Mutation-Induced Changes in the Protein Environment and Site Energies in the (M)L214G Mutant of the Rhodobacter sphaeroides Bacterial Reaction Center.

PubMed

Jankowiak, Ryszard; Rancova, Olga; Chen, Jinhai; Kell, Adam; Saer, Rafael G; Beatty, J Thomas; Abramavicius, Darius

2016-08-18

This work focuses on the low-temperature (5 K) photochemical (transient) hole-burned (HB) spectra within the P870 absorption band, and their theoretical analysis, for the (M)L214G mutant of the photosynthetic Rhodobacter sphaeroides bacterial reaction center (bRC). To provide insight into system-bath interactions of the bacteriochlorophyll a (BChl a) special pair, i.e., P870, in the mutated bRC, the optical line shape function for the P870 band is calculated numerically. On the basis of the modeling studies, we demonstrate that (M)L214G mutation leads to a heterogeneous population of bRCs with modified (increased) total electron-phonon coupling strength of the special pair BChl a and larger inhomogeneous broadening. Specifically, we show that after mutation in the (M)L214G bRC a large fraction (∼50%) of the bacteriopheophytin (HA) chromophores shifts red and the 800 nm absorption band broadens, while the remaining fraction of HA cofactors retains nearly the same site energy as HA in the wild-type bRC. Modeling using these two subpopulations allowed for fits of the absorption and nonresonant (transient) HB spectra of the mutant bRC in the charge neutral, oxidized, and charge-separated states using the Frenkel exciton Hamiltonian, providing new insight into the mutant's complex electronic structure. Although the average (M)L214G mutant quantum efficiency of P(+)QA(-) state formation seems to be altered in comparison with the wild-type bRC, the average electron transfer time (measured via resonant transient HB spectra within the P870 band) was not affected. Thus, mutation in the vicinity of the electron acceptor (HA) does not tune the charge separation dynamics. Finally, quenching of the (M)L214G mutant excited states by P(+) is addressed by persistent HB spectra burned within the B band in chemically oxidized samples.

Sequencing analysis of mutations induced by N-ethyl-N-nitrosourea at different sampling times in mouse bone marrow.

PubMed

Wang, Jianyong; Chen, Tao

2010-03-01

In our previous study (Wang et al., 2004, Toxicol. Sci. 82: 124-128), we observed that the cII gene mutant frequency (MF) in the bone marrow of Big Blue mice showed significant increase as early as day 1, reached the maximum at day 3 and then decreased to a plateau by day 15 after a single dose of carcinogen N-ethyl-N-nitrosourea (ENU) treatment, which is different from the longer mutation manifestation time and the constancy of MFs after reaching their maximum in some other tissues. To determine the mechanism underlying the quick increase in MF and the peak formation in the mutant manifestation, we examined the mutation frequencies and spectra of the ENU-induced mutants collected from different sampling times in this study. The cII mutants from days 1, 3 and 120 after ENU treatment were randomly selected from different animals. The mutation frequencies were 33, 217, 305 and 144 x 10(-6) for control, days 1, 3, and 120, respectively. The mutation spectra at days 1 and 3 were significantly different from that at day 120. Considering that stem cells are responsible for the ultimate MF plateau (day 120) and transit cells are accountable for the earlier MF induction (days 1 or 3) in mouse bone marrow, we conclude that transit cells are much more sensitive to mutation induction than stem cells in mouse bone marrow, which resulted in the specific mutation manifestation induced by ENU.

Computational DNA hole spectroscopy: A new tool to predict mutation hotspots, critical base pairs, and disease ‘driver’ mutations

PubMed Central

Suárez, Martha Y.; Villagrán; Miller, John H.

2015-01-01

We report on a new technique, computational DNA hole spectroscopy, which creates spectra of electron hole probabilities vs. nucleotide position. A hole is a site of positive charge created when an electron is removed. Peaks in the hole spectrum depict sites where holes tend to localize and potentially trigger a base pair mismatch during replication. Our studies of mitochondrial DNA reveal a correlation between L-strand hole spectrum peaks and spikes in the human mutation spectrum. Importantly, we also find that hole peak positions that do not coincide with large variant frequencies often coincide with disease-implicated mutations and/or (for coding DNA) encoded conserved amino acids. This enables combining hole spectra with variant data to identify critical base pairs and potential disease ‘driver’ mutations. Such integration of DNA hole and variance spectra could ultimately prove invaluable for pinpointing critical regions of the vast non-protein-coding genome. An observed asymmetry in correlations, between the spectrum of human mtDNA variations and the L- and H-strand hole spectra, is attributed to asymmetric DNA replication processes that occur for the leading and lagging strands. PMID:26310834

Computational DNA hole spectroscopy: A new tool to predict mutation hotspots, critical base pairs, and disease 'driver' mutations.

PubMed

Villagrán, Martha Y Suárez; Miller, John H

2015-08-27

We report on a new technique, computational DNA hole spectroscopy, which creates spectra of electron hole probabilities vs. nucleotide position. A hole is a site of positive charge created when an electron is removed. Peaks in the hole spectrum depict sites where holes tend to localize and potentially trigger a base pair mismatch during replication. Our studies of mitochondrial DNA reveal a correlation between L-strand hole spectrum peaks and spikes in the human mutation spectrum. Importantly, we also find that hole peak positions that do not coincide with large variant frequencies often coincide with disease-implicated mutations and/or (for coding DNA) encoded conserved amino acids. This enables combining hole spectra with variant data to identify critical base pairs and potential disease 'driver' mutations. Such integration of DNA hole and variance spectra could ultimately prove invaluable for pinpointing critical regions of the vast non-protein-coding genome. An observed asymmetry in correlations, between the spectrum of human mtDNA variations and the L- and H-strand hole spectra, is attributed to asymmetric DNA replication processes that occur for the leading and lagging strands.

Mutagenesis in human cells with accelerated H and Fe ions

NASA Technical Reports Server (NTRS)

Kronenberg, Amy

1994-01-01

The overall goals of this research were to determine the risks of mutation induction and the spectra of mutations induced by energetic protons and iron ions at two loci in human lymphoid cells. During the three year grant period the research has focused in three major areas: (1) the acquisition of sufficient statistics for human TK6 cell mutation experiments using Fe ions (400 MeV/amu), Fe ions (600 MeV/amu) and protons (250 MeV/amu); (2) collection of thymidine kinase- deficient (tk) mutants or hypoxanthine phosphoribosyltransferase deficient (hprt) mutants induced by either Fe 400 MeV/amu, Fe 600 MeV/amu, or H 250 MeV/amu for subsequent molecular analysis; and (3) molecular characterization of mutants isolated after exposure to Fe ions (600 MeV/amu). As a result of the shutdown of the BEVALAC heavy ion accelerator in December 1992, efforts were rearranged somewhat in time to complete our dose-response studies and to complete mutant collections in particular for the Fe ion beams prior to the shutdown. These goals have been achieved. A major effort was placed on collection, re-screening, and archiving of 3 different series of mutants for the various particle beam exposures: tk-ng mutants, tk-sg mutants, and hprt-deficient mutants. Where possible, groups of mutants were isolated for several particle fluences. Comparative analysis of mutation spectra has occured with characterization of the mutation spectrum for hprt-deficient mutants obtained after exposure of TK6 cells to Fe ions (600 MeV/amu) and a series of spontaneous mutants.

Genome-Wide Biases in the Rate and Molecular Spectrum of Spontaneous Mutations in Vibrio cholerae and Vibrio fischeri.

PubMed

Dillon, Marcus M; Sung, Way; Sebra, Robert; Lynch, Michael; Cooper, Vaughn S

2017-01-01

The vast diversity in nucleotide composition and architecture among bacterial genomes may be partly explained by inherent biases in the rates and spectra of spontaneous mutations. Bacterial genomes with multiple chromosomes are relatively unusual but some are relevant to human health, none more so than the causative agent of cholera, Vibrio cholerae Here, we present the genome-wide mutation spectra in wild-type and mismatch repair (MMR) defective backgrounds of two Vibrio species, the low-%GC squid symbiont V. fischeri and the pathogen V. cholerae, collected under conditions that greatly minimize the efficiency of natural selection. In apparent contrast to their high diversity in nature, both wild-type V. fischeri and V. cholerae have among the lowest rates for base-substitution mutations (bpsms) and insertion-deletion mutations (indels) that have been measured, below 10 - 3 /genome/generation. Vibrio fischeri and V. cholerae have distinct mutation spectra, but both are AT-biased and produce a surprising number of multi-nucleotide indels. Furthermore, the loss of a functional MMR system caused the mutation spectra of these species to converge, implying that the MMR system itself contributes to species-specific mutation patterns. Bpsm and indel rates varied among genome regions, but do not explain the more rapid evolutionary rates of genes on chromosome 2, which likely result from weaker purifying selection. More generally, the very low mutation rates of Vibrio species correlate inversely with their immense population sizes and suggest that selection may not only have maximized replication fidelity but also optimized other polygenic traits relative to the constraints of genetic drift. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Mutational spectra of the lacI transgene isolated from Big Blue{reg_sign} mice exposed to three carcinogenic aromatic amines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Staedtler, F.; Locher, F.; Sreenan, G.

1997-10-01

In order to evaluate the in vivo genotoxic potential of three putative genotoxic mouse liver carcinogens, high doses of 4-chloro-o-phenylenediamine, 2-nitro-p-phenylenediamine and 2, 4-diaminotoluene were tested short term in the Big Blue{reg_sign} transgenic mouse mutation assay. Small statistically significant increases in the lacI mutant frequencies in the liver by factors 1.7 to 2.0 were found. A representative number of 347 lacI mutants isolated from liver tissue of male and female animals were analyses by DNA sequencing. The mutational spectra were examined with the Adams-Skopek algorithm. The spontaneous mutational spectra from untreated male and female animals were similar and consistent withmore » spectral Big Blue{reg_sign} control data stored in the lacI database. Most of the background mutations were located in the 5{prime} portion of the coding region of the lacI gene. Single base substitutions were most prominent. G:C to A:T transitions and G:C to T:A transversions occurred predominatly and were preferentially located at CpG sites. Despite the increases observed in the mutant frequencies of the treated animals, the corresponding mutational spectra did not differ from the controls. However, it is possible that certain classes of point mutations were substantially increased but not detected due to the limited number of sequenced mutants. In two animals treated with 2, 4- diaminotoluene unusually high mutant frequencies and the multiple occurrence of certain mutations in the liver was observed. From one of these animals six lacI mutants isolated from colon tissue were all different. Since 2, 4-diaminotoluene was shown to induce liver cell proliferation these results may reflect clonal expansion of single mutated liver cells.« less

EFFECT OF THE ANTIMUTAGENS VANILLIN AND CINNAMALDEHYDE ON THE SPONTANEOUS MUTATION SPECTRA OF SALMONELLA TA104

EPA Science Inventory

Effect of the Antimutagens Vanillin and Cinnamaldehyde on the / Spontaneous Mutation Spectra of Salmonella TAlO4

Vanillin (VAN) and cinnamaldehyde (CIN) are dietary antimutagens that, when added to assay plates, reduced the spontaneous mutant frequency in Salmonella typhi...

Development of potent in vivo mutagenesis plasmids with broad mutational spectra

PubMed Central

Badran, Ahmed H.; Liu, David R.

2015-01-01

Methods to enhance random mutagenesis in cells offer advantages over in vitro mutagenesis, but current in vivo methods suffer from a lack of control, genomic instability, low efficiency and narrow mutational spectra. Using a mechanism-driven approach, we created a potent, inducible, broad-spectrum and vector-based mutagenesis system in E. coli that enhances mutation 322,000-fold over basal levels, surpassing the mutational efficiency and spectra of widely used in vivo and in vitro methods. We demonstrate that this system can be used to evolve antibiotic resistance in wild-type E. coli in <24 h, outperforming chemical mutagens, ultraviolet light and the mutator strain XL1-Red under similar conditions. This system also enables the continuous evolution of T7 RNA polymerase variants capable of initiating transcription using the T3 promoter in <10 h. Our findings enable broad-spectrum mutagenesis of chromosomes, episomes and viruses in vivo, and are applicable to both bacterial and bacteriophage-mediated laboratory evolution platforms. PMID:26443021

Development of potent in vivo mutagenesis plasmids with broad mutational spectra.

PubMed

Badran, Ahmed H; Liu, David R

2015-10-07

Methods to enhance random mutagenesis in cells offer advantages over in vitro mutagenesis, but current in vivo methods suffer from a lack of control, genomic instability, low efficiency and narrow mutational spectra. Using a mechanism-driven approach, we created a potent, inducible, broad-spectrum and vector-based mutagenesis system in E. coli that enhances mutation 322,000-fold over basal levels, surpassing the mutational efficiency and spectra of widely used in vivo and in vitro methods. We demonstrate that this system can be used to evolve antibiotic resistance in wild-type E. coli in <24 h, outperforming chemical mutagens, ultraviolet light and the mutator strain XL1-Red under similar conditions. This system also enables the continuous evolution of T7 RNA polymerase variants capable of initiating transcription using the T3 promoter in <10 h. Our findings enable broad-spectrum mutagenesis of chromosomes, episomes and viruses in vivo, and are applicable to both bacterial and bacteriophage-mediated laboratory evolution platforms.

A missense mutation in the arginine-vasopressin neurophysin-II gene causes autosomal dominant neurohypophyseal diabetes insipidus in a Chinese family.

PubMed

Ye, Dan; Dong, FengQin; Lu, WeiQin; Zhang, Zhe; Lu, XunLiang; Li, ChengJiang; Liu, YanNing

2013-06-01

Familial neurohypophyseal diabetes insipidus, an autosomal dominant disorder, is mostly caused by mutations in the genes that encode AVP or its intracellular binding protein, neurophysin-II. The mutations lead to aberrant preprohormone processing and progressive destruction of AVP-secreting cells, which gradually manifests a progressive polyuria and polydipsia during early childhood, and a disorder of water homeostasis. We characterized the clinical and biochemical features, and sequenced the AVP neurophysin-II(AVP-NPII) gene of the affected individuals with autosomal dominant neurohypophyseal diabetes insipidus(ADNDI)to determine whether this disease was genetically determined. We obtained the histories of eight affected and four unaffected family individuals. The diagnosis of ADNDI was established using a water deprivation test and exogenous AVP administration. For molecular analysis, genomic DNA was extracted and the AVP-NPII gene was amplified using polymerase chain reaction and sequenced. The eight affected individuals showed different spectra of age of onsets (7-15 years) and urine volumes (132-253 ml/kg/24 h). All affected individuals responded to vasopressin administration, with a resolution of symptoms and an increase in urine osmolality by more than 50%. The characteristic hyperintense signal in the posterior pituitary on T1-weighted magnetic resonance imaging was absent in six family members and present in one. Sequencing analysis revealed a missense heterozygous mutation 1516G > T (Gly17Val) in exon 2 of the AVP-NPII gene among the ADNDI individuals. We identified a missense mutation in the AVP-NPII gene and the same mutation showed different spectra of age of onsets and urine volumes in a new Chinese family with ADNDI. The mutation may provide a molecular basis for understanding the characteristics of NPII and add to our knowledge of the pathogenesis of ADNDI, which would allow the presymptomatic diagnosis of asymptomatic subjects. © 2012 John Wiley & Sons Ltd.

General and inducible hypermutation facilitate parallel adaptation in Pseudomonas aeruginosa despite divergent mutation spectra.

PubMed

Weigand, Michael R; Sundin, George W

2012-08-21

The successful growth of hypermutator strains of bacteria contradicts a clear preference for lower mutation rates observed in the microbial world. Whether by general DNA repair deficiency or the inducible action of low-fidelity DNA polymerases, the evolutionary strategies of bacteria include methods of hypermutation. Although both raise mutation rate, general and inducible hypermutation operate through distinct molecular mechanisms and therefore likely impart unique adaptive consequences. Here we compare the influence of general and inducible hypermutation on adaptation in the model organism Pseudomonas aeruginosa PAO1 through experimental evolution. We observed divergent spectra of single base substitutions derived from general and inducible hypermutation by sequencing rpoB in spontaneous rifampicin-resistant (Rif(R)) mutants. Likewise, the pattern of mutation in a draft genome sequence of a derived inducible hypermutator isolate differed from those of general hypermutators reported in the literature. However, following experimental evolution, populations of both mutator types exhibited comparable improvements in fitness across varied conditions that differed from the highly specific adaptation of nonmutators. Our results suggest that despite their unique mutation spectra, general and inducible hypermutation can analogously influence the ecology and adaptation of bacteria, significantly shaping pathogenic populations where hypermutation has been most widely observed.

Electron holes appear to trigger cancer-implicated mutations

NASA Astrophysics Data System (ADS)

Miller, John; Villagran, Martha

Malignant tumors are caused by mutations, which also affect their subsequent growth and evolution. We use a novel approach, computational DNA hole spectroscopy [M.Y. Suarez-Villagran & J.H. Miller, Sci. Rep. 5, 13571 (2015)], to compute spectra of enhanced hole probability based on actual sequence data. A hole is a mobile site of positive charge created when an electron is removed, for example by radiation or contact with a mutagenic agent. Peaks in the hole spectrum depict sites where holes tend to localize and potentially trigger a base pair mismatch during replication. Our studies of reveal a correlation between hole spectrum peaks and spikes in human mutation frequencies. Importantly, we also find that hole peak positions that do not coincide with large variant frequencies often coincide with cancer-implicated mutations and/or (for coding DNA) encoded conserved amino acids. This enables combining hole spectra with variant data to identify critical base pairs and potential cancer `driver' mutations. Such integration of DNA hole and variance spectra could also prove invaluable for pinpointing critical regions, and sites of driver mutations, in the vast non-protein-coding genome. Supported by the State of Texas through the Texas Ctr. for Superconductivity.

COMPARATIVE MUTAGENICITY OF HALOMETHANES AND HALONITROMETHANES IN SALMONELLA TA100: STRUCTURE-ACTIVITY ANALYSIS AND MUTATION SPECTRA. (ERD)

EPA Science Inventory

Halonitromethanes (HNMs) are a recently identified class of disinfection by-products (DPBs) in drinking water that are mutagenic in Salmonella and potent inducers of DNA strand breaks in mammalian cells. Here we compared the mutagenic potencies of the HNMs to those of their halo...

COMPARATIVE MUTAGENICITY OF HALOMETHANES AND HALONITROMETHANES IN SALMONELLA TA100: STRUCTURE-ACTIVITY ANALYSIS AND MUTATION SPECTRA

EPA Science Inventory

Halonitromethanes (HNMs) are a recently identified class of disinfection by-products (DPBs) in drinking water that are mutagenic in Salmonella and potent inducers of DNA strand breaks in mammalian cells. Here we compared the mutagenic potencies of the HNMs to those of their halom...

Spectrum of rhodopsin mutations in Korean patients with retinitis pigmentosa

PubMed Central

Kim, Kwang Joong; Kim, Cinoo; Bok, Jeong; Kim, Kyung-Seon; Lee, Eun-Ju; Park, Sung Pyo; Chung, Hum; Han, Bok-Ghee; Kim, Hyung-Lae; Kimm, Kuchan; Yu, Hyeong Gon

2011-01-01

Purpose To determine the spectrum and frequency of rhodopsin gene (RHO) mutations in Korean patients with retinitis pigmentosa (RP) and to characterize genotype–phenotype correlations in patients with mutations. Methods The RHO mutations were screened by direct sequencing, and mutation prevalence was measured in patients and controls. The impact of missense mutations to RP was predicted by segregation analysis, peptide sequence alignment, and in silico analysis. The severity of disease in patients with the missense mutations was compared by visual acuity, electroretinography, optical coherence tomography, and kinetic visual field testing. Results Five heterozygous mutations were identified in six of 302 probands with RP, including a novel mutation (c.893C>A, p.A298D) and four known mutations (c.50C>T, p.T17M; c.533A>G, p.Y178C; c.888G>T, p.K296N; and c.1040C>T, p.P347L). The allele frequency of missense mutations was measured in 114 ethnically matched controls. p.A298D, newly identified in a sporadic patient, had never been found in controls and was predicted to be pathogenic. Among the patients with the missense mutations, we observed the most severe phenotype in patients with p.P347L, less severe phenotypes in patients with p.Y178C or p.A298D, and a relatively moderate phenotype in a patient with p.T17M. Conclusions The results reveal the spectrum of RHO mutations in Korean RP patients and clinical features that vary according to mutations. Our findings will be useful for understanding these genetic spectra and the genotype–phenotype correlations and will therefore help with predicting disease prognosis and facilitating the development of gene therapy. PMID:21677794

Mutagenesis: Interactions with a parallel universe.

PubMed

Miller, Jeffrey H

Unexpected observations in mutagenesis research have led to a new perspective in this personal reflection based on years of studying mutagenesis. Many mutagens have been thought to operate via a single principal mechanism, with secondary effects usually resulting in only minor changes in the observed mutation frequencies and spectra. For example, we conceive of base analogs as resulting in direct mispairing as their main mechanism of mutagenesis. Recent studies now show that in fact even these simple mutagens can cause very large and unanticipated effects both in mutation frequencies and in the mutational spectra when used in certain pair-wise combinations. Here we characterize this leap in mutation frequencies as a transport to an alternate universe of mutagenesis. Copyright © 2018 Elsevier B.V. All rights reserved.

Impacts of the Callipyge Mutation on Ovine Plasma Metabolites and Muscle Fibre Type

PubMed Central

Li, Juan; Greenwood, Paul L.; Cockett, Noelle E.; Hadfield, Tracy S.; Vuocolo, Tony; Byrne, Keren; White, Jason D.; Tellam, Ross L.; Schirra, Horst Joachim

2014-01-01

The ovine Callipyge mutation causes postnatal muscle hypertrophy localized to the pelvic limbs and torso, as well as body leanness. The mechanism underpinning enhanced muscle mass is unclear, as is the systemic impact of the mutation. Using muscle fibre typing immunohistochemistry, we confirmed muscle specific effects and demonstrated that affected muscles had greater prevalence and hypertrophy of type 2X fast twitch glycolytic fibres and decreased representation of types 1, 2C, 2A and/or 2AX fibres. To investigate potential systemic effects of the mutation, proton NMR spectra of plasma taken from lambs at 8 and 12 weeks of age were measured. Multivariate statistical analysis of plasma metabolite profiles demonstrated effects of development and genotype but not gender. Plasma from Callipyge lambs at 12 weeks of age, but not 8 weeks, was characterized by a metabolic profile consistent with contributions from the affected hypertrophic fast twitch glycolytic muscle fibres. Microarray analysis of the perirenal adipose tissue depot did not reveal a transcriptional effect of the mutation in this tissue. We conclude that there is an indirect systemic effect of the Callipyge mutation in skeletal muscle in the form of changes of blood metabolites, which may contribute to secondary phenotypes such as body leanness. PMID:24937646

Impacts of the Callipyge mutation on ovine plasma metabolites and muscle fibre type.

PubMed

Li, Juan; Greenwood, Paul L; Cockett, Noelle E; Hadfield, Tracy S; Vuocolo, Tony; Byrne, Keren; White, Jason D; Tellam, Ross L; Schirra, Horst Joachim

2014-01-01

The ovine Callipyge mutation causes postnatal muscle hypertrophy localized to the pelvic limbs and torso, as well as body leanness. The mechanism underpinning enhanced muscle mass is unclear, as is the systemic impact of the mutation. Using muscle fibre typing immunohistochemistry, we confirmed muscle specific effects and demonstrated that affected muscles had greater prevalence and hypertrophy of type 2X fast twitch glycolytic fibres and decreased representation of types 1, 2C, 2A and/or 2AX fibres. To investigate potential systemic effects of the mutation, proton NMR spectra of plasma taken from lambs at 8 and 12 weeks of age were measured. Multivariate statistical analysis of plasma metabolite profiles demonstrated effects of development and genotype but not gender. Plasma from Callipyge lambs at 12 weeks of age, but not 8 weeks, was characterized by a metabolic profile consistent with contributions from the affected hypertrophic fast twitch glycolytic muscle fibres. Microarray analysis of the perirenal adipose tissue depot did not reveal a transcriptional effect of the mutation in this tissue. We conclude that there is an indirect systemic effect of the Callipyge mutation in skeletal muscle in the form of changes of blood metabolites, which may contribute to secondary phenotypes such as body leanness.

Induction of mutations by bismuth-212 alpha particles at two genetic loci in human B-lymphoblasts.

PubMed

Metting, N F; Palayoor, S T; Macklis, R M; Atcher, R W; Liber, H L; Little, J B

1992-12-01

The human lymphoblast cell line TK6 was exposed to the alpha-particle-emitting radon daughter 212Bi by adding DTPA-chelated 212Bi directly to the cell suspension. Cytotoxicity and mutagenicity at two genetic loci were measured, and the molecular nature of mutant clones was studied by Southern blot analysis. Induced mutant fractions were 2.5 x 10(-5)/Gy at the hprt locus and 3.75 x 10(-5)/Gy at the tk locus. Molecular analysis of HPRT- mutant DNAs showed a high frequency (69%) of clones with partial or full deletions of the hprt gene among radiation-induced mutants compared with spontaneous mutants (31%). Chi-squared analyses of mutational spectra show a significant difference (P < or = 0.005) between spontaneous mutants and alpha-particle-induced mutants. Comparison with published studies of accelerator-produced heavy-ion exposures of TK6 cells indicates that the induction of mutations at the hprt locus, and perhaps a subset of mutations at the tk locus, is a simple linear function of particle fluence regardless of the ion species or its LET.

Statistical Methods for Identifying Sequence Motifs Affecting Point Mutations

PubMed Central

Zhu, Yicheng; Neeman, Teresa; Yap, Von Bing; Huttley, Gavin A.

2017-01-01

Mutation processes differ between types of point mutation, genomic locations, cells, and biological species. For some point mutations, specific neighboring bases are known to be mechanistically influential. Beyond these cases, numerous questions remain unresolved, including: what are the sequence motifs that affect point mutations? How large are the motifs? Are they strand symmetric? And, do they vary between samples? We present new log-linear models that allow explicit examination of these questions, along with sequence logo style visualization to enable identifying specific motifs. We demonstrate the performance of these methods by analyzing mutation processes in human germline and malignant melanoma. We recapitulate the known CpG effect, and identify novel motifs, including a highly significant motif associated with A→G mutations. We show that major effects of neighbors on germline mutation lie within ±2 of the mutating base. Models are also presented for contrasting the entire mutation spectra (the distribution of the different point mutations). We show the spectra vary significantly between autosomes and X-chromosome, with a difference in T→C transition dominating. Analyses of malignant melanoma confirmed reported characteristic features of this cancer, including statistically significant strand asymmetry, and markedly different neighboring influences. The methods we present are made freely available as a Python library https://bitbucket.org/pycogent3/mutationmotif. PMID:27974498

Mutagenicity of furan in female Big Blue B6C3F1 mice

PubMed Central

Terrell, Ashley N.; Huynh, Mailee; Grill, Alex E.; Kovi, Ramesh C.; O’Sullivan, M. Gerard; Guttenplan, Joseph B.; Ho, Yen-Yi; Peterson, Lisa A.

2014-01-01

Furan is an abundant food and environmental contaminant that is a potent liver carcinogen in rodent models. To determine if furan is genotoxic in vivo, female B6C3F1 Big Blue transgenic mice were treated with 15 mg/kg bw furan by gavage 5 days a week for 6 weeks, or once weekly for 3 weeks. Liver cII trans-gene mutation-frequency and mutation spectra were determined. Furan did not increase the mutation frequency under either treatment condition. In the 6-week treatment regimen, there was a change in the cII transgene mutation-spectrum, with the fraction of GC to AT transitions significantly reduced. The only other significant change was an increase in GC to CG transversions; these represented a minor contribution to the overall mutation spectrum. A much larger furan-dependent shift was observed in the 3-week study. There was a significant increase in transversion mutations, predominantly GC to TA transversions as well as smaller non-significant changes in GC to CG and AT to TA transversions. To determine if these mutations were caused by cis-2-butene-1,4-dial (BDA), a reactive metabolite of furan, the mutagenic activity and the mutation spectrum of BDA was determined in vitro, in Big Blue mouse embryonic fibroblasts. This compound did not increase the cII gene mutation-frequency but caused a substantial increase in AT to CG transversions. This increase, however, lost statistical significance when adjusted for multiple comparisons. Together, these findings suggest that BDA may not be directly responsible for the in-vivo effects of furan on mutational spectra. Histopathological analysis of livers from furan-treated mice revealed that furan induced multifocal, hepatocellular necrosis admixed with reactive leukocytes and pigment-laden Kupffer cells, enhanced oval-cell hyperplasia, and increased hepatocyte mitoses, some of which were atypical. An indirect mechanism of genotoxicity is proposed in which chronic toxicity followed by inflammation and secondary cell proliferation triggers cancer development in furan-exposed rodents. PMID:25344163

Characterization of the mutant spectra of a fish RNA virus within individual hosts during natural infections

USGS Publications Warehouse

Emmenegger, Eveline J.; Troyer, Ryan M.; Kurath, Gael

2003-01-01

Infectious hematopoietic necrosis virus (IHNV) is an RNA virus that causes significant mortalities of salmonids in the Pacific Northwest of North America. RNA virus populations typically contain genetic variants that form a heterogeneous virus pool, referred to as a quasispecies or mutant spectrum. This study characterized the mutant spectra of IHNV populations within individual fish reared in different environmental settings by RT–PCR of genomic viral RNA and determination of partial glycoprotein gene sequences of molecular clones. The diversity of the mutant spectra from ten in vivo populations was low and the average mutation frequencies of duplicate populations did not significantly exceed the background mutation level expected from the methodology. In contrast, two in vitro populations contained variants with an identical mutational hot spot. These results indicated that the mutant spectra of natural IHNV populations is very homogeneous, and does not explain the different magnitudes of genetic diversity observed between the different IHNV genogroups. Overall the mutant frequency of IHNV within its host is one of the lowest reported for RNA viruses.

Similar Mutation Rates but Highly Diverse Mutation Spectra in Ascomycete and Basidiomycete Yeasts.

PubMed

Long, Hongan; Behringer, Megan G; Williams, Emily; Te, Ronald; Lynch, Michael

2016-12-01

Yeast species are extremely diverse and not monophyletic. Because the majority of yeast research focuses on ascomycetes, the mutational determinants of genetic diversity across yeast species are not well understood. By combining mutation-accumulation techniques with whole-genome sequencing, we resolved the genomic mutation rate and spectrum of the oleaginous (oil-producing) ‘red yeast’ Rhodotorula toruloides, the first such study in the fungal phylum Basidiomycota. We find that the mutation spectrum is quite different from what has been observed in all other studied unicellular eukaryotes, but similar to that in most bacteria—a predominance of transitions relative to transversions. Rhodotorula toruloides has a significantly higher A:T→G:C transition rate—possibly elevated by the abundant flanking G/C nucleotides in the GC-rich genome, as well as a much lower G:C→T:A transversion rate. In spite of these striking differences, there are substantial consistencies between R. toruloides and the ascomycete model yeasts: a spontaneous base-substitution mutation rate of 1.90 × 10 −10 per site per cell division as well as an elevated mutation rate at non-methylated 5'CpG3' sites. These results imply the evolution of variable mutation spectra in the face of similar mutation rates in yeasts.

