Genome-wide characterization of microsatellites and marker development in the carcinogenic liver fluke Clonorchis sinensis.

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Nguyen, Thao T B; Arimatsu, Yuji; Hong, Sung-Jong; Brindley, Paul J; Blair, David; Laha, Thewarach; Sripa, Banchob

2015-06-01

Clonorchis sinensis is an important carcinogenic human liver fluke endemic in East and Southeast Asia. There are several conventional molecular markers that have been used for identification and genetic diversity; however, no information about microsatellites of this liver fluke is published so far. We here report microsatellite characterization and marker development for a genetic diversity study in C. sinensis, using a genome-wide bioinformatics approach. Based on our search criteria, a total of 256,990 microsatellites (≥12 base pairs) were identified from a genome database of C. sinensis, with hexanucleotide motif being the most abundant (51%) followed by pentanucleotide (18.3%) and trinucleotide (12.7%). The tetranucleotide, dinucleotide, and mononucleotide motifs accounted for 9.75, 7.63, and 0.14%, respectively. The total length of all microsatellites accounts for 0. 72% of 547 Mb of the whole genome size, and the frequency of microsatellites was found to be one microsatellite in every 2.13 kb of DNA. For the di-, tri-, and tetranucleotide, the repeat numbers redundant are six (28%), four (45%), and three (76%), respectively. The ATC repeat is the most abundant microsatellites followed by AT, AAT, and AC, respectively. Within 40 microsatellite loci developed, 24 microsatellite markers showed potential to differentiate between C. sinensis and Opisthorchis viverrini. Seven out of 24 loci showed to be heterozygous with observed heterozygosity that ranged from 0.467 to 1. Four primer sets could amplify both C. sinensis and O. viverrini DNA with different sizes. This study provides basic information of C. sinensis microsatellites, and the genome-wide markers developed may be a useful tool for the genetic study of C. sinensis.

Frequent alteration of MLL3 frameshift mutations in microsatellite deficient colorectal cancer.

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Yoshiyuki Watanabe

Full Text Available MLL3 is a histone 3-lysine 4 methyltransferase with tumor-suppressor properties that belongs to a family of chromatin regulator genes potentially altered in neoplasia. Mutations in MLL3 were found in a whole genome analysis of colorectal cancer but have not been confirmed by a separate study.We analyzed mutations of coding region and promoter methylation in MLL3 using 126 cases of colorectal cancer. We found two isoforms of MLL3 and DNA sequencing revealed frameshift and other mutations affecting both isoforms of MLL3 in colorectal cancer cells and 19 of 134 (14% primary colorectal samples analyzed. Moreover, frameshift mutations were more common in cases with microsatellite instability (31% both in CRC cell lines and primary tumors. The largest isoform of MLL3 is transcribed from a CpG island-associated promoter that has highly homology with a pseudo-gene on chromosome 22 (psiTPTE22. Using an assay which measured both loci simultaneously we found prominent age related methylation in normal colon (from 21% in individuals less than 25 years old to 56% in individuals older than 70, R = 0.88, p<0.001 and frequent hypermethylation (83% in both CRC cell lines and primary tumors. We next studied the two loci separately and found that age and cancer related methylation was solely a property of the pseudogene CpG island and that the MLL3 loci was unmethylated.We found that frameshift mutations of MLL3 in both CRC cells and primary tumor that were more common in cases with microsatellite instability. Moreover, we have shown CpG island-associated promoter of MLL3 gene has no DNA methylation in CRC cells but also primary tumor and normal colon, and this region has a highly homologous of pseudo gene (psiTPTE22 that was age relate DNA methylation.

Complex MSH2 and MSH6 mutations in hypermutated microsatellite unstable advanced prostate cancer.

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Pritchard, Colin C; Morrissey, Colm; Kumar, Akash; Zhang, Xiaotun; Smith, Christina; Coleman, Ilsa; Salipante, Stephen J; Milbank, Jennifer; Yu, Ming; Grady, William M; Tait, Jonathan F; Corey, Eva; Vessella, Robert L; Walsh, Tom; Shendure, Jay; Nelson, Peter S

2014-09-25

A hypermutated subtype of advanced prostate cancer was recently described, but prevalence and mechanisms have not been well-characterized. Here we find that 12% (7 of 60) of advanced prostate cancers are hypermutated, and that all hypermutated cancers have mismatch repair gene mutations and microsatellite instability (MSI). Mutations are frequently complex MSH2 or MSH6 structural rearrangements rather than MLH1 epigenetic silencing. Our findings identify parallels and differences in the mechanisms of hypermutation in prostate cancer compared with other MSI-associated cancers.

Microsatellite frequencies vary with body mass and body temperature in mammals, suggesting correlated variation in mutation rate

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William Amos

2014-11-01

Full Text Available Substitution rate is often found to correlate with life history traits such as body mass, a predictor of population size and longevity, and body temperature. The underlying mechanism is unclear but most models invoke either natural selection or factors such as generation length that change the number of mutation opportunities per unit time. Here we use published genome sequences from 69 mammals to ask whether life history traits impact another form of genetic mutation, the high rates of predominantly neutral slippage in microsatellites. We find that the length-frequency distributions of three common dinucleotide motifs differ greatly between even closely related species. These frequency differences correlate with body mass and body temperature and can be used to predict the phenotype of an unknown species. Importantly, different length microsatellites show complicated patterns of excess and deficit that cannot be explained by a simple model where species with short generation lengths have experienced more mutations. Instead, the patterns probably require changes in mutation rate that impact alleles of different length to different extents. Body temperature plausibly influences mutation rate by modulating the propensity for slippage. Existing hypotheses struggle to account for a link between body mass and mutation rate. However, body mass correlates inversely with population size, which in turn predicts heterozygosity. We suggest that heterozygote instability, HI, the idea that heterozygous sites show increased mutability, could provide a plausible link between body mass and mutation rate.

A truncating mutation of HDAC2 in human cancers confers resistance to histone deacetylase inhibition

DEFF Research Database (Denmark)

Ropero, S; Fraga, MF; Ballestar, E

2006-01-01

Disruption of histone acetylation patterns is a common feature of cancer cells, but very little is known about its genetic basis. We have identified truncating mutations in one of the primary human histone deacetylases, HDAC2, in sporadic carcinomas with microsatellite instability and in tumors a...... deacetylase inhibitors. As such drugs may serve as therapeutic agents for cancer, our findings support the use of HDAC2 mutational status in future pharmacogenetic treatment of these individuals....

New chloroplast microsatellite markers suitable for assessing genetic diversity of Lolium perenne and other related grass species.

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Diekmann, Kerstin; Hodkinson, Trevor R; Barth, Susanne

2012-11-01

Lolium perenne (perennial ryegrass) is the most important forage grass species of temperate regions. We have previously released the chloroplast genome sequence of L. perenne 'Cashel'. Here nine chloroplast microsatellite markers are published, which were designed based on knowledge about genetically variable regions within the L. perenne chloroplast genome. These markers were successfully used for characterizing the genetic diversity in Lolium and different grass species. Chloroplast genomes of 14 Poaceae taxa were screened for mononucleotide microsatellite repeat regions and primers designed for their amplification from nine loci. The potential of these markers to assess genetic diversity was evaluated on a set of 16 Irish and 15 European L. perenne ecotypes, nine L. perenne cultivars, other Lolium taxa and other grass species. All analysed Poaceae chloroplast genomes contained more than 200 mononucleotide repeats (chloroplast simple sequence repeats, cpSSRs) of at least 7 bp in length, concentrated mainly in the large single copy region of the genome. Nucleotide composition varied considerably among subfamilies (with Pooideae biased towards poly A repeats). The nine new markers distinguish L. perenne from all non-Lolium taxa. TeaCpSSR28 was able to distinguish between all Lolium species and Lolium multiflorum due to an elongation of an A(8) mononucleotide repeat in L. multiflorum. TeaCpSSR31 detected a considerable degree of microsatellite length variation and single nucleotide polymorphism. TeaCpSSR27 revealed variation within some L. perenne accessions due to a 44-bp indel and was hence readily detected by simple agarose gel electrophoresis. Smaller insertion/deletion events or single nucleotide polymorphisms detected by these new markers could be visualized by polyacrylamide gel electrophoresis or DNA sequencing, respectively. The new markers are a valuable tool for plant breeding companies, seed testing agencies and the wider scientific community due to

Does the evolutionary conservation of microsatellite loci imply function?

Energy Technology Data Exchange (ETDEWEB)

Shriver, M.D.; Deka, R.; Ferrell, R.E. [Univ. of Pittsburgh, PA (United States)] [and others

1994-09-01

Microsatellites are highly polymorphic tandem arrays of short (1-6 bp) sequence motifs which have been found widely distributed in the genomes of all eukaryotes. We have analyzed allele frequency data on 16 microsatellite loci typed in the great apes (human, chimp, orangutan, and gorilla). The majority of these loci (13) were isolated from human genomic libraries; three were cloned from chimpanzee genomic DNA. Most of these loci are not only present in all apes species, but are polymorphic with comparable levels of heterozygosity and have alleles which overlap in size. The extent of divergence of allele frequencies among these four species were studies using the stepwise-weighted genetic distance (Dsw), which was previously shown to conform to linearity with evolutionary time since divergence for loci where mutations exist in a stepwise fashion. The phylogenetic tree of the great apes constructed from this distance matrix was consistent with the expected topology, with a high bootstrap confidence (82%) for the human/chimp clade. However, the allele frequency distributions of these species are 10 times more similar to each other than expected when they were calibrated with a conservative estimate of the time since separation of humans and the apes. These results are in agreement with sequence-based surveys of microsatellites which have demonstrated that they are highly (90%) conserved over short periods of evolutionary time (< 10 million years) and moderately (30%) conserved over long periods of evolutionary time (> 60-80 million years). This evolutionary conservation has prompted some authors to speculate that there are functional constraints on microsatellite loci. In contrast, the presence of directional bias of mutations with constraints and/or selection against aberrant sized alleles can explain these results.

RNF43 is mutated less frequently in Lynch Syndrome compared with sporadic microsatellite unstable colorectal cancers.

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Fennell, Lochlan J; Clendenning, Mark; McKeone, Diane M; Jamieson, Saara H; Balachandran, Samanthy; Borowsky, Jennifer; Liu, John; Kawamata, Futoshi; Bond, Catherine E; Rosty, Christophe; Burge, Matthew E; Buchanan, Daniel D; Leggett, Barbara A; Whitehall, Vicki L J

2018-01-01

The WNT signaling pathway is commonly altered during colorectal cancer development. The E3 ubiquitin ligase, RNF43, negatively regulates the WNT signal through increased ubiquitination and subsequent degradation of the Frizzled receptor. RNF43 has recently been reported to harbor frequent truncating frameshift mutations in sporadic microsatellite unstable (MSI) colorectal cancers. This study assesses the relative frequency of RNF43 mutations in hereditary colorectal cancers arising in the setting of Lynch syndrome. The entire coding region of RNF43 was Sanger sequenced in 24 colorectal cancers from 23 patients who either (i) carried a germline mutation in one of the DNA mismatch repair genes (MLH1, MSH6, MSH2, PMS2), or (ii) showed immunohistochemical loss of expression of one or more of the DNA mismatch repair proteins, was BRAF wild type at V600E, were under 60 years of age at diagnosis, and demonstrated no promoter region methylation for MLH1 in tumor DNA. A validation cohort of 44 colorectal cancers from mismatch repair germline mutation carriers from the Australasian Colorectal Cancer Family Registry (ACCFR) were sequenced for the most common truncating mutation hotspots (X117 and X659). RNF43 mutations were found in 9 of 24 (37.5%) Lynch syndrome colorectal cancers. The majority of mutations were frameshift deletions in the G659 G7 repeat tract (29%); 2 cancers (2/24, 8%) from the one patient harbored frameshift mutations at codon R117 (C6 repeat tract) within exon 3. In the ACCFR validation cohort, RNF43 hotspot mutations were identified in 19/44 (43.2%) of samples, which was not significantly different to the initial series. The proportion of mutant RNF43 in Lynch syndrome related colorectal cancers is significantly lower than the previously reported mutation rate found in sporadic MSI colorectal cancers. These findings identify further genetic differences between sporadic and hereditary colorectal cancers. This may be because Lynch Syndrome cancers

Genome-wide survey and analysis of microsatellites in giant panda (Ailuropoda melanoleuca), with a focus on the applications of a novel microsatellite marker system.

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Huang, Jie; Li, Yu-Zhi; Du, Lian-Ming; Yang, Bo; Shen, Fu-Jun; Zhang, He-Min; Zhang, Zhi-He; Zhang, Xiu-Yue; Yue, Bi-Song

2015-02-07

The giant panda (Ailuropoda melanoleuca) is a critically endangered species endemic to China. Microsatellites have been preferred as the most popular molecular markers and proven effective in estimating population size, paternity test, genetic diversity for the critically endangered species. The availability of the giant panda complete genome sequences provided the opportunity to carry out genome-wide scans for all types of microsatellites markers, which now opens the way for the analysis and development of microsatellites in giant panda. By screening the whole genome sequence of giant panda in silico mining, we identified microsatellites in the genome of giant panda and analyzed their frequency and distribution in different genomic regions. Based on our search criteria, a repertoire of 855,058 SSRs was detected, with mono-nucleotides being the most abundant. SSRs were found in all genomic regions and were more abundant in non-coding regions than coding regions. A total of 160 primer pairs were designed to screen for polymorphic microsatellites using the selected tetranucleotide microsatellite sequences. The 51 novel polymorphic tetranucleotide microsatellite loci were discovered based on genotyping blood DNA from 22 captive giant pandas in this study. Finally, a total of 15 markers, which showed good polymorphism, stability, and repetition in faecal samples, were used to establish the novel microsatellite marker system for giant panda. Meanwhile, a genotyping database for Chengdu captive giant pandas (n = 57) were set up using this standardized system. What's more, a universal individual identification method was established and the genetic diversity were analysed in this study as the applications of this marker system. The microsatellite abundance and diversity were characterized in giant panda genomes. A total of 154,677 tetranucleotide microsatellites were identified and 15 of them were discovered as the polymorphic and stable loci. The individual

Abiotic stress leads to somatic and heritable changes in homologous recombination frequency, point mutation frequency and microsatellite stability in Arabidopsis plants

International Nuclear Information System (INIS)

Yao Youli; Kovalchuk, Igor

2011-01-01

In earlier studies, we showed that abiotic stresses, such as ionizing radiation, heavy metals, temperature and water, trigger an increase in homologous recombination frequency (HRF). We also demonstrated that many of these stresses led to inheritance of high-frequency homologous recombination, HRF. Although an increase in recombination frequency is an important indicator of genome rearrangements, it only represents a minor portion of possible stress-induced mutations. Here, we analyzed the influence of heat, cold, drought, flood and UVC abiotic stresses on two major types of mutations in the genome, point mutations and small deletions/insertions. We used two transgenic lines of Arabidopsis thaliana, one allowing an analysis of reversions in a stop codon-containing inactivated β-glucuronidase transgene and another one allowing an analysis of repeat stability in a microsatellite-interrupted β-glucuronidase transgene. The transgenic Arabidopsis line carrying the β-glucuronidase-based homologous recombination substrate was used as a positive control. We showed that the majority of stresses increased the frequency of point mutations, homologous recombination and microsatellite instability in somatic cells, with the frequency of homologous recombination being affected the most. The analysis of transgenerational changes showed an increase in HRF to be the most prominent effect observed in progeny. Significant changes in recombination frequency were observed upon exposure to all types of stress except drought, whereas changes in microsatellite instability were observed upon exposure to UVC, heat and cold. The frequency of point mutations in the progeny of stress-exposed plants was the least affected; an increase in mutation frequency was observed only in the progeny of plants exposed to UVC. We thus conclude that transgenerational changes in genome stability in response to stress primarily involve an increase in recombination frequency.

Role of microsatellite instability in colon cancer

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M. Yu. Fedyanin

2012-01-01

Full Text Available Coloncancer is among leading causes of cancer morbidity and mortality both inRussiaand worldwide. Development of molecular biology lead to decoding of carcinogenesis and tumor progression mechanisms. These processes require accumulation of genetic and epigenetic alterations in a tumor cell.Coloncancer carcinogenesis is characterized by mutations cumulation in genes controlling growth and differentiation of epithelial cells, which leads to their genetic instability. Microsatellite instability is a type of genetic instability characterized by deterioration of mismatch DNA repair. This leads to faster accumulation of mutations in DNA. Loss of mismatch repair mechanism can easily be diagnosed by length of DNA microsatellites. These alterations are termed microsatellite instability. They can be found both in hereditary and sporadic colon cancers. This review covers the questions of microsatellite instability, its prognostic and predictive value in colon cancer.

Secondary mutation in a coding mononucleotide tract in MSH6 causes loss of immunoexpression of MSH6 in colorectal carcinomas with MLH1/PMS2 deficiency.

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Shia, Jinru; Zhang, Liying; Shike, Moshe; Guo, Min; Stadler, Zsofia; Xiong, Xiaoling; Tang, Laura H; Vakiani, Efsevia; Katabi, Nora; Wang, Hangjun; Bacares, Ruben; Ruggeri, Jeanine; Boland, C Richard; Ladanyi, Marc; Klimstra, David S

2013-01-01

Immunohistochemical staining for DNA mismatch repair proteins may be affected by various biological and technical factors. Staining variations that could potentially lead to erroneous interpretations have been recognized. A recently recognized staining variation is the significant reduction of staining for MSH6 in some colorectal carcinomas. The frequency and specific characteristics of this aberrant MSH6 staining pattern, however, have not been well analyzed. In this study of 420 colorectal carcinoma samples obtained from patients fulfilling the Revised Bethesda Guidelines, we detected 9 tumors (2%) showing extremely limited staining for MSH6 with positive staining present in PMS2 protein-deficient carcinomas (n=5, including 1 with a pathogenic mutation in PMS2); and (2) MLH1, PMS2 and MSH2 normal but with chemotherapy or chemoradiation therapy before surgery (n=4). To test our hypothesis that somatic mutation in the coding region microsatellite of the MSH6 gene might be a potential underlying mechanism for such limited MSH6 staining, we evaluated frameshift mutation in a (C)(8) tract in exon 5 of the MSH6 gene in seven tumors that had sufficient DNA for analysis, and detected mutation in four; all four tumors belonged to the MLH1/PMS2-deficient group. In conclusion, our data outline the main scenarios where significant reduction of MSH6 staining is more likely to occur in colorectal carcinoma, and suggest that somatic mutations of the coding region microsatellites of the MSH6 gene is an underlying mechanism for this staining phenomenon in MLH1/PMS2-deficient carcinomas.

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

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

2017-07-01

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

Transforming growth factor beta receptor 2 (TGFBR2 changes sialylation in the microsatellite unstable (MSI Colorectal cancer cell line HCT116.

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Jennifer Lee

Full Text Available Aberrant glycosylation is a common feature of many malignancies including colorectal cancers (CRCs. About 15% of CRC show the microsatellite instability (MSI phenotype that is associated with a high frequency of biallelic frameshift mutations in the A10 coding mononucleotide microsatellite of the transforming growth factor beta receptor 2 (TGFBR2 gene. If and how impaired TGFBR2 signaling in MSI CRC cells affects cell surface glycan pattern is largely unexplored. Here, we used the TGFBR2-deficient MSI colon carcinoma cell line HCT116 as a model system. Stable clones conferring doxycycline (dox-inducible expression of a single copy wildtype TGFBR2 transgene were generated by recombinase-mediated cassette exchange (RMCE. In two independent clones, dox-inducible expression of wildtype TGFBR2 protein and reconstitution of its signaling function was shown. Metabolic labeling experiments using the tritiated sialic acid precursor N-acetyl-D-mannosamine (ManNAc revealed a significant decline (∼30% of its incorporation into newly synthesized sialoglycoproteins in a TGFBR2-dependent manner. In particular, we detected a significant decrease of sialylated ß1-integrin upon reconstituted TGFBR2 signaling which did not influence ß1-integrin protein turnover. Notably, TGFBR2 reconstitution did not affect the transcript levels of any of the known human sialyltransferases when examined by real-time RT- PCR analysis. These results suggest that reconstituted TGFBR2 signaling in an isogenic MSI cell line model system can modulate sialylation of cell surface proteins like ß1-integrin. Moreover, our model system will be suitable to uncover the underlying molecular mechanisms of altered MSI tumor glycobiology.

Colorectal carcinomas with microsatellite instability display a different pattern of target gene mutations according to large bowel site of origin

International Nuclear Information System (INIS)

Pinheiro, Manuela; Fragoso, Maria; Santos, Lúcio; Henrique, Rui; Lopes, Paula; Lopes, Carlos; Lothe, Ragnhild A; Teixeira, Manuel R; Ahlquist, Terje; Danielsen, Stine A; Lind, Guro E; Veiga, Isabel; Pinto, Carla; Costa, Vera; Afonso, Luís; Sousa, Olga

2010-01-01

Only a few studies have addressed the molecular pathways specifically involved in carcinogenesis of the distal colon and rectum. We aimed to identify potential differences among genetic alterations in distal colon and rectal carcinomas as compared to cancers arising elsewhere in the large bowel. Constitutional and tumor DNA from a test series of 37 patients with rectal and 25 patients with sigmoid carcinomas, previously analyzed for microsatellite instability (MSI), was studied for BAX, IGF2R, TGFBR2, MSH3, and MSH6 microsatellite sequence alterations, BRAF and KRAS mutations, and MLH1 promoter methylation. The findings were then compared with those of an independent validation series consisting of 36 MSI-H carcinomas with origin from each of the large bowel regions. Immunohistochemical and germline mutation analyses of the mismatch repair system were performed when appropriate. In the test series, IGFR2 and BAX mutations were present in one and two out of the six distal MSI-H carcinomas, respectively, and no mutations were detected in TGFBR2, MSH3, and MSH6. We confirmed these findings in the validation series, with TGFBR2 and MSH3 microsatellite mutations occurring less frequently in MSI-H rectal and sigmoid carcinomas than in MSI-H colon carcinomas elsewhere (P = 0.00005 and P = 0.0000005, respectively, when considering all MSI-carcinomas of both series). No MLH1 promoter methylation was observed in the MSI-H rectal and sigmoid carcinomas of both series, as compared to 53% found in MSI-H carcinomas from other locations (P = 0.004). KRAS and BRAF mutational frequencies were 19% and 43% in proximal carcinomas and 25% and 17% in rectal/sigmoid carcinomas, respectively. The mechanism and the pattern of genetic changes driving MSI-H carcinogenesis in distal colon and rectum appears to differ from that occurring elsewhere in the colon and further investigation is warranted both in patients with sporadic or hereditary disease

Isocitrate dehydrogenase 1 R132C mutation occurs exclusively in microsatellite stable colorectal cancers with the CpG island methylator phenotype

OpenAIRE

Whitehall, VLJ; Dumenil, TD; McKeone, DM; Bond, CE; Bettington, ML; Buttenshaw, RL; Bowdler, L; Montgomery, GW; Wockner, LF; Leggett, BA

2014-01-01

The CpG Island Methylator Phenotype (CIMP) is fundamental to an important subset of colorectal cancer; however, its cause is unknown. CIMP is associated with microsatellite instability but is also found in BRAF mutant microsatellite stable cancers that are associated with poor prognosis. The isocitrate dehydrogenase 1 (IDH1) gene causes CIMP in glioma due to an activating mutation that produces the 2-hydroxyglutarate oncometabolite. We therefore examined IDH1 alteration as a potential cause o...

A biomimetic redox flow battery based on flavin mononucleotide.

Science.gov (United States)

Orita, Akihiro; Verde, Michael G; Sakai, Masanori; Meng, Ying Shirley

2016-10-21

The versatility in design of redox flow batteries makes them apt to efficiently store energy in large-scale applications at low cost. The discovery of inexpensive organic electroactive materials for use in aqueous flow battery electrolytes is highly attractive, but is thus far limited. Here we report on a flow battery using an aqueous electrolyte based on the sodium salt of flavin mononucleotide. Flavins are highly versatile electroactive molecules, which catalyse a multitude of redox reactions in biological systems. We use nicotinamide (vitamin B3) as a hydrotropic agent to enhance the water solubility of flavin mononucleotide. A redox flow battery using flavin mononucleotide negative and ferrocyanide positive electrolytes in strong base shows stable cycling performance, with over 99% capacity retention over the course of 100 cycles. We hypothesize that this is enabled due to the oxidized and reduced forms of FMN-Na being stabilized by resonance structures.

A biomimetic redox flow battery based on flavin mononucleotide

Science.gov (United States)

Orita, Akihiro; Verde, Michael G.; Sakai, Masanori; Meng, Ying Shirley

2016-10-01

The versatility in design of redox flow batteries makes them apt to efficiently store energy in large-scale applications at low cost. The discovery of inexpensive organic electroactive materials for use in aqueous flow battery electrolytes is highly attractive, but is thus far limited. Here we report on a flow battery using an aqueous electrolyte based on the sodium salt of flavin mononucleotide. Flavins are highly versatile electroactive molecules, which catalyse a multitude of redox reactions in biological systems. We use nicotinamide (vitamin B3) as a hydrotropic agent to enhance the water solubility of flavin mononucleotide. A redox flow battery using flavin mononucleotide negative and ferrocyanide positive electrolytes in strong base shows stable cycling performance, with over 99% capacity retention over the course of 100 cycles. We hypothesize that this is enabled due to the oxidized and reduced forms of FMN-Na being stabilized by resonance structures.

Survey of microsatellite clustering in eight fully sequenced species sheds light on the origin of compound microsatellites

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Lelley Tamas

2008-12-01

Full Text Available Abstract Background Compound microsatellites are a special variation of microsatellites in which two or more individual microsatellites are found directly adjacent to each other. Until now, such composite microsatellites have not been investigated in a comprehensive manner. Results Our in silico survey of microsatellite clustering in genomes of Homo sapiens, Maccaca mulatta, Mus musculus, Rattus norvegicus, Ornithorhynchus anatinus, Gallus gallus, Danio rerio and Drosophila melanogaster revealed an unexpected high abundance of compound microsatellites. About 4 – 25% of all microsatellites could be categorized as compound microsatellites. Compound microsatellites are approximately 15 times more frequent than expected under the assumption of a random distribution of microsatellites. Interestingly, microsatellites do not only tend to cluster but the adjacent repeat types of compound microsatellites have very similar motifs: in most cases (>90% these motifs differ only by a single mutation (base substitution or indel. We propose that the majority of the compound microsatellites originates by duplication of imperfections in a microsatellite tract. This process occurs mostly at the end of a microsatellite, leading to a new repeat type and a potential microsatellite repeat track. Conclusion Our findings suggest a more dynamic picture of microsatellite evolution than previously believed. Imperfections within microsatellites might not only cause the "death" of microsatellites they might also result in their "birth".

Differences in K-ras and mitochondrial DNA mutations and microsatellite instability between colorectal cancers of Vietnamese and Japanese patients.

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Miwata, Tomohiro; Hiyama, Toru; Quach, Duc Trong; Le, Huy Minh; Hua, Ha Ngoc Thi; Oka, Shiro; Tanaka, Shinji; Arihiro, Koji; Chayama, Kazuaki

2014-11-30

The incidence of early-onset (under 50 years of age) colorectal cancer (CRC) in the Vietnamese has been reported to be quite higher than that in the Japanese. To clarify the differences in genetic alterations between Vietnamese and Japanese CRCs, we investigated mutations in K-ras and mitochondrial DNA (mtDNA) and high-frequency microsatellite instability (MSI-H) in the CRCs of Vietnamese and Japanese patients. We enrolled 60 Vietnamese and 233 Japanese patients with invasive CRCs. DNA was extracted from formalin-fixed, paraffin-embedded tissue sections. K-ras mutations were examined with PCR-single-strand conformation polymorphism analysis. mtDNA mutations and MSI-H were examined with microsatellite analysis using D310 and BAT-26, respectively. K-ras mutations were examined in 60 Vietnamese and 45 Japanese CRCs. The frequency of the mutations in the Vietnamese CRCs was significantly higher than that in the Japanese CRCs (8 of 24 [33%] vs 5 of 45 [11%], p =0.048). MSI-H was examined in 60 Vietnamese and 130 Japanese CRCs. The frequency of MSI-H in the Vietnamese CRCs was also significantly higher than that in the Japanese CRCs (6 of 27 [22%] vs 10 of 130 [8%], p =0.030). mtDNA mutations were examined in 60 Vietnamese and 138 Japanese CRCs. The frequency of mtDNA mutations in the Vietnamese CRCs was significantly higher than that in the Japanese CRCs (19 of 44 [43%] vs 11 of 133 [9%], p Vietnamese and Japanese patients. These results indicate that the developmental pathways of CRCs in the Vietnamese may differ from those of CRCs in the Japanese.

BRAF, KRAS and PIK3CA mutations in colorectal serrated polyps and cancer: Primary or secondary genetic events in colorectal carcinogenesis?

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Schmitt Fernando

2008-09-01

Full Text Available Abstract Background BRAF, KRAS and PIK3CA mutations are frequently found in sporadic colorectal cancer (CRC. In contrast to KRAS and PIK3CA mutations, BRAF mutations are associated with tumours harbouring CpG Island methylation phenotype (CIMP, MLH1 methylation and microsatellite instability (MSI. We aimed at determine the frequency of KRAS, BRAF and PIK3CA mutations in the process of colorectal tumourigenesis using a series of colorectal polyps and carcinomas. In the series of polyps CIMP, MLH1 methylation and MSI were also studied. Methods Mutation analyses were performed by PCR/sequencing. Bisulfite treated DNA was used to study CIMP and MLH1 methylation. MSI was detected by pentaplex PCR and Genescan analysis of quasimonomorphic mononucleotide repeats. Chi Square test and Fisher's Exact test were used to perform association studies. Results KRAS, PIK3CA or BRAF occur in 71% of polyps and were mutually exclusive. KRAS mutations occur in 35% of polyps. PIK3CA was found in one of the polyps. V600E BRAF mutations occur in 29% of cases, all of them classified as serrated adenoma. CIMP phenotype occurred in 25% of the polyps and all were mutated for BRAF. MLH1 methylation was not detected and all the polyps were microsatellite stable. The comparison between the frequency of oncogenic mutations in polyps and CRC (MSI and MSS lead us to demonstrate that KRAS and PIK3CA are likely to precede both types of CRC. BRAF mutations are likely to precede MSI carcinomas since the frequency found in serrated polyps is similar to what is found in MSI CRC (P = 0.9112, but statistically different from what is found in microsatellite stable (MSS tumours (P = 0.0191. Conclusion Our results show that BRAF, KRAS and PIK3CA mutations occur prior to malignant transformation demonstrating that these oncogenic alterations are primary genetic events in colorectal carcinogenesis. Further, we show that BRAF mutations occur in association with CIMP phenotype in colorectal

BRAF, KRAS and PIK3CA mutations in colorectal serrated polyps and cancer: Primary or secondary genetic events in colorectal carcinogenesis?

International Nuclear Information System (INIS)

Velho, Sérgia; Moutinho, Cátia; Cirnes, Luís; Albuquerque, Cristina; Hamelin, Richard; Schmitt, Fernando; Carneiro, Fátima; Oliveira, Carla; Seruca, Raquel

2008-01-01

BRAF, KRAS and PIK3CA mutations are frequently found in sporadic colorectal cancer (CRC). In contrast to KRAS and PIK3CA mutations, BRAF mutations are associated with tumours harbouring CpG Island methylation phenotype (CIMP), MLH1 methylation and microsatellite instability (MSI). We aimed at determine the frequency of KRAS, BRAF and PIK3CA mutations in the process of colorectal tumourigenesis using a series of colorectal polyps and carcinomas. In the series of polyps CIMP, MLH1 methylation and MSI were also studied. Mutation analyses were performed by PCR/sequencing. Bisulfite treated DNA was used to study CIMP and MLH1 methylation. MSI was detected by pentaplex PCR and Genescan analysis of quasimonomorphic mononucleotide repeats. Chi Square test and Fisher's Exact test were used to perform association studies. KRAS, PIK3CA or BRAF occur in 71% of polyps and were mutually exclusive. KRAS mutations occur in 35% of polyps. PIK3CA was found in one of the polyps. V600E BRAF mutations occur in 29% of cases, all of them classified as serrated adenoma. CIMP phenotype occurred in 25% of the polyps and all were mutated for BRAF. MLH1 methylation was not detected and all the polyps were microsatellite stable. The comparison between the frequency of oncogenic mutations in polyps and CRC (MSI and MSS) lead us to demonstrate that KRAS and PIK3CA are likely to precede both types of CRC. BRAF mutations are likely to precede MSI carcinomas since the frequency found in serrated polyps is similar to what is found in MSI CRC (P = 0.9112), but statistically different from what is found in microsatellite stable (MSS) tumours (P = 0.0191). Our results show that BRAF, KRAS and PIK3CA mutations occur prior to malignant transformation demonstrating that these oncogenic alterations are primary genetic events in colorectal carcinogenesis. Further, we show that BRAF mutations occur in association with CIMP phenotype in colorectal serrated polyps and verified that colorectal serrated

Microsatellite instability in pediatric high grade glioma is associated with genomic profile and differential target gene inactivation.

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Marta Viana-Pereira

Full Text Available High grade gliomas (HGG are one of the leading causes of cancer-related deaths in children, and there is increasing evidence that pediatric HGG may harbor distinct molecular characteristics compared to adult tumors. We have sought to clarify the role of microsatellite instability (MSI in pediatric versus adult HGG. MSI status was determined in 144 patients (71 pediatric and 73 adults using a well established panel of five quasimonomorphic mononucleotide repeat markers. Expression of MLH1, MSH2, MSH6 and PMS2 was determined by immunohistochemistry, MLH1 was assessed for mutations by direct sequencing and promoter methylation using MS-PCR. DNA copy number profiles were derived using array CGH, and mutations in eighteen MSI target genes studied by multiplex PCR and genotyping. MSI was found in 14/71 (19.7% pediatric cases, significantly more than observed in adults (5/73, 6.8%; p = 0.02, Chi-square test. MLH1 expression was downregulated in 10/13 cases, however no mutations or promoter methylation were found. MSH6 was absent in one pediatric MSI-High tumor, consistent with an inherited mismatch repair deficiency associated with germline MSH6 mutation. MSI was classed as Type A, and associated with a remarkably stable genomic profile. Of the eighteen classic MSI target genes, we identified mutations only in MSH6 and DNAPKcs and described a polymorphism in MRE11 without apparent functional consequences in DNA double strand break detection and repair. This study thus provides evidence for a potential novel molecular pathway in a proportion of gliomas associated with the presence of MSI.

Development, characterization and cross species amplification of polymorphic microsatellite markers from expressed sequence tags of turmeric (Curcuma longa L.).

Science.gov (United States)

Siju, S; Dhanya, K; Syamkumar, S; Sasikumar, B; Sheeja, T E; Bhat, A I; Parthasarathy, V A

2010-02-01

Expressed sequence tags (ESTs) from turmeric (Curcuma longa L.) were used for the screening of type and frequency of Class I (hypervariable) simple sequence repeats (SSRs). A total of 231 microsatellite repeats were detected from 12,593 EST sequences of turmeric after redundancy elimination. The average density of Class I SSRs accounts to one SSR per 17.96 kb of EST. Mononucleotides were the most abundant class of microsatellite repeat in turmeric ESTs followed by trinucleotides. A robust set of 17 polymorphic EST-SSRs were developed and used for evaluating 20 turmeric accessions. The number of alleles detected ranged from 3 to 8 per loci. The developed markers were also evaluated in 13 related species of C. longa confirming high rate (100%) of cross species transferability. The polymorphic microsatellite markers generated from this study could be used for genetic diversity analysis and resolving the taxonomic confusion prevailing in the genus.

Candidate driver genes in microsatellite-unstable colorectal cancer

DEFF Research Database (Denmark)

Alhopuro, Pia; Sammalkorpi, Heli; Niittymäki, Iina

2012-01-01

Defects in the mismatch repair system lead to microsatellite instability (MSI), a feature observed in ∼ 15% of all colorectal cancers (CRCs). Microsatellite mutations that drive tumourigenesis, typically inactivation of tumour suppressors, are selected for and are frequently detected in MSI cancers...

In Silico Retrieving of Opium Poppy (Papaver Somniferum L. Microsatellites

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Masárová Veronika

2015-12-01

Full Text Available Repetitive tandem sequences were retrieved within nucleotide sequences of opium poppy (Papaver somniferum L. genomic DNA available in the GenBank® database. Altogether 538 different microsatellites with the desired length characteristics of tandem repeats have been identified within 450 sequences of opium poppy DNA available in the database. The most frequented were mononucleotide repeats (246; nevertheless, 44 dinucleotide, 148 trinucleotide, 62 tetranucleotide, 28 pentanucleotide and 5 hexanucleotide tandem repeats have also been found. The most abundant were trinucleotide motifs (27.50%, and the most abundant motifs within each group of tandem repeats were TA/AT, TTC/GAA, GGTT/AACC and TTTTA/ TAAAA. Five hexanucleotide repeats contained four different motifs.

Mononucleotide precedes dinucleotide repeat instability during colorectal tumour development in Lynch syndrome patients

NARCIS (Netherlands)

Ferreira, Ana M.; Westers, Helga; Sousa, Sonia; Wu, Ying; Niessen, Renee C.; Olderode-Berends, Maran; van der Sluis, Tineke; Reuvekamp, Peter T. W.; Seruca, Raquel; Kleibeuker, Jan H.; Hollema, Harry; Sijmons, Rolf H.; Hofstra, Robert M. W.

A progressive accumulation of genetic alterations underlies the adenoma-carcinoma sequence of colorectal cancer. This accumulation of mutations is driven by genetic instability, of which there are different types. Microsatellite instability (MSI) is the predominant type present in the tumours of

Irrelevance of microsatellite instability in the epidemiology of sporadic pancreatic ductal adenocarcinoma.

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Luigi Laghi

Full Text Available Pancreatic cancer risk is increased in Lynch syndrome (LS patients with mismatch repair gene defects predisposing to colonic and extracolonic cancers with microsatellite instability (MSI. However, the frequency of MSI pancreatic cancers has never been ascertained in consecutive, unselected clinical series, and their contribution to the sporadic and inherited burden of pancreatic cancer remains to be established. Aims of the study were to determine the prevalence of MSI in surgically resected pancreatic cancers in a multicentric, retrospective study, and to assess the occurrence of pancreatic cancer in LS.MS-status was screened by a panel of 5 mononucleotide repeats (Bat26, Bat25, NR-21, NR-24 and NR-27 in 338 consecutive pancreatic ductal adenocarcinoma (PDAC, resected at two Italian and one German referral centres. The personal history of pancreatic cancer was assessed in an independent set of 58 probands with LS and in 138 first degree relatives who had cancers.Only one PDAC (0.3% showed MSI. This was a medullary type cancer, with hMLH1-deficiency, and no identified germ-line mutation but methylation of hMLH1. Pancreatic cancer occurred in 5 (2.5% LS patients. Histological sampling was available for 2 cases, revealing PDAC in one case and an ampullary cancer in the other one.MSI prevalence is negligible in sporadic, resected PDAC. Differently, the prevalence of pancreatic cancer is 2.5% in LS patients, and cancers other than PDAC may be encountered in this setting. Surveillance for pancreatic cancer should be advised in LS mutation carriers at referral centers.

Perspective on sequence evolution of microsatellite locus (CCGn in Rv0050 gene from Mycobacterium tuberculosis

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Jin Ruiliang

2011-08-01

Full Text Available Abstract Background The mycobacterial genome is inclined to polymerase slippage and a high mutation rate in microsatellite regions due to high GC content and absence of a mismatch repair system. However, the exact molecular mechanisms underlying microsatellite variation have not been fully elucidated. Here, we investigated mutation events in the hyper-variable trinucleotide microsatellite locus MML0050 located in the Rv0050 gene of W-Beijing and non-W-Beijing Mycobacterium tuberculosis strains in order to gain insight into the genomic structure and activity of repeated regions. Results Size analysis indicated the presence of five alleles that differed in length by three base pairs. Moreover, nucleotide gains occurred more frequently than loses in this trinucleotide microsatellite. Mutation frequency was not completely related with the total length, though the relative frequency in the longest allele was remarkably higher than that in the shortest. Sequence analysis was able to detect seven alleles and revealed that point mutations enhanced the level of locus variation. Introduction of an interruptive motif correlated with the total allele length and genetic lineage, rather than the length of the longest stretch of perfect repeats. Finally, the level of locus variation was drastically different between the two genetic lineages. Conclusion The Rv0050 locus encodes the bifunctional penicillin-binding protein ponA1 and is essential to mycobacterial survival. Our investigations of this particularly dynamic genomic region provide insights into the overall mode of microsatellite evolution. Specifically, replication slippage was implicated in the mutational process of this microsatellite and a sequence-based genetic analysis was necessary to determine that point mutation events acted to maintain microsatellite size integrity while providing genomic diversity.

Loss of function JAK1 mutations occur at high frequency in cancers with microsatellite instability and are suggestive of immune evasion.

Science.gov (United States)

Albacker, Lee A; Wu, Jeremy; Smith, Peter; Warmuth, Markus; Stephens, Philip J; Zhu, Ping; Yu, Lihua; Chmielecki, Juliann

2017-01-01

Immune evasion is a well-recognized hallmark of cancer and recent studies with immunotherapy agents have suggested that tumors with increased numbers of neoantigens elicit greater immune responses. We hypothesized that the immune system presents a common selective pressure on high mutation burden tumors and therefore immune evasion mutations would be enriched in high mutation burden tumors. The JAK family of kinases is required for the signaling of a host of immune modulators in tumor, stromal, and immune cells. Therefore, we analyzed alterations in this family for the hypothesized signature of an immune evasion mutation. Here, we searched a database of 61,704 unique solid tumors for alterations in the JAK family kinases (JAK1/2/3, TYK2). We used The Cancer Genome Atlas and Cancer Cell Line Encyclopedia data to confirm and extend our findings by analyzing gene expression patterns. Recurrent frameshift mutations in JAK1 were associated with high mutation burden and microsatellite instability. These mutations occurred in multiple tumor types including endometrial, colorectal, stomach, and prostate carcinomas. Analyzing gene expression signatures in endometrial and stomach adenocarcinomas revealed that tumors with a JAK1 frameshift exhibited reduced expression of interferon response signatures and multiple anti-tumor immune signatures. Importantly, endometrial cancer cell lines exhibited similar gene expression changes that were expected to be tumor cell intrinsic (e.g. interferon response) but not those expected to be tumor cell extrinsic (e.g. NK cells). From these data, we derive two primary conclusions: 1) JAK1 frameshifts are loss of function alterations that represent a potential pan-cancer adaptation to immune responses against tumors with microsatellite instability; 2) The mechanism by which JAK1 loss of function contributes to tumor immune evasion is likely associated with loss of the JAK1-mediated interferon response.

Microsatellite instability analysis in hereditary non-polyposis colon cancer using the Bethesda consensus panel of microsatellite markers in the absence of proband normal tissue

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Dourisboure Ricardo J

2006-01-01

Full Text Available Abstract Background Hereditary non-polyposis colon cancer (HNPCC is an autosomal dominant syndrome predisposing to the early development of various cancers including those of colon, rectum, endometrium, ovarium, small bowel, stomach and urinary tract. HNPCC is caused by germline mutations in the DNA mismatch repair genes, mostly hMSH2 or hMLH1. In this study, we report the analysis for genetic counseling of three first-degree relatives (the mother and two sisters of a male who died of colorectal adenocarcinoma at the age of 23. The family fulfilled strict Amsterdam-I criteria (AC-I with the presence of extracolonic tumors in the extended pedigree. We overcame the difficulty of having a proband post-mortem non-tumor tissue sample for MSI testing by studying the alleles carried by his progenitors. Methods Tumor MSI testing is described as initial screening in both primary and metastasis tumor tissue blocks, using the reference panel of 5 microsatellite markers standardized by the National Cancer Institute (NCI for the screening of HNPCC (BAT-25, BAT-26, D2S123, D5S346 and D17S250. Subsequent mutation analysis of the hMLH1 and hMSH2 genes was performed. Results Three of five microsatellite markers (BAT-25, BAT-26 and D5S346 presented different alleles in the proband's tumor as compared to those inherited from his parents. The tumor was classified as high frequency microsatellite instability (MSI-H. We identified in the HNPCC family a novel germline missense (c.1864C>A mutation in exon 12 of hMSH2 gene, leading to a proline 622 to threonine (p.Pro622Thr amino acid substitution. Conclusion This approach allowed us to establish the tumor MSI status using the NCI recommended panel in the absence of proband's non-tumor tissue and before sequencing the obligate carrier. According to the Human Gene Mutation Database (HGMD and the International Society for Gastrointestinal Hereditary Tumors (InSiGHT Database this is the first report of this mutation.

The mutational profile and infiltration pattern of murine MLH1-/- tumors: concurrences, disparities and cell line establishment for functional analysis.

Science.gov (United States)

Maletzki, Claudia; Beyrich, Franziska; Hühns, Maja; Klar, Ernst; Linnebacher, Michael

2016-08-16

Mice lines homozygous negative for one of the four DNA mismatch repair (MMR) genes (MLH1, MSH2, PMS2, MSH6) were generated as models for MMR deficient (MMR-D) diseases. Clinically, hereditary forms of MMR-D include Lynch syndrome (characterized by a germline MMR gene defect) and constitutional MMR-D, the biallelic form. MMR-D knockout mice may be representative for both diseases. Here, we aimed at characterizing the MLH1-/- model focusing on tumor-immune microenvironment and identification of coding microsatellite mutations in lymphomas and gastrointestinal tumors (GIT).All tumors showed microsatellite instability (MSI) in non-coding mononucleotide markers. Mutational profiling of 26 coding loci in MSI+ GIT and lymphomas revealed instability in half of the microsatellites, two of them (Rfc3 and Rasal2) shared between both entities. MLH1-/- tumors of both entities displayed a similar phenotype (high CD71, FasL, PD-L1 and CTLA-4 expression). Additional immunofluorescence verified the tumors' natural immunosuppressive character (marked CD11b/CD200R infiltration). Vice versa, CD3+ T cells as well as immune checkpoints molecules were detectable, indicative for an active immune microenvironment. For functional analysis, a permanent cell line from an MLH1-/- GIT was established. The newly developed MLH1-/- A7450 cells exhibit stable in vitro growth, strong invasive potential and heterogeneous drug response. Moreover, four additional MSI target genes (Nktr1, C8a, Taf1b, and Lig4) not recognized in the primary were identified in this cell line.Summing up, molecular and immunological mechanisms of MLH1-/- driven carcinogenesis correlate well with clinical features of MMR-D. MLH1-/- knockout mice combine characteristics of Lynch syndrome and constitutional MMR-D, making them suitable models for preclinical research aiming at MMR-D related diseases.

Mutation analysis of the CHK2 gene in breast carcinoma and other cancers

International Nuclear Information System (INIS)

Ingvarsson, Sigurdur; Sigbjornsdottir, Bjarnveig I; Huiping, Chen; Hafsteinsdottir, Sigridur H; Ragnarsson, Gisli; Barkardottir, Rosa B; Arason, Adalgeir; Egilsson, Valgardur; Bergthorsson, Jon TH

2002-01-01

Mutations in the CHK2 gene at chromosome 22q12.1 have been reported in families with Li-Fraumeni syndrome. Chk2 is an effector kinase that is activated in response to DNA damage and is involved in cell-cycle pathways and p53 pathways. We screened 139 breast tumors for loss of heterozygosity at chromosome 22q, using seven microsatellite markers, and screened 119 breast tumors with single-strand conformation polymorphism and DNA sequencing for mutations in the CHK2 gene. Seventy-four of 139 sporadic breast tumors (53%) show loss of heterozygosity with at least one marker. These samples and 45 tumors from individuals carrying the BRCA2 999del5 mutation were screened for mutations in the CHK2 gene. In addition to putative polymorphic regions in short mononucleotide repeats in a non-coding exon and intron 2, a germ line variant (T59K) in the first coding exon was detected. On screening 1172 cancer patients for the T59K sequence variant, it was detected in a total of four breast-cancer patients, two colon-cancer patients, one stomach-cancer patient and one ovary-cancer patient, but not in 452 healthy individuals. A tumor-specific 5' splice site mutation at site +3 in intron 8 (TTgt [a → c]atg) was also detected. We conclude that somatic CHK2 mutations are rare in breast cancer, but our results suggest a tumor suppressor function for CHK2 in a small proportion of breast tumors. Furthermore, our results suggest that the T59K CHK2 sequence variant is a low-penetrance allele with respect to tumor growth

Polymorphic human (CTAT)n microsatellite provides a conserved linkage marker for mouse mutants causing cleft palate, vestibular defects, obesity and ataxia

Energy Technology Data Exchange (ETDEWEB)

Griffith, A.J.; Burgess, D.L.; Kohrman, D. [Univ. of MIchigan, Ann Arbor, MI (United States)] [and others

1994-09-01

The Twirler mutation (Tw) causing cleft palate {plus_minus} cleft lip, vestibular defects and obesity is located within 0.5 cM of an ataxia locus (ax) on mouse chromosome 18. We identified a transgene-induced insertional mutation with vestibular and craniofacial defects that appears to be a new allele of Twirler. Mouse DNA flanking the transgene insertion site was isolated from a cosmid library. An evolutionarily conserved, zoo blot positive cosmid subclone was used to probe a human {lambda} genomic library. From the sequence of a highly homologous human {lambda} clone, we designed STS primers and screened a human P1 library. DNA from two positive P1 clones was hybridized with simple sequence probes, and a (CTAT){sub 12} repeat was detected. Analysis of 62 CEPH parents with primers flanking the repeat identified six alleles containing 9 to 14 copies of the repeat, at frequencies of 0.17, 0.17, 0.17, 0.27, 0.15 and 0.07, respectively. The observed heterozygosity was 49/62 with a calculated PIC value of 0.76. This polymorphic microsatellite marker, designated Umi3, was mapped to the predicted conserved human linkage group by analysis of somatic cell hybrid panels. The anticipated short distance between Umi3 and the disease genes will facilitate detection of linkage in small families. We would like to type appropriate human pedigrees with Umi3 in order to identify patients with inherited disorders homologous to the mouse mutations Twirler and ataxia.

Single-tube tetradecaplex panel of highly polymorphic microsatellite markers hemophilia A.

Science.gov (United States)

Zhao, M; Chen, M; Tan, A S C; Cheah, F S H; Mathew, J; Wong, P C; Chong, S S

2017-07-01

Essentials Preimplantation genetic diagnosis (PGD) of severe hemophilia A relies on linkage analysis. Simultaneous multi-marker screening can simplify selection of informative markers in a couple. We developed a single-tube tetradecaplex panel of polymorphic markers for hemophilia A PGD use. Informative markers can be used for linkage analysis alone or combined with mutation detection. Background It is currently not possible to perform single-cell preimplantation genetic diagnosis (PGD) to directly detect the common inversion mutations of the factor VIII (F8) gene responsible for severe hemophilia A (HEMA). As such, PGD for such inversion carriers relies on indirect analysis of linked polymorphic markers. Objectives To simplify linkage-based PGD of HEMA, we aimed to develop a panel of highly polymorphic microsatellite markers located near the F8 gene that could be simultaneously genotyped in a multiplex-PCR reaction. Methods We assessed the polymorphism of various microsatellite markers located ≤ 1 Mb from F8 in 177 female subjects. Highly polymorphic markers were selected for co-amplification with the AMELX/Y indel dimorphism in a single-tube reaction. Results Thirteen microsatellite markers located within 0.6 Mb of F8 were successfully co-amplified with AMELX/Y in a single-tube reaction. Observed heterozygosities of component markers ranged from 0.43 to 0.84, and ∼70-80% of individuals were heterozygous for ≥ 5 markers. The tetradecaplex panel successfully identified fully informative markers in a couple interested in PGD for HEMA because of an intragenic F8 point mutation, with haplotype phasing established through a carrier daughter. In-vitro fertilization (IVF)-PGD involved single-tube co-amplification of fully informative markers with AMELX/Y and the mutation-containing F8 amplicon, followed by microsatellite analysis and amplicon mutation-site minisequencing analysis. Conclusions The single-tube multiplex-PCR format of this highly polymorphic

POLE somatic mutations in advanced colorectal cancer.

Science.gov (United States)

Guerra, Joana; Pinto, Carla; Pinto, Diana; Pinheiro, Manuela; Silva, Romina; Peixoto, Ana; Rocha, Patrícia; Veiga, Isabel; Santos, Catarina; Santos, Rui; Cabreira, Verónica; Lopes, Paula; Henrique, Rui; Teixeira, Manuel R

2017-12-01

Despite all the knowledge already gathered, the picture of somatic genetic changes in colorectal tumorigenesis is far from complete. Recently, germline and somatic mutations in the exonuclease domain of polymerase epsilon, catalytic subunit (POLE) gene have been reported in a small subset of microsatellite-stable and hypermutated colorectal carcinomas (CRCs), affecting the proofreading activity of the enzyme and leading to misincorporation of bases during DNA replication. To evaluate the role of POLE mutations in colorectal carcinogenesis, namely in advanced CRC, we searched for somatic mutations by Sanger sequencing in tumor DNA samples from 307 cases. Microsatellite instability and mutation analyses of a panel of oncogenes were performed in the tumors harboring POLE mutations. Three heterozygous mutations were found in two tumors, the c.857C>G, p.Pro286Arg, the c.901G>A, p.Asp301Asn, and the c.1376C>T, p.Ser459Phe. Of the POLE-mutated CRCs, one tumor was microsatellite-stable and the other had low microsatellite instability, whereas KRAS and PIK3CA mutations were found in one tumor each. We conclude that POLE somatic mutations exist but are rare in advanced CRC, with further larger studies being necessary to evaluate its biological and clinical implications. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Microsatellite instability and B-type Raf proto-oncogene mutation in colorectal cancer: Clinicopathological characteristics and effects on survival

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Sebnem Batur

2016-11-01

Full Text Available Prognostic significance of microsatellite instability (MSI status and B-type Raf proto-oncogene (BRAF mutation in colorectal cancer is controversial. The aim of this study was to examine the clinical and pathological characteristics associated with microsatellite stability and the effect of MSI and BRAF mutation on the survival of patients with colorectal cancer. The study included 145 colorectal cancer cases. All the patients were examined for DNA mismatch repair (MMR proteins with an immunohistochemical method. Molecular assessment of MSI was available in a subset of 41 patients. In addition, BRAF mutation analysis was performed in 30 cases. Immunohistochemically, MMR deficiency was present in 28 (19.3% patients. Female gender (p = 0.001, lesion size ≥5 cm (p = 0.013, Crohn-like response (p = 0.035, and right-sided localization (p < 0.001 were significantly more frequent among MMR-deficient patients. The overall survival was 44.1 ± 5.1 months (95% confidence interval [CI], 33.7-54.4. Multivariate analyses identified only high tumor grade as an independent predictor of poor overall survival: odd ratio, 6.7 (95% CI 2.1-21.7, p = 0.002. In the subset of patients with available BRAF assessment (n = 30, a negative BRAF status was associated with better survival when compared to a positive BRAF status (36.7 ± 2.1 vs. 34.1 ± 7.2 months, p = 0.048. The sensitivity and specificity of the immunohistochemical method in predicting positive MSI status, with the molecular method as a reference, were 85.7% (95% CI: 56.2%-97.5% and 88.9% (95% CI: 69.7%-97.1%, respectively. BRAF appears to be a significant predictor of a worse outcome in patients with colorectal cancer. Further studies with a large spectrum of clinical and biological variables are warranted.

Ten polymorphic microsatellite markers characterized for ...

Indian Academy of Sciences (India)

MING MING BAO

development of new microsatellite primers is expensive and time-consuming, whereas ... constructing microsatellite-enriched libraries (Guo et al. 2013). Thirteen markers .... Due to the influence of human activities, stocks of this species have ...

Frequent non-reciprocal exchange in microsatellite-containing-DNA-regions of vertebrates

DEFF Research Database (Denmark)

Ziegler, J.O.; Wälther, M.; Linzer, T.R.

2009-01-01

Microsatellites are DNA-fragments containing short repetitive motifs with 2-10 bp. They are highly variable in most species and distributed throughout the whole genome. It is broadly accepted that their high degree of variability is closely associated with mispairing of DNA-strands during...... on stepwise mutation models should be interpreted with caution if no detailed information on the allelic variation of microsatellites is available....

HNPCC: Six new pathogenic mutations

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Epplen Joerg T

2004-06-01

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

Microsatellites for the gynogenetic Amazon molly, Poecilia formosa

Indian Academy of Sciences (India)

Home; Journals; Journal of Genetics; Volume 85; Issue 1. Microsatellites for the gynogenetic Amazon molly, Poecilia formosa: useful tools for detection of mutation rate, ploidy determination and overall genetic diversity. Kathrin P. Lampert Dunja K. Lamatsch Susanne Schories Armin Hopf Francisco J. García De León ...

CpG island methylator phenotype identifies high risk patients among microsatellite stable BRAF mutated colorectal cancers.

Science.gov (United States)

Vedeld, Hege Marie; Merok, Marianne; Jeanmougin, Marine; Danielsen, Stine A; Honne, Hilde; Presthus, Gro Kummeneje; Svindland, Aud; Sjo, Ole H; Hektoen, Merete; Eknaes, Mette; Nesbakken, Arild; Lothe, Ragnhild A; Lind, Guro E

2017-09-01

The prognostic value of CpG island methylator phenotype (CIMP) in colorectal cancer remains unsettled. We aimed to assess the prognostic value of this phenotype analyzing a total of 1126 tumor samples obtained from two Norwegian consecutive colorectal cancer series. CIMP status was determined by analyzing the 5-markers CAGNA1G, IGF2, NEUROG1, RUNX3 and SOCS1 by quantitative methylation specific PCR (qMSP). The effect of CIMP on time to recurrence (TTR) and overall survival (OS) were determined by uni- and multivariate analyses. Subgroup analyses were conducted according to MSI and BRAF mutation status, disease stage, and also age at time of diagnosis (CIMP positive tumors demonstrated significantly shorter TTR and worse OS compared to those with CIMP negative tumors (multivariate hazard ratio [95% CI] 1.86 [1.31-2.63] and 1.89 [1.34-2.65], respectively). In stratified analyses, CIMP tumors showed significantly worse outcome among patients with microsatellite stable (MSS, P CIMP is significantly associated with inferior outcome for colorectal cancer patients, and can stratify the poor prognostic patients with MSS BRAF mutated tumors. © 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.

Engineering Escherichia coli Nicotinic Acid Mononucleotide Adenylyltransferase for Fully Active Amidated NAD Biosynthesis.

Science.gov (United States)

Wang, Xueying; Zhou, Yongjin J; Wang, Lei; Liu, Wujun; Liu, Yuxue; Peng, Chang; Zhao, Zongbao K

2017-07-01

NAD and its reduced form NADH function as essential redox cofactors and have major roles in determining cellular metabolic features. NAD can be synthesized through the deamidated and amidated pathways, for which the key reaction involves adenylylation of nicotinic acid mononucleotide (NaMN) and nicotinamide mononucleotide (NMN), respectively. In Escherichia coli , NAD de novo biosynthesis depends on the protein NadD-catalyzed adenylylation of NaMN to nicotinic acid adenine dinucleotide (NaAD), followed by NAD synthase-catalyzed amidation. In this study, we engineered NadD to favor NMN for improved amidated pathway activity. We designed NadD mutant libraries, screened by a malic enzyme-coupled colorimetric assay, and identified two variants, 11B4 (Y84V/Y118D) and 16D8 (A86W/Y118N), with a high preference for NMN. Whereas in the presence of NMN both variants were capable of enabling the viability of cells of E. coli BW25113-derived NAD-auxotrophic strain YJE003, for which the last step of the deamidated pathway is blocked, the 16D8 expression strain could grow without exogenous NMN and accumulated a higher cellular NAD(H) level than BW25113 in the stationary phase. These mutants established fully active amidated NAD biosynthesis and offered a new opportunity to manipulate NAD metabolism for biocatalysis and metabolic engineering. IMPORTANCE Adenylylation of nicotinic acid mononucleotide (NaMN) and adenylylation of nicotinamide mononucleotide (NMN), respectively, are the key steps in the deamidated and amidated pathways for NAD biosynthesis. In most organisms, canonical NAD biosynthesis follows the deamidated pathway. Here we engineered Escherichia coli NaMN adenylyltransferase to favor NMN and expressed the mutant enzyme in an NAD-auxotrophic E. coli strain that has the last step of the deamidated pathway blocked. The engineered strain survived in M9 medium, which indicated the implementation of a functional amidated pathway for NAD biosynthesis. These results enrich

Genome-wide distribution and organization of microsatellites in plants: an insight into marker development in Brachypodium.

Directory of Open Access Journals (Sweden)

Humira Sonah

Full Text Available Plant genomes are complex and contain large amounts of repetitive DNA including microsatellites that are distributed across entire genomes. Whole genome sequences of several monocot and dicot plants that are available in the public domain provide an opportunity to study the origin, distribution and evolution of microsatellites, and also facilitate the development of new molecular markers. In the present investigation, a genome-wide analysis of microsatellite distribution in monocots (Brachypodium, sorghum and rice and dicots (Arabidopsis, Medicago and Populus was performed. A total of 797,863 simple sequence repeats (SSRs were identified in the whole genome sequences of six plant species. Characterization of these SSRs revealed that mono-nucleotide repeats were the most abundant repeats, and that the frequency of repeats decreased with increase in motif length both in monocots and dicots. However, the frequency of SSRs was higher in dicots than in monocots both for nuclear and chloroplast genomes. Interestingly, GC-rich repeats were the dominant repeats only in monocots, with the majority of them being present in the coding region. These coding GC-rich repeats were found to be involved in different biological processes, predominantly binding activities. In addition, a set of 22,879 SSR markers that were validated by e-PCR were developed and mapped on different chromosomes in Brachypodium for the first time, with a frequency of 101 SSR markers per Mb. Experimental validation of 55 markers showed successful amplification of 80% SSR markers in 16 Brachypodium accessions. An online database 'BraMi' (Brachypodium microsatellite markers of these genome-wide SSR markers was developed and made available in the public domain. The observed differential patterns of SSR marker distribution would be useful for studying microsatellite evolution in a monocot-dicot system. SSR markers developed in this study would be helpful for genomic studies in Brachypodium

Gene expression signatures for colorectal cancer microsatellite status and HNPCC

DEFF Research Database (Denmark)

Kruhøffer, M; Jensen, J L; Laiho, P

2005-01-01

The majority of microsatellite instable (MSI) colorectal cancers are sporadic, but a subset belongs to the syndrome hereditary non-polyposis colorectal cancer (HNPCC). Microsatellite instability is caused by dysfunction of the mismatch repair (MMR) system that leads to a mutator phenotype, and MSI...... of 101 stage II and III colorectal cancers (34 MSI, 67 microsatellite stable (MSS)) using high-density oligonucleotide microarrays. From these data, we constructed a nine-gene signature capable of separating the mismatch repair proficient and deficient tumours. Subsequently, we demonstrated...... is correlated to prognosis and response to chemotherapy. Gene expression signatures as predictive markers are being developed for many cancers, and the identification of a signature for MMR deficiency would be of interest both clinically and biologically. To address this issue, we profiled the gene expression...

