained into the molecular mechanisms activated by these "radioresistance" genes will lead to new therapeutic targets for cervical cancer. PMID:20184742

  3. 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.

  4. 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

  5. Experimental repair of segmental bone defects in rabbits by angiopoietin-1 gene transfected MSCs seeded on porous β-TCP scaffolds.

    PubMed

    Cao, Le; Liu, Xudong; Liu, Shen; Jiang, Yao; Zhang, Xianlong; Zhang, Changqing; Zeng, Bingfang

    2012-07-01

    Segmental bone defect repair remains a clinical and experimental challenge in tissue engineering with increasing focus on angiogenesis in the bone substitutes. The objective of this study was to investigate the osteogenic effects of angiopoietin-1 (Ang-1) gene transfected bone marrow-derived mesenchymal stem cells (MSCs) seeded on porous β-TCP scaffolds. This bone substitute (experimental group) and MSCs/β-TCP compounds (control group) were implanted into 15 mm segmental bone defects of the radii of 30 New Zealand white rabbits, with platelet-rich plasma injected at the same time. Bone regeneration and angiogenesis were assessed by Scanning electron microscope (SEM), X-ray, histology, immunohistology, and biomechanical outcome measurements made on the 2nd, 4th, 8th, and 12th week after the operation. In vitro, the amount of proliferation and differentiation of Ang-1 gene transfected MSCs was found to be gross increased than that of the control groups. In vivo, a significantly increased amount of new bone formation accompanied by active capillary vasculature regeneration was observed in the pores of the scaffolds which had been seeded with Ang-1 gene transfected MSCs, as compared with the control groups. The biomechanical test confirmed the failure load of new born bone was close to normal bone. These results suggest that transfer of gene encoding Ang-1 to MSCs increases their osteogenic properties by enhancing capillary regeneration, thus providing a rich blood supply for new bone formation in segmental bone defects.

  6. Promoter hypermethylation and inactivation of hMLH1, a DNA mismatch repair gene, in head and neck squamous cell carcinoma.

    PubMed

    Liu, Kela; Zuo, Chunlai; Luo, Q Kevin; Suen, James Y; Hanna, Ehab; Fan, Chun-Yang

    2003-03-01

    Head and neck squamous cell carcinoma (HNSCC) is a multistage process during which adverse genetic alterations accumulate resulting in loss of cell cycle control, selective cell overgrowth, and ultimately formation of malignancy. Among various genetic alterations in HNSCC is increased microsatellite instability (MSI). hMLH1 is one of the major mismatch DNA repair genes, the inactivation of which caused increased MSI in a variety of human cancers including HNSCC. While somatic mutation is a major mechanism of the hMLH1 gene inactivation in hereditary form of human cancer, promoter hypermethylation appears to be primarily involved in the inactivation of the hMLH1 gene in sporadic form of human cancers. In the current study, we analyzed 78 cases of HNSCC for hMLH1 protein expression and promoter hypermethylation by IHC and methylation-specific PCR (MSP). Twenty-four of 78 cases (31%) of HNSCC contained markedly reduced levels of the hMLH1 protein. Based on the IHC results, 8 cases without and 8 with hMLH1 protein expression (total of 16) were further analyzed by MSP. Seven of 8 cases (88%) that were negative for the hMLH1 protein displayed promoter hypermethylation, whereas 7 of 7 cases (100%) strongly positive for the protein were free of promoter methylation. This study confirms our previous conclusion that promoter hypermethylation represents a major mechanism of the hMLH1 gene inactivation in HNSCC.

  7. Association Between Single-Nucleotide Polymorphisms in Hormone Metabolism and DNA Repair Genes and Epithelial Ovarian Cancer: Results from Two Australian Studies and an Additional Validation Set

    PubMed Central

    Beesley, Jonathan; Jordan, Susan J.; Spurdle, Amanda B.; Song, Honglin; Ramus, Susan J.; Kjaer, Suzanne Kruger; Hogdall, Estrid; DiCioccio, Richard A.; McGuire, Valerie; Whittemore, Alice S.; Gayther, Simon A.; Pharoah, Paul D.P.; Webb, Penelope M.; Chenevix-Trench, Georgia

    2009-01-01

    Although some high-risk ovarian cancer genes have been identified, it is likely that common low penetrance alleles exist that confer some increase in ovarian cancer risk. We have genotyped nine putative functional single-nucleotide polymorphisms (SNP) in genes involved in steroid hormone synthesis (SRD5A2, CYP19A1, HSB17B1, and HSD17B4) and DNA repair (XRCC2, XRCC3, BRCA2, and RAD52) using two Australian ovarian cancer case-control studies, comprising a total of 1,466 cases and 1,821 controls of Caucasian origin. Genotype frequencies in cases and controls were compared using logistic regression. The only SNP we found to be associated with ovarian cancer risk in both of these two studies was SRD5A2 V89L (rs523349), which showed a significant trend of increasing risk per rare allele (P = 0.00002). We then genotyped another SNP in this gene (rs632148; r2 = 0.945 with V89L) in an attempt to validate this finding in an independent set of 1,479 cases and 2,452 controls from United Kingdom, United States, and Denmark. There was no association between rs632148 and ovarian cancer risk in the validation samples, and overall, there was no significant heterogeneity between the results of the five studies. Further analyses of SNPs in this gene are therefore warranted to determine whether SRD5A2 plays a role in ovarian cancer predisposition. PMID:18086758

  8. Epigenetic Studies Point to DNA Replication/Repair Genes as a Basis for the Heritable Nature of Long Term Complications in Diabetes

    PubMed Central

    Leontovich, Alexey A.; Intine, Robert V.; Sarras, Michael P.

    2016-01-01

    Metabolic memory (MM) is defined as the persistence of diabetic (DM) complications even after glycemic control is pharmacologically achieved. Using a zebrafish diabetic model that induces a MM state, we previously reported that, in this model, tissue dysfunction was of a heritable nature based on cell proliferation studies in limb tissue and this correlated with epigenetic DNA methylation changes that paralleled alterations in gene expression. In the current study, control, DM, and MM excised fin tissues were further analyzed by MeDIP sequencing and microarray techniques. Bioinformatics analysis of the data found that genes of the DNA replication/DNA metabolism process group (with upregulation of the apex1, mcm2, mcm4, orc3, lig1, and dnmt1 genes) were altered in the DM state and these molecular changes continued into MM. Interestingly, DNA methylation changes could be found as far as 6–13 kb upstream of the transcription start site for these genes suggesting potential higher levels of epigenetic control. In conclusion, DNA methylation changes in members of the DNA replication/repair process group best explain the heritable nature of cell proliferation impairment found in the zebrafish DM/MM model. These results are consistent with human diabetic epigenetic studies and provide one explanation for the persistence of long term tissue complications as seen in diabetes. PMID:26981540

  9. Pre-UV-treatment of cells results in enhanced host cell reactivation of a UV damaged reporter gene in CHO-AA8 chinese hamster ovary cells but not in transcription-coupled repair deficient CHO-UV61 cells.

    PubMed

    Liu, Lili; Rainbow, Andrew J

    2004-12-01

    We have used a non-replicating recombinant adenovirus, Ad5MCMVlacZ, which expresses the beta-galactosidase reporter gene, to examine both constitutive and inducible repair of UV-damaged DNA in repair proficient CHO-AA8 Chinese hamster ovary cells and in mutant CHO-UV61 cells which are deficient in the transcription-coupled repair (TCR) pathway of nucleotide excision repair. Host cell reactivation (HCR) of beta-galactosidase activity for UV-irradiated Ad5MCMVlacZ was significantly reduced in non-irradiated CHO-UV61 cells compared to that in non-irradiated CHO-AA8 cells suggesting that repair in the transcribed strand of the UV-damaged reporter gene in untreated cells utilizes TCR. Prior UV-irradiation of cells with low UV fluences resulted in a transient enhancement of HCR for expression of the UV-damaged reporter gene in CHO-AA8 cells but not in TCR deficient CHO-UV61 cells. These results suggest the presence of an inducible DNA pathway in CHO cells that results from an enhancement of TCR or a mechanism that involves the TCR pathway. PMID:16158195

  10. The sir locus of Escherichia coli: a gene involved in SOS-independent repair of mitomycin C-induced DNA damage.

    PubMed

    Kumaresan, K R; Jayaraman, R

    1990-03-01

    A Mud1 (lac Apr) insertion has been isolated in a delta (lac)recA+ lexA3(Ind-)rpoB87 gyrA87 mutant of Escherichia coli resulting in a decrease in mitomycin C tolerance and an increase in post-mitomycin C DNA degradation. The mitomycin C sensitivity of the insertion mutant is not further increased by substituting either the rpoB87 or the gyrA mutation by the respective wild-type alleles. However, when both rpoB87 and gyrA87 mutations are replaced by rpoB+ and gyrA+ the strain becomes hypersensitive to mitomycin C. Inactivation of recA in the insertion mutant has no effect on its mitomycin C sensitivity provided both rpoB87 and gyrA87 are present. When either or both of the mutations is/are replaced by the wild-type allele inactivation of recA renders the strain hypersensitive to mitomycin C. The locus of Mud1 (lac Apr) insertion, designated sir (SOS-independent repair), has been mapped between 57 and 61 min on the E. coli linkage map. Expression of the sir gene seems to be constitutive and not enhanced by mitomycin C. These results are discussed in relation to the SOS-independent repair of mitomycin C-induced DNA damage reported earlier. PMID:2155386

  11. Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair.

    PubMed

    He, Xiangjun; Tan, Chunlai; Wang, Feng; Wang, Yaofeng; Zhou, Rui; Cui, Dexuan; You, Wenxing; Zhao, Hui; Ren, Jianwei; Feng, Bo

    2016-05-19

    CRISPR/Cas9-induced site-specific DNA double-strand breaks (DSBs) can be repaired by homology-directed repair (HDR) or non-homologous end joining (NHEJ) pathways. Extensive efforts have been made to knock-in exogenous DNA to a selected genomic locus in human cells; which, however, has focused on HDR-based strategies and was proven inefficient. Here, we report that NHEJ pathway mediates efficient rejoining of genome and plasmids following CRISPR/Cas9-induced DNA DSBs, and promotes high-efficiency DNA integration in various human cell types. With this homology-independent knock-in strategy, integration of a 4.6 kb promoterless ires-eGFP fragment into the GAPDH locus yielded up to 20% GFP+ cells in somatic LO2 cells, and 1.70% GFP+ cells in human embryonic stem cells (ESCs). Quantitative comparison further demonstrated that the NHEJ-based knock-in is more efficient than HDR-mediated gene targeting in all human cell types examined. These data support that CRISPR/Cas9-induced NHEJ provides a valuable new path for efficient genome editing in human ESCs and somatic cells. PMID:26850641

  12. Tumor Mismatch Repair Immunohistochemistry and DNA MLH1 Methylation Testing of Patients With Endometrial Cancer Diagnosed at Age Younger Than 60 Years Optimizes Triage for Population-Level Germline Mismatch Repair Gene Mutation Testing

    PubMed Central

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

    2014-01-01

    Purpose Clinicopathologic data from a population-based endometrial cancer cohort, unselected for age or family history, were analyzed to determine the optimal scheme for identification of patients with germline mismatch repair (MMR) gene mutations. Patients and Methods Endometrial cancers from 702 patients recruited into the Australian National Endometrial Cancer Study (ANECS) were tested for MMR protein expression using immunohistochemistry (IHC) and for MLH1 gene promoter methylation in MLH1-deficient cases. MMR mutation testing was performed on germline DNA of patients with MMR-protein deficient tumors. Prediction of germline mutation status was compared for combinations of tumor characteristics, age at diagnosis, and various clinical criteria (Amsterdam, Bethesda, Society of Gynecologic Oncology, ANECS). Results Tumor MMR-protein deficiency was detected in 170 (24%) of 702 cases. Germline testing of 158 MMR-deficient cases identified 22 truncating mutations (3% of all cases) and four unclassified variants. Tumor MLH1 methylation was detected in 99 (89%) of 111 cases demonstrating MLH1/PMS2 IHC loss; all were germline MLH1 mutation negative. A combination of MMR IHC plus MLH1 methylation testing in women younger than 60 years of age at diagnosis provided the highest positive predictive value for the identification of mutation carriers at 46% versus ≤ 41% for any other criteria considered. Conclusion Population-level identification of patients with MMR mutation-positive endometrial cancer is optimized by stepwise testing for tumor MMR IHC loss in patients younger than 60 years, tumor MLH1 methylation in individuals with MLH1 IHC loss, and germline mutations in patients exhibiting loss of MSH6, MSH2, or PMS2 or loss of MLH1/PMS2 with absence of MLH1 methylation. PMID:24323032

  13. Predisposing factors and prevention of Clostridium perfringens-associated enteritis.

    PubMed

    Allaart, Janneke G; van Asten, Alphons J A M; Gröne, Andrea

    2013-09-01

    Clostridium perfringens is one of the major causes of intestinal disease in humans and animals. Its pathogenicity is contributed to by the production of a variety of toxins. In addition, predisposing environmental factors are important for the induction of C. perfringens-associated enteritis as shown by infection models. Environmental contamination, gastric and intestinal pH, intestinal microflora, nutrition, concurrent infections, and medical interventions may influence the intestinal colonization, growth, and toxin production by C. perfringens. Prevention of C. perfringens-associated enteritis may be mediated by the use of feed additives like probiotics, prebiotics, organic acids, essential oils, bacteriophages, lysozymes, bacteriocins, and antimicrobial peptides. Here we summarize and discuss published data on the influence of different environmental predisposing factors and preventive measures. Further research should focus on feed composition and feed additives in order to prevent C. perfringens-associated enteritis.