Variation of p53 mutational spectra between carcinoma of the upper and lower respiratory tract.

PubMed

Law, J C; Whiteside, T L; Gollin, S M; Weissfeld, J; El-Ashmawy, L; Srivastava, S; Landreneau, R J; Johnson, J T; Ferrell, R E

1995-07-01

Mutations of the p53 tumor suppressor gene are the most common genetic alterations associated with human cancer. Tumor-associated p53 mutations often show characteristic tissue-specific profiles which may infer environmentally induced mutational mechanisms. The p53 mutational frequency and spectrum were determined for 95 carcinomas of the upper and lower respiratory tract (32 lung and 63 upper respiratory tract). Mutations were identified at a frequency of 30% in upper respiratory tract (URT) tumors and 31% in lung tumors. All 29 identified mutations were single-base substitutions. Comparison of the frequency of specific base substitutions between lung and URT showed a striking difference. Transitions occurred at a frequency of 68% in URT, but only 30% in lung. Mutations involving G:C-->A:T transitions, which are commonly reported in gastric and esophageal tumors, were the most frequently identified alteration in URT (11/19). Mutations involving G:C-->T:A transversions, which were relatively common in lung tumors (3/10) and are representative of tobacco smoke-induced mutations were rare in URT tumors (1/19). Interestingly, G:C-->A:T mutations at CpG sites, which are characteristic of endogenous processes, were observed frequently in URT tumors (9/19) but only rarely in lung tumors (1/10), suggesting that both endogenous and exogenous factors are responsible for the observed differences in mutational spectra between the upper and lower respiratory systems.

The distinct spectra of tumor-associated Apc mutations in mismatch repair-deficient Apc1638N mice define the roles of MSH3 and MSH6 in DNA repair and intestinal tumorigenesis.

PubMed

Kuraguchi, M; Yang, K; Wong, E; Avdievich, E; Fan, K; Kolodner, R D; Lipkin, M; Brown, A M; Kucherlapati, R; Edelmann, W

2001-11-01

In mammalian cells, mismatch recognition has been attributed to two partially redundant heterodimeric protein complexes of MutS homologues, MSH2-MSH3 and MSH2-MSH6. We have conducted a comparative analysis of Msh3 and Msh6 deficiency in mouse intestinal tumorigenesis by generating Apc1638N mice deficient in Msh3, Msh6 or both. We have found that Apc1638N mice defective in Msh6 show reduced survival and a 6-7-fold increase in intestinal tumor multiplicity. In contrast, Msh3-deficient Apc1638N mice showed no difference in survival and intestinal tumor multiplicity as compared with Apc1638N mice. However, when Msh3 deficiency is combined with Msh6 deficiency (Msh3(-/-)Msh6(-/-)Apc1638N), the survival rate of the mice was further reduced compared to Msh6(-/-)Apc(1638N) mice because of a high multiplicity of intestinal tumors at a younger age. Almost 90% of the intestinal tumors from both Msh6(-/-)Apc1638N and Msh3(-/-)Msh6(-/-)Apc1638N mice contained truncation mutations in the wild-type Apc allele. Apc mutations in Msh6(-/-)Apc1638N mice consisted predominantly of base substitutions (93%) creating stop codons, consistent with a major role for Msh6 in the repair of base-base mismatches. However, in Msh3(-/-)Msh6(-/-)Apc1638N tumors, we observed a mixture of base substitutions (46%) and frameshifts (54%), indicating that in Msh6(-/-)Apc1638N mice frameshift mutations in the Apc gene were suppressed by Msh3. Interestingly, all except one of the Apc mutations detected in mismatch repair-deficient intestinal tumors were located upstream of the third 20-amino acid beta-catenin binding repeat and before all of the Ser-Ala-Met-Pro repeats, suggesting that there is selection for loss of multiple domains involved in beta-catenin regulation. Our analysis therefore has revealed distinct mutational spectra and clarified the roles of Msh3 and Msh6 in DNA repair and intestinal tumorigenesis.

Holes influence the mutation spectrum of human mitochondrial DNA

NASA Astrophysics Data System (ADS)

Villagran, Martha; Miller, John

Mutations drive evolution and disease, showing highly non-random patterns of variant frequency vs. nucleotide position. We use computational DNA hole spectroscopy [M.Y. Suarez-Villagran & J.H. Miller, Sci. Rep. 5, 13571 (2015)] to reveal sites of enhanced hole probability in selected regions of human mitochondrial DNA. A hole is a mobile site of positive charge created when an electron is removed, for example by radiation or contact with a mutagenic agent. The hole spectra are quantum mechanically computed using a two-stranded tight binding model of DNA. We observe significant correlation between spectra of hole probabilities and of genetic variation frequencies from the MITOMAP database. These results suggest that hole-enhanced mutation mechanisms exert a substantial, perhaps dominant, influence on mutation patterns in DNA. One example is where a trapped hole induces a hydrogen bond shift, known as tautomerization, which then triggers a base-pair mismatch during replication. Our results deepen overall understanding of sequence specific mutation rates, encompassing both hotspots and cold spots, which drive molecular evolution.

MutSpec: a Galaxy toolbox for streamlined analyses of somatic mutation spectra in human and mouse cancer genomes.

PubMed

Ardin, Maude; Cahais, Vincent; Castells, Xavier; Bouaoun, Liacine; Byrnes, Graham; Herceg, Zdenko; Zavadil, Jiri; Olivier, Magali

2016-04-18

The nature of somatic mutations observed in human tumors at single gene or genome-wide levels can reveal information on past carcinogenic exposures and mutational processes contributing to tumor development. While large amounts of sequencing data are being generated, the associated analysis and interpretation of mutation patterns that may reveal clues about the natural history of cancer present complex and challenging tasks that require advanced bioinformatics skills. To make such analyses accessible to a wider community of researchers with no programming expertise, we have developed within the web-based user-friendly platform Galaxy a first-of-its-kind package called MutSpec. MutSpec includes a set of tools that perform variant annotation and use advanced statistics for the identification of mutation signatures present in cancer genomes and for comparing the obtained signatures with those published in the COSMIC database and other sources. MutSpec offers an accessible framework for building reproducible analysis pipelines, integrating existing methods and scripts developed in-house with publicly available R packages. MutSpec may be used to analyse data from whole-exome, whole-genome or targeted sequencing experiments performed on human or mouse genomes. Results are provided in various formats including rich graphical outputs. An example is presented to illustrate the package functionalities, the straightforward workflow analysis and the richness of the statistics and publication-grade graphics produced by the tool. MutSpec offers an easy-to-use graphical interface embedded in the popular Galaxy platform that can be used by researchers with limited programming or bioinformatics expertise to analyse mutation signatures present in cancer genomes. MutSpec can thus effectively assist in the discovery of complex mutational processes resulting from exogenous and endogenous carcinogenic insults.

Insertion of an SVA element, a nonautonomous retrotransposon, in PMS2 intron 7 as a novel cause of Lynch syndrome.

PubMed

van der Klift, Heleen M; Tops, Carli M; Hes, Frederik J; Devilee, Peter; Wijnen, Juul T

2012-07-01

Heterozygous germline mutations in the mismatch repair gene PMS2 predispose carriers for Lynch syndrome, an autosomal dominant predisposition to cancer. Here, we present a LINE-1-mediated retrotranspositional insertion in PMS2 as a novel mutation type for Lynch syndrome. This insertion, detected with Southern blot analysis in the genomic DNA of the patient, is characterized as a 2.2 kb long 5' truncated SVA_F element. The insertion is not detectable by current diagnostic testing limited to MLPA and direct Sanger sequencing on genomic DNA. The molecular nature of this insertion could only be resolved in RNA from cultured lymphocytes in which nonsense-mediated RNA decay was inhibited. Our report illustrates the technical problems encountered in the detection of this mutation type. Especially large heterozygous insertions will remain unnoticed because of preferential amplification of the smaller wild-type allele in genomic DNA, and are probably underreported in the mutation spectra of autosomal dominant disorders. © 2012 Wiley Periodicals, Inc.

MUTANT FREQUENCY AND MUTATIONAL SPECTRA IN THETK AND HPRT GENES OF N-ETHYL-N-NITROSOUREA TREATED MOUSE LYMPHOMA CELLS

EPA Science Inventory

Abstract

The mouse lymphoma assay (MLA) utilizing the Tk locus is widely used to identify chemical mutagens. The autosomal location of the Tk locus allows for the detection of a wide range of mutational events, from point mutations to chromosome alterations. However, the ...

A novel F11 mutation in a Korean pediatric patient with recurrent epistaxis.

PubMed

Kim, Juwon; Kim, Yoonjung; Shin, Seam; Lyu, Chuhl Joo; Choi, Jong Rak; Lee, Kyung-A

2013-06-01

Congenital FXI deficiency (hemophilia C) is a rare bleeding disorder that has been documented mostly in Ashkenazi Jews. Unlike other hemophilias, bleeding tendency varies considerably among individuals, and FXI deficiency rarely manifests as spontaneous bleeding. FXI deficiency is caused primarily by mutations in the F11 gene. Herein, we report a case of a 10-year-old boy with recurrent nose bleeding due to FXI deficiency who was confirmed to have a novel mutation in F11. A molecular analysis of DNA extracted from peripheral blood collected from the patient [FXI clotting activity (FXI:C): 11%] revealed compound heterozygous mutations, Q226X and L424F, in F11, consistent with the severe disease phenotype of the patient. Pedigree analysis showed that the patient received L424F from his father (FXI:C = 49%) and Q226X from the mother (FXI:C = 48%). The sister (FXI:C = 47%) of the patient only had L424F, presumably inherited from her father. Multiple sequence alignment demonstrated that L424 is highly conserved across mammals, indicating that it is important for the function of FXI. In-silico analysis indicated that replacement of L424 by phenylalanine had a detrimental influence on FXI, consistent with the severe phenotype of the patient. Compilation of FXI deficiency cases in east Asian populations would be of great value because different populations appear to have different F11 mutation spectra.

Determinants of spontaneous mutation in the bacterium Escherichia coli as revealed by whole-genome sequencing

PubMed Central

Foster, Patricia L.; Lee, Heewook; Popodi, Ellen; Townes, Jesse P.; Tang, Haixu

2015-01-01

A complete understanding of evolutionary processes requires that factors determining spontaneous mutation rates and spectra be identified and characterized. Using mutation accumulation followed by whole-genome sequencing, we found that the mutation rates of three widely diverged commensal Escherichia coli strains differ only by about 50%, suggesting that a rate of 1–2 × 10−3 mutations per generation per genome is common for this bacterium. Four major forces are postulated to contribute to spontaneous mutations: intrinsic DNA polymerase errors, endogenously induced DNA damage, DNA damage caused by exogenous agents, and the activities of error-prone polymerases. To determine the relative importance of these factors, we studied 11 strains, each defective for a major DNA repair pathway. The striking result was that only loss of the ability to prevent or repair oxidative DNA damage significantly impacted mutation rates or spectra. These results suggest that, with the exception of oxidative damage, endogenously induced DNA damage does not perturb the overall accuracy of DNA replication in normally growing cells and that repair pathways may exist primarily to defend against exogenously induced DNA damage. The thousands of mutations caused by oxidative damage recovered across the entire genome revealed strong local-sequence biases of these mutations. Specifically, we found that the identity of the 3′ base can affect the mutability of a purine by oxidative damage by as much as eightfold. PMID:26460006

In Vivo Rat T-Lymphocyte Pig-a Assay: Detection and Expansion of Cells Deficient in the GPI-Anchored CD48 Surface Marker for Analysis of Mutation in the Endogenous Pig-a Gene.

PubMed

Dobrovolsky, Vasily N; Revollo, Javier; Petibone, Dayton M; Heflich, Robert H

2017-01-01

The Pig-a assay is being developed as an in vivo gene mutation assay for regulatory safety assessments. The assay is based on detecting mutation in the endogenous Pig-a gene of treated rats by using flow cytometry to measure changes in cell surface markers of peripheral blood cells. Here we present a methodology for demonstrating that phenotypically mutant rat T-cells identified by flow cytometry contain mutations in the Pig-a gene, an important step for validating the assay. In our approach, the mutant phenotype T-cells are sorted into individual wells of 96-well plates and expanded into clones. Subsequent sequencing of genomic DNA from the expanded clones confirms that the Pig-a assay detects exactly what it claims to detect-cells with mutations in the endogenous Pig-a gene. In addition, determining the spectra of Pig-a mutations provides information for better understanding the mutational mechanism of compounds of interest. Our methodology of combining phenotypic antibody labeling, magnetic enrichment, sorting, and single-cell clonal expansion can be used in genotoxicity/mutagenicity studies and in other general immunotoxicology research requiring identification, isolation, and expansion of extremely rare subpopulations of T-cells.

Nondestructive analysis of senescence in mesophyll cells by spectral resolution of protein synthesis-dependent pigment metabolism.

PubMed

Gay, Alan; Thomas, Howard; Roca, María; James, Caron; Taylor, Janet; Rowland, Jem; Ougham, Helen

2008-01-01

* Over 6 d of dark-induced senescence, leaf segments of wild-type Lolium temulentum lost > 96% chlorophyll a + b; leaves from plants containing a staygreen mutation introgressed from Festuca pratensis, which has a lesion in the senescence-associated fragmentation of pigment-proteolipid complexes, retained over 43% of total chlorophyll over the same period. * Mutant segments preferentially retained thylakoid membrane proteins (exemplified by LHCP II) but lost other cellular proteins at the same rate as wild-type tissue. The protein synthesis inhibitor D-MDMP inhibited chlorophyll degradation and partially prevented protein loss in both genotypes, but tissues treated with the ineffective L-stereoisomer were indistinguishable from water controls. * Principal-components analysis of leaf reflectance spectra distinguished between genotypes, time points and D-MDMP treatments, showing the disruption of pigment metabolism during senescence brought about by the staygreen mutation, by inhibition of protein synthesis and by combinations of the two factors. * The build-up of oxidized, dephytylated and phaeo-derivatives of chl a during senescence of staygreen tissue was prevented by D-MDMP and associated with characteristic difference spectra when senescent mutant tissue was compared with wild-type or inhibitor-treated samples. The suitability of senescence as a subject for systems biology approaches is discussed.

Discovery of Genomic Breakpoints Affecting Breast Cancer Progression and Prognosis

DTIC Science & Technology

2010-10-01

mutations compared to those detected by the 5Kbp method alone. Fosmid diTag method also reveals much higher proportion of gene fusions and truncations...observed highly similar structural mutational spectra affecting different sets of genes , pointing to similar histories of genomic instability against... mutations have been identified in non-BRCA1/2 multiethnic breast cancer cases (45,46), no truncating mutation of the RAP80 gene in breast cancer has

Identification of hemoglobin variants by top-down mass spectrometry using selected diagnostic product ions.

PubMed

Coelho Graça, Didia; Hartmer, Ralf; Jabs, Wolfgang; Beris, Photis; Clerici, Lorella; Stoermer, Carsten; Samii, Kaveh; Hochstrasser, Denis; Tsybin, Yury O; Scherl, Alexander; Lescuyer, Pierre

2015-04-01

Hemoglobin disorder diagnosis is a complex procedure combining several analytical steps. Due to the lack of specificity of the currently used protein analysis methods, the identification of uncommon hemoglobin variants (proteoforms) can become a hard task to accomplish. The aim of this work was to develop a mass spectrometry-based approach to quickly identify mutated protein sequences within globin chain variants. To reach this goal, a top-down electron transfer dissociation mass spectrometry method was developed for hemoglobin β chain analysis. A diagnostic product ion list was established with a color code strategy allowing to quickly and specifically localize a mutation in the hemoglobin β chain sequence. The method was applied to the analysis of rare hemoglobin β chain variants and an (A)γ-β fusion protein. The results showed that the developed data analysis process allows fast and reliable interpretation of top-down electron transfer dissociation mass spectra by nonexpert users in the clinical area.

Detrimental Effects of UV-B Radiation in a Xeroderma Pigmentosum-Variant Cell Line

PubMed Central

Herman, Kimberly N.; Toffton, Shannon; McCulloch, Scott D.

2014-01-01

DNA polymerase η (pol η), of the Y-family, is well known for its in vitro DNA lesion bypass ability. The most well-characterized lesion bypassed by this polymerase is the cyclobutane pyrimidine dimer (CPD) caused by ultraviolet (UV) light. Historically, cellular and whole-animal models for this area of research have been conducted using UV-C (λ = 100–280 nm) owing to its ability to generate large quantities of CPDs and also the more structurally distorting 6-4 photoproduct. Although UV-C is useful as a laboratory tool, exposure to these wavelengths is generally very low owing to being filtered by stratospheric ozone. We are interested in the more environmentally relevant wavelength range of UV-B (λ = 280–315 nm) for its role in causing cytotoxicity and mutagenesis. We evaluated these endpoints in both a normal human fibroblast control line and a Xeroderma pigmentosum variant cell line in which the POLH gene contains a truncating point mutation, leading to a nonfunctional polymerase. We demonstrate that UV-B has similar but less striking effects compared to UV-C in both its cytotoxic and its mutagenic effects. Analysis of the mutation spectra after a single dose of UV-B shows that a majority of mutations can be attributed to mutagenic bypass of dipyrimidine sequences. However, we do note additional types of mutations with UV-B that are not previously reported after UV-C exposure. We speculate that these differences are attributed to a change in the spectra of photoproduct lesions rather than other lesions caused by oxidative stress. PMID:24549972

In Silico and In Vitro Investigations of the Mutability of Disease-Causing Missense Mutation Sites in Spermine Synthase

PubMed Central

Zhang, Zhe; Norris, Joy; Schwartz, Charles; Alexov, Emil

2011-01-01

Background Spermine synthase (SMS) is a key enzyme controlling the concentration of spermidine and spermine in the cell. The importance of SMS is manifested by the fact that single missense mutations were found to cause Snyder-Robinson Syndrome (SRS). At the same time, currently there are no non-synonymous single nucleoside polymorphisms, nsSNPs (harmless mutations), found in SMS, which may imply that the SMS does not tolerate amino acid substitutions, i.e. is not mutable. Methodology/Principal Findings To investigate the mutability of the SMS, we carried out in silico analysis and in vitro experiments of the effects of amino acid substitutions at the missense mutation sites (G56, V132 and I150) that have been shown to cause SRS. Our investigation showed that the mutation sites have different degree of mutability depending on their structural micro-environment and involvement in the function and structural integrity of the SMS. It was found that the I150 site does not tolerate any mutation, while V132, despite its key position at the interface of SMS dimer, is quite mutable. The G56 site is in the middle of the spectra, but still quite sensitive to charge residue replacement. Conclusions/Significance The performed analysis showed that mutability depends on the detail of the structural and functional factors and cannot be predicted based on conservation of wild type properties alone. Also, harmless nsSNPs can be expected to occur even at sites at which missense mutations were found to cause diseases. PMID:21647366

French experts report on MUTYH-associated polyposis (MAP).

PubMed

Buecher, Bruno; Bonaïti, Catherine; Buisine, Marie-Pierre; Colas, Chrystelle; Saurin, Jean-Christophe

2012-09-01

Recent years have been characterised by an improvement in our knowledge of genetic determinism of adenomatous polyposes and by the description in 2002 of a new entity called "MUTYH-associated polyposis" (MAP), related to biallelic mutations of this gene. Its autosomal recessive mode of inheritance contrasts with the autosomal dominant inheritance of the classical "familial adenomatous polyposis" (FAP), associated with an APC germline mutation. Although some phenotypic features may be of value to distinguish these two conditions, their clinical "spectra" largely overlap and the differential diagnosis may be difficult. The purpose of this expertise conducted under the auspices of the French Institut National du Cancer (INCa) was to assess the current state of knowledge on MUTYH-associated polyposis and to establish some recommendations in the field of molecular analysis (indications of tests and analysis strategies for affected patients and their relatives) and of clinical management based on available data in the literature, on the results from the French molecular genetics laboratories performing MUTYH analysis and on the opinions of biologists and clinicians experts (genetic counsellors and gastroenterologists). The risk of colorectal cancer among relatives carrying a monoallelic MUTYH mutation was also studied.

Analysis and implications of mutational variation.

PubMed

Keightley, Peter D; Halligan, Daniel L

2009-06-01

Variation from new mutations is important for several questions in quantitative genetics. Key parameters are the genomic mutation rate and the distribution of effects of mutations (DEM), which determine the amount of new quantitative variation that arises per generation from mutation (V(M)). Here, we review methods and empirical results concerning mutation accumulation (MA) experiments that have shed light on properties of mutations affecting quantitative traits. Surprisingly, most data on fitness traits from laboratory assays of MA lines indicate that the DEM is platykurtic in form (i.e., substantially less leptokurtic than an exponential distribution), and imply that most variation is produced by mutations of moderate to large effect. This finding contrasts with results from MA or mutagenesis experiments in which mutational changes to the DNA can be assayed directly, which imply that the vast majority of mutations have very small phenotypic effects, and that the distribution has a leptokurtic form. We compare these findings with recent approaches that attempt to infer the DEM for fitness based on comparing the frequency spectra of segregating nucleotide polymorphisms at putatively neutral and selected sites in population samples. When applied to data for humans and Drosophila, these analyses also indicate that the DEM is strongly leptokurtic. However, by combining the resultant estimates of parameters of the DEM with estimates of the mutation rate per nucleotide, the predicted V(M) for fitness is only a tiny fraction of V(M) observed in MA experiments. This discrepancy can be explained if we postulate that a few deleterious mutations of large effect contribute most of the mutational variation observed in MA experiments and that such mutations segregate at very low frequencies in natural populations, and effectively are never seen in population samples.

Similar Mutation Rates but Highly Diverse Mutation Spectra in Ascomycete and Basidiomycete Yeasts

DTIC Science & Technology

2016-12-24

Te, and Michael Lynch Department of Biology , Indiana University, Bloomington, IN *Corresponding author: E-mail: longhongan@gmail.com. Accepted...GBE The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access...fungal mutation spectrum. Supplementary Material Supplementary data are available at Genome Biology and Evolution online. Acknowledgments This research

Clinical and mutational spectra of 23 Chinese patients with glutaric aciduria type 1.

PubMed

Wang, Qiao; Li, Xiyuan; Ding, Yuan; Liu, Yupeng; Song, Jinqing; Yang, Yanling

2014-10-01

Glutaric aciduria type 1 (GA1) is a rare neurometabolic disorder caused by glutaryl-CoA dehydrogenase deficiency due to GCDH gene mutations. In this study, the clinical presentation and molecular aspects of 23 Chinese patients (11 males and 12 females) were investigated. All patients were diagnosed by elevated urinary glutaric acid and GCDH gene analysis. Protein-restricted diet supplemented with special formula, l-carnitine and GABA analog were initialed after diagnosis. The clinical and biochemical features were analyzed. Mutational analysis of GCDH was conducted. Clinical manifestations of 23 patients varied from asymptomatic to severe encephalopathy, with notable phenotypic differences between siblings with the same mutations. One case was detected by newborn screening, while 22 Cases were diagnosed between the ages of 5 months and 51 years. 29 mutations in GCDH were identified. Among them, 11 were novel, including seven missense mutations (c.406G > T, C.416C > G, c.442G > A, c.640A > G, c.901G > A, c.979G > A, and c.1207C > T), three frameshift mutations (c.873delC, c.1172-1173insT and c.1282-1285ins71) and one nonsense mutation (c.411C > G). In exon 5, c.553G > A and c.148T > C were found in four alleles (8.7%) and three alleles (6.5%) of the patients, respectively. In 23 Chinese patients with GA1, 11 novel GCDH mutations were identified. This may indicate that the genetic profiles of Chinese patients are different from those of other populations. 23 Chinese GA1 patients with varied clinical manifestations have been reported. 11 novel mutations in their GCDH gene were identified, indicating that the genetic profiles of Chinese GA1 patients differ from those of other populations. Copyright © 2013 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

Identification of somatic mutations in non-small cell lung carcinomas using whole-exome sequencing

PubMed Central

Liu, Pengyuan; Morrison, Carl; Wang, Liang; Xiong, Donghai; Vedell, Peter; Cui, Peng; Hua, Xing; Ding, Feng; Lu, Yan; James, Michael; Ebben, John D.; Xu, Haiming; Adjei, Alex A.; Head, Karen; Andrae, Jaime W.; Tschannen, Michael R.; Jacob, Howard; Pan, Jing; Zhang, Qi; Van den Bergh, Francoise; Xiao, Haijie; Lo, Ken C.; Patel, Jigar; Richmond, Todd; Watt, Mary-Anne; Albert, Thomas; Selzer, Rebecca; Anderson, Marshall; Wang, Jiang; Wang, Yian; Starnes, Sandra; Yang, Ping; You, Ming

2012-01-01

Lung cancer is the leading cause of cancer-related death, with non-small cell lung cancer (NSCLC) being the predominant form of the disease. Most lung cancer is caused by the accumulation of genomic alterations due to tobacco exposure. To uncover its mutational landscape, we performed whole-exome sequencing in 31 NSCLCs and their matched normal tissue samples. We identified both common and unique mutation spectra and pathway activation in lung adenocarcinomas and squamous cell carcinomas, two major histologies in NSCLC. In addition to identifying previously known lung cancer genes (TP53, KRAS, EGFR, CDKN2A and RB1), the analysis revealed many genes not previously implicated in this malignancy. Notably, a novel gene CSMD3 was identified as the second most frequently mutated gene (next to TP53) in lung cancer. We further demonstrated that loss of CSMD3 results in increased proliferation of airway epithelial cells. The study provides unprecedented insights into mutational processes, cellular pathways and gene networks associated with lung cancer. Of potential immediate clinical relevance, several highly mutated genes identified in our study are promising druggable targets in cancer therapy including ALK, CTNNA3, DCC, MLL3, PCDHIIX, PIK3C2B, PIK3CG and ROCK2. PMID:22510280

Expanding the Oro-Dental and Mutational Spectra of Kabuki Syndrome and Expression of KMT2D and KDM6A in Human Tooth Germs

PubMed Central

Porntaveetus, Thantrira; Abid, Mushriq F; Theerapanon, Thanakorn; Srichomthong, Chalurmpon; Ohazama, Atsushi; Kawasaki, Katsushige; Kawasaki, Maiko; Suphapeetiporn, Kanya; Sharpe, Paul T.; Shotelersuk, Vorasuk

2018-01-01

Kabuki syndrome is a rare genetic disorder characterized by distinct dysmorphic facial features, intellectual disability, and multiple developmental abnormalities. Despite more than 350 documented cases, the oro-dental spectrum associated with kabuki syndrome and expression of KMT2D (histone-lysine N-methyltransferase 2D) or KDM6A (lysine-specific demethylase 6A) genes in tooth development have not been well defined. Here, we report seven unrelated Thai patients with Kabuki syndrome having congenital absence of teeth, malocclusion, high-arched palate, micrognathia, and deviated tooth shape and size. Exome sequencing successfully identified that six patients were heterozygous for mutations in KMT2D, and one in KDM6A. Six were novel mutations, of which five were in KMT2D and one in KDM6A. They were truncating mutations including four frameshift deletions and two nonsense mutations. The predicted non-functional KMT2D and KDM6A proteins are expected to cause disease by haploinsufficiency. Our study expands oro-dental, medical, and mutational spectra associated with Kabuki syndrome. We also demonstrate for the first time that KMT2D and KDM6A are expressed in the dental epithelium of human tooth germs. PMID:29725259

IDH1 R132H mutation generates a distinct phospholipid metabolite profile in glioma.

PubMed

Esmaeili, Morteza; Hamans, Bob C; Navis, Anna C; van Horssen, Remco; Bathen, Tone F; Gribbestad, Ingrid S; Leenders, William P; Heerschap, Arend

2014-09-01

Many patients with glioma harbor specific mutations in the isocitrate dehydrogenase gene IDH1 that associate with a relatively better prognosis. IDH1-mutated tumors produce the oncometabolite 2-hydroxyglutarate. Because IDH1 also regulates several pathways leading to lipid synthesis, we hypothesized that IDH1-mutant tumors have an altered phospholipid metabolite profile that would impinge on tumor pathobiology. To investigate this hypothesis, we performed (31)P-MRS imaging in mouse xenograft models of four human gliomas, one of which harbored the IDH1-R132H mutation. (31)P-MR spectra from the IDH1-mutant tumor displayed a pattern distinct from that of the three IDH1 wild-type tumors, characterized by decreased levels of phosphoethanolamine and increased levels of glycerophosphocholine. This spectral profile was confirmed by ex vivo analysis of tumor extracts, and it was also observed in human surgical biopsies of IDH1-mutated tumors by (31)P high-resolution magic angle spinning spectroscopy. The specificity of this profile for the IDH1-R132H mutation was established by in vitro (31)P-NMR of extracts of cells overexpressing IDH1 or IDH1-R132H. Overall, our results provide evidence that the IDH1-R132H mutation alters phospholipid metabolism in gliomas involving phosphoethanolamine and glycerophosphocholine. These new noninvasive biomarkers can assist in the identification of the mutation and in research toward novel treatments that target aberrant metabolism in IDH1-mutant glioma. ©2014 American Association for Cancer Research.

Serum peptide expression and treatment responses in patients with advanced non-small-cell lung cancer

PubMed Central

An, Juan; Tang, Chuan-Hao; Wang, Na; Liu, Yi; Lv, Jin; Xu, Bin; Li, Xiao-Yan; Guo, Wan-Feng; Gao, Hong-Jun; He, Kun; Liu, Xiao-Qing

2018-01-01

Epidermal growth factor receptor (EGFR) mutation is an important predictor for response to personalized treatments of patients with advanced non-small-cell lung cancer (NSCLC). However its usage is limited due to the difficult of obtaining tissue specimens. A novel prediction system using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been reported to be a perspective tool in European countries to identify patients who are likely to benefit from EGFR tyrosine kinase inhibitor (TKI) treatment. In the present study, MALDI-TOF MS was used on pretreatment serum samples of patients with advanced non-small-cell lung cancer to discriminate the spectra between disease control and disease progression groups in one cohort of Chinese patients. The candidate features for classification were subsequently validated in a blinded fashion in another set of patients. The correlation between plasma EGFR mutation status and the intensities of representative spectra for classification was evaluated. A total of 103 patients that were treated with EGFR-TKIs were included. It was determined that 8 polypeptides peaks were significant different between the disease control and disease progression group. A total of 6 polypeptides were established in the classification algorithm. The sensitivity of the algorithm to predict treatment responses was 76.2% (16/21) and the specificity was 81.8% (18/22). The accuracy rate of the algorithm was 79.1% (34/43). A total of 3 polypeptides were significantly correlated with EGFR mutations (P=0.04, P=0.03 and P=0.04, respectively). The present study confirmed that MALDI-TOF MS analysis can be used to predict responses to EGFR-TKI treatment of the Asian population where the EGFR mutation status differs from the European population. Furthermore, the expression intensities of the three polypeptides in the classification model were associated with EGFR mutation. PMID:29844828

Lynch Syndrome in high risk Ashkenazi Jews in Israel.

