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Sample records for testing high mutation

  1. High prevalence of BRCA1 stop mutation c.4183C>T in the Tyrolean population: implications for genetic testing.

    Science.gov (United States)

    Pölsler, Laura; Fiegl, Heidi; Wimmer, Katharina; Oberaigner, Willi; Amberger, Albert; Traunfellner, Pia; Morscher, Raphael J; Weber, Ingrid; Fauth, Christine; Wernstedt, Annekatrin; Sperner-Unterweger, Barbara; Oberguggenberger, Anne; Hubalek, Michael; Marth, Christian; Zschocke, Johannes

    2016-02-01

    Screening for founder mutations in BRCA1 and BRCA2 has been discussed as a cost-effective testing strategy in certain populations. In this study, comprehensive BRCA1 and BRCA2 testing was performed in a routine diagnostic setting. The prevalence of the BRCA1 stop mutation c.4183C>T, p.(Gln1395Ter), was determined in unselected breast and ovarian cancer patients from different regions in the Tyrol. Cancer registry data were used to evaluate the impact of this mutation on regional cancer incidence. The mutation c.4183C>T was detected in 30.4% of hereditary BRCA1-associated breast and ovarian cancer patients in our cohort. It was also identified in 4.1% of unselected (26% of unselected triple negative) Tyrolean breast cancer patients and 6.8% of unselected ovarian cancer patients from the Lower Inn Valley (LIV) region. Cancer incidences showed a region-specific increase in age-stratified breast and ovarian cancer risk with standardized incidence ratios of 1.23 and 2.13, respectively. We, thus, report a Tyrolean BRCA1 founder mutation that correlates to a local increase in the breast and ovarian cancer risks. On the basis of its high prevalence, we suggest that targeted genetic analysis should be offered to all women with breast or ovarian cancer and ancestry from the LIV region.

  2. Experiences from treatment-predictive KRAS testing; high mutation frequency in rectal cancers from females and concurrent mutations in the same tumor

    DEFF Research Database (Denmark)

    Jönsson, Mats; Ekstrand, Anna; Edekling, Thomas

    2009-01-01

    . METHODS: We used a real-time PCR based method to determine KRAS mutations in 136 colorectal cancers with mutations identified in 53 (39%) tumors. RESULTS: KRAS mutations were significantly more often found in rectal cancer (21/38, 55%) than in colon cancer (32/98, 33%) (P = 0.02). This finding...... was explained by marked differences mutation rates in female patients who showed mutations in 33% of the colon cancers and in 67% of the rectal cancers (P = 0.01). Concurrent KRAS mutations were identified in three tumors; two colorectal cancers harbored Gly12Asp/Gly13Asp and Gly12Cys/Gly13Asp and a third tumor...... carried Gly12Cys/Gly12Asp in an adenomatous component and additionally acquired Gly12Val in the invasive component. CONCLUSION: The demonstration of a particularly high KRAS mutation frequency among female rectal cancer patients suggests that this subset is the least likely to respond to anti...

  3. Highly Concordant Results Between Immunohistochemistry and Molecular Testing of Mutated V600E BRAF in Primary and Metastatic Melanoma.

    Science.gov (United States)

    Manfredi, Laure; Meyer, Nicolas; Tournier, Emilie; Grand, David; Uro-Coste, Emmanuelle; Rochaix, Philippe; Brousset, Pierre; Lamant, Laurence

    2016-06-15

    This study tested the sensitivity and specificity of VE1 antibody raised against BRAFV600E protein, on 189 melanoma samples, compared with molecular testing. In addition, the therapeutic response to BRAF inhibitors was analysed in 27 patients, according to staining intensity (scored from weak to strong) and pattern (homogeneous or heterogeneous). BRAFV600E status during melanoma progression was evaluated in a cohort of 54 patients with at least paired-samples. High sensitivity (98.6%) and specificity (97.7%) of VE1 were confirmed. During melanoma progression different samples showed concordant phenotypes. Heterogeneous VE1 staining was observed in 28.5% of cases, and progression-free survival was higher in patients with tumour samples displaying such staining. These findings suggest that only VE1-negative tumours would be genotyped to detect other BRAFV600 mutations, and that either primary melanoma or metastasis can be tested using immunohistochemistry, according to the material available.

  4. Careful selection of sample dilution and factor-V-deficient plasma makes the modified activated protein C resistance test highly specific for the factor V Leiden mutation.

    Science.gov (United States)

    de Ronde, H; Bertina, R M

    1999-01-01

    The aim of this study was to evaluate critically the recently modified activated-partial-thromboplastin-time (APTT)-based activated protein C (APC)-resistance tests, which are more specific for the factor V Leiden mutation than the first generation APC-resistance tests. The only modification to these tests is the predilution of the plasma sample in factor-V-deficient plasma. The intended effect of this predilution is to bring the concentrations of all clotting factors, except factor V, to the same normal levels. This, in principle, makes the tests also suitable for assaying the plasma of patients treated with oral anticoagulants and heparin, or of patients with a lupus anticoagulant. However, not every factor-V-deficient plasma is suitable for this application. Because the factor V:factor VIII ratio is important in establishing the APC ratio, the factor-V-deficient plasma should contain a sufficiently high factor VIII concentration. We also found that the optimal dilution to obtain the same APC ratios for patients, whether or not treated with coumarins or heparin, is not the same for each test or factor-V-deficient plasma. We compared two modified APTT-based APC-resistance tests (one developed in our laboratory and one commercial) with respect to their ability to discriminate between carriers and non-carriers of the factor V Leiden mutation. Both modified tests gave complete separation of carriers and non-carriers of the factor V Leiden mutation whether or not they are treated with anticoagulants. This makes these tests very suitable for routine screening.

  5. High Frequency of AML1/RUNX1 Point Mutations in Radiation-Associated Myelodysplastic Syndrome Around Semipalatinsk Nuclear Test Site

    OpenAIRE

    Dinara, ZHARLYGANOVA; Hironori, HARADA; Yuka, HARADA; Sergey, SHINKAREV; Zhaxybay, ZHUMADILOV; Aigul, ZHUNUSOVA; Naylya J., TCHAIZHUNUSOVA; Kazbek N., APSALIKOV; Vadim, KEMAIKIN; Kassym, ZHUMADILOV; Noriyuki, KAWANO; Akiro, KIMURA; Masaharu, HOSHI; State Research Center Institute of Biophysics, Federal Medical Biological Agency; Semipalatinsk State Medical Academy

    2008-01-01

    It is known that bone marrow is a sensitive organ to ionizing radiation, and many patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) have been diagnosed in radiation-treated cases and atomic bomb survivors in Hiroshima and Nagasaki. The AML1/RUNX1 gene has been known to be frequently mutated in MDS/AML patients among atomic bomb survivors and radiation therapy-related MDS/AML patients. In this study, we investigated the AML1 mutations in radiation-exposed patients wi...

  6. Factor V Leiden Mutation and PT 20210 Mutation Test

    Science.gov (United States)

    ... Patient Resources For Health Professionals Subscribe Search Factor V Leiden Mutation and PT 20210 Mutation Send Us ... As Activated Protein C Resistance APC Resistance Factor V R506Q PT G20210A Factor II 20210 Factor II ...

  7. Founder mutations characterise the mutation panorama in 200 Swedish index cases referred for Long QT syndrome genetic testing.

    Science.gov (United States)

    Stattin, Eva-Lena; Boström, Ida Maria; Winbo, Annika; Cederquist, Kristina; Jonasson, Jenni; Jonsson, Björn-Anders; Diamant, Ulla-Britt; Jensen, Steen M; Rydberg, Annika; Norberg, Anna

    2012-10-25

    Long QT syndrome (LQTS) is an inherited arrhythmic disorder characterised by prolongation of the QT interval on ECG, presence of syncope and sudden death. The symptoms in LQTS patients are highly variable, and genotype influences the clinical course. This study aims to report the spectrum of LQTS mutations in a Swedish cohort. Between March 2006 and October 2009, two hundred, unrelated index cases were referred to the Department of Clinical Genetics, Umeå University Hospital, Sweden, for LQTS genetic testing. We scanned five of the LQTS-susceptibility genes (KCNQ1, KCNH2, SCN5A, KCNE1, and KCNE2) for mutations by DHPLC and/or sequencing. We applied MLPA to detect large deletions or duplications in the KCNQ1, KCNH2, SCN5A, KCNE1, and KCNE2 genes. Furthermore, the gene RYR2 was screened in 36 selected LQTS genotype-negative patients to detect cases with the clinically overlapping disease catecholaminergic polymorphic ventricular tachycardia (CPVT). In total, a disease-causing mutation was identified in 103 of the 200 (52%) index cases. Of these, altered exon copy numbers in the KCNH2 gene accounted for 2% of the mutations, whereas a RYR2 mutation accounted for 3% of the mutations. The genotype-positive cases stemmed from 64 distinct mutations, of which 28% were novel to this cohort. The majority of the distinct mutations were found in a single case (80%), whereas 20% of the mutations were observed more than once. Two founder mutations, KCNQ1 p.Y111C and KCNQ1 p.R518*, accounted for 25% of the genotype-positive index cases. Genetic cascade screening of 481 relatives to the 103 index cases with an identified mutation revealed 41% mutation carriers who were at risk of cardiac events such as syncope or sudden unexpected death. In this cohort of Swedish index cases with suspected LQTS, a disease-causing mutation was identified in 52% of the referred patients. Copy number variations explained 2% of the mutations and 3 of 36 selected cases (8%) harboured a mutation in the

  8. Founder mutations characterise the mutation panorama in 200 Swedish index cases referred for Long QT syndrome genetic testing

    Directory of Open Access Journals (Sweden)

    Stattin Eva-Lena

    2012-10-01

    Full Text Available Abstract Background Long QT syndrome (LQTS is an inherited arrhythmic disorder characterised by prolongation of the QT interval on ECG, presence of syncope and sudden death. The symptoms in LQTS patients are highly variable, and genotype influences the clinical course. This study aims to report the spectrum of LQTS mutations in a Swedish cohort. Methods Between March 2006 and October 2009, two hundred, unrelated index cases were referred to the Department of Clinical Genetics, Umeå University Hospital, Sweden, for LQTS genetic testing. We scanned five of the LQTS-susceptibility genes (KCNQ1, KCNH2, SCN5A, KCNE1, and KCNE2 for mutations by DHPLC and/or sequencing. We applied MLPA to detect large deletions or duplications in the KCNQ1, KCNH2, SCN5A, KCNE1, and KCNE2 genes. Furthermore, the gene RYR2 was screened in 36 selected LQTS genotype-negative patients to detect cases with the clinically overlapping disease catecholaminergic polymorphic ventricular tachycardia (CPVT. Results In total, a disease-causing mutation was identified in 103 of the 200 (52% index cases. Of these, altered exon copy numbers in the KCNH2 gene accounted for 2% of the mutations, whereas a RYR2 mutation accounted for 3% of the mutations. The genotype-positive cases stemmed from 64 distinct mutations, of which 28% were novel to this cohort. The majority of the distinct mutations were found in a single case (80%, whereas 20% of the mutations were observed more than once. Two founder mutations, KCNQ1 p.Y111C and KCNQ1 p.R518*, accounted for 25% of the genotype-positive index cases. Genetic cascade screening of 481 relatives to the 103 index cases with an identified mutation revealed 41% mutation carriers who were at risk of cardiac events such as syncope or sudden unexpected death. Conclusion In this cohort of Swedish index cases with suspected LQTS, a disease-causing mutation was identified in 52% of the referred patients. Copy number variations explained 2% of the

  9. Factor V Leiden Mutation and PT 20210 Mutation Test

    Science.gov (United States)

    ... Sex Hormone Binding Globulin (SHBG) Shiga toxin-producing Escherichia coli Sickle Cell Tests Sirolimus Smooth Muscle Antibody (SMA) ... genetics.com . (2001 January 10, Modified). Coagulation Test Descriptions, Factor V Leiden (Activated Protein C Resistance Pcr ...

  10. Population testing for cancer predisposing BRCA1/BRCA2 mutations

    OpenAIRE

    Wardle, J.

    2014-01-01

    Background: Technological advances raise the possibility of systematic population-based genetic testing for cancer-predisposing mutations, but it is uncertain whether benefits outweigh disadvantages. We directly compared the psychological/quality-of-life consequences of such an approach to family history (FH)–based testing. Methods: In a randomized controlled trial of BRCA1/2 gene-mutation testing in the Ashkenazi Jewish (AJ) population, we compared testing all participants in the...

  11. Selective testing for calreticulin gene mutations in patients with splanchnic vein thrombosis: A prospective cohort study.

    Science.gov (United States)

    Poisson, Johanne; Plessier, Aurélie; Kiladjian, Jean-Jacques; Turon, Fanny; Cassinat, Bruno; Andreoli, Annalisa; De Raucourt, Emmanuelle; Goria, Odile; Zekrini, Kamal; Bureau, Christophe; Lorre, Florence; Cervantes, Francisco; Colomer, Dolors; Durand, François; Garcia-Pagan, Juan-Carlos; Casadevall, Nicole; Valla, Dominique-Charles; Rautou, Pierre-Emmanuel; Marzac, Christophe

    2017-09-01

    Myeloproliferative neoplasms (MPN) are the leading cause of splanchnic vein thrombosis (SVT). Janus kinase 2 gene (JAK2) V617F mutations are found in 80 to 90% of patients with SVT and MPN. Mutations of the calreticulin (CALR) gene have also been reported. However, as their prevalence ranges from 0 to 2%, the utility of routine testing is questionable. This study aimed to identify a group of patients with SVT at high risk of harboring CALR mutations and thus requiring this genetic testing. CALR, JAK2 V617F and thrombopoietin receptor gene (MPL) mutations were analysed in a test cohort that included 312 patients with SVT. Criteria to identify patients at high risk of CALR mutations in this test cohort was used and evaluated in a validation cohort that included 209 patients with SVT. In the test cohort, 59 patients had JAK2 V617F , five had CALR and none had MPL mutations. Patients with CALR mutations had higher spleen height and platelet count than patients without these mutations. All patients with CALR mutations had a spleen height ⩾16cm and platelet count >200×10 9 /L. These criteria had a positive predictive value of 56% (5/9) and a negative predictive value of 100% (0/233) for the identification of CALR mutations. In the validation cohort, these criteria had a positive predictive value of 33% (2/6) and a negative predictive value of 99% (1/96). CALR mutations should be tested in patients with SVT, a spleen height ⩾16cm, platelet count >200×10 9 /L, and no JAK2 V617F . This strategy avoids 96% of unnecessary CALR mutations testing. Lay summary: Mutations of the CALR gene are detected in 0 to 2% of patients with SVT, thus the utility of systematic CALR mutation testing to diagnose MPN is questionable. This study demonstrates that CALR mutations testing can be restricted to patients with SVT, a spleen height ⩾16cm, a platelet count >200×10 9 /L, and no JAK2 V617F . This strategy avoids 96% of unnecessary CALR mutations testing. Copyright © 2017 European

  12. Skin prick test reactivity to aeroallergens by filaggrin mutation status

    DEFF Research Database (Denmark)

    Hougaard, M G; Johansen, J D; Linneberg, A

    2014-01-01

    BACKGROUND: Studies have shown that filaggrin gene (FLG) mutations are positively associated with sensitization to aero allergens. We hypothesized that FLG mutations would also have an effect on the mean size of positive skin prick test (SPT) reactions as well as the number of positive reactions....... OBJECTIVE: To investigate the effect of FLG mutations on the mean size and the number of positive SPT reactions, as well as the association with positive specific IgE. METHODS: A random sample of 3335 adults from the general population in Denmark was genotyped for the R501X and 2282del4 mutations in the FLG....... SPT and specific IgE measurements to common aeroallergens were also performed. RESULTS: FLG mutations did not influence the mean size and number of positive SPT reactions. Also, no association was found between FLG mutations and specific IgE measurements. CONCLUSION: Our findings suggest that FLG...

  13. Dealing with the unexpected: consumer responses to direct-access BRCA mutation testing.

    Science.gov (United States)

    Francke, Uta; Dijamco, Cheri; Kiefer, Amy K; Eriksson, Nicholas; Moiseff, Bianca; Tung, Joyce Y; Mountain, Joanna L

    2013-01-01

    Background. Inherited BRCA gene mutations convey a high risk for breast and ovarian cancer, but current guidelines limit BRCA mutation testing to women with early-onset cancer and relatives of mutation-positive cases. Benefits and risks of providing this information directly to consumers are unknown. Methods. To assess and quantify emotional and behavioral reactions of consumers to their 23andMe Personal Genome Service(®) report of three BRCA mutations that are common in Ashkenazi Jews, we invited all 136 BRCA1 and BRCA2 mutation-positive individuals in the 23andMe customer database who had chosen to view their BRCA reports to participate in this IRB-approved study. We also invited 160 mutation-negative customers who were matched for age, sex and ancestry. Semi-structured phone interviews were completed for 32 mutation carriers, 16 women and 16 men, and 31 non-carriers. Questions addressed personal and family history of cancer, decision and timing of viewing the BRCA report, recollection of the result, emotional responses, perception of personal cancer risk, information sharing, and actions taken or planned. Results. Eleven women and 14 men had received the unexpected result that they are carriers of a BRCA1 185delAG or 5382insC, or BRCA2 6174delT mutation. None of them reported extreme anxiety and four experienced moderate anxiety that was transitory. Remarkably, five women and six men described their response as neutral. Most carrier women sought medical advice and four underwent risk-reducing procedures after confirmatory mutation testing. Male carriers realized that their test results implied genetic risk for female relatives, and several of them felt considerably burdened by this fact. Sharing mutation information with family members led to screening of at least 30 relatives and identification of 13 additional carriers. Non-carriers did not report inappropriate actions, such as foregoing cancer screening. All but one of the 32 mutation-positive participants

  14. Dealing with the unexpected: consumer responses to direct-access BRCA mutation testing

    Directory of Open Access Journals (Sweden)

    Uta Francke

    2013-02-01

    Full Text Available Background. Inherited BRCA gene mutations convey a high risk for breast and ovarian cancer, but current guidelines limit BRCA mutation testing to women with early-onset cancer and relatives of mutation-positive cases. Benefits and risks of providing this information directly to consumers are unknown.Methods. To assess and quantify emotional and behavioral reactions of consumers to their 23andMe Personal Genome Service® report of three BRCA mutations that are common in Ashkenazi Jews, we invited all 136 BRCA1 and BRCA2 mutation-positive individuals in the 23andMe customer database who had chosen to view their BRCA reports to participate in this IRB-approved study. We also invited 160 mutation-negative customers who were matched for age, sex and ancestry. Semi-structured phone interviews were completed for 32 mutation carriers, 16 women and 16 men, and 31 non-carriers. Questions addressed personal and family history of cancer, decision and timing of viewing the BRCA report, recollection of the result, emotional responses, perception of personal cancer risk, information sharing, and actions taken or planned.Results. Eleven women and 14 men had received the unexpected result that they are carriers of a BRCA1 185delAG or 5382insC, or BRCA2 6174delT mutation. None of them reported extreme anxiety and four experienced moderate anxiety that was transitory. Remarkably, five women and six men described their response as neutral. Most carrier women sought medical advice and four underwent risk-reducing procedures after confirmatory mutation testing. Male carriers realized that their test results implied genetic risk for female relatives, and several of them felt considerably burdened by this fact. Sharing mutation information with family members led to screening of at least 30 relatives and identification of 13 additional carriers. Non-carriers did not report inappropriate actions, such as foregoing cancer screening. All but one of the 32 mutation

  15. APC mutation spectrum of Norwegian familial adenomatous polyposis families: high ratio of novel mutations.

    Science.gov (United States)

    Andresen, Per Arne; Heimdal, Ketil; Aaberg, Kristin; Eklo, Katrine; Eklo, Kristin; Ariansen, Sarah; Silye, Alexandra; Fausa, Olav; Aabakken, Lars; Aretz, Stefan; Eide, Tor J; Gedde-Dahl, Tobias

    2009-10-01

    Familial adenomatous polyposis (FAP) is an autosomal dominantly inherited disease caused by mutations in the adenomatous polyposis coli (APC) gene. Massive formation of colorectal adenomas, of which some will inevitably develop into adenocarcinomas, is the hallmark of the disease. Characterization of causative APC mutations allows presymptomatic diagnosis, close follow-up and prophylactic intervention in families. To date more than 900 different germline mutations have been characterized worldwide demonstrating allelic heterogeneity. The germline mutation spectrum of APC identified in 69 apparently unrelated Norwegian FAP families are presented and discussed with reference to clinical phenotype and novel mutation rate. Different methods have been used over the years. However, all mutations were confirmed detectable by an implemented denaturing high-performance liquid chromatography screening approach. Multiplex ligation-dependent probe amplification analysis was employed for potential gross rearrangements. Fifty-three distinctive mutations were detected, of which 22 have been detected in Norway exclusively. Except for two major deletion mutations encompassing the entire APC, all mutations resulted in premature truncation of translation caused by non-sense (31%) or change in reading frame (69%). A high ratio of novel APC mutations continues to contribute to APC mutation heterogeneity causing FAP. This is the first comprehensive report of APC germline mutation spectrum in Norway.

  16. Evaluation of BRCA1 and BRCA2 mutation prevalence, risk prediction models and a multistep testing approach in French‐Canadian families with high risk of breast and ovarian cancer

    Science.gov (United States)

    Simard, Jacques; Dumont, Martine; Moisan, Anne‐Marie; Gaborieau, Valérie; Vézina, Hélène; Durocher, Francine; Chiquette, Jocelyne; Plante, Marie; Avard, Denise; Bessette, Paul; Brousseau, Claire; Dorval, Michel; Godard, Béatrice; Houde, Louis; Joly, Yann; Lajoie, Marie‐Andrée; Leblanc, Gilles; Lépine, Jean; Lespérance, Bernard; Malouin, Hélène; Parboosingh, Jillian; Pichette, Roxane; Provencher, Louise; Rhéaume, Josée; Sinnett, Daniel; Samson, Carolle; Simard, Jean‐Claude; Tranchant, Martine; Voyer, Patricia; BRCAs, INHERIT; Easton, Douglas; Tavtigian, Sean V; Knoppers, Bartha‐Maria; Laframboise, Rachel; Bridge, Peter; Goldgar, David

    2007-01-01

    Background and objective In clinical settings with fixed resources allocated to predictive genetic testing for high‐risk cancer predisposition genes, optimal strategies for mutation screening programmes are critically important. These depend on the mutation spectrum found in the population under consideration and the frequency of mutations detected as a function of the personal and family history of cancer, which are both affected by the presence of founder mutations and demographic characteristics of the underlying population. The results of multistep genetic testing for mutations in BRCA1 or BRCA2 in a large series of families with breast cancer in the French‐Canadian population of Quebec, Canada are reported. Methods A total of 256 high‐risk families were ascertained from regional familial cancer clinics throughout the province of Quebec. Initially, families were tested for a panel of specific mutations known to occur in this population. Families in which no mutation was identified were then comprehensively tested. Three algorithms to predict the presence of mutations were evaluated, including the prevalence tables provided by Myriad Genetics Laboratories, the Manchester Scoring System and a logistic regression approach based on the data from this study. Results 8 of the 15 distinct mutations found in 62 BRCA1/BRCA2‐positive families had never been previously reported in this population, whereas 82% carried 1 of the 4 mutations currently observed in ⩾2 families. In the subset of 191 families in which at least 1 affected individual was tested, 29% carried a mutation. Of these 27 BRCA1‐positive and 29 BRCA2‐positive families, 48 (86%) were found to harbour a mutation detected by the initial test. Among the remaining 143 inconclusive families, all 8 families found to have a mutation after complete sequencing had Manchester Scores ⩾18. The logistic regression and Manchester Scores provided equal predictive power, and both were significantly better

  17. Simultaneous mutation detection of three homoeologous genes in wheat by High Resolution Melting analysis and Mutation Surveyor®

    Directory of Open Access Journals (Sweden)

    Vincent Kate

    2009-12-01

    Full Text Available Abstract Background TILLING (Targeting Induced Local Lesions IN Genomes is a powerful tool for reverse genetics, combining traditional chemical mutagenesis with high-throughput PCR-based mutation detection to discover induced mutations that alter protein function. The most popular mutation detection method for TILLING is a mismatch cleavage assay using the endonuclease CelI. For this method, locus-specific PCR is essential. Most wheat genes are present as three similar sequences with high homology in exons and low homology in introns. Locus-specific primers can usually be designed in introns. However, it is sometimes difficult to design locus-specific PCR primers in a conserved region with high homology among the three homoeologous genes, or in a gene lacking introns, or if information on introns is not available. Here we describe a mutation detection method which combines High Resolution Melting (HRM analysis of mixed PCR amplicons containing three homoeologous gene fragments and sequence analysis using Mutation Surveyor® software, aimed at simultaneous detection of mutations in three homoeologous genes. Results We demonstrate that High Resolution Melting (HRM analysis can be used in mutation scans in mixed PCR amplicons containing three homoeologous gene fragments. Combining HRM scanning with sequence analysis using Mutation Surveyor® is sensitive enough to detect a single nucleotide mutation in the heterozygous state in a mixed PCR amplicon containing three homoeoloci. The method was tested and validated in an EMS (ethylmethane sulfonate-treated wheat TILLING population, screening mutations in the carboxyl terminal domain of the Starch Synthase II (SSII gene. Selected identified mutations of interest can be further analysed by cloning to confirm the mutation and determine the genomic origin of the mutation. Conclusion Polyploidy is common in plants. Conserved regions of a gene often represent functional domains and have high sequence

  18. A high frequent BRCA1 founder mutation identified in the Greenlandic population

    DEFF Research Database (Denmark)

    Harboe, Theresa Larriba; Eiberg, Hans; Kern, Peder

    2009-01-01

    Approximately 10% of all breast and ovarian cancers are dominantly inherited and mutations are mainly found in the BRCA 1 and 2 genes. The penetrance of BRCA1 mutations is reported to be between 68 and 92% and confers a 36-92% life time risk of breast cancer. Most mutations in BRCA1 are uniquely...... the clinical relevance of the mutation, we have examined ten breast cancer patients and nine ovarian cancer patients from Greenland for the presence of the p.Cys39Gly mutation. We found three ovarian cancer patients (33%) and one breast cancer patient (10%) carrying the mutation. The high number of women...... carrying a BRCA1 mutation known to trigger the development of potentially lethal diseases leads us to recommend an offer of genetic counselling and test for the mutation to all females of Inuit origin, thereby hopefully preventing a number of breast and ovarian cancer deaths....

  19. Prevalence and clinical association of gene mutations through multiplex mutation testing in patients with NSCLC: results from the ETOP Lungscape Project.

    Science.gov (United States)

    Kerr, K M; Dafni, U; Schulze, K; Thunnissen, E; Bubendorf, L; Hager, H; Finn, S; Biernat, W; Vliegen, L; Losa, J H; Marchetti, A; Cheney, R; Warth, A; Speel, E-J; Blackhall, F; Monkhorst, K; Jantus Lewintre, E; Tischler, V; Clark, C; Bertran-Alamillo, J; Meldgaard, P; Gately, K; Wrona, A; Vandenberghe, P; Felip, E; De Luca, G; Savic, S; Muley, T; Smit, E F; Dingemans, A-M C; Priest, L; Baas, P; Camps, C; Weder, W; Polydoropoulou, V; Geiger, T R; Kammler, R; Sumiyoshi, T; Molina, M A; Shames, D S; Stahel, R A; Peters, S

    2018-01-01

    Reported prevalence of driver gene mutations in non-small-cell lung cancer (NSCLC) is highly variable and clinical correlations are emerging. Using NSCLC biomaterial and clinical data from the European Thoracic Oncology Platform Lungscape iBiobank, we explore the epidemiology of mutations and association to clinicopathologic features and patient outcome (relapse-free survival, time-to-relapse, overall survival). Clinically annotated, resected stage I-III NSCLC FFPE tissue was assessed for gene mutation using a microfluidics-based multiplex PCR platform. Mutant-allele detection sensitivity is >1% for most of the ∼150 (13 genes) mutations covered in the multiplex test. Multiplex testing has been carried out in 2063 (76.2%) of the 2709 Lungscape cases (median follow-up 4.8 years). FFPE samples mostly date from 2005 to 2008, yet recently extracted DNA quality and quantity was generally good. Average DNA yield/case was 2.63 µg; 38 cases (1.4%) failed QC and were excluded from study; 95.1% of included cases allowed the complete panel of mutations to be tested. Most common were KRAS, MET, EGFR and PIK3CA mutations with overall prevalence of 23.0%, 6.8%, 5.4% and 4.9%, respectively. KRAS and EGFR mutations were significantly more frequent in adenocarcinomas: PIK3CA in squamous cell carcinomas. MET mutation prevalence did not differ between histology groups. EGFR mutations were found predominantly in never smokers; KRAS in current/former smokers. For all the above mutations, there was no difference in outcome between mutated and non-mutated cases. Archival FFPE NSCLC material is adequate for multiplex mutation analysis. In this large, predominantly European, clinically annotated stage I-III NSCLC cohort, none of the mutations characterized showed prognostic significance.

  20. Automation of diagnostic genetic testing: mutation detection by cyclic minisequencing.

    Science.gov (United States)

    Alagrund, Katariina; Orpana, Arto K

    2014-01-01

    The rising role of nucleic acid testing in clinical decision making is creating a need for efficient and automated diagnostic nucleic acid test platforms. Clinical use of nucleic acid testing sets demands for shorter turnaround times (TATs), lower production costs and robust, reliable methods that can easily adopt new test panels and is able to run rare tests in random access principle. Here we present a novel home-brew laboratory automation platform for diagnostic mutation testing. This platform is based on the cyclic minisequecing (cMS) and two color near-infrared (NIR) detection. Pipetting is automated using Tecan Freedom EVO pipetting robots and all assays are performed in 384-well micro plate format. The automation platform includes a data processing system, controlling all procedures, and automated patient result reporting to the hospital information system. We have found automated cMS a reliable, inexpensive and robust method for nucleic acid testing for a wide variety of diagnostic tests. The platform is currently in clinical use for over 80 mutations or polymorphisms. Additionally to tests performed from blood samples, the system performs also epigenetic test for the methylation of the MGMT gene promoter, and companion diagnostic tests for analysis of KRAS and BRAF gene mutations from formalin fixed and paraffin embedded tumor samples. Automation of genetic test reporting is found reliable and efficient decreasing the work load of academic personnel.

  1. Semantic Mutation Testing for Multi-Agent Systems

    OpenAIRE

    Huang, Zhan; Alexander, Robert David

    2015-01-01

    This paper introduces semantic mutation testing (SMT) into multiagent systems. SMT is a test assessment technique that makes changes to the interpretation of a program and then examines whether a given test set has the ability to detect each change to the original interpretation. These changes represent possible misunderstandings of how the program is interpreted. SMT is also a technique for assessing the robustness of a program to semantic changes. This paper applies SMT to three rule-based ...

  2. High specificity but low sensitivity of mutation-specific antibodies against EGFR mutations in non-small-cell lung cancer

    DEFF Research Database (Denmark)

    Bondgaard, Anna-Louise; Høgdall, Estrid; Mellemgaard, Anders

    2014-01-01

    Determination of epidermal growth factor receptor (EGFR) mutations has a pivotal impact on treatment of non-small-cell lung cancer (NSCLC). A standardized test has not yet been approved. So far, Sanger DNA sequencing has been widely used. Its rather low sensitivity has led to the development......, and staining score (multipum of intensity (graded 0-3) and percentages (0-100%) of stained tumor cells) was calculated. Positivity was defined as staining score >0. Specificity of exon19 antibody was 98.8% (95% confidence interval=95.9-99.9%) and of exon21 antibody 97.8% (95% confidence interval=94...... positive (immunohistochemistry positive, RT-PCR negative). One false positive exon21 mutation had staining score 300. The EGFR variantIII antibody showed no correlation to EGFR mutation status determined by RT-PCR or to EGFR immunohistochemistry. High specificity of the mutation-specific antibodies...

  3. Current guidelines for BRCA testing of breast cancer patients are insufficient to detect all mutation carriers.

    Science.gov (United States)

    Grindedal, Eli Marie; Heramb, Cecilie; Karsrud, Inga; Ariansen, Sarah Louise; Mæhle, Lovise; Undlien, Dag Erik; Norum, Jan; Schlichting, Ellen

    2017-06-21

    Identification of BRCA mutations in breast cancer (BC) patients influences treatment and survival and may be of importance for their relatives. Testing is often restricted to women fulfilling high-risk criteria. However, there is limited knowledge of the sensitivity of such a strategy, and of the clinical aspects of BC caused by BRCA mutations in less selected BC cohorts. The aim of this report was to address these issues by evaluating the results of BRCA testing of BC patients in South-Eastern Norway. 1371 newly diagnosed BC patients were tested with sequencing and Multi Ligation Probe Amplification (MLPA). Prevalence of mutations was calculated, and BC characteristics among carriers and non-carriers compared. Sensitivity and specificity of common guidelines for BRCA testing to identify carriers was analyzed. Number of identified female mutation positive relatives was evaluated. A pathogenic BRCA mutation was identified in 3.1%. Carriers differed from non-carriers in terms of age at diagnosis, family history, grade, ER/PR-status, triple negativity (TNBC) and Ki67, but not in HER2 and TNM status. One mutation positive female relative was identified per mutation positive BC patient. Using age of onset below 40 or TNBC as criteria for testing identified 32-34% of carriers. Common guidelines for testing identified 45-90%, and testing all below 60 years identified 90%. Thirty-seven percent of carriers had a family history of cancer that would have qualified for predictive BRCA testing. A Variant of Uncertain Significance (VUS) was identified in 4.9%. Mutation positive BC patients differed as a group from mutation negative. However, the commonly used guidelines for testing were insufficient to detect all mutation carriers in the BC cohort. Thirty-seven percent had a family history of cancer that would have qualified for predictive testing before they were diagnosed with BC. Based on our combined observations, we suggest it is time to discuss whether all BC patients

  4. A Novel Class of Tests for the Detection of Mitochondrial DNA–Mutation Involvement in Diseases

    OpenAIRE

    Sun, Fengzhu; Cui, Jing; Gavras, Haralambos; Schwartz, Faina

    2003-01-01

    We develop a novel class of tests to detect mitochondrial DNA (mtDNA)–mutation involvement in complex diseases by the study of affected pedigree members. For a pedigree, affected individuals are first considered and are then connected through their relatives. We construct a reduced pedigree from an original pedigree. Each configuration of a reduced pedigree is given a score, with high scores given to configurations that are consistent with mtDNA-mutation involvement and low scores given to co...

  5. High frequency of SH3TC2 mutations in Czech HMSN I patients.

    Science.gov (United States)

    Laššuthová, P; Mazanec, R; Vondráček, P; Sišková, D; Haberlová, J; Sabová, J; Seeman, P

    2011-10-01

    Charcot-Marie-Tooth (CMT) neuropathy type 4C (CMT4C) is an autosomal recessive (AR), demyelinating neuropathy with early spine deformities caused by mutations in the SH3TC2 gene. To determine the spectrum of SH3TC2 mutations in the Czech population, the entire coding region of SH3TC2 was sequenced in 60 unrelated Czech patients. The prevalent mutation was shown to be the p.Arg954Stop. Therefore, 412 additional patients referred for CMT testing were tested for the presence of p.Arg954Stop only. Of 60 patients in whom the SH3TC2 gene was sequenced, at least one mutation was detected in 13 (21.7%) patients and biallelic pathogenic mutations were detected in 7 (11.6%) patients. Of the 412 patients tested for p.Arg954Stop, the mutation was found in 8 patients (1.94%), 6 were homozygous and 2 were heterozygous. The second causative mutation was detected by sequencing in one of the patients but not in the other. Nine novel sequence variants were detected. Their pathogenicity was further tested in silico and in control samples. Mutations in the SH3TC2 gene are a frequent cause of demyelinating hereditary neuropathy among Czech patients. In total, at least one mutation was found in 21 unrelated patients. CMT4C seems to be the most frequent type of AR CMT and one of the most frequent of all CMT types. Mutation p.Arg954Stop is highly prevalent in the Czech population. Patients with demyelinating neuropathy along with non-dominant mode of inheritance and negative for CMT1A/hereditary neuropathy with liability to pressure palsy should be tested for the presence of the p.Arg954Stop mutation or other mutations in the SH3TC2 gene. © 2011 John Wiley & Sons A/S.

  6. High specificity but low sensitivity of mutation-specific antibodies against EGFR mutations in non-small-cell lung cancer.

    Science.gov (United States)

    Bondgaard, Anna-Louise; Høgdall, Estrid; Mellemgaard, Anders; Skov, Birgit G

    2014-12-01

    Determination of epidermal growth factor receptor (EGFR) mutations has a pivotal impact on treatment of non-small-cell lung cancer (NSCLC). A standardized test has not yet been approved. So far, Sanger DNA sequencing has been widely used. Its rather low sensitivity has led to the development of more sensitive methods including real-time PCR (RT-PCR). Immunohistochemistry with mutation-specific antibodies might be a promising detection method. We evaluated 210 samples with NSCLC from an unselected Caucasian population. Extracted DNA was analyzed for EGFR mutations by RT-PCR (Therascreen EGFR PCR kit, Qiagen, UK; reference method). For immunohistochemistry, antibodies against exon19 deletions (clone 6B6), exon21 mutations (clone 43B2) from Cell Signaling Technology (Boston, USA) and EGFR variantIII (clone 218C9) from Dako (Copenhagen, DK) were applied. Protein expression was evaluated, and staining score (multipum of intensity (graded 0-3) and percentages (0-100%) of stained tumor cells) was calculated. Positivity was defined as staining score >0. Specificity of exon19 antibody was 98.8% (95% confidence interval=95.9-99.9%) and of exon21 antibody 97.8% (95% confidence interval=94.4-99.4%). Sensitivity of exon19 antibody was 63.2% (95% confidence interval=38.4-83.7%) and of exon21 antibody was 80.0% (95% confidence interval=44.4-97.5%). Seven exon19 and four exon21 mutations were false negatives (immunohistochemistry negative, RT-PCR positive). Two exon19 and three exon21 mutations were false positive (immunohistochemistry positive, RT-PCR negative). One false positive exon21 mutation had staining score 300. The EGFR variantIII antibody showed no correlation to EGFR mutation status determined by RT-PCR or to EGFR immunohistochemistry. High specificity of the mutation-specific antibodies was demonstrated. However, sensitivity was low, especially for exon19 deletions, and thus these antibodies cannot yet be used as screening method for EGFR mutations in NSCLC

  7. High voltage test techniques

    CERN Document Server

    Kind, Dieter

    2001-01-01

    The second edition of High Voltage Test Techniques has been completely revised. The present revision takes into account the latest international developments in High Voltage and Measurement technology, making it an essential reference for engineers in the testing field.High Voltage Technology belongs to the traditional area of Electrical Engineering. However, this is not to say that the area has stood still. New insulating materials, computing methods and voltage levels repeatedly pose new problems or open up methods of solution; electromagnetic compatibility (EMC) or components and systems al

  8. Teaching the Fluctuation Test "In Silico" by Using Mutate: A Program to Distinguish between the Adaptive and Spontaneous Mutation Hypotheses

    Science.gov (United States)

    Carvajal-Rodriguez, Antonio

    2012-01-01

    Mutate is a program developed for teaching purposes to impart a virtual laboratory class for undergraduate students of Genetics in Biology. The program emulates the so-called fluctuation test whose aim is to distinguish between spontaneous and adaptive mutation hypotheses in bacteria. The plan is to train students in certain key multidisciplinary…

  9. Clinical utility of TERT promoter mutations and ALK rearrangement in thyroid cancer patients with a high prevalence of the BRAF V600E mutation.

    Science.gov (United States)

    Bae, Ja Seong; Kim, Yourha; Jeon, Sora; Kim, Se Hee; Kim, Tae Jung; Lee, Sohee; Kim, Min-Hee; Lim, Dong Jun; Lee, Youn Soo; Jung, Chan Kwon

    2016-02-09

    Mutations in the TERT promoter, ALK rearrangement, and the BRAF V600E mutation are associated with aggressive clinicopathologic features in thyroid cancers. However, little is known about the impact of TERT promoter mutations and ALK rearrangement in thyroid cancer patients with a high prevalence of BRAF mutations. We performed Sanger sequencing to detect BRAF V600E and TERT promoter mutations and both immunohistochemistry and fluorescence in situ hybridization to identify ALK rearrangement on 243 thyroid cancers. TERT promoter mutations were not present in 192 well-differentiated thyroid carcinomas (WDTC) without distant metastasis or in 9 medullary carcinomas. However, the mutations did occur in 40 % (12/30) of WDTC with distant metastasis, 29 % (2/7) of poorly differentiated carcinomas and 60 % (3/5) of anaplastic carcinomas. ALK rearrangement was not present in all thyroid cancers. The BRAF V600E mutation was more frequently found in WDTC without distant metastasis than in WDTC with distant metastasis (p = 0.007). In the cohort of WDTC with distant metastasis, patients with wild-type BRAF and TERT promoter had a significantly higher response rate after radioiodine therapy (p = 0.024), whereas the BRAF V600E mutation was significantly correlated with progressive disease (p = 0.025). The TERT promoter mutation is an independent predictor for distant metastasis of WDTC, but ALK testing is not useful for clinical decision-making in Korean patients with a high prevalence of the BRAF V600E mutation. Radioiodine therapy for distant metastasis of WDTC is most effective in patients without BRAF V600E and TERT promoter mutations.

  10. Mutations of short tandem repeat loci in cases of paternity testing in Chinese.

    Science.gov (United States)

    Sun, Mao; Zhang, XiaoNan; Wu, Dan; Shen, Qi; Wu, YuanMing; Fu, ShanMin

    2016-09-01

    In order to find out the characteristics of genetic mutations in 15 short tandem repeat (STR) loci, 3734 parentage cases were analyzed using AmpFlSTR Sinofiler kit. The allele source, mutation rate, and mutation rule of the STR loci were determined. Seventy mutations were observed in all cases for paternity testing. Among 15 STR loci, the highest mutation rate was observed in D12S391 (0.21 %), but the D5S818 gene mutation rate was relatively low (0.02 %). One-step mutation cases accounted for 95.7 % of all of the cases monitored. And the mutations in this study mainly showed paternal mutation (64/70). The research results are of great significance for identification and paternity tests and for the improvement of genetic studies on Chinese population in the future.

  11. Mutation Rate at Commonly Used Forensic STR Loci: Paternity Testing Experience

    Directory of Open Access Journals (Sweden)

    Faruk Aşıcıoğlua

    2004-01-01

    Full Text Available Paternity tests are carried out by the analysis of hypervariable short tandem repeat DNA loci. These microsatellite sequences mutate at a higher rate than that of bulk DNA. The occurrence of germline mutations at STR loci posses problems in interpretation of resulting genetic profiles. We recently analyzed 59–159 parent/child allele transfers at 13 microsatellite loci. We identified 12 mutations in 7 microsatellite loci. No mutations were occurred in other 6 loci. The highest mutation rate was observed with 5 mutations at D8S1179 locus at different alleles. The event was always single repeat related. The mutation rate was between 0 and 1.5 x 10-2 per locus per gamete per generation. The mutation event is very crucial for forensic DNA testing and accumulation of STR mutation data is extremely important for genetic profile interpretation.

  12. Extremely High Mutation Rate of HIV-1 In Vivo.

    Directory of Open Access Journals (Sweden)

    José M Cuevas

    Full Text Available Rates of spontaneous mutation critically determine the genetic diversity and evolution of RNA viruses. Although these rates have been characterized in vitro and in cell culture models, they have seldom been determined in vivo for human viruses. Here, we use the intrapatient frequency of premature stop codons to quantify the HIV-1 genome-wide rate of spontaneous mutation in DNA sequences from peripheral blood mononuclear cells. This reveals an extremely high mutation rate of (4.1 ± 1.7 × 10-3 per base per cell, the highest reported for any biological entity. Sequencing of plasma-derived sequences yielded a mutation frequency 44 times lower, indicating that a large fraction of viral genomes are lethally mutated and fail to reach plasma. We show that the HIV-1 reverse transcriptase contributes only 2% of mutations, whereas 98% result from editing by host cytidine deaminases of the A3 family. Hypermutated viral sequences are less abundant in patients showing rapid disease progression compared to normal progressors, highlighting the antiviral role of A3 proteins. However, the amount of A3-mediated editing varies broadly, and we find that low-edited sequences are more abundant among rapid progressors, suggesting that suboptimal A3 activity might enhance HIV-1 genetic diversity and pathogenesis.

  13. High tolerance to simultaneous active-site mutations in TEM-1 beta-lactamase: Distinct mutational paths provide more generalized beta-lactam recognition.

    Science.gov (United States)

    De Wals, Pierre-Yves; Doucet, Nicolas; Pelletier, Joelle N

    2009-01-01

    The diversity in substrate recognition spectra exhibited by various beta-lactamases can result from one or a few mutations in the active-site area. Using Escherichia coli TEM-1 beta-lactamase as a template that efficiently hydrolyses penicillins, we performed site-saturation mutagenesis simultaneously on two opposite faces of the active-site cavity. Residues 104 and 105 as well as 238, 240, and 244 were targeted to verify their combinatorial effects on substrate specificity and enzyme activity and to probe for cooperativity between these residues. Selection for hydrolysis of an extended-spectrum cephalosporin, cefotaxime (CTX), led to the identification of a variety of novel mutational combinations. In vivo survival assays and in vitro characterization demonstrated a general tendency toward increased CTX and decreased penicillin resistance. Although selection was undertaken with CTX, productive binding (K(M)) was improved for all substrates tested, including benzylpenicillin for which catalytic turnover (k(cat)) was reduced. This indicates broadened substrate specificity, resulting in more generalized (or less specialized) variants. In most variants, the G238S mutation largely accounted for the observed properties, with additional mutations acting in an additive fashion to enhance these properties. However, the most efficient variant did not harbor the mutation G238S but combined two neighboring mutations that acted synergistically, also providing a catalytic generalization. Our exploration of concurrent mutations illustrates the high tolerance of the TEM-1 active site to multiple simultaneous mutations and reveals two distinct mutational paths to substrate spectrum diversification.

  14. Competitive amplification of differentially melting amplicons (CADMA improves KRAS hotspot mutation testing in colorectal cancer

    Directory of Open Access Journals (Sweden)

    Kristensen Lasse

    2012-11-01

    Full Text Available Abstract Background Cancer is an extremely heterogeneous group of diseases traditionally categorized according to tissue of origin. However, even among patients with the same cancer subtype the cellular alterations at the molecular level are often very different. Several new therapies targeting specific molecular changes found in individual patients have initiated the era of personalized therapy and significantly improved patient care. In metastatic colorectal cancer (mCRC a selected group of patients with wild-type KRAS respond to antibodies against the epidermal growth factor receptor (EGFR. Testing for KRAS mutations is now required prior to anti-EGFR treatment, however, less sensitive methods based on conventional PCR regularly fail to detect KRAS mutations in clinical samples. Methods We have developed sensitive and specific assays for detection of the seven most common KRAS mutations based on a novel methodology named Competitive Amplification of Differentially Melting Amplicons (CADMA. The clinical applicability of these assays was assessed by analyzing 100 colorectal cancer samples, for which KRAS mutation status has been evaluated by the commercially available TheraScreen® KRAS mutation kit. Results The CADMA assays were sensitive to at least 0.5% mutant alleles in a wild-type background when using 50 nanograms of DNA in the reactions. Consensus between CADMA and the TheraScreen kit was observed in 96% of the colorectal cancer samples. In cases where disagreement was observed the CADMA result could be confirmed by a previously published assay based on TaqMan probes and by fast COLD-PCR followed by Sanger sequencing. Conclusions The high analytical sensitivity and specificity of CADMA may increase diagnostic sensitivity and specificity of KRAS mutation testing in mCRC patients.

  15. Designing a high-throughput somatic mutation profiling panel specifically for gynaecological cancers.

    Directory of Open Access Journals (Sweden)

    Vivian M Spaans

    Full Text Available Somatic mutations play a major role in tumour initiation and progression. The mutation status of a tumour may predict prognosis and guide targeted therapies. The majority of techniques to study oncogenic mutations require high quality and quantity DNA or are analytically challenging. Mass-spectrometry based mutation analysis however is a relatively simple and high-throughput method suitable for formalin-fixed, paraffin-embedded (FFPE tumour material. Targeted gene panels using this technique have been developed for several types of cancer. These current cancer hotspot panels are not focussed on the genes that are most relevant in gynaecological cancers. In this study, we report the design and validation of a novel, mass-spectrometry based panel specifically for gynaecological malignancies and present the frequencies of detected mutations. Using frequency data from the online Catalogue of Somatic Mutations in Cancer, we selected 171 somatic hotspot mutations in the 13 most important genes for gynaecological cancers, being BRAF, CDKN2A, CTNNB1, FBXW7, FGFR2, FGFR3, FOXL2, HRAS, KRAS, NRAS, PIK3CA, PPP2R1A and PTEN. A total of 546 tumours (205 cervical, 227 endometrial, 89 ovarian, and 25 vulvar carcinomas were used to test and validate our panel, and to study the prevalence and spectrum of somatic mutations in these types of cancer. The results were validated by testing duplicate samples and by allele-specific qPCR. The panel presented here using mass-spectrometry shows to be reproducible and high-throughput, and is usefull in FFPE material of low quality and quantity. It provides new possibilities for studying large numbers of gynaecological tumour samples in daily practice, and could be useful in guided therapy selection.

  16. Sensitive KIT D816V mutation analysis of blood as a diagnostic test in mastocytosis

    DEFF Research Database (Denmark)

    Kielsgaard Kristensen, Thomas; Vestergaard, Hanne; Bindslev-Jensen, Carsten

    2014-01-01

    The recent progress in sensitive KIT D816V mutation analysis suggests that mutation analysis of peripheral blood (PB) represents a promising diagnostic test in mastocytosis. However, there is a need for systematic assessment of the analytical sensitivity and specificity of the approach in order...... to establish its value in clinical use. We therefore evaluated sensitive KIT D816V mutation analysis of PB as a diagnostic test in an entire case-series of adults with mastocytosis. We demonstrate for the first time that by using a sufficiently sensitive KIT D816V mutation analysis, it is possible to detect...... the mutation in PB in nearly all adult mastocytosis patients. The mutation was detected in PB in 78 of 83 systemic mastocytosis (94%) and 3 of 4 cutaneous mastocytosis patients (75%). The test was 100% specific as determined by analysis of clinically relevant control patients who all tested negative. Mutation...

  17. High incidence of GJB2 gene mutations among assortatively mating ...

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Genetics; Volume 93; Issue 1. High incidence of GJB2 gene mutations among assortatively mating hearing impaired families in Kerala: future implications. Amritkumar Pavithra Justin Margret Jeffrey Jayasankaran Chandru Arabandi Ramesh C. R. Srikumari Srisailapathy. Research Note Volume ...

  18. Detection of somatic mutations by high-resolution DNA melting (HRM analysis in multiple cancers.

    Directory of Open Access Journals (Sweden)

    Jesus Gonzalez-Bosquet

    Full Text Available Identification of somatic mutations in cancer is a major goal for understanding and monitoring the events related to cancer initiation and progression. High resolution melting (HRM curve analysis represents a fast, post-PCR high-throughput method for scanning somatic sequence alterations in target genes. The aim of this study was to assess the sensitivity and specificity of HRM analysis for tumor mutation screening in a range of tumor samples, which included 216 frozen pediatric small rounded blue-cell tumors as well as 180 paraffin-embedded tumors from breast, endometrial and ovarian cancers (60 of each. HRM analysis was performed in exons of the following candidate genes known to harbor established commonly observed mutations: PIK3CA, ERBB2, KRAS, TP53, EGFR, BRAF, GATA3, and FGFR3. Bi-directional sequencing analysis was used to determine the accuracy of the HRM analysis. For the 39 mutations observed in frozen samples, the sensitivity and specificity of HRM analysis were 97% and 87%, respectively. There were 67 mutation/variants in the paraffin-embedded samples, and the sensitivity and specificity for the HRM analysis were 88% and 80%, respectively. Paraffin-embedded samples require higher quantity of purified DNA for high performance. In summary, HRM analysis is a promising moderate-throughput screening test for mutations among known candidate genomic regions. Although the overall accuracy appears to be better in frozen specimens, somatic alterations were detected in DNA extracted from paraffin-embedded samples.

  19. High Intra- and Inter-Tumoral Heterogeneity of RAS Mutations in Colorectal Cancer

    Directory of Open Access Journals (Sweden)

    Marion Jeantet

    2016-12-01

    Full Text Available Approximately 30% of patients with wild type RAS metastatic colorectal cancer are non-responders to anti-epidermal growth factor receptor monoclonal antibodies (anti-EGFR mAbs, possibly due to undetected tumoral subclones harboring RAS mutations. The aim of this study was to analyze the distribution of RAS mutations in different areas of the primary tumor, metastatic lymph nodes and distant metastasis. A retrospective cohort of 18 patients with a colorectal cancer (CRC was included in the study. Multiregion analysis was performed in 60 spatially separated tumor areas according to the pathological tumor node metastasis (pTNM staging and KRAS, NRAS and BRAF mutations were tested using pyrosequencing. In primary tumors, intra-tumoral heterogeneity for RAS mutation was found in 33% of cases. Inter-tumoral heterogeneity for RAS mutation between primary tumors and metastatic lymph nodes or distant metastasis was found in 36% of cases. Moreover, 28% of tumors had multiple RAS mutated subclones in the same tumor. A high proportion of CRCs presented intra- and/or inter-tumoral heterogeneity, which has relevant clinical implications for anti-EGFR mAbs prescription. These results suggest the need for multiple RAS testing in different parts of the same tumor and/or more sensitive techniques.

  20. High frequency of the HRAS oncogene codon 12 mutation in Macedonian patients with urinary bladder cancer

    Directory of Open Access Journals (Sweden)

    Sasho Panov

    2004-01-01

    Full Text Available Point mutations at codon 12 of the HRAS (v-Ha-ras Harvey rat sarcoma viral oncogene homolog oncogene are one of the best defined and widely studied molecular genetic events in transitional cell carcinoma (TCC of the urinary bladder. The aim of this study was to use the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP analysis of paraffin-embedded tissue-derived DNA to determine the frequency of the HRAS oncogene G ->T codon 12 mutation in TCC patients being treated at the University Urology Clinic in Skopje, Republic of Macedonia. DNA isolated from paraffin-embedded tissue (PET surgically removed TCC specimens of 62 (81.58% out of 76 patients were successfully amplified, the remaining 14 (18.42% showing compromised DNA integrity. The codon 12 mutation of the HRAS oncogene was found in 24 (38.71% out of 62 successfully tested TCC urinary bladder samples. No significant relationship between the mutation frequency and the histopathological grade of tumor differentiation was detected (chi² = 0.044; p = 0.978. The relatively high frequency of mutations found in our study was comparable with some of the previously reported data obtained by this and/or other PCR-based methods. This highly sensitive and specific PCR-RFLP analysis was demonstrated to be a suitable method for the detection of mutations at codon 12 of the HRAS oncogene in PET samples of urinary bladder TCC.

  1. High-stage urachal adenocarcinoma can be associated with microsatellite instability and KRAS mutations.

    Science.gov (United States)

    Sirintrapun, S Joseph; Ward, Martha; Woo, Jennifer; Cimic, Adela

    2014-02-01

    Urachal adenocarcinoma (UAC) is a rare tumor of the urinary bladder, which can show intestinal, mucinous, and signet ring cell histology. The morphology is similar to that of colorectal adenocarcinoma (CAC). Microsatellite instability (MSI), KRAS, and BRAF have been more extensively studied in CAC. What is not known is whether UAC in its morphologic similarity to CAC could show immunohistochemical features of MSI along with KRAS- and BRAF-activating mutations. A retrospective review of institutional archives for UAC cases found 7 cases, all of which were high stage. Most (6/7) of our UAC cases showed evidence of MSI or mutations of KRAS. No cases showed a BRAF mutation at codon 600. Of the cases that demonstrated MSI, 1 showed mutS homolog 2 and mutS homolog 6 loss, and 2 showed PMS2 (postmeiotic segregation increased 2) loss. Of the remaining 4 cases, 3 showed KRAS mutations at codon 12. Our UAC series showed mutual exclusivity of MSI and KRAS mutations. Furthermore, our UAC cases with KRAS mutations showed markedly better overall survival (mean, 101.7 versus 6.5 months; P = .035). Thus, our study justifies ancillary testing for MSI and KRAS in UAC, particularly when there is high-stage and mucinous histology, but a larger multi-institutional accruement of UAC cases is necessary to further validate our novel findings. © 2014.

  2. Exome sequencing identifies ZNF644 mutations in high myopia.

    Directory of Open Access Journals (Sweden)

    Yi Shi

    2011-06-01

    Full Text Available Myopia is the most common ocular disorder worldwide, and high myopia in particular is one of the leading causes of blindness. Genetic factors play a critical role in the development of myopia, especially high myopia. Recently, the exome sequencing approach has been successfully used for the disease gene identification of Mendelian disorders. Here we show a successful application of exome sequencing to identify a gene for an autosomal dominant disorder, and we have identified a gene potentially responsible for high myopia in a monogenic form. We captured exomes of two affected individuals from a Han Chinese family with high myopia and performed sequencing analysis by a second-generation sequencer with a mean coverage of 30× and sufficient depth to call variants at ∼97% of each targeted exome. The shared genetic variants of these two affected individuals in the family being studied were filtered against the 1000 Genomes Project and the dbSNP131 database. A mutation A672G in zinc finger protein 644 isoform 1 (ZNF644 was identified as being related to the phenotype of this family. After we performed sequencing analysis of the exons in the ZNF644 gene in 300 sporadic cases of high myopia, we identified an additional five mutations (I587V, R680G, C699Y, 3'UTR+12 C>G, and 3'UTR+592 G>A in 11 different patients. All these mutations were absent in 600 normal controls. The ZNF644 gene was expressed in human retinal and retinal pigment epithelium (RPE. Given that ZNF644 is predicted to be a transcription factor that may regulate genes involved in eye development, mutation may cause the axial elongation of eyeball found in high myopia patients. Our results suggest that ZNF644 might be a causal gene for high myopia in a monogenic form.

  3. Mutation analysis of BRCA1 and BRCA2 genes in Iranian high risk breast cancer families.

    Science.gov (United States)

    Pietschmann, Andrea; Mehdipour, Parvin; Mehdipour, Parvin; Atri, Morteza; Hofmann, Wera; Hosseini-Asl, S Said; Scherneck, Siegfried; Mundlos, Stefan; Peters, Hartmut

    2005-08-01

    Germline mutations in either BRCA1 or BRCA2 genes are responsible for the majority of hereditary breast and ovarian cancers. At present, over thousand distinct BRCA1 and BRCA2 mutations have been identified. Specific mutations are found to be common within particular populations, resulting from genetic founder effects. To investigate the contribution of germline mutations in these two genes to inherited breast cancer in Iran, we performed BRCA1/BRCA2 mutation analyses in ten Iranian high risk breast cancer families. This is the first study analysing the complete coding sequences of both genes that concerns the Iranian population. BRCA1/BRCA2 mutation detection included sequencing of the coding and the 3' and 5' untranslated regions. To detect large genomic rearrangements in the BRCA1 gene semi-quantitative multiplex PCR was performed. Two pathogenic mutations in the BRCA2 gene were detected: a novel deletion c.4415_4418delAGAA and a previously described insertion c.6033_6034insGT. In addition, one intronic variation g.5075-53C > T and a deletion/insertion g.*381_389del9ins29 in the 3' untranslated region of BRCA1 were found in two of the investigated families. Both sequence alterations were absent in an age matched Iranian control group. The BRCA2 homozygous variation p.N372H, previously associated with an increased risk for developing breast cancer, was not identified in this study. We did not detect large genomic rearrangements in BRCA1 in patients tested negatively for disease causing mutations in both genes by standard sequencing. At present, the BRCA2 mutations c.4415_4418delAGAA and c.6033_6034insGT have not been identified in any investigated population except the Iranian. Whether both mutations are specific for the Iranian population or a special subgroup remains to be investigated in larger studies. The absence of BRCA1 mutations in the analysed families may suggest that penetrance or prevalence of BRCA1 mutations may be lower in Iran.

  4. Mutation analysis of Swedish haemophilia B families - high frequency of unique mutations.

    Science.gov (United States)

    Mårtensson, A; Letelier, A; Halldén, C; Ljung, R

    2016-05-01

    Haemophilia B is caused by a heterogeneous spectrum of mutations. Mutation characterization is important in genetic counselling, prenatal diagnosis and to predict risk of inhibitor development. To study the mutation spectrum, frequency of unique recurrent mutations, genotype-phenotype association and inhibitor development in a population-based study of the complete Swedish haemophilia B population. The study included, facilitated by centralized DNA diagnostics, the complete registered Swedish haemophilia B population (113 families: 47 severe, 22 moderate and 44 mild), each represented by a single patient. Mutation characterization was performed by conventional sequencing of all exons and haplotyping by genotyping of single nucleotide variants and microsatellites. A mutation was found in every family: eight had large deletions, three had small deletions (mutations were found and were predicted to be deleterious. Sixteen mutations (one total gene deletion, 14 substitutions and one acceptor splice site) were present in more than one family. Of the single nucleotide mutations (37/102), 36% arose at CpG sites. Haplotyping of families with identical mutations and present analyses showed that the frequency of unique mutations was at least 65%. Inhibitors developed in 9/47 (19%) patients with severe haemophilia B. The spectrum of haemophilia B mutations reveals at least 65% of the families carry a unique mutation, but with more inhibitor patients than reported internationally, probably as a result of many 'null' mutations. © 2015 John Wiley & Sons Ltd.

  5. Genetic Testing for Long-QT Syndrome Distinguishing Pathogenic Mutations From Benign Variants

    NARCIS (Netherlands)

    Kapa, Suraj; Tester, David J.; Salisbury, Benjamin A.; Harris-Kerr, Carole; Pungliya, Manish S.; Alders, Marielle; Wilde, Arthur A. M.; Ackerman, Michael J.

    2009-01-01

    Background-Genetic testing for long-QT syndrome (LQTS) has diagnostic, prognostic, and therapeutic implications. Hundreds of causative mutations in 12 known LQTS-susceptibility genes have been identified. Genetic testing that includes the 3 most commonly mutated genes is available clinically.

  6. Competitive amplification of differentially melting amplicons (CADMA) enables sensitive and direct detection of all mutation types by high-resolution melting analysis.

    Science.gov (United States)

    Kristensen, Lasse S; Andersen, Gitte B; Hager, Henrik; Hansen, Lise Lotte

    2012-01-01

    Sensitive and specific mutation detection is of particular importance in cancer diagnostics, prognostics, and individualized patient treatment. However, the majority of molecular methodologies that have been developed with the aim of increasing the sensitivity of mutation testing have drawbacks in terms of specificity, convenience, or costs. Here, we have established a new method, Competitive Amplification of Differentially Melting Amplicons (CADMA), which allows very sensitive and specific detection of all mutation types. The principle of the method is to amplify wild-type and mutated sequences simultaneously using a three-primer system. A mutation-specific primer is designed to introduce melting temperature decreasing mutations in the resulting mutated amplicon, while a second overlapping primer is designed to amplify both wild-type and mutated sequences. When combined with a third common primer very sensitive mutation detection becomes possible, when using high-resolution melting (HRM) as detection platform. The introduction of melting temperature decreasing mutations in the mutated amplicon also allows for further mutation enrichment by fast coamplification at lower denaturation temperature PCR (COLD-PCR). For proof-of-concept, we have designed CADMA assays for clinically relevant BRAF, EGFR, KRAS, and PIK3CA mutations, which are sensitive to, between 0.025% and 0.25%, mutated alleles in a wild-type background. In conclusion, CADMA enables highly sensitive and specific mutation detection by HRM analysis. © 2011 Wiley Periodicals, Inc.

  7. Validation of high-resolution DNA melting analysis for mutation scanning of the CDKL5 gene: identification of novel mutations.

    Science.gov (United States)

    Raymond, Laure; Diebold, Bertrand; Leroux, Céline; Maurey, Hélène; Drouin-Garraud, Valérie; Delahaye, Andre; Dulac, Olivier; Metreau, Julia; Melikishvili, Gia; Toutain, Annick; Rivier, François; Bahi-Buisson, Nadia; Bienvenu, Thierry

    2013-01-01

    Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) have been predominantly described in epileptic encephalopathies of female, including infantile spasms with Rett-like features. Up to now, detection of mutations in this gene was made by laborious, expensive and/or time consuming methods. Here, we decided to validate high-resolution melting analysis (HRMA) for mutation scanning of the CDKL5 gene. Firstly, using a large DNA bank consisting to 34 samples carrying different mutations and polymorphisms, we validated our analytical conditions to analyse the different exons and flanking intronic sequences of the CDKL5 gene by HRMA. Secondly, we screened CDKL5 by both HRMA and denaturing high performance liquid chromatography (dHPLC) in a cohort of 135 patients with early-onset seizures. Our results showed that point mutations and small insertions and deletions can be reliably detected by HRMA. Compared to dHPLC, HRMA profiles are more discriminated, thereby decreasing unnecessary sequencing. In this study, we identified eleven novel sequence variations including four pathogenic mutations (2.96% prevalence). HRMA appears cost-effective, easy to set up, highly sensitive, non-toxic and rapid for mutation screening, ideally suited for large genes with heterogeneous mutations located along the whole coding sequence, such as the CDKL5 gene. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Stability Test For Sorghum Mutant Lines Derived From Induced Mutations with Gamma-Ray Irradiation

    Directory of Open Access Journals (Sweden)

    S. Human

    2011-12-01

    Full Text Available Sorghum breeding program had been conducted at the Center for the Application of Isotopes and Radiation Technology, BATAN. Plant genetic variability was increased through induced mutations using gamma-ray irradiation. Through selection process in successive generations, some promising mutant lines had been identified to have good agronomic characteristics with high grain yield. These breeding lines were tested in multi location trials and information of the genotypic stability was obtained to meet the requirements for officially varietal release by the Ministry of Agriculture. A total of 11 sorghum lines and varieties consisting of 8 mutant lines derived from induced mutations (B-100, B-95, B-92, B-83, B-76, B-75, B-69 and Zh-30 and 3 control varieties (Durra, UPCA-S1 and Mandau were included in the experiment. All materials were grown in 10 agro-ecologically different locations namely Gunungkidul, Bantul, Citayam, Garut, Lampung, Bogor, Anyer, Karawaci, Cianjur and Subang. In each location, the local adaptability test was conducted by randomized block design with 3 replications. Data of grain yield was used for evaluating genotypic stability using AMMI approach. Results revealed that sorghum mutation breeding had generated 3 mutant lines (B-100, B-76 and Zh-30 exhibiting grain yield significantly higher than the control varieties. These mutant lines were genetically stable in all locations so that they would be recommended for official release as new sorghum varieties to the Ministry of Agriculture

  9. [Clinical role of BRAF V600E mutation testing in thyroid nodules].

    Science.gov (United States)

    Wan, Hanfeng; Zhang, Bin; Wang, Yong; Xiao, Ting; Guo, Huiqin; Liu, Wensheng; Yan, Dangui; Xu, Zhengang; Tang, Pingzhang

    2014-06-01

    To evaluate the clinical role of BRAF V600E mutation testing in fine-needle aspirates (FNA) of thyroid nodules. This study included 83 nodules in 80 patients who underwent FNA from March 2013 to September 2013. Cytological specimens were collected and BRAF exon 15 was examined by polymerase chain reaction (PCR). DNA sequencing and analysis were performed. Diagnostic performances of cytology and cytology with BRAF V600E mutation analysis were compared according to postoperative pathological diagnosis. The relation of BRAF V600E mutation with clinical factors including sex and age of patients, tumor size, lymph node metastasis, multifocality, and AJCC stage were analyzed. Of 83 nodules, 33 nodules were clinically observed, and 48 nodules underwent surgery, and suggestions of surgery were refused in 2 nodules. Among 48 nodules with surgery, BRAF V600E mutation was found in 25 nodules with histologic confirmation of papillary thyroid carcinoma after thyroidectomy, 13 of the 25 nodules were cytologically diagnosed as carcinoma and 12 were indeterminate. Among the 23 BRAF V600E negative noodles, 5 were cytologically diagnosed as carcinoma, 2 were benign, and 16 were indeterminate; 15 nodules were histologic confirmation of papillary thyroid carcinoma after thyroidectomy, 1 nodule was medullary thyroid carcinoma, and 7 nodules were benign. Biomolecular analysis significantly increased cytology sensitivity for papillary thyroid carcinoma from 43.9% to 73.2% (P DNA sequencing showed that the presence of BRAF V600E mutation was 62.5% in 40 thyroid papillary nodules. There were 16 BRAF-positive nodules (80.0%) among 20 papillary thyroid nodules with extrathyroidal extension, however there were 9 BRAF-negative nodules (45.0%) among 20 papillary thyroid nodules without extrathyroidal extension. Univariate analysis indicated the BRAF V600E mutation was associated with extrathyroidal extension (χ² = 5.227, P = 0.022), but not with sex, age, tumor size, lymph node metastasis

  10. Cost effectiveness of population based BRCA1 founder mutation testing in Sephardi Jewish women.

    Science.gov (United States)

    Patel, Shreeya; Legood, Rosa; Evans, D Gareth; Turnbull, Clare; Antoniou, Antonis C; Menon, Usha; Jacobs, Ian; Manchanda, Ranjit

    2017-12-26

    Population-based BRCA1/BRCA2 founder-mutation testing has been demonstrated as cost-effective compared to family-history(FH) based testing in Ashkenazi Jewish(AJ) women. However, only one of the three AJ BRCA1/BRCA2 founder-mutations (185delAG(c.68_69delAG), 5382insC(c.5266dupC) and 6174delT(c.5946delT)) is found in the Sephardi Jewish(SJ) population (185delAG(c.68_69delAG)) and the overall prevalence of BRCA mutations in the SJ population is accordingly lower (0.7% compared to 2.5% in the AJ population). Cost-effectiveness analyses of BRCA testing have not previously been performed at these lower BRCA prevalence levels seen in SJ. Here we present a cost-effectiveness analysis for UK and US populations comparing population-testing with Clinical-criteria/FH-based testing in SJ women. A Markov model was built comparing the lifetime costs-&-effects of population-based BRCA1-testing with testing using FH-based clinical criteria in SJ women ≥30years. BRCA1-carriers identified were offered MRI/mammograms and risk-reducing surgery. Costs are reported at 2015 prices. Outcomes include breast cancer(BC), ovarian cancer(OC) and excess deaths from heart disease. All costs-&-outcomes are discounted at 3.5%. The time horizon is life-time, and perspective is payer. The incremental-cost-effectiveness-ratio (ICER) per quality-adjusted life-year (QALY) was calculated. Parameter uncertainty was evaluated through one-way and probabilistic-sensitivity-analysis (PSA). Population-testing resulted in gain in life-expectancy of 12months (QALY=1.00). The baseline discounted ICER for UK population-based testing =£67.04/QALY and for US population=$308.42/QALY. Results were robust in the one-way sensitivity analysis. The PSA showed 100% of simulations were cost-effective at £20,000/QALY UK and the $100,000/QALY US WTP thresholds. Scenario analysis showed, population-testing remains cost-effective in UK and US populations even if pre-menopausal oophorectomy does not reduce BC-risk or if

  11. SRSF2-p95 hotspot mutation is highly associated with advanced forms of mastocytosis and mutations in epigenetic regulator genes.

    Science.gov (United States)

    Hanssens, Katia; Brenet, Fabienne; Agopian, Julie; Georgin-Lavialle, Sophie; Damaj, Gandhi; Cabaret, Laure; Chandesris, Maria Olivia; de Sepulveda, Paulo; Hermine, Olivier; Dubreuil, Patrice; Soucie, Erinn

    2014-05-01

    Mastocytosis is a rare and chronic disease with phenotypes ranging from indolent to severe. Prognosis for this disease is variable and very few biomarkers to predict disease evolution or outcome are currently known. We have performed comprehensive screening in our large cohort of mastocytosis patients for mutations previously found in other myeloid diseases and that could serve as prognostic indicators. KIT, SRSF2-P95 and TET2 mutations were by far the most frequent, detected in 81%, 24% and 21% of patients, respectively. Where TET2 and SRSF2-P95 mutation both correlated with advanced disease phenotypes, SRSF2-P95 hotspot mutation was found almost exclusively in patients diagnosed with associated clonal hematologic non-mast cell disease. Statistically, TET2 and SRSF2-P95 mutations were highly associated, suggesting a mechanistic link between these two factors. Finally, analysis of both clonal and sorted cell populations from patients confirms the presence of these mutations in the mast cell component of the disease, suggests an ontological mutation hierarchy and provides evidence for the expansion of multiple clones. This highlights the prognostic potential of such approaches, if applied systematically, for delineating the roles of specific mutations in predisposing and/or driving distinct disease phenotypes.

  12. Reliability of KRAS mutation testing in metastatic colorectal cancer patients across five laboratories

    Directory of Open Access Journals (Sweden)

    Feigelson Heather

    2012-04-01

    Full Text Available Abstract Background Mutations in the KRAS gene are associated with poor response to epidermal growth factor receptor inhibitors used in the treatment of metastatic colorectal cancer. Factors influencing KRAS test results in tumor specimens include: tumor heterogeneity, sample handling, slide preparation, techniques for tumor enrichment, DNA preparation, assay design and sensitivity. We evaluated comparability and consistency of KRAS test results among five laboratories currently being used to determine KRAS mutation status of metastatic colorectal cancer specimens in a large, multi-center observational study. Findings Twenty formalin-fixed paraffin-embedded human colorectal cancer samples from colon resections previously tested for KRAS mutations were selected based on mutation status (6 wild type, 8 codon 12 mutations, and 6 codon 13 mutations. We found good agreement across laboratories despite differences in mutation detection methods. Eighteen of twenty samples (90% were concordant across all five labs. Discordant results are likely not due to laboratory error, but instead to tumor heterogeneity, contamination of the tumor sample with normal tissue, or analytic factors affecting assay sensitivity. Conclusions Our results indicate commercial and academic laboratories provide reliable results for the common KRAS gene mutations at codons 12 and 13 when an adequate percentage of tumor cells is present in the sample.

  13. [Clinical significance and distribution of BRCA genes mutation in sporadic high grade serous ovarian cancer].

    Science.gov (United States)

    Liu, W L; Wang, Z Z; Zhao, J Z; Hou, Y Y; Wu, X X; Li, W; Dong, B; Tong, T T; Guo, Y J

    2017-01-25

    Objective: To investigate the mutations of BRCA genes in sporadic high grade serous ovarian cancer (HGSOC) and study its clinical significance. Methods: Sixty-eight patients between January 2015 and January 2016 from the Affiliated Cancer Hospital of Zhengzhou University were collected who were based on pathological diagnosis of ovarian cancer and had no reported family history, and all patients firstly hospitalized were untreated in other hospitals before. (1) The BRCA genes were detected by next-generation sequencing (NGS) method. (2) The serum tumor markers included carcinoembryonic antigen (CEA), CA(125), CA(199), and human epididymis protein 4 (HE4) were detected by the chemiluminescence methods, and their correlation was analyzed by Pearson linear correlation. Descriptive statistics and comparisons were performed using two-tailed t-tests, Pearson's chi square test, Fisher's exact tests or logistic regression analysis as appropriate to research the clinicopathologic features associated with BRCA mutations, including age, International Federation of Gynecology and Obstetrics (FIGO) stage, platinum-based chemotherapy sensitivity, distant metastases, serum tumor markers (STM) . Results: (1) Fifteen cases (22%, 15/68) BRCA mutations were identified (BRCA1: 11 cases; BRCA2: 4 cases), and four novel mutations were observed. (2) The levels of CEA, CA(199), and HE4 were lower in BRCA mutations compared to that in control group, while no significant differences were found (P>0.05), but the level of CA(125) was much higher in BRCA mutation group than that in controls (t=-3.536, P=0.003). Further linear regression analysis found that there was a significant linear correlation between CA(125) and HE4 group (r=0.494, P0.05), while significant differences were found in CA(125) and sensitivity to platinum-based chemotherapy between the patients with BRCA mutation and wild type (Pmutations in sporadic HGSOC (P=0.007). Conclusion: The combination of CA(125) with BRCA have

  14. Mutations to Less-Preferred Synonymous Codons in a Highly Expressed Gene of Escherichia coli: Fitness and Epistatic Interactions.

    Directory of Open Access Journals (Sweden)

    David J Hauber

    Full Text Available Codon-tRNA coevolution to maximize protein production has been, until recently, the dominant hypothesis to explain codon-usage bias in highly expressed bacterial genes. Two predictions of this hypothesis are 1 selection is weak; and 2 similar silent replacements at different codons should have similar fitness consequence. We used an allele-replacement strategy to change five specific 3rd-codon-position (silent sites in the highly expressed Escherichia coli ribosomal protein gene rplQ from the wild type to a less-preferred alternative. We introduced the five mutations within a 10-codon region. Four of the silent sites were chosen to test the second prediction, with a CTG to CTA mutation being introduced at two closely linked leucine codons and an AAA to AAG mutation being introduced at two closely linked lysine codons. We also introduced a fifth silent mutation, a GTG to GTA mutation at a valine codon in the same genic region. We measured the fitness effect of the individual mutations by competing each single-mutant strain against the parental wild-type strain, using a disrupted form of the araA gene as a selectively neutral phenotypic marker to distinguish between strains in direct competition experiments. Three of the silent mutations had a fitness effect of |s| > 0.02, which is contradictory to the prediction that selection will be weak. The two leucine mutations had significantly different fitness effects, as did the two lysine mutations, contradictory to the prediction that similar mutations at different codons should have similar fitness effects. We also constructed a strain carrying all five silent mutations in combination. Its fitness effect was greater than that predicted from the individual fitness values, suggesting that negative synergistic epistasis acts on the combination allele.

  15. 40 CFR 799.9530 - TSCA in vitro mammalian cell gene mutation test.

    Science.gov (United States)

    2010-07-01

    ..., non-genotoxic mechanisms or mechanisms absent in bacterial cells. (e) Test method—(1) Principle. (i.... and Malling, H.V. Mammalian Cell Genetics. II. Chemical Induction of Specific Locus Mutations in...

  16. Lack of utility of SDHB mutation testing in adrenergic metastatic phaeochromocytoma

    NARCIS (Netherlands)

    Sue, M.; Martucci, V.; Frey, F.; Lenders, J.M.; Timmers, H.J.L.M.; Peczkowska, M.; Prejbisz, A.; Swantje, B.; Bornstein, S.R.; Arlt, W.; Fassnacht, M.; Beuschlein, F.; Robledo, M.; Pacak, K.; Eisenhofer, G.

    2015-01-01

    OBJECTIVE: Testing for succinate dehydrogenase subunit B (SDHB) mutations is recommended in all patients with metastatic phaeochromocytomas and paragangliomas (PPGLs), but may not be required when metastatic disease is accompanied by adrenaline production. This retrospective cohort study aimed to

  17. A rapid MZI-IDA sensor system for EGFR mutation testing in non-small cell lung cancer (NSCLC).

    Science.gov (United States)

    Liu, Qing; Lim, Swee Yin; Soo, Ross A; Park, Mi Kyoung; Shin, Yong

    2015-12-15

    Epidermal growth factor receptor (EGFR) is a non-small-cell lung cancer biomarker, based on which several near-patient-testing methods have been developed and applied to predict treatment response on individual patients. Existing methods for detection of EGFR mutation are costly, labor-intensive and time-consuming. In this paper, we report a novel EGFR mutation testing system, which is based on Mach-Zehnder Interferometer (MZI) sensor and isothermal solid-phase DNA amplification (IDA) technique, called MZI-IDA sensor system. The system can deliver results within 30 min and shows high sensitivity to detect trace amounts of genomic DNA (IDA sensor system is proved to be capable of fast and accurate detection of the L858R mutation of EGFR gene in clinical samples. This may greatly help the clinicians develop an appropriate treatment plan. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. High-Throughput Mutational Analysis of a Twister Ribozyme.

    Science.gov (United States)

    Kobori, Shungo; Yokobayashi, Yohei

    2016-08-22

    Recent discoveries of new classes of self-cleaving ribozymes in diverse organisms have triggered renewed interest in the chemistry and biology of ribozymes. Functional analysis and engineering of ribozymes often involve performing biochemical assays on multiple ribozyme mutants. However, because each ribozyme mutant must be individually prepared and assayed, the number and variety of mutants that can be studied are severely limited. All of the single and double mutants of a twister ribozyme (a total of 10 296 mutants) were generated and assayed for their self-cleaving activity by exploiting deep sequencing to count the numbers of cleaved and uncleaved sequences for every mutant. Interestingly, we found that the ribozyme is highly robust against mutations such that 71 % and 30 % of all single and double mutants, respectively, retain detectable activity under the assay conditions. It was also observed that the structural elements that comprise the ribozyme exhibit distinct sensitivity to mutations. © 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  19. Induction of rice mutations by high hydrostatic pressure.

    Science.gov (United States)

    Zhang, Wei; Liu, Xuncheng; Zheng, Feng; Zeng, Songjun; Wu, Kunlin; da Silva, Jaime A Teixeira; Duan, Jun

    2013-09-01

    High hydrostatic pressure (HHP) is an extreme thermo-physical factor that affects the synthesis of DNA, RNA and proteins and induces mutagenesis in microorganisms. Our previous studies showed that exposure to 25-100 MPa HHP for 12 h retarded the germination and affected the viability of rice (Oryza sativa L.) seeds, increased the tolerance of rice plants to cold stress and altered gene expression patterns in germinating rice seeds. However, the mutagenic effect of HHP on rice remains unknown. In this study, exposure to 25, 50, 75 or 100 MPa for 12 h HHP could efficiently induce variation in rice plants. Furthermore, presoaking time and HHP strength during HHP treatment affected the efficiency of mutation. In addition, the Comet assay revealed that exposure to 25-100 MPa HHP for 12 h induced DNA strand breakage in germinating seeds and may have been the source of mutations. Our results suggest that HHP is a promising physical mutagen in rice breeding. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  20. Deep Mutational Scanning: A Highly Parallel Method to Measure the Effects of Mutation on Protein Function.

    Science.gov (United States)

    Starita, Lea M; Fields, Stanley

    2015-08-03

    Deep mutational scanning is a method that makes use of next-generation sequencing technology to measure in a single experiment the activity of 10(5) or more unique variants of a protein. Because of this depth of mutational coverage, this strategy provides data that can be analyzed to reveal many protein properties. Deep mutational scanning approaches are particularly amenable to being performed in Saccharomyces cerevisiae, given the extensive toolkit of reagents and technologies available for this organism. © 2015 Cold Spring Harbor Laboratory Press.

  1. Use of Exfoliative Specimens and Fine-Needle Aspiration Smears for Mutation Testing in Lung Adenocarcinoma.

    Science.gov (United States)

    Jain, Deepali; Ramachandrappa, Vijayakumar S; Singh, Varsha; Malik, Prabhat S; Madan, Karan; Faruq, Mohammed; Guleria, Randeep

    2017-01-01

    Cytology specimens are considered to be a promising alternative for detecting driver mutations in lung cancer patients. We aimed to explore the suitability and utility of various cytology samples of non-small-cell lung cancer (NSCLC) patients for mutation testing. In addition to mutation detection, the importance of preanalytic factors was discussed. A total of 116 cytology samples including 32 controls comprising pleural effusions, bronchial washings, and direct fine-needle aspiration (FNA) smears were included in the study for the detection of EGFR, KRAS, and Her-2/neu gene mutations. Tumor content was checked by microscopic evaluation. Tumor enrichment was done by scraping direct smears. DNA yield was assessed before selecting the method of mutation detection. Sanger sequencing and real-time PCR-based methods were used. Overall, 20.23% EGFR mutations and 2.74% KRAS mutations were observed in this study. Nondriver genetic polymorphisms were observed in EGFR exon 20 in 37% cases. The coexistence of the EGFR mutation and EGFR polymorphism was seen in 7 cases. DNA yield was better in pleural effusions and bronchial washings. Real-time PCR was used in specimens of low DNA yield. Cytology samples are useful in ascertaining molecular diagnostic information for treatment purposes if they are optimized judiciously in their preanalytic phase. © 2017 S. Karger AG, Basel.

  2. High-throughput Phenotyping of Lung Cancer Somatic Mutations.

    Science.gov (United States)

    Berger, Alice H; Brooks, Angela N; Wu, Xiaoyun; Shrestha, Yashaswi; Chouinard, Candace; Piccioni, Federica; Bagul, Mukta; Kamburov, Atanas; Imielinski, Marcin; Hogstrom, Larson; Zhu, Cong; Yang, Xiaoping; Pantel, Sasha; Sakai, Ryo; Watson, Jacqueline; Kaplan, Nathan; Campbell, Joshua D; Singh, Shantanu; Root, David E; Narayan, Rajiv; Natoli, Ted; Lahr, David L; Tirosh, Itay; Tamayo, Pablo; Getz, Gad; Wong, Bang; Doench, John; Subramanian, Aravind; Golub, Todd R; Meyerson, Matthew; Boehm, Jesse S

    2016-08-08

    Recent genome sequencing efforts have identified millions of somatic mutations in cancer. However, the functional impact of most variants is poorly understood. Here we characterize 194 somatic mutations identified in primary lung adenocarcinomas. We present an expression-based variant-impact phenotyping (eVIP) method that uses gene expression changes to distinguish impactful from neutral somatic mutations. eVIP identified 69% of mutations analyzed as impactful and 31% as functionally neutral. A subset of the impactful mutations induces xenograft tumor formation in mice and/or confers resistance to cellular EGFR inhibition. Among these impactful variants are rare somatic, clinically actionable variants including EGFR S645C, ARAF S214C and S214F, ERBB2 S418T, and multiple BRAF variants, demonstrating that rare mutations can be functionally important in cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. The complete linkage disequilibrium test: a test that points to causative mutations underlying quantitative traits

    Directory of Open Access Journals (Sweden)

    Uleberg Eivind

    2011-05-01

    Full Text Available Abstract Background Genetically, SNP that are in complete linkage disequilibrium with the causative SNP cannot be distinguished from the causative SNP. The Complete Linkage Disequilibrium (CLD test presented here tests whether a SNP is in complete LD with the causative mutation or not. The performance of the CLD test is evaluated in 1000 simulated datasets. Methods The CLD test consists of two steps i.e. analysis I and analysis II. Analysis I consists of an association analysis of the investigated region. The log-likelihood values from analysis I are next ranked in descending order and in analysis II the CLD test evaluates differences in log-likelihood ratios between the best and second best markers. Under the null-hypothesis distribution, the best SNP is in greater LD with the QTL than the second best, while under the alternative-CLD-hypothesis, the best SNP is alike-in-state with the QTL. To find a significance threshold, the test was also performed on data excluding the causative SNP. The 5th, 10th and 50th highest TCLD value from 1000 replicated analyses were used to control the type-I-error rate of the test at p = 0.005, p = 0.01 and p = 0.05, respectively. Results In a situation where the QTL explained 48% of the phenotypic variance analysis I detected a QTL in 994 replicates (p = 0.001, where 972 were positioned in the correct QTL position. When the causative SNP was excluded from the analysis, 714 replicates detected evidence of a QTL (p = 0.001. In analysis II, the CLD test confirmed 280 causative SNP from 1000 simulations (p = 0.05, i.e. power was 28%. When the effect of the QTL was reduced by doubling the error variance, the power of the test reduced relatively little to 23%. When sequence data were used, the power of the test reduced to 16%. All SNP that were confirmed by the CLD test were positioned in the correct QTL position. Conclusions The CLD test can provide evidence for a causative SNP, but its power may be low in situations

  4. EGFR mutation testing using archival-stained smears in non-small cell lung carcinoma.

    Science.gov (United States)

    Ozluk, Y; Firat, P; Yegen, G; Hocaoglu, J; Tas, S; Yilmazbayhan, D

    2017-02-01

    Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown benefits regarding progression-free and overall survival in patients whose tumours show EGFR mutations. Most patients' lung cancer is metastatic when detected. Small tissue samples and cytological materials are widely used in diagnosis. The aim of the present study was to compare the EGFR mutation analysis results between cytology, small biopsies and resections. Archival material for EGFR testing was reviewed. Cell blocks and/or stained smears and tissue blocks were used where appropriate. The tumour cell count and percentage were recorded as well as the DNA content. The influence of TTF-1 immunoreactivity on EGFR testing was also investigated. The study cohort included 300 unpaired specimens of 84 resections, 83 small biopsies and 133 cytological materials. EGFR mutation rates did not differ significantly for cytology, small biopsy and resections (P > 0.05). The higher tumour cell percentage in FNAs than in exfoliative cytology did not affect the EGFR mutation status. EGFR mutation rates were similar when either slides or cell blocks were used. Cytology slides revealed a higher tumour cell content and DNA concentration than the cell blocks. May-Grünwald-Giemsa (MGG)-stained smears had higher rates of the EGFR mutation than the Papanicolaou (Pap)-stained slides (P Cytological materials can be used successfully for mutation analysis in lung cancer. © 2016 John Wiley & Sons Ltd.

  5. High-resolution melting facilitates mutation screening of PYGM in patients with McArdle disease

    DEFF Research Database (Denmark)

    Duno, M.; Quinlivan, R.; Vissing, J.

    2009-01-01

    Mutations in PYGM, encoding the muscle-specific glycogen phosphorylase (myophosphorylase), are responsible for McArdle disease. Among Caucasians, a large proportion of patients are homozygous for the R50X mutation, but other mutations can affect all the 20 exons of PYGM, making mutation detection...... variations. Thirteen of these are pathogenic, and three were classified as polymorphisms. Nine variations had not previously been described. One of the novel mutations, c.2430C > T, was initially predicted to result in a silent G810G change, but cDNA analysis demonstrated that the mutation led to abnormal m...... laborious. We have developed a high-resolution melting (HRM) assay for mutation detection in PYGM. Twelve McArdle patients were investigated, in whom pre-screening had ruled out homozygosity or compound heterozygosity for the two common G205S and R50X mutations. In total, we identified 16 different...

  6. DMSO increases mutation-scanning detection sensitivity in clinical samples using high resolution melting

    Science.gov (United States)

    Song, Chen; Castellanos-Rizaldos, Elena; Bejar, Rafael; Ebert, Benjamin L.; Makrigiorgos, G. Mike

    2016-01-01

    BACKGROUND Mutation scanning provides the simplest, lowest cost method for identifying DNA variations on single PCR amplicons, and it may be performed prior to sequencing to avoid screening of non-informative wild type samples. High resolution melting (HRM) is the most commonly used method for mutation scanning. However, by using PCR-HRM mutations below ≈ 3–10% that can still be clinically significant may often be missed. Therefore, enhancing HRM detection sensitivity is important for mutation scanning and its clinical application. METHODS We used serial dilution of TP53 exon 8 mutation containing cell lines to demonstrate the improvement in detection sensitivity for conventional-PCR-HRM in the presence of DMSO. We also conducted full-COLD-PCR to further enrich low-level mutations prior to HRM±DMSO and employed droplet-digital PCR to derive the optimal conditions for mutation enrichment. Both conventional-PCR-HRM and full-COLD-PCR-HRM ±DMSO were used for mutation scanning in TP53 exon 8 in cancer samples containing known mutations and in myelodysplastic syndrome samples with unknown mutations. Mutations in other genes were also examined. RESULTS The detection sensitivity of PCR-HRM-scanning increases 2–5-fold in the presence of DMSO, depending also on mutation type and sequence context, and can typically detect mutation abundance of about 1%. When mutation enrichment is applied during amplification using full-COLD-PCR and followed by HRM in the presence of DMSO, mutations with 0.2–0.3% mutation abundance in TP53 exon 8 can be detected. CONCLUSIONS DMSO improves HRM mutation scanning sensitivity. When full-COLD-PCR is employed, followed by DMSO-HRM, the overall improvement is about 20-fold as compared to conventional PCR-HRM. PMID:26432802

  7. KRAS mutation testing in borderline ovarian tumors and low-grade ovarian carcinomas with a rapid, fully integrated molecular diagnostic system.

    Science.gov (United States)

    Sadlecki, Pawel; Antosik, Paulina; Grzanka, Dariusz; Grabiec, Marek; Walentowicz-Sadlecka, Malgorzata

    2017-10-01

    Epithelial ovarian neoplasms are a heterogeneous group of tumors, including various malignancies with distinct clinicopathologic and molecular features. Mutations in BRAF and KRAS genes are the most frequent genetic aberrations found in low-grade serous ovarian carcinomas and serous and mucinous borderline tumors. Implementation of targeted therapeutic strategies requires access to highly specific and highly sensitive diagnostic tests for rapid determination of mutation status. One candidate for such test is fully integrated, real-time polymerase chain reaction-based Idylla™ system for quick and simple detection of KRAS mutations in formaldehyde fixed-paraffin embedded tumor samples. The primary aim of this study was to verify whether fully integrated real-time polymerase chain reaction-based Idylla system may be useful in determination of KRAS mutation status in patients with borderline ovarian tumors and low-grade ovarian carcinomas. The study included tissue specimens from 37 patients with histopathologically verified ovarian masses, operated on at the Department of Obstetrics and Gynecology, Nicolaus Copernicus University Collegium Medicum in Bydgoszcz (Poland) between January 2009 and June 2012. Based on histopathological examination of surgical specimens, 30 lesions were classified as low-grade ovarian carcinomas and 7 as borderline ovarian tumors. Seven patients examined with Idylla KRAS Mutation Test tested positive for KRAS mutation. No statistically significant association was found between the incidence of KRAS mutations and histopathological type of ovarian tumors. Mean survival of the study subjects was 48.51 months (range 3-60 months). Presence of KRAS mutation did not exert a significant effect on the duration of survival in our series. Our findings suggest that Idylla KRAS Mutation Test may be a useful tool for rapid detection of KRAS mutations in ovarian tumor tissue.

  8. Genetic mutations in early-onset Parkinson's disease Mexican patients: molecular testing implications.

    Science.gov (United States)

    Monroy-Jaramillo, Nancy; Guerrero-Camacho, Jorge Luis; Rodríguez-Violante, Mayela; Boll-Woehrlen, Marie-Catherine; Yescas-Gómez, Petra; Alonso-Vilatela, María Elisa; López-López, Marisol

    2014-04-01

    Mutations in PARK2, PINK1, and DJ-1 have been associated with autosomal recessive early-onset Parkinson's disease. Here, we report the prevalence of sequence and structural mutations in these three main recessive genes in Mexican Mestizo patients. The complete sequences of these three genes were analyzed by homo/heteroduplex DNA formation and direct sequencing; exon dosage was determined by multiplex ligation-dependent probe amplification and real-time PCR in 127 patients belonging to 122 families and 120 healthy Mexican Mestizo controls. All individuals had been previously screened for the three most common LRRK2 mutations. The presence of two mutations in compound heterozygous or homozygous genotypes was found in 16 unrelated patients, 10 had mutations in PARK2, six in PINK1, and none in DJ-1. Two PARK2-PINK1 and one PARK2-LRRK2 digenic cases were observed. Novel mutations were identified in PARK2 and PINK1 genes, including PINK1 duplication for the first time. Exon dosage deletions were the most frequent mutations in PARK2 (mainly in exons 9 and 12), followed by those in PINK1. The high prevalence of heterozygous mutations in PARK2 (12.3%) and the novel heterozygous and homozygous point mutations in PINK1 observed in familial and sporadic cases from various states of Mexico support the concept that single heterozygous mutations in recessive Parkinson's disease genes play a pathogenic role. These data have important implications for genetic counseling of Mexican Mestizo patients with early-onset Parkinson's disease. The presence of digenic inheritance underscores the importance of studying several genes in this disease. A step-ordered strategy for molecular diagnosis is proposed. © 2014 Wiley Periodicals, Inc.

  9. Prevalence of BRCA1/2 mutations in sporadic breast/ovarian cancer patients and identification of a novel de novo BRCA1 mutation in a patient diagnosed with late onset breast and ovarian cancer: implications for genetic testing.

    Science.gov (United States)

    De Leeneer, Kim; Coene, Ilse; Crombez, Brecht; Simkens, Justine; Van den Broecke, Rudy; Bols, Alain; Stragier, Barbara; Vanhoutte, Ilse; De Paepe, Anne; Poppe, Bruce; Claes, Kathleen

    2012-02-01

    In order to adequately evaluate the clinical relevance of genetic testing in sporadic breast and ovarian cancer patients, we offered comprehensive BRCA1/2 mutation analysis in patients without a family history for the disease. We evaluated the complete coding and splice site regions of BRCA1/2 in 193 sporadic patients. In addition, a de novo mutation was further investigated with ultra deep sequencing and microsatellite marker analysis. In 17 patients (8.8%), a deleterious germline BRCA1/2 mutation was identified. The highest mutation detection ratio (3/7 = 42.9%) was obtained in sporadic patients diagnosed with breast and ovarian cancer after the age of 40. In 21 bilateral breast cancer patients, two mutations were identified (9.5%). Furthermore, 140 sporadic patients with unilateral breast cancer were investigated. Mutations were only identified in patients diagnosed with breast cancer before the age of 40 (12/128 = 9.4% vs. 0/12 with Dx > 40). No mutations were detected in 17 sporadic male breast cancer and 6 ovarian cancer patients. BRCA1 c.3494_3495delTT was identified in a patient diagnosed with breast and ovarian cancer at the age of 52 and 53, respectively, and was proven to have occurred de novo at the paternal allele. Our study shows that the mutation detection probability in specific patient subsets can be significant, therefore mutation analysis should be considered in sporadic patients. As a consequence, a family history for the disease and an early age of onset should not be used as the only criteria for mutation analysis of BRCA1/2. The relatively high mutation detection ratio suggests that the prevalence of BRCA1/2 may be underestimated, especially in sporadic patients who developed breast and ovarian cancer. In addition, although rare, the possibility of a de novo occurrence in a sporadic patient should be considered.

  10. HIV-1 Drug Resistance Mutations: Potential Applications for Point-of-Care Genotypic Resistance Testing.

    Directory of Open Access Journals (Sweden)

    Soo-Yon Rhee

    Full Text Available The increasing prevalence of acquired and transmitted HIV-1 drug resistance is an obstacle to successful antiretroviral therapy (ART in the low- and middle-income countries (LMICs hardest hit by the HIV-1 pandemic. Genotypic drug resistance testing could facilitate the choice of initial ART in areas with rising transmitted drug resistance (TDR and enable care-providers to determine which individuals with virological failure (VF on a first- or second-line ART regimen require a change in treatment. An inexpensive near point-of-care (POC genotypic resistance test would be useful in settings where the resources, capacity, and infrastructure to perform standard genotypic drug resistance testing are limited. Such a test would be particularly useful in conjunction with the POC HIV-1 viral load tests that are currently being introduced in LMICs. A POC genotypic resistance test is likely to involve the use of allele-specific point mutation assays for detecting drug-resistance mutations (DRMs. This study proposes that two major nucleoside reverse transcriptase inhibitor (NRTI-associated DRMs (M184V and K65R and four major NNRTI-associated DRMs (K103N, Y181C, G190A, and V106M would be the most useful for POC genotypic resistance testing in LMIC settings. One or more of these six DRMs was present in 61.2% of analyzed virus sequences from ART-naïve individuals with intermediate or high-level TDR and 98.8% of analyzed virus sequences from individuals on a first-line NRTI/NNRTI-containing regimen with intermediate or high-level acquired drug resistance. The detection of one or more of these DRMs in an ART-naïve individual or in a individual with VF on a first-line NRTI/NNRTI-containing regimen may be considered an indication for a protease inhibitor (PI-containing regimen or closer virological monitoring based on cost-effectiveness or country policy.

  11. Mutagenicity of flavonoids assayed by bacterial reverse mutation (Ames) test

    National Research Council Canada - National Science Library

    Resende, Flavia Aparecida; Vilegas, Wagner; Dos Santos, Lourdes Campaner; Varanda, Eliana Aparecida

    2012-01-01

    The mutagenicity of ten flavonoids was assayed by the Ames test, in Salmonella typhimurium strains TA98, TA100 and TA102, with the aim of establishing hydroxylation pattern-mutagenicity relationship profiles...

  12. Deep Mutational Scanning: Library Construction, Functional Selection, and High-Throughput Sequencing.

    Science.gov (United States)

    Starita, Lea M; Fields, Stanley

    2015-08-03

    Deep mutational scanning is a highly parallel method that uses high-throughput sequencing to track changes in >10(5) protein variants before and after selection to measure the effects of mutations on protein function. Here we outline the stages of a deep mutational scanning experiment, focusing on the construction of libraries of protein sequence variants and the preparation of Illumina sequencing libraries. © 2015 Cold Spring Harbor Laboratory Press.

  13. High frequency of germline p53 mutations in childhood adrenocortical cancer.

    Science.gov (United States)

    Wagner, J; Portwine, C; Rabin, K; Leclerc, J M; Narod, S A; Malkin, D

    1994-11-16

    Adrenocortical carcinoma (ADCC) is a rare childhood cancer, affecting three of 1 million children younger than 16 years old in the United States. ADCC may be found in association with the Li-Fraumeni and Beckwith-Wiedemann syndromes. Children with ADCC are also at substantially increased risk of second primary cancers. Because of these associations, it is believed that the genetic basis for ADCC is stronger than for most childhood malignancies. Germline mutations of the TP53 tumor suppressor gene are associated with cancer predisposition in families with the Li-Fraumeni syndrome as well as in individuals with sporadically occurring component tumors of the syndrome. We investigated the possibility that germline TP53 gene alterations existed in children with ADCC. Sixteen children with ADCC under the age of 18 were identified from searches of medial oncology records at three Canadian hospitals. Eleven of these 16 patients identified were alive. The mean age at diagnosis was 4.8 years (range, 1-17 years). Family histories were obtained for 11 unselected children with ADCC (six girls and five boys). Pathologic confirmation of tumor diagnosis was obtained from the medical records. Using single-strand conformational polymorphism analysis followed by single-strand DNA sequencing, genomic DNA extracted from whole blood was analyzed for the presence of TP53 mutations for six living ADCC patients. Three of six (50%) children were found to carry germline TP53 mutations in exons 5, 6, and 7, respectively. Both wild-type and mutant alleles were identified in all three TP53 sequences, indicating that the patients were heterozygous for germline TP53 mutations. None of these children was from a family with the Li-Fraumeni syndrome. The mutation in one child was shown to be inherited from the mother, who subsequently developed breast cancer. A striking excess of cancer was found in one family of a patient carrying wild-type TP53. Our observation of a high frequency of germline TP53

  14. Contribution of a mutational hot spot to hemoglobin adaptation in high-altitude Andean house wrens.

    Science.gov (United States)

    Galen, Spencer C; Natarajan, Chandrasekhar; Moriyama, Hideaki; Weber, Roy E; Fago, Angela; Benham, Phred M; Chavez, Andrea N; Cheviron, Zachary A; Storz, Jay F; Witt, Christopher C

    2015-11-10

    A key question in evolutionary genetics is why certain mutations or certain types of mutation make disproportionate contributions to adaptive phenotypic evolution. In principle, the preferential fixation of particular mutations could stem directly from variation in the underlying rate of mutation to function-altering alleles. However, the influence of mutation bias on the genetic architecture of phenotypic evolution is difficult to evaluate because data on rates of mutation to function-altering alleles are seldom available. Here, we report the discovery that a single point mutation at a highly mutable site in the β(A)-globin gene has contributed to an evolutionary change in hemoglobin (Hb) function in high-altitude Andean house wrens (Troglodytes aedon). Results of experiments on native Hb variants and engineered, recombinant Hb mutants demonstrate that a nonsynonymous mutation at a CpG dinucleotide in the β(A)-globin gene is responsible for an evolved difference in Hb-O2 affinity between high- and low-altitude house wren populations. Moreover, patterns of genomic differentiation between high- and low-altitude populations suggest that altitudinal differentiation in allele frequencies at the causal amino acid polymorphism reflects a history of spatially varying selection. The experimental results highlight the influence of mutation rate on the genetic basis of phenotypic evolution by demonstrating that a large-effect allele at a highly mutable CpG site has promoted physiological differentiation in blood O2 transport capacity between house wren populations that are native to different elevations.

  15. Mutation breeding of Bacillus subtilis YTB4 with high yield of ...

    African Journals Online (AJOL)

    Helium-neon (He-Ne) laser irradiation is a highly efficient mutation breeding technology and is widely applied to various fields of biological science. Using Bacillus subtilis YTB4 with high yield of multienzyme complex as original strain, mutation breeding was carried out by He-Ne laser irradiation in this study. Based on the ...

  16. Mutation Analysis of SLC26A4 for Pendred Syndrome and Nonsyndromic Hearing Loss by High-Resolution Melting

    Science.gov (United States)

    Chen, Neng; Tranebjærg, Lisbeth; Rendtorff, Nanna Dahl; Schrijver, Iris

    2011-01-01

    Pendred syndrome and DFNB4 (autosomal recessive nonsyndromic congenital deafness, locus 4) are associated with autosomal recessive congenital sensorineural hearing loss and mutations in the SLC26A4 gene. Extensive allelic heterogeneity, however, necessitates analysis of all exons and splice sites to identify mutations for individual patients. Although Sanger sequencing is the gold standard for mutation detection, screening methods supplemented with targeted sequencing can provide a cost-effective alternative. One such method, denaturing high-performance liquid chromatography, was developed for clinical mutation detection in SLC26A4. However, this method inherently cannot distinguish homozygous changes from wild-type sequences. High-resolution melting (HRM), on the other hand, can detect heterozygous and homozygous changes cost-effectively, without any post-PCR modifications. We developed a closed-tube HRM mutation detection method specific for SLC26A4 that can be used in the clinical diagnostic setting. Twenty-eight primer pairs were designed to cover all 21 SLC26A4 exons and splice junction sequences. Using the resulting amplicons, initial HRM analysis detected all 45 variants previously identified by sequencing. Subsequently, a 384-well plate format was designed for up to three patient samples per run. Blinded HRM testing on these plates of patient samples collected over 1 year in a clinical diagnostic laboratory accurately detected all variants identified by sequencing. In conclusion, HRM with targeted sequencing is a reliable, simple, and cost-effective method for SLC26A4 mutation screening and detection. PMID:21704276

  17. High frequency of RPL22 mutations in microsatellite-unstable colorectal and endometrial tumors.

    Science.gov (United States)

    Ferreira, Ana M; Tuominen, Iina; van Dijk-Bos, Krista; Sanjabi, Bahram; van der Sluis, Tineke; van der Zee, Ate G; Hollema, Harry; Zazula, Monika; Sijmons, Rolf H; Aaltonen, Lauri A; Westers, Helga; Hofstra, Robert M W

    2014-12-01

    Ribosomal Protein L22 (RPL22) encodes a protein that is a component of the 60S subunit of the ribosome. Variants in this gene have recently been linked to cancer development. Mutations in an A8 repeat in exon 2 were found in a recent study in 52% of microsatellite-unstable endometrial tumors. These tumors are particularly prone to mutations in repeats due to mismatch repair deficiency. We screened this coding repeat in our collection of microsatellite-unstable endometrial tumors (EC) and colorectal tumors (CRC). We found 50% mutation frequency for EC and 77% mutation frequency for CRC. These results confirm the previous study on the involvement of RPL22 in EC and, more importantly, reports for the first time such high mutation frequency in this gene in colorectal cancer. Furthermore, considering the high mutation frequency found, our data point toward an important role for RPL22 in microsatellite instability carcinogenesis. © 2014 WILEY PERIODICALS, INC.

  18. Mutagenicity of flavonoids assayed by bacterial reverse mutation (Ames) test.

    Science.gov (United States)

    Resende, Flavia Aparecida; Vilegas, Wagner; Dos Santos, Lourdes Campaner; Varanda, Eliana Aparecida

    2012-05-07

    The mutagenicity of ten flavonoids was assayed by the Ames test, in Salmonella typhimurium strains TA98, TA100 and TA102, with the aim of establishing hydroxylation pattern-mutagenicity relationship profiles. The compounds assessed were: quercetin, kaempferol, luteolin, fisetin, chrysin, galangin, flavone, 3-hydroxyflavone, 5-hydroxyflavone and 7-hydroxyflavone. In the Ames assay, quercetin acted directly and its mutagenicity increased with metabolic activation. In the presence of S9 mix, kaempferol and galangin were mutagenic in the TA98 strain and kaempferol showed signs of mutagenicity in the other strains. The absence of hydroxyl groups, as in flavone, only signs of mutagenicity were shown in strain TA102, after metabolization and, among monohydroxylated flavones (3-hydroxyflavone, 5-hydroxyflavone and 7-hydroxyflavone), the presence of hydroxyl groups only resulted in minor changes. Luteolin and fisetin also showed signs of mutagenicity in strain TA102. Finally, chrysin, which has only two hydroxy groups, at the 5-OH and 7-OH positions, also did not induce mutagenic activity in any of the bacterial strains used, under either activation condition. All the flavonoids were tested at concentrations varying from 2.6 to 30.7 nmol/plate for galangin and 12.1 to 225.0 nmol/plate for other flavonoids. In light of the above, it is necessary to clarify the conditions and the mechanisms that mediate the biological effects of flavonoids before treating them as therapeutical agents, since some compounds can be biotransformed into more genotoxic products; as is the case for galangin, kaempferol and quercetin.

  19. Mutagenicity of Flavonoids Assayed by Bacterial Reverse Mutation (Ames Test

    Directory of Open Access Journals (Sweden)

    Eliana Aparecida Varanda

    2012-05-01

    Full Text Available The mutagenicity of ten flavonoids was assayed by the Ames test, in Salmonella typhimurium strains TA98, TA100 and TA102, with the aim of establishing hydroxylation pattern-mutagenicity relationship profiles. The compounds assessed were: quercetin, kaempferol, luteolin, fisetin, chrysin, galangin, flavone, 3-hydroxyflavone, 5-hydroxyflavone and 7-hydroxyflavone. In the Ames assay, quercetin acted directly and its mutagenicity increased with metabolic activation. In the presence of S9 mix, kaempferol and galangin were mutagenic in the TA98 strain and kaempferol showed signs of mutagenicity in the other strains. The absence of hydroxyl groups, as in flavone, only signs of mutagenicity were shown in strain TA102, after metabolization and, among monohydroxylated flavones (3-hydroxyflavone, 5-hydroxyflavone and 7-hydroxyflavone, the presence of hydroxyl groups only resulted in minor changes. Luteolin and fisetin also showed signs of mutagenicity in strain TA102. Finally, chrysin, which has only two hydroxy groups, at the 5-OH and 7-OH positions, also did not induce mutagenic activity in any of the bacterial strains used, under either activation condition. All the flavonoids were tested at concentrations varying from 2.6 to 30.7 nmol/plate for galangin and 12.1 to 225.0 nmol/plate for other flavonoids. In light of the above, it is necessary to clarify the conditions and the mechanisms that mediate the biological effects of flavonoids before treating them as therapeutical agents, since some compounds can be biotransformed into more genotoxic products; as is the case for galangin, kaempferol and quercetin.

  20. Identification of High-Impact cis-Regulatory Mutations Using Transcription Factor Specific Random Forest Models.

    Directory of Open Access Journals (Sweden)

    Dmitry Svetlichnyy

    2015-11-01

    Full Text Available Cancer genomes contain vast amounts of somatic mutations, many of which are passenger mutations not involved in oncogenesis. Whereas driver mutations in protein-coding genes can be distinguished from passenger mutations based on their recurrence, non-coding mutations are usually not recurrent at the same position. Therefore, it is still unclear how to identify cis-regulatory driver mutations, particularly when chromatin data from the same patient is not available, thus relying only on sequence and expression information. Here we use machine-learning methods to predict functional regulatory regions using sequence information alone, and compare the predicted activity of the mutated region with the reference sequence. This way we define the Predicted Regulatory Impact of a Mutation in an Enhancer (PRIME. We find that the recently identified driver mutation in the TAL1 enhancer has a high PRIME score, representing a "gain-of-target" for MYB, whereas the highly recurrent TERT promoter mutation has a surprisingly low PRIME score. We trained Random Forest models for 45 cancer-related transcription factors, and used these to score variations in the HeLa genome and somatic mutations across more than five hundred cancer genomes. Each model predicts only a small fraction of non-coding mutations with a potential impact on the function of the encompassing regulatory region. Nevertheless, as these few candidate driver mutations are often linked to gains in chromatin activity and gene expression, they may contribute to the oncogenic program by altering the expression levels of specific oncogenes and tumor suppressor genes.

  1. Life insurance and genetic test results: a mutation carrier's fight to achieve full cover.

    Science.gov (United States)

    Keogh, Louise A; Otlowski, Margaret F A

    2013-09-02

    Currently, there is debate about life insurance companies' use of genetic information for assessing applicants. In his early 20s, James (pseudonym) was denied full life insurance cover because he revealed that he had discussed genetic testing with a genetic counsellor. He was later tested and found to carry a mutation in the MSH6 gene; after disclosing this, he was denied cover for cancer by two other life insurance companies. Unsatisfied with the insurance companies' risk assessments, and based on his understanding that regular colonoscopy significantly reduced his risk of cancer, James made a complaint to the Australian Human Rights Commission. After informing the third insurance company that he had done so, he was offered full coverage, which suggests that the company did not have actuarial data to justify its decision. This case provides evidence of the high level of initiative and proactivity required for a consumer to achieve a fair result. Few Australians would be in a position to pursue the level of research and advocacy undertaken by James (a professional with scientific training). We call on a collaborative approach between industry, government and researchers to address the issues that James's case raises about genetic testing and life insurance.

  2. Mutation Detection in Patients With Advanced Cancer by Universal Sequencing of Cancer-Related Genes in Tumor and Normal DNA vs Guideline-Based Germline Testing.

    Science.gov (United States)

    Mandelker, Diana; Zhang, Liying; Kemel, Yelena; Stadler, Zsofia K; Joseph, Vijai; Zehir, Ahmet; Pradhan, Nisha; Arnold, Angela; Walsh, Michael F; Li, Yirong; Balakrishnan, Anoop R; Syed, Aijazuddin; Prasad, Meera; Nafa, Khedoudja; Carlo, Maria I; Cadoo, Karen A; Sheehan, Meg; Fleischut, Megan H; Salo-Mullen, Erin; Trottier, Magan; Lipkin, Steven M; Lincoln, Anne; Mukherjee, Semanti; Ravichandran, Vignesh; Cambria, Roy; Galle, Jesse; Abida, Wassim; Arcila, Marcia E; Benayed, Ryma; Shah, Ronak; Yu, Kenneth; Bajorin, Dean F; Coleman, Jonathan A; Leach, Steven D; Lowery, Maeve A; Garcia-Aguilar, Julio; Kantoff, Philip W; Sawyers, Charles L; Dickler, Maura N; Saltz, Leonard; Motzer, Robert J; O'Reilly, Eileen M; Scher, Howard I; Baselga, Jose; Klimstra, David S; Solit, David B; Hyman, David M; Berger, Michael F; Ladanyi, Marc; Robson, Mark E; Offit, Kenneth

    2017-09-05

    Guidelines for cancer genetic testing based on family history may miss clinically actionable genetic changes with established implications for cancer screening or prevention. To determine the proportion and potential clinical implications of inherited variants detected using simultaneous sequencing of the tumor and normal tissue ("tumor-normal sequencing") compared with genetic test results based on current guidelines. From January 2014 until May 2016 at Memorial Sloan Kettering Cancer Center, 10 336 patients consented to tumor DNA sequencing. Since May 2015, 1040 of these patients with advanced cancer were referred by their oncologists for germline analysis of 76 cancer predisposition genes. Patients with clinically actionable inherited mutations whose genetic test results would not have been predicted by published decision rules were identified. Follow-up for potential clinical implications of mutation detection was through May 2017. Tumor and germline sequencing compared with the predicted yield of targeted germline sequencing based on clinical guidelines. Proportion of clinically actionable germline mutations detected by universal tumor-normal sequencing that would not have been detected by guideline-directed testing. Of 1040 patients, the median age was 58 years (interquartile range, 50.5-66 years), 65.3% were male, and 81.3% had stage IV disease at the time of genomic analysis, with prostate, renal, pancreatic, breast, and colon cancer as the most common diagnoses. Of the 1040 patients, 182 (17.5%; 95% CI, 15.3%-19.9%) had clinically actionable mutations conferring cancer susceptibility, including 149 with moderate- to high-penetrance mutations; 101 patients tested (9.7%; 95% CI, 8.1%-11.7%) would not have had these mutations detected using clinical guidelines, including 65 with moderate- to high-penetrance mutations. Frequency of inherited mutations was related to case mix, stage, and founder mutations. Germline findings led to discussion or initiation of

  3. Pitfalls in genetic testing : the story of missed SCN1A mutations

    NARCIS (Netherlands)

    Djémié, Tania; Weckhuysen, Sarah; von Spiczak, Sarah; Carvill, Gemma L; Jaehn, Johanna; Anttonen, Anna-Kaisa; Brilstra, Eva|info:eu-repo/dai/nl/23639195X; Caglayan, Hande S; de Kovel, Carolien G|info:eu-repo/dai/nl/304812129; Depienne, Christel; Gaily, Eija; Gennaro, Elena; Giraldez, Beatriz G; Gormley, Padhraig; Guerrero-López, Rosa; Guerrini, Renzo; Hämäläinen, Eija; Hartmann, Corinna; Hernandez-Hernandez, Laura; Hjalgrim, Helle; Koeleman, Bobby P C|info:eu-repo/dai/nl/157197468; Leguern, Eric; Lehesjoki, Anna-Elina; Lemke, Johannes R; Leu, Costin; Marini, Carla; McMahon, Jacinta M; Mei, Davide; Møller, Rikke S; Muhle, Hiltrud; Myers, Candace T; Nava, Caroline; Serratosa, Jose M; Sisodiya, Sanjay M; Stephani, Ulrich; Striano, Pasquale; van Kempen, Marjan J A|info:eu-repo/dai/nl/118723073; Verbeek, Nienke E|info:eu-repo/dai/nl/304816310; Usluer, Sunay; Zara, Federico; Palotie, Aarno; Mefford, Heather C; Scheffer, Ingrid E; De Jonghe, Peter; Helbig, Ingo; Suls, Arvid

    BACKGROUND: Sanger sequencing, still the standard technique for genetic testing in most diagnostic laboratories and until recently widely used in research, is gradually being complemented by next-generation sequencing (NGS). No single mutation detection technique is however perfect in identifying

  4. SDH Subunit Mutation Status in Saliva: Genetic Testing in Patients with Pheochromocytoma.

    Science.gov (United States)

    Osinga, T E; Xekouki, P; Nambuba, J; Faucz, F R; de la Luz Sierra, M; Links, T P; Kema, I P; Adams, K; Stratakis, C A; van der Horst-Schrivers, A N A; Pacak, K

    2016-04-01

    Germline mutations occur in up to 30-40% of pheochromocytoma/paraganglioma, with mutations in the succinate dehydrogenase (SDH) subunits B (SDHB) and D (SDHD) being the most common. Blood samples are favored for obtaining high quality DNA, however, leukocytes can also be obtained by collecting saliva. The aim of this study was to determine whether SDHB and SDHD gene mutations in patients with pheochromocytoma/paraganglioma could be determined using a salivary sample. Paired blood and salivary samples were collected from 30 patients: 9 SDHB mutation positive, 13 with a SDHD mutation, and 8 without any SDHx mutations. The Oragene DISCOVER kit was used to collect and extract DNA from saliva. Blood DNA was extracted from EDTA blood samples. The DNA purification and concentration were measured by spectrophotometry. The 8 exons of SDHB and the 4 exons of SDHD were amplified and sequenced by PCR-based bidirectional Sanger sequencing. Total DNA yields from blood DNA were similar to those obtained from saliva DNA [mean (±SD) saliva vs. blood DNA concentration 514.6 (±580.8) ng/µl vs. 360.9 (±262.7) ng/µl; p=0.2)]. The purity of the saliva DNA samples was lower than that of blood [mean OD260/OD280 ratio 1.78 (±0.13) vs. 1.87 (±0.04); p=0.001, respectively], indicating more protein contamination in the saliva-extracted DNA. This study shows that salivary DNA collected from patients with pheochromocytoma/paraganglioma is a good alternative for extraction of genomic DNA for its high DNA concentration and acceptable purity and can be used as an alternative to blood derived DNA in screening for SDHB and SDHD mutations. © Georg Thieme Verlag KG Stuttgart · New York.

  5. Interspecific tests of allelism reveal the evolutionary timing and pattern of accumulation of reproductive isolation mutations.

    Science.gov (United States)

    Sherman, Natasha A; Victorine, Anna; Wang, Richard J; Moyle, Leonie C

    2014-09-01

    Despite extensive theory, little is known about the empirical accumulation and evolutionary timing of mutations that contribute to speciation. Here we combined QTL (Quantitative Trait Loci) analyses of reproductive isolation, with information on species evolutionary relationships, to reconstruct the order and timing of mutations contributing to reproductive isolation between three plant (Solanum) species. To evaluate whether reproductive isolation QTL that appear to coincide in more than one species pair are homologous, we used cross-specific tests of allelism and found evidence for both homologous and lineage-specific (non-homologous) alleles at these co-localized loci. These data, along with isolation QTL unique to single species pairs, indicate that >85% of isolation-causing mutations arose later in the history of divergence between species. Phylogenetically explicit analyses of these data support non-linear models of accumulation of hybrid incompatibility, although the specific best-fit model differs between seed (pairwise interactions) and pollen (multi-locus interactions) sterility traits. Our findings corroborate theory that predicts an acceleration ('snowballing') in the accumulation of isolation loci as lineages progressively diverge, and suggest different underlying genetic bases for pollen versus seed sterility. Pollen sterility in particular appears to be due to complex genetic interactions, and we show this is consistent with a snowball model where later arising mutations are more likely to be involved in pairwise or multi-locus interactions that specifically involve ancestral alleles, compared to earlier arising mutations.

  6. Interspecific Tests of Allelism Reveal the Evolutionary Timing and Pattern of Accumulation of Reproductive Isolation Mutations

    Science.gov (United States)

    Sherman, Natasha A.; Victorine, Anna; Wang, Richard J.; Moyle, Leonie C.

    2014-01-01

    Despite extensive theory, little is known about the empirical accumulation and evolutionary timing of mutations that contribute to speciation. Here we combined QTL (Quantitative Trait Loci) analyses of reproductive isolation, with information on species evolutionary relationships, to reconstruct the order and timing of mutations contributing to reproductive isolation between three plant (Solanum) species. To evaluate whether reproductive isolation QTL that appear to coincide in more than one species pair are homologous, we used cross-specific tests of allelism and found evidence for both homologous and lineage-specific (non-homologous) alleles at these co-localized loci. These data, along with isolation QTL unique to single species pairs, indicate that >85% of isolation-causing mutations arose later in the history of divergence between species. Phylogenetically explicit analyses of these data support non-linear models of accumulation of hybrid incompatibility, although the specific best-fit model differs between seed (pairwise interactions) and pollen (multi-locus interactions) sterility traits. Our findings corroborate theory that predicts an acceleration (‘snowballing’) in the accumulation of isolation loci as lineages progressively diverge, and suggest different underlying genetic bases for pollen versus seed sterility. Pollen sterility in particular appears to be due to complex genetic interactions, and we show this is consistent with a snowball model where later arising mutations are more likely to be involved in pairwise or multi-locus interactions that specifically involve ancestral alleles, compared to earlier arising mutations. PMID:25211473

  7. KRAS mutation status is highly homogeneous between areas of the primary tumor and the corresponding metastasis of colorectal adenocarcinomas: one less problem in patient care.

    Science.gov (United States)

    Petaccia de Macedo, Mariana; Melo, Fernanda M; Ribeiro, Heber Salvador C; Marques, Marcio C; Kagohara, Luciane T; Begnami, Maria Dirlei; Neto, Julio C; Ribeiro, Júlia S; Soares, Fernando A; Carraro, Dirce M; Cunha, Isabela W

    2017-01-01

    Background: Mutations in KRAS are negative predictors of the response to anti-EGFR therapies in the treatment of metastatic colorectal cancer. Yet, the ideal tissue to test for KRAS mutation-primary or metastatic-remains unknown, as is the validity of testing only 1 area of the primary tumor. The aim of this study was to determine the heterogeneity of KRAS mutational status between areas of the primary lesion and between paired primary CRC and the corresponding lymph node (LN), liver, and lung metastasis with a high-sensitivity sequencing method. Design: DNA from 2 or 3 areas from the primary tumor and 1 area of metastatic tissue was obtained from formalin-fixed paraffin-embedded specimens from 102 metastatic CRC patients. Mutations in KRAS codons 12, 13, and 61 were analyzed by pyrosequencing. Ninety-one cases had DNA extracted from more than 1 area of the primary tumor. Only 1 patient showed intratumor heterogeneity, which involved KRAS mutation type, not KRAS mutational status. We examined KRAS mutations in 97 primaries and matched metastatic samples, recording 2 discordant cases, representing 2.1% of our cohort of matched samples. Conclusion:KRAS status is highly homogeneous throughout primary CRC tumor areas and consistent between the primary tumor and metastatic tissue in the same patient. Our data suggest that testing KRAS mutations in only 1 area of the primary or metastatic tissue is suitable for predicting the response to anti-EGFR treatment and guiding clinical decisions.

  8. A high frequency of distinct ATM gene mutations in ataxia-telangiectasia

    Energy Technology Data Exchange (ETDEWEB)

    Wright, J.; Teraoka, S.; Concannon, P. [Univ. of Washington School of Medicine, Seattle, WA (United States)] [and others

    1996-10-01

    The clinical features of the autosomal recessive disorder ataxia-telangiectasia (AT) include a progressive cerebellar ataxia, hypersensitivity to ionizing radiation, and an increased susceptibility to malignancies. Epidemiological studies have suggested that AT heterozygotes may also be at increased risk for malignancy, possibly as a consequence of radiation exposure. A gene mutated in AT patients (ATM) has recently been isolated, making mutation screening in both patients and the general population possible. Because of the relatively large size of the ATM gene, the design of screening programs will depend on the types and distribution of mutations in the general population. In this report, we describe 30 mutations identified in a panel of unrelated AT patients and controls. Twenty-five of the 30 were distinct, and most patients were compound heterozygotes. The most frequently detected mutation was found in three different families and had previously been reported in five others. This corresponds to a frequency of 8% of all reported ATM mutations. Twenty-two of the alterations observed would be predicted to lead to protein truncation at sites scattered throughout the molecule. Two fibroblast cell lines, which displayed normal responses to ionizing radiation, also proved to be heterozygous for truncation mutations of ATM. These observations suggest that the carrier frequency of ATM mutations may be sufficiently high to make population screening practical. However, such screening may need to be done prospectively, that is, by searching for new mutations rather than by screening for just those already identified in AT families. 33 refs., 1 fig., 1 tab.

  9. Breast cancer size estimation with MRI in BRCA mutation carriers and other high risk patients

    Energy Technology Data Exchange (ETDEWEB)

    Mann, R.M., E-mail: r.mann@rad.umcn.nl [Radboud University Nijmegen Medical Centre, Department of Radiology, Nijmegen (Netherlands); Bult, P., E-mail: p.bult@path.umcn.nl [Radboud University Nijmegen Medical Centre, Department of Pathology, Nijmegen (Netherlands); Laarhoven, H.W.M. van, E-mail: h.vanlaarhoven@amc.uva.nl [Academic Medical Centre, University of Amsterdam, Department of Medical Oncology, Amsterdam (Netherlands); Radboud University Nijmegen Medical Centre, Department of Medical Oncology, Nijmegen (Netherlands); Span, P.N., E-mail: p.span@rther.umcn.nl [Radboud University Nijmegen Medical Centre, Department of Radiation Oncology, Nijmegen (Netherlands); Schlooz, M., E-mail: m.schlooz@chir.umcn.nl [Radboud University Nijmegen Medical Centre, Department of Surgery, Nijmegen (Netherlands); Veltman, J., E-mail: j.veltman@zgt.nl [Hospital group Twente (ZGT), Department of Radiology, Almelo (Netherlands); Hoogerbrugge, N., E-mail: n.hoogerbrugge@gen.umcn.nl [Radboud University Nijmegen Medical Centre, Department of Human Genetics, Nijmegen (Netherlands)

    2013-09-15

    Objective: To assess the value of breast MRI in size assessment of breast cancers in high risk patients, including those with a BRCA 1 or 2 mutation. Guidelines recommend invariably breast MRI screening for these patients and therapy is thus based on these findings. However, the accuracy of breast MRI for staging purposes is only tested in sporadic cancers. Methods: We assessed concordance of radiologic staging using MRI with histopathology in 49 tumors in 46 high risk patients (23 BRCA1, 12 BRCA2 and 11 Non-BRCA patients). The size of the total tumor area (TTA) was compared to pathology. In invasive carcinomas (n = 45) the size of the largest focus (LF) was also addressed. Results: Correlation of MRI measurements with pathology was 0.862 for TTA and 0.793 for LF. TTA was underestimated in 8(16%), overestimated in 5(10%), and correctly measured in 36(73%) cases. LF was underestimated in 4(9%), overestimated in 5(11%), and correctly measured in 36(80%) cases. Impact of BRCA 1 or 2 mutations on the quality of size estimation was not observed. Conclusions: Tumor size estimation using breast MRI in high risk patients is comparable to its performance in sporadic cancers. Therefore, breast MRI can safely be used for treatment planning.

  10. More breast cancer patients prefer BRCA-mutation testing without prior face-to-face genetic counseling

    NARCIS (Netherlands)

    Sie, A.S.; Zelst-Stams, W.A.G. van; Spruijt, L.; Mensenkamp, A.R.; Ligtenberg, M.J.L.; Brunner, H.G.; Prins, J.B.; Hoogerbrugge, N.

    2014-01-01

    Currently, most breast cancer (BC) patients receive face-to-face genetic counseling (DNA-intake) prior to BRCA-mutation testing, with generic information regarding hereditary BC and BRCA-mutation testing. This prospective study evaluated a novel format: replacing the intake consultation with

  11. Transmission of the P250R mutation of the FGFR3 gene in four generations with highly variable phenotype

    DEFF Research Database (Denmark)

    Hove, Hanne Buciek; Dunø, Morten; Daugaard-Jensen, Jette

    Transmission of the P250R mutation of the FGFR3 gene in four generations with highly variable phenotype.......Transmission of the P250R mutation of the FGFR3 gene in four generations with highly variable phenotype....

  12. Multiplex genomic test of mutation and fusion genes in small biopsy specimen of lung cancer.

    Science.gov (United States)

    Oshita, Fumihiro; Kasajima, Rika; Miyagi, Yohei

    2016-07-01

    We evaluated multiple oncogenic mutations and fusion genes in small specimen obtained by bronchoscopy. Eight patients with lung cancer were recruited, 3 small cell lung cancer, 3 non-small cell lung cancer, 1 adenocarcinoma and 1 squamous cell carcinoma. A median value of extracted RNA and DNA amounts from specimen was 1573 ng (range 367.5 to 8900) and 6700 ng (range 550 to 68000 ng), respectively. We applied amplicon sequencing panels that cover exon regions of 41 genes related to lung tumorigenesis as well as total 61 major variants of ALK, ROS, RET or NTRK1 fusion transcripts. Nineteen of 41 gene mutations were detected in our isolated DNAs of 8 patients. We could detect four to eleven mutations in each specimen; however the mutation combination in each 8 patients were different. The most common genetic alterations were TP53, KMT2D, MET, NOTCH2 and SETD2, which were detected in 4 to 6 patients. We did not detect fusion transcripts of ALK, ROS, RET and NTRK1 in every specimen. In conclusion, multiplex genomic test was performed on small amounts specimen of bronchoscopy biopsy with a 100% success rate. Such testing is considered to be able to assist physicians in matching patients with approved or experimental targeted treatments. © 2016 Old City Publishing, Inc.

  13. Frequency of CDH1 germline mutations in gastric carcinoma coming from high- and low-risk areas: metanalysis and systematic review of the literature

    Directory of Open Access Journals (Sweden)

    Corso Giovanni

    2012-01-01

    Full Text Available Abstract Background The frequency of E-cadherin germline mutations in countries with different incidence rates for gastric carcinoma has not been well established. The goal of this study was to assess the worldwide frequency of CDH1 germline mutations in gastric cancers coming from low- and high-risk areas. Methods English articles using MEDLINE access (from 1998 to 2011. Search terms included CDH1, E-cadherin, germline mutation, gastric cancer, hereditary, familial and diffuse histotype. The study included all E-cadherin germline mutations identified in gastric cancer patients; somatic mutations and germline mutations reported in other tumors were excluded. The method of this study was scheduled in accordance with the "PRISMA statement for reporting systematic reviews and meta-analyses". Countries were classified as low- or middle/high risk-areas for gastric carcinoma incidence. Statistical analysis was performed to correlate the CDH1 mutation frequency with gastric cancer incidence areas. Results A total of 122 E-cadherin germline mutations have been identified; the majority (87.5% occurred in gastric cancers coming from low-risk areas. In high-risk areas, we identified 16 mutations in which missense mutations were predominant. (68.8%. We verified a significant association between the mutation frequency and the gastric cancer risk area (p Conclusions E-cadherin genetic screenings performed in low-risk areas for gastric cancer identified a higher frequency of CDH1 germline mutations. This data could open new approaches in the gastric cancer prevention test; before proposing a proband candidate for the CDH1 genetic screening, geographic variability, alongside the family history should be considered.

  14. Comprehensive RNA Analysis of the NF1 Gene in Classically Affected NF1 Affected Individuals Meeting NIH Criteria has High Sensitivity and Mutation Negative Testing is Reassuring in Isolated Cases With Pigmentary Features Only

    Directory of Open Access Journals (Sweden)

    D.G. Evans

    2016-05-01

    Interpretation: RNA analysis in individuals with presumed NF1 has high sensitivity and includes a small subset with DNET without an NF1 variant. Furthermore negative analysis for NF1/SPRED1 provides strong reassurance to children with ≥6 CAL that they are unlikely to have NF1.

  15. Frameshift Mutations of AKAP9 Gene in Gastric and Colorectal Cancers with High Microsatellite Instability.

    Science.gov (United States)

    Jo, Yun Sol; Kim, Min Sung; Yoo, Nam Jin; Lee, Sug Hyung

    2016-07-01

    A-kinase-anchoring protein 9 (AKAP9) coordinates the cellular location and function of protein kinase A. AKAP9 plays an important role in centrosome duplication, cell cycle progression and maintenance of cell membrane integrity, alterations of which contribute to tumorigenesis. Somatic mutations of AKAP9 gene have been detected in many cancers including gastric (GC) and colorectal cancers (CRC), but the mutation status with respect to microsatellite instability (MSI) has not been reported. In a public database, we found that AKAP9 gene had mononucleotide repeats in the coding sequences that might be mutation targets in the cancers with MSI. We analyzed the mutations in 79 GCs and 124 CRCs including high MSI (MSI-H) and microsatellite stable/low MSI (MSS/MSI-L) cases by single-strand conformation polymorphism analysis and DNA sequencing. Overall, we found AKAP9 frameshift mutations in 4 (11.7 %) GCs and 20 (17.7 %) CRCs with MSI-H (24/113), but not in MSS/MSI-L cancers (0/90) (p < 0.001). In addition, we analyzed intratumoral heterogeneity (ITH) of AKAP9 frameshift mutations in 16 CRCs and found that five CRCs (31.3 %) harbored regional ITH of the AKAP9 frameshift mutations. Our data indicate that AKAP9 gene harbors not only somatic frameshift mutations but also mutational ITH, which together may be features of GC and CRC with MSI-H. Our results also suggest that regional mutation analysis is needed for a better evaluation of mutation status in these tumors to overcome ITH.

  16. BRIP1 loss-of-function mutations confer high risk for familial ovarian cancer, but not familial breast cancer.

    Science.gov (United States)

    Weber-Lassalle, Nana; Hauke, Jan; Ramser, Juliane; Richters, Lisa; Groß, Eva; Blümcke, Britta; Gehrig, Andrea; Kahlert, Anne-Karin; Müller, Clemens R; Hackmann, Karl; Honisch, Ellen; Weber-Lassalle, Konstantin; Niederacher, Dieter; Borde, Julika; Thiele, Holger; Ernst, Corinna; Altmüller, Janine; Neidhardt, Guido; Nürnberg, Peter; Klaschik, Kristina; Schroeder, Christopher; Platzer, Konrad; Volk, Alexander E; Wang-Gohrke, Shan; Just, Walter; Auber, Bernd; Kubisch, Christian; Schmidt, Gunnar; Horvath, Judit; Wappenschmidt, Barbara; Engel, Christoph; Arnold, Norbert; Dworniczak, Bernd; Rhiem, Kerstin; Meindl, Alfons; Schmutzler, Rita K; Hahnen, Eric

    2018-01-24

    Germline mutations in the BRIP1 gene have been described as conferring a moderate risk for ovarian cancer (OC), while the role of BRIP1 in breast cancer (BC) pathogenesis remains controversial. To assess the role of deleterious BRIP1 germline mutations in BC/OC predisposition, 6341 well-characterized index patients with BC, 706 index patients with OC, and 2189 geographically matched female controls were screened for loss-of-function (LoF) mutations and potentially damaging missense variants. All index patients met the inclusion criteria of the German Consortium for Hereditary Breast and Ovarian Cancer for germline testing and tested negative for pathogenic BRCA1/2 variants. BRIP1 LoF mutations confer a high OC risk in familial index patients (odds ratio (OR) = 20.97, 95% confidence interval (CI) = 12.02-36.57, P mutations with familial BC was observed (OR = 1.81 95% CI = 1.00-3.30, P = 0.0623). In the subgroup of familial BC index patients without a family history of OC there was also no apparent association (OR = 1.42, 95% CI = 0.70-2.90, P = 0.3030). In 1027 familial BC index patients with a family history of OC, the BRIP1 mutation prevalence was significantly higher than that observed in controls (OR = 3.59, 95% CI = 1.43-9.01; P = 0.0168). Based on the negative association between BRIP1 LoF mutations and familial BC in the absence of an OC family history, we conclude that the elevated mutation prevalence in the latter cohort was driven by the occurrence of OC in these families. Compared with controls, predicted damaging rare missense variants were significantly more prevalent in OC (P = 0.0014) but not in BC (P = 0.0693) patients. To avoid ambiguous results, studies aimed at assessing the impact of candidate predisposition gene mutations on BC risk might differentiate between BC index patients with an OC family history and those without. In familial cases, we suggest that BRIP1 is a high-risk gene for late

  17. 6 Minute Walk Test in Duchenne MD Patients with Different Mutations: 12 Month Changes

    Science.gov (United States)

    Pane, Marika; Mazzone, Elena S.; Sormani, Maria Pia; Messina, Sonia; Vita, Gian Luca; Fanelli, Lavinia; Berardinelli, Angela; Torrente, Yvan; D'Amico, Adele; Lanzillotta, Valentina; Viggiano, Emanuela; D'Ambrosio, Paola; Cavallaro, Filippo; Frosini, Silvia; Bello, Luca; Bonfiglio, Serena; Scalise, Roberta; De Sanctis, Roberto; Rolle, Enrica; Bianco, Flaviana; Van der Haawue, Marlene; Magri, Francesca; Palermo, Concetta; Rossi, Francesca; Donati, Maria Alice; Alfonsi, Chiara; Sacchini, Michele; Arnoldi, Maria Teresa; Baranello, Giovanni; Mongini, Tiziana; Pini, Antonella; Battini, Roberta; Pegoraro, Elena; Previtali, Stefano C.; Napolitano, Sara; Bruno, Claudio; Politano, Luisa; Comi, Giacomo P.; Bertini, Enrico; Morandi, Lucia; Gualandi, Francesca; Ferlini, Alessandra; Goemans, Nathalie; Mercuri, Eugenio

    2014-01-01

    Objective In the last few years some of the therapeutical approaches for Duchenne muscular dystrophy (DMD) are specifically targeting distinct groups of mutations, such as deletions eligible for skipping of individual exons. The aim of this observational study was to establish whether patients with distinct groups of mutations have different profiles of changes on the 6 minute walk test (6MWT) over a 12 month period. Methods The 6MWT was performed in 191 ambulant DMD boys at baseline and 12 months later. The results were analysed using a test for heterogeneity in order to establish possible differences among different types of mutations (deletions, duplications, point mutations) and among subgroups of deletions eligible to skip individual exons. Results At baseline the 6MWD ranged between 180 and 560,80 metres (mean 378,06, SD 74,13). The 12 month changes ranged between −325 and 175 (mean −10.8 meters, SD 69.2). Although boys with duplications had better results than those with the other types of mutations, the difference was not significant. Similarly, boys eligible for skipping of the exon 44 had better baseline results and less drastic changes than those eligible for skipping exon 45 or 53, but the difference was not significant. Conclusions even if there are some differences among subgroups, the mean 12 month changes in each subgroup were all within a narrow Range: from the mean of the whole DMD cohort. This information will be of help at the time of designing clinical trials with small numbers of eligible patients. PMID:24421885

  18. 6 Minute walk test in Duchenne MD patients with different mutations: 12 month changes.

    Directory of Open Access Journals (Sweden)

    Marika Pane

    Full Text Available In the last few years some of the therapeutical approaches for Duchenne muscular dystrophy (DMD are specifically targeting distinct groups of mutations, such as deletions eligible for skipping of individual exons. The aim of this observational study was to establish whether patients with distinct groups of mutations have different profiles of changes on the 6 minute walk test (6MWT over a 12 month period.The 6MWT was performed in 191 ambulant DMD boys at baseline and 12 months later. The results were analysed using a test for heterogeneity in order to establish possible differences among different types of mutations (deletions, duplications, point mutations and among subgroups of deletions eligible to skip individual exons.At baseline the 6MWD ranged between 180 and 560,80 metres (mean 378,06, SD 74,13. The 12 month changes ranged between -325 and 175 (mean -10.8 meters, SD 69.2. Although boys with duplications had better results than those with the other types of mutations, the difference was not significant. Similarly, boys eligible for skipping of the exon 44 had better baseline results and less drastic changes than those eligible for skipping exon 45 or 53, but the difference was not significant.even if there are some differences among subgroups, the mean 12 month changes in each subgroup were all within a narrow Range: from the mean of the whole DMD cohort. This information will be of help at the time of designing clinical trials with small numbers of eligible patients.

  19. Identification of gene mutation in patients with osteogenesis imperfect using high resolution melting analysis.

    Science.gov (United States)

    Wang, Jianhai; Ren, Xiuzhi; Bai, Xue; Zhang, Tianke; Wang, Yi; Li, Keqiu; Li, Guang

    2015-08-26

    Osteogenesis imperfecta (OI), a congenital bone disorder, is caused by mutations in COL1A1 and COL1A2 genes, leading to deficiency of type I collagen. The high resolution melting (HRM) analysis has been used for detecting mutations, polymorphisms and epigenetic alteration in double-stranded DNAs. This study was to evaluate the potential application of HRM analysis for identifying gene mutations in patients with OI. This study included four children with OI and their parents and fifty normal people as controls. Blood samples were collected for HRM analysis of PCR-amplified exons and flanking DNA sequences of COL1A1 and COL1A2 genes. Direct gene sequencing was performed to validate HRM-identified gene mutations. As compared to controls, HRM analysis of samples form children with OI showed abnormal melting curves in exons 11 and 33-34 of the COL1A1 gene and exons 19 and 48 of the COL1A2 gene, which indicates the presence of heterozygous mutations in COL1A1 and COL1A2 genes. In addition to two known mutations in the COL1A2 gene, c.982G > A and c.3197G > T, sequencing analysis identified two novel mutations in the COL1A1 gene, c.2321delC and c.768dupC mutations, which function as premature stop codons. These results support future studies of applying HRM analysis as a diagnostic approach for OI.

  20. High incidence of activating TERT promoter mutations in meningiomas undergoing malignant progression.

    Science.gov (United States)

    Goutagny, Stéphane; Nault, Jean C; Mallet, Maxime; Henin, Dominique; Rossi, Jessica Z; Kalamarides, Michel

    2014-03-01

    Meningiomas are common central nervous system tumors. The World Health Organization (WHO) defines three grades, predictive of the risk of recurrence. These tumors can relapse frequently and sometimes undergo malignant transformation. Maintenance of telomere length is a key process in malignant progression, and mutations in TERT promoter have recently been identified in various types of cancer. We sequenced the TERT promoter in 85 meningiomas from 73 patients. We found a high incidence of TERT promoter mutations in patients with meningiomas undergoing malignant histological progression (28%, n = 5/18 patients). In this subset of patients with histological progression, TERT promoter mutations were found in both the lowest and the highest grade tumors, and in both NF2-mutated and nonmutated samples. In contrast, one mutation was identified in 35 meningiomas without recurrence or progression, belonging to various histological grades. This sample was an aggressive meningioma in a patient who died shortly after surgery. Interestingly, tumors showing relapse without histological progression were not mutated for TERT promoter (n = 20). Finally, TERT promoter mutations were associated with a marked increase in TERT expression. Thus, TERT promoter mutations are pivotal genetic alterations involved in malignant progression of meningiomas and could be used as a biomarker to identify meningiomas at risk of malignant transformation. © 2013 International Society of Neuropathology.

  1. Epidermal Growth Factor Receptor Mutation in a Patient with Squamous Cell Carcinoma of the Lung: Who Should Be Tested

    Directory of Open Access Journals (Sweden)

    Michael Schwitter

    2013-05-01

    Full Text Available We report the case of a 64-year-old ex-smoker with metastatic poorly differentiated squamous cell carcinoma (SCC of the lung and an epidermal growth factor receptor (EGFR mutation in exon 21 (p.L858R who achieved prolonged clinical benefit from treatment with an EGFR tyrosine kinase inhibitor (TKI. The initial diagnosis of SCC of the lung obtained by bronchoscopic biopsy was based on immunohistochemical staining only with positivity for cytokeratin (CK 5/6 and p63 because morphological diagnosis was not possible. Patients with non-small cell lung cancer (NSCLC, not otherwise specified (NOS favouring SCC are usually not tested for the presence of EGFR mutations, and therefore may not receive EGFR TKI therapy. A bronchoscopic rebiopsy showed small nests of undifferentiated tumour cells with weak immunoreactivity of some tumour cells for CK5/6, p63 and no positivity of some tumour cells for thyroid transcription factor-1. These findings suggested a mixed squamous/glandular immunophenotype that has been missed at the initial biopsy. Our clinical case illustrates the problem of tumour heterogeneity encountered in small bronchoscopic biopsies and the difficulties of evaluating the histological subtype in poorly differentiated carcinomas. Initial bronchoscopy should be performed by an experienced pulmonologist who attempts to obtain sufficient material from different areas of the tumour. In the era of targeted therapy, a remote smoking history in a patient with NOS favouring SCC should also lead to EGFR mutation testing to allow highly effective therapy to be offered to mutation-positive patients.

  2. Use of Mycobacterium smegmatis deficient in ADP-ribosyltransferase as surrogate for Mycobacterium tuberculosis in drug testing and mutation analysis.

    Directory of Open Access Journals (Sweden)

    Priyanka Agrawal

    Full Text Available Rifampicin (Rif is a first line drug used for tuberculosis treatment. However, the emergence of drug resistant strains has necessitated synthesis and testing of newer analogs of Rif. Mycobacterium smegmatis is often used as a surrogate for M. tuberculosis. However, the presence of an ADP ribosyltransferase (Arr in M. smegmatis inactivates Rif, rendering it impractical for screening of Rif analogs or other compounds when used in conjunction with them (Rif/Rif analogs. Rifampicin is also used in studying the role of various DNA repair enzymes by analyzing mutations in RpoB (a subunit of RNA polymerase causing Rif resistance. These analyses use high concentrations of Rif when M. smegmatis is used as model. Here, we have generated M. smegmatis strains by deleting arr (Δarr. The M. smegmatis Δarr strains show minimum inhibitory concentration (MIC for Rif which is similar to that for M. tuberculosis. The MICs for isoniazid, pyrazinamide, ethambutol, ciprofloxacin and streptomycin were essentially unaltered for M. smegmatis Δarr. The growth profiles and mutation spectrum of Δarr and, Δarr combined with ΔudgB (udgB encodes a DNA repair enzyme that excises uracil strains were similar to their counterparts wild-type for arr. However, the mutation spectrum of ΔfpgΔarr strain differed somewhat from that of the Δfpg strain (fpg encodes a DNA repair enzyme that excises 8-oxo-G. Our studies suggest M. smegmatis Δarr strain as an ideal model system in drug testing and mutation spectrum determination in DNA repair studies.

  3. Applications of the method of high resolution melting analysis for diagnosis of Leber's disease and the three primary mutation spectrum of LHON in the Han Chinese population.

    Science.gov (United States)

    Cui, Guanglin; Ding, Hu; Xu, Yujun; Li, Bin; Wang, Dao Wen

    2013-01-01

    Current screening methods, such as single strand conformational polymorphism (SSCP), denaturing high performance liquid chromatography (dHPLC) and direct DNA sequencing that are used for detecting mutation in Leber's hereditary optic neuropathy (LHON) subjects are time consuming and costly. Here we tested high-resolution melt (HRM) analysis for mtDNA primary mutations in LHON patients. In this study, we applied the high resolution melting (HRM) technology to screen mtDNA primary mutations in 50 LHON patients from their peripheral blood. In order to evaluate the reliability of this technique, we compared the results obtained by HRM and direct mtDNA sequencing. We also investigated the spectrum of three most common mtDNA mutations implicated in LHON in the Han Chinese population. The results showed HRM analysis differentiated all of the mtDNA primary mutations and identified 4 additional mtDNA mutations from 50 patients in the blind study. The prevalence of three primary mutations were 11778G>A (87.9%), 14484T>C (6.5%) and 3460G>A (1.7%) in the Han Chinese population. In conclusion, HRM analysis is a rapid, reliable, and low-cost tool for detecting mtDNA primary mutations and has practical applications in molecular genetics. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. Cost-effectiveness of population screening for BRCA mutations in Ashkenazi jewish women compared with family history-based testing.

    Science.gov (United States)

    Manchanda, Ranjit; Legood, Rosa; Burnell, Matthew; McGuire, Alistair; Raikou, Maria; Loggenberg, Kelly; Wardle, Jane; Sanderson, Saskia; Gessler, Sue; Side, Lucy; Balogun, Nyala; Desai, Rakshit; Kumar, Ajith; Dorkins, Huw; Wallis, Yvonne; Chapman, Cyril; Taylor, Rohan; Jacobs, Chris; Tomlinson, Ian; Beller, Uziel; Menon, Usha; Jacobs, Ian

    2015-01-01

    Population-based testing for BRCA1/2 mutations detects the high proportion of carriers not identified by cancer family history (FH)-based testing. We compared the cost-effectiveness of population-based BRCA testing with the standard FH-based approach in Ashkenazi Jewish (AJ) women. A decision-analytic model was developed to compare lifetime costs and effects amongst AJ women in the UK of BRCA founder-mutation testing amongst: 1) all women in the population age 30 years or older and 2) just those with a strong FH (≥10% mutation risk). The model assumes that BRCA carriers are offered risk-reducing salpingo-oophorectomy and annual MRI/mammography screening or risk-reducing mastectomy. Model probabilities utilize the Genetic Cancer Prediction through Population Screening trial/published literature to estimate total costs, effects in terms of quality-adjusted life-years (QALYs), cancer incidence, incremental cost-effectiveness ratio (ICER), and population impact. Costs are reported at 2010 prices. Costs/outcomes were discounted at 3.5%. We used deterministic/probabilistic sensitivity analysis (PSA) to evaluate model uncertainty. Compared with FH-based testing, population-screening saved 0.090 more life-years and 0.101 more QALYs resulting in 33 days' gain in life expectancy. Population screening was found to be cost saving with a baseline-discounted ICER of -£2079/QALY. Population-based screening lowered ovarian and breast cancer incidence by 0.34% and 0.62%. Assuming 71% testing uptake, this leads to 276 fewer ovarian and 508 fewer breast cancer cases. Overall, reduction in treatment costs led to a discounted cost savings of £3.7 million. Deterministic sensitivity analysis and 94% of simulations on PSA (threshold £20000) indicated that population screening is cost-effective, compared with current NHS policy. Population-based screening for BRCA mutations is highly cost-effective compared with an FH-based approach in AJ women age 30 years and older. © The Author

  5. Cost-effectiveness of Population Screening for BRCA Mutations in Ashkenazi Jewish Women Compared With Family History–Based Testing

    Science.gov (United States)

    Manchanda, Ranjit; Legood, Rosa; Burnell, Matthew; McGuire, Alistair; Raikou, Maria; Loggenberg, Kelly; Wardle, Jane; Sanderson, Saskia; Gessler, Sue; Side, Lucy; Balogun, Nyala; Desai, Rakshit; Kumar, Ajith; Dorkins, Huw; Wallis, Yvonne; Chapman, Cyril; Taylor, Rohan; Jacobs, Chris; Tomlinson, Ian; Beller, Uziel; Menon, Usha

    2015-01-01

    Background: Population-based testing for BRCA1/2 mutations detects the high proportion of carriers not identified by cancer family history (FH)–based testing. We compared the cost-effectiveness of population-based BRCA testing with the standard FH-based approach in Ashkenazi Jewish (AJ) women. Methods: A decision-analytic model was developed to compare lifetime costs and effects amongst AJ women in the UK of BRCA founder-mutation testing amongst: 1) all women in the population age 30 years or older and 2) just those with a strong FH (≥10% mutation risk). The model assumes that BRCA carriers are offered risk-reducing salpingo-oophorectomy and annual MRI/mammography screening or risk-reducing mastectomy. Model probabilities utilize the Genetic Cancer Prediction through Population Screening trial/published literature to estimate total costs, effects in terms of quality-adjusted life-years (QALYs), cancer incidence, incremental cost-effectiveness ratio (ICER), and population impact. Costs are reported at 2010 prices. Costs/outcomes were discounted at 3.5%. We used deterministic/probabilistic sensitivity analysis (PSA) to evaluate model uncertainty. Results: Compared with FH-based testing, population-screening saved 0.090 more life-years and 0.101 more QALYs resulting in 33 days’ gain in life expectancy. Population screening was found to be cost saving with a baseline-discounted ICER of -£2079/QALY. Population-based screening lowered ovarian and breast cancer incidence by 0.34% and 0.62%. Assuming 71% testing uptake, this leads to 276 fewer ovarian and 508 fewer breast cancer cases. Overall, reduction in treatment costs led to a discounted cost savings of £3.7 million. Deterministic sensitivity analysis and 94% of simulations on PSA (threshold £20000) indicated that population screening is cost-effective, compared with current NHS policy. Conclusion: Population-based screening for BRCA mutations is highly cost-effective compared with an FH-based approach in AJ

  6. SLC26A4 mutation testing for hearing loss associated with enlargement of the vestibular aqueduct.

    Science.gov (United States)

    Ito, Taku; Muskett, Julie; Chattaraj, Parna; Choi, Byung Yoon; Lee, Kyu Yup; Zalewski, Christopher K; King, Kelly A; Li, Xiangming; Wangemann, Philine; Shawker, Thomas; Brewer, Carmen C; Alper, Seth L; Griffith, Andrew J

    2013-05-28

    Pendred syndrome (PS) is characterized by autosomal recessive inheritance of goiter associated with a defect of iodide organification, hearing loss, enlargement of the vestibular aqueduct (EVA), and mutations of the SLC26A4 gene. However, not all EVA patients have PS or SLC26A4 mutations. Two mutant alleles of SLC26A4 are detected in ¼ of North American or European EVA populations, one mutant allele is detected in another ¼ of patient populations, and no mutations are detected in the other ½. The presence of two mutant alleles of SLC26A4 is associated with abnormal iodide organification, increased thyroid gland volume, increased severity of hearing loss, and bilateral EVA. The presence of a single mutant allele of SLC26A4 is associated with normal iodide organification, normal thyroid gland volume, less severe hearing loss and either bilateral or unilateral EVA. When other underlying correlations are accounted for, the presence of a cochlear malformation or the size of EVA does not have an effect on hearing thresholds. This is consistent with observations of an Slc26a4 mutant mouse model of EVA in which hearing loss is independent of endolymphatic hydrops or inner ear malformations. Segregation analyses of EVA in families suggest that the patients carrying one mutant allele of SLC26A4 have a second, undetected mutant allele of SLC26A4, and the probability of a sibling having EVA is consistent with its segregation as an autosomal recessive trait. Patients without any mutations are an etiologically heterogeneous group in which siblings have a lower probability of having EVA. SLC26A4 mutation testing can provide prognostic information to guide clinical surveillance and management, as well as the probability of EVA affecting a sibling.