  14. Meta-analysis of estrogen response in MCF-7 distinguishes early target genes involved in signaling and cell proliferation from later target genes involved in cell cycle and DNA repair

    PubMed Central

    2011-01-01

    Background Many studies have been published outlining the global effects of 17β-estradiol (E2) on gene expression in human epithelial breast cancer derived MCF-7 cells. These studies show large variation in results, reporting between ~100 and ~1500 genes regulated by E2, with poor overlap. Results We performed a meta-analysis of these expression studies, using the Rank product method to obtain a more accurate and stable list of the differentially expressed genes, and of pathways regulated by E2. We analyzed 9 time-series data sets, concentrating on response at 3-4 hrs (early) and at 24 hrs (late). We found >1000 statistically significant probe sets after correction for multiple testing at 3-4 hrs, and >2000 significant probe sets at 24 hrs. Differentially expressed genes were examined by pathway analysis. This revealed 15 early response pathways, mostly related to cell signaling and proliferation, and 20 late response pathways, mostly related to breast cancer, cell division, DNA repair and recombination. Conclusions Our results confirm that meta-analysis identified more differentially expressed genes than the individual studies, and that these genes act together in networks. These results provide new insight into E2 regulated mechanisms, especially in the context of breast cancer. PMID:21878096

  15. Depressive behavior induced by social isolation of predisposed female rats.

    PubMed

    Zanier-Gomes, Patrícia Helena; de Abreu Silva, Tomaz Eugênio; Zanetti, Guilherme Cia; Benati, Évelyn Raquel; Pinheiro, Nanci Mendes; Murta, Beatriz Martins Tavares; Crema, Virgínia Oliveira

    2015-11-01

    Depression is a mood disorder that is more prevalent in women and has been closely associated with chronic stress. Many models of depression have been suggested that consider different forms of stress. In fact, stress is present in the life of every human being, but only a few develop depression. Accordingly, it seems wrong to consider all stressed animals to be depressed, emphasizing the importance of predisposition for this mood disorder. Based on this finding, we evaluated a predisposition to depressive behavior of female rats on the forced swim test (FST), and the more immobile the animal was during the FST, the more predisposed to depression it was considered to be. Then, animals were subjected to the stress of social isolation for 21 days and were re-evaluated by the FST. The Predisposed/Isolated rats presented higher immobility times. Once all the rats had prior experience in the FST, we calculated an Index of Increase by Isolation, confirming the previous results. Based on this result, we considered the Predisposed/Isolated group as presenting depressive behavior ('Depressed') and the Nonpredisposed/Nonisolated group as the control group ('Nondepressed'). The animals were distributed into 4 new groups: Nondepressed/Vehicle, Nondepressed/Amitriptyline, Depressed/Vehicle, Depressed/Amitriptyline. After 21 days of treatment, only the Depressed/Vehicle group differed from the other 3 groups, demonstrating the efficacy of amitriptyline in treating the depressive behavior of the Depressed animals, validating the model. This study shows that conducting an FST prior to any manipulation can predict predisposition to depressive behavior in female rats and that the social isolation of predisposed animals for 21 days is effective in inducing depressive behavior. This behavior can be considered real depressive behavior because it takes into account predisposition, chronic mild stress, and the prevalent gender.

  16. CITED2 silencing sensitizes cancer cells to cisplatin by inhibiting p53 trans-activation and chromatin relaxation on the ERCC1 DNA repair gene

    PubMed Central

    Liu, Yu-Chin; Chang, Pu-Yuan; Chao, Chuck C.-K.

    2015-01-01

    In this study, we show that silencing of CITED2 using small-hairpin RNA (shCITED2) induced DNA damage and reduction of ERCC1 gene expression in HEK293, HeLa and H1299 cells, even in the absence of cisplatin. In contrast, ectopic expression of ERCC1 significantly reduced intrinsic and induced DNA damage levels, and rescued the effects of CITED2 silencing on cell viability. The effects of CITED2 silencing on DNA repair and cell death were associated with p53 activity. Furthermore, CITED2 silencing caused severe elimination of the p300 protein and markers of relaxed chromatin (acetylated H3 and H4, i.e. H3K9Ac and H3K14Ac) in HEK293 cells. Chromatin immunoprecipitation assays further revealed that DNA damage induced binding of p53 along with H3K9Ac or H3K14Ac at the ERCC1 promoter, an effect which was almost entirely abrogated by silencing of CITED2 or p300. Moreover, lentivirus-based CITED2 silencing sensitized HeLa cell line-derived tumor xenografts to cisplatin in immune-deficient mice. These results demonstrate that CITED2/p300 can be recruited by p53 at the promoter of the repair gene ERCC1 in response to cisplatin-induced DNA damage. The CITED2/p300/p53/ERCC1 pathway is thus involved in the cell response to cisplatin and represents a potential target for cancer therapy. PMID:26384430

  17. CITED2 silencing sensitizes cancer cells to cisplatin by inhibiting p53 trans-activation and chromatin relaxation on the ERCC1 DNA repair gene.

    PubMed

    Liu, Yu-Chin; Chang, Pu-Yuan; Chao, Chuck C-K

    2015-12-15

    In this study, we show that silencing of CITED2 using small-hairpin RNA (shCITED2) induced DNA damage and reduction of ERCC1 gene expression in HEK293, HeLa and H1299 cells, even in the absence of cisplatin. In contrast, ectopic expression of ERCC1 significantly reduced intrinsic and induced DNA damage levels, and rescued the effects of CITED2 silencing on cell viability. The effects of CITED2 silencing on DNA repair and cell death were associated with p53 activity. Furthermore, CITED2 silencing caused severe elimination of the p300 protein and markers of relaxed chromatin (acetylated H3 and H4, i.e. H3K9Ac and H3K14Ac) in HEK293 cells. Chromatin immunoprecipitation assays further revealed that DNA damage induced binding of p53 along with H3K9Ac or H3K14Ac at the ERCC1 promoter, an effect which was almost entirely abrogated by silencing of CITED2 or p300. Moreover, lentivirus-based CITED2 silencing sensitized HeLa cell line-derived tumor xenografts to cisplatin in immune-deficient mice. These results demonstrate that CITED2/p300 can be recruited by p53 at the promoter of the repair gene ERCC1 in response to cisplatin-induced DNA damage. The CITED2/p300/p53/ERCC1 pathway is thus involved in the cell response to cisplatin and represents a potential target for cancer therapy.

  18. Mouse BAZ1A (ACF1) Is Dispensable for Double-Strand Break Repair but Is Essential for Averting Improper Gene Expression during Spermatogenesis

    PubMed Central

    Dowdle, James A.; Mehta, Monika; Kass, Elizabeth M.; Vuong, Bao Q.; Inagaki, Akiko; Egli, Dieter; Jasin, Maria; Keeney, Scott

    2013-01-01

    ATP-dependent chromatin remodelers control DNA access for transcription, recombination, and other processes. Acf1 (also known as BAZ1A in mammals) is a defining subunit of the conserved ISWI-family chromatin remodelers ACF and CHRAC, first purified over 15 years ago from Drosophila melanogaster embryos. Much is known about biochemical properties of ACF and CHRAC, which move nucleosomes in vitro and in vivo to establish ordered chromatin arrays. Genetic studies in yeast, flies and cultured human cells clearly implicate these complexes in transcriptional repression via control of chromatin structures. RNAi experiments in transformed mammalian cells in culture also implicate ACF and CHRAC in DNA damage checkpoints and double-strand break repair. However, their essential in vivo roles in mammals are unknown. Here, we show that Baz1a-knockout mice are viable and able to repair developmentally programmed DNA double-strand breaks in the immune system and germ line, I-SceI endonuclease-induced breaks in primary fibroblasts via homologous recombination, and DNA damage from mitomycin C exposure in vivo. However, Baz1a deficiency causes male-specific sterility in accord with its high expression in male germ cells, where it displays dynamic, stage-specific patterns of chromosomal localization. Sterility is caused by pronounced defects in sperm development, most likely a consequence of massively perturbed gene expression in spermatocytes and round spermatids in the absence of BAZ1A: the normal spermiogenic transcription program is largely intact but more than 900 other genes are mis-regulated, primarily reflecting inappropriate up-regulation. We propose that large-scale changes in chromatin composition that occur during spermatogenesis create a window of vulnerability to promiscuous transcription changes, with an essential function of ACF and/or CHRAC chromatin remodeling activities being to safeguard against these alterations. PMID:24244200

  19. Craniosynostosis repair

    MedlinePlus

    ... will be asleep and will not feel pain. Traditional surgery is called open repair. It includes these ... helps keep the swelling down. Talking, singing, playing music, and telling stories may help soothe your child. ...

  20. The genetic variations in DNA repair genes ERCC2 and XRCC1 were associated with the overall survival of advanced non-small-cell lung cancer patients.

    PubMed

    Wang, Suhan; Wang, Jianzhong; Bai, Yansen; Wang, Qing; Liu, Li; Zhang, Kai; Hong, Xiaohua; Deng, Qifei; Zhang, Xiaomin; He, Meian; Wu, Tangchun; Xu, Ping; Guo, Huan

    2016-09-01

    It was reported that DNA repair can confer cancer cell resistance to therapeutic treatments by activating antiapoptotic cellular defense. We hypothesized that genetic variants of DNA repair genes may be associated with lung cancer prognosis. Seventeen tagging single-nucleotide polymorphism (tagSNPs) selected from 12 DNA repair genes were genotyped in 280 advanced non-small-cell lung cancer (NSCLC) patients by TaqMan assay. The associations of these SNPs and overall survival of advanced NSCLC patients were investigated. Advanced NSCLC patients carrying ERCC2 rs50872 CT+TT genotypes had significantly longer median survival time (MST) and decreased death risk than patients with rs50872 CC genotype [log-rank P = 0.031; adjusted HR(95% CI) = 0.73 (0.55-0.98), P = 0.033]. These effects were mainly seen among younger patients (≤65 years old) [HR(95% CI) = 0.57 (0.37-0.87), P = 0.010], patients without surgery [HR(95% CI) = 0.68 (0.47-0.98), P = 0.036] but with chemotherapy [HR(95% CI) = 0.64 (0.46-0.91), P = 0.012] or radiotherapy [HR(95% CI) = 0.58 (0.38-0.89), P = 0.013]. Meanwhile, compared to advanced NSCLC patients with rs25487 GG genotype, patients carrying XRCC1 rs25487 GA+AA genotypes had significantly shorter MST (MST = 11.7 vs. 16.7, log-rank P = 0.048). In addition, advanced NSCLC patients carrying the ERCC2 rs50872 CC in combination with XRCC1 rs25487 GA+AA genotype had the shortest MST (11.2 month) and highest death risk [HR(95% CI) = 1.70 (1.15-2.52), P = 0.008] when compared with those carrying rs50872 CT+TT and rs25487 GG genotype (MST = 22.0 month). The ERCC2 rs50872 T allele was associated with favorable but XRCC1 rs25487 A allele with bad survival for advanced NSCLC in Chinese population, which may offer novel biomarkers for predicting clinical outcomes.

  1. The association of six polymorphisms of five genes involved in three steps of nucleotide excision repair pathways with hepatocellular cancer risk

    PubMed Central

    Yang, Huai-wei; Sun, Li-ping; Yuan, Yuan

    2016-01-01

    Background Hundreds of single nucleotide polymorphisms (SNPs) of the genes encoding nucleotide excision repair (NER) proteins are involved in every step of the DNA recognition–unwinding–incision process, which may affect cancer risk. However, only a limited number of studies have examined the association of NER SNPs with hepatocellular cancer (HCC) risk. Results In screening stage, single-locus analysis showed that six SNPs in five genes were associated with HCC risk, including three risk SNPs (XPA rs10817938, XPC rs1870134 and ERCC2 rs238417) and three protective SNPs (ERCC1 rs2298881 and rs3212961, and ERCC5 rs873601). In verification stage, only XPC rs1870134 was verified to be associated with HCC risk (P = 4.7 × 10−4). Furthermore, multivariate logistic regression and MDR analysis consistently revealed a gene–gene interaction among ERCC1 rs2298881 and XPC rs1870134 SNPs associated with HCC risk (Pinteraction = 0.023). When analyzing the effect of the positive SNP on the mRNA expression, we found XPC rs1870134 GG genotype which was associated with an increased HCC risk showed lower XPC mRNA expression. Methods This study designed as “screening-verification” experiments and included a total of 1472 participants (570 HCC patients vs. 902 controls). We explored 39 SNPs in eight genes involved in NER Pathways, including XPA, XPC, DDB2, ERCC3, ERCC2, ERCC1, ERCC4 and ERCC5, using Sequenom MassARRAY and KASPar platform. Eighty-six cases of HCC and the neighboring noncancerous tissues were subjected to the measurement of mRNA expression level of the promising gene. Conclusions XPC promoter rs1870134 SNP and SNP-SNP interaction were associated with HCC risk. PMID:26967386

  2. DIMETHYLARSINIC ACID ALTERS EXPRESSION OF OXIDATIVE STRESS AND DNA REPAIR GENES IN A DOSE DEPENDENT MANNER IN THE TRANSITIONAL EPITHELIUM OF THE URINARY BLADDER FROM FEMALE F344 RATS.

    EPA Science Inventory

    Dose-dependent alteration of oxidative stress and DNA repair gene expression by Dimethylarsinic acid [DMA(V)] in transitional epithelium of urinary bladder from female F344 rats.
    Arsenic (As) is a major concern as millions of people are at risk from drinking arsenic contaminat...

  3. DNA repair in Chromobacterium violaceum.

    PubMed

    Duarte, Fábio Teixeira; Carvalho, Fabíola Marques de; Bezerra e Silva, Uaska; Scortecci, Kátia Castanho; Blaha, Carlos Alfredo Galindo; Agnez-Lima, Lucymara Fassarella; Batistuzzo de Medeiros, Silvia Regina

    2004-03-31

    Chromobacterium violaceum is a Gram-negative beta-proteobacterium that inhabits a variety of ecosystems in tropical and subtropical regions, including the water and banks of the Negro River in the Brazilian Amazon. This bacterium has been the subject of extensive study over the last three decades, due to its biotechnological properties, including the characteristic violacein pigment, which has antimicrobial and anti-tumoral activities. C. violaceum promotes the solubilization of gold in a mercury-free process, and has been used in the synthesis of homopolyesters suitable for the production of biodegradable polymers. The complete genome sequence of this organism has been completed by the Brazilian National Genome Project Consortium. The aim of our group was to study the DNA repair genes in this organism, due to their importance in the maintenance of genomic integrity. We identified DNA repair genes involved in different pathways in C. violaceum through a similarity search against known sequences deposited in databases. The phylogenetic analyses were done using programs of the PHILYP package. This analysis revealed various metabolic pathways, including photoreactivation, base excision repair, nucleotide excision repair, mismatch repair, recombinational repair, and the SOS system. The similarity between the C. violaceum sequences and those of Neisserie miningitidis and Ralstonia solanacearum was greater than that between the C. violaceum and Escherichia coli sequences. The peculiarities found in the C. violaceum genome were the absence of LexA, some horizontal transfer events and a large number of repair genes involved with alkyl and oxidative DNA damage.

  4. Gene-Silencing Screen for Mammalian Axon Regeneration Identifies Inpp5f (Sac2) as an Endogenous Suppressor of Repair after Spinal Cord Injury

    PubMed Central

    Zou, Yixiao; Stagi, Massimiliano; Wang, Xingxing; Yigitkanli, Kazim; Siegel, Chad S.; Nakatsu, Fubito; Cafferty, William B. J.

    2015-01-01

    Axonal growth and neuronal rewiring facilitate functional recovery after spinal cord injury. Known interventions that promote neural repair remain limited in their functional efficacy. To understand genetic determinants of mammalian CNS axon regeneration, we completed an unbiased RNAi gene-silencing screen across most phosphatases in the genome. We identified one known and 17 previously unknown phosphatase suppressors of injury-induced CNS axon growth. Silencing Inpp5f (Sac2) leads to robust enhancement of axon regeneration and growth cone reformation. Results from cultured Inpp5f−/− neurons confirm lentiviral shRNA results from the screen. Consistent with the nonoverlapping substrate specificity between Inpp5f and PTEN, rapamycin does not block enhanced regeneration in Inpp5f−/− neurons, implicating mechanisms independent of the PI3K/AKT/mTOR pathway. Inpp5f−/− mice develop normally, but show enhanced anatomical and functional recovery after mid-thoracic dorsal hemisection injury. More serotonergic axons sprout and/or regenerate caudal to the lesion level, and greater numbers of corticospinal tract axons sprout rostral to the lesion. Functionally, Inpp5f-null mice exhibit enhanced recovery of motor functions in both open-field and rotarod tests. This study demonstrates the potential of an unbiased high-throughput functional screen to identify endogenous suppressors of CNS axon growth after injury, and reveals Inpp5f (Sac2) as a novel suppressor of CNS axon repair after spinal cord injury. SIGNIFICANCE STATEMENT The extent of axon regeneration is a critical determinant of neurological recovery from injury, and is extremely limited in the adult mammalian CNS. We describe an unbiased gene-silencing screen that uncovered novel molecules suppressing axonal regeneration. Inpp5f (Sac2) gene deletion promoted recovery from spinal cord injury with no side effects. The mechanism of action is distinct from another lipid phosphatase implicated in regeneration

  5. Pectus excavatum repair

    MedlinePlus

    Funnel chest repair; Chest deformity repair; Sunken chest repair; Cobbler's chest repair; Nuss repair; Ravitch repair ... There are two types of surgery to repair this condition -- open surgery ... surgery is done while the child is in a deep sleep and pain- ...

  6. Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis in Aedes aegypti.

    PubMed

    Basu, Sanjay; Aryan, Azadeh; Overcash, Justin M; Samuel, Glady Hazitha; Anderson, Michelle A E; Dahlem, Timothy J; Myles, Kevin M; Adelman, Zach N

    2015-03-31

    Conventional control strategies for mosquito-borne pathogens such as malaria and dengue are now being complemented by the development of transgenic mosquito strains reprogrammed to generate beneficial phenotypes such as conditional sterility or pathogen resistance. The widespread success of site-specific nucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 in model organisms also suggests that reprogrammable gene drive systems based on these nucleases may be capable of spreading such beneficial phenotypes in wild mosquito populations. Using the mosquito Aedes aegypti, we determined that mutations in the FokI domain used in TALENs to generate obligate heterodimeric complexes substantially and significantly reduce gene editing rates. We found that CRISPR/Cas9-based editing in the mosquito Ae. aegypti is also highly variable, with the majority of guide RNAs unable to generate detectable editing. By first evaluating candidate guide RNAs using a transient embryo assay, we were able to rapidly identify highly effective guide RNAs; focusing germ line-based experiments only on this cohort resulted in consistently high editing rates of 24-90%. Microinjection of double-stranded RNAs targeting ku70 or lig4, both essential components of the end-joining response, increased recombination-based repair in early embryos as determined by plasmid-based reporters. RNAi-based suppression of Ku70 concurrent with embryonic microinjection of site-specific nucleases yielded consistent gene insertion frequencies of 2-3%, similar to traditional transposon- or ΦC31-based integration methods but without the requirement for an initial docking step. These studies should greatly accelerate investigations into mosquito biology, streamline development of transgenic strains for field releases, and simplify the evaluation of novel Cas9-based gene drive systems. PMID:25775608

  7. The contribution of deleterious germline mutations in BRCA1, BRCA2 and the mismatch repair genes to ovarian cancer in the population.

    PubMed

    Song, Honglin; Cicek, Mine S; Dicks, Ed; Harrington, Patricia; Ramus, Susan J; Cunningham, Julie M; Fridley, Brooke L; Tyrer, Jonathan P; Alsop, Jennifer; Jimenez-Linan, Mercedes; Gayther, Simon A; Goode, Ellen L; Pharoah, Paul D P

    2014-09-01

    The aim of this study was to estimate the contribution of deleterious mutations in BRCA1, BRCA2, MLH1, MSH2, MSH6 and PMS2 to invasive epithelial ovarian cancer (EOC) in the population. The coding sequence and splice site boundaries of all six genes were amplified in germline DNA from 2240 invasive EOC cases and 1535 controls. Barcoded fragment libraries were sequenced using the Illumina GAII or HiSeq and sequence data for each subject de-multiplexed prior to interpretation. GATK and Annovar were used for variant detection and annotation. After quality control 2222 cases (99.2%) and 1528 controls (99.5%) were included in the final analysis. We identified 193 EOC cases (8.7%) carrying a deleterious mutation in at least one gene compared with 10 controls (0.65%). Mutations were most frequent in BRCA1 and BRCA2, with 84 EOC cases (3.8%) carrying a BRCA1 mutation and 94 EOC cases (4.2%) carrying a BRCA2 mutation. The combined BRCA1 and BRCA2 mutation prevalence was 11% in high-grade serous disease. Seventeen EOC cases carried a mutation in a mismatch repair gene, including 10 MSH6 mutation carriers (0.45%) and 4 MSH2 mutation carriers (0.18%). At least 1 in 10 women with high-grade serous EOC has a BRCA1 or BRCA2 mutation. The development of next generation sequencing technologies enables rapid mutation screening for multiple susceptibility genes at once, suggesting that routine clinical testing of all incidence cases should be considered.

  8. Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis in Aedes aegypti.

    PubMed

    Basu, Sanjay; Aryan, Azadeh; Overcash, Justin M; Samuel, Glady Hazitha; Anderson, Michelle A E; Dahlem, Timothy J; Myles, Kevin M; Adelman, Zach N

    2015-03-31

    Conventional control strategies for mosquito-borne pathogens such as malaria and dengue are now being complemented by the development of transgenic mosquito strains reprogrammed to generate beneficial phenotypes such as conditional sterility or pathogen resistance. The widespread success of site-specific nucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 in model organisms also suggests that reprogrammable gene drive systems based on these nucleases may be capable of spreading such beneficial phenotypes in wild mosquito populations. Using the mosquito Aedes aegypti, we determined that mutations in the FokI domain used in TALENs to generate obligate heterodimeric complexes substantially and significantly reduce gene editing rates. We found that CRISPR/Cas9-based editing in the mosquito Ae. aegypti is also highly variable, with the majority of guide RNAs unable to generate detectable editing. By first evaluating candidate guide RNAs using a transient embryo assay, we were able to rapidly identify highly effective guide RNAs; focusing germ line-based experiments only on this cohort resulted in consistently high editing rates of 24-90%. Microinjection of double-stranded RNAs targeting ku70 or lig4, both essential components of the end-joining response, increased recombination-based repair in early embryos as determined by plasmid-based reporters. RNAi-based suppression of Ku70 concurrent with embryonic microinjection of site-specific nucleases yielded consistent gene insertion frequencies of 2-3%, similar to traditional transposon- or ΦC31-based integration methods but without the requirement for an initial docking step. These studies should greatly accelerate investigations into mosquito biology, streamline development of transgenic strains for field releases, and simplify the evaluation of novel Cas9-based gene drive systems.

  9. Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis in Aedes aegypti

    PubMed Central

    Basu, Sanjay; Aryan, Azadeh; Overcash, Justin M.; Samuel, Glady Hazitha; Anderson, Michelle A. E.; Dahlem, Timothy J.; Myles, Kevin M.; Adelman, Zach N.

    2015-01-01

    Conventional control strategies for mosquito-borne pathogens such as malaria and dengue are now being complemented by the development of transgenic mosquito strains reprogrammed to generate beneficial phenotypes such as conditional sterility or pathogen resistance. The widespread success of site-specific nucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 in model organisms also suggests that reprogrammable gene drive systems based on these nucleases may be capable of spreading such beneficial phenotypes in wild mosquito populations. Using the mosquito Aedes aegypti, we determined that mutations in the FokI domain used in TALENs to generate obligate heterodimeric complexes substantially and significantly reduce gene editing rates. We found that CRISPR/Cas9-based editing in the mosquito Ae. aegypti is also highly variable, with the majority of guide RNAs unable to generate detectable editing. By first evaluating candidate guide RNAs using a transient embryo assay, we were able to rapidly identify highly effective guide RNAs; focusing germ line-based experiments only on this cohort resulted in consistently high editing rates of 24–90%. Microinjection of double-stranded RNAs targeting ku70 or lig4, both essential components of the end-joining response, increased recombination-based repair in early embryos as determined by plasmid-based reporters. RNAi-based suppression of Ku70 concurrent with embryonic microinjection of site-specific nucleases yielded consistent gene insertion frequencies of 2–3%, similar to traditional transposon- or ΦC31-based integration methods but without the requirement for an initial docking step. These studies should greatly accelerate investigations into mosquito biology, streamline development of transgenic strains for field releases, and simplify the evaluation of novel Cas9-based gene drive systems. PMID:25775608

  10. 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.

  11. The contribution of deleterious germline mutations in BRCA1, BRCA2 and the mismatch repair genes to ovarian cancer in the population

    PubMed Central

    Song, Honglin; Cicek, Mine S.; Dicks, Ed; Harrington, Patricia; Ramus, Susan J.; Cunningham, Julie M.; Fridley, Brooke L.; Tyrer, Jonathan P.; Alsop, Jennifer; Jimenez-Linan, Mercedes; Gayther, Simon A.; Goode, Ellen L.; Pharoah, Paul D.P.

    2014-01-01

    The aim of this study was to estimate the contribution of deleterious mutations in BRCA1, BRCA2, MLH1, MSH2, MSH6 and PMS2 to invasive epithelial ovarian cancer (EOC) in the population. The coding sequence and splice site boundaries of all six genes were amplified in germline DNA from 2240 invasive EOC cases and 1535 controls. Barcoded fragment libraries were sequenced using the Illumina GAII or HiSeq and sequence data for each subject de-multiplexed prior to interpretation. GATK and Annovar were used for variant detection and annotation. After quality control 2222 cases (99.2%) and 1528 controls (99.5%) were included in the final analysis. We identified 193 EOC cases (8.7%) carrying a deleterious mutation in at least one gene compared with 10 controls (0.65%). Mutations were most frequent in BRCA1 and BRCA2, with 84 EOC cases (3.8%) carrying a BRCA1 mutation and 94 EOC cases (4.2%) carrying a BRCA2 mutation. The combined BRCA1 and BRCA2 mutation prevalence was 11% in high-grade serous disease. Seventeen EOC cases carried a mutation in a mismatch repair gene, including 10 MSH6 mutation carriers (0.45%) and 4 MSH2 mutation carriers (0.18%). At least 1 in 10 women with high-grade serous EOC has a BRCA1 or BRCA2 mutation. The development of next generation sequencing technologies enables rapid mutation screening for multiple susceptibility genes at once, suggesting that routine clinical testing of all incidence cases should be considered. PMID:24728189

  12. The Saccharomyces cerevisiae MGT1 DNA repair methyltransferase gene: its promoter and entire coding sequence, regulation and in vivo biological functions.

    PubMed Central

    Xiao, W; Samson, L

    1992-01-01

    We previously cloned a yeast DNA fragment that, when fused with the bacterial lacZ promoter, produced O6-methylguanine DNA repair methyltransferase (MGT1) activity and alkylation resistance in Escherichia coli (Xiao et al., EMBO J. 10,2179). Here we describe the isolation of the entire MGT1 gene and its promoter by sequence directed chromosome integration and walking. The MGT1 promoter was fused to a lacZ reporter gene to study how MGT1 expression is controlled. MGT1 is not induced by alkylating agents, nor is it induced by other DNA damaging agents such as UV light. However, deletion analysis defined an upstream repression sequence, whose removal dramatically increased basal level gene expression. The polypeptide deduced from the complete MGT1 sequence contained 18 more N-terminal amino acids than that previously determined; the role of these 18 amino acids, which harbored a potential nuclear localization signal, was explored. The MGT1 gene was also cloned under the GAL1 promoter, so that MTase levels could be manipulated, and we examined MGT1 function in a MTase deficient yeast strain (mgt1). The extent of resistance to both alkylation-induced mutation and cell killing directly correlated with MTase levels. Finally we show that mgt1 S.cerevisiae has a higher rate of spontaneous mutation than wild type cells, indicating that there is an endogenous source of DNA alkylation damage in these eukaryotic cells and that one of the in vivo roles of MGT1 is to limit spontaneous mutations. PMID:1641326

  13. Celiac Disease in a Predisposed Subject (HLA-DQ2.5) with Coexisting Graves' Disease.

    PubMed

    Hwang, In Kyoung; Kim, Seon Hye; Lee, Unjoo; Chin, Sang Ouk; Rhee, Sang Youl; Oh, Seungjoon; Woo, Jeong Taek; Kim, Sung Woon; Kim, Young Seol; Chon, Suk

    2015-03-27

    Celiac disease is an intestinal autoimmune disorder, triggered by ingestion of a gluten-containing diet in genetically susceptible individuals. The genetic predisposition is related to human leukocyte antigen (HLA) class II genes, especially HLA-DQ2-positive patients. The prevalence of celiac disease has been estimated to be ~1% in Europe and the USA, but it is rarer and/or underdiagnosed in Asia. We report a case of celiac disease in a predisposed patient, with a HLA-DQ2 heterodimer, and Graves' disease that was treated successfully with a gluten-free diet. A 47-year-old woman complained of persistent chronic diarrhea and weight loss over a 9 month period. Results of all serological tests and stool exams were negative. However, the patient was found to carry the HLA DQ2 heterodimer. Symptoms improved after a gluten-free diet was initiated. The patient has been followed and has suffered no recurrence of symptoms while on the gluten-free diet. An overall diagnosis of celiac disease was made in a genetically predisposed patient (HLA-DQ2 heterodimer) with Graves' disease.

  14. The use of SHP-2 gene transduced bone marrow mesenchymal stem cells to promote osteogenic differentiation and bone defect repair in rat.

    PubMed

    Fan, Dapeng; Liu, Shen; Jiang, Shichao; Li, Zhiwei; Mo, Xiumei; Ruan, Hongjiang; Zou, Gang-Ming; Fan, Cunyi

    2016-08-01

    Bone tissue engineering is a promising approach for bone regeneration, in which growth factors play an important role. The tyrosine phosphatase Src-homology region 2-containing protein tyrosine phosphatase 2 (SHP2), encoded by the PTPN11 gene, is essential for the differentiation, proliferation and metabolism of osteoblasts. However, SHP-2 has never been systematically studied for its effect in osteogenesis. We predicted that overexpression of SHP-2 could promote bone marrow-derived mesenchymal stem cell (BMSC)osteogenic differentiation and SHP-2 transduced BMSCs could enhance new bone formation, determined using the following study groups: (1) BMSCs transduced with SHP-2 and induced with osteoblast-inducing liquid (BMSCs/SHP-2/OL); (2) BMSCs transduced with SHP-2 (BMSCs/-SHP-2); (3) BMSCs induced with osteoblast-inducing liquid (BMSCs/OL) and (4) pure BMSCs. Cells were assessed for osteogenic differentiation by quantitative real-time polymerase chain reaction analysis, western blot analysis, alkaline phosphatase activity and alizarin red S staining. For in vivo assessment, cells were combined with beta-tricalcium phosphate scaffolds and transplanted into rat calvarial defects for 8 weeks. Following euthanasia, skull samples were explanted for osteogenic evaluation, including micro-computed tomography measurement, histology and immunohistochemistry staining. SHP-2 and upregulation of its gene promoted BMSC osteogenic differentiation and therefore represents a potential new therapeutic approach to bone repair. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1871-1881, 2016.

  15. Femoral hernia repair

    MedlinePlus

    Femorocele repair; Herniorrhaphy; Hernioplasty - femoral ... During surgery to repair the hernia, the bulging tissue is pushed back in. The weakened area is sewn closed or strengthened. This repair ...

  16. Undescended testicle repair

    MedlinePlus

    Orchidopexy; Inguinal orchidopexy; Orchiopexy; Repair of undescended testicle; Cryptorchidism repair ... first year of life without treatment. Undescended testicle repair surgery is recommended for patients whose testicles do ...

  17. The Saccharomyces Cerevisiae Rad30 Gene, a Homologue of Escherichia Coli Dinb and Umuc, Is DNA Damage Inducible and Functions in a Novel Error-Free Postreplication Repair Mechanism

    PubMed Central

    McDonald, J. P.; Levine, A. S.; Woodgate, R.

    1997-01-01

    Damage-inducible mutagenesis in prokaryotes is largely dependent upon the activity of the UmuD'C-like proteins. Since many DNA repair processes are structurally and/or functionally conserved between prokaryotes and eukaryotes, we investigated the role of RAD30, a previously uncharacterized Saccharomyces cerevisiae DNA repair gene related to the Escherichia coli dinB, umuC and S. cerevisiae REV1 genes, in UV resistance and UV-induced mutagenesis. Similar to its prokaryotic homologues, RAD30 was found to be damage inducible. Like many S. cerevisiae genes involved in error-prone DNA repair, epistasis analysis clearly places RAD30 in the RAD6 group and rad30 mutants display moderate UV sensitivity reminiscent of rev mutants. However, unlike rev mutants, no defect in UV-induced reversion was seen in rad30 strains. While rad6 and rad18 are both epistatic to rad30, no epistasis was observed with rev1, rev3, rev7 or rad5, all of which are members of the RAD6 epistasis group. These findings suggest that RAD30 participates in a novel error-free repair pathway dependent on RAD6 and RAD18, but independent of REV1, REV3, REV7 and RAD5. PMID:9409821

  18. Completion of meiosis in male zebrafish (Danio rerio) despite lack of DNA mismatch repair gene mlh1.

    PubMed

    Leal, Marcelo C; Feitsma, Harma; Cuppen, Edwin; França, Luiz R; Schulz, Rüdiger W

    2008-04-01

    Mlh1 is a member of DNA mismatch repair (MMR) machinery and is also essential for the stabilization of crossovers during the first meiotic division. Recently, we have shown that zebrafish mlh1 mutant males are completely infertile because of a block in metaphase I, whereas females are fertile but have aneuploid progeny. When studying fertility in males in a two-fold more inbred background, we have however observed low numbers of fertilized eggs (approximately 0.4%). Histological examination of the testis has revealed that all spermatogenic stages prior to spermatids (spermatogonia, primary spermatocytes, and secondary spermatocytes) are significantly increased in the mutant, whereas the total weight of spermatids and spermatozoa is highly decreased (1.8 mg in wild-type vs. 0.1 mg in mutants), a result clearly different from our previous study in which outbred males lack secondary spermatocytes or postmeiotic cells. Thus, a delay of both meiotic divisions occurs rather than complete arrest during meiosis I in these males. Eggs fertilized with mutant sperm develop as malformed embryos and are aneuploid making this male phenotype much more similar to that previously described in the mutant females. Therefore, crossovers are still essential for proper meiosis, but meiotic cell divisions can progress without it, suggesting that this mutant is a suitable model for studying the cellular mechanisms of completing meiosis without crossover stabilization. PMID:18247060

  19. Human mediator MED17 subunit plays essential roles in gene regulation by associating with the transcription and DNA repair machineries.

    PubMed

    Kikuchi, Yuko; Umemura, Hiroyasu; Nishitani, Saori; Iida, Satoshi; Fukasawa, Rikiya; Hayashi, Hiroto; Hirose, Yutaka; Tanaka, Aki; Sugasawa, Kaoru; Ohkuma, Yoshiaki

    2015-03-01

    In eukaryotes, holo-Mediator consists of four modules: head, middle, tail, and CDK/Cyclin. The head module performs an essential function involved in regulation of RNA polymerase II (Pol II). We studied the human head module subunit MED17 (hMED17). Recent structural studies showed that yeast MED17 may function as a hinge connecting the neck and movable jaw regions of the head module to the fixed jaw region. Luciferase assays in hMED17-knockdown cells showed that hMED17 supports transcriptional activation, and pulldown assays showed that hMED17 interacted with Pol II and the general transcription factors TFIIB, TBP, TFIIE, and TFIIH. In addition, hMED17 bound to a DNA helicase subunit of TFIIH, XPB, which is essential for both transcription and nucleotide excision repair (NER). Because hMED17 associates with p53 upon UV-C irradiation, we treated human MCF-7 cells with either UV-C or the MDM2 inhibitor Nutlin-3. Both treatments resulted in accumulation of p53 in the nucleus, but hMED17 remained concentrated in the nucleus in response to UV-C. hMED17 colocalized with the NER factors XPB and XPG following UV-C irradiation, and XPG and XPB bound to hMED17 in vitro. These findings suggest that hMED17 may play essential roles in switching between transcription and NER.

  20. Pharmacogenetic Study in Rectal Cancer Patients Treated With Preoperative Chemoradiotherapy: Polymorphisms in Thymidylate Synthase, Epidermal Growth Factor Receptor, GSTP1, and DNA Repair Genes

    SciTech Connect

    Paez, David; Salazar, Juliana; Pare, Laia; Pertriz, Lourdes; Targarona, Eduardo; Rio, Elisabeth del; Barnadas, Agusti; Marcuello, Eugenio; Baiget, Montserrat

    2011-12-01

    Purpose: Several studies have been performed to evaluate the usefulness of neoadjuvant treatment using oxaliplatin and fluoropyrimidines for locally advanced rectal cancer. However, preoperative biomarkers of outcome are lacking. We studied the polymorphisms in thymidylate synthase, epidermal growth factor receptor, glutathione S-transferase pi 1 (GSTP1), and several DNA repair genes to evaluate their usefulness as pharmacogenetic markers in a cohort of 128 rectal cancer patients treated with preoperative chemoradiotherapy. Methods and Materials: Blood samples were obtained from 128 patients with Stage II-III rectal cancer. DNA was extracted from the peripheral blood nucleated cells, and the genotypes were analyzed by polymerase chain reaction amplification and automated sequencing techniques or using a 48.48 dynamic array on the BioMark system. The germline polymorphisms studied were thymidylate synthase, (VNTR/5 Prime UTR, 2R G>C single nucleotide polymorphism [SNP], 3R G>C SNP), epidermal growth factor receptor (Arg497Lys), GSTP1 (Ile105val), excision repair cross-complementing 1 (Asn118Asn, 8092C>A, 19716G>C), X-ray repair cross-complementing group 1 (XRCC1) (Arg194Trp, Arg280His, Arg399Gln), and xeroderma pigmentosum group D (Lys751Gln). The pathologic response, pathologic regression, progression-free survival, and overall survival were evaluated according to each genotype. Results: The Asterisk-Operator 3/ Asterisk-Operator 3 thymidylate synthase genotype was associated with a greater response rate (pathologic complete remission and microfoci residual tumor, 59% in Asterisk-Operator 3/ Asterisk-Operator 3 vs. 35% in Asterisk-Operator 2/ Asterisk-Operator 2 and Asterisk-Operator 2/ Asterisk-Operator 3; p = .013). For the thymidylate synthase genotype, the median progression-free survival was 103 months for the Asterisk-Operator 3/ Asterisk-Operator 3 patients and 84 months for the Asterisk-Operator 2/ Asterisk-Operator 2 and Asterisk-Operator 2/ Asterisk

  1. DNA repair in Mycoplasma gallisepticum

    PubMed Central

    2013-01-01

    Background DNA repair is essential for the maintenance of genome stability in all living beings. Genome size as well as the repertoire and abundance of DNA repair components may vary among prokaryotic species. The bacteria of the Mollicutes class feature a small genome size, absence of a cell wall, and a parasitic lifestyle. A small number of genes make Mollicutes a good model for a “minimal cell” concept. Results In this work we studied the DNA repair system of Mycoplasma gallisepticum on genomic, transcriptional, and proteomic levels. We detected 18 out of 22 members of the DNA repair system on a protein level. We found that abundance of the respective mRNAs is less than one per cell. We studied transcriptional response of DNA repair genes of M. gallisepticum at stress conditions including heat, osmotic, peroxide stresses, tetracycline and ciprofloxacin treatment, stationary phase and heat stress in stationary phase. Conclusions Based on comparative genomic study, we determined that the DNA repair system M. gallisepticum includes a sufficient set of proteins to provide a cell with functional nucleotide and base excision repair and mismatch repair. We identified SOS-response in M. gallisepticum on ciprofloxacin, which is a known SOS-inducer, tetracycline and heat stress in the absence of established regulators. Heat stress was found to be the strongest SOS-inducer. We found that upon transition to stationary phase of culture growth transcription of DNA repair genes decreases dramatically. Heat stress does not induce SOS-response in a stationary phase. PMID:24148612

  2. Activated RecA protein may induce expression of a gene that is not controlled by the LexA repressor and whose function is required for mutagenesis and repair of UV-irradiated bacteriophage lambda

    SciTech Connect

    Calsou, P.; Villaverde, A.; Defais, M.

    1987-10-01

    The activated form of the RecA protein (RecA) is known to be involved in the reactivation and mutagenesis of UV-irradiated bacteriophage lambda and in the expression of the SOS response in Escherichia coli K-12. The expression of the SOS response requires cleavage of the LexA repressor by RecA and the subsequent expression of LexA-controlled genes. The evidence presented here suggests that RecA induces the expression of a gene(s) that is not under LexA control and that is also necessary for maximal repair and mutagenesis of damaged phage. This conclusion is based on the chloramphenicol sensitivity of RecA -dependent repair and mutagenesis of damaged bacteriophage lambda in lexA(Def) hosts.

  3. Ectopic pregnancy and IUDs; incidence, risk rate and predisposing factors.

    PubMed

    Meirik, O; Nygren, K G

    1980-01-01

    During a period of 4 years, 1974-77, in Uppsala county; Sweden, 203 women underwent surgery for ectopic pregnancy with histological proof of the diagnosis. For the female population of fertile age this corresponds to 0.11 ectopics per 100 women 15-44 years of age, or 1.08 per 100 notified pregnancies, or 1.53 per 100 births. Fifty-five of the women with ectopic pregnancy were using an intrauterine device (IUD) (48 a copper-bearing IUD and 7 some other type of device), and 6 women used a low dose progestogen contraceptive. For users of copper-bearing IUDs the risk of an ectopic pregnancy was estimated to be 0.15 per 100 women years. When comparing this latter risk rate with the overall incidence rate of 0.11, it must be observed that the populations forming the denominator in these two rates differ with respect to some crucial characteristics. Nulliparity and predisposing factors were found statistically significantly more often in non-IUD-users with an ectopic pregnancy than in IUD-users. Such predisposing factors may be less prevalent in IUD-users, as in other populations. This may explain why ectopic pregnancy has been found to occur less frequently than theoretically expected among IUD-users. The "ectopic preventing" capacity of the IUD may therefore be considerably lower than has been previously claimed.