PubMed

Goldberg, Yael; Kedar, Inbal; Kariiv, Revital; Halpern, Naama; Plesser, Morasha; Hubert, Ayala; Kaduri, Luna; Sagi, Michal; Lerer, Israela; Abeliovich, Dvorah; Hamburger, Tamar; Nissan, Aviram; Goldshmidt, Hanoch; Solar, Irit; Geva, Ravit; Strul, Hana; Rosner, Guy; Baris, Hagit; Levi, Zohar; Peretz, Tamar

2014-03-01

Lynch Syndrome is caused by mutations in DNA mismatch repair genes. Diagnosis is not always trivial and may be costly. Information regarding incidence, genotype-phenotype correlation, spectrum of mutations and genes involved in specific populations facilitate the diagnostic process and contribute to clinical work-up. To report gene distribution, mutations detected and co-occurrence of related syndromes in a cohort of Ashkenazi Jews in Israel. Patients were identified in dedicated high risk clinics in 3 medical centers in Israel. Diagnostic process followed a multi-step scheme. It included testing for founder mutations, tumor testing, gene sequencing and MLPA. Lynch Syndrome was defined either by positive mutation testing, or by clinical criteria and positive tumor analysis. We report a cohort of 75 Ashkenazi families suspected of Lynch Syndrome. Mutations were identified in 51/75 (68%) families: 38 in MSH2, 9 in MSH6, and 4 in MLH1. 37/51 (73%) of these families carried one of the 3 'Ashkenazi' founder mutations in MSH2 or MSH6. Each of the other 14 families carried a private mutation. 3 (6%) were large deletions. Only 20/51 (39%) families were Amsterdam Criteria positive; 42 (82%) were positive for the Bethesda guidelines and 9 (18%) did not fulfill any Lynch Syndrome criteria. We report C-MMRD and co-occurrence of BRCA and Lynch Syndrome in our cohort. Mutation spectra and gene distribution among Ashkenazi Jews are unique. Three founder Lynch Syndrome mutations are found in 73% families with known mutations. Among the three, MSH2 and MSH6 are the most common. These features affect the phenotype, the diagnostic process, risk estimation, and genetic counseling.

Correlating the vibrational spectra of structurally related molecules: A spectroscopic measure of similarity.

PubMed

Tao, Yunwen; Zou, Wenli; Cremer, Dieter; Kraka, Elfi

2018-03-05

Using catastrophe theory and the concept of a mutation path, an algorithm is developed that leads to the direct correlation of the normal vibrational modes of two structurally related molecules. The mutation path is defined by weighted incremental changes in mass and geometry of the molecules in question, which are successively applied to mutate a molecule into a structurally related molecule and thus continuously converting their normal vibrational spectra from one into the other. Correlation diagrams are generated that accurately relate the normal vibrational modes to each other by utilizing mode-mode overlap criteria and resolving allowed and avoided crossings of vibrational eigenstates. The limitations of normal mode correlation, however, foster the correlation of local vibrational modes, which offer a novel vibrational measure of similarity. It will be shown how this will open new avenues for chemical studies. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Statistical Coupling Analysis-Guided Library Design for the Discovery of Mutant Luciferases.

PubMed

Liu, Mira D; Warner, Elliot A; Morrissey, Charlotte E; Fick, Caitlyn W; Wu, Taia S; Ornelas, Marya Y; Ochoa, Gabriela V; Zhang, Brendan S; Rathbun, Colin M; Porterfield, William B; Prescher, Jennifer A; Leconte, Aaron M

2018-02-06

Directed evolution has proven to be an invaluable tool for protein engineering; however, there is still a need for developing new approaches to continue to improve the efficiency and efficacy of these methods. Here, we demonstrate a new method for library design that applies a previously developed bioinformatic method, Statistical Coupling Analysis (SCA). SCA uses homologous enzymes to identify amino acid positions that are mutable and functionally important and engage in synergistic interactions between amino acids. We use SCA to guide a library of the protein luciferase and demonstrate that, in a single round of selection, we can identify luciferase mutants with several valuable properties. Specifically, we identify luciferase mutants that possess both red-shifted emission spectra and improved stability relative to those of the wild-type enzyme. We also identify luciferase mutants that possess a >50-fold change in specificity for modified luciferins. To understand the mutational origin of these improved mutants, we demonstrate the role of mutations at N229, S239, and G246 in altered function. These studies show that SCA can be used to guide library design and rapidly identify synergistic amino acid mutations from a small library.

Understanding mutagenesis through delineation of mutational signatures in human cancer

DOE PAGES

Petljak, Mia; Alexandrov, Ludmil B.

2016-05-04

Each individual cell within a human body acquires a certain number of somatic mutations during a course of its lifetime. These mutations originate from a wide spectra of both endogenous and exogenous mutational processes that leave distinct patterns of mutations, termed mutational signatures, embedded within the genomes of all cells. In recent years, the vast amount of data produced by sequencing of cancer genomes was coupled with novel mathematical models and computational tools to generate the first comprehensive map of mutational signatures in human cancer. Up to date, >30 distinct mutational signatures have been identified, and etiologies have been proposedmore » for many of them. This paper provides a brief historical background on examination of mutational patterns in human cancer, summarizes the knowledge accumulated since introducing the concept of mutational signatures and discusses their future potential applications and perspectives within the field.« less

THE MUTATION SPECTRA OF DRINKING WATER SAMPLES USING THE BASE-SPECIFIC TA7000 STRAINS OF SALMONELLA IN THE MICROSUSPENSION ASSAY

EPA Science Inventory

Previous studies showed that disinfected drinking water samples gave mutagenic spectra typical of halogenated furanones. In this study, we used the TA7000 base-¿specific Salmonella typhimurium tester strains to characterize water samples from two drinking water treatment plants (...

Molecular basis and clinical presentation of classic galactosemia in a Croatian population.

PubMed

Ramadža, Danijela Petković; Sarnavka, Vladimir; Vuković, Jurica; Fumić, Ksenija; Krželj, Vjekoslav; Lozić, Bernarda; Pušeljić, Silvija; Pereira, Hana; Silva, Maria João; Tavares de Almeida, Isabel; Barić, Ivo; Rivera, Isabel

2018-01-26

Classic galactosemia is an autosomal recessive disorder of galactose metabolism caused by severely decreased activity of galactose-1-phosphate uridylyltransferase (GALT) due to pathogenic mutations in the GALT gene. To date more than 330 mutations have been described, with p.Q188R and p.K285N being the most common in Caucasian populations. Although acute manifestations can be fully avoided by a galactose-restricted diet, chronic complications, such as neurological ones, cannot be prevented in a significant number of patients despite compliance with the dietary treatment. A cohort of 16 galactosemic Croatian patients, including one pair of siblings, was studied. Molecular characterization was performed by direct sequence analysis of the GALT gene. Sixteen patients were analyzed and only four different mutations were detected. As expected, p.Q188R and p.K285N were common, accounting for 40% and 37% of unrelated alleles, respectively. The third mutation accounting for 20% of mutant alleles was p.R123X causing a premature stop codon, is thus considered to be severe, which is in accordance with the phenotype presented by the homozygous patient described here. The fourth mutation p.E271D was found in a single allele. More than half of our patients manifested some chronic neurological complications. This is the first report on mutational and phenotypic spectra of classic galactosemia in Croatia that expands the knowledge on the mutational map of the GALT gene across Europe and reveals the genetic homogeneity of the Croatian population.

Evolution at increased error rate leads to the coexistence of multiple adaptive pathways in an RNA virus.

PubMed

Cabanillas, Laura; Arribas, María; Lázaro, Ester

2013-01-16

When beneficial mutations present in different genomes spread simultaneously in an asexual population, their fixation can be delayed due to competition among them. This interference among mutations is mainly determined by the rate of beneficial mutations, which in turn depends on the population size, the total error rate, and the degree of adaptation of the population. RNA viruses, with their large population sizes and high error rates, are good candidates to present a great extent of interference. To test this hypothesis, in the current study we have investigated whether competition among beneficial mutations was responsible for the prolonged presence of polymorphisms in the mutant spectrum of an RNA virus, the bacteriophage Qβ, evolved during a large number of generations in the presence of the mutagenic nucleoside analogue 5-azacytidine. The analysis of the mutant spectra of bacteriophage Qβ populations evolved at artificially increased error rate shows a large number of polymorphic mutations, some of them with demonstrated selective value. Polymorphisms distributed into several evolutionary lines that can compete among them, making it difficult the emergence of a defined consensus sequence. The presence of accompanying deleterious mutations, the high degree of recurrence of the polymorphic mutations, and the occurrence of epistatic interactions generate a highly complex interference dynamics. Interference among beneficial mutations in bacteriophage Qβ evolved at increased error rate permits the coexistence of multiple adaptive pathways that can provide selective advantages by different molecular mechanisms. In this way, interference can be seen as a positive factor that allows the exploration of the different local maxima that exist in rugged fitness landscapes.

Inactivation and mutation induction in Saccharomyces cerevisiae exposed to simulated sunlight: evaluation of action spectra.

PubMed

Schenk-Meuser, K; Pawlowsky, K; Kiefer, J

1992-07-15

The effectiveness of polychromatic light irradiation was investigated for haploid yeast cells. Inactivation and mutation induction were measured in both a RAD-wildtype strain and an excision-repair defective strain. The behaviour of vegetative "wet" cells was compared to that of dehydrated cells. The aim of the study was to assess the interaction of UVC with other wavelengths in cells of different states of humidity. The irradiation procedure was therefore carried out using a solar simulator either with full spectrum or with a UVC-blocking filter (modified sunlight) added. The results were analysed on the basis of separately determined action spectra. The summation of the efficiency of individual wavelengths was compared to the values obtained from polychromatic irradiation. It is shown that the effects caused by the whole-spectrum irradiation in wet cells can be predicted sufficiently from the calculation, while dried wildtype cells exhibit higher mutation rates. Thus it can be assumed that drying-specific damage leads to lethal and mutagenic lesions which are processed in different ways, causing a synergistic behaviour in mutation induction. Irradiation of vegetative cells with modified sunlight (UVC-) results in less inactivation and lower mutation rates than were calculated. From these results it can be concluded that this antagonistic behaviour is caused by the interaction of near-UV photoproducts.

Topology of evolving, mutagenized viral populations: quasispecies expansion, compression, and operation of negative selection.

PubMed

Ojosnegros, Samuel; Agudo, Rubén; Sierra, Macarena; Briones, Carlos; Sierra, Saleta; González-López, Claudia; Domingo, Esteban; Cristina, Juan

2008-07-17

The molecular events and evolutionary forces underlying lethal mutagenesis of virus (or virus extinction through an excess of mutations) are not well understood. Here we apply for the first time phylogenetic methods and Partition Analysis of Quasispecies (PAQ) to monitor genetic distances and intra-population structures of mutant spectra of foot-and-mouth disease virus (FMDV) quasispecies subjected to mutagenesis by base and nucleoside analogues. Phylogenetic and PAQ analyses have revealed a highly dynamic variation of intrapopulation diversity of FMDV quasispecies. The population diversity first suffers striking expansions in the presence of mutagens and then compressions either when the presence of the mutagenic analogue was discontinued or when a mutation that decreased sensitivity to a mutagen was selected. The pattern of mutations found in the populations was in agreement with the behavior of the corresponding nucleotide analogues with FMDV in vitro. Mutations accumulated at preferred genomic sites, and dn/ds ratios indicate the operation of negative (or purifying) selection in populations subjected to mutagenesis. No evidence of unusually elevated genetic distances has been obtained for FMDV populations approaching extinction. Phylogenetic and PAQ analysis provide adequate procedures to describe the evolution of viral sequences subjected to lethal mutagenesis. These methods define the changes of intra-population structure more precisely than mutation frequencies and Shannon entropies. PAQ is very sensitive to variations of intrapopulation genetic distances. Strong negative (or purifying) selection operates in FMDV populations subjected to enhanced mutagenesis. The quantifications provide evidence that extinction does not imply unusual increases of intrapopulation complexity, in support of the lethal defection model of virus extinction.

Mapping differential interactomes by affinity purification coupled with data-independent mass spectrometry acquisition.

PubMed

Lambert, Jean-Philippe; Ivosev, Gordana; Couzens, Amber L; Larsen, Brett; Taipale, Mikko; Lin, Zhen-Yuan; Zhong, Quan; Lindquist, Susan; Vidal, Marc; Aebersold, Ruedi; Pawson, Tony; Bonner, Ron; Tate, Stephen; Gingras, Anne-Claude

2013-12-01

Characterizing changes in protein-protein interactions associated with sequence variants (e.g., disease-associated mutations or splice forms) or following exposure to drugs, growth factors or hormones is critical to understanding how protein complexes are built, localized and regulated. Affinity purification (AP) coupled with mass spectrometry permits the analysis of protein interactions under near-physiological conditions, yet monitoring interaction changes requires the development of a robust and sensitive quantitative approach, especially for large-scale studies in which cost and time are major considerations. We have coupled AP to data-independent mass spectrometric acquisition (sequential window acquisition of all theoretical spectra, SWATH) and implemented an automated data extraction and statistical analysis pipeline to score modulated interactions. We used AP-SWATH to characterize changes in protein-protein interactions imparted by the HSP90 inhibitor NVP-AUY922 or melanoma-associated mutations in the human kinase CDK4. We show that AP-SWATH is a robust label-free approach to characterize such changes and propose a scalable pipeline for systems biology studies.

Brute-Force Approach for Mass Spectrometry-Based Variant Peptide Identification in Proteogenomics without Personalized Genomic Data

NASA Astrophysics Data System (ADS)

Ivanov, Mark V.; Lobas, Anna A.; Levitsky, Lev I.; Moshkovskii, Sergei A.; Gorshkov, Mikhail V.

2018-02-01

In a proteogenomic approach based on tandem mass spectrometry analysis of proteolytic peptide mixtures, customized exome or RNA-seq databases are employed for identifying protein sequence variants. However, the problem of variant peptide identification without personalized genomic data is important for a variety of applications. Following the recent proposal by Chick et al. (Nat. Biotechnol. 33, 743-749, 2015) on the feasibility of such variant peptide search, we evaluated two available approaches based on the previously suggested "open" search and the "brute-force" strategy. To improve the efficiency of these approaches, we propose an algorithm for exclusion of false variant identifications from the search results involving analysis of modifications mimicking single amino acid substitutions. Also, we propose a de novo based scoring scheme for assessment of identified point mutations. In the scheme, the search engine analyzes y-type fragment ions in MS/MS spectra to confirm the location of the mutation in the variant peptide sequence.

Using circular dichroism collected as a function of temperature to determine the thermodynamics of protein unfolding and binding interactions

PubMed Central

Greenfield, Norma J.

2009-01-01

Circular dichroism (CD) is an excellent spectroscopic technique for following the unfolding and folding of proteins as a function of temperature. One of its principal applications is to determine the effects of mutations and ligands on protein and polypeptide stability If the change in CD as a function of temperature is reversible, analysis of the data may be used to determined the van't Hoff enthalpy (ΔH) and entropy (ΔS) of unfolding, the midpoint of the unfolding transition (TM) and the free energy (ΔG) of unfolding. Binding constants of protein-protein and protein-ligand interactions may also be estimated from the unfolding curves. Analysis of CD spectra obtained as a function of temperature is also useful to determine whether a protein has unfolding intermediates. Measurement of the spectra of five folded proteins and their unfolding curves at a single wavelength takes approximately eight hours. PMID:17406506

Stratospheric ozone loss, ultraviolet effects and action spectroscopy

NASA Astrophysics Data System (ADS)

Coohill, Thomas P.

The major effect of stratospheric ozone loss will be an increase in the amount of ultraviolet radiation reaching the ground. This increase will be entirely contained within the UV-B (290-320nm). How this will impact life on Earth will be determined by the UV-B photobiology of exposed organisms, including humans. One of the analytical methods useful in estimating these effects is Action Spectroscopy (biological effect as a function of wavelength). Carefully constructed action spectra will allow us to partially predict the increase in bio-effect due to additional UV exposure. What effect this has on the organism and the system in which the organism resides is of paramount importance. Suitable action spectra already exist for human skin cancer, human cell mutation and killing, and for one immune response. Comprehensive and widely applicable action spectra for terrestrial and aquatic plant responses are being generated but are not yet suitable for extensive analysis. There is little data available for animals, other than those experiments completed in the laboratory as model systems for human studies. Some polychromatic action spectra have proven useful in determining the possible impact of ozone loss on biological systems. The pitfalls and limits of this approach will be addressed.

Novel near-infrared spectrum analysis tool: Synergy adaptive moving window model based on immune clone algorithm.

PubMed

Wang, Shenghao; Zhang, Yuyan; Cao, Fuyi; Pei, Zhenying; Gao, Xuewei; Zhang, Xu; Zhao, Yong

2018-02-13

This paper presents a novel spectrum analysis tool named synergy adaptive moving window modeling based on immune clone algorithm (SA-MWM-ICA) considering the tedious and inconvenient labor involved in the selection of pre-processing methods and spectral variables by prior experience. In this work, immune clone algorithm is first introduced into the spectrum analysis field as a new optimization strategy, covering the shortage of the relative traditional methods. Based on the working principle of the human immune system, the performance of the quantitative model is regarded as antigen, and a special vector corresponding to the above mentioned antigen is regarded as antibody. The antibody contains a pre-processing method optimization region which is created by 11 decimal digits, and a spectrum variable optimization region which is formed by some moving windows with changeable width and position. A set of original antibodies are created by modeling with this algorithm. After calculating the affinity of these antibodies, those with high affinity will be selected to clone. The regulation for cloning is that the higher the affinity, the more copies will be. In the next step, another import operation named hyper-mutation is applied to the antibodies after cloning. Moreover, the regulation for hyper-mutation is that the lower the affinity, the more possibility will be. Several antibodies with high affinity will be created on the basis of these steps. Groups of simulated dataset, gasoline near-infrared spectra dataset, and soil near-infrared spectra dataset are employed to verify and illustrate the performance of SA-MWM-ICA. Analysis results show that the performance of the quantitative models adopted by SA-MWM-ICA are better especially for structures with relatively complex spectra than traditional models such as partial least squares (PLS), moving window PLS (MWPLS), genetic algorithm PLS (GAPLS), and pretreatment method classification and adjustable parameter changeable size moving window PLS (CA-CSMWPLS). The selected pre-processing methods and spectrum variables are easily explained. The proposed method will converge in few generations and can be used not only for near-infrared spectroscopy analysis but also for other similar spectral analysis, such as infrared spectroscopy. Copyright © 2017 Elsevier B.V. All rights reserved.

CFTR allelic heterogeneity in Mexican patients with cystic fibrosis: implications for molecular screening.

PubMed

Chávez-Saldaña, Margarita; Yokoyama, Emiy; Lezana, José Luis; Carnevale, Alessandra; Macías, Miguel; Vigueras, Rosa M; López, Marisol; Orozco, Lorena

2010-01-01

Cystic fibrosis, the most common autosomal recessive disorder, is caused by defects in the CF transmembrane conductance regulator gene (CFTR) that encodes a chloride channel. To date, over 1,800 mutations have been described related to the causative gene of CF, showing a variable frequency among populations. In a previous extensive analysis of the CFTR locus in 97 Mexican patients, 34 different mutations (75% of CF alleles) were found using several strategies for mutation screening; however, 63% had at least an uncharacterized allele. Despite the combined technologies used, there are still a great number of unknown mutations in the Mexican population. Screening of the CFTR gene to provide additional evidence of the mutational wide spectrum responsible for CF in Mexican patients. In this study, the number of unrelated CF patients was increased to 230, 133 new cases and the 97 previously reported to include 63% with at least an uncharacterized allele. Additional tools were used to improve the detection rate of CF mutations, such as a commercial kit for 36 mutations plus a single chain conformational polymorphism method and DNA sequencing. By using a combination of these strategies we characterized 77.7% of all the CF alleles, resulting in a total of 46 different mutations detected, including the identification of 12 additional mutations (p.R334W, p.A455E, c.3120+1G > A, c.3272-26A > G, c.711+1G > T, p.Q552X, p.W1282X, c.IVS8-5T, p.R1162X and p.R347P, p.D1152H and p.T1036N). Although these 12 mutations have been reported in other populations, they have not yet been reported in Mexican patients. This report shows that Mexico has one of the widest spectra of CFTR mutations worldwide. The knowledge of the ethnic and geographic distribution of CFTR mutations in this population will allow the development of more effective methods for diagnosis and treatment.

Evidence that a burst of DNA depurination in SENCAR mouse skin induces error-prone repair and forms mutations in the H-ras gene.

PubMed

Chakravarti, D; Mailander, P C; Li, K M; Higginbotham, S; Zhang, H L; Gross, M L; Meza, J L; Cavalieri, E L; Rogan, E G

2001-11-29

Treatment of SENCAR mouse skin with dibenzo[a,l]pyrene results in abundant formation of abasic sites that undergo error-prone excision repair, forming oncogenic H-ras mutations in the early preneoplastic period. To examine whether the abundance of abasic sites causes repair infidelity, we treated SENCAR mouse skin with estradiol-3,4-quinone (E(2)-3,4-Q) and determined adduct levels 1 h after treatment, as well as mutation spectra in the H-ras gene between 6 h and 3 days after treatment. E(2)-3,4-Q formed predominantly (> or =99%) the rapidly-depurinating 4-hydroxy estradiol (4-OHE(2))-1-N3Ade adduct and the slower-depurinating 4-OHE(2)-1-N7Gua adduct. Between 6 h and 3 days, E(2)-3,4-Q induced abundant A to G mutations in H-ras DNA, frequently in the context of a 3'-G residue. Using a T.G-DNA glycosylase (TDG)-PCR assay, we determined that the early A to G mutations (6 and 12 h) were in the form of G.T heteroduplexes, suggesting misrepair at A-specific depurination sites. Since G-specific mutations were infrequent in the spectra, it appears that the slow rate of depurination of the N7Gua adducts during active repair may not generate a threshold level of G-specific abasic sites to affect repair fidelity. These results also suggest that E(2)-3,4-Q, a suspected endogenous carcinogen, is a genotoxic compound and could cause mutations.

An Unbiased Genome-Wide View of the Mutation Rate and Spectrum of the Endosymbiotic Bacterium Teredinibacter turnerae.

PubMed

Senra, Marcus V X; Sung, Way; Ackerman, Matthew; Miller, Samuel F; Lynch, Michael; Soares, Carlos Augusto G

2018-03-01

Mutations contribute to genetic variation in all living systems. Thus, precise estimates of mutation rates and spectra across a diversity of organisms are required for a full comprehension of evolution. Here, a mutation-accumulation (MA) assay was carried out on the endosymbiotic bacterium Teredinibacter turnerae. After ∼3,025 generations, base-pair substitutions (BPSs) and insertion-deletion (indel) events were characterized by whole-genome sequencing analysis of 47 independent MA lines, yielding a BPS rate of 1.14 × 10-9 per site per generation and indel rate of 1.55 × 10-10 events per site per generation, which are among the highest within free-living and facultative intracellular bacteria. As in other endosymbionts, a significant bias of BPSs toward A/T and an excess of deletion mutations over insertion mutations are observed for these MA lines. However, even with a deletion bias, the genome remains relatively large (∼5.2 Mb) for an endosymbiotic bacterium. The estimate of the effective population size (Ne) in T. turnerae is quite high and comparable to free-living bacteria (∼4.5 × 107), suggesting that the heavy bottlenecking associated with many endosymbiotic relationships is not prevalent during the life of this endosymbiont. The efficiency of selection scales with increasing Ne and such strong selection may have been operating against the deletion bias, preventing genome erosion. The observed mutation rate in this endosymbiont is of the same order of magnitude of those with similar Ne, consistent with the idea that population size is a primary determinant of mutation-rate evolution within endosymbionts, and that not all endosymbionts have low Ne.

PhosphoSitePlus, 2014: mutations, PTMs and recalibrations

PubMed Central

Hornbeck, Peter V.; Zhang, Bin; Murray, Beth; Kornhauser, Jon M.; Latham, Vaughan; Skrzypek, Elzbieta

2015-01-01

PhosphoSitePlus® (PSP, http://www.phosphosite.org/), a knowledgebase dedicated to mammalian post-translational modifications (PTMs), contains over 330 000 non-redundant PTMs, including phospho, acetyl, ubiquityl and methyl groups. Over 95% of the sites are from mass spectrometry (MS) experiments. In order to improve data reliability, early MS data have been reanalyzed, applying a common standard of analysis across over 1 000 000 spectra. Site assignments with P > 0.05 were filtered out. Two new downloads are available from PSP. The ‘Regulatory sites’ dataset includes curated information about modification sites that regulate downstream cellular processes, molecular functions and protein-protein interactions. The ‘PTMVar’ dataset, an intersect of missense mutations and PTMs from PSP, identifies over 25 000 PTMVars (PTMs Impacted by Variants) that can rewire signaling pathways. The PTMVar data include missense mutations from UniPROTKB, TCGA and other sources that cause over 2000 diseases or syndromes (MIM) and polymorphisms, or are associated with hundreds of cancers. PTMVars include 18 548 phosphorlyation sites, 3412 ubiquitylation sites, 2316 acetylation sites, 685 methylation sites and 245 succinylation sites. PMID:25514926

INTERDISCIPLINARY PHYSICS AND RELATED AREAS OF SCIENCE AND TECHNOLOGY: Relaxation Property and Stability Analysis of the Quasispecies Models

NASA Astrophysics Data System (ADS)

Feng, Xiao-Li; Li, Yu-Xiao; Gu, Jian-Zhong; Zhuo, Yi-Zhong

2009-10-01

The relaxation property of both Eigen model and Crow-Kimura model with a single peak fitness landscape is studied from phase transition point of view. We first analyze the eigenvalue spectra of the replication mutation matrices. For sufficiently long sequences, the almost crossing point between the largest and second-largest eigenvalues locates the error threshold at which critical slowing down behavior appears. We calculate the critical exponent in the limit of infinite sequence lengths and compare it with the result from numerical curve fittings at sufficiently long sequences. We find that for both models the relaxation time diverges with exponent 1 at the error (mutation) threshold point. Results obtained from both methods agree quite well. From the unlimited correlation length feature, the first order phase transition is further confirmed. Finally with linear stability theory, we show that the two model systems are stable for all ranges of mutation rate. The Eigen model is asymptotically stable in terms of mutant classes, and the Crow-Kimura model is completely stable.

Rapid evolution of the human mutation spectrum

PubMed Central

Harris, Kelley; Pritchard, Jonathan K

2017-01-01

DNA is a remarkably precise medium for copying and storing biological information. This high fidelity results from the action of hundreds of genes involved in replication, proofreading, and damage repair. Evolutionary theory suggests that in such a system, selection has limited ability to remove genetic variants that change mutation rates by small amounts or in specific sequence contexts. Consistent with this, using SNV variation as a proxy for mutational input, we report here that mutational spectra differ substantially among species, human continental groups and even some closely related populations. Close examination of one signal, an increased TCC→TTC mutation rate in Europeans, indicates a burst of mutations from about 15,000 to 2000 years ago, perhaps due to the appearance, drift, and ultimate elimination of a genetic modifier of mutation rate. Our results suggest that mutation rates can evolve markedly over short evolutionary timescales and suggest the possibility of mapping mutational modifiers. DOI: http://dx.doi.org/10.7554/eLife.24284.001 PMID:28440220

Evaluation of the metabolic fate of munitions material (TNT & RDX) in plant systems. Initial assessment of plant DNA mutation spectra as a biomarker

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leung, F.; Cataldo, D.A.; Fellows, R.J.

1995-09-01

Munitions material can enter the environment as a result of manufacturing activities and field usage. Predictor methodologies, or biomarkers would enhance evaluation of environmental impacts. The goal of this exploratory study deoxyribonucleic acid (DNA) mutation frequency as a biomarker for munitions exposure. The approach e resolution of an effective repetitive sequence probe for the identification of characteristic mutations, and (2) the development of a testing media [a clonal cell line of carrot (Daucus carota) spension cells]. Commercially available probes demonstrated marginal resolution therefore a low-C{sub o}t library was then constructed. Three colonies from the low-C{sub o}t DNA library were screenedmore » and the DNA isolates sequenced. A suspension culture of carrot (Daucus carota) was developed. A mutation spectra experiment was initiated at a 10-mg TNT/L exposure concentration with the attempt to clone over 1500 single TNT-exposed cells. Over the following six months greater than 98% of the initially isolated cells were unable to survive and produce micro calluses. The remaining calli were too few to be statistically significant and the experiment was terminated. The biomarker concept itself remains to be disproved, but the need for large numbers of uniform clones to differentiate true mutations suggest that more direct techniques using whole tissues need to be developed.« less

Isolation of rpoB mutations causing rifampicin resistance in Bacillus subtilis spores exposed to simulated Martian surface conditions.

PubMed

Perkins, Amy E; Schuerger, Andrew C; Nicholson, Wayne L

2008-12-01

ABSTRACT Bacterial spores are considered prime candidates for Earth-to-Mars transport by natural processes and human spaceflight activities. Previous studies have shown that exposure of Bacillus subtilis spores to ultrahigh vacuum (UHV) characteristic of space both increased the spontaneous mutation rate and altered the spectrum of mutation in various marker genes; but, to date, mutagenesis studies have not been performed on spores exposed to milder low pressures encountered in the martian environment. Mutations to rifampicin-resistance (Rif(R)) were isolated in B. subtilis spores exposed to simulated martian atmosphere (99.9% CO(2), 710 Pa) for 21 days in a Mars Simulation Chamber (MSC) and compared to parallel Earth controls. Exposure in the MSC reduced spore viability by approximately 67% compared to Earth controls, but this decrease was not statistically significant (P = 0.3321). The frequency of mutation to Rif(R) was also not significantly increased in the MSC compared to Earth-exposed spores (P = 0.479). Forty-two and 51 Rif(R) mutant spores were isolated from the MSC- and Earth-exposed controls, respectively. Nucleotide sequencing located the Rif(R) mutations in the rpoB gene encoding the beta subunit of RNA polymerase at residue V135F of the N-cluster and at residues Q469K/L, H482D/P/R/Y, and S487L in Cluster I. No mutations were found in rpoB Clusters II or III. Two new alleles, Q469L and H482D, previously unreported in B. subtilis rpoB, were isolated from spores exposed in the MSC; otherwise, only slight differences were observed in the spectra of spontaneous Rif(R) mutations from spores exposed to Earth vs. the MSC. However, both spectra are distinctly different from Rif(R) mutations previously reported arising from B. subtilis spores exposed to simulated space vacuum.

Topology of evolving, mutagenized viral populations: quasispecies expansion, compression, and operation of negative selection

PubMed Central

2008-01-01

Background The molecular events and evolutionary forces underlying lethal mutagenesis of virus (or virus extinction through an excess of mutations) are not well understood. Here we apply for the first time phylogenetic methods and Partition Analysis of Quasispecies (PAQ) to monitor genetic distances and intra-population structures of mutant spectra of foot-and-mouth disease virus (FMDV) quasispecies subjected to mutagenesis by base and nucleoside analogues. Results Phylogenetic and PAQ analyses have revealed a highly dynamic variation of intrapopulation diversity of FMDV quasispecies. The population diversity first suffers striking expansions in the presence of mutagens and then compressions either when the presence of the mutagenic analogue was discontinued or when a mutation that decreased sensitivity to a mutagen was selected. The pattern of mutations found in the populations was in agreement with the behavior of the corresponding nucleotide analogues with FMDV in vitro. Mutations accumulated at preferred genomic sites, and dn/ds ratios indicate the operation of negative (or purifying) selection in populations subjected to mutagenesis. No evidence of unusually elevated genetic distances has been obtained for FMDV populations approaching extinction. Conclusion Phylogenetic and PAQ analysis provide adequate procedures to describe the evolution of viral sequences subjected to lethal mutagenesis. These methods define the changes of intra-population structure more precisely than mutation frequencies and Shannon entropies. PAQ is very sensitive to variations of intrapopulation genetic distances. Strong negative (or purifying) selection operates in FMDV populations subjected to enhanced mutagenesis. The quantifications provide evidence that extinction does not imply unusual increases of intrapopulation complexity, in support of the lethal defection model of virus extinction. PMID:18637173

[Analysis of the 4th generation outer space bred Angelica dahurica by FTIR spectroscopy].

PubMed

Zhu, Yan-ying; Wu, Peng-le; Liu, Mei-yi; Wang, Zhi-zhou; Guo, Xi-hua; Guan, Ying

2012-03-01

The major components of the 4th generation outer space bred angelica and the ground group were determined and analyzed by Fourier transform infrared spectroscopy (FTIR) and second derivative spectrum, considering the large mutation of the plants with space mutagenesis. The results show that the content of the coumarin (1741 cm(-1)), which is the main active components of the space angelica dahurica increased, and the content of the protein (1 459, 1 419 cm(-1)) and the fat (930 cm(-1)) increased slightly, whereas the content of the starch and the dietary fiber reduced drastically. There are obvious differences between the peak values of the second derivative spectra of the plants, revealing that the outer space angelica dahurica contained amine component at 1 279 cm(-1). Space mutation breeding is favor of breeding angelica with better idiosyncrasy.

Genomic profiles of lung cancer associated with idiopathic pulmonary fibrosis.

PubMed

Hwang, Ji An; Kim, Deokhoon; Chun, Sung-Min; Bae, SooHyun; Song, Joon Seon; Kim, Mi Young; Koo, Hyun Jung; Song, Jin Woo; Kim, Woo Sung; Lee, Jae Cheol; Kim, Hyeong Ryul; Choi, Chang-Min; Jang, Se Jin

2018-01-01

Little is known about the pathogenesis or molecular profiles of idiopathic pulmonary fibrosis-associated lung cancer (IPF-LC). This study was performed to investigate the genomic profiles of IPF-LC and to explore the possibility of defining potential therapeutic targets in IPF-LC. We assessed genomic profiles of IPF-LC by using targeted exome sequencing (OncoPanel version 2) in 35 matched tumour/normal pairs surgically resected between 2004 and 2014. Germline and somatic variant calling was performed with GATK HaplotypeCaller and MuTect with GATK SomaticIndelocator, respectively. Copy number analysis was conducted with CNVkit, with focal events determined by Genomic Identification of Significant Targets in Cancer 2.0, and pathway analysis (KEGG) with DAVID. Germline mutations in TERT (rs2736100, n = 33) and CDKN1A (rs2395655, n = 27) associated with idiopathic pulmonary fibrosis risk were detected in most samples. A total of 410 somatic mutations were identified, with an average of 11.7 per tumour, including 69 synonymous, 177 missense, 17 nonsense, 1 nonstop and 11 splice-site mutations, and 135 small coding indels. Spectra of the somatic mutations revealed predominant C > T transitions despite an extensive smoking history in most patients, suggesting a potential association between APOBEC-related mutagenesis and the development of IPF-LC. TP53 (22/35, 62.9%) and BRAF (6/35, 17.1%) were found to be significantly mutated in IPF-LC. Recurrent focal amplifications in three chromosomal loci (3q26.33, 7q31.2, and 12q14.3) and 9p21.3 deletion were identified, and genes associated with the JAK-STAT signalling pathway were significantly amplified in IPF-LC (P = 0.012). This study demonstrates that IPF-LC is genetically characterized by the presence of somatic mutations reflecting a variety of environmental exposures on the background of specific germline mutations, and is associated with potentially targetable alterations such as BRAF mutations. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Different mechanisms of radiation-induced loss of heterozygosity in two human lymphoid cell lines from a single donor

NASA Technical Reports Server (NTRS)

Wiese, C.; Gauny, S. S.; Liu, W. C.; Cherbonnel-Lasserre, C. L.; Kronenberg, A.

2001-01-01

Allelic loss is an important mutational mechanism in human carcinogenesis. Loss of heterozygosity (LOH) at an autosomal locus is one outcome of the repair of DNA double-strand breaks (DSBs) and can occur by deletion or by mitotic recombination. We report that mitotic recombination between homologous chromosomes occurred in human lymphoid cells exposed to densely ionizing radiation. We used cells derived from the same donor that express either normal TP53 (TK6 cells) or homozygous mutant TP53 (WTK1 cells) to assess the influence of TP53 on radiation-induced mutagenesis. Expression of mutant TP53 (Met 237 Ile) was associated with a small increase in mutation frequencies at the hemizygous HPRT (hypoxanthine phosphoribosyl transferase) locus, but the mutation spectra were unaffected at this locus. In contrast, WTK1 cells (mutant TP53) were 30-fold more susceptible than TK6 cells (wild-type TP53) to radiation-induced mutagenesis at the TK1 (thymidine kinase) locus. Gene dosage analysis combined with microsatellite marker analysis showed that the increase in TK1 mutagenesis in WTK1 cells could be attributed, in part, to mitotic recombination. The microsatellite marker analysis over a 64-cM region on chromosome 17q indicated that the recombinational events could initiate at different positions between the TK1 locus and the centromere. Virtually all of the recombinational LOH events extended beyond the TK1 locus to the most telomeric marker. In general, longer LOH tracts were observed in mutants from WTK1 cells than in mutants from TK6 cells. Taken together, the results demonstrate that the incidence of radi-ation-induced mutations is dependent on the genetic background of the cell at risk, on the locus examined, and on the mechanisms for mutation available at the locus of interest.

Comparative analysis of charged particle-induced autosomal mutations in murine cells and tissues

NASA Astrophysics Data System (ADS)

Kronenberg, Amy; Gauny, Stacey; Turker, Mitchell; Dan, Cristian; Kwoh, Ely

Carcinogenesis requires the accumulation of mutations and most of these mutations of occur on autosomes. This study seeks to determine the effect of the tissue microenvironment on the frequency and types of autosomal mutations in epithelial cells exposed to the types of charged particles in space radiation environments. Epithelial cells are the principal cells at risk for the development of solid cancers in humans. Aprt heterozygous mice from a cross between C57BL/6 and DBA/2 mice (B6D2F1) are used for these studies. The tissue of interest here is the kidney. We evaluated the effects of Fe ion on cytotoxicity, mutant frequency, and mutant spectra in kidney epithelium exposed in vivo. In vitro studies use primary kidney clones from B6D2F1 mice. Animals or cells were exposed to graded doses (0-2 Gy) of 1 GeV/amu Fe ions at the NASA Space Radiation Laboratories at Brookhaven National Laboratory. Animals were given whole body exposure in plexiglas holders. Cells were irradiated in T-75 flasks as monolayers. Cytotoxicity for cells exposed as monolayers were performed immediately post-irradiation. In vitro mutation assays were performed after a 5-6 day expression period post-irradiation, at which time cells were seeded in standard medium supplemented with 2,6 diaminopurine to screen for Aprt mutants. Colony formation was assessed in parallel in standard medium. In contrast, mice were euthanized after 2-4 months post-irradiation (early) or 8-10 months post-irradiation (late) to determine the cytotoxic and mutagenic response to Fe ion irradiation. Once the kidneys were digested, the cytotoxicity and mutation assays were performed using the same methodology employed for cells in vitro. Individual Apr t mutant colonies were collected from separate flasks exposed in vitro to 2 Gy of Fe ions. A similar group of Aprt mutants were collected from separate, un-irradiated flasks Aprt mutant colonies were also collected from individual kidneys for un-irradiated mice and for mice exposed to 2 Gy of Fe ions. Mutant spectra were analyzed via PCR using a series of heterozygous markers along mouse chromosome 8. Cytotoxicity assays were performed immediately after Fe ion exposure of cells from two primary clones. Cells irradiated in vitro demonstrated a dose-dependent decrement in cloning efficiency with no evidence of a shoulder. The results demonstrate the two clones behave similarly (unpaired t-test, p>0.3) with a D0 of 84.3 cGy for the combined data set. Mutation data were obtained using cells from one of the primary clones. In three experiments, we observed a linear dose-response for Aprt mutation with an induced mutant frzction of 1.06 x 10-3 /Gy. Kidney epithelial cells irradiated in vivo and incubated for 2-4 months in situ prior to harvest also showed an exponential reduction of cloning efficiency. Cells harvested 8-10 months postirradiation showed evidence of recovery for doses up to 1.5 Gy, but there was no improvement in cloning efficiency for kidney cells exposed to 2 Gy Fe ions in vivo evaluated at the late time point. Results for Aprt mutation induction in vivo indicated considerable inter-animal variation within each dose group (0, 1.0, 1.5 and 2 Gy). Fe ion exposures were mutagenic to the kidney, even at the lowest dose (p<0.01). A comparison of the mutant frequency results at the two harvest times indicates that the dose response did not vary with incubation time in vivo. Analysis of the pooled data from the 2-4 months harvests and the 8-10 month harvests indicated an increase in mutant frequency of 1.49 fold per Gy (p=0.01, CI 1.11-2.01). Molecular analysis of Aprtdeficient cells collected after a 2 Gy exposure to Fe ions in vitro showed an increased proportion of mutants arising via interstitial deletion or mitotic recombination, with an indication of an increase in chromosome loss. Similar results have been obtained for Aprt mutants isolated from mice exposed to 2 Gy of Fe ions, as compared with mutants collected from sham-irradiated mice. Taken together, the results to date demonstrate that Fe ions are mutagenic to mouse kidney epithelium exposed in vitro assayed at short times post-irradiation and they are also mutagenic to kidney epithelium exposed in vivo. While cytotoxicity is somewhat ameliorated in vivo, toxicity was evident at the highest dose up to 8-10 months post-exposure. A comparison of the Aprt mutant frequency analyses (in vitro vs. in vivo) and mutation spectra analyses (in vitro vs. in vivo) reflect similar trends. Supported by NASA grant T-403X to A. Kronenberg.

Reprint of "pFind-Alioth: A novel unrestricted database search algorithm to improve the interpretation of high-resolution MS/MS data".

PubMed

Chi, Hao; He, Kun; Yang, Bing; Chen, Zhen; Sun, Rui-Xiang; Fan, Sheng-Bo; Zhang, Kun; Liu, Chao; Yuan, Zuo-Fei; Wang, Quan-Hui; Liu, Si-Qi; Dong, Meng-Qiu; He, Si-Min

2015-11-03

Database search is the dominant approach in high-throughput proteomic analysis. However, the interpretation rate of MS/MS spectra is very low in such a restricted mode, which is mainly due to unexpected modifications and irregular digestion types. In this study, we developed a new algorithm called Alioth, to be integrated into the search engine of pFind, for fast and accurate unrestricted database search on high-resolution MS/MS data. An ion index is constructed for both peptide precursors and fragment ions, by which arbitrary digestions and a single site of any modifications and mutations can be searched efficiently. A new re-ranking algorithm is used to distinguish the correct peptide-spectrum matches from random ones. The algorithm is tested on several HCD datasets and the interpretation rate of MS/MS spectra using Alioth is as high as 60%-80%. Peptides from semi- and non-specific digestions, as well as those with unexpected modifications or mutations, can be effectively identified using Alioth and confidently validated using other search engines. The average processing speed of Alioth is 5-10 times faster than some other unrestricted search engines and is comparable to or even faster than the restricted search algorithms tested.This article is part of a Special Issue entitled: Computational Proteomics. Copyright © 2015 Elsevier B.V. All rights reserved.

TP53 mutations in primary breast carcinomas from white and African-Brazilian patients.

PubMed

Nagai, Maria Aparecida; Schaer Barbosa, Helenemarie; Zago, Marco Antonio; Araújo Silva, Wilson; Nishimoto, Inês Nobuko; Salaorni, Sibeli; Guerreiro Costa, Lívia Nery Franco; Silva Araújo, Marcos; Caldas Oliveira, Ana Gabriela; Mourâo Neto, Mário; Brentani, Maria Mitzi

2003-07-01

We have attempted to determine the incidence, nature and clinical significance of TP53 mutation in a group of white (242 cases) and African-Brazilian (52 cases) patients with breast cancer. The interethnic admixture as estimated by STR markers showed that white subjects displayed 67.9+/-0.4%, 25.0+/-1.7% and 7.0%+/-1.6% and the black populations had 34.4+/-1.9%, 56.2+/-1.9 and 9.4+/-2.2% respectively of European, African and Amerindian genes. Clinical parameters such as age, lymph node status and steroid receptors were similar in both groups. African-Brazilian patients presented more advanced lesions. Mutation screening was performed using polymerase chain reaction-single strand conformation analysis followed by sequencing. Compared to whites (13.6%), a relatively high frequency of TP53 mutation was found in blacks (32.7%) (p=0.001). African-Brazilian women have a larger proportion of mutations in exons 5 and 7, whereas white women have more mutations in exon 8. Mutations within exon 4 were found only in tumors of white patients. The spectra of TP53 mutations show that A:T-->G:C nucleotide transversion and G:C-->C:G transition were more common in African-Brazilian women whereas G:C-->T:A transversion occurs very frequently in whites. A high prevalence of G:C-->A:T nucleotide transitions and deletions was detected in both groups. No association was found between p53 gene mutation and tumor or clinical parameters independently of the ethnic group. With a median follow-up of 35.6 months for whites and 43.4 months for the blacks, no differences in overall survival were found. If white patients were stratified according to the type and location of TP53 mutations, patients with mutations affecting amino acids directly involved in DNA or Zn binding displayed a poor prognosis. The pattern of mutations found in our population seems to reflect a base line pattern observed in populations with similar ethnic profile with some modifications, which might be derived from specific etiological factors.

Thailand mutation and variation database (ThaiMUT).

PubMed

Ruangrit, Uttapong; Srikummool, Metawee; Assawamakin, Anunchai; Ngamphiw, Chumpol; Chuechote, Suparat; Thaiprasarnsup, Vilasinee; Agavatpanitch, Gallissara; Pasomsab, Ekawat; Yenchitsomanus, Pa-Thai; Mahasirimongkol, Surakameth; Chantratita, Wasun; Palittapongarnpim, Prasit; Uyyanonvara, Bunyarit; Limwongse, Chanin; Tongsima, Sissades

2008-08-01

With the completion of the human genome project, novel sequencing and genotyping technologies had been utilized to detect mutations. Such mutations have continually been produced at exponential rate by researchers in various communities. Based on the population's mutation spectra, occurrences of Mendelian diseases are different across ethnic groups. A proportion of Mendelian diseases can be observed in some countries at higher rates than others. Recognizing the importance of mutation effects in Thailand, we established a National and Ethnic Mutation Database (NEMDB) for Thai people. This database, named Thailand Mutation and Variation database (ThaiMUT), offers a web-based access to genetic mutation and variation information in Thai population. This NEMDB initiative is an important informatics tool for both research and clinical purposes to retrieve and deposit human variation data. The mutation data cataloged in ThaiMUT database were derived from journal articles available in PubMed and local publications. In addition to collected mutation data, ThaiMUT also records genetic polymorphisms located in drug related genes. ThaiMUT could then provide useful information for clinical mutation screening services for Mendelian diseases and pharmacogenomic researches. ThaiMUT can be publicly accessed from http://gi.biotec.or.th/thaimut.

Exome sequencing of bilateral testicular germ cell tumors suggests independent development lineages.

PubMed

Brabrand, Sigmund; Johannessen, Bjarne; Axcrona, Ulrika; Kraggerud, Sigrid M; Berg, Kaja G; Bakken, Anne C; Bruun, Jarle; Fosså, Sophie D; Lothe, Ragnhild A; Lehne, Gustav; Skotheim, Rolf I

2015-02-01

Intratubular germ cell neoplasia, the precursor of testicular germ cell tumors (TGCTs), is hypothesized to arise during embryogenesis from developmentally arrested primordial germ cells (PGCs) or gonocytes. In early embryonal life, the PGCs migrate from the yolk sac to the dorsal body wall where the cell population separates before colonizing the genital ridges. However, whether the malignant transformation takes place before or after this separation is controversial. We have explored the somatic exome-wide mutational spectra of bilateral TGCT to provide novel insight into the in utero critical time frame of malignant transformation and TGCT pathogenesis. Exome sequencing was performed in five patients with bilateral TGCT (eight tumors), of these three patients in whom both tumors were available (six tumors) and two patients each with only one available tumor (two tumors). Selected loci were explored by Sanger sequencing in 71 patients with bilateral TGCT. From the exome-wide mutational spectra, no identical mutations in any of the three bilateral tumor pairs were identified. Exome sequencing of all eight tumors revealed 87 somatic non-synonymous mutations (median 10 per tumor; range 5-21), some in already known cancer genes such as CIITA, NEB, platelet-derived growth factor receptor α (PDGFRA), and WHSC1. SUPT6H was found recurrently mutated in two tumors. We suggest independent development lineages of bilateral TGCT. Thus, malignant transformation into intratubular germ cell neoplasia is likely to occur after the migration of PGCs. We reveal possible drivers of TGCT pathogenesis, such as mutated PDGFRA, potentially with therapeutic implications for TGCT patients. Copyright © 2014 Neoplasia Press, Inc. Published by Elsevier Inc. All rights reserved.

The Impact of Environmental and Endogenous Damage on Somatic Mutation Load in Human Skin Fibroblasts

PubMed Central

Saini, Natalie; Chan, Kin; Grimm, Sara A.; Dai, Shuangshuang; Fargo, David C.; Kaufmann, William K.; Taylor, Jack A.; Lee, Eunjung; Cortes-Ciriano, Isidro; Park, Peter J.; Schurman, Shepherd H.; Malc, Ewa P.; Mieczkowski, Piotr A.

2016-01-01

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 PMID:27788131

Inference of directional selection and mutation parameters assuming equilibrium.

PubMed

Vogl, Claus; Bergman, Juraj

2015-12-01

In a classical study, Wright (1931) proposed a model for the evolution of a biallelic locus under the influence of mutation, directional selection and drift. He derived the equilibrium distribution of the allelic proportion conditional on the scaled mutation rate, the mutation bias and the scaled strength of directional selection. The equilibrium distribution can be used for inference of these parameters with genome-wide datasets of "site frequency spectra" (SFS). Assuming that the scaled mutation rate is low, Wright's model can be approximated by a boundary-mutation model, where mutations are introduced into the population exclusively from sites fixed for the preferred or unpreferred allelic states. With the boundary-mutation model, inference can be partitioned: (i) the shape of the SFS distribution within the polymorphic region is determined by random drift and directional selection, but not by the mutation parameters, such that inference of the selection parameter relies exclusively on the polymorphic sites in the SFS; (ii) the mutation parameters can be inferred from the amount of polymorphic and monomorphic preferred and unpreferred alleles, conditional on the selection parameter. Herein, we derive maximum likelihood estimators for the mutation and selection parameters in equilibrium and apply the method to simulated SFS data as well as empirical data from a Madagascar population of Drosophila simulans. Copyright © 2015 Elsevier Inc. All rights reserved.

DNA polymerase ι functions in the generation of tandem mutations during somatic hypermutation of antibody genes.

PubMed

Maul, Robert W; MacCarthy, Thomas; Frank, Ekaterina G; Donigan, Katherine A; McLenigan, Mary P; Yang, William; Saribasak, Huseyin; Huston, Donald E; Lange, Sabine S; Woodgate, Roger; Gearhart, Patricia J

2016-08-22

DNA polymerase ι (Pol ι) is an attractive candidate for somatic hypermutation in antibody genes because of its low fidelity. To identify a role for Pol ι, we analyzed mutations in two strains of mice with deficiencies in the enzyme: 129 mice with negligible expression of truncated Pol ι, and knock-in mice that express full-length Pol ι that is catalytically inactive. Both strains had normal frequencies and spectra of mutations in the variable region, indicating that loss of Pol ι did not change overall mutagenesis. We next examined if Pol ι affected tandem mutations generated by another error-prone polymerase, Pol ζ. The frequency of contiguous mutations was analyzed using a novel computational model to determine if they occur during a single DNA transaction or during two independent events. Analyses of 2,000 mutations from both strains indicated that Pol ι-compromised mice lost the tandem signature, whereas C57BL/6 mice accumulated significant amounts of double mutations. The results support a model where Pol ι occasionally accesses the replication fork to generate a first mutation, and Pol ζ extends the mismatch with a second mutation. @2016.

DNA polymerase ι functions in the generation of tandem mutations during somatic hypermutation of antibody genes

PubMed Central

Donigan, Katherine A.; Huston, Donald E.; Lange, Sabine S.

2016-01-01

DNA polymerase ι (Pol ι) is an attractive candidate for somatic hypermutation in antibody genes because of its low fidelity. To identify a role for Pol ι, we analyzed mutations in two strains of mice with deficiencies in the enzyme: 129 mice with negligible expression of truncated Pol ι, and knock-in mice that express full-length Pol ι that is catalytically inactive. Both strains had normal frequencies and spectra of mutations in the variable region, indicating that loss of Pol ι did not change overall mutagenesis. We next examined if Pol ι affected tandem mutations generated by another error-prone polymerase, Pol ζ. The frequency of contiguous mutations was analyzed using a novel computational model to determine if they occur during a single DNA transaction or during two independent events. Analyses of 2,000 mutations from both strains indicated that Pol ι–compromised mice lost the tandem signature, whereas C57BL/6 mice accumulated significant amounts of double mutations. The results support a model where Pol ι occasionally accesses the replication fork to generate a first mutation, and Pol ζ extends the mismatch with a second mutation. PMID:27455952

High Mutation Levels are Compatible with Normal Embryonic Development in Mlh1-Deficient Mice.

PubMed

Fan, Xiaoyan; Li, Yan; Zhang, Yulong; Sang, Meixiang; Cai, Jianhui; Li, Qiaoxia; Ozaki, Toshinori; Ono, Tetsuya; He, Dongwei

2016-10-01

To elucidate the role of the mismatch repair gene Mlh1 in genome instability during the fetal stage, spontaneous mutations were studied in Mlh1-deficient lacZ-transgenic mouse fetuses. Mutation levels were high at 9.5 days post coitum (dpc) and gradually increased during the embryonic stage, after which they remained unchanged. In addition, mutations that were found in brain, liver, spleen, small intestine and thymus showed similar levels and no statistically significant difference was found. The molecular nature of mutations at 12.5 dpc in fetuses of Mlh1 +/+ and Mlh1 -/- mice showed their own unique spectra, suggesting that deletion mutations were the main causes in the deficiency of the Mlh1 gene. Of note, fetuses of irradiated mice exhibited marked differences such as post-implantation loss and Mendelian distribution. Collectively, these results strongly suggest that high mutation ofMlh1 -/- -deficient fetuses has little effect on the fetuses during their early developmental stages, whereas Mlh1 -/- -deficient fetuses from X-ray irradiated mothers are clearly effected.

Clinical Characterization of the Pheochromocytoma and Paraganglioma Susceptibility Genes SDHA, TMEM127, MAX, and SDHAF2 for Gene-Informed Prevention

PubMed Central

Schiavi, Francesca; Ni, Ying; Welander, Jenny; Patocs, Attila; Ngeow, Joanne; Wellner, Ulrich; Malinoc, Angelica; Taschin, Elisa; Barbon, Giovanni; Lanza, Virginia; Söderkvist, Peter; Stenman, Adam; Larsson, Catharina; Svahn, Fredrika; Chen, Jin-Lian; Marquard, Jessica; Fraenkel, Merav; Walter, Martin A.; Peczkowska, Mariola; Prejbisz, Aleksander; Jarzab, Barbara; Hasse-Lazar, Kornelia; Petersenn, Stephan; Moeller, Lars C.; Meyer, Almuth; Reisch, Nicole; Trupka, Arnold; Brase, Christoph; Galiano, Matthias; Preuss, Simon F.; Kwok, Pingling; Lendvai, Nikoletta; Berisha, Gani; Makay, Özer; Boedeker, Carsten C.; Weryha, Georges; Racz, Karoly; Januszewicz, Andrzej; Walz, Martin K.; Gimm, Oliver; Opocher, Giuseppe; Eng, Charis; Neumann, Hartmut P. H.

2017-01-01

Importance Effective cancer prevention is based on accurate molecular diagnosis and results of genetic family screening, genotype-informed risk assessment, and tailored strategies for early diagnosis. The expanding etiology for hereditary pheochromocytomas and paragangliomas has recently included SDHA, TMEM127, MAX, and SDHAF2 as susceptibility genes. Clinical management guidelines for patients with germline mutations in these 4 newly included genes are lacking. Objective To study the clinical spectra and age-related penetrance of individuals with mutations in the SDHA, TMEM127, MAX, and SDHAF2 genes. Design, Setting, and Patients This study analyzed the prospective, longitudinally followed up European-American-Asian Pheochromocytoma-Paraganglioma Registry for prevalence of SDHA, TMEM127, MAX, and SDHAF2 germline mutation carriers from 1993 to 2016. Genetic predictive testing and clinical investigation by imaging from neck to pelvis was offered to mutation-positive registrants and their relatives to clinically characterize the pheochromocytoma/paraganglioma diseases associated with mutations of the 4 new genes. Main Outcomes and Measures Prevalence and spectra of germline mutations in the SDHA, TMEM127, MAX, and SDHAF2 genes were assessed. The clinical features of SDHA, TMEM127, MAX, and SDHAF2 disease were characterized. Results Of 972 unrelated registrants without mutations in the classic pheochromocytoma- and paraganglioma-associated genes (632 female [65.0%] and 340 male [35.0%]; age range, 8-80; mean [SD] age, 41.0 [13.3] years), 58 (6.0%) carried germline mutations of interest, including 29 SDHA, 20 TMEM127, 8 MAX, and 1 SDHAF2. Fifty-three of 58 patients (91%) had familial, multiple, extra-adrenal, and/or malignant tumors and/or were younger than 40 years. Newly uncovered are 7 of 63 (11%) malignant pheochromocytomas and paragangliomas in SDHA and TMEM127 disease. SDHA disease occurred as early as 8 years of age. Extra-adrenal tumors occurred in 28 mutation carriers (48%) and in 23 of 29 SDHA mutation carriers (79%), particularly with head and neck paraganglioma. MAX disease occurred almost exclusively in the adrenal glands with frequently bilateral tumors. Penetrance in the largest subset, SDHA carriers, was 39% at 40 years of age and is statistically different in index patients (45%) vs mutation-carrying relatives (13%; P < .001). Conclusions and Relevance The SDHA, TMEM127, MAX, and SDHAF2 genes may contribute to hereditary pheochromocytoma and paraganglioma. Genetic testing is recommended in patients at clinically high risk if the classic genes are mutation negative. Gene-specific prevention and/or early detection requires regular, systematic whole-body investigation. PMID:28384794

Clinical Characterization of the Pheochromocytoma and Paraganglioma Susceptibility Genes SDHA, TMEM127, MAX, and SDHAF2 for Gene-Informed Prevention.

PubMed

Bausch, Birke; Schiavi, Francesca; Ni, Ying; Welander, Jenny; Patocs, Attila; Ngeow, Joanne; Wellner, Ulrich; Malinoc, Angelica; Taschin, Elisa; Barbon, Giovanni; Lanza, Virginia; Söderkvist, Peter; Stenman, Adam; Larsson, Catharina; Svahn, Fredrika; Chen, Jin-Lian; Marquard, Jessica; Fraenkel, Merav; Walter, Martin A; Peczkowska, Mariola; Prejbisz, Aleksander; Jarzab, Barbara; Hasse-Lazar, Kornelia; Petersenn, Stephan; Moeller, Lars C; Meyer, Almuth; Reisch, Nicole; Trupka, Arnold; Brase, Christoph; Galiano, Matthias; Preuss, Simon F; Kwok, Pingling; Lendvai, Nikoletta; Berisha, Gani; Makay, Özer; Boedeker, Carsten C; Weryha, Georges; Racz, Karoly; Januszewicz, Andrzej; Walz, Martin K; Gimm, Oliver; Opocher, Giuseppe; Eng, Charis; Neumann, Hartmut P H

2017-09-01

Effective cancer prevention is based on accurate molecular diagnosis and results of genetic family screening, genotype-informed risk assessment, and tailored strategies for early diagnosis. The expanding etiology for hereditary pheochromocytomas and paragangliomas has recently included SDHA, TMEM127, MAX, and SDHAF2 as susceptibility genes. Clinical management guidelines for patients with germline mutations in these 4 newly included genes are lacking. To study the clinical spectra and age-related penetrance of individuals with mutations in the SDHA, TMEM127, MAX, and SDHAF2 genes. This study analyzed the prospective, longitudinally followed up European-American-Asian Pheochromocytoma-Paraganglioma Registry for prevalence of SDHA, TMEM127, MAX, and SDHAF2 germline mutation carriers from 1993 to 2016. Genetic predictive testing and clinical investigation by imaging from neck to pelvis was offered to mutation-positive registrants and their relatives to clinically characterize the pheochromocytoma/paraganglioma diseases associated with mutations of the 4 new genes. Prevalence and spectra of germline mutations in the SDHA, TMEM127, MAX, and SDHAF2 genes were assessed. The clinical features of SDHA, TMEM127, MAX, and SDHAF2 disease were characterized. Of 972 unrelated registrants without mutations in the classic pheochromocytoma- and paraganglioma-associated genes (632 female [65.0%] and 340 male [35.0%]; age range, 8-80; mean [SD] age, 41.0 [13.3] years), 58 (6.0%) carried germline mutations of interest, including 29 SDHA, 20 TMEM127, 8 MAX, and 1 SDHAF2. Fifty-three of 58 patients (91%) had familial, multiple, extra-adrenal, and/or malignant tumors and/or were younger than 40 years. Newly uncovered are 7 of 63 (11%) malignant pheochromocytomas and paragangliomas in SDHA and TMEM127 disease. SDHA disease occurred as early as 8 years of age. Extra-adrenal tumors occurred in 28 mutation carriers (48%) and in 23 of 29 SDHA mutation carriers (79%), particularly with head and neck paraganglioma. MAX disease occurred almost exclusively in the adrenal glands with frequently bilateral tumors. Penetrance in the largest subset, SDHA carriers, was 39% at 40 years of age and is statistically different in index patients (45%) vs mutation-carrying relatives (13%; P < .001). The SDHA, TMEM127, MAX, and SDHAF2 genes may contribute to hereditary pheochromocytoma and paraganglioma. Genetic testing is recommended in patients at clinically high risk if the classic genes are mutation negative. Gene-specific prevention and/or early detection requires regular, systematic whole-body investigation.

First biological and dosimetric results of the free flyer biostack experiment AO015 on LDEF

NASA Technical Reports Server (NTRS)

Reitz, G.; Buecker, H.; Facius, R.; Horneck, G.; Schaeffer, M.; Schott, J. U.; Bayonove, J.; Beaujean, R.; Benton, E. V.; Delpoux, M.

1991-01-01

The main objectives of the Biostack Experiment are to study the effectiveness of the structured components of the cosmic radiation to bacterial spores, plant seeds, and animal cysts for a long duration spaceflight and to get dosimetric data such as particle fluences and spectra and total doses for the Long Duration Exposure Facility orbit. The configuration of the experiment packages allows the localization of the trajectory of the particles in each biological layer and to correlate the potential biological impairment or injury with the physical characteristics of the responsible particle. Although the Biostack Experiment was designed for a long duration flight of only nine months, most of the biological systems show a high hatching or germination rate. Some of the first observations are an increase of the mutation rate of embryonic lethals in the second generation of Arabidopsis seeds, somatic mutations, and a reduction of growth rates of corn plants and a reduction of life span of Artemia salina shrimps. The different passive detector systems are also in a good shape and give access to a proper dosimetric analysis. The results are summarized, and some aspects of future analysis are shown.

Surface charge modification increases firefly luciferase rigidity without alteration in bioluminescence spectra.

PubMed

Mortazavi, Mojtaba; Hosseinkhani, Saman

2017-01-01

Protein engineering can provide useful approaches for loop anchoring and mutation of surface-exposed loop residues to Arg for the design of thermostable proteins. In this context and due to the high proportion of surface loops, some of the solvent-exposed residues in the Lampyris turkestanicus luciferase were mutated to Arg. Using the red-emitter mutant luciferase (E354R/Arg356), the single (-Q35R, -I182R, -I232R and -L300R), double (-Q35R/I232R) and triple (-Q35R/I232R/I182R) mutant luciferases were introduced. The relative remaining activity of -I232R, double and triple mutants increased significantly compared to the wild-type at 40°C. The optimal temperature of these mutants increased up to 40°C which were 15°C more than wild-type luciferase. It is anticipated that these mutations increased the local interactions that finally improved the thermostability and optimum temperature of luciferase. It should be noted that Arg substitution at amino acid positions 35, 182 and 232 had no effect on the bioluminescence emission spectra. Furthermore, these mutations have not significantly changed the specific activities of firefly luciferases. Finally, with the use of the homology modeling and molecular docking, the effects of these substitutions were evaluated. In conclusion, this study provides beneficial insights on how the thermal stability of luciferase can be improved by protein engineering for biological applications. Copyright © 2016 Elsevier Inc. All rights reserved.

The Spectrum of Replication Errors in the Absence of Error Correction Assayed Across the Whole Genome of Escherichia coli.

PubMed

Niccum, Brittany A; Lee, Heewook; MohammedIsmail, Wazim; Tang, Haixu; Foster, Patricia L

2018-06-15

When the DNA polymerase that replicates the Escherichia coli chromosome, DNA Pol III, makes an error, there are two primary defenses against mutation: proofreading by the epsilon subunit of the holoenzyme and mismatch repair. In proofreading deficient strains, mismatch repair is partially saturated and the cell's response to DNA damage, the SOS response, may be partially induced. To investigate the nature of replication errors, we used mutation accumulation experiments and whole genome sequencing to determine mutation rates and mutational spectra across the entire chromosome of strains deficient in proofreading, mismatch repair, and the SOS response. We report that a proofreading-deficient strain has a mutation rate 4,000-fold greater than wild-type strains. While the SOS response may be induced in these cells, it does not contribute to the mutational load. Inactivating mismatch repair in a proofreading-deficient strain increases the mutation rate another 1.5-fold. DNA polymerase has a bias for converting G:C to A:T base pairs, but proofreading reduces the impact of these mutations, helping to maintain the genomic G:C content. These findings give an unprecedented view of how polymerase and error-correction pathways work together to maintain E. coli' s low mutation rate of 1 per thousand generations. Copyright © 2018, Genetics.

Autosomal recessive posterior column ataxia with retinitis pigmentosa caused by novel mutations in the FLVCR1 gene.

PubMed

Shaibani, Aziz; Wong, Lee-Jun; Wei Zhang, Victor; Lewis, Richard Alan; Shinawi, Marwan

2015-01-01

Posterior column ataxia with retinitis pigmentosa (PCARP) is an autosomal recessive disorder characterized by severe sensory ataxia, muscle weakness and atrophy, and progressive pigmentary retinopathy. Recently, mutations in the FLVCR1 gene were described in four families with this condition. We investigated the molecular basis and studied the phenotype of PCARP in a new family. The proband is a 33-year-old woman presented with sensory polyneuropathy and retinitis pigmentosa (RP). The constellation of clinical findings with normal metabolic and genetic evaluation, including mitochondrial DNA (mtDNA) analysis and normal levels of phytanic acid and vitamin E, prompted us to seek other causes of our patient's condition. Sequencing of FLVCR1 in the proband and targeted mutation testing in her two affected siblings revealed two novel variants, c.1547G > A (p.R516Q) and c.1593+5_+8delGTAA predicted, respectively, to be highly conserved throughout evolution and affecting the normal splicing, therefore, deleterious. This study supports the pathogenic role of FLVCR1 in PCARP and expands the molecular and clinical spectra of PCARP. We show for the first time that nontransmembrane domain (TMD) mutations in the FLVCR1 can cause PCARP, suggesting different mechanisms for pathogenicity. Our clinical data reveal that impaired sensation can be part of the phenotypic spectrum of PCARP. This study along with previously reported cases suggests that targeted sequencing of the FLVCR1 gene should be considered in patients with severe sensory ataxia, RP, and peripheral sensory neuropathy.

A method for multi-codon scanning mutagenesis of proteins based on asymmetric transposons.

PubMed

Liu, Jia; Cropp, T Ashton

2012-02-01

Random mutagenesis followed by selection or screening is a commonly used strategy to improve protein function. Despite many available methods for random mutagenesis, nearly all generate mutations at the nucleotide level. An ideal mutagenesis method would allow for the generation of 'codon mutations' to change protein sequence with defined or mixed amino acids of choice. Herein we report a method that allows for mutations of one, two or three consecutive codons. Key to this method is the development of a Mu transposon variant with asymmetric terminal sequences. As a demonstration of the method, we performed multi-codon scanning on the gene encoding superfolder GFP (sfGFP). Characterization of 50 randomly chosen clones from each library showed that more than 40% of the mutants in these three libraries contained seamless, in-frame mutations with low site preference. By screening only 500 colonies from each library, we successfully identified several spectra-shift mutations, including a S205D variant that was found to bear a single excitation peak in the UV region.

Resonance Raman spectra of an O2-binding H-NOX domain reveal heme relaxation upon mutation.

PubMed

Tran, Rosalie; Boon, Elizabeth M; Marletta, Michael A; Mathies, Richard A

2009-09-15

Resonance Raman spectra were measured for the wild type Heme-Nitric oxide/OXygen binding domain from Thermoanaerobacter tengcongensis (Tt H-NOX WT) and three other Tt H-NOX proteins containing mutations at key conserved residues to determine the heme conformation in solution. The most dramatic changes in heme conformation occurred in the O2-bound forms, and the single Tt H-NOX P115A mutation was sufficient to generate a significant relaxation of the chromophore. Clear evidence of heme relaxation in the Tt H-NOX I5L, P115A, and I5L/P115A mutants in solution is demonstrated by the observation of reduced resonance Raman intensities for several out-of-plane low frequency modes (e.g., gamma11, gamma12, gamma13, and gamma15) in the 400-750 cm(-1) region known to be sensitive to ruffling and saddling deformations, as well as increased vibrational frequencies for the core heme skeletal stretching modes, nu3, nu2, and nu10. In addition, all three mutants exhibited some degree of heme conformational heterogeneity based on several broad skeletal markers (e.g., nu10) in the high frequency region. These results are comparable to those observed by Olea et al. for Tt H-NOX P115A in crystal form, where four different heme structures were determined from a single unit cell. On the basis of the resonance Raman spectra, it is clear that the actual heme conformation for Tt H-NOX P115A in solution is considerably more relaxed than that of the WT protein, with increased flexibility within the protein pocket, allowing for rapid sampling of alternate conformations.

ATP interacts with the CPVT mutation-associated central domain of the cardiac ryanodine receptor.

PubMed

Blayney, Lynda; Beck, Konrad; MacDonald, Ewan; D'Cruz, Leon; Nomikos, Michail; Griffiths, Julia; Thanassoulas, Angelos; Nounesis, George; Lai, F Anthony

2013-10-01

This study was designed to determine whether the cardiac ryanodine receptor (RyR2) central domain, a region associated with catecholamine polymorphic ventricular tachycardia (CPVT) mutations, interacts with the RyR2 regulators, ATP and the FK506-binding protein 12.6 (FKBP12.6). Wild-type (WT) RyR2 central domain constructs (G(2236)to G(2491)) and those containing the CPVT mutations P2328S and N2386I, were expressed as recombinant proteins. Folding and stability of the proteins were examined by circular dichroism (CD) spectroscopy and guanidine hydrochloride chemical denaturation. The far-UV CD spectra showed a soluble stably-folded protein with WT and mutant proteins exhibiting a similar secondary structure. Chemical denaturation analysis also confirmed a stable protein for both WT and mutant constructs with similar two-state unfolding. ATP and caffeine binding was measured by fluorescence spectroscopy. Both ATP and caffeine bound with an EC50 of ~200-400μM, and the affinity was the same for WT and mutant constructs. Sequence alignment with other ATP binding proteins indicated the RyR2 central domain contains the signature of an ATP binding pocket. Interaction of the central domain with FKBP12.6 was tested by glutaraldehyde cross-linking and no association was found. The RyR2 central domain, expressed as a 'correctly' folded recombinant protein, bound ATP in accord with bioinformatics evidence of conserved ATP binding sequence motifs. An interaction with FKBP12.6 was not evident. CPVT mutations did not disrupt the secondary structure nor binding to ATP. Part of the RyR2 central domain CPVT mutation cluster, can be expressed independently with retention of ATP binding. Copyright © 2013 Elsevier B.V. All rights reserved.

Genome-Wide Estimates of Mutation Rates and Spectrum in Schizosaccharomyces pombe Indicate CpG Sites are Highly Mutagenic Despite the Absence of DNA Methylation

PubMed Central

Behringer, Megan G.; Hall, David W.

2015-01-01

We accumulated mutations for 1952 generations in 79 initially identical, haploid lines of the fission yeast Schizosaccharomyces pombe, and then performed whole-genome sequencing to determine the mutation rates and spectrum. We captured 696 spontaneous mutations across the 79 mutation accumulation (MA) lines. We compared the mutation spectrum and rate to a recently published equivalent experiment on the same species, and to another model ascomycetous yeast, the budding yeast Saccharomyces cerevisiae. While the two species are approximately 600 million years diverged from each other, they share similar life histories, genome size and genomic G/C content. We found that Sc. pombe and S. cerevisiae have similar mutation rates, but Sc. pombe exhibits a stronger insertion bias. Intriguingly, we observed an increased mutation rate at cytosine nucleotides, specifically CpG nucleotides, which is also seen in S. cerevisiae. However, the absence of methylation in Sc. pombe and the pattern of mutation at these sites, primarily C → A as opposed to C → T, strongly suggest that the increased mutation rate is not caused by deamination of methylated cytosines. This result implies that the high mutability of CpG dinucleotides in other species may be caused in part by a methylation-independent mechanism. Many of our findings mirror those seen in the recent study, despite the use of different passaging conditions, indicating that MA is a reliable method for estimating mutation rates and spectra. PMID:26564949

A shifting mutational landscape in 6 nutritional states: Stress-induced mutagenesis as a series of distinct stress input-mutation output relationships.

PubMed

Maharjan, Ram P; Ferenci, Thomas

2017-06-01

Environmental stresses increase genetic variation in bacteria, plants, and human cancer cells. The linkage between various environments and mutational outcomes has not been systematically investigated, however. Here, we established the influence of nutritional stresses commonly found in the biosphere (carbon, phosphate, nitrogen, oxygen, or iron limitation) on both the rate and spectrum of mutations in Escherichia coli. We found that each limitation was associated with a remarkably distinct mutational profile. Overall mutation rates were not always elevated, and nitrogen, iron, and oxygen limitation resulted in major spectral changes but no net increase in rate. Our results thus suggest that stress-induced mutagenesis is a diverse series of stress input-mutation output linkages that is distinct in every condition. Environment-specific spectra resulted in the differential emergence of traits needing particular mutations in these settings. Mutations requiring transpositions were highest under iron and oxygen limitation, whereas base-pair substitutions and indels were highest under phosphate limitation. The unexpected diversity of input-output effects explains some important phenomena in the mutational biases of evolving genomes. The prevalence of bacterial insertion sequence transpositions in the mammalian gut or in anaerobically stored cultures is due to environmentally determined mutation availability. Likewise, the much-discussed genomic bias towards transition base substitutions in evolving genomes can now be explained as an environment-specific output. Altogether, our conclusion is that environments influence genetic variation as well as selection.

A shifting mutational landscape in 6 nutritional states: Stress-induced mutagenesis as a series of distinct stress input–mutation output relationships

PubMed Central

Maharjan, Ram P.

2017-01-01

Environmental stresses increase genetic variation in bacteria, plants, and human cancer cells. The linkage between various environments and mutational outcomes has not been systematically investigated, however. Here, we established the influence of nutritional stresses commonly found in the biosphere (carbon, phosphate, nitrogen, oxygen, or iron limitation) on both the rate and spectrum of mutations in Escherichia coli. We found that each limitation was associated with a remarkably distinct mutational profile. Overall mutation rates were not always elevated, and nitrogen, iron, and oxygen limitation resulted in major spectral changes but no net increase in rate. Our results thus suggest that stress-induced mutagenesis is a diverse series of stress input–mutation output linkages that is distinct in every condition. Environment-specific spectra resulted in the differential emergence of traits needing particular mutations in these settings. Mutations requiring transpositions were highest under iron and oxygen limitation, whereas base-pair substitutions and indels were highest under phosphate limitation. The unexpected diversity of input–output effects explains some important phenomena in the mutational biases of evolving genomes. The prevalence of bacterial insertion sequence transpositions in the mammalian gut or in anaerobically stored cultures is due to environmentally determined mutation availability. Likewise, the much-discussed genomic bias towards transition base substitutions in evolving genomes can now be explained as an environment-specific output. Altogether, our conclusion is that environments influence genetic variation as well as selection. PMID:28594817

Screening mutations in drug-resistant Mycobacterium tuberculosis strains in Yunnan, China.

PubMed

Li, Daoqun; Song, Yuzhu; Zhang, Cheng-Lin; Li, Xiaofei; Xia, Xueshan; Zhang, A-Mei

Drug-resistant tuberculosis (DR-TB), especially multidrug-resistant tuberculosis (MDR-TB), is a serious medical and societal problem in China. The purpose of this study was to evaluate the mutation characteristics of drug-resistant Mycobacterium tuberculosis (M. tuberculosis) isolates in Yunnan, China. Drug susceptibility testing (DST) was performed in 523 clinical M. tuberculosis isolates. Six drug resistance genes (katG, inhA, rpoB, rpsL, embB, and pncA) were selected to screen for mutations. In total, 54 clinical M. tuberculosis strains were identified as drug-resistant by DST, including 18 single drug-resistant (SDR) strains and 36 multidrug-resistant (MDR) strains. Twenty-four types of mutations in five genes (excluding the inhA gene) were screened in forty-one strains. Six novel mutations were identified in this study, including three missense mutations (p.S302R in katG, p.D78G in embB, and p.M1I in pncA), two frameshift mutations (408 ins A and 538-580 del in pncA), and one mutation in a control region (-6 C>T located upstream of rpsL). The mutation frequencies in the hotspot mutation regions in the katG, rpoB, rpsL, embB, and pncA genes were 92.5%, 44.4%, 54.2%, 52.6%, and 37.5%, respectively. The mutation spectra and frequencies seemed somewhat unique in the Yunnan DR-TB strains. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Overlapping spectra of SMAD4 mutations in juvenile polyposis (JP) and JP-HHT syndrome.

PubMed

Gallione, Carol; Aylsworth, Arthur S; Beis, Jill; Berk, Terri; Bernhardt, Barbara; Clark, Robin D; Clericuzio, Carol; Danesino, Cesare; Drautz, Joanne; Fahl, Jeffrey; Fan, Zheng; Faughnan, Marie E; Ganguly, Arupa; Garvie, John; Henderson, Katharine; Kini, Usha; Leedom, Tracey; Ludman, Mark; Lux, Andreas; Maisenbacher, Melissa; Mazzucco, Sara; Olivieri, Carla; Ploos van Amstel, Johannes K; Prigoda-Lee, Nadia; Pyeritz, Reed E; Reardon, Willie; Vandezande, Kirk; Waldman, J Deane; White, Robert I; Williams, Charles A; Marchuk, Douglas A

2010-02-01

Juvenile polyposis (JP) and hereditary hemorrhagic telangiectasia (HHT) are clinically distinct diseases caused by mutations in SMAD4 and BMPR1A (for JP) and endoglin and ALK1 (for HHT). Recently, a combined syndrome of JP-HHT was described that is also caused by mutations in SMAD4. Although both JP and JP-HHT are caused by SMAD4 mutations, a possible genotype:phenotype correlation was noted as all of the SMAD4 mutations in the JP-HHT patients were clustered in the COOH-terminal MH2 domain of the protein. If valid, this correlation would provide a molecular explanation for the phenotypic differences, as well as a pre-symptomatic diagnostic test to distinguish patients at risk for the overlapping but different clinical features of the disorders. In this study, we collected 19 new JP-HHT patients from which we identified 15 additional SMAD4 mutations. We also reviewed the literature for other reports of JP patients with HHT symptoms with confirmed SMAD4 mutations. Our combined results show that although the SMAD4 mutations in JP-HHT patients do show a tendency to cluster in the MH2 domain, mutations in other parts of the gene also cause the combined syndrome. Thus, any mutation in SMAD4 can cause JP-HHT. Any JP patient with a SMAD4 mutation is, therefore, at risk for the visceral manifestations of HHT and any HHT patient with SMAD4 mutation is at risk for early onset gastrointestinal cancer. In conclusion, a patient who tests positive for any SMAD4 mutation must be considered at risk for the combined syndrome of JP-HHT and monitored accordingly. Copyright 2010 Wiley-Liss, Inc.

Contribution of APC and MUTYH mutations to familial adenomatous polyposis susceptibility in Hungary.

PubMed

Papp, Janos; Kovacs, Marietta Eva; Matrai, Zoltan; Orosz, Enikő; Kásler, Miklós; Børresen-Dale, Anne-Lise; Olah, Edith

2016-01-01

Familial adenomatous polyposis (FAP) is a colorectal cancer predisposition syndrome with considerable genetic and phenotypic heterogeneity, defined by the development of multiple adenomas throughout the colorectum. FAP is caused either by monoallelic mutations in the adenomatous polyposis coli gene APC, or by biallelic germline mutations of MUTYH, this latter usually presenting with milder phenotype. The aim of the present study was to characterize the genotype and phenotype of Hungarian FAP patients. Mutation screening of 87 unrelated probands from FAP families (21 of them presented as the attenuated variant of the disease, showing <100 polyps) was performed using DNA sequencing and multiplex ligation-dependent probe amplification. Twenty-four different pathogenic mutations in APC were identified in 65 patients (75 %), including nine cases (37.5 %) with large genomic alterations. Twelve of the point mutations were novel. In addition, APC-negative samples were also tested for MUTYH mutations and we were able to identify biallelic pathogenic mutations in 23 % of these cases (5/22). Correlations between the localization of APC mutations and the clinical manifestations of the disease were observed, cases with a mutation in the codon 1200-1400 region showing earlier age of disease onset (p < 0.003). There were only a few, but definitive dissimilarities between APC- and MUTYH-associated FAP in our cohort: the age at onset of polyposis was significantly delayed for biallelic MUTYH mutation carriers as compared to patients with an APC mutation. Our data represent the first comprehensive study delineating the mutation spectra of both APC and MUTYH in Hungarian FAP families, and underscore the overlap between the clinical characteristics of APC- and MUTYH-associated phenotypes, necessitating a more appropriate clinical characterization of FAP families.

Genome sequencing of mucosal melanomas reveals that they are driven by distinct mechanisms from cutaneous melanoma.

PubMed

Furney, Simon J; Turajlic, Samra; Stamp, Gordon; Nohadani, Mahrokh; Carlisle, Anna; Thomas, J Meirion; Hayes, Andrew; Strauss, Dirk; Gore, Martin; van den Oord, Joost; Larkin, James; Marais, Richard

2013-07-01

Mucosal melanoma displays distinct clinical and epidemiological features compared to cutaneous melanoma. Here we used whole genome and whole exome sequencing to characterize the somatic alterations and mutation spectra in the genomes of ten mucosal melanomas. We observed somatic mutation rates that are considerably lower than occur in sun-exposed cutaneous melanoma, but comparable to the rates seen in cancers not associated with exposure to known mutagens. In particular, the mutation signatures are not indicative of ultraviolet light- or tobacco smoke-induced DNA damage. Genes previously reported as mutated in other cancers were also mutated in mucosal melanoma. Notably, there were substantially more copy number and structural variations in mucosal melanoma than have been reported in cutaneous melanoma. Thus, mucosal and cutaneous melanomas are distinct diseases with discrete genetic features. Our data suggest that different mechanisms underlie the genesis of these diseases and that structural variations play a more important role in mucosal than in cutaneous melanomagenesis. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The determination of complete human mitochondrial DNA sequences in single cells: implications for the study of somatic mitochondrial DNA point mutations

PubMed Central

Taylor, Robert W.; Taylor, Geoffrey A.; Durham, Steve E.; Turnbull, Douglass M.

2001-01-01

Studies of single cells have previously shown intracellular clonal expansion of mitochondrial DNA (mtDNA) mutations to levels that can cause a focal cytochrome c oxidase (COX) defect. Whilst techniques are available to study mtDNA rearrangements at the level of the single cell, recent interest has focused on the possible role of somatic mtDNA point mutations in ageing, neurodegenerative disease and cancer. We have therefore developed a method that permits the reliable determination of the entire mtDNA sequence from single cells without amplifying contaminating, nuclear-embedded pseudogenes. Sequencing and PCR–RFLP analyses of individual COX-negative muscle fibres from a patient with a previously described heteroplasmic COX II (T7587C) mutation indicate that mutant loads as low as 30% can be reliably detected by sequencing. This technique will be particularly useful in identifying the mtDNA mutational spectra in age-related COX-negative cells and will increase our understanding of the pathogenetic mechanisms by which they occur. PMID:11470889

The iron-sulfur cluster of electron transfer flavoprotein-ubiquinone oxidoreductase is the electron acceptor for electron transfer flavoprotein.

PubMed

Swanson, Michael A; Usselman, Robert J; Frerman, Frank E; Eaton, Gareth R; Eaton, Sandra S

2008-08-26

Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) accepts electrons from electron transfer flavoprotein (ETF) and reduces ubiquinone from the ubiquinone pool. It contains one [4Fe-4S] (2+,1+) and one FAD, which are diamagnetic in the isolated oxidized enzyme and can be reduced to paramagnetic forms by enzymatic donors or dithionite. In the porcine protein, threonine 367 is hydrogen bonded to N1 and O2 of the flavin ring of the FAD. The analogous site in Rhodobacter sphaeroides ETF-QO is asparagine 338. Mutations N338T and N338A were introduced into the R. sphaeroides protein by site-directed mutagenesis to determine the impact of hydrogen bonding at this site on redox potentials and activity. The mutations did not alter the optical spectra, EPR g-values, spin-lattice relaxation rates, or the [4Fe-4S] (2+,1+) to FAD point-dipole interspin distances. The mutations had no impact on the reduction potential for the iron-sulfur cluster, which was monitored by changes in the continuous wave EPR signals of the [4Fe-4S] (+) at 15 K. For the FAD semiquinone, significantly different potentials were obtained by monitoring the titration at 100 or 293 K. Based on spectra at 293 K the N338T mutation shifted the first and second midpoint potentials for the FAD from +47 and -30 mV for wild type to -11 and -19 mV, respectively. The N338A mutation decreased the potentials to -37 and -49 mV. Lowering the midpoint potentials resulted in a decrease in the quinone reductase activity and negligible impact on disproportionation of ETF 1e (-) catalyzed by ETF-QO. These observations indicate that the FAD is involved in electron transfer to ubiquinone but not in electron transfer from ETF to ETF-QO. Therefore, the iron-sulfur cluster is the immediate acceptor from ETF.

The Iron-Sulfur Cluster of Electron Transfer Flavoprotein-Ubiquinone Oxidoreductase Is the Electron Acceptor for Electron Transfer Flavoprotein†

PubMed Central

Swanson, Michael A.; Usselman, Robert J.; Frerman, Frank E.; Eaton, Gareth R.; Eaton, Sandra S.

2009-01-01

Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) accepts electrons from electron transfer flavoprotein (ETF) and reduces ubiquinone from the ubiquinone pool. It contains one [4Fe-4S]2+,1+ and one FAD, which are diamagnetic in the isolated oxidized enzyme and can be reduced to paramagnetic forms by enzymatic donors or dithionite. In the porcine protein, threonine 367 is hydrogen bonded to N1 and O2 of the flavin ring of the FAD. The analogous site in Rhodobacter sphaeroides ETF-QO is asparagine 338. Mutations N338T and N338A were introduced into the R. sphaeroides protein by site-directed mutagenesis to determine the impact of hydrogen bonding at this site on redox potentials and activity. The mutations did not alter the optical spectra, EPR g-values, spin-lattice relaxation rates, or the [4Fe-4S]2+,1+ to FAD point-dipole interspin distances. The mutations had no impact on the reduction potential for the iron-sulfur cluster, which was monitored by changes in the continuous wave EPR signals of the [4Fe-4S]+ at 15 K. For the FAD semiquinone, significantly different potentials were obtained by monitoring the titration at 100 or 293 K. Based on spectra at 293 K the N338T mutation shifted the first and second midpoint potentials for the FAD from +47 and -30 mV for wild type to -11 and -19 mV, respectively. The N338A mutation decreased the potentials to -37 and -49 mV. Lowering the midpoint potentials resulted in a decrease in the quinone reductase activity and negligible impact on disproportionation of ETF1e- catalyzed by ETF-QO. These observations indicate that the FAD is involved in electron transfer to ubiquinone but not in electron transfer from ETF to ETF-QO. Therefore, the iron-sulfur cluster is the immediate acceptor from ETF. PMID:9585549

Influence of Electron–Holes on DNA Sequence-Specific Mutation Rates

PubMed Central

Suárez-Villagrán, Martha Y; Azevedo, Ricardo B R; Miller, John H

2018-01-01

Abstract Biases in mutation rate can influence molecular evolution, yielding rates of evolution that vary widely in different parts of the genome and even among neighboring nucleotides. Here, we explore one possible mechanism of influence on sequence-specific mutation rates, the electron–hole, which can localize and potentially trigger a replication mismatch. A hole is a mobile site of positive charge created during one-electron oxidation by, for example, radiation, contact with a mutagenic agent, or oxidative stress. Its quantum wavelike properties cause it to localize at various sites with probabilities that vary widely, by orders of magnitude, and depend strongly on the local sequence. We find significant correlations between hole probabilities and mutation rates within base triplets, observed in published mutation accumulation experiments on four species of bacteria. We have also computed hole probability spectra for hypervariable segment I of the human mtDNA control region, which contains several mutational hotspots, and for heptanucleotides in noncoding regions of the human genome, whose polymorphism levels have recently been reported. We observe significant correlations between hole probabilities, and context-specific mutation and substitution rates. The correlation with hole probability cannot be explained entirely by CpG methylation in the heptanucleotide data. Peaks in hole probability tend to coincide with mutational hotspots, even in mtDNA where CpG methylation is rare. Our results suggest that hole-enhanced mutational mechanisms, such as oxidation-stabilized tautomerization and base deamination, contribute to molecular evolution. PMID:29617801

MUTAGENICITY AND MUTATION SPECTRA OF AIR AND WATER: ROLE OF GSST1-1 AND AKR GENOTYPES

EPA Science Inventory

Recent molecular epidemiology research confirms that an increased risk for bladder cancer is associated with dermal/inhalation exposure to chlorinated water (from bathing/showering and/or swimming), rather than to drinking the water, and that risk is enhanced in people carrying a...

Association between Mutation Spectra and Stable and Unstable DNA Adduct Profiles in Salmonella for Benzo[a]pyrene and Dibenzo[a.l]pyrene

EPA Science Inventory

Benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP) are two polycyclic aromatic hydrocarbons (PAHs) that exhibit distinctly different mutagenicity and carcinogenicity profiles. Although some studies show that these PAHs produce unstable DNA adducts, conflicting data and arguments ha...

Impact of Site-Directed Mutant Luciferase on Quantitative Green and Orange/Red Emission Intensities in Firefly Bioluminescence

NASA Astrophysics Data System (ADS)

Wang, Yu; Akiyama, Hidefumi; Terakado, Kanako; Nakatsu, Toru

2013-08-01

Firefly bioluminescence has attracted great interest because of its high quantum yield and intriguing modifiable colours. Modifications to the structure of the enzyme luciferase can change the emission colour of firefly bioluminescence, and the mechanism of the colour change has been intensively studied by biochemists, structural biologists, optical physicists, and quantum-chemistry theorists. Here, we report on the quantitative spectra of firefly bioluminescence catalysed by wild-type and four site-directed mutant luciferases. While the mutation caused different emission spectra, the spectra differed only in the intensity of the green component (λmax ~ 560 nm). In contrast, the orange (λmax ~ 610 nm) and red (λmax ~ 650 nm) components present in all the spectra were almost unaffected by the modifications to the luciferases and changes in pH. Our results reveal that the intensity of the green component is the unique factor that is influenced by the luciferase structure and other reaction conditions.

Specific UV-induced mutation spectrum in the p53 gene of skin tumors from DNA-repair-deficient xeroderma pigmentosum patients

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dumaz, N.; Drougard, C.; Sarasin, A.

1993-11-15

The UV component of sunlight is the major carcinogen involved in the etiology of skin cancers. The authors have studied the rare, hereditary syndrome xeroderma pigmentosum (XP), which is characterized by a very high incidence of cutaneous tumors on exposed skin at an early age, probably due to a deficiency in excision repair of UV-induced lesions. It is interesting to determine the UV mutation spectrum in XP skin tumors in order to correlate the absence of repair of specific DNA lesions and the initiation of skin tumors. The p53 gene is frequently mutated in human cancers and represents a goodmore » target for studying mutation spectra since there are >100 potential sites for phenotypic mutations. Using reverse transcription-PCR and single-strand conformation polymorphism to analyze >40 XP skin tumors (mainly basal and squamous cell carcinomas), the authors have found that 40% (17 out of 43) contained at least one point mutation on the p53 gene. All the mutations were located at dipyrimidine sites, essentially at CC sequences, which are hot spots for UV-induced DNA lesions. Sixty-one percent of these mutations were tandem CC [yields] TT mutations considered to be unique to UV-induced lesions; these mutations are not observed in internal human tumors. All the mutations, except two, must be due to translesion synthesis of unrepaired dipyrimidine lesions left on the nontranscribed strand. These results show the existence of preferential repair of UV lesions [either pyrimidine dimers or pyrimidine-pyrimidone (6-4) photoproducts] on the transcribed strand in human tissues.« less

Mutational effects of γ-rays and carbon ion beams on Arabidopsis seedlings

PubMed Central

Yoshihara, Ryouhei; Nozawa, Shigeki; Hase, Yoshihiro; Narumi, Issay; Hidema, Jun; Sakamoto, Ayako N.

2013-01-01

To assess the mutational effects of radiation on vigorously proliferating plant tissue, the mutation spectrum was analyzed with Arabidopsis seedlings using the plasmid-rescue method. Transgenic plants containing the Escherichia coli rpsL gene were irradiated with γ-rays and carbon ion beams (320-MeV 12C6+), and mutations in the rpsL gene were analyzed. Mutant frequency increased significantly following irradiation by γ-rays, but not by 320-MeV 12C6+. Mutation spectra showed that both radiations increased the frequency of frameshifts and other mutations, including deletions and insertions, but only γ-rays increased the frequency of total base substitutions. These results suggest that the type of DNA lesions which cause base substitutions were less often induced by 320-MeV 12C6+ than by γ-rays in Arabidopsis seedlings. Furthermore, γ-rays never increased the frequencies of G:C to T:A or A:T to C:G transversions, which are caused by oxidized guanine; 320-MeV 12C6+, however, produced a slight increase in both transversions. Instead, γ-rays produced a significant increase in the frequency of G:C to A:T transitions. These results suggest that 8-oxoguanine has little effect on mutagenesis in Arabidopsis cells. PMID:23728320

Mutations and Deletions in PCDH19 Account for Various Familial or Isolated Epilepsies in Females

PubMed Central

Depienne, Christel; Trouillard, Oriane; Bouteiller, Delphine; Gourfinkel-An, Isabelle; Poirier, Karine; Rivier, François; Berquin, Patrick; Nabbout, Rima; Chaigne, Denys; Steschenko, Dominique; Gautier, Agnès; Hoffman-Zacharska, Dorota; Lannuzel, Annie; Lackmy-Port-Lis, Marilyn; Maurey, Hélène; Dusser, Anne; Bru, Marie; Gilbert-Dussardier, Brigitte; Roubertie, Agathe; Kaminska, Anna; Whalen, Sandra; Mignot, Cyril; Baulac, Stéphanie; Lesca, Gaetan; Arzimanoglou, Alexis; LeGuern, Eric

2011-01-01

Mutations in PCDH19, encoding protocadherin 19 on chromosome X, cause familial epilepsy and mental retardation limited to females or Dravet-like syndrome. Heterozygous females are affected while hemizygous males are spared, this unusual mode of inheritance being probably due to a mechanism called cellular interference. To extend the mutational and clinical spectra associated with PCDH19, we screened 150 unrelated patients (113 females) with febrile and afebrile seizures for mutations or rearrangements in the gene. Fifteen novel point mutations were identified in 15 female patients (6 sporadic and 9 familial cases). In addition, qPCR revealed two whole gene deletions and one partial deletion in 3 sporadic female patients. Clinical features were highly variable but included almost constantly a high sensitivity to fever and clusters of brief seizures. Interestingly, cognitive functions were normal in several family members of 2 families: the familial condition in family 1 was suggestive of Generalized Epilepsy with Febrile Seizures Plus (GEFS+) whereas all three affected females had partial cryptogenic epilepsy. These results show that mutations in PCDH19 are a relatively frequent cause of epilepsy in females and should be considered even in absence of family history and/or mental retardation. © 2010 Wiley-Liss, Inc. PMID:21053371

A single-point mutation in the extreme heat- and pressure-resistant sso7d protein from sulfolobus solfataricus leads to a major rearrangement of the hydrophobic core.

PubMed

Consonni, R; Santomo, L; Fusi, P; Tortora, P; Zetta, L

1999-09-28

Sso7d is a basic 7-kDa DNA-binding protein from Sulfolobus solfataricus, also endowed with ribonuclease activity. The protein consists of a double-stranded antiparallel beta-sheet, onto which an orthogonal triple-stranded antiparallel beta-sheet is packed, and of a small helical stretch at the C-terminus. Furthermore, the two beta-sheets enclose an aromatic cluster displaying a fishbone geometry. We previously cloned the Sso7d-encoding gene, expressed it in Escherichia coli, and produced several single-point mutants, either of residues located in the hydrophobic core or of Trp23, which is exposed to the solvent and plays a major role in DNA binding. The mutation F31A was dramatically destabilizing, with a loss in thermo- and piezostabilities by at least 27 K and 10 kbar, respectively. Here, we report the solution structure of the F31A mutant, which was determined by NMR spectroscopy using 744 distance constraints obtained from analysis of multidimensional spectra in conjunction with simulated annealing protocols. The most remarkable finding is the change in orientation of the Trp23 side chain, which in the wild type is completely exposed to the solvent, whereas in the mutant is largely buried in the aromatic cluster. This prevents the formation of a cavity in the hydrophobic core of the mutant, which would arise in the absence of structural rearrangements. We found additional changes produced by the mutation, notably a strong distortion in the beta-sheets with loss in several hydrogen bonds, increased flexibility of some stretches of the backbone, and some local strains. On one hand, these features may justify the dramatic destabilization provoked by the mutation; on the other hand, they highlight the crucial role of the hydrophobic core in protein stability. To the best of our knowledge, no similar rearrangement has been so far described as a result of a single-point mutation.

Pan-Cancer Analysis of Mutation Hotspots in Protein Domains.

PubMed

Miller, Martin L; Reznik, Ed; Gauthier, Nicholas P; Aksoy, Bülent Arman; Korkut, Anil; Gao, Jianjiong; Ciriello, Giovanni; Schultz, Nikolaus; Sander, Chris

2015-09-23

In cancer genomics, recurrence of mutations in independent tumor samples is a strong indicator of functional impact. However, rare functional mutations can escape detection by recurrence analysis owing to lack of statistical power. We enhance statistical power by extending the notion of recurrence of mutations from single genes to gene families that share homologous protein domains. Domain mutation analysis also sharpens the functional interpretation of the impact of mutations, as domains more succinctly embody function than entire genes. By mapping mutations in 22 different tumor types to equivalent positions in multiple sequence alignments of domains, we confirm well-known functional mutation hotspots, identify uncharacterized rare variants in one gene that are equivalent to well-characterized mutations in another gene, detect previously unknown mutation hotspots, and provide hypotheses about molecular mechanisms and downstream effects of domain mutations. With the rapid expansion of cancer genomics projects, protein domain hotspot analysis will likely provide many more leads linking mutations in proteins to the cancer phenotype. Copyright © 2015 Elsevier Inc. All rights reserved.

Large-mutation spectra induced at hemizygous loci by low-LET radiation: evidence for intrachromosomal proximity effects

NASA Technical Reports Server (NTRS)

Costes, S.; Sachs, R.; Hlatky, L.; Vannais, D.; Waldren, C.; Fouladi, B.; Chatterjee, A. (Principal Investigator)

2001-01-01

A mathematical model is used to analyze mutant spectra for large mutations induced by low-LET radiation. The model equations are based mainly on two-break misrejoining that leads to deletions or translocations. It is assumed, as a working hypothesis, that the initial damage induced by low-LET radiation is located randomly in the genome. Specifically, we analyzed data for two hemizygous loci: CD59- mutants, mainly very large-scale deletions (>3 Mbp), in human-hamster hybrid cells, and data from the literature on those HPRT- mutants which involve at least deletion of the whole gene, and often of additional flanking markers (approximately 50-kbp to approximately 4.4-Mbp deletions). For five data sets, we estimated f, the probability that two given breaks on the same chromosome will misrejoin to make a deletion, as a function of the separation between the breaks. We found that f is larger for nearby breaks than for breaks that are more widely separated; i.e., there is a "proximity effect". For acute irradiation, the values of f determined from the data are consistent with the corresponding break misrejoining parameters found previously in quantitative modeling of chromosome aberrations. The value of f was somewhat smaller for protracted irradiation than for acute irradiation at a given total dose; i.e., the mutation data show a decrease that was smaller than expected for dose protraction by fractionation or low dose rate.

Expanding the clinical and genetic spectra of NKX6-2-related disorder.

PubMed

Baldi, C; Bertoli-Avella, A M; Al-Sannaa, N; Alfadhel, M; Al-Thihli, K; Alameer, S; Elmonairy, A A; Al Shamsi, A M; Abdelrahman, H A; Al-Gazali, L; Shawli, A; Al-Hakami, F; Yavuz, H; Kandaswamy, K K; Rolfs, A; Brandau, O; Bauer, P

2018-05-01

Hypomyelinating leukodystrophies (HLDs) affect the white matter of the central nervous system and manifest as neurological disorders. They are genetically heterogeneous. Very recently, biallelic variants in NKX6-2 have been suggested to cause a novel form of autosomal recessive HLD. Using whole-exome or whole-genome sequencing, we identified the previously reported c.196delC and c.487C>G variants in NKX6-2 in 3 and 2 unrelated index cases, respectively; the novel c.608G>A variant was identified in a sixth patient. All variants were homozygous in affected family members only. Our patients share a primary diagnosis of psychomotor delay, and they show spastic quadriparesis, nystagmus and hypotonia. Seizures and dysmorphic features (observed in 2 families each) represent an addition to the phenotype, while developmental regression (observed in 3 families) appears to be a notable and previously underestimated clinical feature. Our findings extend the clinical and mutational spectra associated with this novel form of HLD. Comparative analysis of our 10 patients and the 15 reported previously did, however, not reveal clear evidence for a genotype-phenotype correlation. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Role of CFTR mutation analysis in the diagnostic algorithm for cystic fibrosis.

PubMed

Ratkiewicz, Michelle; Pastore, Matthew; McCoy, Karen Sharrock; Thompson, Rohan; Hayes, Don; Sheikh, Shahid Ijaz

2017-04-01

The cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation identification is being used with increased frequency to aid in the diagnosis of cystic fibrosis (CF) in those suspected with CF. Aim of this study was to identify diagnostic outcomes when CFTR mutational analysis was used in CF diagnosis. CFTR mutational analysis results were also compared with sweat chloride results. This study was done on all patients at our institution who had CFTR mutation analysis over a sevenyear period since August 2006. A total of 315 patients underwent CFTR mutational analysis. Fifty-one (16.2%) patients had two mutations identified. Among them 32 had positive sweat chloride levels (≥60 mmol/L), while seven had borderline sweat chloride levels (40-59 mmol/L). An additional 70 patients (22.3%) had only one mutation identified. Among them eight had positive sweat chloride levels, and 17 had borderline sweat chloride levels. Fifty-five patients (17.5%) without CFTR mutations had either borderline (n=45) or positive (n=10) sweat chloride results. Three patients with a CF phenotype had negative CFTR analysis but elevated sweat chloride levels. In eighty-three patients (26.4%) CFTR mutational analysis was done without corresponding sweat chloride testing. Although CFTR mutation analysis has improved the diagnostic capability for CF, its use either as the first step or the only test to diagnose CFTR dysfunction should be discouraged and CF diagnostic guidelines need to be followed.

An approximate stationary solution for multi-allele neutral diffusion with low mutation rates.

PubMed

Burden, Conrad J; Tang, Yurong

2016-12-01

We address the problem of determining the stationary distribution of the multi-allelic, neutral-evolution Wright-Fisher model in the diffusion limit. A full solution to this problem for an arbitrary K×K mutation rate matrix involves solving for the stationary solution of a forward Kolmogorov equation over a (K-1)-dimensional simplex, and remains intractable. In most practical situations mutations rates are slow on the scale of the diffusion limit and the solution is heavily concentrated on the corners and edges of the simplex. In this paper we present a practical approximate solution for slow mutation rates in the form of a set of line densities along the edges of the simplex. The method of solution relies on parameterising the general non-reversible rate matrix as the sum of a reversible part and a set of (K-1)(K-2)/2 independent terms corresponding to fluxes of probability along closed paths around faces of the simplex. The solution is potentially a first step in estimating non-reversible evolutionary rate matrices from observed allele frequency spectra. Copyright © 2016 Elsevier Inc. All rights reserved.

[Mechanism of mutant induction in the ade2 gene of diploid Saccharomyces cerevisiae yeasts by ultraviolet rays].

PubMed

Gordenin, D A; Inge-Vechtomov, S G

1981-01-01

Ultraviolet light (UV) at 3000 ergs/mm-2 induces ade2 mutants with a frequency about 10(-4) in wild-type haploid strains of yeast and about 10(-5) in diploid wild-type strains. UV irradiation effectively induced mitotic segregation of ade2 in the heterozygous diploid (the frequency of segregation is 6%). Interallelic complementation and localization spectra are similar for mutations induced both in haploids and diploids. The occurrence of ade2 mutants in diploids correlated with mitotic segregation of the marker his8 which is situated in the same arm of XY chromosome as ade2 is, distal to the centromere. Our data about the frequency of ade2 mutants in diploids and haploids, the frequency of ade2 mitotic segregation, mitotic segregation of other markers and genetic characteristics of ade2 mutations confirm the suggestion that the major mechanism of diploid ade2 mutants appearance is mutation in one of the two ADE2 alleles and consequent mitotic homozygotisation of mutation as a result of mitotic crossingover between ade2 and the centromere.

alphaT244M mutation affects the redox, kinetic, and in vitro folding properties of Paracoccus denitrificans electron transfer flavoprotein.

PubMed

Griffin, K J; Dwyer, T M; Manning, M C; Meyer, J D; Carpenter, J F; Frerman, F E

1997-04-08

Threonine 244 in the alpha subunit of Paracoccus denitrificans transfer flavoprotein (ETF) lies seven residues to the amino terminus of a proposed dinucleotide binding motif for the ADP moiety of the FAD prosthetic group. This residue is highly conserved in the alpha subunits of all known ETFs, and the most frequent pathogenic mutation in human ETF encodes a methionine substitution at the corresponding position, alphaT266. The X-ray crystal structures of human and P. denitrificans ETFs are very similar. The hydroxyl hydrogen and a backbone amide hydrogen of alphaT266 are hydrogen bonded to N(5) and C(4)O of the flavin, respectively, and the corresponding alphaT244 has the same structural role in P. denitrificans ETF. We substituted a methionine for T244 in the alpha subunit of P. denitrificans ETF and expressed the mutant ETF in Escherichia coli. The mutant protein was purified, characterized, and compared with wild type P. denitrificans ETF. The mutation has no significant effect on the global structure of the protein as inferred from visible and near-ultraviolet absorption and circular dichroism spectra, far-ultraviolet circular dichroism spectra, and infrared spectra in 1H2O and 2H2O. Intrinsic fluorescence due to tryptophan of the mutant protein is 60% greater than that of the wild type ETF. This increased tryptophan fluorescence is probably due to a change in the environment of the nearby W239. Tyrosine fluorescence is unchanged in the mutant protein, although two tyrosine residues are close to the site of the mutation. These results indicate that a change in structure is minor and localized. Kinetic constants of the reductive half-reaction of ETF with porcine medium chain acyl-CoA dehydrogenase are unaltered when alphaT244M ETF serves as the substrate; however, the mutant ETF fails to exhibit saturation kinetics when the semiquinone form of the protein is used as the substrate in the disproportionation reaction catalyzed by P. denitrificans electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO). The redox behavior of the mutant ETF was also altered as determined from the equilibrium constant of the disproportionation reaction. The separation of flavin redox potentials between the oxidized/semiquinone couple and semiquinone/hydroquinone couple are -6 mV in the wild type ETF and -27 mV in the mutant ETF. The mutation does not alter the AMP content of the protein, although the extent and fidelity of AMP-dependent, in vitro renaturation of the mutant AMP-free apoETF is reduced by 57% compared to renaturation of wild type apoETF, likely due to the absence of the potential hydrogen bond donor T244.

Structure-functional prediction and analysis of cancer mutation effects in protein kinases.

PubMed

Dixit, Anshuman; Verkhivker, Gennady M

2014-01-01

A central goal of cancer research is to discover and characterize the functional effects of mutated genes that contribute to tumorigenesis. In this study, we provide a detailed structural classification and analysis of functional dynamics for members of protein kinase families that are known to harbor cancer mutations. We also present a systematic computational analysis that combines sequence and structure-based prediction models to characterize the effect of cancer mutations in protein kinases. We focus on the differential effects of activating point mutations that increase protein kinase activity and kinase-inactivating mutations that decrease activity. Mapping of cancer mutations onto the conformational mobility profiles of known crystal structures demonstrated that activating mutations could reduce a steric barrier for the movement from the basal "low" activity state to the "active" state. According to our analysis, the mechanism of activating mutations reflects a combined effect of partial destabilization of the kinase in its inactive state and a concomitant stabilization of its active-like form, which is likely to drive tumorigenesis at some level. Ultimately, the analysis of the evolutionary and structural features of the major cancer-causing mutational hotspot in kinases can also aid in the correlation of kinase mutation effects with clinical outcomes.

The Arrhythmogenic Calmodulin p.Phe142Leu Mutation Impairs C-domain Ca2+ Binding but Not Calmodulin-dependent Inhibition of the Cardiac Ryanodine Receptor*

PubMed Central

Liu, Yingjie; Larsen, Kamilla Taunsig; Nani, Alma; Tian, Xixi; Holt, Christian; Wang, Ruiwu; Fill, Michael

2017-01-01

A number of point mutations in the intracellular Ca2+-sensing protein calmodulin (CaM) are arrhythmogenic, yet their underlying mechanisms are not clear. These mutations generally decrease Ca2+ binding to CaM and impair inhibition of CaM-regulated Ca2+ channels like the cardiac Ca2+ release channel (ryanodine receptor, RyR2), and it appears that attenuated CaM Ca2+ binding correlates with impaired CaM-dependent RyR2 inhibition. Here, we investigated the RyR2 inhibitory action of the CaM p.Phe142Leu mutation (F142L; numbered including the start-Met), which markedly reduces CaM Ca2+ binding. Surprisingly, CaM-F142L had little to no aberrant effect on RyR2-mediated store overload-induced Ca2+ release in HEK293 cells compared with CaM-WT. Furthermore, CaM-F142L enhanced CaM-dependent RyR2 inhibition at the single channel level compared with CaM-WT. This is in stark contrast to the actions of arrhythmogenic CaM mutations N54I, D96V, N98S, and D130G, which all diminish CaM-dependent RyR2 inhibition. Thermodynamic analysis showed that apoCaM-F142L converts an endothermal interaction between CaM and the CaM-binding domain (CaMBD) of RyR2 into an exothermal one. Moreover, NMR spectra revealed that the CaM-F142L-CaMBD interaction is structurally different from that of CaM-WT at low Ca2+. These data indicate a distinct interaction between CaM-F142L and the RyR2 CaMBD, which may explain the stronger CaM-dependent RyR2 inhibition by CaM-F142L, despite its reduced Ca2+ binding. Collectively, these results add to our understanding of CaM-dependent regulation of RyR2 as well as the mechanistic effects of arrhythmogenic CaM mutations. The unique properties of the CaM-F142L mutation may provide novel clues on how to suppress excessive RyR2 Ca2+ release by manipulating the CaM-RyR2 interaction. PMID:27927985

Epidemiology of hereditary ocular disorders.

PubMed

Rosenberg, Thomas

2003-01-01

Molecular genetic evidence has contributed significantly to the understanding of the fundamental molecular biology of the eye in health and disease, but it has also exposed the inadequacy of our traditional clinically based classification of hereditary eye disorders by unravelling significant genetic non-allelic heterogeneity in many eye disorders. Furthermore, our understanding of the epidemiology of hereditary ocular disorders has gained considerably by the establishment of mutation spectra in a rapidly growing number of monogenic eye disorders. In this overview, special emphasis has been put on the growing impact of genetic eye diseases in visual impairment, genetic heterogeneity, and the role of founder mutations for the skewed appearance of certain hereditary ocular disorders in some populations.

The Iron-Sulfur Cluster of Electron Transfer Flavoprotein-ubiquinone Oxidoreductase (ETF-QO) is the Electron Acceptor for Electron Transfer Flavoprotein†

PubMed Central

Swanson, Michael A.; Usselman, Robert J.; Frerman, Frank E.; Eaton, Gareth R.; Eaton, Sandra S.

2011-01-01

Electron-transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) accepts electrons from electron-transfer flavoprotein (ETF) and reduces ubiquinone from the ubiquinone-pool. It contains one [4Fe-4S]2+,1+ and one FAD, which are diamagnetic in the isolated oxidized enzyme and can be reduced to paramagnetic forms by enzymatic donors or dithionite. In the porcine protein, threonine 367 is hydrogen bonded to N1 and O2 of the flavin ring of the FAD. The analogous site in Rhodobacter sphaeroides ETF-QO is asparagine 338. Mutations N338T and N338A were introduced into the R. sphaeroides protein by site-directed mutagenesis to determine the impact of hydrogen bonding at this site on redox potentials and activity. The mutations did not alter the optical spectra, EPR g-values, spin-lattice relaxation rates, or the [4Fe-4S]2+,1+ to FAD point-dipole interspin distances. The mutations had no impact on the reduction potential for the iron-sulfur cluster, which was monitored by changes in the continuous wave EPR signals of the [4Fe-4S]+ at 15 K. For the FAD semiquinone, significantly different potentials were obtained by monitoring the titration at 100 or 293 K. Based on spectra at 293 K the N338T mutation shifted the first and second midpoint potentials for the FAD from +47 mV and −30 mV for wild type to −11 mV and −19 mV, respectively. The N338A mutation decreased the potentials to −37 mV and −49 mV. Lowering the midpoint potentials resulted in a decrease in the quinone reductase activity and negligible impact on disproportionation of ETF1e− catalyzed by ETF-QO. These observations indicate that the FAD is involved in electron transfer to ubiquinone, but not in electron transfer from ETF to ETF-QO. Therefore the iron-sulfur cluster is the immediate acceptor from ETF. PMID:18672901

Mutation analysis of the MECP2 gene in patients of Slavic origin with Rett syndrome: novel mutations and polymorphisms.

PubMed

Zahorakova, Daniela; Rosipal, Robert; Hadac, Jan; Zumrova, Alena; Bzduch, Vladimir; Misovicova, Nadezda; Baxova, Alice; Zeman, Jiri; Martasek, Pavel

2007-01-01

Rett syndrome (RTT), an X-linked dominant neurodevelopmental disorder in females, is caused mainly by de novo mutations in the methyl-CpG-binding protein 2 gene (MECP2). Here we report mutation analysis of the MECP2 gene in 87 patients with RTT from the Czech and Slovak Republics, and Ukraine. The patients, all girls, with classical RTT were investigated for mutations using bi-directional DNA sequencing and conformation sensitive gel electrophoresis analysis of the coding sequence and exon/intron boundaries of the MECP2 gene. Restriction fragment length polymorphism analysis was performed to confirm the mutations that cause the creation or abolition of the restriction site. Mutation-negative cases were subsequently examined by multiple ligation-dependent probe amplification (MLPA) to identify large deletions. Mutation screening revealed 31 different mutations in 68 patients and 12 non-pathogenic polymorphisms. Six mutations have not been previously published: two point mutations (323T>A, 904C>T), three deletions (189_190delGA, 816_832del17, 1069delAGC) and one deletion/inversion (1063_1236del174;1189_1231inv43). MLPA analysis revealed large deletions in two patients. The detection rate was 78.16%. Our results confirm the high frequency of MECP2 mutations in females with RTT and provide data concerning the mutation heterogeneity in the Slavic population.

Base changes in tumour DNA have the power to reveal the causes and evolution of cancer

DOE PAGES

Hollstein, M.; Alexandrov, L. B.; Wild, C. P.; ...

2016-06-06

Next-generation sequencing (NGS) technology has demonstrated that the cancer genomes are peppered with mutations. Although most somatic tumour mutations are unlikely to have any role in the cancer process per se, the spectra of DNA sequence changes in tumour mutation catalogues have the potential to identify the mutagens, and to reveal the mutagenic processes responsible for human cancer. Very recently, a novel approach for data mining of the vast compilations of tumour NGS data succeeded in separating and precisely defining at least 30 distinct patterns of sequence change hidden in mutation databases. At least half of these mutational signatures canmore » be readily assigned to known human carcinogenic exposures or endogenous mechanisms of mutagenesis. A quantum leap in our knowledge of mutagenesis in human cancers has resulted, stimulating a flurry of research activity. We trace here the major findings leading first to the hypothesis that carcinogenic insults leave characteristic imprints on the DNA sequence of tumours, and culminating in empirical evidence from NGS data that well-defined carcinogen mutational signatures are indeed present in tumour genomic DNA from a variety of cancer types. The notion that tumour DNAs can divulge environmental sources of mutation is now a well-accepted fact. This approach to cancer aetiology has also incriminated various endogenous, enzyme-driven processes that increase the somatic mutation load in sporadic cancers. The tasks now confronting the field of molecular epidemiology are to assign mutagenic processes to orphan and newly discovered tumour mutation patterns, and to determine whether avoidable cancer risk factors influence signatures produced by endogenous enzymatic mechanisms. As a result, innovative research with experimental models and exploitation of the geographical heterogeneity in cancer incidence can address these challenges.« less

Mutation spectrum of MSH3-deficient HHUA/chr.2 cells reflects in vivo activity of the MSH3 gene product in mismatch repair.

PubMed

Tauchi, H; Komatsu, K; Ishizaki, K; Yatagai, F; Kato, T

2000-02-14

The endometrial tumor cell line HHUA carries mutations in two mismatch repair (MMR) genes MSH3 and MSH6. We have established an MSH3-deficient HHUA/chr.2 cell line by introducing human chromosome 2, which carries wild-type MSH6 and MSH2 genes, to HHUA cells. Introduction of chromosome 2 to HHUA cells partially restored G:G MMR activity to the cell extract and reduced the frequency of mutation at the hypoxanthine-guanine phosphoribosyltransferase (hprt*) locus to about 3% that of the parental HHUA cells, which is five-fold the frequency in MMR-proficient cells, indicating that the residual mutator activity in HHUA/chr.2 is due to an MSH3-deficiency in these cells. The spectrum of mutations occurring at the HPRT locus of HHUA/chr.2 was determined with 71 spontaneous 6TG(r) clones. Base substitutions and +/-1 bp frameshifts were the major mutational events constituting, respectively, 54% and 42% of the total mutations, and more than 70% of them occurred at A:T sites. A possible explanation for the apparent bias of mutations to A:T sites in HHUA/chr.2 is haploinsufficiency of the MSH6 gene on the transferred chromosome 2. Comparison of the mutation spectra of HHUA/chr.2 with that of the MSH6-deficient HCT-15 cell line [S. Ohzeki, A. Tachibana, K. Tatsumi, T. Kato, Carcinogenesis 18 (1997) 1127-1133.] suggests that in vivo the MutSalpha (MSH2:MSH6) efficiently repairs both mismatch and unpaired extrahelical bases, whereas MutSbeta (MSH2:MSH3) efficiently repairs extrahelical bases and repairs mismatch bases to a limited extent.

Base changes in tumour DNA have the power to reveal the causes and evolution of cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hollstein, M.; Alexandrov, L. B.; Wild, C. P.

Next-generation sequencing (NGS) technology has demonstrated that the cancer genomes are peppered with mutations. Although most somatic tumour mutations are unlikely to have any role in the cancer process per se, the spectra of DNA sequence changes in tumour mutation catalogues have the potential to identify the mutagens, and to reveal the mutagenic processes responsible for human cancer. Very recently, a novel approach for data mining of the vast compilations of tumour NGS data succeeded in separating and precisely defining at least 30 distinct patterns of sequence change hidden in mutation databases. At least half of these mutational signatures canmore » be readily assigned to known human carcinogenic exposures or endogenous mechanisms of mutagenesis. A quantum leap in our knowledge of mutagenesis in human cancers has resulted, stimulating a flurry of research activity. We trace here the major findings leading first to the hypothesis that carcinogenic insults leave characteristic imprints on the DNA sequence of tumours, and culminating in empirical evidence from NGS data that well-defined carcinogen mutational signatures are indeed present in tumour genomic DNA from a variety of cancer types. The notion that tumour DNAs can divulge environmental sources of mutation is now a well-accepted fact. This approach to cancer aetiology has also incriminated various endogenous, enzyme-driven processes that increase the somatic mutation load in sporadic cancers. The tasks now confronting the field of molecular epidemiology are to assign mutagenic processes to orphan and newly discovered tumour mutation patterns, and to determine whether avoidable cancer risk factors influence signatures produced by endogenous enzymatic mechanisms. As a result, innovative research with experimental models and exploitation of the geographical heterogeneity in cancer incidence can address these challenges.« less

Excitonic Energy Landscape of the Y16F Mutant of the Chlorobium tepidum Fenna-Matthews-Olson (FMO) Complex: High Resolution Spectroscopic and Modeling Studies.

PubMed

Khmelnitskiy, Anton; Saer, Rafael G; Blankenship, Robert E; Jankowiak, Ryszard

2018-04-12

We report high-resolution (low-temperature) absorption, emission, and nonresonant/resonant hole-burned (HB) spectra and results of excitonic calculations using a non-Markovian reduced density matrix theory (with an improved algorithm for parameter optimization in heterogeneous samples) obtained for the Y16F mutant of the Fenna-Matthews-Olson (FMO) trimer from the green sulfur bacterium Chlorobium tepidum. We show that the Y16F mutant is a mixture of FMO complexes with three independent low-energy traps (located near 817, 821, and 826 nm), in agreement with measured composite emission and HB spectra. Two of these traps belong to mutated FMO subpopulations characterized by significantly modified low-energy excitonic states. Hamiltonians for the two major subpopulations (Sub 821 and Sub 817 ) provide new insight into extensive changes induced by the single-point mutation in the vicinity of BChl 3 (where tyrosine Y16 was replaced with phenylalanine F16). The average decay time(s) from the higher exciton state(s) in the Y16F mutant depends on frequency and occurs on a picosecond time scale.

Identifying Spectra of Activity and Therapeutic Niches for Ceftazidime-Avibactam and Imipenem-Relebactam against Carbapenem-Resistant Enterobacteriaceae

PubMed Central

Chen, Liang; Samanta, Palash; Shields, Ryan K.; Kreiswirth, Barry N.; Nguyen, M. Hong

2017-01-01

ABSTRACT We determined imipenem, imipenem-relebactam, ceftazidime, and ceftazidime-avibactam MICs against 100 CRE isolates that underwent whole-genome sequencing. Klebsiella pneumoniae carbapenemases (KPCs) were the most common carbapenemases. Forty-six isolates carried extended-spectrum β-lactamases (ESBLs). With the addition of relebactam, imipenem susceptibility increased from 8% to 88%. With the addition of avibactam, ceftazidime susceptibility increased from 0% to 85%. Neither imipenem-relebactam nor ceftazidime-avibactam was active against metallo-β-lactamase (MBL) producers. Ceftazidime-avibactam (but not imipenem-relebactam) was active against OXA-48-like producers, including a strain not harboring any ESBL. Major OmpK36 porin mutations were independently associated with higher imipenem-relebactam MICs (P < 0.0001) and showed a trend toward independent association with higher ceftazidime-avibactam MICs (P = 0.07). The presence of variant KPC-3 was associated with ceftazidime-avibactam resistance (P < 0.0001). In conclusion, imipenem-relebactam and ceftazidime-avibactam had overlapping spectra of activity and niches in which each was superior. Major OmpK36 mutations in KPC-K. pneumoniae may provide a foundation for stepwise emergence of imipenem-relebactam and ceftazidime-avibactam resistance. PMID:28630202

Identifying Spectra of Activity and Therapeutic Niches for Ceftazidime-Avibactam and Imipenem-Relebactam against Carbapenem-Resistant Enterobacteriaceae.

PubMed

Haidar, Ghady; Clancy, Cornelius J; Chen, Liang; Samanta, Palash; Shields, Ryan K; Kreiswirth, Barry N; Nguyen, M Hong

2017-09-01

We determined imipenem, imipenem-relebactam, ceftazidime, and ceftazidime-avibactam MICs against 100 CRE isolates that underwent whole-genome sequencing. Klebsiella pneumoniae carbapenemases (KPCs) were the most common carbapenemases. Forty-six isolates carried extended-spectrum β-lactamases (ESBLs). With the addition of relebactam, imipenem susceptibility increased from 8% to 88%. With the addition of avibactam, ceftazidime susceptibility increased from 0% to 85%. Neither imipenem-relebactam nor ceftazidime-avibactam was active against metallo-β-lactamase (MBL) producers. Ceftazidime-avibactam (but not imipenem-relebactam) was active against OXA-48-like producers, including a strain not harboring any ESBL. Major OmpK36 porin mutations were independently associated with higher imipenem-relebactam MICs ( P < 0.0001) and showed a trend toward independent association with higher ceftazidime-avibactam MICs ( P = 0.07). The presence of variant KPC-3 was associated with ceftazidime-avibactam resistance ( P < 0.0001). In conclusion, imipenem-relebactam and ceftazidime-avibactam had overlapping spectra of activity and niches in which each was superior. Major OmpK36 mutations in KPC- K. pneumoniae may provide a foundation for stepwise emergence of imipenem-relebactam and ceftazidime-avibactam resistance. Copyright © 2017 American Society for Microbiology.

Rapid repair of UVA-induced oxidized purines and persistence of UVB-induced dipyrimidine lesions determine the mutagenicity of sunlight in mouse cells

PubMed Central

Besaratinia, Ahmad; Kim, Sang-in; Pfeifer, Gerd P.

2009-01-01

Despite the predominance of UVA relative to UVB in terrestrial sunlight, solar mutagenesis in humans and rodents is characterized by mutations specific for UVB. We have investigated the kinetics of repair of UVA- and UVB-induced DNA lesions in relation to mutagenicity in transgenic mouse fibroblasts irradiated with equilethal doses of UVA and UVB in comparison to SSL. We have also analyzed mutagenesis-derived carcinogenesis in sunlight-associated human skin cancers by compiling the published data on mutation types found in crucial genes in non-melanoma and melanoma skin cancers. Here, we demonstrate a resistance to repair of UVB-induced CPDs together with rapid removal of UVA-induced oxidized purines in the genome overall and in the cII transgene of SSL-irradiated cells. The spectra of mutation induced by both UVB- and SSL-irradiation in this experimental system are characterized by significant increases in relative frequency of C to T transitions at dipyrimidines, which are the established signature mutation of CPDs. This type of mutation is also the predominant mutation found in human non-melanoma and melanoma tumor samples in the TP53, CDKN2, PTCH, and protein kinase genes. The prevailing role of UVB over UVA in solar mutagenesis in our test system can be ascribed to different kinetics of repair for lesions induced by the respective UV-irradiation. PMID:18326785

Mutation Analysis in Classical Phenylketonuria Patients Followed by Detecting Haplotypes Linked to Some PAH Mutations.

PubMed

Dehghanian, Fatemeh; Silawi, Mohammad; Tabei, Seyed M B

2017-02-01

Deficiency of phenylalanine hydroxylase (PAH) enzyme and elevation of phenylalanine in body fluids cause phenylketonuria (PKU). The gold standard for confirming PKU and PAH deficiency is detecting causal mutations by direct sequencing of the coding exons and splicing involved sequences of the PAH gene. Furthermore, haplotype analysis could be considered as an auxiliary approach for detecting PKU causative mutations before direct sequencing of the PAH gene by making comparisons between prior detected mutation linked-haplotypes and new PKU case haplotypes with undetermined mutations. In this study, 13 unrelated classical PKU patients took part in the study detecting causative mutations. Mutations were identified by polymerase chain reaction (PCR) and direct sequencing in all patients. After that, haplotype analysis was performed by studying VNTR and PAHSTR markers (linked genetic markers of the PAH gene) through application of PCR and capillary electrophoresis (CE). Mutation analysis was performed successfully and the detected mutations were as follows: c.782G>A, c.754C>T, c.842C>G, c.113-115delTCT, c.688G>A, and c.696A>G. Additionally, PAHSTR/VNTR haplotypes were detected to discover haplotypes linked to each mutation. Mutation detection is the best approach for confirming PAH enzyme deficiency in PKU patients. Due to the relatively large size of the PAH gene and high cost of the direct sequencing in developing countries, haplotype analysis could be used before DNA sequencing and mutation detection for a faster and cheaper way via identifying probable mutated exons.

Calreticulin mutation analysis in non-mutated Janus kinase 2 essential thrombocythemia patients in Chiang Mai University: analysis of three methods and clinical correlations.

PubMed

Rattarittamrong, Ekarat; Tantiworawit, Adisak; Kumpunya, Noppamas; Wongtagan, Ornkamon; Tongphung, Ratchanoo; Phusua, Arunee; Chai-Adisaksopha, Chatree; Hantrakool, Sasinee; Rattanathammethee, Thanawat; Norasetthada, Lalita; Charoenkwan, Pimlak; Lekawanvijit, Suree

2018-03-09

The primary objective was to determine the prevalence of calreticulin (CALR) mutation in patients with non-JAK2V617F mutated essential thrombocythemia (ET). The secondary objectives were to evaluate the accuracy of CALR mutation analysis by high-resolution melting (HRM) analysis and real-time polymerase chain reaction (PCR) compared with DNA sequencing and to compare clinical characteristics of CALR mutated and JAK2V617F mutated ET. This was a prospective cohort study involving ET patients registered at Chiang Mai University in the period September 2015-September 2017 who were aged more than 2 years, and did not harbor JAK2V617F mutation. The presence of CALR mutation was established by DNA sequencing, HRM, and real-time PCR for type 1 and type 2 mutation. Clinical data were compared with that from ET patients with mutated JAK2V617F. Twenty-eight patients were enrolled onto the study. CALR mutations were found in 10 patients (35.7%). Three patients had type 1 mutation, 5 patients had type 2 mutation, 1 patient had type 18 mutation, and 1 patients had novel mutations (c.1093 C-G, c.1098_1131 del, c.1135 G-A). HRM could differentiate between the types of mutation in complete agreement with DNA sequencing. Patients with a CALR mutation showed a significantly greater male predominance and had a higher platelet count when compared with 42 JAK2V617F patients. The prevalence of CALR mutation in JAK2V617F-negative ET in this study is 35.7%. HRM is an effective method of detecting CALR mutation and is a more advantageous method of screening for CALR mutation.

Differential analysis between somatic mutation and germline variation profiles reveals cancer-related genes.

PubMed

Przytycki, Pawel F; Singh, Mona

2017-08-25

A major aim of cancer genomics is to pinpoint which somatically mutated genes are involved in tumor initiation and progression. We introduce a new framework for uncovering cancer genes, differential mutation analysis, which compares the mutational profiles of genes across cancer genomes with their natural germline variation across healthy individuals. We present DiffMut, a fast and simple approach for differential mutational analysis, and demonstrate that it is more effective in discovering cancer genes than considerably more sophisticated approaches. We conclude that germline variation across healthy human genomes provides a powerful means for characterizing somatic mutation frequency and identifying cancer driver genes. DiffMut is available at https://github.com/Singh-Lab/Differential-Mutation-Analysis .

Structure-Functional Prediction and Analysis of Cancer Mutation Effects in Protein Kinases

PubMed Central

Dixit, Anshuman; Verkhivker, Gennady M.

2014-01-01

A central goal of cancer research is to discover and characterize the functional effects of mutated genes that contribute to tumorigenesis. In this study, we provide a detailed structural classification and analysis of functional dynamics for members of protein kinase families that are known to harbor cancer mutations. We also present a systematic computational analysis that combines sequence and structure-based prediction models to characterize the effect of cancer mutations in protein kinases. We focus on the differential effects of activating point mutations that increase protein kinase activity and kinase-inactivating mutations that decrease activity. Mapping of cancer mutations onto the conformational mobility profiles of known crystal structures demonstrated that activating mutations could reduce a steric barrier for the movement from the basal “low” activity state to the “active” state. According to our analysis, the mechanism of activating mutations reflects a combined effect of partial destabilization of the kinase in its inactive state and a concomitant stabilization of its active-like form, which is likely to drive tumorigenesis at some level. Ultimately, the analysis of the evolutionary and structural features of the major cancer-causing mutational hotspot in kinases can also aid in the correlation of kinase mutation effects with clinical outcomes. PMID:24817905

Fragment analysis represents a suitable approach for the detection of hotspot c.7541_7542delCT NOTCH1 mutation in chronic lymphocytic leukemia.

PubMed

Vavrova, Eva; Kantorova, Barbara; Vonkova, Barbara; Kabathova, Jitka; Skuhrova-Francova, Hana; Diviskova, Eva; Letocha, Ondrej; Kotaskova, Jana; Brychtova, Yvona; Doubek, Michael; Mayer, Jiri; Pospisilova, Sarka

2017-09-01

The hotspot c.7541_7542delCT NOTCH1 mutation has been proven to have a negative clinical impact in chronic lymphocytic leukemia (CLL). However, an optimal method for its detection has not yet been specified. The aim of our study was to examine the presence of the NOTCH1 mutation in CLL using three commonly used molecular methods. Sanger sequencing, fragment analysis and allele-specific PCR were compared in the detection of the c.7541_7542delCT NOTCH1 mutation in 201 CLL patients. In 7 patients with inconclusive mutational analysis results, the presence of the NOTCH1 mutation was also confirmed using ultra-deep next generation sequencing. The NOTCH1 mutation was detected in 15% (30/201) of examined patients. Only fragment analysis was able to identify all 30 NOTCH1-mutated patients. Sanger sequencing and allele-specific PCR showed a lower detection efficiency, determining 93% (28/30) and 80% (24/30) of the present NOTCH1 mutations, respectively. Considering these three most commonly used methodologies for c.7541_7542delCT NOTCH1 mutation screening in CLL, we defined fragment analysis as the most suitable approach for detecting the hotspot NOTCH1 mutation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Exome Sequencing in 32 Patients with Anophthalmia/Microphthalmia and Developmental Eye Defects

PubMed Central

Slavotinek, Anne M.; Garcia, Sarah T.; Chandratillake, Gemma; Bardakjian, Tanya; Ullah, Ehsan; Wu, Di; Umeda, Kyle; Lao, Richard; Tang, Paul Ling-Fung; Wan, Eunice; Madireddy, Lohith; Lyalina, Svetlana; Mendelsohn, Bryce A.; Dugan, Sarah; Tirch, Jean; Tischler, Reana; Harris, Jason; Clark, Michael J.; Chervitz, Stephen; Patwardhan, Anil; West, John M.; Ursell, Phillip; de Alba Campomanes, Alejandra; Schneider, Adele; Kwok, Pui-yan; Baranzini, Sergio; Chen, Richard O.

2014-01-01

Anophthalmia/microphthalmia (A/M) is a genetically heterogeneous birth defect for which the etiology is unknown in more than 50% of patients. We used exome sequencing with the ACE Exome™ (Personalis, Inc; 18 cases) and UCSF Genomics Core (21 cases) to sequence 28 patients with A/M and four patients with varied developmental eye defects. In the 28 patients with A/M, we identified de novo mutations in three patients (OTX2, p.(Gln91His), RARB, p.Arg387Cys and GDF6, p.Ala249Glu) and inherited mutations in STRA6 in two patients. In patients with developmental eye defects, a female with cataracts and cardiomyopathy had a de novo COL4A1 mutation, p.(Gly773Arg), expanding the phenotype associated with COL4A1 to include cardiomyopathy. A male with a chorioretinal defect, microcephaly, seizures and sensorineural deafness had two PNPT1 mutations, p.(Ala507Ser) and c.401-1G>A, and we describe eye defects associated with this gene for the first time. Exome sequencing was efficient for identifying mutations in pathogenic genes for which there is no clinical testing available and for identifying cases that expand phenotypic spectra, such as the PNPT1 and COL4A1-associated disorders described here. PMID:25457163

Mutational analysis of BRAF and KRAS in ovarian serous borderline (atypical proliferative) tumours and associated peritoneal implants

PubMed Central

Ardighieri, Laura; Zeppernick, Felix; Hannibal, Charlotte G; Vang, Russell; Cope, Leslie; Junge, Jette; Kjaer, Susanne K; Kurman, Robert J; Shih, Ie-Ming

2014-01-01

There is debate as to whether peritoneal implants associated with serous borderline tumours/atypical proliferative serous tumours (SBT/APSTs) of the ovary are derived from the primary ovarian tumour or arise independently in the peritoneum. We analysed 57 SBT/APSTs from 45 patients with advanced-stage disease identified from a nation-wide tumour registry in Denmark. Mutational analysis for hotspots in KRAS and BRAF was successful in 55 APSTs and demonstrated KRAS mutations in 34 (61.8%) and BRAF mutations in eight (14.5%). Mutational analysis was successful in 56 peritoneal implants and revealed KRAS mutations in 34 (60.7%) and BRAF mutations in seven (12.5%). Mutational analysis could not be performed in two primary tumours and in nine implants, either because DNA amplification failed or because there was insufficient tissue for mutational analysis. For these specimens we performed VE1 immunohistochemistry, which was shown to be a specific and sensitive surrogate marker for a V600E BRAF mutation. VE1 staining was positive in one of two APSTs and seven of nine implants. Thus, among 63 implants for which mutation status was known (either by direct mutational analysis or by VE1 immunohistochemistry), 34 (53.9%) had KRAS mutations and 14 (22%) had BRAF mutations, of which identical KRAS mutations were found in 34 (91%) of 37 SBT/APST–implant pairs and identical BRAF mutations in 14 (100%) of 14 SBT/APST–implant pairs. Wild-type KRAS and BRAF (at the loci investigated) were found in 11 (100%) of 11 SBT/APST–implant pairs. Overall concordance of KRAS and BRAF mutations was 95% in 59 of 62 SBT/APST–implant (non-invasive and invasive) pairs (p < 0.00001). This study provides cogent evidence that the vast majority of peritoneal implants, non-invasive and invasive, harbour the identical KRAS or BRAF mutations that are present in the associated SBT/APST, supporting the view that peritoneal implants are derived from the primary ovarian tumour. PMID:24307542

Nuclear Radiation Fields on the Mars Surface: Risk Analysis for Long-term Living Environment

NASA Technical Reports Server (NTRS)

Anderson, Brooke M.; Clowdsley, Martha S.; Qualls, Garry D.; Nealy, John E.

2005-01-01

Mars, our nearest planet outward from the sun, has been targeted for several decades as a prospective site for expanded human habitation. Background space radiation exposures on Mars are expected to be orders of magnitude higher than on Earth. Recent risk analysis procedures based on detailed dosimetric techniques applicable to sensitive human organs have been developed along with experimental data regarding cell mutation rates resulting from exposures to a broad range of particle types and energy spectra. In this context, simulated exposure and subsequent risk for humans in residence on Mars are examined. A conceptual habitat structure, CAD-modeled with duly considered inherent shielding properties, has been implemented. Body self-shielding is evaluated using NASA standard computerized male and female models. The background environment is taken to consist not only of exposure from incident cosmic ray ions and their secondaries, but also include the contribution from secondary neutron fields produced in the tenuous atmosphere and the underlying regolith.

Hyperinsulinism–hyperammonaemia syndrome: novel mutations in the GLUD1 gene and genotype–phenotype correlations

PubMed Central

Kapoor, Ritika R; Flanagan, Sarah E; Fulton, Piers; Chakrapani, Anupam; Chadefaux, Bernadette; Ben-Omran, Tawfeg; Banerjee, Indraneel; Shield, Julian P; Ellard, Sian; Hussain, Khalid

2009-01-01

Background Activating mutations in the GLUD1 gene (which encodes for the intra-mitochondrial enzyme glutamate dehydrogenase, GDH) cause the hyperinsulinism–hyperammonaemia (HI/HA) syndrome. Patients present with HA and leucine-sensitive hypoglycaemia. GDH is regulated by another intra-mitochondrial enzyme sirtuin 4 (SIRT4). Sirt4 knockout mice demonstrate activation of GDH with increased amino acid-stimulated insulin secretion. Objectives To study the genotype–phenotype correlations in patients with GLUD1 mutations. To report the phenotype and functional analysis of a novel mutation (P436L) in the GLUD1 gene associated with the absence of HA. Patients and methods Twenty patients with HI from 16 families had mutational analysis of the GLUD1 gene in view of HA (n=19) or leucine sensitivity (n=1). Patients negative for a GLUD1 mutation had sequence analysis of the SIRT4 gene. Functional analysis of the novel P436L GLUD1 mutation was performed. Results Heterozygous missense mutations were detected in 15 patients with HI/HA, 2 of which are novel (N410D and D451V). In addition, a patient with a normal serum ammonia concentration (21 μmol/l) was heterozygous for a novel missense mutation P436L. Functional analysis of this mutation confirms that it is associated with a loss of GTP inhibition. Seizure disorder was common (43%) in our cohort of patients with a GLUD1 mutation. No mutations in the SIRT4 gene were identified. Conclusion Patients with HI due to mutations in the GLUD1 gene may have normal serum ammonia concentrations. Hence, GLUD1 mutational analysis may be indicated in patients with leucine sensitivity; even in the absence of HA. A high frequency of epilepsy (43%) was observed in our patients with GLUD1 mutations. PMID:19690084

Prediction of the association state of insulin using spectral parameters.

PubMed

Uversky, Vladimir N; Garriques, Liza Nielsen; Millett, Ian S; Frokjaer, Sven; Brange, Jens; Doniach, Sebastian; Fink, Anthony L

2003-04-01

Human insulin exists in different association states, from monomer to hexamer, depending on the conditions. In the presence of zinc the "normal" state is a hexamer. The structural properties of 20 variants of human insulin were studied by near-UV circular dichroism, fluorescence spectroscopy, and small-angle X-ray scattering (SAXS). The mutants showed different degrees of association (monomer, dimers, tetramers, and hexamers) at neutral pH. A correlation was shown between the accessibility of tyrosines to acrylamide quenching and the degree of association of the insulin mutants. The near-UV CD spectra of the insulins were affected by protein association and by mutation-induced structural perturbations. However, the shape and intensity of difference CD spectra, obtained by subtraction of the spectra measured in 20% acetic acid (where all insulin species were monomeric) from the corresponding spectra measured at neutral pH, correlate well with the degree of insulin association. In fact, the near-UV CD difference spectra for monomeric, dimeric, tetrameric, and hexameric insulin are very distinctive, both in terms of intensity and shape. The results show that the spectral properties of the insulins reflect their state of association, and can be used to predict their oligomeric state. Copyright 2003 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 92:847-858, 2003

[Identification of a HPGD mutation in three families affected with primary hypertrophic osteoarthropathy].

PubMed

Zhang, Wanying; Wang, Tao; Huang, Shuaiwu; Zhao, Xiuli

2018-04-10

To detect mutation of HPGD gene among three pedigrees affected with primary hypertrophic osteoarthropathy (PHO) by DNA sequencing and high-resolution melting (HRM) analysis. Genomic DNA was extracted from peripheral blood samples collected from the pedigrees. PCR and direct sequencing were carried out to identify potential mutations of the HPGD gene. Amplicons containing the mutation spot were generated by nested PCR. The products were then subjected to HRM analysis using the HR-1 instrument. Direct sequencing was carried out in family members and healthy individuals to confirm the result of HRM analysis. A homozygous mutation c.310_311delCT was detected in 2 affected probands, while a heterozygous mutation c.310_311delCT was detected in the third proband. HRM analysis of the fragments encompassing HPGD exon 3 showed 3 curve patterns representing three different genotypes, i.e., the wild type, the c.310_311delCT homozygote, and the c.310_311delCT heterozygote. Result of DNA sequencing was consistent with that of the HRM analysis and phenotype of the subjects. The c.310_311delCT mutation may be the most prevalent mutation among Chinese population. HRM analysis has provided an optimized method for genetic testing of HPGD mutation for its simplicity, rapid turnover and high sensitivity.

1H MRS spectroscopy in preclinical autosomal dominant Alzheimer disease.

PubMed

Joe, Elizabeth; Medina, Luis D; Ringman, John M; O'Neill, Joseph

2018-06-16

1 H magnetic resonance spectroscopy (MRS) can reveal changes in brain biochemistry in vivo in humans and has been applied to late onset Alzheimer disease (AD). Carriers of mutations for autosomal dominant Alzheimer disease (ADAD) may show changes in levels of metabolites prior to the onset of clinical symptoms. Proton MR spectra were acquired at 1.5 T for 16 cognitively asymptomatic or mildly symptomatic mutation carriers (CDR < 1) and 11 non-carriers as part of a comprehensive cross-sectional study of preclinical ADAD. Levels of N-acetyl-aspartate+N-acetyl-aspartyl-glutamate (NAA), glutamate/glutamine (Glx), creatine/phosphocreate (Cr), choline (Cho), and myo-inositol (mI) in the left and right anterior cingulate and midline posterior cingulate and precuneus were compared between mutation carriers (MCs) and non-carriers (NCs) using multivariate analysis of variance with age as a covariate. Among MCs, correlations between metabolite levels and time until expected age of dementia diagnosis were calculated. MCs had significantly lower levels of NAA and Glx in the left pregenual anterior cingulate cortex, and lower levels of NAA and higher levels of mI and Cho in the precuneus compared to NCs. Increased levels of mI were seen in these regions in association with increased proximity to expected age of dementia onset. MRS shows effects of ADAD similar to those seen in late onset AD even during the preclinical period including lower levels of NAA and higher levels of mI. These indices of neuronal and glial dysfunction might serve as surrogate outcome measures in prevention studies of putative disease-modifying agents.

High-resolution melting analysis for prenatal diagnosis of beta-thalassemia in northern Thailand.

PubMed

Charoenkwan, Pimlak; Sirichotiyakul, Supatra; Phusua, Arunee; Suanta, Sudjai; Fanhchaksai, Kanda; Sae-Tung, Rattika; Sanguansermsri, Torpong

2017-12-01

High-resolution melting (HRM) analysis is a rapid mutation analysis which assesses the pattern of reduction of fluorescence signal after subjecting the amplified PCR product with saturated fluorescence dye to an increasing temperature. We used HRM analysis for prenatal diagnosis of beta-thalassemia disease in northern Thailand. Five PCR-HRM protocols were used to detect point mutations in five different segments of the beta-globin gene, and one protocol to detect the 3.4 kb beta-globin deletion. We sought to characterize the mutations in carriers and to enable prenatal diagnosis in 126 couples at risk of having a fetus with beta-thalassemia disease. The protocols identified 18 common mutations causing beta-thalassemia, including the rare codon 132 (A-T) mutation. Each mutation showed a specific HRM pattern and all results were in concordance with those from direct DNA sequencing or gap-PCR methods. In cases of beta-thalassemia disease resulting from homozygosity for a mutation or compound heterozygosity for two mutations on the same amplified segment, the HRM patterns were different to those of a single mutation and were specific for each combination. HRM analysis is a simple and useful method for mutation identification in beta-thalassemia carriers and prenatal diagnosis of beta-thalassemia in northern Thailand.

Structural effects of extracellular loop mutations in CFTR helical hairpins.

PubMed

Chang, Yuan-Heng; Stone, Tracy A; Chin, Stephanie; Glibowicka, Mira; Bear, Christine E; Deber, Charles M

2018-05-01

Missense mutations constitute 40% of 2000 cystic fibrosis-phenotypic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) database, yet the precise mechanism as to how a point mutation can render the entire 1480-residue CFTR protein dysfunctional is not well-understood. Here we investigate the structural effects of two CF-phenotypic mutations - glutamic acid to glycine at position 217 (E217G) and glutamine to arginine at position 220 (Q220R) - in the extracellular (ECL2) loop region of human CFTR using helical hairpin constructs derived from transmembrane (TM) helices 3 and 4 of the first membrane domain. We systematically replaced the wild type (WT) residues E217 and Q220 with the subset of missense mutations that could arise through a single nucleotide change in their respective codons. Circular dichroism spectra of E217G revealed that a significant increase in helicity vs. WT arises in the membrane-mimetic environment of sodium dodecylsulfate (SDS) micelles, while this mutant showed a similar gel shift to WT on SDS-PAGE gels. In contrast, the CF-mutant Q220R showed similar helicity but an increased gel shift vs. WT. These structural variations are compared with the maturation levels of the corresponding mutant full-length CFTRs, which we found are reduced to approx. 50% for E217G and 30% for Q220R vs. WT. The overall results with CFTR hairpins illustrate the range of impacts that single mutations can evoke in intramolecular protein-protein and/or protein-lipid interactions - and the levels to which corresponding mutations in full-length CFTR may be flagged by quality control mechanisms during biosynthesis. Copyright © 2018 Elsevier B.V. All rights reserved.

Severe Early-Onset Combined Immunodeficiency due to Heterozygous Gain-of-Function Mutations in STAT1.

PubMed

Baris, Safa; Alroqi, Fayhan; Kiykim, Ayca; Karakoc-Aydiner, Elif; Ogulur, Ismail; Ozen, Ahmet; Charbonnier, Louis-Marie; Bakır, Mustafa; Boztug, Kaan; Chatila, Talal A; Barlan, Isil B

2016-10-01

Loss and gain-of-function (GOF) mutations in human signal transducer and activator of transcription 1 (STAT1) lead to distinct phenotypes. Although recurrent infections are common to both types of STAT1 mutations, GOF mutations are distinguished by chronic mucocutaneous candidiasis and autoimmunity. However, the clinical spectra of STAT1 GOF mutations continue to expand. We here describe two patients with STAT1 GOF mutations presenting early in life with combined immunodeficiency (CID). Clinical data and laboratory findings including immunophenotyping, level of interferon (IFN)-γ/IL-17(+) T cells, interferon-induced STAT1 phosphorylation, and JAK inhibitor assays were evaluated. Sequencing of STAT1 gene was performed by Sanger sequencer. Patient 1 (P1) had persistent oral candidiasis and cytomegalovirus (CMV) infection since 2 months of age and later developed cavitary lung lesions due to Mycobacterium tuberculosis. Patient 2 (P2) presented with oral candidiasis and recurrent pneumonia at 4 months of age and subsequently developed CMV pneumonitis. Both patients suffered heterozygous missense mutations in STAT1, leading to deleterious amino acid substitutions in the DNA binding domain (P1: c.1154C > T; p.T385M; P2. c.971G > T; p.C324F). Circulating CD4(+) T cells of both patients exhibited increased interferon-γ and decreased IL-17 expression as compared to controls. They also exhibited increased IFN-β and -γ-induced STAT1 phosphorylation that was reversed upon treatment with the JAK kinase inhibitor ruxolitinib. STAT1 GOF mutations may present early in life with CID, consistent with the clinical heterogeneity of the disease. JAK kinase inhibitors may potentially be useful in some patients as adjunct therapy pending definitive treatment with bone marrow transplantation.

Common and Rare EGFR and KRAS Mutations in a Dutch Non-Small-Cell Lung Cancer Population and Their Clinical Outcome

PubMed Central

Kerner, Gerald S. M. A.; Schuuring, Ed; Sietsma, Johanna; Hiltermann, Thijo J. N.; Pieterman, Remge M.; de Leede, Gerard P. J.; van Putten, John W. G.; Liesker, Jeroen; Renkema, Tineke E. J.; van Hengel, Peter; Platteel, Inge; Timens, Wim; Groen, Harry J. M.

2013-01-01

Introduction In randomly assigned studies with EGFR TKI only a minor proportion of patients with NSCLC have genetically profiled biopsies. Guidelines provide evidence to perform EGFR and KRAS mutation analysis in non-squamous NSCLC. We explored tumor biopsy quality offered for mutation testing, different mutations distribution, and outcome with EGFR TKI. Patient and Methods Clinical data from 8 regional hospitals were studied for patient and tumor characteristics, treatment and overall survival. Biopsies sent to the central laboratory were evaluated for DNA quality and subsequently analyzed for mutations in exons 18–21 of EGFR and exon 2 of KRAS by bidirectional sequence analysis. Results Tumors from 442 subsequent patients were analyzed. For 74 patients (17%) tumors were unsuitable for mutation analysis. Thirty-eight patients (10.9%) had EGFR mutations with 79% known activating mutations. One hundred eight patients (30%) had functional KRAS mutations. The mutation spectrum was comparable to the Cosmic database. Following treatment in the first or second line with EGFR TKI median overall survival for patients with EGFR (n = 14), KRAS (n = 14) mutations and wild type EGFR/KRAS (n = 31) was not reached, 20 and 9 months, respectively. Conclusion One out of every 6 tumor samples was inadequate for mutation analysis. Patients with EGFR activating mutations treated with EGFR-TKI have the longest survival. PMID:23922984

Theories of Lethal Mutagenesis: From Error Catastrophe to Lethal Defection.

PubMed

Tejero, Héctor; Montero, Francisco; Nuño, Juan Carlos

2016-01-01

RNA viruses get extinct in a process called lethal mutagenesis when subjected to an increase in their mutation rate, for instance, by the action of mutagenic drugs. Several approaches have been proposed to understand this phenomenon. The extinction of RNA viruses by increased mutational pressure was inspired by the concept of the error threshold. The now classic quasispecies model predicts the existence of a limit to the mutation rate beyond which the genetic information of the wild type could not be efficiently transmitted to the next generation. This limit was called the error threshold, and for mutation rates larger than this threshold, the quasispecies was said to enter into error catastrophe. This transition has been assumed to foster the extinction of the whole population. Alternative explanations of lethal mutagenesis have been proposed recently. In the first place, a distinction is made between the error threshold and the extinction threshold, the mutation rate beyond which a population gets extinct. Extinction is explained from the effect the mutation rate has, throughout the mutational load, on the reproductive ability of the whole population. Secondly, lethal defection takes also into account the effect of interactions within mutant spectra, which have been shown to be determinant for the understanding the extinction of RNA virus due to an augmented mutational pressure. Nonetheless, some relevant issues concerning lethal mutagenesis are not completely understood yet, as so survival of the flattest, i.e. the development of resistance to lethal mutagenesis by evolving towards mutationally more robust regions of sequence space, or sublethal mutagenesis, i.e., the increase of the mutation rate below the extinction threshold which may boost the adaptability of RNA virus, increasing their ability to develop resistance to drugs (including mutagens). A better design of antiviral therapies will still require an improvement of our knowledge about lethal mutagenesis.

[Analysis of gene mutation in a Chinese family with Norrie disease].

PubMed

Zhang, Tian-xiao; Zhao, Xiu-li; Hua, Rui; Zhang, Jin-song; Zhang, Xue

2012-09-01

To detect the pathogenic mutation in a Chinese family with Norrie disease. Clinical diagnosis was based on familial history, clinical sign and B ultrasonic examination. Peripheral blood samples were obtained from all available members in a Chinese family with Norrie disease. Genomic DNA was extracted from lymphocytes by the standard SDS-proteinase K-phenol/chloroform method. Two coding exons and all intron-exon boundaries of the NDP gene were PCR amplified using three pairs of primers and subjected to automatic DNA sequence. The causative mutation was confirmed by restriction enzyme analysis and genotyping analysis in all members. Sequence analysis of NDP gene revealed a missense mutation c.220C > T (p.Arg74Cys) in the proband and his mother. Further mutation identification by restriction enzyme analysis and genotyping analysis showed that the proband was homozygote of this mutation. His mother and other four unaffected members (III3, IV4, III5 and II2) were carriers of this mutation. The mutant amino acid located in the C-terminal cystine knot-like domain, which was critical motif for the structure and function of NDP. A NDP missense mutation was identified in a Chinese family with Norrie disease.

The TREAT-NMD DMD Global Database: Analysis of More than 7,000 Duchenne Muscular Dystrophy Mutations

PubMed Central

Bladen, Catherine L; Salgado, David; Monges, Soledad; Foncuberta, Maria E; Kekou, Kyriaki; Kosma, Konstantina; Dawkins, Hugh; Lamont, Leanne; Roy, Anna J; Chamova, Teodora; Guergueltcheva, Velina; Chan, Sophelia; Korngut, Lawrence; Campbell, Craig; Dai, Yi; Wang, Jen; Barišić, Nina; Brabec, Petr; Lahdetie, Jaana; Walter, Maggie C; Schreiber-Katz, Olivia; Karcagi, Veronika; Garami, Marta; Viswanathan, Venkatarman; Bayat, Farhad; Buccella, Filippo; Kimura, En; Koeks, Zaïda; van den Bergen, Janneke C; Rodrigues, Miriam; Roxburgh, Richard; Lusakowska, Anna; Kostera-Pruszczyk, Anna; Zimowski, Janusz; Santos, Rosário; Neagu, Elena; Artemieva, Svetlana; Rasic, Vedrana Milic; Vojinovic, Dina; Posada, Manuel; Bloetzer, Clemens; Jeannet, Pierre-Yves; Joncourt, Franziska; Díaz-Manera, Jordi; Gallardo, Eduard; Karaduman, A Ayşe; Topaloğlu, Haluk; El Sherif, Rasha; Stringer, Angela; Shatillo, Andriy V; Martin, Ann S; Peay, Holly L; Bellgard, Matthew I; Kirschner, Jan; Flanigan, Kevin M; Straub, Volker; Bushby, Kate; Verschuuren, Jan; Aartsma-Rus, Annemieke; Béroud, Christophe; Lochmüller, Hanns

2015-01-01

Analyzing the type and frequency of patient-specific mutations that give rise to Duchenne muscular dystrophy (DMD) is an invaluable tool for diagnostics, basic scientific research, trial planning, and improved clinical care. Locus-specific databases allow for the collection, organization, storage, and analysis of genetic variants of disease. Here, we describe the development and analysis of the TREAT-NMD DMD Global database (http://umd.be/TREAT_DMD/). We analyzed genetic data for 7,149 DMD mutations held within the database. A total of 5,682 large mutations were observed (80% of total mutations), of which 4,894 (86%) were deletions (1 exon or larger) and 784 (14%) were duplications (1 exon or larger). There were 1,445 small mutations (smaller than 1 exon, 20% of all mutations), of which 358 (25%) were small deletions and 132 (9%) small insertions and 199 (14%) affected the splice sites. Point mutations totalled 756 (52% of small mutations) with 726 (50%) nonsense mutations and 30 (2%) missense mutations. Finally, 22 (0.3%) mid-intronic mutations were observed. In addition, mutations were identified within the database that would potentially benefit from novel genetic therapies for DMD including stop codon read-through therapies (10% of total mutations) and exon skipping therapy (80% of deletions and 55% of total mutations). PMID:25604253

Amino acid substitutions enhancing thermostability of Bacillus polymyxa beta-glucosidase A.

PubMed Central

Lopez-Camacho, C; Salgado, J; Lequerica, J L; Madarro, A; Ballestar, E; Franco, L; Polaina, J

1996-01-01

Mutations enhancing the thermostability of beta-glucosidase A of Bacillus polymyxa, a family 1 glycosyl hydrolase, have been obtained after hydroxylamine mutagenesis of a plasmid containing the bglA gene, transformation of Escherichia coli with the mutagenized plasmid, and identification of transformant colonies that showed beta-glucosidase activity after a thermal treatment that inactivated the wild-type enzyme. Two additive mutations have been characterized that cause replacement of glutamate at position 96 by lysine and of methionine at position 416 by isoleucine respectively. The thermoresistant mutant enzymes showed increased resistance to other denaturing agents, such as pH and urea, while their kinetic parameters did not change. CD spectra indicated that the E96K replacement caused an increase in alpha-helix content. The observed effect of the M416I mutation is consistent with the lower content of cysteine and methionine found in family 1 enzymes of thermophilic species compared with similar ones from mesophilic organisms. PMID:8615777

Adaptive changes in HIV-1 subtype C proteins during early infection are driven by changes in HLA-associated immune pressure

PubMed Central

Treurnicht, F.K.; Seoighe, C.; Martin, D.P.; Wood, N.; Abrahams, M-R.; de Assis Rosa, D.; Bredell, H.; Woodman, Z.; Hide, W.; Mlisana, K.; Karim, S Abdool; Gray, C.M.; Williamson, C.

2009-01-01

It is unresolved whether recently transmitted human immunodeficiency viruses (HIV) have genetic features that specifically favour their transmissibility. To identify potential “transmission signatures”, we compared 20 full-length HIV-1 subtype C genomes from primary infections, with 66 sampled from ethnically and geographically matched individuals with chronic infections. Controlling for recombination and phylogenetic relatedness, we identified 39 sites at which amino acid frequency spectra differed significantly between groups. These sites were predominantly located within Env, Pol and Gag (14/39, 9/39 and 6/39 respectively) and were significantly clustered (33/39) within known immunoreactive peptides. Within 6 months of infection we detected reversion-to-consensus mutations at 14 sites and potential CTL escape mutations at seven. Here we provide evidence that frequent reversion mutations probably allows the virus to recover replicative fitness which, together with immune escape driven by the HLA alleles of the new hosts, differentiate sequences from chronic infections from those sampled shortly after transmission. PMID:19913270

Stabilizing Selection, Purifying Selection, and Mutational Bias in Finite Populations

PubMed Central

Charlesworth, Brian

2013-01-01

Genomic traits such as codon usage and the lengths of noncoding sequences may be subject to stabilizing selection rather than purifying selection. Mutations affecting these traits are often biased in one direction. To investigate the potential role of stabilizing selection on genomic traits, the effects of mutational bias on the equilibrium value of a trait under stabilizing selection in a finite population were investigated, using two different mutational models. Numerical results were generated using a matrix method for calculating the probability distribution of variant frequencies at sites affecting the trait, as well as by Monte Carlo simulations. Analytical approximations were also derived, which provided useful insights into the numerical results. A novel conclusion is that the scaled intensity of selection acting on individual variants is nearly independent of the effective population size over a wide range of parameter space and is strongly determined by the logarithm of the mutational bias parameter. This is true even when there is a very small departure of the mean from the optimum, as is usually the case. This implies that studies of the frequency spectra of DNA sequence variants may be unable to distinguish between stabilizing and purifying selection. A similar investigation of purifying selection against deleterious mutations was also carried out. Contrary to previous suggestions, the scaled intensity of purifying selection with synergistic fitness effects is sensitive to population size, which is inconsistent with the general lack of sensitivity of codon usage to effective population size. PMID:23709636

Shifted termination assay (STA) fragment analysis to detect BRAF V600 mutations in papillary thyroid carcinomas

PubMed Central

2013-01-01

Background BRAF mutation is an important diagnostic and prognostic marker in patients with papillary thyroid carcinoma (PTC). To be applicable in clinical laboratories with limited equipment, diverse testing methods are required to detect BRAF mutation. Methods A shifted termination assay (STA) fragment analysis was used to detect common V600 BRAF mutations in 159 PTCs with DNAs extracted from formalin-fixed paraffin-embedded tumor tissue. The results of STA fragment analysis were compared to those of direct sequencing. Serial dilutions of BRAF mutant cell line (SNU-790) were used to calculate limit of detection (LOD). Results BRAF mutations were detected in 119 (74.8%) PTCs by STA fragment analysis. In direct sequencing, BRAF mutations were observed in 118 (74.2%) cases. The results of STA fragment analysis had high correlation with those of direct sequencing (p < 0.00001, κ = 0.98). The LOD of STA fragment analysis and direct sequencing was 6% and 12.5%, respectively. In PTCs with pT3/T4 stages, BRAF mutation was observed in 83.8% of cases. In pT1/T2 carcinomas, BRAF mutation was detected in 65.9% and this difference was statistically significant (p = 0.007). Moreover, BRAF mutation was more frequent in PTCs with extrathyroidal invasion than tumors without extrathyroidal invasion (84.7% versus 62.2%, p = 0.001). To prepare and run the reactions, direct sequencing required 450 minutes while STA fragment analysis needed 290 minutes. Conclusions STA fragment analysis is a simple and sensitive method to detect BRAF V600 mutations in formalin-fixed paraffin-embedded clinical samples. Virtual Slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/5684057089135749 PMID:23883275

Deriving the intermediate spectra and photocycle kinetics from time-resolved difference spectra of bacteriorhodopsin. The simpler case of the recombinant D96N protein

NASA Technical Reports Server (NTRS)

Zimanyi, L.; Lanyi, J. K.

1993-01-01

The bacteriorhodopsin photocycle contains more than five spectrally distinct intermediates, and the complexity of their interconversions has precluded a rigorous solution of the kinetics. A representation of the photocycle of mutated D96N bacteriorhodopsin near neutral pH was given earlier (Varo, G., and J. K. Lanyi. 1991. Biochemistry. 30:5008-5015) as BRhv-->K<==>L<==>M1-->M2--> BR. Here we have reduced a set of time-resolved difference spectra for this simpler system to three base spectra, each assumed to consist of an unknown mixture of the pure K, L, and M difference spectra represented by a 3 x 3 matrix of concentration values between 0 and 1. After generating all allowed sets of spectra for K, L, and M (i.e., M1 + M2) at a 1:50 resolution of the matrix elements, invalid solutions were eliminated progressively in a search based on what is expected, empirically and from the theory of polyene excited states, for rhodopsin spectra. Significantly, the average matrix values changed little after the first and simplest of the search criteria that disallowed negative absorptions and more than one maximum for the M intermediate. We conclude from the statistics that during the search the solutions strongly converged into a narrow region of the multidimensional space of the concentration matrix. The data at three temperatures between 5 and 25 degrees C yielded a single set of spectra for K, L, and M; their fits are consistent with the earlier derived photocycle model for the D96N protein.

Improved quantitative analysis of spectra using a new method of obtaining derivative spectra based on a singular perturbation technique.

PubMed

Li, Zhigang; Wang, Qiaoyun; Lv, Jiangtao; Ma, Zhenhe; Yang, Linjuan

2015-06-01

Spectroscopy is often applied when a rapid quantitative analysis is required, but one challenge is the translation of raw spectra into a final analysis. Derivative spectra are often used as a preliminary preprocessing step to resolve overlapping signals, enhance signal properties, and suppress unwanted spectral features that arise due to non-ideal instrument and sample properties. In this study, to improve quantitative analysis of near-infrared spectra, derivatives of noisy raw spectral data need to be estimated with high accuracy. A new spectral estimator based on singular perturbation technique, called the singular perturbation spectra estimator (SPSE), is presented, and the stability analysis of the estimator is given. Theoretical analysis and simulation experimental results confirm that the derivatives can be estimated with high accuracy using this estimator. Furthermore, the effectiveness of the estimator for processing noisy infrared spectra is evaluated using the analysis of beer spectra. The derivative spectra of the beer and the marzipan are used to build the calibration model using partial least squares (PLS) modeling. The results show that the PLS based on the new estimator can achieve better performance compared with the Savitzky-Golay algorithm and can serve as an alternative choice for quantitative analytical applications.

Core Needle Lung Biopsy Specimens: Adequacy for EGFR and KRAS Mutational Analysis

PubMed Central

Zakowski, Maureen F.; Pao, William; Thornton, Raymond H.; Ladanyi, Marc; Kris, Mark G.; Rusch, Valerie W.; Rizvi, Naiyer A.

2013-01-01

OBJECTIVE The purpose of this study was to prospectively compare the adequacy of core needle biopsy specimens with the adequacy of specimens from resected tissue, the histologic reference standard, for mutational analysis of malignant tumors of the lung. SUBJECTS AND METHODS The first 18 patients enrolled in a phase 2 study of gefitinib for lung cancer in July 2004 through August 2005 underwent CT- or fluoroscopy-guided lung biopsy before the start of gefitinib therapy. Three weeks after gefitinib therapy, the patients underwent lung tumor resection. The results of EGFR and KRAS mutational analysis of the core needle biopsy specimens were compared with those of EGFR and KRAS mutational analysis of the surgical specimens. RESULTS Two specimens were unsatisfactory for mutational analysis. The results of mutational assay results of the other 16 specimens were the same as those of analysis of the surgical specimens obtained an average of 31 days after biopsy. CONCLUSION Biopsy with small (18- to 20-gauge) core needles can yield sufficient and reliable samples for mutational analysis. This technique is likely to become an important tool with the increasing use of pharmacotherapy based on the genetics of specific tumors in individual patients. PMID:20028932

Expert System for Analysis of Spectra in Nuclear Metrology

NASA Astrophysics Data System (ADS)

Petrović, Ivan; Petrović, V.; Krstić, D.; Nikezić, D.; Bočvarski, V.

In this paper is described an expert system (ES) developed in order to enable the analysis of emission spectra, which are obtained by measurements of activities of radioactive elements, i.e., isotopes, actually cesium. In the structure of those spectra exists two parts: first on lower energies, which originates from the Compton effect, and second on higher energies, which contains the photopeak. The aforementioned ES is made to perform analysis of spectra in whole range of energies. Analysis of those spectra is very interesting because of the problem of environmental contamination by radio nuclides.

Collaborative Study for Analysis of High Resolution Infrared Atmospheric Spectra Between NASA Langley Research Center and the University of Denver

NASA Technical Reports Server (NTRS)

Goldman, A.

2002-01-01

The Langley-D.U. collaboration on the analysis of high resolultion infrared atmospheric spectra covered a number of important studies of trace gases identification and quantification from field spectra, and spectral line parameters analysis. The collaborative work included: 1) Quantification and monitoring of trace gases from ground-based spectra available from various locations and seasons and from balloon flights; 2) Identification and preliminary quantification of several isotopic species, including oxygen and Sulfur isotopes; 3) Search for new species on the available spectra, including the use of selective coadding of ground-based spectra for high signal to noise; 4) Update of spectroscopic line parameters, by combining laboratory and atmospheric spectra with theoretical spectroscopy methods; 5) Study of trends and correlations of atmosphere trace constituents; and 6) Algorithms developments, retrievals intercomparisons and automatization of the analysis of NDSC spectra, for both column amounts and vertical profiles.

Analysis of Atmospheric Trace Constituents from High Resolution Infrared Balloon-Borne and Ground-Based Solar Absorption Spectra

NASA Technical Reports Server (NTRS)

Goldman, A.; Murcray, F. J.; Rinsland, C. P.; Blatherwick, R. D.; Murcray, F. H.; Murcray, D. G.

1991-01-01

Recent results and ongoing studies of high resolution solar absorption spectra will be presented. The analysis of these spectra is aimed at the identification and quantification of trace constituents important in atmospheric chemistry of the stratosphere and upper troposphere. Analysis of balloon-borne and ground-based spectra obtained at 0.0025/ cm covering the 700-2200/ cm interval will be presented. Results from ground-based 0.02/ cm solar spectra, from several locations such as Denver, South Pole, M. Loa, and New Zealand will also be shown. The 0.0025/ cm spectra show many new spectroscopic features. The analysis of these spectra, along with corresponding laboratory spectra, improves the spectral line parameters, and thus the accuracy of trace constituents quantification. The combination of the recent balloon flights, with earlier flights data since 1978 at 0.02/ cm resolution, provides trends analysis of several stratospheric trace species. Results for COF2, F22, SF6, and other species will be presented. Analysis of several ground-based solar spectra provides trends for HCl, HF and other species. The retrieval methods used for total column density and altitude distribution for both ground-based and balloon-borne spectra will be presented. These are extended for the analysis of the ground-based spectra to be obtained by the high resolution interferometers of the Network for Detection of Stratospheric Change (NDSC). Progress or the University of Denver studies for the NDSC will be presented. This will include intercomparison of solar spectra and trace gases retrievals obtained from simultaneous scans by the high resolution (0.0025/ cm) interferometers of BRUKER and BOMEM.

Collaborative Study of Analysis of High Resolution Infrared Atmospheric Spectra Between NASA Langley Research Center and the University of Denver

NASA Technical Reports Server (NTRS)

Goldman, Aaron

1999-01-01

The Langley-D.U. collaboration on the analysis of high resolution infrared atmospheric spectra covered a number of important studies of trace gases identification and quantification from field spectra, and spectral line parameters analysis. The collaborative work included: Quantification and monitoring of trace gases from ground-based spectra available from various locations and seasons and from balloon flights. Studies toward identification and quantification of isotopic species, mostly oxygen and Sulfur isotopes. Search for new species on the available spectra. Update of spectroscopic line parameters, by combining laboratory and atmospheric spectra with theoretical spectroscopy methods. Study of trends of atmosphere trace constituents. Algorithms developments, retrievals intercomparisons and automatization of the analysis of NDSC spectra, for both column amounts and vertical profiles.

Exome sequencing in 32 patients with anophthalmia/microphthalmia and developmental eye defects.

PubMed

Slavotinek, A M; Garcia, S T; Chandratillake, G; Bardakjian, T; Ullah, E; Wu, D; Umeda, K; Lao, R; Tang, P L-F; Wan, E; Madireddy, L; Lyalina, S; Mendelsohn, B A; Dugan, S; Tirch, J; Tischler, R; Harris, J; Clark, M J; Chervitz, S; Patwardhan, A; West, J M; Ursell, P; de Alba Campomanes, A; Schneider, A; Kwok, P-Y; Baranzini, S; Chen, R O

2015-11-01

Anophthalmia/microphthalmia (A/M) is a genetically heterogeneous birth defect for which the etiology is unknown in more than 50% of patients. We used exome sequencing with the ACE Exome(TM) (Personalis, Inc; 18 cases) and UCSF Genomics Core (21 cases) to sequence 28 patients with A/M and four patients with varied developmental eye defects. In the 28 patients with A/M, we identified de novo mutations in three patients (OTX2, p.(Gln91His), RARB, p.Arg387Cys and GDF6, p.Ala249Glu) and inherited mutations in STRA6 in two patients. In patients with developmental eye defects, a female with cataracts and cardiomyopathy had a de novo COL4A1 mutation, p.(Gly773Arg), expanding the phenotype associated with COL4A1 to include cardiomyopathy. A male with a chorioretinal defect, microcephaly, seizures and sensorineural deafness had two PNPT1 mutations, p.(Ala507Ser) and c.401-1G>A, and we describe eye defects associated with this gene for the first time. Exome sequencing was efficient for identifying mutations in pathogenic genes for which there is no clinical testing available and for identifying cases that expand phenotypic spectra, such as the PNPT1 and COL4A1-associated disorders described here. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Clinical evaluation and mutational analysis of GALK and GALE genes in patients with galactosemia in Greece: one novel mutation and two rare cases.

PubMed

Schulpis, Kleopatra H; Thodi, Georgia; Iakovou, Konstantinos; Chatzidaki, Maria; Dotsikas, Yannis; Molou, Elina; Triantafylli, Olga; Loukas, Yannis L

2017-07-26

Deficiencies of galactokinase (GALK) and UDP-epimerase (GALE) are implicated with galactose metabolic disorders. The aim of the study was the identification of mutations in GALK and GALE genes and clinical evaluation of patients. Five patients with GALK and five with GALE deficiency were picked up via the Neonatal Screening Program. Additionally, two females, 4 years old, were referred with late diagnosed galactosemia, as rare cases. Mutational analysis was conducted via Sanger sequencing, while in silico analysis tools were utilized for the novel mutation. Psychomotor and speech development tests were performed, as well. The mutation p.Pro28Thr was identified in both alleles in GALK-deficient patients of Roma (gypsy) origin, whereas the novel p.Asn39Ser was detected in two non-Roma patients. In GALE-deficient patients benign and/or likely benign mutations were found. Psychomotor and speech delay were determined in the Roma GALK patients. In each of the late diagnosed females, four mutations were identified in all galactosemia-related genes. The mutational spectrums of GALE- and GALK-deficient patients in Greece are presented for the first time along with a clinical evaluation. Mutational analysis in all galactosemia-related genes of symptomatic patients is highly recommended for future cases.

Novel compound heterozygous mutations in MYO7A in a Chinese family with Usher syndrome type 1.

PubMed

Liu, Fei; Li, Pengcheng; Liu, Ying; Li, Weirong; Wong, Fulton; Du, Rong; Wang, Lei; Li, Chang; Jiang, Fagang; Tang, Zhaohui; Liu, Mugen

2013-01-01

To identify the disease-causing mutation(s) in a Chinese family with autosomal recessive Usher syndrome type 1 (USH1). An ophthalmic examination and an audiometric test were conducted to ascertain the phenotype of two affected siblings. The microsatellite marker D11S937, which is close to the candidate gene MYO7A (USH1B locus), was selected for genotyping. From the DNA of the proband, all coding exons and exon-intron boundaries of MYO7A were sequenced to identify the disease-causing mutation(s). Restriction fragment length polymorphism (RFLP) analysis was performed to exclude the alternative conclusion that the mutations are non-pathogenic rare polymorphisms. Based on severe hearing impairment, unintelligible speech, and retinitis pigmentosa, a clinical diagnosis of Usher syndrome type 1 was made. The genotyping results did not exclude the USH1B locus, which suggested that the MYO7A gene was likely the gene associated with the disease-causing mutation(s) in the family. With direct DNA sequencing of MYO7A, two novel compound heterozygous mutations (c.3742G>A and c.6051+1G>A) of MYO7A were identified in the proband. DNA sequence analysis and RFLP analysis of other family members showed that the mutations cosegregated with the disease. Unaffected members, including the parents, uncle, and sister of the proband, carry only one of the two mutations. The mutations were not present in the controls (100 normal Chinese subjects=200 chromosomes) according to the RFLP analysis. In this study, we identified two novel mutations, c.3742G>A (p.E1248K) and c.6051+1G>A (donor splice site mutation in intron 44), of MYO7A in a Chinese non-consanguineous family with USH1. The mutations cosegregated with the disease and most likely cause the phenotype in the two affected siblings who carry these mutations compound heterozygously. Our finding expands the mutational spectrum of MYO7A.

Fourier-Based Diffraction Analysis of Live Caenorhabditis elegans.

PubMed

Magnes, Jenny; Hastings, Harold M; Raley-Susman, Kathleen M; Alivisatos, Clara; Warner, Adam; Hulsey-Vincent, Miranda

2017-09-13

This manuscript describes how to classify nematodes using temporal far-field diffraction signatures. A single C. elegans is suspended in a water column inside an optical cuvette. A 632 nm continuous wave HeNe laser is directed through the cuvette using front surface mirrors. A significant distance of at least 20-30 cm traveled after the light passes through the cuvette ensures a useful far-field (Fraunhofer) diffraction pattern. The diffraction pattern changes in real time as the nematode swims within the laser beam. The photodiode is placed off-center in the diffraction pattern. The voltage signal from the photodiode is observed in real time and recorded using a digital oscilloscope. This process is repeated for 139 wild type and 108 "roller" C. elegans. Wild type worms exhibit a rapid oscillation pattern in solution. The "roller" worms have a mutation in a key component of the cuticle that interferes with smooth locomotion. Time intervals that are not free of saturation and inactivity are discarded. It is practical to divide each average by its maximum to compare relative intensities. The signal for each worm is Fourier transformed so that the frequency pattern for each worm emerges. The signal for each type of worm is averaged. The averaged Fourier spectra for the wild type and the "roller" C. elegans are distinctly different and reveal that the dynamic worm shapes of the two different worm strains can be distinguished using Fourier analysis. The Fourier spectra of each worm strain match an approximate model using two different binary worm shapes that correspond to locomotory moments. The envelope of the averaged frequency distribution for actual and modeled worms confirms the model matches the data. This method can serve as a baseline for Fourier analysis for many microscopic species, as every microorganism will have its unique Fourier spectrum.

DHPLC-based mutation analysis of ENG and ALK-1 genes in HHT Italian population.

PubMed

Lenato, Gennaro M; Lastella, Patrizia; Di Giacomo, Marilena C; Resta, Nicoletta; Suppressa, Patrizia; Pasculli, Giovanna; Sabbà, Carlo; Guanti, Ginevra

2006-02-01

Hereditary haemorrhagic telangiectasia (HHT or Rendu-Osler-Weber syndrome) is an autosomal dominant disorder characterized by localized angiodysplasia due to mutations in endoglin, ALK-1 gene, and a still unidentified locus. The lack of highly recurrent mutations, locus heterogeneity, and the presence of mutations in almost all coding exons of the two genes makes the screening for mutations time-consuming and costly. In the present study, we developed a DHPLC-based protocol for mutation detection in ALK1 and ENG genes through retrospective analysis of known sequence variants, 20 causative mutations and 11 polymorphisms, and a prospective analysis on 47 probands with unknown mutation. Overall DHPLC analysis identified the causative mutation in 61 out 66 DNA samples (92.4%). We found 31 different mutations in the ALK1 gene, of which 15 are novel, and 20, of which 12 are novel, in the ENG gene, thus providing for the first time the mutational spectrum in a cohort of Italian HHT patients. In addition, we characterized the splicing pattern of ALK1 gene in lymphoblastoid cells, both in normal controls and in two individuals carrying a mutation in the non-invariant -3 position of the acceptor splice site upstream exon 6 (c.626-3C>G). Functional essay demonstrated the existence, also in normal individuals, of a small proportion of ALK1 alternative splicing, due to exon 5 skipping, and the presence of further aberrant splicing isoforms in the individuals carrying the c.626-3C>G mutation. 2006 Wiley-Liss, Inc.

KRAS mutation analysis of washing fluid from endoscopic ultrasound-guided fine needle aspiration improves cytologic diagnosis of pancreatic ductal adenocarcinoma.

PubMed

Park, Joo Kyung; Lee, Yoon Jung; Lee, Jong Kyun; Lee, Kyu Taek; Choi, Yoon-La; Lee, Kwang Hyuck

2017-01-10

EUS-FNA becomes one of the most important diagnostic modalities for PDACs. However, acquired tissue specimens were sometimes insufficient to make a definite cytological diagnosis. On the other hand, KRAS mutation is the most frequently acquired genetic alteration found more than 90% of PDACs. To investigate the way to improve diagnostic accuracy for PDACs using both cytological examination and KRAS mutation analysis would be a great help. Therefore, the aims of this study were to evaluate usefulness of conventional cytological examination combined with KRAS mutation analysis with modified PCR technology to improve the sensitivity and the accuracy. We enrolled 43 patients with solid pancreatic masses and 86 EUS-FNA specimens were obtained. During the EUS-FNA, the needle catheter was flushed with 2 cc of saline and the washed fluid was collected for KRAS mutation analysis for the first 2 passes; PNAClamp™ KRAS Mutation Detection Kit. There were 46 specimens from the 23 PDACs and 40 specimens from the 20 other pancreatic diseases. The sensitivity, specificity and accuracy were as follows; conventional cytopathologic examination: 63%, 100% and 80%; combination of cytopathologic examination and K-ras mutation analysis: 87%, 100% and 93%. Furthermore, KRAS mutation was detected 11 out of 17 PDAC samples whose cytopathology results were inconclusive. KRAS mutation analysis with PNAClamp™ technique using washing fluid from EUS-FNA along with cytological examination may not only improve the diagnostic accuracy of PDACs, but also establish the platform using genetic analysis which would be helpful as diagnostic modality for PDACs.

A protein domain-centric approach for the comparative analysis of human and yeast phenotypically relevant mutations

PubMed Central

2013-01-01

Background The body of disease mutations with known phenotypic relevance continues to increase and is expected to do so even faster with the advent of new experimental techniques such as whole-genome sequencing coupled with disease association studies. However, genomic association studies are limited by the molecular complexity of the phenotype being studied and the population size needed to have adequate statistical power. One way to circumvent this problem, which is critical for the study of rare diseases, is to study the molecular patterns emerging from functional studies of existing disease mutations. Current gene-centric analyses to study mutations in coding regions are limited by their inability to account for the functional modularity of the protein. Previous studies of the functional patterns of known human disease mutations have shown a significant tendency to cluster at protein domain positions, namely position-based domain hotspots of disease mutations. However, the limited number of known disease mutations remains the main factor hindering the advancement of mutation studies at a functional level. In this paper, we address this problem by incorporating mutations known to be disruptive of phenotypes in other species. