Sample records for gene mutation assay

  1. Microelectronic DNA assay for the detection of BRCA1 gene mutations (United States)

    Chen, Hua; Han, Jie; Li, Jun; Meyyappan, Meyya


    Mutations in BRCA1 are characterized by predisposition to breast cancer, ovarian cancer and prostate cancer as well as colon cancer. Prognosis for this cancer survival depends upon the stage at which cancer is diagnosed. Reliable and rapid mutation detection is crucial for the early diagnosis and treatment. We developed an electronic assay for the detection of a representative single nucleotide polymorphism (SNP), deletion and insertion in BRCA1 gene by the microelectronics microarray instrumentation. The assay is rapid, and it takes 30 minutes for the immobilization of target DNA samples, hybridization, washing and readout. The assay is multiplexing since it is carried out at the same temperature and buffer conditions for each step. The assay is also highly specific, as the signal-to-noise ratio is much larger than recommended value (72.86 to 321.05 vs. 5) for homozygotes genotyping, and signal ratio close to the perfect value 1 for heterozygotes genotyping (1.04).

  2. Determining lower limits of detection of digital PCR assays for cancer-related gene mutations

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    Coren A. Milbury


    Full Text Available Digital PCR offers very high sensitivity compared to many other technologies for processing molecular detection assays. Herein, a process is outlined for determining the lower limit of detection (LoD of two droplet-based digital PCR assays for point mutations of the epidermal growth factor receptor (EGFR gene. Hydrolysis probe mutation-detection assays for EGFR p.L858R and p.T790M mutations were characterized in detail. Furthermore, sixteen additional cancer-related mutation assays were explored by the same approach. For the EGFR L8585R assay, the assay sensitivity is extremely good, and thus, the LoD is limited by the amount of amplifiable DNA that is analyzed. With 95% confidence limits, the LoD is one mutant in 180,000 wild-type molecules for the evaluation of 3.3 μg of genomic DNA, and detection of one mutant molecule in over 4 million wild-type molecules was achieved when 70 million copies of DNA were processed. The measured false-positive rate for the EGFR L8585R assay is one in 14 million, which indicates the theoretical LoD if an unlimited amount of DNA is evaluated. For the EFGR T790M assay, the LoD is one mutant in 13,000 for analysis of a 3.3 μg sample of genomic DNA, and the dPCR assay limit sensitivity approaches one mutant in 22,000 wild-type molecules.

  3. Transgenic Animal Mutation Assays

    Institute of Scientific and Technical Information of China (English)

    Tao Chen; Ph.D.D.A.B.T.


    @@ The novel transgenic mouse and rat mutation assays have provided a tool for analyzing in vivo mutation in any tissue, thus permitting the direct comparison of cancer incidence with mutant frequency.

  4. Possible incorrect genotyping of heterozygous factor V Leiden and Prothrombin 20210 gene mutations by the GeneXpert assay. (United States)

    Marturano, Alessandro; Bury, Loredana; Gresele, Paolo


    The GeneXpert analyzer is a hands-off system for the detection of Factor V Leiden and of Prothrombin G20210A (GPRO) gene thrombophilic mutations. Although the system is efficient and easy to use, we report the rare possibility of incorrect genotyping. 1648 samples were evaluated using the GeneXpert HemosIL Factor II and Factor V assay: 1319 were freshly analyzed while 329 were frozen, thawed and diluted with saline prior to analysis to avoid clogging of the instrument syringe. Two samples, both heterozygous, one for the factor V Leiden and the other for the GPRO gene, were incorrectly genotyped as homozygous for the relative mutation. Inspection of the Ct values and amplification curves and genotyping with PCR revealed the correct genotype as heterozygous for factor V Leiden and GPRO mutation. The GeneXpert HemosIL Factor II and Factor V assay is an automated, fast genotyping assay requiring almost no sample manipulation, advantageous characteristics if compared with other PCR-based methods. However, an inattentive use of it can generate incorrect diagnosis. A careful handling of the sample, in particular correct dilution of frozen/thawed samples before analysis, and the inspection of the amplification curves and Ct values are required to avoid artifacts. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Development and validation of a clinical trial patient stratification assay that interrogates 27 mutation sites in MAPK pathway genes.

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    Ken C N Chang

    Full Text Available Somatic mutations identified on genes related to the cancer-developing signaling pathways have drawn attention in the field of personalized medicine in recent years. Treatments developed to target a specific signaling pathway may not be effective when tumor activating mutations occur downstream of the target and bypass the targeted mechanism. For instance, mutations detected in KRAS/BRAF/NRAS genes can lead to EGFR-independent intracellular signaling pathway activation. Most patients with these mutations do not respond well to anti-EGFR treatment. In an effort to detect various mutations in FFPE tissue samples among multiple solid tumor types for patient stratification many mutation assays were evaluated. Since there were more than 30 specific mutations among three targeted RAS/RAF oncogenes that could activate MAPK pathway genes, a custom designed Single Nucleotide Primer Extension (SNPE multiplexing mutation assay was developed and analytically validated as a clinical trial assay. Throughout the process of developing and validating the assay we overcame many technical challenges which include: the designing of PCR primers for FFPE tumor tissue samples versus normal blood samples, designing of probes for detecting consecutive nucleotide double mutations, the kinetics and thermodynamics aspects of probes competition among themselves and against target PCR templates, as well as validating an assay when positive control tumor tissue or cell lines with specific mutations are not available. We used Next Generation sequencing to resolve discordant calls between the SNPE mutation assay and Sanger sequencing. We also applied a triplicate rule to reduce potential false positives and false negatives, and proposed special considerations including pre-define a cut-off percentage for detecting very low mutant copies in the wild-type DNA background.

  6. Development of Multiplex-Mismatch Amplification Mutation-PCR Assay for Simultaneous Detection of Campylobacter jejuni and Mutation in gyrA Gene Related to Fluoroquinolone Resistance. (United States)

    Cui, Mingquan; Wu, Chenbin; Zhang, Peng; Wu, Congming


    Campylobacter jejuni, a foodborne pathogen, is the major cause of enteritis in humans worldwide, however, its increasing resistance to fluoroquinolones reported recently is of a major concern. In the present study, multiplex-mismatch amplification mutation assay-polymerase chain reaction (MMAMA-PCR) was developed for the first time with the aim to quickly identify C. jejuni and to detect the single nucleotide mutation (C-257 to T) frequently observed in gyrA gene, associated with the acquisition of resistance to fluoroquinolones. In this assay, mismatch amplification mutation primers for the detection of gyrA mutation in C. jejuni were coupled with primers for the hip gene encoding for hippuricase and 16S rRNA gene of C. jejuni, respectively, in the multiplex PCR assay. The specificity and accuracy of this method were analyzed by the use of 78 C. jejuni strains with previously confirmed resistance phenotypes and the mutation (C-257 to T) in gyrA gene, as well as 107 clinical isolates of various bacterial species, including 29 C. jejuni isolates. This study indicates that MMAMA-PCR is a promising assay for the rapid identification of C. jejuni with a specific mutation in gyrA gene, responsible for the resistance to fluoroquinolones.

  7. Erythrocyte-based Pig-a gene mutation assay: demonstration of cross-species potential. (United States)

    Phonethepswath, Souk; Bryce, Steven M; Bemis, Jeffrey C; Dertinger, Stephen D


    Glycosylphosphatidylinositol (GPI) anchors attach specific proteins to the cell surface of hematopoietic cells. Of the genes required to form GPI anchors, only Pig-a is located on the X-chromosome. Prior work with rats suggests that the GPI anchor deficient phenotype is a reliable indicator of Pig-a mutation [Bryce et al., Environ. Mol. Mutagen., 49 (2008) 256-264]. The current report extends this line of investigation by describing simplified blood handling procedures, and by testing the assay principle in a second species, Mus musculus. With this method, erythrocytes are isolated, incubated with anti-CD24-PE, and stained with SYTO 13. Flow cytometric analyses quantify GPI anchor-deficient erythrocytes and reticulocytes. After reconstruction experiments with mutant-mimicking cells demonstrated that the analytical performance of the method is high, CD-1 mice were treated on three occasions with 7,12-dimethyl-1,2-benz[a]anthracene (DMBA, 75 mg/kg/day) or ethyl-N-nitrosourea (ENU, 40 mg/kg/day). Two weeks after the final treatment, DMBA-treated mice were found to exhibit markedly elevated frequencies of GPI anchor deficient erythrocytes and reticulocytes. For the ENU experiment, blood specimens were collected at weekly intervals over a 5-week period. Whereas the frequencies of mutant reticulocytes were significantly elevated 1 week after the last administration, the erythrocyte population was unchanged until the second week. Thereafter, both populations exhibited persistently elevated frequencies for the duration of the experiment (mean frequency at termination=310x10(-6) and 523x10(-6) for erythrocyte and reticulocyte populations, respectively). These data provide evidence that Pig-a mutation does not convey an appreciable positive or negative cell survival advantage to affected erythroid progenitors, although they do suggest that affected erythrocytes have a reduced lifespan in circulation. Collectively, accumulated data support the hypothesis that flow cytometric

  8. Benzo[a]pyrene, aflatoxine B₁ and acetaldehyde mutational patterns in TP53 gene using a functional assay: relevance to human cancer aetiology.

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    Vincent Paget

    Full Text Available Mutations in the TP53 gene are the most common alterations in human tumours. TP53 mutational patterns have sometimes been linked to carcinogen exposure. In hepatocellular carcinoma, a specific G>T transversion on codon 249 is classically described as a fingerprint of aflatoxin B(1 exposure. Likewise G>T transversions in codons 157 and 158 have been related to tobacco exposure in human lung cancers. However, controversies remain about the interpretation of TP53 mutational pattern in tumours as the fingerprint of genotoxin exposure. By using a functional assay, the Functional Analysis of Separated Alleles in Yeast (FASAY, the present study depicts the mutational pattern of TP53 in normal human fibroblasts after in vitro exposure to well-known carcinogens: benzo[a]pyrene, aflatoxin B(1 and acetaldehyde. These in vitro patterns of mutations were then compared to those found in human tumours by using the IARC database of TP53 mutations. The results show that the TP53 mutational patterns found in human tumours can be only partly ascribed to genotoxin exposure. A complex interplay between the functional impact of the mutations on p53 phenotype and the cancer natural history may affect these patterns. However, our results strongly support that genotoxins exposure plays a major role in the aetiology of the considered cancers.

  9. Restoration of correct splicing in IVSI-110 mutation of β-globin gene with antisense oligonucleotides: implications and applications in functional assay development

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    Sima Mansoori Derakhshan


    Full Text Available Objective(s: The use of antisense oligonucleotides (AOs to restore normal splicing by blocking the recognition of aberrant splice sites by the spliceosome represents an innovative means of potentially controlling certain inherited disorders affected by aberrant splicing. Selection of the appropriate target site is essential in the success of an AO therapy. In this study, in search for a splice model system to facilitate the evaluation of AOs to redirect defective splicing of IVSI-110 β-globin intron, an EGFP-based IVSI-110 specific cellular reporter assay system has been developed and a number of AOs were tested in this cellular splicing assay. Materials and Methods: A recombinant plasmid (pEGFP/I-110 carrying the EGFP gene interrupted by a mutated human β-globin intron 1 (IVSI-110 was developed and transfected into K562 cells. A number of AOs with a 2’-O-methyl oligoribonucleotide (2’-O-Me backbone system were systematically tested in this cellular splicing assay. Results: The mutation in the intron causes aberrant splicing of EGFP pre-mRNA, preventing translation of EGFP; however, treatment of the cells with specific concentration of a sequence specific 2’-O-Me AO targeted to the aberrant splice site induced correct splicing and resulted in restoring of EGFP activity. Conclusion: This cellular splicing assay provides a novel functional assay system in assessing the cellular delivery efficiency of AOs and therapeutic effect of AOs in restoration of aberrant splicing.

  10. Enzymic colorimetry-based DNA chip: a rapid and accurate assay for detecting mutations for clarithromycin resistance in the 23S rRNA gene of Helicobacter pylori. (United States)

    Xuan, Shi-Hai; Zhou, Yu-Gui; Shao, Bo; Cui, Ya-Lin; Li, Jian; Yin, Hong-Bo; Song, Xiao-Ping; Cong, Hui; Jing, Feng-Xiang; Jin, Qing-Hui; Wang, Hui-Min; Zhou, Jie


    Macrolide drugs, such as clarithromycin (CAM), are a key component of many combination therapies used to eradicate Helicobacter pylori. However, resistance to CAM is increasing in H. pylori and is becoming a serious problem in H. pylori eradication therapy. CAM resistance in H. pylori is mostly due to point mutations (A2142G/C, A2143G) in the peptidyltransferase-encoding region of the 23S rRNA gene. In this study an enzymic colorimetry-based DNA chip was developed to analyse single-nucleotide polymorphisms of the 23S rRNA gene to determine the prevalence of mutations in CAM-related resistance in H. pylori-positive patients. The results of the colorimetric DNA chip were confirmed by direct DNA sequencing. In 63 samples, the incidence of the A2143G mutation was 17.46 % (11/63). The results of the colorimetric DNA chip were concordant with DNA sequencing in 96.83 % of results (61/63). The colorimetric DNA chip could detect wild-type and mutant signals at every site, even at a DNA concentration of 1.53 x 10(2) copies microl(-1). Thus, the colorimetric DNA chip is a reliable assay for rapid and accurate detection of mutations in the 23S rRNA gene of H. pylori that lead to CAM-related resistance, directly from gastric tissues.

  11. Gold-nanorod-based colorimetric and fluorescent approach for sensitive and specific assay of disease-related gene and mutation. (United States)

    Wang, Wenhong; Zhao, Yina; Jin, Yan


    Sensitive and specific detection of disease-related gene and single nucleotide polymorphism (SNP) is of great importance in cancer diagnosis. Here, a colorimetric and fluorescent approach is described for detection of the p53 gene and SNP in homogeneous solution by using gold nanorods (GNRs) as both colorimetric probe and fluorescence quencher. Hairpin oligonucleotide was utilized as DNA probe to ensure highly sequence-specific detection of target DNA. In the presence of target DNA, the formation of DNA duplex greatly changed the electrostatic interaction between GNR and DNAs, leading to an obvious change in fluorescence and colorimetric response. The detection limit of fluorescent and colorimetric assay is 0.26 pM and 0.3 nM, respectively. Both fluorescence and colorimetric strategies were able to effectively discriminate complementary DNA from single-base mismatched DNA, which is meaningful for cancer diagnosis. More important, target DNA can be detected as low as 10 nM by the naked eye. Furthermore, transmission electron microscopy and fluorescence anisotropy measurements demonstrated that the color change as well as fluorescence quenching is ascribed to the DNA hybridization-induced aggregation of GNRs. Therefore, the assay provided a fast, sensitive, cost-effective, and specific sensing platform for detecting disease-related gene and SNP.

  12. 体内Pig-a基因突变试验方法的开发%Progress of in vivo Pig-a gene mutation assay

    Institute of Scientific and Technical Information of China (English)

    张铭; 周长慧; 王庆利; 常艳


    In vivo gene mutation is a key genetic toxicology endpoint. Recently, the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) published genotoxic-guideline, more emphasis will be placed on the results of in vivo genotoxicity testing assays. In the recent years, a new in vivo genotoxicity tests have been developed, especially for the in vivo Pig-a gene mutation assay which is under international validation and potential to be a tool for regulatory safety assessment. This paper mainly reviews characteristics of the in vivo Pig-a gene mutation testing method and its application in the genotoxicity detection of new products.%体内基因突变是遗传毒理试验中非常重要的检测终点.国际人用药物注册技术协调会议(ICH)新版遗传毒性指导原则的出台,对体内遗传毒性检测方法的发展提出了更高的要求.新的检测体细胞突变的体内实验方法即磷脂酰肌醇聚糖A (Pig-a)基因突变试验,有望成为一项新药遗传毒性评价的标准试验.本文简要综述Pig-a体内基因突变试验的特点及其在新品种开发遗传毒性检测中的应用.

  13. Two novel nonradioactive polymerase chain reaction-based assays of dried blood spots, genomic DNA, or whole cells for fast, reliable detection of Z and S mutations in the alpha 1-antitrypsin gene

    DEFF Research Database (Denmark)

    Andresen, B S; Knudsen, I; Jensen, P K;


    Two new nonradioactive polymerase chain reaction (PCR)-based assays for the Z and S mutations in the alpha 1-antitrypsin gene are presented. The assays take advantage of PCR-mediated mutagenesis, creating new diagnostic restriction enzyme sites for unambiguous discrimination between test samples...

  14. Development of an endpoint genotyping assay to detect the Mycoplasma pneumoniae 23S rRNA gene and distinguish the existence of macrolide resistance-associated mutations at position 2063. (United States)

    Suzuki, Yu; Seto, Junji; Shimotai, Yoshitaka; Ikeda, Tatsuya; Yahagi, Kazue; Mizuta, Katsumi; Matsuzaki, Yoko; Hongo, Seiji


    The prevalence of macrolide-resistant Mycoplasma pneumoniae harboring a mutation in the 23S rRNA gene is increasing, and rapid detection assays are needed for clinical management. We developed an endpoint genotyping assay to detect the M. pneumoniae 23S rRNA gene and determine the existence of macrolide resistance-associated mutations at position 2063 (A2063G, A2063T and A2063C mutations). This A2063B genotyping assay detected more than 50 copies/reaction of the M. pneumoniae gene in every nucleotide mutation at position 2063. Of 42 clinical specimens, 3 were positive without mutation, 6 were positive with the A2063G mutation, and 33 were negative. The results were confirmed using nested PCR with the sequencing of the M. pneumoniae 23S rRNA gene, and a high sensitivity (90%), specificity (100%), and coincidence ratio (kappa coefficient=0.93) were obtained. Therefore, the A2063B genotyping assay is useful for the rapid discrimination of macrolide resistance mutations at position 2063.

  15. PRRT2 gene mutations (United States)

    Gardiner, Alice R.; Bhatia, Kailash P.; Stamelou, Maria; Dale, Russell C.; Kurian, Manju A.; Schneider, Susanne A.; Wali, G.M.; Counihan, Tim; Schapira, Anthony H.; Spacey, Sian D.; Valente, Enza-Maria; Silveira-Moriyama, Laura; Teive, Hélio A.G.; Raskin, Salmo; Sander, Josemir W.; Lees, Andrew; Warner, Tom; Kullmann, Dimitri M.; Wood, Nicholas W.; Hanna, Michael


    ABSTRACT Objective: The proline-rich transmembrane protein (PRRT2) gene was recently identified using exome sequencing as the cause of autosomal dominant paroxysmal kinesigenic dyskinesia (PKD) with or without infantile convulsions (IC) (PKD/IC syndrome). Episodic neurologic disorders, such as epilepsy, migraine, and paroxysmal movement disorders, often coexist and are thought to have a shared channel-related etiology. To investigate further the frequency, spectrum, and phenotype of PRRT2 mutations, we analyzed this gene in 3 large series of episodic neurologic disorders with PKD/IC, episodic ataxia (EA), and hemiplegic migraine (HM). Methods: The PRRT2 gene was sequenced in 58 family probands/sporadic individuals with PKD/IC, 182 with EA, 128 with HM, and 475 UK and 96 Asian controls. Results: PRRT2 genetic mutations were identified in 28 out of 58 individuals with PKD/IC (48%), 1/182 individuals with EA, and 1/128 individuals with HM. A number of loss-of-function and coding missense mutations were identified; the most common mutation found was the p.R217Pfs*8 insertion. Males were more frequently affected than females (ratio 52:32). There was a high proportion of PRRT2 mutations found in families and sporadic cases with PKD associated with migraine or HM (10 out of 28). One family had EA with HM and another large family had typical HM alone. Conclusions: This work expands the phenotype of mutations in the PRRT2 gene to include the frequent occurrence of migraine and HM with PKD/IC, and the association of mutations with EA and HM and with familial HM alone. We have also extended the PRRT2 mutation type and frequency in PKD and other episodic neurologic disorders. PMID:23077024

  16. Differential genotoxicity of acrylamide in the micronucleus and Pig-a gene mutation assays in F344 rats and B6C3F1 mice. (United States)

    Hobbs, Cheryl A; Davis, Jeffrey; Shepard, Kim; Chepelev, Nikolai; Friedman, Marvin; Marroni, Dennis; Recio, Leslie


    Acrylamide is used in many industrial processes and is present in a variety of fried and baked foods. In rodent carcinogenicity assays, acrylamide exposure leads to tumour formation at doses lower than those demonstrated to induce genotoxic damage. We evaluated the potential of acrylamide to induce structural DNA damage and gene mutations in rodents using highly sensitive flow cytometric analysis of micronucleus and Pig-a mutant frequencies, respectively. Male F344 rats and B6C3F1 mice were administered acrylamide in drinking water for 30 days at doses spanning and exceeding the range of acrylamide exposure tested in cancer bioassays-top dose of 12.0 and 24.0mg/kg/day in mice and in rats, respectively. A positive control, N-ethyl-N-nitrosourea, was administered at the beginning and end of the study to meet the expression time for the two DNA damage phenotypes. The results of the micronucleus and Pig-a assays were negative and equivocal, respectively, for male rats exposed to acrylamide at the concentrations tested. In contrast, acrylamide induced a dose-dependent increase in micronucleus formation but tested negative in the Pig-a assay in mice. Higher plasma concentrations of glycidamide in mice than rats are hypothesized to explain, at least in part, the differences in the response. Benchmark dose modelling indicates that structural DNA damage as opposed to point mutations is most relevant to the genotoxic mode of action of acrylamide-induced carcinogenicity. Moreover, the lack of genotoxicity detected at acrylamide-induced carcinogenicity in rodents. © The Author 2016. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail:

  17. Detection of the A2058G and A2059G 23S rRNA gene point mutations associated with azithromycin resistance in Treponema pallidum by use of a TaqMan real-time multiplex PCR assay. (United States)

    Chen, Cheng-Yen; Chi, Kai-Hua; Pillay, Allan; Nachamkin, Eli; Su, John R; Ballard, Ronald C


    Macrolide treatment failure in syphilis patients is associated with a single point mutation (either A2058G or A2059G) in both copies of the 23S rRNA gene in Treponema pallidum strains. The conventional method for the detection of both point mutations uses nested PCR combined with restriction enzyme digestions, which is laborious and time-consuming. We initially developed a TaqMan-based real-time duplex PCR assay for detection of the A2058G mutation, and upon discovery of the A2059G mutation, we modified the assay into a triplex format to simultaneously detect both mutations. The point mutations detected by the real-time triplex PCR were confirmed by pyrosequencing. A total of 129 specimens PCR positive for T. pallidum that were obtained from an azithromycin resistance surveillance study conducted in the United States were analyzed. Sixty-six (51.2%) of the 129 samples with the A2058G mutation were identified by both real-time PCR assays. Of the remaining 63 samples that were identified as having a macrolide-susceptible genotype by the duplex PCR assay, 17 (27%) were found to contain the A2059G mutation by the triplex PCR. The proportions of macrolide-susceptible versus -resistant genotypes harboring either the A2058G or the A2059G mutation among the T. pallidum strains were 35.6, 51.2, and 13.2%, respectively. None of the T. pallidum strains examined had both point mutations. The TaqMan-based real-time triplex PCR assay offers an alternative to conventional nested PCR and restriction fragment length polymorphism analyses for the rapid detection of both point mutations associated with macrolide resistance in T. pallidum.


    DEFF Research Database (Denmark)


    The present invention relates to an isolated polynucleotide encoding at least a part of calmodulin and an isolated polypeptide comprising at least a part of a calmodulin protein, wherein the polynucleotide and the polypeptide comprise at least one mutation associated with a cardiac disorder. The ...... the binding of calmodulin to ryanodine receptor 2 and use of such compound in a treatment of an individual having a cardiac disorder. The invention further provides a kit that can be used to detect specific mutations in calmodulin encoding genes....


    DEFF Research Database (Denmark)


    The present invention relates to an isolated polynucleotide encoding at least a part of calmodulin and an isolated polypeptide comprising at least a part of a calmodulin protein, wherein the polynucleotide and the polypeptide comprise at least one mutation associated with a cardiac disorder...... the binding of calmodulin to ryanodine receptor 2 and use of such compound in a treatment of an individual having a cardiac disorder. The invention further provides a kit that can be used to detect specific mutations in calmodulin encoding genes....

  20. Evaluation of CFTR gene mutations in Adana

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    Ozlem Goruroglu Ozturk


    Full Text Available ABSTRACT Objective: Cystic fibrosis is the most common autosomal recessive inherited disorder seen in the white populations. It develops in result of mutations of cystic fibrosis transmembrane regulator (CFTR gene. Rate of these mutations vary in different geographical regions. In this study, we aimed to determine the frequency of CFTR gene mutations in Adana. Methods: DNA samples of 63 subjects (21 women, 42 men who were diagnosed as cystic fibrosis at Balcali Hospital of Cukurova University, were studied for 19 different CFTR mutations by the strip assay method which is based on reverse hybridization. Results: In cystic fibrosis diagnosed patients, 19 mutations were observed of which 9 were homozygous and 10 were heterozygous. ∆F508 frequency was found as 11.9%, and rate of homozygous was found as 66.7%. Mutation frequencies of W1282X and N1303K were found as 2.40% and 4.80% respectively and rate of homozygous mutations were 50% for both. I148T mutation frequency was found as 3.20% and all were heterozygous. For the whole 19 mutations, frequency of mutation in 63 subjects was 22.3%. Conclusion: Detection of CFTR gene mutations by the strip assay method by reverse hybridization is an easy, fast and informative method. However, due to improvability of the common mutations in probable cystic fibrosis patients because of heterogenity in this region, it is still a major problem and does not exclude cystic fibrosis diagnosis. But this problematic issue can be overcome by evaluating the whole exons of CFTR mutations by advanced molecular tecniques. Key words: CFTR, cystic fibrosis, molecular diagnosis, reverse hibridisation [Cukurova Med J 2013; 38(2.000: 202-208

  1. Identification of three novel OA1 gene mutations identified in three families misdiagnosed with congenital nystagmus and carrier status determination by real-time quantitative PCR assay

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    Hamel Christian


    Full Text Available Abstract Background X-linked ocular albinism type 1 (OA1 is caused by mutations in OA1 gene, which encodes a membrane glycoprotein localised to melanosomes. OA1 mainly affects pigment production in the eye, resulting in optic changes associated with albinism including hypopigmentation of the retina, nystagmus, strabismus, foveal hypoplasia, abnormal crossing of the optic fibers and reduced visual acuity. Affected Caucasian males usually appear to have normal skin and hair pigment. Results We identified three previously undescribed mutations consisting of two intragenic deletions (one encompassing exon 6, the other encompassing exons 7–8, and a point mutation (310delG in exon 2. We report the development of a new method for diagnosis of heterozygous deletions in OA1 gene based on measurement of gene copy number using real-time quantitative PCR from genomic DNA. Conclusion The identification of OA1 mutations in families earlier reported as families with hereditary nystagmus indicate that ocular albinism type 1 is probably underdiagnosed. Our method of real-time quantitative PCR of OA1 exons with DMD exon as external standard performed on the LightCycler™ allows quick and accurate carrier-status assessment for at-risk females.

  2. Gene mutations in hepatocellular adenomas

    DEFF Research Database (Denmark)

    Raft, Marie B; Jørgensen, Ernö N; Vainer, Ben


    is associated with bi-allelic mutations in the TCF1 gene and morphologically has marked steatosis. β-catenin activating HCA has increased activity of the Wnt/β-catenin pathway and is associated with possible malignant transformation. Inflammatory HCA is characterized by an oncogene-induced inflammation due....... This review offers an overview of the reported gene mutations associated with hepatocellular adenomas together with a discussion of the diagnostic and prognostic value....

  3. A misleading false-negative result using Neisseria gonorrhoeae opa MGB multiplex PCR assay in patient's rectal sample due to partial mutations of the opa gene. (United States)

    Vahidnia, Ali; van Empel, Pieter Jan; Costa, Sandra; Oud, Rob T N; van der Straaten, Tahar; Bliekendaal, Harry; Spaargaren, Joke


    A 53-year-old homosexual man presented at his general practitioner (GP) practice with a suspicion of sexually transmitted infection. Initial NAAT screening was performed for Chlamydia trachomatis and Neisseria gonorrhoeae. The patient was positive for Neisseria gonorrhoeae both for his urine and rectal sample. The subsequent confirmation test for Neisseria gonorrhoeae by a second laboratory was only confirmed for the urine sample and the rectal sample was negative. We report a case of a potential false-negative diagnosis of Neisseria gonorrhoeae due to mutations of DNA sequence in the probe region of opa-MGB assay of the rectal sample. The patient did not suffer any discomfort as diagnosis of Neisseria gonorrhoeae in his urine sample had already led to treatment by prescribing the patient with Ceftriaxone 500 mg IV dissolved in 1 ml lidocaine 2% and 4 mL saline. The patient also received a prescription for Azithromycin (2x500 mg).

  4. In house reverse membrane hybridisation assay versus GenoType MTBDRplus and their performance to detect mutations in the genes rpoB, katG and inhA

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    Sergio Luiz Montego Ferreira Junior


    Full Text Available Drug-resistant tuberculosis (TB threatens global TB control and is a major public health concern in several countries. We therefore developed a multiplex assay (LINE-TB/MDR that is able to identify the most frequent mutations related to rifampicin (RMP and isoniazid (INH resistance. The assay is based on multiplex polymerase chain reaction, membrane hybridisation and colorimetric detection targeting of rpoB and katG genes, as well as the inhA promoter, which are all known to carry specific mutations associated with multidrug-resistant TB (MDR-TB. The assay was validated on a reference panel of 108 M. tuberculosis isolates that were characterised by the proportion method and by DNA sequencing of the targets. When comparing the performance of LINE-TB/MDR with DNA sequencing, the sensitivity, specificity and agreement were 100%, 100% and 100%, respectively, for RMP and 77.6%, 90.6% and 88.9%, respectively, for INH. Using drug sensibility testing as a reference standard, the performance of LINE-TB/MDR regarding sensitivity, specificity and agreement was 100%, 100% and 100% (95%, respectively, for RMP and 77%, 100% and 88.7% (82.2-95.1, respectively, for INH. LINE-TB/MDR was compared with GenoType MTBDRplus for 65 isolates, resulting in an agreement of 93.6% (86.7-97.5 for RIF and 87.4% (84.3-96.2 for INH. LINE-TB/MDR warrants further clinical validation and may be an affordable alternative for MDR-TB diagnosis.

  5. Validation of a Multiplex Allele-Specific Polymerase Chain Reaction Assay for Detection of KRAS Gene Mutations in Formalin-Fixed, Paraffin-Embedded Tissues from Colorectal Cancer Patients.

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    Sirirat Seekhuntod

    Full Text Available Patients with KRAS mutations do not respond to epidermal growth factor receptor (EGFR inhibitors and fail to benefit from adjuvant chemotherapy. Mutation analysis of KRAS is needed before starting treatment with monoclonal anti-EGFR antibodies in patients with metastatic colorectal cancer (mCRC. The objective of this study is to develop a multiplex allele-specific PCR (MAS-PCR assay to detect KRAS mutations.We developed a single-tube MAS-PCR assay for the detection of seven KRAS mutations (G12D, G12A, G12R, G12C, G12S, G12V, and G13D. We performed MAS-PCR assay analysis for KRAS on DNA isolated from 270 formalin-fixed paraffin-embedded (FFPE colorectal cancer tissues. Sequences of all 270 samples were determined by pyrosequencing. Seven known point-mutation DNA samples diluted with wild-type DNA were assayed to determine the limitation of detection and reproducibility of the MAS-PCR assay.Overall, the results of MAS-PCR assay were in good concordance with pyrosequencing, and only seven discordant samples were found. The MAS-PCR assay reproducibly detected 1 to 2% mutant alleles. The most common mutations were G13D in codon 13 (49.17%, G12D (25.83% and G12V (12.50% in codon 12.The MAS-PCR assay provides a rapid, cost-effective, and reliable diagnostic tool for accurate detection of KRAS mutations in routine FFPE colorectal cancer tissues.

  6. Simple, low-cost molecular assays for TR34/L98H mutations in the cyp51A gene for rapid detection of triazole-resistant Aspergillus fumigatus isolates

    NARCIS (Netherlands)

    Ahmad, S.; Khan, Z.; Hagen, F.; Meis, J.F.G.M.


    Simple, low-cost PCR/PCR-restriction fragment length polymorphism (RFLP) assays targeting cyp51A promoter and codon 98 regions were developed for the detection of triazole-resistant Aspergillus fumigatus strains carrying TR34/L98H mutations. The assays were evaluated using 40 itraconazole-susceptibl

  7. Detecting the spectrum of multigene mutations in non-small cell lung cancer by Snapshot assay

    Institute of Scientific and Technical Information of China (English)

    Jian Su; Xiao-Sui Huang; Yi-Long Wu; Xu-Chao Zhang; She-Juan An; Wen-Zhao Zhong; Ying Huang; Shi-Liang Chen; Hong-Hong Yan; Zhi-Hong Chen; Wei-Bang Guo


    As molecular targets continue to be identified and more targeted inhibitors are developed for personalized treatment of non-small cell lung cancer (NSCLC), multigene mutation determination will be needed for routine oncology practice and for clinical trials. In this study, we evaluated the sensitivity and specificity of multigene mutation testing by using the Snapshot assay in NSCLC. We retrospectively reviewed a cohort of 110 consecutive NSCLC specimens for which epidermal growth factor receptor (EGFR) mutation testing was performed between November 2011 and December 2011 using Sanger sequencing. Using the Snapshot assay, mutation statuses were detected forEGFR, Kirsten rate sarcoma viral oncogene homolog (KRAS), phosphoinositide-3-kinase catalytic alpha polypeptide (PIK3CA), v-Raf murine sarcoma viral oncogene homolog B1 (BRAF), v-ras neuroblastoma viral oncogene homolog (NRAS), dual specificity mitogen activated protein kinase kinase 1 (MEK1), phosphatase and tensin homolog (PTEN), and human epidermal growth factor receptor 2 (HER2) in patient specimens and cellline DNA. Snapshot data were compared to Sanger sequencing data. Of the 110 samples, 51 (46.4%) harbored at least one mutation. The mutation frequency in adenocarcinoma specimens was 55.6%, and the frequencies ofEGFR, KRAS, PIK3CA, PTEN, andMEK1 mutations were 35.5%, 9.1%, 3.6%, 0.9%, and 0.9%, respectively. No mutation was found in theHER2, NRAS, orBRAF genes. Three of the 51 mutant samples harbored double mutations: twoPIK3CA mutations coexisted withKRAS orEGFR mutations, and another KRAS mutation coexisted with aPTEN mutation. Among the 110 samples, 47 were surgical specimens, 60 were biopsy specimens, and 3 were cytological specimens; the corresponding mutation frequencies were 51.1%, 41.7%, and 66.7%, respectively (P = 0.532). Compared to Sanger sequencing, Snapshot specificity was 98.4% and sensitivity was 100% (positive predictive value, 97.9%; negative predictive value, 100%). The Snapshot assay

  8. PFAPA and 12 Common MEFV Gene Mutations Our Clinical Experience. (United States)

    Salehzadeh, Farhad; Vahedi, Maryam; Hosseini-Asl, Saeid; Jahangiri, Sepideh; Habibzadeh, Shahram; Hosseini-Khotbesara, Mahsa


    Marshall Syndrome or PFAPA is an inflammatory periodic disease characterized by periodic fever, aphthous stomatitis, pharyngitis and cervical adenitis. Although PFAPA is an auto inflammatory disease, it doesn't have genetic basis such as other periodic fevers. This study evaluates the 12 common MEFV gene mutations in patients with PFAPA syndrome. 21 patients with PFAPA syndrome who had diagnostic criteria were enrolled in this study and 12 common MEFV gene mutations i.e. P369S, F479L, M680I (G/C), M680I (G/A), I692del, M694V, M694I, K695R, V726A, A744S, R761H, E148Q evaluated. All the patients were screened for MEFV gene mutations by a reverse hybridization assay (FMF Strip Assay, Vienna lab, Vienna, Austria) according to the instructions provided by the manufacturer. Findings : The age of patients was between 6 months to 14 years, and 15 were males. Seven patients had heterozygote and one had compound heterozygote (K695R, V725A) mutation. There were 4 alleles M694V, 3 alleles V726A, 1 allele E148Q and 1 allele K694R. No significant difference existed between mutated patients with non-mutated in symptoms like aphthous and stomatitis, duration of attacks, episodes of fever and response to treatment. Gaslini score test was not helpful to predict the probability of gene mutations. About 30 percent of patients had MEFV gene mutations but these mutations did not play a main role in presentation of PFAPA symptoms.

  9. Mutations in the human TWIST gene. (United States)

    Gripp, K W; Zackai, E H; Stolle, C A


    Saethre-Chotzen syndrome is a relatively common craniosynostosis disorder with autosomal dominant inheritance. Mutations in the TWIST gene have been identified in patients with Saethre-Chotzen syndrome. The TWIST gene product is a transcription factor with DNA binding and helix-loop-helix domains. Numerous missense and nonsense mutations cluster in the functional domains, without any apparent mutational hot spot. Two novel point mutations and one novel polymorphism are included in this review. Large deletions including the TWIST gene have been identified in some patients with learning disabilities or mental retardation, which are not typically part of the Saethre-Chotzen syndrome. Comprehensive studies in patients with the clinical diagnosis of Saethre-Chotzen syndrome have demonstrated a TWIST gene abnormality in about 80%, up to 37% of which may be large deletions [Johnson et al., 1998]. The gene deletions and numerous nonsense mutations are suggestive of haploinsufficiency as the disease-causing mechanism. No genotype phenotype correlation was apparent.





    Somatic cell mutation is able to create genetic variance in a cell population and can induce cancer and tumor when gene mutations took place at repressor gene in controlling cell cycles such as p53 gene. Whereas germline cell mutation can cause genetic disease such as sickle cell anemia, breast cancer, thalassemia, parkinson’s as well as defect of biochemical pathway that influence drug-receptor interaction, which has negative effect and lead to hospitalized of patient. Most of reports mentio...

  11. Genotoxicity assessment of melamine in the in vivo Pig-a mutation assay and in a standard battery of assays. (United States)

    Tu, Honggang; Zhang, Ming; Zhou, Changhui; Wang, Zheng; Huang, Pengcheng; Ou, Hongmei; Chang, Yan


    The genotoxicity of melamine was evaluated with the combined Pig-a mutation/micronucleus assay, the bacterial reverse mutation assay, and the in vitro cytokinesis-block micronucleus assay (CBMN). Five groups of six- to eight-week-old male Sprague-Dawley (SD) rats were given three daily doses of vehicle control (100% pure sesame oil), melamine (500, 1000, and 2000 mg/kg) or positive control (N-ethyl-N-nitrosourea, ENU, 20 mg/kg) by oral gavage. Peripheral blood was sampled pre-dose (day -1) and at time points up to day 60. Pig-a mutant frequencies were determined in total red blood cells (RBCs) and reticulocytes (RETs) as RBC(CD59-) and RET(CD59-) frequencies, on days -1, 15, 29 and 60, and micronucleus frequencies were measured in RETs on day 4. No significant increases in RBC(CD59-) or RET(CD59-) frequencies were observed for the melamine-treated group at any of the time points studied, but the positive control, ENU, induced statistically significant increases compared with the vehicle control. Similar results were obtained in the micronucleus assay. Melamine did not induce statistically significant increases in %MN-RET. In the bacterial reverse mutation assay, melamine was tested from 62.5 to 1000 μg/plate in tester strains TA97a, TA98, TA100, TA102, and TA1535, with and without metabolic activation, and no evidence of toxicity or mutagenicity was observed at any dose tested. In the in vitro CBMN assay, in Chinese hamster ovary (CHO) cells, melamine was tested (75, 150, and 300 μg/mL) in the presence and absence of S9 mix, and no positive increases in the number of cells containing micronuclei were seen. These results suggest that melamine does not exhibit significant genotoxic potential. These data could be valuable for risk assessment purposes and also for further characterizing the new in vivoPig-a gene mutation assay.


    Institute of Scientific and Technical Information of China (English)

    ZENG Ji-bin; SONG Yue; WANG Yi; SHI Yu-yuan


    @@ Genetic alternations, such as mutations caused inactivities of tumor suppressor gene, have been identified in a wide variety of tumors, including osteosarcoma. Osteosarcoma is the most frequent primary malignant bone tumor that occurs in the extremities of young adolescents in most cases. Because of the high frequent occurrence of this type of tumor in hereditary retinoblastoma patients, involvement of the Rb1 gene mutations was suspected in the development of osteosarcoma, and a few reports have shown alternations of the Rb1 gene in osteosarcoma. We studied Rb1 gene mutations in 9 osteosarcoma samples and one cell line (OS 732) to explore the types and mechanism of Rb1 gene mutations in osteosarcoma.

  13. Computational Analysis of PTEN Gene Mutation

    Directory of Open Access Journals (Sweden)

    Siew-Kien Mah


    Full Text Available Post-genomic data can be efficiently analyzed using computational tools. It has the advantage over the biochemical and biophysical methods in term of higher coverage. In this research, we adopted a computational analysis on PTEN gene mutation.  Mutation in PTEN is responsible for many human diseases. The results of this research provide insights into the protein domains of PTEN and the distribution of mutation.

  14. Cooperating gene mutations in childhood acute myeloid leukemia with special reference on mutations of ASXL1, TET2, IDH1, IDH2, and DNMT3A. (United States)

    Liang, Der-Cherng; Liu, Hsi-Che; Yang, Chao-Ping; Jaing, Tang-Her; Hung, Iou-Jih; Yeh, Ting-Chi; Chen, Shih-Hsiang; Hou, Jen-Yin; Huang, Ying-Jung; Shih, Yu-Shu; Huang, Yu-Hui; Lin, Tung-Huei; Shih, Lee-Yung


    Gene mutations involving epigenetic regulators recently have been described in adult acute myeloid leukemia (AML). Similar studies are limited in children. We analyzed gene mutations and cooperation in pediatric AML with special reference on mutated epigenetic regulators. Nineteen gene mutations, including 8 class I genes, 4 class II genes, WT1 and TP53 (class III), and 5 epigenetic regulator genes (class IV), were analyzed in 206 children with de novo AML. Mutational analysis was performed with polymerase chain reaction-based assay followed by direct sequencing. One hundred seventeen of 206 patients (56.8%) had at least one mutation: 51% class I, 13% class II, 6.8% class III, and 5.6% class IV. FLT3-internal tandem duplication was most frequent, and 29% of patients had more than one gene mutation. Two patients carried ASXL1 mutations, both with t(8;21), 2 had DNMT3A mutations, 2 had IDH1 mutations, 1 had IDH2 mutation, and 3 had TET2 mutations. Both patients with IDH1 mutations had AML-M0 subtype and MLL-partial tandem duplication. Cooperating mutations with mutated epigenetic regulators were observed in 8 of 10 patients. We conclude that mutated epigenetic regulators were much less than those in adult AML but with frequent cooperating mutations. ASXL1, TET2, and IDH1 mutations were associated with specific genetic subtypes.

  15. Mutation update for the PORCN gene

    DEFF Research Database (Denmark)

    Lombardi, Maria Paola; Bulk, Saskia; Celli, Jacopo


    Mutations in the PORCN gene were first identified in Goltz-Gorlin syndrome patients in 2007. Since then, several reports have been published describing a large variety of genetic defects resulting in the Goltz-Gorlin syndrome, and mutations or deletions were also reported in angioma serpiginosum,...

  16. Screening for mutations in the uroporphyrinogen decarboxylase gene using denaturing gradient gel electrophoresis

    DEFF Research Database (Denmark)

    Christiansen, L; Ged, C; Hombrados, I


    The two porphyrias, familial porphyria cutanea tarda (fPCT) and hepatoerythropoietic porphyria (HEP), are associated with mutations in the gene encoding the enzyme uroporphyrinogen decarboxylase (UROD). Several mutations, most of which are private, have been identified in HEP and fPCT patients......, confirming the heterogeneity of the underlying genetic defects of these diseases. We have established a denaturing gradient gel electrophoresis (DGGE) assay for mutation detection in the UROD gene, enabling the simultaneous screening for known and unknown mutations. The established assay has proved able...

  17. Assays to Study Consequences of Cytoplasmic Intermediate Filament Mutations: The Case of Epidermal Keratins. (United States)

    Tan, Tong San; Ng, Yi Zhen; Badowski, Cedric; Dang, Tram; Common, John E A; Lacina, Lukas; Szeverényi, Ildikó; Lane, E Birgitte


    The discovery of the causative link between keratin mutations and a growing number of human diseases opened the way for a better understanding of the function of the whole intermediate filament families of cytoskeleton proteins. This chapter describes analytical approaches to identification and interpretation of the consequences of keratin mutations, from the clinical and diagnostic level to cells in tissue culture. Intermediate filament pathologies can be accurately diagnosed from skin biopsies and DNA samples. The Human Intermediate Filament Database collates reported mutations in intermediate filament genes and their diseases, and can help clinicians to establish accurate diagnoses, leading to disease stratification for genetic counseling, optimal care delivery, and future mutation-aligned new therapies. Looking at the best-studied keratinopathy, epidermolysis bullosa simplex, the generation of cell lines mimicking keratinopathies is described, in which tagged mutant keratins facilitate live-cell imaging to make use of today's powerful enhanced light microscopy modalities. Cell stress assays such as cell spreading and cell migration in scratch wound assays can interrogate the consequences of the compromised cytoskeletal network. Application of extrinsic stresses, such as heat, osmotic, or mechanical stress, can enhance the differentiation of mutant keratin cells from wild-type cells. To bring the experiments to the next level, 3D organotypic human cultures can be generated, and even grafted onto the backs of immunodeficient mice for greater in vivo relevance. While development of these assays has focused on mutant K5/K14 cells, the approaches are often applicable to mutations in other intermediate filaments, reinforcing fundamental commonalities in spite of diverse clinical pathologies.

  18. Somatic mutations of APC gene in carcinomas from hereditary non-polyposis colorectal cancer patients

    Institute of Scientific and Technical Information of China (English)

    Jian Huang; Shu Zheng; Shen-Hang Jin; Su-Zhan Zhang


    AIM: To investigate the mutational features of adenomatous polyposis coii (APC) gene and its possible arising mechanism in hereditary non-polyposis colorectal cancers (HNPCC).METHODS: PCR-based In Vitro Synthesized Protein Test (IVSP) assay and sequencing analysis were used to confirm somatic mutations of whole APC gene in 19 HNPCC cases. RESULTS: Eleven cases with 13 mutations were determined to harbor APC mutations. The prevalence of APC mutation was 58%(11/19). The mutations consisted of 9 frameshift and 4 nonsense ones, indicating that there were more frameshift mutations (69%). The frameshift mutations allexhibited deletion or insertion of 1-2 bp and most of them (7/9) happened at simple nucleotide repeat sequences, particularly within (A)n tracts (5/9). All point mutations presented C-to-T transitions at CpG sites. CONCLUSION: Mutations of APC gene were detected in more than half of HNPCC, indicating that its mutation was a common molecular event and might play an important role in the tumorigenesis of HNPCC. Locations of frameshift mutations at simple nucleotide repeat sequences and point mutations at CpG sites suggested that many mutations probably derived from endogenous processes including mismatch repair (MMR) deficiency. Defective MMR might affect the nature of APC mutations in HNPCC and likely occur earlier than APC mutational inactivation in some patients.

  19. Mutational robustness of gene regulatory networks.

    Directory of Open Access Journals (Sweden)

    Aalt D J van Dijk

    Full Text Available Mutational robustness of gene regulatory networks refers to their ability to generate constant biological output upon mutations that change network structure. Such networks contain regulatory interactions (transcription factor-target gene interactions but often also protein-protein interactions between transcription factors. Using computational modeling, we study factors that influence robustness and we infer several network properties governing it. These include the type of mutation, i.e. whether a regulatory interaction or a protein-protein interaction is mutated, and in the case of mutation of a regulatory interaction, the sign of the interaction (activating vs. repressive. In addition, we analyze the effect of combinations of mutations and we compare networks containing monomeric with those containing dimeric transcription factors. Our results are consistent with available data on biological networks, for example based on evolutionary conservation of network features. As a novel and remarkable property, we predict that networks are more robust against mutations in monomer than in dimer transcription factors, a prediction for which analysis of conservation of DNA binding residues in monomeric vs. dimeric transcription factors provides indirect evidence.

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


    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. Postnatal and non-invasive prenatal detection of β-thalassemia mutations based on Taqman genotyping assays (United States)

    Breveglieri, Giulia; Travan, Anna; D’Aversa, Elisabetta; Cosenza, Lucia Carmela; Pellegatti, Patrizia; Guerra, Giovanni; Gambari, Roberto


    The β-thalassemias are genetic disorder caused by more than 200 mutations in the β-globin gene, resulting in a total (β0) or partial (β+) deficit of the globin chain synthesis. The most frequent Mediterranean mutations for β-thalassemia are: β039, β+IVSI-110, β+IVSI-6 and β0IVSI-1. Several molecular techniques for the detection of point mutations have been developed based on the amplification of the DNA target by polymerase chain reaction (PCR), but they could be labor-intensive and technically demanding. On the contrary, TaqMan® genotyping assays are a simple, sensitive and versatile method suitable for the single nucleotide polymorphism (SNP) genotyping affecting the human β-globin gene. Four TaqMan® genotyping assays for the most common β-thalassemia mutations present in the Mediterranean area were designed and validated for the genotype characterization of genomic DNA extracted from 94 subjects comprising 25 healthy donors, 33 healthy carriers and 36 β-thalassemia patients. In addition, 15 specimens at late gestation (21–39 gestational weeks) and 11 at early gestation (5–18 gestational weeks) were collected from pregnant women, and circulating cell-free fetal DNAs were extracted and analyzed with these four genotyping assays. We developed four simple, inexpensive and versatile genotyping assays for the postnatal and prenatal identification of the thalassemia mutations β039, β+IVSI-110, β+IVSI-6, β0IVSI-1. These genotyping assays are able to detect paternally inherited point mutations in the fetus and could be efficiently employed for non-invasive prenatal diagnosis of β-globin gene mutations, starting from the 9th gestational week. PMID:28235086

  2. Rapid detection of common mutations in the arylsulfatase A gene

    Energy Technology Data Exchange (ETDEWEB)

    Coulter-Mackie, M.B. [Univ. of Western Ontario (Canada)]|[CPRI, London, Ontario (Canada)


    Metachromatic leukodystrophy (MLD), an autosomal recessive lysosomal storage disease results from a deficiency of arylsulfatase A activity. This disease is usually fatal within a few years of onset in the pediatric age group. A pseuodeficiency occurs in up to 15% of alleles in the general population which significantly decreases enzyme activity. Although there is no clinical phenotype associated with the pseudodeficiency, the decreased enzyme activity can complicate interpretation of biochemical assay results particularly in the case of potential heterozygous carriers of MLD. Two mutations have been found to be simultaneously associated with the pseudodeficiency: one at a glycosylatin site in exon 6 and one in the polyA addition signal. Another mutation, the `I` allele has been reported in up to 50% of alleles in the severe infantile onset form of MLD. The deleterious mutation in this case is in the +1 position of intron 2. In order to screen for these commonly occurring mutations in the arylsulfatase A gene, a simple combination of PCR amplification from genomic DNA and restriction enzyme digestions was developed for each situation. In the case of the pseuodeficiency mutations, oligonucleotide primers were designed which incorporated a single base mismatch 3 bases upstream from the 3{prime} end of the primer so that the presence of the mutation created new MaeIII restriction site in the case of the glycosylation site or an RsaI site in the case of the polyA site. The `I` allele mutation creates a new MvaI site without the use of mismatches. These tests have successfully detected the mutations in individuals suspected of having the pseudodeficiency on the basis of biochemical assay. The `I` allele was detected in 1 of 16 MLD alleles analyzed.

  3. Targeted gene mutation in Phytophthora spp.

    NARCIS (Netherlands)

    Lamour, K.H.; Finley, L.; Hurtado-Gonzales, O.; Gobena, D.; Tierney, M.; Meijer, H.J.G.


    The genus Phytophthora belongs to the oomycetes and is composed of plant pathogens. Currently, there are no strategies to mutate specific genes for members of this genus. Whole genome sequences are available or being prepared for Phytophthora sojae, P. ramorum, P. infestans, and P. capsici and the d

  4. NFU1 gene mutation and mitochondrial disorders

    Directory of Open Access Journals (Sweden)

    Yasemin G Kurt


    Full Text Available Mitochondrial respiratory chains consist of approximately 100 structural proteins. Thirteen of these structural proteins are encoded by mitochondrial DNA (mtDNA, and the others by nuclear DNA (nDNA. Mutation in any of the mitochondrial structural-protein related genes, regardless of whether they are in the nDNA or mtDNA, might cause mitochondrial disorders. In the recent past, new nuclear genes required for assembly, maintenance, and translation of respiratory chain proteins have been found. Mutation in these genes might also cause mitochondrial disorders (MD. NFU1 gene is one of such genes and has a role in the assembly of iron–sulfur cluster (ISC. ISCs are included in a variety of metalloproteins, such as the ferredoxins, as well as in enzymatic reactions and have been first identified in the oxidation-reduction reactions of mitochondrial electron transport. It is important to be aware of NFU1 gene mutations that may cause severe mitochondrial respiratory chain defects, mitochondrial encephalomyopathies and death, early in life.

  5. Mutagenicity test system based on a reporter gene assay for short-term detection of mutagens (MutaGen assay). (United States)

    Schmid, Claudia; Arndt, Christian; Reifferscheid, Georg


    The construction of a bacterial mutation assay system detecting reversions of base substitutions and frameshifts in tetracycline (tet) and ampicillin resistance genes located on low copy plasmids is described. Frameshift mutations were introduced into repetitive GC-sequences and G-repeats known to be mutagenic hot-spots. Base pair substitutions were inserted in or around the active site of the ampicillinase gene thus generating reversibility of the ampicilline sensitivity. The plasmids carry genes to enable sensitive, fast and specific detection of mutagens in bacteria. MucAB was cloned into the test plasmid to enhance error-prone DNA-repair. The conventional reversion principle has been combined with the luminometric measurement of an inducible reporter gene. The revertants are detected after induction of the beta-galactosidase-producing lacZ-gene either controlled by its natural lac-promotor or by the more stringently repressed (anhydrotetracyclin inducible) tetA promotor. The tester strains containing the tetA/lacZ reporter gene construct can grow in full medium over the complete assay. This test procedure enables screening for mutations within one working day. Incubation for 16 h reveals high sensitivity.

  6. The Wilson disease gene: Haplotypes and mutations

    Energy Technology Data Exchange (ETDEWEB)

    Thomas, G.R.; Roberts, E.A.; Cox, D.W. [Hospital for Sick Children, Toronto (Canada); Walshe, J.M. [Middlesex Hospital, London (United Kingdom)


    Wilson disease (WND) is an autosomal recessive defect of copper transport. The gene involved in WND, located on chromosome 13, has recently been shown to be a putative copper transporting P-type ATPase, designated ATP7B. The gene is highly similar to ATP7A, located on the X chromosome, which is defective in Menkes disease, another disorder of copper transport. We have available for study WND families from Canada (34 families), the United Kingdom (32 families), Japan (4 families), Iceland (3 families) and Hong Kong (2 families). We have utilized four highly polymorphic CA repeat markers (D13S296, D13S301, D13S314 and D13S316) surrounding the ATP7B locus to construct haplotypes in these families. Analysis indicates that there are many unique WND haplotypes not present on normal chromosomes and that there may be a large number of different WND mutations. We have screened the WND patients for mutations in the ATP7B gene. Fifty six patients, representing all of the identified haplotypes, have been screened using single strand conformational polymorphism (SSCP), followed by selective sequencing. To date, 19 mutations and 12 polymorphisms have been identified. All of the changes are nucleotide substitutions or small insertions/deletions and there is no evidence for larger deletions as seen in the similar gene on the X chromosome, ATP7A. Haplotypes of close markers and the ability to detect some of the mutations present in the gene allow for more reliable molecular diagnosis of presymptomatic sibs of WND patients. A reassessment of individuals previously diagnosed in the presymptomatic phase is now required, as we have have identified some heterozygotes who are biochemically indistinguishable from affected homozygotes. The identification of specific mutations will soon allow direct diagnosis of WND patients with a high level of certainty.

  7. Mutations in connexin genes and disease. (United States)

    Pfenniger, Anna; Wohlwend, Annelise; Kwak, Brenda R


    Connexins are a family of transmembrane proteins that are widely expressed in the human body. Connexins play an important role in cell-cell communication and homeostasis in various tissues by forming gap junction channels, which enable a direct passage of ions or metabolites from one cell to another. Twenty-one different connexins are expressed in humans, each having distinct expression patterns and regulation properties. Knowledge on this family of proteins can be gained by making an inventory of mutations and associated diseases in human. PubMed and other relevant databases were searched. In addition, key review articles were screened for relevant original publications. Sections of representative organs were photographed and annotated. The crucial role of connexins is highlighted by the discovery of mutations in connexin genes which cause a variety of disorders such as myelin-related diseases, skin disorders, hearing loss, congenital cataract, or more complex syndromes such as the oculodendrodigital dysplasia. This review systematically addresses current knowledge on mutations in connexin genes and disease, focusing on the correlation between genetic defects, cellular phenotypes and clinical manifestations. The review of diseases caused by mutations in connexin genes highlights the essential nature of connexin function and intercellular communication in tissue homeostasis. © 2010 The Authors. European Journal of Clinical Investigation © 2010 Stichting European Society for Clinical Investigation Journal Foundation.

  8. Recurrent APC gene mutations in Polish FAP families

    Directory of Open Access Journals (Sweden)

    Pławski Andrzej


    Full Text Available Abstract The molecular diagnostics of genetically conditioned disorders is based on the identification of the mutations in the predisposing genes. Hereditary cancer disorders of the gastrointestinal tracts are caused by mutations of the tumour suppressor genes or the DNA repair genes. Occurrence of recurrent mutation allows improvement of molecular diagnostics. The mutation spectrum in the genes causing hereditary forms of colorectal cancers in the Polish population was previously described. In the present work an estimation of the frequency of the recurrent mutations of the APC gene was performed. Eight types of mutations occurred in 19.4% of our FAP families and these constitute 43% of all Polish diagnosed families.

  9. Mutation scanning of peach floral genes

    Directory of Open Access Journals (Sweden)

    Wilde H Dayton


    Full Text Available Abstract Background Mutation scanning technology has been used to develop crop species with improved traits. Modifications that improve screening throughput and sensitivity would facilitate the targeted mutation breeding of crops. Technical innovations for high-resolution melting (HRM analysis are enabling the clinic-based screening for human disease gene polymorphism. We examined the application of two HRM modifications, COLD-PCR and QMC-PCR, to the mutation scanning of genes in peach, Prunus persica. The targeted genes were the putative floral regulators PpAGAMOUS and PpTERMINAL FLOWER I. Results HRM analysis of PpAG and PpTFL1 coding regions in 36 peach cultivars found one polymorphic site in each gene. PpTFL1 and PpAG SNPs were used to examine approaches to increase HRM throughput. Cultivars with SNPs could be reliably detected in pools of twelve genotypes. COLD-PCR was found to increase the sensitivity of HRM analysis of pooled samples, but worked best with small amplicons. Examination of QMC-PCR demonstrated that primary PCR products for further analysis could be produced from variable levels of genomic DNA. Conclusions Natural SNPs in exons of target peach genes were discovered by HRM analysis of cultivars from a southeastern US breeding program. For detecting natural or induced SNPs in larger populations, HRM efficiency can be improved by increasing sample pooling and template production through approaches such as COLD-PCR and QMC-PCR. Technical advances developed to improve clinical diagnostics can play a role in the targeted mutation breeding of crops.

  10. 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


    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

  11. Chromosomal mutagen sensitivity associated with mutations in BRCA genes. (United States)

    Speit, G; Trenz, K


    Chromosomal mutagen sensitivity is a common feature of cells from patients with different kinds of cancer. A portion of breast cancer patients also shows an elevated sensitivity to the induction of chromosome damage in cells exposed to ionizing radiation or chemical mutagens. Segregation analysis in families of patients with breast cancer indicated heritability of mutagen sensitivity. It has therefore been suggested that mutations in low-penetrance genes which are possibly involved in DNA repair predispose a substantial portion of breast cancer patients. Chromosomal mutagen sensitivity has been determined with the G2 chromosome aberration test and the G(0) micronucleus test (MNT). However, there seems to be no clear correlation between the results from the two tests, indicating that the inherited defect leading to enhanced G(0) sensitivity is different from that causing G2 sensitivity. Less than 5% of breast cancer patients have a familial form of the disease due to inherited mutations in the breast cancer susceptibility genes BRCA1 or BRCA2. Heterozygous mutations in BRCA1 or BRCA2 in lymphocytes from women with familial breast cancer are also associated with mutagen sensitivity. Differentiation between mutation carriers and controls seems to be much better with the MNT than with the G2 assay. Mutagen sensitivity was detected with the MNT not only after irradiation but also after treatment with chemical mutagens including various cytostatics. The enhanced formation of micronuclei after exposure of lymphocytes to these substances suggests that different DNA repair pathways are affected by a BRCA1 mutation in accordance with the proposed central role of BRCA1 in maintaining genomic integrity. Mutations in BRCA1 and BRCA2 seem to predispose cells to an increased risk of mutagenesis and transformation after exposure to radiation or cytostatics. This raises a question about potentially increased risks by mammography and cancer therapy in women carrying a mutation in

  12. The Androgen Receptor Gene Mutations Database. (United States)

    Gottlieb, B; Lehvaslaiho, H; Beitel, L K; Lumbroso, R; Pinsky, L; Trifiro, M


    The current version of the androgen receptor (AR) gene mutations database is described. The total number of reported mutations has risen from 272 to 309 in the past year. We have expanded the database: (i) by giving each entry an accession number; (ii) by adding information on the length of polymorphic polyglutamine (polyGln) and polyglycine (polyGly) tracts in exon 1; (iii) by adding information on large gene deletions; (iv) by providing a direct link with a completely searchable database (courtesy EMBL-European Bioinformatics Institute). The addition of the exon 1 polymorphisms is discussed in light of their possible relevance as markers for predisposition to prostate or breast cancer. The database is also available on the internet (http://www.mcgill. ca/androgendb/ ), from EMBL-European Bioinformatics Institute (ftp. ), or as a Macintosh FilemakerPro or Word file (

  13. A 3-plex methylation assay combined with the FGFR3 mutation assay sensitively detects recurrent bladder cancer in voided urine

    DEFF Research Database (Denmark)

    Kandimalla, Raju; Masius, Roy; Beukers, Willemien;


    Purpose: DNA methylation is associated with bladder cancer and these modifications could serve as useful biomarkers. FGFR3 mutations are present in 60% to 70% of non–muscle invasive bladder cancer (NMIBC). Low-grade bladder cancer recurs in more than 50% of patients. The aim of this study......, and nonmalignant urines (n = 130). Results: The 3-plex assay identified recurrent bladder cancer in voided urine with a sensitivity of 74% in the validation set. In combination with the FGFR3 mutation assay, a sensitivity of 79% was reached (specificity of 77%). Sensitivity of FGFR3 and cytology was 52% and 57......%, respectively. Conclusion: The combination of methylation and FGFR3 assays efficiently detects recurrent bladder cancer without the need for stratification of patients regarding methylation/mutation status of the primary tumor. We conclude that the sensitivity of this combination is in the same range...

  14. Hereditary sideroblastic anemia: pathophysiology and gene mutations. (United States)

    Harigae, Hideo; Furuyama, Kazumichi


    Sideroblastic anemia is characterized by anemia with the emergence of ring sideroblasts in the bone marrow. Ring sideroblasts are erythroblasts characterized by iron accumulation in perinuclear mitochondria due to impaired iron utilization. There are two forms of sideroblastic anemia, i.e., inherited and acquired sideroblastic anemia. Inherited sideroblastic anemia is a rare and heterogeneous disease caused by mutations of genes involved in heme biosynthesis, iron-sulfur (Fe-S) cluster biogenesis, or Fe-S cluster transport, and mitochondrial metabolism. The most common inherited sideroblastic anemia is X-linked sideroblastic anemia (XLSA) caused by mutations of the erythroid-specific δ-aminolevulinate synthase gene (ALAS2), which is the first enzyme of heme biosynthesis in erythroid cells. Sideroblastic anemia due to SLC25A38 gene mutations, which is a mitochondrial transporter, is the next most common inherited sideroblastic anemia. Other forms of inherited sideroblastic anemia are very rare, and accompanied by impaired function of organs other than hematopoietic tissue, such as the nervous system, muscle, or exocrine glands due to impaired mitochondrial metabolism. Moreover, there are still significant numbers of cases with genetically undefined inherited sideroblastic anemia. Molecular analysis of these cases will contribute not only to the development of effective treatment, but also to the understanding of mitochondrial iron metabolism.

  15. MAMA-PCR assay for the detection of point mutations associated with high-level erythromycin resistance in Campylobacter jejuni and Campylobacter coli strains. (United States)

    Alonso, Rodrigo; Mateo, Estibaliz; Churruca, Estibaliz; Martinez, Irati; Girbau, Cecilia; Fernández-Astorga, Aurora


    Twenty Campylobacter jejuni and 16 Campylobacter coli strains isolated from humans and food/animals, including 17 isolates resistant to erythromycin, were analyzed. A combined mismatch amplification mutation assay-PCR technique was developed to detect the mutations A 2074 C and A 2075 G in the 23S rRNA gene associated with erythromycin resistance. All high-level erythromycin-resistant strains examined by DNA sequencing carried the transition mutation A 2075 G, whereas no isolate carried the A 2074 C mutation. No mutations were found among the susceptible and low-level erythromycin-resistant strains.

  16. Collodion Baby with TGM1 gene mutation

    Directory of Open Access Journals (Sweden)

    Sharma D


    Full Text Available Deepak Sharma,1 Basudev Gupta,2 Sweta Shastri,3 Aakash Pandita,1 Smita Pawar4 1Department of Neonatology, Fernandez Hospital, Hyderguda, Hyderabad, Andhra Pradesh, 2Department of Pediatrics, Civil Hospital, Palwal, Haryana, 3Department of Pathology, NKP Salve Medical College, Nagpur, Maharashtra, 4Department of Obstetrics and Gynaecology, Fernandez Hospital, Hyderguda, Hyderabad, Andhra Pradesh, IndiaAbstract: Collodion baby (CB is normally diagnosed at the time of birth and refers to a newborn infant that is delivered with a lambskin-like membrane encompassing the total body surface. CB is not a specific disease entity, but is a common phenotype in conditions like harlequin ichthyosis, lamellar ichthyosis, nonbullous congenital ichthyosiform erythroderma, and trichothiodystrophy. We report a CB that was brought to our department and later diagnosed to have TGM1 gene c.984+1G>A mutation. However, it could not be ascertained whether the infant had lamellar ichthyosis or congenital ichthyosiform erythroderma (both having the same mutation. The infant was lost to follow-up.Keywords: cellophane membrane, c.984+1G>A mutation, lamellar ichthyosis, nonbullous congenital ichthyosiform erythroderma, parchment membrane, TGM1 gene

  17. Nitric oxide as a carcinogen. Analysis by yeast functional assay of inactivating p53 mutations induced by nitric oxide

    Energy Technology Data Exchange (ETDEWEB)

    Murata, Jun-Ichi; Tada, Mitsuhiro; Sawamura, Yutaka; Shinohe, Yumiko; Abe, Hiroshi [Laboratory for Molecular Brain Research, Department of Neurosurgery, Hokkaido University School of Medicine, Sapporo (Japan); Iggo, Richard D. [Oncogene group, Swiss Institute for Experimental Cancer Research ISREC, Epalinges (Switzerland)


    We have used a yeast p53 functional assay to study induction of mutations in the p53 tumor suppressor gene by nitric oxide and cytosine methylation. The yeast assay identifies only biologically important p53 mutations. p53 cDNA was treated with the nitric oxide donor sydnonimine, PCR-amplified and transfected into yeast. Sydnonimine produced a significant, dose-dependent increase in C:G->A:T transversions. Many important p53 mutational hotspots are postulated to arise by deamination of methylCpG in tumors. We therefore examined nitric oxide induction of mutations in p53 cDNA methylated by PCR-mediated substitution of 5-methylcytosine for cytosine or by treatment with the SssI CpG methylase. Both methylation procedures increased the baseline mutation rate, and nitric oxide treatment produced a further increase in mutation yield. Sequence analysis showed that methylation alone led to C:G->T:A transitions, whereas nitric oxide treatment simply produced more C:G->A:T transversions. Thus the most important factor in C:G->T:A transition at CpG sites identified in this experimental system is cytosine methylation, consistent with spontaneous conversion of 5-methylcytosine to thymine by deamination

  18. 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


    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.

  19. Gene mutation patterns in patients with minimally differentiated acute myeloid leukemia. (United States)

    Kao, Hsiao-Wen; Liang, Der-Cherng; Wu, Jin-Hou; Kuo, Ming-Chung; Wang, Po-Nan; Yang, Chao-Ping; Shih, Yu-Shu; Lin, Tung-Huei; Huang, Yu-Hui; Shih, Lee-Yung


    Minimally differentiated acute myeloid leukemia (AML-M0) is a rare subtype of AML with poor prognosis. Although genetic alterations are increasingly reported in AML, the gene mutations have not been comprehensively studied in AML-M0. We aimed to examine a wide spectrum of gene mutations in patients with AML-M0 to determine their clinical relevance. Twenty gene mutations including class I, class II, class III of epigenetic regulators (IDH1, IDH2, TET2, DNMT3A, MLL-PTD, ASXL1, and EZH2), and class IV (tumor suppressor genes) were analyzed in 67 patients with AML-M0. Mutational analysis was performed with polymerase chain reaction-based assays followed by direct sequencing. The most frequent gene mutations from our data were FLT3-ITD/FLT3-TKD (28.4%), followed by mutations in IDH1/IDH2 (28.8%), RUNX1 (23.9%), N-RAS/K-RAS (12.3%), TET2 (8.2%), DNMT3A (8.1%), MLL-PTD (7.8%), and ASXL1 (6.3%). Seventy-nine percent (53/67) of patients had at least one gene mutation. Class I genes (49.3%) were the most common mutated genes, which were mutually exclusive. Class III genes of epigenetic regulators were also frequent (43.9%). In multivariate analysis, old age [hazard ratio (HR) 1.029, 95% confidence interval (CI) 1.013-1.044, P=.001) was the independent adverse factor for overall survival, and RUNX1 mutation (HR 2.326, 95% CI 0.978-5.533, P=.056) had a trend toward inferior survival. In conclusion, our study showed a high frequency of FLT3, RUNX1, and IDH mutations in AML-M0, suggesting that these mutations played a role in the pathogenesis and served as potential therapeutic targets in this rare and unfavorable subtype of AML.