  4. The prevalence of predisposing deformity in osteoarthritic hip joints.

    PubMed

    Klit, Jakob; Gosvig, Kasper; Jacobsen, Steffen; Sonne-Holm, Stig; Troelsen, Anders

    2011-01-01

    It is becoming increasingly evident that hip joint deformities may be major contributors to the development of osteoarthritis, and the term 'idiopathic osteoarthritis' may be inappropriate in many cases. Our study cohort was derived from the Copenhagen Osteoarthritis Sub-study, a cross sectional population-based database of 4151 individuals, all of whom had a standard anteroposterior weight-bearing pelvic radiograph taken. Hip joints were classified according to type and degree of deformity. We defined hip osteoarthritis by a minimum joint space width of < or = 2 mm. This cut-off has a significant relationship in both sexes with the clinical presentation. The study cohort which fulfilled these inclusion criteria consisted of 322 females (149 right hips and 173 left hips) and 162 males (77 right hips and 85 left hips) with osteoarthritis. We found an overall prevalence of predisposing hip deformities in females of 62.4% and in males of 78.9%. Minor and major deformities showed the same prevalence. Both sexes had a comparable prevalence of minor and major hip joint deformity, except for pistol grip deformity, which was more prevalent in men. We concluded that 'idiopathic osteoarthritis' is uncommon, and that even minor predisposing deformities are associated with hip osteoarthritis.

  5. Intestinal obstruction repair

    MedlinePlus

    Repair of volvulus; Intestinal volvulus - repair; Bowel obstruction - repair ... Intestinal obstruction repair is done while you are under general anesthesia . This means you are asleep and DO NOT feel pain. ...

  6. Abnormal albumin gene expression is associated with weight loss in immunodeficient/DNA-repair-impaired wasted mice.

    SciTech Connect

    Libertin, C.; Weaver, P.; Mobarhan, S.; Woloschak, G.; Center for Mechanistic Biology and Biotechnology; Loyola Univ.; Loyola Univ.

    1994-01-01

    OBJECTIVE: Mice bearing the autosomal recessive mutation wst express a disease syndrome of immunodeficiency, neurologic dysfunction, increased sensitivity to the killing effects of ionizing radiation, and dramatic weight loss that begins at 21 days of age and progresses until death at 28-32 days of age. Because of the reported association between abnormal liver status and weight loss, we designed experiments to examine expression of a variety of liver-specific genes in wst/wst mice relative to littermates (wst/.) and parental strain (BCF1) controls. METHOD: Animals were individually weighed from ages 21-28 days to determine relative weight comparisons between wst/wst mice and controls. Dot blot hybridizations were set up to quantitate the accumulation of transcripts specific for alpha-fetoprotein, albumin and other liver-specific gene products. RESULTS: These results showed a 67% reduction in albumin mRNA expression in livers derived from wst/wst mice relative to both controls. Expression of alpha-fetoprotein, as well as a variety of other liver-specific genes [secretory component (SC), metallothionein (MT-2), cytochrome P1-450 (Cyt P1-450), transferrin receptor (Tf Rec), tumor necrosis factor (TNF), and immune-associated antigen (Ia)], was unaffected. CONCLUSIONS: These results suggest a relationship between low albumin expression and wasting syndromes in mice. In addition, our data suggest that the wasted mouse may serve as a unique model for subnormal albumin expression.

  7. Motorcycle Repair.

    ERIC Educational Resources Information Center

    Hein, Jim; Bundy, Mike

    This motorcycle repair curriculum guide contains the following ten areas of study: brake systems, clutches, constant mesh transmissions, final drives, suspension, mechanical starting mechanisms, electrical systems, fuel systems, lubrication systems, and overhead camshafts. Each area consists of one or more units of instruction. Each instructional…

  8. Outboard Repair.

    ERIC Educational Resources Information Center

    Hardway, Jack

    This consortium-developed instructor's manual for small engine repair (with focus on outboard motors) consists of the following nine instructional units: electrical remote control assembly, mechanical remote control assembly, tilt assemblies, exhaust housing, propeller and trim tabs, cooling system, mechanical gearcase, electrical gearcase, and…

  9. Snowmobile Repair.

    ERIC Educational Resources Information Center

    Helbling, Wayne

    This guide is designed to provide and/or improve instruction for occupational training in the area of snowmobile repair, and includes eight areas. Each area consists of one or more units of instruction, with each instructional unit including some or all of the following basic components: Performance objectives, suggested activities for teacher and…

  10. Stem cells with FGF4-bFGF fused gene enhances the expression of bFGF and improves myocardial repair in rats

    SciTech Connect

    Chen, Xiang-Qi; Chen, Liang-Long Fan, Lin; Fang, Jun; Chen, Zhao-Yang; Li, Wei-Wei

    2014-04-25

    Highlights: • BFGF exists only in the cytoplasm of live cells. • BFGF cannot be secreted into the extracellular space to promote cell growth. • We combine the secretion-promoting signal peptide of FGF4. • We successfully modified BMSCs with the fused genes of FGF4-bFGF. • We promoted the therapeutic effects of transplanted BMSCs in myocardial infarction. - Abstract: The aim of this study was to investigate whether the modification of bone marrow-derived mesenchymal stem cells (BMSCs) with the fused FGF4 (fibroblast growth factor 4)-bFGF (basic fibroblast growth factor) gene could improve the expression and secretion of BFGF, and increase the efficacies in repairing infarcted myocardium. We used In-Fusion technique to construct recombinant lentiviral vectors containing the individual gene of bFGF, enhanced green fluorescent protein (EGFP), or genes of FGF4-bFGF and EGFP, and then transfected these lentiviruses into rat BMSCs. We conducted an in vitro experiment to compare the secretion of bFGF in BMSCs infected by these lentiviruses and also examined their therapeutic effects in the treatment of myocardial infraction in a rodent study. Sixty rats were tested in the following five conditions: Group-SHAM received only sham operation as controls; Group-AMI received only injection of placebo PBS buffer; Group-BMSC, Group-bFGF and Group-FGF4-bFGF received implantation of BMSCs with empty lentivirus, bFGF lentivirus, and FGF4-bFGF lentivirus, respectively. Our results found out that the transplanted FGF4-bFGF BMSCs had the highest survival rate, and also the highest myocardial expression of bFGF and microvascular density as evidenced by Western blotting and immunohistochemistry, respectively. As compared to other groups, the Group-FGF4-BFGF rats had the lowest myocardial fibrotic fraction, and the highest left ventricular ejection fraction. These results suggest that the modification of BMSCs with the FGF4-bFGF fused gene can not only increase the expression of

  11. Turbine repair process, repaired coating, and repaired turbine component

    SciTech Connect

    Das, Rupak; Delvaux, John McConnell; Garcia-Crespo, Andres Jose

    2015-11-03

    A turbine repair process, a repaired coating, and a repaired turbine component are disclosed. The turbine repair process includes providing a turbine component having a higher-pressure region and a lower-pressure region, introducing particles into the higher-pressure region, and at least partially repairing an opening between the higher-pressure region and the lower-pressure region with at least one of the particles to form a repaired turbine component. The repaired coating includes a silicon material, a ceramic matrix composite material, and a repaired region having the silicon material deposited on and surrounded by the ceramic matrix composite material. The repaired turbine component a ceramic matrix composite layer and a repaired region having silicon material deposited on and surrounded by the ceramic matrix composite material.

  12. Subnormal albumin gene expression is associated with weight loss in immunodeficient/DNA-repair-deficient wasted mice

    SciTech Connect

    Libertin, C.R.; Weaver, P.; Woloschak, G.E. |; Mobarhan, S.

    1993-09-01

    Mice bearing the autosomal recessive mutation wst express a disease syndrome of immunodeficiency, neurologic dysfunction, and increased sensitivity to the killing effects of ionizing radiation. The mice were originally characterized as ``wasted`` because of their dramatic weight loss that begins at 21 days of age and progresses until death at 28-32 days of age. Because of the reported association between abnormal liver status and weight loss, we examined expression of a variety of liver-specific genes in wst/wst 10 mice relative to littermate (wst/{center_dot}) and parental strain (BCF{sub 1}) controls. Interestingly, the results revealed a greater than 67% reduction in albumin mRNA expression in livers derived from wst/wst mice relative to both controls. Expression of alpha-fetoprotein as well as a variety of other liver-specific genes (secretory component, metallothionein, cytochrome P{sub 1}450, transferrin receptor, tumor necrosis factor, and Ia antigen) was unaffected. These results suggest a relationship between low albumin expression and wasting syndromes in mice. In addition, we believe that our data suggest the wasted mouse as a unique model for subnormal albumin expression in humans.

  13. Efficient Generation of Gene-Modified Pigs Harboring Precise Orthologous Human Mutation via CRISPR/Cas9-Induced Homology-Directed Repair in Zygotes.

    PubMed

    Zhou, Xiaoyang; Wang, Lulu; Du, Yinan; Xie, Fei; Li, Liang; Liu, Yu; Liu, Chuanhong; Wang, Shiqiang; Zhang, Shibing; Huang, Xingxu; Wang, Yong; Wei, Hong

    2016-01-01

    Precise genetic mutation of model animals is highly valuable for functional investigation of human mutations. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9)-induced homology-directed repair (HDR) is usually used for precise genetic mutation, being limited by the relatively low efficiency compared with that of non-homologous end joining (NHEJ). Although inhibition of NHEJ was shown to enhance HDR-derived mutation, in this work, without inhibition of NHEJ, we first generated gene-modified pigs harboring precise orthologous human mutation (Sox10 c.A325>T) via CRISPR/Cas9-induced HDR in zygotes using single-strand oligo DNA (ssODN) as template with an efficiency as high as 80%, indicating that pig zygotes exhibited high activities of HDR relative to NHEJ and were highly amendable to genetic mutation via CIRSPR/Cas9-induced HDR. Besides, we found a higher concentration of ssODN remarkably reduced HDR-derived mutation in pig zygotes, suggesting a possible balance for optimal HDR-derived mutation in zygotes between the excessive accessibility to HDR templates and the activities of HDR relative to NHEJ which appeared to be negatively correlated to ssODN concentration. In addition, the HDR-derived mutation, as well as those from NHEJ, extensively integrated into various tissues including gonad of founder pig without detected off-targeting, suggesting CRISPR/Cas9-induced HDR in zygotes is a reliable approach for precise genetic mutation in pigs.

  14. Multivariate analysis of MLH1 c.1664T>C (p.Leu555Pro) mismatch repair gene variant demonstrates its pathogenicity.

    PubMed

    Farrell, M P; Hughes, D J; Drost, M; Wallace, A J; Cummins, R J; Fletcher, T A; Meany, M A; Kay, E W; de Wind, N; Power, D G; Andrews, E J; Green, A J; Gallagher, D J

    2013-12-01

    Genetic testing of an Irish kindred identified an exonic nucleotide substitution c.1664T>C (p.Leu555Pro) in the MLH1 mismatch repair (MMR) gene. This previously unreported variant is classified as a "variant of uncertain significance" (VUS). Immunohistochemical (IHC) analysis and microsatellite instability (MSI) studies, genetic testing, a literature and online MMR mutation database review, in silico phenotype prediction tools, and an in vitro MMR activity assay were used to study the clinical significance of this variant. The MLH1 c.1664T>C (p.Leu555Pro) VUS co-segregated with three cases of classic Lynch syndrome-associated malignancies over two generations, with consistent loss of MLH1 and PMS2 protein expression on IHC, and evidence of the MSI-High mutator phenotype. The leucine at position 555 is well conserved across a number of species, and this novel variant has not been reported as a normal polymorphism in the general population. In silico and in vitro analyses suggest that this variant may have a deleterious effect on the MLH1 protein and abrogate MMR activity. Evidence from clinical, histological, immunohistochemical, and molecular genetic data suggests that MLH1 c.1664T>C (p.Leu555Pro) is likely to be the pathogenic cause of Lynch syndrome in this family.

  15. Genomic diversity of Mycobacterium tuberculosis Beijing strains isolated in Tuscany, Italy, based on large sequence deletions, SNPs in putative DNA repair genes and MIRU-VNTR polymorphisms.

    PubMed

    Garzelli, Carlo; Lari, Nicoletta; Rindi, Laura

    2016-03-01

    The Beijing genotype of Mycobacterium tuberculosis is cause of global concern as it is rapidly spreading worldwide, is considered hypervirulent, and is most often associated to massive spread of MDR/XDR TB, although these epidemiological or pathological properties have not been confirmed for all strains and in all geographic settings. In this paper, to gain new insights into the biogeographical heterogeneity of the Beijing family, we investigated a global sample of Beijing strains (22% from Italian-born, 78% from foreign-born patients) by determining large sequence polymorphism of regions RD105, RD181, RD150 and RD142, single nucleotide polymorphism of putative DNA repair genes mutT4 and mutT2 and MIRU-VNTR profiles based on 11 discriminative loci. We found that, although our sample of Beijing strains showed a considerable genomic heterogeneity, yielding both ancient and recent phylogenetic strains, the prevalent successful Beijing subsets were characterized by deletions of RD105 and RD181 and by one nucleotide substitution in one or both mutT genes. MIRU-VNTR analysis revealed 47 unique patterns and 9 clusters including a total of 33 isolates (41% of total isolates); the relatively high proportion of Italian-born Beijing TB patients, often occurring in mixed clusters, supports the possibility of an ongoing cross-transmission of the Beijing genotype to autochthonous population. High rates of extra-pulmonary localization and drug-resistance, particularly MDR, frequently reported for Beijing strains in other settings, were not observed in our survey. PMID:26597137

  16. 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.

  17. Transcriptional and translational adjustments of psbA gene expression in mature chloroplasts during photoinhibition and subsequent repair of photosystem II.

    PubMed

    Kettunen, R; Pursiheimo, S; Rintamäki, E; Van Wijk, K J; Aro, E M

    1997-07-01

    The D1 reaction centre protein of photosystem II (PSII), encoded by the plastid psbA gene, has the highest turnover rate of all thylakoid proteins, due to light-induced damage to D1. The expression of the psbA gene was studied in chloroplasts of fully developed pea (Pisum sativum L.) leaves during high-light photoinhibitory treatment and subsequent restoration of PSII function at low light. psbA transcript levels were determined and the translational activity was followed by in vivo pulse-labelling, by in vitro translations with intact chloroplasts, and by run-off translations on isolated thylakoid membranes. PSII photochemical efficiency was determined in vivo by monitoring the ratio of variable fluorescence to maximal fluorescence (F(V)/F(M)). Enhanced D1 synthesis in pea leaves, upon a shift first from darkness to growth light and subsequently to high light, was accompanied by a substantial increase in the total number of pshA transcripts and by the accumulation of psbA mRNA x initiation complexes on thylakoid membrane. This suggested that high-light illumination increased the transcriptional activity of the psbA gene in mature leaves, and that enhanced translational initiation of psbA mRNA was followed by docking of the initiation complexes to the thylakoid membrane. The high-light-induced increase in the number of thylakoid-associated psbA mRNA x initiation complexes, occurred in parallel with enhanced in vivo D1 synthesis. This, however, did not result in an enhanced accumulation of D1 translation products in in vitro run-off translations when pea leaves were shifted from growth light to high light. This may suggest that at high light only a portion of thylakoid-associated psbA mRNA can be under translational elongation at a given moment. When the leaves were shifted from high light to low light to allow repair of PSII, thylakoid-associated psbA mRNA was rapidly released from the membrane and the high-light-induced pool of psbA transcripts was degraded. The

  18. [Breast cancer genetics. BRCA1 and BRCA2: the main genes for disease predisposition].

    PubMed

    Ruiz-Flores, P; Calderón-Garcidueñas, A L; Barrera-Saldaña, H A

    2001-01-01

    Breast cancer is among the most common world cancers. In Mexico this neoplasm has been progressively increasing since 1990 and is expected to continue. The risk factors for this disease are age, some reproductive factors, ionizing radiation, contraceptives, obesity and high fat diets, among other factors. The main risk factor for BC is a positive family history. Several families, in which clustering but no mendelian inheritance exists, the BC is due probably to mutations in low penetrance genes and/or environmental factors. In families with autosomal dominant trait, the BRCA1 and BRCA2 genes are frequently mutated. These genes are the two main BC susceptibility genes. BRCA1 predispose to BC and ovarian cancer, while BRCA2 mutations predispose to BC in men and women. Both are long genes, tumor suppressors, functioning in a cell cycle dependent manner, and it is believed that both switch on the transcription of several genes, and participate in DNA repair. The mutations profile of these genes is known in developed countries, while in Latin America their search has just began. A multidisciplinary group most be responsible of the clinical management of patients with mutations in BRCA1 and BRCA2, and the risk assignment and Genetic counseling most be done carefully.

  19. Prenatal TCDD Exposure Predisposes for Mammary Cancer in Rats

    PubMed Central

    Jenkins, Sarah; Rowell, Craig; Wang, Jun; Lamartiniere, Coral A.

    2007-01-01

    Epidemiological data are conflicting in the link between 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure and breast cancer causation. We have hypothesized that timing of exposure to endocrine disruptors, such as TCDD, will alter breast cancer susceptibility. Using a carcinogen induced rat mammary cancer model, we have shown that prenatal exposure to TCDD alters mammary gland differentiation and increases susceptibility for mammary cancer. Investigations into imprinting via DNA methylation mechanisms showed that there were no changes in protein expression in DNA methyltransferases, ER-alpha, ER-beta, GST-pi, or MDGI. Using 2-D gels and mass spectrometry, we have found seven proteins to be differentially regulated, including a decrease in superoxide dismutase 1 (SOD1). Down-regulation of SOD1 could provide an environment ill equipped to deal with subsequent free radical exposure. We conclude that prenatal TCDD can predispose for mammary cancer susceptibility in the adult offspring by altering the mammary proteome. PMID:17157473

  20. Rumen conditions that predispose cattle to pasture bloat.

    PubMed

    Majak, W; Howarth, R E; Cheng, K J; Hall, J W

    1983-08-01

    Rumen contents from the dorsal sac were examined before alfalfa ingestion to determine factors that predispose cattle to pasture bloat. Chlorophyll concentration, buoyancy of particulate matter, and rates of gas production were significantly higher in cattle that subsequently bloated than in those that did not. Higher chlorophyll in bloat cases indicated accumulation of suspended chloroplast particles in the dorsal sac, perhaps due to increased buoyancy of the particulate matter. The higher fermentation rates (in the presence of glucose) suggested that the latent capacity for gas production was due to microbial colonization of suspended feed particles. Chlorophyll 4 h after feeding was also higher in bloated as compared to unbloated animals. In short, the microbial colonization and retention of particulate matter provided active inocula for promoting rapid legume digestion. Consequently, gas production was enhanced when feeding commenced, but the fermentation gases were trapped by the buoyant, frothy ingesta, resulting in the condition of pasture bloat. PMID:6619348

  1. Nucleotide excision repair in humans.

    PubMed

    Spivak, Graciela

    2015-12-01

    The demonstration of DNA damage excision and repair replication by Setlow, Howard-Flanders, Hanawalt and their colleagues in the early 1960s, constituted the discovery of the ubiquitous pathway of nucleotide excision repair (NER). The serial steps in NER are similar in organisms from unicellular bacteria to complex mammals and plants, and involve recognition of lesions, adducts or structures that disrupt the DNA double helix, removal of a short oligonucleotide containing the offending lesion, synthesis of a repair patch copying the opposite undamaged strand, and ligation, to restore the DNA to its original form. The transcription-coupled repair (TCR) subpathway of NER, discovered nearly two decades later, is dedicated to the removal of lesions from the template DNA strands of actively transcribed genes. In this review I will outline the essential factors and complexes involved in NER in humans, and will comment on additional factors and metabolic processes that affect the efficiency of this important process. PMID:26388429

  2. Genetic variant in DNA repair gene GTF2H4 is associated with lung cancer risk: a large-scale analysis of six published GWAS datasets in the TRICL consortium.

    PubMed

    Wang, Meilin; Liu, Hongliang; Liu, Zhensheng; Yi, Xiaohua; Bickeboller, Heike; Hung, Rayjean J; Brennan, Paul; Landi, Maria Teresa; Caporaso, Neil; Christiani, David C; Doherty, Jennifer Anne; Amos, Christopher I; Wei, Qingyi

    2016-09-01

    DNA repair pathways maintain genomic integrity and stability, and dysfunction of DNA repair leads to cancer. We hypothesize that functional genetic variants in DNA repair genes are associated with risk of lung cancer. We performed a large-scale meta-analysis of 123,371 single nucleotide polymorphisms (SNPs) in 169 DNA repair genes obtained from six previously published genome-wide association studies (GWASs) of 12160 lung cancer cases and 16838 controls. We calculated odds ratios (ORs) with 95% confidence intervals (CIs) using the logistic regression model and used the false discovery rate (FDR) method for correction of multiple testing. As a result, 14 SNPs had a significant odds ratio (OR) for lung cancer risk with P FDR < 0.05, of which rs3115672 in MSH5 (OR = 1.20, 95% CI = 1.14-1.27) and rs114596632 in GTF2H4 (OR = 1.19, 95% CI = 1.12-1.25) at 6q21.33 were the most statistically significant (P combined = 3.99×10(-11) and P combined = 5.40×10(-10), respectively). The MSH5 rs3115672, but not GTF2H4 rs114596632, was strongly correlated with MSH5 rs3131379 in that region (r (2) = 1.000 and r (2) = 0.539, respectively) as reported in a previous GWAS. Importantly, however, the GTF2H4 rs114596632 T, but not MSH5 rs3115672 T, allele was significantly associated with both decreased DNA repair capacity phenotype and decreased mRNA expression levels. These provided evidence that functional genetic variants of GTF2H4 confer susceptibility to lung cancer.

  3. Haploinsufficiency of the ESCRT Component HD-PTP Predisposes to Cancer.

    PubMed

    Manteghi, Sanaz; Gingras, Marie-Claude; Kharitidi, Dmitri; Galarneau, Luc; Marques, Maud; Yan, Ming; Cencic, Regina; Robert, Francis; Paquet, Marilène; Witcher, Michael; Pelletier, Jerry; Pause, Arnim

    2016-05-31

    Endosomal sorting complexes required for transport (ESCRT) drive cell surface receptor degradation resulting in attenuation of oncogenic signaling and pointing to a tumor suppressor function. Here, we show that loss of function of an ESCRT protein (HD-PTP encoded by the PTPN23 gene, located on the tumor suppressor gene cluster 3p21.3) drives tumorigenesis in vivo. Indeed, Ptpn23(+/-) loss predisposes mice to sporadic lung adenoma, B cell lymphoma, and promotes Myc-driven lymphoma onset, dissemination, and aggressiveness. Ptpn23(+/-)-derived tumors exhibit an unaltered remaining allele and maintain 50% of HD-PTP expression. Consistent with the role of HD-PTP in attenuation of integrin recycling, cell migration, and invasion, hemizygous Ptpn23(+/-) loss increases integrin β1-dependent B cell lymphoma survival and dissemination. Finally, we reveal frequent PTPN23 deletion and downregulation in human tumors that correlates with poor survival. Altogether, we establish HD-PTP/PTPN23 as a prominent haploinsufficient tumor suppressor gene preventing tumor progression through control of integrin trafficking. PMID:27210750

  4. CDKN2A and BAP1 germline mutations predispose to melanoma and mesothelioma.

    PubMed

    Betti, M; Aspesi, A; Biasi, A; Casalone, E; Ferrante, D; Ogliara, P; Gironi, L C; Giorgione, R; Farinelli, P; Grosso, F; Libener, R; Rosato, S; Turchetti, D; Maffè, A; Casadio, C; Ascoli, V; Dianzani, C; Colombo, E; Piccolini, E; Pavesi, M; Miccoli, S; Mirabelli, D; Bracco, C; Righi, L; Boldorini, R; Papotti, M; Matullo, G; Magnani, C; Pasini, B; Dianzani, I

    2016-08-10

    BAP1 germline mutations predispose to a cancer predisposition syndrome that includes mesothelioma, cutaneous melanoma, uveal melanoma and other cancers. This co-occurrence suggests that these tumors share a common carcinogenic pathway. To evaluate this hypothesis, we studied 40 Italian families with mesothelioma and/or melanoma. The probands were sequenced for BAP1 and for the most common melanoma predisposition genes (i.e. CDKN2A, CDK4, TERT, MITF and POT1) to investigate if these genes may also confer susceptibility to mesothelioma. In two out of six families with both mesothelioma and melanoma we identified either a germline nonsense mutation (c.1153C > T, p.Arg385*) in BAP1 or a recurrent pathogenic germline mutation (c.301G > T, p.Gly101Trp) in CDKN2A. Our study suggests that CDKN2A, in addition to BAP1, could be involved in the melanoma and mesothelioma susceptibility, leading to the rare familial cancer syndromes. It also suggests that these tumors share key steps that drive carcinogenesis and that other genes may be involved in inherited predisposition to malignant mesothelioma and melanoma.

  5. Unusual maintenance of X chromosome inactivation predisposes female lymphocytes for increased expression from the inactive X

    PubMed Central

    Wang, Jianle; Syrett, Camille M.; Kramer, Marianne C.; Basu, Arindam; Atchison, Michael L.; Anguera, Montserrat C.

    2016-01-01

    Females have a greater immunological advantage than men, yet they are more prone to autoimmune disorders. The basis for this sex bias lies in the X chromosome, which contains many immunity-related genes. Female mammals use X chromosome inactivation (XCI) to generate a transcriptionally silent inactive X chromosome (Xi) enriched with heterochromatic modifications and XIST/Xist RNA, which equalizes gene expression between the sexes. Here, we examine the maintenance of XCI in lymphocytes from females in mice and humans. Strikingly, we find that mature naïve T and B cells have dispersed patterns of XIST/Xist RNA, and they lack the typical heterochromatic modifications of the Xi. In vitro activation of lymphocytes triggers the return of XIST/Xist RNA transcripts and some chromatin marks (H3K27me3, ubiquitin-H2A) to the Xi. Single-cell RNA FISH analysis of female T cells revealed that the X-linked immunity genes CD40LG and CXCR3 are biallelically expressed in some cells. Using knockout and knockdown approaches, we find that Xist RNA-binding proteins, YY1 and hnRNPU, are critical for recruitment of XIST/Xist RNA back to the Xi. Furthermore, we examined B cells from patients with systemic lupus erythematosus, an autoimmune disorder with a strong female bias, and observed different XIST RNA localization patterns, evidence of biallelic expression of immunity-related genes, and increased transcription of these genes. We propose that the Xi in female lymphocytes is predisposed to become partially reactivated and to overexpress immunity-related genes, providing the first mechanistic evidence to our knowledge for the enhanced immunity of females and their increased susceptibility for autoimmunity. PMID:27001848

  6. Association between single nucleotide polymorphisms (SNPs) of XRCC2 and XRCC3 homologous recombination repair genes and ovarian cancer in Polish women.

    PubMed

    Michalska, Magdalena M; Samulak, Dariusz; Romanowicz, Hanna; Jabłoński, Filip; Smolarz, Beata

    2016-04-01

    The variability, perceived in DNA repair genes, may be of clinical importance for evaluation of the risk of occurrence of a given type of cancer, its prophylactics and therapy. The aim of the present work was to evaluate associations between the risk of ovarian cancer and polymorphisms in the genes, encoding for two key proteins of homologous recombination: XRCC2 Arg188His (c. 563 G>A; rs3218536) and XRCC3 Thr241Met (c. 722 C>T; rs861539). The study consisted of 700 patients with ovarian cancer and 700 healthy subjects. Analysis of the gene polymorphisms was performed using PCR-RFLP (restriction length fragment polymorphism). We found a statistically significant increase of the 188His allele frequency (OR=4.01; 95% CI=3.40-4.72; p<.0001) of XRCC2 in ovarian cancer compared to healthy controls. There were no differences in the genotype and allele distributions and odds ratios of the XRCC3 Thr241Met polymorphism between patient and control groups. Association of these genetic polymorphisms with histological grading showed increased XRCC2 188Arg/His (OR=33.0; 95% CI=14.51-75.05; p<.0001) and 188His/His genotypes (OR=9.37; 95% CI=4.79-18.32; p<.0001) and XRCC3 241Thr/Met (OR=24.28; 95% CI=12.38-47.61; p<.0001) and 241Met/Met genotype frequencies (OR=17.00; 95% CI=8.42-34.28; p<.0001) in grading 1 (G1) as well as 188His (OR=2.78; 95% CI=2.11-3.69; p<.0001) and 241Met allele overrepresentation (OR=2.59; 95% CI=2.08-3.22; p<.0001) in G1 ovarian patients. Finally, with clinical FIGO staging under evaluation, an increase in XRCC2 188His/His homozygote and 188Arg/His heterozygote frequencies in staging I (SI) and XRCC3 Thr/Met heterozygote frequencies in SI was observed. The obtained results indicate that XRCC2 Arg188His and XRCC3 Thr241Met polymorphisms may be positively associated with the incidence of ovarian carcinoma in the population of Polish women.

  7. Polymorphisms and haplotypes of DNA repair and xenobiotic metabolism genes and risk of DNA damage in Chinese vinyl chloride monomer (VCM)-exposed workers.

    PubMed

    Zhu, Shou-Min; Xia, Zhao-Lin; Wang, Ai-Hong; Ren, Xue-Feng; Jiao, Jie; Zhao, Nai-Qing; Qian, Ji; Jin, Li; Christiani, David C

    2008-05-01

    In this case-control study, we investigated the association between DNA damage and genetic susceptibility among vinyl chloride monomer (VCM)-exposed workers. The cumulative exposure dose of VCM was calculated based on the workers' duration of exposure and the geometric mean concentration of VCM in the workplace. DNA damage to peripheral blood lymphocytes was measured by single cell gel electrophoresis (SCGE) assay, and single nucleotide-polymorphisms (SNPs) in xenobiotic metabolism and DNA repair genes were detected by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods. Univariate analysis showed that the CYP2E1 c1c2/c2c2 and XPD751 Lys/Gln and Gln/Gln genotypes were significantly associated with the levels of DNA damage (P<0.01 and 0.05, respectively). Further logistic regression analysis showed a significant association between CYP2E1 c1c2/c2c2 and DNA damage, and risk of having increased levels of DNA damage was more pronounced in those individuals having XRCC1 194 mutant genotypes and/or XPD751 Lys/Gln and Gln/Gln genotypes. Although most of the XPD and XRCC1 haplotypes did not show any significant difference, the XRCC1 haplotype Trp194-Arg280 was significantly over-represented in the case group (P<0.05; OR 2.09; 95% CI: 1.07-4.06) than in controls. Overall, our data suggest that the genotypes of CYP2E1, XRCC1 194, and XPD 751 were associated with the level of DNA damage and may contribute to individual sensitivity to DNA damage induced by VCM in the workplace.

  8. Polymorphisms in DNA-Repair Genes in a Cohort of Prostate Cancer Patients from Different Areas in Spain: Heterogeneity between Populations as a Confounding Factor in Association Studies

    PubMed Central

    Henríquez-Hernández, Luis Alberto; Valenciano, Almudena; Foro-Arnalot, Palmira; Álvarez-Cubero, María Jesús; Cozar, José Manuel; Suárez-Novo, José Francisco; Castells-Esteve, Manel; Ayala-Gil, Adriana; Fernández-Gonzalo, Pablo; Ferrer, Montse; Guedea, Ferrán; Sancho-Pardo, Gemma; Craven-Bartle, Jordi; Ortiz-Gordillo, María José; Cabrera-Roldán, Patricia; Herrera-Ramos, Estefanía; Lara, Pedro C.

    2013-01-01

    Background Differences in the distribution of genotypes between individuals of the same ethnicity are an important confounder factor commonly undervalued in typical association studies conducted in radiogenomics. Objective To evaluate the genotypic distribution of SNPs in a wide set of Spanish prostate cancer patients for determine the homogeneity of the population and to disclose potential bias. Design, Setting, and Participants A total of 601 prostate cancer patients from Andalusia, Basque Country, Canary and Catalonia were genotyped for 10 SNPs located in 6 different genes associated to DNA repair: XRCC1 (rs25487, rs25489, rs1799782), ERCC2 (rs13181), ERCC1 (rs11615), LIG4 (rs1805388, rs1805386), ATM (rs17503908, rs1800057) and P53 (rs1042522). The SNP genotyping was made in a Biotrove OpenArray® NT Cycler. Outcome Measurements and Statistical Analysis Comparisons of genotypic and allelic frequencies among populations, as well as haplotype analyses were determined using the web-based environment SNPator. Principal component analysis was made using the SnpMatrix and XSnpMatrix classes and methods implemented as an R package. Non-supervised hierarchical cluster of SNP was made using MultiExperiment Viewer. Results and Limitations We observed that genotype distribution of 4 out 10 SNPs was statistically different among the studied populations, showing the greatest differences between Andalusia and Catalonia. These observations were confirmed in cluster analysis, principal component analysis and in the differential distribution of haplotypes among the populations. Because tumor characteristics have not been taken into account, it is possible that some polymorphisms may influence tumor characteristics in the same way that it may pose a risk factor for other disease characteristics. Conclusion Differences in distribution of genotypes within different populations of the same ethnicity could be an important confounding factor responsible for the lack of validation of

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

    PubMed Central

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

    2015-01-01

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

  10. Negative regulation of mutS and mutH repair gene expression by the Hfq and RpoS global regulators of Escherichia coli K-12.

    PubMed Central

    Tsui, H C; Feng, G; Winkler, M E

    1997-01-01

    The MutS, MutL, and MutH proteins play major roles in several DNA repair pathways. We previously reported that the cellular amounts of MutS and MutH decreased by as much as 10-fold in stationary-phase cultures. Consequently, we tested whether the amounts of MutS, MutL, and MutH were regulated by two global regulators, RpoS (sigma38) and Hfq (HF-I [putative RNA chaperone]), which are involved in stationary-phase transition. We report here that mutations in hfq and rpoS reversed the stationary-phase down-regulation of the amounts of MutS and MutH. hfq regulation of the amount of MutS in stationary-phase cultures was mediated by RpoS-dependent and -independent mechanisms, whereas hfq regulation of the amount of MutH was mediated only through RpoS. Consistent with this interpretation, the amount of MutS but not MutH was regulated by Hfq, but not RpoS, in exponentially growing cells. The amount of MutL remained unchanged in rpoS, hfq-1, and rpoS+, hfq+ strains in exponentially growing and stationary-phase cultures and served as a control. The beta-galactosidase activities of single-copy mutS-lacZ operon and gene fusions suggested that hfq regulates mutS posttranscriptionally in exponentially growing cultures. RNase T2 protection assays revealed increased amounts of mutS transcript that are attributed to increased mutS transcript stability in hfq-1 mutants. Lack of Hfq also increased the amounts and stabilities of transcripts initiated from P(miaA) and P1hfqHS, two of the promoters for hfq, suggesting autoregulation, but did not change the half-life of bulk mRNA. These results suggest that the amounts of MutS and MutH may be adjusted in cells subjected to different stress conditions by an RpoS-dependent mechanism. In addition, Hfq directly or indirectly regulates several genes, including mutS, hfq, and miaA, by an RpoS-independent mechanism that destabilizes transcripts. PMID:9393714

  11. Epigenetic reduction of DNA repair in progression to gastrointestinal cancer

    PubMed Central

    Bernstein, Carol; Bernstein, Harris

    2015-01-01

    Deficiencies in DNA repair due to inherited germ-line mutations in DNA repair genes cause increased risk of gastrointestinal (GI) cancer. In sporadic GI cancers, mutations in DNA repair genes are relatively rare. However, epigenetic alterations that reduce expression of DNA repair genes are frequent in sporadic GI cancers. These epigenetic reductions are also found in field defects that give rise to cancers. Reduced DNA repair likely allows excessive DNA damages to accumulate in somatic cells. Then either inaccurate translesion synthesis past the un-repaired DNA damages or error-prone DNA repair can cause mutations. Erroneous DNA repair can also cause epigenetic alterations (i.e., epimutations, transmitted through multiple replication cycles). Some of these mutations and epimutations may cause progression to cancer. Thus, deficient or absent DNA repair is likely an important underlying cause of cancer. Whole genome sequencing of GI cancers show that between thousands to hundreds of thousands of mutations occur in these cancers. Epimutations that reduce DNA repair gene expression and occur early in progression to GI cancers are a likely source of this high genomic instability. Cancer cells deficient in DNA repair are more vulnerable than normal cells to inactivation by DNA damaging agents. Thus, some of the most clinically effective chemotherapeutic agents in cancer treatment are DNA damaging agents, and their effectiveness often depends on deficient DNA repair in cancer cells. Recently, at least 18 DNA repair proteins, each active in one of six DNA repair pathways, were found to be subject to epigenetic reduction of expression in GI cancers. Different DNA repair pathways repair different types of DNA damage. Evaluation of which DNA repair pathway(s) are deficient in particular types of GI cancer and/or particular patients may prove useful in guiding choice of therapeutic agents in cancer therapy. PMID:25987950

  12. Molecular Regulation of UV-Induced DNA Repair

    PubMed Central

    Shah, Palak; He, Yu-Ying

    2014-01-01

    Ultraviolet (UV) radiation from sunlight is a major etiologic factor for skin cancer, the most prevalent cancer in the U.S., as well as premature skin aging. In particular, UVB radiation causes formation of specific DNA damage photoproducts between pyrimidine bases. These DNA damage photoproducts are repaired by a process called nucleotide excision repair, also known as UV-induced DNA repair. When left unrepaired, UVB-induced DNA damage leads to accumulation of mutations, predisposing people to carcinogenesis as well as to premature aging. Genetic loss of nucleotide excision repair leads to severe disorders, namely, xeroderma pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne syndrome (CS), which are associated with predisposition to skin carcinogenesis at a young age as well as developmental and neurological conditions. Regulation of nucleotide excision repair is an attractive avenue to preventing or reversing these detrimental consequences of impaired nucleotide excision repair. Here we review recent studies on molecular mechanisms regulating nucleotide excision repair by extracellular cues and intracellular signaling pathways, with a special focus on the molecular regulation of individual repair factors. PMID:25534312

  13. Are reptiles predisposed to temperature-dependent sex determination?

    PubMed

    Georges, A; Ezaz, T; Quinn, A E; Sarre, S D

    2010-01-01

    Vertebrates show an astonishing array of sex determining mechanisms, including male and female heterogamety, multiple sex chromosome systems, environmental sex determination, parthenogenesis and hermaphroditism. Sex determination in mammals and birds is extraordinarily conservative compared to that of reptiles, amphibians and fish. In this paper, we explore possible explanations for the diversity of sex determining modes in reptiles, and in particular, address the prevalence of reptilian temperature-dependent sex determination (TSD) and its almost haphazard distribution across the reptile phylogeny. We suggest that reptiles are predisposed to evolving TSD from genotypic sex determination (GSD) by virtue of the uniquely variable thermal environment experienced by their embryos during the critical period in which sex is determined. Explicit mechanisms for canalization of sexual phenotype in the face of high thermal variation during development provide a context for thermolability in sex determination at extremes and the raw material for natural selection to move this thermolability into the developmental mainstream when there is a selective advantage to do so. Release of cryptic variation when canalization is challenged and fails at extremes may accelerate evolutionary transitions between GSD and TSD.

  14. Predisposing factors leading to depression in the British Army.

    PubMed

    Finnegan, Alan; Finnegan, Sara; McGee, Paula; Srinivasan, Mike; Simpson, Robin

    Few studies have explored the predisposing factors leading to depression within the British Army, and this qualitative investigation provides a novel approach to advance knowledge in this poorly researched area. Information was provided by army mental health (MH) clinicians, with results aligned to theoretical groupings under the headings of: occupational stressors; macho culture, stigma and bullying; unhappy young soldier; relationships and gender. These issues were influenced by peacetime and operational settings; the support offered by the Army Medical Services and unit command. The results indicate that Army personnel are exposed to multi-factorial stressors that are incremental/accumulative in nature. Soldiers can cope with extreme pressures, often in hostile environments, but often cannot cope with a failing relationship. Officers were worried about the occupational implications of reporting ill, and the negative impact on their career, and might seek support from private civilian agencies, which have potentially dangerous ramifications as they may still deploy. GPs refer female soldiers more frequently for a mental health assessment because women express their emotions more openly then men. Young disillusioned soldiers who want to leave the Army form the main group of personnel accessing mental health support, although often they are not clinically depressed.

  15. Brain aneurysm repair

    MedlinePlus

    ... aneurysm repair; Dissecting aneurysm repair; Endovascular aneurysm repair - brain; Subarachnoid hemorrhage - aneurysm ... Your scalp, skull, and the coverings of the brain are opened. A metal clip is placed at ...

  16. The Intertwined Roles of Transcription and Repair Proteins

    PubMed Central

    Fong, Yick W.; Cattoglio, Claudia; Tjian, Robert

    2014-01-01

    Transcription is apparently risky business. Its intrinsic mutagenic potential must be kept in check by networks of DNA repair factors that monitor the transcription process to repair DNA lesions that could otherwise compromise transcriptional fidelity and genome integrity. Intriguingly, recent studies point to an even more direct function of DNA repair complexes as co-activators of transcription and the unexpected role of “scheduled” DNA damage/repair at gene promoters. Paradoxically, spontaneous DNA double-strand breaks also induce ectopic transcription that is essential for repair. Thus, transcription, DNA damage and repair may be more physically and functionally intertwined than previously appreciated. PMID:24207023

  17. Somatic Overgrowth Predisposes to Seizures in Autism Spectrum Disorders

    PubMed Central

    Brachini, Francesca; Apicella, Fabio; Cosenza, Angela; Ferrari, Anna Rita; Guerrini, Renzo; Muratori, Filippo; Romano, Maria Francesca; Santorelli, Filippo M.; Tancredi, Raffaella; Sicca, Federico

    2013-01-01

    Background Comorbidity of Autism Spectrum Disorders with seizures or abnormal EEG (Autism-Epilepsy Phenotype) suggests shared pathomechanisms, and might be a starting point to identify distinct populations within the clinical complexity of the autistic spectrum. In this study, we tried to assess whether distinct subgroups, having distinctive clinical hallmarks, emerge from this comorbid condition. Methods Two-hundred and six individuals with idiopathic Autism Spectrum Disorders were subgrouped into three experimental classes depending on the presence of seizures and EEG abnormalities. Neurobehavioral, electroclinical and auxological parameters were investigated to identify differences among groups and features which increase the risk of seizures. Our statistical analyses used ANOVA, post-hoc multiple comparisons, and the Chi-squared test to analyze continuous and categorical variables. A correspondence analysis was also used to decompose significant Chi-squared and reduce variables dimensions. Results The high percentage of children with seizures (28.2% of our whole cohort) and EEG abnormalities (64.1%) confirmed that the prevalence of epilepsy in Autism Spectrum Disorders exceeds that of the general population. Seizures were associated with severe intellectual disability, and not with autism severity. Interestingly, tall stature (without macrocephaly) was significantly associated with EEG abnormalities or later onset seizures. However, isolated macrocephaly was equally distributed among groups or associated with early onset seizures when accompanied by tall stature. Conclusions Tall stature seems to be a phenotypic “biomarker” of susceptibility to EEG abnormalities or late epilepsy in Autism Spectrum Disorders and, when concurring with macrocephaly, predisposes to early onset seizures. Growth pattern might act as an endophenotypic marker in Autism-Epilepsy comorbidity, delineating distinct pathophysiological subtypes and addressing personalized diagnostic work

  18. [Speech impairment predisposes to cognitive deterioration in hepatic encephalopathy].

    PubMed

    Meparidze, M M; Kodua, T E; Lashkhi, K S

    2010-04-01

    Hepatic encephalopathy is a reversible neuro-psychiatric syndrome that complicates liver insufficiency. The changes are complex and disorders are detected in digestive and neural systems. Disturbed consciousness and intellectual deterioration, particularly communicative difficulties are observed: speech is slurred, voice monotonous, writing disturbances, amimic face and rigid posture. Difficulties of socialization and tendency to self-isolation are observed. Memory, attention and perception are decreased. We suppose that disorders of cognitive functions are determined by impairment of speech and other communicative abilities. According to the theories of linguistic determinism and linguistic relativity thought categories move through the mould of native language. That means, speech impairment causes misperception of the real world. To confirm this hypothesis we investigated 106 patients with following diseases: peptic ulcer - 46, fatty liver - 30, liver cirrhosis - 19, viral hepatitis - 11 and 19 controls. Brain magnetic resonance tomography was carried out and psychometric tests were performed to patients with symptoms of hepatic encephalopathy. Atrophic changes in frontal, temporal and insular area of brain cortex were revealed in most cases. Those regions are responsible for actor observation, imitation and emotion, i.e. for empathy and sociability. They are very sensitive to the increased levels of ammonia and glutamine. In case of early treatment only slight atrophic changes are presented but without treatment atrophic processes become stable and expressed by impairments of speech and entire communicative ability. Human beings are very much at the mercy of the particular language which has become the medium of expression for their society. They do not live in the objective world alone, or in the world of social activity alone. Accordingly, damage of speech in hepatic encephalopathy is primary and predisposes to cognitive dysfunction. PMID:20495225

  19. gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

    PubMed Central

    Chevrier, Sandy; Boidot, Romain

    2014-01-01

    The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3'-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA. PMID:25350069

  20. gDNA enrichment by a transposase-based technology for NGS analysis of the whole sequence of BRCA1, BRCA2, and 9 genes involved in DNA damage repair.

    PubMed

    Chevrier, Sandy; Boidot, Romain

    2014-10-06

    The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3'-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.

  1. Acute Normal Tissue Reactions in Head-and-Neck Cancer Patients Treated With IMRT: Influence of Dose and Association With Genetic Polymorphisms in DNA DSB Repair Genes

    SciTech Connect

    Werbrouck, Joke Ruyck, Kim de; Duprez, Frederic; Veldeman, Liv; Claes, Kathleen; Eijkeren, Marc van; Boterberg, Tom; Willems, Petra; Vral, Anne; Neve, Wilfried de; Thierens, Hubert

    2009-03-15

    Purpose: To investigate the association between dose-related parameters and polymorphisms in DNA DSB repair genes XRCC3 (c.-1843A>G, c.562-14A>G, c.722C>T), Rad51 (c.-3429G>C, c.-3392G>T), Lig4 (c.26C>T, c.1704T>C), Ku70 (c.-1310C>G), and Ku80 (c.2110-2408G>A) and the occurrence of acute reactions after radiotherapy. Materials and Methods: The study population consisted of 88 intensity-modulated radiation therapy (IMRT)-treated head-and-neck cancer patients. Mucositis, dermatitis, and dysphagia were scored using the Common Terminology Criteria (CTC) for Adverse Events v.3.0 scale. The population was divided into a CTC0-2 and CTC3+ group for the analysis of each acute effect. The influence of the dose on critical structures was analyzed using dose-volume histograms. Genotypes were determined by polymerase chain reaction (PCR) combined with restriction fragment length polymorphism or PCR-single base extension assays. Results: The mean dose (D{sub mean}) to the oral cavity and constrictor pharyngeus (PC) muscles was significantly associated with the development of mucositis and dysphagia, respectively. These parameters were considered confounding factors in the radiogenomics analyses. The XRCC3c.722CT/TT and Ku70c.-1310CG/GG genotypes were significantly associated with the development of severe dysphagia (CTC3+). No association was found between the investigated polymorphisms and the development of mucositis or dermatitis. A risk analysis model for severe dysphagia, which was developed based on the XRCC3c.722CT/TT and Ku70c.-1310CG/GG genotypes and the PC dose, showed a sensitivity of 78.6% and a specificity of 77.6%. Conclusions: The XRCC3c.722C>T and Ku70c.-1310C>G polymorphisms as well as the D{sub mean} to the PC muscles were highly associated with the development of severe dysphagia after IMRT. The prediction model developed using these parameters showed a high sensitivity and specificity.

  2. The Cockayne syndrome group B DNA repair protein as an anti-cancer target.

    PubMed

    Lu, Y; Mani, S; Kandimalla, E R; Yu, D; Agrawal, S; States, J C; Bregman, D B

    2001-12-01

    Cells from individuals with Cockayne syndrome (CS) have a defect in transcription-coupled DNA repair (TCR), which rapidly corrects certain DNA lesions located on the transcribed strand of active genes. Despite this DNA repair defect, individuals with CS (of which there are two complementation groups, CSA and CSB) do not demonstrate an elevated incidence of cancer. Recently, we demonstrated that disruption of the CSB gene reduces the spontaneous tumor rate in cancer predisposed Ink4a/ARF-/- mice as well as causing their embryo fibroblasts to proliferate more slowly and be more sensitive to UV-induced apoptosis. In the present study we characterized phosphorothioate backbone antisense oligodeoxynucleotides (AOs) that reduced the levels of CSB mRNA in A2780/CP70 ovarian carcinoma cells. The AOs caused the cells to proliferate more slowly and made them more sensitive to either cisplatin or oxaliplatin. The AOs also enhanced the cytotoxicity of hydrogen peroxide and gamma-radiation, both of which can induce oxidative DNA lesions, which are subject to TCR. The AOs did not potentiate the cytotoxicity of topotecan, which induces DNA strand breaks. Chemically modified () AOs (MBOs) targeting CSB were able to potentiate the anti-tumor effect of cisplatin against A2780/CP70 tumor xenografts formed in nude mice. The MBOs enabled a non-toxic (3 mg/kg) dose of cisplatin to have the same degree of anti-tumor efficacy as a more toxic (5 mg/kg) cisplatin dose. Collectively, these results suggest that the CSB gene product may be viewed as an anti-cancer target. PMID:11713576

  3. [Studies of the repair of radiation-induced genetic damage in Drosophila]. Annual progress report, September 1, 1990--July 1, 1991

    SciTech Connect

    1991-12-31

    Research is focused on the following areas: characterization of DNA double-strand break repair; using injected oligonucleotides as templates to repair double-strand DNA breaks; analysis of a gene required for postreplication repair; cloning of a gene required for resistance to DNA cross-linking agents; cloning of a gene required for excision repair; cloning of a gene required for X-ray resistance; and transposon tagging DNA repair genes.

  4. Book Repair Manual.

    ERIC Educational Resources Information Center

    Milevski, Robert J.

    1995-01-01

    This book repair manual developed for the Illinois Cooperative Conservation Program includes book structure and book problems, book repair procedures for 4 specific problems, a description of adhesive bindings, a glossary, an annotated list of 11 additional readings, book repair supplies and suppliers, and specifications for book repair kits. (LRW)

  5. Endothelial nitric oxide synthase haplotypes associated with hypertension do not predispose to cardiac hypertrophy.

    PubMed

    Vasconcellos, Vivian; Lacchini, Riccardo; Jacob-Ferreira, Anna L B; Sales, Maria L; Ferreira-Sae, Maria C; Schreiber, Roberto; Nadruz, Wilson; Tanus-Santos, Jose E

    2010-04-01

    Left ventricular hypertrophy (LVH) is a complication that may result from chronic hypertension. While nitric oxide (NO) deficiency has been associated with LVH, inconsistent results have been reported with regards to the association of endothelial NO synthase (eNOS) polymorphisms and LVH in hypertensive patients. This study aims to assess whether eNOS haplotypes are associated with LVH in hypertensive patients. This study included 101 healthy controls and 173 hypertensive patients submitted to echocardiography examination. Genotypes for three eNOS polymorphisms were determined: a single-nucleotide polymorphism in the promoter region (T-786C) and in exon 7 (Glu298Asp), and variable number of tandem repeats in intron 4. We found no significant association between eNOS genotypes and hypertension or with LVH (all p > 0.05). However, while we found two eNOS haplotypes associated with variable risk of hypertension (all p < 0.05), we found no significant associations between eNOS haplotypes and LVH (all p > 0.05), even after adjustment in multiple linear regression analysis. These findings suggest that eNOS haplotypes that have been associated with variable susceptibility to hypertension were not associated with LVH in hypertensive patients. Further studies are necessary to examine whether other genes downstream may interact with eNOS polymorphisms and predispose to LVH in hypertensive patients. PMID:20070154

  6. Polymorphisms in the DNA Repair Gene ERCC2/XPD and Breast Cancer Risk: A HapMap-Based Case–Control Study Among Han Women in a Chinese Less-Developed Area

    PubMed Central

    Wang, Tao; Wang, Haitao; Guo, Hongyun; Yang, Suisheng; Zhu, Gongjian; Guo, Huan; Wang, Lan; Li, Yonghui; Yang, Kai; Li, Haining; Min, Jianping; Li, Xueping; Hu, Qingrong; Wang, Yumei; Liu, Ying; Zhang, Binming; Chen, Xuezhong

    2014-01-01

    Aims: Genetic variations in DNA repair genes may impact repair functions, DNA damage, and breast cancer risk. This study is aimed to assess the associations of genetic polymorphisms in excision repair cross-complementing group 2 (ERCC2) with the risk of developing breast cancer. Materials and Methods: In total, 101 histopathologically confirmed breast cancer cases and 101 age/region-matched healthy controls were genotyped for rs3916840, rs1799793, and rs238416 in ERCC2 by polymerase chain reaction–restriction fragment length polymorphism. Results: The rs238416 heterozygous GA genotype combined with the rs238416 genotypes (GA+AA) showed a significant association with breast cancer susceptibility (corrected p<0.01, odds ratio [OR]=0.29, 95% confidence interval [CI]=0.15–0.54; corrected p<0.01, OR=0.31, 95% CI=0.17–0.56, respectively). The rs238416 GA genotype carriers had a decreased risk of breast cancer. However, we observed no significant association between the rs3916840 and rs1799793 polymorphisms in ERCC2 and breast cancer risk. Moreover, haplotype analysis showed that the ACG haplotype was associated with a significantly decreased risk of breast cancer, whereas the GCG haplotype was associated with a significantly increased risk of breast cancer (corrected p=0.004 and p=0.002, respectively). Multifactor dimensionality reduction analysis demonstrated that the interactions between rs3916840 and rs238416 were significantly synergistic. Conclusion: To the best of our knowledge, this study is the first to demonstrate that the rs238416 heterozygous genotype likely has a higher DNA repair capacity and, thus, can be protective against breast cancer in Chinese Han women. PMID:25117088

  7. POLYMORPHISMS IN THE DNA BASE EXCISION REPAIR GENES APEX1 AND XRCC1 AND LUNG CANCER RISK IN XUAN WEI, CHINA

    EPA Science Inventory

    The lung cancer mortality rate in Xuan Wei County is among the highest in China and has been attributed to exposure to indoor smoky coal emissions that contain very high levels of polycyclic aromatic hydrocarbons (PAHs). Nucleotide excision repair (NER) plays a key role in revers...

  8. 32 CFR 644.389 - Army military-modified predisposal procedures where E.O. 11954 surveys have been made.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 32 National Defense 4 2011-07-01 2011-07-01 false Army military-modified predisposal procedures... (Continued) DEPARTMENT OF THE ARMY (CONTINUED) REAL PROPERTY REAL ESTATE HANDBOOK Disposal Predisposal Action § 644.389 Army military—modified predisposal procedures where E.O. 11954 surveys have been made. (a)...

  9. 32 CFR 644.389 - Army military-modified predisposal procedures where E.O. 11954 surveys have been made.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 32 National Defense 4 2010-07-01 2010-07-01 true Army military-modified predisposal procedures... (Continued) DEPARTMENT OF THE ARMY (CONTINUED) REAL PROPERTY REAL ESTATE HANDBOOK Disposal Predisposal Action § 644.389 Army military—modified predisposal procedures where E.O. 11954 surveys have been made. (a)...

  10. 32 CFR 644.389 - Army military-modified predisposal procedures where E.O. 11954 surveys have been made.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 32 National Defense 4 2013-07-01 2013-07-01 false Army military-modified predisposal procedures... (Continued) DEPARTMENT OF THE ARMY (CONTINUED) REAL PROPERTY REAL ESTATE HANDBOOK Disposal Predisposal Action § 644.389 Army military—modified predisposal procedures where E.O. 11954 surveys have been made. (a)...

  11. 32 CFR 644.389 - Army military-modified predisposal procedures where E.O. 11954 surveys have been made.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 32 National Defense 4 2012-07-01 2011-07-01 true Army military-modified predisposal procedures... (Continued) DEPARTMENT OF THE ARMY (CONTINUED) REAL PROPERTY REAL ESTATE HANDBOOK Disposal Predisposal Action § 644.389 Army military—modified predisposal procedures where E.O. 11954 surveys have been made. (a)...

  12. 32 CFR 644.389 - Army military-modified predisposal procedures where E.O. 11954 surveys have been made.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 32 National Defense 4 2014-07-01 2013-07-01 true Army military-modified predisposal procedures... (Continued) DEPARTMENT OF THE ARMY (CONTINUED) REAL PROPERTY REAL ESTATE HANDBOOK Disposal Predisposal Action § 644.389 Army military—modified predisposal procedures where E.O. 11954 surveys have been made. (a)...

  13. 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 evidenc