  7. Germline BRCA1/2 mutation testing is indicated in every patient with epithelial ovarian cancer : A systematic review

    NARCIS (Netherlands)

    Arts-de Jong, Marieke; de Bock, Geertruida H.; van Asperen, Christi J.; Mourits, Marian J. E.; de Hullu, Joanne A.; Kets, C. Marleen

    The presence of a germline BRCA1/2 mutation improves options for tailored risk-reducing strategies and treatment in both breast and ovarian cancer patients and their relatives. Currently, referral for germline BRCA1/2 mutation testing of women with epithelial ovarian cancer (EOC) varies widely,

  8. Spectrum and prevalence of mutations from the first 2,500 consecutive unrelated patients referred for the FAMILION long QT syndrome genetic test

    NARCIS (Netherlands)

    Kapplinger, Jamie D.; Tester, David J.; Salisbury, Benjamin A.; Carr, Janet L.; Harris-Kerr, Carole; Pollevick, Guido D.; Wilde, Arthur A. M.; Ackerman, Michael J.

    2009-01-01

    BACKGROUND: Long QT syndrome (LQTS) is a potentially lethal, highly treatable cardiac channelopathy for which genetic testing has matured from discovery to translation and now clinical implementation. OBJECTIVES: Here we examine the spectrum and prevalence of mutations found in the first 2,500

  9. Efficient detection of factor IX mutations by denaturing high-performance liquid chromatography in Taiwanese hemophilia B patients, and the identification of two novel mutations

    Directory of Open Access Journals (Sweden)

    Pei-Chin Lin

    2014-04-01

    Full Text Available Hemophilia B (HB is an X-linked recessive disorder characterized by mutations in the clotting factor IX (FIX gene that result in FIX deficiency. Previous studies have shown a wide variation of FIX gene mutations in HB. Although the quality of life in HB has greatly improved mainly because of prophylactic replacement therapy with FIX concentrates, there exists a significant burden on affected families and the medical care system. Accurate detection of FIX gene mutations is critical for genetic counseling and disease prevention in HB. In this study, we used denaturing high-performance liquid chromatography (DHPLC, which has proved to be a highly informative and practical means of detecting mutations, for the molecular diagnosis of our patients with HB. Ten Taiwanese families affected by HB were enrolled. We used the DHPLC technique followed by direct sequencing of suspected segments to detect FIX gene mutations. In all, 11 FIX gene mutations (8 point mutations, 2 small deletions/insertions, and 1 large deletion, including two novel mutations (exon6 c.687–695, del 9 mer and c.460–461, ins T were found. According to the HB pedigrees, 25% and 75% of our patients were defined as familial and sporadic HB cases, respectively. We show that DHPLC is a highly sensitive and cost-effective method for FIX gene analysis and can be used as a convenient system for disease prevention.

  10. Recommendations for spacing of test chemical concentrations in the mouse lymphoma tk mutation assay (MLA)

    Science.gov (United States)

    Kirkland, D J; Clements, J

    1998-07-08

    Recent test guidelines for the mouse lymphoma tk mutation assay (MLA) have highlighted the need to achieve 80-90% reduction in cell survival for a valid, robust assay with toxic chemicals. For many pharmaceuticals, under new ICH recommendations, this may be the only in vitro mammalian cell test that is performed. It was important to discover, therefore, how critical it is to achieve 80-90% toxicity, and how best to select the number and spacing of test concentrations to fulfil this requirement. We analysed data from 121 positive chemicals, provided by nine industrial and commercial laboratories, and found that for 17 chemicals (14%), the response profiles were so steep that using a conventional 2-fold dilution series of test concentrations would have failed to identify the active range (> 90% toxicity at one concentration, and no significant mutation at 50% of this dose), and positive responses would have been missed. Analysis of genotoxicity results in other test systems with these 17 chemicals revealed no differences in overall response profiles from the 104 chemicals that exhibited less steep MLA responses. The MLA results were therefore deemed to be equally biologically relevant. From this analysis, it is recommended that concentration spacing in the MLA needs to be closer than that obtained with a 2-fold dilution series, and a dilution factor where each concentration is 0.75 or 0.8 of the one above is recommended to identify the active range of positive mutagens.

  11. NYX mutations in four families with high myopia with or without CSNB1

    Science.gov (United States)

    Zhou, Lin; Song, Xiusheng; Li, Yin; Li, Hongyan; Dan, Handong

    2015-01-01

    Purpose Mutations in the NYX gene are known to cause complete congenital stationary night blindness (CSNB1), which is always accompanied by high myopia. In this study, we aimed to investigate the association between NYX mutations and high myopia with or without CSNB1. Methods Four Chinese families having high myopia with or without CSNB1 and 96 normal controls were recruited. We searched for mutations in the NYX gene using Sanger sequencing. Further analyses of the detected variations in the available family members were performed, and the frequencies of the detected variations in 96 normal controls were determined to verify our deduction. The effect of each variation on the nyctalopin protein was predicted using online tools. Results Four potential pathogenic variations in the NYX gene were found in four families with high myopia with or without CSNB1. Three of the four variants were novel (c.626G>C; c.121delG; c.335T>C). The previously identified variant, c.529_530delGCinsAT, was found in an isolated highly myopic patient and an affected brother, but the other affected brother did not carry the same variation. Further linkage analyses of this family showed a coinheritance of markers at MYP1. These four mutations were not identified in the 96 normal controls. Conclusions Our study expands the mutation spectrum of NYX for cases of high myopia with CSNB1; however, more evidence is needed to elucidate the pathogenic effects of NYX on isolated high myopia. PMID:25802485

  12. Nonsyndromic retinitis pigmentosa is highly prevalent in the Jerusalem region with a high frequency of founder mutations

    Science.gov (United States)

    Banin, Eyal

    2015-01-01

    Purpose Nonsyndromic retinitis pigmentosa (RP) is the most common inherited retinal degeneration, and prevalence of the disease has been reported in populations of American and European origin with a relatively low consanguinity rate. Our aim was to determine the prevalence of nonsyndromic RP in the Jerusalem region, which has a population of about 1 million individuals with a high rate of consanguinity. Methods The patients’ clinical data included eye exam findings (visual acuity, anterior segment, and funduscopy) as well as electroretinographic (ERG) testing results under scotopic and photopic conditions. Mutation analysis on a subgroup of patients was performed mainly with candidate gene analysis and homozygosity mapping. Results We evaluated the medical records of patients with degenerative retinal diseases residing in the Jerusalem region who were examined over the past 20 years in a large tertiary medical center. A total of 453 individuals affected with nonsyndromic RP were diagnosed at our center, according to funduscopic findings and ERG testing. Based on the estimated population size of 945,000 individuals who reside in the vicinity of Jerusalem, the prevalence of nonsyndromic RP in this region is 1:2,086. The prevalence of RP was higher among Arab Muslims (1:1,798) compared to Jews (1:2,230), mainly due to consanguineous marriages that are more common in the Arab Muslim population. To identify the genetic causes of RP in our cohort, we recruited 383 patients from 183 different families for genetic analysis: 70 with autosomal recessive (AR) inheritance, 15 with autosomal dominant, 86 isolate cases, and 12 with an X-linked inheritance pattern. In 64 (35%) of the families, we identified the genetic cause of the disease, and we revised the inheritance pattern of 20 isolate cases to the AR pattern; 49% of the families in our cohort had AR inheritance. Interestingly, in 42 (66%) of the genetically identified families, the cause of disease was a founder

  13. Nonsyndromic retinitis pigmentosa is highly prevalent in the Jerusalem region with a high frequency of founder mutations.

    Science.gov (United States)

    Sharon, Dror; Banin, Eyal

    2015-01-01

    Nonsyndromic retinitis pigmentosa (RP) is the most common inherited retinal degeneration, and prevalence of the disease has been reported in populations of American and European origin with a relatively low consanguinity rate. Our aim was to determine the prevalence of nonsyndromic RP in the Jerusalem region, which has a population of about 1 million individuals with a high rate of consanguinity. The patients' clinical data included eye exam findings (visual acuity, anterior segment, and funduscopy) as well as electroretinographic (ERG) testing results under scotopic and photopic conditions. Mutation analysis on a subgroup of patients was performed mainly with candidate gene analysis and homozygosity mapping. We evaluated the medical records of patients with degenerative retinal diseases residing in the Jerusalem region who were examined over the past 20 years in a large tertiary medical center. A total of 453 individuals affected with nonsyndromic RP were diagnosed at our center, according to funduscopic findings and ERG testing. Based on the estimated population size of 945,000 individuals who reside in the vicinity of Jerusalem, the prevalence of nonsyndromic RP in this region is 1:2,086. The prevalence of RP was higher among Arab Muslims (1:1,798) compared to Jews (1:2,230), mainly due to consanguineous marriages that are more common in the Arab Muslim population. To identify the genetic causes of RP in our cohort, we recruited 383 patients from 183 different families for genetic analysis: 70 with autosomal recessive (AR) inheritance, 15 with autosomal dominant, 86 isolate cases, and 12 with an X-linked inheritance pattern. In 64 (35%) of the families, we identified the genetic cause of the disease, and we revised the inheritance pattern of 20 isolate cases to the AR pattern; 49% of the families in our cohort had AR inheritance. Interestingly, in 42 (66%) of the genetically identified families, the cause of disease was a founder mutation. Previous studies

  14. High-resolution melting analysis of MED12 mutations in uterine leiomyomas in Chinese patients.

    Science.gov (United States)

    Wang, Hua; Ye, Jun; Qian, Hua; Zhou, Ruifang; Jiang, Jun; Ye, Lihua

    2015-03-01

    Somatic mutations in mediator complex subunit 12 (MED12) have emerged as a critical genetic change in the development of uterine leiomyomas. Studies, however, have focused largely on cohorts consisting of Caucasian patients. In this study, uterine leiomyomas from Chinese patients were examined for MED12 mutations. In addition, polymerase chain reaction (PCR)-based high-resolution melting analysis (HRMA) was compared with direct sequencing as a potentially more sensitive method for the detection of MED12 mutations. Tissue samples with the pathologies of uterine leiomyoma (n=181) and other endometrial diseases (n=157) were collected from Chinese patients at the Taizhou People's Hospital and Taizhou Polytechnic College (Taizhou City, China). Genomic DNA was prepared from all samples. Both PCR-based HRMA and PCR-based direct sequencing were used to detect MED12 mutations. PCR-based HRMA and direct sequencing revealed MED12 mutations in 95/181 (52.5%) and 93/181 (51.4%) uterine leiomyomas, respectively. Nearly half of these mutations (46/93) were found in a single codon, codon 131. The coincidence rate between the two methods was 98.9% (179/181) so that no statistically significant difference was evident in the application of the methodologies (χ(2)=0.011, p=0.916). In addition, MED12 mutations were identified in 1/157 (4.17%) case of other endometrial pathologies by both methods. MED12 mutations were closely associated with the development of uterine leiomyomas, as opposed to other uterine pathologies in Chinese patients, and PCR-based HRMA was found to be a reliable method for the detection of MED12 mutations.

  15. Ultrasonographic prediction of highly aggressive telomerase reverse transcriptase (TERT) promoter-mutated papillary thyroid cancer.

    Science.gov (United States)

    Kim, Tae Hyuk; Ki, Chang-Seok; Hahn, Soo Yeon; Oh, Young Lyun; Jang, Hye Won; Kim, Sun Wook; Chung, Jae Hoon; Shin, Jung Hee

    2017-08-01

    Telomerase reverse transcriptase promoter mutations are found in highly aggressive thyroid malignancies. Our aim was to define the ultrasonographic features of telomerase reverse transcriptase promoter-mutated papillary thyroid cancer and to evaluate their predictive performances. Ultrasonographic findings were reviewed for 185 patients with surgically confirmed papillary thyroid cancer between 1994 and 2004. Genomic DNA to identify telomerase reverse transcriptase promoter mutations was extracted from archived surgical specimens. Logistic regression analysis was performed to compare clinical factors and ultrasonographic findings between telomerase reverse transcriptase promoter-mutated and wild-type papillary thyroid cancers. A telomerase reverse transcriptase promoter mutation was detected in 8.1% (15 of 185) of specimens from papillary thyroid cancer patients with a strong trend toward increasing age. Nonparallel orientation and microlobulated margin were independent ultrasonographic findings for predicting telomerase reverse transcriptase promoter-mutated papillary thyroid cancer in patients over 50 years (odds ratio 5.898, 95% confidence interval 1.092-31.851, P = 0.039 for orientation; odds ratio 5.813, 95% confidence interval 1.320-25.602, P = 0.020 for margin). Prevalence of telomerase reverse transcriptase promoter mutations increased to 50.0% in papillary thyroid cancer patients older than 50 years with both ultrasonographic findings and was 0% in patients without either finding. For identifying telomerase reverse transcriptase promoter-mutated papillary thyroid cancer, ultrasonographic had 64.3% sensitivity, 80.8% specificity, 50.0% positive predictive value and 88.4% negative predictive value. Telomerase reverse transcriptase promoter-mutated papillary thyroid cancer could be suggested by the ultrasonographic features of nonparallel orientation and microlobulated margin in patients older than 50 years. Prebiopsy recognition of this unique

  16. Feasibility of molecular testing in a multicenter study with geographical variation in India: Epidermal growth factor receptor mutation as a model molecular test

    Directory of Open Access Journals (Sweden)

    Kumar Prabhash

    2017-01-01

    Conclusions: Molecular testing at a central laboratory is a feasible option in India. Prevalence of EGFR mutation in Indian NSCLC patients was similar across western and southern centers in India. A statistically significant association between EGFR mutation and gender as well as the smoking status of the patients was observed. Majority of the patients had in-frame deletions in exon 19.

  17. BRCA mutational status, initial disease presentation, and clinical outcome in high-grade serous advanced ovarian cancer: a multicenter study.

    Science.gov (United States)

    Petrillo, Marco; Marchetti, Claudia; De Leo, Rossella; Musella, Angela; Capoluongo, Ettore; Paris, Ida; Benedetti Panici, Pierluigi; Scambia, Giovanni; Fagotti, Anna

    2017-09-01

    In the last decades, there have been several efforts to clarify the role of BRCA mutational status in women with advanced ovarian cancer, demonstrating its role in cancer development, as well as the prognostic significance of BRCA genotype. Our aim is to evaluate the correlation between BRCA mutational status and disease presentation in a large series of advanced high-grade serous ovarian cancer patients. This is a retrospective multicenter study including a consecutive series of newly diagnosed high-grade serous ovarian cancer patients with International Federation of Gynecology and Obstetrics stage IIIC-IV disease, at least 18 months of follow-up time, and tested for BRCA 1/2 germline mutation status. Disease presentation was analyzed using the following variables: laparoscopic predictive index value, incidence of bulky lymph nodes, and ovarian masses. Progression-free survival was defined as the months elapsed from initial diagnosis (staging laparoscopy) and recurrent disease or last follow-up. In all, 324 high-grade serous ovarian cancer patients received BRCA testing, and 273 fulfilled inclusion criteria. BRCA1/2 germline mutations were observed in 107 women (39.2%). No differences were documented according to BRCA mutation status in terms of International Federation of Gynecology and Obstetrics stage, CA125 levels, or presence of ascites. In patients with BRCA1/2 mutations we observed a higher incidence of peritoneal spread without ovarian mass (25.2% vs 13.9%; P value = .018) and of bulky lymph nodes (30.8% vs 17.5%; P value = .010) compared with women showing BRCA1/2 wild type genotype. Furthermore, women with BRCA1/2 mutations showed high peritoneal tumor load (laparoscopic predictive index value ≥8; 42.1% vs 27.1%; P value = .016) more frequently. Focusing on survival, no differences in term of median progression-free survival were observed among women treated with primary debulking surgery and neoadjuvant chemotherapy in the group of patients with

  18. BRCA1-mutated and basal-like breast cancers have similar aCGH profiles and a high incidence of protein truncating TP53 mutations

    Directory of Open Access Journals (Sweden)

    van de Vijver Marc J

    2010-11-01

    Full Text Available Abstract Background Basal-like breast cancers (BLBC are aggressive breast cancers for which, so far, no targeted therapy is available because they typically lack expression of hormone receptors and HER2. Phenotypic features of BLBCs, such as clinical presentation and early age of onset, resemble those of breast tumors from BRCA1-mutation carriers. The genomic instability of BRCA1-mutated tumors can be effectively targeted with DNA-damaging agents and poly-(ADP-ribose polymerase 1 (PARP1 inhibitors. Molecular similarities between BLBCs and BRCA1-mutated tumors may therefore provide predictive markers for therapeutic response of BLBCs. Methods There are several known molecular features characteristic for BRCA1-mutated breast tumors: 1 increased numbers of genomic aberrations, 2 a distinct pattern of genomic aberrations, 3 a high frequency of TP53 mutations and 4 a high incidence of complex, protein-truncating TP53 mutations. We compared the frequency of TP53 mutations and the pattern and amount of genomic aberrations between BRCA1-mutated breast tumors, BLBCs and luminal breast tumors by TP53 gene sequencing and array-based comparative genomics hybridization (aCGH analysis. Results We found that the high incidence of protein truncating TP53 mutations and the pattern and amount of genomic aberrations specific for BRCA1-mutated breast tumors are also characteristic for BLBCs and different from luminal breast tumors. Conclusions Complex, protein truncating TP53 mutations in BRCA1-mutated tumors may be a direct consequence of genomic instability caused by BRCA1 loss, therefore, the presence of these types of TP53 mutations in sporadic BLBCs might be a hallmark of BRCAness and a potential biomarker for sensitivity to PARP inhibition. Also, our data suggest that a small subset of genomic regions may be used to identify BRCA1-like BLBCs. BLBCs share molecular features that were previously found to be specific for BRCA1-mutated breast tumors. These

  19. BRCA1-mutated and basal-like breast cancers have similar aCGH profiles and a high incidence of protein truncating TP53 mutations.

    Science.gov (United States)

    Holstege, Henne; Horlings, Hugo M; Velds, Arno; Langerød, Anita; Børresen-Dale, Anne-Lise; van de Vijver, Marc J; Nederlof, Petra M; Jonkers, Jos

    2010-11-30

    Basal-like breast cancers (BLBC) are aggressive breast cancers for which, so far, no targeted therapy is available because they typically lack expression of hormone receptors and HER2. Phenotypic features of BLBCs, such as clinical presentation and early age of onset, resemble those of breast tumors from BRCA1-mutation carriers. The genomic instability of BRCA1-mutated tumors can be effectively targeted with DNA-damaging agents and poly-(ADP-ribose) polymerase 1 (PARP1) inhibitors. Molecular similarities between BLBCs and BRCA1-mutated tumors may therefore provide predictive markers for therapeutic response of BLBCs. There are several known molecular features characteristic for BRCA1-mutated breast tumors: 1) increased numbers of genomic aberrations, 2) a distinct pattern of genomic aberrations, 3) a high frequency of TP53 mutations and 4) a high incidence of complex, protein-truncating TP53 mutations. We compared the frequency of TP53 mutations and the pattern and amount of genomic aberrations between BRCA1-mutated breast tumors, BLBCs and luminal breast tumors by TP53 gene sequencing and array-based comparative genomics hybridization (aCGH) analysis. We found that the high incidence of protein truncating TP53 mutations and the pattern and amount of genomic aberrations specific for BRCA1-mutated breast tumors are also characteristic for BLBCs and different from luminal breast tumors. Complex, protein truncating TP53 mutations in BRCA1-mutated tumors may be a direct consequence of genomic instability caused by BRCA1 loss, therefore, the presence of these types of TP53 mutations in sporadic BLBCs might be a hallmark of BRCAness and a potential biomarker for sensitivity to PARP inhibition. Also, our data suggest that a small subset of genomic regions may be used to identify BRCA1-like BLBCs. BLBCs share molecular features that were previously found to be specific for BRCA1-mutated breast tumors. These features might be useful for the identification of tumors with

  20. Rapid identification of HBB gene mutations by high-resolution melting analysis.

    Science.gov (United States)

    Shih, Hung-Chang; Er, Tze-Kiong; Chang, Tien-Jye; Chang, Ya-Sian; Liu, Ta-Chih; Chang, Jan-Gowth

    2009-11-01

    This study was undertaken to identify HBB gene mutation. Herein we evaluated high-resolution melting analysis in the identification of HBB mutations. We have successfully established a diagnostic strategy for identifying HBB gene mutations including c.-78A>G, c.-79A>G, c.2T>G, c.79_80insT, c.84_85insC, c.123_124insT, c.125_128delTCTT, c.130 G>T, c.170G>A, c.216_217ins A and c.316-197 C>T from wild-type DNA using HRM analysis. The results of HRM analysis were confirmed by direct DNA sequencing. In summary, we report that HRM analysis is an appealing technique for the identification of HBB mutations. We also believe that HRM can be used as a method for prenatal diagnosis of beta-thalassemia.

  1. High-resolution melting analysis for detection of MYH9 mutations.

    Science.gov (United States)

    Provaznikova, Dana; Kumstyrova, Tereza; Kotlin, Roman; Salaj, Peter; Matoska, Vaclav; Hrachovinova, Ingrid; Rittich, Simon

    2008-09-01

    May-Hegglin anomaly (MHA), Sebastian (SBS), Fechtner (FTNS) and Epstein (EPS) syndromes are rare autosomal dominant disorders with giant platelets and thrombocytopenia. Other manifestations of these disorders are combinations of the presence of granulocyte inclusions and deafness, cataracts and renal failure. Currently, MHA, SBS, FTNS and EPS are considered to be distinct clinical manifestation of a single illness caused by mutations of the MYH9 gene encoding the heavy chain of non-muscle myosin IIA (NMMHC-IIA). As the MYH9 gene has a high number of exons, it takes much time and material to use this method for the detection of MYH9 mutations. Recently, a new method has been introduced for scanning DNA mutations without the need for direct sequencing: high-resolution melting analysis (HRMA). Mutation detection with HRMA relies on the intercalation of the specific dye (LC Green plus) in double-strand DNA and fluorescence monitoring of PCR product melting profiles. In our study, we optimized the conditions and used HRMA for rapid screening of mutations in all MYH9 exons in seven affected individuals from four unrelated families with suspected MYH9 disorders. Samples identified by HRMA as positive for the mutation were analysed by direct sequencing. HRMA saved us over 85% of redundant sequencing.

  2. Optimal selection for BRCA1 and BRCA2 mutation testing using a combination of 'easy to apply' probability models

    National Research Council Canada - National Science Library

    Bodmer, D; Ligtenberg, M.J.L; Hout, A.H. van der; Gloudemans, S; Ansink, K; Oosterwijk-Wakka, J.C; Hoogerbrugge-van der Linden, N

    2006-01-01

    To establish an efficient, reliable and easy to apply risk assessment tool to select families with breast and/or ovarian cancer patients for BRCA mutation testing, using available probability models...

  3. Optimal selection for BRCA1 and BRCA2 mutation testing using a combination of ' easy to apply ' probability models

    National Research Council Canada - National Science Library

    Bodmer, D; Ligtenberg, M. J. L; van der Hout, A. H; Gloudemans, S; Ansink, K; Oosterwijk, J. C; Hoogerbrugge, N

    2006-01-01

    To establish an efficient, reliable and easy to apply risk assessment tool to select families with breast and/or ovarian cancer patients for BRCA mutation testing, using available probability models...

  4. How close are we to standardised extended RAS gene mutation testing? The UK NEQAS evaluation.

    Science.gov (United States)

    Richman, Susan D; Fairley, Jennifer; Butler, Rachel; Deans, Zandra C

    2017-01-01

    Since 2008, KRAS mutation status in exon 2 has been used to predict response to anti-EGFR therapies. Recent evidence has demonstrated that NRAS status is also predictive of response. Several retrospective 'extended RAS' analyses have been performed on clinical trial material. Despite this, are we really moving towards such extended screening practice in reality? Data were analysed from four consecutive UK National External Quality Assessment Service for Molecular Genetics Colorectal cancer External Quality Assessment schemes (during the period 2014-2016), with up to 110 laboratories (worldwide) participating in each scheme. Testing of four or five tumour samples is required per scheme. Laboratories provided information on which codons were routinely screened, and provided genotyping and interpretation results for each sample. At least 85% of laboratories routinely tested KRAS codons 12, 13 and 61. Over the four schemes, an increasing number of laboratories routinely tested KRAS codons 59, 117 and 146. Furthermore, more laboratories were introducing next generation sequencing technologies. The pattern of 'extended testing' was reassuringly similar for NRAS, although fewer laboratories currently test for mutations in this gene. Alarmingly, still only 36.1% and 24.1% of participating laboratories met the ACP Molecular Pathology and Diagnostics Group and American Society of Clinical Oncology guidelines, respectively, for extended RAS testing in the latest assessment. Despite recommendations in the UK and USA on extended RAS testing, there has clearly been, based on these results, a delay in implementation. Inadequate testing results in patients being subjected to harmful treatment regimens, which would not be the case, were routine practice altered, in line with evidence-based guidelines. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  5. Extremely high Tp53 mutation load in esophageal squamous cell carcinoma in Golestan Province, Iran.

    Directory of Open Access Journals (Sweden)

    Behnoush Abedi-Ardekani

    Full Text Available BACKGROUND: Golestan Province in northeastern Iran has one of the highest incidences of esophageal squamous cell carcinoma (ESCC in the world with rates over 50 per 100,000 person-years in both sexes. We have analyzed TP53 mutation patterns in tumors from this high-risk geographic area in search of clues to the mutagenic processes involved in causing ESCC. METHODOLOGY/PRINCIPAL FINDINGS: Biopsies of 119 confirmed ESCC tumor tissue from subjects enrolled in a case-control study conducted in Golestan Province were analyzed by direct sequencing of TP53 exons 2 through 11. Immunohistochemical staining for p53 was carried out using two monoclonal antibodies, DO7 and 1801. A total of 120 TP53 mutations were detected in 107/119 cases (89.9%, including 11 patients with double or triple mutations. The mutation pattern was heterogeneous with infrequent mutations at common TP53 "hotspots" but frequent transversions potentially attributable to environmental carcinogens forming bulky DNA adducts, including 40% at bases known as site of mutagenesis by polycyclic aromatic hydrocarbons (PAHs. Mutations showed different patterns according to the reported temperature of tea consumption, but no variation was observed in relation to ethnicity, tobacco or opium use, and alcoholic beverage consumption or urban versus rural residence. CONCLUSION/SIGNIFICANCE: ESCC tumors in people from Golestan Province show the highest rate of TP53 mutations ever reported in any cancer anywhere. The heterogeneous mutation pattern is highly suggestive of a causative role for multiple environmental carcinogens, including PAHs. The temperature and composition of tea may also influence mutagenesis.

  6. Identification of BRCA1/2 founder mutations in Southern Chinese breast cancer patients using gene sequencing and high resolution DNA melting analysis.

    Directory of Open Access Journals (Sweden)

    Ava Kwong

    Full Text Available BACKGROUND: Ethnic variations in breast cancer epidemiology and genetics have necessitated investigation of the spectra of BRCA1 and BRCA2 mutations in different populations. Knowledge of BRCA mutations in Chinese populations is still largely unknown. We conducted a multi-center study to characterize the spectra of BRCA mutations in Chinese breast and ovarian cancer patients from Southern China. METHODOLOGY/PRINCIPAL FINDINGS: A total of 651 clinically high-risk breast and/or ovarian cancer patients were recruited from the Hong Kong Hereditary Breast Cancer Family Registry from 2007 to 2011. Comprehensive BRCA1 and BRCA2 mutation screening was performed using bi-directional sequencing of all coding exons of BRCA1 and BRCA2. Sequencing results were confirmed by in-house developed full high resolution DNA melting (HRM analysis. Among the 451 probands analyzed, 69 (15.3% deleterious BRCA mutations were identified, comprising 29 in BRCA1 and 40 in BRCA2. The four recurrent BRCA1 mutations (c.470_471delCT, c.3342_3345delAGAA, c.5406+1_5406+3delGTA and c.981_982delAT accounted for 34.5% (10/29 of all BRCA1 mutations in this cohort. The four recurrent BRCA2 mutations (c.2808_2811delACAA, c.3109C>T, c.7436_7805del370 and c.9097_9098insA accounted for 40% (16/40 of all BRCA2 mutations. Haplotype analysis was performed to confirm 1 BRCA1 and 3 BRCA2 mutations are putative founder mutations. Rapid HRM mutation screening for a panel of the founder mutations were developed and validated. CONCLUSION: In this study, our findings suggest that BRCA mutations account for a substantial proportion of hereditary breast/ovarian cancer in Southern Chinese population. Knowing the spectrum and frequency of the founder mutations in this population will assist in the development of a cost-effective rapid screening assay, which in turn facilitates genetic counseling and testing for the purpose of cancer risk assessment.

  7. Identification of BRCA1/2 founder mutations in Southern Chinese breast cancer patients using gene sequencing and high resolution DNA melting analysis.

    Science.gov (United States)

    Kwong, Ava; Ng, Enders Kai On; Wong, Chris Lei Po; Law, Fian Bic Fai; Au, Tommy; Wong, Hong Nei; Kurian, Allison W; West, Dee W; Ford, James M; Ma, Edmond Siu Kwan

    2012-01-01

    Ethnic variations in breast cancer epidemiology and genetics have necessitated investigation of the spectra of BRCA1 and BRCA2 mutations in different populations. Knowledge of BRCA mutations in Chinese populations is still largely unknown. We conducted a multi-center study to characterize the spectra of BRCA mutations in Chinese breast and ovarian cancer patients from Southern China. A total of 651 clinically high-risk breast and/or ovarian cancer patients were recruited from the Hong Kong Hereditary Breast Cancer Family Registry from 2007 to 2011. Comprehensive BRCA1 and BRCA2 mutation screening was performed using bi-directional sequencing of all coding exons of BRCA1 and BRCA2. Sequencing results were confirmed by in-house developed full high resolution DNA melting (HRM) analysis. Among the 451 probands analyzed, 69 (15.3%) deleterious BRCA mutations were identified, comprising 29 in BRCA1 and 40 in BRCA2. The four recurrent BRCA1 mutations (c.470_471delCT, c.3342_3345delAGAA, c.5406+1_5406+3delGTA and c.981_982delAT) accounted for 34.5% (10/29) of all BRCA1 mutations in this cohort. The four recurrent BRCA2 mutations (c.2808_2811delACAA, c.3109C>T, c.7436_7805del370 and c.9097_9098insA) accounted for 40% (16/40) of all BRCA2 mutations. Haplotype analysis was performed to confirm 1 BRCA1 and 3 BRCA2 mutations are putative founder mutations. Rapid HRM mutation screening for a panel of the founder mutations were developed and validated. In this study, our findings suggest that BRCA mutations account for a substantial proportion of hereditary breast/ovarian cancer in Southern Chinese population. Knowing the spectrum and frequency of the founder mutations in this population will assist in the development of a cost-effective rapid screening assay, which in turn facilitates genetic counseling and testing for the purpose of cancer risk assessment.

  8. Lack of Mutagenicity Potential of Periploca sepium Bge. in Bacterial Reverse Mutation (Ames) Test, Chromosomal Aberration and Micronucleus Test in Mice.

    Science.gov (United States)

    Zhang, Mei-Shu; Bang, In-Seok; Park, Cheol Beom

    2012-01-01

    The root barks of Periploca sepium Bge. (P. sepium) has been used in traditional Chinese medicine for healing wounds and treating rheumatoid arthritis. However, toxicity in high-doses was often diagnosed by the presence of many glycosides. The potential mutagenicity of P. sepium was investigated both in vitro and in vivo. This was examined by the bacterial reverse mutation (Ames) test using Escherichia coli WP2uvrA and Salmonella typhimurium strains, such as TA98, TA100, TA1535, and TA1537. Chromosomal aberrations were investigated using Chinese hamster lung cells, and the micronucleus test using mice. P. sepium did not induce mutagenicity in the bacterial test or chromosomal aberrations in Chinese hamster lung cells, although metabolic activation and micronucleated polychromatic erythrocytes were seen in the mice bone marrow cells. Considering these results, it is suggested that P. sepium does not have mutagenic potential under the conditions examined in each study.

  9. Economic Evaluation of Companion Diagnostic Testing for EGFR Mutations and First-Line Targeted Therapy in Advanced Non-Small Cell Lung Cancer Patients in South Korea.

    Science.gov (United States)

    Lim, Eun-A; Lee, Haeyoung; Bae, Eunmi; Lim, Jaeok; Shin, Young Kee; Choi, Sang-Eun

    2016-01-01

    As targeted therapy becomes increasingly important, diagnostic techniques for identifying targeted biomarkers have also become an emerging issue. The study aims to evaluate the cost-effectiveness of treating patients as guided by epidermal growth factor receptor (EGFR) mutation status compared with a no-testing strategy that is the current clinical practice in South Korea. A cost-utility analysis was conducted to compare an EGFR mutation testing strategy with a no-testing strategy from the Korean healthcare payer's perspective. The study population consisted of patients with stage 3b and 4 lung adenocarcinoma. A decision tree model was employed to select the appropriate treatment regimen according to the results of EGFR mutation testing and a Markov model was constructed to simulate disease progression of advanced non-small cell lung cancer. The length of a Markov cycle was one month, and the time horizon was five years (60 cycles). In the base case analysis, the testing strategy was a dominant option. Quality-adjusted life-years gained (QALYs) were 0.556 and 0.635, and total costs were $23,952 USD and $23,334 USD in the no-testing and testing strategy respectively. The sensitivity analyses showed overall robust results. The incremental cost-effectiveness ratios (ICERs) increased when the number of patients to be treated with erlotinib increased, due to the high cost of erlotinib. Treating advanced adenocarcinoma based on EGFR mutation status has beneficial effects and saves the cost compared to no testing strategy in South Korea. However, the cost-effectiveness of EGFR mutation testing was heavily affected by the cost-effectiveness of the targeted therapy.

  10. Economic Evaluation of Companion Diagnostic Testing for EGFR Mutations and First-Line Targeted Therapy in Advanced Non-Small Cell Lung Cancer Patients in South Korea.

    Directory of Open Access Journals (Sweden)

    Eun-A Lim

    Full Text Available As targeted therapy becomes increasingly important, diagnostic techniques for identifying targeted biomarkers have also become an emerging issue. The study aims to evaluate the cost-effectiveness of treating patients as guided by epidermal growth factor receptor (EGFR mutation status compared with a no-testing strategy that is the current clinical practice in South Korea.A cost-utility analysis was conducted to compare an EGFR mutation testing strategy with a no-testing strategy from the Korean healthcare payer's perspective. The study population consisted of patients with stage 3b and 4 lung adenocarcinoma. A decision tree model was employed to select the appropriate treatment regimen according to the results of EGFR mutation testing and a Markov model was constructed to simulate disease progression of advanced non-small cell lung cancer. The length of a Markov cycle was one month, and the time horizon was five years (60 cycles.In the base case analysis, the testing strategy was a dominant option. Quality-adjusted life-years gained (QALYs were 0.556 and 0.635, and total costs were $23,952 USD and $23,334 USD in the no-testing and testing strategy respectively. The sensitivity analyses showed overall robust results. The incremental cost-effectiveness ratios (ICERs increased when the number of patients to be treated with erlotinib increased, due to the high cost of erlotinib.Treating advanced adenocarcinoma based on EGFR mutation status has beneficial effects and saves the cost compared to no testing strategy in South Korea. However, the cost-effectiveness of EGFR mutation testing was heavily affected by the cost-effectiveness of the targeted therapy.

  11. Next generation MUT-MAP, a high-sensitivity high-throughput microfluidics chip-based mutation analysis panel.

    Directory of Open Access Journals (Sweden)

    Erica B Schleifman

    Full Text Available Molecular profiling of tumor tissue to detect alterations, such as oncogenic mutations, plays a vital role in determining treatment options in oncology. Hence, there is an increasing need for a robust and high-throughput technology to detect oncogenic hotspot mutations. Although commercial assays are available to detect genetic alterations in single genes, only a limited amount of tissue is often available from patients, requiring multiplexing to allow for simultaneous detection of mutations in many genes using low DNA input. Even though next-generation sequencing (NGS platforms provide powerful tools for this purpose, they face challenges such as high cost, large DNA input requirement, complex data analysis, and long turnaround times, limiting their use in clinical settings. We report the development of the next generation mutation multi-analyte panel (MUT-MAP, a high-throughput microfluidic, panel for detecting 120 somatic mutations across eleven genes of therapeutic interest (AKT1, BRAF, EGFR, FGFR3, FLT3, HRAS, KIT, KRAS, MET, NRAS, and PIK3CA using allele-specific PCR (AS-PCR and Taqman technology. This mutation panel requires as little as 2 ng of high quality DNA from fresh frozen or 100 ng of DNA from formalin-fixed paraffin-embedded (FFPE tissues. Mutation calls, including an automated data analysis process, have been implemented to run 88 samples per day. Validation of this platform using plasmids showed robust signal and low cross-reactivity in all of the newly added assays and mutation calls in cell line samples were found to be consistent with the Catalogue of Somatic Mutations in Cancer (COSMIC database allowing for direct comparison of our platform to Sanger sequencing. High correlation with NGS when compared to the SuraSeq500 panel run on the Ion Torrent platform in a FFPE dilution experiment showed assay sensitivity down to 0.45%. This multiplexed mutation panel is a valuable tool for high-throughput biomarker discovery in

  12. Perceived versus predicted risks of colorectal cancer and self-reported colonoscopies by members of mismatch repair gene mutation-carrying families who have declined genetic testing.

    Science.gov (United States)

    Flander, Louisa; Speirs-Bridge, Andrew; Rutstein, Alison; Niven, Heather; Win, Aung Ko; Ait Ouakrim, Driss; Hopper, John L; Macrae, Finlay; Keogh, Louise; Gaff, Clara; Jenkins, Mark

    2014-02-01

    People carrying germline mutations in mismatch repair genes are at high risk of colorectal cancer (CRC), yet about half of people from mutation-carrying families decline genetic counselling and/or testing to identify mutation status. We studied the association of quantitative measures of risk perception, risk prediction and self-reported screening colonoscopy in this elusive yet high-risk group. The sample of 26 participants (mean age 43.1 years, 14 women) in the Australasian Colorectal Cancer Family Registry were relatives of mutation carriers; had not been diagnosed with any cancer at the time of recruitment and had declined an invitation to attend genetic counselling and/or testing. A structured elicitation protocol captured perceived CRC risk over the next 10 years. Self-reported colonoscopy screening was elicited during a 45-minute semi-structured interview. Predicted 10-year CRC risk based on age, gender, known mutation status and family history was calculated using "MMRpro." Mean perceived 10-year risk of CRC was 31 % [95 % CI 21, 40], compared with mean predicted risk of 4 % [2, 7] (p independent of age and sex (p = 0.9). Among those reporting any medical advice and any screening colonoscopy (n = 18), those with higher risk perception had less frequent colonoscopy (Pearson's r = 0.49 [0.02, 0.79]). People who decline genetic testing for CRC susceptibility mutations perceive themselves to be at substantially higher risk than they really are. Those with high perceived risk do not undertake screening colonoscopy more often than those who perceive themselves to be at average risk.

  13. High Test Peroxide High Sealing Conical Seal Project

    Data.gov (United States)

    National Aeronautics and Space Administration — High Test Peroxide (HTP) Highly Compatible High Sealing Conical Seals are necessary for ground test operations and space based applications. Current conical seals...

  14. High prevalence of drug-resistance mutations in Plasmodium falciparum and Plasmodium vivax in southern Ethiopia

    Directory of Open Access Journals (Sweden)

    Löscher Thomas

    2006-07-01

    Full Text Available Abstract Background In Ethiopia, malaria is caused by both Plasmodium falciparum and Plasmodium vivax. Drug resistance of P. falciparum to sulfadoxine-pyrimethamine (SP and chloroquine (CQ is frequent and intense in some areas. Methods In 100 patients with uncomplicated malaria from Dilla, southern Ethiopia, P. falciparum dhfr and dhps mutations as well as P. vivax dhfr polymorphisms associated with resistance to SP and P. falciparum pfcrt and pfmdr1 mutations conferring CQ resistance were assessed. Results P. falciparum and P. vivax were observed in 69% and 31% of the patients, respectively. Pfdhfr triple mutations and pfdhfr/pfdhps quintuple mutations occurred in 87% and 86% of P. falciparum isolates, respectively. Pfcrt T76 was seen in all and pfmdr1 Y86 in 81% of P. falciparum. The P. vivax dhfr core mutations N117 and R58 were present in 94% and 74%, respectively. Conclusion These data point to an extraordinarily high frequency of drug-resistance mutations in both P. falciparum and P. vivax in southern Ethiopia, and strongly support that both SP and CQ are inadequate drugs for this region.

  15. High-throughput mutational analysis of TOR1A in primary dystonia.

    Science.gov (United States)

    Xiao, Jianfeng; Bastian, Robert W; Perlmutter, Joel S; Racette, Brad A; Tabbal, Samer D; Karimi, Morvarid; Paniello, Randal C; Blitzer, Andrew; Batish, Sat Dev; Wszolek, Zbigniew K; Uitti, Ryan J; Hedera, Peter; Simon, David K; Tarsy, Daniel; Truong, Daniel D; Frei, Karen P; Pfeiffer, Ronald F; Gong, Suzhen; Zhao, Yu; LeDoux, Mark S

    2009-03-11

    Although the c.904_906delGAG mutation in Exon 5 of TOR1A typically manifests as early-onset generalized dystonia, DYT1 dystonia is genetically and clinically heterogeneous. Recently, another Exon 5 mutation (c.863G>A) has been associated with early-onset generalized dystonia and some DeltaGAG mutation carriers present with late-onset focal dystonia. The aim of this study was to identify TOR1A Exon 5 mutations in a large cohort of subjects with mainly non-generalized primary dystonia. High resolution melting (HRM) was used to examine the entire TOR1A Exon 5 coding sequence in 1014 subjects with primary dystonia (422 spasmodic dysphonia, 285 cervical dystonia, 67 blepharospasm, 41 writer's cramp, 16 oromandibular dystonia, 38 other primary focal dystonia, 112 segmental dystonia, 16 multifocal dystonia, and 17 generalized dystonia) and 250 controls (150 neurologically normal and 100 with other movement disorders). Diagnostic sensitivity and specificity were evaluated in an additional 8 subjects with known DeltaGAG DYT1 dystonia and 88 subjects with DeltaGAG-negative dystonia. HRM of TOR1A Exon 5 showed high (100%) diagnostic sensitivity and specificity. HRM was rapid and economical. HRM reliably differentiated the TOR1A DeltaGAG and c.863G>A mutations. Melting curves were normal in 250/250 controls and 1012/1014 subjects with primary dystonia. The two subjects with shifted melting curves were found to harbor the classic DeltaGAG deletion: 1) a non-Jewish Caucasian female with childhood-onset multifocal dystonia and 2) an Ashkenazi Jewish female with adolescent-onset spasmodic dysphonia. First, HRM is an inexpensive, diagnostically sensitive and specific, high-throughput method for mutation discovery. Second, Exon 5 mutations in TOR1A are rarely associated with non-generalized primary dystonia.

  16. High-throughput mutational analysis of TOR1A in primary dystonia

    Directory of Open Access Journals (Sweden)

    Truong Daniel D

    2009-03-01

    Full Text Available Abstract Background Although the c.904_906delGAG mutation in Exon 5 of TOR1A typically manifests as early-onset generalized dystonia, DYT1 dystonia is genetically and clinically heterogeneous. Recently, another Exon 5 mutation (c.863G>A has been associated with early-onset generalized dystonia and some ΔGAG mutation carriers present with late-onset focal dystonia. The aim of this study was to identify TOR1A Exon 5 mutations in a large cohort of subjects with mainly non-generalized primary dystonia. Methods High resolution melting (HRM was used to examine the entire TOR1A Exon 5 coding sequence in 1014 subjects with primary dystonia (422 spasmodic dysphonia, 285 cervical dystonia, 67 blepharospasm, 41 writer's cramp, 16 oromandibular dystonia, 38 other primary focal dystonia, 112 segmental dystonia, 16 multifocal dystonia, and 17 generalized dystonia and 250 controls (150 neurologically normal and 100 with other movement disorders. Diagnostic sensitivity and specificity were evaluated in an additional 8 subjects with known ΔGAG DYT1 dystonia and 88 subjects with ΔGAG-negative dystonia. Results HRM of TOR1A Exon 5 showed high (100% diagnostic sensitivity and specificity. HRM was rapid and economical. HRM reliably differentiated the TOR1A ΔGAG and c.863G>A mutations. Melting curves were normal in 250/250 controls and 1012/1014 subjects with primary dystonia. The two subjects with shifted melting curves were found to harbor the classic ΔGAG deletion: 1 a non-Jewish Caucasian female with childhood-onset multifocal dystonia and 2 an Ashkenazi Jewish female with adolescent-onset spasmodic dysphonia. Conclusion First, HRM is an inexpensive, diagnostically sensitive and specific, high-throughput method for mutation discovery. Second, Exon 5 mutations in TOR1A are rarely associated with non-generalized primary dystonia.

  17. EGFR mutation testing in lung cancer: a review of available methods and their use for analysis of tumour tissue and cytology samples.

    Science.gov (United States)

    Ellison, Gillian; Zhu, Guanshan; Moulis, Alexandros; Dearden, Simon; Speake, Georgina; McCormack, Rose

    2013-02-01

    Activating mutations in the gene encoding epidermal growth factor receptor (EGFR) can confer sensitivity to EGFR tyrosine kinase inhibitors such as gefitinib in patients with advanced non-small-cell lung cancer. Testing for mutations in EGFR is therefore an important step in the treatment-decision pathway. We reviewed reported methods for EGFR mutation testing in patients with lung cancer, initially focusing on studies involving standard tumour tissue samples. We also evaluated data on the use of cytology samples in order to determine their suitability for EGFR mutation analysis. We searched the MEDLINE database for studies reporting on EGFR mutation testing methods in patients with lung cancer. Various methods have been investigated as potential alternatives to the historical standard for EGFR mutation testing, direct DNA sequencing. Many of these are targeted methods that specifically detect the most common EGFR mutations. The development of targeted mutation testing methods and commercially available test kits has enabled sensitive, rapid and robust analysis of clinical samples. The use of screening methods, subsequent to sample micro dissection, has also ensured that identification of more rare, uncommon mutations is now feasible. Cytology samples including fine needle aspirate and pleural effusion can be used successfully to determine EGFR mutation status provided that sensitive testing methods are employed. Several different testing methods offer a more sensitive alternative to direct sequencing for the detection of common EGFR mutations. Evidence published to date suggests cytology samples are viable alternatives for mutation testing when tumour tissue samples are not available.

  18. High-Altitude Electromagnetic Pulse (HEMP) Testing

    Science.gov (United States)

    2015-07-09

    Final 3. DATES COVERED (From - To) 4. TITLE AND SUBTITLE Test Operations Procedure (TOP) 01-2-620A High-Altitude Electromagnetic Pulse ( HEMP ...planning and execution of testing Army/DOD equipment to determine the effects of Horizontal Component High Altitude Electromagnetic Pulse ( HEMP ...5 2.3 HEMP Pre/Post Test Illuminations ..................................................... 7 3. REQUIRED TEST

  19. MECP2 mutations in Czech patients with Rett syndrome and Rett-like phenotypes: novel mutations, genotype-phenotype correlations and validation of high-resolution melting analysis for mutation scanning.

    Science.gov (United States)

    Zahorakova, Daniela; Lelkova, Petra; Gregor, Vladimir; Magner, Martin; Zeman, Jiri; Martasek, Pavel

    2016-07-01

    Rett syndrome (RTT) is an X-linked neurodevelopmental disorder characterized by developmental regression with loss of motor, communication and social skills, onset of stereotypic hand movements and often seizures. RTT is primarily caused by de novo mutations in the methyl-CpG-binding protein 2 gene (MECP2). We established a high-resolution melting (HRM) technique for mutation scanning of the MECP2 gene and performed analyses in Czech patients with RTT, autism spectrum conditions and intellectual disability with Rett-like features. In the cases with confirmed MECP2 mutations, we determined X-chromosome inactivation (XCI), examined the relationships between genotype and clinical severity and evaluated the modifying influence of XCI. Our results demonstrate that HRM analysis is a reliable method for the detection of point mutations, small deletions and duplications in the MECP2 gene. We identified 29 pathogenic mutations in 75 girls, including four novel mutations: c.155_1189del1035;909_932inv;insC, c.573delC, c.857_858dupAA and c.1163_1200del38. Skewed XCI (ratio >75%) was found in 19.3% of the girls, but no gross divergence in clinical severity was observed. Our findings confirm a high mutation frequency in classic RTT (92%) and a correlation between the MECP2 mutation type and clinical severity. We also demonstrate limitations of XCI in explaining all of the phenotypic differences in RTT.

  20. Protein deimmunization via structure-based design enables efficient epitope deletion at high mutational loads

    Science.gov (United States)

    Salvat, Regina S.; Choi, Yoonjoo; Bishop, Alexandra; Bailey-Kellogg, Chris; Griswold, Karl E.

    2015-01-01

    Anti-drug immune responses are a unique risk factor for biotherapeutics, and undesired immunogenicity can alter pharmacokinetics, compromise drug efficacy, and in some cases even threaten patient safety. To fully capitalize on the promise of biotherapeutics, more efficient and generally applicable protein deimmunization tools are needed. Mutagenic deletion of a protein’s T cell epitopes is one powerful strategy to engineer immunotolerance, but deimmunizing mutations must maintain protein structure and function. Here, EpiSweep, a structure-based protein design and deimmunization algorithm, has been used to produce a panel of seven beta-lactamase drug candidates having 27–47% reductions in predicted epitope content. Despite bearing eight mutations each, all seven engineered enzymes maintained good stability and activity. At the same time, the variants exhibited dramatically reduced interaction with human class II major histocompatibility complex proteins, key regulators of anti-drug immune responses. When compared to 8-mutation designs generated with a sequence-based deimmunization algorithm, the structure-based designs retained greater thermostability and possessed fewer high affinity epitopes, the dominant drivers of anti-biotherapeutic immune responses. These experimental results validate the first structure-based deimmunization algorithm capable of mapping optimal biotherapeutic design space. By designing optimal mutations that reduce immunogenic potential while imparting favorable intramolecular interactions, broadly distributed epitopes may be simultaneously targeted using high mutational loads. PMID:25655032

  1. High frequency of PTEN mutations in nevi and melanomas from xeroderma pigmentosum patients.

    Science.gov (United States)

    Masaki, Taro; Wang, Yun; DiGiovanna, John J; Khan, Sikandar G; Raffeld, Mark; Beltaifa, Senda; Hornyak, Thomas J; Darling, Thomas N; Lee, Chyi-Chia R; Kraemer, Kenneth H

    2014-05-01

    We examined nevi and melanomas in 10 xeroderma pigmentosum (XP) patients with defective DNA repair. The lesions had a lentiginous appearance with markedly increased numbers of melanocytes. Using laser capture microdissection, we performed DNA sequencing of 18 benign and atypical nevi and 75 melanomas (melanoma in situ and invasive melanomas). The nevi had a similar high frequency of PTEN mutations as melanomas [61% (11/18) versus 53% (39/73)]. Both had a very high proportion of UV-type mutations (occurring at adjacent pyrimidines) [91% (10/11) versus 92% (36/39)]. In contrast to melanomas in the general population, the frequency of BRAF mutations (11%, 7/61), NRAS mutations (21%, 13/62), and KIT mutations (21%, 6/28) in XP melanomas was lower than for PTEN. Phospho-S6 immunostaining indicated activation of the mTOR pathway in the atypical nevi and melanomas. Thus, the clinical and histological appearances and the molecular pathology of these UV-related XP nevi and melanomas were different from nevi and melanomas in the general population. © 2014 Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

  2. High Resolution Analysis of Copy Number Mutation in Breast Cancer

    Science.gov (United States)

    2005-05-01

    Pon , in Polysaccharides in Medic- copy number at high resolution throughout the other diseases, we must distinguish abnormal inal Applications, S...was determined to in- leles . In all experiments, there were a total of silico from the human genome sequence as- volve an interchromosomal duplication...well (3), although we do not explore that approach here. PON ) = e -pb o#regular( - )#deviated [1] The negative log likelihood function satisfies an

  3. High-Temperature Test Technology

    Science.gov (United States)

    1987-03-01

    Do any of your facilities have vacuum test capability? YesO No~l If yes, What is the minimum vacuum chamber pressure? What is the maximum allowable...available? YesO N[-- If "yes," please Indicate the following: Vaporizer Superheater Capacity Capacity Max Temperature LH2 LN2 Are gaseous hydrogen...personnel safety? 5. Does the facility have radiant heating capability? YesO NoF- If "yes," please provide the following information: Lamp types Tungsten

  4. Molecular analysis of pyrazinamide resistance in Mycobacterium tuberculosis in Vietnam highlights the high rate of pyrazinamide resistance-associated mutations in clinical isolates.

    Science.gov (United States)

    Huy, Nguyen Quang; Lucie, Contamin; Hoa, Tran Thi Thanh; Hung, Nguyen Van; Lan, Nguyen Thi Ngoc; Son, Nguyen Thai; Nhung, Nguyen Viet; Anh, Dang Duc; Anne-Laure, Bañuls; Van Anh, Nguyen Thi

    2017-10-11

    Pyrazinamide (PZA) is a key antibiotic in current anti-tuberculosis regimens. Although the WHO has stressed the urgent need to obtain data on PZA resistance, in high tuberculosis burden countries, little is known about the level of PZA resistance, the genetic basis of such resistance or its link with Mycobacterium tuberculosis families. In this context, this study assessed PZA resistance through the molecular analysis of 260 Vietnamese M. tuberculosis isolates. First-line drug susceptibility testing, pncA gene sequencing, spoligotyping and mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing were performed. Overall, the pncA mutation frequency was 38.1% (99 out of 260 isolates) but was higher than 72% (89 out of 123 isolates) in multidrug and quadruple-drug resistant isolates. Many different pncA mutations (71 types) were detected, of which 55 have been previously described and 50 were linked to PZA resistance. Among the 16 novel mutations, 14 are likely to be linked to PZA resistance because of their mutation types or codon positions. Genotype analysis revealed that PZA resistance can emerge in any M. tuberculosis cluster or family, although the mutation frequency was the highest in Beijing family isolates (47.7%, 62 out of 130 isolates). These data highlight the high rate of PZA resistance-associated mutations in M. tuberculosis clinical isolates in Vietnam and bring into question the use of PZA for current and future treatment regimens of multidrug-resistant tuberculosis without PZA resistance testing.

  5. The prognostic value of IDH mutations and MGMT promoter status in secondary high-grade gliomas.

    Science.gov (United States)

    Juratli, T A; Kirsch, M; Geiger, K; Klink, B; Leipnitz, E; Pinzer, T; Soucek, S; Schrock, E; Schrok, E; Schackert, G; Krex, D

    2012-12-01

    Reports about the prognostic value of IDH mutations and the promoter region of the O6-Methyl-guanyl-methyl-transferase gene in secondary high-grade gliomas (sHGG) are few in number. We investigated the prognostic value of IDH mutations and methylation of the promoter region of the MGMT gene in 99 patients with sHGG and analyzed the clinical course of those tumors. Patients with sHGG were screened for IDH mutations by direct sequencing, and, for promoter status of MGMT gene, by the methylation-specific polymerase chain reaction. A total of 48 of 99 patients (48.5 %) had secondary anaplastic gliomas (Group 1), while 51 patients had secondary glioblastomas (Group 2). The median survival time after malignant progression of all patients with sHGG and with an IDH mutation was 4 years, which is significantly longer than in patients with wild-type IDH (1.2 years, p = 0.009). Patients' survival was not significantly influenced by the tumors' MGMT promoter status, both in Group 1- 9.7 years vs. 6.1 years, methylated vs. unmethylated promoter (p = 0.330)-as well as in Group 2-1.5 years vs. 1.6 years, methylated versus unmethylated promoter (p = 0.829). In our population, the IDH mutation status was not associated with increased PFS or median survival time in sGBM patients. However, patients with secondary anaplastic glioma and IDH mutation had a significantly improved outcome. In addition, IDH mutations are a more powerful prognostic marker concerning both PFS and MS than the MGMT promoter status in those patients.

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

    Science.gov (United States)

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

    2016-03-01

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

  7. Breast cancer size estimation with MRI in BRCA mutation carriers and other high risk patients

    NARCIS (Netherlands)

    Mann, R. M.; Bult, P.; van Laarhoven, H. W. M.; Span, P. N.; Schlooz, M.; Veltman, J.; Hoogerbrugge, N.

    2013-01-01

    Objective: To assess the value of breast MRI in size assessment of breast cancers in high risk patients, including those with a BRCA 1 or 2 mutation. Guidelines recommend invariably breast MRI screening for these patients and therapy is thus based on these findings. However, the accuracy of breast

  8. high prevalence of the cys282tyr hfe mutation facilitates an improved ...

    African Journals Online (AJOL)

    HIGH PREVALENCE OF THE. CYS282TYR HFE MUTATION. FACILITATES AN IMPROVED. DIAGNOSTIC SERVICE FOR. HEREDITARY HAEMO-. CHROMATOSIS IN SOUTH AFRICA. J Nico P de Villiers, Renate Hillerman, Greetje de Jong. Elzet Langenhoven, Heleen Rossouw, Munro P Marx,. Maritha J Kotze. Objective.

  9. Molecular genetic testing for familial hypercholesterolemia: spectrum of LDL receptor gene mutations in The Netherlands

    NARCIS (Netherlands)

    Lombardi, M. P.; Redeker, E. J.; Defesche, J. C.; Kamerling, S. W.; Trip, M. D.; Mannens, M. M.; Havekes, L. M.; Kastelein, J. J.

    2000-01-01

    Mutations in the LDL receptor are responsible for familial hypercholesterolemia (FH). At present, more than 600 mutations of the LDL receptor gene are known to underlie FH. However, the array of mutations varies considerably in different populations. Therefore, the delineation of essentially all LDL

  10. High Resolution Melting Analysis for Detecting p53 Gene Mutations in Patients with Non-small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Zhihong CHEN

    2011-10-01

    Full Text Available Background and objective It has been proven that p53 gene was related to many human cancers. The mutations in p53 gene play an important role in carcinogensis and mostly happened in exon 5-8. The aim of this study is to establish a high resolution melting (HRM assay to detect p53 mutations from patients with non-small cell lung cancer (NSCLC, to investigate the characteristics of p53 gene mutations, and to analyze the relationship between p53 mutations and evolution regularity of pathogenesis. Methods p53 mutations in exon 5-8 were detected by HRM assay on DNA insolated from 264 NSCLC samples derived from tumor tissues and 54 control samples from pericancerous pulmonary tissues. The mutation samples by the HRM assay were confirmed by sequencing technique. Samples which were positive by HRM but wild type by sequencing were further confirmed by sub-clone and sequencing. Results No mutation was found in 54 pericancerous pulmonary samples by HRM assay. 104 of the 264 tumor tissues demonstrated mutation curves by HRM assay, 102 samples were confirmed by sequencing, including 95 point mutations and 7 frame shift mutations by insertion or deletion. The mutation rate of p53 gene was 39.4%. The mutation rate from exon 5-8 were 11.7%, 8%, 12.5% and 10.6%, respectively and there was no statistically significant difference between them (P=0.35. p53 mutations were significantly more frequent in males than that in females, but not related to the other clinicopathologic characteristics. Conclusion The results indicate that HRM is a sensitive in-tube methodology to detect for mutations in clinical samples. The results suggest that the arising p53 mutations in NSCLC may be due to spontaneous error in DNA synthesis and repair.

  11. Ultra-rare mutation in long-range enhancer predisposes to thyroid carcinoma with high penetrance.

    Directory of Open Access Journals (Sweden)

    Huiling He

    Full Text Available Thyroid cancer shows high heritability but causative genes remain largely unknown. According to a common hypothesis the genetic predisposition to thyroid cancer is highly heterogeneous; being in part due to many different rare alleles. Here we used linkage analysis and targeted deep sequencing to detect a novel single-nucleotide mutation in chromosome 4q32 (4q32A>C in a large pedigree displaying non-medullary thyroid carcinoma (NMTC. This mutation is generally ultra-rare; it was not found in 38 NMTC families, in 2676 sporadic NMTC cases or 2470 controls. The mutation is located in a long-range enhancer element whose ability to bind the transcription factors POU2F and YY1 is significantly impaired, with decreased activity in the presence of the C- allele compared with the wild type A-allele. An enhancer RNA (eRNA is transcribed in thyroid tissue from this region and is greatly downregulated in NMTC tumors. We suggest that this is an example of an ultra-rare mutation predisposing to thyroid cancer with high penetrance.

  12. Ultra-rare mutation in long-range enhancer predisposes to thyroid carcinoma with high penetrance.

    Science.gov (United States)

    He, Huiling; Li, Wei; Wu, Dayong; Nagy, Rebecca; Liyanarachchi, Sandya; Akagi, Keiko; Jendrzejewski, Jaroslaw; Jiao, Hong; Hoag, Kevin; Wen, Bernard; Srinivas, Mukund; Waidyaratne, Gavisha; Wang, Rui; Wojcicka, Anna; Lattimer, Ilene R; Stachlewska, Elzbieta; Czetwertynska, Malgorzata; Dlugosinska, Joanna; Gierlikowski, Wojciech; Ploski, Rafal; Krawczyk, Marek; Jazdzewski, Krystian; Kere, Juha; Symer, David E; Jin, Victor; Wang, Qianben; de la Chapelle, Albert

    2013-01-01

    Thyroid cancer shows high heritability but causative genes remain largely unknown. According to a common hypothesis the genetic predisposition to thyroid cancer is highly heterogeneous; being in part due to many different rare alleles. Here we used linkage analysis and targeted deep sequencing to detect a novel single-nucleotide mutation in chromosome 4q32 (4q32A>C) in a large pedigree displaying non-medullary thyroid carcinoma (NMTC). This mutation is generally ultra-rare; it was not found in 38 NMTC families, in 2676 sporadic NMTC cases or 2470 controls. The mutation is located in a long-range enhancer element whose ability to bind the transcription factors POU2F and YY1 is significantly impaired, with decreased activity in the presence of the C- allele compared with the wild type A-allele. An enhancer RNA (eRNA) is transcribed in thyroid tissue from this region and is greatly downregulated in NMTC tumors. We suggest that this is an example of an ultra-rare mutation predisposing to thyroid cancer with high penetrance.

  13. Highly sensitive and quantitative evaluation of the EGFR T790M mutation by nanofluidic digital PCR.

    Science.gov (United States)

    Iwama, Eiji; Takayama, Koichi; Harada, Taishi; Okamoto, Isamu; Ookubo, Fumihiko; Kishimoto, Junji; Baba, Eishi; Oda, Yoshinao; Nakanishi, Yoichi

    2015-08-21

    The mutation of T790M in EGFR is a major mechanism of resistance to treatment with EGFR-TKIs. Only qualitative detection (presence or absence) of T790M has been described to date, however. Digital PCR (dPCR) analysis has recently been applied to the quantitative detection of target molecules in cancer with high sensitivity. In the present study, 25 tumor samples (13 obtained before and 12 after EGFR-TKI treatment) from 18 NSCLC patients with activating EGFR mutations were evaluated for T790M with dPCR. The ratio of the number of T790M alleles to that of activating mutation alleles (T/A) was determined. dPCR detected T790M in all 25 samples. Although T790M was present in all pre-TKI samples from 13 patients, 10 of these patients had a low T/A ratio and manifested substantial tumor shrinkage during treatment with EGFR-TKIs. In six of seven patients for whom both pre- and post-TKI samples were available, the T/A ratio increased markedly during EGFR-TKI treatment. Highly sensitive dPCR thus detected T790M in all NSCLC patients harboring activating EGFR mutations whether or not they had received EGFR-TKI treatment. Not only highly sensitive but also quantitative detection of T790M is important for evaluation of the contribution of T790M to EGFR-TKI resistance.

  14. The genotoxicity of nitrilotriacetic acid (NTA) in a somatic mutation and recombination test in Drosophila melanogaster.

    Science.gov (United States)

    Zordan, M; Graf, U; Singer, D; Beltrame, C; Dalla Valle, L; Osti, M; Costa, R; Levis, A G

    1991-04-01

    The genotoxicity of a chelating agent, the trisodium salt of nitrilotriacetic acid (NTA), was assessed in a somatic mutation and recombination test (SMART) in Drosophila melanogaster employing the wing hair markers mwh and flr3. The experiments were performed in parallel in two different laboratories (Padua, Italy and Schwerzenbach, Switzerland). The effectively absorbed doses of NTA, which was administered by feeding to larvae, were determined by a sensitive method employing [3H]leucine which allowed individual consumption levels to be measured. The particular pattern of clone induction produced by this compound suggests that NTA is active in inducing mitotic recombination and possibly aneuploidy in somatic cells of Drosophila. This is discussed in relation to the data present in the literature regarding the genotoxicity of NTA in a variety of experimental systems.

  15. Mutation in the seed storage protein kafirin creates a high-value food trait in sorghum.

    Science.gov (United States)

    Wu, Yongrui; Yuan, Lingling; Guo, Xiaomei; Holding, David R; Messing, Joachim

    2013-01-01

    Sustainable food production for the earth's fast-growing population is a major challenge for breeding new high-yielding crops, but enhancing the nutritional quality of staple crops can potentially offset limitations associated with yield increases. Sorghum has immense value as a staple food item for humans in Africa, but it is poorly digested. Although a mutant exhibiting high-protein digestibility and lysine content has market potential, the molecular nature of the mutation is previously unknown. Here, building on knowledge from maize mutants, we take a direct approach and find that the high-digestible sorghum phenotype is tightly linked to a single-point mutation, rendering the signal peptide of a seed storage protein kafirin resistant to processing, indirectly reducing lysine-poor kafirins and thereby increasing lysine-rich proteins in the seeds. These findings indicate that a molecular marker can be used to accelerate introduction of this high nutrition and digestibility trait into different sorghum varieties.

  16. Sporadic autism exomes reveal a highly interconnected protein network of de novo mutations.

    Science.gov (United States)

    O'Roak, Brian J; Vives, Laura; Girirajan, Santhosh; Karakoc, Emre; Krumm, Niklas; Coe, Bradley P; Levy, Roie; Ko, Arthur; Lee, Choli; Smith, Joshua D; Turner, Emily H; Stanaway, Ian B; Vernot, Benjamin; Malig, Maika; Baker, Carl; Reilly, Beau; Akey, Joshua M; Borenstein, Elhanan; Rieder, Mark J; Nickerson, Deborah A; Bernier, Raphael; Shendure, Jay; Eichler, Evan E

    2012-04-04

    It is well established that autism spectrum disorders (ASD) have a strong genetic component; however, for at least 70% of cases, the underlying genetic cause is unknown. Under the hypothesis that de novo mutations underlie a substantial fraction of the risk for developing ASD in families with no previous history of ASD or related phenotypes--so-called sporadic or simplex families--we sequenced all coding regions of the genome (the exome) for parent-child trios exhibiting sporadic ASD, including 189 new trios and 20 that were previously reported. Additionally, we also sequenced the exomes of 50 unaffected siblings corresponding to these new (n = 31) and previously reported trios (n = 19), for a total of 677 individual exomes from 209 families. Here we show that de novo point mutations are overwhelmingly paternal in origin (4:1 bias) and positively correlated with paternal age, consistent with the modest increased risk for children of older fathers to develop ASD. Moreover, 39% (49 of 126) of the most severe or disruptive de novo mutations map to a highly interconnected β-catenin/chromatin remodelling protein network ranked significantly for autism candidate genes. In proband exomes, recurrent protein-altering mutations were observed in two genes: CHD8 and NTNG1. Mutation screening of six candidate genes in 1,703 ASD probands identified additional de novo, protein-altering mutations in GRIN2B, LAMC3 and SCN1A. Combined with copy number variant (CNV) data, these results indicate extreme locus heterogeneity but also provide a target for future discovery, diagnostics and therapeutics.

  17. Preoperative genetic testing impacts surgical decision making in BRCA mutation carriers with breast cancer: a retrospective cohort analysis.

    Science.gov (United States)

    Yadav, Siddhartha; Reeves, Ashley; Campian, Sarah; Sufka, Amy; Zakalik, Dana

    2017-01-01

    The impact of timing of genetic testing on surgical decision making in women with breast cancer and BRCA mutation is not well known. Women who were found to carry a deleterious BRCA mutation and had been diagnosed with breast cancer were identified from a database at Beaumont Health. Women who had received BRCA positive results at least a day prior to their index surgery were considered to be aware of their mutation status prior to surgery. Baseline characteristics and surgical choices were compared between women who were aware of their mutation status prior to surgery and those who were not. Fischer's exact test was used for categorical variables and Mann-Whitney U-Test was used for continuous variables. A total of 220 patients were included in the final analysis, 208 (94.5%) with unilateral breast cancer and 12 (5.5%) with bilateral breast cancer. Out of the 208 patients with unilateral breast cancer, 106 (51.0%) patients were aware of their mutation status prior to index surgery while 102 (49%) were not. A significantly (p < 0.05) higher proportion of women underwent contralateral prophylactic mastectomy in the group that was aware of their mutation status prior to index surgery compared to the group that was not (76.4% vs 14.7%). Our study demonstrates that knowledge of BRCA mutation status impacts surgical decision making in favor of bilateral mastectomy in patients who are aware of their results prior to index surgery. This finding supports the practice of preoperative genetic testing in patients with newly diagnosed breast cancer.

  18. Combining isothermal rolling circle amplification and electrochemiluminescence for highly sensitive point mutation detection

    Science.gov (United States)

    Su, Qiang; Zhou, Xiaoming

    2008-12-01

    Many pathogenic and genetic diseases are associated with changes in the sequence of particular genes. We describe here a rapid and highly efficient assay for the detection of point mutation. This method is a combination of isothermal rolling circle amplification (RCA) and high sensitive electrochemluminescence (ECL) detection. In the design, a circular template generated by ligation upon the recognition of a point mutation on DNA targets was amplified isothermally by the Phi29 polymerase using a biotinylated primer. The elongation products were hybridized with tris (bipyridine) ruthenium (TBR)-tagged probes and detected in a magnetic bead based ECL platform, indicating the mutation occurrence. P53 was chosen as a model for the identification of this method. The method allowed sensitive determination of the P53 mutation from wild-type and mutant samples. The main advantage of RCA-ECL is that it can be performed under isothermal conditions and avoids the generation of false-positive results. Furthermore, ECL provides a faster, more sensitive, and economical option to currently available electrophoresis-based methods.

  19. Novel A14841G mutation is associated with high penetrance of LHON/C4171A family.

    Science.gov (United States)

    Yang, Juhua; Zhu, Yihua; Chen, Lu; Zhang, Hongxing; Tong, Yi; Huang, Dinggou; Zhang, Zhiqiang; Chen, Shi; Han, Xiaoli; Ma, Xu

    2009-09-04

    We report the clinical and genetic characterization of a Chinese LHON family carrying an ND1/C4171A mutation. This family has high penetrance of visual impairment and extremely low frequency of vision recovery, which is in marked contrast to previously reported results for Korean LHON families with the same mutation. Sequence analysis of the complete mtDNA in the partially defined East Asian haplogroup N9a1 revealed the presence of 29 other variants. A novel heteroplasmic A14841G mutation, one of the variants with a serine substituted for a highly conserved asparagine at amino acid 32 of Cytochrome b (Cytb), may play a synergistic role with the C4171A mutation, leading to significantly different clinical manifestations of LHON among these families. The study further confirmed that C4171A was a rare primary LHON mutation, and the mtDNA background could also contribute to the clinical manifestation of the LHON/C4171A mutation.

  20. The high frequency of GJB2 gene mutation c.313_326del14 suggests its possible origin in ancestors of Lithuanian population.

    Science.gov (United States)

    Mikstiene, Violeta; Jakaitiene, Audrone; Byckova, Jekaterina; Gradauskiene, Egle; Preiksaitiene, Egle; Burnyte, Birute; Tumiene, Birute; Matuleviciene, Ausra; Ambrozaityte, Laima; Uktveryte, Ingrida; Domarkiene, Ingrida; Rancelis, Tautvydas; Cimbalistiene, Loreta; Lesinskas, Eugenijus; Kucinskas, Vaidutis; Utkus, Algirdas

    2016-02-19

    Congenital hearing loss (CHL) is diagnosed in 1 - 2 newborns in 1000, genetic factors contribute to two thirds of CHL cases in industrialised countries. Mutations of the GJB2 gene located in the DFNB1 locus (13q11-12) are a major cause of CHL worldwide. The aim of this cross-sectional study was to assess the contribution of the DFNB1 locus containing the GJB2 and GJB6 genes in the development of early onset hearing loss in the affected group of participants, to determine the population-specific mutational profile and DFNB1-related HL burden in Lithuanian population. Clinical data were obtained from a collection of 158 affected participants (146 unrelated probands) with early onset non-syndromic HL. GJB2 and GJB6 gene sequencing and GJB6 gene deletion testing were performed. The data of GJB2 and GJB6 gene sequencing in 98 participants in group of self-reported healthy Lithuanian inhabitants were analysed. Statistic summary, homogeneity tests, and logistic regression analysis were used for the assessment of genotype-phenotype correlation. Our findings show 57.5% of affected participants with two pathogenic GJB2 gene mutations identified. The most prevalent GJB2 mutations were c.35delG, p. (Gly12Valfs*2) (rs80338939) and c.313_326del14, p. (Lys105Glyfs*5) (rs111033253) with allele frequencies 64.7% and 28.3% respectively. GJB6 gene mutations were not identified in the affected group of participants. The statistical analysis revealed significant differences between GJB2(-) and GJB2(+) groups in disease severity (p = 0.001), and family history (p = 0.01). The probability of identification of GJB2 mutations in patients with various HL characteristics was estimated. The carrier rate of GJB2 gene mutations - 7.1% (~1 in 14) was identified in the group of healthy participants and a high frequency of GJB2-related hearing loss was estimated in our population. The results show a very high proportion of GJB2-positive individuals in the research group affected with sensorineural

  1. DHPLC technology for high-throughput detection of mutations in a durum wheat TILLING population.

    Science.gov (United States)

    Colasuonno, Pasqualina; Incerti, Ornella; Lozito, Maria Luisa; Simeone, Rosanna; Gadaleta, Agata; Blanco, Antonio

    2016-02-17

    Durum wheat (Triticum turgidum L.) is a cereal crop widely grown in the Mediterranean regions; the amber grain is mainly used for the production of pasta, couscous and typical breads. Single nucleotide polymorphism (SNP) detection technologies and high-throughput mutation induction represent a new challenge in wheat breeding to identify allelic variation in large populations. The TILLING strategy makes use of traditional chemical mutagenesis followed by screening for single base mismatches to identify novel mutant loci. Although TILLING has been combined to several sensitive pre-screening methods for SNP analysis, most rely on expensive equipment. Recently, a new low cost and time saving DHPLC protocol has been used in molecular human diagnostic to detect unknown mutations. In this work, we developed a new durum wheat TILLING population (cv. Marco Aurelio) using 0.70-0.85% ethyl methane sulfonate (EMS). To investigate the efficiency of the mutagenic treatments, a pilot screening was carried out on 1,140 mutant lines focusing on two target genes (Lycopene epsilon-cyclase, ε-LCY, and Lycopene beta-cyclase, β-LCY) involved in carotenoid metabolism in wheat grains. We simplify the heteroduplex detection by two low cost methods: the enzymatic cleavage (CelI)/agarose gel technique and the denaturing high-performance liquid chromatography (DHPLC). The CelI/agarose gel approach allowed us to identify 31 mutations, whereas the DHPLC procedure detected a total of 46 mutations for both genes. All detected mutations were confirmed by direct sequencing. The estimated overall mutation frequency for the pilot assay by the DHPLC methodology resulted to be of 1/77 kb, representing a high probability to detect interesting mutations in the target genes. We demonstrated the applicability and efficiency of a new strategy for the detection of induced variability. We produced and characterized a new durum wheat TILLING population useful for a better understanding of key gene functions

  2. Test plans of the high temperature test operation at HTTR

    Energy Technology Data Exchange (ETDEWEB)

    Sakaba, Nariaki; Nakagawa, Shigeaki; Takada, Eiji [Japan Atomic Energy Research Inst., Oarai, Ibaraki (Japan). Oarai Research Establishment] [and others

    2003-03-01

    HTTR plans a high temperature test operation as the fifth step of the rise-to-power tests to achieve a reactor outlet coolant temperature of 950 degrees centigrade in the 2003 fiscal year. Since HTTR is the first HTGR in Japan which uses coated particle fuel as its fuel and helium gas as its coolant, it is necessary that the plan of the high temperature test operation is based on the previous rise-to-power tests with a thermal power of 30 MW and a reactor outlet coolant temperature at 850 degrees centigrade. During the high temperature test operation, reactor characteristics, reactor performances and reactor operations are confirmed for the safety and stability of operations. This report describes the evaluation result of the safety confirmations of the fuel, the control rods and the intermediate heat exchanger for the high temperature test operation. Also, problems which were identified during the previous operations are shown with their solution methods. Additionally, there is a discussion on the contents of the high temperature test operation. As a result of this study, it is shown that the HTTR can safely achieve a thermal power of 30 MW with the reactor outlet coolant temperature at 950 degrees centigrade. (author)

  3. The micro-Ames test: A direct comparison of the performance and sensitivities of the standard and 24-well plate versions of the bacterial mutation test.

    Science.gov (United States)

    Proudlock, Raymond; Evans, Kristie

    2016-12-01

    "Ames" bacterial mutation tests are widely performed for evaluation and registration of new materials including industrial chemicals, agrochemicals, medical devices, pharmaceuticals, pharmaceutical impurities and other materials. Tests are used to predict their potential long-term adverse health effects (including carcinogenicity). Given their importance, pre-screening 'miniaturized' versions have been developed which allow higher throughput and use less test material, including the widely-employed 24-well micro-Ames (µAmes) test which uses 20 times less material. However, little quantitative information has been published on the methodology or sensitivity of this system. We describe methods and results used in direct comparisons of the sensitivity of micro and standard systems using the same cultures, formulations, etc. Initial testing utilized the plate incorporation method and, later, the pre-incubation method. In a subsequent phase of testing, a four-way direct comparison was made between the pre-incubation and plate incorporation methods in both systems using some direct-acting mutagens. Tests used only those strain/S9/chemical combinations where a response was expected. Historical control results accumulated during testing are also presented. Spontaneous and induced revertant colony counts for the µAmes system were consistently proportionate and approximately 1/20th those for the standard Ames test. Sensitivities of the two systems were found to be nearly identical in almost all cases for a wide variety of weak and strong inorganic and organic mutagens. Standardized procedures and increased reliability of the estimate of the background revertant frequency in the µAmes system means that the two systems give equivalent results and are expected to be highly predictive of one another. Environ. Mol. Mutagen. 57:687-705, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  4. Distinct evolutionary trajectories of primary high-grade serous ovarian cancers revealed through spatial mutational profiling.

    Science.gov (United States)

    Bashashati, Ali; Ha, Gavin; Tone, Alicia; Ding, Jiarui; Prentice, Leah M; Roth, Andrew; Rosner, Jamie; Shumansky, Karey; Kalloger, Steve; Senz, Janine; Yang, Winnie; McConechy, Melissa; Melnyk, Nataliya; Anglesio, Michael; Luk, Margaret T Y; Tse, Kane; Zeng, Thomas; Moore, Richard; Zhao, Yongjun; Marra, Marco A; Gilks, Blake; Yip, Stephen; Huntsman, David G; McAlpine, Jessica N; Shah, Sohrab P

    2013-09-01

    High-grade serous ovarian cancer (HGSC) is characterized by poor outcome, often attributed to the emergence of treatment-resistant subclones. We sought to measure the degree of genomic diversity within primary, untreated HGSCs to examine the natural state of tumour evolution prior to therapy. We performed exome sequencing, copy number analysis, targeted amplicon deep sequencing and gene expression profiling on 31 spatially and temporally separated HGSC tumour specimens (six patients), including ovarian masses, distant metastases and fallopian tube lesions. We found widespread intratumoural variation in mutation, copy number and gene expression profiles, with key driver alterations in genes present in only a subset of samples (eg PIK3CA, CTNNB1, NF1). On average, only 51.5% of mutations were present in every sample of a given case (range 10.2-91.4%), with TP53 as the only somatic mutation consistently present in all samples. Complex segmental aneuploidies, such as whole-genome doubling, were present in a subset of samples from the same individual, with divergent copy number changes segregating independently of point mutation acquisition. Reconstruction of evolutionary histories showed one patient with mixed HGSC and endometrioid histology, with common aetiologic origin in the fallopian tube and subsequent selection of different driver mutations in the histologically distinct samples. In this patient, we observed mixed cell populations in the early fallopian tube lesion, indicating that diversity arises at early stages of tumourigenesis. Our results revealed that HGSCs exhibit highly individual evolutionary trajectories and diverse genomic tapestries prior to therapy, exposing an essential biological characteristic to inform future design of personalized therapeutic solutions and investigation of drug-resistance mechanisms. © 2013 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

  5. High frequency, spontaneous motA mutations in Campylobacter jejuni strain 81-176.

    Directory of Open Access Journals (Sweden)

    Krystle L Mohawk

    Full Text Available Campylobacter jejuni is an important cause of bacterial diarrhea worldwide. The pathogenesis of C. jejuni is poorly understood and complicated by phase variation of multiple surface structures including lipooligosaccharide, capsule, and flagellum. When C. jejuni strain 81-176 was plated on blood agar for single colonies, the presence of translucent, non-motile colonial variants was noted among the majority of opaque, motile colonies. High-throughput genomic sequencing of two flagellated translucent and two opaque variants as well as the parent strain revealed multiple genetic changes compared to the published genome. However, the only mutated open reading frame common between the two translucent variants and absent from the opaque variants and the parent was motA, encoding a flagellar motor protein. A total of 18 spontaneous motA mutations were found that mapped to four distinct sites in the gene, with only one class of mutation present in a phase variable region. This study exemplifies the mutative/adaptive properties of C. jejuni and demonstrates additional variability in C. jejuni beyond phase variation.

  6. High Test Anxiety among Nursing Students

    Science.gov (United States)

    Driscoll, Richard; Evans, Ginger; Ramsey, Gary; Wheeler, Sara

    2009-01-01

    Nursing programs can be highly stressful, and the investigation was undertaken to see if nursing students are more test anxious than students in other fields. The Westside Test Anxiety Scale has administered to 298 nursing students at two colleges, and to a comparison group of 471 high school and college students. Fully 30% of nursing students…

  7. Automated System Tests High-Power MOSFET's

    Science.gov (United States)

    Huston, Steven W.; Wendt, Isabel O.

    1994-01-01

    Computer-controlled system tests metal-oxide/semiconductor field-effect transistors (MOSFET's) at high voltages and currents. Measures seven parameters characterizing performance of MOSFET, with view toward obtaining early indication MOSFET defective. Use of test system prior to installation of power MOSFET in high-power circuit saves time and money.

  8. Frameshift mutations of a tumor suppressor gene ZNF292 in gastric and colorectal cancers with high microsatellite instability.

    Science.gov (United States)

    Lee, Ju Hwa; Song, Sang Yong; Kim, Min Sung; Yoo, Nam Jin; Lee, Sug Hyung

    2016-07-01

    A transcription factor-encoding gene ZNF292 is considered a candidate tumor suppressor gene (TSG). Its mutations have been identified in cancers from liver, colon, and bone marrow. However, ZNF292 inactivating mutations that might suppress the TSG functions have not been reported in gastric (GC) and colorectal cancers (CRC) with microsatellite instability (MSI). In a public database, we found that ZNF292 gene had mononucleotide repeats in the coding sequences that might be mutation targets in the cancers with MSI. In this study, we analyzed 79 GCs and 124 CRCs including high MSI (MSI-H) and microsatellite stable/low MSI (MSS/MSI-L) cases for the detection of somatic mutations in the repeats. Overall, we identified frameshift mutations of ZNF292 in 3 (8.8%) GCs and 11 (13.9%) CRCs with MSI-H (14/113), but not in MSS/MSI-L cancers (0/90) (p < 0.001). Also, we studied intratumoral heterogeneity (ITH) of the ZNF292 frameshift mutations in 16 CRCs and found that two (12.5%) had regional ITH of the mutations. Our data show that ZNF292 gene harbors not only frameshift mutations but also mutational ITH, which together may be features of GC and CRC with MSI-H. Based on this, the ZNF292 frameshift mutations may possibly contribute to tumorigenesis by altering its TSG functions in GC and CRC. © 2016 APMIS. Published by John Wiley & Sons Ltd.

  9. Development of a High-Resolution Melting Approach for Scanning Beta Globin Gene Point Mutations in the Greek and Other Mediterranean Populations

    Science.gov (United States)

    Chassanidis, Christos; Boutou, Effrossyni; Voskaridou, Ersi; Balassopoulou, Angeliki

    2016-01-01

    Beta-thalassaemia is one of the most common autosomal recessive disorders worldwide. The disease’s high incidence, which is observed in the broader Mediterranean area has led to the establishment of molecular diagnostics’ assays to prevent affected births. Therefore, the development of a reliable, cost-effective and rapid scanning method for β globin gene point mutations, easily adapted to a routine laboratory, is absolutely essential. Here, we describe, for the first time, the development of a High-Resolution Melting Analysis (HRMA) approach, suitable for scanning the particularly heterogeneous beta globin gene mutations present in the Greek population, and thus adaptable to the Mediterranean and other areas where these mutations have been identified. Within this context, β globin gene regions containing mutations frequently identified in the Greek population were divided in ten overlapping amplicons. Our reactions’ setup allowed for the simultaneous amplification of multiple primer sets and partial multiplexing, thereby resulting in significant reduction of the experimental time. DNA samples from β-thalassaemia patients/carriers with defined genotypes were tested. Distinct genotypes displayed distinguishable melting curves, enabling accurate detection of mutations. The described HRMA can be adapted to a high-throughput level. It represents a rapid, simple, cost-effective, reliable, highly feasible and sensitive method for β-thalassaemia gene scanning. PMID:27351925

  10. A population-based study of high-grade gliomas and mutated isocitrate dehydrogenase 1

    DEFF Research Database (Denmark)

    Dahlrot, Rikke H; Kristensen, Bjarne W; Hjelmborg, Jacob

    2012-01-01

    High-grade gliomas have a dismal prognosis, and prognostic factors are needed to optimize treatment algorithms. In this study we identified clinical prognostic factors as well as the prognostic value of isocitrate dehydrogenase 1 (IDH1) status in a population-based group of patients with high......-grade gliomas. Using the Danish Cancer Registry and the Danish Pathology Databank we identified 359 patients: 234 had WHO grade IV gliomas, 58 had WHO grade III gliomas, and 67 were diagnosed clinically. Mutated IDH1 was predominantly observed in oligodendroglial tumors (WHO grade III). Patients with mutated...... IDH1 had a significantly better outcome than patients with wildtype IDH1: 2-year OS 59% and 18%, respectively (HR 0.38, 95% CI 0.21-0.68). However, when adjusting for other prognostic factors, IDH1 status was not a significant independent prognostic factor (HR=0.58, 95% CI 0.32-1.07). Young age...

  11. Telomerase reverse transcriptase promoter mutations in bladder cancer: high frequency across stages, detection in urine, and lack of association with outcome.

    Science.gov (United States)

    Allory, Yves; Beukers, Willemien; Sagrera, Ana; Flández, Marta; Marqués, Miriam; Márquez, Mirari; van der Keur, Kirstin A; Dyrskjot, Lars; Lurkin, Irene; Vermeij, Marcel; Carrato, Alfredo; Lloreta, Josep; Lorente, José A; Carrillo-de Santa Pau, Enrique; Masius, Roy G; Kogevinas, Manolis; Steyerberg, Ewout W; van Tilborg, Angela A G; Abas, Cheno; Orntoft, Torben F; Zuiverloon, Tahlita C M; Malats, Núria; Zwarthoff, Ellen C; Real, Francisco X

    2014-02-01

    Hotspot mutations in the promoter of the gene coding for telomerase reverse transcriptase (TERT) have been described and proposed to activate gene expression. To investigate TERT mutation frequency, spectrum, association with expression and clinical outcome, and potential for detection of recurrences in urine in patients with urothelial bladder cancer (UBC). A set of 111 UBCs of different stages was used to assess TERT promoter mutations by Sanger sequencing and TERT messenger RNA (mRNA) expression by reverse transcription-quantitative polymerase chain reaction. The two most frequent mutations were investigated, using a SNaPshot assay, in an independent set of 184 non-muscle-invasive and 173 muscle-invasive UBC (median follow-up: 53 mo and 21 mo, respectively). Voided urine from patients with suspicion of incident UBC (n=174), or under surveillance after diagnosis of non-muscle-invasive UBC (n=194), was tested using a SNaPshot assay. Association of mutation status with age, sex, tobacco, stage, grade, fibroblast growth factor receptor 3 (FGFR3) mutation, progression-free survival, disease-specific survival, and overall survival. In the two series, 78 of 111 (70%) and 283 of 357 (79%) tumors harbored TERT mutations, C228T being the most frequent substitution (83% for both series). TERT mutations were not associated with clinical or pathologic parameters, but were more frequent among FGFR3 mutant tumors (p=0.0002). There was no association between TERT mutations and mRNA expression (p=0.3). Mutations were not associated with clinical outcome. In urine, TERT mutations had 90% specificity in subjects with hematuria but no bladder tumor, and 73% in recurrence-free UBC patients. The sensitivity was 62% in incident and 42% in recurrent UBC. A limitation of the study is its retrospective nature. Somatic TERT promoter mutations are an early, highly prevalent genetic event in UBC and are not associated with TERT mRNA levels or disease outcomes. A SNaPshot assay in urine may

  12. Computationally optimized deimmunization libraries yield highly mutated enzymes with low immunogenicity and enhanced activity

    Science.gov (United States)

    Salvat, Regina S.; Parker, Andrew S.; Kirsch, Jack R.; Brooks, Seth A.

    2017-01-01

    Therapeutic proteins of wide-ranging function hold great promise for treating disease, but immune surveillance of these macromolecules can drive an antidrug immune response that compromises efficacy and even undermines safety. To eliminate widespread T-cell epitopes in any biotherapeutic and thereby mitigate this key source of detrimental immune recognition, we developed a Pareto optimal deimmunization library design algorithm that optimizes protein libraries to account for the simultaneous effects of combinations of mutations on both molecular function and epitope content. Active variants identified by high-throughput screening are thus inherently likely to be deimmunized. Functional screening of an optimized 10-site library (1,536 variants) of P99 β-lactamase (P99βL), a component of ADEPT cancer therapies, revealed that the population possessed high overall fitness, and comprehensive analysis of peptide–MHC II immunoreactivity showed the population possessed lower average immunogenic potential than the wild-type enzyme. Although similar functional screening of an optimized 30-site library (2.15 × 109 variants) revealed reduced population-wide fitness, numerous individual variants were found to have activity and stability better than the wild type despite bearing 13 or more deimmunizing mutations per enzyme. The immunogenic potential of one highly active and stable 14-mutation variant was assessed further using ex vivo cellular immunoassays, and the variant was found to silence T-cell activation in seven of the eight blood donors who responded strongly to wild-type P99βL. In summary, our multiobjective library-design process readily identified large and mutually compatible sets of epitope-deleting mutations and produced highly active but aggressively deimmunized constructs in only one round of library screening. PMID:28607051

  13. Computationally optimized deimmunization libraries yield highly mutated enzymes with low immunogenicity and enhanced activity.

    Science.gov (United States)

    Salvat, Regina S; Verma, Deeptak; Parker, Andrew S; Kirsch, Jack R; Brooks, Seth A; Bailey-Kellogg, Chris; Griswold, Karl E

    2017-06-27

    Therapeutic proteins of wide-ranging function hold great promise for treating disease, but immune surveillance of these macromolecules can drive an antidrug immune response that compromises efficacy and even undermines safety. To eliminate widespread T-cell epitopes in any biotherapeutic and thereby mitigate this key source of detrimental immune recognition, we developed a Pareto optimal deimmunization library design algorithm that optimizes protein libraries to account for the simultaneous effects of combinations of mutations on both molecular function and epitope content. Active variants identified by high-throughput screening are thus inherently likely to be deimmunized. Functional screening of an optimized 10-site library (1,536 variants) of P99 β-lactamase (P99βL), a component of ADEPT cancer therapies, revealed that the population possessed high overall fitness, and comprehensive analysis of peptide-MHC II immunoreactivity showed the population possessed lower average immunogenic potential than the wild-type enzyme. Although similar functional screening of an optimized 30-site library (2.15 × 10 9 variants) revealed reduced population-wide fitness, numerous individual variants were found to have activity and stability better than the wild type despite bearing 13 or more deimmunizing mutations per enzyme. The immunogenic potential of one highly active and stable 14-mutation variant was assessed further using ex vivo cellular immunoassays, and the variant was found to silence T-cell activation in seven of the eight blood donors who responded strongly to wild-type P99βL. In summary, our multiobjective library-design process readily identified large and mutually compatible sets of epitope-deleting mutations and produced highly active but aggressively deimmunized constructs in only one round of library screening.

  14. BRCAsearch: written pre-test information and BRCA1/2 germline mutation testing in unselected patients with newly diagnosed breast cancer.

    Science.gov (United States)

    Nilsson, Martin P; Törngren, Therese; Henriksson, Karin; Kristoffersson, Ulf; Kvist, Anders; Silfverberg, Barbro; Borg, Åke; Loman, Niklas

    2017-11-21

    To evaluate a simplified method of pre-test information and germline BRCA1/2 mutation testing. In a prospective, single-arm study, comprehensive BRCA1/2 testing was offered to unselected patients with newly diagnosed breast cancer at three hospitals in south Sweden (BRCAsearch, ClinicalTrials.gov Identifier: NCT02557776). Pre-test information was provided by a standardized invitation letter, but the patients could contact a genetic counselor for telephone genetic counseling if they felt a need for that. Noncarriers were informed about the test result through a letter. Mutation carriers were contacted and offered an appointment for in-person post-test genetic counseling. During the period Feb 2, 2015-Aug 26, 2016, eight hundred and eighteen patients were invited to participate in the study. Through Jan 31, 2017, five hundred and forty-two (66.2%) of them consented to analysis of BRCA1 and BRCA2. Eleven pathogenic mutations were found (BRCA1, n = 2; BRCA2, n = 9), corresponding to a mutation prevalence of 2.0%. Six out of 11 fulfilled the Swedish BRCA testing criteria, and 9 out of 11 fulfilled the NCCN testing criteria. None of the BRCA-associated tumors were of the luminal A-like subtype. Very few patients contacted us for telephone genetic counseling or practical questions, suggesting that a majority felt that the written pre-test information was sufficient for them to make a decision on testing. Streamlining the process of pre-test information, genetic testing, and delivery of test results was feasible and was associated with an uptake of genetic testing in 2/3 of the breast cancer patients.

  15. Somatic POLE mutations cause an ultramutated giant cell high-grade glioma subtype with better prognosis.

    Science.gov (United States)

    Erson-Omay, E Zeynep; Çağlayan, Ahmet Okay; Schultz, Nikolaus; Weinhold, Nils; Omay, S Bülent; Özduman, Koray; Köksal, Yavuz; Li, Jie; Serin Harmancı, Akdes; Clark, Victoria; Carrión-Grant, Geneive; Baranoski, Jacob; Çağlar, Caner; Barak, Tanyeri; Coşkun, Süleyman; Baran, Burçin; Köse, Doğan; Sun, Jia; Bakırcıoğlu, Mehmet; Moliterno Günel, Jennifer; Pamir, M Necmettin; Mishra-Gorur, Ketu; Bilguvar, Kaya; Yasuno, Katsuhito; Vortmeyer, Alexander; Huttner, Anita J; Sander, Chris; Günel, Murat

    2015-10-01

    Malignant high-grade gliomas (HGGs), including the most aggressive form, glioblastoma multiforme, show significant clinical and genomic heterogeneity. Despite recent advances, the overall survival of HGGs and their response to treatment remain poor. In order to gain further insight into disease pathophysiology by correlating genomic landscape with clinical behavior, thereby identifying distinct HGG molecular subgroups associated with improved prognosis, we performed a comprehensive genomic analysis. We analyzed and compared 720 exome-sequenced gliomas (136 from Yale, 584 from The Cancer Genome Atlas) based on their genomic, histological, and clinical features. We identified a subgroup of HGGs (6 total, 4 adults and 2 children) that harbored a statistically significantly increased number of somatic mutations (mean = 9257.3 vs 76.2, P = .002). All of these "ultramutated" tumors harbored somatic mutations in the exonuclease domain of the polymerase epsilon gene (POLE), displaying a distinctive genetic profile, characterized by genomic stability and increased C-to-A transversions. Histologically, they all harbored multinucleated giant or bizarre cells, some with predominant infiltrating immune cells. One adult and both pediatric patients carried homozygous germline mutations in the mutS homolog 6 (MSH6) gene. In adults, POLE mutations were observed in patients younger than 40 years and were associated with a longer progression-free survival. We identified a genomically, histologically, and clinically distinct subgroup of HGGs that harbored somatic POLE mutations and carried an improved prognosis. Identification of distinctive molecular and pathological HGG phenotypes has implications not only for improved classification but also for potential targeted treatments. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. Genetic testing in familial AD and FTD: mutation and phenotype spectrum in a Danish cohort

    DEFF Research Database (Denmark)

    Lindquist, S G; Schwartz, M; Batbayli, M

    2009-01-01

    on chromosome 17, the MAPT and the PGRN genes, are associated with autosomal dominant inherited FTD. The aim of this study was to characterize the mutation spectrum and describe genotype-phenotype correlations in families with inherited dementia. The identification of novel mutations and/or atypical genotype-phenotype...

  17. High-Stakes Collaborative Testing: Why Not?

    Science.gov (United States)

    Levine, Ruth E; Borges, Nicole J; Roman, Brenda J B; Carchedi, Lisa R; Townsend, Mark H; Cluver, Jeffrey S; Frank, Julia; Morey, Oma; Haidet, Paul; Thompson, Britta M

    2017-12-08

    Phenomenon: Studies of high-stakes collaborative testing remain sparse, especially in medical education. We explored high-stakes collaborative testing in medical education, looking specifically at the experiences of students in established and newly formed teams. Third-year psychiatry students at 5 medical schools across 6 sites participated, with 4 participating as established team sites and 2 as comparison team sites. For the collaborative test, we used the National Board of Medical Examiners Psychiatry subject test, administering it via a 2-stage process. Students at all sites were randomly selected to participate in a focus group, with 8-10 students per site (N = 49). We also examined quantitative data for additional triangulation. Students described a range of heightened emotions around the collaborative test yet perceived it as valuable regardless if they were in established or newly formed teams. Students described learning about the subject matter, themselves, others, and interpersonal dynamics during collaborative testing. Triangulation of these results via quantitative data supported these themes. Insights: Despite student concerns, high-stakes collaborative tests may be both valuable and feasible. The data suggest that high-stakes tests (tests of learning or summative evaluation) could also become tests for learning or formative evaluation. The paucity of research into this methodology in medical education suggests more research is needed.

  18. Irradiation test of high density Si material

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Man Soon; Choo, Kee Nam; Lee, Chul Yong; Yang, Seong Woo; Shim, Kyue Taek; Park, Sang Jun [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2012-10-15

    The feasibility of irradiation test for the high-density Si material entrusted by Guju Inc. was reviewed. The high density Si material is used for a sealing of the penetration holes of piping at the nuclear power plants. The irradiation test was performed and the density changes between before and after irradiation test were measured. The irradiation tests were performed 2 times for 1 day and 20 days at IP 4 hole of HANARO. The 3 Si specimens irradiated were without flaws and the density changes after irradiation were successfully measured. The result satisfies the requirement of the design specification.

  19. PGD for hereditary breast and ovarian cancer : the route to universal tests for BRCA1 and BRCA2 mutation carriers

    NARCIS (Netherlands)

    Drusedau, Marion; Dreesen, Jos C.; Derks-Smeets, Inge; Coonen, Edith; van Golde, Ron; van Echten-Arends, Jannie; Kastrop, Peter M. M.; Blok, Marinus J.; Gomez-Garcia, Encarna; Geraedts, Joep P.; Smeets, Hubert J.; de Die-Smulders, Christine E.; Paulussen, Aimee D.

    2013-01-01

    Preimplantation Genetic Diagnosis (PGD) is a method of testing in vitro embryos as an alternative to prenatal diagnosis with possible termination of pregnancy in case of an affected child. Recently, PGD for hereditary breast and ovarian cancer caused by BRCA1 and BRCA2 mutations has found its way in

  20. Hereditary nephrotic syndrome: a systematic approach for genetic testing and a review of associated podocyte gene mutations.

    Science.gov (United States)

    Benoit, Geneviève; Machuca, Eduardo; Antignac, Corinne

    2010-09-01

    Several genes have been implicated in genetic forms of nephrotic syndrome occurring in children. It is now known that the phenotypes associated with mutations in these genes display significant variability, rendering genetic testing and counselling a more complex task. This review will focus on the recent clinical findings associated with those genes known to be involved in isolated steroid-resistant nephrotic syndrome in children and, thereby, propose an approach for appropriate mutational screening. The recurrence of proteinuria after transplantation in patients with hereditary forms of nephrotic syndrome will also be discussed.

  1. Non-invasive urine testing of EGFR activating mutation and T790M resistance mutation in non-small cell lung cancer.

    Science.gov (United States)

    Berz, David; Raymond, Victoria M; Garst, Jordan H; Erlander, Mark G

    2015-01-01

    The increasing understanding of non-small cell lung cancer (NSCLC) biology over the last two decades has led to the identification of multiple molecular targets. This led to the development of multiple targeted therapies in the primary and secondary resistance setting and the epidermal growth factor receptor (EGFR) gene remains the most frequently observed molecular target in NSCLC. Tissue biopsies remain the standard for the identification of such EGFR mutations. Obtaining serial tissue biopsies, especially in the secondary resistance setting is associated with multiple medical and logistical challenges. Utilizing circulating tumor DNA (ctDNA) fragments for molecular analysis can overcome these challenges and aid in therapeutic decision-making. Here we present a present a 72-year-old Korean woman with metastatic, EGFR L858R mutated bronchogenic adenocarcinoma. She developed skeletal progression on treatment with first and second generation tyrosine kinase inhibitors (TKIs). Repeated biopsies failed to provide informative molecular test results. A novel urine ctDNA assay was utilized and confirmed T790M positive status. The patient was started on a third generation TKI, which led to a measurable clinical response. Utilization of urine liquid biopsies for EGFR diagnostics are feasible and provided critical clinical information in this patient's case. Urine liquid biopsy represents a viable alternative to tissue biopsy, particularly in the secondary resistance setting, when tissue is not available for molecular testing.

  2. Towards Measuring the Abstractness of State Machines based on Mutation Testing

    Directory of Open Access Journals (Sweden)

    Thomas Baar

    2017-01-01

    Full Text Available Abstract. The notation of state machines is widely adopted as a formalism to describe the behaviour of systems. Usually, multiple state machine models can be developed for the very same software system. Some of these models might turn out to be equivalent, but, in many cases, different state machines describing the same system also differ in their level of abstraction. In this paper, we present an approach to actually measure the abstractness level of state machines w.r.t. a given implemented software system. A state machine is considered to be less abstract when it is conceptionally closer to the implemented system. In our approach, this distance between state machine and implementation is measured by applying coverage criteria known from software mutation testing. Abstractness of state machines can be considered as a new metric. As for other metrics as well, a known value for the abstractness of a given state machine allows to assess its quality in terms of a simple number. In model-based software development projects, the abstract metric can help to prevent model degradation since it can actually measure the semantic distance from the behavioural specification of a system in form of a state machine to the current implementation of the system. In contrast to other metrics for state machines, the abstractness cannot be statically computed based on the state machine’s structure, but requires to execute both state machine and corresponding system implementation. The article is published in the author’s wording. 

  3. High Prevalence of inhA Promoter Mutations among Patients with Drug-Resistant Tuberculosis in KwaZulu-Natal, South Africa.

    Directory of Open Access Journals (Sweden)

    Abraham J Niehaus

    Full Text Available Drug-resistant tuberculosis (TB remains extremely difficult to treat because there are often few remaining active medications and limited diagnostic options to detect resistance. Resistance to isoniazid is typically caused by mutations in either katG or the inhA promoter. inhA mutations confer low-level resistance to isoniazid and cross-resistance to ethionamide while katG mutations confer high-level isoniazid resistance and no cross-resistance. Line Probe Assays (LPAs that detect mutations in katG and inhA are currently performed on all positive TB cultures in KwaZulu-Natal province, South Africa, but the frequency of inhA mutations in drug-resistant TB patients has not been examined.We sought to determine the proportion of patients who could potentially benefit from high-dose isoniazid and who may be resistant to ethionamide. We reviewed 994 LPA (Hain MTBDRplus results at the TB reference laboratory in KwaZulu-Natal to determine the frequency of mutations in either katG or the inhA promoter. We stratified these results by drug-resistance category (i.e., MDR-TB, pre-XDR-TB, and XDR-TB as determined by phenotypic drug-susceptibility testing.Among MDR- and XDR-TB isolates, the prevalence of inhA mutations without a concurrent katG mutation was 14.8% and 10.3% respectively. The prevalence of inhA mutations with OR without a katG mutation was 30.3% and 82.8%, respectively.More than 10% of patients with MDR- and XDR-TB may benefit from high-dose isoniazid. Although ethionamide is empirically included in all MDR- and XDR-TB regimens, nearly a third of MDR-TB patients and a majority of XDR-TB patients likely have resistance to ethionamide. Laboratories performing line probe assays should report specific band patterns so that clinicians may adjust treatment regimens accordingly.

  4. High School Students' Attitudes toward Fitness Testing

    Science.gov (United States)

    Mercier, Kevin; Silverman, Stephen

    2014-01-01

    The purpose of this study was to investigate the attitudes of high school students toward fitness testing. An instrument containing 18 items and four factors measuring student's attitudes toward fitness testing: cognitive, affect-enjoyment, affect-feelings, and affect-teacher was completed by 524 boys and 675 girls (N = 1199). MANOVA indicated…

  5. Optimizing a multiplex high resolution melting curve to diagnose G6PD deficiency based on viangchan and canton mutations

    Directory of Open Access Journals (Sweden)

    Nghia Le Tri

    2016-08-01

    Full Text Available Glucose-6-phosphate dehydrogenase (G6PD deficiency which is caused by mutation on G6PD gene is the most common enzyme disorder in human. There have been 184 discovered mutations among which Viangchan [Val291Met] and Canton mutation [Arg459Leu] are the most common variants in Vietnamese. Due to the severity of this disease, several methods have been devised for diagnostics. However, time-consuming, low sensitivity and expensiveness are major problems of those techniques. Recently, High Resolution Melting (HRM has been developed and proven to be an effective method for DNA genotyping, mutation scanning and sequence matching. Hence, in this study a multiplex HRM has been developed aiming at detecting these two mutations concurrently. At first, a singleplex HRM was designed for each mutation. Then, conditions for these singleplex assay were combined and optimized again in order to get the optimal condition for multiplex HRM. Although this method showed a promising potential with a high accuracy, sensitivity, and specificity, it is impractial to continue developing this method because of the lack of controls. However, the optimized singleplex PCR-HRM in this study still can be used for single mutation detection and serve as the background to develop PCR-HRM for other G6PD mutations in Vietnamese-Kinh population. [Biomed Res Ther 2016; 3(8.000: 757-769

  6. Model and test in a fungus of the probability that beneficial mutations survive drift

    NARCIS (Netherlands)

    Gifford, D.R.; Visser, de J.A.G.M.; Wahl, L.M.

    2013-01-01

    Determining the probability of fixation of beneficial mutations is critically important for building predictive models of adaptive evolution. Despite considerable theoretical work, models of fixation probability have stood untested for nearly a century. However, recent advances in experimental and

  7. Comparison of high-resolution melting analysis with direct sequencing for the detection of recurrent mutations in DNA methyltransferase 3A and isocitrate dehydrogenase 1 and 2 genes in acute myeloid leukemia patients.

    Science.gov (United States)

    Gorniak, Patryk; Ejduk, Anna; Borg, Katarzyna; Makuch-Lasica, Hanna; Nowak, Grazyna; Lech-Maranda, Ewa; Prochorec-Sobieszek, Monika; Warzocha, Krzysztof; Juszczynski, Przemyslaw

    2016-02-01

    Acute myeloid leukemia (AML) cells harbor frequent mutations in genes responsible for epigenetic modifications. Increasing evidence of clinical role of DNMT3A and IDH1/2 mutations highlights the need for a robust and inexpensive test to identify these mutations in routine diagnostic work-up. Herein, we compared routinely used direct sequencing method with high-resolution melting (HRM) assay for screening DNMT3A and IDH1/2 mutations in patients with AML. We show very high concordance between HRM and Sanger sequencing (100% samples for IDH2-R140 and DNMT3-R882 mutations, 99% samples for IDH1-R132 and IDH2-R172 mutations). HRM method reported no false-negative results, suggesting that it can be used for mutations screening. Moreover, HRM displayed much higher sensitivity in comparison with DNA sequencing in all assessed loci. With Sanger sequencing, robust calls were observed when the sample contained 50% of mutant DNA in the background of wild-type DNA. In marked contrast, the detection limit of HRM improved down to 10% of mutated DNA. Given the ubiquitous presence of wild-type DNA background in bone marrow aspirates and clonal variations regarding mutant allele burden, these results favor HRM as a sensitive, specific, labor-, and cost-effective tool for screening and detection of mutations in IDH1/2 and DNMT3A genes in patients with AML. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  8. Adrenoleukodystrophy in Norway: high rate of de novo mutations and age-dependent penetrance.

    Science.gov (United States)

    Horn, Morten A; Retterstøl, Lars; Abdelnoor, Michael; Skjeldal, Ola H; Tallaksen, Chantal M E

    2013-03-01

    To investigate X-linked adrenoleukodystrophy in an unselected population, we performed a population based, cross-sectional prevalence study, supplemented by a retrospective study of deceased subjects. Sixty-three subjects (34 males, 29 females) belonging to 22 kindreds were included. Thirty-nine subjects (13 males, 26 females) were alive, and 24 (21 males, 3 females) were deceased on the prevalence day. The point prevalence of X-linked adrenoleukodystrophy in Norway on July 1, 2011, was 0.8 per 100,000 inhabitants. The incidence at birth in the period 1956-1995 was 1.6 per 100,000 inhabitants. An age-dependent penetrance was observed among males and females, with more severe phenotypes appearing with rising age. Only 5% of deceased males had not developed cerebral leukodystrophy. No female older than 50 years was neurologically intact. Sixteen mutations in the ABCD1 gene were identified. De novo mutations were found in 19% of probands. The frequency of X-linked adrenoleukodystrophy was lower in Norway than reported in the literature. A more severe natural course than previously reported was observed, indicating a need for better follow-up of both male and female patients. Given the high rate of de novo mutations, identification programs such as newborn screening may be required to offer timely treatment to all patients. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Phenotypic switching in Mycoplasma gallisepticum hemadsorption is governed by a high-frequency, reversible point mutation.

    Science.gov (United States)

    Winner, Florian; Markovà, Ivana; Much, Peter; Lugmair, Albin; Siebert-Gulle, Karin; Vogl, Gunther; Rosengarten, Renate; Citti, Christine

    2003-03-01

    Mycoplasma gallisepticum is a flask-shaped organism that commonly induces chronic respiratory disease in chickens and infectious sinusitis in turkeys. Phenotypic switching in M. gallisepticum hemadsorption (HA) was found to correlate with phase variation of the GapA cytadhesin concurrently with that of the CrmA protein, which exhibits cytadhesin-related features and is encoded by a gene located downstream of the gapA gene as part of the same transcription unit. In clones derived from strain R(low), detailed genetic analyses further revealed that on-off switching in GapA expression is governed by a reversible base substitution occurring at the beginning of the gapA structural gene. In HA(-) variants, this event generates a stop codon that results in the premature termination of GapA translation and consequently affects the expression of CrmA. Sequences flanking the mutation spot do not feature any repeated motifs that could account for error-prone mutation via DNA slippage and the exact mechanism underlying this high-frequency mutational event remains to be elucidated. An HA(-) mutant deficient in producing CrmA, mHAD3, was obtained by disrupting the crmA gene by using transposition mutagenesis. Despite a fully functional gapA gene, the amount of GapA detected in this mutant was considerably lower than in HA(+) clonal variants, suggesting that, in absence of CrmA, GapA might be subjected to a higher turnover.

  10. High Resolution Melt analysis for mutation screening in PKD1 and PKD2

    Directory of Open Access Journals (Sweden)

    Fontes Michel

    2011-10-01

    Full Text Available Abstract Background Autosomal dominant polycystic kidney disease (ADPKD is the most common hereditary kidney disorder. It is characterized by focal development and progressive enlargement of renal cysts leading to end-stage renal disease. PKD1 and PKD2 have been implicated in ADPKD pathogenesis but genetic features and the size of PKD1 make genetic diagnosis tedious. Methods We aim to prove that high resolution melt analysis (HRM, a recent technique in molecular biology, can facilitate molecular diagnosis of ADPKD. We screened for mutations in PKD1 and PKD2 with HRM in 37 unrelated patients with ADPKD. Results We identified 440 sequence variants in the 37 patients. One hundred and thirty eight were different. We found 28 pathogenic mutations (25 in PKD1 and 3 in PKD2 within 28 different patients, which is a diagnosis rate of 75% consistent with literature mean direct sequencing diagnosis rate. We describe 52 new sequence variants in PKD1 and two in PKD2. Conclusion HRM analysis is a sensitive and specific method for molecular diagnosis of ADPKD. HRM analysis is also costless and time sparing. Thus, this method is efficient and might be used for mutation pre-screening in ADPKD genes.

  11. High Resolution Melt analysis for mutation screening in PKD1 and PKD2.

    Science.gov (United States)

    Bataille, Stanislas; Berland, Yvon; Fontes, Michel; Burtey, Stéphane

    2011-10-18

    Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disorder. It is characterized by focal development and progressive enlargement of renal cysts leading to end-stage renal disease. PKD1 and PKD2 have been implicated in ADPKD pathogenesis but genetic features and the size of PKD1 make genetic diagnosis tedious. We aim to prove that high resolution melt analysis (HRM), a recent technique in molecular biology, can facilitate molecular diagnosis of ADPKD. We screened for mutations in PKD1 and PKD2 with HRM in 37 unrelated patients with ADPKD. We identified 440 sequence variants in the 37 patients. One hundred and thirty eight were different. We found 28 pathogenic mutations (25 in PKD1 and 3 in PKD2 ) within 28 different patients, which is a diagnosis rate of 75% consistent with literature mean direct sequencing diagnosis rate. We describe 52 new sequence variants in PKD1 and two in PKD2. HRM analysis is a sensitive and specific method for molecular diagnosis of ADPKD. HRM analysis is also costless and time sparing. Thus, this method is efficient and might be used for mutation pre-screening in ADPKD genes.

  12. Activity-enhancing mutations in an E3 ubiquitin ligase identified by high-throughput mutagenesis.

    Science.gov (United States)

    Starita, Lea M; Pruneda, Jonathan N; Lo, Russell S; Fowler, Douglas M; Kim, Helen J; Hiatt, Joseph B; Shendure, Jay; Brzovic, Peter S; Fields, Stanley; Klevit, Rachel E

    2013-04-02

    Although ubiquitination plays a critical role in virtually all cellular processes, mechanistic details of ubiquitin (Ub) transfer are still being defined. To identify the molecular determinants within E3 ligases that modulate activity, we scored each member of a library of nearly 100,000 protein variants of the murine ubiquitination factor E4B (Ube4b) U-box domain for auto-ubiquitination activity in the presence of the E2 UbcH5c. This assay identified mutations that enhance activity both in vitro and in cellular p53 degradation assays. The activity-enhancing mutations fall into two distinct mechanistic classes: One increases the U-box:E2-binding affinity, and the other allosterically stimulates the formation of catalytically active conformations of the E2∼Ub conjugate. The same mutations enhance E3 activity in the presence of another E2, Ube2w, implying a common allosteric mechanism, and therefore the general applicability of our observations to other E3s. A comparison of the E3 activity with the two different E2s identified an additional variant that exhibits E3:E2 specificity. Our results highlight the general utility of high-throughput mutagenesis in delineating the molecular basis of enzyme activity.

  13. High-quality Thermodynamic Data on the Stability Changes of Proteins Upon Single-site Mutations

    Energy Technology Data Exchange (ETDEWEB)

    Pucci, Fabrizio, E-mail: fapucci@ulb.ac.be; Bourgeas, Raphaël, E-mail: rbourgeas@ulb.ac.be; Rooman, Marianne, E-mail: mrooman@ulb.ac.be [Department of BioModeling, BioInformatics and BioProcesses, Université Libre de Bruxelles, CP 165/61, Roosevelt Avenue 50, 1050 Brussels, Belgium and Interuniversity Institute of Bioinformatics in Brussels, CP 263, Triumph Bld, 1050 Brussels (Belgium)

    2016-06-15

    We have set up and manually curated a dataset containing experimental information on the impact of amino acid substitutions in a protein on its thermal stability. It consists of a repository of experimentally measured melting temperatures (T{sub m}) and their changes upon point mutations (ΔT{sub m}) for proteins having a well-resolved x-ray structure. This high-quality dataset is designed for being used for the training or benchmarking of in silico thermal stability prediction methods. It also reports other experimentally measured thermodynamic quantities when available, i.e., the folding enthalpy (ΔH) and heat capacity (ΔC{sub P}) of the wild type proteins and their changes upon mutations (ΔΔH and ΔΔC{sub P}), as well as the change in folding free energy (ΔΔG) at a reference temperature. These data are analyzed in view of improving our insights into the correlation between thermal and thermodynamic stabilities, the asymmetry between the number of stabilizing and destabilizing mutations, and the difference in stabilization potential of thermostable versus mesostable proteins.

  14. Efficacy versus effectiveness of clinical genetic testing criteria for BRCA1 and BRCA2 hereditary mutations in incident breast cancer.

    Science.gov (United States)

    Nilsson, Martin P; Winter, Christof; Kristoffersson, Ulf; Rehn, Martin; Larsson, Christer; Saal, Lao H; Loman, Niklas

    2017-04-01

    Increasing evidence supports the benefit of identifying BRCA1 and BRCA2 germline mutations in early breast cancer. Selection of patients for genetic testing is based on defined criteria taking individual and family history related factors into account. It is important to make a distinction between efficacy and effectiveness of BRCA testing criteria. Efficacy can be defined as the performance under ideal circumstances, whereas effectiveness refers to its real life performance. To allow for an unbiased and detailed evaluation of efficacy and effectiveness of the Swedish BRCA testing criteria, we retrospectively analyzed a prospectively collected cohort of 273 breast cancer patients from the well-characterized, population-based, single-site All Breast Cancer in Malmö (ABiM) study. The patients were diagnosed with breast cancer during the years 2007 through 2009. Out of 20 mutation carriers identified, 13 fulfilled Swedish criteria at time of diagnosis. Thus, the efficacy of these criteria was 65%. Excluding three patients in whom a mutation was already known at time of diagnosis, only 3/17 had been identified in the clinical routine, corresponding to an effectiveness of 18%. Here we detail the reasons why mutation carriers in our cohort were not detected though routine health care. In conclusion, effectiveness of BRCA testing criteria was much lower than efficacy. Our results indicate that current testing criteria and procedures associated with BRCA1 and BRCA2 testing are insufficient. There is room for improvement of their efficacy, but even more so regarding effectiveness. Clinical BRCA testing routines need to be critically revised.

  15. High-voltage engineering and testing

    CERN Document Server

    Ryan, Hugh M

    2013-01-01

    This 3rd edition of High Voltage Engineering Testing describes strategic developments in the field and reflects on how they can best be managed. All the key components of high voltage and distribution systems are covered including electric power networks, UHV and HV. Distribution systems including HVDC and power electronic systems are also considered.

  16. Long QT interval in Turner syndrome--a high prevalence of LQTS gene mutations.

    Directory of Open Access Journals (Sweden)

    Christian Trolle

    Full Text Available QT-interval prolongation of unknown aetiology is common in Turner syndrome. This study set out to explore the presence of known long QT mutations in Turner syndrome and to examine the corrected QT-interval (QTc over time and relate the findings to the Turner syndrome phenotype.Adult women with Turner syndrome (n = 88 were examined thrice and 68 age-matched healthy controls were examined once. QTc was measured by one blinded reader (intra-reader variability: 0.7%, and adjusted for influence of heart rate by Bazett's (bQTc and Hodges's formula (hQTc. The prevalence of mutations in genes related to Long QT syndrome was determined in women with Turner syndrome and a QTc >432.0 milliseconds (ms. Echocardiographic assessment of aortic valve morphology, 24-hour blood pressures and blood samples were done.The mean hQTc in women with Turner syndrome (414.0 ± 25.5 ms compared to controls (390.4 ± 17.8 ms was prolonged (p432 ms, 7 had mutations in major Long QT syndrome genes (SCN5A and KCNH2 and one in a minor Long QT syndrome gene (KCNE2.There is a high prevalence of mutations in the major LQTS genes in women with TS and prolonged QTc. It remains to be settled, whether these findings are related to the unexplained excess mortality in Turner women.NCT00624949. https://register.clinicaltrials.gov/prs/app/action/SelectProtocol/sid/S0001FLI/selectaction/View/ts/3/uid/U000099E.

  17. Two-miRNA classifiers differentiate mutation-negative follicular thyroid carcinomas and follicular thyroid adenomas in fine needle aspirations with high specificity

    DEFF Research Database (Denmark)

    Stokowy, Tomasz; Wojtas, Bartosz; Jarzab, Barbara

    2016-01-01

    Diagnosis of thyroid by fine needle aspiration is challenging for the "indeterminate" category and can be supported by molecular testing. We set out to identify miRNA markers that could be used in a diagnostic setting to improve the discrimination of mutation-negative indeterminate fine needle...... aspirations. miRNA high-throughput sequencing was performed for freshly frozen tissue samples of 19 RAS and PAX8/PPARG mutation-negative follicular thyroid carcinomas, and 23 RAS and PAX8/PPARG mutation-negative follicular adenomas. Differentially expressed miRNAs were validated by quantitative polymerase...... chain reaction in a set of 44 fine needle aspiration samples representing 24 follicular thyroid carcinomas and 20 follicular adenomas. Twenty-six miRNAs characterized by a significant differential expression between follicular thyroid carcinomas and follicular adenomas were identified. Nevertheless...

  18. Patients with Philadelphia-positive leukemia with Y253H or F359V mutation have a high risk of developing new mutations in the setting of dasatinib resistance.

    Science.gov (United States)

    Jiang, Qian; Qin, Ya-Zhen; Lai, Yue-Yun; Jiang, Hao; Wang, Jing; Huang, Xiao-Jun

    2015-07-01

    The treatment outcome and development of new mutations in patients with imatinib- and/or nilotinib-failure Philadelphia chromosome positive (Ph+) leukemia with highly nilotinib-resistant mutations (Y253H, E255K/V and F359V/C) were assessed on dasatinib therapy. A total of 111 patients with Ph+ leukemia were grouped into three cohorts by baseline BCR-ABL kinase domain mutation status: no mutation (n = 44), non-nilotinib-resistant mutations (n = 26) or nilotinib-resistant mutations (n = 41). The frequencies of hematological, cytogenetic and molecular responses and clinical resistance to dasatinib were similar among the three cohorts during dasatinib therapy. In dasatinib-resistant patients, new mutations were most frequently observed in the cohort with nilotinib-resistant mutations (p = 0.001). Multivariate analysis revealed that advanced disease and harboring Y253H or F359V mutation before dasatinib were independent predictors of developing new mutations. We conclude that patients with Ph+ leukemia with Y253H or F359V mutation have a high likelihood of developing new mutations in the setting of dasatinib resistance.

  19. Mutation analysis in {beta}{sub 2-}adrenergic receptor gene by denaturing high performance liquid chromatography (DHPLC)

    Energy Technology Data Exchange (ETDEWEB)

    Park, S.B.; Oh, C.H.; Kim, J.W.; Jang, W.C. [Dankook University, Cheonan (Korea)

    2002-06-01

    We analysed mutation of {beta}{sub 2-}adrenergic receptor gene that controls bronchial asthma by denaturing high performance liquid chromatography (DHPLC) according to ion-pair reversed-phase high performance liquid chromatography (IP-RP-HPLC). We extracted genomic DNA from 50 asthma patients, amplified DNA using PCR, and analysed PCR product by DHPLC. As a result, we obtained that mutation frequency was 15(30%) among 50 cases. Consequently DHPLC mutation detection was confirmed that the result of direct sequencing was coincide exactly. (author). 13 refs., 4 figs.

  20. Prevalence of H63D, S65C and C282Y hereditary hemochromatosis gene mutations in Slovenian population by an improved high-throughput genotyping assay

    Directory of Open Access Journals (Sweden)

    Rupreht Ruth

    2007-11-01

    Full Text Available Abstract Background Hereditary hemochromatosis (HH is a common genetic disease characterized by excessive iron overload that leads to multi-organ failure. Although the most prevalent genotype in HH is homozygosity for C282Y mutation of the HFE gene, two additional mutations, H63D and S65C, appear to be associated with a milder form of HH. The aim of this study was to develop a high-throughput assay for HFE mutations screening based on TaqMan technology and to determine the frequencies of HFE mutations in the Slovenian population. Methods Altogether, 1282 randomly selected blood donors from different Slovenian regions and 21 HH patients were analyzed for the presence of HFE mutations by an in-house developed real-time PCR assay based on TaqMan technology using shorter non-interfering fluorescent single nucleotide polymorphism (SNP-specific MGB probes. The assay was validated by RFLP analysis and DNA sequencing. Results The genotyping assay of the H63D, S65C and C282Y mutations in the HFE gene, based on TaqMan technology proved to be fast, reliable, with a high-throughput capability and 100% concordant with genotypes obtained by RFLP and DNA sequencing. The observed frequency of C282Y homozygotes in the group of HH patients was only 48%, others were of the heterogeneous HFE genotype. Among 1282 blood donors tested, the observed H63D, S65C and C282Y allele frequency were 12.8% (95% confidence interval (CI 11.5 – 14.2%, 1.8% (95% CI 1.4 – 2.5% and 3.6% (95% CI 3.0 – 4.5%, respectively. Approximately 33% of the tested subjects had at least one of the three HH mutations, and 1% of them were C282Y homozygotes or compound heterozygotes C282Y/H63D or C282Y/S65C, presenting an increased risk for iron overload disease. A significant variation in H63D allele frequency was observed for one of the Slovenian regions. Conclusion The improved real-time PCR assay for H63D, S65C and C282Y mutations detection is accurate, fast, cost-efficient and ready for

  1. Prevalence of H63D, S65C and C282Y hereditary hemochromatosis gene mutations in Slovenian population by an improved high-throughput genotyping assay.

    Science.gov (United States)

    Cukjati, Marko; Vaupotic, Tomaz; Rupreht, Ruth; Curin-Serbec, Vladka

    2007-11-23

    Hereditary hemochromatosis (HH) is a common genetic disease characterized by excessive iron overload that leads to multi-organ failure. Although the most prevalent genotype in HH is homozygosity for C282Y mutation of the HFE gene, two additional mutations, H63D and S65C, appear to be associated with a milder form of HH. The aim of this study was to develop a high-throughput assay for HFE mutations screening based on TaqMan technology and to determine the frequencies of HFE mutations in the Slovenian population. Altogether, 1282 randomly selected blood donors from different Slovenian regions and 21 HH patients were analyzed for the presence of HFE mutations by an in-house developed real-time PCR assay based on TaqMan technology using shorter non-interfering fluorescent single nucleotide polymorphism (SNP)-specific MGB probes. The assay was validated by RFLP analysis and DNA sequencing. The genotyping assay of the H63D, S65C and C282Y mutations in the HFE gene, based on TaqMan technology proved to be fast, reliable, with a high-throughput capability and 100% concordant with genotypes obtained by RFLP and DNA sequencing. The observed frequency of C282Y homozygotes in the group of HH patients was only 48%, others were of the heterogeneous HFE genotype. Among 1282 blood donors tested, the observed H63D, S65C and C282Y allele frequency were 12.8% (95% confidence interval (CI) 11.5-14.2%), 1.8% (95% CI 1.4-2.5%) and 3.6% (95% CI 3.0-4.5%), respectively. Approximately 33% of the tested subjects had at least one of the three HH mutations, and 1% of them were C282Y homozygotes or compound heterozygotes C282Y/H63D or C282Y/S65C, presenting an increased risk for iron overload disease. A significant variation in H63D allele frequency was observed for one of the Slovenian regions. The improved real-time PCR assay for H63D, S65C and C282Y mutations detection is accurate, fast, cost-efficient and ready for routine screening and diagnostic procedures. The genotype frequencies in

  2. Biological and clinical evidence for somatic mutations in BRCA1 and BRCA2 as predictive markers for olaparib response in high-grade serous ovarian cancers in the maintenance setting.

    Science.gov (United States)

    Dougherty, Brian A; Lai, Zhongwu; Hodgson, Darren R; Orr, Maria C M; Hawryluk, Matthew; Sun, James; Yelensky, Roman; Spencer, Stuart K; Robertson, Jane D; Ho, Tony W; Fielding, Anitra; Ledermann, Jonathan A; Barrett, J Carl

    2017-07-04

    To gain a better understanding of the role of somatic mutations in olaparib response, next-generation sequencing (NGS) of BRCA1 and BRCA2 was performed as part of a planned retrospective analysis of tumors from a randomized, double-blind, Phase II trial (Study 19; D0810C00019; NCT00753545) in 265 patients with platinum-sensitive high-grade serous ovarian cancer. BRCA1/2 loss-of-function mutations were found in 55% (114/209) of tumors, were mutually exclusive, and demonstrated high concordance with Sanger-sequenced germline mutations in matched blood samples, confirming the accuracy (97%) of tumor BRCA1/2 NGS testing. Additionally, NGS identified somatic mutations absent from germline testing in 10% (20/209) of the patients. Somatic mutations had >80% biallelic inactivation frequency and were predominantly clonal, suggesting that BRCA1/2 loss occurs early in the development of these cancers. Clinical outcomes between placebo- and olaparib-treated patients with somatic BRCA1/2 mutations were similar to those with germline BRCA1/2 mutations, indicating that patients with somatic BRCA1/2 mutations benefit from treatment with olaparib.

  3. The identification of point mutations in Duchenne muscular dystrophy patients by using reverse-transcription PCR and the protein truncation test

    Energy Technology Data Exchange (ETDEWEB)

    Gardner, R.J.; Bobrow, M.; Roberts, R.G. [St. Thomas`s Hospitals, London (United Kingdom)

    1995-08-01

    The protein truncation test (PTT) is a mutation-detection method that monitors the integrity of the open reading frame (ORF). More than 60% of cases of Duchenne muscular dystrophy (DMD) result from gross frameshifting deletions in the dystrophin gene that are detectable by multiplex PCR system. It has become apparent that virtually all of the remaining DMD mutations also disrupt the translational reading frame, making the PTT a logical next step toward a comprehensive strategy for the identification of all DMD mutations. We report here a pilot study involving 22 patients and describe the mutations characterized. These constitute 12 point mutations or small insertions/deletions and 4 gross rearrangements. We also have a remaining five patients in whom there does not appear to be mutation in the ORF. We believe that reverse-transcription-PCR/PTT is an efficient method by which to screen for small mutations in DMD patients with no deletion. 29 refs., 2 figs., 3 tabs.

  4. Novel Sequencing-based Strategies for High-Throughput Discovery of Genetic Mutations Underlying Inherited Antibody Deficiency Disorders

    OpenAIRE

    Wang, Hong-Ying; Jain, Ashish

    2011-01-01

    Human inherited antibody deficiency disorders are generally caused by mutations in genes involved in the pathways regulating B-cell class switch recombination; DNA damage repair; and B-cell development, differentiation, and survival. Sequencing a large set of candidate genes involved in these pathways appears to be a highly efficient way to identify novel mutations. Herein we review several high-throughput sequencing approaches as well as recent improvements in target gene enrichment technolo...

  5. Intratumoral Heterogeneity of Frameshift Mutations in MECOM Gene is Frequent in Colorectal Cancers with High Microsatellite Instability.

    Science.gov (United States)

    Choi, Eun Ji; Kim, Min Sung; Song, Sang Yong; Yoo, Nam Jin; Lee, Sug Hyung

    2017-01-01

    MECOM gene, also known as EVI, encodes a transcriptional regulator involved in hematopoiesis, apoptosis, development and proliferation. In blood system, MECOM is considered an oncogene, but in solid tumors it has both oncogenic and tumor suppressor activities. Low frequent somatic mutations of MECOM have been detected in many cancers including colorectal cancers (CRC), but the mutation status with respect to the microsatellite instability (MSI) has not been studied. There is an A7 mononucleotide repeat in MECOM coding sequences that could be a mutation target in the cancers with MSI. We analyzed the A7 of MECOM in 79 CRCs with high MSI (MSI-H) and 65 microsatellite stable/low MSI (MSS/MSI-L) CRCs by single-strand conformation polymorphism analysis and DNA sequencing. Overall, we found MECOM frameshift mutations in 6 (7.6 %) CRCs with MSI-H, but not in MSS/MSI-L cancers (0/65) (p < 0.025). We also analyzed intratumoral heterogeneity (ITH) of the MECOM frameshift mutation in 16 CRCs and found that four CRCs (25.0 %) harbored regional ITH of the frameshift mutations. Our data indicate that MECOM gene harbors both somatic frameshift mutations and mutational ITH, which together may be features of CRC with MSI-H.

  6. High School Equivalency Testing in Arizona. Forum: Responding to Changes in High School Equivalency Testing

    Science.gov (United States)

    Hart, Sheryl

    2015-01-01

    For decades, the state of Arizona has used the General Educational Development (GED) Test to award the Arizona High School Equivalency (HSE) Diploma, as the GED Test was the only test available, recognized and accepted in the United States as the measure by which adults could demonstrate the educational attainment equivalent to high school…

  7. Genetic testing in familial and young-onset Alzheimer's disease: mutation spectrum in a Serbian cohort.

    Science.gov (United States)

    Dobricic, Valerija; Stefanova, Elka; Jankovic, Milena; Gurunlian, Nicole; Novakovic, Ivana; Hardy, John; Kostic, Vladimir; Guerreiro, Rita

    2012-07-01

    Alzheimer's disease (AD) is the most common form of dementia. To date, more than 200 mutations in three genes have been identified as cause of early-onset autosomal dominant inherited AD. The aim of this study was to characterize the mutation spectrum and describe genotype-phenotype correlations in Serbian patients with positive family history of AD or/and early-onset AD. We performed a genetic screening for mutations in the coding regions of Presenilins 1 and 2 (PSEN1 and PSEN2), as well as exons 16 and 17 of the Amyloid Precursor Protein gene (APP) in a total of 47 patients from Serbia with a clinical diagnosis of familial and/or early-onset AD (mean age at onset of 60.3 years; range 32-77). We found one novel mutation in PSEN1, one novel variant in PSEN2, and three previously described variants, one in each of the analyzed genes. Interestingly, we identified one patient harboring two heterozygous mutations: one in APP (p.L723P) and one in PSEN1 (p.R108Q). Copyright © 2012 Elsevier Inc. All rights reserved.

  8. High-voltage test and measuring techniques

    CERN Document Server

    Hauschild, Wolfgang

    2014-01-01

    It is the intent of this book to combine high-voltage (HV) engineering with HV testing technique and HV measuring technique. Based on long-term experience gained by the authors as lecturer and researcher as well as member in international organizations, such as IEC and CIGRE, the book will reflect the state of the art as well as the future trends in testing and diagnostics of HV equipment to ensure a reliable generation, transmission and distribution of electrical energy. The book is intended not only for experts but also for students in electrical engineering and high-voltage engineering.

  9. High-stakes educational testing and democracy

    DEFF Research Database (Denmark)

    Ydesen, Christian

    2014-01-01

    the field of education. Then a sample of relevant historic case studies are examined in light of these definitions. Among other things, the article concludes that a combination of different evaluation technologies – some formative and some summative – might be the safest way to go from a democratic......This article investigates the relation between high-stakes educational testing and democracy drawn from the experiences of 20th-century high-stakes educational testing practices in the Danish history of education. The article presents various concepts of democracy using leading propositions within...

  10. Multi-gene panel testing for hereditary cancer predisposition in unsolved high-risk breast and ovarian cancer patients.

    Science.gov (United States)

    Crawford, Beth; Adams, Sophie B; Sittler, Taylor; van den Akker, Jeroen; Chan, Salina; Leitner, Ofri; Ryan, Lauren; Gil, Elad; van 't Veer, Laura

    2017-06-01

    Many women with an elevated risk of hereditary breast and ovarian cancer have previously tested negative for pathogenic mutations in BRCA1 and BRCA2. Among them, a subset has hereditary susceptibility to cancer and requires further testing. We sought to identify specific groups who remain at high risk and evaluate whether they should be offered multi-gene panel testing. We tested 300 women on a multi-gene panel who were previously enrolled in a long-term study at UCSF. As part of their long-term care, all previously tested negative for mutations in BRCA1 and BRCA2 either by limited or comprehensive sequencing. Additionally, they met one of the following criteria: (i) personal history of bilateral breast cancer, (ii) personal history of breast cancer and a first or second degree relative with ovarian cancer, and (iii) personal history of ovarian, fallopian tube, or peritoneal carcinoma. Across the three groups, 26 women (9%) had a total of 28 pathogenic mutations associated with hereditary cancer susceptibility, and 23 women (8%) had mutations in genes other than BRCA1 and BRCA2. Ashkenazi Jewish and Hispanic women had elevated pathogenic mutation rates. In addition, two women harbored pathogenic mutations in more than one hereditary predisposition gene. Among women at high risk of breast and ovarian cancer who have previously tested negative for pathogenic BRCA1 and BRCA2 mutations, we identified three groups of women who should be considered for subsequent multi-gene panel testing. The identification of women with multiple pathogenic mutations has important implications for family testing.

  11. Isolation of chromosomal mutations in Drosophila melanogaster by the nondisjunction test

    Energy Technology Data Exchange (ETDEWEB)

    Chadov, B.F.; Ivanov, Yu.N.; Artemova, E.V.

    1986-01-01

    The females carrying the combination of metacentric autosome 2 with two acrocentrics, F(2L) and F(2R), produce aneuploid egg cells 2/F(2L) and F(2R)(2-F(2L) nondisjunction) as well as egg cells 2/F(2R) and F(2L) (2-F(2R) nondisjunction). The frequency of nondisjunction 2-F(2L) sharply increases in the presence of inversion in the left arm of metacentric 2, and the frequency of nondisjunction 2-F(2R) increases when the right arm of the metacentric carries the inversion. The presence of the rearrangement in the left arm can be detected by the unusually high progeny size in the cross between 2/F(2L);F(2R) females, and C(2L);F(2R)/F(2R) males, and the presence of rearrangement in the right arm of the metacentric is inferred from a large progeny in the cross of similar females with F(2L)/F(2L);C(2R) males. The SPx/sup 2//F(2L);F(2R) daughters were obtained from SPx/sup 2//+ males irradiated with the dose of 3000R. Out of these, 972 daughters were individually crossed with F(2L)/F(2L); C(2R) males. Fifty-two cultures with large progeny size were identified by visual observation. Preparations of polytene chromsomes were made from the larvae. Twenty-six cultures had arrangements in 2 R: eight carried rearrangement In (2R), six In(2LR), ten T(2R; 3) and T(2R; X), and two Tp (2). The method can be used effective to detect chromosomal mutations, especially inversions in large samples.

  12. CSF3R T618I is a highly prevalent and specific mutation in chronic neutrophilic leukemia.

    Science.gov (United States)

    Pardanani, A; Lasho, T L; Laborde, R R; Elliott, M; Hanson, C A; Knudson, R A; Ketterling, R P; Maxson, J E; Tyner, J W; Tefferi, A

    2013-09-01

    Truncation mutations of the receptor cytoplasmic domain for colony-stimulating factor 3 (CSF3R) are frequently seen in severe congenital neutropenia, whereas activating missense mutations affecting the extracellular domain (exon 14) have been described in hereditary neutrophilia and chronic neutrophilic leukemia (CNL). In order to clarify mutational frequency, specificity and phenotypic associations, we sequenced CSF3R exons 14-17 in 54 clinically suspected cases of CNL (n=35) or atypical chronic myeloid leukemia (aCML; n=19). Central review of these cases confirmed WHO-defined CNL in 12 patients, monoclonal gammopathy (MG)-associated CNL in 5 and WHO-defined aCML in 9. A total of 14 CSF3R mutations were detected in 13 patients, including 10 with CSF3RT618I (exon 14 mutation, sometimes annotated as CSF3R T595I). CSF3RT618I occurred exclusively in WHO-defined CNL with a mutational frequency of 83% (10 of 12 cases). CSF3R mutations were not seen in aCML or MG-associated CNL. CSF3RT618I was also absent among 170 patients with primary myelofibrosis (PMF; n=76) or chronic myelomonocytic leukemia (CMML; n=94). SETBP1 mutational frequencies in WHO-defined CNL, aCML, CMML and PMF were 33, 0, 7 and 3%, respectively. Four CSF3RT618I-mutated cases co-expressed SETBP1 mutations. We conclude that CSF3RT618I is a highly sensitive and specific molecular marker for CNL and should be incorporated into current diagnostic criteria.

  13. Mutational analysis of 33 autosomal short tandem repeat (STR) loci in southwest Chinese Han population based on trio parentage testing.

    Science.gov (United States)

    Jin, Bo; Su, Qin; Luo, Haibo; Li, Yingbi; Wu, Jin; Yan, Jing; Hou, Yiping; Liang, Weibo; Zhang, Lin

    2016-07-01

    Mutation rates and 95% CI of 33 short tandem repeat (STR) loci (D1S2142, D2S1338, D2S441, D3S1358, D3S1754, D5S818, D6S1043, D7S3048, D7S820, D8S1132, D8S1179, D10S1248, D11S2368, D12S391, D13S1492, D13S317, D13S325, D14S306, D15S659, D16S539, D18S1364, D18S51, D19S433, D20S161, D21S11, D22GATA198B05, CSF1PO, FGA, Penta D, Penta E, TH01, TPOX, and vWA) were investigated through more than 424,000 parent-child meiotic transfers obtained from 10636 trios parentage testing cases in southwest Chinese Han population. Overall, 297, including 292 single-step, 4 double-step and 1 triple-step mutation events were observed. The average mutation rate was 0.70×10(-3). Most of the locus-specific mutation rates (varied from 0.20×10(-3) to 1.96×10(-3)) were lower than the other datasets (pstrict inclusion criteria of large-sized parents/child-trio cases. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  14. Comprehensive molecular diagnosis of 67 Chinese Usher syndrome probands: high rate of ethnicity specific mutations in Chinese USH patients.

    Science.gov (United States)

    Jiang, Lichun; Liang, Xiaofang; Li, Yumei; Wang, Jing; Zaneveld, Jacques Eric; Wang, Hui; Xu, Shan; Wang, Keqing; Wang, Binbin; Chen, Rui; Sui, Ruifang

    2015-09-04

    Usher syndrome (USH) is the most common disease causing combined deafness and blindness. It is predominantly an autosomal recessive genetic disorder with occasionally digenic cases. Molecular diagnosis of USH patients is important for disease management. Few studies have tried to find the genetic cause of USH in Chinese patients. This study was designed to determine the mutation spectrum of Chinese USH patients. We applied next generation sequencing to characterize the mutation spectrum in 67 independent Chinese families with at least one member diagnosed with USH. Blood was collected at Peking Union Medical College Hospital. This cohort is one of the largest USH cohorts reported. We utilized customized panel and whole exome sequencing, variant analysis, Sanger validation and segregation tests to find disease causing mutations in these families. We identified biallelic disease causing mutations in known USH genes in 70 % (49) of our patients. As has been previously reported, MYO7A is the most frequently mutated gene in our USH type I patients while USH2A is the most mutated gene in our USH type II patients. In addition, we identify mutations in CLRN1, DFNB31, GPR98 and PCDH15 for the first time in Chinese USH patients. Together, mutations in CLRN1, DNFB31, GPR98 and PCDH15 account for 11.4 % of disease in our cohort. Interestingly, although the spectrum of disease genes is quite similar between our Chinese patient cohort and other patient cohorts from different (and primarily Caucasian) ethnic backgrounds, the mutations themselves are dramatically different. In particular, 76 % (52/68) of alleles found in this study have never been previously reported. Interestingly, we observed a strong enrichment for severe protein truncating mutations expected to have severe functional consequence on the protein in USH II patients compared to the reported mutation spectrum in RP patients, who often carry partial protein truncating mutations. Our study provides the first

  15. Rapid and inexpensive detection of common HBB gene mutations in Tunisian population by high-resolution melting analysis: implication for molecular diagnosis.

    Science.gov (United States)

    Ouragini, Houyem; Haddad, Faten; Darragi, Imen; Abbes, Salem

    2014-03-01

    In Tunisia, β-thalassemia is a common hereditary disease with a carrying rate of 2.21%. Up to now, detection of responsible mutations was made by laborious, expensive, and/or time consuming methods. The aim of this study is to develop and validate a specific assay for detection of the two most frequent mutations in Tunisian population, the IVS-I-110 (G → A) and Cd39 (C → T) mutations. In this study, we optimize high resolution melting analysis (HRMA) conditions for these mutations, using control DNAs. Then, we evaluate the strength of this methodology by screening a cohort of patients with β-thalassemia. All examined reference DNA samples were unambiguously distinguished from each other. For the blinded test, the results were completely compatible with direct sequencing, performed after the HRMA. As HRMA represents a highly sensitive and high-throughput gene scanning method, it can provide timely diagnosis at low cost for effective clinical management of β-thalassemia.

  16. (Over and) beyond High-Stakes Testing

    Science.gov (United States)

    Duckworth, Angela L.

    2009-01-01

    Sackett, Borneman, and Connelly's article and recent meta-analyses (e.g., Kuncel & Hezlett, 2007) should lay to rest any doubt over whether high-stakes standardized tests predict important academic and professional outcomes--they do. The challenge now is to identify noncognitive individual differences that determine the same outcomes. Noncognitive…

  17. Value of TIRADS, BSRTC and FNA-BRAF V600E mutation analysis in differentiating high-risk thyroid nodules.

    Science.gov (United States)

    Zhang, Yu-zhi; Xu, Ting; Cui, Dai; Li, Xiao; Yao, Qing; Gong, Hai-yan; Liu, Xiao-yun; Chen, Huan-huan; Jiang, Lin; Ye, Xin-hua; Zhang, Zhi-hong; Shen, Mei-ping; Duan, Yu; Yang, Tao; Wu, Xiao-hong

    2015-11-24

    The thyroid imaging reporting and data system (TIRADS) and Bethesda system for reporting thyroid cytopathology (BSRTC) have been used for interpretation of ultrasound and fine-needle aspiration cytology (FNAC) results of thyroid nodules. BRAF(V600E) mutation analysis is a molecular tool in diagnosing thyroid carcinoma. Our objective was to compare the diagnostic value of these methods in differentiating high-risk thyroid nodules. Total 220 patients with high-risk thyroid nodules were recruited in this prospective study. They all underwent ultrasound, FNAC and BRAF(V600E) mutation analysis. The sensitivity and specificity of TIRADS were 73.1% and 88.4%. BSRTC had higher specificity (97.7%) and similar sensitivity (77.6%) compared with TIRADS. The sensitivity and specificity of BRAF(V600E) mutation (85.1%, 100%) were the highest. The combination of BSRTC and BRAF(V600E) mutation analysis significantly increased the efficiency, with 97.8% sensitivity, 97.7% specificity. In patients with BSRTC I-III, the mutation rate of BRAF(V600E) was 64.5% in nodules with TIRADS 4B compared with 8.4% in nodules with TIRADS 3 or 4A (P value in differentiating high-risk thyroid nodules. The TIRADS is useful in selecting high-risk patients for FNAB and patients with BSRTC I-III for BRAF(V600E) mutation analysis.

  18. Mutate Chlorella sp. by nuclear irradiation to fix high concentrations of CO2.

    Science.gov (United States)

    Cheng, Jun; Huang, Yun; Feng, Jia; Sun, Jing; Zhou, Junhu; Cen, Kefa

    2013-05-01

    To improve biomass productivity and CO2 fixation of microalgae under 15% (v/v) CO2 of flue gas, Chlorella species were mutated by nuclear irradiation and domesticated with high concentrations of CO2. The biomass yield of Chlorella pyrenoidosa mutated using 500 Gy of (60)Co γ irradiation increased by 53.1% (to 1.12 g L(-1)) under air bubbling. The mutants were domesticated with gradually increased high concentrations of CO2 [from 0.038% (v/v) to 15% (v/v)], which increased the biomass yield to 2.41 g L(-1). When light transmission and culture mixing in photo-bioreactors were enhanced at 15% (v/v) CO2, the peak growth rate of the domesticated mutant (named Chlorella PY-ZU1) was increased to 0.68 g L(-1) d(-1). When the ratio of gas flow rate (L min(-1)) to 1L of microalgae culture was 0.011, the peak CO2 fixation rate and the efficiency of Chlorella PY-ZU1 were 1.54 g L(-1) d(-1) and 32.7%, respectively. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Identifying Highly Penetrant Disease Causal Mutations Using Next Generation Sequencing: Guide to Whole Process

    Directory of Open Access Journals (Sweden)

    A. Mesut Erzurumluoglu

    2015-01-01

    Full Text Available Recent technological advances have created challenges for geneticists and a need to adapt to a wide range of new bioinformatics tools and an expanding wealth of publicly available data (e.g., mutation databases, and software. This wide range of methods and a diversity of file formats used in sequence analysis is a significant issue, with a considerable amount of time spent before anyone can even attempt to analyse the genetic basis of human disorders. Another point to consider that is although many possess “just enough” knowledge to analyse their data, they do not make full use of the tools and databases that are available and also do not fully understand how their data was created. The primary aim of this review is to document some of the key approaches and provide an analysis schema to make the analysis process more efficient and reliable in the context of discovering highly penetrant causal mutations/genes. This review will also compare the methods used to identify highly penetrant variants when data is obtained from consanguineous individuals as opposed to nonconsanguineous; and when Mendelian disorders are analysed as opposed to common-complex disorders.

  20. Mutation analysis of SLC26A4 for Pendred syndrome and nonsyndromic hearing loss by high-resolution melting

    DEFF Research Database (Denmark)

    Chen, Neng; Tranebjærg, Lisbeth; Rendtorff, Nanna Dahl

    2011-01-01

    to identify mutations for individual patients. Although Sanger sequencing is the gold standard for mutation detection, screening methods supplemented with targeted sequencing can provide a cost-effective alternative. One such method, denaturing high-performance liquid chromatography, was developed...... for clinical mutation detection in SLC26A4. However, this method inherently cannot distinguish homozygous changes from wild-type sequences. High-resolution melting (HRM), on the other hand, can detect heterozygous and homozygous changes cost-effectively, without any post-PCR modifications. We developed...... a closed-tube HRM mutation detection method specific for SLC26A4 that can be used in the clinical diagnostic setting. Twenty-eight primer pairs were designed to cover all 21 SLC26A4 exons and splice junction sequences. Using the resulting amplicons, initial HRM analysis detected all 45 variants previously...

  1. High prevalence of leptin and melanocortin-4 receptor gene mutations in children with severe obesity from Pakistani consanguineous families.

    Science.gov (United States)

    Saeed, Sadia; Butt, Taeed A; Anwer, Mehwish; Arslan, Muhammad; Froguel, Philippe

    2012-05-01

    Recessive or co-dominant single-gene mutations disrupting leptin melanocortin pathway cause severe obesity and hyperphagia. Since Pakistan has a very high rate of consanguinity, therefore, a significantly higher incidence of monogenic obesity is expected in its population. We have assessed the incidence of LEP and MC4R mutations and associated hormonal profiles, in a cohort of randomly selected Pakistani children with early onset of severe obesity. Sixty two unrelated children of consanguineous parents, with a weight-for-age percentile >97 were recruited in the study. Screening for mutations in the coding regions of LEP and MC4R was performed by direct sequencing. Serum hormone concentrations were determined by immunoassay. LEP mutations were found in 16.1% of the probands. Of these, 9 probands carried the homozygous frameshift mutation, G133_VfsX14, whereas one patient had a homozygous mutation involving deletion of 3 base pairs, (I35del). In these probands, leptin levels were very low or undetectable and insulin levels were increased in 33%. Homozygous MC4R mutations, M161T and I316S, identified separately in 2 subjects (3.2%), were associated with severe obesity, hyperphagia, hyperleptinemia and hyperinsulinemia. The heterozygous M161T sibling had normal body weight and hormone levels and the parents were only mildly overweight. Based on genetic analysis of LEP and MC4R genes only, we elucidated genetic causality of severe obesity in 20% of our patients confirming high prevalence of monogenic form of obesity in this consanguineous population. Co-dominancy of MC4R is exacerbated in this group with non-penetrance of obesity in heterozygous loss-of-function MC4R mutation carriers. The sub-ethnic specificity of LEP mutation, G133_VfsX14, suggests a founder effect. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. A Statistical Perspective on Highly Accelerated Testing

    Energy Technology Data Exchange (ETDEWEB)

    Thomas, Edward V. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)

    2015-02-01

    Highly accelerated life testing has been heavily promoted at Sandia (and elsewhere) as a means to rapidly identify product weaknesses caused by flaws in the product's design or manufacturing process. During product development, a small number of units are forced to fail at high stress. The failed units are then examined to determine the root causes of failure. The identification of the root causes of product failures exposed by highly accelerated life testing can instigate changes to the product's design and/or manufacturing process that result in a product with increased reliability. It is widely viewed that this qualitative use of highly accelerated life testing (often associated with the acronym HALT) can be useful. However, highly accelerated life testing has also been proposed as a quantitative means for "demonstrating" the reliability of a product where unreliability is associated with loss of margin via an identified and dominating failure mechanism. It is assumed that the dominant failure mechanism can be accelerated by changing the level of a stress factor that is assumed to be related to the dominant failure mode. In extreme cases, a minimal number of units (often from a pre-production lot) are subjected to a single highly accelerated stress relative to normal use. If no (or, sufficiently few) units fail at this high stress level, some might claim that a certain level of reliability has been demonstrated (relative to normal use conditions). Underlying this claim are assumptions regarding the level of knowledge associated with the relationship between the stress level and the probability of failure. The primary purpose of this document is to discuss (from a statistical perspective) the efficacy of using accelerated life testing protocols (and, in particular, "highly accelerated" protocols) to make quantitative inferences concerning the performance of a product (e.g., reliability) when in fact there is lack-of-knowledge and uncertainty concerning

  3. High resolution mapping and positional cloning of ENU-induced mutations in the Rw region of mouse chromosome 5

    Directory of Open Access Journals (Sweden)

    Schimenti Kerry J

    2010-11-01

    Full Text Available Abstract Background Forward genetic screens in mice provide an unbiased means to identify genes and other functional genetic elements in the genome. Previously, a large scale ENU mutagenesis screen was conducted to query the functional content of a ~50 Mb region of the mouse genome on proximal Chr 5. The majority of phenotypic mutants recovered were embryonic lethals. Results We report the high resolution genetic mapping, complementation analyses, and positional cloning of mutations in the target region. The collection of identified alleles include several with known or presumed functions for which no mutant models have been reported (Tbc1d14, Nol14, Tyms, Cad, Fbxl5, Haus3, and mutations in genes we or others previously reported (Tapt1, Rest, Ugdh, Paxip1, Hmx1, Otoe, Nsun7. We also confirmed the causative nature of a homeotic mutation with a targeted allele, mapped a lethal mutation to a large gene desert, and localized a spermiogenesis mutation to a region in which no annotated genes have coding mutations. The mutation in Tbc1d14 provides the first implication of a critical developmental role for RAB-GAP-mediated protein transport in early embryogenesis. Conclusion This collection of alleles contributes to the goal of assigning biological functions to all known genes, as well as identifying novel functional elements that would be missed by reverse genetic approaches.

  4. Auxotrophic Mutations Reduce Tolerance of Saccharomyces cerevisiae to Very High Levels of Ethanol Stress

    Science.gov (United States)

    Swinnen, Steve; Goovaerts, Annelies; Schaerlaekens, Kristien; Dumortier, Françoise; Verdyck, Pieter; Souvereyns, Kris; Van Zeebroeck, Griet; Foulquié-Moreno, María R.

    2015-01-01

    Very high ethanol tolerance is a distinctive trait of the yeast Saccharomyces cerevisiae with notable ecological and industrial importance. Although many genes have been shown to be required for moderate ethanol tolerance (i.e., 6 to 12%) in laboratory strains, little is known of the much higher ethanol tolerance (i.e., 16 to 20%) in natural and industrial strains. We have analyzed the genetic basis of very high ethanol tolerance in a Brazilian bioethanol production strain by genetic mapping with laboratory strains containing artificially inserted oligonucleotide markers. The first locus contained the ura3Δ0 mutation of the laboratory strain as the causative mutation. Analysis of other auxotrophies also revealed significant linkage for LYS2, LEU2, HIS3, and MET15. Tolerance to only very high ethanol concentrations was reduced by auxotrophies, while the effect was reversed at lower concentrations. Evaluation of other stress conditions showed that the link with auxotrophy is dependent on the type of stress and the type of auxotrophy. When the concentration of the auxotrophic nutrient is close to that limiting growth, more stress factors can inhibit growth of an auxotrophic strain. We show that very high ethanol concentrations inhibit the uptake of leucine more than that of uracil, but the 500-fold-lower uracil uptake activity may explain the strong linkage between uracil auxotrophy and ethanol sensitivity compared to leucine auxotrophy. Since very high concentrations of ethanol inhibit the uptake of auxotrophic nutrients, the active uptake of scarce nutrients may be a major limiting factor for growth under conditions of ethanol stress. PMID:26116212

  5. Baseline mutational patterns and sustained MRD negativity in patients with high-risk smoldering myeloma.

    Science.gov (United States)

    Mailankody, Sham; Kazandjian, Dickran; Korde, Neha; Roschewski, Mark; Manasanch, Elisabet; Bhutani, Manisha; Tageja, Nishant; Kwok, Mary; Zhang, Yong; Zingone, Adriana; Lamy, Laurence; Costello, Rene; Morrison, Candis; Hultcrantz, Malin; Christofferson, Austin; Washington, Megan; Boateng, Martin; Steinberg, Seth M; Stetler-Stevenson, Maryalice; Figg, William D; Papaemmanuil, Elli; Wilson, Wyndham H; Keats, Jonathan J; Landgren, Ola

    2017-10-10

    Early results of a prospective phase 2 clinical trial of carfilzomib, lenalidomide, and dexamethasone followed by lenalidomide maintenance in high-risk smoldering myeloma showed promising results that were previously published. Here, we provide novel insights into the genetic landscape of high-risk smoldering myeloma and information on sustained minimal residual disease (MRD) negativity with an expanded cohort of patients. Eighteen patients with high-risk smoldering myeloma were enrolled between 29 May 2012, and 14 January 2014. We included patients with newly diagnosed multiple myeloma enrolled in a parallel trial who received the same therapy (reference group). The overall response rate was 100%. With median potential follow-up of 43.3 months, 10 (63%) remain in MRD negativity, and the estimated 4-year progression-free and overall survival rates are 71% and 100%, respectively. Importantly, we report differences in mutational patterns in patients with high-risk smoldering myeloma and newly diagnosed multiple myeloma, reflected in a lower frequency of mutations in significant myeloma genes (6.6% vs 45%) and NFKB pathway genes (6.6% vs 25%). Treatment with carfilzomib, lenalidomide, and dexamethasone followed by lenalidomide maintenance was associated with a 100% response rate and 63% MRD negativity with a safety profile consistent with previous reports for this regimen. This study had a small numbers of participants, but there seemed to be important differences in the genetic landscape of patients with high-risk smoldering myeloma and those with newly diagnosed multiple myeloma, suggestive of a more treatment-responsive biology in early disease.

  6. Purging deleterious mutations under self fertilization: paradoxical recovery in fitness with increasing mutation rate in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Levi T Morran

    Full Text Available BACKGROUND: The accumulation of deleterious mutations can drastically reduce population mean fitness. Self-fertilization is thought to be an effective means of purging deleterious mutations. However, widespread linkage disequilibrium generated and maintained by self-fertilization is predicted to reduce the efficacy of purging when mutations are present at multiple loci. METHODOLOGY/PRINCIPAL FINDINGS: We tested the ability of self-fertilizing populations to purge deleterious mutations at multiple loci by exposing obligately self-fertilizing populations of Caenorhabditis elegans to a range of elevated mutation rates and found that mutations accumulated, as evidenced by a reduction in mean fitness, in each population. Therefore, purging in obligate selfing populations is overwhelmed by an increase in mutation rate. Surprisingly, we also found that obligate and predominantly self-fertilizing populations exposed to very high mutation rates exhibited consistently greater fitness than those subject to lesser increases in mutation rate, which contradicts the assumption that increases in mutation rate are negatively correlated with fitness. The high levels of genetic linkage inherent in self-fertilization could drive this fitness increase. CONCLUSIONS: Compensatory mutations can be more frequent under high mutation rates and may alleviate a portion of the fitness lost due to the accumulation of deleterious mutations through epistatic interactions with deleterious mutations. The prolonged maintenance of tightly linked compensatory and deleterious mutations facilitated by self-fertilization may be responsible for the fitness increase as linkage disequilibrium between the compensatory and deleterious mutations preserves their epistatic interaction.

  7. The use of induced mutation combined with crossing in high quality rice breeding

    Energy Technology Data Exchange (ETDEWEB)

    Do Huu At; Bui Huy Thuy; Nguyen Van Bich; Tran Duy Quy [Agricultural Genetics Institute, Division of Genetics and Hybrid Rice Technology, Hanoi (Viet Nam); Nguyen Minh Cong [Hanoi No. 1 Teacher Training Univ., Department of Genetics (Viet Nam)

    2001-03-01

    The high quality rice varieties: Tam thom mutant rice Var., DT17 rice Var, DT21 glutinous rice Var were formed by induced mutation combined with crossing. Tam thom mutant rice Var. lost photosensitivity, could be planted 2 crops/year. DT17 rice Var with high yielding capacity, suitable for growth on lowland in summer crop, is replacing step-by-step Moctuyen rice Var. in North Vietnam. DT21 glutinous rice Var. could be planted 2 crops/year and had short growth duration, average yield was 4.0-4.5 tons/ha. These three ones had good quality, soft and scent cooked rice, suitable for customers and export requirements. Tam thom mutant rice Var. DT17 rice Var., DT21 and glutinous rice Var. were adopted for regional production by Ministry of Agriculture and Rural Development and allowed to be in trial production. (author)

  8. Analysis of KRAS and NRAS Gene Mutations in Arab Asian Children With Acute Leukemia: High Frequency of RAS Mutations in Acute Lymphoblastic Leukemia.

    Science.gov (United States)

    Al-Kzayer, Lika'a Fasih Y; Sakashita, Kazuo; Al-Jadiry, Mazin Faisal; Al-Hadad, Salma Abbas; Ghali, Hasanein Habeeb; Uyen, Le T N; Liu, Tingting; Matsuda, Kazuyuki; Abdulkadhim, Jaafar M H; Al-Shujairi, Tariq Abadi; Matti, Zead Ismael I K; Sughayer, Maher A; Rihani, Rawad; Madanat, Faris F; Inoshita, Toshi; Kamata, Minoru; Koike, Kenichi

    2015-12-01

    KRAS and NRAS gene mutations are frequently observed in childhood leukemia. The objective of this study was to determine the frequency of RAS mutations and the association between RAS mutations and other genetic aberrations in Arab Asian children with acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML). Diagnostic samples of 485 patients (RAS mutations were detected in 86/318 (27%) of ALL cases and 35/167 (21%) of AML cases. The frequency of NRAS mutation was similar to that of KRAS mutation in ALL. Two RAS mutations were detected in nine patients. Among 264 Iraqi patients with ALL, RAS mutation was significantly associated with lower initial white blood cell count. Of 57 patients with chimeric transcripts, only two patients with either TEL-AML1 or E2A-PBX1 had KRAS mutation. The frequency of NRAS mutation was four times higher than that of KRAS mutation in AML. FAB-M4 and M5 subsets were associated with RAS mutation. Among 134 Iraqi patients with AML, 18 patients had RAS mutations and other genetic aberrations. In particular, 9 of 25 (36%) with MLL-rearrangement had RAS mutations. The prevalence of oncogenic RAS mutations was higher among Arab Asian children than in other countries. RAS mutations in AML were found to coexist with other genetic aberrations, particularly MLL rearrangement. © 2015 Wiley Periodicals, Inc.

  9. High temperature and pressure electrochemical test station

    DEFF Research Database (Denmark)

    Chatzichristodoulou, Christodoulos; Allebrod, Frank; Mogensen, Mogens Bjerg

    2013-01-01

    An electrochemical test station capable of operating at pressures up to 100 bars and temperatures up to 400 ◦C has been established. It enables control of the partial pressures and mass flow of O2, N2, H2, CO2, and H2O in a single or dual environment arrangement, measurements with highly corrosive......, to the electrochemical characterization of high temperature and pressure alkaline electrolysis cells and the use of pseudo-reference electrodes for the separation of each electrode contribution. A future perspective of various electrochemical processes and devices that can be developed with the use of the established...

  10. Comparative study of IDH1 mutations in gliomas by high resolution melting analysis, immunohistochemistry and direct DNA sequencing.

    Science.gov (United States)

    Li, Juan; Zhang, Haiyan; Wang, Li; Yang, Chuanhong; Lai, Huangwen; Zhang, Wei; Chen, Xiaodong; Wang, Jie

    2015-09-01

    Patients with glioblastomas with a specific mutation in the isocitrate dehydrogenase 1 (IDH1) gene have a better prognosis than those with gliomas with wild‑type IDH1. IDH1 analysis has become part of the standard diagnostic procedure and a promising tool used for stratification in clinical trials. The present study aimed to compare high resolution melting (HRM) analysis, immunohistochemistry (IHC) and direct DNA sequencing for the detection of IDH mutations in gliomas. Fifty‑one formalin‑fixed paraffin‑embedded tumor samples were selected. For the HRM analysis and direct DNA sequencing, DNA was extracted from the tissues. For IHC, sections were stained with an anti‑IDH1‑R132H specific antibody. The HRM analysis method identified 33 cases of IDH1 gene mutations, and all mutations occurred at the R132H site. There were 33 cases of IDH1 gene mutations found by IHC, which was consistent with that identified using the HRM analysis method. However, only 30 IDH1 samples were confirmed by sequencing, in which mutations occurred at the IDH1 exon 4 R132H site. No mutation was detected in the other three of these 33 cases (two grade II oligodendroglioma and one grade II diffuse astrocytoma) by sequencing, while IHC was positive for IDH1‑R132H. The results showed that the mutation detection rate was not identified to be significantly different (P=0.250) when determined by the HRM analysis method or by direct DNA sequencing, as the concordant rate between the two methods was high (κ=0.866). The HRM analysis method in glioma IDH1 gene mutation detection has advantages of high sensitivity, good repeatability, simple operation and accurate results. It provides a novel method for detecting mutations of the IDH1 gene in paraffin embedded tissue samples of clinical glioma. Related to a small amount of sample, there was no evidence showing that HRM analysis method is superior to IHC. Direct DNA sequencing, HRM analysis and IHC results were consistent; however, HRM and

  11. Heterogeneous Phenotype of Long QT Syndrome Caused by the KCNH2-H562R Mutation: Importance of Familial Genetic Testing.

    Science.gov (United States)

    Muñoz-Esparza, Carmen; García-Molina, Esperanza; Salar-Alcaraz, Mariela; Peñafiel-Verdú, Pablo; Sánchez-Muñoz, Juan J; Martínez Sánchez, Juan; Cabañas-Perianes, Valentín; Valdés Chávarri, Mariano; García Alberola, Arcadio; Gimeno-Blanes, Juan R

    2015-10-01

    Long QT syndrome is an inherited ion channelopathy that leads to syncope and sudden death. Because of the heterogeneous phenotype of this disease, genetic testing is fundamental to detect individuals with concealed long QT syndrome. In this study, we determined the features of a family with 13 carriers of the KCNH2-H562R missense mutation, which affects the pore region of the HERG channel. We identified the KCNH2-H562R mutation in a 65-year-old man with a prolonged QTc interval who had experienced an episode of torsade de pointes. Subsequently, a total of 13 mutation carriers were identified in the family. Carriers (age 48 [26] years; 46% males) underwent clinical evaluation, electrocardiography and echocardiography. The mean (standard deviation) QTc in carriers was 493 (42) ms (3 [23%] showed normal QTc); 6 (46%) had symptoms (4, syncope; 1, sudden death; 1, aborted sudden death [proband]). While under treatment with beta-blockers, 11 of 12 carriers (92%) remained asymptomatic at 5 years of follow-up (1 patient required left cardiac sympathectomy). The QTc shortening with beta-blockers was 50 (37) ms. There was 1 sudden death in a patient who refused treatment. Family study is essential in the interpretation of a genetic testing result. This article describes the heterogeneous and variable phenotype of a large family with the KCNH2-H562R mutation and highlights the role of genetic study for the appropriate identification of at-risk individuals who would benefit from treatment. Copyright © 2014 Sociedad Española de Cardiología. Published by Elsevier España, S.L.U. All rights reserved.

  12. Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells. II. Test validation and interpretation.

    Science.gov (United States)

    Amacher, D E; Paillet, S C; Turner, G N; Ray, V A; Salsburg, D S

    1980-08-01

    The L5178Y Mouse Lymphoma TK assay was studied extensively to determine if this mammalian cell assay for gene mutations at the thymidine kinase (TK) locus could provide valid, interpretable determinations of mutagenic potential, and whether this information is of value in the safety evaluation of chemicals. We first determined that test-derived TFTR mutants were phenotypically stable, possessing little or no thymidine kinase activity as measured by labeled thymidine uptake, but demonstrating 100% cross resistance to bromodeoxyuridine. Common solvent vehicles such as acetone, dimethylsulfoxide and ethanol were shown to produce little cytotoxicity and no mutagenic activity when present at 1% levels. Out of a total of 10 noncarcinogens tested, all were negative when results were analyzed by a 2-sample loge t test on control and treated mutant count means. Of the 13 putative animal carcinogens tested, 10 were positive, 2 were negative (auramine O and sodium phenobarbital), and 1 showed sporadic activity (hydrazine sulfate) in the TK assay on the basis of test-derived t statistics. 2 compounds, 1,2-epoxybutane and ICR 191, which have been described as Ames positive non-carcinogens, were also positive in the TK assay. Although this sampling of a total of 29 compounds is insufficient for precise estimations of expected false-positive or false-negative frequencies, these data indicate the TK assay can be expected to detect a majority of carcinogens as mutagens including some missed by more established point-mutation assays.

  13. TP53 germline mutation testing in 180 families suspected of Li-Fraumeni syndrome: mutation detection rate and relative frequency of cancers in different familial phenotypes.

    NARCIS (Netherlands)

    Ruijs, M.W.; Verhoef, S.; Rookus, M.A.; Pruntel, R.; Hout, A.H. van der; Hogervorst, F.B.L.; Kluijt, I.; Sijmons, R.H.; Aalfs, C.M.; Wagner, A.; Ausems, M.G.E.M.; Hoogerbrugge-van der Linden, N.; Asperen, C.J. van; Gomez Garcia, E.B.; Meijers-Heijboer, H.; Kate, L.P. Ten; Menko, F.H.; Veer, L.J. van 't

    2010-01-01

    BACKGROUND Li-Fraumeni syndrome (LFS) is a rare autosomal dominant cancer predisposition syndrome. Most families fulfilling the classical diagnostic criteria harbour TP53 germline mutations. However, TP53 germline mutations may also occur in less obvious phenotypes. As a result, different criteria

  14. Mosaic and Intronic Mutations in TSC1/TSC2 Explain the Majority of TSC Patients with No Mutation Identified by Conventional Testing.

    Science.gov (United States)

    Tyburczy, Magdalena E; Dies, Kira A; Glass, Jennifer; Camposano, Susana; Chekaluk, Yvonne; Thorner, Aaron R; Lin, Ling; Krueger, Darcy; Franz, David N; Thiele, Elizabeth A; Sahin, Mustafa; Kwiatkowski, David J

    2015-11-01

    Tuberous sclerosis complex (TSC) is an autosomal dominant tumor suppressor gene syndrome due to germline mutations in either TSC1 or TSC2. 10-15% of TSC individuals have no mutation identified (NMI) after thorough conventional molecular diagnostic assessment. 53 TSC subjects who were NMI were studied using next generation sequencing to search for mutations in these genes. Blood/saliva DNA including parental samples were available from all subjects, and skin tumor biopsy DNA was available from six subjects. We identified mutations in 45 of 53 subjects (85%). Mosaicism was observed in the majority (26 of 45, 58%), and intronic mutations were also unusually common, seen in 18 of 45 subjects (40%). Seventeen (38%) mutations were seen at an allele frequency mutation detection, show that analysis of TSC-related tumors can increase the mutation detection rate, indicate that it is not likely that a third TSC gene exists, and enable provision of genetic counseling to the substantial population of TSC individuals who are currently NMI.

  15. A novel loss-of-function mutation in GPR54/KISS1R leads to hypogonadotropic hypogonadism in a highly consanguineous family.

    Science.gov (United States)

    Nimri, Revital; Lebenthal, Yael; Lazar, Liora; Chevrier, Lucie; Phillip, Moshe; Bar, Meytal; Hernandez-Mora, Eva; de Roux, Nicolas; Gat-Yablonski, Galia

    2011-03-01

    The G protein-coupled receptor 54 (GPR54), the kisspeptin receptor, is essential for stimulation of GnRH secretion and induction of puberty. Recently loss-of-function mutations of the GPR54 have been implicated as a cause of isolated idiopathic hypogonadotropic hypogonadism (IHH). The objective of the study was to identify the genetic cause of IHH in a consanguineous pedigree and to characterize the phenotypic features from infancy through early adulthood. In six patients with normosmic IHH belonging to two families of Israeli Muslim-Arab origin highly related to one another, DNA was analyzed for mutations in the GnRHR and GPR54 genes, with functional analysis of the mutation found. The five males underwent comprehensive endocrine evaluation and were under longitudinal follow-up; the one female presented in early adulthood. A new homozygous mutation (c.T815C) in GPR54 leading to a phenylalanine substitution by serine (p.F272S) was detected in all patients. Functional analysis showed an almost complete inhibition of kisspeptin-induced GPR54 signaling and a dramatic decrease of the mutated receptor expression at the cell surface. The males exhibited the same clinical features from infancy to adulthood, characterized by cryptorchidism, a relatively short penis, and no spontaneous pubertal development. The female patient presented at 18 yr with impuberism and primary amenorrhea. Repeated stimulation tests demonstrated complete gonadotropin deficiency throughout follow-up. A novel loss-of-function mutation (p.F272S) in the GPR54 gene is associated with familial normosmic IHH. Underdeveloped external genitalia and impuberism point to the major role of GPR54 in the activation of the gonadotropic axis from intrauterine life to adulthood.

  16. Sensitive High-Resolution Melting Analysis for Screening of KRAS and BRAF Mutations in Iranian Human Metastatic Colorectal Cancers

    Science.gov (United States)

    Niya, Mohammad Hadi Karbalaie; Basi, Ali; Koochak, Aghigh; Tameshkel, Fahimeh Safarnezhad; Rakhshani, Nasser; Zamani, Farhad; Imanzade, Farid; Rezvani, Hamid; sereshki, Mohammad Mahdi Adib; Sohrabi, Masoud Reza

    2016-01-01

    Background: Investigations of methods for detection of mutations have uncovered major weaknesses of direct sequencing and pyrosequencing, with their high costs and low sensitivity in screening for both known and unknown mutations. High resolution melting (HRM) analysis is an alternative tool for the rapid detection of mutations. Here we describe the accuracy of HRM in screening for KRAS and BRAF mutations in metastatic colorectal cancer (mCRCs) samples. Materials and Methods: A total of 1000 mCRC patients in Mehr Hospital, Tehran, Iran, from Feb 2008 to May 2012 were examined for KRAS mutations and 242 of them were selected for further assessment of BRAF mutations by HRM analysis. In order to calculate the sensitivity and specificity, HRM results were checked by pyrosequencing as the golden standard and Dxs Therascreen as a further method. Results: In the total of 1,000 participants, there were 664 (66.4%) with wild type and 336 (33.6%) with mutant codons 12 and/or 13 of the KRAS gene. Among 242 samples randomly checked for the BRAF gene, all were wild type by HRM. Pyrosequencing and Dxs Therascreen results were in line with those of the HRM. In this regard, the sensitivity and specificity of HRM were evaluated as 100%. Conclusion: The findings suggest that the HRM, in comparison with DNA sequencing, is a more appropriate method for precise scanning of KRAS and BRAF mutations. It is also possible to state that HRM may be an attractive technique for the detection of known or unknown somatic mutations in other genes. PMID:28122448

  17. Rate of EGFR mutation testing for patients with nonsquamous non-small-cell lung cancer with implementation of reflex testing by pathologists

    Science.gov (United States)

    Cheema, P.K.; Raphael, S.; El-Maraghi, R.; Li, J.; McClure, R.; Zibdawi, L.; Chan, A.; Victor, J.C.; Dolley, A.; Dziarmaga, A.

    2017-01-01

    Background Testing for mutation of the EGFR (epidermal growth factor receptor) gene is a standard of care for patients with advanced nonsquamous non-small-cell lung cancer (nsclc). To improve timely access to EGFR results, a few centres implemented reflex testing, defined as a request for EGFR testing by the pathologist at the time of a nonsquamous nsclc diagnosis. We evaluated the impact of reflex testing on EGFR testing rates. Methods A retrospective observational review of the Web-based AstraZeneca Canada EGFR Database from 1 April 2010 to 31 March 2014 found centres within Ontario that had requested EGFR testing through the database and that had implemented reflex testing (with at least 2 years’ worth of data, including the pre- and post-implementation period). Results The 7 included centres had requested EGFR tests for 2214 patients. The proportion of pathologists requesting EGFR tests increased after implementation of reflex testing (53% vs. 4%); conversely, the proportion of medical oncologists requesting tests decreased (46% vs. 95%, p testing, the mean number of patients having EGFR testing per centre per month increased significantly [12.6 vs. 4.9 (range: 4.5–14.9), p testing, EGFR testing rates showed a significant monthly increase over time (1.37 more tests per month; 95% confidence interval: 1.19 to 1.55 tests; p testing, because an immediate increase in EGFR test requests was observed with the introduction of reflex testing (p = 0.003), and the overall trend was sustained throughout the post–reflex testing period (p testing for patients with nonsquamous nsclc was successfully implemented at multiple centres and was associated with an increase in EGFR testing. PMID:28270720

  18. Diagnosis of ABCB11 gene mutations in children with intrahepatic cholestasis using high resolution melting analysis and direct sequencing.

    Science.gov (United States)

    Hu, Guorui; He, Ping; Liu, Zhifeng; Chen, Qian; Zheng, Bixia; Zhang, Qihua

    2014-09-01

    Intrahepatic cholestasis represents a heterogeneous group of disorders that begin during childhood, most commonly manifesting as neonatal cholestasis, and lead to ongoing liver dysfunction in children and adults. For children, inherited pathogenic factors of cholestasis have gained increasing attention owing to the rapid development of molecular biology technology. However, these methods have their advantages and disadvantages in terms of simplicity, sensitivity, specificity, time required and expense. In the present study, an effective, sensitive and economical method is recommended, termed high-resolution melting (HRM) analysis and direct sequencing, based on general polymerase chain reaction, to detect mutations in disease‑causing genes. As one type of inherited intrahepatic cholestasis, progressive familial intrahepatic cholestasis type 2 (PFIC2) is caused by pathogenic mutations in the ABCB11 gene, HRM was used to detect mutations in the ABCB11 gene in the present study, and the diagnosis for PFIC2 was made by comprehensive analysis of genetic findings and clinical features. Furthermore, the characteristics of mutations and single nucleotide polymorphisms (SNPs) in the ABCB11 gene were elucidated. A total of 14 types of mutations/polymorphisms were identified in 20 patients from mainland China, including six missense mutations (p.Y337H, p.Y472C, p.R696W, p.Q931P, p.D1131V and p.H1198R), one nonsense mutation (p.R928X) and seven SNPs (p.D36D/rs3815675, p.F90F/rs4148777, p.Y269Y/rs2287616, p.I416I/rs183390670, p.V444A/rs2287622, p.A865V/rs118109635 and p.A1028A/rs497692). Five mutations were novel. The majority of the mutations were different from those detected in other population groups. A total of 4/20 patients (1/5) were diagnosed to be PFIC2 by combining genetic findings with the clinical features. Polymorphisms V444A and A1028A, with an allele frequency of 74.5 and 67.2%, respectively, were highly prevalent in the mainland Chinese subjects. No

  19. Antimicrobial susceptibility testing of Escherichia coli strains isolated from urinary tract infections to fluoroquinolones and detection of gyrA mutations in resistant strains

    Directory of Open Access Journals (Sweden)

    Akbari-Nakhjavani F.

    2007-05-01

    Full Text Available Widespread uses of fluoroquinolones have resulted in increasing incidences of resistance against these agents all over the world. The aim of this study was to assess, susceptibility of Escherichia coli strains from patients with Urinary Tract Infection against common fluoroquinolones and detection of mutations in the gyrA gene. Antimicrobial susceptibility testing of 164 E.coli isolates from patients with UTI, was evaluated by disk agar diffusion (DAD and MIC methods. Polymerase chain reaction of E.coli strains were performed by amplification of Quinolone Resistance Determining Region (QRDR of gyrA gene. PCR products were tested by Conformational Sensitive Gel Electrophoresis (CSGE and those with hetrodublexes were selected and examined by DNA sequencing. According to disc agar diffusion, 49.3% were resistant to nalidixic acid, 41.4% to norfloxacin, 44.5% to ofloxacin and 40.2 % to ciprofloxacin. By Minimal Inhibitory Concentration (MIC testing a high-level of resistance (42.1% to ciprofloxacin was observed. Mutations in codons 83 and 87 in all 81 isolates were positive by CSGE method.

  20. High frequency of additional gene mutations in acute myeloid leukemia with MLL partial tandem duplication: DNMT3A mutation is associated with poor prognosis.

    Science.gov (United States)

    Kao, Hsiao-Wen; Liang, D Cherng; Kuo, Ming-Chung; Wu, Jin-Hou; Dunn, Po; Wang, Po-Nan; Lin, Tung-Liang; Shih, Yu-Shu; Liang, Sung-Tzu; Lin, Tung-Huei; Lai, Chen-Yu; Lin, Chun-Hui; Shih, Lee-Yung

    2015-10-20

    The mutational profiles of acute myeloid leukemia (AML) with partial tandem duplication of mixed-lineage leukemia gene (MLL-PTD) have not been comprehensively studied. We studied 19 gene mutations for 98 patients with MLL-PTD AML to determine the mutation frequency and clinical correlations. MLL-PTD was screened by reverse-transcriptase PCR and confirmed by real-time quantitative PCR. The mutational analyses were performed with PCR-based assays followed by direct sequencing. Gene mutations of signaling pathways occurred in 63.3% of patients, with FLT3-ITD (44.9%) and FLT3-TKD (13.3%) being the most frequent. 66% of patients had gene mutations involving epigenetic regulation, and DNMT3A (32.7%), IDH2 (18.4%), TET2 (18.4%), and IDH1 (10.2%) mutations were most common. Genes of transcription pathways and tumor suppressors accounted for 23.5% and 10.2% of patients. RUNX1 mutation occurred in 23.5% of patients, while none had NPM1 or double CEBPA mutation. 90.8% of MLL-PTD AML patients had at least one additional gene mutation. Of 55 MLL-PTD AML patients who received standard chemotherapy, age older than 50 years and DNMT3A mutation were associated with inferior outcome. In conclusion, gene mutations involving DNA methylation and activated signaling pathway were common co-existed gene mutations. DNMT3A mutation was a poor prognostic factor in MLL-PTD AML.

  1. High resolution melting curve analysis, a rapid and affordable method for mutation analysis in childhood acute myeloid leukemia

    Directory of Open Access Journals (Sweden)

    Yin eLiu

    2014-09-01

    Full Text Available Background: Molecular genetic alterations with prognostic significance have been described in childhood acute myeloid leukemia (AML. The aim of this study was to establish cost-effective techniques to detect mutations of FMS-like tyrosine kinase 3 (FLT3, Nucleophosmin 1 (NPM1, and a partial tandem duplication within the mixed lineage leukemia (MLL-PTD genes in childhood AML. Procedure: Ninety-nine children with newly diagnosed AML were included in this study. We developed a fluoresent dye SYTO-82 based high resolution melting curve (HRM anaylsis to detect FLT3 internal tandem duplication (FLT3-ITD, FLT3 tyrosine kinase domain (FLT3-TKD and NPM1 mutations. MLL-PTD was screened by real-time quantitative PCR. Results: The HRM methodology correlated well with gold standard Sanger sequencing with less cost. Among the 99 patients studied, the FLT3-ITD mutation was associated with significantly worse event free survival (EFS. Patients with the NPM1 mutation had significantly better EFS and overall survival. However, HRM was not sensitive enough for minimal residual disease monitoring. Conclusions: HRM was a rapid and efficient method for screening of FLT3 and NPM1 gene mutations. It was both affordable and accurate, especially in resource underprivileged regions. Our results indicated that HRM could be a useful clinical tool for rapid and cost effective screening of the FLT3 and NPM1 mutations in AML patients.

  2. High prevalence of mutations affecting the splicing process in a Spanish cohort with autosomal dominant retinitis pigmentosa

    Science.gov (United States)

    Ezquerra-Inchausti, Maitane; Barandika, Olatz; Anasagasti, Ander; Irigoyen, Cristina; López de Munain, Adolfo; Ruiz-Ederra, Javier

    2017-01-01

    Retinitis pigmentosa is the most frequent group of inherited retinal dystrophies. It is highly heterogeneous, with more than 80 disease-causing genes 27 of which are known to cause autosomal dominant RP (adRP), having been identified. In this study a total of 29 index cases were ascertained based on a family tree compatible with adRP. A custom panel of 31 adRP genes was analysed by targeted next-generation sequencing using the Ion PGM platform in combination with Sanger sequencing. This allowed us to detect putative disease-causing mutations in 14 out of the 29 (48.28%) families analysed. Remarkably, around 38% of all adRP cases analysed showed mutations affecting the splicing process, mainly due to mutations in genes coding for spliceosome factors (SNRNP200 and PRPF8) but also due to splice-site mutations in RHO. Twelve of the 14 mutations found had been reported previously and two were novel mutations found in PRPF8 in two unrelated patients. In conclusion, our results will lead to more accurate genetic counselling and will contribute to a better characterisation of the disease. In addition, they may have a therapeutic impact in the future given the large number of studies currently underway based on targeted RNA splicing for therapeutic purposes. PMID:28045043

  3. High-throughput mutational analysis of TOR1A in primary dystonia

    OpenAIRE

    Truong Daniel D; Tarsy Daniel; Simon David K; Hedera Peter; Uitti Ryan J; Wszolek Zbigniew K; Batish Sat; Blitzer Andrew; Paniello Randal C; Karimi Morvarid; Tabbal Samer D; Racette Brad A; Perlmutter Joel S; Bastian Robert W; Xiao Jianfeng

    2009-01-01

    Abstract Background Although the c.904_906delGAG mutation in Exon 5 of TOR1A typically manifests as early-onset generalized dystonia, DYT1 dystonia is genetically and clinically heterogeneous. Recently, another Exon 5 mutation (c.863G>A) has been associated with early-onset generalized dystonia and some ΔGAG mutation carriers present with late-onset focal dystonia. The aim of this study was to identify TOR1A Exon 5 mutations in a large cohort of subjects with mainly non-generalized primary dy...

  4. High Frequency of Codon 12 but not Codon 13 and 61 K-ras Gene Mutations in Invasive Ductal Carcinoma of Breast in a South Indian Population.

    Science.gov (United States)

    Sushma, C; Prasad, Shiva; Devi, Rudrama; Murthy, Sudha; Rao, Ts; Naidu, Ck

    2015-01-01

    Ras genes are thought to play an important role in human cancer since they have been found to be activated frequently in several types of tumors including breast cancer, where the overall incidence of K-RAS oncogene activation is 0-10%. Evaluation of K-RAS gene not only for mutational frequency but also for mutation types in this downstream signaling gene pathway is necessary to determine the mechanisms of action. The present study was conducted to test the hypothesis that K-RAS activation is involved in breast cancer risk of south Indian population. A total of 70 paired pathologically confirmed tumor and non-tumor tissues from the same breast cancer patients were analysed for most common K-RAS mutations of codon 12,13 and 61 by polymerase chain reaction followed by restriction digestion and direct nucleotide sequencing method. We found that a high rate of homozygous and heterozygous mutations of codon 12, but not codon 13 and 61, may influence the invasive ductal carcinoma of breast risk in this study. Our study indicated that only codon 12 may be involved in initiating breast carcinogenesis in India.

  5. Screening of WT1 mutations in exon 8 and 9 in children with steroid resistant nephrotic syndrome from a single centre and establishment of a rapid screening assay using high-resolution melting analysis in a clinical setting.

    Science.gov (United States)

    Siji, Annes; Pardeshi, Varsha Chhotusing; Ravindran, Shilpa; Vasudevan, Ambily; Vasudevan, Anil

    2017-01-10

    Mutations in Wilm's tumor 1 (WT1) gene is one of the commonly reported genetic mutations in children with steroid resistant nephrotic syndrome (SRNS). We report the results of direct sequencing of exons 8 and 9 of WT1 gene in 100 children with SRNS from a single centre. We standardized and validated High Resolution Melt (HRM) as a rapid and cost effective screening step to identify individuals with normal sequence and distinguish it from those with a potential mutation. Since only mutation positive samples identified by HRM will be further processed for sequencing it will help in reducing the sequencing burden and speed up the screening process. One hundred SRNS children were screened for WT1 mutations in Exon 8 and 9 using Sanger sequencing. HRM assay was standardized and validated by performing analysis for exon 8 and 9 on 3 healthy control and 5 abnormal variants created by site directed mutagenesis and verified by sequencing. To further test the clinical applicability of the assay, we screened additional 91 samples for HRM testing and performed a blinded assessment. WT1 mutations were not observed in the cohort of children with SRNS. The results of HRM analysis were concordant with the sequencing results. The WT1 gene mutations were not observed in the SRNS cohort indicating it has a low prevalence. We propose applying this simple, rapid and cost effective assay using HRM technique as the first step for screening the WT1 gene hot spot region in a clinical setting.

  6. High voltage testing for the Majorana Demonstrator

    Energy Technology Data Exchange (ETDEWEB)

    Abgrall, N.; Arnquist, Isaac J.; Avignone, F. T.; Barabash, A.; Bertrand, F.; Bradley, A. W.; Brudanin, V.; Busch, Matthew; Buuck, M.; Byram, D.; Caldwell, A. S.; Chan, Yuen-Dat; Christofferson, C. D.; Chu, Pamela M.; Cuesta, C.; Detwiler, Jason A.; Doe, P. J.; Dunagan, C.; Efremenko, Yuri; Ejiri, H.; Elliott, S. R.; Fu, Z.; Galindo-Uribarri, A.; Giovanetti, G. K.; Goett, J.; Green, M. P.; Gruszko, J.; Guinn, I.; Guiseppe, V. E.; Henning, R.; Hoppe, Eric W.; Howard, S.; Howe, M. A.; Jasinski, B. R.; Keeter, K.; Kidd, M. F.; Konovalov, S.; Kouzes, Richard T.; Laferriere, Brian D.; Leon, Jonathan D.; Li, Alexander D.; MacMullin, J.; Martin, R. D.; Massarcyk, R.; Meijer, S. J.; Mertens, S.; Orrell, John L.; O' Shaughnessy, C.; Poon, Alan W.; Radford, D. C.; Rager, J.; Rielage, Keith; Robertson, R. G. H.; Romero Romo, M.; Shanks, B.; Shirchenko, M.; Snyder, N.; Suriano, Anne-Marie E.; Tedeschi, D.; Thompson, Andrew; Ton, K. T.; Trimble, J. E.; Varner, R. L.; Vasilyev, Sergey; Vetter, Kai; Vorren, Kris R.; White, Brandon R.; Wilkerson, J. F.; Wiseman, C.; Xu, W.; Yakushev, E.; Yu, Chang-Hong; Yumatov, V.

    2016-07-01

    The Majorana Collaboration is constructing theMajorana Demonstrator, an ultra-low background, 44-kg modular high-purity Ge (HPGe) detector array to search for neutrinoless double-beta decay in 76Ge. The phenomenon of surface micro-discharge induced by high-voltage has been studied in the context of theMajorana Demonstrator. This effect can damage the front-end electronics or mimic detector signals. To ensure the correct performance, every high-voltage cable and feedthrough must be capable of supplying HPGe detector operating voltages as high as 5 kV without exhibiting discharge. R&D measurements were carried out to understand the testing system and determine the optimum design configuration of the high-voltage path, including different improvements of the cable layout and feedthrough flange model selection. Every cable and feedthrough to be used at the Majorana Demonstrator was characterized and the micro-discharge effects during theMajorana Demonstrator commissioning phase were studied. A stable configuration has been achieved, and the cables and connectors can supply HPGe detector operating voltages without exhibiting discharge.

  7. High frequency of Plasmodium falciparum chloroquine resistance marker (pfcrt T76 mutation) in Yemen: an urgent need to re-examine malaria drug policy.

    Science.gov (United States)

    Al-Mekhlafi, Abdulsalam M; Mahdy, Mohammed A K; Al-Mekhlafi, Hesham M; Azazy, Ahmed A; Fong, Mun Yik

    2011-05-27

    Malaria remains a significant health problem in Yemen with Plasmodium falciparum being the predominant species which is responsible for 90% of the malaria cases. Despite serious concerns regarding increasing drug resistance, chloroquine is still used for the prevention and treatment of malaria in Yemen. This study was carried out to determine the prevalence of choloroquine resistance (CQR) of P. falciparum isolated from Yemen based on the pfcrt T76 mutation. A cross-sectional study was carried out among 511 participants from four governorates in Yemen. Blood samples were screened using microscopic and species-specific nested PCR based on the 18S rRNA gene to detect and identify Plasmodium species. Blood samples positive for P. falciparum were used for detecting the pfcrt T76 mutation using nested-PCR. The prevalence of pfcrt T76 mutation was 81.5% (66 of 81 isolates). Coastal areas/foothills had higher prevalence of pfcrt T76 mutation compared to highland areas (90.5% vs 71.8%) (p = 0.031). The pfcrt T76 mutation had a significant association with parasitaemia (p = 0.045). Univariate analysis shows a significant association of pfcrt T76 mutation with people aged > 10 years (OR = 9, 95% CI = 2.3 - 36.2, p = 0.001), low household income (OR = 5, 95% CI = 1.3 - 19.5, p = 0.027), no insecticide spray (OR = 3.7, 95% CI = 1.16 - 11.86, p = 0.025) and not sleeping under insecticide treated nets (ITNs) (OR = 4.8, 95% CI = 1.38 - 16.78, p = 0.01). Logistic regression model confirmed age > 10 years and low household income as predictors of pfcrt T76 mutation in Yemen P. falciparum isolates. The high prevalence of pfcrt T76 mutation in Yemen could be a predictive marker for the prevalence of P. falciparum CQR. This finding shows the necessity for an in-vivo therapeutic efficacy test for CQ. P. falciparum CQR should be addressed in the national strategy to control malaria.

  8. An epidemiological investigation of a Forkhead box protein E3 founder mutation underlying the high frequency of sclerocornea, aphakia, and microphthalmia in a Mexican village.

    Science.gov (United States)

    Pantoja-Melendez, Carlos; Ali, Manir; Zenteno, Juan C

    2013-01-01

    To investigate the molecular epidemiological basis for the unusually high incidence of sclerocornea, aphakia, and microphthalmia in a village in the Tlaxcala province of central Mexico. A population census was performed in a village to identify all sclerocornea, aphakia, and microphthalmia cases. Molecular analysis of the previously identified Forkhead box protein E3 (FOXE3) mutation, c.292T>C (p.Y98H), was performed with PCR amplification and direct DNA sequencing. In addition, DNA from 405 randomly selected unaffected villagers was analyzed to establish the carrier frequency of the causal mutation. To identify the number of generations since the mutation arose in the village, 17 polymorphic markers distributed in a region of 6 Mb around the mutated locus were genotyped in the affected individuals, followed by DMLE software analysis to calculate mutation age. A total of 22 patients with sclerocornea, aphakia, and microphthalmia were identified in the village, rendering a disease prevalence of 2.52 cases per 1,000 habitants (1 in 397). The FOXE3 homozygous mutation was identified in all 17 affected subjects who consented to molecular analysis. Haplotype analysis indicated that the mutation arose 5.0-6.5 generations ago (approximately 106-138 years). Among the 405 unaffected villagers who were genotyped, ten heterozygote carriers were identified, yielding a population carrier frequency of approximately 1 in 40 and a predicted incidence of affected of 1 in 6,400 based on random marriages between two carriers in the village. This study demonstrates that a cluster of patients with sclerocornea, aphakia, and microphthalmia in a small Mexican village is due to a FOXE3 p.Y98H founder mutation that arose in the village just over a century ago at a time when a population migrated from a nearby village because of land disputes. The actual disease incidence is higher than the calculated predicted value and suggests non-random marriages (i.e., consanguinity) within the

  9. High incidence of protein-truncating TP53 mutations in BRCA1-related breast cancer.

    Science.gov (United States)

    Holstege, Henne; Joosse, Simon A; van Oostrom, Conny Th M; Nederlof, Petra M; de Vries, Annemieke; Jonkers, Jos

    2009-04-15

    Approximately half of all hereditary breast cancers are compromised in their DNA repair mechanisms due to loss of BRCA1 or BRCA2 function. Previous research has found a strong correlation between BRCA mutation and TP53 mutation. However, TP53 mutation status is often indirectly assessed by immunohistochemical staining of accumulated p53 protein. We sequenced TP53 exons 2 to 9 in 21 BRCA1-related breast cancers and 37 sporadic breast tumors. Strikingly, all BRCA1-related breast tumors contained TP53 mutations, whereas only half of these tumors stained positive for p53 accumulation. Positive p53 staining correlates with the presence of TP53 hotspot mutations in both BRCA1-related and sporadic breast tumors. However, whereas the majority of sporadic breast tumors that stained negative for p53 accumulation had wild-type TP53, the majority of BRCA1-associated breast tumors that stained negative for p53 accumulation had protein-truncating TP53 mutations (nonsense, frameshift, and splice mutations). Therefore, the strong selection for p53 loss in BRCA1-related tumors is achieved by an increase of protein-truncating TP53 mutations rather than hotspot mutations. Hence, immunohistochemical detection of TP53 mutation could lead to misdiagnosis in approximately half of all BRCA1-related tumors. The presence of deleterious TP53 mutations in most, if not all, BRCA1-related breast cancers suggests that p53 loss of function is essential for BRCA1-associated tumorigenesis. BRCA1-related tumors may therefore be treated not only with drugs that target BRCA1 deficiency [e.g., poly(ADP-ribose) polymerase inhibitors] but also with drugs that selectively target p53-deficient cells. This raises interesting possibilities for combination therapies against BRCA1-deficient breast cancers and BRCA1-like tumors with homologous recombination deficiency.

  10. Recurrent mutation testing of BRCA1 and BRCA2 in Asian breast cancer patients identify carriers in those with presumed low risk by family history.

    Science.gov (United States)

    Kang, Peter Choon Eng; Phuah, Sze Yee; Sivanandan, Kavitta; Kang, In Nee; Thirthagiri, Eswary; Liu, Jian Jun; Hassan, Norhashimah; Yoon, Sook-Yee; Thong, Meow Keong; Hui, Miao; Hartman, Mikael; Yip, Cheng Har; Mohd Taib, Nur Aishah; Teo, Soo Hwang

    2014-04-01

    Although the breast cancer predisposition genes BRCA1 and BRCA2 were discovered more than 20 years ago, there remains a gap in the availability of genetic counselling and genetic testing in Asian countries because of cost, access and inaccurate reporting of family history of cancer. In order to improve access to testing, we developed a rapid test for recurrent mutations in our Asian populations. In this study, we designed a genotyping assay with 55 BRCA1 and 44 BRCA2 mutations previously identified in Asian studies, and validated this assay in 267 individuals who had previously been tested by full sequencing. We tested the prevalence of these mutations in additional breast cancer cases. Using this genotyping approach, we analysed recurrent mutations in 533 Malaysian breast cancer cases with Chinese and 1 BRCA1 mutation in Indians account for 60, 24 and 20 % of carrier families, respectively. By contrast, haplotype analyses suggest that a recurrent BRCA2 mutation (c.262_263delCT) found in 5 unrelated Malay families has at least 3 distinct haplotypes. Taken together, our data suggests that panel testing may help to identify carriers, particularly Asian BRCA2 carriers, who do not present with a priori strong family history characteristics.

  11. Non-invasive prenatal testing for fetal inheritance of maternal β-thalassaemia mutations using targetted sequencing and relative mutation dosage: a feasibility study.

    Science.gov (United States)

    Xiong, L; Barrett, A N; Hua, R; Ho, Ssy; Jun, L; Chan, Kca; Mei, Z; Choolani, M

    2017-12-06

    To evaluate whether targeted sequencing and relative mutation dosage can be used to diagnose correctly inheritance of maternal β-thalassaemia mutations in cell-free DNA. Feasibility study using samples collected in a prenatal clinic. South East Asia. Couples where both partners were known to be carriers of one of four common β-thalassaemia mutations or an HbE mutation, and therefore at risk of carrying a fetus affected with β-thalassaemia. 49 samples previously identified as having inherited a paternal β-thalassaemia mutation were amplified using nested polymerase chain reaction (PCR), and then sequencing. Relative mutation dosage was used to classify the fetus as having inherited the wild-type or mutant maternal allele. Classification of the fetus as 'unaffected' (if the maternal wild-type allele was inherited) or 'affected' with β-thalassaemia (if the maternal mutant allele was inherited). A classification for inheritance of maternal allele was obtained in 48/49 samples (98.0%). A concordant call was made in 44/48 cases (91.7%): one false-positive and three false-negatives were obtained. Thus, we had an overall sensitivity of 87.5% [95% confidence interval (CI) 67.6-97.3%] and a specificity of 95.8% (95% CI 78.9-99.9%) for inheritance of maternal genotype. RMD for detection of inheritance of maternal β-thalassaemia mutations has potential for clinical use. Our sequential approach could be applied to other single-gene disorders. NIPT for β-thalassaemia achieved using nested-PCR followed by relative mutation dosage. © 2017 Royal College of Obstetricians and Gynaecologists.

  12. high prevalence of the cys282tyr hfe mutation facilitates an improved ...

    African Journals Online (AJOL)

    screened for two common haemochromatosis (HFE) gene mutations. The local frequencies of mutations C282Y and. H63D were determined and the DNA results correlated with biochemical parameters. Setting. Patients were referred from private practitioners, health workers and pathologists for a molecular diagnosis of.

  13. TP53 mutations, tetraploidy and homologous recombination repair defects in early stage high-grade serous ovarian cancer

    Science.gov (United States)

    Chien, Jeremy; Sicotte, Hugues; Fan, Jian-Bing; Humphray, Sean; Cunningham, Julie M.; Kalli, Kimberly R.; Oberg, Ann L.; Hart, Steven N.; Li, Ying; Davila, Jaime I.; Baheti, Saurabh; Wang, Chen; Dietmann, Sabine; Atkinson, Elizabeth J.; Asmann, Yan W.; Bell, Debra A.; Ota, Takayo; Tarabishy, Yaman; Kuang, Rui; Bibikova, Marina; Cheetham, R. Keira; Grocock, Russell J.; Swisher, Elizabeth M.; Peden, John; Bentley, David; Kocher, Jean-Pierre A.; Kaufmann, Scott H.; Hartmann, Lynn C.; Shridhar, Viji; Goode, Ellen L.

    2015-01-01

    To determine early somatic changes in high-grade serous ovarian cancer (HGSOC), we performed whole genome sequencing on a rare collection of 16 low stage HGSOCs. The majority showed extensive structural alterations (one had an ultramutated profile), exhibited high levels of p53 immunoreactivity, and harboured a TP53 mutation, deletion or inactivation. BRCA1 and BRCA2 mutations were observed in two tumors, with nine showing evidence of a homologous recombination (HR) defect. Combined Analysis with The Cancer Genome Atlas (TCGA) indicated that low and late stage HGSOCs have similar mutation and copy number profiles. We also found evidence that deleterious TP53 mutations are the earliest events, followed by deletions or loss of heterozygosity (LOH) of chromosomes carrying TP53, BRCA1 or BRCA2. Inactivation of HR appears to be an early event, as 62.5% of tumours showed a LOH pattern suggestive of HR defects. Three tumours with the highest ploidy had little genome-wide LOH, yet one of these had a homozygous somatic frame-shift BRCA2 mutation, suggesting that some carcinomas begin as tetraploid then descend into diploidy accompanied by genome-wide LOH. Lastly, we found evidence that structural variants (SV) cluster in HGSOC, but are absent in one ultramutated tumor, providing insights into the pathogenesis of low stage HGSOC. PMID:25916844

  14. High heat load test of molybdenum

    Energy Technology Data Exchange (ETDEWEB)

    Tanabe, T. (Faculty of Engineering, Osaka Univ., Suita (Japan)); Fujine, M.; Noguchi, H. (Daido Steel Co. Ltd., Nagoya (Japan)); Yagi, Y.; Hirano, Y.; Shimizu, H. (Electrotechnical Lab., Umezono, Tsukuba (Japan)); Akiba, M.; Araki, M. (Japan Atomic Energy Research Inst., Naka, Ibaraki (Japan)); Kubota, Y.; Miyahara, A. (National Inst. for Fusion Science, Nagoya (Japan))

    1993-03-01

    Three different types of molybdenum, powder metallurgical polycrystalline (PM-Mo), and as-forged polycrystalline and single crystalline of highly purified electron-beam-melted Mo (AFEB-Mo and SCEB-Mo), have been subjected to high heat load test with neutral beam injection (NBI) stands at Japan Atomic Energy Research Institute (JAERI) and National Institute for Fusion Science (NIFS). These materials have also been tested as a movable limiter in a reversed field pinch machine (RFP:TPE-1RM15) in Electrotechnical Laboratory (ETL). The results are summarized as follows. The SCEB-Mo shows the least damage with slight local melting after a very high heat load of 260 MW/m[sup 2] for 250 ms with NBI, while for the PM-Mo the whole irradiated area melt with many craters due to impurity gas evaporation under less heat load (200 ms). All movable limiter heads of the RFP are severely damaged with partial melting. The appearance of the SCEB-Mo limiter after melting is not good and shows the crystalline cleavage. However, SEM observation of the microstructure opposes the surface appearance. In the SCEB-Mo, appreciable recrystallization is not observed and hence no crack is seen to go into the bulk except the crystalline cleavage. In the PM-Mo, on the other hand, the resolidification to columnar grains as well as the recrystallization is apparent, and the cracks not only go along the columnar grains but also separate the recrystallized region from the matrix. In the AFEB-Mo, a slight grain growth occurs and several cracks enter deep along the grain boundaries. Thus the SCEB-Mo is a very nice plasma-facing material if used under the critical heat load for melting. (orig.).

  15. No evidence of BRCA2 mutations in chromosome 13q-linked Utah high-risk prostate cancer pedigrees

    Directory of Open Access Journals (Sweden)

    Allen-Brady Kristina

    2009-05-01

    Full Text Available Abstract Background Germline mutations in the BRCA2 gene have been suggested to account for about 5% of familial prostate cancer; mutations have been reported in 2% of early onset (i.e., ≤ 55 years prostate cancer cases and a segregating founder mutation has been identified in Iceland (999del5. However, the role of BRCA2 in high risk prostate cancer pedigrees remains unclear. Findings We examined the potential involvement of BRCA2 in a set offive high-risk prostate cancer pedigrees in which all prostate cases were no more distantly related than two meioses from another case, and the resulting cluster contained at least four prostate cancer cases. We selected these five pedigrees from a larger dataset of 59 high-risk prostate cancer pedigrees analyzed in a genome-wide linkage screen. Selected pedigrees showed at least nominal linkage evidence to the BRCA2 region on chromosome 13q. We mutation screened all coding regions and intron/exon boundaries of the BRCA2 gene in the youngest prostate cancer case who carried the linked 13q segregating haplotype, as well as in a distantly related haplotype carrier to confirm any segregation. We observed no known protein truncating BRCA2 deleterious mutations. We identified one non-segregating BRCA2 variant of uncertain significance, one non-segregating intronic variant not previously reported, and a number of polymorphisms. Conclusion In this set of high-risk prostate cancer pedigrees with at least nominal linkage evidence to BRCA2, we saw no evidence for segregating BRCA2 protein truncating mutations in heritable prostate cancer.

  16. Perceptions of Ashkenazi Jewish breast cancer patients on genetic testing for mutations in BRCA1 and BRCA2.

    Science.gov (United States)

    Phillips, K A; Warner, E; Meschino, W S; Hunter, J; Abdolell, M; Glendon, G; Andrulis, I L; Goodwin, P J

    2000-05-01

    The perceived benefits and risks of genetic testing may vary between groups of individuals with different cultural, demographic, and family history features. This multicentre study examined the factors that influenced the decision to undergo genetic testing for BRCA1 and BRCA2 in Canadian Jewish women with breast cancer. A self-administered questionnaire was developed and distributed to 134 individuals enrolled in a research-based testing program for Ashkenazi women. The questionnaire assessed demographic, social, and family history parameters, and the influence of medical, family, social, psychological, and cultural/religious factors on decision making about genetic testing. Seventy-six percent of women completed the questionnaire. Forty-one percent of study participants had no family history of breast or ovarian cancer. The most important factors influencing the decision to undergo testing were a desire to contribute to research, potential benefit to other family members, curiosity, and the potential for relief if not found to be a carrier (endorsed by 87, 78, 70, and 60% of participants, respectively). The main perceived risks of undergoing genetic testing related to insurance discrimination, confidentiality, accuracy and interpretability of results, potential impact on marriage prospects for family members, and focus on the Jewish community (endorsed by 28, 24, 30, 17, and 14% of participants, respectively). This study provides novel information on the motivating factors for BRCA1 and BRCA2 mutation testing in Canadian women of Ashkenazi Jewish descent. The focus on altruistic factors and those related to perceived psychological benefits of testing is notable.

  17. KRAS mutation analysis in ovarian samples using a high sensitivity biochip assay

    Directory of Open Access Journals (Sweden)

    Reinthaller Alexander

    2009-04-01

    Full Text Available Abstract Background Mutations in the KRAS gene are one of the most frequent genetic abnormalities in ovarian carcinoma. They are of renewed interest as new epidermal growth factor receptor (EGFR-targeted therapies are being investigated for use in ovarian carcinoma. As KRAS mutations are associated with poor response and resistance to EGFR-targeting drugs, this study was conducted to obtain more information on the spectrum of KRAS mutations in ovarian carcinoma. Methods The presence of KRAS mutations in codon 12 and 13 was analyzed in frozen and formalin-fixed paraffin-embedded (FFPE tissue with a low density biochip platform. 381 malignant (29 borderline malignancy, 270 primary carcinomas, and 82 recurrent carcinomas and 22 benign tissue samples from a total of 394 patients were examined. KRAS mutational status of each sample was correlated with dignity, FIGO stage, grade, histology, and survival. Results KRAS mutations were found in 60 (15% samples with 58 samples deriving from malignant tissue and 2 samples deriving from benign tissue. In 55 (92% samples codon 12 was found to be mutated. Frozen and FFPE samples concurred with respect to KRAS mutation status. Conclusion KRAS mutation is a common event in ovarian cancer primarily in carcinomas of lower grade, lower FIGO stage, and mucinous histotype. The KRAS mutational status is no prognostic factor for patients treated with standard therapy. However, in line with experience from colorectal cancer and non-small-cell-lung cancer (NSCLC, it may be important for prediction of response to EGFR-targeted therapies.

  18. Population testing for cancer predisposing BRCA1/BRCA2 mutations in the Ashkenazi-Jewish community: a randomized controlled trial.

    Science.gov (United States)

    Manchanda, Ranjit; Loggenberg, Kelly; Sanderson, Saskia; Burnell, Matthew; Wardle, Jane; Gessler, Sue; Side, Lucy; Balogun, Nyala; Desai, Rakshit; Kumar, Ajith; Dorkins, Huw; Wallis, Yvonne; Chapman, Cyril; Taylor, Rohan; Jacobs, Chris; Tomlinson, Ian; McGuire, Alistair; Beller, Uziel; Menon, Usha; Jacobs, Ian

    2015-01-01

    Technological advances raise the possibility of systematic population-based genetic testing for cancer-predisposing mutations, but it is uncertain whether benefits outweigh disadvantages. We directly compared the psychological/quality-of-life consequences of such an approach to family history (FH)-based testing. In a randomized controlled trial of BRCA1/2 gene-mutation testing in the Ashkenazi Jewish (AJ) population, we compared testing all participants in the population screening (PS) arm with testing those fulfilling standard FH-based clinical criteria (FH arm). Following a targeted community campaign, AJ participants older than 18 years were recruited by self-referral after pretest genetic counseling. The effects of BRCA1/2 genetic testing on acceptability, psychological impact, and quality-of-life measures were assessed by random effects regression analysis. All statistical tests were two-sided. One thousand, one hundred sixty-eight AJ individuals were counseled, 1042 consented, 1034 were randomly assigned (691 women, 343 men), and 1017 were eligible for analysis. Mean age was 54.3 (SD = 14.66) years. Thirteen BRCA1/2 carriers were identified in the PS arm, nine in the FH arm. Five more carriers were detected among FH-negative FH-arm participants following study completion. There were no statistically significant differences between the FH and PS arms at seven days or three months on measures of anxiety, depression, health anxiety, distress, uncertainty, and quality-of-life. Contrast tests indicated that overall anxiety (P = .0001) and uncertainty (P = .005) associated with genetic testing decreased; positive experience scores increased (P = .0001); quality-of-life and health anxiety did not change with time. Overall, 56% of carriers did not fulfill clinical criteria for genetic testing, and the BRCA1/2 prevalence was 2.45%. Compared with FH-based testing, population-based genetic testing in Ashkenazi Jews doesn't adversely affect short-term psychological

  19. High Temperature Fluoride Salt Test Loop

    Energy Technology Data Exchange (ETDEWEB)

    Aaron, Adam M. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Cunningham, Richard Burns [Univ. of Tennessee, Knoxville, TN (United States); Fugate, David L. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Holcomb, David Eugene [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Kisner, Roger A. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Peretz, Fred J. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Robb, Kevin R. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Wilson, Dane F. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Yoder, Jr, Graydon L. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)

    2015-12-01

    with 3 cm diameter graphite-based fuel pebbles slowly circulating up through the core. Molten salt coolant (FLiBe) at 700°C flows concurrently (at significantly higher velocity) with the pebbles and is used to remove heat generated in the reactor core (approximately 1280 W/pebble), and supply it to a power conversion system. Refueling equipment continuously sorts spent fuel pebbles and replaces spent or damaged pebbles with fresh fuel. By combining greater or fewer numbers of pebble channel assemblies, multiple reactor designs with varying power levels can be offered. The PB-AHTR design is discussed in detail in Reference [1] and is shown schematically in Fig. 1. Fig. 1. PB-AHTR concept (drawing taken from Peterson et al., Design and Development of the Modular PB-AHTR Proceedings of ICApp 08). Pebble behavior within the core is a key issue in proving the viability of this concept. This includes understanding the behavior of the pebbles thermally, hydraulically, and mechanically (quantifying pebble wear characteristics, flow channel wear, etc). The experiment being developed is an initial step in characterizing the pebble behavior under realistic PB-AHTR operating conditions. It focuses on thermal and hydraulic behavior of a static pebble bed using a convective salt loop to provide prototypic fluid conditions to the bed, and a unique inductive heating technique to provide prototypic heating in the pebbles. The facility design is sufficiently versatile to allow a variety of other experimentation to be performed in the future. The facility can accommodate testing of scaled reactor components or sub-components such as flow diodes, salt-to-salt heat exchangers, and improved pump designs as well as testing of refueling equipment, high temperature instrumentation, and other reactor core designs.

  20. High Voltage Testing. Volume 2. Specifications and Test Procedures

    Science.gov (United States)

    1982-08-01

    using the two wattmeter method the wattmeters must be read accurately. Because of the low power factor, the reading of one wattmeter will be negative...each part to be tested - A minimum of two of each type of terminal. 4.12.7.2 Soldering iron method - The test shal l be performed on solder...tested - A minimum of two of each type of terminal. 4.8.2.2 Soldering iron method . The test shall be performed on solder terminations, attached to the

  1. Compound EGFR mutation is frequently detected with co-mutations of actionable genes and associated with poor clinical outcome in lung adenocarcinoma.

    Science.gov (United States)

    Kim, Eun Young; Cho, Eun Na; Park, Heae Surng; Hong, Ji Young; Lim, Seri; Youn, Jong Pil; Hwang, Seung Yong; Chang, Yoon Soo

    2016-01-01

    Compound EGFR mutations, defined as double or multiple mutations in the EGFR tyrosine kinase domain, are frequently detected with advances in sequencing technology but its clinical significance is unclear. This study analyzed 61 cases of EGFR mutation positive lung adenocarcinoma using next-generation sequencing (NGS) based repeated deep sequencing panel of 16 genes that contain actionable mutations and investigated clinical implication of compound EGFR mutations. Compound EGFR mutation was detected in 15 (24.6%) of 61 cases of EGFR mutation-positive lung adenocarcinoma. The majority (12/15) of compound mutations are combination of the atypical mutation and typical mutations such as exon19 deletion, L858R or G719X substitutions, or exon 20 insertion whereas 3 were combinations of rare atypical mutations. The patients with compound mutation showed shorter overall survival than those with simple mutations (83.7 vs. 72.8 mo; P = 0.020, Breslow test). Among the 115 missense mutations discovered in the tested genes, a few number of actionable mutations were detected irrelevant to the subtype of EGFR mutations, including ALK rearrangement, BCL2L11 intron 2 deletion, KRAS c.35G>A, PIK3CA c.1633G>A which are possible target of crizotinib, BH3 mimetics, MEK inhibitors, and PI3K-tyrosine kinase inhibitors, respectively. 31 missense mutations were detected in the cases with simple mutations whereas 84 in those with compound mutation, showing that the cases with compound missense mutation have higher burden of missense mutations (P = 0.001, independent sample t-test). Compound EGFR mutations are detected at a high frequency using NGS-based repeated deep sequencing. Because patients with compound EGFR mutations showed poor clinical outcomes, they should be closely monitored during follow-up.

  2. Predicting cross-reactive immunological material (CRIM) status in Pompe disease using GAA mutations: lessons learned from 10 years of clinical laboratory testing experience.

    Science.gov (United States)

    Bali, Deeksha S; Goldstein, Jennifer L; Banugaria, Suhrad; Dai, Jian; Mackey, Joanne; Rehder, Catherine; Kishnani, Priya S

    2012-02-15

    Enzyme replacement therapy (ERT) for Pompe disease using recombinant acid alpha-glucosidase (rhGAA) has resulted in increased survival although the clinical response is variable. Cross-reactive immunological material (CRIM)-negative status has been recognized as a poor prognostic factor. CRIM-negative patients make no GAA protein and develop sustained high antibody titers to ERT that render the treatment ineffective. Antibody titers are generally low for the majority of CRIM-positive patients and there is typically a better clinical outcome. Because immunomodulation has been found to be most effective in CRIM-negative patients prior to, or shortly after, initiation of ERT, knowledge of CRIM status is important before ERT is begun. We have analyzed 243 patients with infantile Pompe disease using a Western blot method for determining CRIM status and using cultured skin fibroblasts. Sixty-one out of 243 (25.1%) patients tested from various ethnic backgrounds were found to be CRIM-negative. We then correlated the CRIM results with GAA gene mutations where available (52 CRIM-negative and 88 CRIM-positive patients). We found that, in most cases, CRIM status can be predicted from GAA mutations, potentially circumventing the need for invasive skin biopsy and time wasted in culturing cells in the future. Continued studies in this area will help to increase the power of GAA gene mutations in predicting CRIM status as well as possibly identifying CRIM-positive patients who are at risk for developing high antibody titers. Copyright © 2012 Wiley Periodicals, Inc.

  3. Evolutionary Accessibility of Mutational Pathways

    Science.gov (United States)

    Franke, Jasper; Klözer, Alexander; de Visser, J. Arjan G. M.; Krug, Joachim

    2011-01-01

    Functional effects of different mutations are known to combine to the total effect in highly nontrivial ways. For the trait under evolutionary selection (‘fitness’), measured values over all possible combinations of a set of mutations yield a fitness landscape that determines which mutational states can be reached from a given initial genotype. Understanding the accessibility properties of fitness landscapes is conceptually important in answering questions about the predictability and repeatability of evolutionary adaptation. Here we theoretically investigate accessibility of the globally optimal state on a wide variety of model landscapes, including landscapes with tunable ruggedness as well as neutral ‘holey’ landscapes. We define a mutational pathway to be accessible if it contains the minimal number of mutations required to reach the target genotype, and if fitness increases in each mutational step. Under this definition accessibility is high, in the sense that at least one accessible pathway exists with a substantial probability that approaches unity as the dimensionality of the fitness landscape (set by the number of mutational loci) becomes large. At the same time the number of alternative accessible pathways grows without bounds. We test the model predictions against an empirical 8-locus fitness landscape obtained for the filamentous fungus Aspergillus niger. By analyzing subgraphs of the full landscape containing different subsets of mutations, we are able to probe the mutational distance scale in the empirical data. The predicted effect of high accessibility is supported by the empirical data and is very robust, which we argue reflects the generic topology of sequence spaces. Together with the restrictive assumptions that lie in our definition of accessibility, this implies that the globally optimal configuration should be accessible to genome wide evolution, but the repeatability of evolutionary trajectories is limited owing to the presence of a

  4. High-throughput detection of induced mutations and natural variation using KeyPoint technology.

    Directory of Open Access Journals (Sweden)

    Diana Rigola

    Full Text Available Reverse genetics approaches rely on the detection of sequence alterations in target genes to identify allelic variants among mutant or natural populations. Current (pre- screening methods such as TILLING and EcoTILLING are based on the detection of single base mismatches in heteroduplexes using endonucleases such as CEL 1. However, there are drawbacks in the use of endonucleases due to their relatively poor cleavage efficiency and exonuclease activity. Moreover, pre-screening methods do not reveal information about the nature of sequence changes and their possible impact on gene function. We present KeyPoint technology, a high-throughput mutation/polymorphism discovery technique based on massive parallel sequencing of target genes amplified from mutant or natural populations. KeyPoint combines multi-dimensional pooling of large numbers of individual DNA samples and the use of sample identification tags ("sample barcoding" with next-generation sequencing technology. We show the power of KeyPoint by identifying two mutants in the tomato eIF4E gene based on screening more than 3000 M2 families in a single GS FLX sequencing run, and discovery of six haplotypes of tomato eIF4E gene by re-sequencing three amplicons in a subset of 92 tomato lines from the EU-SOL core collection. We propose KeyPoint technology as a broadly applicable amplicon sequencing approach to screen mutant populations or germplasm collections for identification of (novel allelic variation in a high-throughput fashion.

  5. Preappointment testing for BRAF/KIT mutation in advanced melanoma: a model in molecular data delivery for individualized medicine.

    Science.gov (United States)

    Mounajjed, Taofic; Brown, Char L; Stern, Therese K; Bjorheim, Annette M; Bridgeman, Andrew J; Rumilla, Kandelaria M; McWilliams, Robert R; Flotte, Thomas J

    2014-11-01

    The emergence of individualized medicine is driven by developments in precision diagnostics, epitomized by molecular testing. Because treatment decisions are being made based on such molecular data, data management is gaining major importance. Among data management challenges, creating workflow solutions for timely delivery of molecular data has become pivotal. This study aims to design and implement a scalable process that permits preappointment BRAF/KIT mutation analysis in melanoma patients, allowing molecular results necessary for treatment plans to be available before the patient's appointment. Process implementation aims to provide a model for efficient molecular data delivery for individualized medicine. We examined the existing process of BRAF/KIT testing in melanoma patients visiting our institution for oncology consultation. We created 5 working groups, each designing a specific segment of an alternative process that would allow preappointment BRAF/KIT testing and delivery of results. Data were captured and analyzed to evaluate the success of the alternative process. For 1 year, 35 (59%) of 55 patients had prior BRAF/KIT testing. The remaining 20 patients went through the new process of preappointment testing; results were available at the time of appointment for 12 patients (overall preappointment results availability, 85.5%). The overall process averaged 13.4 ± 4.7 days. In conclusion, we describe the successful implementation of a scalable workflow solution that permits preappointment BRAF/KIT mutation analysis and result delivery in melanoma patients. This sets the stage for further applications of this model to other conditions, answering an increasing demand for robust delivery of molecular data for individualized medicine. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Frameshift mutations of tumor suppressor gene EP300 in gastric and colorectal cancers with high microsatellite instability.

    Science.gov (United States)

    Kim, Min Sung; Lee, Sung Hak; Yoo, Nam Jin; Lee, Sug Hyung

    2013-10-01

    Several lines of evidence show that chromatin remodeling is involved in the pathogenesis of disease, including cancer. The E1A-binding protein p300 functions as a histone acetyltransferase and is considered an important modulator of chromatin remodeling. The aim of this study was to explore whether E1A-binding protein p300 is somatically mutated and expressionally altered in gastric and colorectal cancers. By analyzing a public database, we found that E1A-binding protein p300 had mononucleotide repeats in exons 27 and 31 that could be mutation targets in cancers with microsatellite instability. We analyzed mutations in the mononucleotide repeats in 91 gastric and 101 colorectal cancers with high microsatellite instability or stable microsatellite instability by single-strand conformation polymorphism analysis and DNA sequencing. We also analyzed E1A-binding protein p300 expression in gastric and colorectal cancers by immunohistochemistry staining. We found E1A-binding protein p300 frameshift mutations (4 in exon 27 and 3 in exon 31) in 3 gastric and 4 colorectal cancers that were detected exclusively in cancers with high microsatellite instability (7/80). In the immunohistochemistry study, loss of E1A-binding protein p300 expression was identified in 12% and 24% of the gastric and colorectal cancers, respectively, irrespective of microsatellite instability status. Loss was more common in tumors with E1A-binding protein p300 frameshift mutations. Frameshift mutations of E1A-binding protein p300 and its expressional loss may be a feature of gastric and colorectal cancers with high microsatellite instability. These alterations could contribute to cancer pathogenesis by deregulating E1A-binding protein p300-mediated functions. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Combined microsatellite and FGFR3 mutation analysis enables a highly sensitive detection of urothelial cell carcinoma in voided urine

    NARCIS (Netherlands)

    B.W. van Rhijn (Bas); I. Lurkin (Irene); D.K. Chopin; W.J. Kirkels (Wim); J.P. Thiery (Joachim); Th.H. van der Kwast (Theo); F. Radvanyi (Franois); E.C. Zwarthoff (Ellen)

    2003-01-01

    textabstractPURPOSE: Fibroblast growth factor receptor 3 (FGFR3) mutations were reported recently at a high frequency in low-grade urothelial cell carcinoma (UCC). We investigated the feasibility of combining microsatellite analysis (MA) and the FGFR3 status for the detection of

  8. RAD51C germline mutations in breast and ovarian cancer cases from high-risk families.

    Directory of Open Access Journals (Sweden)

    Jessica Clague

    Full Text Available BRCA1 and BRCA2 are the most well-known breast cancer susceptibility genes. Additional genes involved in DNA repair have been identified as predisposing to breast cancer. One such gene, RAD51C, is essential for homologous recombination repair. Several likely pathogenic RAD51C mutations have been identified in BRCA1- and BRCA2-negative breast and ovarian cancer families. We performed complete sequencing of RAD51C in germline DNA of 286 female breast and/or ovarian cancer cases with a family history of breast and ovarian cancers, who had previously tested negative for mutations in BRCA1 and BRCA2. We screened 133 breast cancer cases, 119 ovarian cancer cases, and 34 with both breast and ovarian cancers. Fifteen DNA sequence variants were identified; including four intronic, one 5' UTR, one promoter, three synonymous, and six non-synonymous variants. None were truncating. The in-silico SIFT and Polyphen programs were used to predict possible pathogenicity of the six non-synonomous variants based on sequence conservation. G153D and T287A were predicted to be likely pathogenic. Two additional variants, A126T and R214C alter amino acids in important domains of the protein such that they could be pathogenic. Two-hybrid screening and immunoblot analyses were performed to assess the functionality of these four non-synonomous variants in yeast. The RAD51C-G153D protein displayed no detectable interaction with either XRCC3 or RAD51B, and RAD51C-R214C displayed significantly decreased interaction with both XRCC3 and RAD51B (p<0.001. Immunoblots of RAD51C-Gal4 activation domain fusion peptides showed protein levels of RAD51C-G153D and RAD51C-R214C that were 50% and 60% of the wild-type, respectively. Based on these data, the RAD51C-G153D variant is likely to be pathogenic, while the RAD51C- R214C variant is hypomorphic of uncertain pathogenicity. These results provide further support that RAD51C is a rare breast and ovarian cancer susceptibility gene.

  9. Ultrasonic test of highly stressed gear shafts

    Energy Technology Data Exchange (ETDEWEB)

    Schreiner, T. [Siemens AG, Power Generation, KWU, Muelheim (Germany); Heinrich, W. [Siemens AG, Power Generation, KWU, Berlin (Germany); Achtzehn, J. [Siemens AG, Power Generation, ICVW, Erlangen (Germany); Hensley, H. [Siemens Power Generation (Germany)

    1998-12-31

    In the power plant industry, gears are used for increasingly higher turbine capacities. Efficiency enhancements, particularly for the combined gas and steam turbine process, lead to an increase in stresses, even for high-performance gears. Consequently, the requirements for non-destructive material testing are on the increase as well. At Siemens KWU, high-performance gears are used so far only for gas turbines with lower rating (65 MW) to adapt the gas turbine speed (5413 rpm) to the generator speed (3000 rpm/ 50 Hz or 3600 rpm/60 Hz). The gear train consists of a forged and case-hardened wheel shaft and pinion shaft made of material 17 CrNiMo 6, where the wheel shaft can be either a solid or a hollow shaft. Dimensions are typically 2.3 m length and 1 m diameter. As a rule, pinion shafts are solid. The gear design, calling for an additional torsion shaft turning inside the hollow wheel shaft, can absorb more torsional load surges and is more tolerant of deviations during gear train alignment. This design requires two additional forgings (torsion shaft and hub) and an additional bearing 2 refs.

  10. Development and pilot testing of a decision aid for men considering genetic testing for breast and/or ovarian cancer-related mutations (BRCA1/2).

    Science.gov (United States)

    Juan, Anne S; Wakefield, Claire E; Kasparian, Nadine A; Kirk, Judy; Tyler, Janet; Tucker, Kathy

    2008-12-01

    Despite the fact that both men and women can carry a breast/ovarian cancer-related mutation, the main emphasis in genetic counseling for breast/ovarian cancer-related risk remains on females. This study aimed to develop and pilot a decision aid specifically designed for men with a strong family history of breast and/or ovarian cancer who are considering genetic testing. The decision aid was developed by a multidisciplinary team of experts and a consumer representative. It was then reviewed by 27 men who had previously undergone genetic testing to identify a mutation in a BRCA1 or BRCA2 gene. All men who reviewed the decision aid indicated that they would recommend the booklet to other men in the same situation, and 96% of the sample (n = 26) reported being "very satisfied" or "satisfied" with the information contained in the decision aid. The decision aid was perceived by all participants as "very relevant" or "quite relevant" for men considering genetic testing. Ninety-three percent of men felt that it was easy to weigh the pros and cons of genetic testing with the help of the decision aid. The perceived impact on participants' emotions and understanding of the genetic testing process was also assessed. Several factors may hinder men from effectively weighing up the potential benefits and risks of genetic testing. A greater understanding of these issues may help health professionals to encourage men with a strong family history of breast and/or ovarian cancer to learn about cancer risk and the appropriate management strategies for themselves and their female relatives.

  11. Homogeneous assay based on 52 primer sets to scan for mutations of the ABCA1 gene and its application in genetic analysis of a new patient with familial high-density lipoprotein deficiency syndrome.

    Science.gov (United States)

    Lapicka-Bodzioch, K; Bodzioch, M; Krüll, M; Kielar, D; Probst, M; Kiec, B; Andrikovics, H; Böttcher, A; Hubacek, J; Aslanidis, C; Suttorp, N; Schmitz, G

    2001-07-27

    Familial high-density lipoprotein (HDL)-deficiency syndromes are caused by mutations of the ABCA1 gene, coding for the ATP-binding cassette transporter 1. We have developed a homogeneous assay based on 52 primer sets to amplify all 50 ABCA1 exons and approximately 1 kb of its promoter. The assay allows for convenient amplification of the gene from genomic DNA and easy mutational analysis through automatic sequencing. It obviates the need to use mRNA preparations, which were difficult to handle and posed a risk to miss splice junction or promoter mutations. The application of the test to the molecular analysis of a new patient with familial HDL-deficiency (Tangier disease) led to a discovery of two novel ABCA1 mutations: C2665del and C4457T.

  12. High-speed Stochastic Fatigue Testing

    DEFF Research Database (Denmark)

    Brincker, Rune; Sørensen, John Dalsgaard

    1990-01-01

    testing, quite unacceptable errors are introduced. Usually this problem is solved by running the tests at very low speeds and by editing the load history in order to reduce the duration of the test. In this paper a new method for control of stochastic fatigue tests is proposed. It is based on letting...

  13. Highly sensitive scanning of gene mutations: TaqMan probes as blocking agents

    Directory of Open Access Journals (Sweden)

    I. V. Botezatu

    2016-01-01

    Full Text Available DNA Melting Analysis is very effective in clinical DNA diagnostics: it is simple to perform, high throughput, labor-, time- and cost-effective and is implemented in the “closed tube” format minimizing the risk of samples cross-contamination. Although more sensitive than sequencing by Sanger (mutant allele detection limit is ~5 and ~15 % respectively, it, however, is inferior in this respect to some other, more laborious and expensive methods (in particular, ddPCR (digital droplet PCR. Using the BRAF gene as a prototype, we developed the original version of the DNA melting analysis, based on the ability of TaqMan probes to hamper the primer extension reaction by Taq-polymerase. It is found that the weaker blocking effect on the mutant template, which is due to the mismatch in the probe-DNA heteroduplex, permits enriched amplification of the mutant allele and provides a significant (10-fold or more increase in sensitivity of mutation scanning.

  14. Mutational breeding and genetic engineering in the development of high grain protein content.

    Science.gov (United States)

    Wenefrida, Ida; Utomo, Herry S; Linscombe, Steve D

    2013-12-04

    Cereals are the most important crops in the world for both human consumption and animal feed. Improving their nutritional values, such as high protein content, will have significant implications, from establishing healthy lifestyles to helping remediate malnutrition problems worldwide. Besides providing a source of carbohydrate, grain is also a natural source of dietary fiber, vitamins, minerals, specific oils, and other disease-fighting phytocompounds. Even though cereal grains contain relatively little protein compared to legume seeds, they provide protein for the nutrition of humans and livestock that is about 3 times that of legumes. Most cereal seeds lack a few essential amino acids; therefore, they have imbalanced amino acid profiles. Lysine (Lys), threonine (Thr), methionine (Met), and tryptophan (Trp) are among the most critical and are a limiting factor in many grain crops for human nutrition. Tremendous research has been put into the efforts to improve these essential amino acids. Development of high protein content can be outlined in four different approaches through manipulating seed protein bodies, modulating certain biosynthetic pathways to overproduce essential and limiting amino acids, increasing nitrogen relocation to the grain through the introduction of transgenes, and exploiting new genetic variance. Various technologies have been employed to improve protein content including conventional and mutational breeding, genetic engineering, marker-assisted selection, and genomic analysis. Each approach involves a combination of these technologies. Advancements in nutrigenomics and nutrigenetics continue to improve public knowledge at a rapid pace on the importance of specific aspects of food nutrition for optimum fitness and health. An understanding of the molecular basis for human health and genetic predisposition to certain diseases through human genomes enables individuals to personalize their nutritional requirements. It is critically important

  15. Persistence of HIV-1 Transmitted Drug Resistance Mutations

    Science.gov (United States)

    Castro, Hannah; Pillay, Deenan; Cane, Patricia; Asboe, David; Cambiano, Valentina; Phillips, Andrew; Dunn, David T.; Aitken, Celia; Asboe, David; Webster, Daniel; Cane, Patricia; Castro, Hannah; Chadwick, David; Churchill, Duncan; Clark, Duncan; Collins, Simon; Delpech, Valerie; Geretti, Anna Maria; Goldberg, David; Hale, Antony; Hué, Stéphane; Kaye, Steve; Kellam, Paul; Lazarus, Linda; Leigh-Brown, Andrew; Mackie, Nicola; Orkin, Chloe; Rice, Philip; Pillay, Deenan; Smit, Erasmus; Templeton, Kate; Tilston, Peter; Tong, William; Williams, Ian; Zhang, Hongyi; Zuckerman, Mark; Greatorex, Jane; Wildfire, Adrian; O'Shea, Siobhan; Mullen, Jane; Mbisa, Tamyo; Cox, Alison; Tandy, Richard; Hale, Tony; Fawcett, Tracy; Hopkins, Mark; Ashton, Lynn; Garcia-Diaz, Ana; Shepherd, Jill; Schmid, Matthias L; Payne, Brendan; Chadwick, David; Hay, Phillip; Rice, Phillip; Paynter, Mary; Clark, Duncan; Bibby, David; Kaye, Steve; Kirk, Stuart; MacLean, Alasdair; Aitken, Celia; Gunson, Rory

    2013-01-01

    There are few data on the persistence of individual human immunodeficiency virus type 1 (HIV-1) transmitted drug resistance (TDR) mutations in the absence of selective drug pressure. We studied 313 patients in whom TDR mutations were detected at their first resistance test and who had a subsequent test performed while ART-naive. The rate at which mutations became undetectable was estimated using exponential regression accounting for interval censoring. Most thymidine analogue mutations (TAMs) and T215 revertants (but not T215F/Y) were found to be highly stable, with NNRTI and PI mutations being relatively less persistent. Our estimates are important for informing HIV transmission models. PMID:23904291

  16. High temperature triaxial tests on Rochester shale

    Science.gov (United States)

    Bruijn, Rolf; Burlini, Luigi; Misra, Santanu

    2010-05-01

    Phyllosilicates are one of the major components of the crust, responsible for strength weakening during deformation. High pressure and temperature experiments of natural samples rich in phyllosilicates are needed to test the relevance of proposed weakening mechanisms induced by phyllosilicates, derived from lab experiments on single phase and synthetic polyphase rocks and single crystals. Here, we present the preliminary results of a series of high temperature triaxial tests performed on the illite-rich Rochester Shale (USA - New York) using a Paterson type gas-medium HPT testing machine. Cylindrical samples with homogeneous microstructure and 12-14% porosity were fabricated by cold and hot-isostatically pressing, hot-pressed samples were deformed up to a total shortening of 7.5 to 13%. To study the significance of mica dehydration, iron or copper jackets were used in combination with non-porous or porous spacers. Water content was measured before and after experiments using Karl Fischer Titration (KFT). All experiments show, after yielding at 0.6% strain, rapid hardening in nearly linear fashion until about 4-5% strain, from where stress increases at reducing rates to values at 10% strain, between 400 and 675 MPa, depending on experimental conditions. Neither failure nor steady state however, is achieved within the maximum strain of 13%. Experiments performed under 500 °C and 300 MPa confining pressure show weak strain rate dependence. In addition, iron-jacketed samples appear harder than copper-jacketed ones. At 700 °C samples are 17 to 37% weaker and more sensitive to strain rate than during 500 °C experiments. Although, iron-jacketed samples behave stronger than copper-jacketed ones. By visual inspection, samples appear homogeneously shortened. Preliminary analysis suggests that deformation is mostly accommodated by pore collapse. Although, with finite strain, pore collapse becomes less significant. A temperature, strain rate and jacket material dependent

  17. Genetic counseling and cardiac care in predictively tested hypertrophic cardiomyopathy mutation carriers: The patients' perspective

    NARCIS (Netherlands)

    Christiaans, Imke; Van Langen, Irene M.; Birnie, Erwin; Bonsel, Gouke J.; Wilde, Arthur A. M.; Smets, Ellen M. A.

    2009-01-01

    Hypertrophic cardiomyopathy (HCM) is a common hereditary heart disease associated with sudden cardiac death. Predictive genetic counseling and testing are performed using adapted Huntington guidelines, that is, psychosocial care and time for reflection are not obligatory and the test result can be

  18. Highly frequent mutations in negative regulators of multiple virulence genes in group A streptococcal toxic shock syndrome isolates.

    Directory of Open Access Journals (Sweden)

    Tadayoshi Ikebe

    2010-04-01

    Full Text Available Streptococcal toxic shock syndrome (STSS is a severe invasive infection characterized by the sudden onset of shock and multiorgan failure; it has a high mortality rate. Although a number of studies have attempted to determine the crucial factors behind the onset of STSS, the responsible genes in group A Streptococcus have not been clarified. We previously reported that mutations of csrS/csrR genes, a two-component negative regulator system for multiple virulence genes of Streptococcus pyogenes, are found among the isolates from STSS patients. In the present study, mutations of another negative regulator, rgg, were also found in clinical isolates of STSS patients. The rgg mutants from STSS clinical isolates enhanced lethality and impaired various organs in the mouse models, similar to the csrS mutants, and precluded their being killed by human neutrophils, mainly due to an overproduction of SLO. When we assessed the mutation frequency of csrS, csrR, and rgg genes among S. pyogenes isolates from STSS (164 isolates and non-invasive infections (59 isolates, 57.3% of the STSS isolates had mutations of one or more genes among three genes, while isolates from patients with non-invasive disease had significantly fewer mutations in these genes (1.7%. The results of the present study suggest that mutations in the negative regulators csrS/csrR and rgg of S. pyogenes are crucial factors in the pathogenesis of STSS, as they lead to the overproduction of multiple virulence factors.

  19. A novel frameshift mutation in BLM gene associated with high sister chromatid exchanges (SCE) in heterozygous family members.

    Science.gov (United States)

    Ben Salah, Ghada; Hadj Salem, Ikhlas; Masmoudi, Abderrahmen; Kallabi, Fakhri; Turki, Hamida; Fakhfakh, Faiza; Ayadi, Hamadi; Kamoun, Hassen

    2014-11-01

    The Bloom syndrome (BS) is an autosomic recessive disorder comprising a wide range of abnormalities, including stunted growth, immunodeficiency, sun sensitivity and increased frequency of various types of cancer. Bloom syndrome cells display a high level of genetic instability, including a 10-fold increase in the sister chromatid exchanges (SCE) level. Bloom syndrome arises through mutations in both alleles of the BLM gene, which was identified as a member of the RecQ helicase family. In this study, we screened a Tunisian family with three BS patients. Cytogenetic analysis showed several chromosomal aberrations, and an approximately 14-fold elevated SCE frequency in BS cells. A significant increase in SCE frequency was observed in some family members but not reaching the BS patients values, leading to suggest that this could be due to the heterozygous profile. Microsatellite genotyping using four fluorescent dye-labeled microsatellite markers revealed evidence of linkage to BLM locus and the healthy members, sharing higher SCE frequency, showed heterozygous haplotypes as expected. Additionally, the direct BLM gene sequencing identified a novel homozygous frameshift mutation c.3617-3619delAA (p.K1207fsX9) in BS patients and a heterozygous BLM mutation in the family members with higher SCE frequency. Our findings suggest that this latter mutation likely leads to a reduced BLM activity explaining the homologous recombination repair defect and, therefore, the increase in SCE. Based on the present data, the screening of this mutation could contribute to the rapid diagnosis of BS. The genetic confirmation of the mutation in BLM gene provides crucial information for genetic counseling and prenatal diagnosis.

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

    Science.gov (United States)

    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. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  1. A family-based probabilistic method for capturing de novo mutations from high-throughput short-read sequencing data.

    Science.gov (United States)

    Cartwright, Reed A; Hussin, Julie; Keebler, Jonathan E M; Stone, Eric A; Awadalla, Philip

    2012-01-06

    Recent advances in high-throughput DNA sequencing technologies and associated statistical analyses have enabled in-depth analysis of whole-genome sequences. As this technology is applied to a growing number of individual human genomes, entire families are now being sequenced. Information contained within the pedigree of a sequenced family can be leveraged when inferring the donors' genotypes. The presence of a de novo mutation within the pedigree is indicated by a violation of Mendelian inheritance laws. Here, we present a method for probabilistically inferring genotypes across a pedigree using high-throughput sequencing data and producing the posterior probability of de novo mutation at each genomic site examined. This framework can be used to disentangle the effects of germline and somatic mutational processes and to simultaneously estimate the effect of sequencing error and the initial genetic variation in the population from which the founders of the pedigree arise. This approach is examined in detail through simulations and areas for method improvement are noted. By applying this method to data from members of a well-defined nuclear family with accurate pedigree information, the stage is set to make the most direct estimates of the human mutation rate to date.

  2. A novel mitochondrial DNA m.7507A>G mutation is only pathogenic at high levels of heteroplasmy.

    Science.gov (United States)

    McCann, Beverly Jo; Tuppen, Helen A L; Küsters, Benno; Lammens, Martin; Smeitink, Jan A M; Taylor, Robert W; Rodenburg, Richard J; Wortmann, Saskia B

    2015-03-01

    We present a Dutch family with a novel disease-causing mutation in the mitochondrial tRNA(Ser(UCN)) gene, m.7507A>G. The index patient died during the neonatal period due to cardio-respiratory failure and fatal lactic acidosis. A second patient, his cousin, has severe hearing loss necessitating cochlear implants and progressive exercise intolerance. Laboratory investigations of both patients revealed combined deficiencies of the enzyme complexes of the mitochondrial respiratory chain in several tissues. Reduced levels of fully assembled complexes I and IV in fibroblasts by BN-PAGE associated with (near) homoplasmic levels of the m.7507A>G mutation in several tissues and a severe reduction in the steady-state level of mt-tRNA(Ser(UCN)) in fibroblasts were observed. The novel mitochondrial DNA mutation was shown to segregate with disease; several healthy maternal family members showed high heteroplasmy levels (up to 76 ± 4% in blood and 68 ± 4% in fibroblasts) which did not lead to any alterations in the activities of the enzyme complexes of the respiratory chain in fibroblasts or clinical signs and symptoms. We hereby conclude that the m.7507A>G mutation causes a heterogeneous clinical phenotype and is only pathogenic at very high levels of mtDNA heteroplasmy. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Mitochondrial 12S Ribosomal RNA A1555G Mutation Associated with Cardiomyopathy and Hearing Loss following High-Dose Chemotherapy and Repeated Aminoglycoside Exposure

    DEFF Research Database (Denmark)

    Skou, Anne-Sofie; Tranebjærg, Lisbeth; Jensen, Tim

    2014-01-01

    A 19-month-old girl with the A1555G mitochondrial mutation in the 12S ribosomal RNA gene and acute myelogenous leukemia developed dilated cardiomyopathy and bilateral sensorineural hearing loss before undergoing allogeneic stem cell transplantation. She had received gentamicin during episodes of ...... of febrile neutropenia. Testing for the A1555G mutation is recommended in patients frequently treated with aminoglycosides....

  4. Intra-tumoral Heterogeneity of KRAS and BRAF Mutation Status in Patients with Advanced Colorectal Cancer (aCRC and Cost-Effectiveness of Multiple Sample Testing

    Directory of Open Access Journals (Sweden)

    Susan D. Richman

    2011-01-01

    Full Text Available KRAS mutation status is established as a predictive biomarker of benefit from anti-EGFr therapies. Mutations are normally assessed using DNA extracted from one formalin-fixed, paraffin-embedded (FFPE tumor block. We assessed heterogeneity of KRAS and BRAF mutation status intra-tumorally (multiple blocks from the same primary tumor. We also investigated the utility and efficiency of genotyping a ‘DNA cocktail’ prepared from multiple blocks. We studied 68 consenting patients in two randomized clinical trials. DNA was extracted, from ≥2 primary tumor FFPE blocks per patient. DNA was genotyped by pyrosequencing for KRAS codons 12, 13 and 61 and BRAF codon 600. In patients with heterogeneous mutation status, DNA cocktails were prepared and genotyped. Among 69 primary tumors in 68 patients, 7 (10.1% showed intratumoral heterogeneity; 5 (7.2% at KRAS codons 12, 13 and 2 (2.9% at BRAF codon 600. In patients displaying heterogeneity, the relevant KRAS or BRAF mutation was also identified in ‘DNA cocktail’ samples when including DNA from mutant and wild-type blocks. Heterogeneity is uncommon but not insignificant. Testing DNA from a single block will wrongly assign wild-type status to 10% patients. Testing more than one block, or preferably preparation of a ‘DNA cocktail’ from two or more tumor blocks, improves mutation detection at minimal extra cost.

  5. Implications of using whole genome sequencing to test unselected populations for high risk breast cancer genes: a modelling study.

    Science.gov (United States)

    Warren-Gash, Charlotte; Kroese, Mark; Burton, Hilary; Pharoah, Paul

    2016-01-01

    The decision to test for high risk breast cancer gene mutations is traditionally based on risk scores derived from age, family and personal cancer history. Next generation sequencing technologies such as whole genome sequencing (WGS) make wider population testing more feasible. In the UK's 100,000 Genomes Project, mutations in 16 genes including BRCA1 and BRCA2 are to be actively sought regardless of clinical presentation. The implications of deploying this approach at scale for patients and clinical services are unclear. In this study we aimed to model the effect of using WGS to test an unselected UK population for high risk BRCA1 and BRCA2 gene variants to inform the debate around approaches to secondary genomic findings. We modelled the test performance of WGS for identifying pathogenic BRCA1 and BRCA2 mutations in an unselected hypothetical population of 100,000 UK women, using published literature to derive model input parameters. We calculated analytic and clinical validity, described potential health outcomes and highlighted current areas of uncertainty. We also performed a sensitivity analysis in which we re-ran the model 100,000 times to investigate the effect of varying input parameters. In our models WGS was predicted to identify correctly 93 pathogenic BRCA1 mutations and 151 BRCA2 mutations in 120 and 200 women respectively, resulting in an analytic sensitivity of 75.5-77.5 %. Of 244 women with identified pathogenic mutations, we estimated that 132 (range 121-198) would develop breast cancer, so could potentially be helped by intervention. We also predicted that breast cancer would occur in 41 women (range 36-62) incorrectly identified with no pathogenic mutations and in 12,460 women without BRCA1 or BRCA2 mutations. There was considerable uncertainty about the penetrance of mutations in people without a family history of disease and the appropriate threshold of absolute disease risk for clinical action, which impacts on judgements about the clinical

  6. Identification of BRCA1/2 Founder Mutations in Southern Chinese Breast Cancer Patients Using Gene Sequencing and High Resolution DNA Melting Analysis

    OpenAIRE

    Ava Kwong; Enders Kai On Ng; Chris Lei Po Wong; Fian Bic Fai Law; Tommy Au; Hong Nei Wong; Kurian, Allison W.; West, Dee W.; Ford, James M.; Edmond Siu Kwan Ma

    2012-01-01

    Background: Ethnic variations in breast cancer epidemiology and genetics have necessitated investigation of the spectra of BRCA1 and BRCA2 mutations in different populations. Knowledge of BRCA mutations in Chinese populations is still largely unknown. We conducted a multi-center study to characterize the spectra of BRCA mutations in Chinese breast and ovarian cancer patients from Southern China. Methodology/Principal Findings: A total of 651 clinically high-risk breast and/or ovarian cancer p...

  7. Use of high-dose ganciclovir for the treatment of cytomegalovirus replication in solid organ transplant patients with ganciclovir resistance-inducing mutations.

    Science.gov (United States)

    Gracia-Ahufinger, Irene; Gutiérrez-Aroca, Juan; Cordero, Elisa; Vidal, Elisa; Cantisán, Sara; del Castillo, Domingo; Martín-Gandul, Cecilia; Rivero, Antonio; Torre-Cisneros, Julián

    2013-04-27

    Experience with high-dose ganciclovir for the management of resistant cytomegalovirus (CMV) replication in transplant patients is limited despite its adoption as an effective therapy by some consensus documents. We studied six cases of CMV replication in solid organ transplant patients whose genotypic testing showed mutations associated with different levels of resistance to ganciclovir. All were treated with high-dose intravenous ganciclovir (7.5-10 mg/kg/12 hr) or oral valganciclovir (1350-1800 mg/12 hr) corrected according to creatinine clearance. The virologic response was considered positive if the CMV plasma viral load was undetectable. Safety was evaluated by clinical assessment, including the review of vital signs and laboratory tests. All patients had asymptomatic replication, except one who had digestive disease. Four patients received universal prophylaxis with valganciclovir. Two patients received preemptive therapy with valganciclovir for individual episodes of replication. Two of the six patients received steroid boluses before the episode of replication by resistant CMV. All patients responded to treatment, including those with mutations associated with a high level of ganciclovir resistance. Four patients had neutropenia (ganciclovir/valganciclovir can be an option in the treatment of resistant CMV replication and could be considered an alternative treatment in nonsevere patients for whom the use of foscarnet should be avoided. The toxicity of this regimen does not appear to limit its use.

  8. Use of a DNA-based test for the mutation associated with porcine stress syndrome (malignant hyperthermia) in 10,000 breeding swine.

    Science.gov (United States)

    O'Brien, P J; Shen, H; Cory, C R; Zhang, X

    1993-09-15

    To test the hypothesis that the mutation associated with porcine stress syndrome (PSS; malignant hyperthermia) was present in a large proportion of North American and English swine, a simple and rapid laboratory protocol was used for cost-effective, large-scale diagnosis of susceptibility to PSS. This PSS test was applied to 10,245 breeding swine of various breeds from 129 farms in the United States, Canada, and England. Approximately 1 of 5 swine was a heterozygous carrier of the PSS mutation, with approximately 1% being homozygotes. Prevalence of the PSS mutation was 97% for 58 Pietrain, 35% for 1,962 Landrace, 15% for 718 Duroc, 19% for 720 Large White, 14% for 496 Hampshire, 19% for 1,727 Yorkshire, and 16% for 3,446 crossbred swine. The PPS gene frequencies for these breeds were 0.72, 0.19, 0.08, 0.10, 0.07, 0.10, and 0.09, respectively. In addition to these breeds, we have identified the PSS mutation in Poland China and Berkshire breeds. These gene frequencies were 30 to 75% lower in Canadian swine than in US swine, with the exception of Yorkshires, for which the gene frequency was threefold higher in Canadian swine. English swine were similarly, or more so, affected than were US swine. Accuracy was estimated at > 99%. Cost to perform the test was < $20/animal. Depending on the perceived net balance of deleterious and beneficial effects of the mutation, the PSS test could be used to eradicate the PSS mutation from herds, or for controlled expression of the mutation.

  9. Truncating mutations in SPAST patients are associated with a high rate of psychiatric comorbidities in hereditary spastic paraplegia.

    Science.gov (United States)

    Chelban, Viorica; Tucci, Arianna; Lynch, David S; Polke, James M; Santos, Liana; Jonvik, Hallgeir; Groppa, Stanislav; Wood, Nicholas W; Houlden, Henry

    2017-08-01

    The hereditary spastic paraplegias (HSPs) are a rare and heterogeneous group of neurodegenerative disorders that are clinically characterised by progressive lower limb spasticity. They are classified as either 'pure' or 'complex' where spastic paraplegia is complicated with additional neurological features. Mutations in the spastin gene (SPAST) are the most common cause of HSP and typically present with a pure form. We assessed in detail the phenotypic and genetic spectrum of SPAST-related HSP focused on 118 patients carrying SPAST mutations. This study, one of the largest cohorts of genetically confirmed spastin patients to date, contributes with the discovery of a significant number of novel SPAST mutations. Our data reveal a high rate of complex cases (25%), with psychiatric disorders among the most common comorbidity (10% of all SPASTpatients). Further, we identify a genotype-phenotype correlation between patients carrying loss-of-function mutations in SPAST and the presence of psychiatric disorders. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  10. Long QT interval in Turner syndrome – a high prevalence of LQTS gene mutations

    DEFF Research Database (Denmark)

    Trolle, Christian; Mortensen, Kristian Havmand; Pedersen, Lisbeth Nørum

    Objective: QT interval prolongation of unknown aetiology is common in Turner syndrome (TS). This study set out to explore the presence of known pathogenic long QT (LQT) mutations in TS and to examine the corrected QT interval (QTc) over time and relate the findings to the TS phenotype. Methods......QTc). The prevalence of mutations in genes related to Long QT syndrome (LQTS) was determined in females with TS and a QTc >432.0 milliseconds (ms). Echocardiographic assessment of aortic valve morphology, 24-hour blood pressures and blood samples were done. Results: The mean hQTc in females with TS (414.0±25.5 ms...

  11. Meta-analysis of clinical characteristics of 299 carriers of LMNA gene mutations : do lamin A/C mutations portend a high risk of sudden death?

    NARCIS (Netherlands)

    de Voogt, WG; van der Kooi, AJ; van Tintelen, JP; Bonne, G; Ben Yaou, R; Duboc, D; Rossenbacker, T; Heidbuchel, H; de Visser, M; Crijns, HJGM; Pinto, YM; van Berlo, Jop H.

    This study evaluated common clinical characteristics of patients with lamin A/C gene mutations that cause either isolated dilated cardiornyopathy or dilated cardiomyopathy in association with skeletal muscular dystrophy. We pooled clinical data of all published carriers of lamin A/C gene mutations

  12. HIV-1 Drug Resistance Mutations: Potential Applications for Point-of-Care Genotypic Resistance Testing

    NARCIS (Netherlands)

    Rhee, Soo-Yon; Jordan, Michael R.; Raizes, Elliot; Chua, Arlene; Parkin, Neil; Kantor, Rami; van Zyl, Gert U.; Mukui, Irene; Hosseinipour, Mina C.; Frenkel, Lisa M.; Ndembi, Nicaise; Hamers, Raph L.; Rinke de Wit, Tobias F.; Wallis, Carole L.; Gupta, Ravindra K.; Fokam, Joseph; Zeh, Clement; Schapiro, Jonathan M.; Carmona, Sergio; Katzenstein, David; Tang, Michele; Aghokeng, Avelin F.; de Oliveira, Tulio; Wensing, Annemarie M. J.; Gallant, Joel E.; Wainberg, Mark A.; Richman, Douglas D.; Fitzgibbon, Joseph E.; Schito, Marco; Bertagnolio, Silvia; Yang, Chunfu; Shafer, Robert W.

    2015-01-01

    The increasing prevalence of acquired and transmitted HIV-1 drug resistance is an obstacle to successful antiretroviral therapy (ART) in the low- and middle-income countries (LMICs) hardest hit by the HIV-1 pandemic. Genotypic drug resistance testing could facilitate the choice of initial ART in

  13. HIV-1 drug resistance mutations : Potential applications for point-of-care Genotypic resistance testing

    NARCIS (Netherlands)

    Rhee, Soo Yon; Jordan, Michael R.; Raizes, Elliot; Chua, Arlene; Parkin, Neil; Kantor, Rami; Van Zy, Gert U.; Mukui, Irene; Hosseinipour, Mina C.; Frenkel, Lisa M.; Ndembi, Nicaise; Hamers, Raph L.; De Wit, Tobias F Rinke; Wallis, Carole L.; Gupta, Ravindra K.; Fokam, Joseph; Zeh, Clement; Schapiro, Jonathan M.; Carmona, Sergio; Katzenstein, David; Tang, Michele; Aghokeng, Avelin F.; De Oliveira, Tulio; Wensing, Annemarie M J|info:eu-repo/dai/nl/30817724X; Gallant, Joel E.; Wainberg, Mark A.; Richman, Douglas D.; Fitzgibbon, Joseph E.; Schito, Marco; Bertagnolio, Silvia; Yang, Chunfu; Shafer, Robert W.

    2015-01-01

    The increasing prevalence of acquired and transmitted HIV-1 drug resistance is an obstacle to successful antiretroviral therapy (ART) in the low- and middle-income countries (LMICs) hardest hit by the HIV-1 pandemic. Genotypic drug resistance testing could facilitate the choice of initial ART in

  14. TP53 Mutation Status of Tubo-ovarian and Peritoneal High-grade Serous Carcinoma with a Wild-type p53 Immunostaining Pattern.

    Science.gov (United States)

    Na, Kiyong; Sung, Ji-Youn; Kim, Hyun-Soo

    2017-12-01

    Diffuse and strong nuclear p53 immunoreactivity and a complete lack of p53 expression are regarded as indicative of missense and nonsense mutations, respectively, of the TP53 gene. Tubo-ovarian and peritoneal high-grade serous carcinoma (HGSC) is characterized by aberrant p53 expression induced by a TP53 mutation. However, our experience with some HGSC cases with a wild-type p53 immunostaining pattern led us to comprehensively review previous cases and investigate the TP53 mutational status of the exceptional cases. We analyzed the immunophenotype of 153 cases of HGSC and performed TP53 gene sequencing analysis in those with a wild-type p53 immunostaining pattern. Immunostaining revealed that 109 (71.3%) cases displayed diffuse and strong p53 expression (missense mutation pattern), while 39 (25.5%) had no p53 expression (nonsense mutation pattern). The remaining five cases of HGSC showed a wild-type p53 immunostaining pattern. Direct sequencing analysis revealed that three of these cases harbored nonsense TP53 mutations and two had novel splice site deletions. TP53 mutation is almost invariably present in HGSC, and p53 immunostaining can be used as a surrogate marker of TP53 mutation. In cases with a wild-type p53 immunostaining pattern, direct sequencing for TP53 mutational status can be helpful to confirm the presence of a TP53 mutation. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  15. Distribution of CFTR mutations in Eastern Hungarians: relevance to genetic testing and to the introduction of newborn screening for cystic fibrosis.

    Science.gov (United States)

    Ivady, Gergely; Madar, Laszlo; Nagy, Bela; Gonczi, Ferenc; Ajzner, Eva; Dzsudzsak, Erika; Dvořáková, Lenka; Gombos, Eva; Kappelmayer, Janos; Macek, Milan; Balogh, Istvan

    2011-05-01

    The aim of this study was characterization of an updated distribution of CFTR mutations in a representative cohort of 40 CF patients with the classical form of the disease drawn from Eastern Hungary. Due to the homogeneity of the Hungarian population our data are generally applicable to other regions of the country, including the sizeable diaspora. We utilized the recommended "cascade" CFTR mutation screening approach, initially using a commercial assay, followed by examination of the common "Slavic" deletion CFTRdele2,3(21kb). Subsequently, the entire CFTR coding region of the CFTR gene was sequenced in patients with yet unidentified mutations. The Elucigene CF29(Tm) v2 assay detected 81.25% of all CF causing mutations. An addition of the CFTRdele2,3(21kb) increased the mutation detection rate to 86.25%. DNA sequencing enabled us to identify mutations on 79/80 CF alleles. Mutations [CFTRdele2,3(21kb), p.Gln685ThrfsX4 (2184insA) were found at an unusually high frequency, each comprising 5.00% of all CF alleles. We have identified common CF causing mutations in the Hungarian population with the most common mutations (p.Phe508del, p.Asn1303Lys, CFTRdele2,3(21kb), 2184insA, p.Gly542X, and p.Leu101X), comprising over 93.75% of all CF alleles. Obtained data are applicable to the improvement of DNA diagnostics in Hungary and beyond, and are the necessary prerequisite for the introduction of a nationwide "two tier" CF newborn screening program. Copyright © 2011 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

  16. HNPCC: Six new pathogenic mutations

    Directory of Open Access Journals (Sweden)

    Epplen Joerg T

    2004-06-01

    Full Text Available Abstract Background Hereditary non-polyposis colorectal cancer (HNPCC is an autosomal dominant disease with a high risk for colorectal and endometrial cancer caused by germline mutations in DNA mismatch-repair genes (MMR. HNPCC accounts for approximately 2 to 5% of all colorectal cancers. Here we present 6 novel mutations in the DNA mismatch-repair genes MLH1, MSH2 and MSH6. Methods Patients with clinical diagnosis of HNPCC were counselled. Tumor specimen were analysed for microsatellite instability and immunohistochemistry for MLH1, MSH2 and MSH6 protein was performed. If one of these proteins was not detectable in the tumor mutation analysis of the corresponding gene was carried out. Results We identified 6 frameshift mutations (2 in MLH1, 3 in MSH2, 1 in MSH6 resulting in a premature stop: two mutations in MLH1 (c.2198_2199insAACA [p.N733fsX745], c.2076_2077delTG [p.G693fsX702], three mutations in MSH2 (c.810_811delGT [p.C271fsX282], c.763_766delAGTGinsTT [p.F255fsX282], c.873_876delGACT [p.L292fsX298] and one mutation in MSH6 (c.1421_1422dupTG [p.C475fsX480]. All six tumors tested for microsatellite instability showed high levels of microsatellite instability (MSI-H. Conclusions HNPCC in families with MSH6 germline mutations may show an age of onset that is comparable to this of patients with MLH1 and MSH2 mutations.

  17. High prevalence of DUOX2 mutations in Japanese patients with permanent congenital hypothyroidism or transient hypothyroidism.

    Science.gov (United States)

    Matsuo, Kumihiro; Tanahashi, Yusuke; Mukai, Tokuo; Suzuki, Shigeru; Tajima, Toshihiro; Azuma, Hiroshi; Fujieda, Kenji

    2016-07-01

    Dual oxidase 2 (DUOX2) mutations are a cause of dyshormonogenesis (DH) and have been identified in patients with permanent congenital hypothyroidism (PH) and with transient hypothyroidism (TH). We aimed to elucidate the prevalence and phenotypical variations of DUOX2 mutations. Forty-eight Japanese DH patients were enroled and analysed for sequence variants of DUOX2, DUOXA2, and TPO using polymerase chain reaction-amplified direct sequencing. Fourteen sequence variants of DUOX2, including 10 novel variants, were identified in 11 patients. DUOX2 variants were more prevalent (11/48, 22.9%) than TPO (3/48, 6.3%) (p=0.020). The prevalence of DUOX2 variants in TH was slightly, but not significantly, higher than in PH. Furthermore, one patient had digenic heterozygous sequence variants of both DUOX2 and TPO. Our results suggest that DUOX2 mutations might be the most common cause of both PH and TH, and that phenotypes of these mutations might be milder than those of other causes.

  18. High Frequency of RPL22 Mutations in Microsatellite-Unstable Colorectal and Endometrial Tumors

    NARCIS (Netherlands)

    Monteira Ferreira, Ana M.; Tuominen, Iina; van Dijk-Bos, Krista; Sanjabi, Bahram; van der Sluis, Tineke; van der Zee, Ate G.; Hollema, Harry; Zazula, Monika; Sijmons, Rolf H.; Aaltonen, Lauri A.; Westers, Helga; Hofstra, Robert M. W.

    2014-01-01

    Ribosomal Protein L22 (RPL22) encodes a protein that is a component of the 60S subunit of the ribosome. Variants in this gene have recently been linked to cancer development. Mutations in an A8 repeat in exon 2 were found in a recent study in 52% of microsatellite-unstable endometrial tumors. These

  19. Efficient molecular screening of Lynch syndrome by specific 3' promoter methylation of the MLH1 or BRAF mutation in colorectal cancer with high-frequency microsatellite instability.

    Science.gov (United States)

    Nakagawa, Hitoshi; Nagasaka, Takeshi; Cullings, Harry M; Notohara, Kenji; Hoshijima, Naoko; Young, Joanne; Lynch, Henry T; Tanaka, Noriaki; Matsubara, Nagahide

    2009-06-01

    It is sometimes difficult to diagnose Lynch syndrome by the simple but strict clinical criteria, or even by the definitive genetic testing for causative germline mutation of mismatch repair genes. Thus, some practical and efficient screening strategy to select highly possible Lynch syndrome patients is exceedingly desirable. We performed a comprehensive study to evaluate the methylation status of whole MLH1 promoter region by direct bisulfite sequencing of the entire MLH1 promoter regions on Lynch and non-Lynch colorectal cancers (CRCs). Then, we established a convenient assay to detect methylation in key CpG islands responsible for the silencing of MLH1 expression. We studied the methylation status of MLH1 as well as the CpG island methylator phenotype (CIMP) and immunohistochemical analysis of mismatch repair proteins on 16 cases of Lynch CRC and 19 cases of sporadic CRCs with high-frequency microsatellite instability (MSI-H). Sensitivity to detect Lynch syndrome by MLH1 (CCAAT) methylation was 88% and the specificity was 84%. Positive likelihood ratio (PLR) was 5.5 and negative likelihood ratio (NLR) was 0.15. Sensitivity by mutational analysis of BRAF was 100%, specificity was 84%, PLR was 6.3 and NLR was zero. By CIMP analysis; sensitivity was 88%, specificity was 79%, PLR was 4.2, and NLR was 0.16. BRAF mutation or MLH1 methylation analysis combined with MSI testing could be a good alternative to screen Lynch syndrome patients in a cost effective manner. Although the assay for CIMP status also showed acceptable sensitivity and specificity, it may not be practical because of its rather complicated assay.

  20. Real-Time PCR and High-Resolution Melt Analysis for Rapid Detection of Mycobacterium leprae Drug Resistance Mutations and Strain Types

    Science.gov (United States)

    Li, Wei; Matsuoka, Masanori; Kai, Masanori; Thapa, Pratibha; Khadge, Saraswoti; Hagge, Deanna A.; Brennan, Patrick J.

    2012-01-01

    Drug resistance surveillance and strain typing of Mycobacterium leprae are necessary to investigate ongoing transmission of leprosy in regions of endemicity. To enable wider implementation of these molecular analyses, novel real-time PCR–high-resolution melt (RT-PCR-HRM) assays without allele-specific primers or probes and post-PCR sample handling were developed. For the detection of mutations within drug resistance-determining regions (DRDRs) of folP1, rpoB, and gyrA, targets for dapsone, rifampin, and fluoroquinolones, real-time PCR-HRM assays were developed. Wild-type and drug-resistant mouse footpad-derived strains that included three folP1, two rpoB, and one gyrA mutation types in a reference panel were tested. RT-PCR-HRM correctly distinguished the wild type from the mutant strains. In addition, RT-PCR-HRM analyses aided in recognizing samples with mixed or minor alleles and also a mislabeled sample. When tested in 121 sequence-characterized clinical strains, HRM identified all the folP1 mutants representing two mutation types, including one not within the reference panel. The false positives (PCR inhibition. A second set of RT-PCR-HRM assays for identification of three previously reported single nucleotide polymorphisms (SNPs) that have been used for strain typing were developed and validated in 22 reference and 25 clinical strains. Real-time PCR-HRM is a sensitive, simple, rapid, and high-throughput tool for routine screening known DRDR mutants in new and relapsed cases, SNP typing, and detection of minor mutant alleles in the wild-type background at lower costs than current methods and with the potential for quality control in leprosy investigations. PMID:22170923

  1. ExScalibur: A High-Performance Cloud-Enabled Suite for Whole Exome Germline and Somatic Mutation Identification.

    Science.gov (United States)

    Bao, Riyue; Hernandez, Kyle; Huang, Lei; Kang, Wenjun; Bartom, Elizabeth; Onel, Kenan; Volchenboum, Samuel; Andrade, Jorge

    2015-01-01

    Whole exome sequencing has facilitated the discovery of causal genetic variants associated with human diseases at deep coverage and low cost. In particular, the detection of somatic mutations from tumor/normal pairs has provided insights into the cancer genome. Although there is an abundance of publicly-available software for the detection of germline and somatic variants, concordance is generally limited among variant callers and alignment algorithms. Successful integration of variants detected by multiple methods requires in-depth knowledge of the software, access to high-performance computing resources, and advanced programming techniques. We present ExScalibur, a set of fully automated, highly scalable and modulated pipelines for whole exome data analysis. The suite integrates multiple alignment and variant calling algorithms for the accurate detection of germline and somatic mutations with close to 99% sensitivity and specificity. ExScalibur implements streamlined execution of analytical modules, real-time monitoring of pipeline progress, robust handling of errors and intuitive documentation that allows for increased reproducibility and sharing of results and workflows. It runs on local computers, high-performance computing clusters and cloud environments. In addition, we provide a data analysis report utility to facilitate visualization of the results that offers interactive exploration of quality control files, read alignment and variant calls, assisting downstream customization of potential disease-causing mutations. ExScalibur is open-source and is also available as a public image on Amazon cloud.

  2. Germline and somatic mosaicism for FGFR2 mutation in the mother of a child with Crouzon syndrome: Implications for genetic testing in "paternal age-effect" syndromes.

    Science.gov (United States)

    Goriely, Anne; Lord, Helen; Lim, Jasmine; Johnson, David; Lester, Tracy; Firth, Helen V; Wilkie, Andrew O M

    2010-08-01

    Crouzon syndrome is a dominantly inherited disorder characterized by craniosynostosis and facial dysostosis, caused by mutations in the fibroblast growth factor receptor 2 (FGFR2) gene; it belongs to a class of disorders that mostly arise as de novo mutations and exhibit a near-exclusive paternal origin of mutation and elevated paternal age ("paternal age effect"). However, even if this is the major mode of origin of mutations in paternal age-effect disorders, germline mosaicism may also occur. Here we describe the first molecularly documented evidence of germline and somatic mosaicism for FGFR2 mutation, identified in the mother of a child with Crouzon syndrome caused by a heterozygous c.1007A>G (p.Asp336Gly) substitution. Levels of maternal somatic mosaicism for this mutation, estimated by pyrosequencing, ranged from 3.3% in hair roots to 14.1% in blood. Our observation underlines the importance of parental molecular testing for accurate genetic counseling of the risk of recurrence for Crouzon, and other paternal age-effect syndromes.

  3. High-speed and intercity passenger rail testing strategy.

    Science.gov (United States)

    2013-05-01

    This high-speed and intercity passenger rail (HSIPR) testing strategy addresses the requirements for testing of high-speed train sets and technology before introduction to the North American railroad system. The report documents the results of a surv...

  4. High Power Fiber Laser Test Bed

    Data.gov (United States)

    Federal Laboratory Consortium — This facility, unique within DoD, power-combines numerous cutting-edge fiber-coupled laser diode modules (FCLDM) to integrate pumping of high power rare earth-doped...

  5. Clinical Validation of a Multiplex Kit for RAS Mutations in Colorectal Cancer: Results of the RASKET (RAS KEy Testing) Prospective, Multicenter Study

    Science.gov (United States)

    Yoshino, Takayuki; Muro, Kei; Yamaguchi, Kensei; Nishina, Tomohiro; Denda, Tadamichi; Kudo, Toshihiro; Okamoto, Wataru; Taniguchi, Hiroya; Akagi, Kiwamu; Kajiwara, Takeshi; Hironaka, Shuichi; Satoh, Taroh

    2015-01-01

    Background RAS (KRAS and NRAS) testing is required to predict anti-epidermal growth factor receptor (EGFR) treatment efficacy in metastatic colorectal cancer (CRC). Although direct sequencing (DS) with manual microdissection (MMD) is widely used, a diagnostic kit providing rapid detections of RAS mutations would be clinically beneficial. We evaluated the MEBGENTM RASKET KIT (RASKET KIT), a multiplex assay using PCR-reverse sequence specific oligonucleotide and xMAP® technology to concurrently detect exon 2, 3, and 4 RAS mutations in a short turnaround time (4.5 h/96-specimens). Methods Formalin-fixed paraffin-embedded (FFPE) tissues were obtained from 308 consenting patients with histologically-confirmed CRC at six hospitals in Japan. For the RASKET KIT, we used only 50–100 ng DNA from each FFPE specimen not processed by MMD. The primary endpoint was the concordance rate between RAS mutations identified with the RASKET KIT and two reference assays (DS with MMD and TheraScreen® K-RAS Mutation Kit). As the secondary endpoints, we evaluated the concordance rate between DS and the RASKET KIT for RAS mutations in the wild-type KRAS exon 2 population and the genotyping performance of the RASKET KIT compared with DS. Findings Among 307 analyzable specimens, the reference assays detected 140 (45.6%, 140/307) RAS mutations: 111 KRAS exon 2 and 29 other (minor) RAS mutations. The RASKET KIT detected 143 (46.6%, 143/307) mutations: 114 KRAS exon 2 and 29 minor RAS mutations. The between-method concordance rate was 96.7% (297/307) (95% CI: 94.1–98.4%). Minor RAS mutations were detected in 15.7% (30/191) of the wild-type KRAS exon 2 population (n = 191); the concordance rate was 98.4% (188/191) (95% CI: 95.5–99.7%). The concordance rate of RAS genotyping was 100% (139/139) (95% CI: 97–100%). Interpretation The RASKET KIT provides rapid and precise detections of RAS mutations and consequently, quicker and more effective anti-EGFR therapy for CRC (Study ID: UMIN

  6. Impact of country of birth on genetic testing of metastatic lung adenocarcinomas in France: African women exhibit a mutational spectrum more similar to Asians than to Caucasians.

    Science.gov (United States)

    Saffroy, Raphael; Morère, Jean-François; Bosselut, Nelly; Innominato, Pasquale F; Hamelin, Jocelyne; Trédaniel, Jean; Masse, Sophie; Dussaule-Duchatelle, Véronique; Balaton, André; Validire, Pierre; Guettier, Catherine; Bouchahda, Mohamed; Lemoine, Antoinette

    2017-08-01

    Limited data are available on the prevalence of oncogenic driver mutations in Caucasian populations, and especially in Europeans. To evaluate the targetable mutational spectra in unselected patients with lung adenocarcinoma in routine clinical practice from several French hospitals, using the same molecular platform. Samples from 2,219 consecutive patients with histologically-proven advanced lung adenocarcinoma were centrally analysed at a referenced and certified diagnostic platform in order to test for activating and resistance mutations in EGFR , KRAS , BRAF , ERBB2 and PI3KCA . Demographic and clinical features were retrieved from the medical charts. Multivariate binary logistic regression was used to determine the independent predictive factors for the occurrence of specific mutations, in the whole study population or in selected subgroups. The overall respective incidence of EGFR , KRAS , BRAF , ERBB2 and PI3KCA mutations was 10.5%, 0.9%, 25%, 1.5%, 2.1% and 1.4%, in our study sample including 87.4% white Caucasians, 10.8% Africans and 1.8% Asians; 60.6% men, 30.7% never smoker (median age: 68.3 years). Ethnicity was an independent predictor for EGFR, KRAS and ERBB2 gene abnormalities. In all cases, a significantly higher prevalence of targetable EGFR and ERBB2 , and a lower prevalence of resistance KRAS mutations were observed in African women as compared to African men or Caucasians. In real life conditions of routine genetic testing, we have identified subsets of patients with specific targetable activating somatic mutations according to ethnicity, who could preferentially benefit from anti- EGFR and anti- ERBB2 targeted therapies.

  7. Androgen receptor mutations associated with androgen insensitivity syndrome: a high content analysis approach leading to personalized medicine.

    Directory of Open Access Journals (Sweden)

    Adam T Szafran

    Full Text Available Androgen insensitivity syndrome (AIS is a rare disease associated with inactivating mutations of AR that disrupt male sexual differentiation, and cause a spectrum of phenotypic abnormalities having as a common denominator loss of reproductive viability. No established treatment exists for these conditions, however there are sporadic reports of patients (or recapitulated mutations in cell lines that respond to administration of supraphysiologic doses (or pulses of testosterone or synthetic ligands. Here, we utilize a novel high content analysis (HCA approach to study AR function at the single cell level in genital skin fibroblasts (GSF. We discuss in detail findings in GSF from three historical patients with AIS, which include identification of novel mechanisms of AR malfunction, and the potential ability to utilize HCA for personalized treatment of patients affected by this condition.

  8. High grade leiomyosarcoma of the testes

    Directory of Open Access Journals (Sweden)

    Girish D. Bakhshi

    2011-11-01

    Full Text Available Testicular leiomyosarcoma is a rare tumor. It may arise secondarily following exposure to radiotherapy, chronic inflammation, or usage of high dose anabolic steroids. However, in absence of risk factors, it is rarely seen. Only 15 cases of Primary Intra testicular leiomyosarcoma have been reported in world literature. We present a case of testicular tumor in an elderly male. Preoperative work up showed raised Lactate Dehydrogenase (LDH levels. He underwent high orchidectomy. Histopathology and immunohistochemistry confirmed it to be a primary intra testicular leiomyosarcoma. A brief case report with review of literature is presented.

  9. Accuracy of Hereditary Diffuse Gastric Cancer Testing Criteria and Outcomes in Patients With a Germline Mutation in CDH1

    NARCIS (Netherlands)

    van der Post, Rachel S.; Vogelaar, Ingrid P.; Manders, Peggy; van der Kolk, Lizet E.; Cats, Annemieke; van Hest, Liselotte P.; Sijmons, Rolf; Aalfs, Cora M.; Ausems, Margreet G. E. M.; Garcia, Encarna B. Gomez; Wagner, Anja; Hes, Frederik J.; Arts, Neeltje; Mensenkamp, Arjen R.; van Krieken, J. Han; Hoogerbrugge, Nicoline; Ligtenberg, Marjolijn J. L.

    2015-01-01

    BACKGROUND & AIMS: Germline mutations in the cadherin 1, type 1, E-cadherin gene (CDH1) cause a predisposition to gastric cancer. We evaluated the ability of the internationally accepted hereditary diffuse gastric cancer (HDGC) criteria to identify individuals with pathogenic mutations in CDH1, and

  10. Accuracy of Hereditary Diffuse Gastric Cancer Testing Criteria and Outcomes in Patients With a Germline Mutation in CDH1

    NARCIS (Netherlands)

    Van Der Post, Rachel S.; Vogelaar, Ingrid P.; Manders, Peggy; Van Der Kolk, Lizet E.; Cats, Annemieke; Van Hest, Liselotte P.; Sijmons, Rolf; Aalfs, Cora M.; Ausems, Margreet G E M; Gómez García, Encarna B.; Wagner, Anja; Hes, Frederik J.; Arts, Neeltje; Mensenkamp, Arjen R.; Van Krieken, J. Han; Hoogerbrugge, Nicoline; Ligtenberg, Marjolijn J L

    2015-01-01

    Background & Aims Germline mutations in the cadherin 1, type 1, E-cadherin gene (CDH1) cause a predisposition to gastric cancer. We evaluated the ability of the internationally accepted hereditary diffuse gastric cancer (HDGC) criteria to identify individuals with pathogenic mutations in CDH1, and

  11. A combination of immunohistochemistry and molecular approaches improves highly sensitive detection of BRAF mutations in papillary thyroid cancer.

    Science.gov (United States)

    Martinuzzi, Claudia; Pastorino, Lorenza; Andreotti, Virginia; Garuti, Anna; Minuto, Michele; Fiocca, Roberto; Bianchi-Scarrà, Giovanna; Ghiorzo, Paola; Grillo, Federica; Mastracci, Luca

    2016-09-01

    The optimal method for BRAF mutation detection remains to be determined despite advances in molecular detection techniques. The aim of this study was to compare, against classical Sanger sequencing, the diagnostic performance of two of the most recently developed, highly sensitive methods: BRAF V600E immunohistochemistry (IHC) and peptide nucleic-acid (PNA)-clamp qPCR. BRAF exon 15 mutations were searched in formalin-fixed paraffin-embedded tissues from 86 papillary thyroid carcinoma using the three methods. The limits of detection of Sanger sequencing in borderline or discordant cases were quantified by next generation sequencing. BRAF mutations were found in 74.4 % of cases by PNA, in 71 % of cases by IHC, and in 64 % of cases by Sanger sequencing. Complete concordance for the three methods was observed in 80 % of samples. Better concordance was observed with the combination of two methods, particularly PNA and IHC (59/64) (92 %), while the combination of PNA and Sanger was concordant in 55 cases (86 %). Sensitivity of the three methods was 99 % for PNA, 94.2 % for IHC, and 89.5 % for Sanger. Our data show that IHC could be used as a cost-effective, first-line method for BRAF V600E detection in daily practice, followed by PNA analysis in negative or uninterpretable cases, as the most efficient method. PNA-clamp quantitative PCR is highly sensitive and complementary to IHC as it also recognizes other mutations besides V600E and it is suitable for diagnostic purposes.

  12. Instrumented impact testing at high velocities

    Science.gov (United States)

    Delfosse, Daniel; Pageau, Gilles; Bennett, Roger; Poursartip, Anoush

    Impact loading of carbon fiber-reinforced plastic CFRP) aircraft parts is a major concern. Birds or hailstones striking an aircraft generally have a low mass and a high velocity, whereas typically instrumented impact experiments are performed with a high mass and a low velocity. Our aim has been to build an instrumented impact facility with a low-mass projectile capable of simulating these impact events, since there is evidence that a low-velocity impact will not always result in the same amount or even type of damage as a high-velocity impact. This paper provides a detailed description of the instrumented low-mass impact facility at The University of British Columbia (UBC). A gas gun is used to accelerate the instrumented projectile to impact velocities as high as 50 m/s, corresponding to an energy level of 350 J. The contact force during the impact event is measured by an incorporated load cell. The necessary mathematical operations to determine the real load-displacement curves are outlined, and the results of some impact events at different velocities are shown.

  13. A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species.

    Science.gov (United States)

    Nyerges, Ákos; Csörgő, Bálint; Nagy, István; Bálint, Balázs; Bihari, Péter; Lázár, Viktória; Apjok, Gábor; Umenhoffer, Kinga; Bogos, Balázs; Pósfai, György; Pál, Csaba

    2016-03-01

    Currently available tools for multiplex bacterial genome engineering are optimized for a few laboratory model strains, demand extensive prior modification of the host strain, and lead to the accumulation of numerous off-target modifications. Building on prior development of multiplex automated genome engineering (MAGE), our work addresses these problems in a single framework. Using a dominant-negative mutant protein of the methyl-directed mismatch repair (MMR) system, we achieved a transient suppression of DNA repair in Escherichia coli, which is necessary for efficient oligonucleotide integration. By integrating all necessary components into a broad-host vector, we developed a new workflow we term pORTMAGE. It allows efficient modification of multiple loci, without any observable off-target mutagenesis and prior modification of the host genome. Because of the conserved nature of the bacterial MMR system, pORTMAGE simultaneously allows genome editing and mutant library generation in other biotechnologically and clinically relevant bacterial species. Finally, we applied pORTMAGE to study a set of antibiotic resistance-conferring mutations in Salmonella enterica and E. coli. Despite over 100 million y of divergence between the two species, mutational effects remained generally conserved. In sum, a single transformation of a pORTMAGE plasmid allows bacterial species of interest to become an efficient host for genome engineering. These advances pave the way toward biotechnological and therapeutic applications. Finally, pORTMAGE allows systematic comparison of mutational effects and epistasis across a wide range of bacterial species.

  14. A double EPSPS gene mutation endowing glyphosate resistance shows a remarkably high resistance cost.

    Science.gov (United States)

    Han, Heping; Vila-Aiub, Martin M; Jalaludin, Adam; Yu, Qin; Powles, Stephen B

    2017-12-01

    A novel glyphosate resistance double point mutation (T102I/P106S, TIPS) in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene has been recently identified for the first time only in the weed species Eleusine indica. Quantification of plant resistance cost associated with the TIPS and the often reported glyphosate resistance single P106S mutation was performed. A significant resistance cost (50% in seed number currency) associated with the homozygous TIPS but not the homozygous P106S EPSPS variant was identified in E. indica plants. The resistance cost associated with the TIPS mutation escalated to 85% in plants under resource competition with rice crops. The resistance cost was not detected in nonhomozygous TIPS plants denoting the recessive nature of the cost associated with the TIPS allele. An excess of 11-fold more shikimate and sixfold more quinate in the shikimate pathway was detected in TIPS plants in the absence of glyphosate treatment compared to wild type, whereas no changes in these compounds were observed in P106S plants when compared to wild type. TIPS plants show altered metabolite levels in several other metabolic pathways that may account for the expression of the observed resistance cost. © 2017 John Wiley & Sons Ltd.

  15. RAD51C mutation screening in high-risk patients from Serbian hereditary breast/ovarian cancer families.

    Science.gov (United States)

    Krivokuca, Ana; Yanowski, Kira; Rakobradovic, Jelena; Benitez, Javier; Brankovic-Magic, Mirjana

    2015-01-01

    In 2010 an important finding was published showing that heterozygous mutations in RAD51C were highly penetrant and were able to confer an increased risk for breast and ovarian cancers. The role of possible third high penetrance breast cancer susceptibility gene was assigned to RAD51C. Because of its rising importance in breast cancer development and the lack of information about RAD51C in Slavic populations, our goal was to identify potential population specific mutations in this gene in order to determine more detailed genetic screening strategy and breast cancer risk assessment. The study included 55 females from Serbian hereditary breast/ovarian cancer families negative for sequence alterations and large genomic rearrangements in BRCA1/2 genes. Whole coding region and exon-intron boundaries of RAD51C were analyzed by dHPLC. All mutations were confirmed by Sanger sequencing. SIFT and Polyphen were used to predict possible impact of non-synonymous variants. We found 5 variants in RAD51C including two missense, one intronic, one in the 5'UTR and one variant in the promoter region of the gene. Three detected variants are common - c.1-118G>A (rs16943176, MAF = 0,203); c.1-26C>T (rs12946397, MAF = 0,207) and c.904+34T>C (rs28363318, MAF = 0,186). We detected two missense variants, c.790G>A (p.Gly264Ser) in exon 5 and c.859A>G (p.Thr287Ala) in exon 6. Both of them were previously shown to exhibit reduced protein function but their contribution to cancer risk is still unknown. Although the initial reports implied that RAD51C might be promising candidate for next high penetrance breast cancer susceptibility gene, lack of confirmation suggested that RAD51C mutations are not as common as expected. Our study did not reveal truncating mutations in RAD51C suggesting that other breast cancer susceptibility genes may account for the increased susceptibility in our cohort of high-risk BRCA1/2 negative families.

  16. RAS testing practices and RAS mutation prevalence among patients with metastatic colorectal cancer: results from a Europe-wide survey of pathology centres

    NARCIS (Netherlands)

    Boleij, A.; Tack, V.; Taylor, A.; Kafatos, G.; Jenkins-Anderson, S.; Tembuyser, L.; Dequeker, E.; Krieken, J.H.J.M. van

    2016-01-01

    BACKGROUND: Treatment options for patients with metastatic colorectal cancer (mCRC) include anti-epithelial growth factor therapies, which, in Europe, are indicated in patients with RAS wild-type tumours only and require prior mutation testing of "hot-spot" codons in exons 2, 3 and 4 of KRAS and

  17. Highly sensitive silicon microreactor for catalyst testing

    DEFF Research Database (Denmark)

    Henriksen, Toke Riishøj; Olsen, Jakob Lind; Vesborg, Peter Christian Kjærgaard

    2009-01-01

    by directing the entire gas flow through the catalyst bed to a mass spectrometer, thus ensuring that nearly all reaction products are present in the analyzed gas flow. Although the device can be employed for testing a wide range of catalysts, the primary aim of the design is to allow characterization of model...... catalysts which can only be obtained in small quantities. Such measurements are of significant fundamental interest but are challenging because of the low surface areas involved. The relationship between the reaction zone gas flow and the pressure in the reaction zone is investigated experimentally......, it is found that platinum catalysts with areas as small as 15 mu m(2) are conveniently characterized with the device. (C) 2009 American Institute of Physics. [doi:10.1063/1.3270191]...

  18. Genomic characterization of high-count MBL cases indicates that early detection of driver mutations and subclonal expansion are predictors of adverse clinical outcome.

    Science.gov (United States)

    Barrio, S; Shanafelt, T D; Ojha, J; Chaffee, K G; Secreto, C; Kortüm, K M; Pathangey, S; Van-Dyke, D L; Slager, S L; Fonseca, R; Kay, N E; Braggio, E

    2017-01-01

    High-count monoclonal B-cell lymphocytosis (MBL) is an asymptomatic expansion of clonal B cells in the peripheral blood without other manifestations of chronic lymphocytic leukemia (CLL). Yearly, 1% of MBLs evolve to CLL requiring therapy; thus being critical to understand the biological events that determine which MBLs progress to intermediate/advanced CLL. In this study, we performed targeted deep sequencing on 48 high-count MBLs, 47 of them with 2-4 sequential samples analyzed, exploring the mutation status of 21 driver genes and evaluating clonal evolution. We found somatic non-synonymous mutations in 25 MBLs (52%) at the initial time point analyzed, including 12 (25%) with >1 mutated gene. In cases that subsequently progressed to CLL, mutations were detected 41 months (median) prior to progression. Excepting NOTCH1, TP53 and XPO1, which showed a lower incidence in MBL, genes were mutated with a similar prevalence to CLL, indicating the early origin of most driver mutations in the MBL/CLL continuum. MBLs with mutations at the initial time point analyzed were associated with shorter time-to-treatment (TTT). Furthermore, MBLs showing subclonal expansion of driver mutations on sequential evaluation had shorter progression time to CLL and shorter TTT. These findings support that clonal evolution has prognostic implications already at the pre-malignant MBL stage, anticipating which individuals will progress earlier to CLL.

  19. DEVELOPING COMMUNICATIVE LANGUAGE TESTS FOR SENIOR HIGH SCHOOL

    Directory of Open Access Journals (Sweden)

    Y. M. Harsono

    2005-01-01

    Full Text Available The Communicative Approach of teaching English in senior high school in Indonesia has been adopted since the implementation of The 1984 Curriculum, but the tests–the communicative language tests–(CL Tests have not been developed and used properly. The objective of the study is to develop CL Tests for senior high school. The procedure of conducting the study consists of three major steps, that is, identifying the objectives, developing the test specification, and developing the CL Tests. The development of the CL Tests in detail consists of fifteen sub-steps from determining what language skills tested, selecting the suitable source materials, up to rewriting the CL Tests to be used as CL Tests alternative for senior high school. The results of the test development reveal that there are fifteen CL Tests consisting of three tests of listening, three reading, three speaking, and three writing tests. The whole tests have construct and content validity, no complete evidence of concurrent validity with report marks and semester test scores, high to very high inter-rater reliability, and no complete practicality.

  20. Pulmonary Sarcomatoid Carcinomas Commonly Harbor Either Potentially Targetable Genomic Alterations or High Tumor Mutational Burden as Observed by Comprehensive Genomic Profiling.

    Science.gov (United States)

    Schrock, Alexa B; Li, Shuyu D; Frampton, Garrett M; Suh, James; Braun, Eduardo; Mehra, Ranee; Buck, Steven C; Bufill, Jose A; Peled, Nir; Karim, Nagla Abdel; Hsieh, K Cynthia; Doria, Manuel; Knost, James; Chen, Rong; Ou, Sai-Hong Ignatius; Ross, Jeffrey S; Stephens, Philip J; Fishkin, Paul; Miller, Vincent A; Ali, Siraj M; Halmos, Balazs; Liu, Jane J

    2017-06-01

    Pulmonary sarcomatoid carcinoma (PSC) is a high-grade NSCLC characterized by poor prognosis and resistance to chemotherapy. Development of targeted therapeutic strategies for PSC has been hampered because of limited and inconsistent molecular characterization. Hybrid capture-based comprehensive genomic profiling was performed on DNA from formalin-fixed paraffin-embedded sections of 15,867 NSCLCs, including 125 PSCs (0.8%). Tumor mutational burden (TMB) was calculated from 1.11 megabases (Mb) of sequenced DNA. The median age of the patients with PSC was 67 years (range 32-87), 58% were male, and 78% had stage IV disease. Tumor protein p53 gene (TP53) genomic alterations (GAs) were identified in 74% of cases, which had genomics distinct from TP53 wild-type cases, and 62% featured a GA in KRAS (34%) or one of seven genes currently recommended for testing in the National Comprehensive Cancer Network NSCLC guidelines, including the following: hepatocyte growth factor receptor gene (MET) (13.6%), EGFR (8.8%), BRAF (7.2%), erb-b2 receptor tyrosine kinase 2 gene (HER2) (1.6%), and ret proto-oncogene (RET) (0.8%). MET exon 14 alterations were enriched in PSC (12%) compared with non-PSC NSCLCs (∼3%) (p 20 mutations per Mb) was notably higher than in non-PSC NSCLC (20% versus 14%, p = 0.056). Of nine patients with PSC treated with targeted or immunotherapies, three had partial responses and three had stable disease. Potentially targetable GAs in National Comprehensive Cancer Network NSCLC genes (30%) or intermediate or high TMB (43%, >10 mutations per Mb) were identified in most of the PSC cases. Thus, the use of comprehensive genomic profiling in clinical care may provide important treatment options for a historically poorly characterized and difficult to treat disease. Copyright © 2017 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

  1. A missense mutation in a highly conserved alternate exon of dynamin-1 causes epilepsy in fitful mice.

    Directory of Open Access Journals (Sweden)

    Rebecca M Boumil

    2010-08-01

    Full Text Available Dynamin-1 (Dnm1 encodes a large multimeric GTPase necessary for activity-dependent membrane recycling in neurons, including synaptic vesicle endocytosis. Mice heterozygous for a novel spontaneous Dnm1 mutation--fitful--experience recurrent seizures, and homozygotes have more debilitating, often lethal seizures in addition to severe ataxia and neurosensory deficits. Fitful is a missense mutation in an exon that defines the DNM1a isoform, leaving intact the alternatively spliced exon that encodes DNM1b. The expression of the corresponding alternate transcripts is developmentally regulated, with DNM1b expression highest during early neuronal development and DNM1a expression increasing postnatally with synaptic maturation. Mutant DNM1a does not efficiently self-assemble into higher order complexes known to be necessary for proper dynamin function, and it also interferes with endocytic recycling in cell culture. In mice, the mutation results in defective synaptic transmission characterized by a slower recovery from depression after trains of stimulation. The DNM1a and DNM1b isoform pair is highly conserved in vertebrate evolution, whereas invertebrates have only one isoform. We speculate that the emergence of more specialized forms of DNM1 may be important in organisms with complex neuronal function.

  2. Targeted Sequencing of the Mitochondrial Genome of Women at High Risk of Breast Cancer without Detectable Mutations in BRCA1/2.

    Directory of Open Access Journals (Sweden)

    Sophie Blein

    Full Text Available Breast Cancer is a complex multifactorial disease for which high-penetrance mutations have been identified. Approaches used to date have identified genomic features explaining about 50% of breast cancer heritability. A number of low- to medium penetrance alleles (per-allele odds ratio < 1.5 and 4.0, respectively have been identified, suggesting that the remaining heritability is likely to be explained by the cumulative effect of such alleles and/or by rare high-penetrance alleles. Relatively few studies have specifically explored the mitochondrial genome for variants potentially implicated in breast cancer risk. For these reasons, we propose an exploration of the variability of the mitochondrial genome in individuals diagnosed with breast cancer, having a positive breast cancer family history but testing negative for BRCA1/2 pathogenic mutations. We sequenced the mitochondrial genome of 436 index breast cancer cases from the GENESIS study. As expected, no pathogenic genomic pattern common to the 436 women included in our study was observed. The mitochondrial genes MT-ATP6 and MT-CYB were observed to carry the highest number of variants in the study. The proteins encoded by these genes are involved in the structure of the mitochondrial respiration chain, and variants in these genes may impact reactive oxygen species production contributing to carcinogenesis. More functional and epidemiological studies are needed to further investigate to what extent variants identified may influence familial breast cancer risk.

  3. Targeted Sequencing of the Mitochondrial Genome of Women at High Risk of Breast Cancer without Detectable Mutations in BRCA1/2.

    Science.gov (United States)

    Blein, Sophie; Barjhoux, Laure; Damiola, Francesca; Dondon, Marie-Gabrielle; Eon-Marchais, Séverine; Marcou, Morgane; Caron, Olivier; Lortholary, Alain; Buecher, Bruno; Vennin, Philippe; Berthet, Pascaline; Noguès, Catherine; Lasset, Christine; Gauthier-Villars, Marion; Mazoyer, Sylvie; Stoppa-Lyonnet, Dominique; Andrieu, Nadine; Thomas, Gilles; Sinilnikova, Olga M; Cox, David G

    2015-01-01

    Breast Cancer is a complex multifactorial disease for which high-penetrance mutations have been identified. Approaches used to date have identified genomic features explaining about 50% of breast cancer heritability. A number of low- to medium penetrance alleles (per-allele odds ratio high-penetrance alleles. Relatively few studies have specifically explored the mitochondrial genome for variants potentially implicated in breast cancer risk. For these reasons, we propose an exploration of the variability of the mitochondrial genome in individuals diagnosed with breast cancer, having a positive breast cancer family history but testing negative for BRCA1/2 pathogenic mutations. We sequenced the mitochondrial genome of 436 index breast cancer cases from the GENESIS study. As expected, no pathogenic genomic pattern common to the 436 women included in our study was observed. The mitochondrial genes MT-ATP6 and MT-CYB were observed to carry the highest number of variants in the study. The proteins encoded by these genes are involved in the structure of the mitochondrial respiration chain, and variants in these genes may impact reactive oxygen species production contributing to carcinogenesis. More functional and epidemiological studies are needed to further investigate to what extent variants identified may influence familial breast cancer risk.

  4. Highly adaptive tests for group differences in brain functional connectivity

    Directory of Open Access Journals (Sweden)

    Junghi Kim

    2015-01-01

    The proposed tests combine statistical evidence against a null hypothesis from multiple sources across a range of plausible tuning parameter values reflecting uncertainty with the unknown truth. These highly adaptive tests are not only easy to use, but also high-powered robustly across various scenarios. The usage and advantages of these novel tests are demonstrated on an Alzheimer's disease dataset and simulated data.

  5. Combining COLD-PCR and high-resolution melt analysis for rapid detection of low-level, rifampin-resistant mutations in Mycobacterium tuberculosis.

    Science.gov (United States)

    Pang, Yu; Liu, Guan; Wang, Yufeng; Zheng, Suhua; Zhao, Yan-Lin

    2013-04-01

    Multidrug-resistant Mycobacterium tuberculosis (M. tuberculosis) remains a serious threat to public health. Mutational analysis of the gene encoding the beta subunit of RNA polymerase (rpoB) is an established and widely used surrogate marker for multidrug-resistant tuberculosis (MDR-TB). The rpoB-based drug-resistant assay requires relatively less time to detect drug resistance in M. tuberculosis, yet it fails to detect low-level mutations in wild-type DNA. Here, we describe a low-level mutation detection method that combines co-amplification at lower denaturation temperature polymerase chain reaction (COLD-PCR) with high-resolution melting (HRM) analysis, aimed at detecting low-level, rifampin-resistant mutations in M. tuberculosis. Compared to conventional polymerase chain reaction (PCR), dilution experiments demonstrated a four- to eightfold improvement in selectivity using COLD-PCR/HRM to detect low-level, rifampin-resistant mutations. The mutation detection limit of conventional PCR/HRM was approximately 20%, whereas COLD-PCR/HRM had a mutation detection limit of 2.5%. Using traditional PCR/HRM and DNA sequencing, we found rpoB mutation in 110 rifampin-resistant isolates. The use of COLD-PCR/HRM allowed us to detect 10 low-level, rifampin-resistant mutations in 16 additional drug-resistant isolates. The sensitivity of COLD-PCR/HRM (95.2%) is significantly higher than that of PCR/HRM (87.3%). Our findings demonstrate that combined use of COLD-PCR with HRM can provide a sensitivity of at least 5% in detecting rpoB-mutated populations in a wild-type background, decreasing the delay in drug-resistant TB diagnosis and leading to faster, cheaper, more efficient, and more personalized antibiotic treatment, especially for low-level drug resistance mutations among the excess wild-type DNA. Copyright © 2013 Elsevier B.V. All rights reserved.

  6. Frameshift mutation of a histone methylation-related gene SETD1B and its regional heterogeneity in gastric and colorectal cancers with high microsatellite instability.

    Science.gov (United States)

    Choi, Youn Jin; Oh, Hye Rim; Choi, Mi Ryoung; Gwak, Min; An, Chang Hyeok; Chung, Yeun Jun; Yoo, Nam Jin; Lee, Sug Hyung

    2014-08-01

    Histone methyltransferase (HMT), which catalyzes a histone methylation, is frequently altered in cancers at mutation and expression levels. The aims of this study were to explore whether SETD1B, SETDB2, and SETD2, SET domain-containing HMT genes, are mutated and expressionally altered in gastric (GC) and colorectal cancers (CRC). In a public database, we found that SETD1B, SETDB2, and SETD2 had mononucleotide repeats in coding sequences that might be mutation targets in cancers with microsatellite instability (MSI). We analyzed the mutations in 76 GCs and 93 CRCs and found SETD1B (38.7% of GC and 35.6% of CRC with high MSI [MSI-H]), SETDB2 (11.1% of CRC with MSI-H), and SETD2 frameshift mutations (6.7% of CRC with MSI-H). These mutations were not found in stable MSI/low MSI. In addition, we analyzed intratumoral heterogeneity (ITH) of SETD1B mutation in 6 CRCs and found that 2 CRCs harbored regional ITH of SETD1B. We also analyzed SETD1B expression in GC and CRC by immunohistochemistry. Loss of SETD1B expression was identified in 15% to 55% of the GC and CRC with respect to the MSI status. Of note, the loss of expression was more common in those with SETD1B mutations than those with wild-type SETD1B. We identified alterations of SET domain-containing HMT at various levels (frameshift mutations, genetic ITH, and expression loss), which together might play a role in tumorigenesis of GC and CRC with MSI-H. Our data suggest that mutation analysis in multiple regions is needed for a better evaluation of mutation status in CRC with MSI-H. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. High Rate of Recurrent De Novo Mutations in Developmental and Epileptic Encephalopathies.

    Science.gov (United States)

    Hamdan, Fadi F; Myers, Candace T; Cossette, Patrick; Lemay, Philippe; Spiegelman, Dan; Laporte, Alexandre Dionne; Nassif, Christina; Diallo, Ousmane; Monlong, Jean; Cadieux-Dion, Maxime; Dobrzeniecka, Sylvia; Meloche, Caroline; Retterer, Kyle; Cho, Megan T; Rosenfeld, Jill A; Bi, Weimin; Massicotte, Christine; Miguet, Marguerite; Brunga, Ledia; Regan, Brigid M; Mo, Kelly; Tam, Cory; Schneider, Amy; Hollingsworth, Georgie; FitzPatrick, David R; Donaldson, Alan; Canham, Natalie; Blair, Edward; Kerr, Bronwyn; Fry, Andrew E; Thomas, Rhys H; Shelagh, Joss; Hurst, Jane A; Brittain, Helen; Blyth, Moira; Lebel, Robert Roger; Gerkes, Erica H; Davis-Keppen, Laura; Stein, Quinn; Chung, Wendy K; Dorison, Sara J; Benke, Paul J; Fassi, Emily; Corsten-Janssen, Nicole; Kamsteeg, Erik-Jan; Mau-Them, Frederic T; Bruel, Ange-Line; Verloes, Alain; Õunap, Katrin; Wojcik, Monica H; Albert, Dara V F; Venkateswaran, Sunita; Ware, Tyson; Jones, Dean; Liu, Yu-Chi; Mohammad, Shekeeb S; Bizargity, Peyman; Bacino, Carlos A; Leuzzi, Vincenzo; Martinelli, Simone; Dallapiccola, Bruno; Tartaglia, Marco; Blumkin, Lubov; Wierenga, Klaas J; Purcarin, Gabriela; O'Byrne, James J; Stockler, Sylvia; Lehman, Anna; Keren, Boris; Nougues, Marie-Christine; Mignot, Cyril; Auvin, Stéphane; Nava, Caroline; Hiatt, Susan M; Bebin, Martina; Shao, Yunru; Scaglia, Fernando; Lalani, Seema R; Frye, Richard E; Jarjour, Imad T; Jacques, Stéphanie; Boucher, Renee-Myriam; Riou, Emilie; Srour, Myriam; Carmant, Lionel; Lortie, Anne; Major, Philippe; Diadori, Paola; Dubeau, François; D'Anjou, Guy; Bourque, Guillaume; Berkovic, Samuel F; Sadleir, Lynette G; Campeau, Philippe M; Kibar, Zoha; Lafrenière, Ronald G; Girard, Simon L; Mercimek-Mahmutoglu, Saadet; Boelman, Cyrus; Rouleau, Guy A; Scheffer, Ingrid E; Mefford, Heather C; Andrade, Danielle M; Rossignol, Elsa; Minassian, Berge A; Michaud, Jacques L

    2017-11-02

    Developmental and epileptic encephalopathy (DEE) is a group of conditions characterized by the co-occurrence of epilepsy and intellectual disability (ID), typically with developmental plateauing or regression associated with frequent epileptiform activity. The cause of DEE remains unknown in the majority of cases. We performed whole-genome sequencing (WGS) in 197 individuals with unexplained DEE and pharmaco-resistant seizures and in their unaffected parents. We focused our attention on de novo mutations (DNMs) and identified candidate genes containing such variants. We sought to identify additional subjects with DNMs in these genes by performing targeted sequencing in another series of individuals with DEE and by mining various sequencing datasets. We also performed meta-analyses to document enrichment of DNMs in candidate genes by leveraging our WGS dataset with those of several DEE and ID series. By combining these strategies, we were able to provide a causal link between DEE and the following genes: NTRK2, GABRB2, CLTC, DHDDS, NUS1, RAB11A, GABBR2, and SNAP25. Overall, we established a molecular diagnosis in 63/197 (32%) individuals in our WGS series. The main cause of DEE in these individuals was de novo point mutations (53/63 solved cases), followed by inherited mutations (6/63 solved cases) and de novo CNVs (4/63 solved cases). De novo missense variants explained a larger proportion of individuals in our series than in other series that were primarily ascertained because of ID. Moreover, these DNMs were more frequently recurrent than those identified in ID series. These observations indicate that the genetic landscape of DEE might be different from that of ID without epilepsy. Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  8. Limited copy number-high resolution melting (LCN-HRM) enables the detection and identification by sequencing of low level mutations in cancer biopsies

    National Research Council Canada - National Science Library

    Do, Hongdo; Dobrovic, Alexander

    2009-01-01

    .... High resolution melting (HRM) is more sensitive than sequencing but identification of the mutation is desirable, particularly when it is important to discriminate false positives due to PCR errors or template degradation from true...

  9. Thrombophilic mutations among Southern Iranian patients with sickle cell disease: high prevalence of factor V Leiden.

    Science.gov (United States)

    Rahimi, Zohreh; Vaisi-Raygani, Asad; Nagel, Ronald L; Muniz, Adriana

    2008-06-01

    A hypercoagulable state in sickle cell disease (SCD) and beta thalassemia has been established and thrombosis is an important aspect of the clinical spectrum of sickle cell disease. In a case-control study, the prevalence of factor V Leiden and prothrombin G20210A mutations were investigated among SCD patients from Southern Iran. Patients comprised 60 individuals with SCD; of them 35 were with sickle cell anemia (SS) including 21 males and 14 females aged 17.2+/-8.3 years, 15 were sickle cell trait (AS) consisted of nine males and six females aged 30+/-15.4 years and 10 were sickle/beta thalassemia (S/Thal) (three males and seven females) aged 24.6+/-10.4 years. The control group were 126 apparently healthy individuals (50 males and 76 females) aged 20.1+/-9.8 years. Genotyping was done by polymerase chain reaction restriction fragment-length polymorphism (PCR-RFLP) using Mnl I and Hind III for factor V Leiden and prothrombin G20210A, respectively. Heterozygous factor V Leiden mutation was found in five of 35 (14.3%) SS patients, two of 15 (13.3%) AS individuals, one (a sickle/beta-zero thalassemia patient with IVSII.1 G-->A mutation) of 10 S/Thal patients (10%), and two of 126 (1.6%) control subjects (Psickle cell anemia with odds ratios (OR) of 6.5 (95% confidence intervals [CI] 1.19-35.33, P=0.03) in SS patients. However, increased prevalence of the factor V Leiden in AS individuals and S/Thal patients was not statistically significant compared to controls (OR 3.84, 95% CI 0.49-29.9, P=0.19 and OR 3.77, 95% CI 0.31-45.9, P=0.29, respectively). Our findings indicate a significant correlation between factor V Leiden and sickle cell anemia among Iranian patients. Association between venous thrombophilia and factor V Leiden mutation in Iranians with sickle cell anemia should be further studied.

  10. Test of a High Power Target Design

    CERN Multimedia

    2002-01-01

    %IS343 :\\\\ \\\\ A high power tantalum disc-foil target (RIST) has been developed for the proposed radioactive beam facility, SIRIUS, at the Rutherford Appleton Laboratory. The yield and release characteristics of the RIST target design have been measured at ISOLDE. The results indicate that the yields are at least as good as the best ISOLDE roll-foil targets and that the release curves are significantly faster in most cases. Both targets use 20 -25 $\\mu$m thick foils, but in a different internal geometry.\\\\ \\\\Investigations have continued at ISOLDE with targets having different foil thickness and internal geometries in an attempt to understand the release mechanisms and in particular to maximise the yield of short lived isotopes. A theoretical model has been developed which fits the release curves and gives physical values of the diffusion constants.\\\\ \\\\The latest target is constructed from 2 $\\mu$m thick tantalum foils (mass only 10 mg) and shows very short release times. The yield of $^{11}$Li (half-life of ...

  11. Frameshift mutations of chromosome cohesion-related genes SGOL1 and PDS5B in gastric and colorectal cancers with high microsatellite instability.

    Science.gov (United States)

    Kim, Min Sung; An, Chang Hyeok; Yoo, Nam Jin; Lee, Sug Hyung

    2013-10-01

    Cohesin is a protein complex that regulates chromatid cohesion and plays a role in preventing aneuploidy and maintaining chromosomal stability. SGOL1 encodes a cohesin protector, and PDS5B encodes a regulatory cohesion factor. Both SGOL1 and PDS5B are considered putative tumor suppressor genes. The aim of this study was to explore whether SGOL1 and PDS5B genes are mutated and expressionally altered in gastric and colorectal cancers. A genome database indicated that both genes possessed mononucleotide repeats in coding sequences, which could be mutation targets in cancers with microsatellite instability. We analyzed mutations in 91 gastric cancers and 100 colorectal cancers with high microsatellite instability or stable/low microsatellite instability by single-strand conformation polymorphism analysis and DNA sequencing. We also analyzed SGOL1 and PDS5B expression by immunohistochemistry. Overall, we found 21 SGOL1 frameshift mutations in 21 cases and 18 PDS5B frameshift mutations in 16 cases. SGOL1 and PDS5B frameshift mutations were detected in 26.6% and 20.3%, respectively, of high microsatellite instability but not in stable/low microsatellite instability (0/112). By immunohistochemistry, losses of SGOL1 and PDS5B were identified in 19% to 47% of the gastric and colorectal cancers irrespective of microsatellite instability status. The losses were more common in those with frameshift mutations or high microsatellite instability than those without mutations or high microsatellite instability. The data indicate that frameshift mutations of SGOL1 and PDS5B and the loss of their expression may be a feature of gastric and colorectal cancers with high microsatellite instability. In addition, the data suggest that these alterations might contribute to cancer pathogenesis by deregulating cohesin-related functions. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. High Throughput, High Precision Hot Testing Tool for HBLED Wafer Level Testing

    Energy Technology Data Exchange (ETDEWEB)

    Solarz, Richard [KLA-Tencor Corporation, Milpitas, CA (United States); McCord, Mark [KLA-Tencor Corporation, Milpitas, CA (United States)

    2015-12-31

    The Socrates research effort developed an in depth understanding and demonstrated in a prototype tool new precise methods for teh characterization of color characteristics and flux from individual LEDs for the production of uniform quality lighting. This effort was focused on improving the color quality and consistency of solid state lighting and potentially reducing characterization costs for all LED product types. The patented laser hot testing method was demonstrated to be far more accurate than all current state of the art color and flux characterization methods in use by the solid state lighting industry today. A seperately patented LED grouping method (statistical binning) was demonstrated to be a useful approach to improving utilization of entire lots of large color and flux distributions of manufactured LEDs for high quality color solid-state lighting. At the conclusion of the research in late 2015 the solid-state lighting industry was however generally satisfied with its existing production methods for high quality color products for the small segment of customers that demand it, albeit with added costs.

  13. [Germ-line mutation of BRCA1 in patients with breast and/or ovarian cancer in high risk families in Northern France].

    Science.gov (United States)

    Peyrat, J P; Vennin, P; Hornez, L; Bonneterre, J

    1997-01-01

    The BRCA1 gene modification is responsible for an autosomal dominant syndrome of inherited early onset breast and/or ovarian cancer. This gene is estimated to account for almost half of inherited breast cancers and three quarters of inherited breast/ovarian cancers. This suggests that about 1 out of 500 women may carry BRCA1 mutation. The BRCA1 gene was isolated by positional cloning in 1994. More than 100 different mutations have been found in the germline of affected individuals. We looked by systematic sequencing at BRCA1 germline mutations in 36 patients treated at the Centre Oscar-Lambret for breast and/or ovarian cancer and that belonged to high risk families. We have found 24 mutations: 9 true mutations inducing modifications of the BRCA1 protein (BRCA1+), 5 mutations with unknown consequences on the BRCA1 protein and 10 mutations corresponding to polymorphisms that had been previously described. All the BRCA1+ cases had a HPG3 tumor. The median age of discovery and the receptor positivity percentage are lower in hereditary breast cancer than in the standard population of the breast cancers treated in our center. Consequently, BRCA1 mutations are associated to parameters thought to be of bad prognosis.

  14. Coping with the Stress of High Stakes Testing

    Science.gov (United States)

    Kruger, Louis J.; Wandle, Caroline; Struzziero, Joan

    2007-01-01

    High stakes testing puts considerable pressure on schools, teachers, and students to achieve at high levels. Therefore, how schools and individuals cope with this major source of stress may have important implications for the success of high stakes testing. This article reviews relevant theory and research on stress as they relate to public…

  15. A Comparison Between Denaturing Gradient Gel Electrophoresis and Denaturing High Performance Liquid Chromatography in Detecting Mutations in Genes Associated with Hereditary Non-Polyposis Colorectal Cancer (HNPCC and the Identification of 9 New Mutations Previously Unidentified by DGGE

    Directory of Open Access Journals (Sweden)

    Meldrum Cliff J

    2003-12-01

    Full Text Available Abstract Denaturing high performance liquid chromatography is a relatively new method by which heteroduplex structures formed during the PCR amplification of heterozygote samples can be rapidly identified. The use of this technology for mutation detection in hereditary non-polyposis colorectal cancer (HNPCC has the potential to appreciably shorten the time it takes to analyze genes associated with this disorder. Prior to acceptance of this method for screening genes associated with HNPCC, assessment of the reliability of this method should be performed. In this report we have compared mutation and polymorphism detection by denaturing gradient gel electrophoresis (DGGE with denaturing high performance liquid chromatography (DHPLC in a set of 130 families. All mutations/polymorphisms representing base substitutions, deletions, insertions and a 23 base pair inversion were detected by DHPLC whereas DGGE failed to identify four single base substitutions and a single base pair deletion. In addition, we show that DHPLC has been used for the identification of 5 different mutations in exon 7 of hMSH2 that could not be detected by DGGE. From this study we conclude that DHPLC is a more effective and rapid alternative to the detection of mutations in hMSH2 and hMLH1 with the same or better accuracy than DGGE. Furthermore, this technique offers opportunities for automation, which have not been realised for the majority of other methods of gene analysis.

  16. Microarray-based ultra-high resolution discovery of genomic deletion mutations.

    Science.gov (United States)

    Belfield, Eric J; Brown, Carly; Gan, Xiangchao; Jiang, Caifu; Baban, Dilair; Mithani, Aziz; Mott, Richard; Ragoussis, Jiannis; Harberd, Nicholas P

    2014-03-22

    Oligonucleotide microarray-based comparative genomic hybridization (CGH) offers an attractive possible route for the rapid and cost-effective genome-wide discovery of deletion mutations. CGH typically involves comparison of the hybridization intensities of genomic DNA samples with microarray chip representations of entire genomes, and has widespread potential application in experimental research and medical diagnostics. However, the power to detect small deletions is low. Here we use a graduated series of Arabidopsis thaliana genomic deletion mutations (of sizes ranging from 4 bp to ~5 kb) to optimize CGH-based genomic deletion detection. We show that the power to detect smaller deletions (4, 28 and 104 bp) depends upon oligonucleotide density (essentially the number of genome-representative oligonucleotides on the microarray chip), and determine the oligonucleotide spacings necessary to guarantee detection of deletions of specified size. Our findings will enhance a wide range of research and clinical applications, and in particular will aid in the discovery of genomic deletions in the absence of a priori knowledge of their existence.

  17. Group Differences in Test-Taking Behaviour: An Example from a High-Stakes Testing Program

    Science.gov (United States)

    Stenlund, Tova; Eklöf, Hanna; Lyrén, Per-Erik

    2017-01-01

    This study investigated whether different groups of test-takers vary in their reported test-taking behaviour in a high-stakes test situation. A between-group design (N = 1129) was used to examine whether high and low achievers, as well as females and males, differ in their use of test-taking strategies, and in level of reported test anxiety and…

  18. Predicting Freshman Grade Point Average From College Admissions Test Scores and State High School Test Scores

    OpenAIRE

    Daniel Koretz; Carol Yu; Preeya P. Mbekeani; Meredith Langi; Tasmin Dhaliwal; David Braslow

    2016-01-01

    The current focus on assessing “college and career readiness” raises an empirical question: How do high school tests compare with college admissions tests in predicting performance in college? We explored this using data from the City University of New York and public colleges in Kentucky. These two systems differ in the choice of college admissions test, the stakes for students on the high school test, and demographics. We predicted freshman grade point average (FGPA) from high school GPA an...

  19. Motivation for a High Explosive Testing Program in South Africa

    Science.gov (United States)

    2015-12-04

    1~7JJ!i 5a. DATE: 6a. DATE: 7a. DATE: 8. TITLE: Motivation for a High Explosive Testing Program in South Africa 9. CONTRACT NUMBER: 10...00-00-2015 4. TITLE AND SUBTITLE Motivation for a High Explosive Testing Program in South Africa 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM...600 Raleigh, NC 27605 Contract Number: HDTRA2-11-D-0001 Motivation for a High Explosive Testing Program in South Africa 4

  20. Water Containment Systems for Testing High-Speed Flywheels

    Science.gov (United States)

    Trase, Larry; Thompson, Dennis

    2006-01-01

    Water-filled containers are used as building blocks in a new generation of containment systems for testing high-speed flywheels. Such containment systems are needed to ensure safety by trapping high-speed debris in the event of centrifugal breakup or bearing failure. Traditional containment systems for testing flywheels consist mainly of thick steel rings. The effectiveness of this approach to shielding against high-speed debris was demonstrated in a series of tests.

  1. Latex allergy and filaggrin null mutations

    DEFF Research Database (Denmark)

    Carlsen, Berit C; Meldgaard, Michael; Hamann, Dathan

    2011-01-01

    Objectives Natural rubber latex (NRL) contains over 200 proteins of which 13 have been identified as allergens and the cause of type I latex allergy. Health care workers share a high occupational risk for developing latex allergy. Filaggrin null mutations increase the risk of type I sensitizations...... to aeroallergens and it is possible that filaggrin null mutations also increase the risk of latex allergy. The aim of this paper was to examine the association between filaggrin null mutations and type I latex allergy. Methods Twenty latex allergic and 24 non-latex allergic dentists and dental assistants......, occupationally exposed to latex, were genotyped for filaggrin null mutations R501X and 2282del4. Latex allergy was determined by a positive reaction or a historical positive reaction to a skin prick test with NRL. Results 41 individuals were successfully genotyped. Three individuals were filaggrin mutation...

  2. UV Signature Mutations

    Science.gov (United States)

    2014-01-01

    Sequencing complete tumor genomes and exomes has sparked the cancer field's interest in mutation signatures for identifying the tumor's carcinogen. This review and meta-analysis discusses signatures and their proper use. We first distinguish between a mutagen's canonical mutations – deviations from a random distribution of base changes to create a pattern typical of that mutagen – and the subset of signature mutations, which are unique to that mutagen and permit inference backward from mutations to mutagen. To verify UV signature mutations, we assembled literature datasets on cells exposed to UVC, UVB, UVA, or solar simulator light (SSL) and tested canonical UV mutation features as criteria for clustering datasets. A confirmed UV signature was: ≥60% of mutations are C→T at a dipyrimidine site, with ≥5% CC→TT. Other canonical features such as a bias for mutations on the non-transcribed strand or at the 3' pyrimidine had limited application. The most robust classifier combined these features with criteria for the rarity of non-UV canonical mutations. In addition, several signatures proposed for specific UV wavelengths were limited to specific genes or species; non-signature mutations induced by UV may cause melanoma BRAF mutations; and the mutagen for sunlight-related skin neoplasms may vary between continents. PMID:25354245

  3. BRCA1 and BRCA2 mutational profile and prevalence in hereditary breast and ovarian cancer (HBOC probands from Southern Brazil: Are international testing criteria appropriate for this specific population?

    Directory of Open Access Journals (Sweden)

    Bárbara Alemar

    Full Text Available Germline pathogenic variants in BRCA1 and BRCA2 (BRCA are the main cause of Hereditary Breast and Ovarian Cancer syndrome (HBOC.In this study we evaluated the mutational profile and prevalence of BRCA pathogenic/likely pathogenic variants among probands fulfilling the NCCN HBOC testing criteria. We characterized the clinical profile of these individuals and explored the performance of international testing criteria.A pathogenic/likely pathogenic variant was detected in 19.1% of 418 probands, including seven novel frameshift variants. Variants of uncertain significance were found in 5.7% of individuals. We evaluated 50 testing criteria and mutation probability algorithms. There was a significant odds-ratio (OR for mutation prediction (p ≤ 0.05 for 25 criteria; 14 of these had p ≤ 0.001. Using a cutoff point of four criteria, the sensitivity is 83.8%, and the specificity is 53.5% for being a carrier. The prevalence of pathogenic/likely pathogenic variants for each criterion ranged from 22.1% to 55.6%, and criteria with the highest ORs were those related to triple-negative breast cancer or ovarian cancer.This is the largest study of comprehensive BRCA testing among Brazilians to date, and the first to analyze clinical criteria for genetic testing. Several criteria that are not included in the NCCN achieved a higher predictive value. Identification of the most informative criteria for each population will assist in the development of a rational approach to genetic testing, and will enable the prioritization of high-risk individuals as a first step towards offering testing in low-income countries.

  4. Weaver syndrome and EZH2 mutations: Clarifying the clinical phenotype.

    Science.gov (United States)

    Tatton-Brown, Katrina; Murray, Anne; Hanks, Sandra; Douglas, Jenny; Armstrong, Ruth; Banka, Siddharth; Bird, Lynne M; Clericuzio, Carol L; Cormier-Daire, Valerie; Cushing, Tom; Flinter, Frances; Jacquemont, Marie-Line; Joss, Shelagh; Kinning, Esther; Lynch, Sally Ann; Magee, Alex; McConnell, Vivienne; Medeira, Ana; Ozono, Keiichi; Patton, Michael; Rankin, Julia; Shears, Debbie; Simon, Marleen; Splitt, Miranda; Strenger, Volker; Stuurman, Kyra; Taylor, Clare; Titheradge, Hannah; Van Maldergem, Lionel; Temple, I Karen; Cole, Trevor; Seal, Sheila; Rahman, Nazneen

    2013-12-01

    Weaver syndrome, first described in 1974, is characterized by tall stature, a typical facial appearance, and variable intellectual disability. In 2011, mutations in the histone methyltransferase, EZH2, were shown to cause Weaver syndrome. To date, we have identified 48 individuals with EZH2 mutations. The mutations were primarily missense mutations occurring throughout the gene, with some clustering in the SET domain (12/48). Truncating mutations were uncommon (4/48) and only identified in the final exon, after the SET domain. Through analyses of clinical data and facial photographs of EZH2 mutation-positive individuals, we have shown that the facial features can be subtle and the clinical diagnosis of Weaver syndrome is thus challenging, especially in older individuals. However, tall stature is very common, reported in >90% of affected individuals. Intellectual disability is also common, present in ~80%, but is highly variable and frequently mild. Additional clinical features which may help in stratifying individuals to EZH2 mutation testing include camptodactyly, soft, doughy skin, umbilical hernia, and a low, hoarse cry. Considerable phenotypic overlap between Sotos and Weaver syndromes is also evident. The identification of an EZH2 mutation can therefore provide an objective means of confirming a subtle presentation of Weaver syndrome and/or distinguishing Weaver and Sotos syndromes. As mutation testing becomes increasingly accessible and larger numbers of EZH2 mutation-positive individuals are identified, knowledge of the clinical spectrum and prognostic implications of EZH2 mutations should improve. © 2013 Wiley Periodicals, Inc.

  5. High-Resolution Structures of HIV-1 Reverse Transcriptase/TMC278 Complexes: Strategic Flexibility Explains Potency Against Resistance Mutations

    Energy Technology Data Exchange (ETDEWEB)

    Das,K.; Bauman, J.; Clark, Jr., A.; Frenkel, Y.; Lewi, P.; Shatkin, A.; Hughes, S.; Arnold, E.

    2008-01-01

    TMC278 is a diarylpyrimidine (DAPY) nonnucleoside reverse transcriptase inhibitor (NNRTI) that is highly effective in treating wild-type and drug-resistant HIV-1 infections in clinical trials at relatively low doses ({approx}25-75 mg/day). We have determined the structure of wild-type HIV-1 RT complexed with TMC278 at 1.8 Angstroms resolution, using an RT crystal form engineered by systematic RT mutagenesis. This high-resolution structure reveals that the cyanovinyl group of TMC278 is positioned in a hydrophobic tunnel connecting the NNRTI-binding pocket to the nucleic acid-binding cleft. The crystal structures of TMC278 in complexes with the double mutant K103N/Y181C (2.1 Angstroms ) and L100I/K103N HIV-1 RTs (2.9 Angstroms ) demonstrated that TMC278 adapts to bind mutant RTs. In the K103N/Y181C RT/TMC278 structure, loss of the aromatic ring interaction caused by the Y181C mutation is counterbalanced by interactions between the cyanovinyl group of TMC278 and the aromatic side chain of Y183, which is facilitated by an {approx}1.5 Angstroms shift of the conserved Y183MDD motif. In the L100I/K103N RT/TMC278 structure, the binding mode of TMC278 is significantly altered so that the drug conforms to changes in the binding pocket primarily caused by the L100I mutation. The flexible binding pocket acts as a molecular 'shrink wrap' that makes a shape complementary to the optimized TMC278 in wild-type and drug-resistant forms of HIV-1 RT. The crystal structures provide a better understanding of how the flexibility of an inhibitor can compensate for drug-resistance mutations.

  6. A novel missense mutation in the high mobility group domain of SRY drastically reduces its DNA-binding capacity and causes paternally transmitted 46,XY complete gonadal dysgenesis.

    Science.gov (United States)

    Filges, Isabel; Kunz, Christophe; Miny, Peter; Boesch, Nemya; Szinnai, Gabor; Wenzel, Friedel; Tschudin, Sibil; Zumsteg, Urs; Heinimann, Karl

    2011-10-01

    To investigate the familial segregation, role, and function of a novel SRY missense mutation c.347T>C in two half-sisters affected by 46,XY complete gonadal dysgenesis (CDG) compatible with a successful pregnancy outcome. Phenotypic, mutational, and functional study. Academic research unit. Two half-sisters, their common father, and 100 healthy control individuals. Chromosome, molecular cytogenetic analysis, and Sanger sequencing of the SRY gene in blood lymphocytes of the proband, her affected half-sister, and in inflammatory tissue of the father postmortem. Cloning and expression of high mobility group box carboxy-terminal domains of Sry and electrophoretic mobility shift assay were performed. Not applicable. A novel SRY missense mutation c.347T>C (p.Leu116Ser) was identified in two half-sisters and segregates with the CGD phenotype. It is present in the common healthy father in a mosaic state. Functional analyses demonstrate the pathogenic effect of the mutation by a strong reduction of DNA affinity for the mutant p.Leu116Ser SRY protein. The missense mutation c.347T>C in the high mobility group domain of SRY causes 46,XY CGD. Paternal gonadal mosaicism is likely to explain the familial occurrence of 46,XY CGD suggesting a de novo mutational event during the early stages of embryonic development. This novel mutation is compatible with a successful pregnancy outcome. Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  7. The prognostic impact of K-RAS mutations in adult acute myeloid leukemia patients treated with high-dose cytarabine

    Directory of Open Access Journals (Sweden)

    Ahmad EI

    2011-07-01

    Full Text Available Ebtesam I Ahmad, Heba H Gawish, Nashwa MA Al Azizi, Ashraf M ElhefniClinical Pathology Department, Hematology and Oncology Unit of Internal Medicine Department, Faculty of Medicine, Zagazig University, Sharkia, EgyptBackground: Activating point mutation of the RAS gene has been generally accepted as an oncogenic event in a variety of malignancies. It represents one of the most common genetic alterations in acute myeloid leukemia (AML. However, little is known about its clinical relevance in the treatment outcome for this leukemia.Objective: This study aimed to clarify the biologic and prognostic impact of K-RAS mutations in relation to the dose of cytarabine (ara-C used in postinduction consolidation chemotherapy in adult AML patients.Patients and methods: The study comprised of 71 de novo AML patients with male/female ratio 1.4:1; their ages ranged from 21–59 years with a median of 37 years. They were subjected to full clinical evaluation, routine laboratory investigations, cytogenetic studies by G-banding (Giemsa staining, and K-RAS mutation detection using real-time polymerase chain reaction. The patients were randomized into two groups according to the ara-C dose used in consolidation treatment, the high the dose ara-C (HDAC group receiving 400 mg ara-C and-low-dose ara-C (LDAC group receiving 100 mg ara-C; they were followed over a period of five years.Results: Mutations in the K-RAS gene (mutRAS were detected in 23 patients (32% with the remaining 48 patients (68% having wild-type RAS (wtRAS. The percent of blast cells was significantly lower in mutRAS compared to wtRAS patients (P ≤ 0.001 while M4 subtype of AML and Inv(16 frequencies were significantly higher in mutRAS compared to wtRAS patients (P = 0.015 and (P = 0.003, respectively. The patients were followed up for a median of 43 months (range 11–57 months. There was no significant difference in overall survival (OS between mutRAS and wtRAS (P = 0.326. Within the mut

  8. Gag mutations strongly contribute to HIV-1 resistance to protease inhibitors in highly drug-experienced patients besides compensating for fitness loss.

    Directory of Open Access Journals (Sweden)

    Elisabeth Dam

    2009-03-01

    Full Text Available Human immunodeficiency virus type 1 (HIV-1 resistance to protease inhibitors (PI results from mutations in the viral protease (PR that reduce PI binding but also decrease viral replicative capacity (RC. Additional mutations compensating for the RC loss subsequently accumulate within PR and in Gag substrate cleavage sites. We examined the respective contribution of mutations in PR and Gag to PI resistance and RC and their interdependence using a panel of HIV-1 molecular clones carrying different sequences from six patients who had failed multiple lines of treatment. Mutations in Gag strongly and directly contributed to PI resistance besides compensating for fitness loss. This effect was essentially carried by the C-terminal region of Gag (containing NC-SP2-p6 with little or no contribution from MA, CA, and SP1. The effect of Gag on resistance depended on the presence of cleavage site mutations A431V or I437V in NC-SP2-p6 and correlated with processing of the NC/SP2 cleavage site. By contrast, reverting the A431V or I437V mutation in these highly evolved sequences had little effect on RC. Mutations in the NC-SP2-p6 region of Gag can be dually selected as compensatory and as direct PI resistance mutations, with cleavage at the NC-SP2 site behaving as a rate-limiting step in PI resistance. Further compensatory mutations render viral RC independent of the A431V or I437V mutations while their effect on resistance persists.

  9. First report of Ser653Asn mutation endowing high-level resistance to imazamox in downy brome (Bromus tectorum L.).

    Science.gov (United States)

    Kumar, Vipan; Jha, Prashant

    2017-12-01

    Bromus tectorum L. is one of the most troublesome grass weed species in cropland and non-cropland areas of the northwestern USA. In summer 2016, a B. tectroum accession (R) that survived imazamox at the field-use rate (44 g ha -1 ) in an imidazolinone-tolerant (IMI-tolerant or Clearfield™) winter wheat field was collected from a wheat field in Carter County, MT, USA. The aim of this study was to determine the resistance profile of the B. tectroum R accession to imazamox and other ALS inhibitors, and investigate the mechanism of resistance to imazamox. The R B. tectorum accession had a high-level resistance (110.1-fold) to imazamox (IMI) and low to moderate-levels cross-resistance to pyroxsulam (TP) (4.6-fold) and propoxycarbazone (SCT) (13.9-fold). The R accession was susceptible to sulfosulfuron (SU) and quizalofop and clethodim (ACCase inhibitors), paraquat (PS I inhibitor), glyphosate (EPSPS inhibitor) and glufosinate (GS inhibitor). Sequence analysis of the ALS gene revealed a single, target-site Ser653Asn mutation in R plants. Pretreatment of malathion followed by imazamox at 44 or 88 g ha -1 did not reverse the resistance phenotype. This is the first report of evolution of cross-resistance to ALS-inhibiting herbicides in B. tectorum. A single-point mutation, Ser653Asn, was identified, conferring the high-level resistance to imazamox. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  10. Dialogic Teaching to the High-Stakes Standardised Test?

    Science.gov (United States)

    Segal, Aliza; Snell, Julia; Lefstein, Adam

    2017-01-01

    Within current educational discourse, dialogic pedagogy is diametrically opposed to "teaching to the test", especially the high-stakes standardised test. While dialogic pedagogy is about critical thinking, authenticity and freedom, test preparation evokes all that is narrow, instrumental and cynical in education. In this paper, we argue…

  11. Using high-sensitivity sequencing for the detection of mutations in BTK and PLCγ2 genes in cellular and cell-free DNA and correlation with progression in patients treated with BTK inhibitors.

    Science.gov (United States)

    Albitar, Adam; Ma, Wanlong; DeDios, Ivan; Estella, Jeffrey; Ahn, Inhye; Farooqui, Mohammed; Wiestner, Adrian; Albitar, Maher

    2017-03-14

    Patients with chronic lymphocytic leukemia (CLL) that develop resistance to Bruton tyrosine kinase (BTK) inhibitors are typically positive for mutations in BTK or phospholipase c gamma 2 (PLCγ2). We developed a high sensitivity (HS) assay utilizing wild-type blocking polymerase chain reaction achieved via bridged and locked nucleic acids. We used this high sensitivity assay in combination with Sanger sequencing and next generation sequencing (NGS) and tested cellular DNA and cell-free DNA (cfDNA) from patients with CLL treated with the BTK inhibitor, ibrutinib. We also tested ibrutinib-naïve patients with CLL. HS testing achieved 100x greater sensitivity than Sanger. HS Sanger sequencing was capable of detecting sequencing, plasma cfDNA is more reliable than cellular DNA in detecting mutations. Our studies indicate that wild-type blocking and HS sequencing is necessary for proper and early detection of BTK or PLCγ2 mutations in monitoring patients treated with BTK inhibitors. Furthermore, cfDNA from plasma is very reliable sample-type for testing.

  12. Use of Gene Expression Profiles of Peripheral Blood Lymphocytes to Distinguish BRCA1 Mutation Carriers in High Risk Breast Cancer Families

    Directory of Open Access Journals (Sweden)

    Marie-Laure Vuillaume

    2009-01-01

    Full Text Available Mutations in two major genes, BRCA1 and BRCA2, account for up to 30% of families with hereditary breast cancer. Unfortunately, in most families there is little to indicate which gene should be targeted first for mutation screening, which is labor intensive, time consuming and often prohibitively expensive. As BRCA1 is a tumor suppressor gene involved in various cellular processes, heterozygous mutations could deregulate dependent pathways, such as DNA damage response, and disturb transcriptional activity of genes involved in the downstream signaling cascade. We investigated gene expression profiling in peripheral blood lymphocytes to evaluate this strategy for distinguishing BRCA1 mutation carriers from non-carriers. RNA from whole blood samples of 15 BRCA1 mutation carriers and 15 non-carriers from BRCA1 or BRCA2 families were hybridized to Agilent Technologies Whole Human Genome OligoMicroarrays (4 × 44 K multiplex format containing 41,000 unique human genes and transcripts. Gene expression data were analyzed with Welch’s t-tests and submitted to hierarchical clustering (GeneSpring GX software, Agilent Technologies. Statistical analysis revealed a slight tendency for 133 genes to be differentially expressed between BRCA1 mutation carriers and non-carriers. However, hierarchical clustering of these genes did not accurately discriminate BRCA1 mutation carriers from non-carriers. Expression variation for these genes according to BRCA1 mutation status was weak. In summary, microarray profiling of untreated whole blood does not appear to be informative in identifying breast cancer risk due to BRCA1 mutation.

  13. High Quality Test Pattern Generation and Boolean Satisfiability

    CERN Document Server

    Eggersglüß, Stephan

    2012-01-01

    This book provides an overview of automatic test pattern generation (ATPG) and introduces novel techniques to complement classical ATPG, based on Boolean Satisfiability (SAT).  A fast and highly fault efficient SAT-based ATPG framework is presented which is also able to generate high-quality delay tests such as robust path delay tests, as well as tests with long propagation paths to detect small delay defects. The aim of the techniques and methodologies presented in this book is to improve SAT-based ATPG, in order to make it applicable in industrial practice. Readers will learn to improve the performance and robustness of the overall test generation process, so that the ATPG algorithm reliably will generate test patterns for most targeted faults in acceptable run time to meet the high fault coverage demands of industry. The techniques and improvements presented in this book provide the following advantages: Provides a comprehensive introduction to test generation and Boolean Satisfiability (SAT); Describes a...

  14. The Alzheimer’s Prevention Initiative composite cognitive test score: Sample size estimates for the evaluation of preclinical Alzheimer’s disease treatments in presenilin 1 E280A mutation carriers

    Science.gov (United States)

    Ayutyanont, Napatkamon; Langbaum, Jessica B.; Hendrix, Suzanne B.; Chen, Kewei; Fleisher, Adam S.; Friesenhahn, Michel; Ward, Michael; Aguirre, Camilo; Acosta-Baena, Natalia; Madrigal, Lucìa; Muñoz, Claudia; Tirado, Victoria; Moreno, Sonia; Tariot, Pierre N.; Lopera, Francisco; Reiman, Eric M.

    2014-01-01

    Objective There is a need to identify a cognitive composite that is sensitive to tracking preclinical AD decline to be used as a primary endpoint in treatment trials. Method We capitalized on longitudinal data, collected from 1995 to 2010, from cognitively unimpaired presenilin 1 (PSEN1) E280A mutation carriers from the world’s largest known early-onset autosomal dominant AD (ADAD) kindred to identify a composite cognitive test with the greatest statistical power to track preclinical AD decline and estimate the number of carriers age 30 and older needed to detect a treatment effect in the Alzheimer’s Prevention Initiative’s (API) preclinical AD treatment trial. The mean-to-standard-deviation ratios (MSDRs) of change over time were calculated in a search for the optimal combination of one to seven cognitive tests/sub-tests drawn from the neuropsychological test battery in cognitively unimpaired mutation carriers during a two and five year follow-up period, using data from non-carriers during the same time period to correct for aging and practice effects. Combinations that performed well were then evaluated for robustness across follow-up years, occurrence of selected items within top performing combinations and representation of relevant cognitive domains. Results This optimal test combination included CERAD Word List Recall, CERAD Boston Naming Test (high frequency items), MMSE Orientation to Time, CERAD Constructional Praxis and Ravens Progressive Matrices (Set A) with an MSDR of 1.62. This composite is more sensitive than using either the CERAD Word List Recall (MSDR=0.38) or the entire CERAD-Col battery (MSDR=0.76). A sample size of 75 cognitively normal PSEN1-E280A mutation carriers age 30 and older per treatment arm allows for a detectable treatment effect of 29% in a 60-month trial (80% power, p=0.05). Conclusions We have identified a composite cognitive test score representing multiple cognitive domains that has improved power compared to the most

  15. A novel high-throughput in vivo molecular screen for shade avoidance mutants identifies a novel phyA mutation.

    Science.gov (United States)

    Wang, Xuewen; Roig-Villanova, Irma; Khan, Safina; Shanahan, Hugh; Quail, Peter H; Martinez-Garcia, Jaime F; Devlin, Paul F

    2011-05-01

    The shade avoidance syndrome (SAS) allows plants to anticipate and avoid shading by neighbouring plants by initiating an elongation growth response. The phytochrome photoreceptors are able to detect a reduction in the red:far red ratio in incident light, the result of selective absorption of red and blue wavelengths by proximal vegetation. A shade-responsive luciferase reporter line (PHYB::LUC) was used to carry out a high-throughput screen to identify novel SAS mutants. The dracula 1 (dra1) mutant, that showed no avoidance of shade for the PHYB::LUC response, was the result of a mutation in the PHYA gene. Like previously characterized phyA mutants, dra1 showed a long hypocotyl in far red light and an enhanced hypocotyl elongation response to shade. However, dra1 additionally showed a long hypocotyl in red light. Since phyB levels are relatively unaffected in dra1, this gain-of-function red light phenotype strongly suggests a disruption of phyB signalling. The dra1 mutation, G773E within the phyA PAS2 domain, occurs at a residue absolutely conserved among phyA sequences. The equivalent residue in phyB is absolutely conserved as a threonine. PAS domains are structurally conserved domains involved in molecular interaction. Structural modelling of the dra1 mutation within the phyA PAS2 domain shows some similarity with the structure of the phyB PAS2 domain, suggesting that the interference with phyB signalling may be the result of non-functional mimicry. Hence, it was hypothesized that this PAS2 residue forms a key distinction between the phyA and phyB phytochrome species.

  16. Gene expression profiles of luteal phase fallopian tube epithelium from BRCA mutation carriers resemble high-grade serous carcinoma.

    Science.gov (United States)

    Tone, Alicia A; Begley, Heather; Sharma, Monika; Murphy, Joan; Rosen, Barry; Brown, Theodore J; Shaw, Patricia A

    2008-07-01

    To identify molecular alterations potentially involved in predisposition to adnexal serous carcinoma (SerCa) in the nonmalignant fallopian tube epithelium (FTE) of BRCA1/2 mutation carriers, given recent evidence implicating the distal FTE as a common source for SerCa. We obtained and compared gene expression profiles of laser capture microdissected nonmalignant distal FTE from 12 known BRCA1/2 mutation carriers (FTEb) and 12 control women (FTEn) during the luteal and follicular phase, as well as 13 high-grade tubal and ovarian SerCa. Gene expression profiles of tubal and ovarian SerCa specimens were indistinguishable by unsupervised cluster analysis and significance analysis of microarrays. FTEb samples as a group, and four individual FTEb samples from the luteal phase in particular, clustered closely with SerCa rather than normal control FTE. Differentially expressed genes from these four samples relative to other FTEb samples, as well as differentially expressed genes in all FTEb luteal samples relative to follicular samples, were mapped to the I2D protein-protein interaction database, revealing a complex network affecting signaling pathways previously implicated in tumorigenesis. Two candidates, disabled homolog 2 mitogen-responsive phosphoprotein (DAB2) and Ski-like (SKIL), were further validated by real-time reverse transcription-PCR and tissue arrays. FTEb luteal and SerCa samples expressed higher levels of oncogenic SKIL and decreased levels of tumor suppressor DAB2, relative to FTEb follicular samples. These findings support a common molecular pathway for adnexal SerCa and implicate factors associated with the luteal phase in predisposition to ovarian cancer in BRCA mutation carriers.

  17. Variants of cancer susceptibility genes in Korean BRCA1/2 mutation-negative patients with high risk for hereditary breast cancer

    OpenAIRE

    Park, Ji Soo; Lee, Seung-Tae; Nam, Eun Ji; Han, Jung Woo; Lee, Jung-Yun; Kim, Jieun; Kim, Tae Il; Park, Hyung Seok

    2018-01-01

    Background We evaluated the incidence and spectrum of pathogenic and likely pathogenic variants of cancer susceptibility genes in BRCA1/2 mutation-negative Korean patients with a high risk for hereditary breast cancer using a comprehensive multigene panel that included 35 cancer susceptibility genes. Methods Samples from 120 patients who were negative for BRCA1/2 mutations, but had been diagnosed with breast cancer that was likely hereditary, were prospectively evaluated for the prevalence of...

  18. High frequency of mutations in exon 10 of the porphobilinogen deaminase gene in patients with a CRIM-positive subtype of acute intermittent porphyria

    Energy Technology Data Exchange (ETDEWEB)

    Gu, X.F.; Rooij, F. de; Voortman, G.; Velde, K.T.; Nordmann, Y.; Grandchamp, B.

    1992-09-01

    Acute intermittent porphyria (AIP) is an autosomal dominant disease characterized by a partial deficiency of porphobilinogen (PBG) deaminase. Different subtypes of the disease have been defined, and more than 10 different mutations have been described. The authors focused their study on exon 10, since they previously found that three different mutations were located in this exon and that two of them seemed to be relatively common. They used denaturing gradient gel electrophoresis (DGGE) after in vitro amplification to detect all possible mutations in exon 10 in 41 unrelated AIP patients. In about one-fourth of these patients they could distinguish three abnormal migration patterns, indicating the presence of various mutations. Additional sequencing demonstrated the presence of three different single-base substitutions. Two of these mutations had already been described. A third one consisted of a C-to-T transition located at position 499 of the PBG deaminase mRNA and resulted in an Arg-to-Trp substitution. All three mutations were found in patients with crossreacting immunological material (CRIM)-positive forms of AlP. The high frequency of these mutations make DGGE analysis of exon 10 a useful approach allowing the direct detection of the DNA abnormality in most of the families with the CRIM-positive subtype of AlP. 23 refs., 3 figs., 1 tab.

  19. Reliability of a Computerized Neurocognitive Test in Baseline Concussion Testing of High School Athletes.

    Science.gov (United States)

    MacDonald, James; Duerson, Drew

    2015-07-01

    Baseline assessments using computerized neurocognitive tests are frequently used in the management of sport-related concussions. Such testing is often done on an annual basis in a community setting. Reliability is a fundamental test characteristic that should be established for such tests. Our study examined the test-retest reliability of a computerized neurocognitive test in high school athletes over 1 year. Repeated measures design. Two American high schools. High school athletes (N = 117) participating in American football or soccer during the 2011-2012 and 2012-2013 academic years. All study participants completed 2 baseline computerized neurocognitive tests taken 1 year apart at their respective schools. The test measures performance on 4 cognitive tasks: identification speed (Attention), detection speed (Processing Speed), one card learning accuracy (Learning), and one back speed (Working Memory). Reliability was assessed by measuring the intraclass correlation coefficient (ICC) between the repeated measures of the 4 cognitive tasks. Pearson and Spearman correlation coefficients were calculated as a secondary outcome measure. The measure for identification speed performed best (ICC = 0.672; 95% confidence interval, 0.559-0.760) and the measure for one card learning accuracy performed worst (ICC = 0.401; 95% confidence interval, 0.237-0.542). All tests had marginal or low reliability. In a population of high school athletes, computerized neurocognitive testing performed in a community setting demonstrated low to marginal test-retest reliability on baseline assessments 1 year apart. Further investigation should focus on (1) improving the reliability of individual tasks tested, (2) controlling for external factors that might affect test performance, and (3) identifying the ideal time interval to repeat baseline testing in high school athletes. Computerized neurocognitive tests are used frequently in high school athletes, often within a model of baseline testing

  20. Targeted Next Generation Sequencing Approach in Patients Referred for Silver-Russell Syndrome Testing Increases the Mutation Detection Rate and Provides Decisive Information for Clinical Management.

    Science.gov (United States)

    Meyer, Robert; Soellner, Lukas; Begemann, Matthias; Dicks, Severin; Fekete, György; Rahner, Nils; Zerres, Klaus; Elbracht, Miriam; Eggermann, Thomas

    2017-08-01

    To investigate the contribution of differential diagnoses to the mutation spectrum of patients referred for Silver-Russell syndrome (SRS) testing. Forty-seven patients referred for molecular testing for SRS were examined after exclusion of one of the SRS-associated alterations. After clinical classification, a targeted next generation sequencing approach comprising 25 genes associated with other diagnoses or postulated as SRS candidate genes was performed. By applying the Netchine-Harbinson clinical scoring system, indication for molecular testing for SRS was confirmed in 15 out of 47 patients. In 4 out of these 15 patients, disease-causing variants were found in genes associated with other diagnoses. These patients carried mutations associated with Bloom syndrome, Mulibrey nanism, KBG syndrome, or IGF1R-associated short stature. We could not detect any pathogenic mutation in patients with a negative clinical score. Some of the differential diagnoses detected in the cohort presented here have a major impact on clinical management. Therefore, we emphasize that the molecular defects associated with these clinical pictures should be excluded before the clinical diagnosis "SRS" is made. Finally, we could show that a broad molecular approach including the differential diagnoses of SRS increases the detection rate. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Spontaneous Mutation Rate of Measles Virus: Direct Estimation Based on Mutations Conferring Monoclonal Antibody Resistance

    Science.gov (United States)

    Schrag, Stephanie J.; Rota, Paul A.; Bellini, William J.

    1999-01-01

    High mutation rates typical of RNA viruses often generate a unique viral population structure consisting of a large number of genetic microvariants. In the case of viral pathogens, this can result in rapid evolution of antiviral resistance or vaccine-escape mutants. We determined a direct estimate of the mutation rate of measles virus, the next likely target for global elimination following poliovirus. In a laboratory tissue culture system, we used the fluctuation test method of estimating mutation rate, which involves screening a large number of independent populations initiated by a small number of viruses each for the presence or absence of a particular single point mutation. The mutation we focused on, which can be screened for phenotypically, confers resistance to a monoclonal antibody (MAb 80-III-B2). The entire H gene of a subset of mutants was sequenced to verify that the resistance phenotype was associated with single point mutations. The epitope conferring MAb resistance was further characterized by Western blot analysis. Based on this approach, measles virus was estimated to have a mutation rate of 9 × 10−5 per base per replication and a genomic mutation rate of 1.43 per replication. The mutation rates we estimated for measles virus are comparable to recent in vitro estimates for both poliovirus and vesicular stomatitis virus. In the field, however, measles virus shows marked genetic stability. We briefly discuss the evolutionary implications of these results. PMID:9847306

  2. Acute lymphoblastic leukemia with low hypodiploid/near triploid karyotype is a specific clinical entity and exhibits a very high TP53 mutation frequency of 93%.

    Science.gov (United States)

    Mühlbacher, Verena; Zenger, Melanie; Schnittger, Susanne; Weissmann, Sandra; Kunze, Franziska; Kohlmann, Alexander; Bellos, Frauke; Kern, Wolfgang; Haferlach, Torsten; Haferlach, Claudia

    2014-06-01

    B lymphoblastic leukemia/lymphoma (ALL) are subdivided by the WHO classification into five subgroups defined by specific translocations and two further subgroups defined by the number of chromosomes. The hypodiploid subgroup is heterogeneous and comprises ALL with a chromosome number of karyotype, we performed chromosome banding analysis, FISH, array comparative genomic hybridization, and mutational analyses of FBXW7, NOTCH1, KRAS, NRAS, TP53, and IKZF1 in 29 cases. We observed a nonrandom pattern of chromosome losses, including chromosomes 3, 7, 13, 15, 16, and 17. A deletion encompassing the CDKN2A/B locus was the only recurrent structural abnormality. A duplication of the low hypodiploid karyotype occurred frequently, resulting in a near triploid karyotype based on the definition by merely counting chromosomes but in fact was a very low tetraploid chromosome set. Mutational analyses revealed no mutations in IKZF1, FBXW7, NOTCH1, and KRAS and only one mutation in NRAS. However, we discovered a high frequency of TP53 mutations in 93% (27/29) of cases. In 26/27 cases with TP53 mutation, the second TP53 allele was lost due to monosomy 17. Median overall survival was short (18.5 months), which might be related to the high frequency of TP53 alterations. Therefore, ALL with low hypodiploidy is characterized by a typical pattern of chromosome losses and a remarkably high TP53 mutation frequency. Our data suggest the introduction of a novel WHO entity within the B lymphoblastic leukemia/lymphoma group showing low hypodiploid/very low tetraploid karyotype and concomitant TP53 mutation. Copyright © 2014 Wiley Periodicals, Inc.

  3. Two founder mutations in the SEC23B gene account for the relatively high frequency of CDA II in the Italian population

    Science.gov (United States)

    Russo, Roberta; Gambale, Antonella; Esposito, Maria Rosaria; Serra, Maria Luisa; Troiano, Annaelena; De Maggio, Ilaria; Capasso, Mario; Luzzatto, Lucio; Delaunay, Jean; Tamary, Hannah; Iolascon, Achille

    2011-01-01

    Congenital Dyserythropoietic Anemia type II is an autosomal recessive disorder characterized by unique abnormalities in the differentiation of cells of the erythroid lineage. The vast majority of CDA II cases result from mutations in the SEC23B gene. To date, 53 different causative mutations have been reported in 86 unrelated cases (from the CDA II European Registry), 47 of them Italian. We have now identified SEC23B mutations in 23 additional patients, 17 Italians and 6 non-Italian Europeans. The relative allelic frequency of the mutations was then reassessed in a total of 64 Italian and 45 non-Italian unrelated patients. Two mutations, E109K and R14W, account for over one-half of the cases of CDA II in Italy. Whereas the relative frequency of E109K is similar in Italy and in the rest of Europe (and is also prevalent in Moroccan Jews), the relative frequency of R14W is significantly higher in Italy (26.3% vs. 10.7%). By haplotype analysis we demonstrated that both are founder mutations in the Italian population. By using the DMLE+ program our estimate for the age of the E109K mutation in Italian population is ≈2,200 years; whereas for the R14W mutation it is ≈3,000 years. We hypothesize that E109K may have originated in the Middle East and may have spread in the heyday of the Roman Empire. Instead, R14W may have originated in Southern Italy. The relatively high frequency of the R14W mutation may account for the known increased prevalence of CDA II in Italy. Am. J. Hematol. 86:727–732, 2011. © 2011 Wiley-Liss, Inc. PMID:21850656

  4. A high response rate to liposomal doxorubicin is seen among women with BRCA mutations treated for recurrent epithelial ovarian cancer.

    Science.gov (United States)

    Adams, Sarah F; Marsh, Evelyn B; Elmasri, Wafic; Halberstadt, Steffanie; Vandecker, Stephanie; Sammel, Mary D; Bradbury, Angela R; Daly, Mary; Karlan, Beth; Rubin, Stephen C

    2011-12-01

    Ten percent of ovarian cancer is attributed to hereditary syndromes, most commonly to mutations in the BRCA1 or BRCA2 genes. These cancers are characterized by a prolonged sensitivity to platinum agents in spite of presentation at advanced stages. We hypothesized that women with BRCA-associated ovarian cancer would also show a high response rate to pegylated liposomal doxorubicin (Doxil). A retrospective cohort study was conducted to compare the response rate, progression-free, and overall survival among women with BRCA-associated or sporadic ovarian cancer who were treated with Doxil. A response to Doxil was seen in 13 of 23 patients with BRCA mutations (56.5%; 3 by RECIST criteria and 10 by CA125 levels) compared with only 8 of 41 women with non-hereditary cancers (19.5%; 2 by RECIST criteria and 6 by CA125 levels; p=0.004). This was associated with an improved progression-free and overall survival as measured from the time of Doxil administration. Notably, platinum sensitivity did not directly correlate with a response to Doxil. Women with BRCA-associated ovarian tumors demonstrate a greater sensitivity to cytotoxic therapy with Doxil than has previously been reported in unselected cases. Copyright © 2011 Elsevier Inc. All rights reserved.

  5. Initial antibodies binding to HIV-1 gp41 in acutely infected subjects are polyreactive and highly mutated

    Science.gov (United States)

    Chen, Xi; Munshaw, Supriya; Zhang, Ruijun; Marshall, Dawn J.; Vandergrift, Nathan; Whitesides, John F.; Lu, Xiaozhi; Yu, Jae-Sung; Hwang, Kwan-Ki; Gao, Feng; Markowitz, Martin; Heath, Sonya L.; Bar, Katharine J.; Goepfert, Paul A.; Montefiori, David C.; Shaw, George C.; Alam, S. Munir; Margolis, David M.; Denny, Thomas N.; Boyd, Scott D.; Marshal, Eleanor; Egholm, Michael; Simen, Birgitte B.; Hanczaruk, Bozena; Fire, Andrew Z.; Voss, Gerald; Kelsoe, Garnett; Tomaras, Georgia D.; Moody, M. Anthony; Kepler, Thomas B.

    2011-01-01

    The initial antibody response to HIV-1 is targeted to envelope (Env) gp41, and is nonneutralizing and ineffective in controlling viremia. To understand the origins and characteristics of gp41-binding antibodies produced shortly after HIV-1 transmission, we isolated and studied gp41-reactive plasma cells from subjects acutely infected with HIV-1. The frequencies of somatic mutations were relatively high in these gp41-reactive antibodies. Reverted unmutated ancestors of gp41-reactive antibodies derived from subjects acutely infected with HIV-1 frequently did not react with autologous HIV-1 Env; however, these antibodies were polyreactive and frequently bound to host or bacterial antigens. In one large clonal lineage of gp41-reactive antibodies, reactivity to HIV-1 Env was acquired only after somatic mutations. Polyreactive gp41-binding antibodies were also isolated from uninfected individuals. These data suggest that the majority of gp41-binding antibodies produced after acute HIV-1 infection are cross-reactive responses generated by stimulating memory B cells that have previously been activated by non–HIV-1 antigens. PMID:21987658

  6. Large scale steam valve test: Performance testing of large butterfly valves and full scale high flowrate steam testing

    Energy Technology Data Exchange (ETDEWEB)

    Meadows, J.B.; Robbins, G.E.; Roselius, D.G. [and others

    1995-05-01

    This report presents the results of the design testing of large (36-inch diameter) butterfly valves under high flow conditions. The two butterfly valves were pneumatically operated air-open, air-shut valves (termed valves 1 and 2). These butterfly valves were redesigned to improve their ability to function under high flow conditions. Concern was raised regarding the ability of the butterfly valves to function as required with high flow-induced torque imposed on the valve discs during high steam flow conditions. High flow testing was required to address the flow-induced torque concerns. The valve testing was done using a heavily instrumented piping system. This test program was called the Large Scale Steam Valve Test (LSSVT). The LSSVT program demonstrated that the redesigned valves operated satisfactorily under high flow conditions.

  7. Social aspects of genetic testing for factor V leiden mutation in healthy individuals and their importance for daily practice

    NARCIS (Netherlands)

    Bank, Ivan; Scavenius, Michael P. R. B.; Büller, Harry R.; Middeldorp, Saskia

    2004-01-01

    To explore social aspects of asymptomatic carriership of factor V Leiden mutation (FVL) and how carriers have experienced procedure of screening for FVL, we have performed a qualitative study using semi-structured interviews. Seventeen carriers of FVL without history of venous thromboembolism (VTE)

  8. Population-based study of BRCA1/2 mutations: family history based criteria identify minority of mutation carriers.

    Science.gov (United States)

    Mateju, M; Stribrna, J; Zikan, M; Kleibl, Z; Janatova, M; Kormunda, S; Novotny, J; Soucek, P; Petruzelka, L; Pohlreich, P

    2010-01-01

    The two major susceptibility genes, BRCA1 and BRCA2, are involved in hereditary breast and ovarian cancer syndrome. Early detection of mutation carriers has crucial clinical importance, as it allows identification of women who may benefit from intensive clinical follow-up or prophylactic surgery. Generally accepted inclusion criteria for BRCA1/2 mutation testing are based either upon family history of breast or ovarian cancer or young age at cancer diagnosis. In order to analyze the impact of BRCA1/2 mutations on breast cancer development in the Czech population and to confront the clinical and histopathological data of mutation carriers with current criteria for mutation testing we examined the frequency of mutations in unselected breast cancer cases. Mutational analysis of BRCA1/2 genes performed in 679 unselected female breast cancer patients included all recurrent deleterious alterations previously identified in the Prague area and truncating mutations in the whole exon 11 of BRCA1. Within analyzed gene sequences more than 80% of mutations were identified previously in high-risk patients. A total of 16 breast cancer patients (2.4%) carried a mutation. BRCA1 mutations were identified in 14 (2.1%) whereas BRCA2 in 2 (0.3%) women. Family history of ovarian cancer was a strong predictor of a BRCA1/2 mutation (OR = 8.3; p = 0.01), however, family history of breast cancer was not indicative of carrier status. A significant association between medullary breast cancer and mutation status was observed. Current criteria for BRCA1/2 mutation testing would distinguish only 6 out of 16 (37.5%) carriers identified in our study. Ten breast cancer patients with confirmed BRCA1/2 germ-line mutation exhibited no clinical characteristics that would predict their carrier status. Therefore, we believe that the testing for BRCA1/2 mutations in the Czech Republic may not be restricted only to high-risk patients. Our results indicate that analysis of locally prevalent BRCA1

  9. TOCUSO: Test of Conceptual Understanding on High School Optics Topics

    Science.gov (United States)

    Akarsu, Bayram

    2012-01-01

    Physics educators around the world often need reliable diagnostic materials to measure students' understanding of physics concept in high school. The purpose of this study is to evaluate a new diagnostic tool on High School Optics concept. Test of Conceptual Understanding on High School Optics (TOCUSO) consists of 25 conceptual items that measures…

  10. High-resolution melting analysis of gyrA codon 84 and grlA codon 80 mutations conferring resistance to fluoroquinolones in Staphylococcus pseudintermedius isolates from canine clinical samples.

    Science.gov (United States)

    Loiacono, Monica; Martino, Piera A; Albonico, Francesca; Dell'Orco, Francesca; Ferretti, Manuela; Zanzani, Sergio; Mortarino, Michele

    2017-09-01

    Staphylococcus pseudintermedius is an opportunistic pathogen of dogs and cats. A high-resolution melting analysis (HRMA) protocol was designed and tested on 42 clinical isolates with known fluoroquinolone (FQ) susceptibility and gyrA codon 84 and grlA codon 80 mutation status. The HRMA approach was able to discriminate between FQ-sensitive and FQ-resistant strains and confirmed previous reports that the main mutation site associated with FQ resistance in S. pseudintermedius is located at position 251 (Ser84Leu) of gyrA. Routine, HRMA-based FQ susceptibility profiles may be a valuable tool to guide therapy. The FQ resistance-predictive power of the assay should be tested in a significantly larger number of isolates.

  11. Molecular imaging with (99m)Tc-MIBI and molecular testing for mutations in differentiating benign from malignant follicular neoplasm: a prospective comparison.

    Science.gov (United States)

    Giovanella, L; Campenni, A; Treglia, G; Verburg, F A; Trimboli, P; Ceriani, L; Bongiovanni, M

    2016-06-01

    To compare mutation analysis of cytology specimens and (99m)Tc-MIBI thyroid scintigraphy for differentiating benign from malignant thyroid nodules in patients with a cytological reading of follicular neoplasm. Patients ≥18 years of age with a solitary hypofunctioning thyroid nodule (≥10 mm), normal thyrotropin and calcitonin levels, and a cytological diagnosis of follicular neoplasm were prospectively enrolled. Mutation analysis and (99m)Tc-MIBI scintigraphy were performed and patients were subsequently operated on to confirm or exclude a malignant lesion. Mutations for KRAS, HRAS and NRAS and for BRAF and translocations of PAX8/PPARγ, RET/PTC1 and RET/PTC3 were investigated. Static thyroid scintigraphic images were acquired 10 and 60 min after intravenous injection of 200 MBq of (99m)Tc-MIBI and visually assessed. Additionally, the MIBI washout index was calculated using a semiquantitative method. In our series, 26 % of nodules with a follicular pattern on cytology were malignant with a prevalence of follicular carcinomas. (99m)Tc-MIBI scintigraphy was found to be significantly more accurate (positive likelihood ratio 4.56 for visual assessment and 12.35 for semiquantitative assessment) than mutation analysis (positive likelihood ratio 1.74). A negative (99m)Tc-MIBI scan reliably excluded malignancy. In patients with a thyroid nodule cytologically diagnosed as a follicular proliferation, semiquantitative analysis of (99m)Tc-MIBI scintigraphy should be the preferred method for differentiating benign from malignant nodules. It is superior to molecular testing for the presence of differentiated thyroid cancer-associated mutations in fine-needle aspiration cytology sample material.

  12. Molecular imaging with {sup 99m}Tc-MIBI and molecular testing for mutations in differentiating benign from malignant follicular neoplasm: a prospective comparison

    Energy Technology Data Exchange (ETDEWEB)

    Giovanella, L.; Treglia, G.; Ceriani, L. [Oncology Institute of Southern Switzerland, Department of Nuclear Medicine and Thyroid Centre, Bellinzona (Switzerland); Campenni, A. [Policlinico Universitario, Istituto di Medicina Nucleare, Messina (Italy); Verburg, F.A. [RWTH University Hospital Aachen, Department of Nuclear Medicine, Aachen (Germany); Trimboli, P. [Oncology Institute of Southern Switzerland, Department of Nuclear Medicine and Thyroid Centre, Bellinzona (Switzerland); Ospedale Israelitico, Sezione di Endocrinologia e Diabetologia, Roma (Italy); Bongiovanni, M. [Centre Hopitalier Universitaire Vaudouise, Institut de Pathologie, Lausanne (Switzerland)

    2016-06-15

    To compare mutation analysis of cytology specimens and {sup 99m}Tc-MIBI thyroid scintigraphy for differentiating benign from malignant thyroid nodules in patients with a cytological reading of follicular neoplasm. Patients ≥18 years of age with a solitary hypofunctioning thyroid nodule (≥10 mm), normal thyrotropin and calcitonin levels, and a cytological diagnosis of follicular neoplasm were prospectively enrolled. Mutation analysis and {sup 99m}Tc-MIBI scintigraphy were performed and patients were subsequently operated on to confirm or exclude a malignant lesion. Mutations for KRAS, HRAS and NRAS and for BRAF and translocations of PAX8/PPARγ, RET/PTC1 and RET/PTC3 were investigated. Static thyroid scintigraphic images were acquired 10 and 60 min after intravenous injection of 200 MBq of {sup 99m}Tc-MIBI and visually assessed. Additionally, the MIBI washout index was calculated using a semiquantitative method. In our series, 26 % of nodules with a follicular pattern on cytology were malignant with a prevalence of follicular carcinomas. {sup 99m}Tc-MIBI scintigraphy was found to be significantly more accurate (positive likelihood ratio 4.56 for visual assessment and 12.35 for semiquantitative assessment) than mutation analysis (positive likelihood ratio 1.74). A negative {sup 99m}Tc-MIBI scan reliably excluded malignancy. In patients with a thyroid nodule cytologically diagnosed as a follicular proliferation, semiquantitative analysis of {sup 99m}Tc-MIBI scintigraphy should be the preferred method for differentiating benign from malignant nodules. It is superior to molecular testing for the presence of differentiated thyroid cancer-associated mutations in fine-needle aspiration cytology sample material. (orig.)

  13. Comparison of the mismatch-specific endonuclease method and denaturing high-performance liquid chromatography for the identification of HBB gene mutations

    Science.gov (United States)

    Hung, Chia-Cheng; Su, Yi-Ning; Lin, Chia-Yun; Chang, Yin-Fei; Chang, Chien-Hui; Cheng, Wen-Fang; Chen, Chi-An; Lee, Chien-Nan; Lin, Win-Li

    2008-01-01

    Background Beta-thalassemia is a common autosomal recessive hereditary disease in the Meditertanean, Asia and African areas. Over 600 mutations have been described in the beta-globin (HBB), of which more than 200 are associated with a beta-thalassemia phenotype. Results We used two highly-specific mutation screening methods, mismatch-specific endonuclease and denaturing high-performance liquid chromatography, to identify mutations in the HBB gene. The sensitivity and specificity of these two methods were compared. We successfully distinguished mutations in the HBB gene by the mismatch-specific endonuclease method without need for further assay. This technique had 100% sensitivity and specificity for the study sample. Conclusion Compared to the DHPLC approach, the mismatch-specific endonuclease method allows mutational screening of a large number of samples because of its speed, sensitivity and adaptability to semi-automated systems. These findings demonstrate the feasibility of using the mismatch-specific endonuclease method as a tool for mutation screening. PMID:18694524

  14. Comparison of the mismatch-specific endonuclease method and denaturing high-performance liquid chromatography for the identification of HBB gene mutations

    Directory of Open Access Journals (Sweden)

    Cheng Wen-Fang

    2008-08-01

    Full Text Available Abstract Background Beta-thalassemia is a common autosomal recessive hereditary disease in the Meditertanean, Asia and African areas. Over 600 mutations have been described in the beta-globin (HBB, of which more than 200 are associated with a beta-thalassemia phenotype. Results We used two highly-specific mutation screening methods, mismatch-specific endonuclease and denaturing high-performance liquid chromatography, to identify mutations in the HBB gene. The sensitivity and specificity of these two methods were compared. We successfully distinguished mutations in the HBB gene by the mismatch-specific endonuclease method without need for further assay. This technique had 100% sensitivity and specificity for the study sample. Conclusion Compared to the DHPLC approach, the mismatch-specific endonuclease method allows mutational screening of a large number of samples because of its speed, sensitivity and adaptability to semi-automated systems. These findings demonstrate the feasibility of using the mismatch-specific endonuclease method as a tool for mutation screening.

  15. EGFR Mutation Status in Uighur Lung Adenocarcinoma Patients

    Directory of Open Access Journals (Sweden)

    Li SHAN

    2013-02-01

    Full Text Available Background and objective Epidermal growth factor receptor (EGFR, a transmembrane protein, is a member of the tyrosine kinase family. Gefitinib, an EGFR tyrosine-kinase inhibitors, has shown a high response rate in the treatment of lung cancer in patients with EGFR mutation. However, significant differences in EGFR mutations exist among different ethnic groups. The aim of this study is to investigate the prevalence of EGFR mutations in Uighur lung adenocarcinoma patients by using a rapid and sensitive detection method and to analyze EGFR mutation differences compared with Han lung adenocarcinoma patients. Methods We examined lung adenocarcinoma tissues from 138 patients, including 68 Uighur lung adenocarcinoma patients and 70 Han lung adenocarcinoma patients, for EGFR mutations in exons 18, 19, 20, and 21 by using the amplification refractory mutation system (ARMS PCR method. The mutation differences between Uighur and Han lung adenocarcinoma were compared by using the chi-square test method. Results EGFR mutations were detected in 43 (31.2% of the 138 lung adenocarcinoma patients. EGFR mutations were detected in 11 (16.2% of the 68 Uighur lung adenocarcinoma patients and in 32 (45.7% of the 70 Han lung adenocarcinoma patients. Significant differences were observed in the EGFR mutations between Uighur lung adenocarcinoma patients and Han lung adenocarcinoma patients (P<0.001. Conclusion Our results indicate that the EGFR mutation in Uighur lung adenocarcinoma patients (16.2% is significantly lower than that in Han lung adenocarcinoma patients (45.7%.

  16. Utilization of Emotional Freedom Techniques (EFT) to Reduce Test Anxiety in High Stakes Testing

    Science.gov (United States)

    Mohler, Marie Elaine

    2013-01-01

    There are many reasons a person may fail a high stakes test such as the National Council Licensure Examination for Registered Nurses (NCLEX-RN®). Sleep deprivation, illness, life stressors, knowledge deficit, and test anxiety are some of the common explanations. A student with test anxiety may feel threatened by this evaluation process. This…

  17. Tests of a High Resolution Beam Profile Monitor

    Energy Technology Data Exchange (ETDEWEB)

    Norem, J.

    2004-10-28

    High energy linear colliders require very small beams at the interaction point to produce high luminosities, and these beams must be measured and monitored. We have developed and tested a technique where the profile can be obtained from an extension of pinhole camera optics using thick, single sided collimators and slits. Very high resolutions (a few nm) should be possible. Gamma beams can be obtained from bremsstrahlung, Compton or beamstrahlung radiation. We describe tests of the technique using bremsstrahlung from an 800 MeV electron beam at Bates/MIT, Compton scattered photons from 47 GeV Final Focus Test Beam (FFTB) at SLAC, and other applications, such as linear colliders.

  18. The Childhood Asperger Syndrome Test (CAST): Test-Retest Reliability in a High Scoring Sample

    Science.gov (United States)

    Allison, Carrie; Williams, Jo; Scott, Fiona; Stott, Carol; Bolton, Patrick; Baron-Cohen, Simon; Brayne, Carol

    2007-01-01

    The Childhood Asperger Syndrome Test (CAST) is a 37-item parental self-completion questionnaire designed to screen for high-functioning autism spectrum conditions in epidemiological research. The CAST has previously demonstrated good accuracy for use as a screening test, with high sensitivity in studies with primary school aged children in…

  19. SQSTM1 Mutations and Glaucoma.

    Directory of Open Access Journals (Sweden)

    Todd E Scheetz

    Full Text Available Glaucoma is the most common cause of irreversible blindness worldwide. One subset of glaucoma, normal tension glaucoma (NTG occurs in the absence of high intraocular pressure. Mutations in two genes, optineurin (OPTN and TANK binding kinase 1 (TBK1, cause familial NTG and have known roles in the catabolic cellular process autophagy. TKB1 encodes a kinase that phosphorylates OPTN, an autophagy receptor, which ultimately activates autophagy. The sequestosome (SQSTM1 gene also encodes an autophagy receptor and also is a target of TBK1 phosphorylation. Consequently, we hypothesized that mutations in SQSTM1 may also cause NTG. We tested this hypothesis by searching for glaucoma-causing mutations in a cohort of NTG patients (n = 308 and matched controls (n = 157 using Sanger sequencing. An additional 1098 population control samples were also analyzed using whole exome sequencing. A total of 17 non-synonymous mutations were detected which were not significantly skewed between cases and controls when analyzed separately, or as a group (p > 0.05. These data suggest that SQSTM1 mutations are not a common cause of NTG.

  20. Highly prevalent LIPH founder mutations causing autosomal recessive woolly hair/hypotrichosis in Japan and the genotype/phenotype correlations.

    Directory of Open Access Journals (Sweden)

    Kana Tanahashi

    Full Text Available Mutations in LIPH cause of autosomal recessive woolly hair/hypotrichosis (ARWH, and the 2 missense mutations c.736T>A (p.Cys246Ser and c.742C>A (p.His248Asn are considered prevalent founder mutations for ARWH in the Japanese population. To reveal genotype/phenotype correlations in ARWH cases in Japan and the haplotypes in 14 Japanese patients from 14 unrelated Japanese families. 13 patients had woolly hair, and 1 patient had complete baldness since birth. An LIPH mutation search revealed homozygous c.736T>A mutations in 10 of the patients. Compound heterozygous c.736T>A and c.742C>A mutations were found in 3 of the patients, and homozygous c.742C>A mutation in 1 patient. The phenotype of mild hypotrichosis with woolly hair was restricted to the patients with the homozygous c.736T>A mutation. The severe phenotype of complete baldness was seen in only 1 patient with homozygous c.742C>A. Haplotype analysis revealed that the alleles containing the LIPH c.736T>A mutation had a haplotype identical to that reported previously, although 4 alleles out of 5 chromosomes containing the LIPH c.742C>A mutation had a different haplotype from the previously reported founder allele. These alleles with c.742C>A are thought to be the third founder LIPH mutation causing ARWH. To accurately determine the prevalence of the founder mutations, we investigated allele frequencies of those mutations in 819 Japanese controls. Heterozygous c.736T>A mutations were found in 13 controls (allele frequency: 0.0079; carrier rate: 0.016, and heterozygous c.742C>A mutations were found in 2 controls (allele frequency: 0.0012; carrier rate: 0.0024. In conclusion, this study confirms the more accurate allele frequencies of the pathogenic founder mutations of LIPH and shows that there is a third founder mutation in Japan. In addition, the present findings suggest that the mutation patterns of LIPH might be associated with hypotrichosis severity in ARWH.

  1. Participation of Korean families at high risk for hereditary breast and ovarian cancer in BRCA1/2 genetic testing.

    Science.gov (United States)

    Sun, Young; Kang, Eunyoung; Baek, Hyunnam; Jung, Jaehag; Hwang, Euijun; Koo, Jauk; Kim, Eun-Kyu; Kim, Sung-Won

    2015-06-01

    The aim of our study was to determine the rate of participation in genetic testing, to determine the reasons for non-participation and to identify the factors affecting participation in BRCA genetic testing for high-risk patients. This study was performed through a retrospective review of 804 individuals who underwent genetic counseling for BRCA1/2 gene mutations at Seoul National University Bundang Hospital between July 2003 and September 2012. In total, 728 (90.5%) individuals underwent BRCA1/2 mutation screening after the initial genetic counseling; 88.2% of 647 probands and 100% of 157 family members were screened. In multivariate analysis, family history of breast cancer and younger age were independent variables affecting participation in genetic testing. Of the 132 people who initially declined genetic testing, 58 (43.9%) postponed the decision, 30 (22.7%) needed time to discuss the issue with family members, 22 (16.7%) did not want to know if they had a BRCA1/2 mutation, and 22 (16.7%) declined the test because of financial problems. When analyzing refusal of testing according to the time period before and after the implementation of national health insurance coverage for BRCA1/2 genetic testing, the critical reason given for refusal was different. After insurance coverage, refusal for financial reason was decreased from 61.1 to 9.6%. A family history of breast cancer and a younger age were important factors associated with participation in genetic testing. National health insurance decreased the proportion of individuals who did not participate in testing owing to a financial reason. In genetic counseling, we have to understand these issues and consider several factors that may influence an individual's decision to be tested. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  2. High resolution melting curve analysis targeting the HBB gene mutational hot-spot offers a reliable screening approach for all common as well as most of the rare beta-globin gene mutations in Bangladesh.

    Science.gov (United States)

    Islam, Md Tarikul; Sarkar, Suprovath Kumar; Sultana, Nusrat; Begum, Mst Noorjahan; Bhuyan, Golam Sarower; Talukder, Shezote; Muraduzzaman, A K M; Alauddin, Md; Islam, Mohammad Sazzadul; Biswas, Pritha Promita; Biswas, Aparna; Qadri, Syeda Kashfi; Shirin, Tahmina; Banu, Bilquis; Sadya, Salma; Hussain, Manzoor; Sarwardi, Golam; Khan, Waqar Ahmed; Mannan, Mohammad Abdul; Shekhar, Hossain Uddin; Chowdhury, Emran Kabir; Sajib, Abu Ashfaqur; Akhteruzzaman, Sharif; Qadri, Syed Saleheen; Qadri, Firdausi; Mannoor, Kaiissar

    2018-01-02

    Bangladesh lies in the global thalassemia belt, which has a defined mutational hot-spot in the beta-globin gene. The high carrier frequencies of beta-thalassemia trait and hemoglobin E-trait in Bangladesh necessitate a reliable DNA-based carrier screening approach that could supplement the use of hematological and electrophoretic indices to overcome the barriers of carrier screening. With this view in mind, the study aimed to establish a high resolution melting (HRM) curve-based rapid and reliable mutation screening method targeting the mutational hot-spot of South Asian and Southeast Asian countries that encompasses exon-1 (c.1 - c.92), intron-1 (c.92 + 1 - c.92 + 130) and a portion of exon-2 (c.93 - c.217) of the HBB gene which harbors more than 95% of mutant alleles responsible for beta-thalassemia in Bangladesh. Our HRM approach could successfully differentiate ten beta-globin gene mutations, namely c.79G > A, c.92 + 5G > C, c.126_129delCTTT, c.27_28insG, c.46delT, c.47G > A, c.92G > C, c.92 + 130G > C, c.126delC and c.135delC in heterozygous states from the wild type alleles, implying the significance of the approach for carrier screening as the first three of these mutations account for ~85% of total mutant alleles in Bangladesh. Moreover, different combinations of compound heterozygous mutations were found to generate melt curves that were distinct from the wild type alleles and from one another. Based on the findings, sixteen reference samples were run in parallel to 41 unknown specimens to perform direct genotyping of the beta-thalassemia specimens using HRM. The HRM-based genotyping of the unknown specimens showed 100% consistency with the sequencing result. Targeting the mutational hot-spot, the HRM approach could be successfully applied for screening of beta-thalassemia carriers in Bangladesh as well as in other countries of South Asia and Southeast Asia. The approach could be a useful supplement of hematological and

  3. Effects of dependence in high-dimensional multiple testing problems

    NARCIS (Netherlands)

    Kim, K.I.; van de Wiel, M.A.

    2008-01-01

    Background: We consider effects of dependence among variables of high-dimensional data in multiple hypothesis testing problems, in particular the False Discovery Rate (FDR) control procedures. Recent simulation studies consider only simple correlation structures among variables, which is hardly

  4. Variants of cancer susceptibility genes in Korean BRCA1/2 mutation-negative patients with high risk for hereditary breast cancer.

    Science.gov (United States)

    Park, Ji Soo; Lee, Seung-Tae; Nam, Eun Ji; Han, Jung Woo; Lee, Jung-Yun; Kim, Jieun; Kim, Tae Il; Park, Hyung Seok

    2018-01-16

    We evaluated the incidence and spectrum of pathogenic and likely pathogenic variants of cancer susceptibility genes in BRCA1/2 mutation-negative Korean patients with a high risk for hereditary breast cancer using a comprehensive multigene panel that included 35 cancer susceptibility genes. Samples from 120 patients who were negative for BRCA1/2 mutations, but had been diagnosed with breast cancer that was likely hereditary, were prospectively evaluated for the prevalence of high-penetrance and moderate-penetrance germline mutations. Nine patients (7.5%) had at least one pathogenic or likely pathogenic variant. Ten variants were identified in these patients: TP53 in two patients, PALB2 in three patients, BARD1 in two patients, BRIP1 in two patients, and MRE11A in one patient. We also identified 30 types of 139 variants of unknown significance (VUS). High-penetrance germline mutations, including TP53 and PALB2, tended to occur with high frequency in young (cancer patients (4/19, 21.1%) than in those diagnosed with breast cancer at ≥35 years of age (1/101, 1.0%; p = 0.003). These combined results demonstrate that multigene panels offer an alternative strategy for identifying veiled pathogenic and likely pathogenic mutations in breast cancer susceptibility genes.

  5. Motivating High School Students to Score Proficient on State Tests

    Science.gov (United States)

    Brown, Sarah Lee

    2015-01-01

    The researcher interviewed two groups of eleventh grade students, in a rural Appalachian setting, who tended to score low on the state mandated high stakes/low stakes test to discover their efforts on the test, specifically in reading, and to obtain their opinions concerning the effects of a specific incentive or consequence. Before the eleventh…

  6. Multidimensional Computerized Adaptive Testing for Indonesia Junior High School Biology

    Science.gov (United States)

    Kuo, Bor-Chen; Daud, Muslem; Yang, Chih-Wei

    2015-01-01

    This paper describes a curriculum-based multidimensional computerized adaptive test that was developed for Indonesia junior high school Biology. In adherence to the Indonesian curriculum of different Biology dimensions, 300 items was constructed, and then tested to 2238 students. A multidimensional random coefficients multinomial logit model was…

  7. Distinct Effects of Abelson Kinase Mutations on Myocytes and Neurons in Dissociated Drosophila Embryonic Cultures: Mimicking of High Temperature

    Science.gov (United States)

    Liu, Lijuan; Wu, Chun-Fang

    2014-01-01

    Abelson tyrosine kinase (Abl) is known to regulate axon guidance, muscle development, and cell-cell interaction in vivo. The Drosophila primary culture system offers advantages in exploring the cellular mechanisms mediated by Abl with utilizing various experimental manipulations. Here we demonstrate that single-embryo cultures exhibit stage-dependent characteristics of cellular differentiation and developmental progression in neurons and myocytes, as well as nerve-muscle contacts. In particular, muscle development critically depends on the stage of dissociated embryos. In wild-type (WT) cultures derived from embryos before stage 12, muscle cells remained within cell clusters and were rarely detected. Interestingly, abundant myocytes were spotted in Abl mutant cultures, exhibiting enhanced myocyte movement and fusion, as well as neuron-muscle contacts even in cultures dissociated from younger, stage 10 embryos. Notably, Abl myocytes frequently displayed well-expanded lamellipodia. Conversely, Abl neurons were characterized with fewer large veil-like lamellipodia, but instead had increased numbers of filopodia and darker nodes along neurites. These distinct phenotypes were equally evident in both homo- and hetero-zygous cultures (Abl/Abl vs. Abl/+) of different alleles (Abl1 and Abl4) indicating dominant mutational effects. Strikingly, in WT cultures derived from stage 10 embryos, high temperature (HT) incubation promoted muscle migration and fusion, partially mimicking the advanced muscle development typical of Abl cultures. However, HT enhanced neuronal growth with increased numbers of enlarged lamellipodia, distinct from the characteristic Abl neuronal morphology. Intriguingly, HT incubation also promoted Abl lamellipodia expansion, with a much greater effect on nerve cells than muscle. Our results suggest that Abl is an essential regulator for myocyte and neuron development and that high-temperature incubation partially mimics the faster muscle development

  8. Unusual Reversible Oligomerization of Unfolded Dengue Envelope Protein Domain 3 at High Temperatures and Its Abolition by a Point Mutation.

    Science.gov (United States)

    Saotome, Tomonori; Nakamura, Shigeyoshi; Islam, Mohammad M; Nakazawa, Akiko; Dellarole, Mariano; Arisaka, Fumio; Kidokoro, Shun-Ichi; Kuroda, Yutaka

    2016-08-16

    We report differential scanning calorimetry (DSC) experiments between 10 and 120 °C of Dengue 4 envelope protein domain 3 (DEN4 ED3), a small 107-residue monomeric globular protein domain. The thermal unfolding of DEN4 ED3 was fully reversible and exhibited two peculiar endothermic peaks. AUC (analytical ultracentrifugation) experiments at 25 °C indicated that DEN4 ED3 was monomeric. Detailed thermodynamic analysis indicated that the two endothermic peaks separated with an increasing protein concentration, and global fitting of the DSC curves strongly suggested the presence of unfolded tetramers at temperatures around 80-90 °C, which dissociated to unfolded monomers at even higher temperatures. To further characterize this rare thermal unfolding process, we designed and constructed a DEN4 ED3 variant that would unfold according to a two-state model, typical of globular proteins. We thus substituted Val 380, the most buried residue at the dimeric interface in the protein crystal, with less hydrophobic amino acids (Ala, Ser, Thr, Asn, and Lys). All variants showed a single heat absorption peak, typical of small globular proteins. In particular, the DSC thermogram of DEN4 V380K indicated a two-state reversible thermal unfolding independent of protein concentration, indicating that the high-temperature oligomeric state was successfully abolished by a single mutation. These observations confirmed the standard view that small monomeric globular proteins undergo a two-state unfolding. However, the reversible formation of unfolded oligomers at high temperatures is a truly new phenomenon, which was fully inhibited by an accurately designed single mutation.

  9. High prevalence of impaired glucose homeostasis and myopathy in asymptomatic and oligosymptomatic 3243A>G mitochondrial DNA mutation-positive subjects

    DEFF Research Database (Denmark)

    Frederiksen, A.L.; Jeppesen, T.D.; Vissing, J.

    2009-01-01

    INTRODUCTION: The point mutation of 3243A>G mtDNA is the most frequent cause of mitochondrial diabetes, often presenting as the syndrome maternally inherited diabetes and deafness (MIDD). The mutation may also cause myopathy, ataxia, strokes, ophthalmoplegia, epilepsy, and cardiomyopathy in vario...... correlated with the age for diagnosis of impaired glucose homeostasis and hearing impairment (rho = -0.71 to -0.78; P G mutation carriers should be screened for diabetes and myopathy Udgivelsesdato: 2009/8...... for the different phenotypes associated with this mutation. AIM: The aim of the study was to screen asymptomatic and oligosymptomatic 3243A>G mtDNA carriers for diabetes and myopathy. METHODS: The study is a case-control study. Nineteen adult 3243A>G carriers presumed to be normoglycemic and matched healthy...... controls were subjected to an oral glucose tolerance test. Twenty-six adult 3243A>G carriers with unknown myopathy status and 17 healthy controls had a maximal cycle test and a muscle biopsy performed. The mutation loads were quantified in blood and muscle biopsies and correlated to the clinical...

  10. Two-Sample Tests for High-Dimensional Linear Regression with an Application to Detecting Interactions.

    Science.gov (United States)

    Xia, Yin; Cai, Tianxi; Cai, T Tony

    2018-01-01

    Motivated by applications in genomics, we consider in this paper global and multiple testing for the comparisons of two high-dimensional linear regression models. A procedure for testing the equality of the two regression vectors globally is proposed and shown to be particularly powerful against sparse alternatives. We then introduce a multiple testing procedure for identifying unequal coordinates while controlling the false discovery rate and false discovery proportion. Theoretical justifications are provided to guarantee the validity of the proposed tests and optimality results are established under sparsity assumptions on the regression coefficients. The proposed testing procedures are easy to implement. Numerical properties of the procedures are investigated through simulation and data analysis. The results show that the proposed tests maintain the desired error rates under the null and have good power under the alternative at moderate sample sizes. The procedures are applied to the Framingham Offspring study to investigate the interactions between smoking and cardiovascular related genetic mutations important for an inflammation marker.

  11. A high incidence of WT1 abnormality in bilateral Wilms tumours in Japan, and the penetrance rates in children with WT1 germline mutation.

    Science.gov (United States)

    Kaneko, Y; Okita, H; Haruta, M; Arai, Y; Oue, T; Tanaka, Y; Horie, H; Hinotsu, S; Koshinaga, T; Yoneda, A; Ohtsuka, Y; Taguchi, T; Fukuzawa, M

    2015-03-17

    Bilateral Wilms tumours (BWTs) occur by germline mutation of various predisposing genes; one of which is WT1 whose abnormality was reported in 17-38% of BWTs in Caucasians, whereas no such studies have been conducted in East-Asians. Carriers with WT1 mutations are increasing because of improved survival. Statuses of WT1 and IGF2 were examined in 45 BWTs from 31 patients with WT1 sequencing and SNP array-based genomic analyses. The penetrance rates were estimated in WT1-mutant familial Wilms tumours collected from the present and previous studies. We detected WT1 abnormalities in 25 (81%) of 31 patients and two families, which were included in the penetrance rate analysis of familial Wilms tumour. Of 35 BWTs from the 25 patients, 31 had small homozygous WT1 mutations and uniparental disomy of IGF2, while 4 had large 11p13 deletions with the retention of 11p heterozygosity. The penetrance rate was 100% if children inherited small WT1 mutations from their fathers, and 67% if inherited the mutations from their mothers, or inherited or had de novo 11p13 deletions irrespective of parental origin (P=0.057). The high incidence of WT1 abnormalities in Japanese BWTs sharply contrasts with the lower incidence in Caucasian counterparts, and the penetrance rates should be clarified for genetic counselling of survivors with WT1 mutations.

  12. mRNA-based detection of rare CFTR mutations improves genetic diagnosis of cystic fibrosis in populations with high genetic heterogeneity.

    Science.gov (United States)

    Felício, V; Ramalho, A S; Igreja, S; Amaral, M D

    2017-03-01

    Even with advent of next generation sequencing complete sequencing of large disease-associated genes and intronic regions is economically not feasible. This is the case of cystic fibrosis transmembrane conductance regulator (CFTR), the gene responsible for cystic fibrosis (CF). Yet, to confirm a CF diagnosis, proof of CFTR dysfunction needs to be obtained, namely by the identification of two disease-causing mutations. Moreover, with the advent of mutation-based therapies, genotyping is an essential tool for CF disease management. There is, however, still an unmet need to genotype CF patients by fast, comprehensive and cost-effective approaches, especially in populations with high genetic heterogeneity (and low p.F508del incidence), where CF is now emerging with new diagnosis dilemmas (Brazil, Asia, etc). Herein, we report an innovative mRNA-based approach to identify CFTR mutations in the complete coding and intronic regions. We applied this protocol to genotype individuals with a suspicion of CF and only one or no CFTR mutations identified by routine methods. It successfully detected multiple intronic mutations unlikely to be detected by CFTR exon sequencing. We conclude that this is a rapid, robust and inexpensive method to detect any CFTR coding/intronic mutation (including rare ones) that can be easily used either as primary approach or after routine DNA analysis. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. Epidemiological and Molecular Characterization of a Mexican Population Isolate with High Prevalence of Limb-Girdle Muscular Dystrophy Type 2A Due to a Novel Calpain-3 Mutation.

    Science.gov (United States)

    Pantoja-Melendez, Carlos A; Miranda-Duarte, Antonio; Roque-Ramirez, Bladimir; Zenteno, Juan C

    2017-01-01

    Limb-Girdle Muscular Dystrophy type 2 (LGMD2) is a group of autosomally recessive inherited disorders defined by weakness and wasting of the shoulder and pelvic girdle muscles. In the past, several population isolates with high incidence of LGMD2 arising from founder mutation effects have been identified. The aim of this work is to describe the results of clinical, epidemiologic, and molecular studies performed in a Mexican village segregating numerous cases of LGMD2. A population census was conducted in the village to identify all LGMD affected patients. Molecular analysis included genome wide homozygosity mapping using a 250K SNP Affymetrix microarray followed by PCR amplification and direct nucleotide sequencing of the candidate gene. In addition, DNA from 401 randomly selected unaffected villagers was analyzed to establish the carrier frequency of the LGMD2 causal mutation. A total of 32 LGMD2 patients were identified in the village, rendering a disease prevalence of 4.3 (CI: 2.9-5.9) cases per 1,000 habitants (1 in 232). Genome wide homozygosity mapping revealed that affected individuals shared a 6.6 Mb region of homozygosity at chromosome 15q15. The identified homozygous interval contained CAPN3, the gene responsible for LGMD2 type A (LGMD2A). Direct sequencing of this gene revealed homozygosity for a novel c.348C>A mutation (p.Ala116Asp) in DNA from all 20 affected subjects available for genetic screening, except one which was heterozygous for the mutation. In such patient, a heterozygous c.2362AG>TCATCT deletion/insertion was recognized as the second CAPN3 mutation. Western blot and autocatalytic activity analyses in protein lysates from skeletal muscle biopsy obtained from a p.Ala116Asp homozygous patient suggested that this particular mutation increased the autocatalytic activity of CAPN3. Thirty eigth heterozygotes of the p.Ala116Asp mutation were identified among 401 genotyped unaffected villagers, yielding a population carrier frequency of 1 in 11

  14. Epidemiological and Molecular Characterization of a Mexican Population Isolate with High Prevalence of Limb-Girdle Muscular Dystrophy Type 2A Due to a Novel Calpain-3 Mutation.

    Directory of Open Access Journals (Sweden)

    Carlos A Pantoja-Melendez

    Full Text Available Limb-Girdle Muscular Dystrophy type 2 (LGMD2 is a group of autosomally recessive inherited disorders defined by weakness and wasting of the shoulder and pelvic girdle muscles. In the past, several population isolates with high incidence of LGMD2 arising from founder mutation effects have been identified. The aim of this work is to describe the results of clinical, epidemiologic, and molecular studies performed in a Mexican village segregating numerous cases of LGMD2. A population census was conducted in the village to identify all LGMD affected patients. Molecular analysis included genome wide homozygosity mapping using a 250K SNP Affymetrix microarray followed by PCR amplification and direct nucleotide sequencing of the candidate gene. In addition, DNA from 401 randomly selected unaffected villagers was analyzed to establish the carrier frequency of the LGMD2 causal mutation. A total of 32 LGMD2 patients were identified in the village, rendering a disease prevalence of 4.3 (CI: 2.9-5.9 cases per 1,000 habitants (1 in 232. Genome wide homozygosity mapping revealed that affected individuals shared a 6.6 Mb region of homozygosity at chromosome 15q15. The identified homozygous interval contained CAPN3, the gene responsible for LGMD2 type A (LGMD2A. Direct sequencing of this gene revealed homozygosity for a novel c.348C>A mutation (p.Ala116Asp in DNA from all 20 affected subjects available for genetic screening, except one which was heterozygous for the mutation. In such patient, a heterozygous c.2362AG>TCATCT deletion/insertion was recognized as the second CAPN3 mutation. Western blot and autocatalytic activity analyses in protein lysates from skeletal muscle biopsy obtained from a p.Ala116Asp homozygous patient suggested that this particular mutation increased the autocatalytic activity of CAPN3. Thirty eigth heterozygotes of the p.Ala116Asp mutation were identified among 401 genotyped unaffected villagers, yielding a population carrier frequency

  15. Spectrum and characterisation of BRCA1 and BRCA2 deleterious mutations in high-risk Czech patients with breast and/or ovarian cancer

    Directory of Open Access Journals (Sweden)

    Kosinova Veronika

    2008-05-01

    Full Text Available Abstract Background The incidence of breast cancer has doubled over the past 20 years in the Czech Republic. Hereditary factors may be a cause of young onset, bilateral breast or ovarian cancer, and familial accumulation of the disease. BRCA1 and BRCA2 mutations account for an important fraction of hereditary breast and ovarian cancer cases. One thousand and ten unrelated high-risk probands with breast and/or ovarian cancer were analysed for the presence of a BRCA1 or BRCA2 gene mutation at the Masaryk Memorial Cancer Institute (Czech Republic during 1999–2006. Methods The complete coding sequences and splice sites of both genes were screened, and the presence of large intragenic rearrangements in BRCA1 was verified. Putative splice-site variants were analysed at the cDNA level for their potential to alter mRNA splicing. Results In 294 unrelated families (29.1% of the 1,010 probands pathogenic mutations were identified, with 44 different BRCA1 mutations and 41 different BRCA2 mutations being detected in 204 and 90 unrelated families, respectively. In total, three BRCA1 founder mutations (c.5266dupC; c.3700_3704del5; p.Cys61Gly and two BRCA2 founder mutations (c.7913_7917del5; c.8537_8538del2 represent 52% of all detected mutations in Czech high-risk probands. Nine putative splice-site variants were evaluated at the cDNA level. Three splice-site variants in BRCA1 (c.302-3C>G; c.4185G>A and c.4675+1G>A and six splice-site variants in BRCA2 (c.475G>A; c.476-2>G; c.7007G>A; c.8755-1G>A; c.9117+2T>A and c.9118-2A>G were demonstrated to result in aberrant transcripts and are considered as deleterious mutations. Conclusion This study represents an evaluation of deleterious genetic variants in the BRCA1 and 2 genes in the Czech population. The classification of several splice-site variants as true pathogenic mutations may prove useful for genetic counselling of families with high risk of breast and ovarian cancer.

  16. Secondary variants in individuals undergoing exome sequencing: screening of 572 individuals identifies high-penetrance mutations in cancer-susceptibility genes.

    Science.gov (United States)

    Johnston, Jennifer J; Rubinstein, Wendy S; Facio, Flavia M; Ng, David; Singh, Larry N; Teer, Jamie K; Mullikin, James C; Biesecker, Leslie G

    2012-07-13

    Genome- and exome-sequencing costs are continuing to fall, and many individuals are undergoing these assessments as research participants and patients. The issue of secondary (so-called incidental) findings in exome analysis is controversial, and data are needed on methods of detection and their frequency. We piloted secondary variant detection by analyzing exomes for mutations in cancer-susceptibility syndromes in subjects ascertained for atherosclerosis phenotypes. We performed exome sequencing on 572 ClinSeq participants, and in 37 genes, we interpreted variants that cause high-penetrance cancer syndromes by using an algorithm that filtered results on the basis of mutation type, quality, and frequency and that filtered mutation-database entries on the basis of defined categories of causation. We identified 454 sequence variants that differed from the human reference. Exclusions were made on the basis of sequence quality (26 variants) and high frequency in the cohort (77 variants) or dbSNP (17 variants), leaving 334 variants of potential clinical importance. These were further filtered on the basis of curation of literature reports. Seven participants, four of whom were of Ashkenazi Jewish descent and three of whom did not meet family-history-based referral criteria, had deleterious BRCA1 or BRCA2 mutations. One participant had a deleterious SDHC mutation, which causes paragangliomas. Exome sequencing, coupled with multidisciplinary interpretation, detected clinically important mutations in cancer-susceptibility genes; four of such mutations were in individuals without a significant family history of disease. We conclude that secondary variants of high clinical importance will be detected at an appreciable frequency in exomes, and we suggest that priority be given to the development of more efficient modes of interpretation with trials in larger patient groups. Copyright © 2012 The American Society of Human Genetics. Published by Elsevier Inc. All rights

  17. Fast forward genetics to identify mutations causing a high light tolerant phenotype in Chlamydomonas reinhardtii by whole-genome-sequencing.

    Science.gov (United States)

    Schierenbeck, Lisa; Ries, David; Rogge, Kristin; Grewe, Sabrina; Weisshaar, Bernd; Kruse, Olaf

    2015-02-06

    High light tolerance of microalgae is a desired phenotype for efficient cultivation in large scale production systems under fluctuating outdoor conditions. Outdoor cultivation requires the use of either wild-type or non-GMO derived mutant strains due to safety concerns. The identification and molecular characterization of such mutants derived from untagged forward genetics approaches was limited previously by the tedious and time-consuming methods involving techniques such as classical meiotic mapping. The combination of mapping with next generation sequencing technologies offers alternative strategies to identify genes involved in high light adaptation in untagged mutants. We used the model alga Chlamydomonas reinhardtii in a non-GMO mutation strategy without any preceding crossing step or pooled progeny to identify genes involved in the regulatory processes of high light adaptation. To generate high light tolerant mutants, wildtype cells were mutagenized only to a low extent, followed by a stringent selection. We performed whole-genome sequencing of two independent mutants hit1 and hit2 and the parental wildtype. The availability of a reference genome sequence and the removal of shared bakground variants between the wildtype strain and each mutant, enabled us to identify two single nucleotide polymorphisms within the same gene Cre02.g085050, hereafter called LRS1 (putative Light Response Signaling protein 1). These two independent single amino acid exchanges are both located in the putative WD40 propeller domain of the corresponding protein LRS1. Both mutants exhibited an increased rate of non-photochemical-quenching (NPQ) and an improved resistance against chemically induced reactive oxygen species. In silico analyses revealed homology of LRS1 to the photoregulatory protein COP1 in plants. In this work we identified the nuclear encoded gene LRS1 as an essential factor for high light adaptation in C. reinhardtii. The causative random mutation within this gene was

  18. Selection of the highly replicative and partially multidrug resistant rtS78T HBV polymerase mutation during TDF-ETV combination therapy.

    Science.gov (United States)

    Shirvani-Dastgerdi, Elham; Winer, Benjamin Y; Celià-Terrassa, Toni; Kang, Yibin; Tabernero, David; Yagmur, Eray; Rodríguez-Frías, Francisco; Gregori, Josep; Luedde, Tom; Trautwein, Christian; Ploss, Alexander; Tacke, Frank

    2017-08-01

    mutations in the hepatitis B virus (HBV). We herein report two cases of patients with insufficient response to dual tenofovir and entecavir therapy. Molecular analyses identified a distinct mutation, rtS78T/sC69∗, that abolishes HBsAg detection, enhances replication, sustains exosome-mediated virion secretion and decreases susceptibility to antivirals, thereby representing a potentially high-risk mutation for HBV-infected individuals. Copyright © 2017 European Association for the Study of the Liver. All rights reserved.

  19. Testing the efficiency of high-frequency foreign exchange market

    Directory of Open Access Journals (Sweden)

    Václav Mastný

    2004-01-01

    Full Text Available This paper deals with the efficiency of the high-frequency foreign exchange market. The objective of this paper is to investigate whether standard statistical tests give the same results for time series resampled at intervals of 15.30 and 60 min. The data used for the purpose of this paper contain major currency pairs such as EUR/USD, GBP/USD and JPY/USD. The results of statistical tests indicate that the high frequency intervals (15-minute are not random and should not be considered independent. On the other hand, tests with lower frequency rates (30 and 60 min indicate rising randomness of the market.

  20. Task force for integral test of High Energy nuclear data

    Energy Technology Data Exchange (ETDEWEB)

    Oyama, Yukio [Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan). Tokai Research Establishment

    1998-11-01

    According to completion of the JENDL-High Energy file for neutron nuclear cross sections up to 50 MeV, a task force for integral test of high energy nuclear data was organized to discuss a guide line for integral test activities. A status of existing differential and integral experiments and how to perform such a test were discussed in the task force. Here the purpose and outline of the task force is explained with some future problems raised in discussion among the task member. (author)

  1. Stop High-Stakes Testing: An Appeal to America's Conscience

    Science.gov (United States)

    Johnson, Dale; Johnson, Bonnie; Farenga, Steve; Ness, Daniel

    2007-01-01

    This book is a compelling indictment of the use of high-stakes assessments with punitive consequences in public schools. The authors trace the history of the policy and document the inequities for children of poverty that undergird high-stakes testing practices. Lack of dental and medical care, environmental violence, insufficient school funding,…

  2. Mitochondrial mutations in cancer.

    Science.gov (United States)

    Brandon, M; Baldi, P; Wallace, D C

    2006-08-07

    The metabolism of solid tumors is associated with high lactate production while growing in oxygen (aerobic glycolysis) suggesting that tumors may have defects in mitochondrial function. The mitochondria produce cellular energy by oxidative phosphorylation (OXPHOS), generate reactive oxygen species (ROS) as a by-product, and regulate apoptosis via the mitochondrial permeability transition pore (mtPTP). The mitochondria are assembled from both nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) genes. The mtDNA codes for 37 genes essential of OXPHOS, is present in thousands of copies per cell, and has a very high mutations rate. In humans, severe mtDNA mutations result in multisystem disease, while some functional population-specific polymorphisms appear to have permitted humans to adapt to new environments. Mutations in the nDNA-encoded mitochondrial genes for fumarate hydratase and succinate dehydrogenase have been linked to uterine leiomyomas and paragangliomas, and cancer cells have been shown to induce hexokinase II which harnesses OXPHOS adenosine triphosphate (ATP) production to drive glycolysis. Germline mtDNA mutations at nucleotides 10398 and 16189 have been associated with breast cancer and endometrial cancer. Tumor mtDNA somatic mutations range from severe insertion-deletion and chain termination mutations to mild missense mutations. Surprisingly, of the 190 tumor-specific somatic mtDNA mutations reported, 72% are also mtDNA sequence variants found in the general population. These include 52% of the tumor somatic mRNA missense mutations, 83% of the tRNA mutations, 38% of the rRNA mutations, and 85% of the control region mutations. Some associations might reflect mtDNA sequencing errors, but analysis of several of the tumor-specific somatic missense mutations with population counterparts appear legitimate. Therefore, mtDNA mutations in tumors may fall into two main classes: (1) severe mutations that inhibit OXPHOS, increase ROS production and promote tumor

  3. Androgen receptor functional analyses by high throughput imaging: determination of ligand, cell cycle, and mutation-specific effects.

    Directory of Open Access Journals (Sweden)

    Adam T Szafran

    Full Text Available Understanding how androgen receptor (AR function is modulated by exposure to steroids, growth factors or small molecules can have important mechanistic implications for AR-related disease therapies (e.g., prostate cancer, androgen insensitivity syndrome, AIS, and in the analysis of environmental endocrine disruptors.We report the development of a high throughput (HT image-based assay that quantifies AR subcellular and subnuclear distribution, and transcriptional reporter gene activity on a cell-by-cell basis. Furthermore, simultaneous analysis of DNA content allowed determination of cell cycle position and permitted the analysis of cell cycle dependent changes in AR function in unsynchronized cell populations. Assay quality for EC50 coefficients of variation were 5-24%, with Z' values reaching 0.91. This was achieved by the selective analysis of cells expressing physiological levels of AR, important because minor over-expression resulted in elevated nuclear speckling and decreased transcriptional reporter gene activity. A small screen of AR-binding ligands, including known agonists, antagonists, and endocrine disruptors, demonstrated that nuclear translocation and nuclear "speckling" were linked with transcriptional output, and specific ligands were noted to differentially affect measurements for wild type versus mutant AR, suggesting differing mechanisms of action. HT imaging of patient-derived AIS mutations demonstrated a proof-of-principle personalized medicine approach to rapidly identify ligands capable of restoring multiple AR functions.HT imaging-based multiplex screening will provide a rapid, systems-level analysis of compounds/RNAi that may differentially affect wild type AR or clinically relevant AR mutations.

  4. High resistance of Plasmodium falciparum to sulphadoxine/pyrimethamine in northern Tanzania and the emergence of dhps resistance mutation at Codon 581.

    Science.gov (United States)

    Gesase, Samwel; Gosling, Roly D; Hashim, Ramadhan; Ord, Rosalynn; Naidoo, Inbarani; Madebe, Rashid; Mosha, Jacklin F; Joho, Angel; Mandia, Victor; Mrema, Hedwiga; Mapunda, Ephraim; Savael, Zacharia; Lemnge, Martha; Mosha, Frank W; Greenwood, Brian; Roper, Cally; Chandramohan, Daniel

    2009-01-01

    Sulphadoxine-pyrimethamine (SP) a widely used treatment for uncomplicated malaria and recommended for intermittent preventive treatment of malaria in pregnancy, is being investigated for intermittent preventive treatment of malaria in infants (IPTi). High levels of drug resistance to SP have been reported from north-eastern Tanzania associated with mutations in parasite genes. This study compared the in vivo efficacy of SP in symptomatic 6-59 month children with uncomplicated malaria and in asymptomatic 2-10 month old infants. An open label single arm (SP) standard 28 day in vivo WHO antimalarial efficacy protocol was used in 6 to 59 months old symptomatic children and a modified protocol used in 2 to 10 months old asymptomatic infants. Enrolment was stopped early (87 in the symptomatic and 25 in the asymptomatic studies) due to the high failure rate. Molecular markers were examined for recrudescence, re-infection and markers of drug resistance and a review of literature of studies looking for the 581G dhps mutation was carried out. In symptomatic children PCR-corrected early treatment failure was 38.8% (95% CI 26.8-50.8) and total failures by day 28 were 82.2% (95% CI 72.5-92.0). There was no significant difference in treatment failures between asymptomatic and symptomatic children. 96% of samples carried parasites with mutations at codons 51, 59 and 108 in the dhfr gene and 63% carried a double mutation at codons 437 and 540. 55% carried a third mutation with the addition of a mutation at codon 581 in the dhps gene. This triple: triple haplotype maybe associated with earlier treatment failure. In northern Tanzania SP is a failed drug for treatment and its utility for prophylaxis is doubtful. The study found a new combination of parasite mutations that maybe associated with increased and earlier failure. ClinicalTrials.gov NCT00361114.

  5. Development of Genetic Testing for Fragile X Syndrome and Associated Disorders, and Estimates of the Prevalence of FMR1 Expansion Mutations

    Directory of Open Access Journals (Sweden)

    James N. Macpherson

    2016-11-01

    Full Text Available The identification of a trinucleotide (CGG expansion as the chief mechanism of mutation in Fragile X syndrome in 1991 heralded a new chapter in molecular diagnostic genetics and generated a new perspective on mutational mechanisms in human genetic disease, which rapidly became a central paradigm (“dynamic mutation” as more and more of the common hereditary neurodevelopmental disorders were ascribed to this novel class of mutation. The progressive expansion of a CGG repeat in the FMR1 gene from “premutation” to “full mutation” provided an explanation for the “Sherman paradox,” just as similar expansion mechanisms in other genes explained the phenomenon of “anticipation” in their pathogenesis. Later, FMR1 premutations were unexpectedly found associated with two other distinct phenotypes: primary ovarian insufficiency and tremor-ataxia syndrome. This review will provide a historical perspective on procedures for testing and reporting of Fragile X syndrome and associated disorders, and the population genetics of FMR1 expansions, including estimates of prevalence and the influence of AGG interspersions on the rate and probability of expansion.

  6. Rapid detection of pathological mutations and deletions of the haemoglobin beta gene (HBB) by High Resolution Melting (HRM) analysis and Gene Ratio Analysis Copy Enumeration PCR (GRACE-PCR).

    Science.gov (United States)

    Turner, Andrew; Sasse, Jurgen; Varadi, Aniko

    2016-10-19

    Inherited disorders of haemoglobin are the world's most common genetic diseases, resulting in significant morbidity and mortality. The large number of mutations associated with the haemoglobin beta gene (HBB) makes gene scanning by High Resolution Melting (HRM) PCR an attractive diagnostic approach. However, existing HRM-PCR assays are not able to detect all common point mutations and have only a very limited ability to detect larger gene rearrangements. The aim of the current study was to develop a HBB assay, which can be used as a screening test in highly heterogeneous populations, for detection of both point mutations and larger gene rearrangements. The assay is based on a combination of conventional HRM-PCR and a novel Gene Ratio Analysis Copy Enumeration (GRACE) PCR method. HRM-PCR was extensively optimised, which included the use of an unlabelled probe and incorporation of universal bases into primers to prevent interference from common non-pathological polymorphisms. GRACE-PCR was employed to determine HBB gene copy numbers relative to a reference gene using melt curve analysis to detect rearrangements in the HBB gene. The performance of the assay was evaluated by analysing 410 samples. A total of 44 distinct pathological genotypes were detected. In comparison with reference methods, the assay has a sensitivity of 100 % and a specificity of 98 %. We have developed an assay that detects both point mutations and larger rearrangements of the HBB gene. This assay is quick, sensitive, specific and cost effective making it suitable as an initial screening test that can be used for highly heterogeneous cohorts.

  7. Lack of mutational events of RAS genes in sporadic thyroid cancer but high risk associated with HRAS T81C single nucleotide polymorphism (case-control study).

    Science.gov (United States)

    Khan, Mosin S; Pandith, Arshad A; Ul Hussain, Mahboob; Iqbal, Mohammad; Khan, Nighat P; Wani, Khurshid A; Masoodi, Shariq R; Mudassar, Syed

    2013-02-01

    High incidence of thyroid cancer worldwide indicates the importance of studying genetic alterations that lead to its carcinogenesis. Specific acquired RAS mutations have been found to predominate in different cancers, and HRAS T81C polymorphism has been determined to contribute the risk of various cancers, including thyroid cancer. We screened the exons 1 and 2 of RAS genes (HRAS, KRAS, and NRAS) in 60 consecutive thyroid tissue (tumor and adjacent normal) samples, and a case-control study was also conducted for HRAS T81C polymorphism in HRAS codon 27 using the polymerase chain reaction-restriction fragment length polymorphism to test the genotype distribution of 140 thyroid cancer patients in comparison with 170 cancer-free controls from a Kashmiri population. No mutation was found in any of the thyroid tumor tissue samples, but we frequently detected polymorphism at nucleotide 81 (T > C) in exon 1 of HRAS gene. In HRAS T81C SNP, frequencies of TT, TC, and CC genotypes among cases were 41.4, 38.6, and 20.0 %, while in controls genotype frequencies were 84.1, 11.7, and 4.2 %, respectively. A significant difference was observed in variant allele frequencies (TC + CC) between the cases and controls (58.6 vs. 16 %) with odds ratio = 7.4; confidence interval (CI) = 4.3-12.7 (P RAS genes do not seem to be involved. Contrary to this HRAS T81C SNP of HRAS gene moderately increases thyroid cancer risk with rare allele as a predictive marker for follicular tumors.

  8. Mutations in ABC1 in Tangier disease and familial high-density lipoprotein deficiency

    NARCIS (Netherlands)

    Brooks-Wilson, A.; Marcil, M.; Clee, S. M.; Zhang, L. H.; Roomp, K.; van Dam, M.; Yu, L.; Brewer, C.; Collins, J. A.; Molhuizen, H. O.; Loubser, O.; Ouelette, B. F.; Fichter, K.; Ashbourne-Excoffon, K. J.; Sensen, C. W.; Scherer, S.; Mott, S.; Denis, M.; Martindale, D.; Frohlich, J.; Morgan, K.; Koop, B.; Pimstone, S.; Kastelein, J. J.; Genest, J.; Hayden, M. R.

    1999-01-01

    Genes have a major role in the control of high-density lipoprotein (HDL) cholesterol (HDL-C) levels. Here we have identified two Tangier disease (TD) families, confirmed 9q31 linkage and refined the disease locus to a limited genomic region containing the gene encoding the ATP-binding cassette

  9. The light-hyperresponsive high pigment-2dg mutation of tomato: alterations in the fruit metabolome

    NARCIS (Netherlands)

    Bino, R.J.; Vos, de C.H.; Lieberman, M.; Hall, R.D.; Bovy, A.G.; Jonker, H.H.; Tikunov, Y.M.; Lommen, A.; Moco, S.I.A.; Levin, I.

    2005-01-01

    Overall metabolic modifications between fruit of light-hyperresponsive high-pigment (hp) tomato (Lycopersicon esculentum) mutant plants and isogenic nonmutant (wt) control plants were compared. Targeted metabolite analyses, as well as large-scale nontargeted mass spectrometry (MS)-based metabolite

  10. Mutation breeding of Bacillus subtilis YTB4 with high yield of ...

    African Journals Online (AJOL)

    DR TONUKARI NYEROVWO

    2012-07-17

    Jul 17, 2012 ... activity, bacteria of Bacillus are generally of poor cellulase and amylase activities. Furthermore, there are few reports about the strain of Bacillus of multienzyme complex. In this study, using Bacillus subtilis YTB4 with high yield of multienzyme complex as original strain, we reported our recent attempt to ...

  11. High prevalence of Plasmodium falciparum pfcrt K76T mutation in ...

    African Journals Online (AJOL)

    The high prevalence of sickle cell disease (SCD) and trait in Sub-Saharan Africa coincides with the distribution of Plasmodium falciparum malaria. Due to prolonged heavy use of chloroquine (CQ) as an antimalarial, drug resistance has developed. Many countries including Tanzania abandoned the use of CQ for ...

  12. High prevalence of arterial thrombosis in JAK2 mutated essential thrombocythaemia: independence of the V617F allele burden

    DEFF Research Database (Denmark)

    Larsen, Thomas Stauffer; Pallisgaard, Niels; Møller, Michael Boe

    2008-01-01

    such as higher haemoglobin levels (p=0.02), lower platelet counts (p=0.002) and lower plasma EPO levels (p=0.04). The JAK2 V617F mutation per se but not the mutational load in patients with ET is associated with a PV-like phenotype and a higher prevalence of previous arterial thrombosis. This study adds further...

  13. High dietary intake of sodium selenite does not affect gene mutation frequency in rat colon and liver

    Science.gov (United States)

    Gene mutations have been implicated in the etiology of cancer. In the present study, we utilized Big Blue transgenic rats to evaluate the in vivo mutation frequency of the ' cII gene in rats fed either a Se-deficient (0 µg Se/g diet) or selenium-supplemented diet (2 µg Se/g diet) (n=6 rats/diet) and...

  14. Test Facilities in Support of High Power Electric Propulsion Systems

    Science.gov (United States)

    van Dyke, Melissa; Houts, Mike; Godfroy, Thomas; Dickens, Ricky; Martin, James J.; Salvail, Patrick; Carter, Robert

    2003-01-01

    Successful development of space fission systems requires an extensive program of affordable and realistic testing. In addition to tests related to design/development of the fission system, realistic testing of the actual flight unit must also be performed. If the system is designed to operate within established radiation damage and fuel burn up limits while simultaneously being designed to allow close simulation of heat from fission using resistance heaters, high confidence in fission system performance and lifetime can be attained through non-nuclear testing. Through demonstration of systems concepts (designed by DOE National Laboratories) in relevant environments, this philosophy has been demonstrated through hardware testing in the High Power Propulsion Thermal Simulator (HPPTS). The HPPTS is designed to enable very realistic non-nuclear testing of space fission systems. Ongoing research at the HPPTS is geared towards facilitating research, development, system integration, and system utilization via cooperative efforts with DOE labs, industry, universities, and other NASA centers. Through hardware based design and testing, the HPPTS investigates High Power Electric Propulsion (HPEP) component, subsystem, and integrated system design and performance.

  15. Predicting Freshman Grade Point Average From College Admissions Test Scores and State High School Test Scores

    Directory of Open Access Journals (Sweden)

    Daniel Koretz

    2016-09-01

    Full Text Available The current focus on assessing “college and career readiness” raises an empirical question: How do high school tests compare with college admissions tests in predicting performance in college? We explored this using data from the City University of New York and public colleges in Kentucky. These two systems differ in the choice of college admissions test, the stakes for students on the high school test, and demographics. We predicted freshman grade point average (FGPA from high school GPA and both college admissions and high school tests in mathematics and English. In both systems, the choice of tests had only trivial effects on the aggregate prediction of FGPA. Adding either test to an equation that included the other had only trivial effects on prediction. Although the findings suggest that the choice of test might advantage or disadvantage different students, it had no substantial effect on the over- and underprediction of FGPA for students classified by race-ethnicity or poverty.

  16. Development of laboratory test methods to replace the simulated high-temperature grout fluidity test.

    Science.gov (United States)

    2014-06-01

    This report contains a summary of the research performed to develop a replacement for the high-temperature grout : fluidity (HTGF) test. The HTGF test was employed in the past by FDOT to qualify post-tensioning (PT) grouts for use in : post-tensioned...

  17. Standard guide for corrosion tests in high temperature or high pressure environment, or both

    CERN Document Server

    American Society for Testing and Materials. Philadelphia

    2010-01-01

    1.1 This guide covers procedures, specimens, and equipment for conducting laboratory corrosion tests on metallic materials under conditions of high pressure (HP) or the combination of high temperature and high pressure (HTHP). See for definitions of high pressure and temperature. 1.2 Tests conducted under HP or HTHP by their nature have special requirements. This guide establishes the basic considerations that are necessary when these conditions must be incorporated into laboratory corrosion tests. 1.3 The procedures and methods in this guide are applicable for conducting mass loss corrosion, localized corrosion, and electrochemical tests as well as for use in environmentally induced cracking tests that need to be conducted under HP or HTHP conditions. 1.4 The primary purpose for this guide is to promote consistency of corrosion test results. Furthermore, this guide will aid in the comparison of corrosion data between laboratories or testing organizations that utilize different equipment. 1.5 The values s...

  18. Analysis of Slug Tests in Formations of High Hydraulic Conductivity

    Science.gov (United States)

    Butler, J.J.; Garnett, E.J.; Healey, J.M.

    2003-01-01

    A new procedure is presented for the analysis of slug tests performed in partially penetrating wells in formations of high hydraulic conductivity. This approach is a simple, spreadsheet-based implementation of existing models that can be used for analysis of tests from confined or unconfined aquifers. Field examples of tests exhibiting oscillatory and nonoscillatory behavior are used to illustrate the procedure and to compare results with estimates obtained using alternative approaches. The procedure is considerably simpler than recently proposed methods for this hydrogeologic setting. Although the simplifications required by the approach can introduce error into hydraulic-conductivity estimates, this additional error becomes negligible when appropriate measures are taken in the field. These measures are summarized in a set of practical field guidelines for slug tests in highly permeable aquifers.

  19. High current test facility for superconductors at Saclay

    CERN Document Server

    Berriaud, C; Vieillard, L

    2001-01-01

    A high DC current (100 kA-design) test facility for superconducting material is under realisation. Aluminum stabilised conductor (as for LHC detectors) can be tested Including the stabiliser in a 4.75 T dipole field of 0.8 m length which can be rotated in both cable perpendicular directions. A superconductor transformer creates the high current with a primary current from -200 A to +200 A. The output power useable is 25 kJ so that junctions between cables or conductors can be measured at high current. Samples, with a cross sections up to 12 mm*30 mm, were 0.8 m long and were equipped with soldered cables of 0.4 m length at both ends. To test different samples without warming the dipole magnet, samples are placed in a separate dewar. The conception design is described and the first results without external dipole magnetic field are reported. (9 refs).

  20. [Combining high-risk human papillomavirus DNA test and cytological test to detect early cervical dysplasia].

    Science.gov (United States)

    Qian, De-ying; Cen, Jian-min; Wang, Ding; Zeng, Ren-hai; Lin, Ai-hua; Shu, Yan-hong; Hong, Dan-hua; Huang, Zhi-hong

    2006-01-01

    To assess the value of combining high-risk human papillomavirus (HPV) DNA test and cytological test in detection of early cervical dysplasia. During January 2003 to June 2004, a total of 5210 women were screened by combining high-risk HPV DNA test (hybrid capture II, HC-II) and cytological test (liquid-based ThinPrep cytology test), and the abnormal cytological or HPV DNA findings were further biopsied under the colposcope. The age of the patients was between 17 to 80, the average was 34 +/- 9. Final pathological diagnosis was HPV infection in 890 cases, cervical intraepithelial neoplasia (CIN) I in 83 cases, CIN II in 73 cases, CIN III in 80 cases, invasive cervical cancer in 54 cases, endometrial cancer in 5 cases, vaginal intraepithelial neoplasia in 1 case and cervical tuberculosis in 1 case. Based on the criteria of histology and pathology, the sensitivity, specificity, positive-predictive value and negative-predictive value of high-risk HPV DNA test for detecting all cases of CIN II, III were 92.22%, 74.71%, 5.19% and 99.84% respectively. In detecting all cases of CIN II, III by cytological test, for atypical squamous cell of undetermined signification (ASCUS), the sensitivity, specificity, positive-predictive value and negative-predictive value were 90.00%, 80.34%, 11.94% and 99.63% respectively; for low-grade squamous intraepithelial lesion (LSIL), the sensitivity, specificity, positive-predictive value and negative-predictive value were 70.13%, 91.58%, 11.11% and 99.51% respectively; for high-grade squamous intraepithelial lesion (HSIL), the sensitivity, specificity, positive-predictive value and negative-predictive value were 48.05%, 98.46%, 31.90% and 99.21% respectively. By the combination of high-risk HPV DNA test and cytological test, the sensitivity, specificity, positive-predictive value and negative-predictive value for detecting all cases of CIN II, III were 98.70%, 73.08%, 5.21% and 100.00% respectively. The infection rate of HPV in cervical

  1. Very high cycle fatigue testing of concrete using ultrasonic cycling

    Energy Technology Data Exchange (ETDEWEB)

    Karr, Ulrike; Schuller, Reinhard; Fitzka, Michael; Mayer, Herwig [Univ. of Natural Resources and Life Sciences, Vienna (Austria). Inst. of Physics and Materials Science; Denk, Andreas; Strauss, Alfred [Univ. of Natural Resources and Life Sciences, Vienna (Austria)

    2017-06-01

    The ultrasonic fatigue testing method has been further developed to perform cyclic compression tests with concrete. Cylindrical specimens vibrate in resonance at a frequency of approximately 20 kHz with superimposed compressive static loads. The high testing frequency allows time-saving investigations in the very high cycle fatigue regime. Fatigue tests were carried out on ''Concrete 1'' (compressive strength f{sub c} = 80 MPa) and ''Concrete 2'' (f{sub c} = 107 MPa) under purely compressive loading conditions. Experiments at maximum compressive stresses of 0.44 f{sub c} (Concrete 1) and 0.38 f{sub c} (Concrete 2) delivered specimen failures above 109 cycles, indicating that no fatigue limit exists for concrete below one billion load cycles. Resonance frequency, power required to resonate the specimen and second order harmonics of the vibration are used to monitor fatigue damage in situ. Specimens were scanned by X-ray computed tomography prior to and after testing. Fatigue cracks were produced by ultrasonic cycling in the very high cycle fatigue regime at interfaces of grains as well as in cement. The possibilities as well as limitations of ultrasonic fatigue testing of concrete are discussed.

  2. High Frequency Vibration Based Fatigue Testing of Developmental Alloys

    Science.gov (United States)

    Holycross, Casey M.; Srinivasan, Raghavan; George, Tommy J.; Tamirisakandala, Seshacharyulu; Russ, Stephan M.

    Many fatigue test methods have been previously developed to rapidly evaluate fatigue behavior. This increased test speed can come at some expense, since these methods may require non-standard specimen geometry or increased facility and equipment capability. One such method, developed by George et al, involves a base-excited plate specimen driven into a high frequency bending resonant mode. This resonant mode is of sufficient frequency (typically 1200 to 1700 Hertz) to accumulate 107 cycles in a few hours. One of the main limitations of this test method is that fatigue cracking is almost certainly guaranteed to be surface initiated at regions of high stress. This brings into question the validity of the fatigue test results, as compared to more traditional uniaxial, smooth-bar testing, since high stresses are subjecting only a small volume to fatigue damage. This limitation also brings into question the suitability of this method to screen developmental alloys, should their initiation life be governed by subsurface flaws. However, if applicable, the rapid generation of fatigue data using this method would facilitate faster design iterations, identifying more quickly, material and manufacturing process deficiencies. The developmental alloy used in this study was a powder metallurgy boron-modified Ti-6Al-4V, a new alloy currently being considered for gas turbine engine fan blades. Plate specimens were subjected to fully reversed bending fatigue. Results are compared with existing data from commercially available Ti-6Al-4V using both vibration based and more traditional fatigue test methods.

  3. Does the globally invasive marine angiosperm, Halophila stipulacea, have high genetic diversity or unique mutations?

    Science.gov (United States)

    Chiquillo, K.; Campese, L.; Barber, P. H.; Willette, D. A.

    2016-02-01

    Seagrasses are important primary producers in many marine ecosystems, and support a wide diversity of marine life. However, invasive seagrasses like Halophila stipulacea can have pronounced negative impacts on an ecosystem by displacing native seagrasses and changing the community composition of the reef. Endemic to the Red Sea, Persian Gulf and Indian Ocean, Halophila stipulacea has become invasive in the Mediterranean and Caribbean Seas, presumably as a result of the opening of the Suez Canal and international ship traffic. However, it is unclear why this marine angiosperm has become invasive in parts of its range and not others. It is hypothesized that invasive forms may have evolved rapidly in response to natural selection in new and novel environments. Alternatively, genetic variation of introduced populations may be uniquely suited to thrive in regions where it is invasive. In this study, we use RAD next-generation sequencing to screen thousands of SNPs to investigate the genetic basis of adaptation in both native and invasive populations. We test whether genes under selection in the native range are the same as in the invasive range, or whether new genes have arisen with the invasion of each marine basin. The comparison of SNP frequencies unique among basins and environmental variables will aid in predicting new areas of invasion, assisting in improved management strategies to combat this invasive seagrass.

  4. Development of a high-throughput real-time PCR assay for the detection of the R81T mutation in the nicotinic acetylcholine receptor of neonicotinoid-resistant Myzus persicae.

    Science.gov (United States)

    Puinean, Alin M; Elias, Jan; Slater, Russell; Warren, Anne; Field, Linda M; Williamson, Martin S; Bass, Chris

    2013-02-01

    Myzus persicae is a globally important aphid pest that is mainly controlled through the application of chemical insecticides. Recently, a clone of M. persicae exhibiting control-compromising levels of resistance to neonicotinoid insecticides was described. The resistance of this clone was associated with reduced affinity of imidacloprid for the target site (the nicotinic acetylcholine receptor) as a result of mutation of a key amino acid residue (R81T) in the loop D region of a nAChR β1 subunit. The potent levels of resistance conferred by this mechanism are cause for considerable concern, and the frequency and distribution of the mutation in worldwide populations of M. persicae require careful monitoring. In this study, a high-throughput assay has been developed that allows detection of the mutation in individual aphids. A real-time TaqMan assay to detect the R81T substitution was developed that proved to be sensitive and specific in tests of analytical sensitivity and in a blind genotyping trial of DNA extracted from individual aphids comprising the three possible genotypes. The assay was then used to examine the frequency of the R81T mutation in aphids collected and stored in ethanol from peach orchards in southern France. The R81T frequency varied from 33 to 100% in seven populations from the department of Gard, France. This study describes a rapid and sensitive assay that very effectively detects the R81T mutation in individual aphids. The results also have practical significance for the control of M. persicae in southern France and provide contemporary data to inform current resistance management strategies. Copyright © 2012 Society of Chemical Industry.

  5. High proportion of 22q13 deletions and SHANK3 mutations in Chinese patients with intellectual disability.

    Directory of Open Access Journals (Sweden)

    Xiaohong Gong

    Full Text Available Intellectual disability (ID is a heterogeneous disorder caused by chromosomal abnormalities, monogenic factors and environmental factors. 22q13 deletion syndrome is a genetic disorder characterized by severe ID. Although the frequency of 22q13 deletions in ID is unclear, it is believed to be largely underestimated. To address this issue, we used Affymetrix Human SNP 6.0 array to detect the 22q13 deletions in 234 Chinese unexplained ID patients and 103 controls. After the Quality Control (QC test of raw data, 22q13 deletions were found in four out of 230 cases (1.7%, while absent in parents of the cases and 101 controls. A review of genome-wide microarray studies in ID was performed and the frequency of 22q13 deletions from the literatures was 0.24%, much lower than our report. The overlapping region shared by all 4 cases encompasses the gene SHANK3. A heterozygous de novo nonsense mutation Y1015X of SHANK3 was identified in one ID patient. Cortical neurons were prepared from embryonic mice and were transfected with a control plasmid, shank3 wild-type (WT or mutant plasmids. Overexpression of the Y1015 mutant in neurons significantly affected neurite outgrowth compared with shank3 WT. These findings suggest that 22q13 deletions may be a more frequent cause for Chinese ID patients than previously thought, and the SHANK3 gene is involved in the neurite development.

  6. Identification and characterization of novel loss of function mutations in ATP-binding cassette transporter A1 in patients with low plasma high-density lipoprotein cholesterol

    NARCIS (Netherlands)

    Candini, C.; Schimmel, A. W.; Peter, J.; Bochem, A. E.; Holleboom, A. G.; Vergeer, M.; Dullaart, R. P. F.; Dallinga-Thie, G. M.; Hovingh, G. K.; Khoo, K. L.; Fasano, T.; Bocchi, L.; Calandra, S.; Kuivenhoven, J. A.; Motazacker, M. M.

    2010-01-01

    Objectives: The current literature provides little information on the frequency of mutations in the ATP-binding cassette transporter A1 (ABCA1) in patients with low high-density lipoprotein cholesterol (HDL) levels that are referred to the clinic. In 78 patients with low plasma levels of HDL

  7. Association of loss-of-function mutations in the ABCA1 gene with high-density lipoprotein cholesterol levels and risk of ischemic heart disease

    DEFF Research Database (Denmark)

    Frikke-Schmidt, R.; Nordestgaard, B.G.; Stene, M.C.A.

    2008-01-01

    Context Low levels of high- density lipoprotein ( HDL) cholesterol are inversely related to cardiovascular risk. Whether this is a causal effect is unclear. Objective To determine whether genetically reduced HDL cholesterol due to heterozygosity for 4 loss- of- function mutations in ABCA1 cause i...

  8. A high frequency of XO offspring from X(Paf)Y* male mice: evidence that the Paf mutation involves an inversion spanning the X PAR boundary.

    Science.gov (United States)

    Burgoyne, P S; Evans, E P

    2000-01-01

    It has previously been reported that 19% of the daughters of males carrying the X-linked mutation patchy fur (Paf) are XO with a maternally derived X chromosome. We now report that hemizygous Paf males that also carry the variant Y chromosome Y*, show a much increased XO production ( approximately 40% of daughters). We hypothesize that the Paf mutation is associated with an inversion spanning the pseudoautosomal region (PAR) boundary, and that this leads to preferential crossing over between the resulting inverted region of PAR and an equivalent inverted PAR region within the compound Y* PAR. This would lead to the production of dicentric X and acentric Y products and consequent sex chromosome loss. This interpretation is supported by analysis of the sex chromosome complements at the second meiotic metaphase, which revealed a high incidence of dicentrics. Another curious feature of the Paf mutation is that mice that are homozygous Paf have more hair than mice that are hemizygous Paf. This can be explained if the Paf mutation is a hypomorphic mutation that escapes X inactivation because, unlike the wild type allele, it is now located within the PAR. Copyright 2001 S. Karger AG, Basel

  9. High-Throughput Toxicity Testing: New Strategies for ...

    Science.gov (United States)

    In recent years, the food industry has made progress in improving safety testing methods focused on microbial contaminants in order to promote food safety. However, food industry toxicologists must also assess the safety of food-relevant chemicals including pesticides, direct additives, and food contact substances. With the rapidly growing use of new food additives, as well as innovation in food contact substance development, an interest in exploring the use of high-throughput chemical safety testing approaches has emerged. Currently, the field of toxicology is undergoing a paradigm shift in how chemical hazards can be evaluated. Since there are tens of thousands of chemicals in use, many of which have little to no hazard information and there are limited resources (namely time and money) for testing these chemicals, it is necessary to prioritize which chemicals require further safety testing to better protect human health. Advances in biochemistry and computational toxicology have paved the way for animal-free (in vitro) high-throughput screening which can characterize chemical interactions with highly specific biological processes. Screening approaches are not novel; in fact, quantitative high-throughput screening (qHTS) methods that incorporate dose-response evaluation have been widely used in the pharmaceutical industry. For toxicological evaluation and prioritization, it is the throughput as well as the cost- and time-efficient nature of qHTS that makes it

  10. Limiting the Unintended Consequences of High-Stakes Testing.

    Directory of Open Access Journals (Sweden)

    Stuart S. Yeh

    2005-10-01

    Full Text Available Interviews with 61 teachers and administrators in four Minnesota school districts suggest that, in their judgment, Minnesota's state-mandated tests were well-aligned with curricular priorities and teachers' instructional goals, emphasizing critical thinking as well as competencies needed to pass the Basic Standards exit exam, and avoiding the type of recall item that would require drill and memorization. This result, i n combination with a survey showing that 85 percent of Minnesota teachers support the exit exam, suggests that Minnesota has been unusually successful in designing a high stakes testing system that has garnered teacher support. The success of Minnesota's model suggests that unintended narrowing of the curriculum due to high stakes testing may be avoided if pressure on teachers to narrow the curriculum is reduced through well-designed, well-aligned exams.

  11. Mismatch repair gene mutation spectrum in the Swedish Lynch syndrome population

    DEFF Research Database (Denmark)

    Lagerstedt-Robinson, Kristina; Rohlin, Anna; Aravidis, Christos

    2016-01-01

    Lynch syndrome caused by constitutional mismatch‑repair defects is one of the most common hereditary cancer syndromes with a high risk for colorectal, endometrial, ovarian and urothelial cancer. Lynch syndrome is caused by mutations in the mismatch repair (MMR) genes i.e., MLH1, MSH2, MSH6 and PMS2....... After 20 years of genetic counseling and genetic testing for Lynch syndrome, we have compiled the mutation spectrum in Sweden with the aim to provide a population-based perspective on the contribution from the different MMR genes, the various types of mutations and the influence from founder mutations....... Mutation data were collected on a national basis from all laboratories involved in genetic testing. Mutation analyses were performed using mainly Sanger sequencing and multiplex ligation-dependent probe amplification. A total of 201 unique disease-predisposing MMR gene mutations were identified in 369...

  12. Association of high CD4-positive T cell infiltration with mutations in HLA class II-regulatory genes in microsatellite-unstable colorectal cancer.

    Science.gov (United States)

    Surmann, Eva-Maria; Voigt, Anita Y; Michel, Sara; Bauer, Kathrin; Reuschenbach, Miriam; Ferrone, Soldano; von Knebel Doeberitz, Magnus; Kloor, Matthias

    2015-03-01

    Besides being expressed on professional antigen-presenting cells, HLA class II antigens are expressed on various tumors of non-lymphoid origin, including a subset of colorectal cancers (CRC). Information about the regulation of HLA class II antigen expression is important for a better understanding of their role in the interactions between tumor and immune cells. Whether lack of HLA class II antigen expression in tumors reflects the selective immune destruction of HLA class II antigen-expressing tumor cells is unknown. To address this question, we tested whether lack of HLA class II antigen expression in CRC was associated with immune cell infiltration. We selected microsatellite-unstable (MSI-H) CRC, because they show pronounced tumor antigen-specific immune responses and, in a subset of tumors, lack of HLA class II antigen expression due to mutations inactivating HLA class II-regulatory genes. We examined HLA class II antigen expression, mutations in regulatory genes, and CD4-positive T cell infiltration in 69 MSI-H CRC lesions. Mutations in RFX5, CIITA, and RFXAP were found in 13 (28.9%), 3 (6.7%), and 1 (2.2%) out of 45 HLA class II antigen-negative tumors. CD4-positive tumor-infiltrating lymphocyte counts were significantly higher in HLA class II antigen-negative tumors harboring mutations in HLA class II-regulatory genes (107.4 T cells per 0.25 mm(2)) compared to tumors without mutations (55.5 T cells per 0.25 mm(2), p = 0.008). Our results suggest that the outgrowth of tumor cells lacking HLA class II antigen expression due to mutations of regulatory genes is favored in an environment of dense CD4-positive T cell infiltration.

  13. Intermittent Testing and Training for High-Level Football Players

    DEFF Research Database (Denmark)

    Ingebrigtsen, Jørgen

    players, or if they are not sufficiently specific for the physical football match performance requirements.The results of Study II indicates that the Yo-Yo IR2 test has a high discriminant andconcurrent validity as it both discriminates between players of different within- (top- vs. mid- vs. bottom-teams...... x 35 m, and moderate correlations between Yo-Yo IR2 and IR1 test performances and sprint speed at 20 and 35 m are revealed. Both Study II and III indicate that sub-maximal heart rate (HR) during the Yo-Yo IR tests is related to test performance, and as Study III shows, good reproducibility of sub......-endurance production training is effective and appropriate. The enhanced anaerobic capacities, as expressed through the improved Yo-Yo IR2 performance and acceleration performance, are likely to be of practical significancefor football players as it is shown that top teams have superior capacity in speed...

  14. HLA specificities are associated with prognosis in IGHV-mutated CLL-like high-count monoclonal B cell lymphocytosis.

    Science.gov (United States)

    García-Álvarez, María; Alcoceba, Miguel; López-Parra, Miriam; Puig, Noemí; Antón, Alicia; Balanzategui, Ana; Prieto-Conde, Isabel; Jiménez, Cristina; Sarasquete, María E; Chillón, M Carmen; Gutiérrez, María Laura; Corral, Rocío; Alonso, José María; Queizán, José Antonio; Vidán, Julia; Pardal, Emilia; Peñarrubia, María Jesús; Bastida, José M; García-Sanz, Ramón; Marín, Luis; González, Marcos

    2017-01-01

    Molecular alterations leading progression of asymptomatic CLL-like high-count monoclonal B lymphocytosis (hiMBL) to chronic lymphocytic leukemia (CLL) remain poorly understood. Recently, genome-wide association studies have found 6p21.3, where the human leukocyte antigen (HLA) system is coded, to be a susceptibility risk region for CLL. Previous studies have produced discrepant results regarding the association between HLA and CLL development and outcome, but no studies have been performed on hiMBL. We evaluated the role of HLA class I (-A, -B and -C) and class II (-DRB1 and -DQB1) in hiMBL/CLL susceptibility, hiMBL progression to CLL, and treatment requirement in a large series of 263 patients diagnosed in our center with hiMBL (n = 156) or Binet A CLL (n = 107). No consistent association between HLA specificities and hiMBL or CLL susceptibility was found. With a median follow-up of 7.7 years, 48/156 hiMBLs (33%) evolved to asymptomatic CLLs, while 16 hiMBLs (10%) and 44 CLLs (41%) required treatment. No HLA specificities were found to be significantly associated with hiMBL progression or treatment in the whole cohort. However, within antigen-experienced immunoglobulin heavy-chain (IGHV)-mutated hiMBLs, which represents the highest proportion of hiMBL cases (81%), the presence of HLA-DQB1*03 showed a trend to a higher risk of progression to CLL (60% vs. 26%, P = 0.062). Moreover, HLA-DQB1*02 specificity was associated with a lesser requirement for 15-year treatment (10% vs. 36%, P = 0.012). In conclusion, our results suggest a role for HLA in IGHV-mutated hiMBL prognosis, and are consistent with the growing evidence of the influence of 6p21 on predisposition to CLL. Larger non-biased series are required to enable definitive conclusions to be drawn.

  15. Senior-Loken syndrome: A novel NPHP5 gene mutation in a family from Kuwait

    Directory of Open Access Journals (Sweden)

    Makia J Marafie

    2014-04-01

    Conclusion: Identification of this pathogenic mutation helped in confirmation of the clinical diagnosis and in providing a proper pre-marital genetic counselling and testing for a couple embarking on marriage from this highly consanguineous high-risk family.

  16. Hereditary Diffuse Gastric Cancer Syndrome: CDH1 Mutations and Beyond.

    Science.gov (United States)

    Hansford, Samantha; Kaurah, Pardeep; Li-Chang, Hector; Woo, Michelle; Senz, Janine; Pinheiro, Hugo; Schrader, Kasmintan A; Schaeffer, David F; Shumansky, Karey; Zogopoulos, George; Santos, Teresa Almeida; Claro, Isabel; Carvalho, Joana; Nielsen, Cydney; Padilla, Sarah; Lum, Amy; Talhouk, Aline; Baker-Lange, Katie; Richardson, Sue; Lewis, Ivy; Lindor, Noralane M; Pennell, Erin; MacMillan, Andree; Fernandez, Bridget; Keller, Gisella; Lynch, Henry; Shah, Sohrab P; Guilford, Parry; Gallinger, Steven; Corso, Giovanni; Roviello, Franco; Caldas, Carlos; Oliveira, Carla; Pharoah, Paul D P; Huntsman, David G

    2015-04-01

    E-cadherin (CDH1) is a cancer predisposition gene mutated in families meeting clinically defined hereditary diffuse gastric cancer (HDGC). Reliable estimates of cancer risk and spectrum in germline mutation carriers are essential for management. For families without CDH1 mutations, genetic-based risk stratification has not been possible, resulting in limited clinical options. To derive accurate estimates of gastric and breast cancer risks in CDH1 mutation carriers and determine if germline mutations in other genes are associated with HDGC. Testing for CDH1 germline mutations was performed on 183 index cases meeting clinical criteria for HDGC. Penetrance was derived from 75 mutation-positive families from within this and other cohorts, comprising 3858 probands (353 with gastric cancer and 89 with breast cancer). Germline DNA from 144 HDGC probands lacking CDH1 mutations was screened using multiplexed targeted sequencing for 55 cancer-associated genes. Accurate estimates of gastric and breast cancer risks in CDH1 mutation carriers and the relative contribution of other cancer predisposition genes in familial gastric cancers. Thirty-one distinct pathogenic CDH1 mutations (14 novel) were identified in 34 of 183 index cases (19%). By the age of 80 years, the cumulative incidence of gastric cancer was 70% (95% CI, 59%-80%) for males and 56% (95% CI, 44%-69%) for females, and the risk of breast cancer for females was 42% (95% CI, 23%-68%). In CDH1 mutation-negative index cases, candidate mutations were identified in 16 of 144 probands (11%), including mutations within genes of high and moderate penetrance: CTNNA1, BRCA2, STK11, SDHB, PRSS1, ATM, MSR1, and PALB2. This is the largest reported series of CDH1 mutation carriers, providing more precise estimates of age-associated risks of gastric and breast cancer that will improve counseling of unaffected carriers. In HDGC families lacking CDH1 mutations, testing of CTNNA1 and other tumor suppressor genes should be considered

  17. A New High-Speed, High-Cycle, Gear-Tooth Bending Fatigue Test Capability

    Science.gov (United States)

    Stringer, David B.; Dykas, Brian D.; LaBerge, Kelsen E.; Zakrajsek, Andrew J.; Handschuh, Robert F.

    2011-01-01

    A new high-speed test capability for determining the high cycle bending-fatigue characteristics of gear teeth has been developed. Experiments were performed in the test facility using a standard spur gear test specimens designed for use in NASA Glenn s drive system test facilities. These tests varied in load condition and cycle-rate. The cycle-rate varied from 50 to 1000 Hz. The loads varied from high-stress, low-cycle loads to near infinite life conditions. Over 100 tests were conducted using AISI 9310 steel spur gear specimen. These results were then compared to previous data in the literature for correlation. Additionally, a cycle-rate sensitivity analysis was conducted by grouping the results according to cycle-rate and comparing the data sets. Methods used to study and verify load-path and facility dynamics are also discussed.

  18. Laboratory testing of high energy density capacitors for electric vehicles

    Energy Technology Data Exchange (ETDEWEB)

    Burke, A.F.

    1991-10-01

    Laboratory tests of advanced, high energy density capacitors in the Battery Test Laboratory of the Idaho National Engineering Laboratory have been performed to investigate their suitability for load-leveling the battery in an electric vehicle. Two types of devices were tested -- 3 V, 70 Farad, spiral wound, carbon-based, single cell devices and 20 V, 3. 5 Farad, mixed-oxide, multi-cell bipolar devices. The energy density of the devices, based on energy stored during charge to the rated voltage, was found to be 1--2 Wh/kg, which agreed well with that claimed by the manufacturers. Constant power discharge tests were performed at power densities up to 1500 W/kg. Discharges at higher power densities could have been performed had equipment been available to maintain constant power during discharges of less than one second. It was found that the capacitance of the devices were rate dependent with the rate dependency of the carbon-based devices being higher than that of the mixed-oxide devices. The resistance of both types of devices were relatively low being 20--30 milliohms. Testing done in the study showed that the advanced high energy density capacitors can be charged and discharged over cycles (PSFUDS) which approximate the duty cycle that would be encountered if the devices are used to load-level the battery in an electric vehicle. Thermal tests of the advanced capacitors in an insulated environment using the PSFUDS cycle showed the devices do not overheat with their temperatures increasing only 4--5{degrees}C for tests that lasted 5--7 hours. 7 refs., 33 figs., 11 tabs.

  19. High Temperature Calcination - MACT Upgrade Equipment Pilot Plant Test

    Energy Technology Data Exchange (ETDEWEB)

    Richard D. Boardman; B. H. O& #39; Brien; N. R. Soelberg; S. O. Bates; R. A. Wood; C. St. Michel

    2004-02-01

    About one million gallons of acidic, hazardous, and radioactive sodium-bearing waste are stored in stainless steel tanks at the Idaho Nuclear Technology and Engineering Center (INTEC), which is a major operating facility of the Idaho National Engineering and Environmental Laboratory. Calcination at high-temperature conditions (600 C, with alumina nitrate and calcium nitrate chemical addition to the feed) is one of four options currently being considered by the Department of Energy for treatment of the remaining tank wastes. If calcination is selected for future processing of the sodium-bearing waste, it will be necessary to install new off-gas control equipment in the New Waste Calcining Facility (NWCF) to comply with the Maximum Achievable Control Technology (MACT) standards for hazardous waste combustors and incinerators. This will require, as a minimum, installing a carbon bed to reduce mercury emissions from their current level of up to 7,500 to <45 {micro}g/dscm, and a staged combustor to reduce unburned kerosene fuel in the off-gas discharge to <100 ppm CO and <10 ppm hydrocarbons. The staged combustor will also reduce NOx concentrations of about 35,000 ppm by 90-95%. A pilot-plant calcination test was completed in a newly constructed 15-cm diameter calciner vessel. The pilot-plant facility was equipped with a prototype MACT off-gas control system, including a highly efficient cyclone separator and off-gas quench/venturi scrubber for particulate removal, a staged combustor for unburned hydrocarbon and NOx destruction, and a packed activated carbon bed for mercury removal and residual chloride capture. Pilot-plant testing was performed during a 50-hour system operability test January 14-16, followed by a 100-hour high-temperature calcination pilot-plant calcination run January 19-23. Two flowsheet blends were tested: a 50-hour test with an aluminum-to-alkali metal molar ratio (AAR) of 2.25, and a 50-hour test with an AAR of 1.75. Results of the testing

  20. Variable epitope library carrying heavily mutated survivin-derived CTL epitope variants as a new class of efficient vaccine immunogen tested in a mouse model of breast cancer.

    Science.gov (United States)

    NoeDominguez-Romero, Allan; Zamora-Alvarado, Rubén; Servín-Blanco, Rodolfo; Pérez-Hernández, Erendira G; Castrillon-Rivera, Laura E; Munguia, Maria Elena; Acero, Gonzalo; Govezensky, Tzipe; Gevorkian, Goar; Manoutcharian, Karen

    2014-01-01

    The antigenic variability of tumor cells leading to dynamic changes in cancer epitope landscape along with escape from immune surveillance by down-regulating tumor antigen expression/presentation and immune tolerance are major obstacles for the design of effective vaccines. We have developed a novel concept for immunogen construction based on introduction of massive mutations within the epitopes targeting antigenically variable pathogens and diseases. Previously, we showed that these immunogens carrying large combinatorial libraries of mutated epitope variants, termed as variable epitope libraries (VELs), induce potent, broad and long lasting CD8+IFN-γ+ T-cell response as well as HIV-neutralizing antibodies. In this proof-of-concept study, we tested immunogenic properties and anti-tumor effects of the VELs bearing survivin-derived CTL epitope (GWEPDDNPI) variants in an aggressive metastatic mouse 4T1 breast tumor model. The constructed VELs had complexities of 10,500 and 8,000 individual members, generated as combinatorial M13 phage display and synthetic peptide libraries, respectively, with structural composition GWXPXDXPI, where X is any of 20 natural amino acids. Statistically significant tumor growth inhibition was observed in BALB/c mice immunized with the VELs in both prophylactic and therapeutic settings. Vaccinated mice developed epitope-specific spleen cell and CD8+ IFN-γ+ T-cell responses that recognize more than 50% of the panel of 87 mutated epitope variants, as demonstrated in T-cell proliferation assays and FACS analysis. These data indicate the feasibility of the application of this new class of immunogens based on VEL concept as an alternative approach for the development of molecular vaccines against cancer.

  1. CD and NMR investigation of collagen peptides mimicking a pathological Gly-Ser mutation and a natural interruption in a similar highly charged sequence context.

    Science.gov (United States)

    Sun, Xiuxia; Liu, Songqing; Yu, Wenyuan; Wang, Shaoru; Xiao, Jianxi

    2016-02-01

    Even a single Gly substitution in the triple helix domain of collagen leads to pathological conditions while natural interruptions are suggested to play important functional roles. Two peptides-one mimicking a pathological Gly-Ser substitution (ERSEQ) and the other one modeling a similar natural interruption sequence (DRSER)-are designed to facilitate the comparison for elucidating the molecular basis of their different biological roles. CD and NMR investigation of peptide ERSEQ indicates a reduction of the thermal stability and disruption of hydrogen bonding at the Ser mutation site, providing a structural basis of the OI disease resulting from the Gly-Ser mutation in the highly charged RGE environment. Both CD and NMR real-time folding results indicate that peptide ERSEQ displays a comparatively slower folding rate than peptide DRSER, suggesting that the Gly-Ser mutation may lead to a larger interference in folding than the natural interruption in a similar RSE context. Our studies suggest that unlike the rigid GPO environment, the abundant R(K)GE(D) motif may provide a more flexible sequence environment that better accommodates mutations as well as interruptions, while the electrostatic interactions contribute to its stability. These results shed insight into the molecular features of the highly charged motif and may aid the design of collagen biomimetic peptides containing important biological sites. © 2015 The Protein Society.

  2. Evolution of mutational robustness in an RNA virus.

    Directory of Open Access Journals (Sweden)

    Rebecca Montville

    2005-11-01

    Full Text Available Mutational (genetic robustness is phenotypic constancy in the face of mutational changes to the genome. Robustness is critical to the understanding of evolution because phenotypically expressed genetic variation is the fuel of natural selection. Nonetheless, the evidence for adaptive evolution of mutational robustness in biological populations is controversial. Robustness should be selectively favored when mutation rates are high, a common feature of RNA viruses. However, selection for robustness may be relaxed under virus co-infection because complementation between virus genotypes can buffer mutational effects. We therefore hypothesized that selection for genetic robustness in viruses will be weakened with increasing frequency of co-infection. To test this idea, we used populations of RNA phage phi6 that were experimentally evolved at low and high levels of co-infection and subjected lineages of these viruses to mutation accumulation through population bottlenecking. The data demonstrate that viruses evolved under high co-infection show relatively greater mean magnitude and variance in the fitness changes generated by addition of random mutations, confirming our hypothesis that they experience weakened selection for robustness. Our study further suggests that co-infection of host cells may be advantageous to RNA viruses only in the short term. In addition, we observed higher mutation frequencies in the more robust viruses, indicating that evolution of robustness might foster less-accurate genome replication in RNA viruses.

  3. Testing the mean matrix in high-dimensional transposable data.

    Science.gov (United States)

    Touloumis, Anestis; Tavaré, Simon; Marioni, John C

    2015-03-01

    The structural information in high-dimensional transposable data allows us to write the data recorded for each subject in a matrix such that both the rows and the columns correspond to variables of interest. One important problem is to test the null hypothesis that the mean matrix has a particular structure without ignoring the dependence structure among and/or between the row and column variables. To address this, we develop a generic and computationally inexpensive nonparametric testing procedure to assess the hypothesis that, in each