Coenzyme Recognition and Gene Regulation by a Flavin Mononucleotide Riboswitch

Energy Technology Data Exchange (ETDEWEB)

Serganov, A.; Huang, L; Patel, D

2009-01-01

The biosynthesis of several protein cofactors is subject to feedback regulation by riboswitches. Flavin mononucleotide (FMN)-specific riboswitches also known as RFN elements, direct expression of bacterial genes involved in the biosynthesis and transport of riboflavin (vitamin B2) and related compounds. Here we present the crystal structures of the Fusobacterium nucleatum riboswitch bound to FMN, riboflavin and antibiotic roseoflavin. The FMN riboswitch structure, centred on an FMN-bound six-stem junction, does not fold by collinear stacking of adjacent helices, typical for folding of large RNAs. Rather, it adopts a butterfly-like scaffold, stapled together by opposingly directed but nearly identically folded peripheral domains. FMN is positioned asymmetrically within the junctional site and is specifically bound to RNA through interactions with the isoalloxazine ring chromophore and direct and Mg{sup 2+}-mediated contacts with the phosphate moiety. Our structural data, complemented by binding and footprinting experiments, imply a largely pre-folded tertiary RNA architecture and FMN recognition mediated by conformational transitions within the junctional binding pocket. The inherent plasticity of the FMN-binding pocket and the availability of large openings make the riboswitch an attractive target for structure-based design of FMN-like antimicrobial compounds. Our studies also explain the effects of spontaneous and antibiotic-induced deregulatory mutations and provided molecular insights into FMN-based control of gene expression in normal and riboflavin-overproducing bacterial strains.

A biomimetic redox flow battery based on flavin mononucleotide

OpenAIRE

Orita, A; Verde, MG; Sakai, M; Meng, YS

2016-01-01

The versatility in design of redox flow batteries makes them apt to efficiently store energy in large-scale applications at low cost. The discovery of inexpensive organic electroactive materials for use in aqueous flow battery electrolytes is highly attractive, but is thus far limited. Here we report on a flow battery using an aqueous electrolyte based on the sodium salt of flavin mononucleotide. Flavins are highly versatile electroactive molecules, which catalyse a multitude of redox reactio...

Prospects for cellular mutational assays in human populations

International Nuclear Information System (INIS)

Mendelsohn, M.L.

1984-01-01

Practical, sensitive, and effective human cellular assays for detecting somatic and germinal mutations would have great value in environmental mutagenesis and carcinogenesis studies. Such assays would fill the void between human mutagenicity and the data that exist from short-term tests and from mutagenicity in other species. This paper discusses the following possible human cellular assays: (1) HPRT (hypoxanthine phosphoribosyltransferase) somatic cell mutation based on 6-thioguanine resistance; (2) hemoglobin somatic cell mutation assay; (3) glycophorin somatic cell mutation assay; and (4) LDH-X sperm cell mutation assay. 18 references

Prospects for cellular mutational assays in human populations

Energy Technology Data Exchange (ETDEWEB)

Mendelsohn, M.L.

1984-06-29

Practical, sensitive, and effective human cellular assays for detecting somatic and germinal mutations would have great value in environmental mutagenesis and carcinogenesis studies. Such assays would fill the void between human mutagenicity and the data that exist from short-term tests and from mutagenicity in other species. This paper discusses the following possible human cellular assays: (1) HPRT (hypoxanthine phosphoribosyltransferase) somatic cell mutation based on 6-thioguanine resistance; (2) hemoglobin somatic cell mutation assay; (3) glycophorin somatic cell mutation assay; and (4) LDH-X sperm cell mutation assay. 18 references.

Experimental estimation of mutation rates in a wheat population with a gene genealogy approach.

Science.gov (United States)

Raquin, Anne-Laure; Depaulis, Frantz; Lambert, Amaury; Galic, Nathalie; Brabant, Philippe; Goldringer, Isabelle

2008-08-01

Microsatellite markers are extensively used to evaluate genetic diversity in natural or experimental evolving populations. Their high degree of polymorphism reflects their high mutation rates. Estimates of the mutation rates are therefore necessary when characterizing diversity in populations. As a complement to the classical experimental designs, we propose to use experimental populations, where the initial state is entirely known and some intermediate states have been thoroughly surveyed, thus providing a short timescale estimation together with a large number of cumulated meioses. In this article, we derived four original gene genealogy-based methods to assess mutation rates with limited bias due to relevant model assumptions incorporating the initial state, the number of new alleles, and the genetic effective population size. We studied the evolution of genetic diversity at 21 microsatellite markers, after 15 generations in an experimental wheat population. Compared to the parents, 23 new alleles were found in generation 15 at 9 of the 21 loci studied. We provide evidence that they arose by mutation. Corresponding estimates of the mutation rates ranged from 0 to 4.97 x 10(-3) per generation (i.e., year). Sequences of several alleles revealed that length polymorphism was only due to variation in the core of the microsatellite. Among different microsatellite characteristics, both the motif repeat number and an independent estimation of the Nei diversity were correlated with the novel diversity. Despite a reduced genetic effective size, global diversity at microsatellite markers increased in this population, suggesting that microsatellite diversity should be used with caution as an indicator in biodiversity conservation issues.

Human TRMU encoding the mitochondrial 5-methylaminomethyl-2-thiouridylate-methyltransferase is a putative nuclear modifier gene for the phenotypic expression of the deafness-associated 12S rRNA mutations

International Nuclear Information System (INIS)

Yan Qingfeng; Bykhovskaya, Yelena; Li Ronghua; Mengesha, Emebet; Shohat, Mordechai; Estivill, Xavier; Fischel-Ghodsian, Nathan; Guan Minxin

2006-01-01

Nuclear modifier genes have been proposed to modulate the phenotypic manifestation of human mitochondrial 12S rRNA A1491G mutation associated with deafness in many families world-wide. Here we identified and characterized the putative nuclear modifier gene TRMU encoding a highly conserved mitochondrial protein related to tRNA modification. A 1937 bp TRMU cDNA has been isolated and the genomic organization of TRMU has been elucidated. The human TRMU gene containing 11 exons encodes a 421 residue protein with a strong homology to the TRMU-like proteins of bacteria and other homologs. TRMU is ubiquitously expressed in various tissues, but abundantly in tissues with high metabolic rates including heart, liver, kidney, and brain. Immunofluorescence analysis of human 143B cells expressing TRMU-GFP fusion protein demonstrated that the human Trmu localizes and functions in mitochondrion. Furthermore, we show that in families with the deafness-associated 12S rRNA A1491G mutation there is highly suggestive linkage and linkage disequilibrium between microsatellite markers adjacent to TRMU and the presence of deafness. These observations suggest that human TRMU may modulate the phenotypic manifestation of the deafness-associated mitochondrial 12S rRNA mutations

Fitness loss and germline mutations in barn swallows breeding in Chernobyl

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Ellegren, Hans; Lindgren, Gabriella; Primmer, C.R. [Swedish Univ. of Agricultural Sciences, Animal Breeding and Genetics Dept., Uppsala (Sweden); Moeller, A.P. [Universite Pierre et Marie Curie. Lab. d`Ecologie, Paris, 75 (France)

1997-10-09

The severe nuclear accident at Chernobyl in 1986 resulted in the worst reported accidental exposure of radioactive material to free-living organisms. Short-term effects on human populations inhabiting polluted areas include increased incidence of thyroid cancer, infant leukaemia, and congenital malformations in newborns. Two recent studies have reported, although with some controversy, that germline mutation rates were increased in humans and voles living close to Chernobyl, but little is known about the viability of the organisms affected. Here we report an increased frequency of partial albinism, a morphological aberration associated with a loss of fitness, among barn swallows, Hirundo rustica, breeding close to Chernobyl. Heretability estimates indicate that mutations causing albinism were at least partly of germline origin. Furthermore, evidence for an increased germline mutation rate was obtained from segregation analysis at two hypervariable microsatellite loci, indicating that mutation events in barn swallows from Chernobyl were two- to tenfold higher than in birds from control areas in Ukraine and Italy. (author).

Fitness loss and germline mutations in barn swallows breeding in Chernobyl

International Nuclear Information System (INIS)

Ellegren, Hans; Lindgren, Gabriella; Primmer, C.R.; Moeller, A.P.

1997-01-01

The severe nuclear accident at Chernobyl in 1986 resulted in the worst reported accidental exposure of radioactive material to free-living organisms. Short-term effects on human populations inhabiting polluted areas include increased incidence of thyroid cancer, infant leukaemia, and congenital malformations in newborns. Two recent studies have reported, although with some controversy, that germline mutation rates were increased in humans and voles living close to Chernobyl, but little is known about the viability of the organisms affected. Here we report an increased frequency of partial albinism, a morphological aberration associated with a loss of fitness, among barn swallows, Hirundo rustica, breeding close to Chernobyl. Heretability estimates indicate that mutations causing albinism were at least partly of germline origin. Furthermore, evidence for an increased germline mutation rate was obtained from segregation analysis at two hypervariable microsatellite loci, indicating that mutation events in barn swallows from Chernobyl were two- to tenfold higher than in birds from control areas in Ukraine and Italy. (author)

Detecting microsatellites within genomes: significant variation among algorithms

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Rivals Eric

2007-04-01

Full Text Available Abstract Background Microsatellites are short, tandemly-repeated DNA sequences which are widely distributed among genomes. Their structure, role and evolution can be analyzed based on exhaustive extraction from sequenced genomes. Several dedicated algorithms have been developed for this purpose. Here, we compared the detection efficiency of five of them (TRF, Mreps, Sputnik, STAR, and RepeatMasker. Results Our analysis was first conducted on the human X chromosome, and microsatellite distributions were characterized by microsatellite number, length, and divergence from a pure motif. The algorithms work with user-defined parameters, and we demonstrate that the parameter values chosen can strongly influence microsatellite distributions. The five algorithms were then compared by fixing parameters settings, and the analysis was extended to three other genomes (Saccharomyces cerevisiae, Neurospora crassa and Drosophila melanogaster spanning a wide range of size and structure. Significant differences for all characteristics of microsatellites were observed among algorithms, but not among genomes, for both perfect and imperfect microsatellites. Striking differences were detected for short microsatellites (below 20 bp, regardless of motif. Conclusion Since the algorithm used strongly influences empirical distributions, studies analyzing microsatellite evolution based on a comparison between empirical and theoretical size distributions should therefore be considered with caution. We also discuss why a typological definition of microsatellites limits our capacity to capture their genomic distributions.

Microsatellite instability typing in serum and tissue of patients with colorectal cancer: comparing real time PCR with hybridization probe and high-performance liquid chromatography.

Science.gov (United States)

Mokarram, P; Rismanchi, M; Alizadeh Naeeni, M; Mirab Samiee, S; Paryan, M; Alipour, A; Honardar, Z; Kavousipour, S; Naghibalhossaini, F; Mostafavi-Pour, Z; Monabati, A; Hosseni, S V; Shamsdin, S A

2014-05-01

Allelic variation of BAT-25 (a 25-repeat quasimonomorphic poly T) and BAT-26 (a 26-repeat quasimonomorphic polyA) loci as two mononucleotide microsatellite markers, were analyzed with high-performance liquid chromatography (HPLC) compared with Real-Time PCR using hybridization probes. BAT-26 and BAT-25 markers were used to determine an appropriate screening technique with high sensitivity and specificity to diagnose microsatellite instability (MSI) status in patients with colorectal cancer (CRC). One of the pathways in colorectal tumor genesis is microsatellite instability (MSI+). MSI is detected in about 15% of all CRCs; 3% are of these are associated with Lynch syndrome and the other 12% are caused by sporadic. Colorectal tumors with MSI have distinctive features compared with microsatellite stable tumors. Due to the high percentage of MSI+ CRC in Iran, screening of this type of CRC is imperative. Two markers were analyzed in tissues and sera of 44 normal volunteers and tumor and matched normal mucosal tissues as well as sera of 44 patients with sporadic CRC. The sensitivity and specificity of BAT-26 with real time PCR method (Hybridization probe) were 100% in comparison with sequencing method as the gold standard, while HPLC had a lower sensitivity and specificity. According to HPLC data, BAT-26 was more sensitive than BAT-25 in identifying MSI tumors. Therefore, MSI typing using the BAT-26 hybridization probe method compared to HPLC could be considered as an accurate method for diagnosing MSI in CRC tumors but not in serum circulating DNAs.

In silico characterization of microsatellites in Eucalyptus spp.: abundance, length variation and transposon associations

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Edenilson Rabello

2005-01-01

Full Text Available This study assessed the abundance of microsatellites, or simple sequence repeats (SSR, in 19 Eucalyptus EST libraries from FORESTs, containing cDNA sequences from five species: E. grandis, E. globulus, E. saligna, E. urophylla and E. camaldulensis. Overall, a total of 11,534 SSRs and 8,447 SSR-containing sequences (25.5% of total ESTs were identified, with an average of 1 SSR/2.5 kb when considering all motifs and 1 SSR/3.1 kb when mononucleotides were not included. Dimeric repeats were the most abundant (41.03%, followed by trimerics (36.11% and monomerics (19.59%. The most frequent motifs were A/T (87.24% for monomerics, AG/CT (94.44% for dimerics, CCG/CGG (37.87% for trimerics, AAGG/CCTT (18.75% for tetramerics, AGAGG/CCTCT (14.04% for pentamerics and ACGGCG/CGCCGT (6.30% for hexamerics. According to sequence length, Class II or potentially variable markers were the most commonly found, followed by Class III. Two sequences presented high similarity to previously published Eucalyptus sequences from the NCBI database, EMBRA_72 and EMBRA_122. Local blastn search for transposons did not reveal the presence of any transposable elements with a cut-off value of 10-50. The large number of microsatellites identified will contribute to the refinement of marker-assisted mapping and to the discovery of novel markers for virtually all genes of economic interest.

Replicative DNA polymerase mutations in cancer☆

Science.gov (United States)

Heitzer, Ellen; Tomlinson, Ian

2014-01-01

Three DNA polymerases — Pol α, Pol δ and Pol ɛ — are essential for DNA replication. After initiation of DNA synthesis by Pol α, Pol δ or Pol ɛ take over on the lagging and leading strand respectively. Pol δ and Pol ɛ perform the bulk of replication with very high fidelity, which is ensured by Watson–Crick base pairing and 3′exonuclease (proofreading) activity. Yeast models have shown that mutations in the exonuclease domain of Pol δ and Pol ɛ homologues can cause a mutator phenotype. Recently, we identified germline exonuclease domain mutations (EDMs) in human POLD1 and POLE that predispose to ‘polymerase proofreading associated polyposis’ (PPAP), a disease characterised by multiple colorectal adenomas and carcinoma, with high penetrance and dominant inheritance. Moreover, somatic EDMs in POLE have also been found in sporadic colorectal and endometrial cancers. Tumors with EDMs are microsatellite stable and show an ‘ultramutator’ phenotype, with a dramatic increase in base substitutions. PMID:24583393

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

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

2013-06-01

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

Rediscovering medicinal plants' potential with OMICS: microsatellite survey in expressed sequence tags of eleven traditional plants with potent antidiabetic properties.

Science.gov (United States)

Sahu, Jagajjit; Sen, Priyabrata; Choudhury, Manabendra Dutta; Dehury, Budheswar; Barooah, Madhumita; Modi, Mahendra Kumar; Talukdar, Anupam Das

2014-05-01

Herbal medicines and traditionally used medicinal plants present an untapped potential for novel molecular target discovery using systems science and OMICS biotechnology driven strategies. Since up to 40% of the world's poor people have no access to government health services, traditional and folk medicines are often the only therapeutics available to them. In this vein, North East (NE) India is recognized for its rich bioresources. As part of the Indo-Burma hotspot, it is regarded as an epicenter of biodiversity for several plants having myriad traditional uses, including medicinal use. However, the improvement of these valuable bioresources through molecular breeding strategies, for example, using genic microsatellites or Simple Sequence Repeats (SSRs) or Expressed Sequence Tags (ESTs)-derived SSRs has not been fully utilized in large scale to date. In this study, we identified a total of 47,700 microsatellites from 109,609 ESTs of 11 medicinal plants (pineapple, papaya, noyontara, bitter orange, bermuda brass, ratalu, barbados nut, mango, mulberry, lotus, and guduchi) having proven antidiabetic properties. A total of 58,159 primer pairs were designed for the non-redundant 8060 SSR-positive ESTs and putative functions were assigned to 4483 unique contigs. Among the identified microsatellites, excluding mononucleotide repeats, di-/trinucleotides are predominant, among which repeat motifs of AG/CT and AAG/CTT were most abundant. Similarity search of SSR containing ESTs and antidiabetic gene sequences revealed 11 microsatellites linked to antidiabetic genes in five plants. GO term enrichment analysis revealed a total of 80 enriched GO terms widely distributed in 53 biological processes, 17 molecular functions, and 10 cellular components associated with the 11 markers. The present study therefore provides concrete insights into the frequency and distribution of SSRs in important medicinal resources. The microsatellite markers reported here markedly add to the genetic

Microsatellite marker isolation and development for the giant Pacific Octopus (Enteroctopus dofleini)

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Toussaint, Rebecca K.; Sage, G. Kevin; Talbot, Sandra L.; Scheel, David

2012-01-01

We isolated and developed 18 novel microsatellite markers for the giant Pacific octopus (Enteroctopus dofleini) and examined them for 31 individuals from Prince William Sound (PWS), Alaska. These loci displayed moderate levels of allelic diversity (averaging 11 alleles per locus) and heterozygosity (averaging 65%). Seven loci deviated from Hardy–Weinberg Equilibrium (HWE) due to heterozygote deficiency for the PWS population, although deviations were not observed for all these loci in other populations, suggesting the PWS population is not in mutation-drift equilibrium. These novel microsatellite loci yielded sufficient genetic diversity for potential use in population genetics, individual identification, and parentage studies.

The Norwegian PMS2 founder mutation c.989-1G > T shows high penetrance of microsatellite instable cancers with normal immunohistochemistry.

Science.gov (United States)

Grindedal, Eli Marie; Aarset, Harald; Bjørnevoll, Inga; Røyset, Elin; Mæhle, Lovise; Stormorken, Astrid; Heramb, Cecilie; Medvik, Heidi; Møller, Pål; Sjursen, Wenche

2014-01-01

Using immunohistochemistry (IHC) to select cases for mismatch repair (MMR) genetic testing, we failed to identify a large kindred with the deleterious PMS2 mutation c.989-1G > T. The purpose of the study was to examine the sensitivity of IHC and microsatellite instability-analysis (MSI) to identify carriers of the mutation, and to estimate its penetrance and expressions. All carriers and obligate carriers of the mutation were identified. All cancer diagnoses were confirmed. IHC and MSI-analysis were performed on available tumours. Penetrances of cancers included in the Amsterdam and the Bethesda Criteria, for MSI-high tumours and MSI-high and low tumours were calculated by the Kaplan-Meier algorithm. Probability for co-segregation of the mutation and cancers by chance was 0.000004. Fifty-six carriers or obligate carriers were identified. There was normal staining for PMS2 in 15/18 (83.3%) of tumours included in the AMS1/AMS2/Bethesda criteria. MSI-analysis showed that 15/21 (71.4%) of tumours were MSI-high and 4/21 (19.0%) were MSI-low. Penetrance at 70 years was 30.6% for AMS1 cancers (colorectal cancers), 42.8% for AMS2 cancers, 47.2% for Bethesda cancers, 55.6% for MSI-high and MSI-low cancers and 52.2% for MSI-high cancers. The mutation met class 5 criteria for pathogenicity. IHC was insensitive in detecting tumours caused by the mutation. Penetrance of cancers that displayed MSI was 56% at 70 years. Besides colorectal cancers, the most frequent expressions were carcinoma of the endometrium and breast in females and stomach and prostate in males.

The mutator pathway is a feature of immunodeficiency-related lymphomas

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Duval, Alex; Raphael, Martine; Brennetot, Caroline; Poirel, Helene; Buhard, Olivier; Aubry, Alban; Martin, Antoine; Krimi, Amor; Leblond, Veronique; Gabarre, Jean; Davi, Frederic; Charlotte, Frederic; Berger, Francoise; Gaidano, Gianluca; Capello, Daniela; Canioni, Danielle; Bordessoule, Dominique; Feuillard, Jean; Gaulard, Philippe; Delfau, Marie Helene; Ferlicot, Sophie; Eclache, Virginie; Prevot, Sophie; Guettier, Catherine; Lefevre, Pascale Cornillet; Adotti, Francoise; Hamelin, Richard

2004-01-01

The mutator phenotype caused by defects in the mismatch repair system is observed in a subset of solid neoplasms characterized by widespread microsatellite instability-high (MSI-H). It is known to be very rare in non-Hodgkin lymphomas (NHL), whereas mutator NHL is the most frequent tumor subtype in mismatch repair-deficient mice. By screening a series of 603 human NHL with specific markers of the mutator phenotype, we found here 12 MSI-H cases (12/603, 2%). Of interest, we demonstrated that this phenotype was specifically associated with immunodeficiency-related lymphomas (ID-RL), because it was observed in both posttransplant lymphoproliferative disorders (9/111, 8.1%) and HIV infection-related lymphomas (3/128, 2.3%) but not in a large series of NHL arising in the general population (0/364) (P < 0.0001). The MSI pathway is known to lead to the production of hundreds of abnormal protein neoantigens that are generated in MSI-H neoplasms by frameshift mutations of a number of genes containing coding microsatellite sequences. As expected, MSI-H ID-RL were found to harbor such genetic alterations in 12 target genes with a putative role in lymphomagenesis. The observation that the MSI-H phenotype was restricted to HIV infection-related lymphomas and posttransplant lymphoproliferative disorders suggests the existence of the highly immunogenic mutator pathway as a novel oncogenic process in lymphomagenesis whose role is favored when host immunosurveillance is reduced. Because MSI-H-positive cases were found to be either Epstein-Barr virus-positive or -negative, the mutator pathway should act synergistically or not with this other oncogenic factor, playing an important role in ID-RL. PMID:15047891

Usefulness of microsatellite typing in population genetic studies of Trypanosoma cruzi

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Macedo Andrea M

2001-01-01

Full Text Available Through microsatellite analysis of 53 monoclonal populations of Trypanosoma cruzi, we found a remarkable degree of genetic polymorphism with no single multilocus genotype being observed more than once. The microsatellite profile proved to be stable during 70 generations of the CL Brener clone in culture. The microsatellite profiling presented also high diagnostic sensitivity since DNA amplifications could be achieved with less than 100 fg DNA, corresponding to half parasite total DNA content. Based on these technical attributes the microsatellite assay turns out to be an important tool for direct typing T. cruzi in biological samples. By using this approach we were able to type T. cruzi in feces of artificially infected bugs and in single cells sorted by FACS. The microsatellites have shown to be excellent markers for T. cruzi phylogenetic reconstruction. We used maximum parsimony based on the minimum number of mutational steps to build an unrooted Wagner network, which confirms previous conclusions based on the analysis of the D7 domain of the LSU rDNA gene that T. cruzi is composed by two major groups. We also obtained evidence that strains belonging to rRNA group 2 are subdivided into two genetically distant clusters, and that one of these clusters is more related to rRNA group 1/2. These results suggest different origins for these strains.

Prospects for cellular mutational assays in human populations

International Nuclear Information System (INIS)

Mendelsohn, M.L.

1985-01-01

Practical, sensitive, effective, human cellular assays for detecting somatic and germinal mutations would have great value in environmental mutagenesis and carcinogenesis. When available, such assays should allow us to fill the void between human mutagenicity and the data that exist from short-term tests and from mutagenicity in other species. We will be able to validate the role of somatic mutations in carcinogenesis, to identify environmental factors that affect human germ cells, to integrate the effects of complex mixtures and the environment in the human subject, and to identify people who are hypersusceptible to genetic injury. Human cellular mutational assays, particularly when combined with cytogenetic and heritable mutational tests, promise to play pivotal roles in estimating the risk from low-dose radiation and chemical exposures. These combined methods avoid extrapolations of dose and from species to species, and may be sensitive enough and credible enough to permit politically, socially and scientifically acceptable risk management. 16 references

A panel of microsatellites to individually identify leopards and its application to leopard monitoring in human dominated landscapes

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Selvaraj Velu

2009-12-01

Full Text Available Abstract Background Leopards are the most widely distributed of the large cats, ranging from Africa to the Russian Far East. Because of habitat fragmentation, high human population densities and the inherent adaptability of this species, they now occupy landscapes close to human settlements. As a result, they are the most common species involved in human wildlife conflict in India, necessitating their monitoring. However, their elusive nature makes such monitoring difficult. Recent advances in DNA methods along with non-invasive sampling techniques can be used to monitor populations and individuals across large landscapes including human dominated ones. In this paper, we describe a DNA-based method for leopard individual identification where we used fecal DNA samples to obtain genetic material. Further, we apply our methods to non-invasive samples collected in a human-dominated landscape to estimate the minimum number of leopards in this human-leopard conflict area in Western India. Results In this study, 25 of the 29 tested cross-specific microsatellite markers showed positive amplification in 37 wild-caught leopards. These loci revealed varied levels of polymorphism (four-12 alleles and heterozygosity (0.05-0.79. Combining data on amplification success (including non-invasive samples and locus specific polymorphisms, we showed that eight loci provide a sibling probability of identity of 0.0005, suggesting that this panel can be used to discriminate individuals in the wild. When this microsatellite panel was applied to fecal samples collected from a human-dominated landscape, we identified 7 individuals, with a sibling probability of identity of 0.001. Amplification success of field collected scats was up to 72%, and genotype error ranged from 0-7.4%. Conclusion Our results demonstrated that the selected panel of eight microsatellite loci can conclusively identify leopards from various kinds of biological samples. Our methods can be used to

Evolution of Microsatellite Loci of Tropical and Temperate Anguilla Eels

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Mei-Chen Tseng

2012-04-01

Full Text Available Anguilla eels are divided into temperate and tropical eels, based on their major distributions. The present study collected two temperate eels, Anguilla japonica and Anguilla anguilla, and two tropical eels, Anguilla marmorata and Anguilla bicolor pacifica, to examine two questions: do temperate and tropical Anguilla eels have different genetic polymorphic patterns?; and do temperate Anguilla japonica and Anguilla anguilla have a closer relationship to each other than to tropical eels? In total, 274 sequences were cloned and sequenced from six conserved microsatellite loci to examine polymorphic patterns of these four catadromous eels. Different mutational events, including substitutions, and repeat-unit deletions and insertions, appeared in major regions, while different point mutations were observed in flanking regions. The results implied that parallel patterns of microsatellite sequences occurred within both tropical and temperate freshwater eels. Consensus flanking sequences of six homologous loci from each of the four species were constructed. Genetic distances ranged from 0.044 (Anguilla bicolor pacifica vs. Anguilla marmorata to 0.061 (Anguilla marmorata vs. Anguilla anguilla. The tree topology suggests the hypothesis of Anguilla japonica and Anguilla anguilla being a sister group must be rejected.

Studies of human mutation rates: Progress report

International Nuclear Information System (INIS)

Neel, J.V.

1988-01-01

Progress was recorded between January 1 and July 1, 1987 on a project entitled ''Studies of Human Mutation Rates''. Studies underway include methodology for studying mutation at the DNA level, algorithms for automated analyses of two-dimensional polyacrylamide DNA gels, theoretical and applied population genetics, and studies of mutation frequency in A-bomb survivors

[Microsatellite instability and human papilloma virus genotypes in preneoplastic and neoplastic uterine cervix lesions].

Science.gov (United States)

Roa S, Juan Carlos; Martínez S, Ricardo; Montenegro, Sonia; Roa E, Iván; Capurro V, Italo; Ibacache S, Gilda; Melo A, Angélica

2007-01-01

The association between some specific human papilloma virus (HPV) types and cervix cancer is well known. However, the genetic conditions that favor the development of cervical cancer are less well known. To determine the presence of satellite instability (MSI) in preneoplastic and neoplastic lesions of the cervix and correlate these findings with HPV genotypes. Biopsy samples of cervical lesions were studied. Sixteen had low grade lesions, 22 had high grade lesions and 28 had an epidermoid cancer. Viral types were identified with polymerase chain reaction, dot-blot hybridization and restriction fragment length polymorphism. MSI was determined using a panel of eight highly informative microsatellites. Microsatellite instability in at least one locus was observed in 91, 56 and 69% of low grade lesions, high grade lesions and epidermoid carcinomas, respectively. MSI-High grade, MSI-Low grade instability and microsatellite stability were observed in 5, 60 and 46% of samples, respectively. Two of three samples with high grade instability had HPV 52 genotype. Other viral subtypes had frequencies that ranged from 78% to 100%, with the exception of HPV16 that was present in only 53% of samples with low grade instability. Two thirds of biopsy samples from cervical lesions had MSI, mechanism that can be involved in the first stages of cervical carcinogenesis. The low frequency of high grade instability, its association with HPV52 and the low frequency of HPV16 in samples with low grade instability, suggest different coadjutant mechanisms in cervical carcinogenesis.

Microsatellites with Macro-Influence in Ewing Sarcoma

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Stephen L. Lessnick

2012-07-01

Full Text Available Numerous molecular abnormalities contribute to the genetic derangements involved in tumorigenesis. Chromosomal translocations are a frequent source of these derangements, producing unique fusion proteins with novel oncogenic properties. EWS/ETS fusions in Ewing sarcoma are a prime example of this, resulting in potent chimeric oncoproteins with novel biological properties and a unique transcriptional signature essential for oncogenesis. Recent evidence demonstrates that EWS/FLI, the most common EWS/ETS fusion in Ewing sarcoma, upregulates gene expression using a GGAA microsatellite response element dispersed throughout the human genome. These GGAA microsatellites function as enhancer elements, are sites of epigenetic regulation and are necessary for EWS/FLI DNA binding and upregulation of principal oncogenic targets. An increasing number of GGAA motifs appear to substantially enhance EWS/FLI-mediated gene expression, which has compelling biological implications as these GGAA microsatellites are highly polymorphic within and between ethnically distinct populations. Historically regarded as junk DNA, this emerging evidence clearly demonstrates that microsatellite DNA plays an instrumental role in EWS/FLI-mediated transcriptional regulation and oncogenesis in Ewing sarcoma. This unprecedented role of GGAA microsatellite DNA in Ewing sarcoma provides a unique opportunity to expand our mechanistic understanding of how EWS/ETS fusions influence cancer susceptibility, prognosis and transcriptional regulation.

Replicative DNA polymerase mutations in cancer.

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Heitzer, Ellen; Tomlinson, Ian

2014-02-01

Three DNA polymerases - Pol α, Pol δ and Pol ɛ - are essential for DNA replication. After initiation of DNA synthesis by Pol α, Pol δ or Pol ɛ take over on the lagging and leading strand respectively. Pol δ and Pol ɛ perform the bulk of replication with very high fidelity, which is ensured by Watson-Crick base pairing and 3'exonuclease (proofreading) activity. Yeast models have shown that mutations in the exonuclease domain of Pol δ and Pol ɛ homologues can cause a mutator phenotype. Recently, we identified germline exonuclease domain mutations (EDMs) in human POLD1 and POLE that predispose to 'polymerase proofreading associated polyposis' (PPAP), a disease characterised by multiple colorectal adenomas and carcinoma, with high penetrance and dominant inheritance. Moreover, somatic EDMs in POLE have also been found in sporadic colorectal and endometrial cancers. Tumors with EDMs are microsatellite stable and show an 'ultramutator' phenotype, with a dramatic increase in base substitutions. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Cyclic mononucleotides modulate potassium and calcium flux responses to H2O2 in Arabidopsis roots

KAUST Repository

Ordoñez, Natalia Maria

2014-02-13

Cyclic mononucleotides are messengers in plant stress responses. Here we show that hydrogen peroxide (H2O2) induces rapid net K+-efflux and Ca2+-influx in Arabidopsis roots. Pre-treatment with either 10 μM cAMP or cGMP for 1 or 24 h does significantly reduce net K+-leakage and Ca2+-influx, and in the case of the K+-fluxes, the cell permeant cyclic mononucleotides are more effective. We also examined the effect of 10 μM of the cell permeant 8-Br-cGMP on the Arabidopsis microsomal proteome and noted a specific increase in proteins with a role in stress responses and ion transport, suggesting that cGMP is sufficient to directly and/or indirectly induce complex adaptive changes to cellular stresses induced by H2O2. © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Eighteen polymorphic microsatellites for domestic pigeon Columba ...

Indian Academy of Sciences (India)

certain parasites which cause health problems in humans and domestic animals ... The genomic DNA was isolated using standard protocol as described by ..... panel of polymorphic microsatellite markers in Himalayan monal. Lophophorus ...

Crystal structure of Sus scrofa quinolinate phosphoribosyltransferase in complex with nicotinate mononucleotide.

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Hyung-Seop Youn

Full Text Available We have determined the crystal structure of porcine quinolinate phosphoribosyltransferase (QAPRTase in complex with nicotinate mononucleotide (NAMN, which is the first crystal structure of a mammalian QAPRTase with its reaction product. The structure was determined from protein obtained from the porcine kidney. Because the full protein sequence of porcine QAPRTase was not available in either protein or nucleotide databases, cDNA was synthesized using reverse transcriptase-polymerase chain reaction to determine the porcine QAPRTase amino acid sequence. The crystal structure revealed that porcine QAPRTases have a hexameric structure that is similar to other eukaryotic QAPRTases, such as the human and yeast enzymes. However, the interaction between NAMN and porcine QAPRTase was different from the interaction found in prokaryotic enzymes, such as those of Helicobacter pylori and Mycobacterium tuberculosis. The crystal structure of porcine QAPRTase in complex with NAMN provides a structural framework for understanding the unique properties of the mammalian QAPRTase active site and designing new antibiotics that are selective for the QAPRTases of pathogenic bacteria, such as H. pylori and M. tuberculosis.

A resolution of the mutation load paradox in humans.

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Lesecque, Yann; Keightley, Peter D; Eyre-Walker, Adam

2012-08-01

Current information on the rate of mutation and the fraction of sites in the genome that are subject to selection suggests that each human has received, on average, at least two new harmful mutations from its parents. These mutations were subsequently removed by natural selection through reduced survival or fertility. It has been argued that the mutation load, the proportional reduction in population mean fitness relative to the fitness of an idealized mutation-free individual, allows a theoretical prediction of the proportion of individuals in the population that fail to reproduce as a consequence of these harmful mutations. Application of this theory to humans implies that at least 88% of individuals should fail to reproduce and that each female would need to have more than 16 offspring to maintain population size. This prediction is clearly at odds with the low reproductive excess of human populations. Here, we derive expressions for the fraction of individuals that fail to reproduce as a consequence of recurrent deleterious mutation () for a model in which selection occurs via differences in relative fitness, such as would occur through competition between individuals. We show that is much smaller than the value predicted by comparing fitness to that of a mutation-free genotype. Under the relative fitness model, we show that depends jointly on U and the selective effects of new deleterious mutations and that a species could tolerate 10's or even 100's of new deleterious mutations per genome each generation.

Genetic progression in microsatellite instability high (MSI-H) colon cancers correlates with clinico-pathological parameters: A study of the TGRbetaRII, BAX, hMSH3, hMSH6, IGFIIR and BLM genes.

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Calin, G A; Gafà, R; Tibiletti, M G; Herlea, V; Becheanu, G; Cavazzini, L; Barbanti-Brodano, G; Nenci, I; Negrini, M; Lanza, G

2000-05-20

Colon carcinomas with microsatellite mutator phenotype exhibit specific genetic and clinico-pathological features. This report describes the analysis of 63 "microsatellite instability-high" (MSI-H) tumors for the presence of mutations in microsatellites located in the coding regions (CDRs) of 6 genes: TGFbetaRII, BAX, hMSH3, hMSH6, IGFIIR, and BLM. The following frequencies of mutations were detected: TGFbetaRII (70%), BAX (54%), hMSH3 (36.5%), IGFIIR (22%), hMSH6 (17.5%), and BLM (16%). The overall picture revealed combinations of mutations suggestive of a progressive order of accumulation, with mutations of TGFbetaRII and BAX first, followed by frameshifts in hMSH3, hMSH6, IGFIIR, and BLM. Correlations with 12 clinico-pathological parameters revealed that tumors with frameshifts in 1 or 2 CDRs were significantly better differentiated than tumors with frameshifts in more than 2 CDRs. We also found that mutations in the hMSH3 gene were significantly associated with decreased wall invasiveness and aneuploidy, and frameshifts in the BLM gene were significantly associated with the mucinous histotype. A trend toward an association between hMSH3 and IGFIIR with the medullary and conventional adenocarcinoma histotypes, respectively, was seen. Our results strengthen the concept that mutations in target genes have a role in the tumorigenic process of MSI-H tumors, and indicate that frameshifts in microsatellites located in CDRs occur in a limited number of combinations that could determine distinct clinico-pathological traits. Copyright 2000 Wiley-Liss, Inc.

Molecular Analysis: Microsatellite Instability and Loss of Heterozygosity of Tumor Suppressor Gene in Hereditary Non-Polyposis Colorectal Cancer (HNPCC

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Vesna Hadžiavdić

2009-02-01

Full Text Available HNPCC (Hereditary non-polyposis colorectal cancer development is caused by mutation of genes included in system of mismatch repair genes. The mutation exists at 60% of patients in hMSH2 gene, 30% in hMLH1 and 10% both in hPMS1and hPMS2 genes. RER+ exists in about 90% in hereditary non-polyposis colorectal cancer and about 15-28% in sporadic cancers.The purpose of the study was to determine highly sensitive microsatellite markers which can be fast and efficient way of microsatellite screening for detection of HNPCC patients. Moreover, we have analysed the loss of heterozygosity of tumour suppressor genes which could have the diagnostic value in detection of HPNCC patients.

Cyclic mononucleotides modulate potassium and calcium flux responses to H2O2 in Arabidopsis roots

KAUST Repository

Ordoñ ez, Natalia Maria; Marondedze, Claudius; Thomas, Ludivine; Pasqualini, Stefania; Shabala, Lana; Shabala, Sergey; Gehring, Christoph A

2014-01-01

Cyclic mononucleotides are messengers in plant stress responses. Here we show that hydrogen peroxide (H2O2) induces rapid net K+-efflux and Ca2+-influx in Arabidopsis roots. Pre-treatment with either 10 μM cAMP or cGMP for 1 or 24 h does

Serum antibodies against frameshift peptides in microsatellite unstable colorectal cancer patients with Lynch syndrome.

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Reuschenbach, Miriam; Kloor, Matthias; Morak, Monika; Wentzensen, Nicolas; Germann, Anja; Garbe, Yvette; Tariverdian, Mirjam; Findeisen, Peter; Neumaier, Michael; Holinski-Feder, Elke; von Knebel Doeberitz, Magnus

2010-06-01

High level microsatellite instability (MSI-H) occurs in about 15% of colorectal cancer (CRCs), either as sporadic cancers or in the context of hereditary non-polyposis cancer or Lynch syndrome. In MSI-H CRC, mismatch repair deficiency leads to insertion/deletion mutations at coding microsatellites and thus to the translation of frameshift peptides (FSPs). FSPs are potent inductors of T cell responses in vitro and in vivo. The present study aims at the identification of FSP-specific humoral immune responses in MSI-H CRC and Lynch syndrome. Sera from patients with history of MSI-H CRC (n = 69), healthy Lynch syndrome mutation carriers (n = 31) and healthy controls (n = 52) were analyzed for antibodies against FSPs using peptide ELISA. Reactivities were measured against FSPs derived from genes frequently mutated in MSI-H CRCs, AIM2, TGFBR2, CASP5, TAF1B, ZNF294, and MARCKS. Antibody reactivity against FSPs was significantly higher in MSI-H CRC patients than in healthy controls (P = 0.036, Mann-Whitney) and highest in patients with shortest interval between tumor resection and serum sampling. Humoral immune responses in patients were most frequently directed against FSPs derived from mutated TAF1B (11.6%, 8/69) and TGFBR2 (10.1%, 7/69). Low level FSP-specific antibodies were also detected in healthy mutation carriers. Our results show that antibody responses against FSPs are detectable in MSI-H CRC patients and healthy Lynch syndrome mutation carriers. Based on the high number of defined FSP antigens, measuring FSP-specific humoral immune responses is a highly promising tool for future diagnostic application in MSI-H cancer patients.

Serum antibodies against frameshift peptides in microsatellite unstable colorectal cancer patients with Lynch syndrome

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Reuschenbach, Miriam; Kloor, Matthias; Morak, Monika; Wentzensen, Nicolas; Germann, Anja; Garbe, Yvette; Tariverdian, Mirjam; Findeisen, Peter; Neumaier, Michael; Holinski-Feder, Elke; Doeberitz, Magnus von Knebel

2014-01-01

High level microsatellite instability (MSI-H) occurs in about 15% of colorectal cancer (CRCs), either as sporadic cancers or in the context of hereditary non-polyposis cancer (HNPCC) or Lynch syndrome. In MSI-H CRC, mismatch repair deficiency leads to insertion/deletion mutations at coding microsatellites (cMS) and thus to the translation of frameshift peptides (FSPs). FSPs are potent inductors of T cell responses in vitro and in vivo. The present study aims at the identification of FSP-specific humoral immune responses in MSI-H CRC and Lynch syndrome. Sera from patients with history of MSI-H CRC (n=69), healthy Lynch syndrome mutation carriers (n=31) and healthy controls (n=52) were analyzed for antibodies against FSPs using peptide ELISA. Reactivities were measured against FSPs derived from genes frequently mutated in MSI-H CRCs, AIM2, TGFBR2, CASP5, TAF1B, ZNF294, and MARCKS. Antibody reactivity against FSPs was significantly higher in MSI-H CRC patients than in healthy controls (p=0.036, Mann-Whitney) and highest in patients with shortest interval between tumor resection and serum sampling. Humoral immune responses in patients were most frequently directed against FSPs derived from mutated TAF1B (11.6%, 8/69) and TGFBR2 (10.1%, 7/69). Low level FSP-specific antibodies were also detected in healthy mutation carriers. Our results show that antibody responses against FSPs are detectable in MSI-H CRC patients and healthy Lynch syndrome mutation carriers. Based on the high number of defined FSP antigens, measuring FSP-specific humoral immune responses is a highly promising tool for future diagnostic application in MSI-H cancer patients. PMID:19957108

Two Novel Mutations Associated With Ataxia-Telangiectasia Identified Using an Ion AmpliSeq Inherited Disease Panel

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Maria V. Kuznetsova

2017-10-01

Full Text Available Ataxia-telangiectasia (A-T, or Louis-Bar syndrome, is a rare neurodegenerative disorder associated with immunodeficiency. For families with at least one affected child, timely A-T genotyping during any subsequent pregnancy allows the parents to make an informed decision about whether to continue to term when the fetus is affected. Mutations in the ATM gene, which is 150 kb long, give rise to A-T; more than 600 pathogenic variants in ATM have been characterized since 1990 and new mutations continue to be discovered annually. Therefore, limiting genetic screening to previously known SNPs by PCR or hybridization with microarrays may not identify the specific pathogenic genotype in ATM for a given A-T family. However, recent developments in next-generation sequencing technology offer prompt high-throughput full-length sequencing of genomic fragments of interest. This allows the identification of the whole spectrum of mutations in a gene, including any novel ones. We report two A-T families with affected children and current pregnancies. Both families are consanguineous and originate from Caucasian regions of Russia and Azerbaijan. Before our study, no ATM mutations had been identified in the older children of these families. We used ion semiconductor sequencing and an Ion AmpliSeq™ Inherited Disease Panel to perform complete ATM gene sequencing in a single member of each family. Then we compared the experimentally determined genotype with the affected/normal phenotype distribution in the whole family to provide unambiguous evidence of pathogenic mutations responsible for A-T. A single novel SNP was allocated to each family. In the first case, we found a mononucleotide deletion, and in the second, a mononucleotide insertion. Both mutations lead to truncation of the ATM protein product. Identification of the pathogenic mutation in each family was performed in a timely fashion, allowing the fetuses to be tested and diagnosed. The parents chose to

Retrospective genetic study of germinative mutations in Str loci of individuals potentially exposed to ionizing radiation

International Nuclear Information System (INIS)

Costa, Emilia Oliveira Alves

2010-01-01

The Brazilian radiological accident that occurred in 1987, in Goiania, it was a terrible radiation episode. As a consequence, hundreds of people were contaminated due to the Cesium-137 radiation. Recently, many studies had shown that genome instabilities, such as, mutations, chromosomal aberrations, micronuclei formation and micro satellite instability and a delay on cellular death are usually reported on mammal cells exposed to ionizing radiation, being considered as a manly risk to humans. Mutations can be spontaneous, and the occurrence is dependent on the organism, or, induced, being associated to mutagenic exposition. Ionizing radiations are an example of physical and mutagenic agents that could harm the cell repair and could cause the development of many types of cancer. The evaluation of the biological effects of the ionizing radiation, in somatic and germ line cells, with a consequent determination of the radio-induced mutations, it is extremely important to estimate the genetic risks, manly in population exposed to radiation. The analyses of repetitive DNA sequences have been demonstrated that such sequences are prone to high rates of spontaneous mutations. The minisatellites and microsatellites have been used to demonstrate the induction of germ line mutation rates on mouse, humans, among others organisms. The aim of the present study was to analyze the frequency of microsatellite alterations to determine the mutation rates occurred in germ cells of the parents exposed to the ionizing radiation of the Cesium-137. The studied group was constitute of 10 families of individuals accidentally exposed to Cesium-137 and by the control group constituted by 645 healthy individuals who carried out paternity tests on 2009. We found only one mutation of paternal origin in the D8S1179 locus on the exposed group, being the mutation rate of 0.002. In the control group, we found 01 mutation on D16S539 loei and on D3S1358; 02 mutations on Penta E loeus; 04 mutations on D

Complete-proteome mapping of human influenza A adaptive mutations: implications for human transmissibility of zoonotic strains.

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Miotto, Olivo; Heiny, A T; Albrecht, Randy; García-Sastre, Adolfo; Tan, Tin Wee; August, J Thomas; Brusic, Vladimir

2010-02-03

There is widespread concern that H5N1 avian influenza A viruses will emerge as a pandemic threat, if they become capable of human-to-human (H2H) transmission. Avian strains lack this capability, which suggests that it requires important adaptive mutations. We performed a large-scale comparative analysis of proteins from avian and human strains, to produce a catalogue of mutations associated with H2H transmissibility, and to detect their presence in avian isolates. We constructed a dataset of influenza A protein sequences from 92,343 public database records. Human and avian sequence subsets were compared, using a method based on mutual information, to identify characteristic sites where human isolates present conserved mutations. The resulting catalogue comprises 68 characteristic sites in eight internal proteins. Subtype variability prevented the identification of adaptive mutations in the hemagglutinin and neuraminidase proteins. The high number of sites in the ribonucleoprotein complex suggests interdependence between mutations in multiple proteins. Characteristic sites are often clustered within known functional regions, suggesting their functional roles in cellular processes. By isolating and concatenating characteristic site residues, we defined adaptation signatures, which summarize the adaptive potential of specific isolates. Most adaptive mutations emerged within three decades after the 1918 pandemic, and have remained remarkably stable thereafter. Two lineages with stable internal protein constellations have circulated among humans without reassorting. On the contrary, H5N1 avian and swine viruses reassort frequently, causing both gains and losses of adaptive mutations. Human host adaptation appears to be complex and systemic, involving nearly all influenza proteins. Adaptation signatures suggest that the ability of H5N1 strains to infect humans is related to the presence of an unusually high number of adaptive mutations. However, these mutations appear

Systematic study on genetic and epimutational profile of a cohort of Amsterdam criteria-defined Lynch Syndrome in Singapore.

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Yanqun Liu

Full Text Available BACKGROUND: Germline defects of mismatch repair (MMR genes underlie Lynch Syndrome (LS. We aimed to gain comprehensive genetic and epigenetic profiles of LS families in Singapore, which will facilitate efficient molecular diagnosis of LS in Singapore and the region. METHODS: Fifty nine unrelated families were studied. Mutations in exons, splice-site junctions and promoters of five MMR genes were scanned by high resolution melting assay followed by DNA sequencing, large fragment deletions/duplications and promoter methylation in MLH1, MSH2, MSH6 and PMS2 were evaluated by multiplex ligation-dependent probe amplification. Tumor microsatellite instability (MSI was assessed with five mononucleotide markers and immunohistochemical staining (IHC was also performed. RESULTS: Pathogenic defects, all confined to MLH1 and MSH2, were identified in 17 out of 59 (28.8% families. The mutational spectrum was highly heterogeneous and 28 novel variants were identified. One recurrent mutation in MLH1 (c.793C>T was also observed. 92.9% sensitivity for indication of germline mutations conferred by IHC surpassed 64.3% sensitivity by MSI. Furthermore, 15.6% patients with MSS tumors harbored pathogenic mutations. CONCLUSIONS: Among major ethnic groups in Singapore, all pathogenic germline defects were confined to MLH1 and MSH2. Caution should be applied when the Amsterdam criteria and consensus microsatellite marker panel recommended in the revised Bethesda guidelines are applied to the local context. We recommend a screening strategy for the local LS by starting with tumor IHC and the hotspot mutation testing at MLH1 c.793C>T followed by comprehensive mutation scanning in MLH1 and MSH2 prior to proceeding to other MMR genes.

How much do we know about spontaneous human mutation rates

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Crow, J.F. (Univ. of Wisconsin, Madison, WI (United States))

1993-01-01

The much larger number of cell divisions between zygote and sperm than between zygote and egg, the increased age of fathers of children with new dominant mutations, and the greater evolution rate of pseudogenes on the Y chromosome than of those on autosomes all point to a much higher mutation rate in human males than in females, as first pointed out by Haldane in his classical study of X-linked hemophilia. The age of the father is the main factor determining the human spontaneous mutation rate, and probably the total mutation rate. The total mutation rate in Drosophila males of genes causing minor reduction in viability is at least 0.4 per sperm and may be considerably higher. The great mutation load implied by a rate of [approx] 1 per zygote can be greatly ameliorated by quasi-transition selection. Corresponding data are not available for the human population. The evolution rate of pseudogenes in primates suggests some 10[sup 2] new mutations per zygote. Presumably the overwhelming majority of these are neutral, but even the approximate fraction is not known. Statistical evidence in Drosophilia shows that mutations with minor effects cause about the same heterozygous impairment of fitness as those that are lethal when homozygous. The magnitude of heterozygous effect is such that almost all mutant genes are eliminated as heterozygotes before ever becoming homozygous. Although quantitative data in the human species are lacking, anecdotal information supports the conclusion that partial dominance is the rule here as well. This suggests that if the human mutation rate were increased or decreased, the effects would be spread over a period of 50-100 generations. 31 refs., 3 figs., 2 tabs.

Microsatellite polymorphisms associated with human behavioural and psychological phenotypes including a gene-environment interaction.

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Bagshaw, Andrew T M; Horwood, L John; Fergusson, David M; Gemmell, Neil J; Kennedy, Martin A

2017-02-03

The genetic and environmental influences on human personality and behaviour are a complex matter of ongoing debate. Accumulating evidence indicates that short tandem repeats (STRs) in regulatory regions are good candidates to explain heritability not accessed by genome-wide association studies. We tested for associations between the genotypes of four selected repeats and 18 traits relating to personality, behaviour, cognitive ability and mental health in a well-studied longitudinal birth cohort (n = 458-589) using one way analysis of variance. The repeats were a highly conserved poly-AC microsatellite in the upstream promoter region of the T-box brain 1 (TBR1) gene and three previously studied STRs in the activating enhancer-binding protein 2-beta (AP2-β) and androgen receptor (AR) genes. Where significance was found we used multiple regression to assess the influence of confounding factors. Carriers of the shorter, most common, allele of the AR gene's GGN microsatellite polymorphism had fewer anxiety-related symptoms, which was consistent with previous studies, but in our study this was not significant following Bonferroni correction. No associations with two repeats in the AP2-β gene withstood this correction. A novel finding was that carriers of the minor allele of the TBR1 AC microsatellite were at higher risk of conduct problems in childhood at age 7-9 (p = 0.0007, which did pass Bonferroni correction). Including maternal smoking during pregnancy (MSDP) in models controlling for potentially confounding influences showed that an interaction between TBR1 genotype and MSDP was a significant predictor of conduct problems in childhood and adolescence (p behaviour up to age 25 years (p ≤ 0.02). This interaction remained significant after controlling for possible confounders including maternal age at birth, socio-economic status and education, and offspring birth weight. The potential functional importance of the TBR1 gene's promoter microsatellite

Effects of chronic low level radiation in the population residing in the high level natural radiation area in Kerala, India: employing heritable DNA mutation studies.

Science.gov (United States)

Ahmad, Shazia; Koya, P K M; Seshadri, M

2013-03-18

To study the effect of chronic low level radiation, 4040 meiosis were screened at eight microsatellite and five minisatellite (2485 and 1555 meiosis respectively) marker loci in people residing in high and normal level natural radiation areas of Kerala. Variants in the repeat length of allele were considered as mutants. Mutation rates (expressed as the number of mutations observed in the total number of meiosis) were 6.4×10(-3) (16/2485) and 2.6×10(-3) (4/1555) at microsatellite and minisatellite respectively. The germline microsatellite mutation frequency of father was 1.78 times higher at 7.52×10(-3) (8/1064) compared to 4.22×10(-3) (6/1421) of mother (P=0.292, Fisher's Exact two-sided test). The paternal and maternal mutation rates at minisatellite loci were more or less similar at 2.78×10(-3) (2/719) and 2.39×10(-3) (2/836), respectively (P=1.0, Fisher's Exact two-sided test). Higher but statistically non-significant microsatellite mutation frequency was observed in HLNRA compared to NLNRA (7.25×10(-3) vs 3.64×10(-3); P=0.547). The apparent increase in the mutation rate of microsatellite loci with the increase in radiation dose was also not statistically significant. All the four minisatellite mutation observed were from HLNRA (1198 meiosis) and no mutation was observed among 357 meiosis screened from NLNRA families. All the markers used in the present study were in the non-coding region and hence mutations in these regions may not cause adverse health effects, but the study is important in understanding the effect of chronic low level radiation. Copyright © 2012 Elsevier B.V. All rights reserved.

Polymorphic microsatellites in the human bloodfluke, Schistosoma japonicum, identified using a genomic resource

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Spear Robert

2011-02-01

Full Text Available Abstract Re-emergence of schistosomiasis in regions of China where control programs have ceased requires development of molecular-genetic tools to track gene flow and assess genetic diversity of Schistosoma populations. We identified many microsatellite loci in the draft genome of Schistosoma japonicum using defined search criteria and selected a subset for further analysis. From an initial panel of 50 loci, 20 new microsatellites were selected for eventual optimization and application to a panel of worms from endemic areas. All but one of the selected microsatellites contain simple tri-nucleotide repeats. Moderate to high levels of polymorphism were detected. Numbers of alleles ranged from 6 to 14 and observed heterozygosity was always >0.6. The loci reported here will facilitate high resolution population-genetic studies on schistosomes in re-emergent foci.

KRAS and BRAF Mutation Detection: Is Immunohistochemistry a Possible Alternative to Molecular Biology in Colorectal Cancer?

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Nicolas Piton

2015-01-01

Full Text Available KRAS genotyping is mandatory in metastatic colorectal cancer treatment prior to undertaking antiepidermal growth factor receptor (EGFR monoclonal antibody therapy. BRAF V600E mutation is often present in colorectal carcinoma with CpG island methylator phenotype and microsatellite instability. Currently, KRAS and BRAF evaluation is based on molecular biology techniques such as SNaPshot or Sanger sequencing. As molecular testing is performed on formalin-fixed paraffin-embedded (FFPE samples, immunodetection would appear to be an attractive alternative for detecting mutations. Thus, our objective was to assess the validity of KRAS and BRAF immunodetection of mutations compared with the genotyping reference method in colorectal adenocarcinoma. KRAS and BRAF genotyping was assessed by SNaPshot. A rabbit anti-human KRAS polyclonal antibody was tested on 33 FFPE colorectal tumor samples with known KRAS status. Additionally, a mouse anti-human BRAF monoclonal antibody was tested on 30 FFPE tumor samples with known BRAF status. KRAS immunostaining demonstrated both poor sensitivity (27% and specificity (64% in detecting KRAS mutation. Conversely, BRAF immunohistochemistry showed perfect sensitivity (100% and specificity (100% in detecting V600E mutation. Although molecular biology remains the reference method for detecting KRAS mutation, immunohistochemistry could be an attractive method for detecting BRAF V600E mutation in colorectal cancer.

KRAS and BRAF Mutation Detection: Is Immunohistochemistry a Possible Alternative to Molecular Biology in Colorectal Cancer?

Science.gov (United States)

Borrini, Francesco; Bolognese, Antonio; Lamy, Aude; Sabourin, Jean-Christophe

2015-01-01

KRAS genotyping is mandatory in metastatic colorectal cancer treatment prior to undertaking antiepidermal growth factor receptor (EGFR) monoclonal antibody therapy. BRAF V600E mutation is often present in colorectal carcinoma with CpG island methylator phenotype and microsatellite instability. Currently, KRAS and BRAF evaluation is based on molecular biology techniques such as SNaPshot or Sanger sequencing. As molecular testing is performed on formalin-fixed paraffin-embedded (FFPE) samples, immunodetection would appear to be an attractive alternative for detecting mutations. Thus, our objective was to assess the validity of KRAS and BRAF immunodetection of mutations compared with the genotyping reference method in colorectal adenocarcinoma. KRAS and BRAF genotyping was assessed by SNaPshot. A rabbit anti-human KRAS polyclonal antibody was tested on 33 FFPE colorectal tumor samples with known KRAS status. Additionally, a mouse anti-human BRAF monoclonal antibody was tested on 30 FFPE tumor samples with known BRAF status. KRAS immunostaining demonstrated both poor sensitivity (27%) and specificity (64%) in detecting KRAS mutation. Conversely, BRAF immunohistochemistry showed perfect sensitivity (100%) and specificity (100%) in detecting V600E mutation. Although molecular biology remains the reference method for detecting KRAS mutation, immunohistochemistry could be an attractive method for detecting BRAF V600E mutation in colorectal cancer. PMID:25983749

Mutagenic effect of cadmium on tetranucleotide repeats in human cells

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Slebos, Robbert J.C. [Department of Cancer Biology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232 (United States) and Department of Otolaryngology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232 (United States)]. E-mail: r.slebos@vanderbilt.edu; Li Ming [Department of Biostatistics, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232 (United States); Evjen, Amy N. [Department of Cancer Biology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232 (United States); Coffa, Jordy [Department of Cancer Biology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232 (United States); Shyr, Yu [Department of Biostatistics, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232 (United States); Yarbrough, Wendell G. [Department of Cancer Biology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232 (United States); Department of Otolaryngology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232 (United States)

2006-12-01

Cadmium is a human carcinogen that affects cell proliferation, apoptosis and DNA repair processes that are all important to carcinogenesis. We previously demonstrated that cadmium inhibits DNA mismatch repair (MMR) in yeast cells and in human cell-free extracts (H.W. Jin, A.B. Clark, R.J.C. Slebos, H. Al-Refai, J.A. Taylor, T.A. Kunkel, M.A. Resnick, D.A. Gordenin, Cadmium is a mutagen that acts by inhibiting mismatch repair, Nat. Genet. 34 (3) (2003) 326-329), but cadmium also inhibits DNA excision repair. For this study, we selected a panel of three hypermutable tetranucleotide markers (MycL1, D7S1482 and DXS981) and studied their suitability as readout for the mutagenic effects of cadmium. We used a clonal derivative of the human fibrosarcoma cell line HT1080 to assess mutation levels in microsatellites after cadmium and/or N-methyl-N-nitro-N-nitrosoguanidine (MNNG) exposure to study effects of cadmium in the presence or absence of base damage. Mutations were measured in clonally expanded cells obtained by limiting dilution after exposure to zero dose, 0.5 {mu}M cadmium, 5 nM MNNG or a combination of 0.5 {mu}M cadmium and 5 nM MNNG. Exposure of HT1080-C1 to cadmium led to statistically significant increases in microsatellite mutations, either with or without concurrent exposure to MNNG. A majority of the observed mutant molecules involved 4-nucleotide shifts consistent with DNA slippage mutations that are normally repaired by MMR. These results provide evidence for the mutagenic effects of low, environmentally relevant levels of cadmium in intact human cells and suggest that inhibition of DNA repair is involved.

The molecular anatomy of spontaneous germline mutations in human testes.

Directory of Open Access Journals (Sweden)

Jian Qin

2007-09-01

Full Text Available The frequency of the most common sporadic Apert syndrome mutation (C755G in the human fibroblast growth factor receptor 2 gene (FGFR2 is 100-1,000 times higher than expected from average nucleotide substitution rates based on evolutionary studies and the incidence of human genetic diseases. To determine if this increased frequency was due to the nucleotide site having the properties of a mutation hot spot, or some other explanation, we developed a new experimental approach. We examined the spatial distribution of the frequency of the C755G mutation in the germline by dividing four testes from two normal individuals each into several hundred pieces, and, using a highly sensitive PCR assay, we measured the mutation frequency of each piece. We discovered that each testis was characterized by rare foci with mutation frequencies 10(3 to >10(4 times higher than the rest of the testis regions. Using a model based on what is known about human germline development forced us to reject (p < 10(-6 the idea that the C755G mutation arises more frequently because this nucleotide simply has a higher than average mutation rate (hot spot model. This is true regardless of whether mutation is dependent or independent of cell division. An alternate model was examined where positive selection acts on adult self-renewing Ap spermatogonial cells (SrAp carrying this mutation such that, instead of only replacing themselves, they occasionally produce two SrAp cells. This model could not be rejected given our observed data. Unlike the disease site, similar analysis of C-to-G mutations at a control nucleotide site in one testis pair failed to find any foci with high mutation frequencies. The rejection of the hot spot model and lack of rejection of a selection model for the C755G mutation, along with other data, provides strong support for the proposal that positive selection in the testis can act to increase the frequency of premeiotic germ cells carrying a mutation

Microsatellite Instability Predicts Clinical Outcome in Radiation-Treated Endometrioid Endometrial Cancer

International Nuclear Information System (INIS)

Bilbao, Cristina; Lara, Pedro Carlos; Ramirez, Raquel; Henriquez-Hernandez, Luis Alberto; Rodriguez, German; Falcon, Orlando; Leon, Laureano; Perucho, Manuel

2010-01-01

Purpose: To elucidate whether microsatellite instability (MSI) predicts clinical outcome in radiation-treated endometrioid endometrial cancer (EEC). Methods and Materials: A consecutive series of 93 patients with EEC treated with extrafascial hysterectomy and postoperative radiotherapy was studied. The median clinical follow-up of patients was 138 months, with a maximum of 232 months. Five quasimonomorphic mononucleotide markers (BAT-25, BAT-26, NR21, NR24, and NR27) were used for MSI classification. Results: Twenty-five patients (22%) were classified as MSI. Both in the whole series and in early stages (I and II), univariate analysis showed a significant association between MSI and poorer 10-year local disease-free survival, disease-free survival, and cancer-specific survival. In multivariate analysis, MSI was excluded from the final regression model in the whole series, but in early stages MSI provided additional significant predictive information independent of traditional prognostic and predictive factors (age, stage, grade, and vascular invasion) for disease-free survival (hazard ratio [HR] 3.25, 95% confidence interval [CI] 1.01-10.49; p = 0.048) and cancer-specific survival (HR 4.20, 95% CI 1.23-14.35; p = 0.022) and was marginally significant for local disease-free survival (HR 3.54, 95% CI 0.93-13.46; p = 0.064). Conclusions: These results suggest that MSI may predict radiotherapy response in early-stage EEC.

Microsatellite instability as prognostic marker in bladder tumors: a clinical significance

Directory of Open Access Journals (Sweden)

Mittal RD

2005-01-01

Full Text Available Abstract Background Carcinoma of urinary bladder is one of the leading causes of death in India. Successful treatment of bladder cancer depends on the early detection & specific diagnostic approaches. In the present study, microsatellite instability (MSI has been evaluated as a prognostic marker in patients with superficial urinary bladder cancer in lower urinary tract for determining risk of recurrence. Methods A total of 44 patients with bladder tumors diagnosed with Transitional Cell Carcinomas [TCC] from lower urinary tract were selected for the study. Tumors were staged and graded according to AJCC-UICC (1997 classification and patients were followed with cystoscopy as per the protocol. Polymerase chain reaction (PCR was done to amplify microsatellite sequences at mononucleotide BAT – 26, BAT – 40, TGFβ RII, IGFIIR, hMSH3, BAX and dinucleotide D2S123, D9S283, D9S1851 and D18S58 loci in blood (control and tumor DNA. PCR products were separated on 8% denaturing polyacrylamide gel and visualized by autoradiography. Results MSI was observed in 72.7% of tumors at BAT – 26, BAT – 40, D2S123, D9S283, D9S1851 and D18S58 loci. Good association of MSI was seen with tumor stage and grade. MSI – High (instability at > 30% of loci was frequently observed in high stage (40.6% and high grade (59.4% tumors. Of 24 tumors of Ta-T1 stage with different grades, 11 (9/18 high grade and 2/6 low grade tumors recurred in the mean duration of 36 months. MSI positivity was significantly high in patients who had one or more recurrences (p = 0.02 for high grade and 0.04 for low grade tumors. Conclusions MSI may be an independent prognostic marker for assessing risk of recurrence in superficial tumors irrespective of the grade. Further studies on progression would help in stratifying the patients of T1G3 for early cystectomy vs bladder preservation protocol.

Étude de l'impact des microARNs sur la carcinogenèse des cancers colorectaux instables sur les séquences répétées microsatellites du génome

OpenAIRE

El-Murr , Nizar

2014-01-01

MSI tumor progression (Microsatellite Instability) is depicted as a multistage process that results from mutations generated by a process of genetic instability affecting mostly DNA tandem repeats (known as microsatellites). These mutations contribute to tumorigenesis when they disrupt the function of oncogenes or tumor suppressor genes. As a phenotypic trait, MSI is the consequence of DNA mismatch repair inactivation (MMR). This work focused on the role microRNAs might play in MSI tumorigene...

Genetic structure of earthworm populations at a regional scale: inferences from mitochondrial and microsatellite molecular markers in Aporrectodea icterica (Savigny 1826.

Directory of Open Access Journals (Sweden)

Magally Torres-Leguizamon

Full Text Available Despite the fundamental role that soil invertebrates (e.g. earthworms play in soil ecosystems, the magnitude of their spatial genetic variation is still largely unknown and only a few studies have investigated the population genetic structure of these organisms. Here, we investigated the genetic structure of seven populations of a common endogeic earthworm (Aporrectodea icterica sampled in northern France to explore how historical species range changes, microevolutionary processes and human activities interact in shaping genetic variation at a regional scale. Because combining markers with distinct modes of inheritance can provide extra, complementary information on gene flow, we compared the patterns of genetic structure revealed using nuclear (7 microsatellite loci and mitochondrial markers (COI. Both types of markers indicated low genetic polymorphism compared to other earthworm species, a result that can be attributed to ancient bottlenecks, for instance due to species isolation in southern refugia during the ice ages with subsequent expansion toward northern Europe. Historical events can also be responsible for the existence of two divergent, but randomly interbreeding mitochondrial lineages within all study populations. In addition, the comparison of observed heterozygosity among microsatellite loci and heterozygosity expected under mutation-drift equilibrium suggested a recent decrease in effective size in some populations that could be due to contemporary events such as habitat fragmentation. The absence of relationship between geographic and genetic distances estimated from microsatellite allele frequency data also suggested that dispersal is haphazard and that human activities favour passive dispersal among geographically distant populations.

In Silico Mining of Microsatellites in Coding Sequences of the Date Palm (Arecaceae Genome, Characterization, and Transferability

Directory of Open Access Journals (Sweden)

Frédérique Aberlenc-Bertossi

2014-01-01

Full Text Available Premise of the study: To complement existing sets of primarily dinucleotide microsatellite loci from noncoding sequences of date palm, we developed primers for tri- and hexanucleotide microsatellite loci identified within genes. Due to their conserved genomic locations, the primers should be useful in other palm taxa, and their utility was tested in seven other Phoenix species and in Chamaerops, Livistona, and Hyphaene. Methods and Results: Tandem repeat motifs of 3–6 bp were searched using a simple sequence repeat (SSR–pipeline package in coding portions of the date palm draft genome sequence. Fifteen loci produced highly consistent amplification, intraspecific polymorphisms, and stepwise mutation patterns. Conclusions: These microsatellite loci showed sufficient levels of variability and transferability to make them useful for population genetic, selection signature, and interspecific gene flow studies in Phoenix and other Coryphoideae genera.

Urinary Tract Cancer in Lynch Syndrome; Increased Risk in Carriers of MSH2 Mutations

DEFF Research Database (Denmark)

Joost, Patrick; Therkildsen, Christina; Dominguez-Valentin, Mev

2015-01-01

and microsatellite instability in 23% of the tumors. Mutations in MSH2 were overrepresented (73%), and MSH2 mutation carriers were at a significantly increased risk of developing urinary tract cancer compared with individuals with mutations in MLH1 or MSH6. CONCLUSION: Cancers of the upper urinary tract...

Detection of duplicates among repatriated Nordic spring barley (Hordeum vulgare L. s.l.) accessions using agronomic and morphological descriptors and microsatellite markers

DEFF Research Database (Denmark)

Lund, Birgitte; Ortiz, Rodomiro; von Bothmer, Roland

2013-01-01

on agronomic and morphological discriminators to detect genetic heterogeneity, although with reduced sensitivity compared with microsatellite markers. These results also suggest that grouping ensuing from either approach could reflect distinct patterns of diversity (due to different mutation rates...... their use with results from previous research with microsatellite markers. These accessions were initially grouped into 36 potential duplicates according to passport data but further analysis with microsatellites reduce them to 22 genetically homogeneous groups. The analysis with 26 agronomic...... and morphological descriptors of putative Nordic spring barley accessions from nine gene banks was compared with a previous study with microsatellites. Each agronomic and morphological descriptor was weighed relative to its genetic determination with the aim of reducing the effect of environmental errors on genetic...

Molecular key to understand the gastric cancer biology in elderly patients-The role of microsatellite instability.

Science.gov (United States)

Polom, Karol; Marrelli, Daniele; Roviello, Giandomenico; Pascale, Valeria; Voglino, Costantino; Rho, Henry; Marini, Mario; Macchiarelli, Raffaele; Roviello, Franco

2017-03-01

Microsatellite instability (MSI) in gastric cancer (GC) is associated with older age. We present the clinicopathological results of younger and older patients with MSI GC. We analyzed 472 patients with GC. MSI analysis was done on fresh frozen tissue using five quasimonomorphic mononucleotide repeats: NR-21, NR-24, NR-27, BAT-25, and BAR-26. Clinical and pathological analysis was performed for different age groups. We observed better survival in elderly MSI GC patients compared to younger patients. The percentage of MSI GC increases gradually with increasing age, accounting for 48% of patients over the age of 85 years. A difference in survival was seen between MSI and MSS groups of patients older than 65 years, while no statistical difference was seen for younger groups. Multivariate analysis revealed that MSI status has a significant prognostic factor in patients aged over 70 years (MSS vs. MSI; HR 1.82, P = 0.013). MSI is an important prognostic factor above all in elderly GC patients. It is associated with favorable prognosis and may help in planning different approaches to treatment in this subgroup. J. Surg. Oncol. 2017;115:344-350. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Tempo and mode of genomic mutations unveil human evolutionary history.

Science.gov (United States)

Hara, Yuichiro

2015-01-01

Mutations that have occurred in human genomes provide insight into various aspects of evolutionary history such as speciation events and degrees of natural selection. Comparing genome sequences between human and great apes or among humans is a feasible approach for inferring human evolutionary history. Recent advances in high-throughput or so-called 'next-generation' DNA sequencing technologies have enabled the sequencing of thousands of individual human genomes, as well as a variety of reference genomes of hominids, many of which are publicly available. These sequence data can help to unveil the detailed demographic history of the lineage leading to humans as well as the explosion of modern human population size in the last several thousand years. In addition, high-throughput sequencing illustrates the tempo and mode of de novo mutations, which are producing human genetic variation at this moment. Pedigree-based human genome sequencing has shown that mutation rates vary significantly across the human genome. These studies have also provided an improved timescale of human evolution, because the mutation rate estimated from pedigree analysis is half that estimated from traditional analyses based on molecular phylogeny. Because of the dramatic reduction in sequencing cost, sequencing on-demand samples designed for specific studies is now also becoming popular. To produce data of sufficient quality to meet the requirements of the study, it is necessary to set an explicit sequencing plan that includes the choice of sample collection methods, sequencing platforms, and number of sequence reads.

Genetic diversity and geographical dispersal in grapevine clones revealed by microsatellite markers.

Science.gov (United States)

Moncada, Ximena; Pelsy, Frédérique; Merdinoglu, Didier; Hinrichsen, Patricio

2006-11-01

Intravarietal genetic diversification associated with geographical dispersal of a vegetatively propagated species was studied using grapevine Vitis vinifera L. 'Cabernet Sauvignon' as a model. Fifty-nine clonal samples obtained from 7 countries (France, Chile, Spain, Australia, Hungary, USA, and Italy) were analyzed using 84 microsatellite markers. Eighteen polymorphic microsatellite loci (21.4%) were detected, finding 22 different genotypes in the population analyzed with a genetic similarity of over 97%. The presence of chimeric clones was evidenced at locus VMC5g7 by means of a segregation analysis of descendants by self-pollination of a triallelic Chilean clone and by somatic embryogenesis analysis, showing a mutation in L2 cell layer. Only 2 clones (obtained from France and Australia) presented the ancestral genotype, and the most divergent genotype was exhibited by another French clone, which had accumulated 5 somatic mutations. The 2 largest populations considered (from France and Chile) showed a clear divergency in the polymorphisms detected. These antecedents enabled the tracing of geographical dispersal with a phylogenetic hypothesis supporting France as the center of origin of diversification of Cabernet Sauvignon. The results obtained could help to explain diversification processes in other grapevine cultivars. The possibility that this kind of genetic variability occurs in other vegetatively propagated species is discussed, focusing on possible fingerprinting applications.

Microsatellite analysis of Saccharomyces uvarum diversity.

Science.gov (United States)

Masneuf-Pomarede, Isabelle; Salin, Franck; Börlin, Marine; Coton, Emmanuel; Coton, Monika; Jeune, Christine Le; Legras, Jean-Luc

2016-03-01

Considered as a sister species of Saccharomyces cerevisiae, S. uvarum is, to a lesser extent, an interesting species for fundamental and applied research studies. Despite its potential interest as a new gene pool for fermenting agents, the intraspecific molecular genetic diversity of this species is still poorly investigated. In this study, we report the use of nine microsatellite markers to describe S. uvarum genetic diversity and population structure among 108 isolates from various geographical and substrate origins (wine, cider and natural sources). Our combined microsatellite markers set allowed differentiating 89 genotypes. In contrast to S. cerevisiae genetic diversity, wild and human origin isolates were intertwined. A total of 75% of strains were proven to be homozygotes and estimated heterozygosity suggests a selfing rate above 0.95 for the different population tested here. From this point of view, the S. uvarum life cycle appears to be more closely related to S. paradoxus or S. cerevisiae of natural resources than S. cerevisiae wine isolates. Population structure could not be correlated to distinct geographic or technological origins, suggesting lower differentiation that may result from a large exchange between human and natural populations mediated by insects or human activities. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Mutations and binding sites of human transcription factors

KAUST Repository

Kamanu, Frederick Kinyua

2012-06-01

Mutations in any genome may lead to phenotype characteristics that determine ability of an individual to cope with adaptation to environmental challenges. In studies of human biology, among the most interesting ones are phenotype characteristics that determine responses to drug treatments, response to infections, or predisposition to specific inherited diseases. Most of the research in this field has been focused on the studies of mutation effects on the final gene products, peptides, and their alterations. Considerably less attention was given to the mutations that may affect regulatory mechanism(s) of gene expression, although these may also affect the phenotype characteristics. In this study we make a pilot analysis of mutations observed in the regulatory regions of 24,667 human RefSeq genes. Our study reveals that out of eight studied mutation types, insertions are the only one that in a statistically significant manner alters predicted transcription factor binding sites (TFBSs). We also find that 25 families of TFBSs have been altered by mutations in a statistically significant manner in the promoter regions we considered. Moreover, we find that the related transcription factors are, for example, prominent in processes related to intracellular signaling; cell fate; morphogenesis of organs and epithelium; development of urogenital system, epithelium, and tube; neuron fate commitment. Our study highlights the significance of studying mutations within the genes regulatory regions and opens way for further detailed investigations on this topic, particularly on the downstream affected pathways. 2012 Kamanu, Medvedeva, Schaefer, Jankovic, Archer and Bajic.

Somatic mutations in mismatch repair genes in sporadic gastric carcinomas are not a cause but a consequence of the mutator phenotype

NARCIS (Netherlands)

Pinto, Mafalda; Wub, Ying; Mensink, Rob G. J.; Cirnes, Luis; Seruca, Raquel; Hofstra, Robert M. W.

2008-01-01

In hereditary nonpolyposis colorectal cancer (HNPCC), patients' mismatch repair (MMR) gene mutations cause MMR deficiency, leading to microsatellite instability (MSI-H). MSI-H is also found in a substantial fraction of sporadic gastric carcinomas (SGC), mainly due to MLH1 promoter hypermethylation,

Studies of human mutation rates, December 1, 1985--November 30, 1986

International Nuclear Information System (INIS)

Neel, J.V.

1985-01-01

This program seeks to quantify native human mutation rates and to determine how man's activities may affect these rates. The program is divided into six tasks, i.e. The American Indian mutation rate, monitoring populations for frequency of mutation by electrophoresis of blood proteins, application of molecular biological approaches to the detection and study of mutational events in human populations, development of two-dimensional electrophoresis for identification of mutant proteins, co-operative program with the Radiation Effects Research Foundation in Hiroshima and Nagasaki, Japan, and statistical problems associated with the estimation of mutation rates. Progress of each of the above tasks is related in detail. (DT)

Imprints from genetic drift and mutation imply relative divergence times across marine transition zones in a Pan European small pelagic fish (Sprattus sprattus)

DEFF Research Database (Denmark)

Limborg, Morten; Hanel, R.; Debes, P.

2012-01-01

.) by combining inference from both mtDNA and microsatellite genetic markers throughout the species’ distribution. We compared effects from genetic drift and mutation for both genetic markers in shaping genetic differentiation across four transition zones. Microsatellite markers revealed significant isolation...... by distance and a complex population structure across the species0 distribution (overall yST¼0.038, Po0.01). Across transition zones markers indicated larger effects of genetic drift over mutations in the northern distribution of sprat contrasting a stronger relative impact of mutation in the species...

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

Science.gov (United States)

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

2009-08-01

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

N3 and O2 Protonated Conformers of the Cytosine Mononucleotides Coexist in the Gas Phase

Science.gov (United States)

Wu, R. R.; Hamlow, L. A.; He, C. C.; Nei, Y.-w.; Berden, G.; Oomens, J.; Rodgers, M. T.

2017-08-01

The gas-phase conformations of the protonated forms of the DNA and RNA cytosine mononucleotides, [pdCyd+H]+ and [pCyd+H]+, are examined by infrared multiple photon dissociation (IRMPD) action spectroscopy over the IR fingerprint and hydrogen-stretching regions complemented by electronic structure calculations. The low-energy conformations of [pdCyd+H]+ and [pCyd+H]+ and their relative stabilities are computed at the B3LYP/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) and MP2(full)/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) levels of theory. Comparisons of the measured IRMPD action spectra and B3LYP/6-311+G(d,p) linear IR spectra computed for the low-energy conformers allow the conformers present in the experiments to be determined. Similar to that found in previous IRMPD action spectroscopy studies of the protonated forms of the cytosine nucleosides, [dCyd+H]+ and [Cyd+H]+, both N3 and O2 protonated cytosine mononucleotides exhibiting an anti orientation of cytosine are found to coexist in the experimental population. The 2'-hydroxyl substituent does not significantly influence the most stable conformations of [pCyd+H]+ versus those of [pdCyd+H]+, as the IRMPD spectral profiles of [pdCyd+H]+ and [pCyd+H]+ are similar. However, the presence of the 2'-hydroxyl substituent does influence the relative intensities of the measured IRMPD bands. Comparisons to IRMPD spectroscopy studies of the deprotonated forms of the cytosine mononucleotides, [pdCyd-H]- and [pCyd-H]-, provide insight into the effects of protonation versus deprotonation on the conformational features of the nucleobase and sugar moieties. Likewise, comparisons to results of IRMPD spectroscopy studies of the protonated cytosine nucleosides provide insight into the influence of the phosphate moiety on structure. Comparison with previous ion mobility results shows the superiority of IRMPD spectroscopy for distinguishing various protonation sites.

Identification of 10 882 porcine microsatellite sequences and virtual mapping of 4528 of these sequences

DEFF Research Database (Denmark)

Karlskov-Mortensen, Peter; Hu, Z.L.; Gorodkin, Jan

2007-01-01

the human genome (BLAST cut-off threshold = 1 x 10-5). All microsatellite sequences placed on the comparative map are accessible at http://www.animalgenome.org/QTLdb/pig.html . These sequences increase the number of identified microsatellites in the porcine genome by several orders of magnitude...

Short template switch events explain mutation clusters in the human genome.

Science.gov (United States)

Löytynoja, Ari; Goldman, Nick

2017-06-01

Resequencing efforts are uncovering the extent of genetic variation in humans and provide data to study the evolutionary processes shaping our genome. One recurring puzzle in both intra- and inter-species studies is the high frequency of complex mutations comprising multiple nearby base substitutions or insertion-deletions. We devised a generalized mutation model of template switching during replication that extends existing models of genome rearrangement and used this to study the role of template switch events in the origin of short mutation clusters. Applied to the human genome, our model detects thousands of template switch events during the evolution of human and chimp from their common ancestor and hundreds of events between two independently sequenced human genomes. Although many of these are consistent with a template switch mechanism previously proposed for bacteria, our model also identifies new types of mutations that create short inversions, some flanked by paired inverted repeats. The local template switch process can create numerous complex mutation patterns, including hairpin loop structures, and explains multinucleotide mutations and compensatory substitutions without invoking positive selection, speculative mechanisms, or implausible coincidence. Clustered sequence differences are challenging for current mapping and variant calling methods, and we show that many erroneous variant annotations exist in human reference data. Local template switch events may have been neglected as an explanation for complex mutations because of biases in commonly used analyses. Incorporation of our model into reference-based analysis pipelines and comparisons of de novo assembled genomes will lead to improved understanding of genome variation and evolution. © 2017 Löytynoja and Goldman; Published by Cold Spring Harbor Laboratory Press.

Microsatellite instability in bladder cancer

DEFF Research Database (Denmark)

Gonzalez-Zulueta, M; Ruppert, J M; Tokino, K

1993-01-01

Somatic instability at microsatellite repeats was detected in 6 of 200 transitional cell carcinomas of the bladder. Instabilities were apparent as changes in (GT)n repeat lengths on human chromosome 9 for four tumors and as alterations in a (CAG)n repeat in the androgen receptor gene on the X...... or larger (> 2 base pairs) alterations in repeat length. All six tumors were low stage (Ta-T1), suggesting that these alterations can occur early in bladder tumorigenesis....

Mutation of miRNA target sequences during human evolution

DEFF Research Database (Denmark)

Gardner, Paul P; Vinther, Jeppe

2008-01-01

It has long-been hypothesized that changes in non-protein-coding genes and the regulatory sequences controlling expression could undergo positive selection. Here we identify 402 putative microRNA (miRNA) target sequences that have been mutated specifically in the human lineage and show that genes...... containing such deletions are more highly expressed than their mouse orthologs. Our findings indicate that some miRNA target mutations are fixed by positive selection and might have been involved in the evolution of human-specific traits....

A novel approach for characterizing microsatellite instability in cancer cells.

Directory of Open Access Journals (Sweden)

Yuheng Lu

Full Text Available Microsatellite instability (MSI is characterized by the expansion or contraction of DNA repeat tracts as a consequence of DNA mismatch repair deficiency (MMRD. Accurate detection of MSI in cancer cells is important since MSI is associated with several cancer subtypes and can help inform therapeutic decisions. Although experimental assays have been developed to detect MSI, they typically depend on a small number of known microsatellite loci or mismatch repair genes and have limited reliability. Here, we report a novel genome-wide approach for MSI detection based on the global detection of insertions and deletions (indels in microsatellites found in expressed genes. Our large-scale analyses of 20 cancer cell lines and 123 normal individuals revealed striking indel features associated with MSI: there is a significant increase of short microsatellite deletions in MSI samples compared to microsatellite stable (MSS ones, suggesting a mechanistic bias of repair efficiency between insertions and deletions in normal human cells. By incorporating this observation into our MSI scoring metric, we show that our approach can correctly distinguish between MSI and MSS cancer cell lines. Moreover, when we applied this approach to primal tumor samples, our metric is also well consistent with diagnosed MSI status. Thus, our study offers new insight into DNA mismatch repair system, and also provides a novel MSI diagnosis method for clinical oncology with better reliability.

Photo-induced reduction of flavin mononucleotide in aqueous solutions

International Nuclear Information System (INIS)

Song, S.-H.; Dick, B.; Penzkofer, A.

2007-01-01

The photo-induced reduction of flavin mononucleotide (FMN) in aqueous solutions is studied by absorption spectra measurement under aerobic and anaerobic conditions. Samples without exogenous reducing agent and with the exogenous reducing agents ethylene-diamine-tetraacetic acid (EDTA) and dithiothreitol (DTT) are investigated. Under anaerobic conditions the photo-induced reduction with and without reducing agents is irreversible. Under aerobic conditions the photo-reduction without added reducing agent is small compared to the photo-degradation, and the photo-reduction of FMN by the reducing agents is reversible (re-oxidation in the dark). During photo-excitation of FMN the dissolved oxygen is consumed by singlet oxygen formation and subsequent chemical reaction. After light switch-off slow re-oxidation (slow absorption recovery) occurs due to air in-diffusion from surface. EDTA degradation by FMN excitation leads to oxygen scavenging. The quantum efficiencies of photo-reduction under aerobic and anaerobic conditions are determined. The re-oxidation of reduced FMN under aerobic conditions and due to air injection is investigated

Training manual on the analysis of microsatellite repeats in human DNA for diagnostic applications

International Nuclear Information System (INIS)

Ioannou, P.

1998-01-01

The recent discovery that simple sequence length polymorphisms (SSLPs), or microsatellites, are highly polymorphic has provided a rich source of genetic markers for the development of high-resolution maps. SSLPs are ideal markers because they are widely distributed throughout eukrayotic genomes and can be efficiently analyzed using the polymerase chain reaction (PCR). The recent development of moderate-resolution maps of both human and mouse genomes built entirely with SSLPs reflects the rapid conversion from manual Southern blot-based markers to semi-automated PCR-amplified markers during the last few years. Furthermore, these markers can also be used as 'sequence-tagged sites' (STS) in physical maps and provide a direct connection between the genetic and physical maps of eukaryotic chromosomes

Radiation in relation to mutation rate, mutational damage and human ill-health

International Nuclear Information System (INIS)

Roberts, P.B.

1976-09-01

The effect of radiation in increasing the frequency of gene mutations is now reasonably understood. We discuss first how an increase in the mutation rate is reflected in the mutational damage expressed in populations. It is shown that the mutational damage, assessed by the loss of fitness in a population or the number of eventual gene extinctions, is equal to the number of new mutations arising per generation or the mutation rate. In a population of stable size, a dose of 1 rem given to 10 6 people leads to roughly 600 gene extinctions when summed over all ensuing generations if the dose is applied to only one generation; this number of extinctions will occur in each succeeding generation if the dose is given to every generation. However, the concept of genetic extinction, although quantifiable, is of limited value in assessing radiation risks since its impact on human ill-health is very speculative. In particular, no estimate can be made of the total cost of effects which are minor in each individual in which they arise, but which, because they are so minor, persist in the population for many generations. The best current estimate is for 14-140 obvious defects in the first few generations following exposure of 10 6 people to a dose of 1 rem. (auth.)

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

DEFF Research Database (Denmark)

Bodo, Sahra; Colas, Chrystelle; Buhard, Olivier

2015-01-01

BACKGROUND & AIMS: Patients with bi-allelic germline mutations in mismatch repair (MMR) genes (MLH1, MSH2, MSH6, or PMS2) develop a rare but severe variant of Lynch syndrome called constitutional MMR deficiency (CMMRD). This syndrome is characterized by early-onset colorectal cancers, lymphomas...... or leukemias, and brain tumors. There is no satisfactory method for diagnosis of CMMRD because screens for mutations in MMR genes are noninformative for 30% of patients. MMR-deficient cancer cells are resistant to genotoxic agents and have microsatellite instability (MSI), due to accumulation of errors...

Nonsense mutations in the human β-globin gene affect mRNA metabolism

International Nuclear Information System (INIS)

Baserga, S.J.; Benz, E.J. Jr.

1988-01-01

A number of premature translation termination mutations (nonsense mutations) have been described in the human α- and β-globin genes. Studies on mRNA isolated from patients with β 0 -thalassemia have shown that for both the β-17 and the β-39 mutations less than normal levels of β-globin mRNA accumulate in peripheral blood cells. (The codon at which the mutation occurs designates the name of the mutation; there are 146 codons in human β-globin mRNA). In vitro studies using the cloned β-39 gene have reproduced this effect in a heterologous transfection system and have suggested that the defect resides in intranuclear metabolism. The authors have asked if this phenomenon of decreased mRNA accumulation is a general property of nonsense mutations and if the effect depends on the location or the type of mutation. Toward this end, they have studied the effect of five nonsense mutations and two missense mutations on the expression of human β-globin mRNA in a heterologous transfection system. In all cases studied, the presence of a translation termination codon correlates with a decrease in the steady-state level of mRNA. The data suggest that the metabolism of a mammalian mRNA is affected by the presence of a mutation that affects translation

The Human Gene Mutation Database: building a comprehensive mutation repository for clinical and molecular genetics, diagnostic testing and personalized genomic medicine.

Science.gov (United States)

Stenson, Peter D; Mort, Matthew; Ball, Edward V; Shaw, Katy; Phillips, Andrew; Cooper, David N

2014-01-01

The Human Gene Mutation Database (HGMD®) is a comprehensive collection of germline mutations in nuclear genes that underlie, or are associated with, human inherited disease. By June 2013, the database contained over 141,000 different lesions detected in over 5,700 different genes, with new mutation entries currently accumulating at a rate exceeding 10,000 per annum. HGMD was originally established in 1996 for the scientific study of mutational mechanisms in human genes. However, it has since acquired a much broader utility as a central unified disease-oriented mutation repository utilized by human molecular geneticists, genome scientists, molecular biologists, clinicians and genetic counsellors as well as by those specializing in biopharmaceuticals, bioinformatics and personalized genomics. The public version of HGMD (http://www.hgmd.org) is freely available to registered users from academic institutions/non-profit organizations whilst the subscription version (HGMD Professional) is available to academic, clinical and commercial users under license via BIOBASE GmbH.

Genetic characterization of Kenai brown bears (Ursus arctos): Microsatellite and mitochondrial DNA control region variation in brown bears of the Kenai Peninsula, south central Alaska

Science.gov (United States)

Jackson, J.V.; Talbot, S.L.; Farley, S.

2008-01-01

We collected data from 20 biparentally inherited microsatellite loci, and nucleotide sequence from the maternally inherited mitochondrial DNA (mtDNA) control region, to determine levels of genetic variation of the brown bears (Ursus arctos L., 1758) of the Kenai Peninsula, south central Alaska. Nuclear genetic variation was similar to that observed in other Alaskan peninsular populations. We detected no significant inbreeding and found no evidence of population substructuring on the Kenai Peninsula. We observed a genetic signature of a bottleneck under the infinite alleles model (IAM), but not under the stepwise mutation model (SMM) or the two-phase model (TPM) of microsatellite mutation. Kenai brown bears have lower levels of mtDNA haplotypic diversity relative to most other brown bear populations in Alaska. ?? 2008 NRC.

Law-medicine interfacing: patenting of human genes and mutations.

Science.gov (United States)

Fialho, Arsenio M; Chakrabarty, Ananda M

2011-08-01

Mutations, Single Nucleotide Polymorphisms (SNPs), deletions and genetic rearrangements in specific genes in the human genome account for not only our physical characteristics and behavior, but can lead to many in-born and acquired diseases. Such changes in the genome can also predispose people to cancers, as well as significantly affect the metabolism and efficacy of many drugs, resulting in some cases in acute toxicity to the drug. The testing of the presence of such genetic mutations and rearrangements is of great practical and commercial value, leading many of these genes and their mutations/deletions and genetic rearrangements to be patented. A recent decision by a judge in the Federal District Court in the Southern District of New York, has created major uncertainties, based on the revocation of BRCA1 and BRCA2 gene patents, in the eligibility of all human and presumably other gene patents. This article argues that while patents on BRCA1 and BRCA2 genes could be challenged based on a lack of utility, the patenting of the mutations and genetic rearrangements is of great importance to further development and commercialization of genetic tests that can save human lives and prevent suffering, and should be allowed.

Antibody-based screening for hereditary nonpolyposis colorectal carcinoma compared with microsatellite analysis and sequencing

DEFF Research Database (Denmark)

Christensen, Mariann; Katballe, Niels; Wikman, Friedrik

2002-01-01

BACKGROUND: Germline mutations in the DNA mismatch repair genes, MSH2, MLH1, and others are associated with hereditary nonpolyposis colorectal cancer (HNPCC). Due to the high costs of sequencing, cheaper screening methods are needed to identify HNPCC cases. Ideally, these methods should have a high...... carcinoma of whom 11 met the Amsterdam criteria and 31 were suspected to belong to HNPCC families. Thirty-five patients were examined by microsatellite analysis, 40 by immunohistochemical staining, and in 31 patients both the MLH1 and MSH2 genes were sequenced. RESULTS: Ninety-two percent of patients...... the three methods was found in 74 % of the tumors. CONCLUSIONS: The authors suggest that immunohistochemistry should be used in combination with microsatellite analysis to prescreen suspected HNPCC patients for the selection of cases where sequencing of the MLH1 and MSH2 mismatch repair genes is indicated....

Characterization of new Schistosoma mansoni microsatellite loci in sequences obtained from public DNA databases and microsatellite enriched genomic libraries

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Rodrigues NB

2002-01-01

Full Text Available In the last decade microsatellites have become one of the most useful genetic markers used in a large number of organisms due to their abundance and high level of polymorphism. Microsatellites have been used for individual identification, paternity tests, forensic studies and population genetics. Data on microsatellite abundance comes preferentially from microsatellite enriched libraries and DNA sequence databases. We have conducted a search in GenBank of more than 16,000 Schistosoma mansoni ESTs and 42,000 BAC sequences. In addition, we obtained 300 sequences from CA and AT microsatellite enriched genomic libraries. The sequences were searched for simple repeats using the RepeatMasker software. Of 16,022 ESTs, we detected 481 (3% sequences that contained 622 microsatellites (434 perfect, 164 imperfect and 24 compounds. Of the 481 ESTs, 194 were grouped in 63 clusters containing 2 to 15 ESTs per cluster. Polymorphisms were observed in 16 clusters. The 287 remaining ESTs were orphan sequences. Of the 42,017 BAC end sequences, 1,598 (3.8% contained microsatellites (2,335 perfect, 287 imperfect and 79 compounds. The 1,598 BAC end sequences 80 were grouped into 17 clusters containing 3 to 17 BAC end sequences per cluster. Microsatellites were present in 67 out of 300 sequences from microsatellite enriched libraries (55 perfect, 38 imperfect and 15 compounds. From all of the observed loci 55 were selected for having the longest perfect repeats and flanking regions that allowed the design of primers for PCR amplification. Additionally we describe two new polymorphic microsatellite loci.

BRAF mutation-specific promoter methylation of FOX genes in colorectal cancer

NARCIS (Netherlands)

E.H.J. van Roon (Eddy); A. Boot (Arnoud); A.A. Dihal (Ashwin); R.F. Ernst (Robert); T. van Wezel (Tom); H. Morreau (Hans); J.M. Boer (Judith)

2013-01-01

textabstractBackground: Cancer-specific hypermethylation of (promoter) CpG islands is common during the tumorigenesis of colon cancer. Although associations between certain genetic aberrations, such as BRAF mutation and microsatellite instability, and the CpG island methylator phenotype (CIMP), have

Improved Detection of Microsatellite Instability in Early Colorectal Lesions.

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Jeffery W Bacher

Full Text Available Microsatellite instability (MSI occurs in over 90% of Lynch syndrome cancers and is considered a hallmark of the disease. MSI is an early event in colon tumor development, but screening polyps for MSI remains controversial because of reduced sensitivity compared to more advanced neoplasms. To increase sensitivity, we investigated the use of a novel type of marker consisting of long mononucleotide repeat (LMR tracts. Adenomas from 160 patients, ranging in age from 29-55 years old, were screened for MSI using the new markers and compared with current marker panels and immunohistochemistry standards. Overall, 15 tumors were scored as MSI-High using the LMRs compared to 9 for the NCI panel and 8 for the MSI Analysis System (Promega. This difference represents at least a 1.7-fold increase in detection of MSI-High lesions over currently available markers. Moreover, the number of MSI-positive markers per sample and the size of allelic changes were significantly greater with the LMRs (p = 0.001, which increased confidence in MSI classification. The overall sensitivity and specificity of the LMR panel for detection of mismatch repair deficient lesions were 100% and 96%, respectively. In comparison, the sensitivity and specificity of the MSI Analysis System were 67% and 100%; and for the NCI panel, 75% and 97%. The difference in sensitivity between the LMR panel and the other panels was statistically significant (p<0.001. The increased sensitivity for detection of MSI-High phenotype in early colorectal lesions with the new LMR markers indicates that MSI screening for the early detection of Lynch syndrome might be feasible.

Microsatellite analysis of Fasciola spp. in Egypt.

Science.gov (United States)

Dar, Yasser; Amer, Said; Courtioux, Bertrand; Dreyfuss, Gilles

2011-12-01

Recently, the topic of diversity in Fasciola population in Egypt is controversial. The present study was performed to study the genetic diversity of isolated flukes based on microsatellites markers. Fasciola worms were collected from different hosts and geographical locations in Egypt. Control samples of Fasciola hepatica from France as well as Fasciola gigantica from Cameroon were included in the study. Collected flukes were identified morphologically and subjected for analysis using four microsatellite markers. Results of microsatellite profile (FM1 and FM2) proved that both species of Fasciola are distributed in Egypt irrespective of geographical location and host. Nevertheless, the microsatellite profile of some analyzed loci (FM2 and FM3) proved that Egyptian flukes showed more alleles compared to the reference ones. Differences of microsatellite profile in Egyptian isolates than that of corresponding reference samples indicate the remarkable diversity of these isolates. The present results highlighted the utility of microsatellite profile to discriminate between Fasciola species and to elucidate the diversity within the species. To our knowledge, this is the first time to study microsatellite polymorphism in Fasciola populations in Egypt.

Three mutations switch H7N9 influenza to human-type receptor specificity

Energy Technology Data Exchange (ETDEWEB)

de Vries, Robert P.; Peng, Wenjie; Grant, Oliver C.; Thompson, Andrew J.; Zhu, Xueyong; Bouwman, Kim M.; de la Pena, Alba T. Torrents; van Breemen, Marielle J.; Ambepitiya Wickramasinghe, Iresha N.; de Haan, Cornelis A. M.; Yu, Wenli; McBride, Ryan; Sanders, Rogier W.; Woods, Robert J.; Verheije, Monique H.; Wilson, Ian A.; Paulson, James C.; Fernandez-Sesma, Ana

2017-06-15

The avian H7N9 influenza outbreak in 2013 resulted from an unprecedented incidence of influenza transmission to humans from infected poultry. The majority of human H7N9 isolates contained a hemagglutinin (HA) mutation (Q226L) that has previously been associated with a switch in receptor specificity from avian-type (NeuAcα2-3Gal) to human-type (NeuAcα2-6Gal), as documented for the avian progenitors of the 1957 (H2N2) and 1968 (H3N2) human influenza pandemic viruses. While this raised concern that the H7N9 virus was adapting to humans, the mutation was not sufficient to switch the receptor specificity of H7N9, and has not resulted in sustained transmission in humans. To determine if the H7 HA was capable of acquiring human-type receptor specificity, we conducted mutation analyses. Remarkably, three amino acid mutations conferred a switch in specificity for human-type receptors that resembled the specificity of the 2009 human H1 pandemic virus, and promoted binding to human trachea epithelial cells.

Three mutations switch H7N9 influenza to human-type receptor specificity.

Directory of Open Access Journals (Sweden)

Robert P de Vries

2017-06-01

Full Text Available The avian H7N9 influenza outbreak in 2013 resulted from an unprecedented incidence of influenza transmission to humans from infected poultry. The majority of human H7N9 isolates contained a hemagglutinin (HA mutation (Q226L that has previously been associated with a switch in receptor specificity from avian-type (NeuAcα2-3Gal to human-type (NeuAcα2-6Gal, as documented for the avian progenitors of the 1957 (H2N2 and 1968 (H3N2 human influenza pandemic viruses. While this raised concern that the H7N9 virus was adapting to humans, the mutation was not sufficient to switch the receptor specificity of H7N9, and has not resulted in sustained transmission in humans. To determine if the H7 HA was capable of acquiring human-type receptor specificity, we conducted mutation analyses. Remarkably, three amino acid mutations conferred a switch in specificity for human-type receptors that resembled the specificity of the 2009 human H1 pandemic virus, and promoted binding to human trachea epithelial cells.

Generation, Release, and Uptake of the NAD Precursor Nicotinic Acid Riboside by Human Cells.

Science.gov (United States)

Kulikova, Veronika; Shabalin, Konstantin; Nerinovski, Kirill; Dölle, Christian; Niere, Marc; Yakimov, Alexander; Redpath, Philip; Khodorkovskiy, Mikhail; Migaud, Marie E; Ziegler, Mathias; Nikiforov, Andrey

2015-11-06

NAD is essential for cellular metabolism and has a key role in various signaling pathways in human cells. To ensure proper control of vital reactions, NAD must be permanently resynthesized. Nicotinamide and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR) are the major precursors for NAD biosynthesis in humans. In this study, we explored whether the ribosides NR and NAR can be generated in human cells. We demonstrate that purified, recombinant human cytosolic 5'-nucleotidases (5'-NTs) CN-II and CN-III, but not CN-IA, can dephosphorylate the mononucleotides nicotinamide mononucleotide and nicotinic acid mononucleotide (NAMN) and thus catalyze NR and NAR formation in vitro. Similar to their counterpart from yeast, Sdt1, the human 5'-NTs require high (millimolar) concentrations of nicotinamide mononucleotide or NAMN for efficient catalysis. Overexpression of FLAG-tagged CN-II and CN-III in HEK293 and HepG2 cells resulted in the formation and release of NAR. However, NAR accumulation in the culture medium of these cells was only detectable under conditions that led to increased NAMN production from nicotinic acid. The amount of NAR released from cells engineered for increased NAMN production was sufficient to maintain viability of surrounding cells unable to use any other NAD precursor. Moreover, we found that untransfected HeLa cells produce and release sufficient amounts of NAR and NR under normal culture conditions. Collectively, our results indicate that cytosolic 5'-NTs participate in the conversion of NAD precursors and establish NR and NAR as integral constituents of human NAD metabolism. In addition, they point to the possibility that different cell types might facilitate each other's NAD supply by providing alternative precursors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Microsatellite typing of clinical and environmental Cryptococcus neoformans var. grubii isolates from Cuba shows multiple genetic lineages.

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Maria-Teresa Illnait-Zaragozi

Full Text Available BACKGROUND: Human cryptococcal infections have been associated with bird droppings as a likely source of infection. Studies toward the local and global epidemiology of Cryptococcus spp. have been hampered by the lack of rapid, discriminatory, and exchangeable molecular typing methods. METHODOLOGY/PRINCIPAL FINDINGS: We selected nine microsatellite markers for high-resolution fingerprinting from the genome of C. neoformans var. grubii. This panel of markers was applied to a collection of clinical (n = 122 and environmental (n = 68; from pigeon guano C. neoformans var. grubii isolates from Cuba. All markers proved to be polymorphic. The average number of alleles per marker was 9 (range 5-51. A total of 104 genotypes could be distinguished. The discriminatory power of this panel of markers was 0.993. Multiple clusters of related genotypes could be discriminated that differed in only one or two microsatellite markers. These clusters were assigned as microsatellite complexes. The majority of environmental isolates (>70% fell into 1 microsatellite complex containing only few clinical isolates (49 environmental versus 2 clinical. Clinical isolates were segregated over multiple microsatellite complexes. CONCLUSIONS/SIGNIFICANCE: A large genotypic variation exists in C. neoformans var. grubii. The genotypic segregation between clinical and environmental isolates from pigeon guano suggests additional source(s of human cryptococcal infections. The selected panel of microsatellite markers is an excellent tool to study the epidemiology of C. neoformans var. grubii.

DRUMS: a human disease related unique gene mutation search engine.

Science.gov (United States)

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

2011-10-01

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 http://www.scbit.org/glif/new/drums/index.html. © 2011 Wiley-Liss, Inc.

Use of human tissue to assess the oncogenic activity of melanoma-associated mutations.

Science.gov (United States)

Chudnovsky, Yakov; Adams, Amy E; Robbins, Paul B; Lin, Qun; Khavari, Paul A

2005-07-01

Multiple genetic alterations occur in melanoma, a lethal skin malignancy of increasing incidence. These include mutations that activate Ras and two of its effector cascades, Raf and phosphoinositide 3-kinase (PI3K). Induction of Ras and Raf can be caused by active N-Ras and B-Raf mutants as well as by gene amplification. Activation of PI3K pathway components occurs by PTEN loss and by AKT3 amplification. Melanomas also commonly show impairment of the p16(INK4A)-CDK4-Rb and ARF-HDM2-p53 tumor suppressor pathways. CDKN2A mutations can produce p16(INK4A) and ARF protein loss. Rb bypass can also occur through activating CDK4 mutations as well as by CDK4 amplification. In addition to ARF deletion, p53 pathway disruption can result from dominant negative TP53 mutations. TERT amplification also occurs in melanoma. The extent to which these mutations can induce human melanocytic neoplasia is unknown. Here we characterize pathways sufficient to generate human melanocytic neoplasia and show that genetically altered human tissue facilitates functional analysis of mutations observed in human tumors.

Clinical and biochemical function of polymorphic NR0B1 GGAA-microsatellites in Ewing sarcoma: a report from the Children's Oncology Group.

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Michael J Monument

Full Text Available The genetics involved in Ewing sarcoma susceptibility and prognosis are poorly understood. EWS/FLI and related EWS/ETS chimeras upregulate numerous gene targets via promoter-based GGAA-microsatellite response elements. These microsatellites are highly polymorphic in humans, and preliminary evidence suggests EWS/FLI-mediated gene expression is highly dependent on the number of GGAA motifs within the microsatellite.Here we sought to examine the polymorphic spectrum of a GGAA-microsatellite within the NR0B1 promoter (a critical EWS/FLI target in primary Ewing sarcoma tumors, and characterize how this polymorphism influences gene expression and clinical outcomes.A complex, bimodal pattern of EWS/FLI-mediated gene expression was observed across a wide range of GGAA motifs, with maximal expression observed in constructs containing 20-26 GGAA motifs. Relative to white European and African controls, the NR0B1 GGAA-microsatellite in tumor cells demonstrated a strong bias for haplotypes containing 21-25 GGAA motifs suggesting a relationship between microsatellite function and disease susceptibility. This selection bias was not a product of microsatellite instability in tumor samples, nor was there a correlation between NR0B1 GGAA-microsatellite polymorphisms and survival outcomes.These data suggest that GGAA-microsatellite polymorphisms observed in human populations modulate EWS/FLI-mediated gene expression and may influence disease susceptibility in Ewing sarcoma.

Segmenting the human genome based on states of neutral genetic divergence.

Science.gov (United States)

Kuruppumullage Don, Prabhani; Ananda, Guruprasad; Chiaromonte, Francesca; Makova, Kateryna D

2013-09-03

Many studies have demonstrated that divergence levels generated by different mutation types vary and covary across the human genome. To improve our still-incomplete understanding of the mechanistic basis of this phenomenon, we analyze several mutation types simultaneously, anchoring their variation to specific regions of the genome. Using hidden Markov models on insertion, deletion, nucleotide substitution, and microsatellite divergence estimates inferred from human-orangutan alignments of neutrally evolving genomic sequences, we segment the human genome into regions corresponding to different divergence states--each uniquely characterized by specific combinations of divergence levels. We then parsed the mutagenic contributions of various biochemical processes associating divergence states with a broad range of genomic landscape features. We find that high divergence states inhabit guanine- and cytosine (GC)-rich, highly recombining subtelomeric regions; low divergence states cover inner parts of autosomes; chromosome X forms its own state with lowest divergence; and a state of elevated microsatellite mutability is interspersed across the genome. These general trends are mirrored in human diversity data from the 1000 Genomes Project, and departures from them highlight the evolutionary history of primate chromosomes. We also find that genes and noncoding functional marks [annotations from the Encyclopedia of DNA Elements (ENCODE)] are concentrated in high divergence states. Our results provide a powerful tool for biomedical data analysis: segmentations can be used to screen personal genome variants--including those associated with cancer and other diseases--and to improve computational predictions of noncoding functional elements.

KRAS mutations and CDKN2A promoter methylation show an interactive adverse effect on survival and predict recurrence of rectal cancer.

Science.gov (United States)

Kohonen-Corish, Maija R J; Tseung, Jason; Chan, Charles; Currey, Nicola; Dent, Owen F; Clarke, Stephen; Bokey, Les; Chapuis, Pierre H

2014-06-15

Colonic and rectal cancers differ in their clinicopathologic features and treatment strategies. Molecular markers such as gene methylation, microsatellite instability and KRAS mutations, are becoming increasingly important in guiding treatment decisions in colorectal cancer. However, their association with clinicopathologic variables and utility in the management of rectal cancer is still poorly understood. We analyzed CDKN2A gene methylation, CpG island methylator phenotype (CIMP), microsatellite instability and KRAS/BRAF mutations in a cohort of 381 rectal cancers with extensive clinical follow-up data. BRAF mutations (2%), CIMP-high (4%) and microsatellite instability-high (2%) were rare, whereas KRAS mutations (39%), CDKN2A methylation (20%) and CIMP-low (25%) were more common. Only CDKN2A methylation and KRAS mutations showed an association with poor overall survival but these did not remain significant when analyzed with other clinicopathologic factors. In contrast, this prognostic effect was strengthened by the joint presence of CDKN2A methylation and KRAS mutations, which independently predicted recurrence of cancer and was associated with poor overall and cancer-specific survival. This study has identified a subgroup of more aggressive rectal cancers that may arise through the KRAS-p16 pathway. It has been previously shown that an interaction of p16 deficiency and oncogenic KRAS promotes carcinogenesis in the mouse and is characterized by loss of oncogene-induced senescence. These findings may provide avenues for the discovery of new treatments in rectal cancer. © 2013 UICC.

Colorectal cancer among young native Indonesians: A clinicopathological and molecular assessment on microsatellite instability

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Aru W. Sudoyo

2010-11-01

Full Text Available Aim: To obtain clinicopathological characteristics of colorectal cancer among young native Indonesians and to assess MLH1, MSH2, and SMAD4 protein expressions, comparing them with a matched population of colorectal cancer patients aged 60 years old and older.Methods: Medical records of colorectal cancer patients aged 40 years or younger and 60 years or older from several hospitals in three Indonesian cities – Jakarta, Makassar, and Bandung - were reviewed. The “native” ethnic groups were selected from those originating from Java, Makassar (South Celebes,  Minangkabau (West Sumatra. Ethnicity of 121 colorectal  carcinoma patients was confirmed by fulfilling requirements in a questionnaire. Tumor specimens of those patients underwent evaluation for histopathology, tumor grading as well as  immunohistochemical analysis to assess MLH1, MSH2 protein expressions to detect microsatellite instability mutation pathway and SMAD4 protein expression to reconfirm that the specimens were not microsatellite instability origin.Results: There were 121 colorectal carcinoma cases of Sundanese, Javanese, Macassarese and Minangkabau ethnic group. This study indicated that colorectal cancer has statistically different grade (p = 0.001 between the young and the older patients. Immunohistochemical staining for MSH2 protein and MLH1 were done for 92 and 97 specimens respectively. There was no significant difference between the expressions of MLH1 and MSH2 on tumor grading, indicated there was no correlation between microsatellite instability and tumor grading in this study.Conclusion: Colorectal cancer in young native Indonesian patients (40 years old or less was not different in clinicopathological characteristics compared to older patients (60 years old or more in similar ethnic groups. There was also no difference in MSH2 and MLH1 protein expressions, important indicators of microsatellite instability and. (Med J Indones 2010; 19:245-51Keywords: colorectal

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

Science.gov (United States)

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

2017-08-01

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

Microsatellite characterization of Cimarron Uruguayo dogs

OpenAIRE

Gagliardi,Rosa; Silvia,Llambí; García,Cristina; Arruga,María Victoria

2011-01-01

Various genetic markers, including microsatellites, have been used to analyze the genetic polymorphism and heterozygosity in canine breeds. In this work, we used nine microsatellite markers to investigate the genetic variability in Cimarron Uruguayo dogs, the only officially recognized native canine breed in Uruguay. DNA from 30 Cimarron Uruguayo dogs from northeastern and southern Uruguay was analyzed. The allelic frequencies for each micro-satellite, the genetic variability and the consangu...

Context-dependent interpretation of the prognostic value of BRAF and KRAS mutations in colorectal cancer

International Nuclear Information System (INIS)

Popovici, Vlad; Budinska, Eva; Bosman, Fred T; Tejpar, Sabine; Roth, Arnaud D; Delorenzi, Mauro

2013-01-01

The mutation status of the BRAF and KRAS genes has been proposed as prognostic biomarker in colorectal cancer. Of them, only the BRAF V600E mutation has been validated independently as prognostic for overall survival and survival after relapse, while the prognostic value of KRAS mutation is still unclear. We investigated the prognostic value of BRAF and KRAS mutations in various contexts defined by stratifications of the patient population. We retrospectively analyzed a cohort of patients with stage II and III colorectal cancer from the PETACC-3 clinical trial (N = 1,423), by assessing the prognostic value of the BRAF and KRAS mutations in subpopulations defined by all possible combinations of the following clinico-pathological variables: T stage, N stage, tumor site, tumor grade and microsatellite instability status. In each such subpopulation, the prognostic value was assessed by log rank test for three endpoints: overall survival, relapse-free survival, and survival after relapse. The significance level was set to 0.01 for Bonferroni-adjusted p-values, and a second threshold for a trend towards statistical significance was set at 0.05 for unadjusted p-values. The significance of the interactions was tested by Wald test, with significance level of 0.05. In stage II-III colorectal cancer, BRAF mutation was confirmed a marker of poor survival only in subpopulations involving microsatellite stable and left-sided tumors, with higher effects than in the whole population. There was no evidence for prognostic value in microsatellite instable or right-sided tumor groups. We found that BRAF was also prognostic for relapse-free survival in some subpopulations. We found no evidence that KRAS mutations had prognostic value, although a trend was observed in some stratifications. We also show evidence of heterogeneity in survival of patients with BRAF V600E mutation. The BRAF mutation represents an additional risk factor only in some subpopulations of colorectal cancers, in

Abundance and Characterization of Perfect Microsatellites on the Cattle Y Chromosome.

Science.gov (United States)

Ma, Zhi-Jie

2017-07-03

Microsatellites or simple sequence repeats (SSRs) are found in most organisms and play an important role in genomic organization and function. To characterize the abundance of SSRs (1-6 base-pairs [bp]) on the cattle Y chromsome, the relative frequency and density of perfect or uninterrupted SSRs based on the published Y chromosome sequence were examined. A total of 17,273 perfect SSRs were found, with total length of 324.78 kb, indicating that approximately 0.75% of the cattle Y chromosome sequence (43.30 Mb) comprises perfect SSRs, with an average length of 18.80 bp. The relative frequency and density were 398.92 loci/Mb and 7500.62 bp/Mb, respectively. The proportions of the six classes of perfect SSRs were highly variable on the cattle Y chromosome. Mononucleotide repeats had a total number of 8073 (46.74%) and an average length of 15.45 bp, and were the most abundant SSRs class, while the percentages of di-, tetra-, tri-, penta-, and hexa-nucleotide repeats were 22.86%, 11.98%, 11.58%, 6.65%, and 0.19%, respectively. Different classes of SSRs varied in their repeat number, with the highest being 42 for dinucleotides. Results reveal that repeat categories A, AC, AT, AAC, AGC, GTTT, CTTT, ATTT, and AACTG predominate on the Y chromosome. This study provides insight into the organization of cattle Y chromosome repetitive DNA, as well as information useful for developing more polymorphic cattle Y-chromosome-specific SSRs.

Variation of mutational burden in healthy human tissues suggests non-random strand segregation and allows measuring somatic mutation rates.

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Werner, Benjamin; Sottoriva, Andrea

2018-06-01

The immortal strand hypothesis poses that stem cells could produce differentiated progeny while conserving the original template strand, thus avoiding accumulating somatic mutations. However, quantitating the extent of non-random DNA strand segregation in human stem cells remains difficult in vivo. Here we show that the change of the mean and variance of the mutational burden with age in healthy human tissues allows estimating strand segregation probabilities and somatic mutation rates. We analysed deep sequencing data from healthy human colon, small intestine, liver, skin and brain. We found highly effective non-random DNA strand segregation in all adult tissues (mean strand segregation probability: 0.98, standard error bounds (0.97,0.99)). In contrast, non-random strand segregation efficiency is reduced to 0.87 (0.78,0.88) in neural tissue during early development, suggesting stem cell pool expansions due to symmetric self-renewal. Healthy somatic mutation rates differed across tissue types, ranging from 3.5 × 10-9/bp/division in small intestine to 1.6 × 10-7/bp/division in skin.

Variation of mutational burden in healthy human tissues suggests non-random strand segregation and allows measuring somatic mutation rates.

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Benjamin Werner

2018-06-01

Full Text Available The immortal strand hypothesis poses that stem cells could produce differentiated progeny while conserving the original template strand, thus avoiding accumulating somatic mutations. However, quantitating the extent of non-random DNA strand segregation in human stem cells remains difficult in vivo. Here we show that the change of the mean and variance of the mutational burden with age in healthy human tissues allows estimating strand segregation probabilities and somatic mutation rates. We analysed deep sequencing data from healthy human colon, small intestine, liver, skin and brain. We found highly effective non-random DNA strand segregation in all adult tissues (mean strand segregation probability: 0.98, standard error bounds (0.97,0.99. In contrast, non-random strand segregation efficiency is reduced to 0.87 (0.78,0.88 in neural tissue during early development, suggesting stem cell pool expansions due to symmetric self-renewal. Healthy somatic mutation rates differed across tissue types, ranging from 3.5 × 10-9/bp/division in small intestine to 1.6 × 10-7/bp/division in skin.

Genome-wide characterization of microsatelittes and marker development in the carcinogenic liver fluke Clonorchis sinensis

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Nguyen, Thao T.B.; Arimatsu, Yuji; Hong, Sung-Jong; Brindley, Paul J.; Blair, David; Laha, Thewarach; Sripa, Banchob

2015-01-01

Clonorchis sinensis is an important carcinogenic human liver fluke endemic in East and Southeast Asia. There are several conventional molecular markers have been used for identification and genetic diversity, however, no information about microsatellites of this liver fluke published so far. We here report microsatellite characterization and marker development for genetic diversity study in C. sinensis using genome-wide bioinformatics approach. Based on our search criteria, a total of 256,990 microsatellites (≥ 12 base pairs) were identified from genome database of C. sinensis with hexa-nucleotide motif being the most abundant (51%) followed by penta-nucleotide (18.3%) and tri-nucleotide (12.7%). The tetra-nucleotide, di-nucleotide and mononucleotide motifs accounted for 9.75 %, 7.63% and 0.14%, respectively. The total length of all microsatellites accounts for 0. 72 % of 547 Mb of the whole genome size and the frequency of microsatellites were found to be one microsatellite in every 2.13 kb of DNA. For the di-, tri, and tetra-nucleotide, the repeat numbers redundant are six (28%), four (45%) and three (76%), respectively. The ATC repeat is the most abundant microsatellites followed by AT, AAT and AC, respectively. Within 40 microsatellite loci developed, 24 microsatellite markers showed potential to differentiate between C. sinensis and O. viverrini. Seven out of 24 loci showed heterozygous with observed heterozygosity ranged from 0.467 to 1. Four-primer sets could amplify both C. sinensis and O. viverrini DNA with different sizes. This study provides basic information of C. sinensis microsatellites and the genome-wide markers developed may be a useful tool for genetic study of C. sinensis. PMID:25782682

The rate of spontaneous mutations in human myeloid cells

International Nuclear Information System (INIS)

Araten, David J.; Krejci, Ondrej; DiTata, Kimberly; Wunderlich, Mark; Sanders, Katie J.; Zamechek, Leah; Mulloy, James C.

2013-01-01

Highlights: • We provide the first measurement of the mutation rate (μ) in human myeloid cells. • μ is measured to be 3.6–23 × 10 −7 per cell division. • The AML-ETO and MLL-AF9 fusions do not seem to increase μ. • Cooperating mutations in NRAS, FLT3 and p53 not seem to increase μ. • Hypermutability may be required to explain leukemogenesis. - Abstract: The mutation rate (μ) is likely to be a key parameter in leukemogenesis, but historically, it has been difficult to measure in humans. The PIG-A gene has some advantages for the detection of spontaneous mutations because it is X-linked, and therefore only one mutation is required to disrupt its function. Furthermore, the PIG-A-null phenotype is readily detected by flow cytometry. Using PIG-A, we have now provided the first in vitro measurement of μ in myeloid cells, using cultures of CD34+ cells that are transduced with either the AML-ETO or the MLL-AF9 fusion genes and expanded with cytokines. For the AML-ETO cultures, the median μ value was ∼9.4 × 10 −7 (range ∼3.6–23 × 10 −7 ) per cell division. In contrast, few spontaneous mutations were observed in the MLL-AF9 cultures. Knockdown of p53 or introduction of mutant NRAS or FLT3 alleles did not have much of an effect on μ. Based on these data, we provide a model to predict whether hypermutability must occur in the process of leukemogenesis

Two truncating USH3A mutations, including one novel, in a German family with Usher syndrome.

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Ebermann, Inga; Wilke, Robert; Lauhoff, Thomas; Lübben, Dirk; Zrenner, Eberhart; Bolz, Hanno Jörn

2007-08-30

To identify the genetic defect in a German family with Usher syndrome (USH) and linkage to the USH3A locus. DNA samples of five family members (both parents and the three patients) were genotyped with polymorphic microsatellite markers specific for eight USH genes. Three affected family members underwent detailed ocular and audiologic characterization. Symptoms in the patients were compatible with Usher syndrome and show intrafamilial variation, for both hearing loss (ranging from severe to profound with non-linear progression) and vision. Genotyping of microsatellite markers for the different USH loci was in line with a defect in the USH3A gene on chromosome 3q25. Sequence analysis of the USH3A gene revealed two truncating mutations; c.149_152delCAGGinsTGTCCAAT, which has been described previously, and a novel mutation, c.502_503insA, segregating with the phenotype. To date, only 11 USH3A mutations have been described. This is the first description of a German family with USH due to USH3A mutations, including one novel. Our findings indicate that also in the Central European population, USH3A mutations should be considered in cases of USH.

Aneuploidy in immortalized human mesenchymal stem cells with non-random loss of chromosome 13 in culture.

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Takeuchi, Masao; Takeuchi, Kikuko; Ozawa, Yutaka; Kohara, Akihiro; Mizusawa, Hiroshi

2009-01-01

Aneuploidy (an abnormal number of chromosomes) is commonly observed in most human cancer cells, highlighting the need to examine chromosomal instability in tumorigenesis. Previously, the immortalized human mesenchymal stem cell line UE6E7T-3 was shown to undergo a preferential loss of one copy of chromosome 13 after prolonged culture. Here, the loss of chromosome 13 was found to be caused by chromosome missegregation during mitosis, which involved unequal segregation, exclusion of the misaligned chromosome 13 on the metaphase plate, and trapping of chromosome 13 in the midbody region, as observed by fluorescence in situ hybridization. Near-diploid aneuploidy, not tetraploidy, was the direct result. The loss of chromosome 13 was non-random, and was detected by analysis of microsatellites and single nucleotide polymorphism-based loss of heterozygosity (LOH). Of the five microsatellite loci on chromosome 13, four loci showed microsatellite instability at an early stage in culture, and LOH was apparent at a late stage in culture. These results suggest that the microsatellite mutations cause changes in centromere integrity provoking loss of this chromosome in the UE6E7T-3 cell line. Thus, these results support the use of this cell line as a useful model for understanding the mechanism of aneuploid formation in cell cultures.

[Analysis of genetic polymorphisms and mutations of 20 frequently used STR loci among ethnic Hans from Henan].

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Wang, Hongdan; Kang, Bing; Gao, Yue; Huo, Xiaodong; Li, Tao; Guo, Qiannan; Zhu, Bofeng; Liao, Shixiu

2017-04-10

To study the genetic polymorphisms and mutations of 20 frequently used autosomal microsatellites among ethnic Hans from Henan. Peripheral blood samples of 2604 individuals were collected. DNA was amplified and genotyped using a PowerPlex(TM) 21 system. The frequencies, forensic parameters and mutation rates of the 20 short tandem repeat (STR) loci were analyzed. A total of 323 alleles were found in this population and the allelic frequencies have ranged from 0.0003 to 0.5144. Except for D3S1358, TH01 and TPOX, mutations have been found in all of the remaining 17 STR loci, which totaled 47, with mutation rates ranging from 0 to 3.46 × 10 -3 . The 20 STR loci selected by the PowerPlex(TM) 21 system are highly polymorphic among ethnic Hans from Henan, and may be of great value in forensic and human population studies. As no similar study has been carried out previously, above results may be of great value for individual discrimination and paternal testing.

Prospects for DNA methods to measure human heritable mutation rates

International Nuclear Information System (INIS)

Mendelsohn, M.L.

1985-01-01

A workshop cosponsored by ICPEMC and the US Department of Energy was held in Alta, Utah, December 9-13, 1984 to examine the extent to which DNA-oriented methods might provide new approaches to the important but intractable problem of measuring mutation rates in control and exposed human populations. The workshop identified and analyzed six DNA methods for detection of human heritable mutation, including several created at the meeting, and concluded that none of the methods combine sufficient feasibility and efficiency to be recommended for general application. 8 refs

Detection of mismatch repair gene germline mutation carrier among Chinese population with colorectal cancer

International Nuclear Information System (INIS)

Jin, Hei-Ying; Zhao, Ronghua; Liu, Xiufang; Li, Vicky Ka Ming; Ding, Yijiang; Yang, Bolin; Geng, Jianxiang; Lai, Rensheng; Ding, Shuqing; Ni, Min

2008-01-01

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant syndrome. The National Cancer Institute (NCI) has recommended the Revised Bethesda guidelines for screening HNPCC. There has been a great deal of research on the value of these tests in other countries. However, literature about the Chinese population is scarce. Our objective is to detect and study microsatellite instability (MSI) and mismatch repair (MMR) gene germline mutation carriers among a Chinese population with colorectal cancer. In 146 prospectively recruited consecutive patients with clinically proven colorectal cancer, MSI carriers were identified by analysis of tumor tissue using multiplex fluorescence polymerase chain reaction (PCR) using the NCI recommended panel and classified into microsatellite instability-low (MSI-L), microsatellite instability-high (MSI-H) and microsatellite stable (MSS) groups. Immunohistochemical staining for MSH2, MSH6 and MLH1 on tissue microarrays (TMAs) was performed, and methylation of the MLH1 promoter was analyzed by quantitative methylation specific PCR (MSP). Germline mutation analysis of blood samples was performed for MSH2, MSH6 and MLH1 genes. Thirty-four out of the 146 colorectal cancers (CRCs, 23.2%) were MSI, including 19 MSI-H CRCs and 15 MSI-L CRCS. Negative staining for MSH2 was found in 8 CRCs, negative staining for MSH6 was found in 6 CRCs. One MSI-H CRC was negative for both MSH6 and MSH2. Seventeen CRCs stained negatively for MLH1. MLH1 promoter methylation was determined in 34 MSI CRCs. Hypermethylation of the MLH1 promoter occurred in 14 (73.7%) out of 19 MSI-H CRCs and 5 (33.3%) out of 15 MSI-L CRCs. Among the 34 MSI carriers and one MSS CRC with MLH1 negative staining, 8 had a MMR gene germline mutation, which accounted for 23.5% of all MSI colorectal cancers and 5.5% of all the colorectal cancers. Five patients harbored MSH2 germline mutations, and three patients harbored MSH6 germline mutations. None of the patients had an MLH

Functionally relevant microsatellites in sugarcane unigenes

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Singh Nagendra K

2010-11-01

Full Text Available Abstract Background Unigene sequences constitute a rich source of functionally relevant microsatellites. The present study was undertaken to mine the microsatellites in the available unigene sequences of sugarcane for understanding their constitution in the expressed genic component of its complex polyploid/aneuploid genome, assessing their functional significance in silico, determining the extent of allelic diversity at the microsatellite loci and for evaluating their utility in large-scale genotyping applications in sugarcane. Results The average frequency of perfect microsatellite was 1/10.9 kb, while it was 1/44.3 kb for the long and hypervariable class I repeats. GC-rich trinucleotides coding for alanine and the GA-rich dinucleotides were the most abundant microsatellite classes. Out of 15,594 unigenes mined in the study, 767 contained microsatellite repeats and for 672 of these putative functions were determined in silico. The microsatellite repeats were found in the functional domains of proteins encoded by 364 unigenes. Its significance was assessed by establishing the structure-function relationship for the beta-amylase and protein kinase encoding unigenes having repeats in the catalytic domains. A total of 726 allelic variants (7.42 alleles per locus with different repeat lengths were captured precisely for a set of 47 fluorescent dye labeled primers in 36 sugarcane genotypes and five cereal species using the automated fragment analysis system, which suggested the utility of designed primers for rapid, large-scale and high-throughput genotyping applications in sugarcane. Pair-wise similarity ranging from 0.33 to 0.84 with an average of 0.40 revealed a broad genetic base of the Indian varieties in respect of functionally relevant regions of the large and complex sugarcane genome. Conclusion Microsatellite repeats were present in 4.92% of sugarcane unigenes, for most (87.6% of which functions were determined in silico. High level of

Novel NTRK1 mutations cause hereditary sensory and autonomic neuropathy type IV: demonstration of a founder mutation in the Turkish population.

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Tüysüz, Beyhan; Bayrakli, Fatih; DiLuna, Michael L; Bilguvar, Kaya; Bayri, Yasar; Yalcinkaya, Cengiz; Bursali, Aysegul; Ozdamar, Elif; Korkmaz, Baris; Mason, Christopher E; Ozturk, Ali K; Lifton, Richard P; State, Matthew W; Gunel, Murat

2008-05-01

Hereditary sensory and autonomic neuropathy type IV (HSAN IV), or congenital insensitivity to pain with anhidrosis, is an autosomal recessive disorder characterized by insensitivity to noxious stimuli, anhidrosis from deinnervated sweat glands, and delayed mental and motor development. Mutations in the neurotrophic tyrosine kinase receptor type 1 (NTRK1), a receptor in the neurotrophin signaling pathway phosphorylated in response to nerve growth factor, are associated with this disorder. We identified six families from Northern Central Turkey with HSAN IV. We screened the NTRK1 gene for mutations in these families. Microsatellite and single nucleotide polymorphism (SNP) markers on the Affymetrix 250K chip platform were used to determine the haplotypes for three families harboring the same mutation. Screening for mutations in the NTRK1 gene demonstrated one novel frameshift mutation, two novel nonsense mutations, and three unrelated kindreds with the same splice-site mutation. Genotyping of the three families with the identical splice-site mutation revealed that they share the same haplotype. This report broadens the spectrum of mutations in NTRK1 that cause HSAN IV and demonstrates a founder mutation in the Turkish population.

Positive selection pressure introduces secondary mutations at Gag cleavage sites in human immunodeficiency virus type 1 harboring major protease resistance mutations

DEFF Research Database (Denmark)

Banke, S.; Lillemark, M.R.; Gerstoft, J.

2009-01-01

Human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) specifically target the HIV-1 protease enzyme. Mutations in the enzyme can result in PI resistance (termed PI mutations); however, mutations in the HIV-1 gag region, the substrate for the protease enzyme, might also lead to PI ...

Immortalized human myotonic dystrophy muscle cell lines to assess therapeutic compounds

OpenAIRE

Arandel, Ludovic; Polay Espinoza, Micaela; Matloka, Magdalena; Bazinet, Audrey; De Dea Diniz, Damily; Naouar, Na?ra; Rau, Fr?d?rique; Jollet, Arnaud; Edom-Vovard, Fr?d?rique; Mamchaoui, Kamel; Tarnopolsky, Mark; Puymirat, Jack; Battail, Christophe; Boland, Anne; Deleuze, Jean-Francois

2017-01-01

International audience; Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are autosomal dominant neuromuscular diseases caused by microsatellite expansions and belong to the family of RNA-dominant disorders. Availability of cellular models in which the DM mutation is expressed within its natural context is essential to facilitate efforts to identify new therapeutic compounds. Here, we generated immortalized DM1 and DM2 human muscle cell lines that display nuclear RNA aggregates of expanded rep...

Solving the mystery of human sleep schedules one mutation at a time.

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Hallows, William C; Ptáček, Louis J; Fu, Ying-Hui

2013-01-01

Sleep behavior remains one of the most enigmatic areas of life. The unanswered questions range from "why do we sleep?" to "how we can improve sleep in today's society?" Identification of mutations responsible for altered circadian regulation of human sleep lead to unique opportunities for probing these territories. In this review, we summarize causative circadian mutations found from familial genetic studies to date. We also describe how these mutations mechanistically affect circadian function and lead to altered sleep behaviors, including shifted or shortening of sleep patterns. In addition, we discuss how the investigation of mutations can not only expand our understanding of the molecular mechanisms regulating the circadian clock and sleep duration, but also bridge the pathways between clock/sleep and other human physiological conditions and ailments such as metabolic regulation and migraine headaches.

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

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

2009-06-01

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

Computational Mining and Genome Wide Distribution of Microsatellite in Fusarium oxysporum f. sp. lycopersici

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Sudheer KUMAR

2012-11-01

Full Text Available Simple sequence repeat (SSR is currently the most preferred molecular marker system owing to their highly desirable properties viz., abundance, hyper-variability, and suitability for high-throughput analysis. Hence, in present study an attempt was made to mine and analyze microsatellite dynamics in whole genome of Fusarium oxysporum f. sp. lycopersici. The distribution pattern of different SSR motifs provides the evidence of greater accumulation of tetra-nucleotide (3837 repeats followed by tri-nucleotide (3367 repeats. Maximum frequency distribution in coding region was shown by mono-nucleotide SSR motifs (34.8%, where as minimum frequency is observed for penta-nucleotide SSR (0.87%. Highest relative abundance (1023 SSR/Mb and density of SSRs (114.46 bp/Mb were observed on chromosome 1, while least density of SSR motifs was recorded on chromosome 11 (7.40 bp/Mb and 12 (7.41 bp/Mb, respectively. Maximum trinucleotide (34.24% motifs code for glutamic acid (GAA while GT/CT were the most frequent repeat of dinucleotide SSRs. Most common and highly repeated SSR motifs were identified as (A64, (T48, (GT24, (GAA31, (TTTC24, (TTTCT28 and (AACCAG27. Overall, the generated information may serve as baseline information for developing SSR markers that could find applications in genomic analysis of F. oxysporum f. sp. lycopersici for better understanding of evolution, diversity analysis, population genetics, race identification and acquisition of new virulence.

Mutation at the Human D1S80 Minisatellite Locus

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Kuppareddi Balamurugan

2012-01-01

Full Text Available Little is known about the general biology of minisatellites. The purpose of this study is to examine repeat mutations from the D1S80 minisatellite locus by sequence analysis to elucidate the mutational process at this locus. This is a highly polymorphic minisatellite locus, located in the subtelomeric region of chromosome 1. We have analyzed 90,000 human germline transmission events and found seven (7 mutations at this locus. The D1S80 alleles of the parentage trio, the child, mother, and the alleged father were sequenced and the origin of the mutation was determined. Using American Association of Blood Banks (AABB guidelines, we found a male mutation rate of 1.04×10-4 and a female mutation rate of 5.18×10-5 with an overall mutation rate of approximately 7.77×10-5. Also, in this study, we found that the identified mutations are in close proximity to the center of the repeat array rather than at the ends of the repeat array. Several studies have examined the mutational mechanisms of the minisatellites according to infinite allele model (IAM and the one-step stepwise mutation model (SMM. In this study, we found that this locus fits into the one-step mutation model (SMM mechanism in six out of seven instances similar to STR loci.

Somatic gene mutation in the human in relation to radiation risk

International Nuclear Information System (INIS)

Mendelsohn, M.L.

1992-01-01

This report discusses the measurement of somatic gene-mutation frequencies in the human. We ask the following questions. How well can they be measured? Do they respond to radiation? Can they also function as a dosimeter? What do they tell us about the somatic mutation theory of carcinogenesis?

Different mutations of the human c-mpl gene indicate distinct haematopoietic diseases.

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He, Xin; Chen, Zhigang; Jiang, Yangyan; Qiu, Xi; Zhao, Xiaoying

2013-01-25

The human c-mpl gene (MPL) plays an important role in the development of megakaryocytes and platelets as well as the self-renewal of haematopoietic stem cells. However, numerous MPL mutations have been identified in haematopoietic diseases. These mutations alter the normal regulatory mechanisms and lead to autonomous activation or signalling deficiencies. In this review, we summarise 59 different MPL mutations and classify these mutations into four different groups according to the associated diseases and mutation rates. Using this classification, we clearly distinguish four diverse types of MPL mutations and obtain a deep understand of their clinical significance. This will prove to be useful for both disease diagnosis and the design of individual therapy regimens based on the type of MPL mutations.

MutHTP: Mutations in Human Transmembrane Proteins.

Science.gov (United States)

A, Kulandaisamy; S, Binny Priya; R, Sakthivel; Tarnovskaya, Svetlana; Bizin, Ilya; Hönigschmid, Peter; Frishman, Dmitrij; Gromiha, M Michael

2018-02-01

We have developed a novel database, MutHTP, which contains information on 183395 disease-associated and 17827 neutral mutations in human transmembrane proteins. For each mutation site MutHTP provides a description of its location with respect to the membrane protein topology, structural environment (if available) and functional features. Comprehensive visualization, search, display and download options are available. The database is publicly available at http://www.iitm.ac.in/bioinfo/MutHTP/. The website is implemented using HTML, PHP and javascript and supports recent versions of all major browsers, such as Firefox, Chrome and Opera. gromiha@iitm.ac.in. Supplementary data are available at Bioinformatics online. © The Author (2018). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

PKD2 mutation in an Iranian autosomal dominant polycystic kidney disease family with misleading linkage analysis data

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Mona Entezam

2016-06-01

Conclusion: Although analysis of additive informative polymorphic markers can overcome the misleading haplotype data, it is limited because of the lack of other highly polymorphic microsatellite markers closer to the gene. Direct mutation screening can identify the causative mutation in the apparently unlinked pedigree; moreover, it is the only approach to achieve the confirmed diagnosis in individuals with equivocal imaging results.

Retrospective genetic study of germinative mutations in Str loci of individuals potentially exposed to ionizing radiation;Estudo genetico retrospectivo de mutacoes germinativas em Loci Str de individuos potencialmente expostos a radiacao ionizante

Energy Technology Data Exchange (ETDEWEB)

Costa, Emilia Oliveira Alves

2010-07-01

The Brazilian radiological accident that occurred in 1987, in Goiania, it was a terrible radiation episode. As a consequence, hundreds of people were contaminated due to the Cesium-137 radiation. Recently, many studies had shown that genome instabilities, such as, mutations, chromosomal aberrations, micronuclei formation and micro satellite instability and a delay on cellular death are usually reported on mammal cells exposed to ionizing radiation, being considered as a manly risk to humans. Mutations can be spontaneous, and the occurrence is dependent on the organism, or, induced, being associated to mutagenic exposition. Ionizing radiations are an example of physical and mutagenic agents that could harm the cell repair and could cause the development of many types of cancer. The evaluation of the biological effects of the ionizing radiation, in somatic and germ line cells, with a consequent determination of the radio-induced mutations, it is extremely important to estimate the genetic risks, manly in population exposed to radiation. The analyses of repetitive DNA sequences have been demonstrated that such sequences are prone to high rates of spontaneous mutations. The minisatellites and microsatellites have been used to demonstrate the induction of germ line mutation rates on mouse, humans, among others organisms. The aim of the present study was to analyze the frequency of microsatellite alterations to determine the mutation rates occurred in germ cells of the parents exposed to the ionizing radiation of the Cesium-137. The studied group was constitute of 10 families of individuals accidentally exposed to Cesium-137 and by the control group constituted by 645 healthy individuals who carried out paternity tests on 2009. We found only one mutation of paternal origin in the D8S1179 locus on the exposed group, being the mutation rate of 0.002. In the control group, we found 01 mutation on D16S539 loei and on D3S1358; 02 mutations on Penta E loeus; 04 mutations on D

Development of a human somatic mutation detection method--GPA assay

International Nuclear Information System (INIS)

Mao Jianping; Dong Yan; Liu Bin; Lin Ruxian; Sun Zhixian

2000-01-01

Objective: To study the damage to human body caused by environmental radiation, and supervise the somatic mutations. Methods: Three monoclonal antibodies specific to M-type(3G4), N-type(6A8), and MN-type (3C5) of glycophorin A, respectively, were prepared. Fluorescence or biotin conjugated antibodies were bound specifically to formalin and/or dimethyl suber-imidate fixed erythrocytes. M, MN and N type cells were divided by cytometry to demonstrate the erythrocyte mutation characteristics (MN→MO, MM, NO, NN) and give out the variant frequency. Results: 1Wa, 1Wb and 2Wa methods of GPA assay were developed. Erythrocytes of MN type individuals could be separated to normal and single locus variant groups by 1W methods and they could be sorted as normal (MN), single gene deletion mutants (MO, NO), homozygous mutants (MM, NN) cell groups by 2Wa method. Conclusion: The assay is applicable to evaluating the frequency of variant erythrocytes from human somatic mutation

Microsatellite Instability Occurs Rarely in Patients with Cholangiocarcinoma: A Retrospective Study from a German Tertiary Care Hospital

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Ria Winkelmann

2018-05-01

Full Text Available Immune-modulating therapy is a promising therapy for patients with cholangiocarcinoma (CCA. Microsatellite instability (MSI might be a favorable predictor for treatment response, but comprehensive data on the prevalence of MSI in CCA are missing. The aim of the current study was to determine the prevalence of MSI in a German tertiary care hospital. Formalin-fixed paraffin-embedded tissue samples, obtained in the study period from 2007 to 2015 from patients with CCA undergoing surgical resection with curative intention at Johann Wolfgang Goethe University hospital, were examined. All samples were investigated immunohistochemically for the presence of MSI (expression of MLH1, PMS2, MSH2, and MSH6 as well as by pentaplex polymerase chain reaction for five quasimonomorphic mononucleotide repeats (BAT-25, BAT-26, NR-21, NR-22, and NR-24. In total, 102 patients were included, presenting intrahepatic (n = 35, 34.3%, perihilar (n = 42, 41.2%, and distal CCA (n = 25, 24.5%. In the immunohistochemical analysis, no loss of expression of DNA repair enzymes was observed. In the PCR-based analysis, one out of 102 patients was found to be MSI-high and one out of 102 was found to be MSI-low. Thus, MSI seems to appear rarely in CCA in Germany. This should be considered when planning immune-modulating therapy trials for patients with CCA.

Identification of geographically distributed sub-populations of Leishmania (Leishmania major by microsatellite analysis

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Schwenkenbecher Jan

2008-06-01

Full Text Available Abstract Background Leishmania (Leishmania major, one of the agents causing cutaneous leishmaniasis (CL in humans, is widely distributed in the Old World where different species of wild rodent and phlebotomine sand fly serve as animal reservoir hosts and vectors, respectively. Despite this, strains of L. (L. major isolated from many different sources over many years have proved to be relatively uniform. To investigate the population structure of the species highly polymorphic microsatellite markers were employed for greater discrimination among it's otherwise closely related strains, an approach applied successfully to other species of Leishmania. Results Multilocus Microsatellite Typing (MLMT based on 10 different microsatellite markers was applied to 106 strains of L. (L. major from different regions where it is endemic. On applying a Bayesian model-based approach, three main populations were identified, corresponding to three separate geographical regions: Central Asia (CA; the Middle East (ME; and Africa (AF. This was congruent with phylogenetic reconstructions based on genetic distances. Re-analysis separated each of the populations into two sub-populations. The two African sub-populations did not correlate well with strains' geographical origin. Strains falling into the sub-populations CA and ME did mostly group according to their place of isolation although some anomalies were seen, probably, owing to human migration. Conclusion The model- and distance-based analyses of the microsatellite data exposed three main populations of L. (L. major, Central Asia, the Middle East and Africa, each of which separated into two sub-populations. This probably correlates with the different species of rodent host.

Albinism in the american mink (Neovison vison) is associated with a tyrosinase nonsense mutation

DEFF Research Database (Denmark)

Anistoroaei, Razvan Marian; Fredholm, Merete; Christensen, Knud

2008-01-01

Albino phenotypes are documented in various species including the American mink. In other species the albino phenotypes are associated with tyrosinase (TYR) gene mutations; therefore TYR was considered the candidate gene for albinism in mink. Four microsatellite markers were chosen in the prodicted...

Clonal mutations in primary human glial tumors: evidence in support of the mutator hypothesis

International Nuclear Information System (INIS)

Misra, Anjan; Chattopadhyay, Parthaprasad; Chosdol, Kunzang; Sarkar, Chitra; Mahapatra, Ashok K; Sinha, Subrata

2007-01-01

A verifiable consequence of the mutator hypothesis is that even low grade neoplasms would accumulate a large number of mutations that do not influence the tumor phenotype (clonal mutations). In this study, we have attempted to quantify the number of clonal mutations in primary human gliomas of astrocytic cell origin. These alterations were identified in tumor tissue, microscopically confirmed to have over 70% neoplastic cells. Random Amplified Polymorphic DNA (RAPD) analysis was performed using a set of fifteen 10-mer primers of arbitrary but definite sequences in 17 WHO grade II astrocytomas (low grade diffuse astrocytoma or DA) and 16 WHO grade IV astrocytomas (Glioblastoma Multiforme or GBM). The RAPD profile of the tumor tissue was compared with that of the leucocyte DNA of the same patient and alteration(s) scored. A quantitative estimate of the overall genomic changes in these tumors was obtained by 2 different modes of calculation. The overall change in the tumors was estimated to be 4.24% in DA and 2.29% in GBM by one method and 11.96% and 6.03% in DA and GBM respectively by the other. The difference between high and lower grade tumors was statistically significant by both methods. This study demonstrates the presence of extensive clonal mutations in gliomas, more in lower grade. This is consistent with our earlier work demonstrating that technique like RAPD analysis, unbiased for locus, is able to demonstrate more intra-tumor genetic heterogeneity in lower grade gliomas compared to higher grade. The results support the mutator hypothesis proposed by Loeb

Microsatellites grant more stable flanking genes

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Joukhadar Reem

2012-10-01

Full Text Available Abstract Background Microsatellites, or simple sequence repeats (SSRs, are DNA sequences that include tandem copies of specific sequences no longer than six bases. SSRs are ubiquitous in all genomes and highly mutable. Presentation of the hypothesis Results from previous studies suggest that flanking regions of SSR are exhibit high stability in a wide range of organisms. We hypothesized that the SSRs ability to discard weak DNA polymerases could be responsible for this unusual stability. . When the weak polymerases are being decayed over SSRs, the flanking sequences would have higher opportunity to be replicated by more stable DNA polymerases. We present evidence of the molecular basis of our hypothesis. Testing the hypothesis The hypothesis could be tested by examining the activity of DNA polymerase during and after a number of PCRs. The PCR reactions should be run with the same SSR locus possessing differences in the SSR length. The hypothesis could also be tested by comparing the mutational rate of a transferred gene between two transformations. The first one has a naked T-DNA (transferred DNA, while the second one has the same T-DNA flanked with two SSRs. Implications of the hypothesis In any transformation experiment, flanking the T-DNA fragment with SSR sequences would result in more stably transferred genes. This process would decrease the unpredictable risks that may occur because of the mutational pressure on this foreign segment.

The Genetic Relationship between Leishmania aethiopica and Leishmania tropica Revealed by Comparing Microsatellite Profiles.

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Krayter, Lena; Schnur, Lionel F; Schönian, Gabriele

2015-01-01

Leishmania (Leishmania) aethiopica and L. (L.) tropica cause cutaneous leishmaniases and appear to be related. L. aethiopica is geographically restricted to Ethiopia and Kenya; L. tropica is widely dispersed from the Eastern Mediterranean, through the Middle East into eastern India and in north, east and south Africa. Their phylogenetic inter-relationship is only partially revealed. Some studies indicate a close relationship. Here, eight strains of L. aethiopica were characterized genetically and compared with 156 strains of L. tropica from most of the latter species' geographical range to discern the closeness. Twelve unlinked microsatellite markers previously used to genotype strains of L. tropica were successfully applied to the eight strains of L. aethiopica and their microsatellite profiles were compared to those of 156 strains of L. tropica from various geographical locations that were isolated from human cases of cutaneous and visceral leishmaniasis, hyraxes and sand fly vectors. All the microsatellite profiles were subjected to various analytical algorithms: Bayesian statistics, distance-based and factorial correspondence analysis, revealing: (i) the species L. aethiopica, though geographically restricted, is genetically very heterogeneous; (ii) the strains of L. aethiopica formed a distinct genetic cluster; and (iii) strains of L. aethiopica are closely related to strains of L. tropica and more so to the African ones, although, by factorial correspondence analysis, clearly separate from them. The successful application of the 12 microsatellite markers, originally considered species-specific for the species L. tropica, to strains of L. aethiopica confirmed the close relationship between these two species. The Bayesian and distance-based methods clustered the strains of L. aethiopica among African strains of L. tropica, while the factorial correspondence analysis indicated a clear separation between the two species. There was no correlation between

Promoting Cas9 degradation reduces mosaic mutations in non-human primate embryos

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Tu, Zhuchi; Yang, Weili; Yan, Sen; Yin, An; Gao, Jinquan; Liu, Xudong; Zheng, Yinghui; Zheng, Jiezhao; Li, Zhujun; Yang, Su; Li, Shihua; Guo, Xiangyu; Li, Xiao-Jiang

2017-01-01

CRISPR-Cas9 is a powerful new tool for genome editing, but this technique creates mosaic mutations that affect the efficiency and precision of its ability to edit the genome. Reducing mosaic mutations is particularly important for gene therapy and precision genome editing. Although the mechanisms underlying the CRSIPR/Cas9-mediated mosaic mutations remain elusive, the prolonged expression and activity of Cas9 in embryos could contribute to mosaicism in DNA mutations. Here we report that tagging Cas9 with ubiquitin-proteasomal degradation signals can facilitate the degradation of Cas9 in non-human primate embryos. Using embryo-splitting approach, we found that shortening the half-life of Cas9 in fertilized zygotes reduces mosaic mutations and increases its ability to modify genomes in non-human primate embryos. Also, injection of modified Cas9 in one-cell embryos leads to live monkeys with the targeted gene modifications. Our findings suggest that modifying Cas9 activity can be an effective strategy to enhance precision genome editing. PMID:28155910

Toward fully automated genotyping: Genotyping microsatellite markers by deconvolution

Energy Technology Data Exchange (ETDEWEB)

Perlin, M.W.; Lancia, G.; See-Kiong, Ng [Carnegie Mellon Univ., Pittsburgh, PA (United States)

1995-11-01

Dense genetic linkage maps have been constructed for the human and mouse genomes, with average densities of 2.9 cM and 0.35 cM, respectively. These genetic maps are crucial for mapping both Mendelian and complex traits and are useful in clinical genetic diagnosis. Current maps are largely comprised of abundant, easily assayed, and highly polymorphic PCR-based microsatellite markers, primarily dinucleotide (CA){sub n} repeats. One key limitation of these length polymorphisms is the PCR stutter (or slippage) artifact that introduces additional stutter bands. With two (or more) closely spaced alleles, the stutter bands overlap, and it is difficult to accurately determine the correct alleles; this stutter phenomenon has all but precluded full automation, since a human must visually inspect the allele data. We describe here novel deconvolution methods for accurate genotyping that mathematically remove PCR stutter artifact from microsatellite markers. These methods overcome the manual interpretation bottleneck and thereby enable full automation of genetic map construction and use. New functionalities, including the pooling of DNAs and the pooling of markers, are described that may greatly reduce the associated experimentation requirements. 32 refs., 5 figs., 3 tabs.

Molecular analysis of formaldehyde-induced mutations in human lymphoblasts and E. coli

International Nuclear Information System (INIS)

Crosby, R.M.; Richardson, K.K.; Craft, T.R.; Benforado, K.B.; Liber, H.L.; Skopek, T.R.

1988-01-01

The molecular nature of formaldehyde (HCHO)-induced mutations was studied in both human lymphoblasts and E. coli. Thirty HPRT - human lymphoblast colonies induced by eight repetitive 150 μM HCHO treatments were characterized by Southern blot analysis. Fourteen of these mutants (47%) had visible deletions of some or all of the X-linked HPRT bands, indicating that HCHO can induce large losses of DNA in human lymphoblasts. In E. coli., DNA alterations induced by HCHO were characterized with use of the xanthine guanine phosphoribosyl transferase (gpt) gene as the genetic target. Exposure of E. coli to 4 mM HCHO for 1 hr induced large insertions (41%), large deletions (18%), and point mutations (41%). Dideoxy DNA sequencing revealed that most of the point mutations were transversions at GC base pairs. In contrast, exposure of E. coli to 40 mM HCHO for 1 hr produced 92% point mutations, 62% of which were transitions at a single AT base pair in the gene. Therefore, HCHO is capable of producing different genetic alterations in E. coli at different concentrations, suggesting fundamental differences in the mutagenic mechanisms operating at the two concentrations used. Naked pSV2gpt plasmid DNA was exposed to 3.3 or 10 mM HCHO and transformed into E. coli. Most of the resulting mutations were frameshifts, again suggesting a different mutagenic mechanism

Genetic mutation analysis of human gastric adenocarcinomas using ion torrent sequencing platform.

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Zhi Xu

Full Text Available Gastric cancer is the one of the major causes of cancer-related death, especially in Asia. Gastric adenocarcinoma, the most common type of gastric cancer, is heterogeneous and its incidence and cause varies widely with geographical regions, gender, ethnicity, and diet. Since unique mutations have been observed in individual human cancer samples, identification and characterization of the molecular alterations underlying individual gastric adenocarcinomas is a critical step for developing more effective, personalized therapies. Until recently, identifying genetic mutations on an individual basis by DNA sequencing remained a daunting task. Recent advances in new next-generation DNA sequencing technologies, such as the semiconductor-based Ion Torrent sequencing platform, makes DNA sequencing cheaper, faster, and more reliable. In this study, we aim to identify genetic mutations in the genes which are targeted by drugs in clinical use or are under development in individual human gastric adenocarcinoma samples using Ion Torrent sequencing. We sequenced 737 loci from 45 cancer-related genes in 238 human gastric adenocarcinoma samples using the Ion Torrent Ampliseq Cancer Panel. The sequencing analysis revealed a high occurrence of mutations along the TP53 locus (9.7% in our sample set. Thus, this study indicates the utility of a cost and time efficient tool such as Ion Torrent sequencing to screen cancer mutations for the development of personalized cancer therapy.

Somatic mutation profiles of MSI and MSS colorectal cancer identified by whole exome next generation sequencing and bioinformatics analysis.

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Bernd Timmermann

Full Text Available BACKGROUND: Colorectal cancer (CRC is with approximately 1 million cases the third most common cancer worldwide. Extensive research is ongoing to decipher the underlying genetic patterns with the hope to improve early cancer diagnosis and treatment. In this direction, the recent progress in next generation sequencing technologies has revolutionized the field of cancer genomics. However, one caveat of these studies remains the large amount of genetic variations identified and their interpretation. METHODOLOGY/PRINCIPAL FINDINGS: Here we present the first work on whole exome NGS of primary colon cancers. We performed 454 whole exome pyrosequencing of tumor as well as adjacent not affected normal colonic tissue from microsatellite stable (MSS and microsatellite instable (MSI colon cancer patients and identified more than 50,000 small nucleotide variations for each tissue. According to predictions based on MSS and MSI pathomechanisms we identified eight times more somatic non-synonymous variations in MSI cancers than in MSS and we were able to reproduce the result in four additional CRCs. Our bioinformatics filtering approach narrowed down the rate of most significant mutations to 359 for MSI and 45 for MSS CRCs with predicted altered protein functions. In both CRCs, MSI and MSS, we found somatic mutations in the intracellular kinase domain of bone morphogenetic protein receptor 1A, BMPR1A, a gene where so far germline mutations are associated with juvenile polyposis syndrome, and show that the mutations functionally impair the protein function. CONCLUSIONS/SIGNIFICANCE: We conclude that with deep sequencing of tumor exomes one may be able to predict the microsatellite status of CRC and in addition identify potentially clinically relevant mutations.

DNA Fingerprinting of Olive Varieties by Microsatellite Markers

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Dunja Bandelj

2002-01-01

Full Text Available Microsatellites combine several features of an ultimate molecular marker and they are used increasingly in various plant genetic studies and applications. In this work we report on the utilisation of fourteen previously developed olive microsatellite markers for the identification and differentiation of a set of nineteen olive varieties. All analysed microsatellite markers revealed a high level of polymorphism that allowed unique genotyping of the examined varieties. Ninety-six alleles were detected at all 14 loci, which multiplied into a large number of observed genotypes, giving high discrimination value for varietal identification. A minimum number of three microsatellite markers was chosen for the rapid and unambiguous varietal identification of nineteen olive varieties and only two markers were sufficient for differentiation of five local varieties. DNA fingerprints of olive cultivars by means of microsatellites provided meaningful data, which can be extended by additional olive varieties or new microsatellites and used for accurate inter-laboratory comparison. The data obtained can be used for the varietal survey and construction of a database of all olive varieties grown in Slovenia providing also additional genetic information on the agronomic and quality characteristics of the olive varieties.

Identification of somatic mutations in postmortem human brains by whole genome sequencing and their implications for psychiatric disorders.

Science.gov (United States)

Nishioka, Masaki; Bundo, Miki; Ueda, Junko; Katsuoka, Fumiki; Sato, Yukuto; Kuroki, Yoko; Ishii, Takao; Ukai, Wataru; Murayama, Shigeo; Hashimoto, Eri; Nagasaki, Masao; Yasuda, Jun; Kasai, Kiyoto; Kato, Tadafumi; Iwamoto, Kazuya

2018-04-01

Somatic mutations in the human brain are hypothesized to contribute to the functional diversity of brain cells as well as the pathophysiology of neuropsychiatric diseases. However, there are still few reports on somatic mutations in non-neoplastic human brain tissues. This study attempted to unveil the landscape of somatic mutations in the human brain. We explored the landscape of somatic mutations in human brain tissues derived from three individuals with no neuropsychiatric diseases by whole-genome deep sequencing at a depth of around 100. The candidate mutations underwent multi-layered filtering, and were validated by ultra-deep target amplicon sequencing at a depth of around 200 000. Thirty-one somatic mutations were identified in the human brain, demonstrating the utility of whole-genome sequencing of bulk brain tissue. The mutations were enriched in neuron-expressed genes, and two-thirds of the identified somatic single nucleotide variants in the brain tissues were cytosine-to-thymine transitions, half of which were in CpG dinucleotides. Our developed filtering and validation approaches will be useful to identify somatic mutations in the human brain. The vulnerability of neuron-expressed genes to mutational events suggests their potential relevance to neuropsychiatric diseases. © 2017 The Authors. Psychiatry and Clinical Neurosciences published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Psychiatry and Neurology.

Mutations in LRRC50 predispose zebrafish and humans to seminomas.

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Sander G Basten

2013-04-01

Full Text Available Seminoma is a subclass of human testicular germ cell tumors (TGCT, the most frequently observed cancer in young men with a rising incidence. Here we describe the identification of a novel gene predisposing specifically to seminoma formation in a vertebrate model organism. Zebrafish carrying a heterozygous nonsense mutation in Leucine-Rich Repeat Containing protein 50 (lrrc50 also called dnaaf1, associated previously with ciliary function, are found to be highly susceptible to the formation of seminomas. Genotyping of these zebrafish tumors shows loss of heterozygosity (LOH of the wild-type lrrc50 allele in 44.4% of tumor samples, correlating with tumor progression. In humans we identified heterozygous germline LRRC50 mutations in two different pedigrees with a family history of seminomas, resulting in a nonsense Arg488* change and a missense Thr590Met change, which show reduced expression of the wild-type allele in seminomas. Zebrafish in vivo complementation studies indicate the Thr590Met to be a loss-of-function mutation. Moreover, we show that a pathogenic Gln307Glu change is significantly enriched in individuals with seminoma tumors (13% of our cohort. Together, our study introduces an animal model for seminoma and suggests LRRC50 to be a novel tumor suppressor implicated in human seminoma pathogenesis.

Parkinson's disease-related LRRK2 G2019S mutation results from independent mutational events in humans.

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Lesage, Suzanne; Patin, Etienne; Condroyer, Christel; Leutenegger, Anne-Louise; Lohmann, Ebba; Giladi, Nir; Bar-Shira, Anat; Belarbi, Soraya; Hecham, Nassima; Pollak, Pierre; Ouvrard-Hernandez, Anne-Marie; Bardien, Soraya; Carr, Jonathan; Benhassine, Traki; Tomiyama, Hiroyuki; Pirkevi, Caroline; Hamadouche, Tarik; Cazeneuve, Cécile; Basak, A Nazli; Hattori, Nobutaka; Dürr, Alexandra; Tazir, Meriem; Orr-Urtreger, Avi; Quintana-Murci, Lluis; Brice, Alexis

2010-05-15

Mutations in the leucine-rich-repeat kinase 2 (LRRK2) gene have been identified in families with autosomal dominant Parkinson's disease (PD) and in sporadic cases; the G2019S mutation is the single most frequent. Intriguingly, the frequency of this mutation in PD patients varies greatly among ethnic groups and geographic origins: it is present at <0.1% in East Asia, approximately 2% in European-descent patients and can reach frequencies of up to 15-40% in PD Ashkenazi Jews and North African Arabs. To ascertain the evolutionary dynamics of the G2019S mutation in different populations, we genotyped 74 markers spanning a 16 Mb genomic region around G2019S, in 191 individuals carrying the mutation from 126 families of different origins. Sixty-seven families were of North-African Arab origin, 18 were of North/Western European descent, 37 were of Jewish origin, mostly from Eastern Europe, one was from Japan, one from Turkey and two were of mixed origins. We found the G2019S mutation on three different haplotypes. Network analyses of the three carrier haplotypes showed that G2019S arose independently at least twice in humans. In addition, the population distribution of the intra-allelic diversity of the most widespread carrier haplotype, together with estimations of the age of G2019S determined by two different methods, suggests that one of the founding G2019S mutational events occurred in the Near East at least 4000 years ago.

Clonal mutations in primary human glial tumors: evidence in support of the mutator hypothesis

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Sarkar Chitra

2007-10-01

Full Text Available Abstract Background A verifiable consequence of the mutator hypothesis is that even low grade neoplasms would accumulate a large number of mutations that do not influence the tumor phenotype (clonal mutations. In this study, we have attempted to quantify the number of clonal mutations in primary human gliomas of astrocytic cell origin. These alterations were identified in tumor tissue, microscopically confirmed to have over 70% neoplastic cells. Methods Random Amplified Polymorphic DNA (RAPD analysis was performed using a set of fifteen 10-mer primers of arbitrary but definite sequences in 17 WHO grade II astrocytomas (low grade diffuse astrocytoma or DA and 16 WHO grade IV astrocytomas (Glioblastoma Multiforme or GBM. The RAPD profile of the tumor tissue was compared with that of the leucocyte DNA of the same patient and alteration(s scored. A quantitative estimate of the overall genomic changes in these tumors was obtained by 2 different modes of calculation. Results The overall change in the tumors was estimated to be 4.24% in DA and 2.29% in GBM by one method and 11.96% and 6.03% in DA and GBM respectively by the other. The difference between high and lower grade tumors was statistically significant by both methods. Conclusion This study demonstrates the presence of extensive clonal mutations in gliomas, more in lower grade. This is consistent with our earlier work demonstrating that technique like RAPD analysis, unbiased for locus, is able to demonstrate more intra-tumor genetic heterogeneity in lower grade gliomas compared to higher grade. The results support the mutator hypothesis proposed by Loeb.

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

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

2017-06-20

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

Novel Polymerase Gene Mutations for Human Adaptation in Clinical Isolates of Avian H5N1 Influenza Viruses.

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Yasuha Arai

2016-04-01

Full Text Available A major determinant in the change of the avian influenza virus host range to humans is the E627K substitution in the PB2 polymerase protein. However, the polymerase activity of avian influenza viruses with a single PB2-E627K mutation is still lower than that of seasonal human influenza viruses, implying that avian viruses require polymerase mutations in addition to PB2-627K for human adaptation. Here, we used a database search of H5N1 clade 2.2.1 virus sequences with the PB2-627K mutation to identify other polymerase adaptation mutations that have been selected in infected patients. Several of the mutations identified acted cooperatively with PB2-627K to increase viral growth in human airway epithelial cells and mouse lungs. These mutations were in multiple domains of the polymerase complex other than the PB2-627 domain, highlighting a complicated avian-to-human adaptation pathway of avian influenza viruses. Thus, H5N1 viruses could rapidly acquire multiple polymerase mutations that function cooperatively with PB2-627K in infected patients for optimal human adaptation.

Functional Studies of Missense TREM2 Mutations in Human Stem Cell-Derived Microglia

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Philip W. Brownjohn

2018-04-01

Full Text Available Summary: The derivation of microglia from human stem cells provides systems for understanding microglial biology and enables functional studies of disease-causing mutations. We describe a robust method for the derivation of human microglia from stem cells, which are phenotypically and functionally comparable with primary microglia. We used stem cell-derived microglia to study the consequences of missense mutations in the microglial-expressed protein triggering receptor expressed on myeloid cells 2 (TREM2, which are causal for frontotemporal dementia-like syndrome and Nasu-Hakola disease. We find that mutant TREM2 accumulates in its immature form, does not undergo typical proteolysis, and is not trafficked to the plasma membrane. However, in the absence of plasma membrane TREM2, microglia differentiate normally, respond to stimulation with lipopolysaccharide, and are phagocytically competent. These data indicate that dementia-associated TREM2 mutations have subtle effects on microglia biology, consistent with the adult onset of disease in individuals with these mutations. : Brownjohn and colleagues report methods to generate microglia from induced pluripotent human stem cells, which they demonstrate are highly similar to cultured primary human microglia. Microglia differentiated from patient-derived stem cells carrying neurological disease-causing mutations in the TREM2 receptor differentiate normally and respond appropriately to pathogenic stimuli, despite the absence of functional TREM2 receptor on the plasma membrane. Keywords: dementia, microglia, TREM2, Nasu-Hakola disease, frontotemporal dementia, iPSC-microglia, neuroinflammation

Attenuated familial adenomatous polyposis and Muir-Torre syndrome linked to compound biallelic constitutional MYH gene mutations.

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Ponti, G; Ponz de Leon, M; Maffei, S; Pedroni, M; Losi, L; Di Gregorio, C; Gismondi, V; Scarselli, A; Benatti, P; Roncari, B; Seidenari, S; Pellacani, G; Varotti, C; Prete, E; Varesco, L; Roncucci, L

2005-11-01

Attenuated familial adenomatous polyposis and Muir-Torre syndrome linked to compound biallelic constitutional MYH gene mutations.Peculiar dermatologic manifestations are present in several heritable gastrointestinal disorders. Muir-Torre syndrome (MTS) is a genodermatosis whose peculiar feature is the presence of sebaceous gland tumors associated with visceral malignancies. We describe one patient in whom multiple sebaceous gland tumors were associated with early onset colon and thyroid cancers and attenuated polyposis coli. Her family history was positive for colonic adenomas. She had a daughter presenting with yellow papules in the forehead region developed in the late infancy. Skin and visceral neoplasms were tested for microsatellite instability and immunohistochemical status of mismatch repair (MMR), APC and MYH proteins. The proband colon and skin tumors were microsatellite stable and showed normal expression of MMR proteins. Cytoplasmic expression of MYH protein was revealed in colonic cancer cells. Compound heterozygosity due to biallelic mutations in MYH, R168H and 379delC, was identified in the proband. The 11-year-old daughter was carrier of the monoallelic constitutional mutation 379delC in the MYH gene; in the sister, the R168H MYH gene mutation was detected. This report presents an interesting case of association between MYH-associated polyposis and sebaceous gland tumors. These findings suggest that patients with MTS phenotype that include colonic polyposis should be screened for MYH gene mutations.

The pathological phenotype of colon cancer with microsatellite instability

DEFF Research Database (Denmark)

Andersen, Helene Schou; Bertelsen, Claus Anders; Henriksen, Rikke

2016-01-01

proteins: pMLH1, pMSH2, pMSH6 and pPMS2 for the determination of microsatellite instability. Microsatellite instability was defined as deficient expression of one or more of these proteins. RESULTS: Of the 833 patients, 177 had microsatellite instable tumours (21%). Using multivariable logistic regression...

Mutation Rate Variation is a Primary Determinant of the Distribution of Allele Frequencies in Humans.

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Arbel Harpak

2016-12-01

Full Text Available The site frequency spectrum (SFS has long been used to study demographic history and natural selection. Here, we extend this summary by examining the SFS conditional on the alleles found at the same site in other species. We refer to this extension as the "phylogenetically-conditioned SFS" or cSFS. Using recent large-sample data from the Exome Aggregation Consortium (ExAC, combined with primate genome sequences, we find that human variants that occurred independently in closely related primate lineages are at higher frequencies in humans than variants with parallel substitutions in more distant primates. We show that this effect is largely due to sites with elevated mutation rates causing significant departures from the widely-used infinite sites mutation model. Our analysis also suggests substantial variation in mutation rates even among mutations involving the same nucleotide changes. In summary, we show that variable mutation rates are key determinants of the SFS in humans.

Prediction of phenotypes of missense mutations in human proteins from biological assemblies.

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Wei, Qiong; Xu, Qifang; Dunbrack, Roland L

2013-02-01

Single nucleotide polymorphisms (SNPs) are the most frequent variation in the human genome. Nonsynonymous SNPs that lead to missense mutations can be neutral or deleterious, and several computational methods have been presented that predict the phenotype of human missense mutations. These methods use sequence-based and structure-based features in various combinations, relying on different statistical distributions of these features for deleterious and neutral mutations. One structure-based feature that has not been studied significantly is the accessible surface area within biologically relevant oligomeric assemblies. These assemblies are different from the crystallographic asymmetric unit for more than half of X-ray crystal structures. We find that mutations in the core of proteins or in the interfaces in biological assemblies are significantly more likely to be disease-associated than those on the surface of the biological assemblies. For structures with more than one protein in the biological assembly (whether the same sequence or different), we find the accessible surface area from biological assemblies provides a statistically significant improvement in prediction over the accessible surface area of monomers from protein crystal structures (P = 6e-5). When adding this information to sequence-based features such as the difference between wildtype and mutant position-specific profile scores, the improvement from biological assemblies is statistically significant but much smaller (P = 0.018). Combining this information with sequence-based features in a support vector machine leads to 82% accuracy on a balanced dataset of 50% disease-associated mutations from SwissVar and 50% neutral mutations from human/primate sequence differences in orthologous proteins. Copyright © 2012 Wiley Periodicals, Inc.

A homozygous missense mutation in human KLOTHO causes severe tumoral calcinosis

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Ichikawa, Shoji; Imel, Erik A.; Kreiter, Mary L.; Yu, Xijie; Mackenzie, Donald S.; Sorenson, Andrea H.; Goetz, Regina; Mohammadi, Moosa; White, Kenneth E.; Econs, Michael J.

2007-01-01

Familial tumoral calcinosis is characterized by ectopic calcifications and hyperphosphatemia due to inactivating mutations in FGF23 or UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3). Herein we report a homozygous missense mutation (H193R) in the KLOTHO (KL) gene of a 13-year-old girl who presented with severe tumoral calcinosis with dural and carotid artery calcifications. This patient exhibited defects in mineral ion homeostasis with marked hyperphosphatemia and hypercalcemia as well as elevated serum levels of parathyroid hormone and FGF23. Mapping of H193R mutation onto the crystal structure of myrosinase, a plant homolog of KL, revealed that this histidine residue was at the base of the deep catalytic cleft and mutation of this histidine to arginine should destabilize the putative glycosidase domain (KL1) of KL, thereby attenuating production of membrane-bound and secreted KL. Indeed, compared with wild-type KL, expression and secretion of H193R KL were markedly reduced in vitro, resulting in diminished ability of FGF23 to signal via its cognate FGF receptors. Taken together, our findings provide what we believe to be the first evidence that loss-of-function mutations in human KL impair FGF23 bioactivity, underscoring the essential role of KL in FGF23-mediated phosphate and vitamin D homeostasis in humans. PMID:17710231

Identification of a Gypsy SHOX mutation (p.A170P) in Léri-Weill dyschondrosteosis and Langer mesomelic dysplasia.

Science.gov (United States)

Barca-Tierno, Verónica; Aza-Carmona, Miriam; Barroso, Eva; Heine-Suner, Damia; Azmanov, Dimitar; Rosell, Jordi; Ezquieta, Begoña; Montané, Lucia Sentchordi; Vendrell, Teresa; Cruz, Jaime; Santos, Fernando; Rodríguez, José Ignacio; Pozo, Jesús; Argente, Jesús; Kalaydjieva, Luba; Gracía, Ricardo; Campos-Barros, Angel; Benito-Sanz, Sara; Heath, Karen E

2011-12-01

We report the clinical and molecular characteristics of 12 Spanish families with multiple members affected with Léri-Weill dyschondrosteosis (LWD) or Langer mesomelic dysplasia (LMD), who present the SHOX (short stature homeobox gene) mutation p.A170P (c.508G>C) in heterozygosity or homozygosity, respectively. In all studied families, the A170P mutation co-segregated with the fully penetrant phenotype of mesomelic limb shortening and Madelung deformity. A shared haplotype around SHOX was observed by microsatellite analysis, confirming the presence of a common ancestor, probably of Gypsy origin, as 11 of the families were of this ethnic group. Mutation screening in 359 Eastern-European Gypsies failed to identify any carriers. For the first time, we have shown SHOX expression in the human growth plate of a 22-week LMD fetus, homozygous for the A170P mutation. Although the mutant SHOX protein was expressed in all zones of the growth plate, the chondrocyte columns in the proliferative zone were disorganized with the chondrocytes occurring in smaller columnal clusters. We have also identified a novel mutation at the same residue, c. 509C>A (p.A170D), in two unrelated Spanish LWD families, which similar to A170P mutation impedes nuclear localization of SHOX. In conclusion, we have identified A170P as the first frequent SHOX mutation in Gypsy LWD and LMD individuals.

Genetic architecture and bottleneck analyses of Salem Black goat breed based on microsatellite markers

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A. K. Thiruvenkadan

2014-09-01

Full Text Available Aim: The present study was undertaken in Salem Black goat population for genetic analysis at molecular level to exploit the breed for planning sustainable improvement, conservation and utilization, which subsequently can improve the livelihood of its stakeholders. Materials and Methods: Genomic DNA was isolated from blood samples of 50 unrelated Salem Black goats with typical phenotypic features in several villages in the breeding tract and the genetic characterization and bottleneck analysis in Salem Black goat was done using 25 microsatellite markers as recommended by the Food and Agricultural Organization, Rome, Italy. The basic measures of genetic variation were computed using bioinformatic software. To evaluate the Salem Black goats for mutation drift equilibrium, three tests were performed under three different mutation models, viz., infinite allele model (IAM, stepwise mutation model (SMM and two-phase model (TPM and the observed gene diversity (He and expected equilibrium gene diversity (Heq were estimated under different models of microsatellite evolution. Results: The study revealed that the observed number of alleles ranged from 4 (ETH10, ILSTS008 to 17 (BM64444 with a total of 213 alleles and mean of 10.14±0.83 alleles across loci. The overall observed heterozygosity, expected heterozygosity, inbreeding estimate and polymorphism information content values were 0.631±0.041, 0.820±0.024, 0.233±0.044 and 0.786±0.023 respectively indicating high genetic diversity. The average observed gene diversities (He pooled over different markers was 0.829±0.024 and the average expected gene diversities under IAM, TPM and SMM models were 0.769±0.026, 0.808±0.024 and 0.837±0.020 respectively. The number of loci found to exhibit gene diversity excess under IAM, TPM and SMM models were 18, 17 and 12 respectively. Conclusion: All the three statistical tests, viz., sign test, standardized differences test and Wilcoxon sign rank test, revealed

Radiation-induced dominant skeletal mutations in mice: mutation rate, characteristics, and usefulness in estimating genetic hazard to humans from radiation

International Nuclear Information System (INIS)

Selby, P.B.

1979-01-01

The work discussed in this paper represents a major advance in the difficult task of trying to estimate the effects that an increase in mutation frequency would have on human health. Male mice were bred to three females prior to being killed and skeleton studies made. Guidelines were instituted for checking progeny mutations. Surprising results showed a mutation frequency of 1.4% per gamete where none would have been expected. It is now clear that mice can be greatly deformed without showing external effects

Subsets of microsatellite-unstable colorectal cancers exhibit discordance between the CpG island methylator phenotype and MLH1 methylation status.

Science.gov (United States)

Kim, Jung H; Rhee, Ye-Y; Bae, Jeong-M; Kwon, Hyeong-J; Cho, Nam-Y; Kim, Mi J; Kang, Gyeong H

2013-07-01

Although the presence of MLH1 methylation in microsatellite-unstable colorectal cancer generally indicates involvement of the CpG island methylator phenotype (CIMP) in the development of the tumor, these two conditions do not always correlate. A minority of microsatellite-unstable colorectal cancers exhibit discordance between CIMP and MLH1 methylation statuses. However, the clinicopathological features of such microsatellite-unstable colorectal cancers with discrepant MLH1 methylation and CIMP statuses remain poorly studied. Microsatellite-unstable colorectal cancers (n=220) were analyzed for CIMP and MLH1 methylation statuses using the MethyLight assay. Based on the combinatorial CIMP and MLH1 methylation statuses, the microsatellite-unstable colorectal cancers were grouped into four subtypes (CIMP-high (CIMP-H) MLH1 methylation-positive (MLH1m+), CIMP-H MLH1 methylation-negative, CIMP-low/0 (CIMP-L/0) MLH1m+, and CIMP-L/0 MLH1 methylation-negative), which were compared in terms of their associations with clinicopathological and molecular features. The CIMP-L/0 MLH1 methylation-negative and CIMP-H MLH1m+ subtypes were predominant, comprising 63.6 and 24.1% of total microsatellite-unstable colorectal cancers, respectively. The discordant subtypes, CIMP-H MLH1 methylation-negative and CIMP-L/0 MLH1m+, were found in 5 and 7% of microsatellite-unstable colorectal cancers, respectively. The CIMP-H MLH1 methylation-negative subtype exhibited elevated incidence rates in male patients and was associated with larger tumor size, more frequent loss of MSH2 expression, increased frequency of KRAS mutation, and advanced cancer stage. The CIMP-L/0 MLH1m+ subtype was associated with onset at an earlier age, a predominance of MLH1 loss, and earlier cancer stage. None of the CIMP-L/0 MLH1m+ subtype patients succumbed to death during the follow-up. Our findings suggest that the discordant subtypes of colorectal cancers exhibit distinct clinicopathological and molecular features

Functional modules, mutational load and human genetic disease.

Science.gov (United States)

Zaghloul, Norann A; Katsanis, Nicholas

2010-04-01

The ability to generate a massive amount of sequencing and genotyping data is transforming the study of human genetic disorders. Driven by such innovation, it is likely that whole exome and whole-genome resequencing will replace regionally focused approaches for gene discovery and clinical testing in the next few years. However, this opportunity brings a significant interpretative challenge to assigning function and phenotypic variance to common and rare alleles. Understanding the effect of individual mutations in the context of the remaining genomic variation represents a major challenge to our interpretation of disease. Here, we discuss the challenges of assigning mutation functionality and, drawing from the examples of ciliopathies as well as cohesinopathies and channelopathies, discuss possibilities for the functional modularization of the human genome. Functional modularization in addition to the development of physiologically relevant assays to test allele functionality will accelerate our understanding of disease architecture and enable the use of genome-wide sequence data for disease diagnosis and phenotypic prediction in individuals. Copyright 2010 Elsevier Ltd. All rights reserved.

The IARC TP53 mutation database: a resource for studying the significance of TP53 mutations in human cancers

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Magali Olivier

2007-02-01

Full Text Available

The tumor suppressor gene TP53 is frequently inactivated by gene mutations in many types of human sporadic cancers, and inherited TP53 mutations predispose to a wide spectrum of early-onset tumors (Li-Fraumeni et Li-Fraumenilike Syndromes. All TP53 gene variations (somatic and germline mutations, as well as polymorphisms that are reported in the scientific literature or in SNP databases are compiled in the IARC TP53 Database. This database provides structured data and analysis tools to study mutation patterns in human cancers and cell-lines and to investigate the clinical impact of mutations. It contains annotations related to the clinical and pathological characteristics of tumors, as well as the demographics and carcinogen exposure of patients. The IARC TP53 web site (http://www-p53.iarc.fr/ provides a search interface for the core database and includes a comprehensive user guide, a slideshow on TP53 mutations in human cancer, protocols and references for sequencing TP53 gene, and links to relevant publications and bioinformatics databases. The database interface allows download of entire data sets and propose various tools for the selection, analysis and downloads of specific sets of data according to user's query.

Recently, new annotations on the functional properties of mutant p53 proteins have been integrated in this database. Indeed, the most frequent TP53 alterations observed in cancers (75% are missense mutations that result in the production of a mutant protein that differ from the wildtype by one single amino-acid. The characterization of the biological activities of these mutant proteins is thus very important. Over the last ten years, a great amount of systematic data has been generated from experimental assays performed in

NRK1 controls nicotinamide mononucleotide and nicotinamide riboside metabolism in mammalian cells.

Science.gov (United States)

Ratajczak, Joanna; Joffraud, Magali; Trammell, Samuel A J; Ras, Rosa; Canela, Núria; Boutant, Marie; Kulkarni, Sameer S; Rodrigues, Marcelo; Redpath, Philip; Migaud, Marie E; Auwerx, Johan; Yanes, Oscar; Brenner, Charles; Cantó, Carles

2016-10-11

NAD + is a vital redox cofactor and a substrate required for activity of various enzyme families, including sirtuins and poly(ADP-ribose) polymerases. Supplementation with NAD + precursors, such as nicotinamide mononucleotide (NMN) or nicotinamide riboside (NR), protects against metabolic disease, neurodegenerative disorders and age-related physiological decline in mammals. Here we show that nicotinamide riboside kinase 1 (NRK1) is necessary and rate-limiting for the use of exogenous NR and NMN for NAD + synthesis. Using genetic gain- and loss-of-function models, we further demonstrate that the role of NRK1 in driving NAD + synthesis from other NAD + precursors, such as nicotinamide or nicotinic acid, is dispensable. Using stable isotope-labelled compounds, we confirm NMN is metabolized extracellularly to NR that is then taken up by the cell and converted into NAD + . Our results indicate that mammalian cells require conversion of extracellular NMN to NR for cellular uptake and NAD + synthesis, explaining the overlapping metabolic effects observed with the two compounds.

Multiplexed microsatellite recovery using massively parallel sequencing

Science.gov (United States)

Jennings, T.N.; Knaus, B.J.; Mullins, T.D.; Haig, S.M.; Cronn, R.C.

2011-01-01

Conservation and management of natural populations requires accurate and inexpensive genotyping methods. Traditional microsatellite, or simple sequence repeat (SSR), marker analysis remains a popular genotyping method because of the comparatively low cost of marker development, ease of analysis and high power of genotype discrimination. With the availability of massively parallel sequencing (MPS), it is now possible to sequence microsatellite-enriched genomic libraries in multiplex pools. To test this approach, we prepared seven microsatellite-enriched, barcoded genomic libraries from diverse taxa (two conifer trees, five birds) and sequenced these on one lane of the Illumina Genome Analyzer using paired-end 80-bp reads. In this experiment, we screened 6.1 million sequences and identified 356958 unique microreads that contained di- or trinucleotide microsatellites. Examination of four species shows that our conversion rate from raw sequences to polymorphic markers compares favourably to Sanger- and 454-based methods. The advantage of multiplexed MPS is that the staggering capacity of modern microread sequencing is spread across many libraries; this reduces sample preparation and sequencing costs to less than $400 (USD) per species. This price is sufficiently low that microsatellite libraries could be prepared and sequenced for all 1373 organisms listed as 'threatened' and 'endangered' in the United States for under $0.5M (USD).

MLH1-Silenced and Non-Silenced Subgroups of Hypermutated Colorectal Carcinomas Have Distinct Mutational Landscapes

Science.gov (United States)

Donehower, Lawrence A.; Creighton, Chad J.; Schultz, Nikolaus; Shinbrot, Eve; Chang, Kyle; Gunaratne, Preethi H.; Muzny, Donna; Sander, Chris; Hamilton, Stanley R.; Gibbs, Richard A.; Wheeler, David

2014-01-01

Approximately 15% of colorectal carcinomas (CRC) exhibit a hypermutated genotype accompanied by high levels of microsatellite instability (MSI-H) and defects in DNA mismatch repair. These tumors, unlike the majority of colorectal carcinomas, are often diploid, exhibit frequent epigenetic silencing of the MLH1 DNA mismatch repair gene, and have a better clinical prognosis. As an adjunct study to The Cancer Genome Atlas consortium that recently analyzed 224 colorectal cancers by whole exome sequencing, we compared the 35 CRC (15.6%) with a hypermutated genotype to those with a non-hypermutated genotype. We found that 22 (63%) of hypermutated CRC exhibited transcriptional silencing of the MLH1 gene, a high frequency of BRAF V600E gene mutations and infrequent APC and KRAS mutations, a mutational pattern significantly different from their non-hypermutated counterparts. However, the remaining 13 (37%) hypermutated CRC lacked MLH1 silencing, contained tumors with the highest mutation rates (“ultramutated” CRC), and exhibited higher incidences of APC and KRAS mutations, but infrequent BRAF mutations. These patterns were confirmed in an independent validation set of 250 exome-sequenced CRC. Analysis of mRNA and microRNA expression signatures revealed that hypermutated CRC with MLH1 silencing had greatly reduced levels of WNT signaling and increased BRAF signaling relative non-hypermutated CRC. Our findings suggest that hypermutated CRC include one subgroup with fundamentally different pathways to malignancy than the majority of CRC. Examination of MLH1 expression status and frequencies of APC, KRAS, and BRAF mutation in CRC may provide a useful diagnostic tool that could supplement the standard microsatellite instability assays and influence therapeutic decisions. PMID:22899370

Microsatellite markers: An important fingerprinting tool for ...

African Journals Online (AJOL)

Microsatellites are simple sequence repeats (SSR) of 1-6 nucleotides. They appear to be ubiquitous in higher organisms, both in animal and plant genomes and involving repetitive as well as unique sequences, although the frequency of microsatellites varies between species. They are abundant, dispersed throughout the ...

Microsatellite markers: An important fingerprinting tool for ...

African Journals Online (AJOL)

ajl yemi

2011-08-01

Aug 1, 2011 ... Microsatellites are simple sequence repeats (SSR) of 1-6 nucleotides. They appear to be ubiquitous in higher organisms, both in animal and plant genomes and involving repetitive as well as unique sequences, although the frequency of microsatellites varies between species. They are abundant,.

Novel ENU-Induced Mutation in Tbx6 Causes Dominant Spondylocostal Dysostosis-Like Vertebral Malformations in the Rat.

Science.gov (United States)

Abe, Koichiro; Takamatsu, Nobuhiko; Ishikawa, Kumiko; Tsurumi, Toshiko; Tanimoto, Sho; Sakurai, Yukina; Lisse, Thomas S; Lisse, Thomas; Imai, Kenji; Serikawa, Tadao; Mashimo, Tomoji

2015-01-01

Congenital vertebral malformations caused by embryonic segmentation defects are relatively common in humans and domestic animals. Although reverse genetics approaches in mice have provided information on the molecular mechanisms of embryonic somite segmentation, hypothesis-driven approaches cannot adequately reflect human dysmorphology within the population. In a N-ethyl-N-nitrosourea (ENU) mutagenesis project in Kyoto, the Oune mutant rat strain was isolated due to a short and kinked caudal vertebra phenotype. Skeletal staining of heterozygous rats showed partial loss of the cervical vertebrae as well as hemivertebrae and fused vertebral blocks in lumbar and sacral vertebrae. In homozygous embryos, severe displacement of the whole vertebrae was observed. The Oune locus was genetically mapped to rat chromosome 1 using 202 backcross animals and 50 genome-wide microsatellite markers. Subsequently, a miss-sense mutation in the Tbx6 gene was identified in the critical region. Although the mutation is located within the T-box domain near a predicted dimmer-interface, in vitro experiments revealed that the Tbx6 variant retains normal DNA binding ability and translational efficiency. However, the variant has decreased transcriptional activation potential in response to Notch-mediated signaling. Recently, it was reported that a dominant type of familial spondylocostal dysostosis is caused by a stoploss mutation in TBX6. Thus, we propose that partial dysfunction of Tbx6 leads to similar congenital vertebral malformations in both humans and rats. The Oune strain could be a unique animal model for dominant spondylocostal dysostosis and is useful for molecular dissection of the pathology of congenital vertebral malformations in humans.

Novel ENU-Induced Mutation in Tbx6 Causes Dominant Spondylocostal Dysostosis-Like Vertebral Malformations in the Rat.

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Koichiro Abe

Full Text Available Congenital vertebral malformations caused by embryonic segmentation defects are relatively common in humans and domestic animals. Although reverse genetics approaches in mice have provided information on the molecular mechanisms of embryonic somite segmentation, hypothesis-driven approaches cannot adequately reflect human dysmorphology within the population. In a N-ethyl-N-nitrosourea (ENU mutagenesis project in Kyoto, the Oune mutant rat strain was isolated due to a short and kinked caudal vertebra phenotype. Skeletal staining of heterozygous rats showed partial loss of the cervical vertebrae as well as hemivertebrae and fused vertebral blocks in lumbar and sacral vertebrae. In homozygous embryos, severe displacement of the whole vertebrae was observed. The Oune locus was genetically mapped to rat chromosome 1 using 202 backcross animals and 50 genome-wide microsatellite markers. Subsequently, a miss-sense mutation in the Tbx6 gene was identified in the critical region. Although the mutation is located within the T-box domain near a predicted dimmer-interface, in vitro experiments revealed that the Tbx6 variant retains normal DNA binding ability and translational efficiency. However, the variant has decreased transcriptional activation potential in response to Notch-mediated signaling. Recently, it was reported that a dominant type of familial spondylocostal dysostosis is caused by a stoploss mutation in TBX6. Thus, we propose that partial dysfunction of Tbx6 leads to similar congenital vertebral malformations in both humans and rats. The Oune strain could be a unique animal model for dominant spondylocostal dysostosis and is useful for molecular dissection of the pathology of congenital vertebral malformations in humans.

Structure-based assessment of disease-related mutations in human voltage-gated sodium channels

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Weiyun Huang

2017-02-01

Full Text Available ABSTRACT Voltage-gated sodium (Nav channels are essential for the rapid upstroke of action potentials and the propagation of electrical signals in nerves and muscles. Defects of Nav channels are associated with a variety of channelopathies. More than 1000 disease-related mutations have been identified in Nav channels, with Nav1.1 and Nav1.5 each harboring more than 400 mutations. Nav channels represent major targets for a wide array of neurotoxins and drugs. Atomic structures of Nav channels are required to understand their function and disease mechanisms. The recently determined atomic structure of the rabbit voltage-gated calcium (Cav channel Cav1.1 provides a template for homology-based structural modeling of the evolutionarily related Nav channels. In this Resource article, we summarized all the reported disease-related mutations in human Nav channels, generated a homologous model of human Nav1.7, and structurally mapped disease-associated mutations. Before the determination of structures of human Nav channels, the analysis presented here serves as the base framework for mechanistic investigation of Nav channelopathies and for potential structure-based drug discovery.

Structure-based assessment of disease-related mutations in human voltage-gated sodium channels.

Science.gov (United States)

Huang, Weiyun; Liu, Minhao; Yan, S Frank; Yan, Nieng

2017-06-01

Voltage-gated sodium (Na v ) channels are essential for the rapid upstroke of action potentials and the propagation of electrical signals in nerves and muscles. Defects of Na v channels are associated with a variety of channelopathies. More than 1000 disease-related mutations have been identified in Na v channels, with Na v 1.1 and Na v 1.5 each harboring more than 400 mutations. Na v channels represent major targets for a wide array of neurotoxins and drugs. Atomic structures of Na v channels are required to understand their function and disease mechanisms. The recently determined atomic structure of the rabbit voltage-gated calcium (Ca v ) channel Ca v 1.1 provides a template for homology-based structural modeling of the evolutionarily related Na v channels. In this Resource article, we summarized all the reported disease-related mutations in human Na v channels, generated a homologous model of human Na v 1.7, and structurally mapped disease-associated mutations. Before the determination of structures of human Na v channels, the analysis presented here serves as the base framework for mechanistic investigation of Na v channelopathies and for potential structure-based drug discovery.

Germline mutation rates in families residing in high level natural radiation areas of Kerala coast in southwest India

International Nuclear Information System (INIS)

Das, Birajalaxmi; Ghosh, Anu; Ahmad, Shazia; Saini, DivyaIakshmi; Chauhan, P.S.; Seshadri, M.

2010-01-01

For this study, 200 nuclear families have been analyzed using over 40 mini- and microsatellite markers. Cord blood samples for the child and peripheral blood samples for the parent(s) were collected in EDTA vacuutainers from the hospital units located in High Level Natural Radiation Areas (HLNRA) and Normal Level Natural Radiation Areas (NLNRA). Both the parents of the newborn were exposed to the background dose. The families were grouped into four distinct dose groups - NLNRA group 5.00 mGy/year. An overall mutation rate of 2.08 X 10 -3 per cell per generation was observed for NLNRA and 2.12 X 10 -3 per cell per generation for HLNRA families. No radiation induced dose response was observed for the stratified groups. Thus, this study shows that mutation rates at mini- and microsatellites in the off springs of the parents living in the high background radiation areas of Kerala does not vary with radiation exposure. This is the first report to understand germline mutation rates at hypervariable loci in families residing in high level natural radiation areas of the world

No significant role for beta tubulin mutations and mismatch repair defects in ovarian cancer resistance to paclitaxel/cisplatin

International Nuclear Information System (INIS)

Mesquita, Bárbara; Veiga, Isabel; Pereira, Deolinda; Tavares, Ana; Pinto, Isabel M; Pinto, Carla; Teixeira, Manuel R; Castedo, Sérgio

2005-01-01

The mechanisms of chemoresistance in ovarian cancer patients remain largely to be elucidated. Paclitaxel/cisplatin combination is the standard chemotherapeutic treatment for this disease, although some patients do not respond to therapy. Our goals were to investigate whether TUBB mutations and mismatch repair defects underlie paclitaxel and cisplatin resistance. Thirty-four patients with primary ovarian carcinomas (26 serous and eight clear cell carcinomas) treated with paclitaxel/cisplatin were analysed. TUBB exon 4 was analysed by nested PCR after a first round PCR using intronic primers. Microsatellite analysis was performed with the quasimonomorphic markers BAT 26 and BAT 34. Twenty-two of the 34 ovarian cancers (64.7%) presented residual tumour after surgery, seven of which (7/22; 31.8%) were shown to be chemoresistant (five serous and two clear cell tumours). Sequence analysis did not find any mutation in TUBB exon 4. Microsatellite instability was not detected in any of the ovarian carcinomas. We conclude that TUBB exon 4 mutations and mismatch repair defects do not play a significant role in paclitaxel/cisplatin resistance

Fidelity and Mutational Spectrum of Pfu DNA Polymerase on a Human Mitochondrial DNA Sequence

Science.gov (United States)

André, Paulo; Kim, Andrea; Khrapko, Konstantin; Thilly, William G.

1997-01-01

The study of rare genetic changes in human tissues requires specialized techniques. Point mutations at fractions at or below 10−6 must be observed to discover even the most prominent features of the point mutational spectrum. PCR permits the increase in number of mutant copies but does so at the expense of creating many additional mutations or “PCR noise”. Thus, each DNA sequence studied must be characterized with regard to the DNA polymerase and conditions used to avoid interpreting a PCR-generated mutation as one arising in human tissue. The thermostable DNA polymerase derived from Pyrococcus furiosus designated Pfu has the highest fidelity of any DNA thermostable polymerase studied to date, and this property recommends it for analyses of tissue mutational spectra. Here, we apply constant denaturant capillary electrophoresis (CDCE) to separate and isolate the products of DNA amplification. This new strategy permitted direct enumeration and identification of point mutations created by Pfu DNA polymerase in a 96-bp low melting domain of a human mitochondrial sequence despite the very low mutant fractions generated in the PCR process. This sequence, containing part of the tRNA glycine and NADH dehydrogenase subunit 3 genes, is the target of our studies of mitochondrial mutagenesis in human cells and tissues. Incorrectly synthesized sequences were separated from the wild type as mutant/wild-type heteroduplexes by sequential enrichment on CDCE. An artificially constructed mutant was used as an internal standard to permit calculation of the mutant fraction. Our study found that the average error rate (mutations per base pair duplication) of Pfu was 6.5 × 10−7, and five of its more frequent mutations (hot spots) consisted of three transversions (GC → TA, AT → TA, and AT → CG), one transition (AT → GC), and one 1-bp deletion (in an AAAAAA sequence). To achieve an even higher sensitivity, the amount of Pfu-induced mutants must be

Fidelity and mutational spectrum of Pfu DNA polymerase on a human mitochondrial DNA sequence.

Science.gov (United States)

André, P; Kim, A; Khrapko, K; Thilly, W G

1997-08-01

The study of rare genetic changes in human tissues requires specialized techniques. Point mutations at fractions at or below 10(-6) must be observed to discover even the most prominent features of the point mutational spectrum. PCR permits the increase in number of mutant copies but does so at the expense of creating many additional mutations or "PCR noise". Thus, each DNA sequence studied must be characterized with regard to the DNA polymerase and conditions used to avoid interpreting a PCR-generated mutation as one arising in human tissue. The thermostable DNA polymerase derived from Pyrococcus furiosus designated Pfu has the highest fidelity of any DNA thermostable polymerase studied to date, and this property recommends it for analyses of tissue mutational spectra. Here, we apply constant denaturant capillary electrophoresis (CDCE) to separate and isolate the products of DNA amplification. This new strategy permitted direct enumeration and identification of point mutations created by Pfu DNA polymerase in a 96-bp low melting domain of a human mitochondrial sequence despite the very low mutant fractions generated in the PCR process. This sequence, containing part of the tRNA glycine and NADH dehydrogenase subunit 3 genes, is the target of our studies of mitochondrial mutagenesis in human cells and tissues. Incorrectly synthesized sequences were separated from the wild type as mutant/wild-type heteroduplexes by sequential enrichment on CDCE. An artificially constructed mutant was used as an internal standard to permit calculation of the mutant fraction. Our study found that the average error rate (mutations per base pair duplication) of Pfu was 6.5 x 10(-7), and five of its more frequent mutations (hot spots) consisted of three transversions (GC-->TA, AT-->TA, and AT-->CG), one transition (AT-->GC), and one 1-bp deletion (in an AAAAAA sequence). To achieve an even higher sensitivity, the amount of Pfu-induced mutants must be reduced.

Microsatellite Polymorphisms Adjacent to the Oxytocin Receptor Gene in Domestic Cats: Association with Personality?

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Minori Arahori

2017-12-01

Full Text Available A growing number of studies have explored the oxytocin system in humans and non-human animals, and some have found important genetic polymorphisms in the oxytocin receptor gene (OXTR associated with the bonding system, social behaviors, and personality in several species. Although single nucleotide polymorphisms in OXTR have been well-examined in various species, microsatellites (or short tandem repeats adjacent to OXTR have rarely been studied, despite some suggestions that microsatellite polymorphisms near genes might play a role in genetic transcription and translation. In this study, we surveyed microsatellites in the upstream, intron, and downstream regions of OXTR in domestic cats (Felis catus. We succeeded in amplifying 5 out of 10 regions, and recognized these five regions as polymorphic. We compared allele frequencies in these five regions between mongrel cats in Japan (n = 100 and cats of 10 pure breeds (n = 40. There were significant differences in allele frequencies between the two populations in all microsatellite regions. Additionally, the owners of mongrel cats answered a comprehensive personality questionnaire, and factor analysis extracted four factors (Openness, Friendliness, Roughness, and Neuroticism. We examined the association between the microsatellite genotypes, age, sex, neutering status, and personality scores. Compared to their counterparts, younger cats tended to score higher on Openness, male cats scored higher on Friendliness, and female and neutered cats scored higher on Roughness. When we divided the sample into three groups depending on the length of alleles, we found a marginally significant association between Friendliness and MS3. Additionally, we found a sex-mediated effect of genotypes in MS4 on Friendliness, resulting in different effects on females and males. Our findings that mongrel cats had longer alleles in MS3 and MS4 than purebred cats, and that those cats tended to score higher on Friendliness

Isolation and Characterization of Microsatellite Markers in Beilschmiedia roxburghiana (Lauraceae

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Lu Liu

2013-07-01

Full Text Available Premise of the study: Although there are as many as 250 species in the genus Beilschmiedia, their genetic diversity has been poorly investigated. Our objective was to develop microsatellite markers for B. roxburghiana to study its genetic diversity for the sustainable management of this species. Methods and Results: Using the microsatellite-enriched library and PCR-based screening method, 22 microsatellite markers were developed and 10 showed high polymorphism in a population. The number of alleles per locus for these 10 microsatellites ranged from five to 19. The observed and expected heterozygosities ranged from 0.298 to 1.000 and from 0.314 to 0.878, respectively. Conclusions: Our results from the 10 highly polymorphic microsatellites indicate that the principal reproductive mode of B. roxburghiana is clonal in the studied population. These microsatellites will facilitate further studies on genetic diversity and structure in B. roxburghiana.

Microsatellite Markers for Russian Olive (Elaeagnus angustifolia; Elaeagnaceae

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John F. Gaskin

2013-09-01

Full Text Available Premise of the study: Microsatellite markers were developed for the plant species Elaeagnus angustifolia to assist in future investigations of genetic variability in its native and invasive ranges and the precise origins of the United States/Canada invasion. Methods and Results: Eleven polymorphic microsatellite markers were developed. The number of alleles observed for each locus ranged from three to 11. Conclusions: These microsatellites have sufficient potential variability to define population structure and origins of the Russian olive invasion.

Patterns of microsatellite evolution inferred from the Helianthus ...

Indian Academy of Sciences (India)

2014-08-21

Aug 21, 2014 ... selection has favoured the maintenance of microsatellites in these genes over others. This study shows that .... Materials and methods. Data collection ... sequences and different microsatellite motifs should code for specific ...

Undermethylated DNA as a source of microsatellites from a conifer genome.

Science.gov (United States)

Zhou, Y; Bui, T; Auckland, L D; Williams, C G

2002-02-01

Developing microsatellites from the large, highly duplicated conifer genome requires special tools. To improve the efficiency of developing Pinus taeda L. microsatellites, undermethylated (UM) DNA fragments were used to construct a microsatellite-enriched copy library. A methylation-sensitive restriction enzyme, McrBC, was used to enrich for UM DNA before library construction. Digested DNA fragments larger than 9 kb were then excised and digested with RsaI and used to construct nine dinucleotide and trinucleotide libraries. A total of 1016 microsatellite-positive clones were detected among 11 904 clones and 620 of these were unique. Of 245 primer sets that produced a PCR product, 113 could be developed as UM microsatellite markers and 70 were polymorphic. Inheritance and marker informativeness were tested for a random sample of 36 polymorphic markers using a three-generation outbred pedigree. Thirty-one microsatellites (86%) had single-locus inheritance despite the highly duplicated nature of the P. taeda genome. Nineteen UM microsatellites had highly informative intercross mating type configurations. Allele number and frequency were estimated for eleven UM microsatellites using a population survey. Allele numbers for these UM microsatellites ranged from 3 to 12 with an average of 5.7 alleles/locus. Frequencies for the 63 alleles were mostly in the low-common range; only 14 of the 63 were in the rare allele (q < 0.05) class. Enriching for UM DNA was an efficient method for developing polymorphic microsatellites from a large plant genome.

Localization of the homolog of a mouse craniofacial mutant to human chromosome 18q11 and evaluation of linkage to human CLP and CPO

Energy Technology Data Exchange (ETDEWEB)

Griffith, A.J.; Burgess, D.L.; Kohrman, D.C.; Yu, J. [Univ. of Michigan, Ann Arbor, MI (United States)] [and others

1996-06-15

The transgene-induced mutation 9257 and the spontaneous mutation twirler cause craniofacial and inner ear malformations and are located on mouse chromosome 18 near the ataxia locus ax. To map the human homolog of 9257, a probe from the transgene insertion site was used to screen a human genomic library. Analysis of a cross-hybridizing human clone identified a 3-kb conserved sequence block that does not appear to contain protein coding sequence. Analysis of somatic cell hybrid panels assigned the human locus to 18q11. The polymorphic microsatellite markers D18S1001 and D18S1002 were isolated from the human locus and mapped by linkage analysis using the CEPH pedigrees. The 9257 locus maps close to the centromeres of human chromosome 18q and mouse chromosome 18 at the proximal end of a conserved linkage group. To evaluate the role of this locus in human craniofacial disorders, linkage to D18S1002 was tested in 11 families with autosomal dominant nonsyndromic cleft lip and palate and 3 families with autosomal dominant cleft palate only. Obligatory recombinants were observed in 8 of the families, and negative lod scores from the other families indicated that these disorders are not linked to the chromosome 18 loci. 23 refs., 4 figs., 2 tabs.

Stability characteristics and structural properties of single- and double-walled boron-nitride nanotubes under physical adsorption of Flavin mononucleotide (FMN) in aqueous environment using molecular dynamics simulations

International Nuclear Information System (INIS)

Ansari, R.; Ajori, S.; Ameri, A.

2016-01-01

Graphical abstract: Structural properties and stability characteristics of single- and double-walled boron-nitride nanotubes functionalized with Flavin mononucleotide (FMN) in aqueous environment are investigated employing molecular dynamics simulations. - Highlights: • Structural and buckling analysis of boron-nitride nanotubes under physical adsorption of Flavin mononucleotide (FMN). • Gyration radius increases linearly as the weight percentage of FMN increases. • Presence of water molecules results in more expansion of FMN around BNNTs. • Critical buckling force of functionalized BNNTs is higher than that of pure BNNTs. • The critical strain of functionalized BNNTs is found to be lower than that of pure ones. - Abstract: The non-cytotoxic properties of Boron-nitride nanotubes (BNNTs) and the ability of stable interaction with biomolecules make them so promising for biological applications. In this research, molecular dynamics (MD) simulations are performed to investigate the structural properties and stability characteristics of single- and double-walled BNNTs under physical adsorption of Flavin mononucleotide (FMN) in vacuum and aqueous environments. According to the simulation results, gyration radius increases by rising the weight percentage of FMN. Also, the results demonstrate that critical buckling force of functionalized BNNTs increases in vacuum. Moreover, it is observed that by increasing the weight percentage of FMN, critical force of functionalized BNNTs rises. By contrast, critical strain reduces by functionalization of BNNTs in vacuum. Considering the aqueous environment, it is observed that gyration radius and critical buckling force of functionalized BNNTs increase more considerably than those of functionalized BNNTs in vacuum, whereas the critical strains approximately remain unchanged.

Mutations in the FMN domain modulate MCD spectra of the heme site in the oxygenase domain of inducible nitric oxide synthase.

Science.gov (United States)

Sempombe, Joseph; Elmore, Bradley O; Sun, Xi; Dupont, Andrea; Ghosh, Dipak K; Guillemette, J Guy; Kirk, Martin L; Feng, Changjian

2009-05-27

The nitric oxide synthase (NOS) output state for NO production is a complex of the flavin mononucleotide (FMN)-binding domain and the heme domain, and thereby it facilitates the interdomain electron transfer from the FMN to the catalytic heme site. Emerging evidence suggests that interdomain FMN-heme interactions are important in the formation of the output state because they guide the docking of the FMN domain to the heme domain. In this study, notable effects of mutations in the adjacent FMN domain on the heme structure in a human iNOS bidomain oxygenase/FMN construct have been observed by using low-temperature magnetic circular dichroism (MCD) spectroscopy. The comparative MCD study of wild-type and mutant proteins clearly indicates that a properly docked FMN domain contributes to the observed L-Arg perturbation of the heme MCD spectrum in the wild-type protein and that the conserved surface residues in the FMN domain (E546 and E603) play key roles in facilitating a productive alignment of the FMN and heme domains in iNOS.

Induced mutations for human welfare through agriculture

International Nuclear Information System (INIS)

Patil, S.A.

2009-01-01

pulses and oil seeds. The Groundnut varieties of BARC have spread on more than one million hectares and are providing additional income of more than five hundred crores annually on a continuing basis. These examples prove beyond doubt that induced mutations have played a vital role for human welfare through agriculture

Microsatellite and single nucleotide polymorphisms in the β-globin locus control region-hypersensitive Site 2: SPECIFICITY of Tunisian βs chromosomes.

Science.gov (United States)

Ben Mustapha, Maha; Moumni, Imen; Zorai, Amine; Douzi, Kaïs; Ghanem, Abderraouf; Abbes, Salem

2012-01-01

The diversity of sickle cell disease severity is attributed to several cis acting factors, among them the single nucleotide polymorphisms (SNPs) and (AT) rich region in the β-locus control region (β-LCR). This contains five DNase I hypersensitive sites (HS) located 6 to 22 kb upstream to the ϵ gene. The most important of these is the HS2 (5' β-LCR-HS2), characterized by the presence of three different SNPs and a microsatellite region known to be in association with β(S) chromosomes in various populations. The aim of this study was to present the molecular investigation of the 5' β-LCR-HS2 site in normal and sickle cell disease individuals in order to determine if there is any correlation or specificity between these molecular markers, the β(S) Tunisian chromosomes and phenotypical expression of sickle cell disease. One hundred and twenty-four chromosomes from Tunisian individuals (49 β(S) carriers and 13 normal individuals) were screened by polymerase chain reaction (PCR) and sequencing for the polymorphic short tandem microsatellite repeats (AT)(X)N(12)(AT)(Y) and the three SNPs (rs7119428, rs9736333 and rs60240093) of the 5' β-LCR-HS2. Twelve configurations of the microsatellite motif were found with an ancestral configuration elaborated by ClustalW software. Normal and mutated alleles were observed at the homozygous and heterozygous states for the three SNPs. Correlation between microsatellites and SNPs suggests that mutant SNP alleles were mainly associated, in the homozygous sickle cell disease phenotype, with the (AT)(8)N(12)GT(AT)(7) configuration, whereas, normal SNP alleles were associated with the (AT)(X)N(12)(AT)(11) configurations in normal β(A) chromosomes. The correlation of these various configurations with Hb F expression was also investigated. The principal component analysis (PCA) showed the correlation between the homozygous sickle cell disease phenotype, mutated SNP alleles and the Benin microsatellite configuration (AT)(8)N(12)GT

Microsatellites in wheat and their applications

International Nuclear Information System (INIS)

Stephenson, P.; Bryan, G.J.; Kirby, J.; Gale, M.D.

1998-01-01

The development of large panels of simply analyzable genetic markers for diversity studies and tagging, agronomically important genes in hexaploid bread wheat is an important goal in applied cereal genetic research. We have isolated and sequenced over two-hundred clones containing microsatellites from the wheat genome, and have tested 150 primer pairs for genetic polymorphism using a panel of ten wheat varieties, including the parents of our main mapping cross. A total of 125 loci were detected by 82 primer pairs, of which 105 loci from 63 primer pairs can be unequivocally allocated to one of the wheat chromosomes. A relatively low frequency of the loci detected are from the D-genome (24%). Generally, the microsatellites show high levels of genetic polymorphism and an average 3.5 alleles per locus with an average polymorphism information content (PIC) value of 0.5. The observed levels of polymorphism are positively correlated with the length of the microsatellite repeats. A high proportion, approximately one half, of primer pairs designed to detect simple sequence repeat (SSR) variation in wheat do not generate the expected amplification products and, more significantly, often generate unresolvable Polymerase Chain Reaction (PCR) products. In general our results agree closely with those obtained from other recent studies using microsatellites in plants. (author)

Characterization of ultraviolet light-induced diphtheria toxin-resistant mutations in normal and Xeroderma pigmentosum human fibroblasts

International Nuclear Information System (INIS)

Glover, T.W.

1979-01-01

Quantitative mutagenesis studies in human cells have been severely limited by the lack of reliable genetic markers. Experiments were therefore performed to develop and characterize a better quantitative mutation assay for human cells. The uv-induction of diphtheria toxin resistant (DT/sup r/) mutations in normal and excision repair defective xeroderma pigmentosum (XP) fibroblasts has been quantitatively characterized. A concentration of diphtheria toxin to use in the selection of resistant mutants was determined whereby DT/sup r/ cells are cross-resistant to Pseudomonas aeurginosa exotoxin A, indicating mutants have altered elongation factor-2 (EF-2) which is not susceptible to ADP-ribosylation by either toxin. Results of this study indicate that XP fibroblasts have higher uv-induced mutation frequencies per unit uv-dose but similar frequencies per unit survival compared to normal cells as measured using a new genetic marker for quantitative mutagenesis. Furthermore, these results support a prediction of the mutation theory of cancer, namely, that cells from individuals with certain human syndromes that predispose the individual to cancer will have higher induced mutation frequencies than cells from non-susceptible individuals. This newly characterized genetic marker should be useful in quantitative mutagenesis studies in human cells

A new world monkey microsatellite (ap74) highly conserved in primates

International Nuclear Information System (INIS)

Oklander, Luciana Ines; Steinberg, Eliana Ruth; Dolores Mudry, Marta

2012-01-01

Given their great variability, microsatellites or STRS became the most commonly used genetic markers over the last 15 years. The analysis of these markers requires minimum quantities of DNA, allowing the use of noninvasive samples, such as feces or hair. We amplified the microsatellite ap74 in blood and hair samples in order to analyze the levels of genomic conservation among a wide range of primates including: lemur catta, alouatta caraya, ateles belzebuth, ateles chamek, pan troglodytes, papio sp., and Homo sapiens. in all cases we obtained amplification products that exhibited similar size both in monkeys and human (oscillating between 126 and 176 bp), except in the lemur where the detected fragment presented a size of approximately 1000 bp. the analysis of the nucleotide sequences permitted the evaluation of the molecular modifications experienced during the evolutionary process in primates.

Mutational landscape of the human Y chromosome-linked genes ...

Indian Academy of Sciences (India)

Mutational landscape of the human Y chromosome-linked genes and loci in patients with hypogonadism. Deepali Pathak, Sandeep Kumar Yadav, Leena Rawal and Sher Ali. J. Genet. 94, 677–687. Table 1. Details showing age, sex, karyotype, clinical features and diagnosis results of the patients with H. Hormone profile.

Homozygous PMS2 deletion causes a severe colorectal cancer and multiple adenoma phenotype without extraintestinal cancer.

Science.gov (United States)

Will, Olivia; Carvajal-Carmona, Luis G; Gorman, Patricia; Howarth, Kimberley M; Jones, Angela M; Polanco-Echeverry, Guadalupe M; Chinaleong, Jo-Anne; Günther, Thomas; Silver, Andrew; Clark, Susan K; Tomlinson, Ian

2007-02-01

We report a patient of Indian descent with parental consanguinity, who developed 10 carcinomas and 35 adenomatous polyps at age 23 and duodenal adenocarcinoma at age 25. He also had dysmorphic features, mental retardation, and café-au-lait spots but no brain tumor. We aimed to establish his molecular diagnosis. Germ-line screening for APC and MYH/MUTYH mutations was normal as was immunohistochemistry for MLH1 and MSH2 proteins. Investigation by array-comparative genomic hybridization revealed deletion of a small region on chromosome 7. Using polymerase chain reaction, this region was refined to a 400-kilobase deletion, which included exons 9-15 of the PMS2 gene, and all coding regions of oncomodulin, TRIAD3, and FSCN1. The deletion was confirmed as homozygous, and both parents were carriers. Immunohistochemistry showed absent PMS2 expression in all tumors and normal tissue. Most tumors showed microsatellite instability, more marked at dinucleotide than mononucleotide repeats. The tumors harbored no somatic mutations in APC, BRAF, AXIN2, or beta-catenin, but KRAS2 and TGFBR2 mutations were found. Our patient represents a novel phenotype for homozygous PMS2 mutation and perhaps the most severe colorectal cancer phenotype-in terms of numbers of malignancies at an early age-described to date. PMS2 mutations-and perhaps other homozygous mismatch repair mutations-should be considered in any patient presenting with multiple gastrointestinal tumors, since our patient could not be distinguished clinically from cases with attenuated familial adenomatous polyposis or MUTYH-associated polyposis.

EuMicroSatdb: A database for microsatellites in the sequenced genomes of eukaryotes

Directory of Open Access Journals (Sweden)

Grover Atul

2007-07-01

Full Text Available Abstract Background Microsatellites have immense utility as molecular markers in different fields like genome characterization and mapping, phylogeny and evolutionary biology. Existing microsatellite databases are of limited utility for experimental and computational biologists with regard to their content and information output. EuMicroSatdb (Eukaryotic MicroSatellite database http://ipu.ac.in/usbt/EuMicroSatdb.htm is a web based relational database for easy and efficient positional mining of microsatellites from sequenced eukaryotic genomes. Description A user friendly web interface has been developed for microsatellite data retrieval using Active Server Pages (ASP. The backend database codes for data extraction and assembly have been written using Perl based scripts and C++. Precise need based microsatellites data retrieval is possible using different input parameters like microsatellite type (simple perfect or compound perfect, repeat unit length (mono- to hexa-nucleotide, repeat number, microsatellite length and chromosomal location in the genome. Furthermore, information about clustering of different microsatellites in the genome can also be retrieved. Finally, to facilitate primer designing for PCR amplification of any desired microsatellite locus, 200 bp upstream and downstream sequences are provided. Conclusion The database allows easy systematic retrieval of comprehensive information about simple and compound microsatellites, microsatellite clusters and their locus coordinates in 31 sequenced eukaryotic genomes. The information content of the database is useful in different areas of research like gene tagging, genome mapping, population genetics, germplasm characterization and in understanding microsatellite dynamics in eukaryotic genomes.

Single genome retrieval of context-dependent variability in mutation rates for human germline.

Science.gov (United States)

Sahakyan, Aleksandr B; Balasubramanian, Shankar

2017-01-13

Accurate knowledge of the core components of substitution rates is of vital importance to understand genome evolution and dynamics. By performing a single-genome and direct analysis of 39,894 retrotransposon remnants, we reveal sequence context-dependent germline nucleotide substitution rates for the human genome. The rates are characterised through rate constants in a time-domain, and are made available through a dedicated program (Trek) and a stand-alone database. Due to the nature of the method design and the imposed stringency criteria, we expect our rate constants to be good estimates for the rates of spontaneous mutations. Benefiting from such data, we study the short-range nucleotide (up to 7-mer) organisation and the germline basal substitution propensity (BSP) profile of the human genome; characterise novel, CpG-independent, substitution prone and resistant motifs; confirm a decreased tendency of moieties with low BSP to undergo somatic mutations in a number of cancer types; and, produce a Trek-based estimate of the overall mutation rate in human. The extended set of rate constants we report may enrich our resources and help advance our understanding of genome dynamics and evolution, with possible implications for the role of spontaneous mutations in the emergence of pathological genotypes and neutral evolution of proteomes.

MUTATIONS OF THE SMARCB1 GENE IN HUMAN CANCERS

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D. S. Mikhaylenko

2016-01-01

Full Text Available In the recent years, the full exome sequencing helped to reveal a  set of mutations in the genes that are not oncogenes or tumor suppressor genes by definition, but play an important role in carcinogenesis and encode proteins involved in chromatin remodeling. Among chromatin remodeling systems, which operate through the ATP-dependent mechanism, the complex SWI/ SNF attracts the great attention. The complex consists of the catalytic ATPase (SMARCA2/4, a group of conservative core subunits (SMARCB1, SMARCC1/2, and variant subunits. Abnormalities in the genes coding for each of these components have been identified as driver mutations in various human tumors. The SMARCB1 gene is of interest for practical oncogenetics, with its typical genotype-phenotype correlations. Germinal inactivating mutations (frameshift insertions/deletions, full deletions of the gene, nonsense mutations lead to development of rhabdoid tumors in the kidneys and the brain in children in their first years of life, or even in utero. These tumors are highly malignant (Rhabdoid Tumor Predisposition Syndrome 1 – RTPS1. If a mutation carrier survives his/hers four years of life without manifestation RTPS1 with a missense mutation or has the mutation in the "hot spot" of the first or the last exon, then he/she will not develop rhabdoid tumors, but after 20 years of life, shwannomatosis may develop as multiple benign tumors of peripheral nerves. Finally, some point mutations in the exons 8–9 can result in Coffin-Siris syndrome characterized by mental retardation and developmental disorders, but no neoplasms. In this regard, rational referral of patients for direct DNA diagnostics of each of the described disease entities plays an important role, based on respective minimal criteria, as well as necessity of further development of NGS technologies (full genome and full exome sequencing that are able to sequence not only individual exons, but all candidate genes of the

The Arctic Alzheimer mutation enhances sensitivity to toxic stress in human neuroblastoma cells

DEFF Research Database (Denmark)

Sennvik, Kristina; Nilsberth, Camilla; Stenh, Charlotte

2002-01-01

The E693G (Arctic) mutation of the amyloid precursor protein was recently found to lead to early-onset Alzheimer's disease in a Swedish family. In the present study, we report that the Arctic mutation decreases cell viability in human neuroblastoma cells. The cell viability, as measured by the MTT...... their secretion of beta-secretase cleaved amyloid precursor protein. The enhanced sensitivity to toxic stress in cells with the Arctic mutation most likely contributes to the pathogenic pathway leading to Alzheimer's disease....

Informative genomic microsatellite markers for efficient genotyping applications in sugarcane.

Science.gov (United States)

Parida, Swarup K; Kalia, Sanjay K; Kaul, Sunita; Dalal, Vivek; Hemaprabha, G; Selvi, Athiappan; Pandit, Awadhesh; Singh, Archana; Gaikwad, Kishor; Sharma, Tilak R; Srivastava, Prem Shankar; Singh, Nagendra K; Mohapatra, Trilochan

2009-01-01

Genomic microsatellite markers are capable of revealing high degree of polymorphism. Sugarcane (Saccharum sp.), having a complex polyploid genome requires more number of such informative markers for various applications in genetics and breeding. With the objective of generating a large set of microsatellite markers designated as Sugarcane Enriched Genomic MicroSatellite (SEGMS), 6,318 clones from genomic libraries of two hybrid sugarcane cultivars enriched with 18 different microsatellite repeat-motifs were sequenced to generate 4.16 Mb high-quality sequences. Microsatellites were identified in 1,261 of the 5,742 non-redundant clones that accounted for 22% enrichment of the libraries. Retro-transposon association was observed for 23.1% of the identified microsatellites. The utility of the microsatellite containing genomic sequences were demonstrated by higher primer designing potential (90%) and PCR amplification efficiency (87.4%). A total of 1,315 markers including 567 class I microsatellite markers were designed and placed in the public domain for unrestricted use. The level of polymorphism detected by these markers among sugarcane species, genera, and varieties was 88.6%, while cross-transferability rate was 93.2% within Saccharum complex and 25% to cereals. Cloning and sequencing of size variant amplicons revealed that the variation in the number of repeat-units was the main source of SEGMS fragment length polymorphism. High level of polymorphism and wide range of genetic diversity (0.16-0.82 with an average of 0.44) assayed with the SEGMS markers suggested their usefulness in various genotyping applications in sugarcane.

Ovarian metastasis from uveal melanoma with MLH1/PMS2 protein loss in a patient with germline MLH1 mutated Lynch syndrome: consequence or coincidence?

Science.gov (United States)

Lobo, João; Pinto, Carla; Freitas, Micaela; Pinheiro, Manuela; Vizcaino, Rámon; Oliva, Esther; Teixeira, Manuel R; Jerónimo, Carmen; Bartosch, Carla

2017-03-01

Currently, uveal melanoma is not considered within the Lynch syndrome tumor spectrum. However, there are studies suggesting a contribution of microsatellite instability in sporadic uveal melanoma tumorigenesis. We report a 45-year-old woman who was referred for genetic counseling due to a family history of Lynch syndrome caused by a MLH1 mutation. She originally underwent enucleation of the right eye secondary to a uveal spindle cell melanoma diagnosed at age 25. The tumor recurred 22 years later presenting as an ovarian metastasis and concurrently a microscopic endometrial endometrioid carcinoma, grade 1/3 was diagnosed. Subsequent studies highlighted that the uveal melanoma showed high microsatellite instability and loss of MLH1 and PMS2 protein expression, with no MLH1 promoter methylation or BRAF mutation. Additionally, a GNAQ mutation was found. We conclude that our patient's uveal melanoma is most likely related to MLH1 germline mutation and thus Lynch syndrome related. To the best of our knowledge, this is the first report of uveal melanoma showing MLH1/PMS2 protein loss in the context of Lynch syndrome.

Distinct Contributions of Replication and Transcription to Mutation Rate Variation of Human Genomes

KAUST Repository

Cui, Peng; Ding, Feng; Lin, Qiang; Zhang, Lingfang; Li, Ang; Zhang, Zhang; Hu, Songnian; Yu, Jun

2012-01-01

Here, we evaluate the contribution of two major biological processes—DNA replication and transcription—to mutation rate variation in human genomes. Based on analysis of the public human tissue transcriptomics data, high-resolution replicating map of Hela cells and dbSNP data, we present significant correlations between expression breadth, replication time in local regions and SNP density. SNP density of tissue-specific (TS) genes is significantly higher than that of housekeeping (HK) genes. TS genes tend to locate in late-replicating genomic regions and genes in such regions have a higher SNP density compared to those in early-replication regions. In addition, SNP density is found to be positively correlated with expression level among HK genes. We conclude that the process of DNA replication generates stronger mutational pressure than transcription-associated biological processes do, resulting in an increase of mutation rate in TS genes while having weaker effects on HK genes. In contrast, transcription-associated processes are mainly responsible for the accumulation of mutations in highly-expressed HK genes.

Distinct Contributions of Replication and Transcription to Mutation Rate Variation of Human Genomes

KAUST Repository

Cui, Peng

2012-03-23

Here, we evaluate the contribution of two major biological processes—DNA replication and transcription—to mutation rate variation in human genomes. Based on analysis of the public human tissue transcriptomics data, high-resolution replicating map of Hela cells and dbSNP data, we present significant correlations between expression breadth, replication time in local regions and SNP density. SNP density of tissue-specific (TS) genes is significantly higher than that of housekeeping (HK) genes. TS genes tend to locate in late-replicating genomic regions and genes in such regions have a higher SNP density compared to those in early-replication regions. In addition, SNP density is found to be positively correlated with expression level among HK genes. We conclude that the process of DNA replication generates stronger mutational pressure than transcription-associated biological processes do, resulting in an increase of mutation rate in TS genes while having weaker effects on HK genes. In contrast, transcription-associated processes are mainly responsible for the accumulation of mutations in highly-expressed HK genes.

Whole genome sequencing of mutation accumulation lines reveals a low mutation rate in the social amoeba Dictyostelium discoideum.

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Gerda Saxer

Full Text Available Spontaneous mutations play a central role in evolution. Despite their importance, mutation rates are some of the most elusive parameters to measure in evolutionary biology. The combination of mutation accumulation (MA experiments and whole-genome sequencing now makes it possible to estimate mutation rates by directly observing new mutations at the molecular level across the whole genome. We performed an MA experiment with the social amoeba Dictyostelium discoideum and sequenced the genomes of three randomly chosen lines using high-throughput sequencing to estimate the spontaneous mutation rate in this model organism. The mitochondrial mutation rate of 6.76×10(-9, with a Poisson confidence interval of 4.1×10(-9 - 9.5×10(-9, per nucleotide per generation is slightly lower than estimates for other taxa. The mutation rate estimate for the nuclear DNA of 2.9×10(-11, with a Poisson confidence interval ranging from 7.4×10(-13 to 1.6×10(-10, is the lowest reported for any eukaryote. These results are consistent with low microsatellite mutation rates previously observed in D. discoideum and low levels of genetic variation observed in wild D. discoideum populations. In addition, D. discoideum has been shown to be quite resistant to DNA damage, which suggests an efficient DNA-repair mechanism that could be an adaptation to life in soil and frequent exposure to intracellular and extracellular mutagenic compounds. The social aspect of the life cycle of D. discoideum and a large portion of the genome under relaxed selection during vegetative growth could also select for a low mutation rate. This hypothesis is supported by a significantly lower mutation rate per cell division in multicellular eukaryotes compared with unicellular eukaryotes.

Colorectal carcinomas in MUTYH-associated polyposis display histopathological similarities to microsatellite unstable carcinomas

International Nuclear Information System (INIS)

Nielsen, Maartje; Hes, Frederik J; Morreau, Hans; Miranda, Noel FCC de; Puijenbroek, Marjo van; Jordanova, Ekaterina S; Middeldorp, Anneke; Wezel, Tom van; Eijk, Ronald van; Tops, Carli MJ; Vasen, Hans FA

2009-01-01

MUTYH-associated polyposis (MAP) is a recessively inherited disorder which predisposes biallelic carriers for a high risk of polyposis and colorectal carcinoma (CRC). Since about one third of the biallelic MAP patients in population based CRC series has no adenomas, this study aimed to identify specific clinicopathological characteristics of MAP CRCs and compare these with reported data on sporadic and Lynch CRCs. From 44 MAP patients who developed ≥ 1 CRCs, 42 of 58 tumours were analyzed histologically and 35 immunohistochemically for p53 and beta-catenin. Cell densities of CD3, CD8, CD57, and granzyme B positive lymphocytes were determined. KRAS2, the mutation cluster region (MCR) of APC, p53, and SMAD4 were analyzed for somatic mutations. MAP CRCs frequently localized to the proximal colon (69%, 40/58), were mucinous in 21% (9/42), and had a conspicuous Crohn's like infiltrate reaction in 33% (13/40); all of these parameters occurred at a higher rate than reported for sporadic CRCs. Tumour infiltrating lymphocytes (TILs) were also highly prevalent in MAP CRCs. Somatic APC MCR mutations occurred in 14% (5/36) while 64% (23/36) had KRAS2 mutations (22/23 c.34G>T). G>T tranversions were found in p53 and SMAD4, although the relative frequency compared to other mutations was low. MAP CRCs show some similarities to micro-satellite unstable cancers, with a preferential proximal location, a high rate of mucinous histotype and increased presence of TILs. These features should direct the practicing pathologist towards a MAP aetiology of CRC as an alternative for a mismatch repair deficient cause. High frequent G>T transversions in APC and KRAS2 (mutated in early tumour development) but not in P53 and SMAD4 (implicated in tumour progression) might indicate a predominant MUTYH effect in early carcinogenesis

Associations between mutations and a VNTR in the human phenylalanine hydroxylase gene

Energy Technology Data Exchange (ETDEWEB)

Goltsov, A.A.; Eisensmith, R.C.; Woo, S.L.C. (Baylor College of Medicine, Houston, TX (United States)); Konecki, D.S.; Lichter-Konecki, U.

1992-09-01

The HindIII RFLP in the human phenylalanine hydroxylase (PAH) gene is caused by the presence of an AT-rich (70%) minisatellite region. This region contains various multiples of 30-bp tandem repeats and is located 3 kb downstream of the final exon of the gene. PCR-mediated amplification of this region from haplotyped PAH chromosomes indicates that the previously reported 4.0-kb HindIII allele contains three of these repeats, while the 4.4-kb HindIII allele contains 12 of these repeats. The 4.2-kb HindIII fragment can contain six, seven, eight, or nine copies of this repeat. These variations permit more detailed analysis of mutant haplotypes 1, 5, 6, and, possibly, others. Kindred analysis in phenylketonuria families demonstrates Mendelian segregation of these VNTR alleles, as well as associations between theses alleles and certain PAH mutations. The R261Q mutation, associated with haplotype 1, is associated almost exclusively with an allele containing eight repeats; the R408W mutation, when occurring on a haplotype 1 background, may also be associated with the eight-repeat VNTR allele. Other PAH mutations associated with haplotype 1, R252W and P281L, do not appear to segregate with specific VNTR alleles. The IVS-10 mutation, when associated with haplotype 6, is associated exclusively with an allele containing seven repeats. The combined use of this VNTR system and the existing RFLP haplotype system will increase the performance of prenatal diagnostic tests based on haplotype analysis. In addition, this VNTR may prove useful in studies concerning the origins and distributions of PAH mutations in different human populations. 32 refs., 3 figs., 3 tabs.

Genetic variability of Taenia solium cysticerci recovered from experimentally infected pigs and from naturally infected pigs using microsatellite markers.

Science.gov (United States)

Pajuelo, Mónica J; Eguiluz, María; Roncal, Elisa; Quiñones-García, Stefany; Clipman, Steven J; Calcina, Juan; Gavidia, Cesar M; Sheen, Patricia; Garcia, Hector H; Gilman, Robert H; Gonzalez, Armando E; Zimic, Mirko

2017-12-01

The adult Taenia solium, the pork tapeworm, usually lives as a single worm in the small intestine of humans, its only known definitive host. Mechanisms of genetic variation in T. solium are poorly understood. Using three microsatellite markers previously reported [1], this study explored the genetic variability of T. solium from cysts recovered from experimentally infected pigs. It then explored the genetic epidemiology and transmission in naturally infected pigs and adult tapeworms recovered from human carriers from an endemic rural community in Peru. In an initial study on experimental infection, two groups of three piglets were each infected with proglottids from one of two genetically different tapeworms for each of the microsatellites. After 7 weeks, pigs were slaughtered and necropsy performed. Thirty-six (92.3%) out of 39 cysts originated from one tapeworm, and 27 (100%) out of 27 cysts from the other had exactly the same genotype as the parental tapeworm. This suggests that the microsatellite markers may be a useful tool for studying the transmission of T. solium. In the second study, we analyzed the genetic variation of T. solium in cysts recovered from eight naturally infected pigs, and from adult tapeworms recovered from four human carriers; they showed genetic variability. Four pigs had cysts with only one genotype, and four pigs had cysts with two different genotypes, suggesting that multiple infections of genetically distinct parental tapeworms are possible. Six pigs harbored cysts with a genotype corresponding to one of the identified tapeworms from the human carriers. In the dendrogram, cysts appeared to cluster within the corresponding pigs as well as with the geographical origin, but this association was not statistically significant. We conclude that genotyping of microsatellite size polymorphisms is a potentially important tool to trace the spread of infection and pinpoint sources of infection as pigs spread cysts with a shared parental genotype.

Genetic variability of Taenia solium cysticerci recovered from experimentally infected pigs and from naturally infected pigs using microsatellite markers.

Directory of Open Access Journals (Sweden)

Mónica J Pajuelo

2017-12-01

Full Text Available The adult Taenia solium, the pork tapeworm, usually lives as a single worm in the small intestine of humans, its only known definitive host. Mechanisms of genetic variation in T. solium are poorly understood. Using three microsatellite markers previously reported [1], this study explored the genetic variability of T. solium from cysts recovered from experimentally infected pigs. It then explored the genetic epidemiology and transmission in naturally infected pigs and adult tapeworms recovered from human carriers from an endemic rural community in Peru. In an initial study on experimental infection, two groups of three piglets were each infected with proglottids from one of two genetically different tapeworms for each of the microsatellites. After 7 weeks, pigs were slaughtered and necropsy performed. Thirty-six (92.3% out of 39 cysts originated from one tapeworm, and 27 (100% out of 27 cysts from the other had exactly the same genotype as the parental tapeworm. This suggests that the microsatellite markers may be a useful tool for studying the transmission of T. solium. In the second study, we analyzed the genetic variation of T. solium in cysts recovered from eight naturally infected pigs, and from adult tapeworms recovered from four human carriers; they showed genetic variability. Four pigs had cysts with only one genotype, and four pigs had cysts with two different genotypes, suggesting that multiple infections of genetically distinct parental tapeworms are possible. Six pigs harbored cysts with a genotype corresponding to one of the identified tapeworms from the human carriers. In the dendrogram, cysts appeared to cluster within the corresponding pigs as well as with the geographical origin, but this association was not statistically significant. We conclude that genotyping of microsatellite size polymorphisms is a potentially important tool to trace the spread of infection and pinpoint sources of infection as pigs spread cysts with a shared

Microsatellite primers for fungus-growing ants

DEFF Research Database (Denmark)

Villesen, Palle; Gertsch, P J; Boomsma, JJ

2002-01-01

We isolated five polymorphic microsatellite loci from a library of two thousand recombinant clones of two fungus-growing ant species, Cyphomyrmex longiscapus and Trachymyrmex cf. zeteki. Amplification and heterozygosity were tested in five species of higher attine ants using both the newly...... developed primers and earlier published primers that were developed for fungus-growing ants. A total of 20 variable microsatellite loci, developed for six different species of fungus-growing ants, are now available for studying the population genetics and colony kin-structure of these ants....

Microsatellite Primers for Fungus-Growing Ants

DEFF Research Database (Denmark)

Villesen Fredsted, Palle; Gertsch, Pia J.; Boomsma, Jacobus Jan (Koos)

2002-01-01

We isolated five polymorphic microsatellite loci from a library of two thousand recombinant clones of two fungus-growing ant species, Cyphomyrmex longiscapus and Trachymyrmex cf. zeteki. Amplification and heterozygosity were tested in five species of higher attine ants using both the newly...... developed primers and earlier published primers that were developed for fungus-growing ants. A total of 20 variable microsatellite loci, developed for six different species of fungus-growing ants, are now available for studying the population genetics and colony kin-structure of these ants....

Microsatellite analysis of the genetic relationships between wild

Indian Academy of Sciences (India)

In the present study, we isolated 11 microsatellite DNA markers, and analysed the genetic diversity and differentiation between cultured stocks and wild populations of the giant grouper originating from the South China Sea. A total of 390 alleles at 11 microsatellite loci were detected in 130 individuals from five different ...

The F309S mutation increases factor VIII secretion in human cell line

Directory of Open Access Journals (Sweden)

Daianne Maciely Carvalho Fantacini

2016-06-01

Full Text Available ABSTRACT OBJECTIVES: The capacity of a human cell line to secrete recombinant factor VIII with a F309S point mutation was investigated, as was the effect of the addition of chemical chaperones (betaine and sodium-4-phenylbutyrate on the secretion of factor VIII. METHODS: This work used a vector with a F309S mutation in the A1 domain to investigate FVIII production in the HEK 293 human cell line. Factor VIII activity was measured by chromogenic assay. Furthermore, the effects of chemical drugs on the culture were evaluated. RESULTS: The addition of the F309S mutation to a previously described FVIII variant increased FVIII secretion by 4.5 fold. Moreover, the addition of betaine or sodium-4-phenylbutyrate increased the secretion rate of FVIIIΔB proteins in HEK 293 cells, but the same effect was not seen for FVIIIΔB-F309S indicating that all the recombinant protein produced had been efficiently secreted. CONCLUSION: Bioengineering factor VIII expressed in human cells may lead to an efficient production of recombinant factor VIII and contribute toward low-cost coagulation factor replacement therapy for hemophilia A. FVIII-F309S produced in human cells can be effective in vivo.

Mutation and Expression of the DCC Gene in Human Lung Cancer

Directory of Open Access Journals (Sweden)

Takashi Kohno

2000-07-01

Full Text Available Chromosome 18q is frequently deleted in lung cancers, a common region of 18q deletions was mapped to chromosome 18g21. Since the DCC candidate tumor suppressor gene has been mapped in this region, mutation and expression of the DCC gene were examined in 46 lung cancer cell lines, consisting of 14 small cell lung carcinomas (SCLCs and 32 non-small cell lung carcinomas (NSCLCs, to elucidate the pathogenetic significance of DCC alterations in human lung carcinogenesis. A heterozygous missense mutation was detected in a NSCLC cell line, Ma26, while homozygous deletion was not detected in any of the cell lines. The DCC gene was expressed in 11 (24% of the 46 cell lines, the incidence of DCC expression was significantly higher in SCLCs (7/14, 50% than in NSCLCs (4/32, 13% (P = .01, Fisher's exact test. Therefore, genetic alterations of DCC are infrequent; however, the levels of DCC expression vary among lung cancer cells, in particular, between SCLCs and NSCLCs. The present result does not implicate DCC as a specific mutational target of 18q deletions in human lung cancer; however, it suggests that DCC is a potential target of inactivation by genetic defects including intron or promoter mutations and/or epigenetic alterations. The present result also suggests that DCC expression is associated with some properties of SCLCs, such as a neuroendocrine (NE feature.

A mitochondrial cytochrome b mutation causing severe respiratory chain enzyme deficiency in humans and yeast.

NARCIS (Netherlands)

Blakely, E.L.; Mitchell, A.L.; Fisher, N.; Meunier, B.; Nijtmans, L.G.J.; Schaefer, A.M.; Jackson, M.J.; Turnbull, D.M.; Taylor, R.W.

2005-01-01

Whereas the majority of disease-related mitochondrial DNA mutations exhibit significant biochemical and clinical heterogeneity, mutations within the mitochondrially encoded human cytochrome b gene (MTCYB) are almost exclusively associated with isolated complex III deficiency in muscle and a clinical

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

Science.gov (United States)

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

2008-06-01

A 14-year-old male presented with a T4 sigmoid adenocarcinoma, PMS2 protein and high frequency microsatellite instability. Germline analysis identified biallelic PMS2 missense mutations. A new cancer syndrome caused by biallelic mutations in the mismatch repair genes, including PMS2, is now emerging and is characterized by café-au-lait macules, colonic polyps and a distinctive tumor spectrum. (c) 2007 Wiley-Liss, Inc.

Genetic changes of MLH1 and MSH2 genes could explain constant findings on microsatellite instability in intracranial meningioma.

Science.gov (United States)

Pećina-Šlaus, Nives; Kafka, Anja; Bukovac, Anja; Vladušić, Tomislav; Tomas, Davor; Hrašćan, Reno

2017-07-01

Postreplicative mismatch repair safeguards the stability of our genome. The defects in its functioning will give rise to microsatellite instability. In this study, 50 meningiomas were investigated for microsatellite instability. Two major mismatch repair genes, MLH1 and MSH2, were analyzed using microsatellite markers D1S1611 and BAT26 amplified by polymerase chain reaction and visualized by gel electrophoresis on high-resolution gels. Furthermore, genes DVL3 (D3S1262), AXIN1 (D16S3399), and CDH1 (D16S752) were also investigated for microsatellite instability. Our study revealed constant presence of microsatellite instability in meningioma patients when compared to their autologous blood DNA. Altogether 38% of meningiomas showed microsatellite instability at one microsatellite locus, 16% on two, and 13.3% on three loci. The percent of detected microsatellite instability for MSH2 gene was 14%, and for MLH1, it was 26%, for DVL3 22.9%, for AXIN1 17.8%, and for CDH1 8.3%. Since markers also allowed for the detection of loss of heterozygosity, gross deletions of MLH1 gene were found in 24% of meningiomas. Genetic changes between MLH1 and MSH2 were significantly positively correlated (p = 0.032). We also noted a positive correlation between genetic changes of MSH2 and DVL3 genes (p = 0.034). No significant associations were observed when MLH1 or MSH2 was tested against specific histopathological meningioma subtype or World Health Organization grade. However, genetic changes in DVL3 were strongly associated with anaplastic histology of meningioma (χ 2  = 9.14; p = 0.01). Our study contributes to better understanding of the genetic profile of human intracranial meningiomas and suggests that meningiomas harbor defective cellular DNA mismatch repair mechanisms.

Genetic variability and bottleneck analyses of Kanni adu goat population using microsatellite markers [Indian Veterinary Journal, 2015, 92(3): 24-27

International Nuclear Information System (INIS)

Thiruvenkadan, R.; Jeyakumar, M.; Saravana, R.; Periasamy, K.

2016-01-01

Full text: The genetic characterization and bottleneck analysis in Kodi Adu goat was done using 25 FAO recommended microsatellite markers. The mean observed number of alleles and polymorphism information content (PIC) were estimated to be 11.52±-0.95 and 0.817±0.023 respectively. The mean observed and expected equilibrium hyterozygosities were 0.660±0.045 and 0.846±0.018 respectively. The mean expected equilibrium gene diversity across 21 microsatellite loci under TAM, SMM and TPM were 0.793±0.028, 0.854±0.023 and 0.827±0.026 respectively. All the three statistical tests revealed significant deviation of Kodi Adu goats from mutation-drift equilibrium under the IAM and TPM model. The mode shift analysis supported the results under SMM indicating the absence of genetic bottleneck in the recent past in Kodi Adu goats. (author)

A COLQ missense mutation in Labrador Retrievers having congenital myasthenic syndrome.

Directory of Open Access Journals (Sweden)

Caitlin J Rinz

Full Text Available Congenital myasthenic syndromes (CMSs are heterogeneous neuromuscular disorders characterized by skeletal muscle weakness caused by disruption of signal transmission across the neuromuscular junction (NMJ. CMSs are rarely encountered in veterinary medicine, and causative mutations have only been identified in Old Danish Pointing Dogs and Brahman cattle to date. Herein, we characterize a novel CMS in 2 Labrador Retriever littermates with an early onset of marked generalized muscle weakness. Because the sire and dam share 2 recent common ancestors, CMS is likely the result of recessive alleles inherited identical by descent (IBD. Genome-wide SNP profiles generated from the Illumina HD array for 9 nuclear family members were used to determine genomic inheritance patterns in chromosomal regions encompassing 18 functional candidate genes. SNP haplotypes spanning 3 genes were consistent with autosomal recessive transmission, and microsatellite data showed that only the segment encompassing COLQ was inherited IBD. COLQ encodes the collagenous tail of acetylcholinesterase, the enzyme responsible for termination of signal transduction in the NMJ. Sequences from COLQ revealed a variant in exon 14 (c.1010T>C that results in the substitution of a conserved amino acid (I337T within the C-terminal domain. Both affected puppies were homozygous for this variant, and 16 relatives were heterozygous, while 288 unrelated Labrador Retrievers and 112 dogs of other breeds were wild-type. A recent study in which 2 human CMS patients were found to be homozygous for an identical COLQ mutation (c.1010T>C; I337T provides further evidence that this mutation is pathogenic. This report describes the first COLQ mutation in canine CMS and demonstrates the utility of SNP profiles from nuclear family members for the identification of private mutations.

Canine and human gastrointestinal stromal tumors display similar mutations in c-KIT exon 11

International Nuclear Information System (INIS)

Gregory-Bryson, Emmalena; Bartlett, Elizabeth; Kiupel, Matti; Hayes, Schantel; Yuzbasiyan-Gurkan, Vilma

2010-01-01

Gastrointestinal stromal tumors (GISTs) are common mesenchymal neoplasms in the gastrointestinal tract of humans and dogs. Little is known about the pathogenesis of these tumors. This study evaluated the role of c-KIT in canine GISTs; specifically, we investigated activating mutations in exons 8, 9, 11, 13, and 17 of c-KIT and exons 12, 14, and 18 of platelet-derived growth factor receptor, alpha polypeptide (PDGFRA), all of which have been implicated in human GISTs. Seventeen canine GISTs all confirmed to be positive for KIT immunostaining were studied. Exons 8, 9, 11, 13 and 17 of c-KIT and exons 12, 14, and 18 of PDGFRA, were amplified from DNA isolated from formalin-fixed paraffin-embedded samples. Of these seventeen cases, six amplicons of exon 11 of c-KIT showed aberrant bands on gel electrophoresis. Sequencing of these amplicons revealed heterozygous in-frame deletions in six cases. The mutations include two different but overlapping six base pair deletions. Exons 8, 9, 13, and 17 of c-KIT and exons 12, 14, and 18 of PDGFRA had no abnormalities detected by electrophoresis and sequencing did not reveal any mutations, other than synonymous single nucleotide polymorphisms (SNPs) found in exon 11 of c-KIT and exons 12 and 14 of PDGFRA. The deletion mutations detected in canine GISTs are similar to those previously found in the juxtamembrane domain of c-KIT in canine cutaneous mast cell tumors in our laboratory as well as to those reported in human GISTs. Interestingly, none of the other c-KIT or PDGFRA exons showed any abnormalities in our cases. This finding underlines the critical importance of c-KIT in the pathophysiology of canine GISTs. The expression of KIT and the identification of these activating mutations in c-KIT implicate KIT in the pathogenesis of these tumors. Our results indicate that mutations in c-KIT may be of prognostic significance and that targeting KIT may be a rational approach to treatment of these malignant tumors. This study further

Development and characterization of polymorphic microsatellite markers in Dysosma pleiantha (Berberidaceae).

Science.gov (United States)

Guan, Bi-Cai; Gong, Xi; Zhou, Shi-Liang

2011-08-01

The development of compound microsatellite markers was conducted in Dysosma pleiantha to investigate genetic diversity and population genetic structure of this threatened medicinal plant. Using the compound microsatellite marker technique, 14 microsatellite markers that were successfully amplified showed polymorphism when tested on 38 individuals from three populations in eastern China. Overall, the number of alleles per locus ranged from 2 to 14, with an average of 7.71 alleles per locus. These results indicate that these microsatellite markers are adequate for detecting and characterizing population genetic structure and genetic diversity in Dysosma pleiantha.

Genomic characterization of EmsB microsatellite loci in Echinococcus multilocularis.

Science.gov (United States)

Valot, Benoît; Knapp, Jenny; Umhang, Gérald; Grenouillet, Frédéric; Millon, Laurence

2015-06-01

EmsB is a molecular marker applied to Echinococcus multilocularis genotyping studies. This marker has largely been used to investigate the epidemiology of the parasite in different endemic foci. The present study has lifted the veil on the genetic structure of this microsatellite. By in silico analysis on the E. multilocularis genome the microsatellite was described in about 40 copies on the chromosome 5 of the parasite. Similar structure was found in the relative parasite Echinococcus granulosus, where the microsatellite was firstly described. The present study completes the first investigations made on the EmsB microsatellite origins and confirms the reliability of this highly discriminant molecular marker. Copyright © 2015 Elsevier B.V. All rights reserved.

Eguchipsammia fistula Microsatellite Development and Population Analysis

KAUST Repository

Mughal, Mehreen

2012-12-01

Deep water corals are an understudied yet biologically important and fragile ecosystem under threat from recent increasing temperatures and high carbon dioxide emissions. Using 454 sequencing, we develop 14 new microsatellite markers for the deep water coral Eguchipsammia fistula, collected from the Red Sea but found in deep water coral ecosystems globally. We tested these microsatellite primers on 26 samples of this coral collected from a single population. Results show that these corals are highly clonal within this population stemming from a high level of asexual reproduction. Mitochondrial studies back up microsatellite findings of high levels of genetic similarity. CO1, ND1 and ATP6 mitochondrial sequences of E. fistula and 11 other coral species were used to build phylogenetic trees which grouped E. fistula with shallow water coral Porites rather than deep sea L. Petusa.

genetic diversity of microsatellit

Indian Academy of Sciences (India)

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with inhabit inland dry gypsum soils, coastal cliffs and salt marshes (Palacios .... Twenty microsatellite loci generated a total of 117 alleles in the 102 samples of 6 .... self-incompatibility is thought to take important part in the maintenance of the ...

Synchronous lung tumours in a patient with metachronous colorectal carcinoma and a germline MSH2 mutation.

LENUS (Irish Health Repository)

Canney, A

2012-02-01

Mutations of DNA mismatch repair genes are characterised by microsatellite instability and are implicated in carcinogenesis. This mutation susceptible phenotype has been extensively studied in patients with hereditary non-polyposis colon carcinoma, but little is known of the contribution of such mutations in other tumour types, particularly non-small-cell lung carcinoma. This report describes the occurrence of two synchronous lung tumours, one mimicking a metastatic colon carcinoma, in a male patient with a history of metachronous colonic carcinoma. Immunohistochemistry supported a pulmonary origin for both lesions. Mismatch repair protein immunohistochemistry showed loss of MSH2 and MSH6 expression in both colonic tumours and in one lung tumour showing enteric differentiation. Subsequent mutational analysis demonstrated a deleterious germline mutation of the MSH2 mismatch repair gene. The significance of these findings and the practical diagnostic difficulties encountered in this case are discussed.

Unique mutational profile associated with a loss of TDG expression in the rectal cancer of a patient with a constitutional PMS2 deficiency.

Science.gov (United States)

Vasovcak, P; Krepelova, A; Menigatti, M; Puchmajerova, A; Skapa, P; Augustinakova, A; Amann, G; Wernstedt, A; Jiricny, J; Marra, G; Wimmer, K

2012-07-01

Cells with DNA repair defects have increased genomic instability and are more likely to acquire secondary mutations that bring about cellular transformation. We describe the frequency and spectrum of somatic mutations involving several tumor suppressor genes in the rectal carcinoma of a 13-year-old girl harboring biallelic, germline mutations in the DNA mismatch repair gene PMS2. Apart from microsatellite instability, the tumor DNA contained a number of C:G→T:A or G:C→A:T transitions in CpG dinucleotides, which often result through spontaneous deamination of cytosine or 5-methylcytosine. Four DNA glycosylases, UNG2, SMUG1, MBD4 and TDG, are involved in the repair of these deamination events. We identified a heterozygous missense mutation in TDG, which was associated with TDG protein loss in the tumor. The CpGs mutated in this patient's tumor are generally methylated in normal colonic mucosa. Thus, it is highly likely that loss of TDG contributed to the supermutator phenotype and that most of the point mutations were caused by deamination of 5-methylcytosine to thymine, which remained uncorrected owing to the TDG deficiency. This case provides the first in vivo evidence of the key role of TDG in protecting the human genome against the deleterious effects of 5-methylcytosine deamination. Copyright © 2012 Elsevier B.V. All rights reserved.

Familial colorectal cancer, can it be identified by microsatellite instability and chromosomal instability? - A case-control study

DEFF Research Database (Denmark)

Sunde, Lone; Bisgaard, Marie Luise; Soll-Johanning, Helle

2009-01-01

(Chromosome INstability=LOH (loss of heterozygosity) and/or DNA-aneuploidy (abnormal nuclear DNA contents)) could be used as predictors of familial CRC. Formalin-fixed tissue from 97 patients with CRC (29 patients with 2 or more affected first-degree relatives (="cases"), 29 matched CRC controls without......Colonoscopy is recommended for persons with a familial risk of colorectal cancer (CRC). A familial risk is identified by a family history with CRC and/or predisposing mutation(s). However, such information may not be available. We analysed whether MSI (MicroSatellite Instability) and/or CIN...... a family history, and 39 relatives to cases) were analysed for MSI and CIN. In this small case-control study, no significant differences in the frequencies of MSI and CIN were observed between cases with a family history and their controls without a family history. MSI+;CIN- was observed in 6/29 cases...

Microsatellites in varied arenas of research

Directory of Open Access Journals (Sweden)

K S Remya

2010-01-01

Full Text Available Microsatellites known as simple-sequence repeats (SSRs or short-tandem repeats (STRs, represent specific sequences of DNA consisting of tandemly repeated units of one to six nucleotides. The repetitive nature of microsatellites makes them particularly prone to grow or shrink in length and these changes can have both good and bad consequences for the organisms that possess them. They are responsible for various neurological diseases and hence the same cause is now utilized for the early detection of various diseases, such as, Schizophrenia and Bipolar Disorder, Congenital generalized Hypertrichosis, Asthma, and Bronchial Hyperresponsiveness. These agents are widely used for forensic identification and relatedness testing, and are predominant genetic markers in this area of application. The application of microsatellites is an extending web and covers the varied scenarios of science, such as, conservation biology, plant genetics, and population studies. At present, researches are progressing round the globe to extend the use of these genetic repeaters to unmask the hidden genetic secrets behind the creation of the world.

The neurological mouse mutations jittery and hesitant are allelic and map to the region of mouse chromosome 10 homologous to 19p13.3

Energy Technology Data Exchange (ETDEWEB)

Kapfhamer, D.; Sufalko, D.; Warren, S. [Univ. of Michigan, Ann Arbor, MI (United States)] [and others

1996-08-01

Jittery (ji) is a recessive mouse mutation on Chromosome 10 characterized by progressive ataxic gait, dystonic movements, spontaneus seizures, and death by dehydration/starvation before fertility. Recently, a viable neurological recessive mutation, hesitant, was discovered. It is characterized by hesitant, uncoordinated movements, exaggerated stepping of the hind limbs, and reduced fertility in males. In a complementation test and by genetic mapping we have shown here that hesitant and jittery are allelic. Using several large intersubspecific backcrosses and intercrosses we have genetically mapped ji near the marker Amh and microsatellite markers D10Mit7, D10Mit21, and D10Mit23. The linked region of mouse Chromosome 10 is homologous to human 19p13.3, to which several human ataxia loci have recently been mapped. By excluding genes that map to human 21q22.3 (Pfkl) and 12q23 (Nfyb), we conclude that jittery is not likely to be a genetic mouse model for human Unverricht-Lundborg progressive myoclonus epilepsy (EPM1) on 21q22.3 nor for spinocerebellar ataxia II (SCA2) on 12q22-q24. The closely linked markers presented here will facilitate positional cloning of the ji gene. 31 refs., 2 figs.

Nine microsatellite loci developed from the octocoral, Paragorgia arborea

Science.gov (United States)

Coykendall, D. Katharine; Morrison, Cheryl L.

2015-01-01

Paragorgia arborea, or bubblegum coral, occurs in continental slope habitats worldwide, which are increasingly threatened by human activities such as energy development and fisheries practices. From 101 putative loci screened, nine microsatellite markers were developed from samples taken from Baltimore canyon in the western North Atlantic Ocean. The number of alleles ranged from two to thirteen per locus and each displayed equilibrium. These nuclear resources will help further research on population connectivity in threatened coral species where mitochondrial markers are known to lack fine-scale genetic diversity.

Rich RNA Structure Landscapes Revealed by Mutate-and-Map Analysis.

Directory of Open Access Journals (Sweden)

Pablo Cordero

2015-11-01

Full Text Available Landscapes exhibiting multiple secondary structures arise in natural RNA molecules that modulate gene expression, protein synthesis, and viral infection [corrected]. We report herein that high-throughput chemical experiments can isolate an RNA's multiple alternative secondary structures as they are stabilized by systematic mutagenesis (mutate-and-map, M2 and that a computational algorithm, REEFFIT, enables unbiased reconstruction of these states' structures and populations. In an in silico benchmark on non-coding RNAs with complex landscapes, M2-REEFFIT recovers 95% of RNA helices present with at least 25% population while maintaining a low false discovery rate (10% and conservative error estimates. In experimental benchmarks, M2-REEFFIT recovers the structure landscapes of a 35-nt MedLoop hairpin, a 110-nt 16S rRNA four-way junction with an excited state, a 25-nt bistable hairpin, and a 112-nt three-state adenine riboswitch with its expression platform, molecules whose characterization previously required expert mutational analysis and specialized NMR or chemical mapping experiments. With this validation, M2-REEFFIT enabled tests of whether artificial RNA sequences might exhibit complex landscapes in the absence of explicit design. An artificial flavin mononucleotide riboswitch and a randomly generated RNA sequence are found to interconvert between three or more states, including structures for which there was no design, but that could be stabilized through mutations. These results highlight the likely pervasiveness of rich landscapes with multiple secondary structures in both natural and artificial RNAs and demonstrate an automated chemical/computational route for their empirical characterization.

A fluorescence study of the molecular interactions of harmane with the nucleobases, their nucleosides and mononucleotides.

Science.gov (United States)

Balón, M; Muñoz, M A; Carmona, C; Guardado, P; Galán, M

1999-07-19

Fluorescence binding studies of harmane to the elemental components of the nucleic acids were undertaken to investigate the origin of the interaction between the drug and DNA. Most of the tested substrates have been found to induce hypochromism in the absorption spectrum of harmane and to quench its fluorescence. The quenching process induced by the nucleobases and their nucleosides is mainly due to the formation of ground state 1:1 complexes. However, in the case of the mononucleotides a dynamic quenching component is also observed. This quenching component is likely due to the excited state interaction of harmane with the phosphate group of the nucleotides. UV-vis spectral changes and quenching measurements have been used to quantify the ground state association constants of the complexes and the quenching rate constants.

Improving solubility and refolding efficiency of human V(H)s by a novel mutational approach.

Science.gov (United States)

Tanha, Jamshid; Nguyen, Thanh-Dung; Ng, Andy; Ryan, Shannon; Ni, Feng; Mackenzie, Roger

2006-11-01

The antibody V(H) domains of camelids tend to be soluble and to resist aggregation, in contrast to human V(H) domains. For immunotherapy, attempts have therefore been made to improve the properties of human V(H)s by camelization of a small set of framework residues. Here, we have identified through sequence comparison of well-folded llama V(H) domains an alternative set of residues (not typically camelid) for mutation. Thus, the solubility and thermal refolding efficiency of a typical human V(H), derived from the human antibody BT32/A6, were improved by introduction of two mutations in framework region (FR) 1 and 4 to generate BT32/A6.L1. Three more mutations in FR3 of BT32/A6.L1 further improved the thermal refolding efficiency while retaining solubility and cooperative melting profiles. To demonstrate practical utility, BT32/A6.L1 was used to construct a phage display library from which were isolated human V(H)s with good antigen binding activity and solubility. The engineered human V(H) domains described here may be useful for immunotherapy, due to their expected low immunogenicity, and in applications involving transient high temperatures, due to their efficient refolding after thermal denaturation.

Parkin Mutations Reduce the Complexity of Neuronal Processes in iPSC-derived Human Neurons

Science.gov (United States)

Ren, Yong; Jiang, Houbo; Hu, Zhixing; Fan, Kevin; Wang, Jun; Janoschka, Stephen; Wang, Xiaomin; Ge, Shaoyu; Feng, Jian

2015-01-01

Parkinson’s disease (PD) is characterized by the degeneration of nigral dopaminergic (DA) neurons and non-DA neurons in many parts of the brain. Mutations of parkin, an E3 ubiquitin ligase that strongly binds to microtubules, are the most frequent cause of recessively inherited Parkinson’s disease. The lack of robust PD phenotype in parkin knockout mice suggests a unique vulnerability of human neurons to parkin mutations. Here, we show that the complexity of neuronal processes as measured by total neurite length, number of terminals, number of branch points and Sholl analysis, was greatly reduced in induced pluripotent stem cell (iPSC)-derived TH+ or TH− neurons from PD patients with parkin mutations. Consistent with these, microtubule stability was significantly decreased by parkin mutations in iPSC-derived neurons. Overexpression of parkin, but not its PD-linked mutant nor GFP, restored the complexity of neuronal processes and the stability of microtubules. Consistent with these, the microtubule-depolymerizing agent colchicine mimicked the effect of parkin mutations by decreasing neurite length and complexity in control neurons while the microtubule-stabilizing drug taxol mimicked the effect of parkin overexpression by enhancing the morphology of parkin-deficient neurons. The results suggest that parkin maintains the morphological complexity of human neurons by stabilizing microtubules. PMID:25332110

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

Science.gov (United States)

Metcalf, Alexander M; Spurdle, Amanda B

2014-03-01

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

Signatures of mutational processes in human cancer

NARCIS (Netherlands)

Alexandrov, L.B.; Nik-Zainal, S.; Wedge, D.C.; Aparicio, S.A.; Behjati, S.; Biankin, A.V.; Bignell, G.R.; Bolli, N.; Borg, A.; Borresen-Dale, A.L.; Boyault, S.; Burkhardt, B.; Butler, A.P.; Caldas, C.; Davies, H.R.; Desmedt, C.; Eils, R.; Eyfjord, J.E.; Foekens, J.A.; Greaves, M.; Hosoda, F.; Hutter, B.; Ilicic, T.; Imbeaud, S.; Imielinsk, M.; Jager, N.; Jones, D.T.; Knappskog, S.; Kool, M.; Lakhani, S.R.; Lopez-Otin, C.; Martin, S.; Munshi, N.C.; Nakamura, H.; Northcott, P.A.; Pajic, M.; Papaemmanuil, E.; Paradiso, A.; Pearson, J.V.; Puente, X.S.; Raine, K.; Ramakrishna, M.; Richardson, A.L.; Richter, J.; Rosenstiel, P.; Schlesner, M.; Schumacher, T.N.; Span, P.N.; Teague, J.W.; Totoki, Y.; Tutt, A.N.; Valdes-Mas, R.; Buuren, M.M. van; Veer, L. van 't; Vincent-Salomon, A.; Waddell, N.; Yates, L.R.; Zucman-Rossi, J.; Futreal, P.A.; McDermott, U.; Lichter, P.; Meyerson, M.; Grimmond, S.M.; Siebert, R.; Campo, E.; Shibata, T.; Pfister, S.M.; Campbell, P.J.; Stratton, M.R.; Schlooz-Vries, M.S.; Tol, J.J. van; Laarhoven, H.W. van; Sweep, F.C.; Bult, P.; et al.,

2013-01-01

All cancers are caused by somatic mutations; however, understanding of the biological processes generating these mutations is limited. The catalogue of somatic mutations from a cancer genome bears the signatures of the mutational processes that have been operative. Here we analysed 4,938,362

Human germline hedgehog pathway mutations predispose to fatty liver.

Science.gov (United States)

Guillen-Sacoto, Maria J; Martinez, Ariel F; Abe, Yu; Kruszka, Paul; Weiss, Karin; Everson, Joshua L; Bataller, Ramon; Kleiner, David E; Ward, Jerrold M; Sulik, Kathleen K; Lipinski, Robert J; Solomon, Benjamin D; Muenke, Maximilian

2017-10-01

Non-alcoholic fatty liver disease (NAFLD) is the most common form of liver disease. Activation of hedgehog (Hh) signaling has been implicated in the progression of NAFLD and proposed as a therapeutic target; however, the effects of Hh signaling inhibition have not been studied in humans with germline mutations that affect this pathway. Patients with holoprosencephaly (HPE), a disorder associated with germline mutations disrupting Sonic hedgehog (SHH) signaling, were clinically evaluated for NAFLD. A combined mouse model of Hh signaling attenuation (Gli2 heterozygous null: Gli2 +/- ) and diet-induced NAFLD was used to examine aspects of NAFLD and hepatic gene expression profiles, including molecular markers of hepatic fibrosis and inflammation. Patients with HPE had a higher prevalence of liver steatosis compared to the general population, independent of obesity. Exposure of Gli2 +/- mice to fatty liver-inducing diets resulted in increased liver steatosis compared to wild-type mice. Similar to humans, this effect was independent of obesity in the mutant mice and was associated with decreased expression of pro-fibrotic and pro-inflammatory genes, and increased expression of PPARγ, a potent anti-fibrogenic and anti-inflammatory regulator. Interestingly, tumor suppressors p53 and p16INK4 were found to be downregulated in the Gli2 +/- mice exposed to a high-fat diet. Our results indicate that germline mutations disrupting Hh signaling promotes liver steatosis, independent of obesity, with reduced fibrosis. While Hh signaling inhibition has been associated with a better NAFLD prognosis, further studies are required to evaluate the long-term effects of mutations affecting this pathway. Lay summary: Non-alcoholic fatty liver disease (NAFLD) is characterized by excess fat deposition in the liver predominantly due to high calorie intake and a sedentary lifestyle. NAFLD progression is usually accompanied by activation of the Sonic hedgehog (SHH) pathway leading to fibrous

Drosophila studies support a role for a presynaptic synaptotagmin mutation in a human congenital myasthenic syndrome.

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Mallory C Shields

Full Text Available During chemical transmission, the function of synaptic proteins must be coordinated to efficiently release neurotransmitter. Synaptotagmin 2, the Ca2+ sensor for fast, synchronized neurotransmitter release at the human neuromuscular junction, has recently been implicated in a dominantly inherited congenital myasthenic syndrome associated with a non-progressive motor neuropathy. In one family, a proline residue within the C2B Ca2+-binding pocket of synaptotagmin is replaced by a leucine. The functional significance of this residue has not been investigated previously. Here we show that in silico modeling predicts disruption of the C2B Ca2+-binding pocket, and we examine the in vivo effects of the homologous mutation in Drosophila. When expressed in the absence of native synaptotagmin, this mutation is lethal, demonstrating for the first time that this residue plays a critical role in synaptotagmin function. To achieve expression similar to human patients, the mutation is expressed in flies carrying one copy of the wild type synaptotagmin gene. We now show that Drosophila carrying this mutation developed neurological and behavioral manifestations similar to those of human patients and provide insight into the mechanisms underlying these deficits. Our Drosophila studies support a role for this synaptotagmin point mutation in disease etiology.

Development of polymorphic microsatellite loci for Iranian river buffalo

African Journals Online (AJOL)

STORAGESEVER

2009-12-15

Dec 15, 2009 ... defining the functional composition of their genome. Microsatellites have been .... species such as fishes (Hsu et al., 2004; Sanches and. JR, 2006; Chung-Jian ... Phylogenetic study of the Iranian buffalo by microsatellite ... Molecular Ecology Laboratory, Department of Biological Sciences,. University of Hull ...

Analysis of APC mutation in human ameloblastoma and clinical significance.

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Li, Ning; Liu, Bing; Sui, Chengguang; Jiang, Youhong

2016-01-01

As a highly conserved signaling pathway, Wnt/β-catenin signal transduction pathway plays an important role in many processes. Either in the occurrence or development of tumor, activation of this pathway takes an important place. APC inhibits Wnt/β-catenin pathway to regulate cell proliferation and differentiation. This study aimed to investigate the function of cancer suppressor gene. PCR amplification and sequencing method was used to analyze APC mutations of human clinical specimens. The pathological specimens were collected for PCR and clear electrophoretic bands were obtained after electrophoresis. The gene sequence obtained after purification and sequencing analysis was compared with the known APC gene sequence (NM_000038.5). Base mutations at APC 1543 (T → C), APC-4564 (G → A), APC-5353 (T → G), APC-5550 (T → A) and APC-5969 (G → A) locus existed in 22 (27.5 %), 12 (15 %), 5 (6.25 %), 13 (16.25 %) and 12 patients (15 %), respectively. Gene mutations existed in ameloblastoma, and the mutation loci were 1543 locus (T → C), 4564 locus (G → A), 5353 locus (T → G), 5550 locus (T → A) and 5969 locus (G → A) 15 %, respectively. APC mutation plays a certain role in monitoring the tumor malignant degree as it may indicate the transition process of ameloblastoma malignant phenotype.

Cross-comparison of the genome sequences from human, chimpanzee, Neanderthal and a Denisovan hominin identifies novel potentially compensated mutations

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Zhang Guojie

2011-07-01

Full Text Available Abstract The recent publication of the draft genome sequences of the Neanderthal and a ~50,000-year-old archaic hominin from Denisova Cave in southern Siberia has ushered in a new age in molecular archaeology. We previously cross-compared the human, chimpanzee and Neanderthal genome sequences with respect to a set of disease-causing/disease-associated missense and regulatory mutations (Human Gene Mutation Database and succeeded in identifying genetic variants which, although apparently pathogenic in humans, may represent a 'compensated' wild-type state in at least one of the other two species. Here, in an attempt to identify further 'potentially compensated mutations' (PCMs of interest, we have compared our dataset of disease-causing/disease-associated mutations with their corresponding nucleotide positions in the Denisovan hominin, Neanderthal and chimpanzee genomes. Of the 15 human putatively disease-causing mutations that were found to be compensated in chimpanzee, Denisovan or Neanderthal, only a solitary F5 variant (Val1736Met was specific to the Denisovan. In humans, this missense mutation is associated with activated protein C resistance and an increased risk of thromboembolism and recurrent miscarriage. It is unclear at this juncture whether this variant was indeed a PCM in the Denisovan or whether it could instead have been associated with disease in this ancient hominin.

Novel tetra-nucleotide microsatellite DNA markers for assessing the evolutionary genetics and demographics of Northern Snakehead (Channa argus) invading North America

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King, Timothy L.; Johnson, Robin L.

2011-01-01

We document the isolation and characterization of 19 tetra-nucleotide microsatellite DNA markers in northern snakehead (Channa argus) fish that recently colonized Meadow Lake, New York City, New York. These markers displayed moderate levels of allelic diversity (averaging 6.8 alleles/locus) and heterozygosity (averaging 74.2%). Demographic analyses suggested that the Meadow Lake collection has not achieved mutation-drift equilibrium. These results were consistent with instances of deviations from Hardy–Weinberg equilibrium and the presence of some linkage disequilibrium. A comparison of individual pair-wise distances suggested the presence of multiple differentiated groups of related individuals. Results of all analyses are consistent with a pattern of multiple, recent introductions. The microsatellite markers developed for C. argus yielded sufficient genetic diversity to potentially: (1) delineate kinship; (2) elucidate fine-scale population structure; (3) define management (eradication) units; (4) estimate dispersal rates; (5) estimate population sizes; and (6) provide unique demographic perspectives of control or eradication effectiveness.

Multilocus Microsatellite Typing reveals intra-focal genetic diversity among strains of Leishmania tropica in Chichaoua Province, Morocco.

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Krayter, Lena; Alam, Mohammad Zahangir; Rhajaoui, Mohamed; Schnur, Lionel F; Schönian, Gabriele

2014-12-01

In Morocco, cutaneous leishmaniasis (CL) caused by Leishmania (L.) tropica is a major public health threat. Strains of this species have been shown to display considerable serological, biochemical, molecular biological and genetic heterogeneity; and Multilocus Enzyme Electrophoresis (MLEE), has shown that in many countries including Morocco heterogenic variants of L. tropica can co-exist in single geographical foci. Here, the microsatellite profiles discerned by MLMT of nine Moroccan strains of L. tropica isolated in 2000 from human cases of CL from Chichaoua Province were compared to those of nine Moroccan strains of L. tropica isolated between 1988 and 1990 from human cases of CL from Marrakech Province, and also to those of 147 strains of L. tropica isolated at different times from different worldwide geographical locations within the range of distribution of the species. Several programs, each employing a different algorithm, were used for population genetic analysis. The strains from each of the two Moroccan foci separated into two phylogenetic clusters independent of their geographical origin. Genetic diversity and heterogeneity existed in both foci, which are geographically close to each other. This intra-focal distribution of genetic variants of L. tropica is not considered owing to in situ mutation. Rather, it is proposed to be explained by the importation of pre-existing variants of L. tropica into Morocco. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Sinai and Norfa chicken diversity revealed by microsatellite markers ...

African Journals Online (AJOL)

The present study aimed to outline the population differentiation of Sinai and Norfa chicken, native to Egypt, with microsatellite markers. Twenty microsatellite loci recommended by the Food and Agriculture Organization (FAO) were used. Fifty eight birds were sampled (29 for each strain: 12 males + 17 females). Data were ...

Using Next Generation RAD Sequencing to Isolate Multispecies Microsatellites for Pilosocereus (Cactaceae.

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Isabel A S Bonatelli

Full Text Available Microsatellite markers (also known as SSRs, Simple Sequence Repeats are widely used in plant science and are among the most informative molecular markers for population genetic investigations, but the development of such markers presents substantial challenges. In this report, we discuss how next generation sequencing can replace the cloning, Sanger sequencing, identification of polymorphic loci, and testing cross-amplification that were previously required to develop microsatellites. We report the development of a large set of microsatellite markers for five species of the Neotropical cactus genus Pilosocereus using a restriction-site-associated DNA sequencing (RAD-seq on a Roche 454 platform. We identified an average of 165 microsatellites per individual, with the absolute numbers across individuals proportional to the sequence reads obtained per individual. Frequency distribution of the repeat units was similar in the five species, with shorter motifs such as di- and trinucleotide being the most abundant repeats. In addition, we provide 72 microsatellites that could be potentially amplified in the sampled species and 22 polymorphic microsatellites validated in two populations of the species Pilosocereus machrisii. Although low coverage sequencing among individuals was observed for most of the loci, which we suggest to be more related to the nature of the microsatellite markers and the possible bias inserted by the restriction enzymes than to the genome size, our work demonstrates that an NGS approach is an efficient method to isolate multispecies microsatellites even in non-model organisms.

Using Next Generation RAD Sequencing to Isolate Multispecies Microsatellites for Pilosocereus (Cactaceae).

Science.gov (United States)

Bonatelli, Isabel A S; Carstens, Bryan C; Moraes, Evandro M

2015-01-01

Microsatellite markers (also known as SSRs, Simple Sequence Repeats) are widely used in plant science and are among the most informative molecular markers for population genetic investigations, but the development of such markers presents substantial challenges. In this report, we discuss how next generation sequencing can replace the cloning, Sanger sequencing, identification of polymorphic loci, and testing cross-amplification that were previously required to develop microsatellites. We report the development of a large set of microsatellite markers for five species of the Neotropical cactus genus Pilosocereus using a restriction-site-associated DNA sequencing (RAD-seq) on a Roche 454 platform. We identified an average of 165 microsatellites per individual, with the absolute numbers across individuals proportional to the sequence reads obtained per individual. Frequency distribution of the repeat units was similar in the five species, with shorter motifs such as di- and trinucleotide being the most abundant repeats. In addition, we provide 72 microsatellites that could be potentially amplified in the sampled species and 22 polymorphic microsatellites validated in two populations of the species Pilosocereus machrisii. Although low coverage sequencing among individuals was observed for most of the loci, which we suggest to be more related to the nature of the microsatellite markers and the possible bias inserted by the restriction enzymes than to the genome size, our work demonstrates that an NGS approach is an efficient method to isolate multispecies microsatellites even in non-model organisms.

Flavin mononucleotide (FMN)-based fluorescent protein (FbFP) as reporter for gene expression in the anaerobe Bacteroides fragilis.

Science.gov (United States)

Lobo, Leandro A; Smith, Charles J; Rocha, Edson R

2011-04-01

In this study, we show the expression of flavin mononucleotide-based fluorescent protein (FbFP) BS2 as a marker for gene expression in the opportunistic human anaerobic pathogen Bacteroides fragilis. Bacteroides fragilis 638R strain carrying osu∷bs2 constructs showed inducible fluorescence following addition of maltose anaerobically compared with nonfluorescent cells under glucose-repressed conditions. Bacteria carrying ahpC∷bs2 or dps∷bs2 constructs were fluorescent following induction by oxygen compared with nonfluorescent cells from the anaerobic control cultures. In addition, when these transcriptional fusion constructs were mobilized into B. fragilis IB263, a constitutive peroxide response strain, fluorescent BS2, was detected in both anaerobic and aerobic cultures, confirming the unique properties of the FbFP BS2 to yield fluorescent signal in B. fragilis in the presence and in the absence of oxygen. Moreover, intracellular expression of BS2 was also detected when cell culture monolayers of J774.1 macrophages were incubated with B. fragilis ahpC∷bs2 or dps∷bs2 strains within an anaerobic chamber. This suggests that ahpC and dps are induced following internalization by macrophages. Thus, we show that BS2 is a suitable tool for the detection of gene expression in obligate anaerobic bacteria in in vivo studies. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Microsatellite Instability Use in Mismatch Repair Gene Sequence Variant Classification

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Bryony A. Thompson

2015-03-01

Full Text Available Inherited mutations in the DNA mismatch repair genes (MMR can cause MMR deficiency and increased susceptibility to colorectal and endometrial cancer. Microsatellite instability (MSI is the defining molecular signature of MMR deficiency. The clinical classification of identified MMR gene sequence variants has a direct impact on the management of patients and their families. For a significant proportion of cases sequence variants of uncertain clinical significance (also known as unclassified variants are identified, constituting a challenge for genetic counselling and clinical management of families. The effect on protein function of these variants is difficult to interpret. The presence or absence of MSI in tumours can aid in determining the pathogenicity of associated unclassified MMR gene variants. However, there are some considerations that need to be taken into account when using MSI for variant interpretation. The use of MSI and other tumour characteristics in MMR gene sequence variant classification will be explored in this review.

A microsatellite linkage map of Drosophila mojavensis

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Schully Sheri

2004-05-01

Full Text Available Abstract Background Drosophila mojavensis has been a model system for genetic studies of ecological adaptation and speciation. However, despite its use for over half a century, no linkage map has been produced for this species or its close relatives. Results We have developed and mapped 90 microsatellites in D. mojavensis, and we present a detailed recombinational linkage map of 34 of these microsatellites. A slight excess of repetitive sequence was observed on the X-chromosome relative to the autosomes, and the linkage groups have a greater recombinational length than the homologous D. melanogaster chromosome arms. We also confirmed the conservation of Muller's elements in 23 sequences between D. melanogaster and D. mojavensis. Conclusions The microsatellite primer sequences and localizations are presented here and made available to the public. This map will facilitate future quantitative trait locus mapping studies of phenotypes involved in adaptation or reproductive isolation using this species.

Polymorphism and association of microsatellite SJ01 with birth ...

African Journals Online (AJOL)

PRECIOUS

2009-12-15

Dec 15, 2009 ... 2College of Life Science, Linyi Normal University, Linyi 276005, China. Accepted 9 November, 2009. Myostatin is a negative regulator of animal skeletal muscle development and SJ01 is a microsatellite locus flanking porcine myostatin gene. In the present study, the polymorphism of microsatellite SJ01 in.

The carcinogenic air pollutant 3-nitrobenzanthrone induces GC to TA transversion mutations in human p53 sequences.

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vom Brocke, Jochen; Krais, Annette; Whibley, Catherine; Hollstein, Monica C; Schmeiser, Heinz H

2009-01-01

3-Nitrobenzanthrone (3-NBA) is a potent mutagen and a suspected human carcinogen present in particulate matter of diesel exhaust and ambient air pollution. Employing an assay with human p53 knock-in (Hupki) murine embryonic fibroblasts (HUFs), we examined p53 mutations induced by 3-NBA and its active metabolite, N-hydroxy-3-aminobenzanthrone (N-OH-3-ABA). Twenty-nine immortalized cultures (cell lines) from 89 HUF primary cultures exposed at passage 1 for 5 days to 2 microM 3-NBA harboured 22 different mutations in the human DNA-binding domain sequence of the Hupki p53 tumour suppressor gene. The most frequently observed mutation was GC to TA transversion (46%), corroborating previous mutation studies with 3-NBA, and consistent with the presence of persistent 3-NBA-guanosine adducts found in DNA of exposed rodents. Six of the transversions found solely in 3-NBA-treated HUFs have not been detected thus far in untreated HUFs, but have been found repeatedly in human lung tumours. (32)P-post-labelling adduct analysis of DNA from HUF cells treated with 2 microM 3-NBA for 5 days showed a pattern similar to that found in vivo, indicating the metabolic competence of HUF cells to metabolize 3-NBA to electrophilic intermediates. Total DNA binding was 160 +/- 56 per 10(7) normal nucleotides with N(2)-guanosine being the major adduct. In contrast, identical treatment with N-OH-3-ABA resulted in a 100-fold lower level of specific DNA adducts and no carcinogen-specific mutation pattern in the Hupki assay. This indicates that the level of DNA adduct formation by the mutagen is critical to obtain specific mutation spectra in the assay. Our results are consistent with previous experiments in Muta Mouse and are compatible with the possibility that diesel exhaust exposure contributes to mutation load in humans and to lung cancer risk.

Toward Microsatellite Based Space Situational Awareness

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Scott, L.; Wallace, B.; Sale, M.; Thorsteinson, S.

2013-09-01

The NEOSSat microsatellite is a dual mission space telescope which will perform asteroid detection and Space Situational Awareness (SSA) observation experiments on deep space, earth orbiting objects. NEOSSat was launched on 25 February 2013 into a 800 dawn-dusk sun synchronous orbit and is currently undergoing satellite commissioning. The microsatellite consists of a small aperture optical telescope, GPS receiver, high performance attitude control system, and stray light rejection baffle designed to reject stray light from the Sun while searching for asteroids with elongations 45 degrees along the ecliptic. The SSA experimental mission, referred to as HEOSS (High Earth Orbit Space Surveillance), will focus on objects in deep space orbits. The HEOSS mission objective is to evaluate the utility of microsatellites to perform catalog maintenance observations of resident space objects in a manner consistent with the needs of the Canadian Forces. The advantages of placing a space surveillance sensor in low Earth orbit are that the observer can conduct observations without the day-night interruption cycle experienced by ground based telescopes, the telescope is insensitive to adverse weather and the system has visibility to deep space resident space objects which are not normally visible from ground based sensors. Also, from a photometric standpoint, the microsatellite is able to conduct observations on objects with a rapidly changing observer position. The possibility of spin axis estimation on geostationary satellites may be possible and an experiment characterize spin axis of distant resident space objects is being planned. Also, HEOSS offers the ability to conduct observations of satellites at high phase angles which can potentially extend the trackable portion of space in which deep space objects' orbits can be monitored. In this paper we describe the HEOSS SSA experimental data processing system and the preliminary findings of the catalog maintenance experiments

Development of Microsatellite Markers for the Korean Mussel, Mytilus coruscus (Mytilidae Using Next-Generation Sequencing

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Hye Suck An

2012-08-01

Full Text Available Mytilus coruscus (family Mytilidae is one of the most important marine shellfish species in Korea. During the past few decades, this species has become endangered due to the loss of habitats and overfishing. Despite this species’ importance, information on its genetic background is scarce. In this study, we developed microsatellite markers for M. coruscus using next-generation sequencing. A total of 263,900 raw reads were obtained from a quarter-plate run on the 454 GS-FLX titanium platform, and 176,327 unique sequences were generated with an average length of 381 bp; 2569 (1.45% sequences contained a minimum of five di- to tetra-nucleotide repeat motifs. Of the 51 loci screened, 46 were amplified successfully, and 22 were polymorphic among 30 individuals, with seven of trinucleotide repeats and three of tetranucleotide repeats. All loci exhibited high genetic variability, with an average of 17.32 alleles per locus, and the mean observed and expected heterozygosities were 0.67 and 0.90, respectively. In addition, cross-amplification was tested for all 22 loci in another congener species, M. galloprovincialis. None of the primer pairs resulted in effective amplification, which might be due to their high mutation rates. Our work demonstrated the utility of next-generation 454 sequencing as a method for the rapid and cost-effective identification of microsatellites. The high degree of polymorphism exhibited by the 22 newly developed microsatellites will be useful in future conservation genetic studies of this species.

Microsatellite marker analysis of the genetic variability in Hanoverian Hounds.

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Lüpke, L; Distl, O

2005-04-01

Genetic variability of the dog breed Hanoverian Hound was analysed using a set of 16 microsatellites. The sample of 92 dogs was representative for the total current population [n=334, inbreeding coefficient 9.2%, relationship coefficient 11.2%] with respect to the level and distribution of the inbreeding and relationship coefficients. All microsatellites used were in Hardy-Weinberg equilibrium. The average number of alleles was 6.4. The average observed heterozygosity (H(O)) was slightly higher than the expected heterozygosity (H(E)). Dinucleotide microsatellites exhibited lower polymorphism information content (PIC) than tetranucleotide microsatellites (0.52 versus 0.66). The average PIC was 0.61. The individual inbreeding coefficient was negatively related to the average H(O) of all microsatellites, whereas the proportion of genes from introducing of Hanoverian Hounds from abroad showed no relationships to H(O). We found that the genetic variability in the Hanoverian Hounds analysed here was unexpectedly higher than that previously published for dog breeds of similar population size. Even in dog breeds of larger population size heterogyzosity was seldom higher than that observed here. The rather high genetic variability as quantified by polymorphic microsatellites in Hanoverian Hounds may be due to a large genetic variation in the founder animals of this breed and to the fact that this genetic diversity could be maintained despite genetic bottlenecks experienced by this breed in the 1920s and 1950s and despite the presence of high inbreeding and relationship coefficients for more than 50 years.

Novel Polymorphic Multilocus Microsatellite Markers to Distinguish Candida tropicalis Isolates.

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Xin Fan

Full Text Available Candida tropicalis is an important pathogen. Here we developed and evaluated a polymorphic multilocus microsatellite scheme employing novel genetic markers for genotyping of C. tropicalis. Using 10 isolates from 10 unique (separate patients to screen over 4000 tandem repeats from the C. tropicalis genome (strain MYA-3404, six new candidate microsatellite loci (ctm1, ctm3, ctm8, ctm18, ctm24 and ctm26 were selected according to amplification success, observed polymorphisms and stability of flanking regions by preliminary testing. Two known microsatellite loci CT14 and URA3 were also studied. The 6-locus scheme was then tested against a set of 82 different isolates from 32 patients. Microsatellite genotypes of isolates from the same patient (two to five isolates per patient were identical. The six loci produced eight to 17 allele types and identified 11 to 24 genotypes amongst 32 patients' isolates, achieving a discriminatory power (DP of 0.76 to 0.97 (versus 0.78 for both CT14 and URA3 loci, respectively. Testing of a combination of only three loci, ctm1, ctm3 and ctm24, also achieved maximum typing efficiency (DP = 0.99, 29 genotypes. The microsatellite typing scheme had good correlation compared with pulsed-field gel electrophoresis, although was slightly less discriminatory. The new six-locus microsatellite typing scheme is a potentially valuable tool for genotyping and investigating microevolution of C. tropicalis.

Genealogical and evolutionary inference with the human Y chromosome.

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Stumpf, M P; Goldstein, D B

2001-03-02

Population genetics has emerged as a powerful tool for unraveling human history. In addition to the study of mitochondrial and autosomal DNA, attention has recently focused on Y-chromosome variation. Ambiguities and inaccuracies in data analysis, however, pose an important obstacle to further development of the field. Here we review the methods available for genealogical inference using Y-chromosome data. Approaches can be divided into those that do and those that do not use an explicit population model in genealogical inference. We describe the strengths and weaknesses of these model-based and model-free approaches, as well as difficulties associated with the mutation process that affect both methods. In the case of genealogical inference using microsatellite loci, we use coalescent simulations to show that relatively simple generalizations of the mutation process can greatly increase the accuracy of genealogical inference. Because model-free and model-based approaches have different biases and limitations, we conclude that there is considerable benefit in the continued use of both types of approaches.

The CDC Hemophilia B mutation project mutation list: a new online resource.

Science.gov (United States)

Li, Tengguo; Miller, Connie H; Payne, Amanda B; Craig Hooper, W

2013-11-01

Hemophilia B (HB) is caused by mutations in the human gene F9. The mutation type plays a pivotal role in genetic counseling and prediction of inhibitor development. To help the HB community understand the molecular etiology of HB, we have developed a listing of all F9 mutations that are reported to cause HB based on the literature and existing databases. The Centers for Disease Control and Prevention (CDC) Hemophilia B Mutation Project (CHBMP) mutation list is compiled in an easily accessible format of Microsoft Excel and contains 1083 unique mutations that are reported to cause HB. Each mutation is identified using Human Genome Variation Society (HGVS) nomenclature standards. The mutation types and the predicted changes in amino acids, if applicable, are also provided. Related information including the location of mutation, severity of HB, the presence of inhibitor, and original publication reference are listed as well. Therefore, our mutation list provides an easily accessible resource for genetic counselors and HB researchers to predict inhibitors. The CHBMP mutation list is freely accessible at http://www.cdc.gov/hemophiliamutations.

Trinucleotide repeat microsatellite markers for Black Poplar (Populus nigra L.)

NARCIS (Netherlands)

Smulders, M.J.M.; Schoot, van der J.; Arens, P.; Vosman, B.

2001-01-01

Using an enrichment procedure, we have cloned microsatellite repeats from black poplar (Populus nigra L.) and developed primers for microsatellite marker analysis. Ten primer pairs, mostly for trinucleotide repeats, produced polymorphic fragments in P. nigra. Some of them also showed amplification

Genetics and epigenetics of small bowel adenocarcinoma: the interactions of CIN, MSI, and CIMP.

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Warth, Arne; Kloor, Matthias; Schirmacher, Peter; Bläker, Hendrik

2011-04-01

Characterization of tumor genetics and epigenetics allows to stratify a tumor entity according to molecular pathways and may shed light on the interactions of different types of DNA alterations during tumorigenesis. Small intestinal adenocarcinoma is rare, and to date the interrelation of genomic instability and epigenetics has not been investigated in this tumor type. We therefore analyzed 37 primary small bowel carcinomas with known microsatellite instability and KRAS status for chromosomal instability using comparative genomic hybridization, for the presence of aberrant methylation (CpG island methylation phenotype) by methylation-specific polymerase chain reaction, and for BRAF mutations. Chromosomal instability was detected in 22 of 37 (59%) tumors (3 of 9 microsatellite instable, and 19 of 28 microsatellite stable carcinomas). Nine carcinomas (24%) were microsatellite and chromosomally stable. High-level DNA methylation was found in 16% of chromosomal instable tumors and in 44% of both microsatellite instable and microsatellite and chromosomally stable carcinomas. KRAS was mutated in 55, 0, and 10% of chromosomal instable, microsatellite instable, and microsatellite and chromosomally stable tumors, respectively whereas the frequencies of BRAF mutations were 6% for chromosomal instable and 22% for both microsatellite instable and microsatellite and chromosomally stable carcinomas. In conclusion, in this study we show that chromosomal instable carcinomas of the small intestine are distinguished from microsatellite instable and microsatellite and chromosomally stable tumors by a high frequency of KRAS mutations, low frequencies of CpG island methylation phenotype, and BRAF mutations. In microsatellite instable and microsatellite and chromosomally stable cancers, CpG island methylation phenotype and BRAF/KRAS mutations are similarly distributed, indicating common mechanisms of tumor initiation or progression in their molecular pathogenesis.

Detection of microsatellite instability but not truncating APC mutations in gastric adenocarcinomas in Brazilian patients

OpenAIRE

Bevilacqua Roberta A.U.; Corvello Cassandra M.; Duarte Ana Paula; Simpson Andrew J.G.

2000-01-01

A crucial role for the adenomatous polyposis colonic (APC) gene in colorectal carcinogenesis has been conclusively established, but, the role of APC in gastric tumors remains controversial. APC mutations have been detected at a relatively high frequency in gastric tumors of Japanese patients, yet such mutations have been reported to be extremely rare in British patients and patients from north-central-Italy. We here report the analysis of 40 primary sporadic gastric adenocarcinomas and 35 pri...

Analyses of carnivore microsatellites and their intimate association with tRNA-derived SINEs.

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López-Giráldez, Francesc; Andrés, Olga; Domingo-Roura, Xavier; Bosch, Montserrat

2006-10-23

The popularity of microsatellites has greatly increased in the last decade on account of their many applications. However, little is currently understood about the factors that influence their genesis and distribution among and within species genomes. In this work, we analyzed carnivore microsatellite clones from GenBank to study their association with interspersed repeats and elucidate the role of the latter in microsatellite genesis and distribution. We constructed a comprehensive carnivore microsatellite database comprising 1236 clones from GenBank. Thirty-three species of 11 out of 12 carnivore families were represented, although two distantly related species, the domestic dog and cat, were clearly overrepresented. Of these clones, 330 contained tRNALys-derived SINEs and 357 contained other interspersed repeats. Our rough estimates of tRNA SINE copies per haploid genome were much higher than published ones. Our results also revealed a distinct juxtaposition of AG and A-rich repeats and tRNALys-derived SINEs suggesting their coevolution. Both microsatellites arose repeatedly in two regions of the interspersed repeat. Moreover, microsatellites associated with tRNALys-derived SINEs showed the highest complexity and less potential instability. Our results suggest that tRNALys-derived SINEs are a significant source for microsatellite generation in carnivores, especially for AG and A-rich repeat motifs. These observations indicate two modes of microsatellite generation: the expansion and variation of pre-existing tandem repeats and the conversion of sequences with high cryptic simplicity into a repeat array; mechanisms which are not specific to tRNALys-derived SINEs. Microsatellite and interspersed repeat coevolution could also explain different distribution of repeat types among and within species genomes.Finally, due to their higher complexity and lower potential informative content of microsatellites associated with tRNALys-derived SINEs, we recommend avoiding

Analyses of carnivore microsatellites and their intimate association with tRNA-derived SINEs

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Bosch Montserrat

2006-10-01

Full Text Available Abstract Background The popularity of microsatellites has greatly increased in the last decade on account of their many applications. However, little is currently understood about the factors that influence their genesis and distribution among and within species genomes. In this work, we analyzed carnivore microsatellite clones from GenBank to study their association with interspersed repeats and elucidate the role of the latter in microsatellite genesis and distribution. Results We constructed a comprehensive carnivore microsatellite database comprising 1236 clones from GenBank. Thirty-three species of 11 out of 12 carnivore families were represented, although two distantly related species, the domestic dog and cat, were clearly overrepresented. Of these clones, 330 contained tRNALys-derived SINEs and 357 contained other interspersed repeats. Our rough estimates of tRNA SINE copies per haploid genome were much higher than published ones. Our results also revealed a distinct juxtaposition of AG and A-rich repeats and tRNALys-derived SINEs suggesting their coevolution. Both microsatellites arose repeatedly in two regions of the insterspersed repeat. Moreover, microsatellites associated with tRNALys-derived SINEs showed the highest complexity and less potential instability. Conclusion Our results suggest that tRNALys-derived SINEs are a significant source for microsatellite generation in carnivores, especially for AG and A-rich repeat motifs. These observations indicate two modes of microsatellite generation: the expansion and variation of pre-existing tandem repeats and the conversion of sequences with high cryptic simplicity into a repeat array; mechanisms which are not specific to tRNALys-derived SINEs. Microsatellite and interspersed repeat coevolution could also explain different distribution of repeat types among and within species genomes. Finally, due to their higher complexity and lower potential informative content of microsatellites

Determination of somatic mutations in human erythrocytes by cytometry

International Nuclear Information System (INIS)

Jensen, R.H.; Langlois, R.G.; Bigbee, W.L.

1985-01-01

Flow cytometric assays of human erythrocytes labeled with monoclonal antibodies specific for glycophorin A were used to enumerate variant cells that appear in peripheral blood as a result of somatic gene-loss mutations in erythrocyte precursor cells. The assay was performed on erythrocytes from 10 oncology patients who had received at least one treatment from radiation or mutagenic chemotherapy at least 3 weeks before being assayed. The patients were suffering from many different malignancies (e.g., breast, renal, bone, colon and lung), and were treated with several different mutagenic therapeutics (e.g., cisplatinum, adriamycin, daunomycin, or cyclophosphamide). The frequency of these variant cells is an indication of the amount of mutagenic damage accumulated in the individual's erythropoietic cell population. Comparing these results to HPRT clonogenic assays, we find similar baseline frequencies of somatic mutation as well as similar correlation with mutagenic exposures. 9 refs., 3 figs., 1 tab

Determination of somatic mutations in human erythrocytes by cytometry

Energy Technology Data Exchange (ETDEWEB)

Jensen, R.H.; Langlois, R.G.; Bigbee, W.L.

1985-06-21

Flow cytometric assays of human erythrocytes labeled with monoclonal antibodies specific for glycophorin A were used to enumerate variant cells that appear in peripheral blood as a result of somatic gene-loss mutations in erythrocyte precursor cells. The assay was performed on erythrocytes from 10 oncology patients who had received at least one treatment from radiation or mutagenic chemotherapy at least 3 weeks before being assayed. The patients were suffering from many different malignancies (e.g., breast, renal, bone, colon and lung), and were treated with several different mutagenic therapeutics (e.g., cisplatinum, adriamycin, daunomycin, or cyclophosphamide). The frequency of these variant cells is an indication of the amount of mutagenic damage accumulated in the individual's erythropoietic cell population. Comparing these results to HPRT clonogenic assays, we find similar baseline frequencies of somatic mutation as well as similar correlation with mutagenic exposures. 9 refs., 3 figs., 1 tab.

Genome-scale mutational signatures of aflatoxin in cells, mice, and human tumors

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Huang, Mi Ni; Yu, Willie; Teoh, Wei Wei; Ardin, Maude; Jusakul, Apinya; Ng, Alvin Wei Tian; Boot, Arnoud; Abedi-Ardekani, Behnoush; Villar, Stephanie; Myint, Swe Swe; Othman, Rashidah; Poon, Song Ling; Heguy, Adriana; Olivier, Magali; Hollstein, Monica; Tan, Patrick; Teh, Bin Tean; Sabapathy, Kanaga; Zavadil, Jiri; Rozen, Steven G.

2017-01-01

Aflatoxin B1 (AFB1) is a mutagen and IARC (International Agency for Research on Cancer) Group 1 carcinogen that causes hepatocellular carcinoma (HCC). Here, we present the first whole-genome data on the mutational signatures of AFB1 exposure from a total of >40,000 mutations in four experimental systems: two different human cell lines, in liver tumors in wild-type mice, and in mice that carried a hepatitis B surface antigen transgene—this to model the multiplicative effects of aflatoxin exposure and hepatitis B in causing HCC. AFB1 mutational signatures from all four experimental systems were remarkably similar. We integrated the experimental mutational signatures with data from newly sequenced HCCs from Qidong County, China, a region of well-studied aflatoxin exposure. This indicated that COSMIC mutational signature 24, previously hypothesized to stem from aflatoxin exposure, indeed likely represents AFB1 exposure, possibly combined with other exposures. Among published somatic mutation data, we found evidence of AFB1 exposure in 0.7% of HCCs treated in North America, 1% of HCCs from Japan, but 16% of HCCs from Hong Kong. Thus, aflatoxin exposure apparently remains a substantial public health issue in some areas. This aspect of our study exemplifies the promise of future widespread resequencing of tumor genomes in providing new insights into the contribution of mutagenic exposures to cancer incidence. PMID:28739859

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

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Erich Heidenreich

2016-12-01

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].

Microsatellite markers of water buffalo, Bubalus bubalis - development, characterisation and linkage disequilibrium studies

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Vaidhegi R

2009-10-01

Full Text Available Abstract Background Microsatellite markers are highly polymorphic and widely used in genome mapping and population genetic studies in livestock species. River buffalo, Bubalus bubalis is an economically important livestock species, though only a limited number of microsatellite markers have been reported thus far in this species. Results In the present study, using two different approaches 571 microsatellite markers have been characterized for water buffalo. Of the 571 microsatellite markers, 498 were polymorphic with average heterozygosity of 0.51 on a panel of 24 unrelated buffalo. Fisher exact test was used to detect LD between the marker pairs. Among the 137550 pairs of marker combination, 14.58% pairs showed significant LD (P Conclusion The high conservation of cattle microsatellite loci in water buffalo promises the usefulness of the cattle microsatellites markers on buffalo. The polymorphic markers characterised in this study will contribute to genetic linkage and radiation hybrid mapping of water buffalo and population genetic studies.

Distinct pattern of TP53 mutations in human immunodeficiency virus-related head and neck squamous cell carcinoma.

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Gleber-Netto, Frederico O; Zhao, Mei; Trivedi, Sanchit; Wang, Jiping; Jasser, Samar; McDowell, Christina; Kadara, Humam; Zhang, Jiexin; Wang, Jing; William, William N; Lee, J Jack; Nguyen, Minh Ly; Pai, Sara I; Walline, Heather M; Shin, Dong M; Ferris, Robert L; Carey, Thomas E; Myers, Jeffrey N; Pickering, Curtis R

2018-01-01

Human immunodeficiency virus-infected individuals (HIVIIs) have a higher incidence of head and neck squamous cell carcinoma (HNSCC), and clinical and histopathological differences have been observed in their tumors in comparison with those of HNSCC patients without a human immunodeficiency virus (HIV) infection. The reasons for these differences are not clear, and molecular differences between HIV-related HNSCC and non-HIV-related HNSCC may exist. This study compared the mutational patterns of HIV-related HNSCC and non-HIV-related HNSCC. The DNA of 20 samples of HIV-related HNSCCs and 32 samples of non-HIV-related HNSCCs was sequenced. DNA libraries covering exons of 18 genes frequently mutated in HNSCC (AJUBA, CASP8, CCND1, CDKN2A, EGFR, FAT1, FBXW7, HLA-A, HRAS, KEAP1, NFE2L2, NOTCH1, NOTCH2, NSD1, PIK3CA, TGFBR2, TP53, and TP63) were prepared and sequenced on an Ion Personal Genome Machine sequencer. DNA sequencing data were analyzed with Ion Reporter software. The human papillomavirus (HPV) status of the tumor samples was assessed with in situ hybridization, the MassARRAY HPV multiplex polymerase chain reaction assay, and p16 immunostaining. Mutation calls were compared among the studied groups. HIV-related HNSCC revealed a distinct pattern of mutations in comparison with non-HIV-related HNSCC. TP53 mutation frequencies were significantly lower in HIV-related HNSCC. Mutations in HIV+ patients tended to be TpC>T nucleotide changes for all mutated genes but especially for TP53. HNSCC in HIVIIs presents a distinct pattern of genetic mutations, particularly in the TP53 gene. HIV-related HNSCC may have a distinct biology, and an effect of the HIV virus on the pathogenesis of these tumors should not be ruled out. Cancer 2018;124:84-94. © 2017 American Cancer Society. © 2017 American Cancer Society.

Characterization and transferability of microsatellite markers of the cultivated peanut (Arachis hypogaea

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Palmieri Dario A

2007-02-01

Full Text Available Abstract Background The genus Arachis includes Arachis hypogaea (cultivated peanut and wild species that are used in peanut breeding or as forage. Molecular markers have been employed in several studies of this genus, but microsatellite markers have only been used in few investigations. Microsatellites are very informative and are useful to assess genetic variability, analyze mating systems and in genetic mapping. The objectives of this study were to develop A. hypogaea microsatellite loci and to evaluate the transferability of these markers to other Arachis species. Results Thirteen loci were isolated and characterized using 16 accessions of A. hypogaea. The level of variation found in A. hypogaea using microsatellites was higher than with other markers. Cross-transferability of the markers was also high. Sequencing of the fragments amplified using the primer pair Ah11 from 17 wild Arachis species showed that almost all wild species had similar repeated sequence to the one observed in A. hypogaea. Sequence data suggested that there is no correlation between taxonomic relationship of a wild species to A. hypogaea and the number of repeats found in its microsatellite loci. Conclusion These results show that microsatellite primer pairs from A. hypogaea have multiple uses. A higher level of variation among A. hypogaea accessions can be detected using microsatellite markers in comparison to other markers, such as RFLP, RAPD and AFLP. The microsatellite primers of A. hypogaea showed a very high rate of transferability to other species of the genus. These primer pairs provide important tools to evaluate the genetic variability and to assess the mating system in Arachis species.

Isolation of novel microsatellites using FIASCO by dual probe enrichment from Jatropha curcas L. and study on genetic equilibrium and diversity of Indian population revealed by isolated microsatellites

KAUST Repository

Pamidimarri, D. V N N Sudheer

2010-03-11

Jatropha curcas L. belongs to family Euphorbiaceae, native to South America attained significant importance for its seed oil which can be converted to biodiesel, a renewable energy source alternative to conventional petrodiesel. Very few attempts were made to isolate novel microsatellite markers and assessment of the extent of genetic equilibrium and diversity that exists in J. curcas. Therefore, the present investigation was undertaken to isolate the novel microsatellites and access genetic equilibrium, diversity that exists among 44 diverse germplasm collected from distinct geographical areas in India using isolated microsatellites. The overall efficiency of the enrichment of microsatellite by dual probe in the present study found to be 54% and among the sequences obtained the percentage of sequences having suitable flanking regions for the primer designing was found to be 89.58%. The mean co-efficient of genetic similarity (CGS) was found to be 0.97. The overall diversity obtained by microsatellites was found to be low in comparison with the diversity reported by multilocus markers systems observed in earlier studies; however, the good allele polymorphism was observed. The overall dendrogram of microsatellite analysis resulted in random clustering of germplasm and not in accordance to geographical area of collection. The present study, diversity analysis using microsatellite markers concludes the low genetic diversity and genetic disequlibrium of J. curcas in India and will provide pavement for further intra-population studies on narrow geographical areas to understand the population genetic structure, phylogeography and molecular ecological studies. The germplasm characterized, and the microsatellite markers isolated and characterized in the present study can be employed efficiently in breeding programs for genetic improvement of the species through marker assisted selection and QTL analysis, for further genetic resource management and help in making the J

Insights into the mutation-induced HHH syndrome from modeling human mitochondrial ornithine transporter-1.

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Jing-Fang Wang

Full Text Available Human mitochondrial ornithine transporter-1 is reported in coupling with the hyperornithinemia-hyperammonemia-homocitrullinuria (HHH syndrome, which is a rare autosomal recessive disorder. For in-depth understanding of the molecular mechanism of the disease, it is crucially important to acquire the 3D structure of human mitochondrial ornithine transporter-1. Since no such structure is available in the current protein structure database, we have developed it via computational approaches based on the recent NMR structure of human mitochondrial uncoupling protein (Berardi MJ, Chou JJ, et al. Nature 2011, 476:109-113. Subsequently, we docked the ligand L-ornithine into the computational structure to search for the favorable binding mode. It was observed that the binding interaction for the most favorable binding mode is featured by six remarkable hydrogen bonds between the receptor and ligand, and that the most favorable binding mode shared the same ligand-binding site with most of the homologous mitochondrial carriers from different organisms, implying that the ligand-binding sites are quite conservative in the mitochondrial carriers family although their sequences similarity is very low with 20% or so. Moreover, according to our structural analysis, the relationship between the disease-causing mutations of human mitochondrial ornithine transporter-1 and the HHH syndrome can be classified into the following three categories: (i the mutation occurs in the pseudo-repeat regions so as to change the region of the protein closer to the mitochondrial matrix; (ii the mutation is directly affecting the substrate binding pocket so as to reduce the substrate binding affinity; (iii the mutation is located in the structural region closer to the intermembrane space that can significantly break the salt bridge networks of the protein. These findings may provide useful insights for in-depth understanding of the molecular mechanism of the HHH syndrome and

Does aerobic exercises induce mtDNA mutation in human blood ...

African Journals Online (AJOL)

The aim of this study was to determine the effect of eight weeks aerobic training on mitochondrial DNA (mtDNA) mutation in human blood leucocytes. Twenty untrained healthy students (training group: n =10, age = 20.7±1.5 yrs, weight = 67.7±10 kg, BF% = 17.5±7.35 & control group: n =10, age = 21±1.3 yrs, weight ...

The Ventasso Horse: genetic characterization by microsatellites markers

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M. Blasi

2010-04-01

Full Text Available The genetic structure of Ventasso Horse (VH was investigated using 12 microsatellites. The analyses were carried out on 117 VH individuals and the results were compared with those obtained analysing 11 other breeds reared in Italy. All microsatellites were polymorphic in VH and in the other breeds. A total of 124 alleles (from 6 to 19 alleles per microsatellite were detected. Average heterozygosity was 0.743 in VH and ranged from 0.613 to 0.759 in the other breeds. The mean FST value had an average value of 0.0932. Genetic distances were calculated using Nei’s standard genetic distance (Ds. The smallest Ds values were found between VH and Anglo-Arab, Thoroughbred, Maremmano and Lipizzan horse breeds. Phylogenetic trees constructed using neighbour-joining method showed two clear separate clusters: the first includes Bardigiano, Haflinger and Italian Heavy Draught Horse, the second contains the other 9 breeds.

Mice overexpressing both non-mutated human SOD1 and mutated SOD1G93A genes: a competent experimental model for studying iron metabolism in amyotrophic lateral sclerosis

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Anna eGajowiak

2016-01-01

Full Text Available Amyotrophic lateral sclerosis (ALS is a progressive neurodegenerative disease characterized by degeneration and loss of motor neurons in the spinal cord, brainstem and motor cortex. Up to 10% of ALS cases are inherited (familial, fALS and associated with mutations, frequently in the superoxide dismutase 1 (SOD1 gene. Rodent transgenic models of ALS are often used to elucidate a complex pathogenesis of this disease. Of importance, both ALS patients and animals carrying mutated human SOD1 gene show symptoms of oxidative stress and iron metabolism misregulation. The aim of our study was to characterize changes in iron metabolism in one of the most commonly used models of ALS – transgenic mice overexpressing human mutated SOD1G93A gene. We analyzed the expression of iron-related genes in asymptomatic, 2-month old and symptomatic, 4-month old SOD1G93A mice. In parallel, respective age-matched mice overexpressing human non-mutated SOD1 transgene and control mice were analyzed. We demonstrate that the overexpression of both SOD1 and SOD1G93A genes account for a substantial increase in SOD1 protein levels and activity in selected tissues and that not all the changes in iron metabolism genes expression are specific for the overexpression of the mutated form of SOD1.

Human Metabolic Enzymes Deficiency: A Genetic Mutation Based Approach

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Swati Chaturvedi

2016-01-01

Full Text Available One of the extreme challenges in biology is to ameliorate the understanding of the mechanisms which emphasize metabolic enzyme deficiency (MED and how these pretend to have influence on human health. However, it has been manifested that MED could be either inherited as inborn error of metabolism (IEM or acquired, which carries a high risk of interrupted biochemical reactions. Enzyme deficiency results in accumulation of toxic compounds that may disrupt normal organ functions and cause failure in producing crucial biological compounds and other intermediates. The MED related disorders cover widespread clinical presentations and can involve almost any organ system. To sum up the causal factors of almost all the MED-associated disorders, we decided to embark on a less traveled but nonetheless relevant direction, by focusing our attention on associated gene family products, regulation of their expression, genetic mutation, and mutation types. In addition, the review also outlines the clinical presentations as well as diagnostic and therapeutic approaches.

Development of microsatellite loci in Scrophularia incisa (Scrophulariaceae) and cross-amplification in congeneric species.

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Wang, Rui-Hong; Chen, Chuan; Ma, Qing; Li, Pan; Fu, Cheng-Xin

2014-02-01

To elucidate the population genetics and phylogeography of Scrophularia incisa, microsatellite primers were developed. We also applied these microsatellite markers to its closely related species S. dentata and S. kiriloviana. • Using the compound microsatellite marker technique, 12 microsatellite primers were identified in S. incisa. The number of alleles ranged from 14 to 26 when assessed in 78 individuals from four populations. With high cross-species transferability, these primers also amplified in S. dentata and S. kiriloviana. • These results indicate that these microsatellite markers are adequate for detecting and characterizing population genetic structure in the Chinese species of sect. Tomiophyllum at fine and range-wide geographical scales.

G protein-coupled receptor mutations and human genetic disease.

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Thompson, Miles D; Hendy, Geoffrey N; Percy, Maire E; Bichet, Daniel G; Cole, David E C

2014-01-01

Genetic variations in G protein-coupled receptor genes (GPCRs) disrupt GPCR function in a wide variety of human genetic diseases. In vitro strategies and animal models have been used to identify the molecular pathologies underlying naturally occurring GPCR mutations. Inactive, overactive, or constitutively active receptors have been identified that result in pathology. These receptor variants may alter ligand binding, G protein coupling, receptor desensitization and receptor recycling. Receptor systems discussed include rhodopsin, thyrotropin, parathyroid hormone, melanocortin, follicle-stimulating hormone (FSH), luteinizing hormone, gonadotropin-releasing hormone (GNRHR), adrenocorticotropic hormone, vasopressin, endothelin-β, purinergic, and the G protein associated with asthma (GPRA or neuropeptide S receptor 1 (NPSR1)). The role of activating and inactivating calcium-sensing receptor (CaSR) mutations is discussed in detail with respect to familial hypocalciuric hypercalcemia (FHH) and autosomal dominant hypocalemia (ADH). The CASR mutations have been associated with epilepsy. Diseases caused by the genetic disruption of GPCR functions are discussed in the context of their potential to be selectively targeted by drugs that rescue altered receptors. Examples of drugs developed as a result of targeting GPCRs mutated in disease include: calcimimetics and calcilytics, therapeutics targeting melanocortin receptors in obesity, interventions that alter GNRHR loss from the cell surface in idiopathic hypogonadotropic hypogonadism and novel drugs that might rescue the P2RY12 receptor congenital bleeding phenotype. De-orphanization projects have identified novel disease-associated receptors, such as NPSR1 and GPR35. The identification of variants in these receptors provides genetic reagents useful in drug screens. Discussion of the variety of GPCRs that are disrupted in monogenic Mendelian disorders provides the basis for examining the significance of common

Enhanced Human-Type Receptor Binding by Ferret-Transmissible H5N1 with a K193T Mutation.

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Peng, Wenjie; Bouwman, Kim M; McBride, Ryan; Grant, Oliver C; Woods, Robert J; Verheije, Monique H; Paulson, James C; de Vries, Robert P

2018-05-15

All human influenza pandemics have originated from avian influenza viruses. Although multiple changes are needed for an avian virus to be able to transmit between humans, binding to human-type receptors is essential. Several research groups have reported mutations in H5N1 viruses that exhibit specificity for human-type receptors and promote respiratory droplet transmission between ferrets. Upon detailed analysis, we have found that these mutants exhibit significant differences in fine receptor specificity compared to human H1N1 and H3N2 and retain avian-type receptor binding. We have recently shown that human influenza viruses preferentially bind to α2-6-sialylated branched N-linked glycans, where the sialic acids on each branch can bind to receptor sites on two protomers of the same hemagglutinin (HA) trimer. In this binding mode, the glycan projects over the 190 helix at the top of the receptor-binding pocket, which in H5N1 would create a stearic clash with lysine at position 193. Thus, we hypothesized that a K193T mutation would improve binding to branched N-linked receptors. Indeed, the addition of the K193T mutation to the H5 HA of a respiratory-droplet-transmissible virus dramatically improves both binding to human trachea epithelial cells and specificity for extended α2-6-sialylated N-linked glycans recognized by human influenza viruses. IMPORTANCE Infections by avian H5N1 viruses are associated with a high mortality rate in several species, including humans. Fortunately, H5N1 viruses do not transmit between humans because they do not bind to human-type receptors. In 2012, three seminal papers have shown how these viruses can be engineered to transmit between ferrets, the human model for influenza virus infection. Receptor binding, among others, was changed, and the viruses now bind to human-type receptors. Receptor specificity was still markedly different compared to that of human influenza viruses. Here we report an additional mutation in ferret

Somatic mutations of the histone H3K27 demethylase, UTX, in human cancer

Science.gov (United States)

van Haaften, Gijs; Dalgliesh, Gillian L; Davies, Helen; Chen, Lina; Bignell, Graham; Greenman, Chris; Edkins, Sarah; Hardy, Claire; O’Meara, Sarah; Teague, Jon; Butler, Adam; Hinton, Jonathan; Latimer, Calli; Andrews, Jenny; Barthorpe, Syd; Beare, Dave; Buck, Gemma; Campbell, Peter J; Cole, Jennifer; Dunmore, Rebecca; Forbes, Simon; Jia, Mingming; Jones, David; Kok, Chai Yin; Leroy, Catherine; Lin, Meng-Lay; McBride, David J; Maddison, Mark; Maquire, Simon; McLay, Kirsten; Menzies, Andrew; Mironenko, Tatiana; Lee, Mulderrig; Mudie, Laura; Pleasance, Erin; Shepherd, Rebecca; Smith, Raffaella; Stebbings, Lucy; Stephens, Philip; Tang, Gurpreet; Tarpey, Patrick S; Turner, Rachel; Turrell, Kelly; Varian, Jennifer; West, Sofie; Widaa, Sara; Wray, Paul; Collins, V Peter; Ichimura, Koichi; Law, Simon; Wong, John; Yuen, Siu Tsan; Leung, Suet Yi; Tonon, Giovanni; DePinho, Ronald A; Tai, Yu-Tzu; Anderson, Kenneth C; Kahnoski, Richard J.; Massie, Aaron; Khoo, Sok Kean; Teh, Bin Tean; Stratton, Michael R; Futreal, P Andrew

2010-01-01

Somatically acquired epigenetic changes are present in many cancers. Epigenetic regulation is maintained via post-translational modifications of core histones. Here, we describe inactivating somatic mutations in the histone lysine demethylase, UTX, pointing to histone H3 lysine methylation deregulation in multiple tumour types. UTX reintroduction into cancer cells with inactivating UTX mutations resulted in slowing of proliferation and marked transcriptional changes. These data identify UTX as a new human cancer gene. PMID:19330029

Microsatellite Primers in the Lichen Symbiotic Alga Trebouxia decolorans (Trebouxiophyceae

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Francesco Dal Grande

2013-03-01

Full Text Available Premise of the study: Polymorphic microsatellite markers were developed for the symbiotic green alga Trebouxia decolorans to study fine-scale population structure and clonal diversity. Methods and Results: Using Illumina pyrosequencing, 20 microsatellite primer sets were developed for T. decolorans. The primer sets were tested on 43 individuals sampled from four subpopulations in Germany. The primers amplified di-, tri-, and tetranucleotide repeats with three to 15 alleles per locus, and the unbiased haploid diversity per locus ranged from 0.636 to 0.821. Conclusions: The identified microsatellite markers will be useful to study the genetic diversity, dispersal, and reproductive mode of this common lichen photobiont.

Mutations Associated with Functional Disorder of Xanthine Oxidoreductase and Hereditary Xanthinuria in Humans

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Takeshi Nishino

2012-11-01

Full Text Available Xanthine oxidoreductase (XOR catalyzes the conversion of hypoxanthine to xanthine and xanthine to uric acid with concomitant reduction of either NAD+ or O2. The enzyme is a target of drugs to treat hyperuricemia, gout and reactive oxygen-related diseases. Human diseases associated with genetically determined dysfunction of XOR are termed xanthinuria, because of the excretion of xanthine in urine. Xanthinuria is classified into two subtypes, type I and type II. Type I xanthinuria involves XOR deficiency due to genetic defect of XOR, whereas type II xanthinuria involves dual deficiency of XOR and aldehyde oxidase (AO, a molybdoflavo enzyme similar to XOR due to genetic defect in the molybdenum cofactor sulfurase. Molybdenum cofactor deficiency is associated with triple deficiency of XOR, AO and sulfite oxidase, due to defective synthesis of molybdopterin, which is a precursor of molybdenum cofactor for all three enzymes. The present review focuses on mutation or chemical modification studies of mammalian XOR, as well as on XOR mutations identified in humans, aimed at understanding the reaction mechanism of XOR and the relevance of mutated XORs as models to estimate the possible side effects of clinical application of XOR inhibitors.

Benzo[a]pyrene, Aflatoxine B1 and Acetaldehyde Mutational Patterns in TP53 Gene Using a Functional Assay: Relevance to Human Cancer Aetiology

Science.gov (United States)

Paget, Vincent; Lechevrel, Mathilde; André, Véronique; Le Goff, Jérémie; Pottier, Didier; Billet, Sylvain; Garçon, Guillaume; Shirali, Pirouz; Sichel, François

2012-01-01

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 B1 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 B1 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. PMID:22319594

Development of Microsatellite Loci in Scrophularia incisa (Scrophulariaceae and Cross-Amplification in Congeneric Species

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Rui-Hong Wang

2014-02-01

Full Text Available Premise of the study: To elucidate the population genetics and phylogeography of Scrophularia incisa, microsatellite primers were developed. We also applied these microsatellite markers to its closely related species S. dentata and S. kiriloviana. Methods and Results: Using the compound microsatellite marker technique, 12 microsatellite primers were identified in S. incisa. The number of alleles ranged from 14 to 26 when assessed in 78 individuals from four populations. With high cross-species transferability, these primers also amplified in S. dentata and S. kiriloviana. Conclusions: These results indicate that these microsatellite markers are adequate for detecting and characterizing population genetic structure in the Chinese species of sect. Tomiophyllum at fine and range-wide geographical scales.

Microsatellite profiles as a basis for intellectual property protection in grape

NARCIS (Netherlands)

Ibanez, J.; Eeuwijk, van F.A.

2003-01-01

The use of microsatellite analysis in a forensic procedure for establishing infringement on plant breeders¿ rights in vegetatively propagated crops was evaluated. A reference collection of 45 seedless grape varieties was chosen as reference collection. Matching probabilities of grape microsatellite

Constraints on Allele size at microsatellite loci : Implications for genetic differentiation

NARCIS (Netherlands)

Nauta, M.J.; Weissing, F.J.

Microsatellites are promising genetic markers for studying the demographic structure and phylogenetic history of populations. We present theoretical arguments indicating that the usefulness of microsatellite data for these purposes may be limited to a short time perspective and to relatively small

Novel and cross-species microsatellite markers for parentage analysis in Sanderling

NARCIS (Netherlands)

Luttikhuizen, P.C.; Bol, A.; Witte, H.; van Bleijswijk, J.; Haddrath, O.; Baker, A.J.; Piersma, T.; Reneerkens, J.; Piersma, T.

2011-01-01

We isolated and tested six novel microsatellite loci in Sanderling (Calidris alba) from Greenland for paternity analyses. In addition, we tested 11 already published microsatellite markers which were originally developed for the congeneric species, the Pectoral Sandpiper (C. melanotos). All loci

Modeling the Mutational and Phenotypic Landscapes of Pelizaeus-Merzbacher Disease with Human iPSC-Derived Oligodendrocytes

DEFF Research Database (Denmark)

Nevin, Zachary S.; Factor, Daniel C.; Karl, Robert T.

2017-01-01

in humans. Attempts to identify a common pathogenic process underlying PMD have been complicated by an incomplete understanding of PLP1 dysfunction and limited access to primary human oligodendrocytes. To address this, we generated panels of human induced pluripotent stem cells (hiPSCs) and hi...... individual and shared defects in PLP1 mRNA expression and splicing, oligodendrocyte progenitor development, and oligodendrocyte morphology and capacity for myelination. These observations enabled classification of PMD subgroups by cell-intrinsic phenotypes and identified a subset of mutations for targeted...... treatment approaches for subsets of individuals. More broadly, this study demonstrates the versatility of a hiPSC-based panel spanning the mutational heterogeneity within a single disease and establishes a widely applicable platform for genotype-phenotype correlation and drug screening in any human myelin...

Radiation-induced mutation at minisatellite loci

International Nuclear Information System (INIS)

Dubrova, Y.E.; Nesterov, V.N.; Krouchinsky, N.G.

1997-01-01

We are studying the radiation-induced increase of mutation rate in minisatellite loci in mice and humans. Minisatellite mutations were scored by multilocus DNA fingerprint analysis in the progeny of γ-irradiated and non-irradiated mice. The frequency of mutation in offspring of irradiated males was 1.7 higher that in the control group. Germline mutation at human minisatellite loci was studied among children born in heavily polluted areas of the Mogilev district of Belarus after the Chernobyl accident and in a control population. The frequency of mutation assayed both by DNA fingerprinting and by eight single locus probes was found to be two times higher in the exposed families than in the control group. Furthermore, mutation rate was correlated with the parental radiation dose for chronic exposure 137 Cs, consistent with radiation-induction of germline mutation. The potential use of minisatellites in monitoring germline mutation in humans will be discussed

Molecular and biochemical characterization of a unique mutation in CCS, the human copper chaperone to superoxide dismutase.

Science.gov (United States)

Huppke, Peter; Brendel, Cornelia; Korenke, Georg Christoph; Marquardt, Iris; Donsante, Anthony; Yi, Ling; Hicks, Julia D; Steinbach, Peter J; Wilson, Callum; Elpeleg, Orly; Møller, Lisbeth Birk; Christodoulou, John; Kaler, Stephen G; Gärtner, Jutta

2012-08-01

Copper (Cu) is a trace metal that readily gains and donates electrons, a property that renders it desirable as an enzyme cofactor but dangerous as a source of free radicals. To regulate cellular Cu metabolism, an elaborate system of chaperones and transporters has evolved, although no human Cu chaperone mutations have been described to date. We describe a child from a consanguineous family who inherited homozygous mutations in the SLC33A1, encoding an acetyl CoA transporter, and in CCS, encoding the Cu chaperone for superoxide dismutase. The CCS mutation, p.Arg163Trp, predicts substitution of a highly conserved arginine residue at position 163, with tryptophan in domain II of CCS, which interacts directly with superoxide dismutase 1 (SOD1). Biochemical analyses of the patient's fibroblasts, mammalian cell transfections, immunoprecipitation assays, and Lys7Δ (CCS homolog) yeast complementation support the pathogenicity of the mutation. Expression of CCS was reduced and binding of CCS to SOD1 impaired. As a result, this mutation causes reduced SOD1 activity and may impair other mechanisms important for normal Cu homeostasis. CCS-Arg163Trp represents the primary example of a human mutation in a gene coding for a Cu chaperone. © 2012 Wiley Periodicals, Inc.

Genetic influence of radiation measured by the effect on the mutation rate of human minisatellite genes

International Nuclear Information System (INIS)

Kodaira, Mieko

2002-01-01

Human minisatellite genes are composed from 0.1-30 kb with a high frequency of polymorphism. The genes exist in mammalian genomes and mice's ones are well studied after irradiation of their gonad cells by X-ray and γ-ray. Following five reports concerning the significant and/or insignificant increases of the mutation rate of the genes post A-bomb exposure, Chernobyl accident and nuclear weapons test in Semipalatinsk are reviewed and discussed on the subject number, exposed dose, problems of the control group, regions examined of loci and exposure conditions. Genetic influences of radiation examined by the author's facility are not recognized in the mutation rate (3.21% vs 4.94% in the control) of minisatellite genes in children of A-bomb survivors and their parents. The mutation rates are 4.27 vs 2.52% (positive influence) and 4.2-6.01% vs 3.5-6.34% in Chernobyl, and 4.3 (parents) and 3.8% (F 1 ) vs 2.5% (positive). Mutation of human minisatellite genes can be an important measure of genetic influences at the medical level. (K.H.)

Simple sequence repeats in mycobacterial genomes

Indian Academy of Sciences (India)

Prakash

2006-12-18

Dec 18, 2006 ... mutation rates have been observed in microsatellites when they are cultured ...... which are falling in the microsatellite rich or poor region are given in the ... regulation of microsatellite evolution (birth, mutation and death).

HMSRP Hawaiian Monk Seal Microsatellite Genotypes

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — Currently ~2,400 Hawaiian monk seal specimens have been analyzed genetically, providing genotypes at 18 microsatellite loci. These data are organized by individual,...

Use of microsatellites to evaluate genetic diversity and species relationships in the genus Lycopersicon

NARCIS (Netherlands)

Alvarez, A.; Wiel, van de C.; Smulders, M.J.M.; Vosman, B.

2001-01-01

In order to determine how informative a set of microsatellites from tomato is across the genus Lycopersicon, 17 microsatellite loci, derived from regions in and around genes, were tested on 31 accessions comprising the nine species of the genus. The microsatellite polymorphisms were used to estimate

Further evidence for elevated human minisatellite mutation rate in Belarus eight years after the Chernobyl accident

International Nuclear Information System (INIS)

Dubrova, Yuri E.; Buard, Jerome; Jeffreys, Alec J.; Nesterov, Valeri N.; Krouchinsky, Nicolay G.; Ostapenko, Vladislav A.; Vergnaud, Gilles; Giraudeau, Fabienne

1997-01-01

Analysis of germline mutation rate at human minisatellites among children born in areas of the Mogilev district of Belarus heavily polluted after the Chernobyl accident has been extended, both by recruiting more families from the affected region and by using five additional minisatellite probes, including multi-locus probe 33.6 and four hypervariable single-locus probes. These additional data confirmed a twofold higher mutation rate in exposed families compared with non-irradiated families from the United Kingdom. An elevated rate was seen at all three independent sets of minisatellites (detected separately by multi-locus probes 33.15, 33.6 and six single-locus probes), indicating a generalised increase in minisatellite germline mutation rate in the Belarus families. Within the Belarus cohort, mutation rate was significantly greater in families with higher parental radiation dose estimated for chronic external and internal exposure to caesium-137, consistent with radiation induction of germline mutation. The spectra of mutation seen in the unexposed and exposed families were indistinguishable, suggesting that increased mutation observed over multiple loci arises indirectly by some mechanism that enhances spontaneous minisatellite mutation

Screening of seven microsatellite markers for litter size in Xinong ...

African Journals Online (AJOL)

Jane

2011-08-08

Aug 8, 2011 ... microsatellite loci. The number of effective alleles (Ne), polymorphism information content (PIC) and average heterozygosity (He) were the highest at OarFCB11 and the lowest at OarAE129 in Xinong. Saanen dairy goat. The analysis of the effect of the six polymorphisms microsatellite loci on the litter size of ...

Analysis of simple sequence repeats in the Gaeumannomyces graminis var. tritici genome and the development of microsatellite markers.

Science.gov (United States)

Li, Wei; Feng, Yanxia; Sun, Haiyan; Deng, Yuanyu; Yu, Hanshou; Chen, Huaigu

2014-11-01

Understanding the genetic structure of Gaeumannomyces graminis var. tritici is essential for the establishment of efficient disease control strategies. It is becoming clear that microsatellites, or simple sequence repeats (SSRs), play an important role in genome organization and phenotypic diversity, and are a large source of genetic markers for population genetics and meiotic maps. In this study, we examined the G. graminis var. tritici genome (1) to analyze its pattern of SSRs, (2) to compare it with other plant pathogenic filamentous fungi, such as Magnaporthe oryzae and M. poae, and (3) to identify new polymorphic SSR markers for genetic diversity. The G. graminis var. tritici genome was rich in SSRs; a total 13,650 SSRs have been identified with mononucleotides being the most common motifs. In coding regions, the densities of tri- and hexanucleotides were significantly higher than in noncoding regions. The di-, tri-, tetra, penta, and hexanucleotide repeats in the G. graminis var. tritici genome were more abundant than the same repeats in M. oryzae and M. poae. From 115 devised primers, 39 SSRs are polymorphic with G. graminis var. tritici isolates, and 8 primers were randomly selected to analyze 116 isolates from China. The number of alleles varied from 2 to 7 and the expected heterozygosity (He) from 0.499 to 0.837. In conclusion, SSRs developed in this study were highly polymorphic, and our analysis indicated that G. graminis var. tritici is a species with high genetic diversity. The results provide a pioneering report for several applications, such as the assessment of population structure and genetic diversity of G. graminis var. tritici.

Ancient and recent positive selection transformed opioid cis-regulation in humans.

Directory of Open Access Journals (Sweden)

Matthew V Rockman

2005-12-01

Full Text Available Changes in the cis-regulation of neural genes likely contributed to the evolution of our species' unique attributes, but evidence of a role for natural selection has been lacking. We found that positive natural selection altered the cis-regulation of human prodynorphin, the precursor molecule for a suite of endogenous opioids and neuropeptides with critical roles in regulating perception, behavior, and memory. Independent lines of phylogenetic and population genetic evidence support a history of selective sweeps driving the evolution of the human prodynorphin promoter. In experimental assays of chimpanzee-human hybrid promoters, the selected sequence increases transcriptional inducibility. The evidence for a change in the response of the brain's natural opioids to inductive stimuli points to potential human-specific characteristics favored during evolution. In addition, the pattern of linked nucleotide and microsatellite variation among and within modern human populations suggests that recent selection, subsequent to the fixation of the human-specific mutations and the peopling of the globe, has favored different prodynorphin cis-regulatory alleles in different parts of the world.

FANCJ suppresses microsatellite instability and lymphomagenesis independent of the Fanconi anemia pathway.

Science.gov (United States)

Matsuzaki, Kenichiro; Borel, Valerie; Adelman, Carrie A; Schindler, Detlev; Boulton, Simon J

2015-12-15

Microsatellites are short tandem repeat sequences that are highly prone to expansion/contraction due to their propensity to form non-B-form DNA structures, which hinder DNA polymerases and provoke template slippage. Although error correction by mismatch repair plays a key role in preventing microsatellite instability (MSI), which is a hallmark of Lynch syndrome, activities must also exist that unwind secondary structures to facilitate replication fidelity. Here, we report that Fancj helicase-deficient mice, while phenotypically resembling Fanconi anemia (FA), are also hypersensitive to replication inhibitors and predisposed to lymphoma. Whereas metabolism of G4-DNA structures is largely unaffected in Fancj(-/-) mice, high levels of spontaneous MSI occur, which is exacerbated by replication inhibition. In contrast, MSI is not observed in Fancd2(-/-) mice but is prevalent in human FA-J patients. Together, these data implicate FANCJ as a key factor required to counteract MSI, which is functionally distinct from its role in the FA pathway. © 2015 Matsuzaki et al.; Published by Cold Spring Harbor Laboratory Press.

Structure of human POFUT1, its requirement in ligand-independent oncogenic Notch signaling, and functional effects of Dowling-Degos mutations

Energy Technology Data Exchange (ETDEWEB)

McMillan, Brian J.; Zimmerman, Brandon; Egan, Emily D.; Lofgren, Michael; Xu, Xiang; Hesser, Anthony; Blacklow, Stephen C.

2017-03-17

Protein O-fucosyltransferase-1 (POFUT1), which transfers fucose residues to acceptor sites on serine and threonine residues of epidermal growth factor-like repeats of recipient proteins, is essential for Notch signal transduction in mammals. Here, we examine the consequences of POFUT1 loss on the oncogenic signaling associated with certain leukemia-associated mutations of human Notch1, report the structures of human POFUT1 in free and GDP-fucose bound states, and assess the effects of Dowling-Degos mutations on human POFUT1 function. CRISPR-mediated knockout of POFUT1 in U2OS cells suppresses both normal Notch1 signaling, and the ligand-independent signaling associated with leukemogenic mutations of Notch1. Normal and oncogenic signaling are rescued by wild-type POFUT1 but rescue is impaired by an active-site R240A mutation. The overall structure of the human enzyme closely resembles that of the Caenorhabditis elegans protein, with an overall backbone RMSD of 0.93 Å, despite primary sequence identity of only 39% in the mature protein. GDP-fucose binding to the human enzyme induces limited backbone conformational movement, though the side chains of R43 and D244 reorient to make direct contact with the fucose moiety in the complex. The reported Dowling-Degos mutations of POFUT1, except for M262T, fail to rescue Notch1 signaling efficiently in the CRISPR-engineered POFUT1^-/- background. Together, these studies identify POFUT1 as a potential target for cancers driven by Notch1 mutations and provide a structural roadmap for its inhibition.

Published sequences do not support transfer of oseltamivir resistance mutations from avian to human influenza A virus strains.

Science.gov (United States)

Norberg, Peter; Lindh, Magnus; Olofsson, Sigvard

2015-03-28

Tamiflu (oseltamivir phosphate ester, OE) is a widely used antiviral active against influenza A virus. Its active metabolite, oseltamivir carboxylate (OC), is chemically stable and secreted into wastewater treatment plants. OC contamination of natural habitats of waterfowl might induce OC resistance in influenza viruses persistently infecting waterfowl, and lead to transfer of OC-resistance from avian to human influenza. The aim of this study was to evaluate whether such has occurred. A genomics approach including phylogenetic analysis and probability calculations for homologous recombination was applied on altogether 19,755 neuraminidase (N1 and N2) genes from virus sampled in humans and birds, with and without resistance mutations. No evidence for transfer of OE resistance mutations from avian to human N genes was obtained, and events suggesting recombination between human and avian influenza virus variants could not be traced in the sequence material studied. The results indicate that resistance in influenza viruses infecting humans is due to the selection pressure posed by the global OE administration in humans rather than transfer from avian influenza A virus strains carrying mutations induced by environmental exposure to OC.

Computational analysis of histidine mutations on the structural stability of human tyrosinases leading to albinism insurgence.

Science.gov (United States)

Hassan, Mubashir; Abbas, Qamar; Raza, Hussain; Moustafa, Ahmed A; Seo, Sung-Yum

2017-07-25

Misfolding and structural alteration in proteins lead to serious malfunctions and cause various diseases in humans. Mutations at the active binding site in tyrosinase impair structural stability and cause lethal albinism by abolishing copper binding. To evaluate the histidine mutational effect, all mutated structures were built using homology modelling. The protein sequence was retrieved from the UniProt database, and 3D models of original and mutated human tyrosinase sequences were predicted by changing the residual positions within the target sequence separately. Structural and mutational analyses were performed to interpret the significance of mutated residues (N 180 , R 202 , Q 202 , R 211 , Y 363 , R 367 , Y 367 and D 390 ) at the active binding site of tyrosinases. CSpritz analysis depicted that 23.25% residues actively participate in the instability of tyrosinase. The accuracy of predicted models was confirmed through online servers ProSA-web, ERRAT score and VERIFY 3D values. The theoretical pI and GRAVY generated results also showed the accuracy of the predicted models. The CCA negative correlation results depicted that the replacement of mutated residues at His within the active binding site disturbs the structural stability of tyrosinases. The predicted CCA scores of Tyr 367 (-0.079) and Q/R 202 (0.032) revealed that both mutations have more potential to disturb the structural stability. MD simulation analyses of all predicted models justified that Gln 202 , Arg 202 , Tyr 367 and D 390 replacement made the protein structures more susceptible to destabilization. Mutational results showed that the replacement of His with Q/R 202 and Y/R 363 has a lethal effect and may cause melanin associated diseases such as OCA1. Taken together, our computational analysis depicts that the mutated residues such as Q/R 202 and Y/R 363 actively participate in instability and misfolding of tyrosinases, which may govern OCA1 through disturbing the melanin biosynthetic pathway.

A novel mutation of PAX3 in a Chinese family with Waardenburg syndrome.

Science.gov (United States)

Qin, Wei; Shu, Anli; Qian, Xueqing; Gao, Jianjun; Xing, Qinghe; Zhang, Juan; Zheng, Yonglan; Li, Xingwang; Li, Sheng; Feng, Guoyin; He, Lin

2006-08-28

The molecular characterization of 34 members of a Chinese family, with 22 members in four generations, affected with Waardenburg syndrome (WS1). A detailed family history and clinical data were collected. A genome-wide scan by two-point linkage analysis using more than 400 microsatellite markers in combination with haplotype analysis was performed. Mutation screening was carried out in the candidate gene by sequencing of amplified products. A maximum two-point lod score of 6.53 at theta = 0.00 was obtained with marker D2S2248. Haplotype analysis placed the WS1 locus to a 45.74 cM region between D2S117 and D2S206, in close proximity to the PAX3 gene on chromosome 2q35. Mutation screening in PAX3 identified a 701T > C mutation which converted a highly conserved Leu to Pro. This nucleotide alteration was neither seen in unaffected members of the family nor found in 50 unrelated control subjects. The present study identified a novel 701T > C mutation in PAX3. The mutation observed in this family highlights the phenotypic heterogeneity of the disorder.

Cytosine arabinoside enhancement of gamma irradiation induced mutations in human T-lymphocytes

International Nuclear Information System (INIS)

O'Neill, J.P.; Sullivan, L.M.; Hunter, T.C.; Nicklas, J.A.

1991-01-01

The frequency of 6-thioguanine resistant (TGr) mutants induced in human G0 phase T-lymphocytes by 200 cGy of gamma irradiation is greatly enhanced by incubation with cytosine arabinoside (ara-C) after irradiation. The mutant frequency increased with increasing incubation time in ara-C for up to 2 hr. This mutation induction required a phenotypic expression time of 5-8 days mass culture growth, similar to that found with mutants induced by 300 cGy of irradiation alone. Southern blot analysis of 40 isolated mutant clones revealed 8 independent mutations by T-cell receptor (TCR) gene rearrangement patterns. Four of these eight showed hprt gene structural alterations (0.50). An alternative method to allow phenotypic expression was developed to minimize the isolation of hprt/TCR sibling mutants. The use of in situ expression in the microtiter dish wells resulted in the isolation of 17 independent mutations in 19 mutant clones. Ten of these 17 mutations showed hprt structural alterations (0.59). The high fraction of mutations involving structural alterations detected by Southern blot analysis is consistent with the known induction of chromosome aberrations by irradiation plus ara-C treatment. We propose that both the increase in Mf and the increase in the incidence of hprt gene structural alterations are due to the accumulation of strand breaks in repairing regions of DNA under these conditions of ara-C induced inhibition of repair. We further propose that upon release of the ara-C inhibition, these repairing regions can interact to yield both gene mutations and chromosome aberrations

VHL Frameshift Mutation as Target of Nonsense-Mediated mRNA Decay in Drosophila melanogaster and Human HEK293 Cell Line

Directory of Open Access Journals (Sweden)

Lucia Micale

2009-01-01

Full Text Available There are many well-studied examples of human phenotypes resulting from nonsense or frameshift mutations that are modulated by Nonsense-Mediated mRNA Decay (NMD, a process that typically degrades transcripts containing premature termination codons (PTCs in order to prevent translation of unnecessary or aberrant transcripts. Different types of germline mutations in the VHL gene cause the von Hippel-Lindau disease, a dominantly inherited familial cancer syndrome with a marked phenotypic variability and age-dependent penetrance. By generating the Drosophila UAS:Upf1D45B line we showed the possible involvement of NMD mechanism in the modulation of the c.172delG frameshift mutation located in the exon 1 of Vhl gene. Further, by Quantitative Real-time PCR (QPCR we demonstrated that the corresponding c.163delG human mutation is targeted by NMD in human HEK 293 cells. The UAS:Upf1D45B line represents a useful system to identify novel substrates of NMD pathway in Drosophila melanogaster. Finally, we suggest the possible role of NMD on the regulation of VHL mutations.

Diphtheria toxin resistance in human lymphocytes and lymphoblasts in the in vivo somatic cell mutation test

International Nuclear Information System (INIS)

Tomkins, D.J.; Wei, L.; Laurie, K.E.

1985-01-01

It has been shown that circulating peripheral blood lymphocytes can be used for the enumeration of 6-thioguanine-resistant cells that presumably arise by mutation in vivo. This somatic cell mutation test has been studied in lymphocytes from human populations exposed to known mutagens and/or carcinogens. The sensitivity of the test could be further enhanced by including other gene markers, since there is evidence for locus-specific differences in response to mutagens. Resistance to diphtheria toxin (Dip/sup r/) seemed like a potential marker to incorporate into the test because the mutation acts codominantly, can readily be selected in human diploid fibroblasts and Chinese hamster cells with no evidence for cell density or cross-feeding effects, and can be assayed for in nondividing cells by measuring protein synthesis inhibition. Blood samples were collected from seven individuals, and fresh, cryopreserved, or Epstein-Barr virus (EBV)-transformed lymphocytes were tested for continued DNA synthesis ( 3 H-thymidine, autoradiography) or protein synthesis ( 35 S-methionine, scintillation counting). Both fresh and cryopreserved lymphocytes, stimulated to divide with phytohemagglutinin (PHA), continued to synthesize DNA in the presence of high doses of diphtheria toxin (DT). Similarly, both dividing (PHA-stimulated) and nondividing fresh lymphocytes carried on significant levels of protein synthesis even 68 hr after exposure to 100 flocculating units (LF)/ml DT. The results suggest that human T and B lymphocytes may not be as sensitive to DT protein synthesis inhibition as human fibroblast and Chinese hamster cells. For this reason, Dip/sup r/ may not be a suitable marker for the somatic cell mutation test

TP53 mutation and human papilloma virus status of oral squamous cell carcinomas in young adult patients

NARCIS (Netherlands)

Braakhuis, B.J.M.; Rietbergen, M.M.; Buijze, M.; Snijders, P.J.F.; Bloemena, E.; Brakenhoff, R.H.; Leemans, C.R.

2014-01-01

Objective Little is known about the molecular carcinogenesis of oral squamous cell carcinoma (OSCC) in young adult patients. The aim of this study was to investigate the detailed TP53 mutation and human papilloma virus (HPV) status of OSCC in patients, younger than 45 years. Methods TP53 mutations

Erratum Haldane and the first estimates of the human mutation rate

Indian Academy of Sciences (India)

Published on the Web: 1 December 2008. Erratum. Haldane and the first estimates of the human mutation rate. (A commentary on J.B.S. Haldane 1935 J. Genet. 31, 317–326; reprinted in volume 83, 235–244 as a J. Genet. classic). Michael W. Nachman. J. Genet. 83, 231–233. Page 1, right column, para 1, line 6 from ...

Characterization of the canine desmin (DES) gene and evaluation as a candidate gene for dilated cardiomyopathy in the Dobermann.

Science.gov (United States)

Stabej, Polona; Imholz, Sandra; Versteeg, Serge A; Zijlstra, Carla; Stokhof, Arnold A; Domanjko-Petric, Aleksandra; Leegwater, Peter A J; van Oost, Bernard A

2004-10-13

Canine-dilated cardiomyopathy (DCM) in dogs is a disease of the myocardium associated with dilatation and impaired contraction of the ventricles and is suspected to have a genetic cause. A missense mutation in the desmin gene (DES) causes DCM in a human family. Human DCM closely resembles the canine disease. In the present study, we evaluated whether DES gene mutations are responsible for DCM in Dobermann dogs. We have isolated bacterial artificial chromosome clones (BACs) containing the canine DES gene and determined the chromosomal location by fluorescence in situ hybridization (FISH). Using data deposited in the NCBI trace archive and GenBank, the canine DES gene DNA sequence was assembled and seven single nucleotide polymorphisms (SNPs) were identified. From the canine DES gene BAC clones, a polymorphic microsatellite marker was isolated. The microsatellite marker and four informative desmin SNPs were typed in a Dobermann family with frequent DCM occurrence, but the disease phenotype did not associate with a desmin haplotype. We concluded that mutations in the DES gene do not play a role in Dobermann DCM. Availability of the microsatellite marker, SNPs and DNA sequence reported in this study enable fast evaluation of the DES gene as a DCM candidate gene in other dog breeds with DCM occurrence.

Visualizing the origins of selfish de novo mutations in individual seminiferous tubules of human testes.

Science.gov (United States)

Maher, Geoffrey J; McGowan, Simon J; Giannoulatou, Eleni; Verrill, Clare; Goriely, Anne; Wilkie, Andrew O M

2016-03-01

De novo point mutations arise predominantly in the male germline and increase in frequency with age, but it has not previously been possible to locate specific, identifiable mutations directly within the seminiferous tubules of human testes. Using microdissection of tubules exhibiting altered expression of the spermatogonial markers MAGEA4, FGFR3, and phospho-AKT, whole genome amplification, and DNA sequencing, we establish an in situ strategy for discovery and analysis of pathogenic de novo mutations. In 14 testes from men aged 39-90 y, we identified 11 distinct gain-of-function mutations in five genes (fibroblast growth factor receptors FGFR2 and FGFR3, tyrosine phosphatase PTPN11, and RAS oncogene homologs HRAS and KRAS) from 16 of 22 tubules analyzed; all mutations have known associations with severe diseases, ranging from congenital or perinatal lethal disorders to somatically acquired cancers. These results support proposed selfish selection of spermatogonial mutations affecting growth factor receptor-RAS signaling, highlight its prevalence in older men, and enable direct visualization of the microscopic anatomy of elongated mutant clones.

Reciprocal mouse and human limb phenotypes caused by gain- and loss-of-function mutations affecting Lmbr1.

OpenAIRE

Clark, R M; Marker, P C; Roessler, E; Dutra, A; Schimenti, J C; Muenke, M; Kingsley, D M

2001-01-01

The major locus for dominant preaxial polydactyly in humans has been mapped to 7q36. In mice the dominant Hemimelic extra toes (Hx) and Hammertoe (Hm) mutations map to a homologous chromosomal region and cause similar limb defects. The Lmbr1 gene is entirely within the small critical intervals recently defined for both the mouse and human mutations and is misexpressed at the exact time that the mouse Hx phenotype becomes apparent during limb development. This result suggests that Lmbr1 may un...

FISH mapping of microsatellite loci from Drosophila subobscura and its comparison to related species.

Science.gov (United States)

Santos, Josiane; Serra, Lluis; Solé, Elisabet; Pascual, Marta

2010-02-01

Microsatellites are highly polymorphic markers that are distributed through all the genome being more abundant in non-coding regions. Whether they are neutral or under selection, these markers if localized can be used as co-dominant molecular markers to explore the dynamics of the evolutionary processes. Their cytological localization can allow identifying genes under selection, inferring recombination from a genomic point of view, or screening for the genomic reorganizations occurring during the evolution of a lineage, among others. In this paper, we report for the first time the localization of microsatellite loci by fluorescent in situ hybridization on Drosophila polytene chromosomes. In Drosophila subobscura, 72 dinucleotide microsatellite loci were localized by fluorescent in situ hybridization yielding unique hybridization signals. In the sex chromosome, microsatellite distribution was not uniform and its density was higher than in autosomes. We identified homologous segments to the sequence flanking the microsatellite loci by browsing the genome sequence of Drosophila pseudoobscura and Drosophila melanogaster. Their localization supports the conservation of Muller's chromosomal elements among Drosophila species and the existence of multiple intrachromosomal rearrangements within each evolutionary lineage. Finally, the lack of microsatellite repeats in the homologous D. melanogaster sequences suggests convergent evolution for high microsatellite density in the distal part of the X chromosome.

Bi-allelic Mutations in PKD1L1 Are Associated with Laterality Defects in Humans.

Science.gov (United States)

Vetrini, Francesco; D'Alessandro, Lisa C A; Akdemir, Zeynep C; Braxton, Alicia; Azamian, Mahshid S; Eldomery, Mohammad K; Miller, Kathryn; Kois, Chelsea; Sack, Virginia; Shur, Natasha; Rijhsinghani, Asha; Chandarana, Jignesh; Ding, Yan; Holtzman, Judy; Jhangiani, Shalini N; Muzny, Donna M; Gibbs, Richard A; Eng, Christine M; Hanchard, Neil A; Harel, Tamar; Rosenfeld, Jill A; Belmont, John W; Lupski, James R; Yang, Yaping

2016-10-06

Disruption of the establishment of left-right (L-R) asymmetry leads to situs anomalies ranging from situs inversus totalis (SIT) to situs ambiguus (heterotaxy). The genetic causes of laterality defects in humans are highly heterogeneous. Via whole-exome sequencing (WES), we identified homozygous mutations in PKD1L1 from three affected individuals in two unrelated families. PKD1L1 encodes a polycystin-1-like protein and its loss of function is known to cause laterality defects in mouse and medaka fish models. Family 1 had one fetus and one deceased child with heterotaxy and complex congenital heart malformations. WES identified a homozygous splicing mutation, c.6473+2_6473+3delTG, which disrupts the invariant splice donor site in intron 42, in both affected individuals. In the second family, a homozygous c.5072G>C (p.Cys1691Ser) missense mutation was detected in an individual with SIT and congenital heart disease. The p.Cys1691Ser substitution affects a highly conserved cysteine residue and is predicted by molecular modeling to disrupt a disulfide bridge essential for the proper folding of the G protein-coupled receptor proteolytic site (GPS) motif. Damaging effects associated with substitutions of this conserved cysteine residue in the GPS motif have also been reported in other genes, namely GPR56, BAI3, and PKD1 in human and lat-1 in C. elegans, further supporting the likely pathogenicity of p.Cys1691Ser in PKD1L1. The identification of bi-allelic PKD1L1 mutations recapitulates previous findings regarding phenotypic consequences of loss of function of the orthologous genes in mice and medaka fish and further expands our understanding of genetic contributions to laterality defects in humans. Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Albinism-causing mutations in recombinant human tyrosinase alter intrinsic enzymatic activity.

Science.gov (United States)

Dolinska, Monika B; Kovaleva, Elena; Backlund, Peter; Wingfield, Paul T; Brooks, Brian P; Sergeev, Yuri V

2014-01-01

Tyrosinase (TYR) catalyzes the rate-limiting, first step in melanin production and its gene (TYR) is mutated in many cases of oculocutaneous albinism (OCA1), an autosomal recessive cause of childhood blindness. Patients with reduced TYR activity are classified as OCA1B; some OCA1B mutations are temperature-sensitive. Therapeutic research for OCA1 has been hampered, in part, by the absence of purified, active, recombinant wild-type and mutant human enzymes. The intra-melanosomal domain of human tyrosinase (residues 19-469) and two OCA1B related temperature-sensitive mutants, R422Q and R422W were expressed in insect cells and produced in T. ni larvae. The short trans-membrane fragment was deleted to avoid potential protein insolubility, while preserving all other functional features of the enzymes. Purified tyrosinase was obtained with a yield of >1 mg per 10 g of larval biomass. The protein was a monomeric glycoenzyme with maximum enzyme activity at 37°C and neutral pH. The two purified mutants when compared to the wild-type protein were less active and temperature sensitive. These differences are associated with conformational perturbations in secondary structure. The intramelanosomal domains of recombinant wild-type and mutant human tyrosinases are soluble monomeric glycoproteins with activities which mirror their in vivo function. This advance allows for the structure - function analyses of different mutant TYR proteins and correlation with their corresponding human phenotypes; it also provides an important tool to discover drugs that may improve tyrosinase activity and treat OCA1.

Albinism-causing mutations in recombinant human tyrosinase alter intrinsic enzymatic activity.

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Monika B Dolinska

Full Text Available Tyrosinase (TYR catalyzes the rate-limiting, first step in melanin production and its gene (TYR is mutated in many cases of oculocutaneous albinism (OCA1, an autosomal recessive cause of childhood blindness. Patients with reduced TYR activity are classified as OCA1B; some OCA1B mutations are temperature-sensitive. Therapeutic research for OCA1 has been hampered, in part, by the absence of purified, active, recombinant wild-type and mutant human enzymes.The intra-melanosomal domain of human tyrosinase (residues 19-469 and two OCA1B related temperature-sensitive mutants, R422Q and R422W were expressed in insect cells and produced in T. ni larvae. The short trans-membrane fragment was deleted to avoid potential protein insolubility, while preserving all other functional features of the enzymes. Purified tyrosinase was obtained with a yield of >1 mg per 10 g of larval biomass. The protein was a monomeric glycoenzyme with maximum enzyme activity at 37°C and neutral pH. The two purified mutants when compared to the wild-type protein were less active and temperature sensitive. These differences are associated with conformational perturbations in secondary structure.The intramelanosomal domains of recombinant wild-type and mutant human tyrosinases are soluble monomeric glycoproteins with activities which mirror their in vivo function. This advance allows for the structure - function analyses of different mutant TYR proteins and correlation with their corresponding human phenotypes; it also provides an important tool to discover drugs that may improve tyrosinase activity and treat OCA1.

Identification of novel microsatellite markers preimplantation genetic diagnosis of beta-thalassemia.

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Chen, Min; Tan, Arnold S C; Cheah, Felicia S H; Saw, Eugene E L; Chong, Samuel S

2015-12-01

Beta (β)-thalassemia is one of the most common monogenic diseases worldwide. Affected pregnancies can be avoided through preimplantation genetic diagnosis (PGD), which commonly involves customized assays to detect the different combinations of β-globin (HBB) gene mutations present in couples, in conjunction with linkage analysis of flanking microsatellite markers. Currently, the limited number of reported closely linked markers hampers their utility in indirect linkage-based PGD for this disorder. To increase the available markers closely flanking the HBB gene, an in silico search was performed to identify all markers within 1 Mb flanking the HBB gene. Fifteen markers with potentially high polymorphism information content (PIC) and heterozygosity values were selected and optimized into a single-tube pentadecaplex PCR panel. Allele frequencies and polymorphism and heterozygosity indices of each marker were assessed in five populations. A total of 238 alleles were observed from the 15 markers. PIC was >0.7 for all markers, with expected heterozygosity and observed heterozygosity values ranging from 0.74 to 0.90 and 0.72 to 0.88, respectively. Greater than 99% of individuals were heterozygous for at least seven markers, with at least two heterozygous markers on either side of the HBB gene. The pentadecaplex marker assay also performed reliably on single cells either directly or after whole genome amplification, thus validating its use in standalone linkage-based β-thalassemia PGD or in conjunction with HBB mutation detection. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Origin of Somatic Mutations in β-Catenin versus Adenomatous Polyposis Coli in Colon Cancer: Random Mutagenesis in Animal Models versus Nonrandom Mutagenesis in Humans.

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Yang, Da; Zhang, Min; Gold, Barry

2017-07-17

Wnt signaling is compromised early in the development of human colorectal cancer (CRC) due to truncating nonsense mutations in adenomatous polyposis coli (APC). CRC induced by chemical carcinogens, such as heterocyclic aromatic amines and azoxymethane, in mice also involves dysregulation of Wnt signaling but via activating missense mutations in the β-catenin oncogene despite the fact that genetically modified mice harboring an inactive APC allele efficiently develop CRC. In contrast, activating mutations in β-catenin are rarely observed in human CRC. Dysregulation of the Wnt signaling pathway by the two distinct mechanisms reveals insights into the etiology of human CRC. On the basis of calculations related to DNA adduct levels produced in mouse CRC models using mutagens, and the number of stem cells in the mouse colon, we show that two nonsense mutations required for biallelic disruption of APC are statistically unlikely to produce CRC in experiments using small numbers of mice. We calculate that an activating mutation in one allele near the critical GSK3β phosphorylation site on β-catenin is >10 5 -times more likely to produce CRC by random mutagenesis due to chemicals than inactivating two alleles in APC, yet it does not occur in humans. Therefore, the mutagenesis mechanism in human CRC cannot be random. We explain that nonsense APC mutations predominate in human CRC because of deamination at 5-methylcytosine at CGA and CAG codons, coupled with the number of human colonic stem cells and lifespan. Our analyses, including a comparison of mutation type and age at CRC diagnosis in U.S. and Chinese patients, also indicate that APC mutations in CRC are not due to environmental mutagens that randomly damage DNA.

Disheveled hair and ear (Dhe, a spontaneous mouse Lmna mutation modeling human laminopathies.

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Paul R Odgren

Full Text Available BACKGROUND: Investigations of naturally-occurring mutations in animal models provide important insights and valuable disease models. Lamins A and C, along with lamin B, are type V intermediate filament proteins which constitute the proteinaceous boundary of the nucleus. LMNA mutations in humans cause a wide range of phenotypes, collectively termed laminopathies. To identify the mutation and investigate the phenotype of a spontaneous, semi-dominant mutation that we have named Disheveled hair and ear (Dhe, which causes a sparse coat and small external ears in heterozygotes and lethality in homozygotes by postnatal day 10. FINDINGS: Genetic mapping identified a point mutation in the Lmna gene, causing a single amino acid change, L52R, in the coiled coil rod domain of lamin A and C proteins. Cranial sutures in Dhe/+ mice failed to close. Gene expression for collagen types I and III in sutures was deficient. Skulls were small and disproportionate. Skeletons of Dhe/+ mice were hypomineralized and total body fat was deficient in males. In homozygotes, skin and oral mucosae were dysplastic and ulcerated. Nuclear morphometry of cultured cells revealed gene dose-dependent blebbing and wrinkling. CONCLUSION: Dhe mice should provide a useful new model for investigations of the pathogenesis of laminopathies.

Primary Mucinous Adenocarcinoma of the Vermiform Appendix with High Grade Microsatellite Instability

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Moritz Komm, Michaela Kronawitter-Fesl, Marcus Kremer, Ludwig Lutz, Elke Holinski-Feder, Reinhard Kopp

2011-01-01

Full Text Available Primary adenocarcinoma of the vermiform appendix is a rare entity and is frequently discovered by the pathologist following appendectomy for suspected appendicitis.We present a 42-year-old male with primary mucinous adenocarcinoma of the appendix initially presenting symptoms of acute appendicitis. Histological investigation of the appendectomy specimen showed a mucinous adenocarcinoma and the patient was treated by secondary right hemicolectomy giving the final histopathological classification of an UICC IIIC tumor. Since the patient fulfills the revised Bethesda criteria analysis of immunoreactivity of DNA mismatch repair proteins was performed showing loss of MLH1 and MSH2 expression associated with high microsatellite instability (MSI-H, not yet reported for primary mucinous appendiceal carcinoma. Further genetic analysis for DNA mismatch repair gene mutations were negative. The patient received intensified adjuvant chemotherapy according to the FOLFOX-4-scheme, since MSI-H colorectal carcinomas might show lower response rates following standard 5-FU-based adjuvant chemotherapy.

Chromosomal localization of microsatellite loci in Drosophila mediopunctata

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Renato Cavasini

2015-03-01

Full Text Available Drosophila mediopunctata has been used as a model organism for genetics and evolutionary studies in the last three decades. A linkage map with 48 microsatellite loci recently published for this species showed five syntenic groups, which had their homology determined to Drosophila melanogaster chromosomes. Then, by inference, each of the groups was associated with one of the five major chromosomes of D. mediopunctata. Our objective was to carry out a genetic (chromosomal analysis to increase the number of available loci with known chromosomal location. We made a simultaneous analysis of visible mutant phenotypes and microsatellite genotypes in a backcross of a standard strain and a mutant strain, which had each major autosome marked. Hence, we could establish the chromosomal location of seventeen loci; including one from each of the five major linkage groups previously published, and twelve new loci. Our results were congruent with the previous location and they open new possibilities to future work integrating microsatellites, chromosomal inversions, and genetic determinants of physiological and morphological variation.

Loss of CDX2 Expression and Microsatellite Instability Are Prominent Features of Large Cell Minimally Differentiated Carcinomas of the Colon

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Hinoi, Takao; Tani, Masachika; Lucas, Peter C.; Caca, Karel; Dunn, Rodney L.; Macri¶, Ettore; Loda¶, Massimo; Appelman, Henry D.; Cho, Kathleen R.; Fearon, Eric R.

2001-01-01

Most large bowel cancers are moderately to well-differentiated adenocarcinomas comprised chiefly or entirely of glands lined by tall columnar cells. We have identified a subset of poorly differentiated colon carcinomas with a distinctive histopathological appearance that we term large cell minimally differentiated carcinomas (LCMDCs). These tumors likely include a group of poorly differentiated carcinomas previously described by others as medullary adenocarcinomas. To better understand the pathogenesis of these uncommon neoplasms, we compared molecular features of 15 LCMDCs to those present in 25 differentiated adenocarcinomas (DACs) of the colon. Tumors were examined for alterations commonly seen in typical colorectal carcinomas, including increased p53 and β-catenin immunoreactivity, K-ras gene mutations, microsatellite instability, and loss of heterozygosity of markers on chromosomes 5q, 17p, and 18q. In addition, tumors were evaluated by immunohistochemistry for CDX2, a homeobox protein whose expression in normal adult tissues is restricted to intestinal and colonic epithelium. Markedly reduced or absent CDX2 expression was noted in 13 of 15 (87%) LCMDCs, whereas only 1 of the 25 (4%) DACs showed reduced CDX2 expression (P < 0.001). Nine of 15 (60%) LCMDCs had the high-frequency microsatellite instability phenotype, but only 2 of 25 (8%) DACs had the high-frequency microsatellite instability phenotype (P = 0.002). Our findings provide support for the hypothesis that the molecular pathogenesis of LCMDCs is distinct from that of most DACs. CDX2 alterations and DNA mismatch repair defects have particularly prominent roles in the development of LCMDCs. PMID:11733373

Age and sex effects on human mutation rates. An old problem with new complexities

International Nuclear Information System (INIS)

Crow, James F.

2006-01-01

Base substitution mutations are far more common in human males than in females, and the frequency increases with paternal age. Both can be accounted for by the greater number of pre-meiotic cell divisions in males, especially old ones. In contrast, small deletions do not show any important age effect and occur with approximately equal frequency in the two sexes. Mutations in most genes include both types, and the sex and paternal age effect depends on the proportion of the two types. A few traits, of which Apert Syndrome is best understood, are mutation hot spots with all the mutations occurring in one or two codons, usually at one nucleotide. They occur with very high frequency almost exclusively in males and the frequency increases rapidly with paternal age. It has been suggested that the mutant cells have a selective advantage in the male germ-line prior to meiosis. Evidence for this surprising, but important, hypothesis is discussed. A possible mechanism is the conversion of asymmetrical stem-cell divisions into symmetric ones. Some traits with complex etiology show a slight paternal age effect. There is also a short discussion of the high deleterious mutation rate and the role of sexual reproduction in reducing the consequent mutation load. (author)

Microsatellite instability and the association with plasma homocysteine and thymidylate synthase in colorectal cancer

DEFF Research Database (Denmark)

Jensen, Lars Henrik; Lindebjerg, Jan; Crüger, Dorthe G.

2008-01-01

, carcinoembryonic antigen, vitamin B12, and folate. Microsatellite instability of tumors was associated with higher levels of plasma homocysteine (p = 0.008) and higher protein expression of thymidylate synthase (p ... factors. CEA was not associated with neither homocysteine nor microsatellite instability. The data suggests that there is a more pronounced methyl unit deficiency in microsatellite instable tumors....

Characterization of pathogenic germline mutations in human Protein Kinases

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Orengo Christine A

2011-07-01

Full Text Available Abstract Background Protein Kinases are a superfamily of proteins involved in crucial cellular processes such as cell cycle regulation and signal transduction. Accordingly, they play an important role in cancer biology. To contribute to the study of the relation between kinases and disease we compared pathogenic mutations to neutral mutations as an extension to our previous analysis of cancer somatic mutations. First, we analyzed native and mutant proteins in terms of amino acid composition. Secondly, mutations were characterized according to their potential structural effects and finally, we assessed the location of the different classes of polymorphisms with respect to kinase-relevant positions in terms of subfamily specificity, conservation, accessibility and functional sites. Results Pathogenic Protein Kinase mutations perturb essential aspects of protein function, including disruption of substrate binding and/or effector recognition at family-specific positions. Interestingly these mutations in Protein Kinases display a tendency to avoid structurally relevant positions, what represents a significant difference with respect to the average distribution of pathogenic mutations in other protein families. Conclusions Disease-associated mutations display sound differences with respect to neutral mutations: several amino acids are specific of each mutation type, different structural properties characterize each class and the distribution of pathogenic mutations within the consensus structure of the Protein Kinase domain is substantially different to that for non-pathogenic mutations. This preferential distribution confirms previous observations about the functional and structural distribution of the controversial cancer driver and passenger somatic mutations and their use as a proxy for the study of the involvement of somatic mutations in cancer development.

Microsatellite analyses of the trout of northwest Mexico

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Nielsen, J.L.; Sage, G.K.

2001-01-01

The trout of northwest Mexico represent an undescribed group of fish considered part of the Oncorhynchus mykiss (Pacific trout) complex of species and subspecies. Recent genetic studies have shown these fish to have important genetic diversity and a unique evolutionary history when compared to coastal rainbow trout. Increased levels of allelic diversity have been found in this species at the southern extent of its range. In this study we describe the trout in the Sierra Madre Occidental from the rios Yaqui, Mayo, Casas Grandes and de Bavispe, and their relationship to the more southern distribution of Mexican golden trout (O. chrysogaster) using 11 microsatellite loci. Microsatellite allelic diversity in Mexican trout was high with a mean of 6.6 alleles/locus, average heterozygosity = 0.35, and a mean Fst = 0.43 for all loci combined. Microsatellite data were congruent with previously published mtDNA results showing unique panmictic population structure in the Rio Yaqui trout that differs from Pacific coastal trout and Mexican golden trout. These data also add support for the theory of headwaters transfer of trout across the Continental Divide from tributaries of the Rio de Bavispe into the Rio Casas Grandes. Rio Mayo trout share a close genetic relationship to trout in Rio Yaqui, but sample sizes from the Rio Mayo prevent significant comparisons in this study. Microsatellite analyses show significant allelic frequency differences between Rio Yaqui trout and O. chrysogaster in Sinaloa and Durango Mexico, adding further support for a unique evolutionary status for this group of northwestern Mexican trout.

Mutations to PB2 and NP proteins of an avian influenza virus combine to confer efficient growth in primary human respiratory cells.

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Danzy, Shamika; Studdard, Lydia R; Manicassamy, Balaji; Solorzano, Alicia; Marshall, Nicolle; García-Sastre, Adolfo; Steel, John; Lowen, Anice C

2014-11-01

Influenza pandemics occur when influenza A viruses (IAV) adapted to other host species enter humans and spread through the population. Pandemics are relatively rare due to host restriction of IAV: strains adapted to nonhuman species do not readily infect, replicate in, or transmit among humans. IAV can overcome host restriction through reassortment or adaptive evolution, and these are mechanisms by which pandemic strains arise in nature. To identify mutations that facilitate growth of avian IAV in humans, we have adapted influenza A/duck/Alberta/35/1976 (H1N1) (dk/AB/76) virus to a high-growth phenotype in differentiated human tracheo-bronchial epithelial (HTBE) cells. Following 10 serial passages of three independent lineages, the bulk populations showed similar growth in HTBE cells to that of a human seasonal virus. The coding changes present in six clonal isolates were determined. The majority of changes were located in the polymerase complex and nucleoprotein (NP), and all isolates carried mutations in the PB2 627 domain and regions of NP thought to interact with PB2. Using reverse genetics, the impact on growth and polymerase activity of individual and paired mutations in PB2 and NP was evaluated. The results indicate that coupling of the mammalian-adaptive mutation PB2 E627K or Q591K to selected mutations in NP further augments the growth of the corresponding viruses. In addition, minimal combinations of three (PB2 Q236H, E627K, and NP N309K) or two (PB2 Q591K and NP S50G) mutations were sufficient to recapitulate the efficient growth in HTBE cells of dk/AB/76 viruses isolated after 10 passages in this substrate. Influenza A viruses adapted to birds do not typically grow well in humans. However, as has been seen recently with H5N1 and H7N9 subtype viruses, productive and virulent infection of humans with avian influenza viruses can occur. The ability of avian influenza viruses to adapt to new host species is a consequence of their high mutation rate that

Gain-of-function R225W mutation in human AMPKgamma(3 causing increased glycogen and decreased triglyceride in skeletal muscle.

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Sheila R Costford

Full Text Available BACKGROUND: AMP-activated protein kinase (AMPK is a heterotrimeric enzyme that is evolutionarily conserved from yeast to mammals and functions to maintain cellular and whole body energy homeostasis. Studies in experimental animals demonstrate that activation of AMPK in skeletal muscle protects against insulin resistance, type 2 diabetes and obesity. The regulatory gamma(3 subunit of AMPK is expressed exclusively in skeletal muscle; however, its importance in controlling overall AMPK activity is unknown. While evidence is emerging that gamma subunit mutations interfere specifically with AMP activation, there remains some controversy regarding the impact of gamma subunit mutations. Here we report the first gain-of-function mutation in the muscle-specific regulatory gamma(3 subunit in humans. METHODS AND FINDINGS: We sequenced the exons and splice junctions of the AMPK gamma(3 gene (PRKAG3 in 761 obese and 759 lean individuals, identifying 87 sequence variants including a novel R225W mutation in subjects from two unrelated families. The gamma(3 R225W mutation is homologous in location to the gamma(2R302Q mutation in patients with Wolf-Parkinson-White syndrome and to the gamma(3R225Q mutation originally linked to an increase in muscle glycogen content in purebred Hampshire Rendement Napole (RN- pigs. We demonstrate in differentiated muscle satellite cells obtained from the vastus lateralis of R225W carriers that the mutation is associated with an approximate doubling of both basal and AMP-activated AMPK activities. Moreover, subjects bearing the R225W mutation exhibit a approximately 90% increase of skeletal muscle glycogen content and a approximately 30% decrease in intramuscular triglyceride (IMTG. CONCLUSIONS: We have identified for the first time a mutation in the skeletal muscle-specific regulatory gamma(3 subunit of AMPK in humans. The gamma(3R225W mutation has significant functional effects as demonstrated by increases in basal and AMP

Clinical significance of the BRAFV600E mutation in Asian patients with colorectal cancer.

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Cheng, Hou-Hsuan; Lin, Jen-Kou; Chen, Wei-Shone; Jiang, Jeng-Kai; Yang, Shung-Haur; Chang, Shih-Ching

2018-06-04

To investigate the clinicopathological features and prognostic significance of the BRAFV600E mutation in Asian patients with colorectal cancer. We retrospectively reviewed the medical records of 1969 patients with colorectal cancer admitted to Taipei Veterans General Hospital for surgical treatment between 2000 and 2013. The measured endpoint was overall survival after surgery. The prognostic value of the BRAFV600E mutation was analyzed using the log-rank test and Cox regression analysis. The BRAFV600E mutation was detected in 106 (5.4%) patients and associated with female gender, abnormal cancer antigen (CA)19-9 at diagnosis, microsatellite status, right-sided primary tumors, mucinous histology, poor differentiation, and lymphovascular invasion. Metastatic patterns were more common in non-regional lymph node metastasis (20.8 vs. 7.4%, p = 0.06) and peritoneal seeding (41. vs. 21.2%, p = 0.04). Mutations were not prognostic in the overall survival of the entire study group but only in specific patients: age < 65, normal carcinoembryonic antigen at diagnosis, and stage IV disease. The BRAFV600E mutation was associated with distinct clinicopathological features and metastatic patterns. The overall survival rate was lower in selected colorectal patients with the BRAFV600E mutation.

Microsatellite Primer Development for Post Oak, Quercus stellata (Fagaceae

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Warren B. Chatwin

2014-10-01

Full Text Available Premise of the study: The American Cross Timbers forest ecosystem runs from southeastern Kansas to Central Texas and is primarily composed of post oak (Quercus stellata. This old-growth forest currently occupies only about 2% of its ancestral range. To facilitate genetic research on this species, we developed microsatellite primers specific to post oak from reduced genomic libraries. Methods and Results: Two Q. stellata individuals, sampled from the northern and southern range of the post oak forest, were subject to genomic reduction and 454 pyrosequencing. Bioinformatic analysis identified putative microsatellites from which 12 polymorphic primer sets were screened on three populations. The number of alleles observed ranged from five to 20 across all populations, while observed and expected heterozygosity values ranged from 0.05 to 0.833 and 0.236 to 0.893, respectively, within individual populations. Conclusions: We report the development of microsatellite markers, specific to post oak, to aid the study of genetic diversity and population structure of extant forest remnants.

Development of microsatellite loci in Artocarpus altilis (Moraceae) and cross-amplification in congeneric species.

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Witherup, Colby; Ragone, Diane; Wiesner-Hanks, Tyr; Irish, Brian; Scheffler, Brian; Simpson, Sheron; Zee, Francis; Zuberi, M Iqbal; Zerega, Nyree J C

2013-07-01

Microsatellite loci were isolated and characterized from enriched genomic libraries of Artocarpus altilis (breadfruit) and tested in four Artocarpus species and one hybrid. The microsatellite markers provide new tools for further studies in Artocarpus. • A total of 25 microsatellite loci were evaluated across four Artocarpus species and one hybrid. Twenty-one microsatellite loci were evaluated on A. altilis (241), A. camansi (34), A. mariannensis (15), and A. altilis × mariannensis (64) samples. Nine of those loci plus four additional loci were evaluated on A. heterophyllus (jackfruit, 426) samples. All loci are polymorphic for at least one species. The average number of alleles ranges from two to nine within taxa. • These microsatellite primers will facilitate further studies on the genetic structure and evolutionary and domestication history of Artocarpus species. They will aid in cultivar identification and establishing germplasm conservation strategies for breadfruit and jackfruit.

Analysis of beta-catenin, Ki-ras, and microsatellite stability in azoxymethane-induced colon tumors of BDIX/Orl Ico rats

DEFF Research Database (Denmark)

Sørensen, Nanna Møller; Kobaek-Larsen, Morten; Bonne, Anita

2003-01-01

The aim of the study reported here was to investigate whether the azoxymethane (AOM)-induced colon cancer rat model mimics the human situation with regard to microsatellite stability, changes in expression of beta-catenin, and/or changes in the sequence of the proto-oncogene Ki-ras. Colon cancer ...

Molecular and biochemical characterization of a unique mutation in CCS, the human copper chaperone to superoxide dismutase

DEFF Research Database (Denmark)

Huppke, Peter; Brendel, Cornelia; Korenke, Georg Christoph

2012-01-01

chaperone mutations have been described to date. We describe a child from a consanguineous family who inherited homozygous mutations in the SLC33A1, encoding an acetyl CoA transporter, and in CCS, encoding the Cu chaperone for superoxide dismutase. The CCS mutation, p.Arg163Trp, predicts substitution...... of a highly conserved arginine residue at position 163, with tryptophan in domain II of CCS, which interacts directly with superoxide dismutase 1 (SOD1). Biochemical analyses of the patient's fibroblasts, mammalian cell transfections, immunoprecipitation assays, and Lys7Δ (CCS homolog) yeast complementation...... support the pathogenicity of the mutation. Expression of CCS was reduced and binding of CCS to SOD1 impaired. As a result, this mutation causes reduced SOD1 activity and may impair other mechanisms important for normal Cu homeostasis. CCS-Arg163Trp represents the primary example of a human mutation...

Development of Microsatellites in Labisia pumila (Myrsinaceae, an Economically Important Malaysian Herb

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Lee Hong Tnah

2014-06-01

Full Text Available Premise of the study: The exploitation of Labisia pumila for commercial demand is gradually increasing. It is therefore important that conservation is prioritized to ensure sustainable utilization. We developed microsatellites for L. pumila var. alata and evaluated their polymorphism across var. alata, var. pumila, and var. lanceolata. Methods and Results: Ten polymorphic microsatellites of L. pumila were developed using the magnetic bead hybridization selection approach. A total of 84, 48, and 66 alleles were observed in L. pumila var. alata, var. pumila, and var. lanceolata, respectively. The species is likely a tetraploid, with the majority of the loci exhibiting up to four alleles per individual. Conclusions: This is the first report on the development of microsatellites in L. pumila. The microsatellites will provide a good basis for investigating the population genetics of the species and will serve as a useful tool for DNA profiling.

Eucalyptus microsatellites mined in silico

Indian Academy of Sciences (India)

https://www.ias.ac.in/article/fulltext/jgen/087/01/0021-0025 ... Eucalyptus is an important short rotation pulpy woody plant, grown widely in the tropics. ... In this study, in silico analysis of 15,285 sequences representing partial and full-length mRNA from Eucalyptus species for their use in developing SSRs or microsatellites ...

Evolutionary dynamics of microsatellite distribution in plants: insight from the comparison of sequenced brassica, Arabidopsis and other angiosperm species.

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Jiaqin Shi

Full Text Available Despite their ubiquity and functional importance, microsatellites have been largely ignored in comparative genomics, mostly due to the lack of genomic information. In the current study, microsatellite distribution was characterized and compared in the whole genomes and both the coding and non-coding DNA sequences of the sequenced Brassica, Arabidopsis and other angiosperm species to investigate their evolutionary dynamics in plants. The variation in the microsatellite frequencies of these angiosperm species was much smaller than those for their microsatellite numbers and genome sizes, suggesting that microsatellite frequency may be relatively stable in plants. The microsatellite frequencies of these angiosperm species were significantly negatively correlated with both their genome sizes and transposable elements contents. The pattern of microsatellite distribution may differ according to the different genomic regions (such as coding and non-coding sequences. The observed differences in many important microsatellite characteristics (especially the distribution with respect to motif length, type and repeat number of these angiosperm species were generally accordant with their phylogenetic distance, which suggested that the evolutionary dynamics of microsatellite distribution may be generally consistent with plant divergence/evolution. Importantly, by comparing these microsatellite characteristics (especially the distribution with respect to motif type the angiosperm species (aside from a few species all clustered into two obviously different groups that were largely represented by monocots and dicots, suggesting a complex and generally dichotomous evolutionary pattern of microsatellite distribution in angiosperms. Polyploidy may lead to a slight increase in microsatellite frequency in the coding sequences and a significant decrease in microsatellite frequency in the whole genome/non-coding sequences, but have little effect on the microsatellite

Evolutionary Dynamics of Microsatellite Distribution in Plants: Insight from the Comparison of Sequenced Brassica, Arabidopsis and Other Angiosperm Species

Science.gov (United States)

Shi, Jiaqin; Huang, Shunmou; Fu, Donghui; Yu, Jinyin; Wang, Xinfa; Hua, Wei; Liu, Shengyi; Liu, Guihua; Wang, Hanzhong

2013-01-01

Despite their ubiquity and functional importance, microsatellites have been largely ignored in comparative genomics, mostly due to the lack of genomic information. In the current study, microsatellite distribution was characterized and compared in the whole genomes and both the coding and non-coding DNA sequences of the sequenced Brassica, Arabidopsis and other angiosperm species to investigate their evolutionary dynamics in plants. The variation in the microsatellite frequencies of these angiosperm species was much smaller than those for their microsatellite numbers and genome sizes, suggesting that microsatellite frequency may be relatively stable in plants. The microsatellite frequencies of these angiosperm species were significantly negatively correlated with both their genome sizes and transposable elements contents. The pattern of microsatellite distribution may differ according to the different genomic regions (such as coding and non-coding sequences). The observed differences in many important microsatellite characteristics (especially the distribution with respect to motif length, type and repeat number) of these angiosperm species were generally accordant with their phylogenetic distance, which suggested that the evolutionary dynamics of microsatellite distribution may be generally consistent with plant divergence/evolution. Importantly, by comparing these microsatellite characteristics (especially the distribution with respect to motif type) the angiosperm species (aside from a few species) all clustered into two obviously different groups that were largely represented by monocots and dicots, suggesting a complex and generally dichotomous evolutionary pattern of microsatellite distribution in angiosperms. Polyploidy may lead to a slight increase in microsatellite frequency in the coding sequences and a significant decrease in microsatellite frequency in the whole genome/non-coding sequences, but have little effect on the microsatellite distribution with

Genome-wide microsatellite characterization and marker development in the sequenced Brassica crop species.

Science.gov (United States)

Shi, Jiaqin; Huang, Shunmou; Zhan, Jiepeng; Yu, Jingyin; Wang, Xinfa; Hua, Wei; Liu, Shengyi; Liu, Guihua; Wang, Hanzhong

2014-02-01

Although much research has been conducted, the pattern of microsatellite distribution has remained ambiguous, and the development/utilization of microsatellite markers has still been limited/inefficient in Brassica, due to the lack of genome sequences. In view of this, we conducted genome-wide microsatellite characterization and marker development in three recently sequenced Brassica crops: Brassica rapa, Brassica oleracea and Brassica napus. The analysed microsatellite characteristics of these Brassica species were highly similar or almost identical, which suggests that the pattern of microsatellite distribution is likely conservative in Brassica. The genomic distribution of microsatellites was highly non-uniform and positively or negatively correlated with genes or transposable elements, respectively. Of the total of 115 869, 185 662 and 356 522 simple sequence repeat (SSR) markers developed with high frequencies (408.2, 343.8 and 356.2 per Mb or one every 2.45, 2.91 and 2.81 kb, respectively), most represented new SSR markers, the majority had determined physical positions, and a large number were genic or putative single-locus SSR markers. We also constructed a comprehensive database for the newly developed SSR markers, which was integrated with public Brassica SSR markers and annotated genome components. The genome-wide SSR markers developed in this study provide a useful tool to extend the annotated genome resources of sequenced Brassica species to genetic study/breeding in different Brassica species.

Immunoassays for riboflavin and flavin mononucleotide using antibodies specific to d-ribitol and d-ribitol-5-phosphate.

Science.gov (United States)

Ravi, G; Venkatesh, Yeldur P

2017-06-01

Riboflavin (vitamin B 2 ), a water-soluble vitamin, plays a key role in maintaining human health. Though, numerous methods have been reported for the determination of total riboflavin (TRF) content in foods and biological samples, very few methods are reported for quantifying riboflavin and its coenzymes [flavin mononucleotide (FMN); flavin adenine dinucleotide (FAD)] individually. Recently, we have demonstrated that antibodies specific to d-ribitol and d-ribitol-5-phosphate also recognize riboflavin and FMN, respectively, and not vice-versa. In this study, we have evaluated these two antibodies for the analysis of riboflavin and FMN by indirect competitive ELISA (icELISA) in selected foods and pharmaceuticals. Under the optimal assay conditions, 50% inhibition concentration (IC 50 ) and limit of detection (LOD, IC 10 ) were 3.41ng/mL and 0.02ng/mL for riboflavin, and 7.84ng/mL and 0.24ng/mL for FMN, respectively, with detectable concentration range between 0.1 and 100ng of analytes and riboflavin and FMN) from the same food samples showed variation in their values compared to TRF, and were in good agreement with values obtained from HPLC and AOAC methods. Further, spiking and recovery analysis of food samples and pharmaceuticals showed no significant matrix effects. The immunoassays were validated in terms of accuracy and precision using inter- and intra-assays. The immunoassays developed in this study are sensitive and appears feasible for screening a large number of samples in the quantification of riboflavin and FMN in various biological samples, pharmaceuticals and natural/processed foods. Copyright © 2017 Elsevier B.V. All rights reserved.

Crystallization and preliminary crystallographic studies of human septin 1 with site-directed mutations

Energy Technology Data Exchange (ETDEWEB)

Hu, Hao [Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Yu, Wen-bo [State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433 (China); Li, Shu-xing [Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Ding, Xiang-ming; Yu, Long [State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433 (China); Bi, Ru-Chang [Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China)

2006-02-01

The homogeneity of septin 1 has been improved by site-directed mutation of serine residues and only a small alteration in the secondary structure is observed to arise from the mutations. Crystals of the septin 1 mutant were grown and diffraction data were collected to 2.5 Å resolution. Septin 1 is a member of an evolutionarily conserved family of GTP-binding and filament-forming proteins named septins, which function in diverse processes including cytokinasis, vesicle trafficking, apoptosis, remodelling of the cytoskeleton, infection, neurodegeneration and neoplasia. Human septin 1 has been expressed and purified, but suffers from severe aggregation. Studies have shown that septin 1 with site-directed mutations of five serine residues (Ser19, Ser206, Ser307, Ser312 and Ser315) has a much lower degree of aggregation and better structural homogeneity and that the mutations cause only slight perturbations in the secondary structure of septin 1. This septin 1 mutant was crystallized and diffraction data were collected to 2.5 Å resolution. The space group is P422, with unit-cell parameters a = b = 106.028, c = 137.852 Å.

The observed human sperm mutation frequency cannot explain the achondroplasia paternal age effect

Science.gov (United States)

Tiemann-Boege, Irene; Navidi, William; Grewal, Raji; Cohn, Dan; Eskenazi, Brenda; Wyrobek, Andrew J.; Arnheim, Norman

2002-01-01

The lifelong spermatogonial stem cell divisions unique to male germ cell production are thought to contribute to a higher mutation frequency in males. The fact that certain de novo human genetic conditions (e.g., achondroplasia) increase in incidence with the age of the father is consistent with this idea. Although it is assumed that the paternal age effect is the result of an increasing frequency of mutant sperm as a man grows older, no direct molecular measurement of the germ-line mutation frequency has been made to confirm this hypothesis. Using sperm DNA from donors of different ages, we determined the frequency of the nucleotide substitution in the fibroblast growth factor receptor 3 (FGFR3) gene that causes achondroplasia. Surprisingly, the magnitude of the increase in mutation frequency with age appears insufficient to explain why older fathers have a greater chance of having a child with this condition. A number of alternatives may explain this discrepancy, including selection for sperm that carry the mutation or an age-dependent increase in premutagenic lesions that remain unrepaired in sperm and are inefficiently detected by the PCR assay. PMID:12397172

Crystallization and preliminary crystallographic studies of human septin 1 with site-directed mutations

International Nuclear Information System (INIS)

Hu, Hao; Yu, Wen-bo; Li, Shu-xing; Ding, Xiang-ming; Yu, Long; Bi, Ru-Chang

2006-01-01

The homogeneity of septin 1 has been improved by site-directed mutation of serine residues and only a small alteration in the secondary structure is observed to arise from the mutations. Crystals of the septin 1 mutant were grown and diffraction data were collected to 2.5 Å resolution. Septin 1 is a member of an evolutionarily conserved family of GTP-binding and filament-forming proteins named septins, which function in diverse processes including cytokinasis, vesicle trafficking, apoptosis, remodelling of the cytoskeleton, infection, neurodegeneration and neoplasia. Human septin 1 has been expressed and purified, but suffers from severe aggregation. Studies have shown that septin 1 with site-directed mutations of five serine residues (Ser19, Ser206, Ser307, Ser312 and Ser315) has a much lower degree of aggregation and better structural homogeneity and that the mutations cause only slight perturbations in the secondary structure of septin 1. This septin 1 mutant was crystallized and diffraction data were collected to 2.5 Å resolution. The space group is P422, with unit-cell parameters a = b = 106.028, c = 137.852 Å

mRNA/microRNA gene expression profile in microsatellite unstable colorectal cancer

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Calin George A

2007-08-01

Full Text Available Abstract Background Colorectal cancer develops through two main genetic instability pathways characterized by distinct pathologic features and clinical outcome. Results We investigated colon cancer samples (23 characterized by microsatellite stability, MSS, and 16 by high microsatellite instability, MSI-H for genome-wide expression of microRNA (miRNA and mRNA. Based on combined miRNA and mRNA gene expression, a molecular signature consisting of twenty seven differentially expressed genes, inclusive of 8 miRNAs, could correctly distinguish MSI-H versus MSS colon cancer samples. Among the differentially expressed miRNAs, various members of the oncogenic miR-17-92 family were significantly up-regulated in MSS cancers. The majority of protein coding genes were also up-regulated in MSS cancers. Their functional classification revealed that they were most frequently associated with cell cycle, DNA replication, recombination, repair, gastrointestinal disease and immune response. Conclusion This is the first report that indicates the existence of differences in miRNA expression between MSS versus MSI-H colorectal cancers. In addition, the work suggests that the combination of mRNA/miRNA expression signatures may represent a general approach for improving bio-molecular classification of human cancer.

Mutations in Human Tubulin Proximal to the Kinesin-Binding Site Alter Dynamic Instability at Microtubule Plus- and Minus-Ends

Energy Technology Data Exchange (ETDEWEB)

Ti, Shih-Chieh; Pamula, Melissa C.; Howes, Stuart C.; Duellberg, Christian; Cade, Nicholas I.; Kleiner, Ralph E.; Forth, Scott; Surrey, Thomas; Nogales, Eva; Kapoor, Tarun M.

2016-04-01

The assembly of microtubule-based cellular structures depends on regulated tubulin polymerization and directional transport. In this research, we have purified and characterized tubulin heterodimers that have human β-tubulin isotype III (TUBB3), as well as heterodimers with one of two β-tubulin mutations (D417H or R262H). Both point mutations are proximal to the kinesin-binding site and have been linked to an ocular motility disorder in humans. Compared to wild-type, microtubules with these mutations have decreased catastrophe frequencies and increased average lifetimes of plus- and minus-end-stabilizing caps. Importantly, the D417H mutation does not alter microtubule lattice structure or Mal3 binding to growing filaments. Instead, this mutation reduces the affinity of tubulin for TOG domains and colchicine, suggesting that the distribution of tubulin heterodimer conformations is changed. Together, our findings reveal how residues on the surface of microtubules, distal from the GTP-hydrolysis site and inter-subunit contacts, can alter polymerization dynamics at the plus- and minus-ends of microtubules.

The significance of microsatellite instability in colorectal cancer after controlling for clinicopathological factors.

Science.gov (United States)

Kang, Sanghee; Na, Younghyun; Joung, Sung Yup; Lee, Sun Il; Oh, Sang Cheul; Min, Byung Wook

2018-03-01

The colorectal cancer (CRC) patients with microsatellite instability (MSI) have distinct clinicopathological characteristics consisting of factors predicting positive and negative outcomes, such as a high lymph node harvest and poor differentiation. In this study, we measured the value of MSI as a prognostic factor after controlling for these discrepant factors. A total of 603 patients who underwent curative surgery for stages I to III colorectal cancer were enrolled. The patients were divided into microsatellite instability high (MSI-H) and microsatellite stable/microsatellite instability low (MSS/MSI-L) groups. Propensity score matching was used to match clinicopathological factors between the 2 groups. MSI-H patients had a high lymph node harvest (median: 31.0 vs 23.0, P controlling for pathological characteristics, MSI-H could be a potent prognostic factor regarding patient survival.

Mutational profile of GNAQQ209 in human tumors.

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Simona Lamba

Full Text Available BACKGROUND: Frequent somatic mutations have recently been identified in the ras-like domain of the heterotrimeric G protein alpha-subunit (GNAQ in blue naevi 83%, malignant blue naevi (50% and ocular melanoma of the uvea (46%. The mutations exclusively affect codon 209 and result in GNAQ constitutive activation which, in turn, acts as a dominant oncogene. METHODOLOGY: To assess if the mutations are present in other tumor types we performed a systematic mutational profile of the GNAQ exon 5 in a panel of 922 neoplasms, including glioblastoma, gastrointestinal stromal tumors (GIST, acute myeloid leukemia (AML, blue naevi, skin melanoma, bladder, breast, colorectal, lung, ovarian, pancreas, and thyroid carcinomas. PRINCIPAL FINDINGS: We detected the previously reported mutations in 6/13 (46% blue naevi. Changes affecting Q209 were not found in any of the other tumors. Our data indicate that the occurrence of GNAQ mutations display a unique pattern being present in a subset of melanocytic tumors but not in malignancies of glial, epithelial and stromal origin analyzed in this study.

Development of Microsatellite Loci in Artocarpus altilis (Moraceae and Cross-Amplification in Congeneric Species

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Colby Witherup

2013-07-01

Full Text Available Premise of the study: Microsatellite loci were isolated and characterized from enriched genomic libraries of Artocarpus altilis (breadfruit and tested in four Artocarpus species and one hybrid. The microsatellite markers provide new tools for further studies in Artocarpus. Methods and Results: A total of 25 microsatellite loci were evaluated across four Artocarpus species and one hybrid. Twenty-one microsatellite loci were evaluated on A. altilis (241, A. camansi (34, A. mariannensis (15, and A. altilis × mariannensis (64 samples. Nine of those loci plus four additional loci were evaluated on A. heterophyllus (jackfruit, 426 samples. All loci are polymorphic for at least one species. The average number of alleles ranges from two to nine within taxa. Conclusions: These microsatellite primers will facilitate further studies on the genetic structure and evolutionary and domestication history of Artocarpus species. They will aid in cultivar identification and establishing germplasm conservation strategies for breadfruit and jackfruit.

Single-Cell Analysis of Human Pancreas Reveals Transcriptional Signatures of Aging and Somatic Mutation Patterns.

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Enge, Martin; Arda, H Efsun; Mignardi, Marco; Beausang, John; Bottino, Rita; Kim, Seung K; Quake, Stephen R

2017-10-05

As organisms age, cells accumulate genetic and epigenetic errors that eventually lead to impaired organ function or catastrophic transformation such as cancer. Because aging reflects a stochastic process of increasing disorder, cells in an organ will be individually affected in different ways, thus rendering bulk analyses of postmitotic adult cells difficult to interpret. Here, we directly measure the effects of aging in human tissue by performing single-cell transcriptome analysis of 2,544 human pancreas cells from eight donors spanning six decades of life. We find that islet endocrine cells from older donors display increased levels of transcriptional noise and potential fate drift. By determining the mutational history of individual cells, we uncover a novel mutational signature in healthy aging endocrine cells. Our results demonstrate the feasibility of using single-cell RNA sequencing (RNA-seq) data from primary cells to derive insights into genetic and transcriptional processes that operate on aging human tissue. Copyright © 2017 Elsevier Inc. All rights reserved.

Development of Multiplex Microsatellite PCR Panels for the Seagrass Thalassia hemprichii (Hydrocharitaceae

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Kor-jent van Dijk

2014-11-01

Full Text Available Premise of the study: New microsatellites were developed for the seagrass Thalassia hemprichii (Hydrocharitaceae, a long-lived seagrass species that is found throughout the shallow waters of tropical and subtropical Indo-West Pacific. Three multiplex PCR panels were designed utilizing new and previously developed markers, resulting in a toolkit for generating a 16-locus genotype. Methods and Results: Through the use of microsatellite enrichment and next-generation sequencing, 16 new, validated, polymorphic microsatellite markers were isolated. Diversity was between two and four alleles per locus totaling 36 alleles. These markers, plus previously developed microsatellite markers for T. hemprichii and T. testudinum, were tested for suitability in multiplex PCR panels. Conclusions: The generation of an easily replicated suite of multiplex panels of codominant molecular markers will allow for high-resolution and detailed genetic structure analysis and clonality assessment with minimal genotyping costs. We suggest the establishment of a T. hemprichii primer convention for the unification of future data sets.

Mining microsatellites in the peach genome: development of new long-core SSR markers for genetic analyses in five Prunus species.

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Dettori, Maria Teresa; Micali, Sabrina; Giovinazzi, Jessica; Scalabrin, Simone; Verde, Ignazio; Cipriani, Guido

2015-01-01

A wide inventory of molecular markers is nowadays available for individual fingerprinting. Microsatellites, or simple sequence repeats (SSRs), play a relevant role due to their relatively ease of use, their abundance in the plant genomes, and their co-dominant nature, together with the availability of primer sequences in many important agricultural crops. Microsatellites with long-core motifs are more easily scored and were adopted long ago in human genetics but they were developed only in few crops, and Prunus species are not among them. In the present work the peach whole-genome sequence was used to select 216 SSRs containing long-core motifs with tri-, tetra- and penta-nucleotide repeats. Microsatellite primer pairs were designed and tested for polymorphism in the five diploid Prunus species of economic relevance (almond, apricot, Japanese plum, peach and sweet cherry). A set of 26 microsatellite markers covering all the eight chromosomes, was also selected and used in the molecular characterization, population genetics and structure analyses of a representative sample of the five diploid Prunus species, assessing their transportability and effectiveness. The combined probability of identity between two random individuals for the whole set of 26 SSRs was quite low, ranging from 2.30 × 10(-7) in peach to 9.48 × 10(-10) in almond, confirming the usefulness of the proposed set for fingerprinting analyses in Prunus species.

Microsatellite loci isolated from the scleractinian coral, Acropora nobilis.

Science.gov (United States)

Isomura, Naoko; Hidaka, Michio

2008-05-01

We report the isolation and characterization of eight microsatellite loci from the scleractinian coral, Acropora nobilis. The microsatellite loci were obtained using compound SSR primers or an enrichment protocol. All the loci were polymorphic with four to eight alleles per locus and observed heterozygosities ranging from 0.22 to 0.76. Some of the primers developed for the two congeners, Acropora palmata and Acropora millepora were applicable to A. nobilis. These loci are useful for studying the connectivity among A. nobilis populations in Okinawa, southern Japan. © 2007 The Authors.

Microsatellite D21D210 (GT-12) allele frequencies in sporadic Alzheimer's disease

International Nuclear Information System (INIS)

Lannfelt, L.; Lilius, L.; Viitanen, M.; Winblad, B.; Basun, H.; Houlden, H.; Rossor, M.; Hardy, J.

1995-01-01

Four disease-causing mutations have so far been described in the amyloid precursor protein gene on chromosome 21 in familial early-onset Alzheimer's disease. Linkage analysis with a fourteen-allele microsatellite at D21S210 named GT-12 has proven useful in the elucidation of amyloid presursor protein gene involvement in Alzheimer's disease families, as it is closely linked to the gene. Most cases of Alzheimer's disease are thought to be sporadic and not familial. However, evidence from earlier studies suggests an important genetic contribution also in sporadic cases, where gene-environment interaction may contribute to the disease. We have determined frequencies of the GT-12 alleles in 78 Swedish and 49 British sporadic Alzheimer's disease cases and 104 healthy elderly control subjects, to investigate if the disease associates with a particular genotype in GT-12. However, no differences in allele frequencies were observed between any of the groups. (au) (26 refs.)

Analysis of mutations in the human HPRT gene induced by accelerated heavy-ion irradiation

International Nuclear Information System (INIS)

Kagawa, Yasuhiro; Yatagai, Fumio; Hanaoka, Fumio; Suzuki, Masao; Kase, Youko; Kobayashi, Akiko; Hirano, Masahiko; Kato, Takesi; Watanabe, Masami.

1995-01-01

Multiplex PCR analysis of HPRT(-) mutations in human embryo (HE) cells induced by 230 keV/μm carbon-ion irradiation showed no large deletion around the exon regions of the locus gene in contrast to the irradiations at different LETs. To identify these mutations, the sequence alterations in a cDNA of hprt gene were determined for 18 mutant clones in this study. Missing of exon 6 was the most frequent mutational event (10 clones), and missing of both exons 6 and 8 was next most frequent event (6 clones), then base substitutions (2 clones). These characteristics were not seen in a similar analysis of spontaneous mutations, which showed base substitution (5 clones), frameshift (2 clones), missing of both exons 2 and 3 (2 clones), and a single unidentified clone. Direct sequencing and restriction enzyme digestion of the genomic DNA of the mutants which showed missing of exons 6 and 8 in the cDNA, supports the possibility that they were induced by aberrant mRNA splicing. (author)

Crystallization and preliminary X-ray crystallographic analysis of quinolinate phosphoribosyltransferase from porcine kidney in complex with nicotinate mononucleotide

International Nuclear Information System (INIS)

Youn, Hyung-Seop; Kim, Mun-Kyoung; Kang, Gil Bu; Kim, Tae Gyun; An, Jun Yop; Lee, Jung-Gyu; Park, Kyoung Ryoung; Lee, Youngjin; Fukuoka, Shin-Ichi; Eom, Soo Hyun

2012-01-01

Crystals of S. scrofa quinolinate phosphoribosyltransferase purified from porcine kidney in complex with nicotinate mononucleotidewere obtained and diffraction data were collected and processed to 2.1 Å resolution. Quinolinate phosphoribosyltransferase (QAPRTase) is a key enzyme in NAD biosynthesis; it catalyzes the formation of nicotinate mononucleotide (NAMN) from quinolinate and 5-phosphoribosyl-1-pyrophosphate. In order to elucidate the mechanism of NAMN biosynthesis, crystals of Sus scrofa QAPRTase (Ss-QAPRTase) purified from porcine kidney in complex with NAMN were obtained and diffraction data were collected and processed to 2.1 Å resolution. The Ss-QAPRTase–NAMN cocrystals belonged to space group P321, with unit-cell parameters a = 119.1, b = 119.1, c = 93.7 Å, γ = 120.0°. The Matthews coefficient and the solvent content were estimated as 3.10 Å 3 Da –1 and 60.3%, respectively, assuming the presence of two molecules in the asymmetric unit

Nuclear import of human MLH1, PMS2, and MutLalpha: redundancy is the key.

Science.gov (United States)

Leong, Vivian; Lorenowicz, Jessica; Kozij, Natalie; Guarné, Alba

2009-08-01

DNA mismatch repair maintains genomic stability by correcting errors that have escaped polymerase proofreading. Defects on mismatch repair genes lead to an increased mutation rate, microsatellite instability and predisposition to human non-polyposis colorectal cancer (HNPCC). Human MutLalpha is a heterodimer formed by the interaction of MLH1 and PMS2 that coordinates a series of key events in mismatch repair. It has been proposed that nuclear import of MutLalpha may be the first regulatory step on the activation of the mismatch repair pathway. Using confocal microscopy and mismatch repair deficient cells, we have identified the sequence determinants that drive nuclear import of human MLH1, PMS2, and MutLalpha. Transient transfection of the individual proteins reveals that MLH1 has a bipartite and PMS2 has a single monopartite nuclear localization signal. Although dimerization is not required for nuclear localization, the MutLalpha heterodimer is imported more efficiently than the MLH1 or PMS2 monomers. Interestingly, the bipartite localization signal of MLH1 can direct import of MutLalpha even when PMS2 encompasses a mutated localization signal. Hence we conclude that the presence of redundant nuclear localization signals guarantees nuclear transport of MutLalpha and, consequently, efficient mismatch repair.

Direct Measurements of Human Colon Crypt Stem Cell Niche Genetic Fidelity: The Role of Chance in Non-Darwinian Mutation Selection

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Haeyoun eKang

2013-10-01

Full Text Available Perfect human stem cell genetic fidelity would prevent aging and cancer. However, perfection would be difficult to achieve, and aging is universal and cancers common. A hypothesis is that because mutations are inevitable over a human lifetime, downstream mechanisms have evolved to manage the deleterious effects of beneficial and lethal mutations. In the colon, a crypt stem cell architecture reduces the number of mitotic cells at risk for mutation accumulation, and multiple niche stem cells ensure that a lethal mutation within any single stem cell does not lead to crypt death. In addition, the architecture of the colon crypt stem cell niche may harness probability or chance to randomly discard many beneficial mutations that might lead to cancer. An analysis of somatic chromosome copy number alterations (CNAs reveals a lack of perfect fidelity in individual normal human crypts, with age-related increases and higher frequencies in ulcerative colitis, a proliferative, inflammatory disease. The age-related increase in somatic CNAs appears consistent with relatively normal replication error and cell division rates. Surprisingly, and similar to point mutations in cancer genomes, the types of crypt mutations were more consistent with random fixation rather than selection. In theory, a simple non-Darwinian way to nullify selection is to reduce the size of the reproducing population. Fates are more determined by chance rather than selection in very small populations, and therefore selection may be minimized within small crypt niches. The desired effect is that many beneficial mutations that might lead to cancer are randomly lost by drift rather than fixed by selection. The subdivision of the colon into multiple very small stem cell niches may trade Darwinian evolution for non-Darwinian somatic cell evolution, capitulating to aging but reducing cancer risks.

Isolation and Characterization of Polymorphic Microsatellite Loci from Metapenaeopsis barbata Using PCR-Based Isolation of Microsatellite Arrays (PIMA)

Science.gov (United States)

Chiang, Tzen-Yuh; Tzeng, Tzong-Der; Lin, Hung-Du; Cho, Ching-Ju; Lin, Feng-Jiau

2012-01-01

The red-spot prawn, Metapenaeopsis barbata, is a commercially important, widely distributed demersal species in the Indo-West Pacific Ocean. Overfishing has made its populations decline in the past decade. To study conservation genetics, eight polymorphic microsatellite loci were isolated. Genetic characteristics of the SSR (simple sequence repeat) fingerprints were estimated in 61 individuals from adjacent seas of Taiwan and China. The number of alleles, ranging from 2 to 4, as well as observed and expected heterozygosities in populations, ranging from 0.048 to 0.538, and 0.048 and 0.654, respectively, were detected. No deviation from Hardy–Weinberg expectations was detected at either locus. No significant linkage disequilibrium was detected in locus pairs. The polymorphic microsatellite loci will be useful for investigations of the genetic variation, population structure, and conservation genetics of this species. PMID:22489123

Novel mutations in scavenger receptor BI associated with high HDL cholesterol in humans

NARCIS (Netherlands)

Brunham, Liam R.; Tietjen, Ian; Bochem, Andrea E.; Singaraja, Roshni R.; Franchini, Patrick L.; Radomski, Chris; Mattice, Maryanne; Legendre, Annick; Hovingh, G. Kees; Kastelein, John J. P.; Hayden, Michael R.

2011-01-01

The scavenger receptor class B, member 1 (SR-BI), is a key cellular receptor for high-density lipoprotein (HDL) in mice, but its relevance to human physiology has not been well established. Recently a family was reported with a mutation in the gene encoding SR-BI and high HDL cholesterol (HDL-C).

Microsatellite loci development in mycoheterotrophic Corallorhiza maculata with amplification in C. mertensiana

Science.gov (United States)

Sarah E. Hopkins; D. Lee. Taylor

2011-01-01

Microsatellite primers were developed for the first time in the species Corallorhiza maculata, a nonphotosynthetic orchid that is becoming a model for studying mycorrhizal specificity. Eight polymorphic microsatellite markers were developed using an enrichment and cloning protocol. The number of alleles for each locus ranged from two to seven. The...

Tracing Asian Seabass Individuals to Single Fish Farms Using Microsatellites

Science.gov (United States)

Yue, Gen Hua; Xia, Jun Hong; Liu, Peng; Liu, Feng; Sun, Fei; Lin, Grace

2012-01-01

Traceability through physical labels is well established, but it is not highly reliable as physical labels can be easily changed or lost. Application of DNA markers to the traceability of food plays an increasingly important role for consumer protection and confidence building. In this study, we tested the efficiency of 16 polymorphic microsatellites and their combinations for tracing 368 fish to four populations where they originated. Using the maximum likelihood and Bayesian methods, three most efficient microsatellites were required to assign over 95% of fish to the correct populations. Selection of markers based on the assignment score estimated with the software WHICHLOCI was most effective in choosing markers for individual assignment, followed by the selection based on the allele number of individual markers. By combining rapid DNA extraction, and high-throughput genotyping of selected microsatellites, it is possible to conduct routine genetic traceability with high accuracy in Asian seabass. PMID:23285169

Eguchipsammia fistula Microsatellite Development and Population Analysis

KAUST Repository

Mughal, Mehreen

2012-01-01

Deep water corals are an understudied yet biologically important and fragile ecosystem under threat from recent increasing temperatures and high carbon dioxide emissions. Using 454 sequencing, we develop 14 new microsatellite markers for the deep

Research Note Development and characterization of microsatellite ...

Indian Academy of Sciences (India)

Srirama R

2School of Biosciences and Technology, VIT University, Vellore 632014, India. 7. 8 ... microsatellite markers developed can be used for studying the population ... characterized by diverse growth forms, including shrubs, trees, and annual or ...

Genetic Variation and Population Structure in Jamunapari Goats Using Microsatellites, Mitochondrial DNA, and Milk Protein Genes

Science.gov (United States)

Rout, P. K.; Thangraj, K.; Mandal, A.; Roy, R.

2012-01-01

Jamunapari, a dairy goat breed of India, has been gradually declining in numbers in its home tract over the years. We have analysed genetic variation and population history in Jamunapari goats based on 17 microsatellite loci, 2 milk protein loci, mitochondrial hypervariable region I (HVRI) sequencing, and three Y-chromosomal gene sequencing. We used the mitochondrial DNA (mtDNA) mismatch distribution, microsatellite data, and bottleneck tests to infer the population history and demography. The mean number of alleles per locus was 9.0 indicating that the allelic variation was high in all the loci and the mean heterozygosity was 0.769 at nuclear loci. Although the population size is smaller than 8,000 individuals, the amount of variability both in terms of allelic richness and gene diversity was high in all the microsatellite loci except ILST 005. The gene diversity and effective number of alleles at milk protein loci were higher than the 10 other Indian goat breeds that they were compared to. Mismatch analysis was carried out and the analysis revealed that the population curve was unimodal indicating the expansion of population. The genetic diversity of Y-chromosome genes was low in the present study. The observed mean M ratio in the population was above the critical significance value (Mc) and close to one indicating that it has maintained a slowly changing population size. The mode-shift test did not detect any distortion of allele frequency and the heterozygosity excess method showed that there was no significant departure from mutation-drift equilibrium detected in the population. However, the effects of genetic bottlenecks were observed in some loci due to decreased heterozygosity and lower level of M ratio. There were two observed genetic subdivisions in the population supporting the observations of farmers in different areas. This base line information on genetic diversity, bottleneck analysis, and mismatch analysis was obtained to assist the conservation

Novel and cross-species microsatellite markers for parentage analysis in Sanderling Calidris alba

NARCIS (Netherlands)

Luttikhuizen, Pieternella C.; Bol, Anneke; Witte, Harry; van Bleijswijk, Judith; Haddrath, Oliver; Baker, Allan J.; Piersma, Theunis; Reneerkens, Jeroen

We isolated and tested six novel microsatellite loci in Sanderling (Calidris alba) from Greenland for paternity analyses. In addition, we tested 11 already published microsatellite markers which were originally developed for the congeneric species, the Pectoral Sandpiper (C. melanotos). All loci

YCL047C/POF1 is a novel nicotinamide mononucleotide adenylyltransferase (NMNAT) in Saccharomyces cerevisiae.

Science.gov (United States)

Kato, Michiko; Lin, Su-Ju

2014-05-30

NAD(+) is an essential metabolic cofactor involved in various cellular biochemical processes. Nicotinamide riboside (NR) is an endogenously produced key pyridine metabolite that plays important roles in the maintenance of NAD(+) pool. Using a NR-specific cell-based screen, we identified mutants that exhibit altered NR release phenotype. Yeast cells lacking the ORF YCL047C/POF1 release considerably more NR compared with wild type, suggesting that POF1 plays an important role in NR/NAD(+) metabolism. The amino acid sequence of Pof1 indicates that it is a putative nicotinamide mononucleotide adenylyltransferase (NMNAT). Unlike other yeast NMNATs, Pof1 exhibits NMN-specific adenylyltransferase activity. Deletion of POF1 significantly lowers NAD(+) levels and decreases the efficiency of NR utilization, resistance to oxidative stress, and NR-induced life span extension. We also show that NR is constantly produced by multiple nucleotidases and that the intracellular NR pools are likely to be compartmentalized, which contributes to the regulation of NAD(+) homeostasis. Our findings may contribute to the understanding of the molecular basis and regulation of NAD(+) metabolism in higher eukaryotes. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

PIK3CA Mutation in Colorectal Cancer: Relationship with Genetic and Epigenetic Alterations

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Katsuhiko Nosho

2008-06-01

Full Text Available Somatic PIK3CA mutations are often present in colorectal cancer. Mutant PIK3CA activates AKT signaling, which up-regulates fatty acid synthase (FASN. Microsatellite instability (MSI and CpG island methylator phenotype (CIMP are important molecular classifiers in colorectal cancer. However, the relationship between PIK3CA mutation, MSI and CIMP remains uncertain. Using Pyrosequencing technology, we detected PIK3CA mutations in 91 (15% of 590 population-based colorectal cancers. To determine CIMP status, we quantified DNA methylation in eight CIMP-specific promoters [CACNA1G, CDKN2A (p16, CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1] by real-time polymerase chain reaction (MethyLight. PIK3CA mutation was significantly associated with mucinous tumors [P = .0002; odds ratio (OR = 2.44], KRAS mutation (P < .0001; OR = 2.68, CIMP-high (P = .03; OR = 2.08, phospho–ribosomal protein S6 expression (P = .002; OR = 2.19, and FASN expression (P = .02; OR = 1.85 and inversely with p53 expression (P = .01; OR = 0.54 and β-catenin (CTNNB1 alteration (P = .004; OR = 0.43. In addition, PIK3CA G-to-A mutations were associated with MGMT loss (P = .001; OR = 3.24 but not with MGMT promoter methylation. In conclusion, PIK3CA mutation is significantly associated with other key molecular events in colorectal cancer, and MGMT loss likely contributes to the development of PIK3CA G>A mutation. In addition, Pyrosequencing is useful in detecting PIK3CA mutation in archival paraffin tumor tissue. PIK3CA mutational data further emphasize heterogeneity of colorectal cancer at the molecular level.

Phylogeography of haplotypes of five microsatellites located in a low-recombination region of the X chromosome: studies worldwide and in Brazilian populations.

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Pereira, Rinaldo Wellerson; Pena, Sérgio D J

2006-01-01

We studied five microsatellites (DXS995, DXS8076, DXS8114, DXS1002 and DXS1050) located in a region of very low recombination rate in the long arm of the human X chromosome (Xq13.3-Xq21.3). No recombination was seen in 291 meioses in CEPH families. To test whether haplotypes composed of the five microsatellites could differentiate among distinct human continental populations, we studied an international panel containing 72 males from Africa, Europe, Asia and the America. Haplotypic diversity was very high within these groups and no haplotypes were shared among them. This led to the hope that we might be able to identify continent-specific lineages. However, in a median joining network there was no clear discrimination of the different continental groups. We then tested whether we could identify X chromosomal lineages from different continental origins in Brazilians. We typed 180 white Brazilians from four different geographical regions and examined their proportions of haplotype sharing with Africans, Asians, Europeans and Amerindians. No phylogeographical patterns emerged from the data. Moreover, there were several instances of the same haplotype being shared by many (and in one instance all) groups, suggesting that recombination might be occurring. We thus studied pairwise the level of linkage disequilibrium (LD) between the microsatellites. No detectable linkage disequilibrium between the most external loci DXS995 and DXS1050 was observed. Thus, even though recombination may be absent on short time spans, as seen in the CEPH pedigrees, on a long term basis it occurs often enough to dissipate all linkage disequilibrium. On the other hand, we observed very strong linkage disequilibrium between the pairs DXS995/DXS8076 and DXS1002/DXS8114, raising the possibility of resequencing the segment between them to identify single nucleotide polymorphisms (SNPs) in their intervals. The combination of X-linked microsatellites and SNPs in strong linkage disequilibrium might

Genetic diversity analysis of Cuban traditional rice (Oryza sativa L. varieties based on microsatellite markers

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Alba Alvarez

2007-01-01

Full Text Available Microsatellite polymorphism was studied in a sample of 39 traditional rice (Oryza sativa L. varieties and 11 improved varieties widely planted in Cuba. The study was aimed at assessing the extent of genetic variation in traditional and improved varieties and to establish their genetic relationship for breeding purposes. Heterozygosity was analyzed at each microsatellite loci and for each genotype using 10 microsatellite primer pairs. Between varieties genetic relationship was estimated. The number of alleles per microsatellite loci was 4 to 8, averaging 6.6 alleles per locus. Higher heterozygosity (H was found in traditional varieties (H TV = 0.72 than in improved varieties (H IV = 0.42, and 68% of the total microsatellite alleles were found exclusively in the traditional varieties. Genetic diversity, represented by cluster analysis, indicated three different genetic groups based on their origin. Genetic relationship estimates based on the proportion of microsatellite loci with shared alleles indicated that the majority of traditional varieties were poorly related to the improved varieties. We also discuss the more efficient use of the available genetic diversity in future programs involving genetic crosses.

Survey of microsatellite DNA in pine

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Craig S. Echt; P. May-Marquardt

1997-01-01

A large insert genomic library from eastern white pine (Pinus strobus) was probed for the microsatellite motifs (AC)n and (AG)n, all 10 trinucleotide motifs, and 22 of the 33 possible tetranucleotide motifs. For comparison with a species from a different subgenus, a loblolly pine (Pinus taeda) genomic...

MSH3-deficiency initiates EMAST without oncogenic transformation of human colon epithelial cells.

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Christoph Campregher

Full Text Available BACKGROUND/AIM: Elevated microsatellite instability at selected tetranucleotide repeats (EMAST is a genetic signature in certain cases of sporadic colorectal cancer and has been linked to MSH3-deficiency. It is currently controversial whether EMAST is associated with oncogenic properties in humans, specifically as cancer development in Msh3-deficient mice is not enhanced. However, a mutator phenotype is different between species as the genetic positions of repetitive sequences are not conserved. Here we studied the molecular effects of human MSH3-deficiency. METHODS: HCT116 and HCT116+chr3 (both MSH3-deficient and primary human colon epithelial cells (HCEC, MSH3-wildtype were stably transfected with an EGFP-based reporter plasmid for the detection of frameshift mutations within an [AAAG]17 repeat. MSH3 was silenced by shRNA and changes in protein expression were analyzed by shotgun proteomics. Colony forming assay was used to determine oncogenic transformation and double strand breaks (DSBs were assessed by Comet assay. RESULTS: Despite differential MLH1 expression, both HCT116 and HCT116+chr3 cells displayed comparable high mutation rates (about 4×10(-4 at [AAAG]17 repeats. Silencing of MSH3 in HCECs leads to a remarkable increased frameshift mutations in [AAAG]17 repeats whereas [CA]13 repeats were less affected. Upon MSH3-silencing, significant changes in the expression of 202 proteins were detected. Pathway analysis revealed overexpression of proteins involved in double strand break repair (MRE11 and RAD50, apoptosis, L1 recycling, and repression of proteins involved in metabolism, tRNA aminoacylation, and gene expression. MSH3-silencing did not induce oncogenic transformation and DSBs increased 2-fold. CONCLUSIONS: MSH3-deficiency in human colon epithelial cells results in EMAST, formation of DSBs and significant changes of the proteome but lacks oncogenic transformation. Thus, MSH3-deficiency alone is unlikely to drive human colon

Mutation rate heterogeneity and the generation of allele diversity at the human minisatellite MS205 (D16S309).

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May, C A; Jeffreys, A J; Armour, J A

1996-11-01

Many tandemly repeated minisatellite loci display extreme levels of length variation as a consequence of high rates of spontaneous germline mutation altering repeat copy number. Direct screening for new allele lengths by small-pool PCR has shown that instability at the human minisatellite locus MS205 (D16S309) is largely germline specific and usually results in the gain or loss of just a few repeat units. Structural analysis of the order of variant repeats has shown that these events occur preferentially at one end of the tandem array and can result in complex rearrangements including the inter-allelic transfer of repeat units. In contrast, putative mutants recovered from somatic DNA occur at a substantially lower rate and are simple and non-polar in nature. Germline mutation rates vary considerably between alleles, consistent with regulation occurring in cis. Although examination of DNA sequence polymorphisms immediately flanking the minisatellite reveals no definitive associations with germline mutation rate variation, differences in rate may be paralleled by changes in mutation spectrum. These findings help to explain the diversity of MS205 allele structures in modern humans and suggest a common mutation pathway with some other minisatellites.

Carcinoma microsatellite instability status as a predictor of benefit from fluorouracil-based adjuvant chemotherapy for stage II rectal cancer.

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Yang, Liu; Sun, Yan; Huang, Xin-En; Yu, Dong-Sheng; Zhou, Jian-Nong; Zhou, Xin; Li, Dong-Zheng; Guan, Xin

2015-01-01

Rectal cancers with high microsatellite-instable have clinical and pathological features that differentiate them from microsatellite-stable or low- frequency carcinomas, which was studied rarely in stage II rectal cancer, promoting the present investigation of the usefulness of microsatellite-instability status as a predictor of the benefit of adjuvant chemotherapy with fluorouracil in stage II rectal cancer. Data of 460 patients who underwent primary anterior resection with a double stapling technique for rectal carcinoma at a single institution from 2008 to 2012 were retrospectively collected. All patients experienced a total mesorectal excision (TME) operation. Survival analysis were analyzed using the Cox regression method. Five-year rate of disease-free survival (DFS) was noted in 390 (84.8%) of 460 patients with stage II rectal cancer. Of 460 tissue specimens, 97 (21.1%) exhibited high-frequency microsatellite instability. Median age of the patients was 65 (50-71) and 185 (40.2%) were male. After univariate and multivariate analysis, microsatellite instability (p= 0.001), female sex (pchemotherapy (pchemotherapy, those cancers displaying high-frequency microsatellite instability had a better 5-year rate of DFS than tumors exhibiting microsatellite stability or low-frequency instability (HR, 13.61 [95% CI, 1.88 to 99.28]; p= 0.010), while in 259 patients who received adjuvant chemotherapy, there was no DFS difference between the two groups (p= 0.145). Furthermore, patients exhibiting microsatellite stability or low-frequency instability who received adjuvant chemotherapy had a better 5-year rate of DFS than patients did not (HR, 5.16 [95% CI, 2.90 to 9.18]; pchemotherapy and gender. Fluorouracil-based adjuvant chemotherapy benefits patients of stage II rectal cancer with microsatellite-stable or low microsatellite-instable, but not those with high microsatellite- instable. Additionally, free of adjuvant chemotherapy, carcinomas with high microsatellite

Successful development of microsatellite markers in a challenging species: the horizontal borer Austroplatypus incompertus (Coleoptera: Curculionidae).

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Smith, S; Joss, T; Stow, A

2011-10-01

The analysis of microsatellite loci has allowed significant advances in evolutionary biology and pest management. However, until very recently, the potential benefits have been compromised by the high costs of developing these neutral markers. High-throughput sequencing provides a solution to this problem. We describe the development of 13 microsatellite markers for the eusocial ambrosia beetle, Austroplatypus incompertus, a significant pest of forests in southeast Australia. The frequency of microsatellite repeats in the genome of A. incompertus was determined to be low, and previous attempts at microsatellite isolation using a traditional genomic library were problematic. Here, we utilised two protocols, microsatellite-enriched genomic library construction and high-throughput 454 sequencing and characterised 13 loci which were polymorphic among 32 individuals. Numbers of alleles per locus ranged from 2 to 17, and observed and expected heterozygosities from 0.344 to 0.767 and from 0.507 to 0.860, respectively. These microsatellites have the resolution required to analyse fine-scale colony and population genetic structure. Our work demonstrates the utility of next-generation 454 sequencing as a method for rapid and cost-effective acquisition of microsatellites where other techniques have failed, or for taxa where marker development has historically been both complicated and expensive.

Molecular Detection of Bladder Cancer by Fluorescence Microsatellite Analysis and an Automated Genetic Analyzing System

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Sarel Halachmi

2007-01-01

Full Text Available To investigate the ability of an automated fluorescent analyzing system to detect microsatellite alterations, in patients with bladder cancer. We investigated 11 with pathology proven bladder Transitional Cell Carcinoma (TCC for microsatellite alterations in blood, urine, and tumor biopsies. DNA was prepared by standard methods from blood, urine and resected tumor specimens, and was used for microsatellite analysis. After the primers were fluorescent labeled, amplification of the DNA was performed with PCR. The PCR products were placed into the automated genetic analyser (ABI Prism 310, Perkin Elmer, USA and were subjected to fluorescent scanning with argon ion laser beams. The fluorescent signal intensity measured by the genetic analyzer measured the product size in terms of base pairs. We found loss of heterozygocity (LOH or microsatellite alterations (a loss or gain of nucleotides, which alter the original normal locus size in all the patients by using fluorescent microsatellite analysis and an automated analyzing system. In each case the genetic changes found in urine samples were identical to those found in the resected tumor sample. The studies demonstrated the ability to detect bladder tumor non-invasively by fluorescent microsatellite analysis of urine samples. Our study supports the worldwide trend for the search of non-invasive methods to detect bladder cancer. We have overcome major obstacles that prevented the clinical use of an experimental system. With our new tested system microsatellite analysis can be done cheaper, faster, easier and with higher scientific accuracy.

The Impact of Environmental and Endogenous Damage on Somatic Mutation Load in Human Skin Fibroblasts.

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Natalie Saini

2016-10-01

Full Text Available Accumulation of somatic changes, due to environmental and endogenous lesions, in the human genome is associated with aging and cancer. Understanding the impacts of these processes on mutagenesis is fundamental to understanding the etiology, and improving the prognosis and prevention of cancers and other genetic diseases. Previous methods relying on either the generation of induced pluripotent stem cells, or sequencing of single-cell genomes were inherently error-prone and did not allow independent validation of the mutations. In the current study we eliminated these potential sources of error by high coverage genome sequencing of single-cell derived clonal fibroblast lineages, obtained after minimal propagation in culture, prepared from skin biopsies of two healthy adult humans. We report here accurate measurement of genome-wide magnitude and spectra of mutations accrued in skin fibroblasts of healthy adult humans. We found that every cell contains at least one chromosomal rearrangement and 600–13,000 base substitutions. The spectra and correlation of base substitutions with epigenomic features resemble many cancers. Moreover, because biopsies were taken from body parts differing by sun exposure, we can delineate the precise contributions of environmental and endogenous factors to the accrual of genetic changes within the same individual. We show here that UV-induced and endogenous DNA damage can have a comparable impact on the somatic mutation loads in skin fibroblasts. Trial Registration: ClinicalTrials.gov NCT01087307.

Colon and rectal cancer survival by tumor location and microsatellite instability: the Colon Cancer Family Registry.

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Phipps, Amanda I; Lindor, Noralane M; Jenkins, Mark A; Baron, John A; Win, Aung Ko; Gallinger, Steven; Gryfe, Robert; Newcomb, Polly A

2013-08-01

Cancers in the proximal colon, distal colon, and rectum are frequently studied together; however, there are biological differences in cancers across these sites, particularly in the prevalence of microsatellite instability. We assessed the differences in survival by colon or rectal cancer site, considering the contribution of microsatellite instability to such differences. This is a population-based prospective cohort study for cancer survival. This study was conducted within the Colon Cancer Family Registry, an international consortium. Participants were identified from population-based cancer registries in the United States, Canada, and Australia. Information on tumor site, microsatellite instability, and survival after diagnosis was available for 3284 men and women diagnosed with incident invasive colon or rectal cancer between 1997 and 2002, with ages at diagnosis ranging from 18 to 74. Cox regression was used to calculate hazard ratios for the association between all-cause mortality and tumor location, overall and by microsatellite instability status. Distal colon (HR, 0.59; 95% CI, 0.49-0.71) and rectal cancers (HR, 0.68; 95% CI, 0.57-0.81) were associated with lower mortality than proximal colon cancer overall. Compared specifically with patients with proximal colon cancer exhibiting no/low microsatellite instability, patients with distal colon and rectal cancers experienced lower mortality, regardless of microsatellite instability status; patients with proximal colon cancer exhibiting high microsatellite instability had the lowest mortality. Study limitations include the absence of stage at diagnosis and cause-of-death information for all but a subset of study participants. Some patient groups defined jointly by tumor site and microsatellite instability status are subject to small numbers. Proximal colon cancer survival differs from survival for distal colon and rectal cancer in a manner apparently dependent on microsatellite instability status. These

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

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Heema Hewitson

2016-03-01

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.

Hyper-radiation sensitivity of murine scid mutation and mapping of the human homologue HYRC1 gene

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Komatsu, Kenshi; Ohta, Tohru; Niikawa, Norio; Okumura, Yutaka; Kubota, Nobuo.

1994-01-01

The murine severe combined immunodeficient mutation (scid) is characterized by a lack of both B and T cells, due to a defect in lymphoid variable-(diversity)-joining(V(D)J) rearrangement. Scid cells are highly sensitive to both radiation-induced killing and chromosomal aberrations. Present experiments also demonstrated the high sensitivity of scid cells to killing, because of a deficient repair of double strand breaks(DSB). Scid cells can repair only 60% of radiation-induced DSB for 3 hours, while normal cells repair 85% of the DSB. Significantly reduced Do and n values were obtained from survival curves of scid cells and were similar to ataxia-telangiectasia(AT) cells (a unique human disease conferring whole body radiosensitivity). However, the kinetics of DNA synthesis after irradiation were different between the two cell types. In contrast with the radioresistant DNA synthesis of AT cells, DNA synthesis of scid cells was markedly inhibited after irradiation. The existence of different mutations was also supported by evidence of complementation in somatic cell hybrids between scid cells and AT cells. Using these hybrid cells, fragments of human chromosome 8 were introduced into scid cells HPRT mutant via X-irradiation and somatic cell fusion. The resulting hybrid clones contained human DNA fragment(s) which complemented the hyper-radiosensitivity of the scid cells. Alu-PCR products from these hybrids were used for chromosome painting using the technique of chromosome in situ suppression hybridization, allowing assignment of the human HYRC1 (hyper-radiosensitivity of murine scid mutation, complementing 1) gene, a candidate for a V(D)J recombinant gene, to human chromosome 8q11. (author)