  20. Genotoxicity of nano/microparticles in in vitro micronuclei, in vivo comet and mutation assay systems

    Directory of Open Access Journals (Sweden)

    Fukumori Nobutaka


    Full Text Available Abstract Background Recently, manufactured nano/microparticles such as fullerenes (C60, carbon black (CB and ceramic fiber are being widely used because of their desirable properties in industrial, medical and cosmetic fields. However, there are few data on these particles in mammalian mutagenesis and carcinogenesis. To examine genotoxic effects by C60, CB and kaolin, an in vitro micronuclei (MN test was conducted with human lung cancer cell line, A549 cells. In addition, DNA damage and mutations were analyzed by in vivo assay systems using male C57BL/6J or gpt delta transgenic mice which were intratracheally instilled with single or multiple doses of 0.2 mg per animal of particles. Results In in vitro genotoxic analysis, increased MN frequencies were observed in A549 cells treated with C60, CB and kaolin in a dose-dependent manner. These three nano/microparticles also induced DNA damage in the lungs of C57BL/6J mice measured by comet assay. Moreover, single or multiple instillations of C60 and kaolin, increased either or both of gpt and Spi- mutant frequencies in the lungs of gpt delta transgenic mice. Mutation spectra analysis showed transversions were predominant, and more than 60% of the base substitutions occurred at G:C base pairs in the gpt genes. The G:C to C:G transversion was commonly increased by these particle instillations. Conclusion Manufactured nano/microparticles, CB, C60 and kaolin, were shown to be genotoxic in in vitro and in vivo assay systems.

  1. Pathogenic mutations of nuclear genes associated with mitochondrial disorders

    Institute of Scientific and Technical Information of China (English)

    Xiaoyu Zhu; Xuerui Peng; Min-Xin Guan; Qingfeng Yan


    Mitochondrial disorders are clinical phenotypes associated with mitochondrial dysfunction, which can be caused by mutations in mitochondrial DNA (mtDNA) or nuclear genes. In this review, we summarized the pathogenic mutations of nuclear genes associated with mitochondrial disorders. These nuclear genes encode, components of mitochondrial translational machinery and structural subunits and assembly factors of the oxidative phosphorylation, that complex. The molecular mechanisms, that nuclear modifier genes modulate the phenotypic expression of mtDNA mutations, are discussed in detail.

  2. Mutations induced by monofunctional and bifunctional phosphoramide mustards in supF tRNA gene. (United States)

    Mudipalli, A; Maccubbin, A E; Nadadur, S S; Struck, R F; Gurtoo, H L


    The relative mutagenicity, nature of the mutations and the sequence specificity of mutations induced by the bifunctional alkylating agent, phosphoramide mustard (PM) and a monofunctional derivative, dechloroethyl phosphoramide mustard (dePM), were analyzed by the Ames test and by an in vitro shuttle vector mutagenesis assay. Both PM and dePM increased the mutation frequency above background in either assay. However, on an equimolar basis, dePM was less mutagenic than PM. In the in vitro shuttle vector mutagenesis assay, sequencing demonstrated that about 40% of the mutant plasmids contained more than one mutation in the supF tRNA gene segment of the plasmid. About 70% of the mutations observed in dePM-treated plasmids were single base substitutions with A:T and G:C base pairs being mutated at equivalent rates. In contrast, only about 50% of the mutations observed in PM-treated plasmids were single base substitutions, 80% of which involved G:C base pairs. Single base deletions and insertions were found in approximately equal proportions with both compounds; however, these lesions were in greater abundance in PM-treated plasmids. Putative hot-spots for mutation in the supF tRNA gene included base pairs at position 102 and 110 for PM and positions 170 and 171 for dePM.


    Institute of Scientific and Technical Information of China (English)

    卞留贯; 孙青芳; 沈建康; 赵卫国; 罗其中


    Objective To analyze the mutation of NF2 gene (exon 2,4,6 and 13) in schwannomas. Methods The NF2 gene mutation in 36 schwannomas were observed by PCR-SSCP and DNA sequence. The proliferative index of schwannoma was detected by immunohistochemistry. Results We found 13 mutations in 36 schwannomas, including 6 deletion or insertion resulting in a frameshift, 2 nonsense mutations, 2 missense mutations, and 3 alterations affecting acceptor or donor of splicing sites in E4,E6,E13. The proliferative index of schwannomas with mutation were significantly higher than those without mutation (P< 0.05). Conclusion NF2 gene mutation is the frequent event in the tumorigenesis of schwannomas, and there is some correlation between the mutation and clinical behavior(tumor proliferation).

  4. Comparative Evaluation of Sloppy Molecular Beacon and Dual-Labeled Probe Melting Temperature Assays to Identify Mutations in Mycobacterium tuberculosis Resulting in Rifampin, Fluoroquinolone and Aminoglycoside Resistance (United States)

    Lee, Jong Seok; Via, Laura E.; Barry, Clifton E.; Alland, David; Chakravorty, Soumitesh


    Several molecular assays to detect resistance to Rifampin, the Fluoroquinolones, and Aminoglycosides in Mycobacterium tuberculosis (M. tuberculosis) have been recently described. A systematic approach for comparing these assays in the laboratory is needed in order to determine the relative advantage of each assay and to decide which ones should be advanced to evaluation. We performed an analytic comparison of a Sloppy Molecular Beacon (SMB) melting temperature (Tm) assay and a Dual labeled probe (DLP) Tm assay. Both assays targeted the M. tuberculosis rpoB, gyrA, rrs genes and the eis promoter region. The sensitivity and specificity to detect mutations, analytic limit of detection (LOD) and the detection of heteroresistance were tested using a panel of 56 clinical DNA samples from drug resistant M. tuberculosis strains. Both SMB and DLP assays detected 29/29 (100%) samples with rpoB RRDR mutations and 3/3 (100%) samples with eis promoter mutations correctly. The SMB assay detected all 17/17 gyrA mutants and 22/22 rrs mutants, while the DLP assay detected 16/17 (94%) gyrA mutants and 12/22 (55%) rrs mutants. Both assays showed comparable LODs for detecting rpoB and eis mutations; however, the SMB assay LODs were at least two logs better for detecting wild type and mutants in gyrA and rrs targets. The SMB assay was also moderately better at detecting heteroresistance. In summary, both assays appeared to be promising methods to detect drug resistance associated mutations in M. tuberculosis; however, the relative advantage of each assay varied under each test condition. PMID:25938476

  5. Comparative Evaluation of Sloppy Molecular Beacon and Dual-Labeled Probe Melting Temperature Assays to Identify Mutations in Mycobacterium tuberculosis Resulting in Rifampin, Fluoroquinolone and Aminoglycoside Resistance.

    Directory of Open Access Journals (Sweden)

    Sandy S Roh

    Full Text Available Several molecular assays to detect resistance to Rifampin, the Fluoroquinolones, and Aminoglycosides in Mycobacterium tuberculosis (M. tuberculosis have been recently described. A systematic approach for comparing these assays in the laboratory is needed in order to determine the relative advantage of each assay and to decide which ones should be advanced to evaluation. We performed an analytic comparison of a Sloppy Molecular Beacon (SMB melting temperature (Tm assay and a Dual labeled probe (DLP Tm assay. Both assays targeted the M. tuberculosis rpoB, gyrA, rrs genes and the eis promoter region. The sensitivity and specificity to detect mutations, analytic limit of detection (LOD and the detection of heteroresistance were tested using a panel of 56 clinical DNA samples from drug resistant M. tuberculosis strains. Both SMB and DLP assays detected 29/29 (100% samples with rpoB RRDR mutations and 3/3 (100% samples with eis promoter mutations correctly. The SMB assay detected all 17/17 gyrA mutants and 22/22 rrs mutants, while the DLP assay detected 16/17 (94% gyrA mutants and 12/22 (55% rrs mutants. Both assays showed comparable LODs for detecting rpoB and eis mutations; however, the SMB assay LODs were at least two logs better for detecting wild type and mutants in gyrA and rrs targets. The SMB assay was also moderately better at detecting heteroresistance. In summary, both assays appeared to be promising methods to detect drug resistance associated mutations in M. tuberculosis; however, the relative advantage of each assay varied under each test condition.

  6. Role of embCAB gene mutations in ethambutol resistance in Mycobacterium tuberculosis isolates from India. (United States)

    Jadaun, G P S; Das, Ram; Upadhyay, Prashant; Chauhan, D S; Sharma, V D; Katoch, V M


    In the present study, ethambutol (EMB) resistance-associated mutations were characterised in the embCAB genes of clinical isolates of Mycobacterium tuberculosis (MTB) collected in India. Thirty MTB isolates were tested for their susceptibility to first-line antitubercular drugs using the Löwenstein-Jensen proportion method, and EMB minimum inhibitory concentrations of MTB isolates were determined by the resazurin microtitre assay. Sequencing of various regions of the embCAB genes was performed to identify EMB resistance-associated mutations. Mutations of embB306 were detected in 15 of 23 EMB-resistant MTB isolates. Three EMB-resistant isolates had mutations at codon 270 of the embC gene, two of which also harboured embB306 mutations. No mutation was identified in the embA gene. All seven EMB-sensitive MTB isolates had the wild-type embCAB sequence. In summary, embB306 mutations were associated with EMB resistance, and mutation at codon 270 of the embC gene may contribute to high-level EMB resistance in some MTB isolates.

  7. 40 CFR 79.68 - Salmonella typhimurium reverse mutation assay. (United States)


    ... emissions from fuel or additive/base fuel mixtures are, first, filtered to trap particulate matter and, then... extract from both the filtered particulates and the resin-trapped organics. Assays are conducted using... fraction prepared from the livers of rodents treated with enzyme-inducing agents, such as Aroclor 1254. For...

  8. Automated DNA mutation detection using universal conditions direct sequencing: application to ten muscular dystrophy genes

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    Wu Bai-Lin


    Full Text Available Abstract Background One of the most common and efficient methods for detecting mutations in genes is PCR amplification followed by direct sequencing. Until recently, the process of designing PCR assays has been to focus on individual assay parameters rather than concentrating on matching conditions for a set of assays. Primers for each individual assay were selected based on location and sequence concerns. The two primer sequences were then iteratively adjusted to make the individual assays work properly. This generally resulted in groups of assays with different annealing temperatures that required the use of multiple thermal cyclers or multiple passes in a single thermal cycler making diagnostic testing time-consuming, laborious and expensive. These factors have severely hampered diagnostic testing services, leaving many families without an answer for the exact cause of a familial genetic disease. A search of GeneTests for sequencing analysis of the entire coding sequence for genes that are known to cause muscular dystrophies returns only a small list of laboratories that perform comprehensive gene panels. The hypothesis for the study was that a complete set of universal assays can be designed to amplify and sequence any gene or family of genes using computer aided design tools. If true, this would allow automation and optimization of the mutation detection process resulting in reduced cost and increased throughput. Results An automated process has been developed for the detection of deletions, duplications/insertions and point mutations in any gene or family of genes and has been applied to ten genes known to bear mutations that cause muscular dystrophy: DMD; CAV3; CAPN3; FKRP; TRIM32; LMNA; SGCA; SGCB; SGCG; SGCD. Using this process, mutations have been found in five DMD patients and four LGMD patients (one in the FKRP gene, one in the CAV3 gene, and two likely causative heterozygous pairs of variations in the CAPN3 gene of two other

  9. A Mutagenesis Assay for Reporter Gene Screening Using Partially Degenerate Oligonucleotides of the Tandems NNT and NNC

    Directory of Open Access Journals (Sweden)

    Huifen Xu


    Full Text Available Not all proteins are tolerable to mutations. Whether a specific protein can be a mutable target is of importance in the biotechnology and pharmaceutical industry. This study reported a novel mutagenesis assay using tandem NNT and NNC oligonucleotides to test the mutability of a candidate gene. These two tandem oligonucleotides avoid the risk of forming nonsense mutations and render flexibility of truncating or expanding the insertion size. As a reporter gene, ZeoR (zeocin resistance gene was confirmed to have a high tolerance for mutagenesis by this new assay.

  10. "ATM Gene Mutations Detection in Iranian Ataxia-Telangiectasia Patients "


    Toshio MiyawakiMohammad Hossein Sanati; Behnaz Bayat; Ahmad Aleyasin; Hasti Atashi Shirazi; "Anna Isaian; Abolhassan Farhoudi; Mostafa Moin


    Ataxia-Telangiectasia (AT) is an autosomal recessive disorder involving cerebellar degeneration, immunodeficiency, radiation sensitivity and cancer predisposition. The ATM gene on human chromosome 11q22.3 has recently been identified as the gene responsible for ataxia-telangiectasia (AT). The gene mutated in AT, which has been designated as the ATM gene, encodes a large protein kinase with a PI-3 kinase-related domain. More than 100 mutations are broadly distributed throughout the ATM gene. T...

  11. Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations (United States)

    Martin, Dorrelyn P.; Miya, Jharna; Reeser, Julie W.; Roychowdhury, Sameek


    RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding RNAs. Standard RNAseq requires 30 – 100 million sequencing reads and can include multiple RNA products such as mRNA and noncoding RNAs. We demonstrate how targeted RNAseq (capture) permits a focused study on selected RNA products using a desktop sequencer. RNAseq capture can characterize unannotated, low, or transiently expressed transcripts that may otherwise be missed using traditional RNAseq methods. Here we describe the extraction of RNA from cell lines, ribosomal RNA depletion, cDNA synthesis, preparation of barcoded libraries, hybridization and capture of targeted transcripts and multiplex sequencing on a desktop sequencer. We also outline the computational analysis pipeline, which includes quality control assessment, alignment, fusion detection, gene expression quantification and identification of single nucleotide variants. This assay allows for targeted transcript sequencing to characterize gene expression, gene fusions, and mutations. PMID:27585245

  12. Detecting mutation pattern of drug-resistant Mycobacterium tuberculosis isolates in Himachal Pradesh using GenoType® MTBDRplus assay

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    C Thakur


    Full Text Available Context: Tuberculosis (TB is a major public health problem in India and a principal cause of death in adults, especially among the economically productive age group. India accounts for one-fifth of the global burden of TB. It is estimated that about 40% of Indian population is infected with TB bacillus. The GenoType® MTBDRplus molecular method allows rapid diagnosis of the clinical samples and detection of the most common mutations in the genes associated with rifampicin (R and isoniazid (H resistance. Aims: To study the drug resistance and mutational patterns in multidrug-resistant (MDR suspects clinical strains using GenoType® MTBDRplus assay. Subjects and Methods: A total of 770 sputum samples of the MDR-TB suspects were included in this study, which were received at Intermediate Reference Laboratory, Government TB Sanatorium, Dharampur, Solan, Himachal Pradesh from the Designated Microscopy Centres of Himachal Pradesh for the culture and susceptibility testing. All the 521 Mycobacterium tuberculosis complex (MTBC strains were subjected to GenoType® MTBDRplus (HAIN Lifescience assay to detect molecular resistance pattern to first line anti-tubercular drugs (isoniazid and rifampicin. Results: Of 770 samples, 556 (72.20% were from male and 214 (27.80% were from female. Among the 521 MTBC strains, 19.76% were found to be MDR and mono-resistance to isoniazid and rifampicin was detected in 8.63% and 6.14% strains respectively. About 74.81%, 76.35% and 5.40% strains harboured known mutation in rpoB, katG and inhA genes respectively. Conclusions: In rpoB gene, the most common mutation is associated with S531 L region. The GenoType® MTBDRplus assay is a rapid test for the detection of the most common mutations in MDR-TB strains. In our study, unknown rpoB gene mutations were found in 25.18% strains that may further be detected by gene sequencing.

  13. A novel mutation in the DSPP gene associated with dentinogenesis imperfecta type II. (United States)

    Lee, S-K; Lee, K-E; Jeon, D; Lee, G; Lee, H; Shin, C-U; Jung, Y-J; Lee, S-H; Hahn, S-H; Kim, J-W


    Hereditary dentin defects are divided into dentinogenesis imperfecta and dentin dysplasia. We identified a family segregating severe dentinogenesis imperfecta. The kindred spanned four generations and showed an autosomal-dominant pattern of inheritance. The proband was a child presenting with a severely affected primary dentition, with wide-open pulp chambers and multiple pulp exposures, resembling a DGI type III (DGI-III) pattern. We hypothesized that a mutation in the DSPP gene is responsible for this severe phenotype. Mutational analyses revealed a novel mutation (c.53T>A, p.V18D) near the intron-exon boundary in the third exon of the DSPP gene. We analyzed the effect of the mutation by means of an in vitro splicing assay, which revealed that the mutation did not affect pre-mRNA splicing. Further studies are needed for a better understanding of the nature of the disease and the development of an appropriate treatment strategy.

  14. [Some behavioral features in Drosophila melanogaster lines carrying a flamenco gene mutation]. (United States)

    Subocheva, E A; Romanova, L G; Romanova, N I; Kim, A I


    Olfactory sensitivity and locomotor activity was assayed in Drosophila melanogaster strains carrying a mutation of the flamenco gene, which controls transposition of the mobile genetic element 4 (MGE4) retrotransposon the gypsy mobile element. A change in olfactory sensitivity was detected. The reaction to the odor of acetic acid was inverted in flies of the mutator strain (MS), which carried the flam mutation and active MGE4 copies and were characterized by genetic instability. Flies of the genetically unstable strains displayed a lower locomotor activity. The behavioral changes in MS flies can be explained by the pleiotropic effect of the flam mutation or by insertion mutations which arise in behavior genes as a result of genome destabilization by MGE4.

  15. RAS gene mutations in acute and chronic myelocytic leukemias, chronic myeloproliferative disorders, and myelodysplastic syndromes

    Energy Technology Data Exchange (ETDEWEB)

    Janssen, J.W.G.; Steenvoorden, A.C.M.; Lyons, J.; Anger, B.; Boehlke, J.U.; Bos, J.L.; Seliger, H.; Bartram, C.R.


    The authors report on investigations aimed at detecting mutated RAS genes in a variety of preleukemic disorders and leukemias of myeloid origin. DNA transfection analyses (tumorigenicity assay) and hybridization to mutation-specific oligonucleotide probes established NRAS mutations in codon 12 or 61 of 4/9 acute myelocytic leukemias (AML) and three AML lines. Leukemic cells of another AML patient showed HRAS gene activation. By using a rapid and sensitive dot-blot screening procedure based on the combination of in vitro amplification of RAS-specific sequences and oligonucleotide hybridization they additionally screened 15 myelodysplastic syndromes, 26 Philadelphia chromosome-positive chronic myelocytic leukemias in chronic or acute phase, and 19 other chronic myeloproliferative disorders. A mutation within NRAS codon 12 could thus be demonstrated in a patient with idiopathic myelofibrosis and in another with chronic myelomonocytic leukemia. Moreover, mutated NRAS sequences were detected in lymphocytes, in granulocytes, as well as in monocytes/macrophages of the latter case.

  16. "Deafness –Associated Connexin 26 Gene (GJB2 Mutations in Iranian Population"

    Directory of Open Access Journals (Sweden)

    M Hashemzadeh Chaleshtori


    Full Text Available Mutations in the GJB2 gene at the DFNB1 locus on chromosome 13q12 are associated with autosomal recessive non syndromic hearing loss (ARNSHL in many populations. A single mutation, at position 35 (35delG accounts for approximately 30-63% of mutations in white populations with a carrier frequency of 1.5-2.5% in most European, North American and Mediterranean populations. In this study we have investigated the prevalence of the GJB2 gene mutations using direct sequencing in 43 presumed ARNSHL subjects from 34 families in an Iranian population. Eleven different genetic variants were identified. GJB2-related deafness mutations (35delG, 235delC, W24X, R184P and IVS1+1G>A were found in 9 of 34 families (26.5%. The 35delG was the most common mutation found in 5 of 34 families (14.7%. We found one novel variant (–3517G>A in the upstream region to the gene. The mutation frequency found in this study is lower than other ethnic groups with European ancestry, but it is indicating that mutation in GJB2 in Iranian population has contribution to ARNSHL. We have also developed a simple and accurate nested PCR assay to screen the 35delG mutation in 250 unrelated unaffected Iranian individual (controls. No 35delG heterozygous was found in the control population.

  17. Mutated Genes in Schizophrenia Map to Brain Networks (United States)

    ... Matters NIH Research Matters August 12, 2013 Mutated Genes in Schizophrenia Map to Brain Networks Schizophrenia networks in the ... in People with Serious Mental Illness Clues for Schizophrenia in Rare Gene Glitch Recognizing Schizophrenia: Seeking Clues to a Difficult ...

  18. Gene mutations of acute myeloid leukemia in the genome era. (United States)

    Naoe, Tomoki; Kiyoi, Hitoshi


    Ten years ago, gene mutations found in acute myeloid leukemia (AML) were conceptually grouped into class I mutation, which causes constitutive activation of intracellular signals that contribute to the growth and survival, and class II mutation, which blocks differentiation and/or enhance self-renewal by altered transcription factors. A cooperative model between two classes of mutations has been suggested by murine experiments and partly supported by epidemiological findings. In the last 5 years, comprehensive genomic analysis proceeded to find new gene mutations, which are found in the epigenome-associated enzymes and the molecules never noticed so far. These new mutations apparently increase the complexity and heterogeneity of AML. Although a long list of gene mutations might have been compiled, the entire picture of molecular pathogenesis in AML remains to be elucidated because gene rearrangement, gene copy number, DNA methylation and expression profiles are not fully studied in conjunction with gene mutations. Comprehensive genome research will deepen the understanding of AML to promote the development of new classification and treatment. This review focuses on gene mutations that were recently discovered by genome sequencing.

  19. Preliminary Report of Molecular Detection of Retinoblastoma Gene Mutations

    Institute of Scientific and Technical Information of China (English)


    To develop gene diagnosis for retinoblastoma predisposition, it is necessary to disclose the retinoblastoma gene mutations or deletions in detail. Genomic DNA from tumor and peripheral white blood cells in 33 patients with retinoblastoma was detected with 3.8kb probe derived from 3' end of retinoblastoma gene cDNA. The gene abnormalities, including deletion, partial deletion and rearrangement, were found in 18 patients. Further research will be aimed at microdeletions or mutations for those patients wti...

  20. A new scintillation proximity assay-based approach for the detection of KRAS mutations

    Energy Technology Data Exchange (ETDEWEB)

    Lee, So-Young; Lim, Jae-Cheong; Cho, Eun-Ha; Jung, Sung-Hee [Korea Atomic Energy Research Institute (KAERI), Daejeon (Korea, Republic of). Radioisotope Research Div.


    KRAS is very commonly mutated resulting in a constitutively activated protein, which is independent of epidermal growth factor receptor (EGFR) ligand binding and resistant to anti-EGFR therapy. Although KRAS is frequently studied, there is still no uniform standard for detecting of KRAS mutations. In this report, a new scintillation proximity assay-based approach is described that determines the relative affinities of wild-type and mutated KRAS to the anti-KRAS antibody. We performed in vitro experiments using normal human colonic cells (CCD18Co), KRAS wild type (Caco-2) and KRAS mutant (HCT 116) cell lines to determine the relative affinities of wild type or mutated KRAS toward an anti-KRAS monoclonal antibody. The process consists of two primary steps: immunoprecipitation from cell lysate to enrich the KRAS protein and the scintillation proximity assay of the immunoprecipitant to determine the relative affinity against the antibody. A fixed concentration of cell lysates was purified by the immunoprecipitation method. The expressions of the KRAS protein in all cell lines was quantitatively confirmed by western blot analysis. For the scintillation proximity assay, the KRAS standard protein was radiolabeled with {sup 125}I by a simple mixing process in the iodogen tube immediately at room temperature immediately before use. The obtained CPM (count per minute) values of were used to calculate the KRAS concentration using purified KRAS as the standard. The calculated relative affinities of 7 μg of Caco-2 and HCT 116 immunoprecipitants for the anti-KRAS antibody were 77 and 0%, respectively. The newly developed scintillation proximity assay-based strategy determines the relative affinities of wild-type or mutated KRAS towards the anti-KRAS monoclonal antibody. This determination can help distinguish mutated KRAS from the wild type protein. The new SPA based approach for detecting KRAS mutations is applicable to many other cancer-related mutations.

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

    Directory of Open Access Journals (Sweden)

    Eliana Aparecida Varanda


    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.

  2. Splice Site Mutations in the ATP7A Gene

    DEFF Research Database (Denmark)

    Skjørringe, Tina; Tümer, Zeynep; Møller, Lisbeth Birk


    Menkes disease (MD) is caused by mutations in the ATP7A gene. We describe 33 novel splice site mutations detected in patients with MD or the milder phenotypic form, Occipital Horn Syndrome. We review these 33 mutations together with 28 previously published splice site mutations. We investigate 12...... mutations for their effect on the mRNA transcript in vivo. Transcriptional data from another 16 mutations were collected from the literature. The theoretical consequences of splice site mutations, predicted with the bioinformatics tool Human Splice Finder, were investigated and evaluated in relation...... to in vivo results. Ninety-six percent of the mutations identified in 45 patients with classical MD were predicted to have a significant effect on splicing, which concurs with the absence of any detectable wild-type transcript in all 19 patients investigated in vivo. Sixty-seven percent of the mutations...

  3. Mutations in the Motile Cilia Gene DNAAF1 Are Associated with Neural Tube Defects in Humans

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    Chunyue Miao


    Full Text Available Neural tube defects (NTDs are severe malformations of the central nervous system caused by complex genetic and environmental factors. Among genes involved in NTD, cilia-related genes have been well defined and found to be essential for the completion of neural tube closure (NTC. We have carried out next-generation sequencing on target genes in 373 NTDs and 222 healthy controls, and discovered eight disease-specific rare mutations in cilia-related gene DNAAF1. DNAAF1 plays a central role in cytoplasmic preassembly of distinct dynein-arm complexes, and is expressed in some key tissues involved in neural system development, such as neural tube, floor plate, embryonic node, and brain ependyma epithelial cells in zebrafish and mouse. Therefore, we evaluated the expression and functions of mutations in DNAAF1 in transfected cells to analyze the potential correlation of these mutants to NTDs in humans. One rare frameshift mutation (p.Gln341Argfs*10 resulted in significantly diminished DNAAF1 protein expression, compared to the wild type. Another mutation, p.Lys231Gln, disrupted cytoplasmic preassembly of the dynein-arm complexes in cellular assay. Furthermore, results from NanoString assay on mRNA from NTD samples indicated that DNAAF1 mutants altered the expression level of NTC-related genes. Altogether, these findings suggest that the rare mutations in DNAAF1 may contribute to the susceptibility for NTDs in humans.

  4. A missense mutation (Q279R in the Fumarylacetoacetate Hydrolase gene, responsible for hereditary tyrosinemia, acts as a splicing mutation

    Directory of Open Access Journals (Sweden)

    Baklouti Faouzi


    Full Text Available Abstract Background Tyrosinemia type I, the most severe disease of the tyrosine catabolic pathway is caused by a deficiency in fumarylacetoacetate hydrolase (FAH. A patient showing few of the symptoms associated with the disease, was found to be a compound heterozygote for a splice mutation, IVS6-1g->t, and a putative missense mutation, Q279R. Analysis of FAH expression in liver sections obtained after resection for hepatocellular carcinoma revealed a mosaic pattern of expression. No FAH was found in tumor regions while a healthy region contained enzyme-expressing nodules. Results Analysis of DNA from a FAH expressing region showed that the expression of the protein was due to correction of the Q279R mutation. RT-PCR was used to assess if Q279R RNA was produced in the liver cells and in fibroblasts from the patient. Normal mRNA was found in the liver region where the mutation had reverted while splicing intermediates were found in non-expressing regions suggesting that the Q279R mutation acted as a splicing mutation in vivo. Sequence of transcripts showed skipping of exon 8 alone or together with exon 9. Using minigenes in transfection assays, the Q279R mutation was shown to induce skipping of exon 9 when placed in a constitutive splicing environment. Conclusion These data suggest that the putative missense mutation Q279R in the FAH gene acts as a splicing mutation in vivo. Moreover FAH expression can be partially restored in certain liver cells as a result of a reversion of the Q279R mutation and expansion of the corrected cells.

  5. Novel recurrently mutated genes and a prognostic mutation signature in colorectal cancer (United States)

    Yu, Jun; Wu, William K K; Li, Xiangchun; He, Jun; Li, Xiao-Xing; Ng, Simon S M; Yu, Chang; Gao, Zhibo; Yang, Jie; Li, Miao; Wang, Qiaoxiu; Liang, Qiaoyi; Pan, Yi; Tong, Joanna H; To, Ka F; Wong, Nathalie; Zhang, Ning; Chen, Jie; Lu, Youyong; Lai, Paul B S; Chan, Francis K L; Li, Yingrui; Kung, Hsiang-Fu; Yang, Huanming; Wang, Jun; Sung, Joseph J Y


    Background Characterisation of colorectal cancer (CRC) genomes by next-generation sequencing has led to the discovery of novel recurrently mutated genes. Nevertheless, genomic data has not yet been used for CRC prognostication. Objective To identify recurrent somatic mutations with prognostic significance in patients with CRC. Method Exome sequencing was performed to identify somatic mutations in tumour tissues of 22 patients with CRC, followed by validation of 187 recurrent and pathway-related genes using targeted capture sequencing in additional 160 cases. Results Seven significantly mutated genes, including four reported (APC, TP53, KRAS and SMAD4) and three novel recurrently mutated genes (CDH10, FAT4 and DOCK2), exhibited high mutation prevalence (6–14% for novel cancer genes) and higher-than-expected number of non-silent mutations in our CRC cohort. For prognostication, a five-gene-signature (CDH10, COL6A3, SMAD4, TMEM132D, VCAN) was devised, in which mutation(s) in one or more of these genes was significantly associated with better overall survival independent of tumor-node-metastasis (TNM) staging. The median survival time was 80.4 months in the mutant group versus 42.4 months in the wild type group (p=0.0051). The prognostic significance of this signature was successfully verified using the data set from the Cancer Genome Atlas study. Conclusions The application of next-generation sequencing has led to the identification of three novel significantly mutated genes in CRC and a mutation signature that predicts survival outcomes for stratifying patients with CRC independent of TNM staging. PMID:24951259

  6. Novel assay of competitively differentiated polymerase chain reaction for screening point mutation of hepatitis B virus

    Institute of Scientific and Technical Information of China (English)

    Xiao-Mou Peng; Xue-Juan Chen; Jian-Guo Li; Lin Gu; Yang-Su Huang; Zhi-Liang Gao


    AIM: Point mutation, one of the commonest gene mutations,is the most important molecular pathogenesis of cancer and chronic infection. The commonest methods for detection of point mutation are based on polymerase chain reaction (PCR). These techniques, however, cannot be used in large scale screening since they are neither accurate nor simple.For this reason, this study established a novel method of competitively differentiated PCR (CD-PCR) for screening point mutation in clinical practice.METHODS: Two competitively differentiated primers for mutant-type and wild-type templates respectively with an identically complemented region in 3′ end except for last 2base pairs and a different non-complemented region in 5′end were designed. Thus, competitive amplification might be carried out at a lower annealing temperature at first, and then differentiated amplification at a higher annealing temperature when primers could not combine with initial templates. The amplification was performed in one-tube.The products of CD-PCR were detected using microplate hybridization assay. CD-PCR was evaluated by detecting G1896A variant of hepatitis B virus (HBV) in form of recombinant plasmids and in sera from patients with hepatitis B, and compared with allele-specific PCR (AS-PCR) and competitive AS-PCR.RESULTS: CD-PCR was successfully established. It could clearly distinguish wild-type and mutant-type plasmid DNA of G1896A variant when the amount of plasmid DNA was between 102-108copies/reaction, while for AS-PCR and competitive AS-PCR, the DNA amount was between 102-104copies/reaction. CD-PCR could detect one copy of G1896A variant among 10-100 copies of wild-type plasmid DNA. The specificity of CD-PCR was higher than those of AS-PCR and competitive AS-PCR in the detection of HBV G1896A variant in sera from patients with hepatitis B. CD-PCR was independent of the amount of HBV DNA in serum. HBV G1896A variant was more often found in HBeAg (-) patients with a lower level of

  7. A new mutation (1062 del 16) of iduronate-2-sulfatase gene from a Chinese patient with Hunter syndrome

    Institute of Scientific and Technical Information of China (English)


    Objective: To identify the mutations of iduronate-2-sulfatase (IDS) gene, to reveal its mutation features, and to establish a basis for genetic counseling and prenatal gene diagnosis of Hunter syndrome. Methods: Urine glycosaminoglycans (GAGs) assay, PCR and DNA sequencing were performed to detect mutation of IDS gene of the patient and his parents. Results:The result showed that the patient was: DS(++), HS(++), KS(-), CS(-), and that both of his parents were negative. A frame-shift deletion mutation (1062 del 16) was identified in exon 7 of the patient's IDS gene. His parents'genotypes were normal. Conclusion: The patient's mutation was not inherited by his parents but a novel one. The mutation probably altered the primary structure and tertiary structure of IDS enzyme protein remarkably and lowered the activity of IDS enzyme greatly. Therefore it is supposed to be the direct cause of the disorder.

  8. Detection of EGFR mutations in plasma and biopsies from non-small cell lung cancer patients by allele-specific PCR assays

    DEFF Research Database (Denmark)

    Weber, Britta; Meldgaard, Peter; Hager, Henrik


    BACKGROUND: Lung cancer patients with mutations in the epidermal growth factor receptor (EGFR) are primary candidates for EGFR-targeted therapy. Reliable analyses of such mutations have previously been possible only in tumour tissue. Here, we demonstrate that mutations can be detected in plasma...... samples with allele-specific PCR assays. METHODS: Pairs of the diagnostic biopsy and plasma obtained just prior to start of erlotinib treatment were collected from 199 patients with adenocarcinoma of non-small-cell lung cancer. DNA from both sample types was isolated and examined for the presence...... identified in 24/199 (12%) plasma samples and 28/196 (14%) biopsy samples, and 17/196 (9%) matched pairs contained the same mutation. Six EGFR mutations were present only in plasma samples but not in the biopsy samples. The overall concordance of the EGFR gene mutations detected in plasma and biopsy tissue...

  9. Rapid assay of A2058T-mutated 23S rRNA allelic profiles associated with high-level macrolide resistance in Moraxella catarrhalis. (United States)

    Saito, Ryoichi; Kasai, Ayako; Ogihara, Shinji; Yamada, Kageto; Tao, Kazuyuki


    We report on a restriction fragment-length polymorphism (HpyCH4III) assay for profile analysis of 23S rRNA gene A2058T-mutated alleles associated with high-level macrolide resistance in Moraxella catarrhalis. Our assay results were supported by DNA sequencing analysis, allowed for simultaneous testing of many strains, and produced results from pure-cultured colonies within 4 h.

  10. Three new BLM gene mutations associated with Bloom syndrome. (United States)

    Amor-Guéret, Mounira; Dubois-d'Enghien, Catherine; Laugé, Anthony; Onclercq-Delic, Rosine; Barakat, Abdelhamid; Chadli, Elbekkay; Bousfiha, Ahmed Aziz; Benjelloun, Meriem; Flori, Elisabeth; Doray, Bérénice; Laugel, Vincent; Lourenço, Maria Teresa; Gonçalves, Rui; Sousa, Silvia; Couturier, Jérôme; Stoppa-Lyonnet, Dominique


    Bloom's syndrome (BS) is a rare autosomal recessive disease predisposing patients to all types of cancers affecting the general population. BS cells display a high level of genetic instability, including a 10-fold increase in the rate of sister chromatid exchanges, currently the only objective criterion for BS diagnosis. We have developed a method for screening the BLM gene for mutations based on direct genomic DNA sequencing. A questionnaire based on clinical information, cytogenetic features, and family history was addressed to physicians prescribing BS genetic screening, with the aim of confirming or guiding diagnosis. We report here four BLM gene mutations, three of which have not been described before. Three of the mutations are frameshift mutations, and the fourth is a nonsense mutation. All these mutations introduce a stop codon, and may therefore be considered to have deleterious biological effect. This approach should make it possible to identify new mutations and to correlate them with clinical information.

  11. Applications of homemade kit in mutation detection of genes

    Institute of Scientific and Technical Information of China (English)

    ZHAO Chunxia; XU Guowang; SHI Xianzhe; MA Jianmei; ZHANG Yan; L(U) Shen; YANG Qing


    Several methods of mutation detection, such as single-strand conformation polymorphism (SSCP), tandem SSCP/heteroduplex analysis and SNaPshot analysis were developed using homemade kit on ABI 310 genetic analyzer, and were successfully applied to mutation detection of 31 colorectal tumor samples. The sieving capability of homemade kit and commercial kit were compared, results demonstrate that homemade kit has higher resolution and shorter analysis time. In clinical tumor samples, 26% K-ras (exon 1) and 24% p53 (exons 7-8) were found to have mutations, and all mutations were single point variations. A majority of mutations occurred in one gene, only 1 tumor contained alterations in the two genes, which indicates that development of colorectal cancer lies on alternate pathways, and may correlate with different gene mutations.

  12. A T7 Endonuclease I Assay to Detect Talen-Mediated Targeted Mutation of HBV cccDNA. (United States)

    Bloom, Kristie; Ely, Abdullah; Arbuthnot, Patrick


    Gene editing using designer nucleases is now widely used in many fields of molecular biology. The technology is being developed for the treatment of viral infections such as persistant hepatitis B virus (HBV). The replication intermediate of HBV comprising covalently closed circular DNA (cccDNA) is stable and resistant to available licensed antiviral agents. Advancing gene editing as a means of introducing targeted mutations into cccDNA thus potentially offers the means to cure infection by the virus. Essentially, targeted mutations are initiated by intracellular DNA cleavage, then error-prone nonhomologous end joining results in insertions and deletions (indels) at intended sites. Characterization of these mutations is crucial to confirm activity of potentially therapeutic nucleases. A convenient tool for evaluation of the efficiency of target cleavage is the single strand-specific endonuclease, T7EI. Assays employing this enzyme entail initial amplification of DNA encompassing the targeted region. Thereafter the amplicons are denatured and reannealed to allow hybridization between indel-containing and wild-type sequences. Heteroduplexes that contain mismatched regions are susceptible to action by T7EI and cleavage of the hybrid amplicons may be used as an indicator of efficiency of designer nucleases. The protocol described here provides a method of isolating cccDNA from transfected HepG2.2.15 cells and evaluation of the efficiency of mutation by a transcription activator-like effector nuclease that targets the surface open reading frame of HBV.

  13. Androgen receptor gene mutation, rearrangement, polymorphism. (United States)

    Eisermann, Kurtis; Wang, Dan; Jing, Yifeng; Pascal, Laura E; Wang, Zhou


    Genetic aberrations of the androgen receptor (AR) caused by mutations, rearrangements, and polymorphisms result in a mutant receptor that has varied functions compared to wild type AR. To date, over 1,000 mutations have been reported in the AR with most of these being associated with androgen insensitivity syndrome (AIS). While mutations of AR associated with prostate cancer occur less often in early stage localized disease, mutations in castration-resistant prostate cancer (CRPC) patients treated with anti-androgens occur more frequently with 10-30% of these patients having some form of mutation in the AR. Resistance to anti-androgen therapy usually results from gain-of-function mutations in the LBD such as is seen with bicalutamide and more recently with enzalutamide (MDV3100). Thus, it is crucial to investigate these new AR mutations arising from drug resistance to anti-androgens and other small molecule pharmacological agents.

  14. Comparison of somatic mutation frequency among immunoglobulin genes. (United States)

    Motoyama, N; Miwa, T; Suzuki, Y; Okada, H; Azuma, T


    We analyzed the frequency of somatic mutation in immunoglobulin genes from hybridomas that secrete anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) monoclonal antibodies. A high frequency of mutation (3.3-4.4%) was observed in both the rearranged VH186.2 and V lambda 1 genes, indicating that somatic mutation occurs with similar frequency in these genes in spite of the absence of an intron enhancer in lambda 1 chain genes. In contrast to the high frequency in J-C introns, only two nucleotide substitutions occurred at positions -462 and -555 in the 5' noncoding region in one of the lambda 1-chain genes and in none of the other three so far studied. Since a similar low frequency of somatic mutation was observed in the 5' noncoding region of inactive lambda 2-chain genes rendered inactive because of incorrect rearrangement, this region may not be a target or alternatively, may be protected from the mutator system. We observed a low frequency of nucleotide substitution in unrearranged V lambda 1 genes (approximately 1/15 that of rearranged genes). Together with previous results (Azuma T., N. Motoyama, L. Fields, and D. Loh, 1993. Int. Immunol. 5:121), these findings suggest that the 5' noncoding region, which contains the promoter element, provides a signal for the somatic mutator system and that rearrangement, which brings the promoter into close proximity to the enhancer element, should increase mutation efficiency.

  15. C-kit gene mutation in human gastrointestinal stromal tumors

    Institute of Scientific and Technical Information of China (English)

    Ying-Yong Hou; Ai-Hua Zheng; Tai-Ming Zhang; Wen-Zhong Hou; Jian Wang; Xiang Du; Xiong-Zeng Zhu; Yun-Shan Tan; Meng-Hong Sun; Yong-Kun Wei; Jian-Fang Xu; Shao-Hua Lu; Su-Jie A-Ke-Su; Yan-Nan Zhou; Feng Gao


    AIM: To investigate the significance of c-kit gene mutation in gastrointestinal stromal tumors (GIST).METHODS: Fifty two cases of GIST and 28 cases of other tumors were examined. DNA samples were extracted from paraffin sections and fresh blocks. Exons 11, 9 and 13 of the c-kit gene were amplified by PCR and sequenced.RESULTS: Mutations of exon 11 were found in 14 of 25 malignant GISTs (56%), mutations of exon 11 of the c-kit gene were revealed in 2 of 19 borderline GISTs (10.5%),and no mutation was found in benign tumors. The mutation rate showed significant difference (X2=14.39, P<0.01)between malignant and benign GISTs. Most of mutations consisted of the in-frame deletion or replication from 3 to 48 bp in heterozygous and homozygous fashions, None of the mutations disrupted the downstream reading frame of the gene. Point mutations and frame deletions were most frequently observed at codons 550-560, but duplications were most concentrated at codons 570-585. No mutations of exons 9 and 13 were revealed in GISTs, Neither c-kit gene expression nor gene mutations were found in 3 leiomyomas, 8 leiomyosarcomas, 2 schwannomas, 2malignant peripheral nerve sheath tumors, 2 intraabdominal fibromatoses, 2 malignant fibrous histiocytomas and 9 adenocarcinomas.CONCLUSION: C-kit gene mutations occur preferentially in malignant GISTs and might be a clinically useful adjunct marker in the evaluation of GISTs and can help to differentiate GISTs from other mesenchymal tumors of gastrointestinal tract, such as smooth muscle tumors,schwannomas, etc.

  16. MEFV Gene Profile in Northwest of Iran, Twelve Common MEFV Gene Mutations Analysis in 216 Patients with Familial Mediterranean Fever

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    Farhad Salehzadeh


    Full Text Available Familial Mediterranean Fever (FMF is a hereditary autoinflammatory disease with autosomal recessive inheritance pattern often seen around the Mediterranean Sea. It is characterized by recurrent episodes of fever and polyserositis and rash. Recently, MEFV gene analysis determines the definitive diagnosis of FMF. In this study, we analyzed 12 MEFV gene mutations in more than 200 FMF patients, previously diagnosed by Tel-Hashomer clinical criteria, in northwest of Iran, located in the proximity of the Mediterranean Sea. In the northwest of Iran (Ardabil, 216 patients with FMF diagnosis, based on Tel-Hashomer criteria, referred to the genetic laboratory to be tested for the following mutations; P369S, F479L, M680I(G/C, M680I(G/A, I692del, M694V, M694I, K695R, V726A, A744S, R761H, E148Q. All patients were screened for MEFV gene mutations by a reverse hybridization assay (FMF Strip Assay, Vienna lab, Vienna, Austria according to manufacturer’s instructions. Among these FMF patients, no mutation was detected in 51 (23/62% patients, but 165 (76/38% patients had one or two mutations, 33 patients (15/28% homozygous, 86 patients (39/81% compound heterozygous and 46 patients (21/29% were heterozygous. The most common mutations were M694V (23/61%, V726A (11/11% and E148Q (9/95% respectively. MEFV gene mutations showed similarities and dissimilarities in different ethnic groups, while it is common among Arabs and Armenians genotype. Since common 12 MEFV gene analysis could not detect up to 50% of our patients, who had FMF on the basis of clinical Tel-Hashomer criteria, clinical criteria is still the best way in the diagnosis of FMF in this area.

  17. Novel KRAS gene mutations in sporadic colorectal cancer.

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    Walid M Naser

    Full Text Available In this article, we report 7 novel KRAS gene mutations discovered while retrospectively studying the prevalence and pattern of KRAS mutations in cancerous tissue obtained from 56 Saudi sporadic colorectal cancer patients from the Eastern Province.Genomic DNA was extracted from formalin-fixed, paraffin-embedded cancerous and noncancerous colorectal tissues. Successful and specific PCR products were then bi-directionally sequenced to detect exon 4 mutations while Mutector II Detection Kits were used for identifying mutations in codons 12, 13 and 61. The functional impact of the novel mutations was assessed using bioinformatics tools and molecular modeling.KRAS gene mutations were detected in the cancer tissue of 24 cases (42.85%. Of these, 11 had exon 4 mutations (19.64%. They harbored 8 different mutations all of which except two altered the KRAS protein amino acid sequence and all except one were novel as revealed by COSMIC database. The detected novel mutations were found to be somatic. One mutation is predicted to be benign. The remaining mutations are predicted to cause substantial changes in the protein structure. Of these, the Q150X nonsense mutation is the second truncating mutation to be reported in colorectal cancer in the literature.Our discovery of novel exon 4 KRAS mutations that are, so far, unique to Saudi colorectal cancer patients may be attributed to environmental factors and/or racial/ethnic variations due to genetic differences. Alternatively, it may be related to paucity of clinical studies on mutations other than those in codons 12, 13, 61 and 146. Further KRAS testing on a large number of patients of various ethnicities, particularly beyond the most common hotspot alleles in exons 2 and 3 is needed to assess the prevalence and explore the exact prognostic and predictive significance of the discovered novel mutations as well as their possible role in colorectal carcinogenesis.

  18. The sequence spectrum of frameshift reversions obtained with a novel adaptive mutation assay in Saccharomyces cerevisiae

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    Erich Heidenreich


    Full Text Available Research on the mechanisms of adaptive mutagenesis in resting, i.e. non-replicating cells relies on appropriate mutation assays. Here we provide a novel procedure for the detection of frameshift-reverting mutations in yeast. Proliferation of non-reverted cells in this assay is suppressed by the lack of a fermentable carbon source. The test allele was constructed in a way that the reversions mimic microsatellite instability, a condition often found in cancer cells. We show the cell numbers during these starvation conditions and provide a DNA sequence spectrum of a representative set of revertants. The data in this article support the publication "Glucose starvation as a selective tool for the study of adaptive mutations in Saccharomyces cerevisiae" (Heidenreich and Steinboeck, 2016 [1].

  19. Mutation analysis and prenatal diagnosis of EXT1 gene mutations in Chinese patients with multiple osteochondromas

    Institute of Scientific and Technical Information of China (English)

    ZHU Hai-yan; HU Ya-li; YANG Ying; WU Xing; ZHU Rui-fang; ZHU Xiang-yu; DUAN Hong-lei; ZHANG Ying; ZHOU Jin-yong


    Background Multiple osteochondromas (MO), an inherited autosomal dominant disorder, is characterized by the presence of multiple exostoses on the long bones. MO is caused by mutations in the EXT1 or EXT2 genes which encode glycosyltransferases implicated in heparin sulfate biosynthesis.Methods In this study, efforts were made to identify the underlying disease-causing mutations in patients from two MO families in China.Results Two novel EXT1 gene mutations were identified and no mutation was found in EXT2 gene. The mutation c.497T>A in exon 1 of the EXT1 gene was cosegregated with the disease phenotype in family 1 and formed a stop codon at amino acid site 166. The fetus of the proband was diagnosed negative. In family 2, the mutation c. 1430-1431delCC in exon 6 of the EXT1 gene would cause frameshift and introduce a premature stop codon after the reading frame being open for 42 amino acids. The fetus of this family inherited this mutation from the father.Conclusions Mutation analysis of two MO families in this study demonstrates its further application in MO genetic counseling and prenatal diagnosis.

  20. PARK1 gene mutation of autosomal dominant Parkinson's disease family

    Institute of Scientific and Technical Information of China (English)

    Ligang Jiang; Guohua Hu; Qiuhui Chen; Ying Zhang; Xinyu Hu; Jia Fan; Lifeng Liu; Rui Guo; Yajuan Sun; Yixhi Zhang


    Studies have shown that PARK1 gene is associated with the autosomal dominant inheritance of Parkinson's disease.PARK1 gene contains two mutation sites, namely Ala30Pro and AIa53Thr, which are located on exons 3 and 4, respectively.However, the genetic loci of the pathogenic genes remain unclear.In this study, blood samples were collected from 11 members of a family with high prevalence of Parkinson's disease, including four affected cases, five suspected cases,and two non-affected cases.Point mutation screening of common mutation sites on PARK1 gene exon 4 was conducted using PCR, to determine the genetic loci of the causative gene for Parkinson's disease.Gene identification and sequencing results showed that a T base deletion mutation was observed in the PARK1 gene exon 4 of all 11 collected samples.It was confirmed that the PARKf gene exon 4 gene mutation is an important pathogenic mutation for Parkinson's disease.

  1. DRUMS: a human disease related unique gene mutation search engine. (United States)

    Li, Zuofeng; Liu, Xingnan; Wen, Jingran; Xu, Ye; Zhao, Xin; Li, Xuan; Liu, Lei; Zhang, Xiaoyan


    With the completion of the human genome project and the development of new methods for gene variant detection, the integration of mutation data and its phenotypic consequences has become more important than ever. Among all available resources, locus-specific databases (LSDBs) curate one or more specific genes' mutation data along with high-quality phenotypes. Although some genotype-phenotype data from LSDB have been integrated into central databases little effort has been made to integrate all these data by a search engine approach. In this work, we have developed disease related unique gene mutation search engine (DRUMS), a search engine for human disease related unique gene mutation as a convenient tool for biologists or physicians to retrieve gene variant and related phenotype information. Gene variant and phenotype information were stored in a gene-centred relational database. Moreover, the relationships between mutations and diseases were indexed by the uniform resource identifier from LSDB, or another central database. By querying DRUMS, users can access the most popular mutation databases under one interface. DRUMS could be treated as a domain specific search engine. By using web crawling, indexing, and searching technologies, it provides a competitively efficient interface for searching and retrieving mutation data and their relationships to diseases. The present system is freely accessible at

  2. Droplet digital polymerase chain reaction assay for screening of ESR1 mutations in 325 breast cancer specimens. (United States)

    Takeshita, Takashi; Yamamoto, Yutaka; Yamamoto-Ibusuki, Mutsuko; Inao, Toko; Sueta, Aiko; Fujiwara, Saori; Omoto, Yoko; Iwase, Hirotaka


    Droplet digital polymerase chain reaction (ddPCR), which could perform thousands of PCRs on a nanoliter scale simultaneously, would be an attractive method to massive parallel sequencing for identifying and studying the significance of low-frequency rare mutations. Recent evidence has shown that the key potential mechanisms of the failure of aromatase inhibitors-based therapy involve identifying activating mutations affecting the ligand-binding domain of the ESR1 gene. Therefore, the detection of ESR1 mutations may be useful as a biomarker predicting an effect of the treatment. We aimed to develop a ddPCR-based method for the sensitive detection of ESR1 mutations in 325 breast cancer specimens, in which 270 primary and 55 estrogen receptor-positive (ER+) metastatic breast cancer (MBC) specimens. Our ddPCR assay could detect the ESR1 mutant molecules with low concentration of 0.25 copies/μL. According to the selected cutoff, ESR1 mutations occurred in 7 (2.5%) of 270 primary breast cancer specimens and in 11 (20%) of 55 ER+ MBC specimens. Among the 11 MBC specimens, 5 specimens (45.5%) had the most common ESR1 mutation, Y537S, 4 specimens (36.3%) each had D538G, Y537N, and Y537C. Interestingly, 2 patients had 2 ESR1 mutations, Y537N/D538G and Y537S/Y537C, and 2 patients had 3 ESR1 mutations, Y537S/Y537N/D538G. Biopsy was performed in heterochrony in 8 women twice. In 8 women, 4 women had primary breast cancer and MBC specimens and 4 women had 2 specimens when treatment was failure. Four of these 8 women acquired ESR1 mutation, whereas no ESR1 mutation could be identified at first biopsy. ddPCR technique could be a promising tool for the next-generation sequencing-free precise detection of ESR1 mutations in endocrine therapy resistant cases and may assist in determining the treatment strategy.

  3. High Resolution Melting Analysis for Rapid Mutation Screening in Gyrase and Topoisomerase IV Genes in Quinolone-Resistant Salmonella enterica

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    Soo Tein Ngoi


    Full Text Available The increased Salmonella resistance to quinolones and fluoroquinolones is a public health concern in the Southeast Asian region. The objective of this study is to develop a high resolution melt curve (HRM assay to rapidly screen for mutations in quinolone-resistant determining region (QRDR of gyrase and topoisomerase IV genes. DNA sequencing was performed on 62 Salmonella strains to identify mutations in the QRDR of gyrA, gyrB, parC, and parE genes. Mutations were detected in QRDR of gyrA (n=52; S83F, S83Y, S83I, D87G, D87Y, and D87N and parE (n=1; M438I. Salmonella strains with mutations within QRDR of gyrA are generally more resistant to nalidixic acid (MIC 16>256 μg/mL. Mutations were uncommon within the QRDR of gyrB, parC, and parE genes. In the HRM assay, mutants can be distinguished from the wild-type strains based on the transition of melt curves, which is more prominent when the profiles are displayed in difference plot. In conclusion, HRM analysis allows for rapid screening for mutations at the QRDRs of gyrase and topoisomerase IV genes in Salmonella. This assay markedly reduced the sequencing effort involved in mutational studies of quinolone-resistance genes.

  4. Functionomics of NCC mutations in Gitelman syndrome using a novel mammalian cell-based activity assay. (United States)

    Valdez-Flores, Marco A; Vargas-Poussou, Rosa; Verkaart, Sjoerd; Tutakhel, Omar A Z; Valdez-Ortiz, Angel; Blanchard, Anne; Treard, Cyrielle; Hoenderop, Joost G J; Bindels, René J M; Jeleń, Sabina


    Gitelman syndrome (GS) is an autosomal recessive salt-wasting tubular disorder resulting from loss-of-function mutations in the thiazide-sensitive NaCl cotransporter (NCC). Functional analysis of these mutations has been limited to the use of Xenopus laevis oocytes. The aim of the present study was, therefore, to analyze the functional consequences of NCC mutations in a mammalian cell-based assay, followed by analysis of mutated NCC protein expression as well as glycosylation and phosphorylation profiles using human embryonic kidney (HEK) 293 cells. NCC activity was assessed with a novel assay based on thiazide-sensitive iodide uptake in HEK293 cells expressing wild-type or mutant NCC (N59I, R83W, I360T, C421Y, G463R, G731R, L859P, or R861C). All mutations caused a significantly lower NCC activity. Immunoblot analysis of the HEK293 cells revealed that 1) all NCC mutants have decreased NCC protein expression; 2) mutant N59I, R83W, I360T, C421Y, G463R, and L859P have decreased NCC abundance at the plasma membrane; 3) mutants C421Y and L859P display impaired NCC glycosylation; and 4) mutants N59I, R83W, C421Y, C731R, and L859P show affected NCC phosphorylation. In conclusion, we developed a mammalian cell-based assay in which NCC activity assessment together with a profiling of mutated protein processing aid our understanding of the pathogenic mechanism of the NCC mutations. Copyright © 2016 the American Physiological Society.

  5. Mitochondrial gene mutations and type 2 diabetes in Chinese families

    Institute of Scientific and Technical Information of China (English)

    LI Ming-zhen; YU De-min; YU Pei; LIU De-min; WANG Kun; TANG Xin-zhi


    Background Numerous mitochondrial DNA mutations are significantly correlated with development of diabetes. This study investigated mitochondrial gene, point mutations in patients with type 2 diabetes and their families. Methods Unrelated patients with type 2 diabetes(n=826)were randomly recruited; unrelated and nondiabetic subjects (n=637)served as controls. The clinical and biochemical data of the participants were collected. Total genome was extracted from peripheral leucocytes. Polymerase chain reaction, restriction fragment length polymorphism (PCR-RFLP)and clonig techniques were used to screen mitochondrial genes including np3316,np3394 and np3426 in the ND1 region and np3243 in the tRNALeu (UUR). Results In 39 diabetics with one or more mitochondrial gene point mutations, the prevalence(4.7%,39/826)of mtDNA mutations was higher than that(0.7%,5/637)in the controls. The identical mutation was found in 23 of 43 tested members from three pedigrees. Affected family members presented with variable clinical features ranging from normal glucose tolerance to impaired glucose tolerance (IGT)(n=2),impaired fasting glucose(IFG)(n=1)to type 2 diabetes (n=13)with 3 family members suffering from hearing loss. Conclusions Type 2 diabetes in China is associated with several mitochondrial gene mutations. Aged patients with diabetic family history had a higher prevalence of mutation and various clinical pictures. Mitochondrial gene mutation might be one of the genetic factors contributing to diabetic familial clustering.

  6. A naturally occurring mutation within the probe-binding region compromises a molecular-based West Nile virus surveillance assay for mosquito pools (Diptera: Culicidae). (United States)

    Brault, Aaron C; Fang, Ying; Dannen, Maureen; Anishchenko, Michael; Reisen, William K


    A naturally occurring mutation was detected within the probe binding region targeting the envelope gene sequence of West Nile virus used in real-time polymerase chain reaction assays to test mosquito pools and other samples. A single C-->T transition 6nt from the 5' end of the 16mer in the envelope gene probe-binding region at genomic position 1,194 reduced assay sensitivity. The mutation first was detected in 2009 and persisted at a low prevalence into 2011. The mutation caused a 0.4% false negative error rate during 2011. These data emphasized the importance of confirmational testing and redundancy in surveillance systems relying on highly specific nucleic acid detection platforms.

  7. Molecular Analysis of CYP21A2 Gene Mutations among Iraqi Patients with Congenital Adrenal Hyperplasia

    Directory of Open Access Journals (Sweden)

    Ruqayah G. Y. Al-Obaidi


    Full Text Available Congenital adrenal hyperplasia is a group of autosomal recessive disorders. The most frequent one is 21-hydroxylase deficiency. Analyzing CYP21A2 gene mutations was so far not reported in Iraq. This work aims to analyze the spectrum and frequency of CYP21A2 mutations among Iraqi CAH patients. Sixty-two children were recruited from the Pediatric Endocrine Consultation Clinic, Children Welfare Teaching Hospital, Baghdad, Iraq, from September 2014 till June 2015. Their ages ranged between one day and 15 years. They presented with salt wasting, simple virilization, or pseudoprecocious puberty. Cytogenetic study was performed for cases with ambiguous genitalia. Molecular analysis of CYP21A2 gene was done using the CAH StripAssay (ViennaLab Diagnostics for detection of 11 point mutations and >50% of large gene deletions/conversions. Mutations were found in 42 (67.7% patients; 31 (50% patients were homozygotes, 9 (14.5% were heterozygotes, and 2 (3.2% were compound heterozygotes with 3 mutations, while 20 (32.3% patients had none of the tested mutations. The most frequently detected mutations were large gene deletions/conversions found in 12 (19.4% patients, followed by I2Splice and Q318X in 8 (12.9% patients each, I172N in 5 (8.1% patients, and V281L in 4 (6.5% patients. Del 8 bp, P453S, and R483P were each found in one (1.6% and complex alleles were found in 2 (3.2%. Four point mutations (P30L, Cluster E6, L307 frameshift, and R356W were not identified in any patient. In conclusion, gene deletions/conversions and 7 point mutations were recorded in varying proportions, the former being the commonest, generally similar to what was reported in regional countries.

  8. Pyridoxine responsiveness in novel mutations of the PNPO gene



    OBJECTIVE: To determine whether patients with pyridoxine-responsive seizures but normal biomarkers for antiquitin deficiency and normal sequencing of the ALDH7A1 gene may have PNPO mutations. METHODS: We sequenced the PNPO gene in 31 patients who fulfilled the above-mentioned criteria. RESULTS: We were able to identify 11 patients carrying 3 novel mutations of the PNPO gene. In 6 families, a homozygous missense mutation p.Arg225His in exon 7 was identified, while 1 family was compound h...

  9. Novel strategies for comprehensive mutation screening of the APC gene. (United States)

    Wachsmannova, L; Mego, M; Stevurkova, V; Zajac, V; Ciernikova, S


    Colorectal cancer is the 4th most common cause of cancer related deaths worldwide and new possibilities in accurate diagnosis and targeted treatment are highly required. Mutations in adenomatous polyposis coli (APC) gene play a pivotal role in adenoma-carcinoma pathway of colorectal tumorigenesis. The quarter century from its´ first cloning, APC became one of the most frequently mutated, known driver genes in colorectal cancer. Intensive routine molecular testing of APC has brought the benefits for patients with family history of polyposis or colorectal cancer. Nevertheless, multiple mutational disease-causing mechanisms make the genetic testing still challenging. This minireview is focused on implementation of novel APC mutation screening diagnostic strategies for polyposis families according to the current findings. A further understanding and improved algorithms may help to increase the mutation detection rate. APC germline mutations achieve close to 100% penetrance, so more comprehensive approach followed by preventive and therapeutic strategies might reflect in decrease in burden of colorectal cancer.

  10. Update of the androgen receptor gene mutations database. (United States)

    Gottlieb, B; Beitel, L K; Lumbroso, R; Pinsky, L; Trifiro, M


    The current version of the androgen receptor (AR) gene mutations database is described. The total number of reported mutations has risen from 309 to 374 during the past year. We have expanded the database by adding information on AR-interacting proteins; and we have improved the database by identifying those mutation entries that have been updated. Mutations of unknown significance have now been reported in both the 5' and 3' untranslated regions of the AR gene, and in individuals who are somatic mosaics constitutionally. In addition, single nucleotide polymorphisms, including silent mutations, have been discovered in normal individuals and in individuals with male infertility. A mutation hotspot associated with prostatic cancer has been identified in exon 5. The database is available on the internet (, from EMBL-European Bioinformatics Institute (, or as a Macintosh FilemakerPro or Word file (

  11. Amelogenesis Imperfecta and Screening of Mutation in Amelogenin Gene

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    Fernanda Veronese Oliveira


    Full Text Available The aim of this study was to report the clinical findings and the screening of mutations of amelogenin gene of a 7-year-old boy with amelogenesis imperfecta (AI. The genomic DNA was extracted from saliva of patient and his family, followed by PCR and direct DNA sequencing. The c.261C>T mutation was found in samples of mother, father, and brother, but the mutation was not found in the sequence of the patient. This mutation is a silent mutation and a single-nucleotide polymorphism (rs2106416. Thus, it is suggested that the mutation found was not related to the clinical presence of AI. Further research is necessary to examine larger number of patients and genes related to AI.

  12. Ferredoxin Gene Mutation in Iranian Trichomonas Vaginalis Isolates

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    Soudabeh Heidari


    Full Text Available Background: Trichomonas vaginalis causes trichomoniasis and metronidazole is its chosen drug for treatment. Ferredoxin has role in electron transport and carbohydrate metabolism and the conversion of an inactive form of metronidazole (CO to its active form (CPR. Ferredoxin gene mutations reduce gene expression and increase its resistance to metronidazole. In this study, the frequency of ferredoxin gene mutations in clinical isolates of T.vaginalis in Tehran has been studied.Methods: Forty six clinical T. vaginalis isolates of vaginal secretions and urine sediment were collected from Tehran Province since 2011 till 2012. DNA was extracted and ferredoxin gene was amplified by PCR technique. The ferredoxin gene PCR products were sequenced to determine gene mutations.Results: In four isolates (8.69% point mutation at nucleotide position -239 (the translation start codon of the ferredoxin gene were detected in which adenosine were converted to thymine.Conclusion: Mutation at nucleotide -239 ferredoxin gene reduces translational regulatory protein’s binding affinity which concludes reduction of ferredoxin expression. For this reduction, decrease in activity and decrease in metronidazole drug delivery into the cells occur. Mutations in these four isolates may lead to resistance of them to metronidazole.

  13. Mutational screening of the RB1 gene in Italian patients with retinoblastoma reveals 11 novel mutations. (United States)

    Sampieri, Katia; Hadjistilianou, Theodora; Mari, Francesca; Speciale, Caterina; Mencarelli, Maria Antonietta; Cetta, Francesco; Manoukian, Siranoush; Peissel, Bernard; Giachino, Daniela; Pasini, Barbara; Acquaviva, Antonio; Caporossi, Aldo; Frezzotti, Renato; Renieri, Alessandra; Bruttini, Mirella


    Retinoblastoma (RB, OMIM#180200) is the most common intraocular tumour in infancy and early childhood. Constituent mutations in the RB1 gene predispose individuals to RB development. We performed a mutational screening of the RB1 gene in Italian patients affected by RB referred to the Medical Genetics of the University of Siena. In 35 unrelated patients, we identified germline RB1 mutations in 6 out of 9 familial cases (66%) and in 7 out of 26 with no family history of RB (27%). Using the single-strand conformational polymorphism (SSCP) technique, 11 novel mutations were detected, including 3 nonsense, 5 frameshift and 4 splice-site mutations. Only two of these mutations (1 splice site and 1 missense) were previously reported. The mutation spectrum reflects the published literature, encompassing predominately nonsense or frameshift and splicing mutations. RB1 germline mutation was detected in 37% of our cases. Gross rearrangements outside the investigated region, altered DNA methylation, or mutations in non-coding regions, may be the cause of disease in the remainder of the patients. Some cases, e.g. a case of incomplete penetrance, or variable expressivity ranging from retinoma to multiple tumours, are discussed in detail. In addition, a case of pre-conception genetic counselling resolved by rescue of banked cordonal blood of the affected deceased child is described.

  14. Diverse growth hormone receptor gene mutations in Laron syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Berg, M.A.; Francke, U. (Stanford Univ. School of Medicine, CA (United States)); Gracia, R.; Rosenbloom, A.; Toledo, S.P.A. (Univ. Autonoma, Madrid (Spain)); Chernausek, S. (Children' s Hospital Medical Center, Cincinnati, OH (United States)); Guevara-Aguirre, J. (Institute of Endocrinology, Metabolism, and Reproduction, Quito (Ecuador)); Hopp, M. (Univ. of Witwatersrand, Johannesburg (South Africa)); Rosenbloom, A.; Argente, J. (Univ. of Florida, Gainesville (United States)); Toledo, S.P.A. (Univ. of Sao Paulo (Brazil))


    To better understand the molecular genetic basis and genetic epidemiology of Laron syndrome (growth-hormone insensitivity syndrome), the authors analysed the growth-hormone receptor (GHR) genes of seven unrelated affected individuals from the United States, South America, Europe, and Africa. They amplified all nine GHR gene exons and splice junctions from these individuals by PCR and screened the products for mutations by using denaturing gradient gel electrophoresis (DGGE). They identified a single GHR gene fragment with abnormal DGGE results for each affected individual, sequenced this fragment, and, in each case, identified a mutation likely to cause Laron syndrome, including two nonsense mutations (R43X and R217X), two splice-junction mutations, (189-1 G to T and 71+1 G to A), and two frameshift mutations (46 del TT and 230 del TA or AT). Only one of these mutations, R43X, has been previously reported. Using haplotype analysis, they determined that this mutation, which involves a CpG dinucleotide hot spot, likely arose as a separate event in this case, relative to the two prior reports of R43X. Aside from R43X, the mutations identified are unique to patients from particular geographic regions. Ten GHR gene mutations have now been described in this disorder. The authors conclude that Laron syndrome is caused by diverse GHR gene mutations, including deletions, RNA processing defects, translational stop codons, and missense codons. All the identified mutations involve the extracellular domain of the receptor, and most are unique to particular families or geographic areas. 35 refs., 3 figs., 1 tab.

  15. Social Health Insurance-Based Simultaneous Screening for 154 Mutations in 19 Deafness Genes Efficiently Identified Causative Mutations in Japanese Hearing Loss Patients. (United States)

    Mori, Kentaro; Moteki, Hideaki; Miyagawa, Maiko; Nishio, Shin-Ya; Usami, Shin-Ichi


    Sensorineural hearing loss is one of the most common neurosensory disorders in humans. The incidence of SNHL is estimated to be 1 in 500-1000 newborns. In more than half of these patients, the hearing loss is associated with genetic causes. In Japan, genetic testing for the patients with SNHL using the Invader assay to screen for 46 mutations in 13 deafness genes was approved by the Ministry of Health, Labour and Welfare for inclusion in social health insurance coverage in 2012. Furthermore, from August 2015, this genetic testing has been expanded to screen for 154 mutations in 19 deafness genes using targeted genomic enrichment with massively parallel DNA sequencing combined with the Invader assay and TaqMan genotyping. For this study we analyzed 717 unrelated Japanese hearing loss patients. The total allele frequency of 154 mutations in 19 deafness genes was 32.64% (468/1434) and the total numbers of cases associated with at least one mutation was 44.07% (316/717). Among these, we were able to diagnose 212 (30%) patients, indicating that the present screening could efficiently identify causative mutations in hearing loss patients. It is noteworthy that 27 patients (3.8%) had coexistent multiple mutations in different genes. Five of these 27 patients (0.7%, 5/717 overall) were diagnosed with genetic hearing loss affected by concomitant with responsible mutations in more than two different genes. For patients identified with multiple mutations in different genes, it is necessary to consider that several genes might have an impact on their phenotypes.

  16. No effect of high fat diet-induced obesity on spontaneous reporter gene mutations in gpt delta mice. (United States)

    Takasu, Shinji; Ishii, Yuji; Matsushita, Kohei; Kuroda, Ken; Kijima, Aki; Kodama, Yukio; Ogawa, Kumiko; Umemura, Takashi


    A large number of epidemiological studies have demonstrated that obesity is a risk factor for several human cancers. Several animal studies using rodents with diet-induced or genetic obesity have also demonstrated that obesity can promote tumor development. However, the effects of obesity on the early stages of carcinogenesis, and especially on the spontaneous occurrence of somatic gene mutations, remain unclear. To investigate the effects of obesity on the rate of spontaneous gene mutations, we performed reporter gene mutation assays in liver, kidney, and colon, organs in which obesity appears to be associated with cancer development on the basis of epidemiological or animal studies, in mice with high fat diet (HFD)-induced obesity. Six-week-old male and female C57BL/6 gpt delta mice were fed HFD or standard diet (STD) for 13 or 26 weeks. At the end of the experiments, reporter gene mutation assays of liver, kidney, and colon were performed. Final body weights and serum leptin levels of male and female mice fed HFD for 13 or 26 weeks were significantly increased compared with corresponding STD-fed groups. Reporter gene mutation assays of liver, kidney, and colon revealed that there were no significant differences in gpt or Spi- mutant frequencies between STD- and HFD-fed mice in either the 13-week or 26-week groups. These results indicate that HFD treatment and consequent obesity does not appear to influence the spontaneous occurrence of somatic gene mutations.

  17. Founder mutations in BRCA1 and BRCA2 genes. (United States)

    Ferla, R; Calò, V; Cascio, S; Rinaldi, G; Badalamenti, G; Carreca, I; Surmacz, E; Colucci, G; Bazan, V; Russo, A


    BRCA1 and BRCA2 germline mutations contribute to a significant number of familial and hereditary breast and/or ovarian cancers. The proportion of high-risk families with breast and/or ovarian cancer cases due to mutations in these tumor suppressor genes varies widely among populations. In some population, a wide spectrum of different mutations in both genes are present, whereas in other groups specific mutations in BRCA1 and BRCA2 have been reported with high frequency. Most of these mutations are prevalent in restricted populations as consequence of a founder effect. The comparison of haplotypes between families with the same mutation can distinguish whether high-frequency alleles derive from an older or more recent single mutational event or whether they have arisen independently more than once. Here, we review some of the most well-known and significant examples of founder mutations in BRCA genes found in European and non-European populations. In conclusion, the identification of the ethnic group of families undergoing genetic counseling enables the geneticist and oncologist to make more specific choices, leading to simplify the clinical approach to genetic testing carried out on members of high-risk families. Futhermore, the high frequency of founder mutations, allowing to analyze a large number of cases, might provide accurate information regarding their penetrance.

  18. Gene Mutation Patterns in Patients with Minimally Differentiated Acute Myeloid Leukemia

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    Hsiao-Wen Kao


    Full Text Available Minimally differentiated acute myeloid leukemia (AML-M0 is a rare subtype of AML with poor prognosis. Although genetic alterations are increasingly reported in AML, the gene mutations have not been comprehensively studied in AML-M0. We aimed to examine a wide spectrum of gene mutations in patients with AML-M0 to determine their clinical relevance. Twenty gene mutations including class I, class II, class III of epigenetic regulators (IDH1, IDH2, TET2, DNMT3A, MLL-PTD, ASXL1, and EZH2, and class IV (tumor suppressor genes were analyzed in 67 patients with AML-M0. Mutational analysis was performed with polymerase chain reaction–based assays followed by direct sequencing. The most frequent gene mutations from our data were FLT3-ITD/FLT3-TKD (28.4%, followed by mutations in IDH1/IDH2 (28.8%, RUNX1 (23.9%, N-RAS/K-RAS (12.3%, TET2 (8.2%, DNMT3A (8.1%, MLL-PTD (7.8%, and ASXL1 (6.3%. Seventy-nine percent (53/67 of patients had at least one gene mutation. Class I genes (49.3% were the most common mutated genes, which were mutually exclusive. Class III genes of epigenetic regulators were also frequent (43.9%. In multivariate analysis, old age [hazard ratio (HR 1.029, 95% confidence interval (CI 1.013-1.044, P = .001 was the independent adverse factor for overall survival, and RUNX1 mutation (HR 2.326, 95% CI 0.978-5.533, P = .056 had a trend toward inferior survival. In conclusion, our study showed a high frequency of FLT3, RUNX1, and IDH mutations in AML-M0, suggesting that these mutations played a role in the pathogenesis and served as potential therapeutic targets in this rare and unfavorable subtype of AML.

  19. Insulin gene mutations as a cause of permanent neonatal diabetes. (United States)

    Støy, Julie; Edghill, Emma L; Flanagan, Sarah E; Ye, Honggang; Paz, Veronica P; Pluzhnikov, Anna; Below, Jennifer E; Hayes, M Geoffrey; Cox, Nancy J; Lipkind, Gregory M; Lipton, Rebecca B; Greeley, Siri Atma W; Patch, Ann-Marie; Ellard, Sian; Steiner, Donald F; Hattersley, Andrew T; Philipson, Louis H; Bell, Graeme I


    We report 10 heterozygous mutations in the human insulin gene in 16 probands with neonatal diabetes. A combination of linkage and a candidate gene approach in a family with four diabetic members led to the identification of the initial INS gene mutation. The mutations are inherited in an autosomal dominant manner in this and two other small families whereas the mutations in the other 13 patients are de novo. Diabetes presented in probands at a median age of 9 weeks, usually with diabetic ketoacidosis or marked hyperglycemia, was not associated with beta cell autoantibodies, and was treated from diagnosis with insulin. The mutations are in critical regions of the preproinsulin molecule, and we predict that they prevent normal folding and progression of proinsulin in the insulin secretory pathway. The abnormally folded proinsulin molecule may induce the unfolded protein response and undergo degradation in the endoplasmic reticulum, leading to severe endoplasmic reticulum stress and potentially beta cell death by apoptosis. This process has been described in both the Akita and Munich mouse models that have dominant-acting missense mutations in the Ins2 gene, leading to loss of beta cell function and mass. One of the human mutations we report here is identical to that in the Akita mouse. The identification of insulin mutations as a cause of neonatal diabetes will facilitate the diagnosis and possibly, in time, treatment of this disorder.

  20. Mutations in the hemochromatosis gene (HFE) and stroke

    NARCIS (Netherlands)

    O.T. Njajou (Omer); M. Hollander (Monika); P.J. Koudstaal (Peter Jan); A. Hofman (Albert); C.M. van Duijn (Cock); J.C.M. Witteman (Jacqueline); M.M.B. Breteler (Monique)


    textabstractBACKGROUND AND PURPOSE: Increased serum iron is found to be a risk factor for stroke. Carriers of HFE C282Y and H63D mutations have elevated serum iron levels and may have an increased risk for stroke. We studied the association between HFE gene mutations, carotid atherosclerosis, and

  1. Analysis of Androgen Receptor Gene Mutations in female with infertility

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    Soyar Sari


    Full Text Available Background : Infertility is a multifactorial disease. Hormonal disorders and genetic factors are important in female infertility. Development and maturation of ovulation are depending on the molecular signaling pathways in response to androgens. Over hundreds of mutations leading to resistance gene function in androgen receptor (AR has been recorded. One of them is polymorphic region 5'UTR. Thus regarding to the role of androgen receptor in infertility, the aim of the present study was to investigate the association between gene mutations AR and infertility in Iranian women Materials and Methods: In this study of 50 infertile women and 80 healthy women as a control, blood samples were taken. After extraction of DNA, PCR method was used to determine the AR gene mutations. Results: In the present study in '5UTR area at position +25 androgen receptor gene a T nucleotide deletion was observed. , therefore single nucleotide mutations did not change in the androgen receptor gene expression, so indicates the lack of communication between the AR gene mutations in the promoter region of 23 to 214+ in women with infertility. According to the results of this study are significant differences between the two groups of patients and healthy women was not found (P=0.5. Conclusion: Results indicated no correlation between mutations in the promoter region of 23 to 214+ AR genes in the population studied women with infertility

  2. Simulation of gene evolution under directional mutational pressure (United States)

    Dudkiewicz, Małgorzata; Mackiewicz, Paweł; Kowalczuk, Maria; Mackiewicz, Dorota; Nowicka, Aleksandra; Polak, Natalia; Smolarczyk, Kamila; Banaszak, Joanna; R. Dudek, Mirosław; Cebrat, Stanisław


    The two main mechanisms generating the genetic diversity, mutation and recombination, have random character but they are biased which has an effect on the generation of asymmetry in the bacterial chromosome structure and in the protein coding sequences. Thus, like in a case of two chiral molecules-the two possible orientations of a gene in relation to the topology of a chromosome are not equivalent. Assuming that the sequence of a gene may oscillate only between certain limits of its structural composition means that the gene could be forced out of these limits by the directional mutation pressure, in the course of evolution. The probability of the event depends on the time the gene stays under the same mutation pressure. Inversion of the gene changes the directional mutational pressure to the reciprocal one and hence it changes the distance of the gene to its lower and upper bound of the structural tolerance. Using Monte Carlo methods we were able to simulate the evolution of genes under experimentally found mutational pressure, assuming simple mechanisms of selection. We found that the mutation and recombination should work in accordance to lower their negative effects on the function of the products of coding sequences.

  3. [Obesity based on mutation of genes involved in energy balance]. (United States)

    Hainerová, I


    Within the last decade an intensive research led to an identification of several genes which are involved in a regulation of energy balance. In most cases, carriers of these gene mutations do not exhibit further characteristic phenotypic features except for a severe obesity. Obesity based on mutation of one gene product is called monogenic obesity. Mutations in genes for leptin, leptin receptor, proopiomelanocortin, prohormone convertase 1, melanocortin 4 and 3 receptor disrupt the physiological humoral signalization between peripheral signals and the hypothalamic centres of satiety and hunger. Defects of all above mentioned genes lead to phenotype of abnormal eating behaviour followed by a development of severe early-onset obesity. Mutations of melanocortin 4 receptor gene represent the most common cause of monogenic obesity because they are detected in almost 6 % children with early-onset severe obesity. Mutations of the other genes involved in energy homeostasis are very rare. Although these mutations are sporadic we assume that further research of monogenic forms of obesity might lead to our understanding of physiology and pathophysiology of regulation of the energy homeostasis and eating behaviour. Additionally, they may open new approach to the management of eating behaviour and to the treatment of obesity.

  4. Prognostic impact of mismatch repair genes germline defects in colorectal cancer patients: are all mutations equal? (United States)

    Maccaroni, Elena; Bracci, Raffaella; Giampieri, Riccardo; Bianchi, Francesca; Belvederesi, Laura; Brugiati, Cristiana; Pagliaretta, Silvia; Del Prete, Michela; Scartozzi, Mario; Cascinu, Stefano


    Background Lynch syndrome (LS) is the most common hereditary colorectal cancer (CRC) syndrome, caused by germline mutations in MisMatch Repair (MMR) genes, particularly in MLH1, MSH2 and MSH6. Patients with LS seem to have a more favourable prognosis than those with sporadic CRC, although the prognostic impact of different mutation types is unknown. Aim of our study is to compare survival outcomes of different types of MMR mutations in patients with LS-related CRC. Methods 302 CRC patients were prospectively selected on the basis of Amsterdam or Revised Bethesda criteria to undergo genetic testing: direct sequencing of DNA and MLPA were used to examine the entire MLH1, MSH2 and MSH6 coding sequence. Patients were classified as mutation-positive or negative according to the genetic testing result. Results A deleterious MMR mutation was found in 38/302 patients. Median overall survival (OS) was significantly higher in mutation-positive vs mutation-negative patients (102.6 vs 77.7 months, HR:0.63, 95%CI:0.46–0.89, p = 0.0083). Different types of mutation were significantly related with OS: missense or splicing-site mutations were associated with better OS compared with rearrangement, frameshift or non-sense mutations (132.5 vs 82.5 months, HR:0.46, 95%CI:0.16–0.82, p = 0.0153). Conclusions Our study confirms improved OS for LS-patients compared with mutation-negative CRC patients. In addition, not all mutations could be considered equal: the better prognosis in CRC patients with MMR pathogenic missense or splicing site mutation could be due to different functional activity of the encoded MMR protein. This matter should be investigated by use of functional assays in the future. PMID:26485756

  5. Keratin 9 gene mutations in epidermolytic palmoplantar keratoderma (EPPK). (United States)

    Reis, A; Hennies, H C; Langbein, L; Digweed, M; Mischke, D; Drechsler, M; Schröck, E; Royer-Pokora, B; Franke, W W; Sperling, K


    We have isolated the gene for human type I keratin 9 (KRT9) and localised it to chromosome 17q21. Patients with epidermolytic palmoplantar keratoderma (EPPK), an autosomal dominant skin disease, were investigated. Three KRT9 mutations, N160K, R162Q, and R162W, were identified. All the mutations are in the highly conserved coil 1A of the rod domain, thought to be important for heterodimerisation. R162W was detected in five unrelated families and affects the corresponding residue in the keratin 14 and keratin 10 genes that is also altered in cases of epidermolysis bullosa simplex and generalised epidermolytic hyperkeratosis, respectively. These findings provide further evidence that mutations in keratin genes may cause epidermolysis and hyperkeratosis and that hyperkeratosis of palms and soles may be caused by different mutations in the KRT9 gene.

  6. Identification of novel mutations in HEXA gene in children affected with Tay Sachs disease from India.

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    Mehul Mistri

    Full Text Available Tay Sachs disease (TSD is a neurodegenerative disorder due to β-hexosaminidase A deficiency caused by mutations in the HEXA gene. The mutations leading to Tay Sachs disease in India are yet unknown. We aimed to determine mutations leading to TSD in India by complete sequencing of the HEXA gene. The clinical inclusion criteria included neuroregression, seizures, exaggerated startle reflex, macrocephaly, cherry red spot on fundus examination and spasticity. Neuroimaging criteria included thalamic hyperdensities on CT scan/T1W images of MRI of the brain. Biochemical criteria included deficiency of hexosaminidase A (less than 2% of total hexosaminidase activity for infantile patients. Total leukocyte hexosaminidase activity was assayed by 4-methylumbelliferyl-N-acetyl-β-D-glucosamine lysis and hexosaminidase A activity was assayed by heat inactivation method and 4-methylumbelliferyl-N-acetyl-β-D-glucosamine-6-sulphate lysis method. The exons and exon-intron boundaries of the HEXA gene were bidirectionally sequenced using an automated sequencer. Mutations were confirmed in parents and looked up in public databases. In silico analysis for mutations was carried out using SIFT, Polyphen2, MutationT@ster and Accelrys Discovery Studio softwares. Fifteen families were included in the study. We identified six novel missense mutations, c.340 G>A (p.E114K, c.964 G>A (p.D322N, c.964 G>T (p.D322Y, c.1178C>G (p.R393P and c.1385A>T (p.E462V, c.1432 G>A (p.G478R and two previously reported mutations. c.1277_1278insTATC and c.508C>T (p.R170W. The mutation p.E462V was found in six unrelated families from Gujarat indicating a founder effect. A previously known splice site mutation c.805+1 G>C and another intronic mutation c.672+30 T>G of unknown significance were also identified. Mutations could not be identified in one family. We conclude that TSD patients from Gujarat should be screened for the common mutation p.E462V.

  7. Identification of novel mutations in HEXA gene in children affected with Tay Sachs disease from India. (United States)

    Mistri, Mehul; Tamhankar, Parag M; Sheth, Frenny; Sanghavi, Daksha; Kondurkar, Pratima; Patil, Swapnil; Idicula-Thomas, Susan; Gupta, Sarita; Sheth, Jayesh


    Tay Sachs disease (TSD) is a neurodegenerative disorder due to β-hexosaminidase A deficiency caused by mutations in the HEXA gene. The mutations leading to Tay Sachs disease in India are yet unknown. We aimed to determine mutations leading to TSD in India by complete sequencing of the HEXA gene. The clinical inclusion criteria included neuroregression, seizures, exaggerated startle reflex, macrocephaly, cherry red spot on fundus examination and spasticity. Neuroimaging criteria included thalamic hyperdensities on CT scan/T1W images of MRI of the brain. Biochemical criteria included deficiency of hexosaminidase A (less than 2% of total hexosaminidase activity for infantile patients). Total leukocyte hexosaminidase activity was assayed by 4-methylumbelliferyl-N-acetyl-β-D-glucosamine lysis and hexosaminidase A activity was assayed by heat inactivation method and 4-methylumbelliferyl-N-acetyl-β-D-glucosamine-6-sulphate lysis method. The exons and exon-intron boundaries of the HEXA gene were bidirectionally sequenced using an automated sequencer. Mutations were confirmed in parents and looked up in public databases. In silico analysis for mutations was carried out using SIFT, Polyphen2, MutationT@ster and Accelrys Discovery Studio softwares. Fifteen families were included in the study. We identified six novel missense mutations, c.340 G>A (p.E114K), c.964 G>A (p.D322N), c.964 G>T (p.D322Y), c.1178C>G (p.R393P) and c.1385A>T (p.E462V), c.1432 G>A (p.G478R) and two previously reported mutations. c.1277_1278insTATC and c.508C>T (p.R170W). The mutation p.E462V was found in six unrelated families from Gujarat indicating a founder effect. A previously known splice site mutation c.805+1 G>C and another intronic mutation c.672+30 T>G of unknown significance were also identified. Mutations could not be identified in one family. We conclude that TSD patients from Gujarat should be screened for the common mutation p.E462V.

  8. 应用探针熔解分析法检测结核分枝杆菌rpoB基因突变%Analyzing the mutations of rpoB gene in Mycobacterium tuberculosis clinical isolates by probe melting analysis assay

    Institute of Scientific and Technical Information of China (English)

    牛建军; 张轶; 温慧欣; 刘歆; 胡思玉; 李庆阁


    Objective To evaluate the clinical performance of a probe melting analysis ( PMA)-based real-time PCR detection kit in rapid detection of rifampin-resistant mutations in Mycobacterium tuberculosis (MTB). Methods The specificity of the assay was evaluated by detecting 37 non-tuberculous mycobacteria (NTM) ,and the detection limit of the method was evaluated by genomic DNA of a standard strain H37Rv. Finally, 962 clinical isolates were analyzed with the PMA assay by detecting mutations in rifampin resistance-determining region (RRDR) of rpoB gene, and results were verified with DNA sequencing. Results Among 37 NTM strains,three strains showed drug resistant mutation signals. The PMA method could detect down to 30 bacteria per reaction. Sample analysis showed that 186 of 962 isolates were mutants,751 isolates were wild type and 25 isolates failed to give amplification signals. Among the mutant samples detected, 112 samples from November 2009 to April 2010 were further analyzed by sequencing, as well as 200 wild-type samples. The results showed a complete agreement with the PMA assay except for 5 samples failed in sequence analysis. Conclusion The PMA assay is rapid, accurate and easy-to-use, and thus can be used for detection of rifampin-resistant in clinical isolate samples.%目的 研究结核分枝杆菌利福平耐药突变实时PCR检测试剂盒(探针熔解分析)的临床应用价值.方法 用37株非结核分枝杆菌考察该方法的特异性,用菌株H37Rv考察其检测限,并检测962份结核分枝杆菌培养标本的rpoB基因利福平耐药决定区突变,检测结果经测序验证.结果 37株非结核分枝杆菌中仅有3株出现耐药突变峰,检测限考察结果表明该方法每反应可重复检出30个菌.用该试剂盒检测962份标本,检出突变株186份,野生株751份,扩增失败标本25份.选取2009年11月以后的标本中试剂盒检出为突变的标本112份,并数字表法随机选取200份试剂盒检出为阴性

  9. Rapid detection of DNMT3A R882 mutations in hematologic malignancies using a novel bead-based suspension assay with BNA(NC probes.

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    Velizar Shivarov

    Full Text Available Mutations in the human DNA methyl transferase 3A (DNMT3A gene are recurrently identified in several hematologic malignancies such as Philadelphia chromosome-negative myeloproliferative neoplasms (MPN, myelodysplastic syndromes (MDS, MPN/MDS overlap syndromes and acute myeloid leukemia (AML. They have been shown to confer worse prognosis in some of these entities. Notably, about 2/3 of these mutations are missense mutations in codon R882 of the gene. We aimed at the development and validation of a novel easily applicable in routine practice method for quantitative detection of the DNMT3A p.R882C/H/R/S mutations bead-based suspension assay. Initial testing on plasmid constructs showed excellent performance of BNA(NC-modified probes with an optimal hybridization temperature of 66°C. The method appeared to be quantitative and showed sensitivity of 2.5% for different mutant alleles, making it significantly superior to direct sequencing. The assay was further validated on plasmid standards at different ratios between wild type and mutant alleles and on clinical samples from 120 patients with known or suspected myeloid malignancies. This is the first report on the quantitative detection of DNMT3A R882 mutations using bead-based suspension assay with BNA(NC-modified probes. Our data showed that it could be successfully implemented in the diagnostic work-up for patients with myeloid malignancies, as it is rapid, easy and reliable in terms of specificity and sensitivity.

  10. Mutations in the AXIN1 gene in advanced prostate cancer

    DEFF Research Database (Denmark)

    Yardy, George W; Bicknell, David C; Wilding, Jennifer L;


    The Wnt signalling pathway directs aspects of embryogenesis and is thought to contribute to maintenance of certain stem cell populations. Disruption of the pathway has been observed in many different tumour types. In bowel, stomach, and endometrial cancer, this is usually due to mutation of genes...... encoding Wnt pathway components APC or beta-catenin. Such mutations are rare in hepatocellular carcinomas and medulloblastomas with Wnt pathway dysfunction, and there, mutation in genes for other Wnt molecules, such as Axin, is more frequently found....

  11. First molecular analysis of F8 gene in algeria: identification of two novel mutations. (United States)

    Abdi, Meriem; Zemani-Fodil, Faouzia; Fodil, Mostefa; Aberkane, Meriem Samia; Touhami, Hadj; Saidi-Mehtar, Nadhira; Costa, Catherine; Boudjema, Abdallah


    The aim of this study was to detect the genetic alterations in the Factor 8 gene in 26 patients from Western Algeria. We detected the presence of "intron 22 inversion" with long-range polymerase chain reaction (PCR). Negative patients for this inversion were analyzed for "intron 1 inversion" using multiplex PCR. Patients who were negative for both inversions were analyzed using a direct sequencing. Deleterious effects of novel mutations on protein were assayed with bioinformatics tools. Causing mutations were identified in 85.71% of the families, including 11 "intron 22 inversion," 1 "intron 1 inversion," and 6 different point mutations (2 nonsense, 1 splice site, and 3 missense mutations). Among these mutations, c.2189G > A (p.Cys711Tyr) and c.5219+1G>T are novel. This is the first study that reports spectrum of mutations in the Factor 8 gene in the Western Algerian population. Knowledge of these mutations is important for genetic counseling and medical care of affected families.

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

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    Reinthaller Alexander


    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.

  13. Parkin gene mutations in younger onset Parkinson's disease

    Institute of Scientific and Technical Information of China (English)

    Piu Chan; Hua Bai; Rong Chen; J Willian Langston


    Objective': To screen for exonic and point mutations in the Parkin gene in both Chinese and American Caucasian younger onset Parkinson's disease (YOPD) patients.Background: Recently, the Autosomal recessive juvenile parkinsonism (ARJP) gene was first mapped to chromosome 6q25.2-27 and was late cloned and designated as Parkin. A wide variety of mutations, including homozygous exonic deletions and point mutations,have been found in at least more than 50 ARJP families of Japanese, European and Jewish origins. However, the distribution of Parkin gene mutations is not known in the Chinese and American Caucasians, It is also not clear how frequent the Parkin gene mutations occur in YOPD patients. Method and Material: Twenty-one Chinese subjects were selected from 121 Chinese PD inpatients who were admitted to the Xuanwu Hospital in Beijing between August of 1998 and April of 1999 and had an onset before age 51. Thirty-eight American subjects were PD patients with an onset before age 41 from the Tissue Bank of the Parkinson′s Institute at California. Homozygous exonic deletion and point mutations in all 12 exons of the Parkin gene were screened using PCR, SSCP and direct sequencing methods. Mutations identified by sequencing were further confirmed by restriction enzyme digestion. Results: Five different types of homozygous deletion mutations (exons 1, 4, 6, 7 and 12) were found in 7 out of 21 Chinese cases but none of the 37 American Caucasian patients in all 12 exons of Parkin gene. One novel and four polymorphic mutationswere found in the American Caucasian YOPD cases.Conclusion: our results suggest that homozygous exonic deletions in the Parkin gene may account for a significant amount of YOPD in the Chinese but not in the American Caucasian YOPD.

  14. Methods for transient assay of gene function in floral tissues

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    Pathirana Nilangani N


    Full Text Available Abstract Background There is considerable interest in rapid assays or screening systems for assigning gene function. However, analysis of gene function in the flowers of some species is restricted due to the difficulty of producing stably transformed transgenic plants. As a result, experimental approaches based on transient gene expression assays are frequently used. Biolistics has long been used for transient over-expression of genes of interest, but has not been exploited for gene silencing studies. Agrobacterium-infiltration has also been used, but the focus primarily has been on the transient transformation of leaf tissue. Results Two constructs, one expressing an inverted repeat of the Antirrhinum majus (Antirrhinum chalcone synthase gene (CHS and the other an inverted repeat of the Antirrhinum transcription factor gene Rosea1, were shown to effectively induce CHS and Rosea1 gene silencing, respectively, when introduced biolistically into petal tissue of Antirrhinum flowers developing in vitro. A high-throughput vector expressing the Antirrhinum CHS gene attached to an inverted repeat of the nos terminator was also shown to be effective. Silencing spread systemically to create large zones of petal tissue lacking pigmentation, with transmission of the silenced state spreading both laterally within the affected epidermal cell layer and into lower cell layers, including the epidermis of the other petal surface. Transient Agrobacterium-mediated transformation of petal tissue of tobacco and petunia flowers in situ or detached was also achieved, using expression of the reporter genes GUS and GFP to visualise transgene expression. Conclusion We demonstrate the feasibility of using biolistics-based transient RNAi, and transient transformation of petal tissue via Agrobacterium infiltration to study gene function in petals. We have also produced a vector for high throughput gene silencing studies, incorporating the option of using T-A cloning to


    Institute of Scientific and Technical Information of China (English)

    CHANG Liang; ZHONG Su; ZHAO Nan; LIU Ping; ZHAO Yangyu; QIAO Jie


    Objective The study is to identify the carrier rate of common deafness mutation in Chinese pregnant women via detecting deafness gene mutations with gene chip. Methods The pregnant women in obstetric clinic without hearing impairment and hearing disorders family history were selected. The informed consent was signed. Peripheral blood was taken to extract genom-ic DNA. Application of genetic deafness gene chip for detecting 9 mutational hot spot of the most common 4 Chinese deafness genes, namely GJB2 (35delG,176del16bp, 235delC, 299delAT), GJB3 (C538T) ,SLC26A4 ( IVS72A>G, A2168G) and mito-chondrial DNA 12S rRNA (A1555G, C1494T) . Further genetic testing were provided to the spouses and newborns of the screened carriers. Results Peripheral blood of 430 pregnant women were detected,detection of deafness gene mutation carri-ers in 24 cases(4.2%), including 13 cases of the GJB2 heterozygous mutation, 3 cases of SLC26A4 heterozygous mutation, 1 cases of GJB3 heterozygous mutation, and 1 case of mitochondrial 12S rRNA mutation. 18 spouses and 17 newborns took fur-ther genetic tests, and 6 newborns inherited the mutation from their mother. Conclusion The common deafness genes muta-tion has a high carrier rate in pregnant women group,235delC and IVS7-2A>G heterozygous mutations are common.

  16. A novel PTEN gene promoter mutation and untypical Cowden syndrome

    Institute of Scientific and Technical Information of China (English)

    Chen Liu; Guangbing Li; Rongrong Chen; Xiaobo Yang; Xue Zhao; Haitao Zhao


    Cowden syndrome (CS),an autosomal dominant disorder,is one of a spectrum of clinical disorders that have been linked to germline mutations in the phosphatase and tensin homolog (PTEN) gene.Although 70-80% of patients with CS have an identifiable germline PTEN mutation,the clinical diagnosis presents many challenges because of the phenotypic and genotypic variations.In the present study,we sequenced the exons and the promoter of PTEN gene,mutations and variations in the promoter and exons were identified,and a PTEN protein expression negative region was determined by immunohistochemistry (IHC).In conclusion,a novel promoter mutation we found in PTEN gene may turn off PTEN protein expression occasionally,leading to the disorder of PTEN and untypical CS manifestations.

  17. Phenotypic consequences of a novel SCO2 gene mutation. (United States)

    Verdijk, Rob M; de Krijger, Ronald; Schoonderwoerd, Kees; Tiranti, Valeria; Smeets, Hubert; Govaerts, Lutgarde C P; de Coo, René


    SCO2 is a cytochrome c oxidase (COX) assembly gene. Mutations in the SCO2 gene have been associated with fatal infantile cardioencephalomyopathy. We report on the phenotype of a novel SCO2 mutation in two siblings with fatal infantile cardioencephalomyopathy. The index patient died of heart failure at 25 days of age. Muscle biopsy was performed for histology and biochemical study of the oxidative phosphorylation system complexes. The entire coding region of the SCO2 gene was sequenced. Autopsy was performed on the index patient and on a female sibling delivered at 23 weeks of gestation following termination of pregnancy during which amniocentesis and genetic testing had been performed. Muscle biopsy and biochemical analysis of heart and skeletal muscle detected a severe isolated COX-IV deficiency. Pathologic findings in both patients confirmed hypertrophic cardiomyopathy. Sequencing of the SCO2 gene showed compound heterozygous mutation; the common E140K mutation and a novel W36X nonsense mutation. Newborns with a combination of hypotonia and cardiomyopathy should be evaluated for multiple congenital anomaly syndromes, inborn errors of metabolism and mitochondrial derangements, and may require extensive diagnostic testing. Mutations in the SCO2 gene are a cause of prenatal-onset hypertrophic cardiomyopathy.

  18. Mutations in the rpoB and katG genes leading to drug resistance in Mycobacterium tuberculosis in Latvia. (United States)

    Tracevska, T; Jansone, I; Broka, L; Marga, O; Baumanis, V


    To characterize the genetic basis of drug resistance in Mycobacterium tuberculosis in Latvia, mutations involved in rifampin (rpoB gene) and isoniazid (katG gene) resistance in DNA from 19 drug-susceptible and 51 multidrug-resistant M. tuberculosis complex isolates were analyzed. The most frequent rpoB gene mutations found by the Line Probe assay were the S531L (14 of 34 isolates), D516V (7 of 34), H526D (4 of 34), and D516Y plus P535S (4 of 34) mutations. Direct sequencing of seven isolates with unclear results from Line Probe assay showed the presence of the L533P mutation and the Q510H plus H526Y (1 of 34) and D516V plus P535S (4 of 34) double mutations, neither of which has been described previously. Single-strand conformation polymorphism analysis showed strand mobility differences between the rifampin-susceptible and -resistant samples for the D516V, H526D, and D516Y plus P535S mutations but not for the S531L mutation. Nucleotide substitution at codon 315 (AGC-->ACC) of the katG gene was found in 48 of 51 multidrug-resistant samples by sequencing. Furthermore, katG gene restriction fragment length polymorphism analysis with endonuclease AciI confirmed the nucleotide change in codon 315.

  19. Sensitive detection of point mutation by electrochemiluminescence and DNA ligase-based assay (United States)

    Zhou, Huijuan; Wu, Baoyan


    The technology of single-base mutation detection plays an increasingly important role in diagnosis and prognosis of genetic-based diseases. Here we reported a new method for the analysis of point mutations in genomic DNA through the integration of allele-specific oligonucleotide ligation assay (OLA) with magnetic beads-based electrochemiluminescence (ECL) detection scheme. In this assay the tris(bipyridine) ruthenium (TBR) labeled probe and the biotinylated probe are designed to perfectly complementary to the mutant target, thus a ligation can be generated between those two probes by Taq DNA Ligase in the presence of mutant target. If there is an allele mismatch, the ligation does not take place. The ligation products are then captured onto streptavidin-coated paramagnetic beads, and detected by measuring the ECL signal of the TBR label. Results showed that the new method held a low detection limit down to 10 fmol and was successfully applied in the identification of point mutations from ASTC-α-1, PANC-1 and normal cell lines in codon 273 of TP53 oncogene. In summary, this method provides a sensitive, cost-effective and easy operation approach for point mutation detection.

  20. Real-time fluorescence PCR-molecular beacon assay for detecting mutations in rpoB genes of rifampin-resistant Mycobacterium tuberculosis%实时荧光PCR分子信标检测耐利福平结核分枝杆菌印rpoB基因

    Institute of Scientific and Technical Information of China (English)

    孙桂芝; 高铁杰; 钟镐镐; 康丽军; 张治国; 衡万杰; 吴秉铨; 刘威


    Objective To establish a rapid method to detect mutations in rpoB genes of rifampin-resistant Mycobacterium tubereulosis in dinical specimens using Real-time fluorescence PCR molecular beacon assay.Methods 174 strains of Mvcobacterium tuberculosis clinical isolates were analyzed using real-time fluorescence PCR molecular beacon assay foilowed with DNA sequencing while 12 strains of NTM and 4 strains of bacteria other than Mycobacterium tuberculosis were used as the contrast.Results Eighty-two 89.1 of 92 rifampin (RIF)-resistant strains and 3 of 82 RIF-sensitive strains were found to harbor mutation in the rpoB gene using real-time fluorescence PCR-molecular beacon assay.The specificity, sensitivity,and accuracy of this assay were 96.3%,89.1%,and 92.5%,respectively-Eithty-three of 92 RIF-resistant strains and 1 of 82 RIF-sensitive strains were found to harbor mutation in the rpoB gene using the direct DNA sequencing.The specificity,sensitivity,and accuracy of the direct DNA sequencing were 98.8,90.2%,and 94.2%,respectively.As compared with real-time PCR molecular beacon assay,171 of 174(98.3%)strains of myeobactefium tuberculosis clinical isolates had the salne results.Conclusion Real-time fluorescence PCR-molecular beacon assay can be used as a rapid screen method to detect RIF-resistant isolates.%目的 应用实时荧光PCR分子信标技术,建立快速检测临床标本中结核分枝杆菌利福平rpoB相关耐药突变点方法,探讨其缩短耐药实验报告时间的临床应用价值.方法 以分枝杆菌药物敏感性实验绝对浓度法为标准,12株非结核分枝杆菌、4株非分枝杆菌作对照,对174例结核患者临床分离株应用实时荧光PCR分子信标方法,检测利福平rpoB核心区域的耐药突变点并将结果与直接测序进行比较.结果 (1)实时荧光PCR分子信标方法:82例结核分枝杆菌利福平敏感菌株中,3例发生rpoB基因突变,特异度为96.3%;92例结核分枝杆菌利福平耐药菌株中,82

  1. Four novel mutations in the lactase gene (LCT underlying congenital lactase deficiency (CLD

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    Höglund Pia


    Full Text Available Abstract Background Congenital lactase deficiency (CLD is a severe gastrointestinal disorder of newborns. The diagnosis is challenging and based on clinical symptoms and low lactase activity in intestinal biopsy specimens. The disease is enriched in Finland but is also present in other parts of the world. Mutations encoding the lactase (LCT gene have recently been shown to underlie CLD. The purpose of this study was to identify new mutations underlying CLD in patients with different ethnic origins, and to increase awareness of this disease so that the patients could be sought out and treated correctly. Methods Disaccharidase activities in intestinal biopsy specimens were assayed and the coding region of LCT was sequenced from five patients from Europe with clinical features compatible with CLD. In the analysis and prediction of mutations the following programs: ClustalW, Blosum62, PolyPhen, SIFT and Panther PSEC were used. Results Four novel mutations in the LCT gene were identified. A single nucleotide substitution leading to an amino acid change S688P in exon 7 and E1612X in exon 12 were present in a patient of Italian origin. Five base deletion V565fsX567 leading to a stop codon in exon 6 was found in one and a substitution R1587H in exon 12 from another Finnish patient. Both Finnish patients were heterozygous for the Finnish founder mutation Y1390X. The previously reported mutation G1363S was found in a homozygous state in two siblings of Turkish origin. Conclusion This is the first report of CLD mutations in patients living outside Finland. It seems that disease is more common than previously thought. All mutations in the LCT gene lead to a similar phenotype despite the location and/or type of mutation.

  2. Screening for Mutations in the TBX1 Gene on Chromosome 22q11.2 in Schizophrenia

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    Lieh-Yung Ping


    Full Text Available A higher-than-expected frequency of schizophrenia in patients with 22q11.2 deletion syndrome suggests that chromosome 22q11.2 harbors the responsive genes related to the pathophysiology of schizophrenia. The TBX1 gene, which maps to the region on chromosome 22q11.2, plays a vital role in neuronal functions. Haploinsufficiency of the TBX1 gene is associated with schizophrenia endophenotype. This study aimed to investigate whether the TBX1 gene is associated with schizophrenia. We searched for mutations in the TBX1 gene in 652 patients with schizophrenia and 567 control subjects using a re-sequencing method and conducted a reporter gene assay. We identified six SNPs and 25 rare mutations with no association with schizophrenia from Taiwan. Notably, we identified two rare schizophrenia-specific mutations (c.-123G>C and c.-11delC located at 5′ UTR of the TBX1 gene. The reporter gene assay showed that c.-123C significantly decreased promoter activity, while c.-11delC increased promoter activity compared with the wild-type. Our findings suggest that the TBX1 gene is unlikely a major susceptible gene for schizophrenia in an ethnic Chinese population for Taiwan, but a few rare mutations in the TBX1 gene may contribute to the pathogenesis of schizophrenia in some patients.

  3. AB125. Neonatal diabetes mellitus due to insulin gene mutation (United States)

    Can, Ngoc Thi Bich; Vu, Dung Chi; Bui, Thao Phuong; Nguyen, Khanh Ngoc; Nguyen, Dat Phu; Craig, Maria; Ellard, Sian; Nguyen, Hoan Thi


    Background and objective Insulin (INS) gene mutations that cause permanent neonatal diabetes mellitus change single protein building blocks (amino acids) in the protein sequence. These mutations are believed to disrupt the cleavage of the proinsulin chain or the binding of the A and B chains to form insulin, leading to impaired blood sugar control. At least ten mutations in the INS gene have been identified in people with permanent neonatal diabetes mellitus. To describe clinical features and laboratory manifestations of patients with INS gene mutation and to evaluate outcome of management. Methods Clinical features, biochemical finding, mutation analysis and management outcome of six cases from six unrelated families were study. All exons of INS gene were amplified from genomic DNA and directly sequenced. Results Six cases (three girls and three boys) onset at 129.2±128.8 days of age (median 101.5 days) with gestation age of 37.3±3.0 weeks, birth weight of 2,816.6±767.8 g. Five out of six patients admitted with the feature of diabetic ketoacidosis with pH of 7.04±0.22; plasma glucose levels were 34.3±12.7 mmoL/L, HbA1C of 9.75%±3.5%. Mutation analysis of the INS gene showed: heterozygous for a novel missense mutation (c.127T > A; C43S) in exon 2 in one case; heterozygous for a splicing mutation c.188-31G > A in intron 2 in two cases; heterozygous for a missense mutation c.286T > C in exon 3 in one case; heterozygous for a missense mutation c.265C > T [p.Arg89Cys (p.R89C)] in exon 3 in two cases. After 19.2±13.4 months of insulin treatment, 4/5 patients have normal development with DQ 80-100%, HbA1C of 6.85%±0.49%, quite normal blood glucose levels. The case with c.127T > A mutation treated with insulin for 14 years has physical development delay, poor blood glucose control with HbA1C of 11.4%. Conclusions It is important to perform screening gene mutation for patients with diabetes diagnosed before 6 months of age to control blood glucose and follow up the

  4. From minisatellites and genes: When do germinal mutations occur

    Energy Technology Data Exchange (ETDEWEB)

    Mohrenweiser, H. [Lawrence Livermore National Lab., CA (United States)


    Utilization of molecular techniques has provided insight into the molecular techniques has provided insight into the molecular techniques has provided insight into the molecular character and origins of spontaneous and induced germinal mutations. Review of the variants and disease loci suggests differences among loci in the frequency of nucleotide substitutions and more complex events. Mechanistic features associated with the alterations in DNA structure are observed in each variant class. The spectrum of mutations identified reflects the gene structure and the selective pressure generating disease phenotypes, and the techniques employed to screen for variation. Locus specificity in spectra has the potential to compromise estimates of increases in germinal gene mutation rates. Recent studies have identified mosaicism, rather than de novo mutation, as the explanation for the non-traditional pattern of inheritance of disease in some families. Mosaicism is a concern for studies of induced mutation rates as it reflects embryonic exposure of the parent of the proband. This is in contrast to the {open_quotes}normal expectation{close_quotes} that induced mutations result from parental exposure to genotoxins in the environment. Observations suggest that the germ cell stage sensitivity may reflect interaction of the mutagen and the loci screened. The mosaicism and germ cell stage issues, in conjunction with incomplete ascertainment of mutational events, increase the complexity of efforts to estimate induced germinal mutation rates and associated health consequences in populations exposed to genotoxic agents.

  5. Somatic mutation of immunoglobulin VH6 genes in human infants (United States)

    Ridings, J; Dinan, L; Williams, R; Roberton, D; Zola, H


    Infants respond to antigen by making antibody that is generally of low affinity for antigen. Somatic hypermutation of immunoglobulin genes, and selection of cells expressing mutations with improved affinity for antigen, are the molecular and cellular processes underlying the maturation of antibody affinity. We have reported previously that neonates and infants up to 2 months of age, including individuals undergoing strong immunological challenge, show very few mutated VH6 sequences, with low mutation frequencies in mutated sequences, and little evidence of selection. We have now examined immunoglobulin genes from healthy infants between 2 and 10 months old for mutation and evidence of selection. In this age group, the proportion of VH6 sequences which are mutated and the mutation frequency in mutated sequences increase with age. There is evidence of selection from 6 months old. These results indicate that the process of affinity maturation, which depends on cognate T–B cell interaction and functional germinal centres, is approaching maturity from 6 months old. PMID:9764600

  6. Mutation analysis of the preproghrelin gene

    DEFF Research Database (Denmark)

    Larsen, Lesli H; Gjesing, Anette P; Sørensen, Thorkild I A;


    To investigate the preproghrelin gene for variants and their association with obesity and type 2 diabetes.......To investigate the preproghrelin gene for variants and their association with obesity and type 2 diabetes....

  7. Mutations of FUS gene in sporadic amyotrophic lateral sclerosis. (United States)

    Corrado, Lucia; Del Bo, Roberto; Castellotti, Barbara; Ratti, Antonia; Cereda, Cristina; Penco, Silvana; Sorarù, Gianni; Carlomagno, Yari; Ghezzi, Serena; Pensato, Viviana; Colombrita, Claudia; Gagliardi, Stella; Cozzi, Lorena; Orsetti, Valeria; Mancuso, Michelangelo; Siciliano, Gabriele; Mazzini, Letizia; Comi, Giacomo Pietro; Gellera, Cinzia; Ceroni, Mauro; D'Alfonso, Sandra; Silani, Vincenzo


    Mutations in the FUS gene have recently been discovered to be a major cause of familial amyotrophic lateral sclerosis (FALS). To determine the identity and frequency of FUS gene mutations in a large cohort of Italian patients enriched in sporadic cases (SALS). Exons 5, 6, 14 and 15 of the FUS gene were screened for mutations in 1009 patients (45 FALS and 964 SALS). The genetic analysis was extended to the entire coding sequence of FUS in all the FALS and 293 of the SALS patients. Seven missense mutations (p.G191S, p.R216C, p.G225V, p.G230C, p.R234C, p.G507D and p.R521C) were identified in nine patients (seven SALS and two FALS), and none in 500 healthy Italian controls. All mutations are novel except for the p.R521C mutation identified in one SALS and one FALS case. Both patients showed a similar unusual presentation, with proximal, mostly symmetrical, upper limb weakness, with neck and axial involvement. With the exception of p.G507D and p.R521C, the mutations identified in SALS patients are all localised in the glycine-rich region encoded by exon 6. In addition, eight different in-frame deletions in two polyglycine motifs were detected, the frequency of which was not significantly different in patients and controls. The results show that FUS missense mutations are present in 0.7% of Italian SALS cases, and confirm the previous mutational frequency reported in FALS (4.4%). An unusual proximal and axial clinical presentation seems to be associated with the presence of the p.R521C mutation.

  8. Mutation analysis of novel human liver-related putative tumor suppressor gene in hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Cheng Liao; Tsai-Ping Li; Mu-Jun Zhao; Jing Zhao; Hai Song; Pascal Pineau; Agnès Marchio; Anne Dejean; Pierre Tiollais; Hong-Yang Wang


    AIM: To find the point mutations meaningful for inactivationof liver-related putative tumor suppressor gene (LPTS) gene,a human novel liver-related putative tumor suppressor geneand telomerase inhibitor in hepatocellular carcinoma.METHODS: The entire coding sequence of LPTS genewas examined for mutations by single strand conformationpolymorphism (SSCP) assay and PCR products directsequencing in 56 liver cancer cell lines, 7 ovarian cancerand 7 head & neck tumor cell lines and 70 pairs of HCCtissues samples. The cDNA fragment coding for the mostfrequent mutant protein was subcloned into GST fusionexpression vector. The product was expressed in E. coliand purified by glutathione-agarose column. Telomericrepeat amplification protocol (TRAP) assays wereperformed to study the effect of point mutation totelomerase inhibitory activity.RESULTS: SSCP gels showed the abnormal shifting bandsand DNA sequencing found that there were 5 differentmutations and/or polymorphisms in 12 tumor cell lineslocated at exon2, exon5 and exon7. The main alterationswere A(778)A/G and A(880)T in exon7. The change in siteof 778 could not be found in HCC tissue samples, while themutation in position 880 was seen in 7 (10 %) cases. Themutation in the site of 880 had no effect on telomeraseinhibitory activity.CONCLUSION: Alterations identified in this study arepolymorphisms of LPTS gene. LPTS mutations occur in HCCbut are infrequent and of little effect on the telomeraseinhibitory function of the protein. Epigenetics, such asmethylation, acetylation, may play the key role in inactivationof LPTS.

  9. Identifying cancer genes from cancer mutation profiles by cancer functions

    Institute of Scientific and Technical Information of China (English)


    It is of great importance to identify new cancer genes from the data of large scale genome screenings of gene mutations in cancers. Considering the alternations of some essential functions are indispensable for oncogenesis, we define them as cancer functions and select, as their approximations, a group of detailed functions in GO (Gene Ontology) highly enriched with known cancer genes. To evaluate the efficiency of using cancer functions as features to identify cancer genes, we define, in the screened genes, the known protein kinase cancer genes as gold standard positives and the other kinase genes as gold standard negatives. The results show that cancer associated functions are more efficient in identifying cancer genes than the selection pressure feature. Furthermore, combining cancer functions with the number of non-silent mutations can generate more reliable positive predictions. Finally, with precision 0.42, we suggest a list of 46 kinase genes as candidate cancer genes which are annotated to cancer functions and carry at least 3 non-silent mutations.

  10. TINF2 Gene Mutation in a Patient with Pulmonary Fibrosis

    NARCIS (Netherlands)

    Hoffman, T W; van der Vis, J J; van Oosterhout, M F M; van Es, H W; van Kessel, D A; Grutters, J C; van Moorsel, C H M


    Pulmonary fibrosis is a frequent manifestation of telomere syndromes. Telomere gene mutations are found in up to 25% and 3% of patients with familial disease and sporadic disease, respectively. The telomere gene TINF2 encodes an eponymous protein that is part of the shelterin complex, a complex invo

  11. Hemochromatosis (HFE gene mutations in Brazilian chronic hemodialysis patients

    Directory of Open Access Journals (Sweden)

    F.V. Perícole


    Full Text Available Patients with chronic renal insufficiency (CRI have reduced hemoglobin levels, mostly as a result of decreased kidney production of erythropoietin, but the relation between renal insufficiency and the magnitude of hemoglobin reduction has not been well defined. Hereditary hemochromatosis is an inherited disorder of iron metabolism. The importance of the association of hemochromatosis with treatment for anemia among patients with CRI has not been well described. We analyzed the frequency of the C282Y and H63D mutations in the HFE gene in 201 Brazilian individuals with CRI undergoing hemodialysis. The analysis of the effects of HFE mutations on iron metabolism and anemia with biochemical parameters was possible in 118 patients of this study (hemoglobin, hematocrit, ferritin levels, transferrin saturation, and serum iron. A C282Y heterozygous mutation was found in 7/201 (3.4% and H63D homozygous and heterozygous mutation were found in 2/201 (1.0% and 46/201 (22.9%, respectively. The allelic frequencies of the HFE mutations (0.017 for C282Y mutation and 0.124 for H63D mutation did not differ between patients with CRI and healthy controls. Regarding the biochemical parameters, no differences were observed between HFE heterozygous and mutation-negative patients, although ferritin levels were not higher among patients with the H63D mutation (P = 0.08. From what we observed in our study, C282Y/H63D HFE gene mutations are not related to degrees of anemia or iron stores in CRI patients receiving intravenous iron supplementation (P > 0.10. Nevertheless, the present data suggest that the H63D mutation may have an important function as a modulating factor of iron overload in these patients.

  12. Mutational analysis of DBD*--a unique antileukemic gene sequence. (United States)

    Ji, Yan-shan; Johnson, Betty H; Webb, M Scott; Thompson, E Brad


    DBD* is a novel gene encoding an 89 amino acid peptide that is constitutively lethal to leukemic cells. DBD* was derived from the DNA binding domain of the human glucocorticoid receptor by a frameshift that replaces the final 21 C-terminal amino acids of the domain. Previous studies suggested that DBD* no longer acted as the natural DNA binding domain. To confirm and extend these results, we mutated DBD* in 29 single amino acid positions, critical for the function in the native domain or of possible functional significance in the novel 21 amino acid C-terminal sequence. Steroid-resistant leukemic ICR-27-4 cells were transiently transfected by electroporation with each of the 29 mutants. Cell kill was evaluated by trypan blue dye exclusion, a WST-1 tetrazolium-based assay for cell respiration, propidium iodide exclusion, and Hoechst 33258 staining of chromatin. Eleven of the 29 point mutants increased, whereas four decreased antileukemic activity. The remainder had no effect on activity. The nonconcordances between these effects and native DNA binding domain function strongly suggest that the lethality of DBD* is distinct from that of the glucocorticoid receptor. Transfections of fragments of DBD* showed that optimal activity localized to the sequence for its C-terminal 32 amino acids.

  13. Effects of genetic mutations and chemical exposures on Caenorhabditis elegans feeding: evaluation of a novel, high-throughput screening assay.

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    Windy A Boyd

    Full Text Available BACKGROUND: Government agencies have defined a need to reduce, refine or replace current mammalian-based bioassays with testing methods that use alternative species. Invertebrate species, such as Caenorhabditis elegans, provide an attractive option because of their short life cycles, inexpensive maintenance, and high degree of evolutionary conservation with higher eukaryotes. The C. elegans pharynx is a favorable model for studying neuromuscular function, and the effects of chemicals on neuromuscular activity, i.e., feeding. Current feeding methodologies, however, are labor intensive and only semi-quantitative. METHODOLOGY/PRINCIPAL FINDINGS: Here a high-throughput assay is described that uses flow cytometry to measure C. elegans feeding by determining the size and intestinal fluorescence of hundreds of nematodes after exposure to fluorescent-labeled microspheres. This assay was validated by quantifying fluorescence in feeding-defective C. elegans (eat mutants, and by exposing wild-type nematodes to the neuroactive compounds, serotonin and arecoline. The eat mutations previously determined to cause slow pumping rates exhibited the lowest feeding levels with our assay. Concentration-dependent increases in feeding levels after serotonin exposures were dependent on food availability, while feeding levels decreased in arecoline-exposed nematodes regardless of the presence of food. The effects of the environmental contaminants, cadmium chloride and chlorpyrifos, on wild-type C. elegans feeding were then used to demonstrate an application of the feeding assay. Cadmium exposures above 200 microM led to a sharp drop in feeding levels. Feeding of chlorpyrifos-exposed nematodes decreased in a concentration-dependent fashion with an EC(50 of 2 microM. CONCLUSIONS/SIGNIFICANCE: The C. elegans fluorescence microsphere feeding assay is a rapid, reliable method for the assessment of neurotoxic effects of pharmaceutical drugs, industrial chemicals or

  14. Mutational analysis of NOTCH1, 2, 3 and 4 genes in common solid cancers and acute leukemias. (United States)

    Lee, Sung Hak; Jeong, Eun Goo; Yoo, Nam Jin; Lee, Sug Hyung


    NOTCH proteins (NOTCH1, NOTCH2, NOTCH3 and NOTCH4) play crucial roles in embryonic development. Also, mounting evidence indicates that NOTCH contributes to the pathogenesis of hematopoietic and solid malignancies. Recent studies reported a high incidence of gain-of-function mutations of the NOTCH1 gene in T-cell acute lymphoblastic leukemias (ALL). To see whether NOTCH1 mutation occurs in other malignancies, we analyzed NOTCH1 for the detection of somatic mutations in 334 malignancies, including 48 lung, 48 breast, 48 colorectal and 48 gastric carcinomas, and 142 acute leukemias (105 acute myelogenous leukemias, 32 B-ALLs and 4 T-ALLs) by single-strand conformation polymorphism assay. Also, to see whether other NOTCH genes harbor somatic mutations, we analyzed NOTCH2, NOTCH3 and NOTCH4 genes in the same tissue samples. Overall, we detected three NOTCH mutations in the cancers, which consisted of one NOTCH1 mutation in the T-ALLs (25.0%), one NOTCH2 mutation in the breast carcinomas (2.1%), and one NOTCH3 mutation in the colorectal carcinomas (2.0%). There was no NOTCH mutation in other malignancies analyzed. Our data indicate that NOTCH1 is mutated in T-ALL, but not in other common human cancers, and that NOTCH2, NOTCH3 and NOTH4 genes are rarely mutated in common human cancers. Despite the importance of NOTCH activation in many types of human cancers, mutation of NOTCH genes, except for NOTCH1 mutation in T-ALL, may not play an important role in the tumorigenesis of common cancers.

  15. Splice site mutations in the ATP7A gene.

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    Tina Skjørringe

    Full Text Available Menkes disease (MD is caused by mutations in the ATP7A gene. We describe 33 novel splice site mutations detected in patients with MD or the milder phenotypic form, Occipital Horn Syndrome. We review these 33 mutations together with 28 previously published splice site mutations. We investigate 12 mutations for their effect on the mRNA transcript in vivo. Transcriptional data from another 16 mutations were collected from the literature. The theoretical consequences of splice site mutations, predicted with the bioinformatics tool Human Splice Finder, were investigated and evaluated in relation to in vivo results. Ninety-six percent of the mutations identified in 45 patients with classical MD were predicted to have a significant effect on splicing, which concurs with the absence of any detectable wild-type transcript in all 19 patients investigated in vivo. Sixty-seven percent of the mutations identified in 12 patients with milder phenotypes were predicted to have no significant effect on splicing, which concurs with the presence of wild-type transcript in 7 out of 9 patients investigated in vivo. Both the in silico predictions and the in vivo results support the hypothesis previously suggested by us and others, that the presence of some wild-type transcript is correlated to a milder phenotype.

  16. Early detection of multi-drug resistance and common mutations in Mycobacterium tuberculosis isolates from Delhi using GenoType MTBDRplus assay

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    R Singhal


    Full Text Available Purpose: There is scarcity of prevalence data of multi-drug-resistant tuberculosis (MDR-TB data and common mutations responsible in North India. This study aimed to detect MDR-TB among MDR-TB suspects from Delhi and mutation patterns using GenoType MTBDRplus assay. Materials and Methods: All MDR suspects in five districts of New Delhi were referred to the laboratory from 1 st October 2011 to 31 st December 2012 as per criterion defined by Programmatic Management of Drug Resistant Tuberculosis (PMDT. GenoType MTBDRplus assay was performed on 2182 samples or cultures and mutations in the rpoB gene for rifampicin (RIF and katG and inhA genes for isoniazid (INH were analyzed. Results: A total of 366 (16.8% MDR-TB cases were diagnosed. MDR rate was found to be 32%, 16.6% and 10.2% during criterion A, B and C respectively. The most common mutation detected for RIF was S531L (59.0% and for INH was S315T1 (88.3%. Mutations S531L and S315T1 occurred significantly higher in MDR strains as compared to RIF mono-resistant and INH mono-resistant strains, respectively. Average laboratory turn-around time (TAT for dispatch of result to districts for test conducted on samples was 4.4 days. Conclusion: GenoType MTBDRplus is a useful assay for rapid detection of MDR-TB. The common mutations for RIF and INH were similar to those seen in other regions. However, mutations determining MDR strains and mono-resistant strains differed significantly for both RIF and INH.

  17. Gene mutation-based and specific therapies in precision medicine. (United States)

    Wang, Xiangdong


    Precision medicine has been initiated and gains more and more attention from preclinical and clinical scientists. A number of key elements or critical parts in precision medicine have been described and emphasized to establish a systems understanding of precision medicine. The principle of precision medicine is to treat patients on the basis of genetic alterations after gene mutations are identified, although questions and challenges still remain before clinical application. Therapeutic strategies of precision medicine should be considered according to gene mutation, after biological and functional mechanisms of mutated gene expression or epigenetics, or the correspondent protein, are clearly validated. It is time to explore and develop a strategy to target and correct mutated genes by direct elimination, restoration, correction or repair of mutated sequences/genes. Nevertheless, there are still numerous challenges to integrating widespread genomic testing into individual cancer therapies and into decision making for one or another treatment. There are wide-ranging and complex issues to be solved before precision medicine becomes clinical reality. Thus, the precision medicine can be considered as an extension and part of clinical and translational medicine, a new alternative of clinical therapies and strategies, and have an important impact on disease cures and patient prognoses.

  18. Molecular evaluation of a novel missense mutation & an insertional truncating mutation in SUMF1 gene

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    Udhaya H Kotecha


    Full Text Available Background & objectives: Multiple suphphatase deficiency (MSD is an autosomal recessive disorder affecting the post translational activation of all enzymes of the sulphatase family. To date, approximately 30 different mutations have been identified in the causative gene, sulfatase modifying factor 1 (SUMF1. We describe here the mutation analysis of a case of MSD. Methods: The proband was a four year old boy with developmental delay followed by neuroregression. He had coarse facies, appendicular hypertonia, truncal ataxia and ichthyosis limited to both lower limbs. Radiographs showed dysostosis multiplex. Clinical suspicion of MSD was confirmed by enzyme analysis of four enzymes of the sulphatase group. Results: The patient was compound heterozygote for a c.451A>G (p.K151E substitution in exon 3 and a single base insertion mutation (c.690_691 InsT in exon 5 in the SUMF1 gene. The bioinformatic analysis of the missense mutation revealed no apparent effect on the overall structure. However, the mutated 151-amino acid residue was found to be adjacent to the substrate binding and the active site residues, thereby affecting the substrate binding and/or catalytic activity, resulting in almost complete loss of enzyme function. Conclusions: The two mutations identified in the present case were novel. This is perhaps the first report of an insertion mutation in SUMF1 causing premature truncation of the protein.

  19. Dihydropteroate synthase gene mutations in Pneumocystis and sulfa resistance

    DEFF Research Database (Denmark)

    Huang, Laurence; Crothers, Kristina; Atzori, Chiara


    Pneumocystis pneumonia (PCP) remains a major cause of illness and death in HIV-infected persons. Sulfa drugs, trimethoprim-sulfamethoxazole (TMP-SMX) and dapsone are mainstays of PCP treatment and prophylaxis. While prophylaxis has reduced the incidence of PCP, its use has raised concerns about...... in the dihydropteroate synthase (DHPS) gene. Similar mutations have been observed in P. jirovecii. Studies have consistently demonstrated a significant association between the use of sulfa drugs for PCP prophylaxis and DHPS gene mutations. Whether these mutations confer resistance to TMP-SMX or dapsone plus trimethoprim...... for PCP treatment remains unclear. We review studies of DHPS mutations in P. jirovecii and summarize the evidence for resistance to sulfamethoxazole and dapsone....

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

    DEFF Research Database (Denmark)

    Heinen, Christopher D; Juel Rasmussen, Lene


    provided an important experimental tool for studying the functional consequences of VUS. However, beyond this repair assay, a number of other experimental methods have been developed that allow us to test the effect of a VUS on discrete biochemical steps or other aspects of MMR function. Here, we describe......ABSTRACT: With the discovery that the hereditary cancer susceptibility disease Lynch syndrome (LS) is caused by deleterious germline mutations in the DNA mismatch repair (MMR) genes nearly 20 years ago, genetic testing can now be used to diagnose this disorder in patients. A definitive diagnosis...

  1. Profiling critical cancer gene mutations in clinical tumor samples.

    Directory of Open Access Journals (Sweden)

    Laura E MacConaill

    Full Text Available BACKGROUND: Detection of critical cancer gene mutations in clinical tumor specimens may predict patient outcomes and inform treatment options; however, high-throughput mutation profiling remains underdeveloped as a diagnostic approach. We report the implementation of a genotyping and validation algorithm that enables robust tumor mutation profiling in the clinical setting. METHODOLOGY: We developed and implemented an optimized mutation profiling platform ("OncoMap" to interrogate approximately 400 mutations in 33 known oncogenes and tumor suppressors, many of which are known to predict response or resistance to targeted therapies. The performance of OncoMap was analyzed using DNA derived from both frozen and FFPE clinical material in a diverse set of cancer types. A subsequent in-depth analysis was conducted on histologically and clinically annotated pediatric gliomas. The sensitivity and specificity of OncoMap were 93.8% and 100% in fresh frozen tissue; and 89.3% and 99.4% in FFPE-derived DNA. We detected known mutations at the expected frequencies in common cancers, as well as novel mutations in adult and pediatric cancers that are likely to predict heightened response or resistance to existing or developmental cancer therapies. OncoMap profiles also support a new molecular stratification of pediatric low-grade gliomas based on BRAF mutations that may have immediate clinical impact. CONCLUSIONS: Our results demonstrate the clinical feasibility of high-throughput mutation profiling to query a large panel of "actionable" cancer gene mutations. In the future, this type of approach may be incorporated into both cancer epidemiologic studies and clinical decision making to specify the use of many targeted anticancer agents.

  2. A new mutation site in the AIRE gene causes autoimmune polyendocrine syndrome type 1. (United States)

    Zhu, Wufei; Hu, Zhen; Liao, Xiangyu; Chen, Xing; Huang, Wenrong; Zhong, Yu; Zeng, Zhaoyang


    Autoimmune polyendocrine syndrome type 1 (APS-1, OMIM 2403000) is a rare autosomal recessive disease that is caused by autoimmune regulator (AIRE). The main symptoms of APS-1 are chronic mucocutaneous candidiasis, autoimmune adrenocortical insufficiency (Addison's disease) and hypoparathyroidism. We collected APS-1 cases and analysed them. The AIRE genes of the patient and his family members were sequenced to identify whether the APS-1 patient had an AIRE mutation. We discovered a mutation site (c.206A>C) that had never before been reported in the AIRE gene located in exon 2 of the AIRE gene. This homogyzous mutation caused a substitution of the 69th amino acid of the AIRE protein from glutamine to proline (p.Q69P). A yeast two-hybrid assay, which was used to analyse the homodimerization properties of the mutant AIRE protein, showed that the mutant AIRE protein could not interact with the normal AIRE protein. Flow cytometry and RT-qPCR analyses indicated that the new mutation site could decrease the expression levels of the AIRE, glutamic acid decarboxylase 65 (GAD65) and tryptophan hydroxylase-1 (TPH1) proteins to affect central immune tolerance. In conclusion, our research has shown that the new mutation site (c.206A>C) may influence the homodimerization and expression levels and other aspects of the AIRE protein. It may also impact the expression levels of tissue-restricted antigens (TRAs), leading to a series of autoimmune diseases.

  3. [Mutations in the gene encoding filaggrin cause ichthyosis vulgaris]. (United States)

    Prasad, Sumangali Chandra; Rasmussen, Kirsten; Bygum, Anette


    Ichthyosis vulgaris is a common genetic skin disorder with an estimated prevalence of 1:250 caused by mutations in the gene encoding filaggrin. This disorder manifests itself within the first year of life and is clinically characterized by dry, scaly skin, keratosis pilaris, palmar hyperlinearity and atopic manifestations. Patients with a severe phenotype are homozygous or compound heterozygous for the mutations, whereas heterozygous patients show mild disease, suggesting semidominant inheritance with incomplete penetrance. We present a patient with classic severe ichthyosis vulgaris, atopic eczema and two loss-of-function mutations.

  4. BRCA1 Gene Mutations in Chinese Families with Breast Cancer

    Institute of Scientific and Technical Information of China (English)

    Yurong Shi; Chenbin Li; Ruifang Niu; Xishan Hao; Xiangcheng Zhi; Liansheng Ning


    OBJECTIVE To investigate the frequency of BRCA1 gene mutations in breast cancer families in China.METHODS Genomic DNA was obtained by conventional techniques from the peripheral blood mononuclear cells collected from 94 persons derived from 45 breast cancer families. All participants gave written informed consent. The mutations in the BRCA1 gene were detected by the polymerase chain reaction and single stranded conformation polymorphism(PCR-SSCP). Then , the samples of interest were sent for direct DNA sequencing.RESULTS No mutation sites were found in exon 2 or 20 by DNA sequencing.Eight sites were found in exon 11 such as 2201C>T (Ser694Ser),3232A>G(Glu 1038Gly), 2201C >A/G (Ser694Arg), 2731C >T (Pro871Leu),2086A >T(Asn591lle) and three sites of 1584G>T (Glu424Stop). Three mutation sites were found in exon 16 which included 5106A >G (Met1663Val),5208delT(Stop 1639) and 4956A>G (Ser 1613Gly).CONCLUSION These mutation sites may be related to breast cancer, but more investigation is needed to determine whether the mutation sites are hot spots of mutations in Chinese familial breast cancer patients.

  5. TINF2 Gene Mutation in a Patient with Pulmonary Fibrosis

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    T. W. Hoffman


    Full Text Available Pulmonary fibrosis is a frequent manifestation of telomere syndromes. Telomere gene mutations are found in up to 25% and 3% of patients with familial disease and sporadic disease, respectively. The telomere gene TINF2 encodes an eponymous protein that is part of the shelterin complex, a complex involved in telomere protection and maintenance. A TINF2 gene mutation was recently reported in a family with pulmonary fibrosis. We identified a heterozygous Ser245Tyr mutation in the TINF2 gene of previously healthy female patient that presented with progressive cough due to pulmonary fibrosis as well as panhypogammaglobulinemia at age 52. Retrospective multidisciplinary evaluation classified her as a case of possible idiopathic pulmonary fibrosis. Telomere length-measurement indicated normal telomere length in the peripheral blood compartment. This is the first report of a TINF2 mutation in a patient with sporadic pulmonary fibrosis, which represents another association between TINF2 mutations and this disease. Furthermore, this case underlines the importance of telomere dysfunction and not telomere length alone in telomere syndromes and draws attention to hypogammaglobulinemia as a manifestation of telomere syndromes.

  6. Mutations in many genes affect aggressive behavior in Drosophila melanogaster

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    Zwarts Liesbeth


    Full Text Available Abstract Background Aggressive behavior in animals is important for survival and reproduction. Identifying the underlying genes and environmental contexts that affect aggressive behavior is important for understanding the evolutionary forces that maintain variation for aggressive behavior in natural populations, and to develop therapeutic interventions to modulate extreme levels of aggressive behavior in humans. While the role of neurotransmitters and a few other molecules in mediating and modulating levels of aggression is well established, it is likely that many additional genetic pathways remain undiscovered. Drosophila melanogaster has recently been established as an excellent model organism for studying the genetic basis of aggressive behavior. Here, we present the results of a screen of 170 Drosophila P-element insertional mutations for quantitative differences in aggressive behavior from their co-isogenic control line. Results We identified 59 mutations in 57 genes that affect aggressive behavior, none of which had been previously implicated to affect aggression. Thirty-two of these mutants exhibited increased aggression, while 27 lines were less aggressive than the control. Many of the genes affect the development and function of the nervous system, and are thus plausibly relevant to the execution of complex behaviors. Others affect basic cellular and metabolic processes, or are mutations in computationally predicted genes for which aggressive behavior is the first biological annotation. Most of the mutations had pleiotropic effects on other complex traits. We characterized nine of these mutations in greater detail by assessing transcript levels throughout development, morphological changes in the mushroom bodies, and restoration of control levels of aggression in revertant alleles. All of the P-element insertions affected the tagged genes, and had pleiotropic effects on brain morphology. Conclusion This study reveals that many more

  7. Androgen receptor gene mutations in hormone-refractory prostate cancer. (United States)

    Wallén, M J; Linja, M; Kaartinen, K; Schleutker, J; Visakorpi, T


    Prostate cancer is considered to be one of the most hormone-dependent human malignancies. As a key mediator of hormonal response, the androgen receptor (AR) is believed to have an important role in the progression of prostate cancer. Mutations in the coding region of the AR gene have been found in both untreated and hormone-refractory prostate cancer, but the frequency of such mutations at different stages of the disease is poorly documented and even contradictory results have been published. In the present study, the frequency of AR gene mutations was determined in 30 locally recurrent and two metastatic hormone-refractory prostate tumours using the polymerase chain reaction (PCR), non-radioactive single strand conformation polymorphism (SSCP), and sequencing. The length of the polymorphic CAG repeat, which is inversely correlated with the ability of the AR to activate transcription, was also analysed as well as the GGC repeat. Twelve samples were known to contain an AR gene amplification. Altogether, one point mutation (Gly(674)-->Ala) and one microsatellite mutation (CAG(20)-->CAG(18)) were found, both in cancers containing the AR gene amplification. The mean lengths of the polymorphic CAG and GGC repeats were similar to those observed in the normal population. These results favour the view that mutations in the AR gene are rare in hormone-refractory prostate cancer and do not play an important role, at least, in local relapse. Instead, the amplification and consequent overexpression of the wild-type AR gene seem to be the most common alteration involving the AR in hormone-refractory prostate cancer.

  8. Analysis of gene mutations associated with isoniazid, rifampicin and ethambutol resistance among Mycobacterium tuberculosis isolates from Ethiopia (United States)


    Background The emergence of drug resistance is one of the most important threats to tuberculosis control programs. This study was aimed to analyze the frequency of gene mutations associated with resistance to isoniazid (INH), rifampicin (RMP) and ethambutol (EMB) among Mycobacterium tuberculosis isolates from Northwest Ethiopia, and to assess the performance of the GenoType® MTBDRplus and GenoType® MTBDRsl assays as compared to the BacT/ALERT 3D system. Methods Two hundred sixty Mycobacterium tuberculosis isolates from smear positive tuberculosis patients diagnosed between March 2009 and July 2009 were included in this study. Drug susceptibility tests were performed in the Institute of Medical Microbiology and Epidemiology of Infectious Diseases, University Hospital of Leipzig, Germany. Results Of 260 isolates, mutations conferring resistance to INH, RMP, or EMB were detected in 35, 15, and 8 isolates, respectively, while multidrug resistance (MDR) was present in 13 of the isolates. Of 35 INH resistant strains, 33 had mutations in the katG gene at Ser315Thr 1 and two strains had mutation in the inhA gene at C15T. Among 15 RMP resistant isolates, 11 had rpoB gene mutation at Ser531Leu, one at His526Asp, and three strains had mutations only at the wild type probes. Of 8 EMB resistant strains, two had mutations in the embB gene at Met306Ile, one at Met306Val, and five strains had mutations only at the wild type probes. The GenoType® MTBDRplus assay had a sensitivity of 92% and specificity of 99% for INH resistance, and 100% sensitivity and specificity to detect RMP resistance and MDR. The GenoType® MTBDRsl assay had a sensitivity of 42% and specificity of 100% for EMB resistance. Conclusion The dominance of single gene mutations associated with the resistance to INH and RMP was observed in the codon 315 of the katG gene and codon 531 of the rpoB gene, respectively. The GenoType® MTBDRplus assay is a sensitive and specific tool for diagnosis of resistance to INH

  9. Analysis of gene mutations associated with isoniazid, rifampicin and ethambutol resistance among Mycobacterium tuberculosis isolates from Ethiopia

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    Tessema Belay


    Full Text Available Abstract Background The emergence of drug resistance is one of the most important threats to tuberculosis control programs. This study was aimed to analyze the frequency of gene mutations associated with resistance to isoniazid (INH, rifampicin (RMP and ethambutol (EMB among Mycobacterium tuberculosis isolates from Northwest Ethiopia, and to assess the performance of the GenoType® MTBDRplus and GenoType® MTBDRsl assays as compared to the BacT/ALERT 3D system. Methods Two hundred sixty Mycobacterium tuberculosis isolates from smear positive tuberculosis patients diagnosed between March 2009 and July 2009 were included in this study. Drug susceptibility tests were performed in the Institute of Medical Microbiology and Epidemiology of Infectious Diseases, University Hospital of Leipzig, Germany. Results Of 260 isolates, mutations conferring resistance to INH, RMP, or EMB were detected in 35, 15, and 8 isolates, respectively, while multidrug resistance (MDR was present in 13 of the isolates. Of 35 INH resistant strains, 33 had mutations in the katG gene at Ser315Thr 1 and two strains had mutation in the inhA gene at C15T. Among 15 RMP resistant isolates, 11 had rpoB gene mutation at Ser531Leu, one at His526Asp, and three strains had mutations only at the wild type probes. Of 8 EMB resistant strains, two had mutations in the embB gene at Met306Ile, one at Met306Val, and five strains had mutations only at the wild type probes. The GenoType® MTBDRplus assay had a sensitivity of 92% and specificity of 99% for INH resistance, and 100% sensitivity and specificity to detect RMP resistance and MDR. The GenoType® MTBDRsl assay had a sensitivity of 42% and specificity of 100% for EMB resistance. Conclusion The dominance of single gene mutations associated with the resistance to INH and RMP was observed in the codon 315 of the katG gene and codon 531 of the rpoB gene, respectively. The GenoType® MTBDRplus assay is a sensitive and specific tool for

  10. Detection of Rare Beta Globin Gene Mutations in Northwestern Iran

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    M Haghi


    Full Text Available Introduction: Recent molecular studies on Iranian β-thalassemia genes revealed the presence of eight common mutations associated with thalassemia. Although these mutations are frequent, there are other rare and unknown mutations that can create large problems in designing preventive programs. We detected and explained the common mutations in north-western Iran previously and detection of the rare and unknown mutations could be useful in diagnosis and design of future preventive programs. Methods: In this study, 5ml peripheral blood from 20 Azari- β-thalassemia patients whose mutation was not revealed in the previous study was collected and DNA extraction was done by isopropanol and proteinase k method. Initially, samples were examined for the rare mutations: Codon6, Codon16, Codon41/42, Codon36/37, -88 and Codon22 by ARMS – PCR techniques and then the unknown cases were directly sequenced. Results: According to our results, Codon15(TGG-TGA, Codon16(-C, Codon36/37(-T, IVSII-848(C-A, IVSII-745(C-G, -28(A-C( and Codon25/26(+T were recognized and added to the spectrom of beta globin gene mutations in Azerbaijan and Iran. Also, we detected four SNP sites: 5’UTR+20(C-T, Codon2 (CAC-CAT , IVSII-16(C-G and IVSII-666(T-C in β-thalassemia genes. Conclusion: Our results could be useful for developing molecular screening plans and help prenatal diagnosis of beta thalassemia in Azerbaijan , Iran and other neighboring countries.

  11. Gene-specific function prediction for non-synonymous mutations in monogenic diabetes genes.

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    Quan Li

    Full Text Available The rapid progress of genomic technologies has been providing new opportunities to address the need of maturity-onset diabetes of the young (MODY molecular diagnosis. However, whether a new mutation causes MODY can be questionable. A number of in silico methods have been developed to predict functional effects of rare human mutations. The purpose of this study is to compare the performance of different bioinformatics methods in the functional prediction of nonsynonymous mutations in each MODY gene, and provides reference matrices to assist the molecular diagnosis of MODY. Our study showed that the prediction scores by different methods of the diabetes mutations were highly correlated, but were more complimentary than replacement to each other. The available in silico methods for the prediction of diabetes mutations had varied performances across different genes. Applying gene-specific thresholds defined by this study may be able to increase the performance of in silico prediction of disease-causing mutations.

  12. Mutations in the perform gene in children with hemophagocytic lymphohistiocytosis

    Institute of Scientific and Technical Information of China (English)

    LU Gen; XIE Zheng-de; SHEN Kun-ling; YE Ling-jun; WU Run-hui; LIU Chun-yan; JIN Ying-kang; YANG Shuang


    Background Recent studies have reported germline mutations in the perforin gene (PRF1) in some types of hemophagocytic lymphohistiocytosis (HLH). However, the prevalence of PRF1 mutations in HLH in Chinese pediatric patients has not been extensively studied. The aim of this study was to investigate the prevalence of mutations and sequence variations in the PRF1 gene in Chinese pediatric patients with HLH.Methods Polymerase chain reaction (PCR) was performed with five pairs of primers for the coding exons and the flanking intron sequences of PRF1. Sequencing of PCR products was subsequently applied in 30 pediatric patients with HLH and in 50 controls.Results Three heterozygous mutations in a coding region were found, which resulted in amino acid changes (C102F, S108N and T450M) in three patients. These mutations were not detected in control subjects. One patient had compound heterozygous mutations (S108N and T450M) in PRF1 as the background defect, and documented familial HLH type 2 (FHL2). One synonymous sequence variant (Q540Q) was observed in one patient but not in the controls. Two SNPs (A274A, H300H) in the coding region were detected in HLH patients and controls, but without differences in the heterozygosity rate between the two groups (P>0.05 for all comparisons).Conclusions We have identified three patients with three heterozygous missense mutations in PRF1; two of those three mutations (C102F and S108N) have so far been found only from Chinese patients. These findings are useful in evaluating the prevalence of PRF1 mutations in Chinese pediatric patients with HLH, and to correlate their genotype with phenotype. Some patients without familial history probably have primary HLH, which should be suspected even beyond the usual age range.

  13. Single-Tube Mutation Scanning of The Epidermal Growth Factor Receptor Gene Using Multiplex LATE-PCR and Lights-On/Lights-Off Probes (United States)

    Tetrault, Shana M.; Rice, John E.; Wangh, Lawrence J.; Sanchez, J. Aquiles


    Background Numerous mutations in exons 18-21 of the epidermal growth factor receptor (EGFR) gene determine the response of many patients with non-small cell lung carcinoma (NSCLC) to anti-EGFR tyrosine kinase inhibitors (TKIs). This paper describes a single closed-tube assay for simultaneous mutational scanning of EGFR exons 18-21. Methods The assay first co-amplifies all four exons as separate single-stranded DNA products using Linear-After-The-Exponential (LATE)-PCR. The amplicons are then interrogated at endpoint along their length using sets of Lights-On/Lights-Off probes of a different color for each exon. The four resulting fluorescent signatures are unique for each underlying DNA sequence. Every mutation in a target potentially alters its unique fluorescent signature thereby revealing the presence of the mutation. Results The assay readily detects mutations which cause sensitivity or resistance to TKIs and can distinguish these clinically important genetic changes from silent mutations which have no impact on protein function. The assay identifies as little as 5% mutant sequences in mixtures of normal DNA and mutant DNA prepared from cancer cell lines. Proof-of-principle experiments demonstrate mutation identification in formalin-fixed, paraffin-embedded NSCLC biopsies. Conclusion The LATE-PCR EGFR assay described here represents a new type of highly informative, single-tube diagnostic test for mutational scanning of multiple gene coding regions and/or multiple gene targets for personalized cancer therapies. PMID:25411647

  14. E2F1 somatic mutation within miRNA target site impairs gene regulation in colorectal cancer. (United States)

    Lopes-Ramos, Camila M; Barros, Bruna P; Koyama, Fernanda C; Carpinetti, Paola A; Pezuk, Julia; Doimo, Nayara T S; Habr-Gama, Angelita; Perez, Rodrigo O; Parmigiani, Raphael B


    Genetic studies have largely concentrated on the impact of somatic mutations found in coding regions, and have neglected mutations outside of these. However, 3' untranslated regions (3' UTR) mutations can also disrupt or create miRNA target sites, and trigger oncogene activation or tumor suppressor inactivation. We used next-generation sequencing to widely screen for genetic alterations within predicted miRNA target sites of oncogenes associated with colorectal cancer, and evaluated the functional impact of a new somatic mutation. Target sequencing of 47 genes was performed for 29 primary colorectal tumor samples. For 71 independent samples, Sanger methodology was used to screen for E2F1 mutations in miRNA predicted target sites, and the functional impact of these mutations was evaluated by luciferase reporter assays. We identified germline and somatic alterations in E2F1. Of the 100 samples evaluated, 3 had germline alterations at the MIR205-5p target site, while one had a somatic mutation at MIR136-5p target site. E2F1 gene expression was similar between normal and tumor tissues bearing the germline alteration; however, expression was increased 4-fold in tumor tissue that harbored a somatic mutation compared to that in normal tissue. Luciferase reporter assays revealed both germline and somatic alterations increased E2F1 activity relative to wild-type E2F1. We demonstrated that somatic mutation within E2F1:MIR136-5p target site impairs miRNA-mediated regulation and leads to increased gene activity. We conclude that somatic mutations that disrupt miRNA target sites have the potential to impact gene regulation, highlighting an important mechanism of oncogene activation.

  15. Altered Chromosomal Positioning, Compaction, and Gene Expression with a Lamin A/C Gene Mutation (United States)

    Abuisneineh, Fida; Fahrenbach, John P.; Zhang, Yuan; MacLeod, Heather; Dellefave, Lisa; Pytel, Peter; Selig, Sara; Labno, Christine M.; Reddy, Karen; Singh, Harinder; McNally, Elizabeth


    Background Lamins A and C, encoded by the LMNA gene, are filamentous proteins that form the core scaffold of the nuclear lamina. Dominant LMNA gene mutations cause multiple human diseases including cardiac and skeletal myopathies. The nuclear lamina is thought to regulate gene expression by its direct interaction with chromatin. LMNA gene mutations may mediate disease by disrupting normal gene expression. Methods/Findings To investigate the hypothesis that mutant lamin A/C changes the lamina's ability to interact with chromatin, we studied gene misexpression resulting from the cardiomyopathic LMNA E161K mutation and correlated this with changes in chromosome positioning. We identified clusters of misexpressed genes and examined the nuclear positioning of two such genomic clusters, each harboring genes relevant to striated muscle disease including LMO7 and MBNL2. Both gene clusters were found to be more centrally positioned in LMNA-mutant nuclei. Additionally, these loci were less compacted. In LMNA mutant heart and fibroblasts, we found that chromosome 13 had a disproportionately high fraction of misexpressed genes. Using three-dimensional fluorescence in situ hybridization we found that the entire territory of chromosome 13 was displaced towards the center of the nucleus in LMNA mutant fibroblasts. Additional cardiomyopathic LMNA gene mutations were also shown to have abnormal positioning of chromosome 13, although in the opposite direction. Conclusions These data support a model in which LMNA mutations perturb the intranuclear positioning and compaction of chromosomal domains and provide a mechanism by which gene expression may be altered. PMID:21179469

  16. Altered chromosomal positioning, compaction, and gene expression with a lamin A/C gene mutation.

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    Stephanie K Mewborn

    Full Text Available BACKGROUND: Lamins A and C, encoded by the LMNA gene, are filamentous proteins that form the core scaffold of the nuclear lamina. Dominant LMNA gene mutations cause multiple human diseases including cardiac and skeletal myopathies. The nuclear lamina is thought to regulate gene expression by its direct interaction with chromatin. LMNA gene mutations may mediate disease by disrupting normal gene expression. METHODS/FINDINGS: To investigate the hypothesis that mutant lamin A/C changes the lamina's ability to interact with chromatin, we studied gene misexpression resulting from the cardiomyopathic LMNA E161K mutation and correlated this with changes in chromosome positioning. We identified clusters of misexpressed genes and examined the nuclear positioning of two such genomic clusters, each harboring genes relevant to striated muscle disease including LMO7 and MBNL2. Both gene clusters were found to be more centrally positioned in LMNA-mutant nuclei. Additionally, these loci were less compacted. In LMNA mutant heart and fibroblasts, we found that chromosome 13 had a disproportionately high fraction of misexpressed genes. Using three-dimensional fluorescence in situ hybridization we found that the entire territory of chromosome 13 was displaced towards the center of the nucleus in LMNA mutant fibroblasts. Additional cardiomyopathic LMNA gene mutations were also shown to have abnormal positioning of chromosome 13, although in the opposite direction. CONCLUSIONS: These data support a model in which LMNA mutations perturb the intranuclear positioning and compaction of chromosomal domains and provide a mechanism by which gene expression may be altered.

  17. Use of GenoType® MTBDRplus assay to assess drug resistance and mutation patterns of multidrug-resistant tuberculosis isolates in northern India

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    A K Maurya


    Full Text Available Purpose: The emergence and spread of multidrug-resistant tuberculosis (MDR-TB is a major public health problem. The diagnosis of MDR-TB is of paramount importance in establishing appropriate clinical management and infection control measures. The aim of this study was to evaluate drug resistance and mutational patterns in clinical isolates MDR-TB by GenoType® MTBDRplus assay. Material and Methods: A total of 350 non-repeated sputum specimens were collected from highly suspected drug-resistant pulmonary tuberculosis (PTB cases; which were processed by microscopy, culture, differentiation and first line drug susceptibility testing (DST using BacT/ALERT 3D system. Results: Among a total of 125 mycobacterium tuberculosis complex (MTBC strains, readable results were obtained from 120 (96% strains by GenoType® MTBDRplus assay. Only 45 MDR-TB isolates were analysed for the performance, frequency and mutational patterns by GenoType® MTBDRplus assay. The sensitivity of the GenoType® MDRTBplus assay for detecting individual resistance to rifampicin (RIF, isoniazid (INH and multidrug resistance was found to be 95.8%, 96.3% and 97.7%, respectively. Mutation in codon S531L of the rpoB gene and codon S315T1 of katG genes were dominated in MDR-TB strains, respectively (P < 0.05. Conclusions: The GenoType® MTBDRplus assay is highly sensitive with short turnaround times and a rapid test for the detection of the most common mutations conferring resistance in MDR-TB strains that can readily be included in a routine laboratory workflow.

  18. Management of Individuals With a Mutation in the Ataxia Telangiectasia Mutated Gene. (United States)

    Mahon, Suzanne M


    Advances in genetic testing have led to the identification of multiple genes associated with a hereditary risk for developing breast and other cancers. One such gene is the ataxia telangiectasia mutated (ATM) gene, which is available on many genetic panels offered to individuals with suspected hereditary risk. Genetic testing can often lead to improved understanding and clarification of risk for developing cancer, as well as allow affected individuals to make informed choices about management, including the adoption of primary prevention strategies and more aggressive screening than typically recommended in the general population. This article provides an overview of the role of mutations in the ATM gene in developing malignancies, along with emerging research on treatment implications based on genetic testing results.

  19. An inherited LMNA gene mutation in atypical Progeria syndrome. (United States)

    Doubaj, Yassamine; De Sandre-Giovannoli, Annachiara; Vera, Esteves-Vieira; Navarro, Claire Laure; Elalaoui, Siham Chafai; Tajir, Mariam; Lévy, Nicolas; Sefiani, Abdelaziz


    Hutchinson-Gilford Progeria syndrome (HGPS) is a rare genetic disorder, characterized by several clinical features that begin in early childhood, recalling an accelerated aging process. The diagnosis of HGPS is based on the recognition of common clinical features and detection of the recurrent heterozygous c.1824C>T (p.Gly608Gly) mutation within exon 11 in the Lamin A/C encoding gene (LMNA). Besides "typical HGPS," several "atypical progeria" syndromes (APS) have been described, in a clinical spectrum ranging from mandibuloacral dysplasia to atypical Werner syndrome. These patients's clinical features include progeroid manifestations, such as short stature, prominent nose, premature graying of hair, partial alopecia, skin atrophy, lipodystrophy, skeletal anomalies, such as mandibular hypoplasia and acroosteolyses, and in some cases severe atherosclerosis with metabolic complications. APS are due in several cases to de novo heterozygous LMNA mutations other than the p.Gly608Gly, or due to homozygous BAFN1 mutations in Nestor-Guillermo Progeria syndrome (NGPS). We report here and discuss the observation of a non-consanguineous Moroccan patient presenting with atypical progeria. The molecular studies showed the heterozygous mutation c.412G>A (p.Glu138Lys) of the LMNA gene. This mutation, previously reported as a de novo mutation, was inherited from the apparently healthy father who showed a somatic cell mosaicism.

  20. Mutation of RET gene in Chinese patients with Hirschsprung's disease

    Institute of Scientific and Technical Information of China (English)

    Ji-Cheng Li; Shi-Ping Ding; Ying Song; Min-Ju Li


    AIM: To investigate the pathogenic mechanism of Hirschsprung's disease (HD) at the molecular level and to elucidate the relationship between RET oncogene and Chinese patients with HD.METHODS: Exon 13 of RET oncogene from 20 unrelated HD patients was analyzed with polymerase chain reactionsingle strand conformation polymorphism (PCR-SSCP). The positive amplifying products were then sequenced. According to the results of SSCP and DNA sequence, SSCP was done as well for the samples from the family other members of some cases with mutated RET gene.RESULTS: SSCP analysis indicated that mobility abnormality existed in 4 unrelated HD patients. Direct DNA sequence analysis identified a missense mutation, T to G at the nucleotide 18 888 and a frameshift mutation at the nucleotide 18 926 insG. In a HD family, the sicked child and his father were the same heterozygous missense mutation (T to G at nucleotide 18 888).CONCLUSION: Among Chinese HD patients, RET gene mutations may exist in considerable proportion with different patterns. These new discoveries indicate that RET mutations may play an important role in the pathogenesis of unrelated HD in the Chinese population. PCR-SSCP combined with DNA sequence can be used as a tool in the genetic diagnosis of HD.

  1. Mutations of the p16 gene in gliomas. (United States)

    Kyritsis, A P; Zhang, B; Zhang, W; Xiao, M; Takeshima, H; Bondy, M L; Cunningham, J E; Levin, V A; Bruner, J


    In the present study we investigated the frequency of p16 gene exon 2 mutations in 35 malignant gliomas, using either direct sequencing of the PCR products or cloning into the pCRII vector and sequencing of the cloned PCR products. No mutations were detected during direct sequencing of the PCR products. However, after sequencing of individual clones, we found multiple mutations in 5 tumors involving codons 73(GCC to ACC, Ala to Thr), 76 (GCC to GTC, Ala to Val), 85(GCT to ACT, Ala to Thr), 98(CAC to TAC, His to Tyr), 102 (GCG to GTG, Ala to Val), 106 (GTG to ATG, Val to Met), 107 (CGC to TGC, Arg to Cys), 127 (GCA to GTA, Ala to Val), 128 (CGG to TGG, Arg to Trp) and 136 (GGC to GAC, Gly to Asp). Mutations were found only in glioblastomas and were either C to T or G to A transitions. Each mutation was detected in a small percentage of tumor cells (1.3-22%) using individual colony sequencing and southern hybridization with mutant oligonucleotides, consistent with the heterogenous cell population of glioblastomas. The presence of p16 gene mutations only in glioblastomas suggests that they are late events in glioma development.

  2. Ethnic disparity in 21-hydroxylase gene mutations identified in Pakistani congenital adrenal hyperplasia patients

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    Jabbar Abdul


    Full Text Available Abstract Background Congenital adrenal hyperplasia (CAH is a group of autosomal recessive disorders caused by defects in the steroid 21 hydroxylase gene (CYP21A2. We studied the spectrum of mutations in CYP21A2 gene in a multi-ethnic population in Pakistan to explore the genetics of CAH. Methods A cross sectional study was conducted for the identification of mutations CYP21A2 and their phenotypic associations in CAH using ARMS-PCR assay. Results Overall, 29 patients were analyzed for nine different mutations. The group consisted of two major forms of CAH including 17 salt wasters and 12 simple virilizers. There were 14 phenotypic males and 15 females representing all the major ethnic groups of Pakistan. Parental consanguinity was reported in 65% cases and was equally distributed in the major ethnic groups. Among 58 chromosomes analyzed, mutations were identified in 45 (78.6% chromosomes. The most frequent mutation was I2 splice (27% followed by Ile173Asn (26%, Arg 357 Trp (19%, Gln319stop, 16% and Leu308InsT (12%, whereas Val282Leu was not observed in this study. Homozygosity was seen in 44% and heterozygosity in 34% cases. I2 splice mutation was found to be associated with SW in the homozygous. The Ile173Asn mutation was identified in both SW and SV forms. Moreover, Arg357Trp manifested SW in compound heterozygous state. Conclusion Our study showed that CAH exists in our population with ethnic difference in the prevalence of mutations examined.

  3. Functional Analysis of Thyroid Peroxidase Gene Mutations Detected in Patients with Thyroid Dyshormonogenesis

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    Srikanta Guria


    Full Text Available Thyroid peroxidase (TPO is the key enzyme in the biosynthesis of thyroid hormones. We aimed to identify the spectrum of mutations in the TPO gene leading to hypothyroidism in the population of West Bengal to establish the genetic etiology of the disease. 200 hypothyroid patients (case and their corresponding sex and age matched 200 normal individuals (control were screened depending on their clinical manifestations. Genomic DNA was isolated from peripheral blood samples and TPO gene (Exon 7 to Exon 14 was amplified by PCR. The PCR products were subjected to sequencing to identify mutations. Single nucleotide changes such as Glu 641 Lys, Asp 668 Asn, Thr 725 Pro, Asp 620 Asn, Ser 398 Thr, and Ala 373 Ser were found. Changes in the TPO were assayed in vitro to compare mutant and wild-type activities. Five mutants were enzymatically inactive in the guaiacol and iodide assays. This is a strong indication that the mutations are present at crucial positions of the TPO gene, resulting in inactivated TPO. The results of this study may help to develop a genetic screening protocol for goiter and hypothyroidism in the population of West Bengal.

  4. [Gliomas and BRCA genes mutations: fortuitous association or imputability?]. (United States)

    Girardstein-Boccara, Laura; Mari, Véronique; Met-Domestici, Marie; Burel-Vandenbos, Fanny; Berthet, Pascaline; Paquis, Philippe; Frenay, Marc Paul; Lebrun-Frenay, Christine


    BRCA is a tumor suppressor gene implicated in the major mechanisms of cellular stability in every type of cell. Its mutations are described in numerous cancers, mainly breast and ovarian in women. It was also found an increase of lifetime risk of pancreas, colon, prostate cancer or lymphoma in men carriers. We report the cases of two female patients aged 40 and 58-years-old female patients and one 35-years-old male patient, with brain or medullar gliomas, carriers of a germline mutation of BRCA gene. Those gliomas were particularly aggressive and were not responding to the standard treatment, with chemo and radiotherapy. The very unusual characteristics in location and evolutive profile of these central nervous system tumors raise the question of a genetical underlying mechanism, maybe linked to the BRCA gene mutation that carry these patients. In addition, a non-fortuitous association between germline mutation of BRCA and occurrence of a glioma can be evoked according to the embryological, epidemiological and biomolecular findings noted in the literature. Other clinical and experimental studies are necessary to precise the physiopathological link existing between BRCA mutations and the occurrence of a glioma; this could have therapeutical and clinical implications in the future.

  5. Glaucoma and Cytochrome P4501B1 Gene Mutations

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    Mukesh Tanwar


    Full Text Available Developmental anomalies of the ocular anterior chamber angle may lead to an incomplete development of the structures that form the conventional aqueous outflow pathway. Thus, disorders that present with such dysfunction tend to be associated with glaucoma. Among them, Axenfeld-Rieger (ARS malformation is a rare clinical entity with an estimated prevalence of one in every 200,000 individuals. The changes in eye morphogenesis in ARS are highly penetrant and are associated with 50% risk of development of glaucoma. Mutations in the cytochrome P4501B1 (CYP1B1 gene have been reported to be associated with primary congenital glaucoma and other forms of glaucoma and mutations in pituitary homeobox 2 (PITX2 gene have been identified in ARS in various studies. This case was negative for PITX2 mutations and compound heterozygote for CYP1B1 mutations. Clinical manifestations of this patient include bilateral elevated intraocular pressure (>40 mmHg with increased corneal diameter (>14 mm and corneal opacity. Patient also had iridocorneal adhesions, anteriorly displaced Schwalbe line, anterior insertion of iris, broad nasal bridge and protruding umbilicus. This is the first study from north India reporting CYP1B1 mutations in Axenfeld-Rieger syndrome with bilateral buphthalmos and early onset glaucoma. Result of this study supports the role of CYP1B1 as a causative gene in ASD disorders and its role in oculogenesis.

  6. Mutator gene and hereditary non-polyposis colorectal cancer (United States)

    de la Chapelle, Albert; Vogelstein, Bert; Kinzler, Kenneth W.


    The human MSH2 gene, responsible for hereditary non-polyposis colorectal cancer, was identified by virtue of its homology to the MutS class of genes, which are involved in DNA mismatch repair. The sequence of cDNA clones of the human gene are provided, and the sequence of the gene can be used to demonstrate the existence of germ line mutations in hereditary non-polyposis colorectal cancer (HNPCC) kindreds, as well as in replication error.sup.+ (RER.sup.+) tumor cells.

  7. Common filaggrin gene mutations and risk of cervical cancer

    DEFF Research Database (Denmark)

    Bager, Peter; Wohlfahrt, Jan; Sørensen, Erik


    BACKGROUND: As carriers of filaggrin gene (FLG) mutations may have a compromised cervical mucosal barrier against human papillomavirus infection, our primary objective was to study their risk of cervical cancer. METHODS: We genotyped 586 cervical cancer patients for the two most common FLG...... mutations, R501X and 2282del4, using blood from the Copenhagen Hospital Biobank, Denmark. Controls (n = 8050) were genotyped in previous population-based studies. Information on cervical cancer, mortality and emigration were obtained from national registers. Odds ratios (OR) were estimated by logistic...... and stratification by cancer stage. RESULTS: The primary results showed that FLG mutations were not associated with the risk of cervical cancer (6.3% of cases and 7.7% of controls were carriers; OR adjusted 0.81, 95% CI 0.57-1.14; OR adjusted+ weighted 0.96, 95% CI 0.58-1.57). Among cases, FLG mutations increased...

  8. Comprehensive TP53-denaturing gradient gel electrophoresis mutation detection assay also applicable to archival paraffin-embedded tissue

    NARCIS (Netherlands)

    Hayes, V M; Bleeker, W; Verlind, E; Timmer, T; Karrenbeld, A; Plukker, J T; Marx, M P; Hofstra, R M; Buys, C H


    A comprehensive mutation detection assay is described for the entire coding region and all splice site junctions of TP53. The assay is based on denaturing gradient gel electrophoresis, which follows either multiplex polymerase chain reaction (PCR) applied to DNA extracted from fresh or frozen tissue

  9. Pathogenic gene linkage analysis and DMP1 gene mutation assay in dentinogenesis imperfecta type Ⅱ%遗传性牙本质发育不全Ⅱ型家系致病基因的连锁分析及DMP1的突变检测

    Institute of Scientific and Technical Information of China (English)

    王英; 史俊南; 仇学明; 肖尚喜; 孔祥银


    目的:对中国江苏淮阴一个遗传性牙本质发育不全Ⅱ型家系的致病基因进行定位,同时对该疾病的候选基因之一DMP1进行突变检测。方法:用位于4q21区域的7个微卫星位点对家系进行遗传连锁分析,并通过聚合酶链反应-单链构象多态分析(PCR-SSCP)和DNA测序方法对DMP1基因进行突变检测。结果:所选的7个位点中,除D4S451和D4S1534之外,最大Lod值Zmax均大于3(θ=0);PCR-SSCP和测序结果显示DMP1 Exon 2~6均无突变。结论:遗传性牙本质发育不全Ⅱ型与位于4q21区域的微卫星位点GATA62A11、DSP、DMP1、SPP1和D4S1563连锁;排除了DMP1做为该疾病致病基因的可能性。%AIM:To study the location of pathogenic gene in dentinogenesis imperfecta type Ⅱ,and to screen the mutation of DMP1 gene. METHODS: The families were analyzed with linkage studies using 7 highly polymorphic microsatellite DNA markers in the region of 4q21. PCR-SSCP and DNA sequencing were employed to detect the mutation of DMP1. RESULT: The maximum Lod scroes of the 7 markers were: D4S451,Zmax=2.76(θ=0.1); D4S1534,Zmax=1.65(θ=0); GATA62A11,Zmax=7.63(θ=0); DSP,Zmax=6.06(θ=0); DMP1,Zmax=8.24(θ=0); SPP1,Zmax=8.39(θ=0); D4S1563,Zmax=7.34(θ=0) respectively. No mutation was detected in DMP1. CONCLUSION: The pathogenic gene of dentinogenesis imperfecta type Ⅱ is linked to GATA62A11,DSP,DMP1,SPP1 and D4S1563 in 4q21; excluding the DMP1 gene from a causative role in the pathogenesis of DGI-Ⅱ.

  10. Mechanistic study on the nuclear modifier gene MSS1 mutation suppressing neomycin sensitivity of the mitochondrial 15S rRNA C1477G mutation in Saccharomyces cerevisiae. (United States)

    Zhou, Qiyin; Wang, Wei; He, Xiangyu; Zhu, Xiaoyu; Shen, Yaoyao; Yu, Zhe; Wang, Xuexiang; Qi, Xuchen; Zhang, Xuan; Fan, Mingjie; Dai, Yu; Yang, Shuxu; Yan, Qingfeng


    The phenotypic manifestation of mitochondrial DNA (mtDNA) mutations can be modulated by nuclear genes and environmental factors. However, neither the interaction among these factors nor their underlying mechanisms are well understood. The yeast Saccharomyces cerevisiae mtDNA 15S rRNA C1477G mutation (PR) corresponds to the human 12S rRNA A1555G mutation. Here we report that a nuclear modifier gene mss1 mutation suppresses the neomycin-sensitivity phenotype of a yeast C1477G mutant in fermentable YPD medium. Functional assays show that the mitochondrial function of the yeast C1477G mutant was impaired severely in YPD medium with neomycin. Moreover, the mss1 mutation led to a significant increase in the steady-state level of HAP5 (heme activated protein), which greatly up-regulated the expression of glycolytic transcription factors RAP1, GCR1, and GCR2 and thus stimulated glycolysis. Furthermore, the high expression of the key glycolytic enzyme genes HXK2, PFK1 and PYK1 indicated that enhanced glycolysis not only compensated for the ATP reduction from oxidative phosphorylation (OXPHOS) in mitochondria, but also ensured the growth of the mss1(PR) mutant in YPD medium with neomycin. This study advances our understanding of the phenotypic manifestation of mtDNA mutations.

  11. Diverse growth hormone receptor gene mutations in Laron syndrome.


    Berg, M.A.; Argente, J.; Chernausek, S; Gracia, R.; Guevara-Aguirre, J; Hopp, M; Pérez-Jurado, L; Rosenbloom, A; Toledo,S.P.; Francke, U.


    To better understand the molecular genetic basis and genetic epidemiology of Laron syndrome (growth-hormone insensitivity syndrome), we analyzed the growth-hormone receptor (GHR) genes of seven unrelated affected individuals from the United States, South America, Europe, and Africa. We amplified all nine GHR gene exons and splice junctions from these individuals by PCR and screened the products for mutations by using denaturing gradient gel electrophoresis (DGGE). We identified a single GHR g...

  12. Mu Opioid Receptor Gene: New Point Mutations in Opioid Addicts


    Dinarvand, Amin; Goodarzi, Ali; Vousooghi, Nasim; Hashemi, Mehrdad; Dinarvand, Rasoul; Ostadzadeh, Fahimeh; Khoshzaban, Ahad; Zarrindast, Mohammad-Reza


    Introduction Association between single-nucleotide polymorphisms (SNPs) in mu opioid receptor gene and drug addiction has been shown in various studies. Here, we have evaluated the existence of polymorphisms in exon 3 of this gene in Iranian population and investigated the possible association between these mutations and opioid addiction. Methods 79 opioid-dependent subjects (55 males, 24 females) and 134 non-addict or control individuals (74 males, 60 females) participated in the study. Geno...


    Institute of Scientific and Technical Information of China (English)

    ZHANG He-long; WANG Wen-liang; CUI Da-xiang


    @@ Lung cancer is a common malignant tumor, which has ahigh incidence and mortality rate. Therefore, it is necessary to seek a new method for the diagnosis, especially the early diagnosis of lung cancer. The development of molecular biology makes the gene diagnosis of lung cancer possible.PCR-SSCP was applied to detect p53 gene mutation of lung cancer patients' sputum cells and we have achieved good results.

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

    Metcalf, Alexander M; Spurdle, Amanda B


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

  15. Mutations in the pericentrin (PCNT) gene cause primordial dwarfism.

    NARCIS (Netherlands)

    Rauch, A.; Thiel, C.T.; Schindler, D.; Wick, U.; Crow, Y.J.; Ekici, A.B.; Essen, A.J. van; Goecke, T.O.; Al-Gazali, L.; Chrzanowska, K.H.; Zweier, C.; Brunner, H.G.; Becker, K.; Curry, C.J.; Dallapiccola, B.; Devriendt, K.; Dorfler, A.; Kinning, E.; Megarbane, A.; Meinecke, P.; Semple, R.K.; Spranger, S.; Toutain, A.; Trembath, R.C.; Voss, E.; Wilson, L.; Hennekam, R.C.M.; Zegher, F. de; Dorr, H.G.; Reis, A.


    Fundamental processes influencing human growth can be revealed by studying extreme short stature. Using genetic linkage analysis, we find that biallelic loss-of-function mutations in the centrosomal pericentrin (PCNT) gene on chromosome 21q22.3 cause microcephalic osteodysplastic primordial dwarfism

  16. Mutations in the pericentrin (PCNT) gene cause primordial dwarfism

    NARCIS (Netherlands)

    Rauch, Anita; Thiel, Christian T.; Schindler, Detlev; Wick, Ursula; Crow, Yanick J.; Ekici, Arif B.; van Essen, Anthonie J.; Goecke, Timm O.; Al-Gazali, Lihadh; Chrzanowska, Krystyna H.; Zweier, Christiane; Brunner, Han G.; Becker, Kristin; Curry, Cynthia J.; Dallapiccola, Bruno; Devriendt, Koenraad; Doerfler, Arnd; Kinning, Esther; Megarbane, Andre; Meinecke, Peter; Semple, Robert K.; Spranger, Stephanie; Toutain, Annick; Trembath, Richard C.; Voss, Egbert; Wilson, Louise; Hennekam, Raoul; de Zegher, Francis; Doerr, Helmuth-Guenther; Reis, Andre


    Fundamental processes influencing human growth can be revealed by studying extreme short stature. Using genetic linkage analysis, we find that biallelic loss- of- function mutations in the centrosomal pericentrin ( PCNT) gene on chromosome 21q22.3 cause microcephalic osteodysplastic primordial

  17. Cathepsin K gene mutations and 1q21 haplotypes in at patients with pycnodysostosis in an outbred population

    DEFF Research Database (Denmark)

    Haagerup, Annette; Hertz, Jens Michael; Christensen, M F


    patients were homozygous for 926T > C. In the remaining two families patients were heterozygous for 926T > C and 121-1G > A in one case, and for 926T > C and 236G > A in the other case. Assays using genomic DNA were developed for all three mutations. We tested 150 healthy control persons and observed......The molecular genetics of the autosomal recessive disorder pycnodysostosis was studied in five independent families from an outbred Caucasian population. We found two new mutations and one recently described mutation in the cathepsin K gene by sequencing DNA from eight patients with pycnodysostosis...

  18. Germline Mutations in Predisposition Genes in Pediatric Cancer (United States)

    Edmonson, Michael N.; Gruber, Tanja A.; Easton, John; Hedges, Dale; Ma, Xiaotu; Zhou, Xin; Yergeau, Donald A.; Wilkinson, Mark R.; Vadodaria, Bhavin; Chen, Xiang; McGee, Rose B.; Hines-Dowell, Stacy; Nuccio, Regina; Quinn, Emily; Shurtleff, Sheila A.; Rusch, Michael; Patel, Aman; Becksfort, Jared B.; Wang, Shuoguo; Weaver, Meaghann S.; Ding, Li; Mardis, Elaine R.; Wilson, Richard K.; Gajjar, Amar; Ellison, David W.; Pappo, Alberto S.; Pui, Ching-Hon; Downing, James R.


    BACKGROUND The prevalence and spectrum of predisposing mutations among children and adolescents with cancer are largely unknown. Knowledge of such mutations may improve the understanding of tumorigenesis, direct patient care, and enable genetic counseling of patients and families. METHODS In 1120 patients younger than 20 years of age, we sequenced the whole genomes (in 595 patients), whole exomes (in 456), or both (in 69). We analyzed the DNA sequences of 565 genes, including 60 that have been associated with autosomal dominant cancer-predisposition syndromes, for the presence of germline mutations. The pathogenicity of the mutations was determined by a panel of medical experts with the use of cancer-specific and locus-specific genetic databases, the medical literature, computational predictions, and second hits identified in the tumor genome. The same approach was used to analyze data from 966 persons who did not have known cancer in the 1000 Genomes Project, and a similar approach was used to analyze data from an autism study (from 515 persons with autism and 208 persons without autism). RESULTS Mutations that were deemed to be pathogenic or probably pathogenic were identified in 95 patients with cancer (8.5%), as compared with 1.1% of the persons in the 1000 Genomes Project and 0.6% of the participants in the autism study. The most commonly mutated genes in the affected patients were TP53 (in 50 patients), APC (in 6), BRCA2 (in 6), NF1 (in 4), PMS2 (in 4), RB1 (in 3), and RUNX1 (in 3). A total of 18 additional patients had protein-truncating mutations in tumor-suppressor genes. Of the 58 patients with a predisposing mutation and available information on family history, 23 (40%) had a family history of cancer. CONCLUSIONS Germline mutations in cancer-predisposing genes were identified in 8.5% of the children and adolescents with cancer. Family history did not predict the presence of an underlying predisposition syndrome in most patients. (Funded by the American

  19. Mutations of the tyrosinase gene produce autosomal recessive ocular albinism

    Energy Technology Data Exchange (ETDEWEB)

    King, R.A.; Summers, C.G.; Oetting, W.S. [Univ. of Minnesota, Minneapolis, MN (United States)] [and others


    Albinism has historically been divided into ocular (OA) and oculocutaneous (OCA) types based on the presence or absence of clinically apparent skin and hair involvement in an individual with the ocular features of albinism. The major genes for OCA include the tyrosinase gene in OCA1 and the P gene in OCA2. X-linked and autosomal recessive OA have been described and the responsible genes have not been identified. We now present six Caucasian individuals who have the phenotype of autosomal recessive OA but who have OCA1 as shown by the presence of mutations of the tyrosinase. They had white or very light hair and white skin at birth, and cutaneous pigment developed in the first decade of life. At ages ranging from 1.5-23 years, hair color was dark blond to light brown. The skin had generalized pigment and well developed tan was present on the exposed arm and face skin of four. Iris pigment was present and iris translucency varied. Molecular analysis of the tyrosinase gene, using PCR amplification and direct di-deoxy sequencing showed the following mutations: E398Z/E398Q, P406S/g346a, R402E/T373K, ?/D383N, and H211N/T373K. The homozygous individual was not from a known consanguineous mating. T373K is the most common tyrosinase gene mutation in our laboratory. Three of these mutations are associated with a total loss of tyrosinase activity (g346a splice-site, T373K, and D383N), while four are associated with residual enzyme activity (H211N, R402E, E398Q, and P406S). These studies show that mutations of the tyrosinase gene can produce the phenotype of autosomal recessive OA in an individual who has normal amounts of cutaneous pigment and the ability to tan after birth. This extends the phenotypic range of OCA1 to normal cutaneous pigment after early childhood, and suggest that mutations of the tyrosinase gene account for a significant number of individuals with autosomal recessive OA.

  20. An extended set of yeast-based functional assays accurately identifies human disease mutations (United States)

    Sun, Song; Yang, Fan; Tan, Guihong; Costanzo, Michael; Oughtred, Rose; Hirschman, Jodi; Theesfeld, Chandra L.; Bansal, Pritpal; Sahni, Nidhi; Yi, Song; Yu, Analyn; Tyagi, Tanya; Tie, Cathy; Hill, David E.; Vidal, Marc; Andrews, Brenda J.; Boone, Charles; Dolinski, Kara; Roth, Frederick P.


    We can now routinely identify coding variants within individual human genomes. A pressing challenge is to determine which variants disrupt the function of disease-associated genes. Both experimental and computational methods exist to predict pathogenicity of human genetic variation. However, a systematic performance comparison between them has been lacking. Therefore, we developed and exploited a panel of 26 yeast-based functional complementation assays to measure the impact of 179 variants (101 disease- and 78 non-disease-associated variants) from 22 human disease genes. Using the resulting reference standard, we show that experimental functional assays in a 1-billion-year diverged model organism can identify pathogenic alleles with significantly higher precision and specificity than current computational methods. PMID:26975778

  1. Allele-specific real-time PCR testing for minor HIV-1 drug resistance mutations: assay preparation and application to reveal dynamic of mutations in vivo

    Institute of Scientific and Technical Information of China (English)

    GUO Dong-xing; LI Jing-yun; LI Han-ping; LI Lin; ZHUANG Dao-min; JIAO Li-yan; WANG Zheng; BAO Zuo-yi; LIU Si-yang; LIU Yong-jian


    Background It is very important for the clinical management to test for minor HIV-1 resistance mutations accurately and sensitively. The conventional genotypic assays of HIV drug resistance detection based on sequencing can only discriminate the mutations which present in more than 20%-30%. The aim of this study was to evaluate allele-specific real-time PCR (ASPCR) to detect the resistance-related mutations located at positions 103, 184 and 215.Methods We developed the allele-specific PCR assay, using the most common drug resistance mutations in Chinese AIDS patients, K103N, M184V/I, T215F/Y as a model system. The standards were constructed by cloning the wild-type and mutant DNA fragments into the T-vector. We designed specific primers to discriminate mutant templates in the real-time PCR using SYBR green as a fluorescence reporter. And then we evaluated the ASPCR assay and tested 140clinical samples using this method.Results The sensitivities of ASPCR assay were 0.04% for K103N, 0.30% for M1841, 0.40% for M184V, 0.03% for T215F and 0.02% for T215Y. The intra-assay and inter-assay coefficients of variation were less than 0.42. One hundred and forty plasma samples were tested by ASPCR and dynamic resistance curves of ten patients were obtained.Conclusions Drug resistance emerged half a year after the start of antiretroviral therapy. The mutation of T215Yemerged 1 to 1.5 years after starting treatment and then increased rapidly. The ASPCR assay we developed was a sensitive, accurate and rapid method to detect the minor HIV-1 variants and it can provide earlier and more drug-resistance information for HIV research and AIDS antiretroviral therapy.

  2. Effects of mutated replicase and movement protein genes on attenuation of tobacco mosaic virus

    Institute of Scientific and Technical Information of China (English)

    杨恭; 邱并生; 魏军亚; 刘广超


    Our previous reports showed that one opal mutation (UGA) and one ochre mutation (UAA) respectively located in the replicase and movement protein (MP) genes of the attenuated tomato mosaic virus K(ToMV-K) contribute to the viral attenuation. To explore a wider application of this attenuation pattern to other plant viruses, we have constructed three mutants which respectively contain one opal mutation of the replicase gene and/or one ochre mutation of the MP using PCR-mediated site-directed mutagenesis from a virulent tobacco mosaic virus isolated from China (TMV-Cv). Plant infection performed by in vitro transcripts revealed that the MP truncated mutant TMV-Cvmp and the replicase-MP truncated mutant TMV-Cvrase-mp were infectious on both local lesion (Nicotiana tabacum cv. Xanthi NC) and systemic (N. tabacum cv. K326) host plants, while the replicase truncated mutant TMV-Cvrase was non-infectious. The K326 plant infected by TMV- Cvrease-mp displayed only a little mild mosaic. By electronic microscopy (EM), plant re-inoculation, RNA Dot-blot, RT-PCR and sequencing we demonstrated that the progeny viruses of TMV-Cvmp and TMV-Cvrease-mp shared similar morphological character with TMV-Cv, owned the abilities to infect, replicate and propagate in the assayed plants, and maintained the mutated sites during infection. These data showed that both the opal and the ochre mutations are able to cooperatively induce the attenuated phenotypes of TMV-Cvrase-mp on plants, indicating that the mutation pattern of ToMV-K could be used to attenuate other virulent plant viruses.

  3. Sjögren-Larsson syndrome: novel mutations in the ALDH3A2 gene in a French cohort. (United States)

    Sarret, Catherine; Rigal, Mélanie; Vaurs-Barrière, Catherine; Dorboz, Imen; Eymard-Pierre, Eléonore; Combes, Patricia; Giraud, Geneviève; Wanders, Ronald J A; Afenjar, Alexandra; Francannet, Christine; Boespflug-Tanguy, Odile


    Sjogren-Larsson syndrome (SLS) is a rare autosomal recessive disorder characterized by ichthyosis, spastic di- or tetraplegia and mental retardation due a defect of the fatty aldehyde dehydrogenase (FALDH), related to mutations in the ALDH3A2 gene. In this study, we screened a French cohort of patients with Sjögren-Larsson syndrome (SLS) for mutations in the ALDH3A2 gene. The five unrelated patients with typical SLS all present mutations in this gene. Three novel mutations were identified whereas three other ones were previously described. We also realized functional analyses at the mRNA level for two splice site mutations to study their deleterious consequences. Two of the previously described mutations had already been identified in the same region of Europe, suggesting a putative founder effect. We suggest that, (1) when clinical and MR features are present, direct sequencing of the ALDH3A2 gene in SLS is of particular interest without necessity of a skin biopsy for enzymatic assay in order to propose genetic counsel and (2) identification of mutations already described in the same population with putative founder effects may simplify genetic analysis in this context.

  4. Mutational Analysis of the Wilms' Tumor (WTI) Gene. (United States)

    King-Underwood, L; Pritchard-Jones, K


    Mutations of the Wilms' tumor (WT1) gene have been shown to underlie a proportion of cases of Wilms' tumor, an embryonal kidney cancer occurring mainly in childhood. The WTl gene comprtses ten exons spanning approx 50 kb of genomrc DNA. The messenger RNA is approx 3 kb in length and encodes a zinc finger protein. The four zinc fingers, which he at the C-terminal end of the protein, are encoded by separate exons 7-10. The 5' end of the gene is extremely GC-rich, with areas approaching a 70% GC content. This makes this region difficult to amplify in polymerase chain reactions.

  5. Mutations in MODY Genes Are not Common Cause of Early-Onset Type 2 Diabetes in Mexican Families

    Directory of Open Access Journals (Sweden)

    Bravo-Ríos LE


    Full Text Available CONTEXT: Maturity-onset diabetes of the young (MODY is a monogenic form of diabetes mellitus characterized by autosomal dominant inheritance, early age of onset and a primary insulin secretion defect. Certain MODY gene sequence variants may be involved in polygenic forms of type 2 diabetes. OBJECTIVE: We assessed the contribution of MODY genes to the etiology of type 2 early-onset diabetes in 23 Mexican families, including five with apparently autosomal dominant inheritance. PATIENTS: Twenty-three unrelated Mexican families with early-onset type 2 diabetes previously screened for the presence of glucokinase mutations, were studied. DESIGN: We screened MODY genes for sequence variants by PCR-SSCP analysis and automated sequencing. We performed a functional analysis of the HNF-1alpha P379H recombinant protein in vitro in both HeLa and RINm5f beta-cell lines. MAIN OUTCOME MEASURES: MODY gene mutation screening and P379H mutant protein transactivation assay. RESULTS: No mutations were detected in the HNF-4alpha, IPF-1, NEUROD1 or HNF-1beta genes in any of the families studied. A new mutation (P379H of the HNF-1alpha gene was identified in one MODY family. RINm5f and HeLa cell transfection assays revealed decreased transactivation activity of the mutant protein on the human insulin promoter. CONCLUSIONS: All known MODY genes were screened for abnormalities in this cohort of early-onset diabetes families which included 5 MODY pedigrees. We identified a new HNF-1alpha MODY mutation (P379H and demonstrated that it reduces the transactivation potential of the mutant protein on the human insulin promoter. No other mutation was identified in this cohort indicating that abnormalities in MODY genes are generally not a common cause of early-onset diabetes and this includes MODY families in Mexico.

  6. Generation of KCL025 research grade human embryonic stem cell line carrying a mutation in NF1 gene

    Directory of Open Access Journals (Sweden)

    Heema Hewitson


    Full Text Available The KCL025 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation in the NF1 gene encoding neurofibromin (c.3739–3742 ΔTTTG. Mutations in this gene have been linked to neurofibromatosis type 1, juvenile myelomonocytic leukemia and Watson syndrome. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays.

  7. Incidence in diverse pig populations of an IGF2 mutation with potential influence on meat quality and quantity: An assay based on real time PCR (RT-PCR). (United States)

    Carrodeguas, José Alberto; Burgos, Carmen; Moreno, Carlos; Sánchez, Ana Cristina; Ventanas, Sonia; Tarrafeta, Luis; Barcelona, José Antonio; López, Maria Otilia; Oria, Rosa; López-Buesa, Pascual


    IGF2, insulin-like growth factor 2, is implicated in myogenesis and lean meat content. A mutation in a single base (A for G substitution) of the gene for IGF2 (position 3072 in intron 3) has been recently described as the cause of a major QTL effect on muscle growth in pigs [Van Laere, A. S, Nguyen, M., Braunschweig, M., Nezer, C., Collete, C., & Moreau, L. et al. (2003). Nature, 425, 832-836]. We describe here a rapid assay based on real time PCR (RT-PCR) to detect this mutation. We have evaluated the incidence of the mutation in commercial pig crosses, in three populations of purebred Iberian or Iberian×Duroc crosses, and in cured meat products and wild boars. The incidence of the mutation varies among these groups. Penetrance of the A mutation is about 80% in the commercial population. Purebred Iberian pigs were all homozygous G/G whereas crosses of Iberian pigs were heterozygous (90%) or homozygous A/A (10%). The implications of this gene for the selection of Iberian pigs are discussed.

  8. Presymptomatic breast cancer in Egypt: role of BRCA1 and BRCA2 tumor suppressor genes mutations detection

    Directory of Open Access Journals (Sweden)

    Hashishe Mervat M


    Full Text Available Abstract Background Breast cancer is one of the most common diseases affecting women. Inherited susceptibility genes, BRCA1 and BRCA2, are considered in breast, ovarian and other common cancers etiology. BRCA1 and BRCA2 genes have been identified that confer a high degree of breast cancer risk. Objective Our study was performed to identify germline mutations in some exons of BRCA1 and BRCA2 genes for the early detection of presymptomatic breast cancer in females. Methods This study was applied on Egyptian healthy females who first degree relatives to those, with or without a family history, infected with breast cancer. Sixty breast cancer patients, derived from 60 families, were selected for molecular genetic testing of BRCA1 and BRCA2 genes. The study also included 120 healthy first degree female relatives of the patients, either sisters and/or daughters, for early detection of presymptomatic breast cancer mutation carriers. Genomic DNA was extracted from peripheral blood lymphocytes of all the studied subjects. Universal primers were used to amplify four regions of the BRCA1 gene (exons 2,8,13 and 22 and one region (exon 9 of BRCA2 gene using specific PCR. The polymerase chain reaction was carried out. Single strand conformation polymorphism assay and heteroduplex analysis were used to screen for mutations in the studied exons. In addition, DNA sequencing of the normal and mutated exons were performed. Results Mutations in both BRCA1 and BRCA2 genes were detected in 86.7% of the families. Current study indicates that 60% of these families were attributable to BRCA1 mutations, while 26.7% of them were attributable to BRCA2 mutations. Results showed that four mutations were detected in the BRCA1 gene, while one mutation was detected in the BRCA2 gene. Asymptomatic relatives, 80(67% out of total 120, were mutation carriers. Conclusions BRCA1 and BRCA2 genes mutations are responsible for a significant proportion of breast cancer. BRCA mutations

  9. Transposon-induced nuclear mutations that alter chloroplast gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Barkan, A.


    The goal of this project is to use mutant phenotypes as a guide to nuclear genes that determine the timing and localization of chloroplast development The immediate goals are to identify nuclear mutants with defects in chloroplast gene expression from maize lines harboring active Mu transposons; characterize their phenotypes to determine the precise defect in gene expression; clone several of the most interesting mutations by exploiting the transposon tag; and use the clones to further define the roles of these genes in modulating chloroplast gene expression. Three mutants were described earlier that had global defects in chloroplast gene expression. We have found that two of these mutations are allelic. Both alleles have global defects in chloroplast translation initiation, as revealed by the failure to assemble chloroplast mRNAs into polysomes. We have isolated and characterized three new mutants from Mu lines that have novel defects in chloroplast RNA metabolism. We are now ready to begin the task of cloning several of these genes, by using the Mu transposon tag.

  10. Reduced rates of gene loss, gene silencing, and gene mutation in Dnmt1-deficient embryonic stem cells

    NARCIS (Netherlands)

    Chan, M.F.; van Amerongen, R.; Nijjar, T.; Cuppen, E.; Jones, P.A.; Laird, P.W.


    Tumor suppressor gene inactivation is a crucial event in oncogenesis. Gene inactivation mechanisms include events resulting in loss of heterozygosity (LOH), gene mutation, and transcriptional silencing. The contribution of each of these different pathways varies among tumor suppressor genes and by c

  11. A systematic study of gene mutations in urothelial carcinoma; inactivating mutations in TSC2 and PIK3R1.

    Directory of Open Access Journals (Sweden)

    Gottfrid Sjödahl

    Full Text Available BACKGROUND: Urothelial carcinoma (UC is characterized by frequent gene mutations of which activating mutations in FGFR3 are the most frequent. Several downstream targets of FGFR3 are also mutated in UC, e.g., PIK3CA, AKT1, and RAS. Most mutation studies of UCs have been focused on single or a few genes at the time or been performed on small sample series. This has limited the possibility to investigate co-occurrence of mutations. METHODOLOGY/PRINCIPAL FINDINGS: We performed mutation analyses of 16 genes, FGFR3, PIK3CA, PIK3R1 PTEN, AKT1, KRAS, HRAS, NRAS, BRAF, ARAF, RAF1, TSC1, TSC2, APC, CTNNB1, and TP53, in 145 cases of UC. We show that FGFR3 and PIK3CA mutations are positively associated. In addition, we identified PIK3R1 as a target for mutations. We demonstrate a negative association at borderline significance between FGFR3 and RAS mutations, and show that these mutations are not strictly mutually exclusive. We show that mutations in BRAF, ARAF, RAF1 rarely occurs in UC. Our data emphasize the possible importance of APC signaling as 6% of the investigated tumors either showed inactivating APC or activating CTNNB1 mutations. TSC1, as well as TSC2, that constitute the mTOR regulatory tuberous sclerosis complex were found to be mutated at a combined frequency of 15%. CONCLUSIONS/SIGNIFICANCE: Our data demonstrate a significant association between FGFR3 and PIK3CA mutations in UC. Moreover, the identification of mutations in PIK3R1 further emphasizes the importance of the PI3-kinase pathway in UC. The presence of TSC2 mutations, in addition to TSC1 mutations, underlines the involvement of mTOR signaling in UC.

  12. Functional characterization of BRCA1 gene variants by mini-gene splicing assay

    DEFF Research Database (Denmark)

    Steffensen, Ane Y; Dandanell, Mette; Jønson, Lars


    Mutational screening of the breast cancer susceptibility gene BRCA1 leads to the identification of numerous pathogenic variants such as frameshift and nonsense variants, as well as large genomic rearrangements. The screening moreover identifies a large number of variants, for example, missense...

  13. Analysis of gene mutations and clinical features in elderly patients with melanoma

    Institute of Scientific and Technical Information of China (English)



    Objective To investigate the gene mutation status in Chinese elderly patients with melanoma and to explore the correlation of gene mutation with clinical characteristics and prognosis.Methods Melanoma tissue samples from Chinese elderly patients were analyzed for gene mutations of KIT,BRAF and NRAS in genomic DNA by polymerase chain reaction (PCR) amplification and Sanger sequencing.The correlations of gene mutations with clinicopathologic features and prognosis were statistically

  14. Filaggrin Gene Mutations and Risk of Basal Cell Carcinoma

    DEFF Research Database (Denmark)

    Kaae, Jesper Rabølle; Thyssen, J P; Johansen, J D


    Basal cell carcinoma (BCC) is prevalent in lightly-pigmented Europeans. While ultraviolet (UV) radiation is an important risk factor, genetic predispositions to BCC have also been identified (1) . Atopic dermatitis (AD), a condition with a heritability that reaches 71-84%, might increase the risk...... of BCC (2) . Loss-of-function mutations in the filaggrin gene (FLG) are observed in approximately 10% of Northern Europeans and are strongly associated with AD (3) . FLG mutations lead to reduced epidermal filaggrin protein and metabolite levels, including the chromophore trans-urocanic acid (UCA) (4......) . Mice with knockdown of filaggrin, or lack of functional histidase, show decreased epidermal trans-UCA levels and increased UVB-induced skin damage (5) . FLG mutation carriers also have 10% increased serum vitamin D levels suggesting increased penetration of UVB (6) . We evaluated the prevalence of FLG...

  15. De novo mutations in ataxin-2 gene and ALS risk.

    Directory of Open Access Journals (Sweden)

    José Miguel Laffita-Mesa

    Full Text Available Pathogenic CAG repeat expansion in the ataxin-2 gene (ATXN2 is the genetic cause of spinocerebellar ataxia type 2 (SCA2. Recently, it has been associated with Parkinsonism and increased genetic risk for amyotrophic lateral sclerosis (ALS. Here we report the association of de novo mutations in ATXN2 with autosomal dominant ALS. These findings support our previous conjectures based on population studies on the role of large normal ATXN2 alleles as the source for new mutations being involved in neurodegenerative pathologies associated with CAG expansions. The de novo mutations expanded from ALS/SCA2 non-risk alleles as proven by meta-analysis method. The ALS risk was associated with SCA2 alleles as well as with intermediate CAG lengths in the ATXN2. Higher risk for ALS was associated with pathogenic CAG repeat as revealed by meta-analysis.

  16. Molecular analysis of the most prevalent mutations of the FANCA and FANCC genes in Brazilian patients with Fanconi anaemia

    Directory of Open Access Journals (Sweden)

    David Enrique Aguilar Rodriguez


    Full Text Available Fanconi anaemia (FA is a recessive autosomal disease determined by mutations in genes of at least eleven complementation groups, with distinct distributions in different populations. As far as we know, there are no reports regarding the molecular characterisation of the disease in unselected FA patients in Brazil. OBECTIVE: This study aimed to investigate the most prevalent mutations of FANCA and FANCC genes in Brazilian patients with FA. METHODS: Genomic DNA obtained from 22 racially and ethnically diverse unrelated FA patients (mean age ± SD: 14.0 ± 7.8 years; 10 male, 12 female; 14 white, 8 black was analysed by polymerase chain reaction and restriction site assays for identification of FANCA (delta3788-3790 and FANCC (delta322G, IVS4+4A -> T, W22X, L496R, R548X, Q13X, R185X, and L554P gene mutations. RESULTS: Mutations in FANCA and FANCC genes were identified in 6 (27.3% and 14 (63.6% out of 22 patients, respectively. The disease could not be attributed to the tested mutations in the two remaining patients enrolled in the study (9.1%. The registry of the two most prevalent gene abnormalities (delta3788-3790 and IVS4 + 4 -> T revealed that they were present in 18.2% and 15.9% of the FA alleles, respectively. Additional FANCC gene mutations were found in the study, with the following prevalence: delta322G (11.4%, W22X (9.1%, Q13X (2.3%, L554P (2.3%, and R548X (2.3% of total FA alleles. CONCLUSION: These results suggest that mutations of FANCA and FANCC genes are the most prevalent mutations among FA patients in Brazil.

  17. Pure myopathy with enlarged mitochondria associated to a new mutation in MTND2 gene

    Directory of Open Access Journals (Sweden)

    Alice Zanolini


    Full Text Available To date, only few mutations in the mitochondrial DNA (mtDNA-encoded ND2 subunit of Complex I have been reported, usually presenting a severe phenotype characterized by early onset encephalomyopathy and early death. In this report, we describe a new mutation in the MTND2 gene in a 21-year-old man with a mild myopathic phenotype characterized by exercise intolerance and increased plasma lactate at rest. Electromyography and brain NMR were normal, and no cardiac involvement was present. Muscle biopsy showed a massive presence of ragged red – COX-positive fibres, with enlarged mitochondria containing osmiophilic inclusions. Biochemical assays revealed a severe isolated complex I deficiency. We identified a novel, heteroplasmic mutation m.4831G>A in the MTND2 gene, causing the p.Gly121Asp substitution in the ND2 protein. The mutation was present in the 95% of mitochondrial genomes from patient's muscle tissue, at a lower level in cells from the urinary tract and at a lowest level in lymphocytes from patient's blood; the base substitution was absent in fibroblasts and in the tissues from proband's healthy mother and brother. The specific skeletal muscle tissue involvement can explain the childhood-onset and the relatively benign, exclusively myopathic course of the disease.

  18. Point mutations in the tyrosine aminotransferase gene in tyrosinemia type II. (United States)

    Natt, E; Kida, K; Odievre, M; Di Rocco, M; Scherer, G


    Tyrosinemia type II (Richner-Hanhart syndrome, RHS) is a disease of autosomal recessive inheritance characterized by keratitis, palmoplantar hyperkeratosis, mental retardation, and elevated blood tyrosine levels. The disease results from deficiency in hepatic tyrosine aminotransferase (TAT; L-tyrosine:2-oxoglutarate aminotransferase, EC, a 454-amino acid protein encoded by a gene with 12 exons. To identify the causative mutations in five TAT alleles cloned from three RHS patients, chimeric genes constructed from normal and mutant TAT alleles were tested in directing TAT activity in a transient expression assay. DNA sequence analysis of the regions identified as nonfunctional revealed six different point mutations. Three RHS alleles have nonsense mutations at codons 57, 223, and 417, respectively. One "complex" RHS allele carries a GT----GG splice donor mutation in intron 8 together with a Gly----Val substitution at amino acid 362. A new splice acceptor site in intron 2 of the fifth RHS allele leads to a shift in reading frame.

  19. 21 CFR 866.5900 - Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation detection system. (United States)


    ... regulator (CFTR) gene mutation detection system. 866.5900 Section 866.5900 Food and Drugs FOOD AND DRUG... DEVICES Immunological Test Systems § 866.5900 Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation detection system. (a) Identification. The CFTR gene mutation detection system is a...

  20. Molecular screening of pituitary adenomas for gene mutations and rearrangements

    Energy Technology Data Exchange (ETDEWEB)

    Herman, V.; Drazin, N.Z.; Gonskey, R.; Melmed, S. (Cedars-Sinai Medical Center, Los Angeles, CA (United States))


    Although pituitary tumors arise as benign monoclonal neoplasms, genetic alterations have not readily been identified in these adenomas. The authors studied restriction fragment abnormalities involving the GH gene locus, and mutations in the p53 and H-, K-, and N-ras genes in 22 human GH cell adenomas. Twenty two nonsecretory adenomas were also examined for p53 and ras gene mutations. Seven prolactinoma DNA samples were tested for deletions in the multiple endocrine neoplasia-1 (MEN-1) locus, as well as for rearrangements in the hst gene, a member of the fibroblast growth factor family. In DNA from GH-cell adenomas, identical GH restriction patterns were detected in both pituitary and lymphocyte DNA in all patients and in one patient with a mixed GH-TSH cell adenoma. Using polymerase chain reaction (PCR)-single stranded conformation polymorphism analysis, no mutations were detected in exons 5, 6, 7 and 8 of the p53 gene in GH cell adenomas nor in 22 nonsecretory adenomas. Codons 12/13 and 61 of H-ras, K-ras, and N-ras genes were also intact on GH cell adenomas and in nonsecretory adenomas. Site-specific probes for chromosome 11q13 including, PYGM, D11S146, and INT2 were used in 7 sporadic PRL-secreting adenomas to detect deletions of the MEN-1 locus on chromosome 11. One patient was identified with a loss of 11p, and the remaining 6 patients did not demonstrate loss of heterozygosity in the pituitary 11q13 locus, compared to lymphocyte DNA. None of these patients demonstrated hst gene rearrangements which also maps to this locus. These results show that p53 and ras gene mutations are not common events in the pathogenesis of acromegaly and nonsecretory tumors. Although hst gene rearrangements and deletions of 11q13 are not associated with sporadic PRl-cell adenoma formation, a single patient was detected with a partial loss of chromosome 11, including the putative MEN-1 site. 31 refs., 5 figs., 2 tabs.

  1. Three faces of recombination activating gene 1 (RAG1) mutations. (United States)

    Patiroglu, Turkan; Akar, Himmet Haluk; Van Der Burg, Mirjam


    Severe combined immune deficiency (SCID) is a group of genetic disorder associated with development of T- and/or B-lymphocytes. Recombination-activating genes (RAG1/2) play a critical role on VDJ recombination process that leads to the production of a broad T-cell receptor (TCR) and B-cell receptor (BCR) repertoire in the development of T and B cells. RAG1/2 genes mutations result in various forms of primary immunodeficiency, ranging from classic SCID to Omenn syndrome (OS) to atypical SCID with such as granuloma formation and autoimmunity. Herein, we reported 4 patients with RAG1 deficiency: classic SCID was seen in two patients who presented with recurrent pneumonia and chronic diarrhoea, and failure to thrive. OS was observed in one patient who presented with chronic diarrhoea, skin rash, recurrent lower respiratory infections, and atypical SCID was seen in one patient who presented with Pyoderma gangrenosum (PG) and had novel RAG1 mutation.

  2. A new spontaneous mouse mutation in the Kcne1 gene. (United States)

    Letts, V A; Valenzuela, A; Dunbar, C; Zheng, Q Y; Johnson, K R; Frankel, W N


    A new mouse mutant, punk rocker (allele symbol Kcne1(pkr)), arose spontaneously on a C57BL/10J inbred strain background and is characterized by a distinctive head-tossing, circling, and ataxic phenotype. It is also profoundly and bilaterally deaf. The mutation resides in the Kcne1 gene on Chromosome (Chr) 16 and has been identified as a single base change within the coding region of the third exon. The C to T nucleotide substitution causes an arginine to be altered to a termination codon at amino acid position 67, and predictably this will result in a significantly truncated protein product. The Kcne1(pkr) mutant represents the first spontaneous mouse model for the human disorder, Jervell and Lange-Nielsen syndrome, associated with mutations in the homologous KCNE1 gene on human Chr 21.

  3. Gene quantification by the NanoGene assay is resistant to inhibition by humic acids. (United States)

    Kim, Gha-Young; Wang, Xiaofang; Ahn, Hosang; Son, Ahjeong


    NanoGene assay is a magnetic bead and quantum dot nanoparticles based gene quantification assay. It relies on a set of probe and signaling probe DNAs to capture the target DNA via hybridization. We have demonstrated the inhibition resistance of the NanoGene assay using humic acids laden genomic DNA (gDNA). At 1 μg of humic acid per mL, quantitiative PCR (qPCR) was inhibited to 0% of its quantification capability whereas NanoGene assay was able to maintain more than 60% of its quantification capability. To further increase the inhibition resistance of NanoGene assay at high concentration of humic acids, we have identified the specific mechanisms that are responsible for the inhibition. We examined five potential mechanisms with which the humic acids can partially inhibit our NanoGene assay. The mechanisms examined were (1) adsorption of humic acids on the particle surface; (2) particle aggregation induced by humic acids; (3) fluorescence quenching of quantum dots by humic acids during hybridization; (4) humic acids mimicking of target DNA; and (5) nonspecific binding between humic acids and target gDNA. The investigation showed that no adsorption of humic acids onto the particles' surface was observed for the humic acids' concentration. Particle aggregation and fluorescence quenching were also negligible. Humic acids also did not mimic the target gDNA except 1000 μg of humic acids per mL and hence should not contribute to the partial inhibition. Four of the above mechanisms were not related to the inhibition effect of humic acids particularly at the environmentally relevant concentrations (captured by the probe and signaling DNA.

  4. Relationship between the Mutation of IRS-1 Gene and β3-adrenergic Receptor Gene

    Institute of Scientific and Technical Information of China (English)

    丁国宪; 沈捷; 陈家伟


    Objective To explore the relationship between the mutation of Insulin receptor substrate-1 ( IRS-1) gene and β3-adrenergic receptor (β3-AR) gene associated with insulin resistance, to further elucidate the etiology and pathogenesis of type 2 DM, hypertension and coronary heart disease. Methods 281 Chinese subjects are divided into three groups according to the oral glucose tolerance test (OGTT), The subjects were genotyped for the codon 64 of β3-AR gene, the codon 972 of IRS-1 gene polymorphisms by applying polymerase chain reaction (PCR) restriction fragment-length polymorphisms (RFLP) screening. Results Our study found that there was significantly increased frequency of IRS-1 gene mutation in IGT subjects and type 2 DM patients (P<0.05, 0.01, respectively), increased frequency of β3-AR gene mutation in type 2 DM patients (P<0.01), compared with NGT subjects. After adjusted for age, sex and plasma glucose, the level of insulin was significantly correlated with polymorphism of IRS-1 gene and β3-AR gene (P<0.001 in all ) by multiple regression analysis. In the models of Logistic regression, type 2 DM is closely related to age and family history (OR=3.1966, 1.4670; P=0.0272, 0.009; respectively), and to the polymorphism of β3-AR gene (OR=1.7380, P=0.0356), but not related to the polymorphism of IRS-1 gene. Conclusions These results suggest that mutation of IRS-1 gene may be the risk factor for insulin resistance, whereas mutation of β3-AR gene may be a common risk factor for insulin resistance, obesity, type 2 DM and hypertension.

  5. Co-inheritance of novel ATRX gene mutation and globin (α & β) gene mutations in transfusion dependent beta-thalassemia patients. (United States)

    Al-Nafie, Awatif N; Borgio, J Francis; AbdulAzeez, Sayed; Al-Suliman, Ahmed M; Qaw, Fuad S; Naserullah, Zaki A; Al-Jarrash, Sana; Al-Madan, Mohammed S; Al-Ali, Rudaynah A; AlKhalifah, Mohammed A; Al-Muhanna, Fahad; Steinberg, Martin H; Al-Ali, Amein K


    α-Thalassemia X-linked mental retardation syndrome is a rare inherited intellectual disability disorder due to mutations in the ATRX gene. In our previous study of the prevalence of β-thalassemia mutations in the Eastern Province of Saudi Arabia, we confirmed the widespread coinheritance of α-thalassemia mutation. Some of these subjects have a family history of mental retardation, the cause of which is unknown. Therefore, we investigated the presence or absence of mutations in the ATRX gene in these patients. Three exons of the ATRX gene and their flanking regions were directly sequenced. Only four female transfusion dependent β-thalassemia patients were found to be carriers of a novel mutation in the ATRX gene. Two of the ATRX gene mutations, c.623delA and c.848T>C were present in patients homozygous for IVS I-5(G→C) and homozygous for Cd39(C → T) β-thalassemia mutation, respectively. While the other two that were located in the intronic region (flanking regions), were present in patients homozygous for Cd39(C → T) β-thalassemia mutation. The two subjects with the mutations in the coding region had family members with mental retardation, which suggests that the novel frame shift mutation and the missense mutation at coding region of ATRX gene are involved in ATRX syndrome.

  6. Myostatin gene mutated mice induced with tale nucleases. (United States)

    Zhou, Fangfang; Sun, Ruilin; Chen, Hongyan; Fei, Jian; Lu, Daru


    Myostain gene (MSTN) is expressed primarily in skeletal muscle, and negatively regulates skeletal muscle mass; it has been suggested that mice with MSTN inhibition have reduced adiposity and improved insulin sensitivity. Therefore, it is important to establish a fast and effective gene editing method. In this report, we established the myostatin mutated-mouse model by microinjection of Transcription Activator-Like Effector Nucleases (TALENs) mRNA within the mouse fertilized oocytes and achieved high rates of mutagenesis of the mouse MSTN in C57BL/6J. Six of 45 born mice carried target mutations and we appointed one as the parental mating with wild mouse to produce the F1 and backcross to produce the F2 generation. All the mutations of the mice were examined quickly and efficiently by high-resolution melting curve analysis (HRMA) and then verified by direct sequencing. We obtained the homozygous of the F2 generation which transmitted the mutant alleles to the progeny with 100% efficiency. Mutant mice exhibited increases in muscle mass comparable to those observed in wild-type mice. Therefore, combining TALEN-mediated gene targeting with HRMA technology is a superior method of constructing genetically modified mice through microinjection in the mouse fertilized oocytes with high efficiency and short time of selection.

  7. Gene augmentation for adRP mutations in RHO. (United States)

    Lewin, Alfred S; Rossmiller, Brian; Mao, Haoyu


    Mutations in the gene for rhodopsin, RHO, cause autosomal dominant retinitis pigmentosa, a disease characterized by death of rod photoreceptor cells. At the end stage, when most rods are gone, cones die too, taking central vision with them. One goal of gene therapy, therefore, is to preserve central vision by promoting rod survival in the vicinity of the macula. Dominance in RHO mutations is associated with two phenomena: interference with the function of normal rhodopsin and intrinsic toxicity of the mutant protein. In the case of interference, increased production of the wild-type protein may be therapeutic, but in the case of toxicity, suppression of the mutant protein may also be needed. RHO augmentation has made use of advances in gene delivery to the retina using adeno-associated virus (AAV). Several strategies have been developed for suppression of rhodopsin expression, but because of the heterogeneity of RHO mutations they are not specific for the mutant allele: They suppress both mutant and wild-type RHO. Experiments in autosomal dominant retinitis pigmentosa (adRP) mouse models suggest that both RHO augmentation and supplementation plus suppression preserve the survival of rod cells. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

  8. A novel frameshift mutation of POU4F3 gene associated with autosomal dominant non-syndromic hearing loss

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Hee Keun [Department of Biology, College of Natural Sciences, Kyungpook National University, Daegu (Korea, Republic of); Park, Hong-Joon [Soree Ear Clinic, Seoul (Korea, Republic of); Lee, Kyu-Yup [Vestibulocochlear Research Center, College of Medicine, Wonkwang University, Iksan (Korea, Republic of); Park, Rekil, E-mail: [Department of Otorhinolaryngology-Head and Neck Surgery, College of Medicine, Kyungpook National University, Daegu (Korea, Republic of); Kim, Un-Kyung, E-mail: [Department of Biology, College of Natural Sciences, Kyungpook National University, Daegu (Korea, Republic of)


    Autosomal dominant mutations in the transcription factor POU4F3 gene are associated with non-syndromic hearing loss in humans; however, there have been few reports of mutations in this gene worldwide. We performed a mutation analysis of the POU4F3 gene in 42 unrelated Koreans with autosomal dominant non-syndromic hearing loss, identifying a novel 14-bp deletion mutation in exon 2 (c.662del14) in one patient. Audiometric examination revealed severe bilateral sensorineural hearing loss in this patient. The novel mutation led to a truncated protein that lacked both functional POU domains. We further investigated the functional distinction between wild-type and mutant POU4F3 proteins using in vitro assays. The wild-type protein was completely localized in the nucleus, while the truncation of protein seriously affected its nuclear localization. In addition, the mutant failed to activate reporter gene expression. This is the first report of a POU4F3 mutation in Asia, and moreover our data suggest that further investigation will need to delineate ethnicity-specific genetic background for autosomal dominant non-syndromic hearing loss within Asian populations.

  9. Mutational analysis of the promoter and the coding region of the 5-HT1A gene

    Energy Technology Data Exchange (ETDEWEB)

    Erdmann, J.; Noethen, M.M.; Shimron-Abarbanell, D. [Univ. of Bonn (Germany)] [and others


    Disturbances of serotonergic pathways have been implicated in many neuropsychiatric disorders. Serotonin (5HT) receptors can be subdivided into at least three major families (5HT1, 5HT2, and 5HT3). Five human 5HT1 receptor subtypes have been cloned, namely 1A, 1D{alpha}, 1D{beta}, 1E, and 1F. Of these, the 5HT1A receptor is the best characterized subtype. In the present study we sought to identify genetic variation in the 5HT1A receptor gene which through alteration of protein function or level of expression might contribute to the genetics of neuropsychiatric diseases. The coding region and the 5{prime} promoter region of the 5HT1A gene from 159 unrelated subjects (45 schizophrenic, 46 bipolar affective, and 43 patients with Tourette`s syndrome, as well as 25 controls) were analyzed using SSCA. SSCA revealed the presence of two mutations both located in the coding region of the 5HT1A receptor gene. The first mutation is a rare silent C{r_arrow}T substitution at nucleotide position 549. The second mutation is characterized by a base pair substitution (A{r_arrow}G) at the first position of codon 28 and results in an amino acid exchange (Ile{r_arrow}Val). Since Val28 was found only in a single schizophrenic patient and in none of the other patients or controls, we decided to extend our samples and to use a restriction assay for screening a further 74 schizophrenic, 95 bipolar affective, and 49 patients with Tourette`s syndrome, as well as 185 controls, for the presence of the mutation. In total, the mutation was found in 2 schizophrenic patients, in 3 bipolars, in 1 Tourette patient, and in 5 controls. To our knowledge the Ile-28-Val substitution reported here is the first natural occuring molecular variant which has been identified for a serotonin receptor so far.

  10. The Ag-195 (C®G mutation in hereditary persistence of fetal hemoglobin is not associated with activation of a reporter gene in vitro

    Directory of Open Access Journals (Sweden)

    Schreiber R.


    Full Text Available Hereditary persistence of fetal hemoglobin is an uncommon, benign disorder in which the expression of gamma-globin genes persists into adult life. Several point mutations have been associated with the increased gamma-globin gene promoter activity. We evaluated the -195 (C->G mutation by a functional in vitro assay based on the luciferase reporter gene system. The results indicated that the increased promoter activity observed in vivo could not be reproduced in vitro under the conditions employed, suggesting that other factors may be involved in the overexpression of the gamma-globin gene containing the -195 (C->G mutation. Furthermore, this is the first time that the -195 (C->G mutation of the Agamma-globin gene has been evaluated by in vitro gene expression.

  11. Adenovirus with DNA Packaging Gene Mutations Increased Virus Release (United States)

    Wechman, Stephen L.; Rao, Xiao-Mei; McMasters, Kelly M.; Zhou, Heshan Sam


    Adenoviruses (Ads) have been extensively manipulated for the development of cancer selective replication, leading to cancer cell death or oncolysis. Clinical studies using E1-modified oncolytic Ads have shown that this therapeutic platform was safe, but with limited efficacy, indicating the necessity of targeting other viral genes for manipulation. To improve the therapeutic efficacy of oncolytic Ads, we treated the entire Ad genome repeatedly with UV-light and have isolated AdUV which efficiently lyses cancer cells as reported previously (Wechman, S. L. et al. Development of an Oncolytic Adenovirus with Enhanced Spread Ability through Repeated UV Irradiation and Cancer Selection. Viruses 2016, 8, 6). In this report, we show that no mutations were observed in the early genes (E1 or E4) of AdUV while several mutations were observed within the Ad late genes which have structural or viral DNA packaging functions. This study also reported the increased release of AdUV from cancer cells. In this study, we found that AdUV inhibits tumor growth following intratumoral injection. These results indicate the potentially significant role of the viral late genes, in particular the DNA packaging genes, to enhance Ad oncolysis. PMID:27999391

  12. Multiple gene mutations, not the type of mutation, are the modifier of left ventricle hypertrophy in patients with hypertrophic cardiomyopathy. (United States)

    Zou, Yubao; Wang, Jizheng; Liu, Xuan; Wang, Yilu; Chen, Yi; Sun, Kai; Gao, Shuo; Zhang, Channa; Wang, Zhimin; Zhang, Yin; Feng, Xinxing; Song, Ying; Wu, Yajie; Zhang, Hongju; Jia, Lei; Wang, Hu; Wang, Dong; Yan, Chaowu; Lu, Minjie; Zhou, Xianliang; Song, Lei; Hui, Rutai


    Genotype-phenotype correlation of hypertrophic cardiomyopathy (HCM) has been challenging because of the genetic and clinical heterogeneity. To determine the mutation profile of Chinese patients with HCM and to correlate genotypes with phenotypes, we performed a systematic mutation screening of the eight most commonly mutated genes encoding sarcomere proteins in 200 unrelated Chinese adult patients using direct DNA sequencing. A total of 98 mutations were identified in 102 mutation carriers. The frequency of mutations in MYH7, MYBPC3, TNNT2 and TNNI3 was 26.0, 18.0, 4.0 and 3.5 % respectively. Among the 200 genotyped HCM patients, 83 harbored a single mutation, and 19 (9.5 %) harbored multiple mutations. The number of mutations was positively correlated with the maximum wall thickness. We found that neither particular gene nor specific mutation was correlated to clinical phenotype. In summary, the frequency of multiple mutations was greater in Chinese HCM patients than in the Caucasian population. Multiple mutations in sarcomere protein may be a risk factor for left ventricular wall thickness.

  13. Clinical characteristics and prognosis of acute myeloid leukemia associated with DNA-methylation regulatory gene mutations. (United States)

    Ryotokuji, Takeshi; Yamaguchi, Hiroki; Ueki, Toshimitsu; Usuki, Kensuke; Kurosawa, Saiko; Kobayashi, Yutaka; Kawata, Eri; Tajika, Kenji; Gomi, Seiji; Kanda, Junya; Kobayashi, Anna; Omori, Ikuko; Marumo, Atsushi; Fujiwara, Yusuke; Yui, Shunsuke; Terada, Kazuki; Fukunaga, Keiko; Hirakawa, Tsuneaki; Arai, Kunihito; Kitano, Tomoaki; Kosaka, Fumiko; Tamai, Hayato; Nakayama, Kazutaka; Wakita, Satoshi; Fukuda, Takahiro; Inokuchi, Koiti


    In recent years, it has been reported that the frequency of DNA-methylation regulatory gene mutations - mutations of the genes that regulate gene expression through DNA methylation - is high in acute myeloid leukemia. The objective of the present study was to elucidate the clinical characteristics and prognosis of acute myeloid leukemia with associated DNA-methylation regulatory gene mutation. We studied 308 patients with acute myeloid leukemia. DNA-methylation regulatory gene mutations were observed in 135 of the 308 cases (43.8%). Acute myeloid leukemia associated with a DNA-methylation regulatory gene mutation was more frequent in older patients (Pgene mutation was an unfavorable prognostic factor for overall survival in the whole cohort (P=0.0018), in patients aged ≤70 years, in patients with intermediate cytogenetic risk, and in FLT3-ITD-negative patients (P=0.0409). Among the patients with DNA-methylation regulatory gene mutations, 26.7% were found to have two or more such mutations and prognosis worsened with increasing number of mutations. In multivariate analysis DNA-methylation regulatory gene mutation was an independent unfavorable prognostic factor for overall survival (P=0.0424). However, patients with a DNA-methylation regulatory gene mutation who underwent allogeneic stem cell transplantation in first remission had a significantly better prognosis than those who did not undergo such transplantation (P=0.0254). Our study establishes that DNA-methylation regulatory gene mutation is an important unfavorable prognostic factor in acute myeloid leukemia.

  14. Combining gene mutation with gene expression data improves outcome prediction in myelodysplastic syndromes (United States)

    Gerstung, Moritz; Pellagatti, Andrea; Malcovati, Luca; Giagounidis, Aristoteles; Porta, Matteo G Della; Jädersten, Martin; Dolatshad, Hamid; Verma, Amit; Cross, Nicholas C. P.; Vyas, Paresh; Killick, Sally; Hellström-Lindberg, Eva; Cazzola, Mario; Papaemmanuil, Elli; Campbell, Peter J.; Boultwood, Jacqueline


    Cancer is a genetic disease, but two patients rarely have identical genotypes. Similarly, patients differ in their clinicopathological parameters, but how genotypic and phenotypic heterogeneity are interconnected is not well understood. Here we build statistical models to disentangle the effect of 12 recurrently mutated genes and 4 cytogenetic alterations on gene expression, diagnostic clinical variables and outcome in 124 patients with myelodysplastic syndromes. Overall, one or more genetic lesions correlate with expression levels of ~20% of all genes, explaining 20–65% of observed expression variability. Differential expression patterns vary between mutations and reflect the underlying biology, such as aberrant polycomb repression for ASXL1 and EZH2 mutations or perturbed gene dosage for copy-number changes. In predicting survival, genomic, transcriptomic and diagnostic clinical variables all have utility, with the largest contribution from the transcriptome. Similar observations are made on the TCGA acute myeloid leukaemia cohort, confirming the general trends reported here. PMID:25574665

  15. Detection of EGFR Mutations by TaqMan Mutation Detection Assays Powered by Competitive Allele-Specific TaqMan PCR Technology

    Directory of Open Access Journals (Sweden)

    Cristin Roma


    Full Text Available Epidermal growth factor receptor (EGFR mutations in non-small-cell lung cancer (NSCLC are predictive of response to treatment with tyrosine kinase inhibitors. Competitive Allele-Specific TaqMan PCR (castPCR is a highly sensitive and specific technology. EGFR mutations were assessed by TaqMan Mutation Detection Assays (TMDA based on castPCR technology in 64 tumor samples: a training set of 30 NSCLC and 6 colorectal carcinoma (CRC samples and a validation set of 28 NSCLC cases. The sensitivity and specificity of this method were compared with routine diagnostic techniques including direct sequencing and the EGFR Therascreen RGQ kit. Analysis of the training set allowed the identification of the threshold value for data analysis (0.2; the maximum cycle threshold (Ct=37; and the cut-off ΔCt value (7 for the EGFR TMDA. By using these parameters, castPCR technology identified both training and validation set EGFR mutations with similar frequency as compared with the Therascreen kit. Sequencing detected rare mutations that are not identified by either castPCR or Therascreen, but in samples with low tumor cell content it failed to detect common mutations that were revealed by real-time PCR based methods. In conclusion, our data suggest that castPCR is highly sensitive and specific to detect EGFR mutations in NSCLC clinical samples.

  16. A network of cancer genes with co-occurring and anti-co-occurring mutations.

    Directory of Open Access Journals (Sweden)

    Qinghua Cui

    Full Text Available Certain cancer genes contribute to tumorigenesis in a manner of either co-occurring or mutually exclusive (anti-co-occurring mutations; however, the global picture of when, where and how these functional interactions occur remains unclear. This study presents a systems biology approach for this purpose. After applying this method to cancer gene mutation data generated from large-scale and whole genome sequencing of cancer samples, a network of cancer genes with co-occurring and anti-co-occurring mutations was constructed. Analysis of this network revealed that genes with co-occurring mutations prefer direct signaling transductions and that the interaction relations among cancer genes in the network are related with their functional similarity. It was also revealed that genes with co-occurring mutations tend to have similar mutation frequencies, whereas genes with anti-co-occurring mutations tend to have different mutation frequencies. Moreover, genes with more exons tend to have more co-occurring mutations with other genes, and genes having lower local coherent network structures tend to have higher mutation frequency. The network showed two complementary modules that have distinct functions and have different roles in tumorigenesis. This study presented a framework for the analysis of cancer genome sequencing outputs. The presented data and uncovered patterns are helpful for understanding the contribution of gene mutations to tumorigenesis and valuable in the identification of key biomarkers and drug targets for cancer.

  17. Progranulin gene mutation with an unusual clinical and neuropathologic presentation. (United States)

    Wider, Christian; Uitti, Ryan J; Wszolek, Zbigniew K; Fang, John Y; Josephs, Keith A; Baker, Matthew C; Rademakers, Rosa; Hutton, Michael L; Dickson, Dennis W


    Progranulin gene (PGRN) mutations cause frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). Patients usually present with a frontotemporal dementia syndrome and have prominent atrophy and neuronal loss in frontal and temporal cortices and the striatum, with neuronal intranuclear and cytoplasmic inclusions. Clinical, neuropathological, and genetic studies are reported on an individual with PGRN mutation and her family members. We describe a patient with a PGRN c.26C>A mutation who presented with progressive stuttering dysarthria, oculomotor abnormalities, choreic buccolingual movements, and mild parkinsonism. Two other family members were affected, one with a behavioral variant frontotemporal dementia syndrome, the other with a diagnosis of probable Alzheimer's disease. At autopsy there was no neuronal loss in the cortex or medial temporal lobe structures, but there was striatal gliosis. Immunohistochemistry for ubiquitin and TDP-43 revealed neuronal cytoplasmic and intranuclear inclusions as well as neurites. This study further expands the clinical and pathological spectrum of PGRN mutations, and suggests the diagnosis could be missed in some individuals with atypical presentations.

  18. Patient with FMF and Triple MEFV Gene Mutations. (United States)

    Salehzadeh, Farhad; Fathi, Afshin


    Familial Mediterranean fever (FMF) is the most common auto-inflammatory disease with monogenic (MEditerranean FeVer -MEFV- gene) inherited pattern. It mainly affects ethnic groups living along the eastern Mediterranean Sea: Turks, Sephardic Jews, Armenians, and Arabs [1]. Today FMF is not rare disease in other Mediterranean ethnicities, such as Greeks, Italians, and Iranians. Here we report a child with complex allele mutations E148Q/V726A/R761H, whilst, whose mother showed E148Q/V726A and his father had R761H/wt in analysis. The severity of the disease and genotype-phenotype correlation of patient showed no significant differences with his mother and other patients with the same two mutations, V726A/R761H, E148Q/V726A, and E148Q/R761H. This type of mutation is the first report of triple mutations in FMF patients with no specific phenotype correlation.

  19. Mismatch repair genes of Streptococcus pneumoniae: HexA confers a mutator phenotype in Escherichia coli by negative complementation. (United States)

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


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

  20. Cystic fibrosis gene mutations: evaluation and assessment of disease severity

    Directory of Open Access Journals (Sweden)

    Vallières E


    Full Text Available Emilie Vallières, Joseph Stuart ElbornCystic Fibrosis and Airways Microbiology Research Group, Queens University Belfast, Belfast, UKAbstract: The cystic fibrosis transmembrane regulator (CFTR gene encodes an ion channel transporter, the CFTR protein. Since its identification in 1989, more than 1,900 sequence variants have been reported, resulting in a wide spectrum of clinical phenotypes. Cystic fibrosis (CF is associated with many CFTR mutants and there is a continuum of disease severity observed. Recent advances in fundamental research have increased our understanding of the consequent molecular defect arising from CF mutations. This knowledge has resulted in the development of CF-specific therapies, targeting either the genetic or the molecular defect. CF care, previously focused on symptom control, is therefore moving toward a "stratified" or "precision" therapeutic approach. This review outlines normal CFTR physiology, the proposed pathologic mechanism underlying CF associated-lung injury, classification of CF mutations, and the CF-specific therapies recently approved or in clinical trials.Keywords: cystic fibrosis, gene mutations, disease severity, evaluation, assessment

  1. BSE case associated with prion protein gene mutation.

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    Jürgen A Richt

    Full Text Available Bovine spongiform encephalopathy (BSE is a transmissible spongiform encephalopathy (TSE of cattle and was first detected in 1986 in the United Kingdom. It is the most likely cause of variant Creutzfeldt-Jakob disease (CJD in humans. The origin of BSE remains an enigma. Here we report an H-type BSE case associated with the novel mutation E211K within the prion protein gene (Prnp. Sequence analysis revealed that the animal with H-type BSE was heterozygous at Prnp nucleotides 631 through 633. An identical pathogenic mutation at the homologous codon position (E200K in the human Prnp has been described as the most common cause of genetic CJD. This finding represents the first report of a confirmed case of BSE with a potential pathogenic mutation within the bovine Prnp gene. A recent epidemiological study revealed that the K211 allele was not detected in 6062 cattle from commercial beef processing plants and 42 cattle breeds, indicating an extremely low prevalence of the E211K variant (less than 1 in 2000 in cattle.

  2. IL7R and RAG1/2 genes mutations/polymorphisms in patients with SCID. (United States)

    Safaei, Sepideh; Pourpak, Zahra; Moin, Mostafa; Houshmand, Massoud


    SCID disorder is major failure of the immune system, usually genetic. The aim of this study was on mutations detection of RAG1, RAG2, and IL7RG genes in SCID cases. Mutation detection was performed by PCR sequencing. Our results indicated that 13 mutations were found through cases which include 4 mutations in IL7R gene (T661I, I138V, T56A, C57W), 7 mutations in RAG1 (W896X, W204R, M324V, T731I, M1006V, K820R, and R249H), and 2 mutations in RAG2 gene (R229W, ΔT251).

  3. Dysferlin Gene Mutation Spectrum in a Large Cohort of Chinese Patients with Dysferlinopathy

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    Su-Qin Jin


    Conclusions: DYSF mutations in Chinese patients clustered in the N-terminal region of the gene. Exonic rearrangements were found in 23% of patients with only one pathogenic mutation identified by Sanger sequencing or NGS. The novel mutations found in this study greatly expanded the mutational spectrum of dysferlinopathy.

  4. [MEFV gene mutation spectrum in familial Mediterranean fever (FMF) : a single center study in the Aegean region of Turkey]. (United States)

    Coker, I; Colak, A; Yolcu, I; Türkön, H; Nalbantoglu, S M


    Familial Mediterranean fever (FMF), an autosomal recessive autoinflammatory disorder, is characterized by recurrent, self-limiting fever and serositis which is frequently seen in Mediterranean populations. In this study, we retrospectively evaluated the MEFV gene mutation distribution of 883 citizens of the Aegean region with preliminary diagnosis of FMF who were referred to the Tepecik Research and Education Hospital's Tissue Typing and Molecular Diagnostic Laboratory (Izmir, Turkey) between 2006 and 2009. The FMF Strip Assay® (ViennaLab Diagnostics, Vienna, Austria) was used to determine the 12 most common MEFV gene mutations in patients prediagnosed with FMF. Allelic frequencies of the major mutations in the mutation positive groups, including M694V, E148Q, M680I(G>C), and V726A, accounted for 48.4, 16.5, 13.5, and 9.7%, respectively. The M694V mutation was found to be the most common mutation among FMF patients in the Aegean region, which is in accordance with mutation studies reported from other regions of the country and different ethnic populations. An English full-text version of this article is available at SpringerLink as supplemental.

  5. Evaluation of IFN-γ polymorphism+874 T/A in patients with recurrent tonsillitis by PCR real time mismatch amplification mutation assay (MAMA real time PCR). (United States)

    Bergallo, Massimiliano; Gambarino, Stefano; Loiacono, Elisa; Vergano, Luca; Galliano, Ilaria; Montanari, Paola; Astegiano, Sara; Tavormina, Paolo; Tovo, Pier-Angelo


    Interferon gamma (IFN-γ) is an important cytokine that plays a crucial role in the balance between normal and pathological immune response. Defect of IFN-γ can give a predisposition to infectious disease, autoimmune pathologies and tumours. Different polymorphisms in this gene have been described, in particular the single nucleotide polymorphism (SNP)+874∗T/A that may affect IFN-γ gene expression. Several techniques can be used for the detection of SNPs. In this work two PCR Real Time assays were developed, an Amplification Refractory Mutation System (ARMS) and a Mismatch Amplification Mutation Assay (MAMA). Twenty-seven samples from patients (tonsillectomy) and 85 from donor's blood bank were considered. As a result, 78/85 controls (91.7%) and 25/27 patients (92.6%) were heterozygosis, considering the ARMS-PCR; 55/85 (64.7%) and 14/27 (51.9%) were heterozygosis using MAMA-PCR assay. Fourteen of 85 (16.5%) and 8/27 (29.6%) were homozygosis A, 16/85 (18.8%) and 5/27 (18.5%) presented homozygosis T, taking into account the MAMA-PCR. There are statistically difference between the two assay with p<0.0001 at Chi-square test. Our preliminary data suggest that tonsillectomy patients had a statistical trend to possess the low IFN-γ polymorphism when compared with control subject (p=0.3) but is not statistically significant. In conclusion the Real time MAMA-PCR assay has several advantages over other SNP identification techniques such as rapidity, reliability, easily to perform in one working day and applicable in clinical molecular diagnostic laboratories, although sequencing remains the gold standard.

  6. Two double heterozygous mutations in the F7 gene show different manifestations. (United States)

    Nagaizumi, Keiko; Inaba, Hiroshi; Suzuki, Takashi; Hatta, Yoshihiro; Hagiwara, Takeshi; Amano, Kagehiro; Arai, Morio; Fukutake, Katsuyuki


    We sequenced the factor VII gene (F7) in two unrelated Japanese patients with factor VII (FVII) deficiency. In the first (an asymptomatic 46-year-old man with FVII activity and antigen levels of 1.2% and 21% of normal respectively), novel E25K and H348Q mutations were identified in the doubly heterozygous state. In transiently transfected HEK293 cells, the level of FVII-E25K mutant activity in the culture media was significantly lower than that of FVII wild type, whereas the antigen levels of both proteins were similar. This suggests that the E25K mutation is associated with a dysfunctional FVII molecule. In the second patient (a 47-year-old woman with FVII activity and antigen levels of less than 1% and 6% respectively), an IVS4+1 mutation and a novel -96C to T transition were detected in the double heterozygous state. In electrophoretic mobility shift assays, the -96T mutation was shown to disrupt binding of Sp1.

  7. Data mining approach to predict BRCA1 gene mutation

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    Olegas Niakšu


    Full Text Available Breast cancer is the most frequent women cancer form and one of the leading mortality causes among women around the world. Patients with pathological mutation of a BRCA gene have 65% lifelong breast cancer probability. It is known that such patients have different cause of illness. In this study, we have proposed a new approach for the prediction of BRCA mutation carriers by methodically applying knowledge discovery steps and utilizing data mining methods. An alternative BRCA risk assessment model has been created utilizing decision tree classifier model. The biggest challenge was a very small size and imbalanced nature of the initial dataset, which have been collected by clinicians during 4 years of clinical trial. Iterative optimization of initial dataset, optimal algorithms selection and their parameterization have resulted in higher classifier model performance, with acceptable prediction accuracy for the clinical usage. In this study, three data mining problems have been analyzed using eleven data mining algorithms.

  8. [Cytogenetic abnormalities and gene mutations in myeloid leukemia]. (United States)

    Kato, Naoko; Kitamura, Toshio


    Myeloid leukemia is a clinically and genetically heterogeneous disease. Cytogenetic studies have revealed specific chromosomal abnormalities, such as translocations, and inversions. Fusion proteins derived from these abnormalities were identified in various subtypes of leukemia. Because most of these fusion proteins were not sufficient to induce leukemia by themselves in mouse models, additional oncogenic events have been thought to be necessary for leukemogenesis. Recently, a hypothesis called "two-hit model" for leukemia has been proposed. Two broad classes of mutations that proliferative or survival advantage of hematopoietic progenitors and impaired differentiation are required for inducing leukemia. In this article, we summarize some typical chromosomal abnormalities or gene mutations associated with myeloid leukemia on the basis of this hypothesis.

  9. Mutation Analysis of the LH Receptor Gene in Leydig Cell Adenoma and Hyperplasia and Functional and Biochemical Studies of Activating Mutations of the LH Receptor Gene

    NARCIS (Netherlands)

    Boot, Annemieke M.; Lumbroso, Serge; Verhoef-Post, Miriam; Richter-Unruh, Annette; Looijenga, Leendert H. J.; Funaro, Ada; Beishuizen, Auke; van Marle, Andre; Drop, Stenvert L. S.; Themmen, Axel P. N.


    Context: Germline and somatic activating mutations in the LH receptor (LHR) gene have been reported. Objective: Our objective was to perform mutation analysis of the LHR gene of patients with Leydig cell adenoma or hyperplasia. Functional studies were conducted to compare the D578H-LHR mutant with t

  10. Analysis of AGXT gene mutation in primary hyperoxaluria type I family

    Institute of Scientific and Technical Information of China (English)



    Objective To describe the clinical characteristics,and to analyze the AGXT gene mutation in three siblings with primary hyperoxaluria typeⅠ(PHI).Methods AGXT gene mutation was analyzed by direct sequencing analysis in this family,and the minor allele status was also tested.One hundred unrelated healthy subjects were also analyzed as controls.Results Three mutations in

  11. Comparison of T7E1 and surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases. (United States)

    Vouillot, Léna; Thélie, Aurore; Pollet, Nicolas


    Genome editing using engineered nucleases is used for targeted mutagenesis. But because genome editing does not target all loci with similar efficiencies, the mutation hit-rate at a given locus needs to be evaluated. The analysis of mutants obtained using engineered nucleases requires specific methods for mutation detection, and the enzyme mismatch cleavage method is used commonly for this purpose. This method uses enzymes that cleave heteroduplex DNA at mismatches and extrahelical loops formed by single or multiple nucleotides. Bacteriophage resolvases and single-stranded nucleases are used commonly in the assay but have not been compared side-by-side on mutations obtained by engineered nucleases. We present the first comparison of the sensitivity of T7E1 and Surveyor EMC assays on deletions and point mutations obtained by zinc finger nuclease targeting in frog embryos. We report the mutation detection limits and efficiencies of T7E1 and Surveyor. In addition, we find that T7E1 outperforms the Surveyor nuclease in terms of sensitivity with deletion substrates, whereas Surveyor is better for detecting single nucleotide changes. We conclude that T7E1 is the preferred enzyme to scan mutations triggered by engineered nucleases.

  12. Detection of multi-drug resistance & characterization of mutations in Mycobacterium tuberculosis isolates from North- Eastern States of India using GenoType MTBDRplus assay

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    Ritu Singhal


    Full Text Available Background & objectives: Information on drug resistance tuberculosis is sparse from North-East (N-E States of India. We undertook this study to detect multi-drug resistant tuberculosis (MDR-TB among MDR-TB suspects, and common mutations among MDR-TB cases using GenoType MTBDRplus. Methods: All MDR suspect patients deposited sputum samples to peripheral designated microscopy centres (DMC in North-East States. The district TB officers (DTOs facilitated the transport of samples collected during January 2012 to August 2012 to our laboratory. The line probe assay to detect common mutations in the rpoB gene for rifampicin (RIF and katG and inhA genes for isoniazid (INH, respectively was performed on 339 samples or cultures. Results: A total of 553 sputum samples from MDR suspects were received of which, 181 (32.7% isolates were found to be multi-drug resistant. Missing WT8 along with mutation in codon S531L was commonest pattern for rifampicin resistant isolates (65.1% and missing WT along with mutations in codon S315T1 of katG gene was commonest pattern for isoniazid resistant isolates (86.2%. Average turn-around time for dispatch of LPA result to these States from cultures and samples was 23.4 and 5.2 days, respectively. Interpretations & conclusions: The MDR-TB among MDR-TB suspects in North-Eastern States of India was found to be 32.7 per cent. The common mutations obtained for RIF and INH in the region were mostly similar to those reported earlier.

  13. Novel point mutation in the uroporphyrinogen III synthase gene causes congenital erythropoietic porphyria of a Japanese family. (United States)

    Takamura, N; Hombrados, I; Tanigawa, K; Namba, H; Nagayama, Y; de Verneuil, H; Yamashita, S


    The molecular basis of the uroporphyrinogen III synthase (UROIIIS) deficiency was investigated in a member of a Japanese family. This defect in heme biosynthesis is responsible for a rare autosomal recessive disease: congenital erythropoietic porphyria (CEP) or Günther's disease. The patient was homozygous for a novel missense mutation: a G to T transition of nucleotide 7 that predicted a valine to phenylalanine substitution at residue 3 (V3F). The parents were heterozygous for the same mutation. The loss of UROIIIS activity was verified by an in vitro assay system. The corresponding mutated protein was expressed in Escherichia coli and no residual activity was observed. Further studies are needed to determine whether the mutations of the UROIIIS gene (UROS) have a specific profile in Japan compared to European or American countries.

  14. Molecular Analysis of Ciprofloxacin Resistance Mechanisms in Malaysian ESBL-Producing Klebsiella pneumoniae Isolates and Development of Mismatch Amplification Mutation Assays (MAMA) for Rapid Detection of gyrA and parC Mutations (United States)

    Mohd Yusof, Mohd Yasim; Tay, Sun Tee


    Ninety-three Malaysian extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae isolates were investigated for ciprofloxacin resistance. Two mismatch amplification mutation (MAMA) assays were developed and used to facilitate rapid detection of gyrA and parC mutations. The isolates were also screened for plasmid-mediated quinolone resistance (PMQR) genes including aac(6′)-Ib-cr, qepA, and qnr. Ciprofloxacin resistance (MICs 4– ≥ 32 μg/mL) was noted in 34 (37%) isolates, of which 33 isolates had multiple mutations either in gyrA alone (n = 1) or in both gyrA and parC regions (n = 32). aac(6′)-Ib-cr was the most common PMQR gene detected in this study (n = 61), followed by qnrB and qnrS (n = 55 and 1, resp.). Low-level ciprofloxacin resistance (MICs 1-2 μg/mL) was noted in 40 (43%) isolates carrying qnrB accompanied by either aac(6′)-Ib-cr (n = 34) or a single gyrA 83 mutation (n = 6). Ciprofloxacin resistance was significantly associated with the presence of multiple mutations in gyrA and parC regions. While the isolates harbouring gyrA and/or parC alteration were distributed into 11 PFGE clusters, no specific clusters were associated with isolates carrying PMQR genes. The high prevalence of ciprofloxacin resistance amongst the Malaysian ESBL-producing K. pneumoniae isolates suggests the need for more effective infection control measures to limit the spread of these resistant organisms in the hospital. PMID:24860827

  15. Insight into norfloxacin resistance of Acinetobacter oleivorans DR1: target gene mutation, persister, and RNA-Seq analyses. (United States)

    Kim, Jisun; Noh, Jaemin; Park, Woojun


    Antibiotic resistance of soilborne Acinetobacter species has been poorly explored. In this study, norfloxacin resistance of a soil bacterium, Acinetobacter oleivorans DR1, was investigated. The frequencies of mutant appearance of all tested non-pathogenic Acinetobacter strains were lower than those of pathogenic strains under minimum inhibitory concentration (MIC). When the quinolone-resistance-determining region of the gyrA gene was examined, only one mutant (His78Asn) out of 10 resistant variants had a mutation. Whole transcriptome analysis using a RNA-Seq demonstrated that genes involved in SOS response and DNA repair were significantly up-regulated by norfloxacin. Determining the MICs of survival cells after norfloxacin treatment confirmed some of those cells were indeed persister cells. Ten colonies, randomly selected from among those that survived in the presence of norfloxacin, did not exhibit increased MIC. Thus, both the low mutation frequency of the target gene and SOS response under norfloxacin suggested that persister formation might contribute to the resistance of DR1 against norfloxacin. The persister frequency increased without a change in MIC when stationary phase cells, low growth rates conditions, and growth-deficient dnaJ mutant were used. Taken together, our comprehensive approach, which included mutational analysis of the target gene, persister formation assays, and RNA sequencing, indicated that DR1 survival when exposed to norfloxacin is related not only to target gene mutation but also to persister formation, possibly through up-regulation of the SOS response and DNA repair genes.

  16. Mutations of p53 gene exons 4-8 in human esophageal cancer

    Institute of Scientific and Technical Information of China (English)

    Li-Ya Li; Jin-Tian Tang; Li-Qun Jia; Pei-Wen Li


    AIM: To characterize the tumor suppressor gene p53 mutations in exon 4, esophageal cancer and adjacent noncancerous tissues.METHODS: We performed p53 (exons 4-8) gene mutation analysis on 24 surgically resected human esophageal cancer specimens by PCR, single-strand conformation polymorphism, and DNA sequencing. RESULTS: p53 gene mutations were detected in 9 of 22 (40.9%) esophageal cancer specimens and 10 of 17 (58.8%) adjacent non-cancerous tissues. Eight of sixteen (50.0%) point mutations detected were G-A transitions and 9 of 18 (50.0%) p53 gene mutations occurred in exon 4 in esophageal cancer specimens. Only 1 of 11 mutations detected was G-A transition and 4 of 11 (36.4%) p53 gene mutations occurred in exon 4 in adjacent non-cancerous tissues.CONCLUSION: Mutation of p53 gene in exon 4 may play an important role in development of esophageal cancer. The observation of p53 gene mutation in adjacent noncancerous tissues suggests that p53 gene mutation may be an early event in esophageal carcinogenesis. Some clinical factors, including age, sex, pre-operation therapy and location of tumors, do not influence p53 gene mutation rates.

  17. Multiplex real-time PCR melting curve assay to detect drug-resistant mutations of Mycobacterium tuberculosis. (United States)

    Luo, Tao; Jiang, Lili; Sun, Weiming; Fu, G; Mei, Jian; Gao, Qian


    Early diagnosis of drug-resistant Mycobacterium tuberculosis is urgently needed to optimize treatment regimens and to prevent the transmission of resistant strains. Real-time PCR assays have been developed to detect drug resistance rapidly, but none of them have been widely applied due to their complexity, high cost, or requirement for advanced instruments. In this study, we developed a real-time PCR method based on melting curve analysis of dually labeled probes. Six probes targeting the rpoB 81-bp core region, katG315, the inhA promoter, the ahpC promoter, and embB306 were designed and validated with clinical isolates. First, 10 multidrug-resistant (MDR) strains with a wide mutation spectrum were used to analyze the melting temperature (T(m)) deviations of different mutations by single real-time PCR. All mutations can be detected by significant T(m) reductions compared to the wild type. Then, three duplex real-time PCRs, with two probes in each, were developed to detect mutations in 158 MDR isolates. Comparison of the results with the sequencing data showed that all mutations covered by the six probes were detected with 100% sensitivity and 100% specificity. Our method provided a new way to rapidly detect drug-resistant mutations in M. tuberculosis. Compared to other real-time PCR methods, we use fewer probes, which are labeled with the same fluorophore, guaranteeing that this assay can be used for detection in a single fluorescent channel or can be run on single-channel instruments. In conclusion, we have developed a widely applicable real-time PCR assay to detect drug-resistant mutations in M. tuberculosis.

  18. Diaphanous gene mutation affects spiral cleavage and chirality in snails (United States)

    Kuroda, Reiko; Fujikura, Kohei; Abe, Masanori; Hosoiri, Yuji; Asakawa, Shuichi; Shimizu, Miho; Umeda, Shin; Ichikawa, Futaba; Takahashi, Hiromi


    L-R (left and right) symmetry breaking during embryogenesis and the establishment of asymmetric body plan are key issues in developmental biology, but the onset including the handedness-determining gene locus still remains unknown. Using pure dextral (DD) and sinistral (dd) strains of the pond snail Lymnaea stagnalis as well as its F2 through to F10 backcrossed lines, the single handedness-determining-gene locus was mapped by genetic linkage analysis, BAC cloning and chromosome walking. We have identified the actin-related diaphanous gene Lsdia1 as the strongest candidate. Although the cDNA and derived amino acid sequences of the tandemly duplicated Lsdia1 and Lsdia2 genes are very similar, we could discriminate the two genes/proteins in our molecular biology experiments. The Lsdia1 gene of the sinistral strain carries a frameshift mutation that abrogates full-length LsDia1 protein expression. In the dextral strain, it is already translated prior to oviposition. Expression of Lsdia1 (only in the dextral strain) and Lsdia2 (in both chirality) decreases after the 1-cell stage, with no asymmetric localization throughout. The evolutionary relationships among body handedness, SD/SI (spiral deformation/spindle inclination) at the third cleavage, and expression of diaphanous proteins are discussed in comparison with three other pond snails (L. peregra, Physa acuta and Indoplanorbis exustus). PMID:27708420

  19. Mutation analysis of the WFS1 gene in seven Danish Wolfram syndrome families; four new mutations identified

    DEFF Research Database (Denmark)

    Hansen, Lars; Eiberg, Hans Rudolf Lytchoff; Barrett, Timothy


    Wolfram syndrome (WS) is a neuro-degenerative autosomal recessive (AR) disorder (OMIM #222300) caused by mutations in the WFS1 gene on 4p16.1. More than 120 mutations have been identified in WFS1 associated with AR WS, as well as autosomal dominant nonsyndromic low-frequency sensorineural hearing...

  20. Generation of KCL035 research grade human embryonic stem cell line carrying a mutation in HBB gene

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    Heema Hewitson


    Full Text Available The KCL035 human embryonic stem cell line was derived from an embryo donated for research that carried a mutation in the HBB gene, which is linked to the β-thalassemia syndrome. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays.

  1. α-Hexylcinnamaldehyde inhibits the genotoxicity of environmental pollutants in the bacterial reverse mutation assay. (United States)

    Di Giacomo, Silvia; Mazzanti, Gabriela; Sarpietro, Maria Grazia; Di Sotto, Antonella


    The antimutagenicity of α-hexylcinnamaldehyde (1), a semisynthetic and more stable derivative of cinnamaldehyde, was evaluated against common environmental pollutants in the bacterial reverse mutation assay. The pre-, co-, and post-treatment protocols were applied to assess the involvement of desmutagenic and/or bioantimutagenic mechanisms. Compound 1 (9-900 μM) produced a strong antimutagenicity (>40% inhibition) in the Salmonella typhimurium TA98 strain against the nitroarenes 2-nitrofluorene and 1-nitropyrene in almost all experimental conditions. A strong inhibition was also reached against the nitroarene 1,8-dinitropyrene and the arylamine 2-aminoanthracene in the cotreatment at the highest concentrations tested. In order to evaluate if an inhibition of bacterial nitroreductase (NR) and O-acetyltransferase (OAT) could be involved in the antimutagenicity of 1 against nitroarenes, the substance was further tested against 1-nitropyrene (activated by both NR and OAT) in TA98NR and TA98 1,8-DNP strains (lacking the NR and OAT enzymes, respectively). Although both desmutagenic and bioantimutagenic mechanisms appear mostly involved in the antimutagenicity of 1, based on data obtained in the TA98NR strain, applying the pretreatment protocol, compound 1 seems to act as an inhibitor of the OAT-mediated mutagen bioactivation. These results provide justification for further studies on 1 as a possible chemopreventive agent.

  2. Novel chloride channel gene mutations in two unrelated Chinese families with myotonia congenita

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    Gao Feng


    Full Text Available Myotonia congenita (MC is a genetic disease characterized by mutations in the muscle chloride channel gene (CLCN1. To date, approximately 130 different mutations on the CLCN1 gene have been identified. However, most of the studies have focused on Caucasians, and reports on CLCN1 mutations in Chinese population are rare. This study investigated the mutation of CLCN1 in two Chinese families with MC. Direct sequencing of the CLCN1 gene revealed a heterozygous mutation (892G>A, resulting in A298T in one family and a compound heterozygous mutations (782A>G, resulting in Y261C; 1679T>C, resulting in M560T in the other family, None of the 100 normal controls had these mutations. Our findings add more to the available information on the CLCN1 mutation spectrum, and provide a valuable reference for studying the mutation types and inheritance pattern of CLCN1 in the Chinese population.

  3. Familial adenomatous polyposis associated APC gene mutation - A case study

    Directory of Open Access Journals (Sweden)

    Avinash Bardia1, Santosh K. Tiwari1, Sandeep K. Vishwakarma1, Md. Aejaz Habeeb1, Pratibha Nallari2, Aleem A. Khan1


    Full Text Available Familial adenomatous polyposis (FAP is an autosomal dominant condition characterized by diffuse intestinal polyposis, specific gene mutation, and predisposition for developing colon cancer. Left untreated, patients with FAP will develop colorectal carcinoma during early adulthood. Hence, early detection and surgical intervention are of the utmost importance. Colectomy is required and may include an ileal pouch with ileo-anal anastomosis, which eli-minates the colon and rectal disease while preserving fecal continence and avoidance of a permanent ileostomy. We report a case of colorectal cancer along with FAP showed features consistent with adenomatous polyposis coli and no evidence of malignancy was seen after the surgery.


    Directory of Open Access Journals (Sweden)

    M. Yu. Fedyanin


    Full Text Available Colon cancer represents a heterogenous disease group, which differ by cancerogenesis mechanisms, molecular alterations, prognosis and treatment possibilities. In modern clinical practice assessment of KRAS and NRAS genes status is already necessary in order to prescribe anti-EGFR treatment for metastatic disease. A separate poor prognosis group are patients with BRAF mutation. In this review we will focus on biological features of BRAF-mutant colorectal cancer, its epidemiology, clinical features on different stages, treatment choice and promising new treatment possibilities.

  5. Designing, optimization and validation of tetra-primer ARMS PCR protocol for genotyping mutations in caprine Fec genes. (United States)

    Ahlawat, Sonika; Sharma, Rekha; Maitra, A; Roy, Manoranjan; Tantia, M S


    New, quick, and inexpensive methods for genotyping novel caprine Fec gene polymorphisms through tetra-primer ARMS PCR were developed in the present investigation. Single nucleotide polymorphism (SNP) genotyping needs to be attempted to establish association between the identified mutations and traits of economic importance. In the current study, we have successfully genotyped three new SNPs identified in caprine fecundity genes viz. T(-242)C (BMPR1B), G1189A (GDF9) and G735A (BMP15). Tetra-primer ARMS PCR protocol was optimized and validated for these SNPs with short turn-around time and costs. The optimized techniques were tested on 158 random samples of Black Bengal goat breed. Samples with known genotypes for the described genes, previously tested in duplicate using the sequencing methods, were employed for validation of the assay. Upon validation, complete concordance was observed between the tetra-primer ARMS PCR assays and the sequencing results. These results highlight the ability of tetra-primer ARMS PCR in genotyping of mutations in Fec genes. Any associated SNP could be used to accelerate the improvement of goat reproductive traits by identifying high prolific animals at an early stage of life. Our results provide direct evidence that tetra-primer ARMS-PCR is a rapid, reliable, and cost-effective method for SNP genotyping of mutations in caprine Fec genes.

  6. Designing, optimization and validation of tetra-primer ARMS PCR protocol for genotyping mutations in caprine Fec genes

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    Sonika Ahlawat


    Full Text Available New, quick, and inexpensive methods for genotyping novel caprine Fec gene polymorphisms through tetra-primer ARMS PCR were developed in the present investigation. Single nucleotide polymorphism (SNP genotyping needs to be attempted to establish association between the identified mutations and traits of economic importance. In the current study, we have successfully genotyped three new SNPs identified in caprine fecundity genes viz. T(-242C (BMPR1B, G1189A (GDF9 and G735A (BMP15. Tetra-primer ARMS PCR protocol was optimized and validated for these SNPs with short turn-around time and costs. The optimized techniques were tested on 158 random samples of Black Bengal goat breed. Samples with known genotypes for the described genes, previously tested in duplicate using the sequencing methods, were employed for validation of the assay. Upon validation, complete concordance was observed between the tetra-primer ARMS PCR assays and the sequencing results. These results highlight the ability of tetra-primer ARMS PCR in genotyping of mutations in Fec genes. Any associated SNP could be used to accelerate the improvement of goat reproductive traits by identifying high prolific animals at an early stage of life. Our results provide direct evidence that tetra-primer ARMS-PCR is a rapid, reliable, and cost-effective method for SNP genotyping of mutations in caprine Fec genes.

  7. NGS-Based Assay for the Identification of Individuals Carrying Recessive Genetic Mutations in Reproductive Medicine. (United States)

    Abulí, Anna; Boada, Montserrat; Rodríguez-Santiago, Benjamín; Coroleu, Buenaventura; Veiga, Anna; Armengol, Lluís; Barri, Pedro N; Pérez-Jurado, Luis A; Estivill, Xavier


    Next-generation sequencing (NGS) has the capacity of carrier screening in gamete donation (GD) programs. We have developed and validated an NGS carrier-screening test (qCarrier test) that includes 200 genes associated with 368 disorders (277 autosomal recessive and 37 X-linked). Carrier screening is performed on oocyte donation candidates and the male partner of oocyte recipient. Carriers of X-linked conditions are excluded from the GD program, whereas donors are chosen who do not carry mutations for the same gene/disease as the recipients. The validation phase showed a high sensitivity (>99% sensitivity) detecting all single-nucleotide variants, 13 indels, and 25 copy-number variants included in the validation set. A total of 1,301 individuals were analysed with the qCarrier test, including 483 candidate oocyte donors and 635 receptor couples, 105 females receiving sperm donation, and 39 couples seeking pregnancy. We identified 56% of individuals who are carriers for at least one genetic condition and 1.7% of female donors who were excluded from the program due to a carrier state of X-linked conditions. Globally, 3% of a priori assigned donations had a high reproductive risk that could be minimized after testing. Genetic counselling at different stages is essential for helping to facilitate a successful and healthy pregnancy.

  8. Exome Sequencing Reveals Cubilin Mutation as a Single-Gene Cause of Proteinuria


    Ovunc, Bugsu; Otto, Edgar A.; Vega-Warner, Virginia; Saisawat, Pawaree; Ashraf, Shazia; Ramaswami, Gokul; Fathy, Hanan M.; Schoeb, Dominik; Chernin, Gil; Lyons, Robert H.; Engin YILMAZ; Hildebrandt, Friedhelm


    In two siblings of consanguineous parents with intermittent nephrotic-range proteinuria, we identified a homozygous deleterious frameshift mutation in the gene CUBN, which encodes cubulin, using exome capture and massively parallel re-sequencing. The mutation segregated with affected members of this family and was absent from 92 healthy individuals, thereby identifying a recessive mutation in CUBN as the single-gene cause of proteinuria in this sibship. Cubulin mutations cause a hereditary fo...

  9. Epidural Analgesia with Ropivacaine during Labour in a Patient with a SCN5A Gene Mutation

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    A. L. M. J. van der Knijff-van Dortmont


    Full Text Available SCN5A gene mutations can lead to ion channel defects which can cause cardiac conduction disturbances. In the presence of specific ECG characteristics, this mutation is called Brugada syndrome. Many drugs are associated with adverse events, making anesthesia in patients with SCN5A gene mutations or Brugada syndrome challenging. In this case report, we describe a pregnant patient with this mutation who received epidural analgesia using low dose ropivacaine and sufentanil during labour.

  10. A mutation in a functional Sp1 binding site of the telomerase RNA gene (hTERC promoter in a patient with Paroxysmal Nocturnal Haemoglobinuria

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    Mason Philip J


    Full Text Available Abstract Background Mutations in the gene coding for the RNA component of telomerase, hTERC, have been found in autosomal dominant dyskeratosis congenita (DC and aplastic anemia. Paroxysmal nocturnal hemoglobinuria (PNH is a clonal blood disorder associated with aplastic anemia and characterized by the presence of one or more clones of blood cells lacking glycosylphosphatidylinositol (GPI anchored proteins due to a somatic mutation in the PIGA gene. Methods We searched for mutations in DNA extracted from PNH patients by amplification of the hTERC gene and denaturing high performance liquid chromatography (dHPLC. After a mutation was found in a potential transcription factor binding site in one patient electrophoretic mobility shift assays were used to detect binding of transcription factors to that site. The effect of the mutation on the function of the promoter was tested by transient transfection constructs in which the promoter is used to drive a reporter gene. Results Here we report the finding of a novel promoter mutation (-99C->G in the hTERC gene in a patient with PNH. The mutation disrupts an Sp1 binding site and destroys its ability to bind Sp1. Transient transfection assays show that mutations in this hTERC site including C-99G cause either up- or down-regulation of promoter activity and suggest that the site regulates core promoter activity in a context dependent manner in cancer cells. Conclusions These data are the first report of an hTERC promoter mutation from a patient sample which can modulate core promoter activity in vitro, raising the possibility that the mutation may affect the transcription of the gene in hematopoietic stem cells in vivo, and that dysregulation of telomerase may play a role in the development of bone marrow failure and the evolution of PNH clones.

  11. A novel Italian presenilin 2 gene mutation with prevalent behavioral phenotype. (United States)

    Marcon, Gabriella; Di Fede, Giuseppe; Giaccone, Giorgio; Rossi, Giacomina; Giovagnoli, Anna Rita; Maccagnano, Elio; Tagliavini, Fabrizio


    Presenilin mutations are the main cause of familial Alzheimer's disease. So far, more than 160 mutations in the Presenilin 1 gene (PSEN1) and approximately 10 mutations in the homologous Presenilin 2 gene (PSEN2) have been identified. Some PSEN1 mutations are associated with a phenotype fulfilling the clinical criteria of frontotemporal dementia. In PSEN2, T122P and M239V mutations presented with severe behavioral disturbances. We describe an Italian patient with a novel PSEN2 mutation (Y231C) who showed behavioral abnormalities and language impairment as presenting symptoms, with later involvement of other cognitive abilities, particularly of posterior functions.

  12. Analysis of FUS gene mutation in familial amyotrophic lateral sclerosis within an Italian cohort. (United States)

    Ticozzi, N; Silani, V; LeClerc, A L; Keagle, P; Gellera, C; Ratti, A; Taroni, F; Kwiatkowski, T J; McKenna-Yasek, D M; Sapp, P C; Brown, R H; Landers, J E


    Mutations in the FUS gene on chromosome 16 have been recently discovered as a cause of familial amyotrophic lateral sclerosis (FALS). This study determined the frequency and identities of FUS gene mutations in a cohort of Italian patients with FALS. We screened all 15 coding exons of FUS for mutations in 94 Italian patients with FALS. We identified 4 distinct missense mutations in 5 patients; 2 were novel. The mutations were not present in 376 healthy Italian controls and thus are likely to be pathogenic. Our results demonstrate that FUS mutations cause approximately 4% of familial amyotrophic lateral sclerosis cases in the Italian population.

  13. No mutations found in exons of TP53, H-RAS and K-RAS genes in liver of male Wistar rats submitted to a medium-term chemical carcinogenesis assay Ausência de mutações em éxons dos genes TP53, H-RAS e K-RAS em fígado de ratos wistar submetidos a ensaio de carcinogênese química de média duração

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    Erick da Cruz Castelli


    Full Text Available The standard protocol to evaluate the carcinogenic potential of chemicals is the long-term bioassay in rodents, not performed in developing countries due to its high cost and complex operational procedures. Our laboratory has established an alternative medium-term bioassay in Wistar rats, also called DMBDD assay, based on the paradigm initiation/promotion of chemical carcinogenesis. This method was accepted by the Brazilian Environment Agency (IBAMA as an official source of evidence of carcinogenicity. The aim of this study was to evaluate alterations in exons 5 to 8 of the tumor suppressor gene TP53 and exons 1 and 2 of oncogenes K-RAS and H-RAS in neoplastic and preneoplastic hepatic lesions observed in DMBDD assay. The characterization of these alterations may contribute to the recognition of patterns of damage in critical genes, as well as to suggest mechanisms of action of the compounds tested in the protocol. Sixty male Wistar rats were separated into 3 groups: the first was treated with no chemicals; the second received five initiating agents and the third received initiation followed by phenobarbital. Liver DNA samples (obtained from formalin-fixed and paraffin-embedded tissues after histological analysis were evaluated by the non-isotopic PCR-SSCP technique. No changes in any analyzed exons were detected by the PCR-SSCP banding pattern in all experimental groups. This result suggests that liver mutations in exons 5 to 8 of TP53 and exons 1 and 2 of H-RAS and K-RAS are not among the early molecular alterations occurring in the hepatic carcinogenesis process induced by the DMBDD protocol in male Wistar rats.O teste padrão para identificar o potencial cancerígeno de compostos químicos é o estudo de longa duração em roedores, não realizado no Brasil. Nosso laboratório estabeleceu um teste alternativo de média duração (ensaio DMBDD, baseado no paradigma iniciação-promoção da carcinogênese química, adotado pelo Instituto

  14. Mutations in the gene region of hepatitis B virus genotype in Turkish patients

    Indian Academy of Sciences (India)

    Mehmet Özaslan; Ersan Özaslan; Arzu Barsgan; Mehmet Koruk


    The gene region of the hepatitis B virus (HBV) is responsible for the expression of surface antigens and includes the ‘a’-determinant region. Thus, mutation(s) in this region would afford HBV variants a distinct survival advantage, permitting the mutant virus to escape from the immune system. The aim of this study was to search for mutations of the gene region in different patient groups infected with genotype variants of HBV, and to analyse the biological significance of these mutations. Moreover, we investigated gene mutation inductance among family members. Forty HBV-DNA-positive patients were determined among 132 hepatitis B surface antigen (HbsAg) carriers by the first stage of seminested PCR. Genotypes and subtypes were established by sequencing of the amplified S gene regions. Variants were compared with original sequences of these serotypes, and mutations were identified. All variants were designated as genotype and subtype ayw3. Ten kinds of point mutations were identified within the region. The highest rates of mutation were found in chronic hepatitis patients and their family members. The amino acid mutations 125 (M → T) and 127 (T → P) were found on the first loop of ‘a’-determinant. The other consequence was mutation inductance in a family member. We found some mutations in the S gene region known to be stable and observed that some of these mutations affected gene expression.

  15. Identification of transforming hepatitis B virus S gene nonsense mutations derived from freely replicative viruses in hepatocellular carcinoma.

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    Shiu-Feng Huang

    Full Text Available BACKGROUND & AIMS: The correlation between chronic hepatitis B virus (HBV infection and hepatocellular carcinoma (HCC has been well-established. But the roles of viral factor remain uncertain. Only HBV X gene and nonsense mutations of S gene (C-terminal truncation of HBV surface protein have been demonstrated to have transforming activity. Whether they play a significant role in hepatocarcinogenesis is still uncertain. METHODS: Twenty-five HBV-related HCC patients were positive for hepatitis B core antigen (HBcAg in the cancerous parts of their HCC liver tissues by immunohistochemistry studies, and had available tissue for whole HBV genome sequence analysis. The results were compared with 25 gender and age-matched HBcAg negative HCCs. Plasmids encoding HBV S gene nonsense mutations identified from HBcAg (+ HCC tissue were constructed to investigate their cell proliferation, transformation activity and the oncogenic potentials by xenograft study and in vivo migration assay. RESULTS: HBcAg (+ HCC patients were significantly associated with cirrhosis and small tumor size (≦2 cm when compared with HBcAg (- HCC patients. Southern blot analyses revealed freely replicative forms of HBV in the cancerous parts of HBcAg(+ HCC. Three nonsense mutations of S gene (sL95*, sW182*, and sL216* were identified in the HBcAg(+ HCC tumor tissues. sW182* and sL216* were recurrently found in the 25 HBcAg (- HCC tumor tissue, too. Functional studies of the above 3 non-sense mutations all demonstrated higher cell proliferation activities and transformation abilities than wild type S, especially sW182*. Tumorigenicity analysis by xenograft experiments and in vitro migration assay showed potent oncogenic activity of sW182* mutant. CONCLUSIONS: This study has demonstrated potent oncogenic activity of nonsense mutations of HBV S gene, suggesting they may play an important role in hepatocarcinogenesis.

  16. Exome mutation burden predicts clinical outcome in ovarian cancer carrying mutated BRCA1 and BRCA2 genes

    DEFF Research Database (Denmark)

    Birkbak, Nicolai Juul; Kochupurakkal, Bose; Gonzalez-Izarzugaza, Jose Maria;


    Reliable biomarkers predicting resistance or sensitivity to anti-cancer therapy are critical for oncologists to select proper therapeutic drugs in individual cancer patients. Ovarian and breast cancer patients carrying germline mutations in BRCA1 or BRCA2 genes are often sensitive to DNA damaging...... drugs and relative to non-mutation carriers present a favorable clinical outcome following therapy. Genome sequencing studies have shown a high number of mutations in the tumor genome in patients carrying BRCA1 or BRCA2 mutations (mBRCA). The present study used exome-sequencing and SNP 6 array data...... had either germlines or somatic mutations of BRCA1 or BRCA2 genes. The results revealed that the Nmut was significantly lower in the chemotherapy-resistant mBRCA HGSOC defined by progression within 6 months after completion of first line platinum-based chemotherapy. We found a significant association...

  17. GJB2 (Cx26) gene mutations in Chinese patients with congenital sensorineural deafness and a report of one novel mutation

    Institute of Scientific and Technical Information of China (English)

    肖自安; 谢鼎华


    Background Mutations in GJB2 gene are a major cause of autosomal recessive congenital hearing loss and the cause in some rare cases of the autosomal dominant form. The purpose of This study was to investigate the frequency and the features of GJB2 mutations in the Chinese patients with congenital sensorineural deafness. Methods Using PCR amplifying the entire coding region of GJB2 gene and direct DNA sequencing to analyze mutations in this gene among unrelated 69 cases with autosomal recessive congenital nonsyndromic deafness and 27 cases of dominant congenital deafness and 35 sporadic cases. We also detected mutations in GJB2 in 100 control subjects with normal hearing.Results 17.4% (12/69) of the probands in the autosomal recessive, 7.4% (2/27) of dominant families and 5.7% (2/35) of the sporadic congenital deafness patients had deafness-causing mutations in GJB2, respectively. Nine types of the mutations in GJB2 were detected in the recessive and sporadic group. They consisted of five types of polymorphism, and four types of deafness-causing mutation with homozygous 35delG in 1 sporadic (1/35), and 235delC frameshift mutation in 1 sporadic (homozygotes) and 10 recessive patients (2 heterozygotes and 8 homozygotes), and homozygous 442G→A missense mutation and homozygous 465T→A nonsense mutation in 1 different recessive proband, respectively. The 465T→A that related to recessive deafness was a novel mutation found by this study. The homozygous (10/69, 14.5%) and the heterozygous (2/69, 2.9%) GJB2 mutation in the recessive patients (12/69, 17.4%) and the homozygotes in the sporadic patient (2/35, 5.7%) all had congenital severe to profound sensorineural hearing loss. 511G→A missense mutation and 299-300delAT frameshift mutation were found in two autosomal dominant congenital deafness families (2/27, 7.4%). The total mutation frequency of GJB2 was 12.2% (16/131) in the Chinese patients with congenital sensorineural deafness and 235delC was the most common

  18. Comparison of Xpert MTB/RIF Assay and GenoType MTBDRplus DNA Probes for Detection of Mutations Associated with Rifampicin Resistance in Mycobacterium tuberculosis.

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    Arfatur Rahman

    Full Text Available GeneXpert MTB/RIF (Xpert and Genotype MTBDRplus (DRplus are two World Health Organization (WHO endorsed probe based molecular drug susceptibility testing (DST methods for rapid diagnosis of drug resistant tuberculosis. Both methods target the same 81 bp Rifampicin Resistance Determining Region (RRDR of bacterial RNA polymerase β subunit (rpoB for detection of Rifampicin (RIF resistance associated mutations using DNA probes. So there is a correspondence of the probes of each other and expected similarity of probe binding.We analyzed 92 sputum specimens by Xpert, DRplus and LJ proportion method (LJ-DST. We compared molecular DSTs with gold standard LJ-DST. We wanted to see the agreement level of two molecular methods for detection of RIF resistance associated mutations. The 81bp RRDR region of rpoB gene of discrepant cases between the two molecular methods was sequenced by Sanger sequencing.The agreement of Xpert and DRplus with LJ-DST for detection of RIF susceptibility was found to be 93.5% and 92.4%, respectively. We also found 92.4% overall agreement of two molecular methods for the detection of RIF susceptibility. A total of 84 out of 92 samples (91.3% had agreement on the molecular locus of RRDR mutation by DRplus and Xpert. Sanger sequencing of 81bp RRDR revealed that Xpert probes detected seven of eight discrepant cases correctly and DRplus was erroneous in all the eight cases.Although the overall concordance with LJ-DST was similar for both Xpert and DRplus assay, Xpert demonstrated more accuracy in the detection of RIF susceptibility for discrepant isolates compared with DRplus. This observation would be helpful for the improvement of probe based detection of drug resistance associated mutations especially rpoB mutation in M. tuberculosis.

  19. GNAS gene mutation may be present only transiently during colorectal tumorigenesis (United States)

    Zauber, Peter; Marotta, Stephen P; Sabbath-Solitare, Marlene


    Mutations of the gene GNAS have been shown to activate the adenylate cyclase gene and lead to constitutive cAMP signaling. Several preliminary reports have suggested a role for GNAS gene mutations during colorectal carcinogenesis, particularly mucinous carcinomas. The aim of this study was to clarify the incidence of GNAS mutations in adenomas (tubular, tubulovillous, and villous), carcinomas with residual adenoma, and carcinomas, and to relate these findings to mutations of the KRAS gene and to the mucinous status of the tumors. We used standard PCR techniques and direct gene sequencing to evaluate tumors for gene mutations. No GNAS mutations were identified in 25 tubular adenomas, but were present in 6.4% of tubulovillous adenomas and 11.2% of villous adenomas. A GNAS mutation was found in 9.7% of the benign portion of carcinoma with residual adenoma, but in none of 86 carcinomas. A similar trend was seen for KRAS mutation across the five groups of tumors. GNAS mutations may function as an important driver mutation during certain phases of colorectal carcinogenesis, but may then be lost once the biological advantage gained by the mutated gene is no longer necessary to sustain or advance tumor development. PMID:27186325

  20. Mutations in the glucose-6-phosphatase gene that cause glycogen storage disease type 1a

    Energy Technology Data Exchange (ETDEWEB)

    Chou, J.Y.; Lei, K.J.; Shelly, L.L. [National Institutes of Health, Bethesda, MD (United States)


    Glycogen storage disease (GSD) type la (von Gierke disease) is caused by the deficiency of glucose-6-phosphatase (G6Pase), the key enzyme in glucose homeostasis. The disease presents with clinical manifestations of severe hypoglycemia, hepatomegaly, growth retardation, lactic acidemia, hyperlipidemia, and hyperuricemia. We have succeeded in isolating a murine G6Pase cDNA from a normal mouse liver cDNA library by differentially screening method. We then isolated the human G6Pase cDNA and gene. To date, we have characterized the G6Pase genes of twelve GSD type la patients and uncovered a total of six different mutations. The mutations are comprised of R83C (an Arg at codon 83 to a Cys), Q347X (a Gly at codon 347 to a stop codon), 459insTA (a two basepair insertion at nucleotide 459 yielding a truncated G6Pase of 129 residues), R295C (an Arg at codon 295 to a Cys), G222R (a Gly at codon 222 to an Arg) and {delta}F327 (a codon deletion for Phe-327 at nucleotides 1058 to 1060). The relative incidences of these mutations are 37.5% (R83C), 33.3% (Q347X), 16.6% (459insTA), 4.2% (G222R), 4.2% (R295C) and 4.2% ({delta}F327). Site-directed mutagenesis and transient expression assays demonstrated that the R83C, Q347X, R295C, and {delta}F327 mutations abolished whereas the G222R mutation greatly reduced G6Pase activity. We further characterized the structure-function requirements of amino acids 83, 222, and 295 in G6Pase catalysis. The identification of mutations in GSD type la patients has unequivocally established the molecular basis of the type la disorder. Knowledge of the mutations may be applied to prenatal diagnosis and opens the way for developing and evaluating new therapeutic approaches.

  1. Phenotype and clinical course in a family with a new de novo Twinkle gene mutation

    DEFF Research Database (Denmark)

    Jeppesen, Tina Dysgaard; Schwartz, M.; Colding-Jorgensen, E.;


    The Twinkle gene product is important for mtDNA replication. Only a few reports have investigated the clinically effect of mutations in this gene. We describe a new de novo mutation (1110C > A) in the PEO1 gene in a mother and her two sons. The mother had progressive ophthalmoplegia, limb weakness...

  2. Social Health Insurance-Based Simultaneous Screening for 154 Mutations in 19 Deafness Genes Efficiently Identified Causative Mutations in Japanese Hearing Loss Patients


    Mori, Kentaro; Moteki, Hideaki; Miyagawa, Maiko; Nishio, Shin-Ya; Usami, Shin-ichi


    Sensorineural hearing loss is one of the most common neurosensory disorders in humans. The incidence of SNHL is estimated to be 1 in 500–1000 newborns. In more than half of these patients, the hearing loss is associated with genetic causes. In Japan, genetic testing for the patients with SNHL using the Invader assay to screen for 46 mutations in 13 deafness genes was approved by the Ministry of Health, Labour and Welfare for inclusion in social health insurance coverage in 2012. Furthermore, ...

  3. Missense mutations of the fibrillin-1 gene in two Chinese patients with severe Marfan syndrome

    Institute of Scientific and Technical Information of China (English)


    Objective To describe two Chinese patients with severe forms of Marfan syndrome and to report findings of mutational analysis of the fibrillin-1 (FBN1) gene.Methods Two Chinese patients were studied, one suffering from Marfan syndrome of infantile onset and the other of neonatal onset. Their clinical features were described. Mutational analysis of the FBN1 gene was performed using polymerase chain reaction (PCR) technique and direct sequencing of exons 23 - 32,where the mutational hotspots for severe forms of Marfan syndrome are located.Results Two missense mutations were successfully identified, a G3037A transition and an A3083T transversion, the latter being an unreported mutation.Conclusion Taking advantage of the clustering phenomenon of mutations in severe forms of Marfan syndrome, one can identify FBN1 mutations in these patients by first screening the mutational hotspots,thus reducing the effort that would otherwise be much greater because of the size of the gene.

  4. A study of familial Mediterranean Fever (MEFV) gene mutations in Egyptian children with type 1 diabetes mellitus. (United States)

    Anwar, Ghada Mohammad; Fouad, Hanan M; Abd El-Hamid, Amal; Mahmoud, Faten; Musa, Noha; Lotfi, Hala; Salah, Nermine


    An association of type 1 DM and familial Mediterranean fever (FMF) has been newly reported in the medical literature. The aim of the present work was to investigate frequency of MEFV gene mutations in Egyptian children with type 1 diabetes mellitus. Forty five children with type 1 DM were screened for Mediterranean Fever (MEFV) gene mutation. Forty one healthy control subjects were included. Identification of FMF gene mutation was done based on polymerase chain reaction (PCR) and reverse hybridization. The assay covers 12 mutations in the FMF gene: E148Q - P369S - F479L - M680I (G/C) - M680I (G/A) - I692del - M694V - M694I - K695R-V726A - A744S and R761H. Among the screened diabetics, the overall frequency of MEFV gene mutations was 42.2% and among the control group it was 34.1% with no significant difference. Fourteen out of 45 diabetic children (31.1%) were heterozygous (E148Q in 7 children, A744S in 3 children, V726A in 2 children, M680I (G/C) in 1 child and P369S in1 child), while 5 children (11.1%) were compound heterozygous (M694V/M694I in 2 children, E148Q/K695R mutations in 1 child, E148Q/M694I in 1 child and E148Q/V726A in 1 child). The control group showed heterozygous mutation in 34.1% of cases (E148Q mutation in 14.6%, V726A in 12.2%, M680I (G/C) in 4.9% and M694V in 2.4%). No significant difference in mutation frequency between diabetic and non-diabetic children. We have high carrier rate of MEFV gene mutations among Egyptian population probably due to high consanguinity. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  5. Relationship between mutations of mitochondrial DNA ND1 gene and type 2 diabetes

    Institute of Scientific and Technical Information of China (English)

    于珮; 于德民; 刘德敏; 王琨; 汤新之


    Background Recent studies have indicated that many mutations in mitochondrial (mt)DNA NDI gene region are related to diabetes mellitus. In this study we explored the relationship between various mtDNA ND1 gene mutations and type 2 diabetes mellitus (DM) among Chinese. Methods Using PCR restriction fragment length polymorphism (PCR-RFLP) analysis and gene sequencing, 4 spots of mtDNA (nt3243, nt3316, nt3394, nt3426) were screened in 478 diabetics and 430 non-diabetic subjects.Results In diabetic group, there were 13 carriers (2.72%)of 3316 G→A mutation,12 (2.51%) of 3394 T→C mutation and 2 (0.42%) of 3426A→G mutation. In controls, only 3394 T→C mutation was observed in 2 subjects (0.47%). There was significant difference in the frequency of 3316 and 3394 mutation between two groups (P<0.05, respectively). More subjects with mitochondrial DNA ND1 gene mutations had DM family history and greater tendency of maternal inheritance when compared to those patients without mutation in diabetic group(P<0.01). A 3426 mutation diabetic pedigree was studied, and we found 12 maternal members in the family had the same mutation. Conclusion mtDNA ND1 gene mutations at nt3316 (G→A), nt3394 (T→C) and 3426 (A→G) might contribute to the pathogenesis of DM with other genetic factors and environment factors.

  6. Mutation Analysis in the BRCA1 Gene in Chinese Breast Cancer Families

    Institute of Scientific and Technical Information of China (English)

    WUZhengyan; ZHENLinlin; FANPing


    Objective: To study the mutation of BRCA1 gene in Chinese breast cancer families. Methods:Fifteen families were selected, involving 41 members, consisting of 23 breast cancer patients. Using poly-merase chain reaction and single stranded conformation polymorphism (PCR-SSCP), and subsequent DNA sequencing, the mutation of BRCA1 genes were analyzed. Results: Four mutations were found in all fam-ilies, and the proportion of mutation was 26.7% (4/15) in breast cancer families. One of the 4 mutations was 2228 insC, resulting in chain termination at codon 711. The remaining 3 mutations were 1884A→T and 3232A→G, resulting in single amino acid change respectively. Conclusion: BRCA1 is a breast cancer susceptibility gene. The relatively low proportion and frequency of BRCA1 mutations in our study hints additional BRCA genes existed.

  7. Detection of ATP2C1 Gene Mutation in Familial Benign Chronic Pemphigus

    Institute of Scientific and Technical Information of China (English)


    The ATP2C1 gene mutation in one case of familial benign chronic pemphigus was investigated.One patient was diagnosed as familial benign chronic pemphigus by pathology, ultrastructral examination and clinical features. Genomic DNA was extracted from blood samples. Mutation of ATP2C1 gene was detected by polymerase chain reaction (PCR) and DNA sequencing. The results showed that deletion mutation was detected in ATP2C1 gene in this patient, which was 2374delTTTG. No mutation was found in the family members and normal individuals. It was concluded that the 2374delTTTG mutation in ATP2C1 gene was the specific mutation for the clinical phenotype for this patient and was a de novo mutation.

  8. A de novo mutation in CYP21A2 gene in a case of in vitro fertilization

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    Roseane Lopes da Silva-Grecco


    Full Text Available Congenital adrenal hyperplasia, one of the most frequent autosome recessive disorders, is caused by defects in steroidogenic enzymes involved in the cortisol biosynthesis. Approximately 95% of the cases are caused by abnormal function of the 21-hydroxylase enzyme. This deficiency leads to androgen excess, consequently, to virilization and rapid somatic growth with accelerated skeletal maturation. Mutations in CYP21A2 are responsible for different forms of 21-hydroxylase deficiency. Mild impairment in the enzymatic activity causes the non-classic or late-onset congenital adrenal hyperplasia that is observed with a prevalence of 1 in 1000 subjects in different populations. The present paper describes a de novo mutation that occurred in the paternal meiosis. The child, who was conceived by in vitro fertilization, presented with precocious puberty and diagnosed with non-classical 21-hydroxylase deficiency. DNA sequencing showed the compound heterozygosis for a de novo CYP21A1P/A2 chimeric gene and the p.Val281Leu mutation inherited from her mother, who was heterozygous for the mutation. The chimeric gene showed pseudogene-derived sequence from 5′-end to intron 3 and CYP21A2 sequences from intron 3 to 3′-end of the gene. Sequencing analysis of the father did not show any mutation. The multiplex ligation-dependent probe amplification (MLPA assay did not indicate loss of DNA discarding gene deletion but confirmed the chimeric gene. In addition, supernumerary copies of CYP21A1P were observed for both parents and for the affect child. Since paternity has been confirmed, those results suggest that a de novo large gene conversion in the paternal meiosis could have occurred by misalignment of alleles bearing different copy numbers of genes in CYP21 locus.

  9. Melt analysis of mismatch amplification mutation assays (Melt-MAMA: a functional study of a cost-effective SNP genotyping assay in bacterial models.

    Directory of Open Access Journals (Sweden)

    Dawn N Birdsell

    Full Text Available Single nucleotide polymorphisms (SNPs are abundant in genomes of all species and biologically informative markers extensively used across broad scientific disciplines. Newly identified SNP markers are publicly available at an ever-increasing rate due to advancements in sequencing technologies. Efficient, cost-effective SNP genotyping methods to screen sample populations are in great demand in well-equipped laboratories, but also in developing world situations. Dual Probe TaqMan assays are robust but can be cost-prohibitive and require specialized equipment. The Mismatch Amplification Mutation Assay, coupled with melt analysis (Melt-MAMA, is flexible, efficient and cost-effective. However, Melt-MAMA traditionally suffers from high rates of assay design failures and knowledge gaps on assay robustness and sensitivity. In this study, we identified strategies that improved the success of Melt-MAMA. We examined the performance of 185 Melt-MAMAs across eight different pathogens using various optimization parameters. We evaluated the effects of genome size and %GC content on assay development. When used collectively, specific strategies markedly improved the rate of successful assays at the first design attempt from ~50% to ~80%. We observed that Melt-MAMA accurately genotypes across a broad DNA range (~100 ng to ~0.1 pg. Genomic size and %GC content influence the rate of successful assay design in an independent manner. Finally, we demonstrated the versatility of these assays by the creation of a duplex Melt-MAMA real-time PCR (two SNPs and conversion to a size-based genotyping system, which uses agarose gel electrophoresis. Melt-MAMA is comparable to Dual Probe TaqMan assays in terms of design success rate and accuracy. Although sensitivity is less robust than Dual Probe TaqMan assays, Melt-MAMA is superior in terms of cost-effectiveness, speed of development and versatility. We detail the parameters most important for the successful application of

  10. Identification and glycerol-induced correction of misfolding mutations in the X-linked mental retardation gene CASK.

    Directory of Open Access Journals (Sweden)

    Leslie E W LaConte

    Full Text Available The overwhelming amount of available genomic sequence variation information demands a streamlined approach to examine known pathogenic mutations of any given protein. Here we seek to outline a strategy to easily classify pathogenic missense mutations that cause protein misfolding and are thus good candidates for chaperone-based therapeutic strategies, using previously identified mutations in the gene CASK. We applied a battery of bioinformatics algorithms designed to predict potential impact on protein structure to five pathogenic missense mutations in the protein CASK that have been shown to underlie pathologies ranging from X-linked mental retardation to autism spectrum disorder. A successful classification of the mutations as damaging was not consistently achieved despite the known pathogenicity. In addition to the bioinformatics analyses, we performed molecular modeling and phylogenetic comparisons. Finally, we developed a simple high-throughput imaging assay to measure the misfolding propensity of the CASK mutants in situ. Our data suggests that a phylogenetic analysis may be a robust method for predicting structurally damaging mutations in CASK. Mutations in two evolutionarily invariant residues (Y728C and W919R exhibited a strong propensity to misfold and form visible aggregates in the cytosolic milieu. The remaining mutations (R28L, Y268H, and P396S showed no evidence of aggregation and maintained their interactions with known CASK binding partners liprin-α3 Mint-1, and Veli, indicating an intact structure. Intriguingly, the protein aggregation caused by the Y728C and W919R mutations was reversed by treating the cells with a chemical chaperone (glycerol, providing a possible therapeutic strategy for treating structural mutations in CASK in the future.

  11. Comparison of quantitative PCR assays for Escherichia coli targeting ribosomal RNA and single copy genes (United States)

    Aims: Compare specificity and sensitivity of quantitative PCR (qPCR) assays targeting single and multi-copy gene regions of Escherichia coli. Methods and Results: A previously reported assay targeting the uidA gene (uidA405) was used as the basis for comparing the taxono...

  12. Mutations in snail family genes enhance craniosynostosis of Twist1 haplo-insufficient mice: implications for Saethre-Chotzen Syndrome. (United States)

    Oram, Kathleen F; Gridley, Thomas


    In Drosophila, mutations in the Twist gene interact with mutations in the Snail gene. We show that the mouse Twist1 mutation interacts with Snai1 and Snai2 mutations to enhance aberrant cranial suture fusion, demonstrating that genetic interactions between genes of the Twist and Snail families have been conserved during evolution.

  13. Mutations in Snail Family Genes Enhance Craniosynostosis of Twist1 Haplo-insufficient Mice: Implications for Saethre-Chotzen Syndrome



    In Drosophila, mutations in the Twist gene interact with mutations in the Snail gene. We show that the mouse Twist1 mutation interacts with Snai1 and Snai2 mutations to enhance aberrant cranial suture fusion, demonstrating that genetic interactions between genes of the Twist and Snail families have been conserved during evolution.

  14. A LTR copia retrotransposon and Mutator transposons interrupt Pgip genes in cultivated and wild wheats. (United States)

    Di Giovanni, Michela; Cenci, Alberto; Janni, Michela; D'Ovidio, Renato


    Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat (LRR) proteins involved in plant defence. Wheat pgip genes have been isolated from the B (Tapgip1) and D (Tapgip2) genomes, and now we report the identification of pgip genes from the A genomes of wild and cultivated wheats. By Southern blots and sequence analysis of BAC clones we demonstrated that wheat contains a single copy pgip gene per genome and the one from the A genome, pgip3, is inactivated by the insertion of a long terminal repeat copia retrotranspon within the fourth LRR. We demonstrated also that this retrotransposon insertion is present in Triticum urartu and all the polyploidy wheats assayed, but is absent in T. monococcum (Tmpgip3), suggesting that this insertion took place after the divergence between T. monococcum and T. urartu, but before the formation of the polyploid wheats. We identified also two independent insertion events of new Class II transposable elements, Vacuna, belonging to the Mutator superfamily, that interrupted the Tdipgip1 gene of T. turgidum ssp. dicoccoides. The occurrence of these transposons within the coding region of Tdipgip1 facilitated the mapping of the Pgip locus in the pericentric region of the short arm of chromosome group 7. We speculate that the inactivation of pgip genes are tolerated because of redundancy of PGIP activities in the wheat genome.


    Directory of Open Access Journals (Sweden)

    I Wayan Ari Sumardika


    Full Text Available Normal 0 false false false EN-US X-NONE X-NONE The management of individual who has a genetic predisposition for breast cancer requires careful planning. It is estimated that 5-10% of breast cancer in Western countries is a hereditary breast cancer and 80-90% of them is the result of BRCA1 and BRCA2 genes mutations. The individual with BRCA1 and BRCA2 gene mutations have a high risk for experiencing breast cancer and other types of cancer, especially ovarian cancer. Although there are some differences, management of patients with hereditary breast cancer in principle is equal to management of non-hereditary breast cancer. Contra lateral mastectomy surgery and/or oophorectomy may be considered as initial therapy. The uses of breast conserving surgery in patients with BRCA-positive status are still controversial because of the risk of recurrence on ipsilateral breast, so did the use of ionization radiation modalities. Post surgery follow up is an important aspect in the management of patients with mutations of these genes in which follow up aims to find local recurrence, secondary breast cancer, contra lateral breast cancer as early as possible /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin-top:0in; mso-para-margin-right:0in; mso-para-margin-bottom:10.0pt; mso-para-margin-left:0in; line-height:115%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}

  16. Multi-allele genotyping platform for the simultaneous detection of mutations in the Wilson disease related ATP7B gene. (United States)

    Amvrosiadou, Maria; Petropoulou, Margarita; Poulou, Myrto; Tzetis, Maria; Kanavakis, Emmanuel; Christopoulos, Theodore K; Ioannou, Penelope C


    Wilson's disease is an inherited disorder of copper transport in the hepatocytes with a wide range of genotype and phenotype characteristics. Mutations in the ATP7B gene are responsible for the disease. Approximately, over 500 mutations in the ATP7B gene have been described to date. We report a method for the simultaneous detection of the ten most common ATP7B gene mutations in Greek patients. The method comprises 3 simple steps: (i) multiplex PCR amplification of fragments in the ATP7B gene flanking the mutations (ii) multiplex primer extension reaction of the unpurified amplification products using allele-specific primers and (iii) visual detection of the primer extension reaction products within minutes by means of dry-reagent multi-allele dipstick assay using anti-biotin conjugated gold nanoparticles. Optimization studies on the efficiency and specificity of the PEXT reaction were performed. The method was evaluated by genotyping 46 DNA samples of known genotype and 34 blind samples. The results were fully concordant with those obtained by reference methods. The method is simple, rapid, cost-effective and it does not require specialized instrumentation or highly qualified personnel.

  17. Amelogenesis Imperfecta: 1 Family, 2 Phenotypes, and 2 Mutated Genes. (United States)

    Prasad, M K; Laouina, S; El Alloussi, M; Dollfus, H; Bloch-Zupan, A


    Amelogenesis imperfecta (AI) is a clinically and genetically heterogeneous group of diseases characterized by enamel defects. The authors have identified a large consanguineous Moroccan family segregating different clinical subtypes of hypoplastic and hypomineralized AI in different individuals within the family. Using targeted next-generation sequencing, the authors identified a novel heterozygous nonsense mutation in COL17A1 (c.1873C>T, p.R625*) segregating with hypoplastic AI and a novel homozygous 8-bp deletion in C4orf26 (c.39_46del, p.Cys14Glyfs*18) segregating with hypomineralized-hypoplastic AI in this family. This study highlights the phenotypic and genotypic heterogeneity of AI that can exist even within a single consanguineous family. Furthermore, the identification of novel mutations in COL17A1 and C4orf26 and their correlation with distinct AI phenotypes can contribute to a better understanding of the pathophysiology of AI and the contribution of these genes to amelogenesis.

  18. Carriers of filaggrin gene (FLG) mutations avoid professional exposure to irritants in adulthood

    DEFF Research Database (Denmark)

    Bandier, Josefine; Ross-Hansen, Katrine; Carlsen, Berit C


    Loss-of-function mutations in the filaggrin gene (FLG) are associated with xerosis, atopic dermatitis, and early onset of hand eczema. Irritant exposure is a risk factor for occupational hand eczema, and FLG mutations increase the risk of occupational irritant contact dermatitis on the hands...... in hospital cohorts. It is unknown whether FLG mutations affect the level of irritant exposure....

  19. Characterization of two novel missense mutations in the AQP2 gene causing nephrogenic diabetes insipidus.

    NARCIS (Netherlands)

    Iolascon, A.; Aglio, V.; Tamma, G.; D'Apolito, M.; Addabbo, F.; Procino, G.; Simonetti, M.C.; Montini, G.; Gesualdo, L.; Debler, E.W.; Svelto, M.; Valenti, G.


    Here, we report the aquaporin 2 (AQP2) mutational analysis of a patient with nephrogenic diabetes insipidus heterozygote due to two novel missense mutations. Direct sequencing of DNA in the male patient revealed that he was compound heterozygote for two mutations in the AQP2 gene: a thymine-to-adeni

  20. Emerging Trend of Mutation Profile of rpoB Gene in MDR Tuberculosis, North India. (United States)

    Makadia, Jemil S; Jain, Anju; Patra, Surajeet Kumar; Sherwal, B L; Khanna, Ashwani


    The present study was conducted on North Indian population to observe rpoB gene mutation profile in multidrug resistant Mycobacterium tuberculosis. This was an observational study. 30 cases of MDR-TB proven by culture and drug sensitivity were selected. DNA sequencing of 81 bp (codon 507-533) long RRDR of Mycobacterium tuberculosis was done to detect the sites of mutation. Out of 30 cases, 24 showed a single mutation in the RRDR region of rpoB gene in which 16 (53.33 %) showed mutation in codon 531(TCG→TTG), 5 cases (16.66 %) showed mutation in codon 526(CAC→TAC), mutation in codon 516(GAC→GTC, AAC) was present in 3 cases (10 %). It was also observed that mutation in more than one codon was present in 4 cases (13.33 %), which included deletion at codon 509(AGC→-GC), mutation at 513(CAA→CTA), 516, 526, 529(CGA→CTA) and 531. No mutation was detected in RRDR in 2 cases (6.66 %). Our finding of 13.33 % cases with multiple sites of mutation in RRDR region is in contrast to earlier studies done in North India which showed single mutation detected in RRDR of rpoB gene that highlights the emerging change in the trend of mutation profile of rpoB gene in rifampicin resistant Mycobacterium tuberculosis.

  1. Murine muscular dystrophy caused by a mutation in the laminin alpha 2 (Lama2) gene

    DEFF Research Database (Denmark)

    Xu, H; Wu, X R; Wewer, U M;


    The classic murine muscular dystrophy strain, dy, was first described almost 40 years ago. We have identified the molecular basis of an allele of dy, called dy2J, by detecting a mutation in the laminin alpha 2 chain gene--the first identified mutation in laminin-2. The G to A mutation in a splice...

  2. Comprehensive and accurate mutation scanning of the CFTR gene by two-dimensional DNA electrophoresis

    NARCIS (Netherlands)

    Wu, Y; Hofstra, RMW; Scheffer, H; Uitterlinden, AG; Mullaart, E; Buys, CHCM; Vijg, J


    The large number of possible disease causing mutations in the 27 exons of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has severely limited direct diagnosis of cystic fibrosis (CF) patients and carriers by mutation detection. Here we show that in principle testing for mutation

  3. Identification of a novel lethal fibrillin-1 gene mutation in a Chinese Marfan family and correlation of 3'fibrillin-1 gene mutations with phenotype

    Institute of Scientific and Technical Information of China (English)

    GAO Ling-gen; ZHANG Lin; SONG Lei; WANG Hu; CHANG Qian; WU Yong-bo; HUI Ru-tai; ZHOU Xian-liang


    Background Mutations in the fibrillin-1 gene have been identified in patients with Marfan syndrome (MFS). This study aimed to identify the molecular defects in the fibrillin-1 gene in a Chinese family with Marfan syndrome, accompanied by aortic aneurysms/dissection.Methods Two patients and one non-carrier in the family underwent complete physical, ophthalmic, and cardiovascular examinations. Genomic DNA was extracted from leukocytes of venous blood of these individuals in the family as well as 50 healthy normal controls. Polymerase chain reaction amplification and direct sequencing of all 65 coding exons of fibrillin-1 gene were analyzed.Results We found a novel mutation (c.8547T>G, p. Tyr2849X) in exon 65 of fibrillin-1 gene in a Chinese proband with Marfan syndrome, accompanied by aortic aneurysms/dissection. Sudden death at a young age of affected members was seen due to aortic aneurysms/dissection. By evaluating genotype-phenotype correlations of patients with mutations in the 3'end of fibrillin-1 gene (exons 64 and 65), we also found that the presence of nonsense mutations occurring in exons 64 and 65 appeared to be an indicator of early-onset aortic risk and sudden death.Conclusions These results expand the mutation spectrum of fibrillin-1 gene and help in the study of the molecular pathogenesis of Marfan syndrome, indicating that mutations occurring in the 3' end of fibrillin-1 gene may play an independent functional role in the pathogenesis of Marfan syndrome.

  4. Effect of TRPV1 gene mutation on bronchial asthma in children before and after treatment. (United States)

    Chen, Chuan-Liang; Li, Hong; Xing, Xiao-Hong; Guan, Hai-Shan; Zhang, Jian-Hua; Zhao, Jun-Wu


    Bronchial asthma is a worldwide disease with high incidence. It not only harms children's physical and mental health, but it also brings a heavy burden to their families as well as the society. However, the trigger and pathogenesis of the disease remain unclear. This study aimed to analyze TRPV1 gene mutation and expression of cytokines in children with acute bronchial asthma before and after treatment, thus providing theoretical guidance for the diagnosis and treatment of bronchial asthma in children. Real-time quantitative polymerase chain reaction was adopted to detect TRPV1 mRNA expression level and enzyme-linked immuno sorbent assay was used to detect the serum total IgE level, eosinophil (EOS) number, IL-4, IL-5, and interferon (IFN) gamma levels in peripheral venous blood of children in the healthy control group and asthma group before and after treatment. Logistic regression analysis was applied to analyze the most essential factor inducing bronchial asthma in children. TRPV1 mRNA level of peripheral blood in the asthma group was higher than that in the control group before treatment (p asthma in children. TRPV1 gene mutation was closely related to bronchial asthma in children, which provided a theoretical basis for the treatment and prognosis of children with bronchial asthma.


    NARCIS (Netherlands)

    Valdez-Flores, M.A.; Vargas-Poussou, R.; Verkaart, S.; Tutakhel, O.A.Z.; Valdez-Ortiz, A.; Blanchard, A.; Treard, C.; Hoenderop, J.G.J.; Bindels, R.J.M.; Jelen, S.


    Gitelman syndrome (GS) is an autosomal recessive salt-wasting tubular disorder resulting from loss-of-function mutations in the thiazide-sensitive NaCl cotransporter (NCC). Functional analysis of these mutations has been limited to the use of Xenopus laevis oocytes. The aim of the present study was,

  6. The role of the ND5 gene in LHON: characterization of a new, heteroplasmic LHON mutation. (United States)

    Mayorov, Vladimir; Biousse, Valerie; Newman, Nancy J; Brown, Michael D


    Leber's hereditary optic neuropathy (LHON) causes central vision loss from bilateral optic neuropathy. Although 13 mitochondrial DNA (mtDNA) mutations are strongly associated with LHON, only three account for roughly 90% of cases and thus are found in multiple independent LHON families. The remaining LHON mutations are rare. Here, we describe the clinical and genetic characterization of a new LHON mtDNA mutation. The 12848T mutation alters a highly conserved amino acid in the ND5 complex I gene, is not found in controls, and is heteroplasmic. Despite ND5 being the largest of the mtDNA complex I genes, ND5 mutations are quite rare in LHON.

  7. Development of RNA-FISH Assay for Detection of Oncogenic FGFR3-TACC3 Fusion Genes in FFPE Samples (United States)

    Kojima, Takahiro; Nishimura, Kouichi; Kandori, Shuya; Kawahara, Takashi; Yoshino, Takayuki; Ueno, Satoshi; Iizumi, Yuichi; Mitsuzuka, Koji; Arai, Yoichi; Tsuruta, Hiroshi; Habuchi, Tomonori; Kobayashi, Takashi; Matsui, Yoshiyuki; Ogawa, Osamu; Sugimoto, Mikio; Kakehi, Yoshiyuki; Nagumo, Yoshiyuki; Tsutsumi, Masakazu; Oikawa, Takehiro; Kikuchi, Koji; Nishiyama, Hiroyuki


    Introduction and Objectives Oncogenic FGFR3-TACC3 fusions and FGFR3 mutations are target candidates for small molecule inhibitors in bladder cancer (BC). Because FGFR3 and TACC3 genes are located very closely on chromosome 4p16.3, detection of the fusion by DNA-FISH (fluorescent in situ hybridization) is not a feasible option. In this study, we developed a novel RNA-FISH assay using branched DNA probe to detect FGFR3-TACC3 fusions in formaldehyde-fixed paraffin-embedded (FFPE) human BC samples. Materials and Methods The RNA-FISH assay was developed and validated using a mouse xenograft model with human BC cell lines. Next, we assessed the consistency of the RNA-FISH assay using 104 human BC samples. In this study, primary BC tissues were stored as frozen and FFPE tissues. FGFR3-TACC3 fusions were independently detected in FFPE sections by the RNA-FISH assay and in frozen tissues by RT-PCR. We also analyzed the presence of FGFR3 mutations by targeted sequencing of genomic DNA extracted from deparaffinized FFPE sections. Results FGFR3-TACC3 fusion transcripts were identified by RNA-FISH and RT-PCR in mouse xenograft FFPE tissues using the human BC cell lines RT112 and RT4. These cell lines have been reported to be fusion-positive. Signals for FGFR3-TACC3 fusions by RNA-FISH were positive in 2/60 (3%) of non-muscle-invasive BC (NMIBC) and 2/44 (5%) muscle-invasive BC (MIBC) patients. The results of RT-PCR of all 104 patients were identical to those of RNA-FISH. FGFR3 mutations were detected in 27/60 (45%) NMIBC and 8/44 (18%) MIBC patients. Except for one NMIBC patient, FGFR3 mutation and FGFR3-TACC3 fusion were mutually exclusive. Conclusions We developed an RNA-FISH assay for detection of the FGFR3-TACC3 fusion in FFPE samples of human BC tissues. Screening for not only FGFR3 mutations, but also for FGFR3-TACC3 fusion transcripts has the potential to identify additional patients that can be treated with FGFR inhibitors. PMID:27930669

  8. De Novo Mutation in the SCN5A Gene Associated with Brugada Syndrome

    Directory of Open Access Journals (Sweden)

    Lumin Wang


    Full Text Available Background: Brugada syndrome (BrS is a genetically determined cardiac electrical disorder, characterized by typical electrocardiography (ECG alterations, and it is an arrhythmogenic syndrome that may lead to sudden cardiac death. The most common genotype found among BrS patients is caused by mutations in the SCN5A gene, which lead to a loss of function of the cardiac sodium (Na+ channel (Nav1.5 by different mechanisms. Methods: The assay of confocal laser microscopy and western blot were used to identify the expression and location of L812Q at the cell surface. Characterization of Nav1.5 L812Q mutant Na+ channels was text by patch-clamp recordings, and the PHYRE2 server was used to build a model for human Nav1.5 channel. Results: Here, we report that a novel missense SCN5A mutation, L812Q, localized in the DII-S4 transmembrane region of the Nav1.5 channel protein, was identified in an index patient who showed a typical BrS type-1 ECG phenotype. The mutation was absent in the patient's parents and brother. Heterologous expression of the wild-type (WT and L812Q mutant Nav1.5 channels in human embryonic kidney cells (HEK293 cells reveals that the mutation results in a reduction of Na+ current density as well as ∼20 mV hyperpolarizing shift of the voltage dependence of inactivation. The voltage dependence of activation and the time course for recovery from inactivation are not affected by the mutation. The hyperpolarizing shift of the voltage dependence of inactivation caused a reduction of the Na+ window current as well. In addition, western blot and confocal laser microscopy imaging experiments showed that the mutation causes fewer channel to be expressed at the membrane than WT channel. A large proportion of the mutant channels are retained in the cytoplasm, probably in the endoplasmic reticulum. Conclusion: The decrease of channel expression, hyperpolarizing shift of voltage dependence of inactivation, and a decline of Na+ window current

  9. Mutation analysis of codons 345 and 347 of rhodopsin gene in Indian retinitis pigmentosa patients

    Indian Academy of Sciences (India)

    Madhurima Dikshit; Rakhi Agarwal


    More than 100 mutations have been reported till date in the rhodopsin gene in patients with retinitis pigmentosa. The present study was undertaken to detect the reported rhodopsin gene point mutations in Indian retinitis pigmentosa patients. We looked for presence or absence of codon 345 and 347 mutations in exon 5 of the gene using the technique of allele-specific polymerase chain reaction by designing primers for each mutation. We have examined 100 patients from 76 families irrespective of genetic categories. Surprisingly, in our sample the very widely reported highly frequent mutations of codon 347 (P → S/A/R/Q/L/T) were absent while the codon 345 mutation V → M was seen in three cases in one family (autosomal dominant form) and in one sporadic case (total two families). This is the first report on codon 345 and 347 mutation in Indian retinitis pigmentosa subjects.

  10. A dynamic sandwich assay on magnetic beads for selective detection of single-nucleotide mutations at room temperature. (United States)

    Wang, Junxiu; Xiong, Guoliang; Ma, Liang; Wang, Shihui; Zhou, Xu; Wang, Lei; Xiao, Lehui; Su, Xin; Yu, Changyuan


    Single-nucleotide mutation (SNM) has proven to be associated with a variety of human diseases. Development of reliable methods for the detection of SNM is crucial for molecular diagnosis and personalized medicine. The sandwich assays are widely used tools for detecting nucleic acid biomarkers due to their low cost and rapid signaling. However, the poor hybridization specificity of signal probe at room temperature hampers the discrimination of mutant and wild type. Here, we demonstrate a dynamic sandwich assay on magnetic beads for SNM detection based on the transient binding between signal probe and target. By taking the advantage of mismatch sensitive thermodynamics of transient DNA binding, the dynamic sandwich assay exhibits high discrimination factor for mutant with a broad range of salt concentration at room temperature. The beads used in this assay serve as a tool for separation, and might be helpful to enhance SNM selectivity. Flexible design of signal probe and facile magnetic separation allow multiple-mode downstream analysis including colorimetric detection and isothermal amplification. With this method, BRAF mutations in the genomic DNA extracted from cancer cell lines were tested, allowing sensitive detection of SNM at very low abundances (0.1-0.5% mutant/wild type). Copyright © 2017 Elsevier B.V. All rights reserved.


    Institute of Scientific and Technical Information of China (English)

    任晓庆; 黄定九; 黄钢; 王利民


    Objective To investigate the relationship between the radiation dose and the HPRT gene locus mutation in rat smooth muscle cells, and provide the molecular basis for prevention of restenosis after percutaneous transluminal coronary angioplasty (PTCA).MethodsThe smooth muscle cells cultured in vitro were irradiated by radionuclide 188Re in different doses. HPRT gene mutation colonies were selected and isolated by 6 thioguanine. Analysis of mutation in exon 7/8 of HPRT gene were accomplished by polymerase chain reaction and single strand conformation polymorphism.ResultsThe HPRT gene mutation frequency of rat smooth muscle cells that were irradiated by radionuclide 188Re ranged from 5.5×10-6 to 13×10-6. Of 91 HPRT gene mutation colonies, 13(14.3%) contained exon 7/8 deletion and 15(16.5%) had point mutation. The exon 7/8 mutation frequency was 30.8%. There were significant relationships between radiation dose and mutation frequency of HPRT gene and exon 7/8.ConclusionThe DNA damage and gene mutation induced by radiation has positive relationship with radiation dose, and is a basis of proliferation inhibition and apoptosis of smooth muscle cells.

  12. From Acute to Chronic Pancreatitis: The Role of Mutations in the Pancreatic Secretory Trypsin Inhibitor Gene


    Hirota M; Kuwata K; Ohmuraya M; Ogawa M


    Pancreatic secretory trypsin inhibitor (PSTI) is a potent natural inhibitor of trypsin. We proposed the hypothesis that, if the function of the PSTI is impaired by its genetic mutation, trypsin may easily promote autodigestion causing pancreatitis and we performed a mutational analysis of the PSTI gene in patients with pancreatitis. Two exonic mutations (N34S and R67C) were thought to be associated with a predisposition to pancreatitis. The N34S mutation was co-segregated with two intronic mu...

  13. Profile of TP53 gene mutations in sinonasal cancer

    DEFF Research Database (Denmark)

    Holmila, Reetta; Bornholdt, Jette; Suitiala, Tuula


    %) frameshift or nonsense mutations, and 36 (23%) intronic or silent mutations. In coding region, the most common base change detected was C-->T transition (43/125; 34% of base changes in the coding region). G-->T transversions occurred at a frequency of 10% (12/125), which is less than reported in mutation...... not been reported before as frequently mutated in head and neck cancer or human cancer in general. About half of all tumours with TP53 mutations carried more than one mutation. Interestingly, 86% (19/22) of the silent mutations detected had occurred in tumours with multiple mutations.......Genetic alterations underlying the development of the cancer of the nose and paranasal sinuses (sinonasal cancer, SNC), a rare cancer that can be included in the group of head and neck cancers, are still largely unknown. We recently reported that TP53 mutations are a common feature of SNC...

  14. Prenatal Diagnosis in a Family of TNFRSF11A (RANK Gene Mutation Detection: A Case Report

    Directory of Open Access Journals (Sweden)

    Mutlu Karkucak


    Full Text Available Autosomal recessive osteoporosis (ARO is a severe disease causing death usually at infancy or childhood. RANKL coded by TNFSF11 gene and RANK coded by TNFRSF11A gene are important proteins for osteoclast maturation and it is indicated that mutation on these genes plays an important role for ARO development. It is reported in this article that c.508 A→G homozygote mutation (pArg170Gly is observed in TNFRSF11A gene of 2 children of consanguineous couple. Mutation analysis performed on CVS material during the next pregnancy revealed heterozygous mutation in the fetus. The pregnancy was continued to term and a healthy boy was delivered. Prenatal mutation analysis is important for diseases with known mutations to relieve parental anxiety and provide genetic counselling for the family.

  15. SRD5A2 gene mutations--a population-based review. (United States)

    Samtani, Ratika; Bajpai, Minu; Ghosh, P K; Saraswathy, K N


    Knowledge of steroid 5 alpha-reductase type 2 (SRD5A2) gene mutations is expanding, and its role has been implicated in various disease susceptibilities concerning reproductive health. Extensive research has revealed the tendency for specific SRD5A2 gene mutations to be passed along certain racial, ethnic and geographically isolated groups, which suggests population specificity of these mutations. The review provides evidence of variation in the mutational spectrum of the SRD5A2 gene leading to population-specific high prevalence of characteristic disease or phenotypic expression.

  16. Permanent neonatal diabetes mellitus caused by a novel mutation in the KCNJ11 gene. (United States)

    Doneray, Hakan; Houghton, Jayne; Tekgunduz, Kadir Serafettin; Balkir, Ferat; Caner, Ibrahim


    Mutations in the KCNJ11 gene are responsible for the majority of permanent neonatal diabetes mellitus (PNDM) cases. Some mutations in this gene, including p.Q52R, are associated with the developmental delay, epilepsy, neonatal diabetes (DEND) syndrome. We describe a patient with PNDM who had no neurological finding although she was determined to have a novel mutation (p.Q52L) in the same residue of the KCNJ11 as in the previously reported cases with DEND syndrome. This case suggests that not all Q52 mutations in the KCNJ11 gene are necessarily related to DEND syndrome.

  17. Mutations in the dihydropteroate synthase gene of Pneumocystis jiroveci isolates from Portuguese patients with Pneumocystis pneumonia

    DEFF Research Database (Denmark)

    Costa, M C; Helweg-Larsen, J; Lundgren, Bettina


    The aim of this study was to evaluate the frequency of mutations of the P. jiroveci dihydropteroate synthase (DHPS) gene in an immunocompromised Portuguese population and to investigate the possible association between DHPS mutations and sulpha exposure. In the studied population, DHPS gene...... mutations were not significantly more frequent in patients exposed to sulpha drugs compared with patients not exposed (P=0.390). The results of this study suggest that DHPS gene mutations are frequent in the Portuguese immunocompromised population but do not seem associated with previous sulpha exposure...

  18. Accuracy of Reverse Dot-Blot PCR in Detection of Different β-Globin Gene Mutations. (United States)

    El-Fadaly, N; Abd-Elhameed, A; Abd-Elbar, E; El-Shanshory, M


    Prevention programs for β-thalassemia based on molecular diagnosis of heterozygous carriers and/or patients require the use of reliable mutation screening methods. The aim of this study was to compare between direct DNA sequencing, and reverse dot-blot PCR in detection of different β-globin gene mutations in Egyptian children with β-thalassemia. Forty children with β-thalassemia were subjected to mutation analysis, performed by both direct DNA sequencing and β-globin Strip Assay MED™ (based on reverse dot-blot PCR). The most frequent mutant alleles detected by reverse dot-blot PCR were; IVSI-110 G>A (31.25 %), IVS I-6 T > C (21.25 %), and IVS I-1 G>A (20 %). Relatively less frequent mutant alleles detected by reverse dot-blot PCR were "IVSII-1 G>A (5 %), IVSII-745 C>G (5 %), IVSII-848 C>A (2.5 %), IVSI-5 G>C (2.5 %), -87 C>G(2.5 %), and cd39 C>T (2.5 %)", While the genotypes of three patients (6 alleles 7.5 %) were not detected by reverse dot-blot PCR. Mutant alleles detected by direct DNA sequencing were the same as reverse dot-blot PCR method except it revealed the genotypes of 3 undetected patients (one patient was homozygous IVSI-110 G>A, and two patients were homozygous IVS I-1 G>A. Sensitivity of the reverse dot-blot PCR was 92.5 % when compared to direct DNA sequencing for detecting β-thalassemia mutations. Our results therefore suggest that, direct DNA sequencing may be preferred over reverse dot-blot PCR in critical diagnostic situations like genetic counseling for prenatal diagnosis.

  19. A mutation in the mouse ttc26 gene leads to impaired hedgehog signaling.

    Directory of Open Access Journals (Sweden)

    Ruth E Swiderski


    Full Text Available The phenotype of the spontaneous mutant mouse hop-sterile (hop is characterized by a hopping gait, polydactyly, hydrocephalus, and male sterility. Previous analyses of the hop mouse revealed a deficiency of inner dynein arms in motile cilia and a lack of sperm flagella, potentially accounting for the hydrocephalus and male sterility. The etiology of the other phenotypes and the location of the hop mutation remained unexplored. Here we show that the hop mutation is located in the Ttc26 gene and impairs Hedgehog (Hh signaling. Expression analysis showed that this mutation led to dramatically reduced levels of the Ttc26 protein, and protein-protein interaction assays demonstrated that wild-type Ttc26 binds directly to the Ift46 subunit of Intraflagellar Transport (IFT complex B. Although IFT is required for ciliogenesis, the Ttc26 defect did not result in a decrease in the number or length of primary cilia. Nevertheless, Hh signaling was reduced in the hop mouse, as revealed by impaired activation of Gli transcription factors in embryonic fibroblasts and abnormal patterning of the neural tube. Unlike the previously characterized mutations that affect IFT complex B, hop did not interfere with Hh-induced accumulation of Gli at the tip of the primary cilium, but rather with the subsequent dissociation of Gli from its negative regulator, Sufu. Our analysis of the hop mouse line provides novel insights into Hh signaling, demonstrating that Ttc26 is necessary for efficient coupling between the accumulation of Gli at the ciliary tip and its dissociation from Sufu.

  20. A Unique Profile of Adenomatous Polyposis Coli Gene Mutations in Iranian Patients Suffering Sporadic Colorectal Cancer

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    Mojtaba Hasanpour


    Full Text Available Objective: Colorectal cancer (CRC is one of the most common and aggressive cancers worldwide. The majority of CRC cases are sporadic that caused by somatic mutations. The Adenomatous Polyposis Coli (APC; OMIM 611731 is a tumor suppressor gene of Wnt pathway and is frequently mutated in CRC cases. This study was designed to investigate the spectrum of APC gene mutations in Iranian patients with sporadic colorectal cancer. Materials and Methods: In this descriptive study, Tumor and normal tissue samples were obtained from thirty randomly selected and unrelated sporadic CRC patients. We examined the hotspot region of the APC gene in all patients. Our mutation detection method was direct DNA sequencing. Results: We found a total of 8 different APC mutations, including two nonsense mutations (c.4099C>T and c.4348C>T, two missense mutations (c.3236C>G and c.3527C>T and four frame shift mutations (c.2804dupA, c.4317delT, c.4464_4471delATTACATT and c.4468_4469dupCA. The c.3236C>G and c.4468_4469dupCA are novel mutations. The overall frequency of APC mutation was 26.7% (8 of 30 patients. Conclusion: This mutation rate is lower in comparison with previous studies from other countries. The findings of present study demonstrate a different APC mutation spectrum in CRC patients of Iranian origin compared with other populations.

  1. [Mutation analysis of the pathogenic gene in a Chinese family with hereditary hemochromatosis]. (United States)

    Yuanfeng, Li; Hongxing, Zhang; Haitao, Zhang; Xiaobo, Peng; Lili, Bai; Fuchu, He; Zewu, Qiu; Gangqiao, Zhou


    Hereditary hemochromatosis (HHC) is a rare autosomal recessive disorder. We recruited a consanguineous Chinese family including the proband with HHC and other four members without HHC. Using whole-exome sequencing, we identified two homozygous mutations (c.G18C [p.Q6H] and c.GC962_963AA [p.C321X]) in the hemojuvelin gene (HJV) in the proband with HHC. No mutation was found in other four previously identified HHC related genes, HAMP, TFR2, FPN and HFE. The functional impact of p.Q6H mutation is weak whereas p.C321X, a premature termination mutation, results in a truncated HJV protein, which lacks the glycosylphosphatidylinositol (GPI) anchor domain. In addition to the mutations in HJV, other 12 homozygous mutations were identified in this patient. However, none of these mutations showed strong damaging impact and the mutated genes are not related to iron metabolism. Our in-house data further demonstrated that p.C321X is absent in the general Chinese population, suggesting that the homozygous mutation p.C321X in HJV is causative in the patient with HHC. Accordingly, all of the four members without HHC from the same family carried wild-type alleles or heterozygous mutations, but not the homozygous mutation in this site. Thus, we found for the first time that the homozygous mutation p.C321X in HJV can result in HHC, which will help genetic diagnosis and prenatal counseling for HHC.

  2. Mal de Meleda in Indonesia: Mutations in the SLURP1 gene appear to be ubiquitous. (United States)

    Taylor, Jessica A; Bondavalli, Davide; Monif, Mastura; Yap, Lee Mei; Winship, Ingrid


    Mal de Meleda is a rare autosomal recessive genodermatosis caused by mutations in the ARS B (SLURP1) gene, with possible founder effects in the Mediterranean and Adriatic regions. We report an affected individual from Indonesia without known consanguinity in the family, suggesting that SLURP1 gene mutations are ubiquitous. Recognition of the phenotype can be confirmed by genetic testing, thus facilitating genetic counselling.

  3. Clinical course of cone dystrophy caused by mutations in the RPGR gene

    NARCIS (Netherlands)

    Thiadens, A.A.H.J.; Soerjoesing, G.G.; Florijn, R.J.; Tjiam, A.G.; Hollander, A.I. den; Born, L.I. van den; Riemslag, F.C.; Bergen, A.A.B.; Klaver, C.C.


    BACKGROUND: Mutations in the RPGR gene predominantly cause rod photoreceptor disorders with a large variability in clinical course. In this report, we describe two families with mutations in this gene and cone involvement. METHODS: We investigated an X-linked cone dystrophy family (1) with 25

  4. Clinical course of cone dystrophy caused by mutations in the RPGR gene

    NARCIS (Netherlands)

    A.A.H.J. Thiadens (Alberta); G.G. Soerjoesing (Gyan); R.J. Florijn; A.G. Tjiam; A.I. Hollander (Anneke); L.I. van den Born (Ingeborgh); F.C.C. Riemslag (Frans); A.A.B. Bergen (Arthur); C.C.W. Klaver (Caroline)


    textabstractBackground: Mutations in the RPGR gene predominantly cause rod photoreceptor disorders with a large variability in clinical course. In this report, we describe two families with mutations in this gene and cone involvement. Methods: We investigated an X-linked cone dystrophy family (1)

  5. Mutation of the planar cell polarity gene VANGL1 in adolescent idiopathic scoliosis

    DEFF Research Database (Denmark)

    Andersen, Malene Rask; Farooq, Muhammad; Rasmussen, Karen Koefoed


    STUDY DESIGN: Mutation analysis of a candidate disease gene in a cohort of patients with moderate to severe Adolescent idiopathic scoliosis (AIS). OBJECTIVE: To investigate if damaging mutations in the planar cell polarity gene VANGL1 could be identified in AIS patients. SUMMARY OF BACKGROUND DAT...

  6. Test Feasibility of Next-Generation Sequencing Assays in Clinical Mutation Detection of Small Biopsy and Fine Needle Aspiration Specimens. (United States)

    Zheng, Gang; Tsai, Harrison; Tseng, Li-Hui; Illei, Peter; Gocke, Christopher D; Eshleman, James R; Netto, George; Lin, Ming-Tseh


    To evaluate preanalytic factors contributing to failure of next-generation sequencing (NGS) assays. AmpliSeq Cancer Hotspot Panel was conducted in 1,121 of 1,152 formalin-fixed paraffin-embedded tissues submitted to a clinical laboratory, including 493 small biopsy or fine needle aspiration (FNA) specimens (44%) and 25 metastatic bone specimens (2.2%). Single nucleotide mutations and/or insertion/deletion mutations were detected in 702 specimens. Thirty-eight specimens (3.4%) were reported as "no results" due to NGS assay failure. Higher failure rates were observed in specimens submitted for lung cancer panel and melanoma panel (3.1% and 3.7% vs 1.0% colorectal cancer panel), metastatic bone specimens (36% vs 2.6% nonbone specimens), referred specimens (5.0% vs 1.8% in-house specimens), and small biopsy and FNA specimens (5.8% and 3.1% vs 0.7% resection/excision specimens). Test feasibility was higher in in-house specimens than referred specimens (99.1% vs 96.9% in resection specimens, 94.4% vs 87.3% in small biopsy specimens, and 94.3% vs 58.8% in FNA specimens). NGS assays demonstrated clinical utility in solid tumor specimens, including those taken by biopsy or FNA. Preanalytic factors identified by this study that may contribute to NGS assay failure highlight the need for pathologists to revisit tissue processing protocols in order to better optimize cancer mutational profiling. © American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail:

  7. Clinical study of DMD gene point mutation causing Becker muscular dystrophy

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    Ji-qing CAO


    Full Text Available Background  DMD gene point mutation, mainly nonsense mutation, always cause the most severe Duchenne muscular dystrophy (DMD. However, we also observed some cases of Becker muscular dystrophy (BMD carrying DMD point mutation. This paper aims to explore the mechanism of DMD point mutation causing BMD, in order to enhance the understanding of mutation types of BMD.  Methods  Sequence analysis was performed in 11 cases of BMD confirmed by typical clinical manifestations and muscle biopsy. The exon of DMD gene was detected non-deletion or duplication by multiplex ligation-dependent probe amplification (MLPA.  Results  Eleven patients carried 10 mutation types without mutational hotspot. Six patients carried nonsense mutations [c.5002G>T, p.(Glu1668X; c.1615C > T, p.(Arg539X; c.7105G > T, p.(Glu2369X; c.5287C > T, p.(Arg1763X; c.9284T > G, p.(Leu3095X]. One patient carried missense mutation [c.5234G > A, p.(Arg1745His]. Two patients carried frameshift mutations (c.10231dupT, c.10491delC. Two patients carried splicing site mutations (c.4518 + 3A > T, c.649 + 2T > C.  Conclusions  DMD gene point mutation may result in BMD with mild clinical symptoms. When clinical manifestations suggest the possibility of BMD and MLPA reveals non?deletion or duplication mutation of DMD gene, BMD should be considered. Study on the mechanism of DMD point mutation causing BMD is very important for gene therapy of DMD. DOI: 10.3969/j.issn.1672-6731.2015.06.005

  8. Dysferlin Gene Mutation Spectrum in a Large Cohort of Chinese Patients with Dysferlinopathy (United States)

    Jin, Su-Qin; Yu, Meng; Zhang, Wei; Lyu, He; Yuan, Yun; Wang, Zhao-Xia


    Background: Dysferlinopathy is caused by mutations in the dysferlin (DYSF) gene. Here, we described the genetic features of a large cohort of Chinese patients with this disease. Methods: Eighty-nine index patients were included in the study. DYSF gene analysis was performed by Sanger sequencing in 41 patients and targeted next generation sequencing (NGS) in 48 patients. Multiplex ligation-dependent probe amplification (MLPA) was performed to detect exon duplication/deletion in patients with only one pathogenic mutation. Results: Among the 89 index patients, 79 patients were demonstrated to carry two disease-causing (73 cases) or possibly disease-causing mutations (6 cases), including 26 patients with homozygous mutations. We identified 105 different mutations, including 59 novel ones. Notably, in 13 patients in whom only one pathogenic mutation was initially found by Sanger sequencing or NGS, 3 were further identified to carry exon deletions by MLPA. The mutations identified in this study appeared to cluster in the N-terminal region. Mutation types included missense mutations (30.06%), nonsense mutations (17.18%), frameshift mutations (30.67%), in-frame deletions (2.45%), intronic mutations (17.79%), and exonic rearrangement (1.84%). No genotype-phenotype correlation was identified. Conclusions: DYSF mutations in Chinese patients clustered in the N-terminal region of the gene. Exonic rearrangements were found in 23% of patients with only one pathogenic mutation identified by Sanger sequencing or NGS. The novel mutations found in this study greatly expanded the mutational spectrum of dysferlinopathy. PMID:27647186

  9. Dysferlin Gene Mutation Spectrum in a Large Cohort of Chinese Patients with Dysferlinopathy

    Institute of Scientific and Technical Information of China (English)

    Su-Qin Jin; Meng Yu; Wei Zhang; He Lyu; Yun Yuan; Zhao-Xia Wang


    Background:Dysferlinopathy is caused by mutations in the dysferlin (DYSF) gene.Here,we described the genetic features of a large cohort of Chinese patients with this disease.Methods:Eighty-nine index patients were included in the study.DYSF gene analysis was performed by Sanger sequencing in 41 patients and targeted next generation sequencing (NGS) in 48 patients.Multiplex ligation-dependent probe amplification (MLPA) was performed to detect exon duplication/deletion in patients with only one pathogenic mutation.Results:Among the 89 index patients,79 patients were demonstrated to carry two disease-causing (73 cases) or possibly disease-causing mutations (6 cases),including 26 patients with homozygous mutations.We identified 105 different mutations,including 59 novel ones.Notably,in 13 patients in whom only one pathogenic mutation was initially found by Sanger sequencing or NGS,3 were further identified to carry exon deletions by MLPA.The mutations identified in this study appeared to cluster in the N-terminal region.Mutation types included missense mutations (30.06%),nonsense mutations (1 7.18%),frameshift mutations (30.67%),in-frame deletions (2.45%),intronic mutations (17.79%),and exonic rearrangement (1.84%).No genotype-phenotype correlation was identified.Conclusions:DYSF mutations in Chinese patients clustered in the N-terminal region of the gene.Exonic rearrangements were found in 23% of patients with only one pathogenic mutation identified by Sanger sequencing or NGS.The novel mutations found in this study greatly expanded the mutational spectrum of dysferlinopathy.

  10. Mutation analysis of presenilin-1 gene in Alzheimer’s disease patients and the effects of its mutation on expression of presenilin-1 and amyloid precursor protein

    Institute of Scientific and Technical Information of China (English)



    Objective To analyze the presenilin-1(PS-1) gene mutations in Alzheimer’s disease(AD) patients and investigate the influence of the initiation codon mutation on the mRNA expression of PS-1 and amyloid precursor protein

  11. Applications of homemade kit in mutation detection of genes

    Institute of Scientific and Technical Information of China (English)

    ZHAO; Chunxia


    [1]Orita, M., Iwahana, H., Kanazawa, H. et al., Detection of poly morphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms, Proc. Natl. Acad. Sci., 1989, 86:2766-2770.[2]Hongyo, T., Buzard, G. S., Calvert, R. J. et al., Cold SSCP: a simple, rapid and non-radioactive method for optimized single-strand conformation polymorphism analyses, Nucleic Acids Res., 1993, 21: 3637-3642.[3]Kutach, L. S., Bolshakov, S., Ananthaswamy, H. N., Detection of mutations and polymorphisms in the p53 tumor suppressor gene by single-strand conformation polymorphism analysis, Electrophoresis, 1999, 20: 1204-1210.[4]Kozlowski, P., Krzyzosiak, W. J., Combined SSCP/duplex analysis by capillary electrophoresis for more efficient mutation detection, Nucleic Acids Res., 2001, 29( 14): E71.[5]Turner, D., Choudhury, F., Reynard, M. et al., Typing of multiple single nucleotide polymorphisms in cytokine and receptor genes using SNaPshot, Human Immunology, 2002, 63: 508-513.[6]Sudor, J., Barbier, V., Thirot, S. et al., New block-copolymer thermoassociating matrices for DNA sequencing: Effect of molecular sreucture on rheology and resolution, Electrophoresis, 2001, 22: 720-728.[7]Barbier, V., Viovy, J. L., Advanced polymers for DNA separation, Curr. Opin. Biotechnol., 2003, 14: 51-57.[8]Ugaz, V. M., Lin, R., Srivastava, N. et al., A versatile microfabri cated platform for electrophoresis of double- and single-stranded DNA, Electrophoresis, 2003, 24:151-157.[9]Lassiter, S. J., Stryjewski, W., Owens, C. V. et al., Optimization of sequencing conditions using near-infrared lifetime identification methods in capillary gel electrophoresis, Electrophoresis, 2002, 23: 1480-1489.[10]Chang, H. T., Yeung, E. S., Poly(ethyleneoxide) for high-resolution and high-speed separation of DNA by capillary electrophoresis, J. Chromatogr. B, 1995, 669: 113-123.[11]Gao, Q., Yeung, E. S., A matrix for DNA separation: genotyping and sequencing using

  12. De novo mutations in synaptic transmission genes including DNM1 cause epileptic encephalopathies

    DEFF Research Database (Denmark)


    analyzed exome-sequencing data of 356 trios with the "classical" epileptic encephalopathies, infantile spasms and Lennox Gastaut syndrome, including 264 trios previously analyzed by the Epi4K/EPGP consortium. In this expanded cohort, we find 429 de novo mutations, including de novo mutations in DNM1...... = 8.2 × 10(-4)), supporting a prominent role for de novo mutations in epileptic encephalopathies. We bring statistical evidence that mutations in DNM1 cause epileptic encephalopathy, find suggestive evidence for a role of three additional genes, and show that at least 12% of analyzed individuals have...... an identifiable causal de novo mutation. Strikingly, 75% of mutations in these probands are predicted to disrupt a protein involved in regulating synaptic transmission, and there is a significant enrichment of de novo mutations in genes in this pathway in the entire cohort as well. These findings emphasize...

  13. De novo mutation of keratin 9 gene in two Taiwanese patients with epidermolytic palmoplantar keratoderma. (United States)

    Yang, Mei-Hui; Lee, Julia Yu-Yun; Lin, Jeng-Hsien; Chao, Sheau-Chiou


    Epidermolytic palmoplantar keratoderma (EPPK) is an autosomal dominant hereditary disorder of keratinization. Recent molecular studies have shown that EPPK is caused by mutations in keratin 9 gene (K9). We report 2 unrelated sporadic cases of EPPK in Taiwanese, confirmed by histopathology and electron microscopy. A de novo mutation with a C to T transition at the first nucleotide of codon 162 in K9 was detected in both patients, but not in their parents. The mutation is expected to result in an arginine to tryptophan substitution (R162W) in the beginning region of the alpha-helical 1A domain of K9. Mutations in this region could disrupt keratin filament assembly, leading to degeneration or cytolysis of keratinocytes. Mutations of this arginine codon (R162W, R162Q) are common in pedigrees with EPPK. Our mutation analysis suggests that codon 162 in K9 gene is an important hot spot for mutation in EPPK.

  14. A novel missense adenine nucleotide translocator-1 gene mutation in a Greek adPEO family. (United States)

    Napoli, L; Bordoni, A; Zeviani, M; Hadjigeorgiou, G M; Sciacco, M; Tiranti, V; Terentiou, A; Moggio, M; Papadimitriou, A; Scarlato, G; Comi, G P


    Autosomal dominant progressive external ophthalmoplegia (adPEO) is caused by mutations in at least three different genes: ANT1 (chromosome 4q34-35), TWINKLE, and POLG. The ANT1 gene encodes the adenine nucleotide translocator-1 (ANT1). We identified a heterozygous T293C mutation of the ANT1 gene in a Greek family with adPEO. The resulting leucine to proline substitution likely modifies the secondary structure of the ANT1 protein. ANT1 gene mutations may account for adPEO in families with different ethnic backgrounds.

  15. Frequency of Thrombophilic Gene Mutations in Patients with Deep Vein Thrombosis and in Women with Recurrent Pregnancy Loss

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    Elgari Mahmoud Mohamed


    Full Text Available Thrombophilia may be anticipated by single or combined hereditary defects in encoding genes factor V, Prothrombin, and MTHFR. The aim of this study was to determine the prevalence and associated risks of V Leiden (G1691A, Prothrombin (G20210A, and MTHFR (C677T mutations in Saudi women with Deep Vein Thrombosis (DVT and women with recurrent pregnancy loss (RPL. Protein C and protein S activity were measured to determine combined effects, if any. We examined 60 women with a history of DVT and 60 with RPL, extracted DNA from EDTA blood and determined three mutations by using multiplex PCR reactions followed by Strip Assay KIT. Pro C Global assay was used to determine the cutoff value [PCATNR = 0.80]. Protein C/S chromogenic assay was used to estimate protein C and S percentages. Frequency of Factor V Leiden G/A genotype in patients with DVT 7 (11.6% had a significant association for DVT χ2 (OR = 5.1, P = 0.03. In women with RPL the three mutations did not show any significant association, levels of Protein C, protein S and PCAT-NR in patient groups not different from controls (P > 0.05. In conclusion, we recommend expanding on these data to provide larger-scale studies.

  16. Exome sequencing reveals AMER1 as a frequently mutated gene in colorectal cancer (United States)

    Sanz-Pamplona, Rebeca; Lopez-Doriga, Adriana; Paré-Brunet, Laia; Lázaro, Kira; Bellido, Fernando; Alonso, M. Henar; Aussó, Susanna; Guinó, Elisabet; Beltrán, Sergi; Castro-Giner, Francesc; Gut, Marta; Sanjuan, Xavier; Closa, Adria; Cordero, David; Morón-Duran, Francisco D.; Soriano, Antonio; Salazar, Ramón; Valle, Laura; Moreno, Victor


    PURPOSE Somatic mutations occur at early stages of adenoma and accumulate throughout colorectal cancer (CRC) progression. The aim of this study was to characterize the mutational landscape of stage II tumors and to search for novel recurrent mutations likely implicated in CRC tumorigenesis. DESIGN The exomic DNA of 42 stage II, microsatellite stable, colon tumors and their paired mucosae were sequenced. Other molecular data available in the discovery dataset (gene expression, methylation, and CNV) was used to further characterize these tumors. Additional datasets comprising 553 CRC samples were used to validate the discovered mutations. RESULTS As a result, 4,886 somatic single nucleotide variants (SNVs) were found. Almost all SNVs were private changes, with few mutations shared by more than one tumor, thus revealing tumor-specific mutational landscapes. Nevertheless, these diverse mutations converged into common cellular pathways such as cell cycle or apoptosis. Among this mutational heterogeneity, variants resulting in early stop-codons in the AMER1 (also known as FAM123B or WTX) gene emerged as recurrent mutations in CRC. Loses of AMER1 by other mechanisms apart from mutations such as methylation and copy number aberrations were also found. Tumors lacking this tumor suppressor gene exhibited a mesenchymal phenotype characterized by inhibition of the canonical Wnt pathway. CONCLUSION In silico and experimental validation in independent datasets confirmed the existence of functional mutations in AMER1 in approximately 10% of analyzed CRC tumors. Moreover, these tumors exhibited a characteristic phenotype. PMID:26071483

  17. Tyrosine kinase domain mutations of EGFR gene in head and neck squamous cell carcinoma (United States)

    Vatte, Chittibabu; Al Amri, Ali M; Cyrus, Cyril; Chathoth, Shahanas; Acharya, Sadananda; Hashim, Tariq Mohammad; Al Ali, Zhara; Alshreadah, Saleh Tawfeeq; Alsayyah, Ahmed; Al-Ali, Amein K


    Background Epidermal growth factor receptor (EGFR) is a commonly altered gene that is identified in various cancers, including head and neck squamous cell carcinoma (HNSCC). Therefore, EGFR is a promising molecular marker targeted by monoclonal antibodies and small molecule inhibitors targeting the tyrosine kinase (TK) domain. Objective The objective of this study was to investigate the spectrum of mutations in exons 18, 19, 20, and 21 of the EGFR gene in HNSCC patients. Materials and methods This retrospective study included 47 confirmed HNSCC cases. Mutations in the TK domain, exons 18, 19, 20, and 21 of the EGFR gene, were detected by Scorpion® chemistry and ARMS® technologies on Rotor-Gene Q real-time polymerase chain reaction. Results The tumors exhibited EGFR-TK domain mutations in 57% of cases. Four cases of T790M mutations were reported for the first time among HNSCC patients. Out of the total mutations, L861Q (exon 21), exon 20 insertions and deletions of exon 19 accounted for the majority of mutations (21%, 19%, and 17%, respectively). EGFR mutation status was correlated with the higher grade (P=0.026) and advanced stage (P=0.034) of HNSCC tumors. Conclusion Higher frequency of EGFR-TK domain mutations together with the presence of the T790M mutation suggests that identification of these mutations might streamline the therapy and provide a better prognosis in HNSCC cases. PMID:28352186

  18. ATM function and its relationship with ATM gene mutations in chronic lymphocytic leukemia with the recurrent deletion (11q22.3-23.2). (United States)

    Jiang, Y; Chen, H-C; Su, X; Thompson, P A; Liu, X; Do, K-A; Wierda, W; Keating, M J; Plunkett, W


    Approximately 10-20% of chronic lymphocytic leukemia (CLL) patients exhibit del(11q22-23) before treatment, this cohort increases to over 40% upon progression following chemoimmunotherapy. The coding sequence of the DNA damage response gene, ataxia-telangiectasia-mutated (ATM), is contained in this deletion. The residual ATM allele is frequently mutated, suggesting a relationship between gene function and clinical response. To investigate this possibility, we sought to develop and validate an assay for the function of ATM protein in these patients. SMC1 (structural maintenance of chromosomes 1) and KAP1 (KRAB-associated protein 1) were found to be unique substrates of ATM kinase by immunoblot detection following ionizing radiation. Using a pool of eight fluorescence in situ hybridization-negative CLL samples as a standard, the phosphorylation of SMC1 and KAP1 from 46 del (11q22-23) samples was analyzed using normal mixture model-based clustering. This identified 13 samples (28%) that were deficient in ATM function. Targeted sequencing of the ATM gene of these samples, with reference to genomic DNA, revealed 12 somatic mutations and 15 germline mutations in these samples. No strong correlation was observed between ATM mutation and function. Therefore, mutation status may not be taken as an indicator of ATM function. Rather, a direct assay of the kinase activity should be used in the development of therapies.

  19. IL7R and RAG1/2 Genes Mutations/Polymorphisms in Patients SCID

    Directory of Open Access Journals (Sweden)

    Sepideh Safaei


    Full Text Available SCID disorder is major failure of the immune system, usually genetic. The aim of this study was on mutations detection of RAG1, RAG2, and IL7RG genes in SCID cases. Mutation detection was performed by PCR sequencing.Our results  indicated  that 13  mutations  were found  through  cases which  include 4 mutations in IL7R gene (T661I, I138V, T56A, C57W, 7 mutations in RAG1 (W896X, W204R, M324V, T731I, M1006V, K820R, and R249H, and 2 mutations in RAG2 gene (R229W, ΔT251.

  20. Rare mutations of the DMBT1 gene in human astrocytic gliomas

    DEFF Research Database (Denmark)

    Mueller, Wolf; Mollenhauer, Jan; Stockhammer, Florian


    The Deleted in Malignant Brain Tumors 1 gene (DMBT1) has been proposed as a tumor suppressor gene candidate in human brain tumors, based on the observation of homozygous deletions affecting the DMBT1 region or part of the gene. In order to support this hypothesis, we performed a mutational analysis...... of the entire coding region of DMBT1, employing SSCP analysis and direct DNA sequencing in a series of 79 astrocytic gliomas. Five somatic mutations were detected. Two mutations, one of which resulted in an amino acid exchange, occurred in glioblastomas. One pilocytic astrocytoma carried two missense mutations...... and another pilocytic astrocytoma contained a somatic mutation, not affecting the presumed protein. In addition, 21 of the 27 single nucleotide polymorphisms identified in this study have not been recognized previously. The data indicate, that small mutations are not a frequent finding in gliomas....

  1. Frequencies of the Common Mefv Gene Mutations in Adiyaman, Southeast Anatolia, Turkey

    Directory of Open Access Journals (Sweden)

    Korkmaz D. T.


    Full Text Available Familial Mediterranean fever (FMF is an autosomal recessive disorder characterized by fever and serosal inflammation. The reasons for the disorder are mutations in the Mediterranean fever (MEFV gene; the most common of which are M694V, M680I, M694I and V726A. In this study, we aimed to screen these common mutations of the MEFV gene and then determine the prevalence of FMF according to these mutations in Adıyaman, Southeast Anatolia, Turkey. Seven hundred and sixty-seven healthy individuals from the region of Adıyaman participated in the study. Polymerase chain reaction-amplification refractory mutation system (PCR-ARMS methods were used to determine the common mutations of the MEFV gene. Twenty-six (3.9% individuals had only one mutation in the MEFV gene, 25 individuals were heterozygous and one person was homozygous for the V726A mutation (0.15%. In the present study, the V726A mutation (50.0% was the most frequent, followed by M694V (38.5%, M680I (7.7% and M694I (3.8%. It was seen that the carrier rate was very low and the prevalence of FMF was 0.15%, according to the common mutations of the MEFV gene in Adıyaman, Southeast Anatolia, Turkey.

  2. Glucocerebrosidase gene L444P mutation is a risk factor for Parkinson's disease in Chinese population. (United States)

    Sun, Qi-Ying; Guo, Ji-Feng; Wang, Lei; Yu, Ren-He; Zuo, Xing; Yao, Ling-Yan; Pan, Qian; Xia, Kun; Tang, Bei-Sha


    An association between mutations in the glucocerebrosidase (GBA) gene and Parkinson's disease (PD) has been reported in several populations. We searched for four common GBA mutations (L444P, F213I, R353W, and N370S) in 402 Chinese PD patients and 413 age- and sex-matched controls. In the PD cohort, 11 patients were found carrying a heterozygous GBA mutation and all of them had the L444P mutation. Heterozygous GBA mutations were detected none in controls. The GBA gene L444P mutation was detected at a significantly higher frequency among PD patients (11/402 = 2.74%), when compared with the control group (0/413): P = 0.0007. To evaluate the possible role of the GBA gene L444P mutation in PD in Ashkenazi Jewish and non-Jewish populations, we conducted a meta-analysis on the topic. In the Chinese population, the GBA gene L444P mutation was detected at a significantly higher frequency among PD patients, when compared with the control group: Z = 3.83, P = 0.0001, OR = 8.42, confidence interval = 95%, 2.83-25.06. In the non-Jewish populations, the difference was obviously significant: Z = 5.76, P < 0.00001, OR = 8.82, confidence interval = 95%, 4.21-18.48. The results suggest that the GBA gene L444P mutation appears to be a risk factor for PD in Chinese population.

  3. Detection of BRAF mutation in Chinese tumor patients using a highly sensitive antibody immunohistochemistry assay (United States)

    Qiu, Tian; Lu, Haizhen; Guo, Lei; Huang, Wenting; Ling, Yun; Shan, Ling; Li, Wenbin; Ying, Jianming; Lv, Ning


    BRAF mutations can be found in various solid tumors. But accurate and reliable screening for BRAF mutation that is compatible for clinical application is not yet available. In this study, we used an automated immunohistochemistry (IHC) staining coupled with mouse monoclonal anti-BRAF V600E (VE1) primary antibody to screen the BRAF V600E mutation in 779 tumor cases, including 611 colorectal carcinomas (CRC), 127 papillary thyroid carcinomas (PTC) and 41 malignant melanomas. Among the 779 cases, 150 cases were positive for BRAF (V600E) staining, including 38 (of 611, 6%) CRCs, 102 (of 127, 80%) PTCs and 10 (of 41, 24%) malignant melanomas. Sanger sequencing and real-time PCR confirmed the sensitivity and specificity of IHC staining for the V600E mutation are 100% and 99%, respectively. Therefore, our study demonstrates that the fully automated IHC is a reliable tool to determine BRAF mutation status in CRC, PTC and melanoma and can be used for routine clinical screen.

  4. Additive effect of mutations in LDLR and PCSK9 genes on the phenotype of familial hypercholesterolemia. (United States)

    Pisciotta, Livia; Priore Oliva, Claudio; Cefalù, Angelo Baldassare; Noto, Davide; Bellocchio, Antonella; Fresa, Raffaele; Cantafora, Alfredo; Patel, Dilip; Averna, Maurizio; Tarugi, Patrizia; Calandra, Sebastiano; Bertolini, Stefano


    Patients homozygous or compound heterozygous for LDLR mutations or double heterozygous for LDLR and apo B R3500Q mutation have higher LDL-C levels, more extensive xanthomatosis and more severe premature coronary disease (pCAD) than simple heterozygotes for mutations in either these genes or for missense mutations in PCSK9 gene. It is not known whether combined mutations in LDLR and PKCS9 are associated with such a severe phenotype. We sequenced Apo B and PCSK9 genes in two patients with the clinical diagnosis of homozygous FH who were heterozygous for LDLR gene mutations. Proband Z.P. (LDL-C 13.39 mmol/L and pCAD) was heterozygous for an LDLR mutation (p.E228K) inherited from her father (LDL-C 8.07 mmol/L) and a PCSK9 mutation (p.R496W) from her mother (LDL-C 5.58 mmol/L). Proband L.R. and her sister (LDL-C 11.51 and 10.47 mmol/L, xanthomatosis and carotid atherosclerosis) were heterozygous for an LDLR mutation (p.Y419X) inherited from their mother (LDL-C 6.54 mmol/L) and a PCSK9 mutation (p.N425S) probably from their deceased father. The LDL-C levels in double heterozygotes of these two families were 56 and 44% higher than those found in simple heterozygotes for the two LDLR mutations, respectively. The two PCSK9 mutations are novel and were not found in 110 controls and 80 patients with co-dominant hypercholesterolemia. These observations indicate that rare missense mutations of PCSK9 may worsen the clinical phenotype of patients carrying LDLR mutations.


    Institute of Scientific and Technical Information of China (English)


    Objective: To determine the feasibility of detecting p53 gene mutations for early diagnosis of lung cancer using the samples from bronchoscopic examination. Methods: Point mutations of the exon 5-8 of p53 gene were detected in 85 bronchoscopic samples of 35 patients suspected to be lung cancer using silver staining PCR-SSCP. Results: p53 gene mutations were founded in 10 of 35 patients(28.6%). The incidence of p53 gene mutations (14.9%) was obviously higher than the cytological positive incidence(2.9%) in samples of sputum, bronchoalveolar lavage and brush, especially for the sputum(27.7%). In the bronchoscopic biopsy specimens, the incidence of p53 gene mutations (12.5%) was lower than that of pathologic positive result (50.0%). However, in view of all the bronchoscopic samples, there was no statistically difference between cytopathologic positive results (11.8%) and the incidence of p53 gene mutations (14.1%). Although the p53 mutations were most common in the samples from the patients bronchoscopically manifested as neoplasm compared with other manifestations, there was no statistical difference. It is valuable to notice that 3 patients with p53 gene mutation merely presented as bronchial inflammation in bronchoscope. Conclusion: Results indicated that the value of detecting p53 gene mutation for the diagnosis of lung cancer using the bronchoscopic samples was more superior to cytological examination and detection of p53 gene mutations in post-bronchoscopic sputum was easy and effective, may be used as a valuable method for early diagnosis of lung cancer.

  6. Relationship between periodontal destruction and gene mutations in patients with familial Mediterranean fever. (United States)

    Sezer, Ufuk; Şenyurt, Süleyman Ziya; Özdemir, Eda Çetin; Zengin, Orhan; Üstün, Kemal; Erciyas, Kamile; Kısacık, Bünyamin; Onat, Ahmet Mesut


    Recent studies have shown that genetic factors involved in the host responses might determine the disease severity for both familial Mediterranean fever (FMF) and periodontitis. The present study aimed to investigate the relationship of FMF with periodontitis and to search for the potential association between periodontitis and MEFV gene missense variations in patients with FMF. The study consisted of 97 FMF patients and 34 healthy volunteers. FMF patients were classified according to the kind of MEFV gene mutation: (1) patients with homozygous M694V gene mutation, (2) patients with heterozygous M694V gene mutation, and (3) patients with MEFV gene different mutations. Gingival Index (GI), Plaque Index (PI), probing pocket depth (PD), and clinical attachment level (CAL) were measured in all participants. The results of multivariate logistic regression showed a highly significant association between homozygous M694V gene mutation and periodontitis in FMF patients (p < 0.05). After adjusting for potential confounders (smoking, body weight, age, and gender), FMF patients with homozygous M694V gene mutation were 3.51 (1.08-11.45) times more likely to present periodontitis than the other FMF patients. These results indicate that the presence of homozygous M694V gene mutation seems to increase the risk for periodontitis in FMF patients.

  7. Mutational analysis of the HGSNAT gene in Italian patients with mucopolysaccharidosis IIIC (Sanfilippo C syndrome). Mutation in brief #959. Online. (United States)

    Fedele, Anthony Olind; Filocamo, Mirella; Di Rocco, Maja; Sersale, Giovanna; Lübke, Torben; di Natale, Paola; Cosma, Maria Pia; Ballabio, Andrea


    Mucopolysaccharidosis (MPS) describes any inherited lysosomal storage disorder resulting from an inability to catabolize glycosaminoglycans. MPS III (or Sanfilippo syndrome) is an autosomal recessive disease caused by a failure to degrade heparan sulphate. There are four subtypes of MPS III, each categorized by a deficiency in a specific enzyme involved in the heparan sulphate degradation pathway. The genes mutated in three of these (MPS IIIA, MPS IIIB, and MPS IIID) have been cloned for some time. However, only very recently has the gene for MPS IIIC (heparin acetyl CoA: alpha-glucosaminide N-acetyltransferase, or HGSNAT) been identified. Its product (previously termed transmembrane protein 76, or TMEM76) has little sequence similarity to other proteins of known function, although it is well conserved among all species. In this study, a group of MPS IIIC patients, who are mainly of Italian origin, have been clinically characterized. Furthermore, mutational analysis of the HGSNAT gene in these patients resulted in the identification of nine alleles, of which eight are novel. Three splice-site mutations, three frameshift deletions resulting in premature stop codons, one nonsense mutation, and two missense mutations were identified. The latter are of particular interest as they are located in regions which are predicted to be of functional significance. This research will aid in determining the molecular basis of HGSNAT protein function, and the mechanisms underlying MPS IIIC.

  8. A novel pyrosequencing assay for the detection of neuraminidase inhibitor resistance-conferring mutations among clinical isolates of avian H7N9 influenza virus. (United States)

    Qi, Yuhua; Fan, Huan; Qi, Xian; Zhu, Zheng; Guo, Xiling; Chen, Yin; Ge, Yiyue; Zhao, Kangchen; Wu, Tao; Li, Yan; Shan, Yunfeng; Zhou, Minghao; Shi, Zhiyang; Wang, Hua; Cui, Lunbiao


    A novel reassortant avian influenza A virus (H7N9) emerged in humans in Eastern China in late February 2013. All virus strains were resistant to adamantanes (amantadine and rimantadine), but susceptible to neuraminidase inhibitors (NAIs) (oseltamivir and zanamivir). One strain (A/shanghai/1/2013) contained the R294K substitution in the neuraminidase (NA) gene, indicating resistance to oseltamivir. Pyrosequencing has proven to be a useful tool in the surveillance of drug resistance in influenza A viruses. Here, we describe a reverse transcription (RT)-PCR assay coupled with pyrosequencing to identify the NA residues E120, H276, and R294 (N9 numbering) of H7N9 viruses. A total of 43 specimens (26 clinical samples and 17 isolates) were tested. Only one isolate containing the E120V heterogenic mutation was detected by pyrosequencing and confirmed by Sanger sequencing. However, this mutation was not detected in the original clinical specimen. Since virus isolation might lead to the selection of variants that might not fully represent the virus population in the clinical specimens, we suggest that using pyrosequencing to detect NAI resistance in H7N9 viruses directly from clinical specimens rather than from cultured isolates. No cross-reactions with other types of influenza virus and respiratory tract viruses were found, and this assay has a sensitivity of 100 copies of synthetic RNA for all three codons. The high sensitivity and specificity of the assay should be sufficient for the detection of positive clinical specimens. In this study, we provide a rapid and reliable method for the characterization of NAI resistance in H7N9 viruses.

  9. 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....

  10. Familial Mediterranean fever gene mutations in the inner northern region of Turkey and genotype-phenotype correlation in children. (United States)

    Yilmaz, Resul; Ozer, Samet; Ozyurt, Huseyin; Erkorkmaz, Unal; Sahin, Semsettin


    Familial Mediterranean fever (FMF) is an autosomal recessive disorder characterised by recurrent episodes of fever, polyserositis and rash. The aim of this study was to determine the most common mutations and clinical features, and their relationships. The medical records of 78 patients were evaluated retrospectively. All of the patients had been diagnosed with FMF according to Tel Hashomer criteria between January 2005 and May 2008 in general paediatric clinics of the School of Medicine at Gaziosmanpasa University. Twelve mutations were detected in the 78 patients by polymerase chain reaction-enzyme-linked immunosorbent assay. The patients were classified into three groups according to allele status. The most prominent clinical symptoms were abdominal pain (95%), fever (90%), arthritis (33%) and pleuritis (31%). Seventeen different genotypes were identified. The mutations were homozygous in 25 (32%) patients, compound heterozygous in 28 (36%) patients and heterozygous in 22 (28%) patients. No mutation was detected in three (4%) patients. The most frequent mutations were M694V (55%), M680I (16%), E148Q (10%) and P369S (4%). The mean symptom severity score was highest in the homozygous group, and high levels of C-reactive protein were also detected in this group. In addition to clinical criteria, molecular studies for detecting disease-causing mutations are needed to establish the diagnosis of FMF. FMF patients who were homozygous for MEFV gene mutations had a higher symptom severity score and higher incidence of appendectomy. The broad spectrum of mutations may reflect intercultural interactions of ethnic groups in Anatolia. Nation-wide studies may help to determine the relationships among demographic, clinical and genetic features of FMF.

  11. Spectrum of MECP2 gene mutations in a cohort of Indian patients with Rett syndrome: report of two novel mutations. (United States)

    Das, Dhanjit Kumar; Raha, Sarbani; Sanghavi, Daksha; Maitra, Anurupa; Udani, Vrajesh


    Rett syndrome (RTT) is an X-linked neurodevelopmental disorder, primarily affecting females and characterized by developmental regression, epilepsy, stereotypical hand movements, and motor abnormalities. Its prevalence is about 1 in 10,000 female births. Rett syndrome is caused by mutations within methyl CpG-binding protein 2 (MECP2) gene. Over 270 individual nucleotide changes which cause pathogenic mutations have been reported. However, eight most commonly occurring missense and nonsense mutations account for almost 70% of all patients. We screened 90 individuals with Rett syndrome phenotype. A total of 19 different MECP2 mutations and polymorphisms were identified in 27 patients. Of the 19 mutations, we identified 7 (37%) frameshift, 6 (31%) nonsense, 14 (74%) missense mutations and one duplication (5%). The most frequent pathogenic changes were: missense p.T158M (11%), p.R133C (7.4%), and p.R306C (7.4%) and nonsense p.R168X (11%), p.R255X (7.4%) mutations. We have identified two novel mutations namely p.385-388delPLPP present in atypical patients and p.Glu290AlafsX38 present in a classical patient of Rett syndrome. Sequence homology for p.385-388delPLPP mutation revealed that these 4 amino acids were conserved across mammalian species. This indicated the importance of these 4 amino acids in structure and function of the protein. A novel variant p.T479T has also been identified in a patient with atypical Rett syndrome. A total of 62 (69%) patients remained without molecular genetics diagnosis that necessitates further search for mutations in other genes like CDKL5 and FOXG1 that are known to cause Rett phenotype. The majority of mutations are detected in exon 4 and only one mutation was present in exon 3. Therefore, our study suggests the need for screening exon 4 of MECP2 as first line of diagnosis in these patients.

  12. Design and validation of a next generation sequencing assay for hereditary BRCA1 and BRCA2 mutation testing

    Directory of Open Access Journals (Sweden)

    Hyunseok P. Kang


    Full Text Available Hereditary breast and ovarian cancer syndrome, caused by a germline pathogenic variant in the BRCA1 or BRCA2 (BRCA1/2 genes, is characterized by an increased risk for breast, ovarian, pancreatic and other cancers. Identification of those who have a BRCA1/2 mutation is important so that they can take advantage of genetic counseling, screening, and potentially life-saving prevention strategies. We describe the design and analytic validation of the Counsyl Inherited Cancer Screen, a next-generation-sequencing-based test to detect pathogenic variation in the BRCA1 and BRCA2 genes. We demonstrate that the test is capable of detecting single-nucleotide variants (SNVs, short insertions and deletions (indels, and copy-number variants (CNVs, also known as large rearrangements with zero errors over a 114-sample validation set consisting of samples from cell lines and deidentified patient samples, including 36 samples with BRCA1/2pathogenic germline mutations.

  13. [Analysis of DIAPH3 gene mutation in a boy with autism spectrum disorder]. (United States)

    Xie, Jiang; Li, Hua; Zhu, Hua; Huang, Li; Li, Hongxia; Zhang, Xiling; Zhou, Yongmei; Zhou, Qiang; Xu, Wenming


    To analyze the clinical manifestations and gene mutation of a 6 year old boy with autism spectrum disorders (ASD). Peripheral blood of the boy and his parents were subjected to genetic testing. The patient was diagnosed with typical autism. Exome sequencing has identified mutations of four candidate genes, namely TUT1, DIAPH3, REELIN and SETD2, which were confirmed with Sanger sequencing. Analysis of family members confirmed that the missense mutations of DIAPH3 and SETD2 genes were of de novo origin. Missense mutations of DIAPH3 and SETD2 genes may have contributed to the risk of ASD. Disrupted neurogenesis associated with such mutations may have been the underlying mechanism for ASD.

  14. Somatic frameshift mutations in the Bloom syndrome BLM gene are frequent in sporadic gastric carcinomas with microsatellite mutator phenotype

    Directory of Open Access Journals (Sweden)

    Matei Irina


    Full Text Available Abstract Background Genomic instability has been reported at microsatellite tracts in few coding sequences. We have shown that the Bloom syndrome BLM gene may be a target of microsatelliteinstability (MSI in a short poly-adenine repeat located in its coding region. To further characterize the involvement of BLM in tumorigenesis, we have investigated mutations in nine genes containing coding microsatellites in microsatellite mutator phenotype (MMP positive and negative gastric carcinomas (GCs. Methods We analyzed 50 gastric carcinomas (GCs for mutations in the BLM poly(A tract aswell as in the coding microsatellites of the TGFβ1-RII, IGFIIR, hMSH3, hMSH6, BAX, WRN, RECQL and CBL genes. Results BLM mutations were found in 27% of MMP+ GCs (4/15 cases but not in any of the MMP negative GCs (0/35 cases. The frequency of mutations in the other eight coding regions microsatellite was the following: TGFβ1-RII (60 %, BAX (27%, hMSH6 (20%,hMSH3 (13%, CBL (13%, IGFIIR (7%, RECQL (0% and WRN (0%. Mutations in BLM appear to be more frequently associated with frameshifts in BAX and in hMSH6and/or hMSH3. Tumors with BLM alterations present a higher frequency of unstable mono- and trinucleotide repeats located in coding regions as compared with mutator phenotype tumors without BLM frameshifts. Conclusions BLM frameshifts are frequent alterations in GCs specifically associated with MMP+tumors. We suggest that BLM loss of function by MSI may increase the genetic instability of a pre-existent unstable genotype in gastric tumors.

  15. Isolated renal vein thrombosis associated with MTHFR-1298 and PAI-1 4G gene mutations. (United States)

    Cinemre, Hakan; Bilir, Cemil; Akdemir, Nermin


    Isolated renal vein thrombosis is very rare without the presence of nephrotic syndrome. It is more common in the newborns and infants. Whereas major risk factors in adults are the procoagulant states such as protein C or S deficiency, factor V Leiden mutation, primary or secondary antiphospholipid syndrome, severe hypothyroidism, and trauma. Here, we report a case of isolated renal vein thrombosis associated with MTHFR-1298 and PAI-1 4G gene mutations. It should be noted that the presence of MTHFR-1298 and PAI-1 4G gene mutations together might be one of the examples of genetic mutation combinations that increase the likelihood of a thrombotic event.

  16. Amelogenin signal peptide mutation: Correlation between mutations in the amelogenin gene (AMGX) and manifestations of X-linked amelogenesis imperfecta

    Energy Technology Data Exchange (ETDEWEB)

    Lagerstroem-Fermer, M.; Nilsson, M.; Pettersson, U. [Univ. of Uppsala (Sweden)] [and others


    Formation of tooth enamel is a poorly understood biological process. In this study the authors describe a 9-bp deletion in exon 2 of the amelogenin gene (AMGX) causing X-linked hypoplastic amelogenesis imperfecta, a disease characterized by defective enamel. The mutation results in the loss of 3 amino acids and exchange of 1 in the signal peptide of the amelogenin protein. This deletion in the signal peptide probably interferes with translocation of the amelogenin protein during synthesis, resulting in the thin enamel observed in affected members of the family. The authors compare this mutation to a previously reported mutation in the amelogenin gene that causes a different disease phenotype. The study illustrates that molecular analysis can help explain the various manifestations of a tooth disorder and thereby provide insights into the mechanisms of tooth enamel formation. 16 refs., 2 figs., 1 tab.

  17. Multi-Center Evaluation of the Fully Automated PCR-Based Idylla™ KRAS Mutation Assay for Rapid KRAS Mutation Status Determination on Formalin-Fixed Paraffin-Embedded Tissue of Human Colorectal Cancer

    DEFF Research Database (Denmark)

    Solassol, Jérôme; Vendrell, Julie; Märkl, Bruno


    sample-to-result solution. This test enables qualitative detection of 21 mutations in codons 12, 13, 59, 61, 117, and 146 of the KRAS oncogene being clinically relevant according to the latest clinical guidelines. Here, the performance of the Idylla™ KRAS Mutation Assay, for Research Use Only...

  18. Burkitt's lymphoma is a malignancy of mature B cells expressing somatically mutated V region genes.


    Klein, U.; Klein, G.; Ehlin-Henriksson, B.; Rajewsky, K.; Küppers, R.


    BACKGROUND: The developmental stage from which stems the malignant B cell population in Burkitt's lymphoma (BL) is unclear. An approach to answering this question is provided by the sequence analysis of rear-ranged immunoglobulin (Ig) variable region (V) genes from BL for evidence of somatic mutations, together with a phenotypic characterization. As somatic hypermutation of Ig V region genes occurs in germinal center B cells, somatically mutated Ig genes are found in germinal center B cells a...

  19. FAM20A Gene Mutation: Amelogenesis or Ectopic Mineralization?

    Directory of Open Access Journals (Sweden)

    Guilhem Lignon


    Full Text Available Background and objective:FAM20A gene mutations result in enamel renal syndrome (ERS associated with amelogenesis imperfecta (AI, nephrocalcinosis, gingival fibromatosis, and impaired tooth eruption. FAM20A would control the phosphorylation of enamel peptides and thus enamel mineralization. Here, we characterized the structure and chemical composition of unerupted tooth enamel from ERS patients and healthy subjects.Methods: Tooth sections were analyzed by Scanning Electron Microscopy (SEM, Energy Dispersive Spectroscopy (EDS, X-Ray Diffraction (XRD, and X-Ray Fluorescence (XRF.Results: SEM revealed that prisms were restricted to the inner-most enamel zones. The bulk of the mineralized matter covering the crown was formed by layers with varying electron-densities organized into lamellae and micronodules. Tissue porosity progressively increased at the periphery, ending with loose and unfused nanonodules also observed in the adjoining soft tissues. Thus, the enamel layer covering the dentin in all ERS patients (except a limited layer of enamel at the dentino-enamel junction displayed an ultrastructural globular pattern similar to one observed in ectopic mineralization of soft tissue, notably in the gingiva of Fam20a knockout mice. XRD analysis confirmed the existence of alterations in crystallinity and composition (vs. sound enamel. XRF identified lower levels of calcium and phosphorus in ERS enamel. Finally, EDS confirmed the reduced amount of calcium in ERS enamel, which appeared similar to dentin.Conclusion: This study suggests that, after an initial normal start to amelogenesis, the bulk of the tissue covering coronal dentin would be formed by different mechanisms based on nano- to micro-nodule aggregation. This evocated ectopic mineralization process is known to intervene in several soft tissues in FAM20A gene mutant.

  20. Comparative analysis of dideoxy sequencing,the KRAS StripAssay and pyrosequencing for detection of KRAS mutation

    Institute of Scientific and Technical Information of China (English)


    AIM:To compare the differences between dideoxy sequencing/KRAS StripAssay/pyrosequencing for detection of KRAS mutation in Chinese colorectal cancer (CRC) patients.METHODS:Formalin-f ixed, paraff in-embedded (FFPE) samples with tumor cells ≥ 50% were collected from 100 Chinese CRC patients at Beijing Cancer Hospital. After the extraction of genome DNA from FFPE samples, fragments contained codons 12 and 13 of KRAS exon 2 were amplified by polymerase chain reaction and analyzed by dideoxy sequencing, the KRA...

  1. Mutations in inhibin and activin genes associated with human disease. (United States)

    Shelling, Andrew N


    Inhibins and activins are members of the transforming growth factor (TGFβ) superfamily, that includes the TGFβs, inhibins and activins, bone morphogenetic proteins (BMPs) and growth and differentiation factors (GDFs). The family members are expressed throughout the human body, and are involved in the regulation of a range of important functions. The precise regulation of the TGFβ pathways is critical, and mutations of individual molecules or even minor alterations of signalling will have a significant affect on function, that may lead to development of disease or predisposition to the development of disease. The inhibins and activins regulate aspects of the male and female reproductive system, therefore, it is not surprising that most of the diseases associated with abnormalities of the inhibin and activin genes are focused on reproductive disorders and reproductive cancers. In this review, I highlight the role of genetic variants in the development of conditions such as premature ovarian failure, pre-eclampsia, and various reproductive cancers. Given the recent advances in human genetic research, such as genome wide association studies and next generation sequencing, it is likely that inhibins and activins will be shown to play more important roles in a range of human genetic diseases in the future.

  2. TET2 gene mutation is unfavorable prognostic factor in cytogenetically normal acute myeloid leukemia patients with NPM1+ and FLT3-ITD - mutations. (United States)

    Tian, Xiaopeng; Xu, Yang; Yin, Jia; Tian, Hong; Chen, Suning; Wu, Depei; Sun, Aining


    Cytogenetically normal acute myeloid leukemia (cn-AML) is a group of heterogeneous diseases. Gene mutations are increasingly used to assess the prognosis of cn-AML patients and guide risk-adapted treatment. In the present study, we analyzed the molecular genetics characteristics of 373 adult cn-AML patients and explored the relationship between TET2 gene mutations or different genetic mutation patterns and prognosis. We found that 16.1 % of patients had TET2 mutations, 31.6 % had FLT3 internal tandem duplications (ITDs), 6.2 % had FLT3 tyrosine kinase domain mutations, 2.4 % had c-KIT mutations, 37.8 % had NPM1 mutations, 11.3 % had WT1 mutations, 5.9 % had RUNX1 mutations, 11.5 % had ASXL1 mutations, 3.8 % had MLL-PTDs, 7.8 % had IDH1 mutations, 7.8 % had NRAS mutations, 12.3 % had IDH2 mutations, 1.6 % had EZH2 mutations, and 14.7 % had DNMT3A mutations, while none had CBL mutations. Gene mutations were detected in 76.94 % (287/373) of all patients. In the NPM1m(+) patients, those with TET2 mutations were associated with a shorter median overall survival (OS) as compared to TET2 wild-type (wt) patients (9.9 vs. 27.0 months, respectively; P = 0.023); Interestingly, the TET2 mutation was identified as an unfavorable prognostic factor and was closely associated with a shorter median OS as compared to TET2-wt (9.5 vs. 32.2 months, respectively; P = 0.013) in the NPM1m(+)/FLT3-ITDm(-) patient group. Thus, identification of TET2 combined with classic NPM1 and FLT3-ITD mutations allowed us to stratify cn-AML into distinct subtypes.


    Directory of Open Access Journals (Sweden)

    Petar Babović


    Full Text Available TP53 tumor suppressor gene mutations are the most frequent genetic alterations in human cancer affecting a specific gene. The occurrence of TP53 mutations is considerably influenced by cancer-initiating events, such as DNA damage, the aftermath of which is the promotion of cancer development through the loss of anti-proliferative activities, including apoptosis and cellular senescence. Over 27.000 TP53 gene mutations have been discovered and found in more than 50% of human cancers. The most frequent alterations are the point mutations with a single base substitution in gene segment encoding for DNA-binding domaine of p53 molecule, leading to the production of mutant protein that differs from the wild-type protein by one amino acid (missense mutations usually causing the change in tertiary structure of gene product, thus preventing p53 to bind to DNA and activate transcription of target genes. The result of the mutations may also be the proteins with new, abnormal functions, and the ability to modulate expression of genes responsible for neoangiogenesis, resistance to chemotherapeutics and prevention of tumor initiation and promotion. In such circumstances, not only the mutant TP53 loses its tumor suppressive function, but acquires oncogenic potential and becomes an active participant in the neoplastic transformation of the cell.Vast heterogeneity of mutations and methodological approaches in p53 status assessment represent the main difficulties in rapid and effective integration of basic p53 research into clinical practice.

  4. Mutation spectrum of the TYR and SLC45A2 genes in patients with oculocutaneous albinism. (United States)

    Ko, Jung Min; Yang, Jung-Ah; Jeong, Seon-Yong; Kim, Hyon-Ju


    Oculocutaneous albinism (OCA) is a group of inherited disorders characterized by defective melanin biosynthesis. OCA1, the most common and severe form, is caused by mutations in the tyrosinase (TYR) gene. OCA4, caused by mutations in the SLC45A2 gene, has frequently been reported in the Japanese population. To determine the mutational spectrum in Korean OCA patients, 12 patients were recruited. The samples were first screened for TYR mutations, and negative samples were screened for SLC45A2 mutations. OCA1 was confirmed in 8 of 12 (66.7%) patients, and OCA4 was diagnosed in 1 (8.3%) patient. In the OCA1 patients, a total of 6 distinct TYR mutations were found in 15 of 16 (93.8%) alleles, all of which had been previously reported. Out of the 6 alleles, c.929insC was the most frequently detected (31.3%), and was mainly associated with OCA1A phenotypes. Other TYR mutations identified included c.1037-7T>A/c.1037-10delTT, p.D383N, p.R77Q and p.R299H. These largely overlapped with mutations found in Japanese and Chinese patients. The SLC45A2 gene analysis identified 1 novel mutation, p.D93N, in 1 patient. This study has provided information on the mutation spectrum in Korean OCA patients, and allows us to estimate the relative frequencies of OCA1 and OCA4 in Korea.

  5. Next-generation sequencing identifies novel CACNA1A gene mutations in episodic ataxia type 2. (United States)

    Maksemous, Neven; Roy, Bishakha; Smith, Robert A; Griffiths, Lyn R


    Episodic Ataxia type 2 (EA2) is a rare autosomal dominantly inherited neurological disorder characterized by recurrent disabling imbalance, vertigo, and episodes of ataxia lasting minutes to hours. EA2 is caused most often by loss of function mutations of the calcium channel gene CACNA1A. In addition to EA2, mutations in CACNA1A are responsible for two other allelic disorders: familial hemiplegic migraine type 1 (FHM1) and spinocerebellar ataxia type 6 (SCA6). Herein, we have utilized next-generation sequencing (NGS) to screen the coding sequence, exon-intron boundaries, and Untranslated Regions (UTRs) of five genes where mutation is known to produce symptoms related to EA2, including CACNA1A. We performed this screening in a group of 31 unrelated patients with EA2 symptoms. Both novel and known mutations were detected through NGS technology, and confirmed through Sanger sequencing. Genetic testing showed in total 15 mutation bearing patients (48%), of which nine were novel mutations (6 missense and 3 small frameshift deletion mutations) and six known mutations (4 missense and 2 nonsense).These results demonstrate the efficiency of our NGS-panel for detecting known and novel mutations for EA2 in the CACNA1A gene, also identifying a novel missense mutation in ATP1A2 which is not a normal target for EA2 screening.

  6. A novel mutation in the HEXA gene specific to Tay-Sachs disease carriers of Jewish Iraqi origin. (United States)

    Karpati, M; Peleg, L; Gazit, E; Akstein, E; Goldman, B


    An increased frequency of carriers of 1:140, as defined by reduced hexosaminidase A (HexA) activity, was observed among Iraqi Jews participating in the Tay-Sachs disease (TSD) carrier detection program. Prior to this finding, TSD among Jews had been restricted to those of Eastern European (Ashkenazi) and Moroccan descent with carrier frequencies of 1:29 and 1:110 for Jews of Ashkenazi and Moroccan extraction, respectively. A general, pan-ethnic frequency of approximately 1:280 has been observed among other Jewish Israeli populations. Analysis of 48 DNA samples from Iraqi Jews suspected, by enzymatic assay, to be carriers revealed a total of five mutations, one of which was novel. In nine carriers (19%), a known mutation typical to either Ashkenazi or Moroccan Jews was identified. DeltaF304/ 305 was detected in four individuals, and + 1278TATC in three. G269S and R170Q each appeared in a single person. The new mutation, G749T, resulting in a substitution of glycine to valine at position 250 has been found in 19 of the DNA samples (40%). This mutation was not detected among 100 non-carrier, Iraqi Jews and 65 Ashkenazi enzymatically determined carriers. Aside from Ashkenazi and Moroccan Jews, a specific mutation in the HEXA gene has now also been identified in Jews of Iraqi descent.

  7. Aminoglycosides, but not PTC124 (Ataluren), rescue nonsense mutations in the leptin receptor and in luciferase reporter genes. (United States)

    Bolze, Florian; Mocek, Sabine; Zimmermann, Anika; Klingenspor, Martin


    In rare cases, monogenetic obesity is caused by nonsense mutations in genes regulating energy balance. A key factor herein is the leptin receptor. Here, we focus on leptin receptor nonsense variants causing obesity, namely the human W31X, murine Y333X and rat Y763X mutations, and explored their susceptibilities to aminoglycoside and PTC124 mediated translational read-through in vitro. In a luciferase based assay, all mutations - when analysed within the mouse receptor - were prone to aminoglycoside mediated nonsense suppression with the highest susceptibility for W31X, followed by Y763X and Y333X. For the latter, the corresponding rodent models appear valuable for in vivo experiments. When W31X was studied in the human receptor, its superior read-through susceptibility - initially observed in the mouse receptor - was eliminated, likely due to the different nucleotide context surrounding the mutation in the two orthologues. The impact of the surrounding context on the read-through opens the possibility to discover novel sequence elements influencing nonsense suppression. As an alternative to toxic aminoglycosides, PTC124 was indicated as a superior nonsense suppressor but inconsistent data concerning its read-through activity are reported. PTC124 failed to rescue W31X as well as different nonsense mutated luciferase reporters, thus, challenging its ability to induce translational read-through.

  8. Expression and mutation of c-kit gene in gastrointestinal stromal tumors

    Institute of Scientific and Technical Information of China (English)

    Fei Feng; Xiao-Hong Liu; Qiang Xie; Wei-Qiang Liu; Cheng-Guang Bai; Da-Lie Ma


    AIM: To investigate the expression and mutation of c-kit gene and its correlation with the clinical pathology and prognosis of gastrointestinal stromal tumors (GISTs).METHODS: A total of 94 cases of GISTs, 10 leiomyomas and 2 schwannomas were studied for the expression of KIT by immunohistochemistry. The c-kit gene mutations in exon 11 of these specimens were detected by PCR-SSCP technique.RESULTS: Of the 94 cases of GISTs, 91 (96.8 %) expressed the KIT protein. Leiomyomas and schwannomas were negative for KIT. The c-kit gene mutations of exon 11 were found in 38 out of the 94 cases of GISTs (40.4 %). The mutations involved point mutations (Va1560-Asp, Ile563-Met),del 557-559 and 579ins12. No mutations were detectable in benign GISTs, leiomyomas or schwannomas. The patients with mutation-positive GISTs showed more frequent recurrences, invasion and metastasis in adjacent tissues than those with mutation-negative ones.CONCLUSION: KIT is a useful marker for diagnosis of GISTs.Mutation of the c-kit gene may play a significant role in the pathogenesis of GISTs and may be associated with poor prognosis in patients with GISTs.

  9. Congenital nephrogenic diabetes insipidus with a novel mutation in the aquaporin 2 gene. (United States)

    Park, Youn Jong; Baik, Haing Woon; Cheong, Hae Il; Kang, Ju Hyung


    Congenital nephrogenic diabetes insipidus (CNDI) is a rare disorder caused by mutations of the arginine vasopressin (AVP) V2 receptor or aquaporin 2 (AQP2) genes. The current study presented the case of CNDI in a 1-month-old male with a novel mutation in the AQP2 gene. The patient was referred due to the occurrence of hypernatremia and mild-intermittent fever since birth. An AVP stimulation test was compatible with CNDI as there was no significant response to desmopressin. Molecular genetic analysis demonstrated two mutations in exon 1 of the AQP2 gene: C to T transition, which resulted in a missense mutation of (108)Thr (ACG) to Met (ATG); and a 127, 128 delCA, which resulted in a deletion mutation of glutamine in position 43 at codon CAG as the first affected amino acid, with the new reading frame endign in a termination codon at position 62. The molecular genetic analysis of the parents showed that the missense mutation was inherited maternally and the deletion mutation was inherited paternally. The parents showed no signs or symptoms of CNDI, indicating autosomal recessive inheritance. The (108)Thr (ACG) to Met (ATG) mutation was confirmed as a novel mutation. Therefore, the molecular identification of the AQP2 gene has clinical significance, as early recognition of CNDI in infants that show only non-specific symptoms, can be facilitated. Thus, repeated episodes of dehydration, which may cause physical and mental retardation can be avoided.

  10. Pyrosequencing-Based Assays for Rapid Detection of HER2 and HER3 Mutations in Clinical Samples Uncover an E332E Mutation Affecting HER3 in Retroperitoneal Leiomyosarcoma. (United States)

    González-Alonso, Paula; Chamizo, Cristina; Moreno, Víctor; Madoz-Gúrpide, Juan; Carvajal, Nerea; Daoud, Lina; Zazo, Sandra; Martín-Aparicio, Ester; Cristóbal, Ion; Rincón, Raúl; García-Foncillas, Jesús; Rojo, Federico


    Mutations in Human Epidermal Growth Factor Receptors (HER) are associated with poor prognosis of several types of solid tumors. Although HER-mutation detection methods are currently available, such as Next-Generation Sequencing (NGS), alternative pyrosequencing allow the rapid characterization of specific mutations. We developed specific PCR-based pyrosequencing assays for identification of most prevalent HER2 and HER3 mutations, including S310F/Y, R678Q, L755M/P/S/W, V777A/L/M, 774-776 insertion, and V842I mutations in HER2, as well as M91I, V104M/L, D297N/V/Y, and E332E/K mutations in HER3. We tested 85 Formalin Fixed and Paraffin Embbeded (FFPE) samples and we detected three HER2-V842I mutations in colorectal carcinoma (CRC), ovarian carcinoma, and pancreatic carcinoma patients, respectively, and a HER2-L755M mutation in a CRC specimen. We also determined the presence of a HER3-E332K mutation in an urothelial carcinoma sample, and two HER3-D297Y mutations, in both gastric adenocarcinoma and CRC specimens. The D297Y mutation was previously detected in breast and gastric tumors, but not in CRC. Moreover, we found a not-previously-described HER3-E332E synonymous mutation in a retroperitoneal leiomyosarcoma patient. The pyrosequencing assays presented here allow the detection and characterization of specific HER2 and HER3 mutations. These pyrosequencing assays might be implemented in routine diagnosis for molecular characterization of HER2/HER3 receptors as an alternative to complex NGS approaches.

  11. A new PKLR gene mutation in the R-type promoter region affects the gene transcription causing pyruvate kinase deficiency. (United States)

    Manco, L; Ribeiro, M L; Máximo, V; Almeida, H; Costa, A; Freitas, O; Barbot, J; Abade, A; Tamagnini, G


    Mutations in the PKLR gene responsible for pyruvate kinase (PK)-deficient anaemia are mainly located in the coding regions: 11 are in the splicing sites and, recently, three mutations have been described in the promoter region. We now report a novel point mutation A-->G on nucleotide 72, upstream from the initiation codon of the PKLR gene, in four Portuguese PK-deficient patients. This new regulatory mutation occurs within the most proximal of the four GATA motifs (GATA-A element) in the R-type promoter region. In two patients who were homozygous for this mutation, a semiquantitative reverse transcription polymerase chain reaction (PCR) procedure was used to evaluate the amount of R-PK mRNA transcript in the reticulocytes. The mRNA level was about five times lower than in normal controls, demonstrating that the PKLR gene transcription is severely affected, most probably because the -72A-->G point mutation disables the binding of the erythroid transcription factor GATA-1 to the GATA-A element. Supporting these data, the two patients homozygous for the -72A-->G mutation had severe haemolytic anaemia and were transfusion dependent until splenectomy. Two other patients who were compound heterozygous for this mutation and the previously described missense mutation 1456C-->T had a mild condition.

  12. Mutations in genes involved in nonsense mediated decay ameliorate the phenotype of sel-12 mutants with amber stop mutations in Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Aubert Sylvie


    Full Text Available Abstract Background Presenilin proteins are part of a complex of proteins that can cleave many type I transmembrane proteins, including Notch Receptors and the Amyloid Precursor Protein, in the middle of the transmembrane domain. Dominant mutations in the human presenilin genes PS1 and PS2 lead to Familial Alzheimer's disease. Mutations in the Caenorhabditis elegans sel-12 presenilin gene cause a highly penetrant egg-laying defect due to reduction of signalling through the lin-12/Notch receptor. Mutations in six spr genes (for suppressor of presenilin are known to strongly suppress sel-12. Mutations in most strong spr genes suppress sel-12 by de-repressing the transcription of the largely functionally equivalent hop-1 presenilin gene. However, how mutations in the spr-2 gene suppress sel-12 is unknown. Results We show that spr-2 mutations increase the levels of sel-12 transcripts with Premature translation Termination Codons (PTCs in embryos and L1 larvae. mRNA transcripts from sel-12 alleles with PTCs undergo degradation by a process known as Nonsense Mediated Decay (NMD. However, spr-2 mutations do not appear to affect NMD. Mutations in the smg genes, which are required for NMD, can restore sel-12(PTC transcript levels and ameliorate the phenotype of sel-12 mutants with amber PTCs. However, the phenotypic suppression of sel-12 by smg genes is nowhere near as strong as the effect of previously characterized spr mutations including spr-2. Consistent with this, we have identified only two mutations in smg genes among the more than 100 spr mutations recovered in genetic screens. Conclusion spr-2 mutations do not suppress sel-12 by affecting NMD of sel-12(PTC transcripts and appear to have a novel mechanism of suppression. The fact that mutations in smg genes can ameliorate the phenotype of sel-12 alleles with amber PTCs suggests that some read-through of sel-12(amber alleles occurs in smg backgrounds.

  13. Detection of mutation in embB gene of Mycobacterium tuberculosis from clinical isolates of tuberculous patients in China by means of reverse-dot blot hybridization

    Institute of Scientific and Technical Information of China (English)



    The relationship between embB mutation of Mycobacterium tuberculosis and ethambutol (EMB) resistance of the clinical isolates of tuberculous patients in China was investigated by reversedot blot hybridization (RDBH) in addition to evaluating the clinical value with application of PCR-RD-BH technique to detect EMB resistance. In the present study, the genotypes of the 258 bp fragments of embB genes from 196 clinical isolates of M. tuberculosis were analysed with RDBH and DNA sequencing. It was demonstrated that 60 out of 91 phenotypically EMB-resistant isolates (65.9%) showed 5 types of missense mutations at codon 306 of embB gene, resulting in the replacement of the Met residue of the wild type strain with Val, Ile or Leu residues. In these mutations, the GTP mutation (38/91,41.8% ) and the ATA mutation ( 16/91, 17.6% ) were the most encountered genotypes. The embB mutation at codon 306 could also be found in 69 isolates of phenotypically EMB-sensitive but resistant to other anti-tuberculous drugs, but no such gene mutation could be found in 36 strains of drug-sensitive isolates. Meanwhile, the concordance with the results of DNA sequencing for one wide-type probe and 5 probes for specific mutations was 100%. It was concluded that the EMB-resistance occurring in most M. tuberculosis is due to appearance of embB mutation at codon 306, and the PCR-RDBH assay was proved to be a rapid, simple and reliable method for the detection of gene mutations, which might be a good alternative for the drug-resistance screening.


    Institute of Scientific and Technical Information of China (English)

    苏鹏程; 李子禹; 张连海; 万文徽; 任晖; 张桂国; 王怡; 邓国仁; 季加孚


    Objective: To investigated p53 gene mutation in plasma of gastric cancer patients. Methods: DNA extracted from plasma and matched tumor and tumor-adjacent non-cancerous tissues of 96 gastric cancer patients, and DNA from 20 healthy volunteers were studied. Exon 5, 6, 7, and 8 of p53 were amplified by Polymerase Chain Reaction (PCR). The mutation status was analyzed by denaturing high-performance liquid chromatography (DHPLC), followed by direct sequencing of cases with aberrant chromatographic patterns. Results: Heterozygous mutations of p53 gene were detected in 19.9% (19/96) of primary tumor tissues and 5.2% (5/96) of corresponding plasma. All p53 gene mutations detected in plasma DNA consisted with mutations in the matched primary tumor samples. Neither the tumor-adjacent gastric mucosa tissues nor control plasma from healthy volunteers showed p53 gene mutation. No correlation was found between p53 mutation status and clinicopathological features of gastric cancer patients. Conclusion: p53 gene mutation in plasma can be detected in tissues and plasma of gastric cancer patients, which could be applied in screening and surveillance of this disease.

  15. [Identification of novel pathogenic gene mutations in pediatric acute myeloid leukemia by whole-exome resequencing]. (United States)

    Shiba, Norio


    A new class of gene mutations, identified in the pathogenesis of adult acute myeloid leukemia (AML), includes DNMT3A, IDH1/2, TET2 and EZH2. However, these mutations are rare in pediatric AML cases, indicating that pathogeneses differ between adult and pediatric forms of AML. Meanwhile, the recent development of massively parallel sequencing technologies has provided a new opportunity to discover genetic changes across entire genomes or proteincoding sequences. In order to reveal a complete registry of gene mutations, we performed whole exome resequencing of paired tumor-normal specimens from 19 pediatric AML cases using Illumina HiSeq 2000. In total, 80 somatic mutations or 4.2 mutations per sample were identified. Many of the recurrent mutations identified in this study involved previously reported targets in AML, such as FLT3, CEBPA, KIT, CBL, NRAS, WT1 and EZH2. On the other hand, several genes were newly identified in the current study, including BCORL1 and major cohesin components such as SMC3 and RAD21. Whole exome resequencing revealed a complex array of gene mutations in pediatric AML genomes. Our results indicate that a subset of pediatric AML represents a discrete entity that could be discriminated from its adult counterpart, in terms of the spectrum of gene mutations.

  16. Mutations in low-density lipoprotein receptor gene as a cause of hypercholesterolemia in Taiwan. (United States)

    Chiu, Chih-Yang; Wu, Yi-Chi; Jenq, Shwu-Fen; Jap, Tjin-Shing


    Familial hypercholesterolemia (FH) is inherited as an autosomal dominant trait that has been associated with more than 920 different mutations in the low-density lipoprotein receptor (LDLR) gene. To characterize LDLR gene mutations in the Chinese of Han descent with FH, we isolated genomic DNA from peripheral blood samples of 20 affected subjects and 50 healthy subjects with no family history of hypercholesterolemia. We used polymerase chain reaction and long polymerase chain reaction to amplify the 18 coding exons and the minimal promoter of the LDLR gene, and subjected amplicons to direct sequence analysis. We identified 6 mutations in LDLR gene, including heterozygous missense mutations I420T (ATC-->ACC), C660W (TGC-->TGG), H562Y (CAC-->TAC), and A606T (GCC-->ACC), and a heterozygous and a homozygous mutation in codon P664L (CCG-->CTG) as well as a homozygous large deletion of exons 6 to 8. The FH homozygotes manifested generalized xanthomatosis. One of the mutations we identified (C660W) was novel. In conclusion, we identified 5 missense mutations and 1 large deletion in LDLR gene, including 1 novel mutation in Han Chinese with FH in Taiwan.

  17. Generation of KCL018 research grade human embryonic stem cell line carrying a mutation in the DMPK gene

    Directory of Open Access Journals (Sweden)

    Cristian Miere


    Full Text Available The KCL018 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting one allele of the DMPK gene encoding the dystrophia myotonica protein kinase (2200 trinucleotide repeats; 14 for the normal allele. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays.

  18. Generation of KCL028 research grade human embryonic stem cell line carrying a mutation in the HTT gene

    Directory of Open Access Journals (Sweden)

    Laureen Jacquet


    Full Text Available The KCL028 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting one allele of the HTT gene encoding huntingtin (43 trinucleotide repeats; 21 for the normal allele. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro and in vivo assays.

  19. False-positive rifampicin resistance on Xpert® MTB/RIF caused by a silent mutation in the rpoB gene. (United States)

    Mathys, V; van de Vyvere, M; de Droogh, E; Soetaert, K; Groenen, G


    The Xpert® MTB/RIF assay detects the presence of Mycobacterium tuberculosis and its resistance to rifampicin (RMP) directly in sputum samples. Discrepant results were observed in a case of smear-positive pulmonary tuberculosis that was Xpert-resistant but phenotypically susceptible to RMP. Complementary investigations (repeat Xpert, Genotype®MTBDRplus assay and sequencing of the rpoB gene) revealed the presence of a silent mutation in the rpoB gene, leading to the conclusion of a false-positive Xpert result. As misinterpretation of Xpert results may lead to inappropriate treatment, the presence of rpoB mutations should be confirmed by sequencing the rpoB gene.

  20. Phenylalanine hydroxylase gene mutations in the United States: Report from the maternal PKU collaborative study

    Energy Technology Data Exchange (ETDEWEB)

    Guldberg, P.; Henriksen, K.F.; Guettler, F. [John F. Kennedy Inst., Glostrup (Denmark)] [and others


    The major cause of hyperphenylalaninemia is mutations in the gene encoding phenylalanine hydroxylase (PAH). The known mutations have been identified primarily in European patients. The purpose of this study was to determine the spectrum of mutations responsible for PAH deficiency in the United States. One hundred forty-nine patients enrolled in the Maternal PKU Collaborative Study were subjects for clinical and molecular investigations. PAH gene mutations associated with phenylketonuria (PKU) or mild hyperphenylalaninemia (MHP) were identified on 279 of 294 independent mutant chromosomes, a diagnostic efficiency of 95%. The spectrum is composed of 71 different mutations, including 47 missense mutations, 11 splice mutations, 5 nonsense mutations, and 8 microdeletions. Sixteen previously unreported mutations were identified. Among the novel mutations, five were found in patients with MHP, and the remainder were found in patients with PKU. The most common mutations were R408W, IVS12nt1g{r_arrow}a, and Y414C, accounting for 18.7%, 7.8% and 5.4% of the mutant chromosomes, respectively. Thirteen mutations had relative frequencies of 1%-5%, and 55 mutations each had frequencies {le}1%. The mutational spectrum corresponded to that observed for the European ancestry of the U.S. population. To evaluate the extent of allelic variation at the PAH locus within the United States in comparison with other populations, we used allele frequencies to calculate the homozygosity for 11 populations where >90% ascertainment has been obtained. The United States was shown to contain one of the most heterogeneous populations, with homozygosity values similar to Sicily and ethnically mixed sample populations in Europe. The extent of allelic heterogeneity must be a major determining factor in the choice of mutation-detection methodology for molecular diagnosis in PAH deficiency. 47 refs., 1 fig., 5 tabs.

  1. LHON/MELAS overlap syndrome associated with a mitochondrial MTND1 gene mutation. (United States)

    Blakely, Emma L; de Silva, Rajith; King, Andrew; Schwarzer, Verena; Harrower, Tim; Dawidek, Gervase; Turnbull, Douglass M; Taylor, Robert W


    Pathogenic point mutations in the mitochondrial MTND1 gene have previously been described in association with two distinct clinical phenotypes -- Leber hereditary optic neuropathy (LHON) and mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS). Here we report the first heteroplasmic mitochondrial DNA (mtDNA) point mutation (3376G>A) in the MTND1 gene associated with an overlap syndrome comprising the clinical features of both LHON and MELAS. Muscle histochemistry revealed subtle mitochondrial abnormalities, while biochemical analysis showed an isolated complex I deficiency. Our findings serve to highlight the growing importance of mutations in mitochondrial complex I structural genes in MELAS and its associated overlap syndromes.

  2. A new gene, EVC2, is mutated in Ellis-van Creveld syndrome. (United States)

    Galdzicka, M; Patnala, S; Hirshman, M G; Cai, J-F; Nitowsky, H; Egeland, J A; Ginns, E I


    Ellis-van Creveld syndrome (EvC; MIM 225500) is an autosomal recessive chondrodysplastic dwarfism. Thus far, the identified mutations in the EVC gene located on chromosome 4p16 have only accounted for illness in a small proportion of affected individuals. In this report we describe a novel gene, EVC2, that is mutated in an Ashkenazi individual with EvC syndrome. Our findings demonstrate for the first time that the heterogeneity observed in this disorder is not solely the result of mutations in a single gene.

  3. [The significance of the epigenetics modifying gene mutations in acute myeloid leukemia]. (United States)

    Wakita, Satoshi; Yamaguchi, Hiroki


    In recent years, recurrent somatic mutations in genes encoding proteins involved in DNA methylation and demethylation, and in histone modifications have been reported in myeloid malignancies. Large clinical correlative studies are beginning to clear the clinical importance, prevalence, and potential prognostic significance of these epigenetics modifying gene mutations. Additionally, recent studies shedding light on the role of epigenetics in the pathogenesis of myeloid malignancies has prompted increased interest in development of novel therapies which target DNA and histone posttranslational modifications. In this review, we summarize the current understanding of the epigenetics modifying gene mutation, discuss how contribute to its pathogenesis and clinical feature in AML.

  4. A novel mutation in the calcium channel gene in a family with hypokalemic periodic paralysis. (United States)

    Hirano, Makito; Kokunai, Yosuke; Nagai, Asami; Nakamura, Yusaku; Saigoh, Kazumasa; Kusunoki, Susumu; Takahashi, Masanori P


    Hypokalemic periodic paralysis (HypoPP) type 1 is an autosomal dominant disease caused by mutations in the Ca(V)1.1 calcium channel encoded by the CACNA1S gene. Only seven mutations have been found since the discovery of the causative gene in 1994. We describe a patient with HypoPP who had a high serum potassium concentration after recovery from a recent paralysis, which complicated the correct diagnosis. This patient and other affected family members had a novel mutation, p.Arg900Gly, in the CACNA1S gene. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. L712V mutation in the androgen receptor gene causes complete androgen insensitivity syndrome due to severe loss of androgen function. (United States)

    Rajender, Singh; Gupta, Nalini J; Chakrabarty, Baidyanath; Singh, Lalji; Thangaraj, Kumarasamy


    Inability to respond to the circulating androgens is named as androgen insensitivity syndrome (AIS). Mutations in the androgen receptor (AR) gene are the most common cause of AIS. A cause and effect relationship between some of these mutations and the AIS phenotype has been proven by in vitro studies. Several other mutations have been identified, but need to be functionally validated for pathogenicity. Screening of the AR mutations upon presumptive diagnosis of AIS is recommended. We analyzed a case of complete androgen insensitivity syndrome (CAIS) for mutations in the AR gene. Sequencing of the entire coding region revealed C>G mutation (CTT-GTT) at codon 712 (position according to the NCBI database) in exon 4 of the gene, resulting in replacement of leucine with valine in the ligand-binding domain of the AR protein. No incidence of this mutation was observed in 230 normal male individuals analyzed for comparison. In vitro androgen binding and transactivation assays using mutant clone showed approximately 71% loss of ligand binding and about 76% loss of transactivation function. We conclude that CAIS in this individual was due to L712V substitution in the androgen receptor protein.

  6. New mutations in MAPT gene causing frontotemporal lobar degeneration: biochemical and structural characterization. (United States)

    Rossi, Giacomina; Bastone, Antonio; Piccoli, Elena; Mazzoleni, Giulia; Morbin, Michela; Uggetti, Andrea; Giaccone, Giorgio; Sperber, Sarah; Beeg, Marten; Salmona, Mario; Tagliavini, Fabrizio


    Frontotemporal lobar degeneration (FTLD) can be sporadic or familial. The genes encoding the microtubule-associated protein tau (MAPT) and progranulin (GRN) are the most relevant genes so far known causing the hereditary forms. Following genetic screening of patients affected by FTLD, we identified 2 new MAPT mutations, P364S and G366R, the former in a sporadic case. In the study we report the clinical and genetic features of the patients carrying these mutations, and the functional effects of the mutations, analyzed in vitro in order to investigate their pathogenic character. Both mutations resulted in reduced ability of tau to promote microtubule polymerization; the P364S protein variant also showed a high propensity to aggregate into filaments. These results suggest a high probability that these mutations are pathogenic. Our findings highlight the importance of genetic analysis also in sporadic forms of FTLD, and the role of in vitro studies to evaluate the pathologic features of new mutations.

  7. Association of mutations in the hemochromatosis gene with shorter life expectancy

    DEFF Research Database (Denmark)

    Bathum, L; Christiansen, L; Nybo, H;


    BACKGROUND: To investigate whether the frequency of carriers of mutations in the HFE gene associated with hereditary hemochromatosis diminishes with age as an indication that HFE mutations are associated with increased mortality. It is of value in the debate concerning screening for hereditary...... hemochromatosis to determine the significance of heterozygosity. METHODS: Genotyping for mutations in exons 2 and 4 of the HFE gene using denaturing gradient gel electrophoresis in 1784 participants aged 45 to 100 years from 4 population-based studies: all 183 centenarians from the Danish Centenarian Study, 601...... heterozygotes for the C282Y mutation-the mutation most often associated with hereditary hemochromatosis-was found. This was significant for the whole population (P=.005) and for women (P=.004) but not for men (P=.26). A group of 599 participants was screened for mutations in exon 2, and there was no variation...

  8. Mutations in the Kv1.5 channel gene KCNA5 in cardiac arrest patients

    DEFF Research Database (Denmark)

    Nielsen, Nathalie H; Winkel, Bo G; Kanters, Jørgen K


    identified the point mutations P91L and E33V in the KCNA5 gene encoding the Kv1.5 potassium channel that has not previously been associated with arrhythmia. We functionally characterized the mutations in HEK293 cells. The mutated channels behaved similarly to the wild-type with respect to biophysical......Mutations in one of the ion channels shaping the cardiac action potential can lead to action potential prolongation. However, only in a minority of cardiac arrest cases mutations in the known arrhythmia-related genes can be identified. In two patients with arrhythmia and cardiac arrest, we...... characteristics and drug sensitivity. Both patients also carried a D85N polymorphism in KCNE1, which was neither found to influence the Kv1.5 nor the Kv7.1 channel activity. We conclude that although the two N-terminal Kv1.5 mutations did not show any apparent electrophysiological phenotype, it is possible...

  9. Keratin 9 gene mutational heterogeneity in patients with epidermolytic palmoplantar keratoderma. (United States)

    Hennies, H C; Zehender, D; Kunze, J; Küster, W; Reis, A


    Mutations in the human keratin 9 gene have recently been shown to be involved in the etiology of palmoplantar keratoderma (PPK). We have investigated eleven unrelated German kindreds with the epidermolytic variant of PPK (EPPK) for mutations in the keratin 9 gene. We have identified two novel mutations, M156V and Q171P, both in the coil 1A segment of keratin 9. Mutation M156V was detected in two unrelated patients with EPPK, and mutation Q171P was shown to cosegregate with the disease in a large four-generation family. These findings confirm the functional importance of coil 1A integrity for heterodimerisation in keratins and for intermediate filament assembly. Our results provide further evidence for mutational heterogeneity in EPPK, and for the involvement of keratins in diseases of hyperkeratinisation and epidermolysis.


    Institute of Scientific and Technical Information of China (English)

    刘世喜; 林代诚; 洪邦泰; 黄光琦


    In order to study the ahered molecular events during laryngeal carcinogenesis and elucidate the role of Ha-ras oncogene amplification and mutation, we have examined their profile by polymerase chain reaction (PCR) and selective oligonucleoride hybridization. We analyzed the mutational status of codon 12 of Ha-ras in 22 laryngeal carcinomas and 10 normal tissues, and found that 7 of 22 laryngeal carcinomas con-tained a Ha-ras mutation at codon 12. The frequency of mutation was 32%. None of the normal tissues re-vealed mutation. Moreover, no amplification was found in cancers when compared to the normal. Our findings indicated that the aefivmed Ha-ras gene existed in laryngeal carcinoma, and activation of the Ha-ras gene by mutation at codon 12 might play a key role in laryngeal carcinogenesis.

  11. Spectrum of NSD1 gene mutations in southern Chinese patients with Sotos syndrome

    Institute of Scientific and Technical Information of China (English)

    Tony M.F. Tong; Edgar W.L. Hau; Ivan F.M. Lo; Daniel H.C. Chan; Stephen T.S. Lam


    Background Sotos syndrome is an overgrowth syndrome with characteristic facial gestalt and mental retardation of variable severity. Haploinsufficiency of the NSD1 gene has been implicated as the major cause of Sotos syndrome, with a predominance of microdeletions reported in Japanese patients. This study was conducted to investigate into the spectrum of NSD1 gene mutations in southern Chinese patients with Sotos syndrome. Methods Thirty-six Chinese patients with Sotos syndrome and two patients with Weaver syndrome were subject to molecular testing. Results NSD1 gene mutations were detected in 26 (72%) Sotos patients. Microdeletion was found in only 3 patients, while the other 23 had point mutations (6 frameshift, 8 nonsense, 2 spice site, and 7 missense). Of these, 19 mutations were never reported. NSD1 gene mutations were not found in the two patients with Weaver syndrome. Conclusions Most cases of Sotos syndrome are caused by NSD1 gene defects, but the spectrum of mutations is different from that of Japanese patients. Genotype-phenotype correlation showed that patients with microdeletions might be more prone to congenital heart disease but less likely to have somatic overgrowth. The two patients with Weaver syndrome were not found to have NSD1 gene mutations, but the number was too small for any conclusion to be drawn.

  12. APC and K-ras gene mutation in aberrant crypt foci of human colon

    Institute of Scientific and Technical Information of China (English)

    Ping Yuan; Meng Hong Sun; Jin Sheng Zhang; Xiong Zeng Zhu; Da Ren Shi


    AIM To study the genetic alteration in ACF andto define the possibility that ACF may be a veryearly morphological lesion with molecularchanges, and to explore the relationshipbetween ACF and colorectal adenoma evencarcinoma.METHODS DNA from 35 CRC, 15 adenomas, 34ACF and 10 normal mucus was isolated by meansof microdissection. Direct gene sequencing of K-ras gene including codon 12, 13 and 61 as well asthe mutation cluster region (MCR) of APC genewas performed.RESULTS K-ras gene mutation frequency inACF, adenoma and carcinoma was 17.6% (6/34), 13.3% (2/ 15), and 14.3% (5/ 35)respectively, showing no difference ( P > 0.05)in K-fas gene mutation among three pathologicprocedures. The K-ras gene mutation inadenoma, carcinoma and 4 ACF restricted incodon 12 (GGT→GAT), but the other 2 mutationsfrom ACF located in codon 13 (GGC→GAC). K-res gene mutation was found more frequently inolder patients and patients with polypoidcancer. No mutation in codon 61 was found in thethree tissue types. Mutation rate of APO gene inadenoma and carcinoma was 22.9% (8/35) and26.7% (4/ 15), which was higher than ACF(2.9%) (P < 0.05). APC gene mutation incarcinoma was not correlated with age ofpatients, location, size and differentiation oftumor.CONCLUSION ACF might be a very earlymorphological lesion in the tumorogenesis ofcolorectal tumor. The morphological feature andgene mutation status was different in ACF andadenoma. ACF is possibly putative"microadenoma" that might be the precursor ofadenoma. In addition, the development of asubgroup of colorectal carcinomas mightundergo a way of "normal epithelium→ ACF→carcinomas".

  13. Novel mutations of PRSS1 gene in patients with pancreatic cancer among Han population

    Institute of Scientific and Technical Information of China (English)

    ZENG Kai; LIU Qi-cai; LIN Jian-hua; LIN Xin-hua; ZHUANG Ze-hao; GAO Feng; OU Qi-shui


    Background A high mortality rate of pancreatic cancer becomes a bottleneck for further treatment with long-term efficacy. It is urgent to find a new mean to predict the early onset of pancreatic cancer accurately. The authors hypothesized that genetic variants of cationic trypsinogen (PRSS1) gene could affect trypsin expression/function and result in abnormal activation of protease activated receptor-2 (PAR-2), then lead to pancreatic cancer. The aim of this study was to elaborate some novel mutations of PRSS1 gene in the patients with pancreatic cancer.Methods Totally 156 patients with pancreatic cancer and 220 unrelated individuals as controls were enrolled in this study. The mutations of PRSS1 gene were analyzed by direct sequencing. K-ras Mutation Detection Kit was used to find the general k-ras gene disorder in the pancreatic cancer tissue. Then the clinical data were collected and analyzed simultaneously.Results There were two patients who carried novel mutations which was IVS 3 +157 G>C of PRSS1 gene in peripheral blood specimens and pancreatic cancer tissue. What's more, it was surprising to find a novel complicated mutation of exon 3 in PRSS1 gene (c.409 A>G and c.416 C>T) in another young patient. The complicated mutation made No. 135 and No.137 amino acid transfer from Thr to Ala and Thr to Met respectively. No any mutation was found in the normal controls while no mutations of k-ras gene were detected in the three patients.Conclusion Mutations of PRSS1 gene may be an important factor of pancreatic cancer.

  14. Novel and recurrent LDLR gene mutations in Pakistani hypercholesterolemia patients

    NARCIS (Netherlands)

    Ahmed, W.; Ajmal, M.; Sadeque, A.; Whittall, R.A.; Rafiq, S.; Putt, W.; Khawaja, A.; Imtiaz, F.; Ahmed, N.; Azam, M.; Humphries, S.E.; Qamar, R.


    The majority of patients with the autosomal dominant disorder familial hypercholesterolemia (FH) carry novel mutations in the low density lipoprotein receptor (LDLR) that is involved in cholesterol regulation. In different populations the spectrum of mutations identified is quite different and to da

  15. Three patients with middle-age-onset hemochromatosis caused by novel mutations in the hemojuvelin gene. (United States)

    Koyama, Chizu; Hayashi, Hisao; Wakusawa, Shinya; Ueno, Toshio; Yano, Motoyoshi; Katano, Yoshiaki; Goto, Hidemi; Kidokoro, Ryuichi


    Hemochromatosis is a genetically heterogeneous condition. Mutations in the recently described hemojuvelin gene were found in patients with juvenile hemochromatosis, who usually manifest clinical signs of iron overload, including cardiomyopathy and hypogonadism, in their teens and early 20s. In this report, we describe three Japanese patients who showed typical clinical and hepatic histological damage compatible with hemochromatosis at around 50 years of age. Genetic analyses showed that all three patients carried mutations in the hemojuvelin gene. The first patient was homozygous for a novel mutation (745G > C [D249H]), and the second and third patients from the same family were homozygous for another novel mutation (934C > T [Q312X]). No mutations in their HFE, hepcidin, transferrin receptor 2, or ferroportin genes were found. One patient had chronic infection with Helicobacter pylori. The age at initial presentation of hemojuvelin-hemochromatosis occurs over a wider range than previously described.

  16. A novel mutation in the HSPD1 gene in a patient with hereditary spastic paraplegia

    DEFF Research Database (Denmark)

    Hansen, Jakob; Svenstrup, Kirsten; Ang, Debbie


    A mutation in the HSPD1 gene has previously been associated with an autosomal dominant form of spastic paraplegia in a French family. HSPD1 encodes heat shock protein 60, a molecular chaperone involved in folding and quality control of mitochondrial proteins. In the present work we have investiga......A mutation in the HSPD1 gene has previously been associated with an autosomal dominant form of spastic paraplegia in a French family. HSPD1 encodes heat shock protein 60, a molecular chaperone involved in folding and quality control of mitochondrial proteins. In the present work we have...... investigated 23 Danish index patients with hereditary spastic paraplegia (HSP) for mutations in the HSPD1 gene. One patient was found to be heterozygous for a c.1381C > G missense mutation encoding the mutant heat shock protein 60 p.Gln461Glu. The mutation was also present in two unaffected brothers...

  17. AIRE gene mutations and autoantibodies to interferon omega in patients with chronic hypoparathyroidism without APECED. (United States)

    Cervato, Sara; Morlin, Luca; Albergoni, Maria Paola; Masiero, Stefano; Greggio, Nella; Meossi, Cristiano; Chen, Shu; del Pilar Larosa, Maria; Furmaniak, Jadwiga; Rees Smith, Bernard; Alimohammadi, Mohammad; Kämpe, Olle; Valenzise, Mariella; Betterle, Corrado


    To assess autoimmune regulator (AIRE) gene mutations, class II HLA haplotypes, and organ- or non-organ-specific autoantibodies in patients with chronic hypoparathyroidism (CH) without associated Addison's disease (AD) or chronic candidiasis (CC). Twenty-four patients who had CH without AD or CC were included in the study. AIRE gene mutations in all 14 exons were studied using PCR in 24 patients, 105 healthy controls and 15 first-degree relatives of CH patients with AIRE mutations. Human leucocyte antigens (HLA) were determined for all 24 patients and 105 healthy controls. Autoantibodies to a range of antigens including NACHT leucine-rich-repeat protein-5 (NALP5) and interferon omega (IFNω) were tested in all 24 patients. AIRE gene mutations were found in 6 of 24 (25%) patients, all females, and this was significantly higher (P AIRE mutations found in healthy controls (2/105). Three patients (12·5%) had homozygous AIRE mutations characteristic of Autoimmune-Poly-Endocrinopathy-Candidiasis-Ectodermal-Dystrophy and all three were also positive for IFNω-autoantibodies. Three patients (12·5%) had heterozygous AIRE mutations; two of these were novel mutations. One of the patients with heterozygous AIRE mutations was positive for both NACHT leucine-rich-repeat protein 5 and IFNω autoantibodies. Heterozygous AIRE mutations were found in 10 of 15 first-degree relatives of CH patients with AIRE mutations, although none was affected by CH. Class II HLA haplotypes were not statistically different in patients with CH compared to healthy controls. Analysis of AIRE gene mutations together with serum autoantibody profile should be helpful in the assessment of patients with CH, in particular young women with associated autoimmune diseases. © 2010 Blackwell Publishing Ltd.

  18. Further Evidence of Mutational Heterogeneity of the XPC Gene in Tunisian Families: A Spectrum of Private and Ethnic Specific Mutations

    Directory of Open Access Journals (Sweden)

    Mariem Ben Rekaya


    Full Text Available Xeroderma Pigmentosum (XP is a rare recessive autosomal cancer prone disease, characterized by UV hypersensitivity and early appearance of cutaneous and ocular malignancies. We investigated four unrelated patients suspected to be XP-C. To confirm linkage to XPC gene, genotyping and direct sequencing of XPC gene were performed. Pathogenic effect of novel mutations was confirmed by reverse Transciptase PCR. Mutation screening revealed the presence of two novel mutations g.18246G>A and g.18810G>T in the XPC gene (NG_011763.1. The first is present in one patient XP50NEF, but the second is present in three unrelated patients (XP16KEB, XP28SFA, and XP45GB. These 3 patients are from three different cities of Southern Tunisia and bear the same haplotype, suggesting a founder effect. Reverse Transciptase PCR revealed the absence of the XPC mRNA. In Tunisia, as observed in an other severe genodermatosis, the mutational spectrum of XP-C group seems to be homogeneous with some clusters of heterogeneity that should be taken into account to improve molecular diagnosis of this disease.

  19. Evaluation of frequency of kirsten rat sarcoma gene mutations in Iranian colorectal cancer

    Directory of Open Access Journals (Sweden)

    Fatemeh Roudbari


    Full Text Available Background: Kirsten rat sarcoma (KRAS gene is a target of genetic alterations which are diagnostic and prognostic biomarkers in patients with metastatic colorectal cancer who are treated with monoclonal anti-EGFR antibodies such as cetuximab and panitumumab. KRAS mutations are seen in 35-42% of patients with colorectal cancer. The high frequency of these mutations in colorectal cancer represents their high potential as a biomarker in early diagnosis of cancer. This study was done to evaluate the frequency of KRAS gene mutations in a small population of Iranian patients suffering from colorectal cancer.   Methods: 50 formalin-fixed paraffin-embedded tissue blocks with colorectal cancer (CRC, already confirmed by histopathology and immunohistochemistry testing, were received to Payvand Clinical and Specialty Laboratory, Tehran, from across the country in 2015. DNA was extracted from the tissue blocks and its quality was then evaluated. The reverse dot blotting method was used to evaluate KRAS gene mutations. Results: KRAS mutations were found in 42% of the study patients. 30% and 12% of the mutations were found in codon 12 and codon 13, respectively. Moreover, no mutation was found in codon 61. Results also showed that the most frequency of samples examined belonged to male with 68% (average age of 56 years old and then to female with 32% (median age of 54.8 years old. Conclusion: This study was performed to evaluate the frequency of KRAS gene mutations in Iranian colorectal cancer patients. According to the study results, the frequency of KRAS mutations was consistent with that of other countries, reported in previous studies. The high prevalence of these mutations in patients with colorectal cancer indicates the important role of these genes in this group of patients. Thus, the presence of these mutations can be used as a suitable biomarker for evaluation of response to targeted therapies in patients suffering from colorectal cancer.

  20. Prognostic implication of N-RAS gene mutations in Egyptian adult acute myeloid leukemia. (United States)

    Elghannam, Doaa M; Abousamra, Nashwa Khayrat; Shahin, Doaa A; Goda, Enas F; Azzam, Hanan; Azmy, Emad; El-Din, Manal Salah; El-Refaei, Mohamed F


    The pathogenesis of acute myeloid leukemia (AML) involves the cooperation of mutations promoting proliferation/survival and those impairing differentiation. Point mutations of the N-RAS gene are the most frequent somatic mutations causing aberrant signal-transduction in acute myeloid leukemia (AML). The aim of the present work is to study the frequency and prognostic significance of N-RAS gene mutations (N-RASmut) in de novo Egyptian adult AML. Bone marrow specimens from 150 patients with de novo acute myeloid leukemia and controls were analyzed by genomic PCR-SSCP at codons 12, 13 (exon 1), and 61 (exon 2) for N-RAS mutations. In 12.7% (19/150) AML cases, N-RAS gene mutations were found and were observed more frequently in the FAB subtype M4eo (P = 0.028) and with codon 12, 13 (14 of 19; 73.7%). Patients with N-RAS mutation had a significant lower peripheral and marrow blasts (P = 0.004, P = 0.03) and clinical outcome did not improve more than in patients without mutation. In patients with N-RAS gene mutation vs. those without, complete remission rate was (63.2% vs. 56.5%; P = 0.46), resistant disease (15.8% vs. 23.6%; P = 0.51), three years overall survival (44% vs 42%; P= 0.85) and disease free survival (42.1% vs. 38.9%, P = 0.74). Multivariate analysis showed that age was the strongest unfavorable factor for overall survival (relative risk [RR], 1.9; P = 0.002), followed by cytogenetics (P = 0.004). FAB types, N-RAS mutation and leukocytosis were the least important. In conclusion, the frequency and spectrum of N-RAS gene mutation differ between biologically distinct subtypes of AML but do not significantly influence prognosis and clinical outcome in patients with AML.

  1. Mutations in Two Independent Genes Lead to Suppression of the Shoot Apical Meristem in Maize1 (United States)

    Pilu, Roberto; Consonni, Gabriella; Busti, Elena; MacCabe, Andrew P.; Giulini, Anna; Dolfini, Silvana; Gavazzi, Giuseppe


    The shoot apical meristem (SAM), initially formed during embryogenesis, gives rise to the aboveground portion of the maize (Zea mays) plant. The shootless phenotype (sml) described here is caused by disruption of SAM formation due to the synergistic interaction of mutations at two genetic loci. Seedlings must be homozygous for both sml (shootmeristemless), and the unlinked dgr (distorted growth) loci for a SAM-less phenotype to occur. Seedlings mutant only for sml are impaired in their morphogenesis to different extents, whereas the dgr mutation alone does not have a recognisable phenotype. Thus, dgr can be envisaged as being a dominant modifier of sml and the 12 (normal):3 (distorted growth):1 (shoot meristemless) segregation observed in the F2 of the double heterozygote is the result of the interaction between the sml and dgr genes. Other segregation patterns were also observed in the F2, suggesting instability of the dgr gene. Efforts to rescue mutant embryos by growth on media enriched with hormones have been unsuccessful so far. However, mutant roots grow normally on medium supplemented with kinetin at a concentration that suppresses wild-type root elongation, suggesting possible involvement of the mutant in the reception or transduction of the kinetin signal or transport of the hormone. The shootless mutant appears to be a valuable tool with which to investigate the organization of the shoot meristem in monocots as well as a means to assay the origins and relationships between organs such as the scutellum, the coleoptile, and leaves that are initiated during the embryogenic process. PMID:11842154

  2. Confirmation of the mitochondrial ND1 gene mutation G3635A as a primary LHON mutation. (United States)

    Yang, Juhua; Zhu, Yihua; Tong, Yi; Chen, Lu; Liu, Lijuan; Zhang, Zhiqiang; Wang, Xiaoyan; Huang, Dinggou; Qiu, Wentong; Zhuang, Shuliu; Ma, Xu


    We report the clinical and genetic characterization of two Chinese LHON families who do not carry the primary LHON-mutations. Mitochondrial genome sequence analysis revealed the presence of a homoplasmic ND1 G3635A mutation in both families. In Family LHON-001, 31 other variants belonging to the East Asian haplogroup R11a were identified and in Family LHON-019, 37 other variants belonging to the East Asian haplogroup D4g were determined. The ND1 G3635A mutation changes the conversed serine110 residue to asparagine. This mutation has been previously described in a single Russian LHON family and has been suggested to contribute to increased LHON expressivity. In addition, a mutation in cytochrome c oxidase subunit II at C7868T (COII/L95F) may act in synergy with G3635A, increasing LHON expressivity in Family LHON-001, which had a higher level of LHON penetrance than Family LHON-019. In summary, the G3635A mutation is confirmed as a rare primary pathogenic mutation for LHON.

  3. Xenobiotic transporters: ascribing function from gene knockout and mutation studies. (United States)

    Klaassen, Curtis D; Lu, Hong


    Transporter-mediated absorption, secretion, and reabsorption of chemicals are increasingly recognized as important determinants in the biological activities of many xenobiotics. In recent years, the rapid progress in generating and characterizing mice with targeted deletion of transporters has greatly increased our knowledge of the functions of transporters in the pharmacokinetics/toxicokinetics of xenobiotics. In this introduction, we focus on functions of transporters learned from experiments on knockout mice as well as humans and rodents with natural mutations of these transporters. We limit our discussion to transporters that either directly transport xenobiotics or are important in biliary excretion or cellular defenses, namely multidrug resistance, multidrug resistance-associated proteins, breast cancer resistance protein, organic anion transporting polypeptides, organic anion transporters, organic cation transporters, nucleoside transporters, peptide transporters, bile acid transporters, cholesterol transporters, and phospholipid transporters, as well as metal transporters. Efflux transporters in intestine, liver, kidney, brain, testes, and placenta can efflux xenobiotics out of cells and serve as barriers against the entrance of xenobiotics into cells, whereas many xenobiotics enter the biological system via uptake transporters. The functional importance of a given transporter in each tissue depends on its substrate specificity, expression level, and the presence/absence of other transporters with overlapping substrate preferences. Nevertheless, a transporter may affect a tissue independent of its local expression by altering systemic metabolism. Further studies on the gene regulation and function of transporters, as well as the interrelationship between transporters and phase I/II xenobiotic-metabolizing enzymes, will provide a complete framework for developing novel strategies to protect us from xenobiotic insults.

  4. Mutations in planar cell polarity gene SCRIB are associated with spina bifida.

    Directory of Open Access Journals (Sweden)

    Yunping Lei

    Full Text Available Neural tube defects (NTDs (OMIM #182940 including anencephaly, spina bifida and craniorachischisis, are severe congenital malformations that affect 0.5-1 in 1,000 live births in the United States, with varying prevalence around the world. Mutations in planar cell polarity (PCP genes are believed to cause a variety of NTDs in both mice and humans. SCRIB is a PCP-associated gene. Mice that are homozygous for the Scrib p.I285K and circletail (Crc mutations, present with the most severe form of NTDs, namely craniorachischisis. A recent study reported that mutations in SCRIB were associated with craniorachischisis in humans, but whether SCRIB mutations contribute to increased spina bifida risk is still unknown. We sequenced the SCRIB gene in 192 infants with spina bifida and 190 healthy controls. Among the spina bifida patients, we identified five novel missense mutations that were predicted-to-be-deleterious by the PolyPhen software. Of these five mutations, three of them (p.P1043L, p.P1332L, p.L1520R significantly affected the subcellular localization of SCRIB. In addition, we demonstrated that the craniorachischisis mouse line-90 mutation I285K, also affected SCRIB subcellular localization. In contrast, only one novel missense mutation (p.A1257T was detected in control samples, and it was predicted to be benign. This study demonstrated that rare deleterious mutations of SCRIB may contribute to the multifactorial risk for human spina bifida.

  5. Recessive mutations in the INS gene result in neonatal diabetes through reduced insulin biosynthesis. (United States)

    Garin, Intza; Edghill, Emma L; Akerman, Ildem; Rubio-Cabezas, Oscar; Rica, Itxaso; Locke, Jonathan M; Maestro, Miguel Angel; Alshaikh, Adnan; Bundak, Ruveyde; del Castillo, Gabriel; Deeb, Asma; Deiss, Dorothee; Fernandez, Juan M; Godbole, Koumudi; Hussain, Khalid; O'Connell, Michele; Klupa, Thomasz; Kolouskova, Stanislava; Mohsin, Fauzia; Perlman, Kusiel; Sumnik, Zdenek; Rial, Jose M; Ugarte, Estibaliz; Vasanthi, Thiruvengadam; Johnstone, Karen; Flanagan, Sarah E; Martínez, Rosa; Castaño, Carlos; Patch, Ann-Marie; Fernández-Rebollo, Eduardo; Raile, Klemens; Morgan, Noel; Harries, Lorna W; Castaño, Luis; Ellard, Sian; Ferrer, Jorge; Perez de Nanclares, Guiomar; Hattersley, Andrew T


    Heterozygous coding mutations in the INS gene that encodes preproinsulin were recently shown to be an important cause of permanent neonatal diabetes. These dominantly acting mutations prevent normal folding of proinsulin, which leads to beta-cell death through endoplasmic reticulum stress and apoptosis. We now report 10 different recessive INS mutations in 15 probands with neonatal diabetes. Functional studies showed that recessive mutations resulted in diabetes because of decreased insulin biosynthesis through distinct mechanisms, including gene deletion, lack of the translation initiation signal, and altered mRNA stability because of the disruption of a polyadenylation signal. A subset of recessive mutations caused abnormal INS transcription, including the deletion of the C1 and E1 cis regulatory elements, or three different single base-pair substitutions in a CC dinucleotide sequence located between E1 and A1 elements. In keeping with an earlier and more severe beta-cell defect, patients with recessive INS mutations had a lower birth weight (-3.2 SD score vs. -2.0 SD score) and were diagnosed earlier (median 1 week vs. 10 weeks) compared to those with dominant INS mutations. Mutations in the insulin gene can therefore result in neonatal diabetes as a result of two contrasting pathogenic mechanisms. Moreover, the recessively inherited mutations provide a genetic demonstration of the essential role of multiple sequence elements that regulate the biosynthesis of insulin in man.

  6. Chromosomal Instability, Aneuploidy, and Gene Mutations in Human Sporadic Colorectal Adenomas

    Directory of Open Access Journals (Sweden)

    Walter Giaretti


    Full Text Available Whether in vivo specific gene mutations lead to chromosomal instability (CIN and aneuploidy or viceversa is so far not proven. We hypothesized that aneuploidy among human sporadic colorectal adenomas and KRAS2 and APC mutations were not independent. Additionally, we investigated if 1p34–36 deletions by dual target FISH were associated with aneuploidy. Among 116 adenomas, 29 were DNA aneuploid by flow cytometry (25% and 29 were KRAS2 mutated (25%. KRAS2 mutations were associated with aneuploidy (P=0.02. However, while G–C and G–T transversions were strongly associated with DNA aneuploidy (P=0.007, G–A transitions were not. Within a second series of 61 adenomas, we found, instead, that APC mutational status and aneuploidy by flow cytometry were not associated. However, a statistically significant association was found with specific APC mutations, i.e., occurring in the mutation cluster region (MCR, codons 1200–1500 or downstream (P=0.016. Finally, the correlation of 1p34–36 deletions with flow cytometric and FISH detected aneuploidy was also significant (P=0.01. Specific KRAS2 and APC mutations and loss of genes in the 1p34–36 region appear associated with aneuploidy suggesting that these events are not independent and may cooperate in inducing human sporadic colorectal adenomas. A cause effect relationship between gene mutations and aneuploidy remains, however, to be demonstrated.

  7. Mutated human androgen receptor gene detected in a prostatic cancer patient is also activated by estradiol

    Energy Technology Data Exchange (ETDEWEB)

    Elo, J.P.; Kvist, L.; Leinonen, K.; Isomaa, V. [Univ. of Oulu (Finland)] [and others


    Androgens are necessary for the development of prostatic cancer. The mechanisms by which the originally androgen-dependent prostatic cancer cells are relieved of the requirement to use androgen for their growth are largely unknown. The human prostatic cancer cell line LNCaP has been shown to contain a point mutation in the human androgen receptor gene (hAR), suggesting that changes in the hAR may contribute to the abnormal hormone response of prostatic cells. To search for point mutations in the hAR, we used single strand conformation polymorphism analysis and a polymerase chain reaction direct sequencing method to screen 23 prostatic cancer specimens from untreated patients, 6 prostatic cancer specimens from treated patients, and 11 benign prostatic hyperplasia specimens. One mutation was identified in DNA isolated from prostatic cancer tissue, and the mutation was also detected in the leukocyte DNA of the patient and his offspring. The mutation changed codon 726 in exon E from arginine to leucine and was a germ line mutation. The mutation we found in exon E of the hAR gene does not alter the ligand binding specificity of the AR, but the mutated receptor was activated by estradiol to a significantly greater extent than the wild-type receptor. The AR gene mutation described in this study might be one explanation for the altered biological activity of prostatic cancer. 36 refs., 4 figs.


    Chou, Annie; Dekker, Nusi; Jordan, Richard C.K.


    Objective Activating mutations in the fibroblast growth factor receptor 3 (FGFR3) gene are responsible for several craniosynostosis and chondrodysplasia syndromes as well as some human cancers including bladder and cervical carcinoma. Despite a high frequency in some benign skin disorders, FGFR3 mutations have not been reported in cutaneous malignancies. Actinic cheilitis (AC) is a sun-induced premalignancy affecting the lower lip that frequently progresses to squamous cell carcinoma (SCC). The objective of this study was to determine if FGFR3 gene mutations are present in AC and SCC of the lip. Study Design DNA was extracted and purified from micro-dissected, formalin-fixed, paraffin-embedded tissue sections of 20 cases of AC and SCC arising in AC. Exons 7, 15, and 17 were PCR amplified and direct sequenced. Results Four novel somatic mutations in the FGFR3 gene were identified: exon 7 mutation 742C→T (amino acid change R248C), exon 15 mutations 1850A→G (D617G) and 1888G→A (V630M), and exon 17 mutation 2056G→A (E686K). Grade of dysplasia did not correlate with presence of mutations. Conclusion The frequency of FGFR3 receptor mutations suggests a functional role for the FGFR3 receptor in the development of epithelial disorders and perhaps a change may contribute to the pathogenesis of some AC and SCC. PMID:19327639


    A commercially available DNA hydribization assay (Gene-trak , Framingham, MA. USA) was compared with the EC-MUG procedure for the detection of Escherichia coli in water. The gene probe gave positive responses for pure cultures of E. coli 0157:H7, E. fergusonii, Shigella sonnei, S...

  10. An Undergraduate Laboratory Class Using CRISPR/Cas9 Technology to Mutate Drosophila Genes (United States)

    Adame, Vanesa; Chapapas, Holly; Cisneros, Marilyn; Deaton, Carol; Deichmann, Sophia; Gadek, Chauncey; Lovato, TyAnna L.; Chechenova, Maria B.; Guerin, Paul; Cripps, Richard M.


    CRISPR/Cas9 genome editing technology is used in the manipulation of genome sequences and gene expression. Because of the ease and rapidity with which genes can be mutated using CRISPR/Cas9, we sought to determine if a single-semester undergraduate class could be successfully taught, wherein students isolate mutants for specific genes using…

  11. CYP1B1 and myocilin gene mutations in Egyptian patients with ...

    African Journals Online (AJOL)

    Mahmoud R. Fassad


    Aug 9, 2016 ... in CYP1B1 gene (G61E, R368H) and one mutation in MYOC gene (Gln48His) using polymerase .... IOP between the 2 eyes [11], or if successive examination under ... coding region of MYOC gene encompassing Gln48His muta- tion was ..... CYP1B1 involvement in the molecular basis of primary congenital.

  12. An Undergraduate Laboratory Class Using CRISPR/Cas9 Technology to Mutate Drosophila Genes (United States)

    Adame, Vanesa; Chapapas, Holly; Cisneros, Marilyn; Deaton, Carol; Deichmann, Sophia; Gadek, Chauncey; Lovato, TyAnna L.; Chechenova, Maria B.; Guerin, Paul; Cripps, Richard M.


    CRISPR/Cas9 genome editing technology is used in the manipulation of genome sequences and gene expression. Because of the ease and rapidity with which genes can be mutated using CRISPR/Cas9, we sought to determine if a single-semester undergraduate class could be successfully taught, wherein students isolate mutants for specific genes using…

  13. Targeted Next Generation Sequencing as a Reliable Diagnostic Assay for the Detection of Somatic Mutations in Tumours Using Minimal DNA Amounts from Formalin Fixed Paraffin Embedded Material.

    Directory of Open Access Journals (Sweden)

    Wendy W J de Leng

    Full Text Available Targeted Next Generation Sequencing (NGS offers a way to implement testing of multiple genetic aberrations in diagnostic pathology practice, which is necessary for personalized cancer treatment. However, no standards regarding input material have been defined. This study therefore aimed to determine the effect of the type of input material (e.g. formalin fixed paraffin embedded (FFPE versus fresh frozen (FF tissue on NGS derived results. Moreover, this study aimed to explore a standardized analysis pipeline to support consistent clinical decision-making.We used the Ion Torrent PGM sequencing platform in combination with the Ion AmpliSeq Cancer Hotspot Panel v2 to sequence frequently mutated regions in 50 cancer related genes, and validated the NGS detected variants in 250 FFPE samples using standard diagnostic assays. Next, 386 tumour samples were sequenced to explore the effect of input material on variant detection variables. For variant calling, Ion Torrent analysis software was supplemented with additional variant annotation and filtering.Both FFPE and FF tissue could be sequenced reliably with a sensitivity of 99.1%. Validation showed a 98.5% concordance between NGS and conventional sequencing techniques, where NGS provided both the advantage of low input DNA concentration and the detection of low-frequency variants. The reliability of mutation analysis could be further improved with manual inspection of sequence data.Targeted NGS can be reliably implemented in cancer diagnostics using both FFPE and FF tissue when using appropriate analysis settings, even with low input DNA.

  14. Mutations in the Gene PRRT2 Cause Paroxysmal Kinesigenic Dyskinesia with Infantile Convulsions

    NARCIS (Netherlands)

    Lee, Hsien-Yang; Huang, Yong; Bruneau, Nadine; Roll, Patrice; Roberson, Elisha D. O.; Hermann, Mark; Quinn, Emily; Maas, James; Edwards, Robert; Ashizawa, Tetsuo; Baykan, Betul; Bhatia, Kailash; Bressman, Susan; Bruno, Michiko K.; Brunt, Ewout R.; Caraballo, Roberto; Echenne, Bernard; Fejerman, Natalio; Frucht, Steve; Gurnett, Christina A.; Hirsch, Edouard; Houlden, Henry; Jankovic, Joseph; Lee, Wei-Ling; Lynch, David R.; Mohammed, Shehla; Mueller, Ulrich; Nespeca, Mark P.; Renner, David; Rochette, Jacques; Rudolf, Gabrielle; Saiki, Shinji; Soong, Bing-Wen; Swoboda, Kathryn J.; Tucker, Sam; Wood, Nicholas; Hanna, Michael; Bowcock, Anne M.; Szepetowski, Pierre; Fu, Ying-Hui; Ptacek, Louis J.


    Paroxysmal kinesigenic dyskinesia with infantile convulsions (PKD/IC) is an episodic movement disorder with autosomal-dominant inheritance and high penetrance, but the causative genetic mutation is unknown. We have now identified four truncating mutations involving the gene PRRT2 in the vast majorit

  15. Mutations in the Gene PRRT2 Cause Paroxysmal Kinesigenic Dyskinesia with Infantile Convulsions

    NARCIS (Netherlands)

    Lee, Hsien-Yang; Huang, Yong; Bruneau, Nadine; Roll, Patrice; Roberson, Elisha D. O.; Hermann, Mark; Quinn, Emily; Maas, James; Edwards, Robert; Ashizawa, Tetsuo; Baykan, Betul; Bhatia, Kailash; Bressman, Susan; Bruno, Michiko K.; Brunt, Ewout R.; Caraballo, Roberto; Echenne, Bernard; Fejerman, Natalio; Frucht, Steve; Gurnett, Christina A.; Hirsch, Edouard; Houlden, Henry; Jankovic, Joseph; Lee, Wei-Ling; Lynch, David R.; Mohammed, Shehla; Mueller, Ulrich; Nespeca, Mark P.; Renner, David; Rochette, Jacques; Rudolf, Gabrielle; Saiki, Shinji; Soong, Bing-Wen; Swoboda, Kathryn J.; Tucker, Sam; Wood, Nicholas; Hanna, Michael; Bowcock, Anne M.; Szepetowski, Pierre; Fu, Ying-Hui; Ptacek, Louis J.


    Paroxysmal kinesigenic dyskinesia with infantile convulsions (PKD/IC) is an episodic movement disorder with autosomal-dominant inheritance and high penetrance, but the causative genetic mutation is unknown. We have now identified four truncating mutations involving the gene PRRT2 in the vast majorit

  16. A novel mutation of the fibrillin-1 gene in a newborn with severe Marfan syndrome. (United States)

    Kochilas, L; Gundogan, F; Atalay, M; Bliss, J M; Vatta, M; Pena, L S; Abuelo, D


    Marfan syndrome in the neonatal age represents a severe early and commonly lethal manifestation of Marfan syndrome, which is caused by mutations in the gene encoding fibrillin-1 (FBN1). Here, we report a newborn with severe Marfan syndrome and a novel mutation involving cysteine substitution within one of the epidermal growth factor-like domains of FBN1.

  17. Genetic basis of cystinosis in Tunisian patients: Identification of novel mutation in CTNS gene

    Directory of Open Access Journals (Sweden)

    Latifa Chkioua


    This study expands the mutational and population spectrum of NC, representing the first molecular diagnosis of NC in Tunisian population. The mutation screening of the CTNS gene was used for prenatal diagnosis to prevent and/or limit this inheritable disease in our country where the families are particularly large and have a high rate of consanguinity.

  18. 40 CFR 799.9530 - TSCA in vitro mammalian cell gene mutation test. (United States)


    ... several base pairs in the DNA. Forward mutation is a gene mutation from the parental type to the mutant... multiple base pairs in the DNA molecule. Mutant frequency is the number of mutant cells observed divided by... suffered the most extensive genetic damage have prolonged doubling times and thus form small colonies....

  19. Mutations in genes encoding subunits of RNA polymerases I and III cause Treacher Collins syndrome.

    NARCIS (Netherlands)

    Dauwerse, J.G.; Dixon, J.; Seland, S.; Ruivenkamp, C.A.; Haeringen, A. van; Hoefsloot, L.H.; Peters, D.J.; Boers, A.C.; Daumer-Haas, C.; Maiwald, R.; Zweier, C.; Kerr, B.; Cobo, A.M.; Toral, J.F.; Hoogeboom, A.J.M.; Lohmann, D.R.; Hehr, U.; Dixon, M.J.; Breuning, M.H.; Wieczorek, D.


    We identified a deletion of a gene encoding a subunit of RNA polymerases I and III, POLR1D, in an individual with Treacher Collins syndrome (TCS). Subsequently, we detected 20 additional heterozygous mutations of POLR1D in 252 individuals with TCS. Furthermore, we discovered mutations in both allele

  20. Mutation patterns of 16 genes in primary and secondary acute myeloid leukemia (AML with normal cytogenetics.

    Directory of Open Access Journals (Sweden)

    Marta Fernandez-Mercado

    Full Text Available Acute myeloid leukemia patients with normal cytogenetics (CN-AML account for almost half of AML cases. We aimed to study the frequency and relationship of a wide range of genes previously reported as mutated in AML (ASXL1, NPM1, FLT3, TET2, IDH1/2, RUNX1, DNMT3A, NRAS, JAK2, WT1, CBL, SF3B1, TP53, KRAS and MPL in a series of 84 CN-AML cases. The most frequently mutated genes in primary cases were NPM1 (60.8% and FLT3 (50.0%, and in secondary cases ASXL1 (48.5% and TET2 (30.3%. We showed that 85% of CN-AML patients have mutations in at least one of ASXL1, NPM1, FLT3, TET2, IDH1/2 and/or RUNX1. Serial samples from 19 MDS/CMML cases that progressed to AML were analyzed for ASXL1/TET2/IDH1/2 mutations; seventeen cases presented mutations of at least one of these genes. However, there was no consistent pattern in mutation acquisition during disease progression. This report concerns the analysis of the largest number of gene mutations in CN-AML studied to date, and provides insight into the mutational profile of CN-AML.

  1. Mutation patterns of 16 genes in primary and secondary acute myeloid leukemia (AML) with normal cytogenetics. (United States)

    Fernandez-Mercado, Marta; Yip, Bon Ham; Pellagatti, Andrea; Davies, Carwyn; Larrayoz, María José; Kondo, Toshinori; Pérez, Cristina; Killick, Sally; McDonald, Emma-Jane; Odero, María Dolores; Agirre, Xabier; Prósper, Felipe; Calasanz, María José; Wainscoat, James S; Boultwood, Jacqueline


    Acute myeloid leukemia patients with normal cytogenetics (CN-AML) account for almost half of AML cases. We aimed to study the frequency and relationship of a wide range of genes previously reported as mutated in AML (ASXL1, NPM1, FLT3, TET2, IDH1/2, RUNX1, DNMT3A, NRAS, JAK2, WT1, CBL, SF3B1, TP53, KRAS and MPL) in a series of 84 CN-AML cases. The most frequently mutated genes in primary cases were NPM1 (60.8%) and FLT3 (50.0%), and in secondary cases ASXL1 (48.5%) and TET2 (30.3%). We showed that 85% of CN-AML patients have mutations in at least one of ASXL1, NPM1, FLT3, TET2, IDH1/2 and/or RUNX1. Serial samples from 19 MDS/CMML cases that progressed to AML were analyzed for ASXL1/TET2/IDH1/2 mutations; seventeen cases presented mutations of at least one of these genes. However, there was no consistent pattern in mutation acquisition during disease progression. This report concerns the analysis of the largest number of gene mutations in CN-AML studied to date, and provides insight into the mutational profile of CN-AML.

  2. Detection of KRAS gene mutation and its clinical significance in colorectal adenocarcinoma

    Institute of Scientific and Technical Information of China (English)



    Objective To explore the clinical significance of KRAS mutation detection in colorectal adenocarcinoma. Methods Paraffin-embedded tissue specimens were obtained from 440 patients with colorectal adenocarcinoma. The genomic DNA was extracted. Mutations of exon 2 of KRAS gene were examined by PCR and

  3. Screening for apolipoprotein E gene mutations in 4 patients with lipoprotein glomerulopathy

    Institute of Scientific and Technical Information of China (English)



    Objective To study the mutations of apolipoprotein E (apoE) gene in 4 Chinese lipoprotein glomerulopathy (LPG) patients and their family members, and to investigate the pathogenesis of LPG. Methods Urinalysis was performed on the family members of two patients, and they were screened for the level of serum creatinine, serum lipid and serum lipoprotein. The mutation of apoE

  4. Mutation of the BRAF Genes in Non-small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Zhimin HUANG


    Full Text Available BRAF mutations have been found to be a driver mutation and maybe a therapy target in patients with non-small cell lung cancer. This article reviews the current understanding of BRAF gene, its structure, expression, the signal pathway, as well as its relationship with cancer especially the targeted therapies for non-small cell lung cancer.

  5. [Mutational analysis of the MECP2 gene by direct sequencing in Hungarian patients with Rett syndrome

    NARCIS (Netherlands)

    Karteszi, J.; Hollody, K.; Bene, J.; Morava, E.; Hadzsiev, K.; Czako, M.; Melegh, B.; Kosztolanyi, G.Y.


    INTRODUCTION: Rett syndrome is an X-linked neurodevelopmental disorder characterized by loss of acquired skills and stereotypical hand movements. Mutations in the gene encoding methyl-CpG-binding protein 2 have been identified as cause of Rett syndrome in 1999. AIM: The authors initialized mutation

  6. Mutations in the nebulin gene in a child with nemaline (rod) myopathy. (United States)

    Kapoor, Seema; Singh, Ankur; Lehtokari, Vilma-Lotta; Wallgren-Pettersson, Carina; Batra, Vineeta Vijay


    Nemaline myopathy, also called rod myopathy, is a relatively common congenital myopathy and probably second in incidence only to central core disease. The mainstay of diagnosis is histopathology, but detection of the causative mutation is mandatory for determining the mode of inheritance and for prenatal diagnosis. The authors report two siblings with nemaline myopathy caused by mutations in the nebulin gene.

  7. Suppressor of cytokine signaling 1 gene mutation status as a prognostic biomarker in classical Hodgkin lymphoma. (United States)

    Lennerz, Jochen K; Hoffmann, Karl; Bubolz, Anna-Maria; Lessel, Davor; Welke, Claudia; Rüther, Nele; Viardot, Andreas; Möller, Peter


    Suppressor of cytokine signaling 1 (SOCS1) mutations are among the most frequent somatic mutations in classical Hodgkin lymphoma (cHL), yet their prognostic relevance in cHL is unexplored. Here, we performed laser-capture microdissection of Hodgkin/Reed-Sternberg (HRS) cells from tumor samples in a cohort of 105 cHL patients. Full-length SOCS1 gene sequencing showed mutations in 61% of all cases (n = 64/105). Affected DNA-motifs and mutation pattern suggest that many of these SOCS1 mutations are the result of aberrant somatic hypermutation and we confirmed expression of mutant alleles at the RNA level. Contingency analysis showed no significant differences of patient-characteristics with HRS-cells containing mutant vs. wild-type SOCS1. By predicted mutational consequence, mutations can be separated into those with non-truncating point mutations ('minor' n = 49/64 = 77%) and those with length alteration ('major'; n = 15/64 = 23%). Subgroups did not differ in clinicopathological characteristics; however, patients with HRS-cells that contained SOCS1 major mutations suffered from early relapse and significantly shorter overall survival (P = 0.03). The SOCS1 major status retained prognostic significance in uni-(P = 0.016) and multivariate analyses (P = 0.005). Together, our data indicate that the SOCS1 mutation type qualifies as a single-gene prognostic biomarker in cHL.

  8. A Patient With Desmoid Tumors and Familial FAP Having Frame Shift Mutation of the APC Gene

    Directory of Open Access Journals (Sweden)

    Sanambar Sadighi


    Full Text Available Desmoids tumors, characterized by monoclonal proliferation of myofibroblasts, could occur in 5-10% of patients with familial adenomatous polyposis (FAP as an extra-colonic manifestation of the disease. FAP can develop when there is a germ-line mutation in the adenomatous polyposis coli gene. Although mild or attenuated FAP may follow mutations in 5΄ extreme of the gene, it is more likely that 3΄ extreme mutations haveamore severe manifestation of thedisease. A 28-year-old woman was admitted to the Cancer Institute of Iran with an abdominal painful mass. She had strong family history of FAP and underwent prophylactic total colectomy. Pre-operative CT scans revealed a large mass. Microscopic observation showed diffuse fibroblast cell infiltration of the adjacent tissue structures. Peripheral blood DNA extraction followed by adenomatous polyposis coli gene exon by exon sequencing was performed to investigate the mutation in adenomatous polyposis coli gene. Analysis of DNA sequencing demonstrated a mutation of 4 bpdeletions at codon 1309-1310 of the exon 16 of adenomatous polyposis coli gene sequence which was repeated in 3 members of the family. Some of them had desmoid tumor without classical FAP history. Even when there is no familial history of adenomatous polyposis, the adenomatous polyposis coli gene mutation should be investigated in cases of familial desmoids tumors for a suitable prevention. The 3΄ extreme of the adenomatous polyposis coli gene is still the best likely location in such families.

  9. Mutation analysis of the NRXN1 gene in autism spectrum disorders

    Directory of Open Access Journals (Sweden)

    Onay H


    Full Text Available The aim of this study was to identify the sequence mutations in the Neurexin 1 (NRXN1 gene that has been considered as one of the strong candidate genes. A total of 30 children and adolescents (aged 3-18 with non syndromic autism were enrolled this study. Sequencing of the coding exons and the exon-intron boundaries of the NRXN1 gene was performed. Two known mutations were described in two different cases. Heterozygous S14L was determined in one patient and heterozygous L748I was determined in another patient. The S14L and L748I mutations have been described in the patients with autism before. Both of these mutations were inherited from their father. In this study, two of 30 (6.7% autism spectrum disorder (ASD patients carrying NRXN1 gene mutations were detected. It indicates that variants in the NRXN1 gene might confer a risk of developing nonsyndromic ASD. However, due to the reduced penetrance in the gene, the causal role of the NRXN1 gene mutations must be evaluated carefully in all cases.

  10. Novel mutations in the NRL gene and associated clinical findings in patients with dominant retinitis pigmentosa. (United States)

    DeAngelis, Margaret M; Grimsby, Jonna L; Sandberg, Michael A; Berson, Eliot L; Dryja, Thaddeus P


    To search for mutations in the neural retina leucine zipper (NRL) gene in patients with dominant retinitis pigmentosa and to compare the severity of disease in these patients with that observed previously in patients with dominant rhodopsin mutations. Single-strand conformation analysis was used to survey 189 unrelated patients for mutations. The available relatives of index patients with mutations were also evaluated. In our clinical examination of patients, we measured visual acuity, final dark-adaptation threshold equivalent visual field diameter, and electroretinogram amplitudes among other parameters of visual function. We compared the clinical findings with those obtained earlier from similar evaluations of a group of 39 patients with the dominant rhodopsin mutation Pro23His and a group of 25 patients with the dominant rhodopsin mutation Pro347Leu. We identified 3 novel missense mutations in a total of 4 unrelated patients with dominant retinitis pigmentosa: Ser50Pro, Ser50Leu (2 patients), and Pro51Thr. Each mutation cosegregated with dominant retinitis pigmentosa. None of these mutations were found among 91 unrelated control individuals. The visual acuities among the 4 index patients and 3 relatives with NRL mutations who were clinically evaluated ranged from 20/20 (in a 9-year-old patient) to 20/200 (in a 73-year-old patient). All patients had bone-spicule pigment deposits in their fundi. Average rod-plus-cone and cone-isolated electroretinogram amplitudes were both decreased by 99% or more compared with normal amplitudes. The dark-adaptation thresholds, equivalent visual field diameters, and electroretinogram amplitudes (all corrected for age and refractive error) indicated that the disease caused by the NRL mutations was more severe than that caused by the dominant rhodopsin mutation Pro23His and was similar in severity to that produced by the rhodopsin mutation Pro347Leu. The 3 novel NRL mutations we discovered bring the total number of reported

  11. Prevalence and significance of MEFV gene mutations in patients with gouty arthritis. (United States)

    Karaarslan, Ahmet; Kobak, Senol; Kaya, Işın; Intepe, Nazım; Orman, Mehmet; Berdelı, Afig


    Gouty arthritis is a chronic erosive autoinflammatory disease. Pyrin has anti-inflammatory effects in the regulation of inflammasome and is encoded by the MEFV gene. The relationship between different rheumatic diseases and the MEFV gene mutations was demonstrated. The aim of this study was to determine the frequency of MEFV gene mutations in patients with gouty arthritis and identify a possible correlation with disease phenotype. Ninety-three patients with gouty arthritis and 102 healthy controls, compatible with age, gender and ethnicity, were included in the study. MEFV gene mutations were investigated by PCR method. Out of 93 patients with gouty arthritis, 36 (38.7 %) showed MEFV gene mutations carriage, whereas 20.6 % in healthy control group. Distribution of mutations identified in patients with gouty arthritis was as; R202Q in 18 (19.3 %), E148Q in 5 (5.4 %), K695R in 4 (4.3 %), M680I in 2 (2.1 %), V726A in 2 (2.1 %), P369S in 2 (2.1 %), R408Q in 2 (2.1 %), M694 V in 1 (1.1 %), respectively. Three patients were identified with compound heterozygosity. Distribution of MEFV gene mutations carriage in healthy controls was; E148Q in 11 (10.7 %), M694 V in 2 (1.9 %), M694I in 1 (0.9 %), M680I in 2 (1.9 %), V726A in 1 (0.9 %), A744S in 1 (0.9 %), K695R in 2 (1.9 %), and P369S in 1 (0.9 %) patients, respectively. Higher MEFV gene mutations carrier frequency was observed in patients with gouty arthritis, compared with the control group (p = 0.009). Heterozygous R202Q was the most common mutation detected in patients with gouty arthritis, while heterozygous E148Q in healthy control group. Statistically significant difference was not detected between clinical findings of gouty arthritis and the MEFV gene mutations (p > 0.05). We determined higher prevalence of MEFV gene mutations in patients with gouty arthritis compared with the healthy control group. The most frequently detected mutation was heterozygous R202Q, whereas E148Q in healthy

  12. Red blood cell PK deficiency: An update of PK-LR gene mutation database. (United States)

    Canu, Giulia; De Bonis, Maria; Minucci, Angelo; Capoluongo, Ettore


    Pyruvate kinase (PK) deficiency is known as being the most common cause of chronic nonspherocytic hemolytic anemia (CNSHA). Clinical PK deficiency is transmitted as an autosomal recessive trait, that can segregate neither in homozygous or in a compound heterozygous modality, respectively. Two PK genes are present in mammals: the pyruvate kinase liver and red blood cells (PK-LR) and the pyruvate kinase muscle (PK-M), of which only the first encodes for the isoenzymes normally expressed in the red blood cells (R-type) and in the liver (L-type). Several reports have been published describing a large variety of genetic defects in PK-LR gene associated to CNSHA. Herein, we present a review of about 250 published mutations and six polymorphisms in PK-LR gene with the corresponding clinical and molecular data. We consulted the PubMed website for searching mutations and papers, along with two main databases: the Leiden Open Variation Database (LOVD, and Human Gene Mutation Database (HGMD, for selecting, reviewing and listing the annotated PK-LR gene mutations present in literature. This paper is aimed to provide useful information to clinicians and laboratory professionals regarding overall reported PK-LR gene mutations, also giving the opportunity to harmonize data regarding PK-deficient individuals.

  13. Seven mutations in the human insulin gene linked to permanent neonatal/infancy-onset diabetes mellitus

    DEFF Research Database (Denmark)

    Colombo, Carlo; Porzio, Ottavia; Liu, Ming;


    Permanent neonatal diabetes mellitus (PNDM) is a rare disorder usually presenting within 6 months of birth. Although several genes have been linked to this disorder, in almost half the cases documented in Italy, the genetic cause remains unknown. Because the Akita mouse bearing a mutation...... in the Ins2 gene exhibits PNDM associated with pancreatic beta cell apoptosis, we sequenced the human insulin gene in PNDM subjects with unidentified mutations. We discovered 7 heterozygous mutations in 10 unrelated probands. In 8 of these patients, insulin secretion was detectable at diabetes onset...... of endoplasmic reticulum stress, and with increased apoptosis. Similarly transfected INS-1E insulinoma cells had diminished viability compared with those expressing WT proinsulin. In conclusion, we find that mutations in the insulin gene that promote proinsulin misfolding may cause PNDM....

  14. Splicing factor gene mutations in the myelodysplastic syndromes: impact on disease phenotype and therapeutic applications. (United States)

    Pellagatti, Andrea; Boultwood, Jacqueline


    Splicing factor gene mutations are the most frequent mutations found in patients with the myeloid malignancy myelodysplastic syndrome (MDS), suggesting that spliceosomal dysfunction plays a major role in disease pathogenesis. The aberrantly spliced target genes and deregulated cellular pathways associated with the commonly mutated splicing factor genes in MDS (SF3B1, SRSF2 and U2AF1) are being identified, illuminating the molecular mechanisms underlying MDS. Emerging data from mouse modeling studies indicate that the presence of splicing factor gene mutations can lead to bone marrow hematopoietic stem/myeloid progenitor cell expansion, impaired hematopoiesis and dysplastic differentiation that are hallmarks of MDS. Importantly, recent evidence suggests that spliceosome inhibitors and splicing modulators may have therapeutic value in the treatment of splicing factor mutant myeloid malignancies.

  15. Methods for the identification of mutations in the human phenylalanine hydroxylase gene using DNA probes

    Energy Technology Data Exchange (ETDEWEB)

    Woo, S.L.C.; Dilella, A.G.


    This patent describes a method of detecting a mutation in a phenylalanine hydroxylase gene of human genomic DNA. Also described is an automated method of detecting PKU affected, PKU helerozgotes and normals in fetal to adult human samples.

  16. Mutation screening of mismatch repair gene Mlh3 in familial esophageal cancer

    Institute of Scientific and Technical Information of China (English)

    Hong-Xu Liu; Yu Li; Xue-Dong Jiang; Hong-Nian Yin; Lin Zhang; Yu Wang; Jun Yang


    AIM: To shed light on the possible role of mismatch repair gene Mlh3 in familial esophageal cancer (FEC).METHODS: A total of 66 members from 10 families suggestive of a genetic predisposition to hereditary esophageal cancer were screened for germline mutations in Mlh3 with denaturing high performance liquid chromatography (DHPLC), a newly developed method of comparative sequencing based on heteroduplex detection. For all samples exhibiting abnormal DHPLC profiles,sequence changes were evaluated by cycle sequencing.For any mutation in family members, we conducted a segregation study to compare its prevalence in sporadic esophageal cancer patients and normal controls.RESULTS: Exons of Mlh3 in all samples were successfully examined. Overall, 4 missense mutations and 3 polymorphisms were identified in 4 families. Mlh3 missense mutations in families 9 and 10 might be pathogenic, but had a reduced penetrance. While in families 1 and 7,there was no sufficient evidence supporting the monogenic explanations of esophageal cancers in families.The mutations were found in 33% of high-risk families and 50% of low-risk families.CONCLUSION: Mlh3 is a high risk gene with a reduced penetrance in some families. However, it acts as a low risk gene for esophageal cancer in most families. Mutations of Mlh3 may work together with other genes in an accumulated manner and result in an increased risk of esophageal tumor. DHPLC is a robust and sensitive technique for screening gene mutations.

  17. Quantification of Atlantic salmon type-I interferon using an Mx1 promoter reporter gene assay. (United States)

    Johansen, Audny; Collet, Bertrand; Sandaker, Elin; Secombes, Christopher J; Jørgensen, Jorunn B


    We here describe an assay for the detection of interferon-like activity in Atlantic salmon based on the transient transfection of chinook salmon embryo cells (CHSE-214 cells) with a rainbow trout Mx1 promoter linked to a luciferase reporter. A beta-galactosidase gene under the control of a constitutively expressed beta-actin promoter was used as a transfection standard, and luciferase and beta gal expression were measured by a commercially available kit. Interferon containing supernatants from poly I:C- or CpG-stimulated leucocytes added to transfected CHSE-cells induced high luciferase expression (>60-fold induction compared to supernatants from non-stimulated cells). There was no response to supernatants from LPS- and ConA/PMA-stimulated leucocytes, demonstrating the specificity for type I interferon-like activity. Duplicate samples analysed using a cell protection assay for detection of antiviral activity correlated well with levels obtained by the Mx1 promoter reporter gene assay (R2=0.97), confirming the reporter assay as a reliable substitute for the standard antiviral assay. The Mx reporter gene assay also has advantages in terms of sensitivity, high dynamic range and reliability over the conventional cell protection assay.

  18. Mutational analysis of BMP15 and GDF9 as candidate genes for premature ovarian failure. (United States)

    Chand, Ashwini L; Ponnampalam, Anna P; Harris, Sarah E; Winship, Ingrid M; Shelling, Andrew N


    Mutational screening of the bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) genes in a population with premature ovarian failure (POF) identified no new mutations. However, three single nucleotide polymorphisms in the BMP15 gene, two in the 5' untranslated region (31T>G and 71C>G) and another in exon 1 (387G>A), were found to be common in both POF and control groups.

  19. Analysis of hemochromatosis gene mutations in 52 consecutive patients with polycythemia vera. (United States)

    Franchini, Massimo; de Matteis, Giovanna; Federici, Francesca; Solero, Pietro; Veneri, Dino


    A literature review reports increased erythrocyte indices [hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin (MCH), MCH concentration] in subjects with hereditary hemochromatosis (HH). We, therefore, screened 52 consecutive patients with polycythemia vera for 12 HH gene mutations, comparing iron status and red cell parameters between patients positive or negative for HH gene mutations. Our results support the evidence that there is no association between these two conditions.

  20. Adverse events in families with hypertrophic or dilated cardiomyopathy and mutations in the MYBPC3 gene

    Directory of Open Access Journals (Sweden)

    Lehrke Stephanie


    Full Text Available Abstract Background Mutations in MYBPC3 encoding myosin binding protein C belong to the most frequent causes of hypertrophic cardiomyopathy (HCM and may also lead to dilated cardiomyopathy (DCM. MYBPC3 mutations initially were considered to cause a benign form of HCM. The aim of this study was to examine the clinical outcome of patients and their relatives with 18 different MYBPC3 mutations. Methods 87 patients with HCM and 71 patients with DCM were screened for MYBPC3 mutations by denaturing gradient gel electrophoresis and sequencing. Close relatives of mutation carriers were genotyped for the respective mutation. Relatives with mutation were then evaluated by echocardiography and magnetic resonance imaging. A detailed family history regarding adverse clinical events was recorded. Results In 16 HCM (18.4% and two DCM (2.8% index patients a mutation was detected. Seven mutations were novel. Mutation carriers exhibited no additional mutations in genes MYH7, TNNT2, TNNI3, ACTC and TPM1. Including relatives of twelve families, a total number of 42 mutation carriers was identified of which eleven (26.2% had at least one adverse event. Considering the twelve families and six single patients with mutations, 45 individuals with cardiomyopathy and nine with borderline phenotype were identified. Among the 45 patients, 23 (51.1% suffered from an adverse event. In eleven patients of seven families an unexplained sudden death was reported at the age between 13 and 67 years. Stroke or a transient ischemic attack occurred in six patients of five families. At least one adverse event occurred in eleven of twelve families. Conclusion MYBPC3 mutations can be associated with cardiac events such as progressive heart failure, stroke and sudden death even at younger age. Therefore, patients with MYBPC3 mutations require thorough clinical risk assessment.

  1. Mutational characteristics of ANK1 and SPTB genes in hereditary spherocytosis. (United States)

    Park, J; Jeong, D-C; Yoo, J; Jang, W; Chae, H; Kim, J; Kwon, A; Choi, H; Lee, J W; Chung, N-G; Kim, M; Kim, Y


    The aim of this study was to describe the mutational characteristics in Korean hereditary spherocytosis (HS) patients. Relevant literatures including genetically confirmed cases with well-documented clinical summaries and relevant information were also reviewed to investigate the mutational gene- or domain-specific laboratory and clinical association. Twenty-five HS patients carried one heterozygous mutation of ANK1 (n = 13) or SPTB (n = 12) but not in SPTA1, SLC4A1, or EPB42. Deleterious mutations including frameshift, nonsense, and splice site mutations were identified in 91% (21/23), and non-hotspot mutations were dispersed across multiple exons. Genotype-phenotype correlation was clarified after combined analysis of the cases and the literature review; anemia was most severe in HS patients with mutations on the ANK1 spectrin-binding domain (p < 0.05), and SPTB mutations in HS patients spared the tetramerization domain in which mutations of hereditary elliptocytosis and pyropoikilocytosis are located. Splenectomy (17/75) was more frequent in ANK1 mutant HS (32%) than in HS with SPTB mutation (10%) (p = 0.028). Aplastic crisis occurred in 32.0% of the patients (8/25; 3 ANK1 and 5 SPTB), and parvovirus B19 was detected in 88%. The study clarifies ANK1 or SPTB mutational characteristics in HS Korean patients. The genetic association of laboratory and clinical aspects suggests comprehensive considerations for genetic-based management of HS.

  2. Immunohistochemical NF1 analysis does not predict NF1 gene mutation status in pheochromocytoma. (United States)

    Stenman, Adam; Svahn, Fredrika; Welander, Jenny; Gustavson, Boel; Söderkvist, Peter; Gimm, Oliver; Juhlin, C Christofer


    Pheochromocytomas (PCCs) are tumors originating from the adrenal medulla displaying a diverse genetic background. While most PCCs are sporadic, about 40 % of the tumors have been associated with constitutional mutations in one of at least 14 known susceptibility genes. As 25 % of sporadic PCCs harbor somatic neurofibromin 1 gene (NF1) mutations, NF1 has been established as the most recurrently mutated gene in PCCs. To be able to pinpoint NF1-related pheochromocytoma (PCC) disease in clinical practice could facilitate the detection of familial cases, but the large size of the NF1 gene makes standard DNA sequencing methods cumbersome. The aim of this study was to examine whether mutations in the NF1 gene could be predicted by immunohistochemistry as a method to identify cases for further genetic characterization. Sixty-seven PCCs obtained from 67 unselected patients for which the somatic and constitutional mutational status of NF1 was known (49 NF1 wild type, 18 NF1 mutated) were investigated for NF1 protein immunoreactivity, and the results were correlated to clinical and genetic data. NF1 immunoreactivity was absent in the majority of the PCCs (44/67; 66 %), including 13 out of 18 cases (72 %) with a somatic or constitutional NF1 mutation. However, only a minority of the NF1 wild-type PCCs (18/49; 37 %) displayed retained NF1 immunoreactivity, thereby diminishing the specificity of the method. We conclude that NF1 immunohistochemistry alone is not a sufficient method to distinguish between NF1-mutated and non-mutated PCCs. In the clinical context, genetic screening therefore remains the most reliable tool to detect NF1-mutated PCCs.

  3. First report of a de novo germline mutation in the MLH1 gene

    Institute of Scientific and Technical Information of China (English)

    Rein P Stulp; Yvonne J Vos; Bart Mol; Arend Karrenbeld; Monique de Raad; Huub JC van der Mijle; Rolf H Sijmons


    Hereditary non-polyposis colorectal carcinoma (HNPCC)is an autosomal dominant disorder associated with colorectal and endometrial cancer and a range of other tumor types. Germline mutations in the DNA mismatch repair (MMR) genes, particularly MLH1, MSH2, and MSH6, underlie this disorder. The vast majority of these HNPCC-associated mutations have been proven,or assumed, given the family history of cancer, to be transmitted through several generations. To the best of our knowledge, only a single case of a de novo germline MMR gene mutation (in MSH2) has been reported till now. Here, we report a patient with a de novo mutation in MLH1. We identified a MLH1 Q701X truncating mutation in the blood lymphocytes of a male who had been diagnosed with rectal cancer at the age of 35. His family history of cancer was negative for the first- and second-degree relatives. The mutation could not be detected in the patient's parents and sibling and paternity was confirmed with a set of highly polymorphic markers. Non-penetrance and small family size is the common explanation of verified negative family histories of cancer in patients with a germline MMR gene mutation. However, in addition to some cases explained by non-paternity, de novo germline mutations should be considered as a possible explanation as well. As guidelines that stress not to restrict MMR gene mutation testing to patients with a positive family history are more widely introduced, more cases of de novo MMR gene germline mutations may be revealed.

  4. Utilization of Gene Mapping and Candidate Gene Mutation Screening for Diagnosing Clinically Equivocal Conditions:A Norrie Disease Case Study

    Institute of Scientific and Technical Information of China (English)

    Vasiliki Chini; Danai Stambouli; Florina Mihaela Nedelea; George Alexandru Filipescu; Diana Mina; Marios Kambouris; Hatem El-Shanti


    Prenatal diagnosis was requested for an undiagnosed eye disease showing X-linked inheritance in a family. No medical records existed for the affected family members..Mapping of the X chromosome and candidate gene mutation screening i-dentified a c.C267A[p.F89L] mutation in NPD previously de-scribed as possibly causing Norrie disease..The detection of the c.C267A[p.F89L] variant in another unrelated family con-firms the pathogenic nature of the mutation for the Norrie dis-ease phenotype. Gene mapping, haplotype analysis, and can-didate gene screening have been previously utilized in research applications but were applied here in a diagnostic setting due to the scarcity of available clinical information..The clinical diagnosis and mutation identification were critical for provid-ing proper genetic counseling and prenatal diagnosis for this family.

  5. Simple multiplex PCR assay for identification of Beijing family Mycobacterium tuberculosis isolates with a lineage-specific mutation in Rv0679c. (United States)

    Nakajima, Chie; Tamaru, Aki; Rahim, Zeaur; Poudel, Ajay; Maharjan, Bhagwan; Khin Saw Aye; Ling, Hong; Hattori, Toshio; Iwamoto, Tomotada; Fukushima, Yukari; Suzuki, Haruka; Suzuki, Yasuhiko; Matsuba, Takashi


    The Beijing genotype of Mycobacterium tuberculosis is known to be a worldwide epidemic clade. It is suggested to be a possibly resistant clone against BCG vaccination and is also suggested to be highly pathogenic and prone to becoming drug resistant. Thus, monitoring the prevalence of this lineage seems to be important for the proper control of tuberculosis. The Rv0679c protein of M. tuberculosis has been predicted to be one of the outer membrane proteins and is suggested to contribute to host cell invasion. Here, we conducted a sequence analysis of the Rv0679c gene using clinical isolates and found that a single nucleotide polymorphism, C to G at position 426, can be observed only in the isolates that are identified as members of the Beijing genotype family. Here, we developed a simple multiplex PCR assay to detect this point mutation and applied it to 619 clinical isolates. The method successfully distinguished Beijing lineage clones from non-Beijing strains with 100% accuracy. This simple, quick, and cost-effective multiplex PCR assay can be used for a survey or for monitoring the prevalence of Beijing genotype M. tuberculosis strains.

  6. [Comparing performance of "TB-BIOCHIP", "Xpert MTB/RIF" and "genotype MTBDRplus" assays for fast identification of mutations in the Mycobacterium tuberculosis complex in sputum from TB patients]. (United States)

    Nosova, E Iu; Krasnova, M A; Galkina, K Iu; Makarova, M V; Litvinov, V I; Moroz, A M


    The frequency of mutations causing drug resistance in MTB isolates were studied in the respiratory material obtained from TB-patients in the Moscow Region. In izoniazid-resistant isolates, the most prevalent mutation was found to be the Ser315Thr substitution in the katG gene (15.8%) whereas the most frequent mutations in multidrug-resistant isolates were Ser531Leu and Ser315Thr in the rpoB and katG genes (26.3%), or a combination of these two substitutions with a T15 mutation in the inhA gene (5.3%). We compared performance of three molecular assays--"TB-BIOCHIP" ("BIOCHIP-IMB", Ltd, Russia), Xpert MTB/RIF ("Cepheid", USA) and GenoType MTBDRplus ("Hain Life-science", Germany), with the efficiency of luminescent microscopy, and phenotypic drug-suscepibility testing in an automated system BACTEC MGIT 960 (Becton, Disckinson and Company, USA). Xpert MTB/RIF, TB-BIOCHIP and GenoType MTBDRplus detected MTB in sputum in 92, 78 and 49% of all culture-positive cases, respectively. The agreement between standard cultural data and molecular DST results for Xpert MTB/RIF (resistance towards rifampicin), for TB-BIOCHIP and GenoType MTBDRplus (resistance towards rifampicin and izoniazid) amounted to 100, 97 and 100% respectively. Summing up, Xpert MTB/RIF was concluded to be the most efficient assay for primary detection of MTB, whereas the TB-BIOCHIP was shown to be the only molecular assay sensitive enough for simultaneous detection of MTB DNA and for revealing multidrug resistance in sputum (i.e. resistance to both first-line anti-TB drugs, rifampicin and izoniazid).

  7. Mutational analysis of the LDL receptor and APOB genes in Mexican individuals with autosomal dominant hypercholesterolemia. (United States)

    Vaca, Gerardo; Vàzquez, Alejandra; Magaña, Marìa Teresa; Ramìrez, Marìa Lourdes; Dàvalos, Ingrid P; Martìnez, Esperanza; Marìn, Bertha; Carrillo, Gabriela


    The goal of this project was to identify families with autosomal dominant hypercholesterolemia (ADH) to facilitate early detection and treatment and to provide genetic counselling as well as to approximate the mutational diversity of ADH in Mexico. Mutational analysis of the LDLR and APOB genes in 62 index cases with a clinical and/or biochemical diagnosis of ADH was performed. Twenty-five mutations (24 LDLR, 1 APOB) were identified in 38 index cases. A total of 162 individuals with ADH were identified using familial segregation analysis performed in 269 relatives of the index cases. In addition, a novel PCSK9 mutation, c.1850 C>A (p.Ala617Asp), was detected. The LDLR mutations showed the following characteristics: (1) four mutations are novel: c.695 -1G>T, c.1034_1035insA, c.1586 G>A, c.2264_2273del; (2) the most common mutations were c.682 G>A (FH-Mexico), c.1055 G>A (FH-Mexico 2), and c.1090 T>C (FH-Mexico 3); (3) five mutations were identified in 3 or more apparently unrelated probands; (4) three mutations were observed in a true homozygous state; and (5) four index cases were compound heterozygous, and one was a carrier of two mutations in the same allele. These results suggest that, in Mexico, ADH exhibits allelic heterogeneity with 5 relatively common LDLR mutations and that mutations in the APOB gene are not a common cause of ADH. This knowledge is important for the genotype-phenotype correlation and for optimising both cholesterol lowering therapies and mutational analysis protocols. In addition, these data contribute to the understanding of the molecular basis of ADH in Mexico.

  8. Detection of six novel mutations in WASP gene in fifteen Iranian Wiskott-Aldrich patients. (United States)

    Safaei, Sepideh; Fazlollahi, Mohammad Reza; Houshmand, Masoud; Hamidieh, Amir Ali; Bemanian, Mohammad Hassan; Alavi, Samin; Mousavi, Farideh; Pourpak, Zahra; Moin, Mostafa


    Wiskott-Aldrich syndrome (WAS) is a life-threatening X-linked recessive immunodeficiency disease described as a clinical triad of thrombocytopenia, eczema, and recurrent infections, caused by mutations of the WAS protein (WASP) gene. The milder form of this disease is X-linked thrombocytopenia (XLT) that presents only as platelet abnormalities. Mutation analysis for 15 boys with Wiskott-Aldrich syndrome was performed. Five previously reported mutations and six novel mutations including G8X, R41X, D283E, P412fsX446, E464X, and AfsX358 were detected.

  9. Screening for germline mutations in the neurofibromatosis type 2 (NF2) gene in NF2 patients

    Energy Technology Data Exchange (ETDEWEB)

    Andermann, A.A.; Ruttledge, M.H.; Rangaratnam, A. [McGill Univ. and Montreal General Hospital Research Institute, Quebec (Canada)] [and others


    Neurofibromatosis type 2 (NF2) is an autosomal dominant disease with over 95% penetrance which predisposes gene carriers to develop multiple tumors of the central nervous system. The NF2 gene is a putative tumor suppressor gene which was previously mapped to the long arm of chromosome 22, and has recently been identified, using positional cloning techniques. The gene encodes a protein, schwannomin (SCH), which is highly homologous to the band 4.1 protein family. In an attempt to identify and characterize mutations which lead to the manifestation of the disease, we have used single strand conformation analysis (SSCA) to screen for germline mutations in all 17 exons of the NF2 gene in 59 unrelated NF2 patients, representing both familial and new mutations. A total of 27 migration abnormalities was found in 26 patients. Using direct sequencing analysis, the majority of these variants were found to result in nonsense, splice-site or frameshift mutations. Mutations identified in familial NF2 patients segregate in the family, and may prove to be useful tools for a simple and direct SSCA-based technique of presymptomatic or prenatal diagnosis in relatives of patients with NF2. This may be of particular importance in children of patients who have new mutations in the NF2 gene, where linkage analysis may not be feasible.

  10. Prenatal diagnosis based on HPRT1 gene mutation in a Lesch-Nyhan family. (United States)

    Liu, N; Zhuo, Z-H; Wang, H-L; Kong, X-D; Shi, H-R; Wu, Q-H; Jiang, M


    We explored the feasibility of applying gene diagnosis in prenatal diagnosis by analysis of hypoxanthine-guanine phosphoribosyltransferase-1 (HPRT1) gene mutation in a Chinese Lesch-Nyhan family. A homozygous mutation of p.R170X (c.508C>T) in HPRT1 gene was detected in the proband, and a heterozygous mutation of p.R170X was detected in his mother. This mutation failed to be found in the 50 unrelated healthy individuals. Prenatal diagnosis indicated that the foetus was male and also carried p.R170X (c.508C>T) mutation, same as the proband. Parents of the foetus decided termination of pregnancy, and the result of gene analysis for the aborted tissue was consistent with that of prenatal diagnosis. We can see that Lesch-Nyhan syndrome (LNS) is caused by non-sense mutation p.R170X(c.508C>T)in HPRT1 gene in this family. Prenatal gene diagnosis is a valid strategy to prevent LNS because it can avoid the birth of LNS foetuses.

  11. NMD Microarray Analysis for Rapid Genome-Wide Screen of Mutated Genes in Cancer

    Directory of Open Access Journals (Sweden)

    Maija Wolf


    Full Text Available Gene mutations play a critical role in cancer development and progression, and their identification offers possibilities for accurate diagnostics and therapeutic targeting. Finding genes undergoing mutations is challenging and slow, even in the post-genomic era. A new approach was recently developed by Noensie and Dietz to prioritize and focus the search, making use of nonsense-mediated mRNA decay (NMD inhibition and microarray analysis (NMD microarrays in the identification of transcripts containing nonsense mutations. We combined NMD microarrays with array-based CGH (comparative genomic hybridization in order to identify inactivation of tumor suppressor genes in cancer. Such a “mutatomics” screening of prostate cancer cell lines led to the identification of inactivating mutations in the EPHB2 gene. Up to 8% of metastatic uncultured prostate cancers also showed mutations of this gene whose loss of function may confer loss of tissue architecture. NMD microarray analysis could turn out to be a powerful research method to identify novel mutated genes in cancer cell lines, providing targets that could then be further investigated for their clinical relevance and therapeutic potential.

  12. Analysis of GPR101 and AIP genes mutations in acromegaly: a multicentric study. (United States)

    Ferraù, Francesco; Romeo, P D; Puglisi, S; Ragonese, M; Torre, M L; Scaroni, C; Occhi, G; De Menis, E; Arnaldi, G; Trimarchi, F; Cannavò, S


    This multicentric study aimed to investigate the prevalence of the G protein-coupled receptor 101 (GPR101) p.E308D variant and aryl hydrocarbon receptor interacting protein (AIP) gene mutations in a representative cohort of Italian patients with acromegaly. 215 patients with GH-secreting pituitary adenomas, referred to 4 Italian referral centres for pituitary diseases, have been included. Three cases of gigantism were present. Five cases were classified as FIPA. All the patients have been screened for germline AIP gene mutations and GPR101 gene p.E308D variant. Heterozygous AIP gene variants have been found in 7 patients (3.2 %). Five patients carried an AIP mutation (2.3 %; 4 females): 3 patients harboured the p.R3O4Q mutation, one had the p.R304* mutation and the last one the IVS3+1G>A mutation. The prevalence of AIP mutations was 3.3 % and 2.8 % when considering only the patients diagnosed when they were <30 or <40-year old, respectively. Furthermore, 2.0 % of the patients with a pituitary macroadenoma and 4.2 % of patients resistant to somatostatin analogues treatment were found to harbour an AIP gene mutation. None of the patients was found to carry the GPR101 p.E308D variant. The prevalence of AIP gene mutations among our sporadic and familial acromegaly cases was similar to that one reported in previous studies, but lower when considering only the cases diagnosed before 40 years of age. The GPR101 p.E308D change is unlikely to have a role in somatotroph adenomas tumorigenesis, since none of our sporadic or familial patients tested positive for this variant.

  13. Three novel PHEX gene mutations in four Chinese families with X-linked dominant hypophosphatemic rickets

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Qing-lin [Department of Orthopedic Surgery, Shanghai Jiao Tong University Affiliated Sixth People' s Hospital, Shanghai 200233 (China); Xu, Jia [Department of Orthopedic Surgery, Shanghai Jiao Tong University Affiliated Sixth People' s Hospital, Shanghai 200233 (China); Metabolic Bone Disease and Genetic Research Unit, Department of Osteoporosis and Bone Diseases, Shanghai Jiao Tong University Affiliated Sixth People' s Hospital, Shanghai 200233 (China); Medical College of Soochow University, Suzhou, Jiangsu province 215000 (China); Zhang, Zeng [Department of Orthopedic Surgery, Shanghai Jiao Tong University Affiliated Sixth People' s Hospital, Shanghai 200233 (China); Metabolic Bone Disease and Genetic Research Unit, Department of Osteoporosis and Bone Diseases, Shanghai Jiao Tong University Affiliated Sixth People' s Hospital, Shanghai 200233 (China); He, Jin-wei [Metabolic Bone Disease and Genetic Research Unit, Department of Osteoporosis and Bone Diseases, Shanghai Jiao Tong University Affiliated Sixth People' s Hospital, Shanghai 200233 (China); Lu, Lian-song [Department of Orthopedic Surgery, Shanghai Jiao Tong University Affiliated Sixth People' s Hospital, Shanghai 200233 (China); Medical College of Soochow University, Suzhou, Jiangsu province 215000 (China); Fu, Wen-zhen [Metabolic Bone Disease and Genetic Research Unit, Department of Osteoporosis and Bone Diseases, Shanghai Jiao Tong University Affiliated Sixth People' s Hospital, Shanghai 200233 (China); Zhang, Zhen-lin, E-mail: [Metabolic Bone Disease and Genetic Research Unit, Department of Osteoporosis and Bone Diseases, Shanghai Jiao Tong University Affiliated Sixth People' s Hospital, Shanghai 200233 (China)


    Highlights: Black-Right-Pointing-Pointer In our study, all of the patients were of Han Chinese ethnicity, which were rarely reported. Black-Right-Pointing-Pointer We identified three novel PHEX gene mutations in four unrelated families with XLH. Black-Right-Pointing-Pointer We found that the relationship between the phenotype and genotype of the PHEX gene was not invariant. Black-Right-Pointing-Pointer We found that two PHEX gene sites, p.534 and p.731, were conserved. -- Abstract: Background: X-linked hypophosphatemia (XLH), the most common form of inherited rickets, is a dominant disorder that is characterized by renal phosphate wasting with hypophosphatemia, abnormal bone mineralization, short stature, and rachitic manifestations. The related gene with inactivating mutations associated with XLH has been identified as PHEX, which is a phosphate-regulating gene with homologies to endopeptidases on the X chromosome. In this study, a variety of PHEX mutations were identified in four Chinese families with XLH. Methods: We investigated four unrelated Chinese families who exhibited typical features of XLH by using PCR to analyze mutations that were then sequenced. The laboratory and radiological investigations were conducted simultaneously. Results: Three novel mutations were found in these four families: one frameshift mutation, c.2033dupT in exon 20, resulting in p.T679H; one nonsense mutation, c.1294A > T in exon 11, resulting in p.K432X; and one missense mutation, c.2192T > C in exon 22, resulting in p.F731S. Conclusions: We found that the PHEX gene mutations were responsible for XLH in these Chinese families. Our findings are useful for understanding the genetic basis of Chinese patients with XLH.

  14. New mutation detection system of repackaged λ gt11 DNA containing LacZ gene

    Institute of Scientific and Technical Information of China (English)

    LIU Yong; CAO Jia; WU Tao; YANG Lu-jun; SUN Hua-ming; YANG Ming-jie; QIAN Ping


    Objective: To establish a reformative detection system which has sound ability of providing information on molecular mutagenesis spectrum and the specificity of detection system of repackaged λ phage.Methods: LacZ gene, as mutational target gene and reporter gene, was applied into the detection system.The λ gt11 DNA treated with ENU (1-ethyl-1-nitrosourea) and 9-AA (9-aminoacridine) was repackaged in vitro. The packaged λ phage was then grown in E. coli Y1090 on a selective plate containing X-gel and IPTG. The survival and mutation frequencies were determined by counting the clear-plaque and blue-plaque,and the molecular mutation mechanism was studied by extracting and sequencing the LacZ gene of mutants.Results: The survival of repackaged λ phages treated with 9-AA and ENU apparently decreased in consistent dose-dependence. The mutation frequency of clear-plaque mutants showed a linear dose-related increase. The predominant mutations induced by 9-AA were ±1 frameshift mutation, and 9-AA induced -1 frameshift was much more effective than induced + 1 frameshift. 9-AA also induced substitutions with transversions more common. ENU-induced mutations were chiefly occurred at G: C sites. Substitutions induced by ENU were mainly G: C→A: T, G: C→C: G and A: T→T: A transversion. Conclusion: Mutation detection system of λgt11 DNA containing LacZ gene is proven better than that of λDNA without LacZ gene. The combination of survival, mutant frequency and sequence spectrum can not only increase the sensitivity and specificity of the new method, but also provide a better understanding of the molecular mechanism of mutation for ultimate extrapolation to risk assessment.

  15. Frequent mutation of histone-modifying genes in non-Hodgkin lymphoma | Office of Cancer Genomics (United States)

    In a recent Nature article, Morin et al. uncovered a novel role for chromatin modification in driving the progression of two non-Hodgkin lymphomas (NHLs), follicular lymphoma and diffuse large B-cell lymphoma. Through DNA and RNA sequencing of 117 tumor samples and 10 assorted cell lines, the authors identified and validated 109 genes with multiple mutations in these B-cell NHLs. Of the 109 genes, several genes not previously linked to lymphoma demonstrated positive selection for mutation including two genes involved in histone modification, MLL2 and MEF2B.

  16. Allele Specific Locked Nucleic Acid Quantitative PCR (ASLNAqPCR): An Accurate and Cost-Effective Assay to Diagnose and Quantify KRAS and BRAF Mutation (United States)

    Morandi, Luca; de Biase, Dario; Visani, Michela; Cesari, Valentina; De Maglio, Giovanna; Pizzolitto, Stefano; Pession, Annalisa; Tallini, Giovanni


    The use of tyrosine kinase inhibitors (TKIs) requires the testing for hot spot mutations of the molecular effectors downstream the membrane-bound tyrosine kinases since their wild type status is expected for response to TKI therapy. We report a novel assay that we have called Allele Specific Locked Nucleic Acid quantitative PCR (ASLNAqPCR). The assay uses LNA-modified allele specific primers and LNA-modified beacon probes to increase sensitivity, specificity and to accurately quantify mutations. We designed primers specific for codon 12/13 KRAS mutations and BRAF V600E, and validated the assay with 300 routine samples from a variety of sources, including cytology specimens. All were analyzed by ASLNAqPCR and Sanger sequencing. Discordant cases were pyrosequenced. ASLNAqPCR correctly identified BRAF and KRAS mutations in all discordant cases and all had a mutated/wild type DNA ratio below the analytical sensitivity of the Sanger method. ASLNAqPCR was 100% specific with greater accuracy, positive and negative predictive values compared with Sanger sequencing. The analytical sensitivity of ASLNAqPCR is 0.1%, allowing quantification of mutated DNA in small neoplastic cell clones. ASLNAqPCR can be performed in any laboratory with real-time PCR equipment, is very cost-effective and can easily be adapted to detect hot spot mutations in other oncogenes. PMID:22558339

  17. Nonsense suppressor therapies rescue peroxisome lipid metabolism and assembly in cells from patients with specific PEX gene mutations (United States)

    Dranchak, Patricia K.; Di Pietro, Erminia; Snowden, Ann; Oesch, Nathan; Braverman, Nancy E.; Steinberg, Steven J.; Hacia, Joseph G.


    Peroxisome biogenesis disorders (PBDs) are multisystemic autosomal recessive disorders resulting from mutations in PEX genes required for normal peroxisome assembly and metabolic activities. Here, we evaluated the potential effectiveness of aminoglycoside G418 (geneticin) and PTC124 (ataluren) nonsense suppression therapies for the treatment of PBD patients with disease-causing nonsense mutations. PBD patient skin fibroblasts producing stable PEX2 or PEX12 nonsense transcripts and Chinese hamster ovary (CHO) cells with a Pex2 nonsense allele all showed dramatic improvements in peroxisomal very long chain fatty acid catabolism and plasmalogen biosynthesis in response to G418 treatments. Cell imaging assays provided complementary confirmatory evidence of improved peroxisome assembly in G418-treated patient fibroblasts. In contrast, we observed no appreciable rescue of peroxisome lipid metabolism or assembly for any patient fibroblast or CHO cell culture treated with various doses of PTC124. Additionally, PTC124 did not show measurable nonsense suppression in immunoblot assays that directly evaluated the read-through of PEX7 nonsense alleles found in PBD patients with rhizomelic chondrodysplasia punctata type 1 (RCDP1). Overall, our results support the continued development of safe and effective nonsense suppressor therapies that could benefit a significant subset of individuals with PBDs. Furthermore, we suggest that the described cell culture assay systems could be useful for evaluating and screening for novel nonsense suppressor therapies. PMID:21465523

  18. Mutations in the promoter region of the aldolase B gene that cause hereditary fructose intolerance. (United States)

    Coffee, Erin M; Tolan, Dean R


    Hereditary fructose intolerance (HFI) is a potentially fatal inherited metabolic disease caused by a deficiency of aldolase B activity in the liver and kidney. Over 40 disease-causing mutations are known in the protein-coding region of ALDOB. Mutations upstream of the protein-coding portion of ALDOB are reported here for the first time. DNA sequence analysis of 61 HFI patients revealed single base mutations in the promoter, intronic enhancer, and the first exon, which is entirely untranslated. One mutation, g.-132G>A, is located within the promoter at an evolutionarily conserved nucleotide within a transcription factor-binding site. A second mutation, IVS1+1G>C, is at the donor splice site of the first exon. In vitro electrophoretic mobility shift assays show a decrease in nuclear extract-protein binding at the g.-132G>A mutant site. The promoter mutation results in decreased transcription using luciferase reporter plasmids. Analysis of cDNA from cells transfected with plasmids harboring the IVS1+1G>C mutation results in aberrant splicing leading to complete retention of the first intron (~5 kb). The IVS1+1G>C splicing mutation results in loss of luciferase activity from a reporter plasmid. These novel mutations in ALDOB represent 2% of alleles in American HFI patients, with IVS1+1G>C representing a significantly higher allele frequency (6%) among HFI patients of Hispanic and African-American ethnicity.

  19. MGB probe assay for rapid detection of mtDNA11778 mutation in the Chinese LHON patients by real-time PCR

    Institute of Scientific and Technical Information of China (English)

    Jian-yong WANG; Yang-shun GU; Jing WANG; Yi TONG; Ying WANG; Jun-bing SHAO; Ming QI


    Objective:Leber's hereditary optic neuropathY (LHON)is a maternally inherited degeneration of the optic nerve caused by point mutations of mitochondrial DNA(mtDNA).Many unsolved questions regarding the penetrance and pathophysiological mechanism of LHON demand efficient and reliable mutation testing.This study aims to develop a minor groove binder(MGB) probe assay for rapid detection of mtDNA11778 mutation and heteroplasmy in Chinese LHON patients by real-time polymerase chain reaction(PCR).Methods:Forty-eight patients suspected of having LHON and their maternal relatives underwent a molecular genetic evaluation,with 20 normal individuals as a control group at the same time.A real-time PCR involving two MGB probes was used to detect the mtDNA 11778 mutation and heteroplasmy.A linear standard curve was obtained by pUCmLHONG and pUCmLHONA clones.Results:All 48 LHON patients and their matemal relatives were positive for mtDNA 11778 mutation in our assay,27 heteroplasmic and 21 homoplasmic.Eighteen cases did not show an occurrence of the disease,while 9 developed the disease among the 27 heteroplasmic mutation cases.Eleven did not show an occurrence of the disease,while 10 cases developed the disease among 21 homoplasmic mutation cases.There was a significant difierence in the incidence between the heteroplasmic and the homoplasmic mutation types.The time needed for running a real-time PCR assay was only 80 min.Conclusion:This real-time PCR assay is a rapid,reliable method for mtDNA mutation detection as well as heteroplasmy quantification.Detecting this ratio is very important for predicting phenotypic expression of unaffected carriers.

  20. AZT-related mutation Lys70Arg in reverse transcriptase of human immunodeficiency virus type 1 confers decrease in susceptibility to ddATP in in vitro RT inhibition assay. (United States)

    Sharma, P L; Chatis, P A; Dogon, A L; Mayers, D L; McCutchan, F E; Page, C; Crumpacker, C S


    The genetic basis for didanosine (ddl) resistance in human immunodeficiency virus (HIV-1) has previously been shown to be commonly associated with a Leu to Val change at codon 74 in the HIV-1 RT gene. In this study sequential viral isolates were analyzed from five patients with prior zidovudine (AZT) use who received 6 to 16 months of ddl therapy. Following ddl therapy, viral isolates exhibited an increased AZT susceptibility and decreased ddl susceptibility. Sequence and nested PCR analysis of the HIV-1 RT gene revealed that two viral isolates contained the Leu to Val change at codon 74, and three other isolates with reduced susceptibility to ddl each contained changes at codons 65, 70, and 72. Site-directed mutagenesis was employed to insert specific mutations in RT gene of proviral clone pNL4-3. Analysis of virion-associated reverse transcriptase activity indicated that the Lys70Arg mutation resulted in an enzyme with 2- to 4-fold decreased susceptibility to ddATP. Statistical analysis of the inhibitory concentration for RT activity between pNL4-3 and mutant Lys70Arg viruses obtained in three independent RT inhibition assays was significant (P = 0.05) by student t test paired analysis. Drug susceptibility assays on the virus with Lys70Arg mutation showed a marginal decrease in susceptibility to ddl (1.5- to 2-fold) and about 4- to 6-fold decrease in susceptibility to AZT. Mutations Lys65Glu and Arg72Ser resulted in an impaired RT with greatly diminished functional RT activity. The AZT-associa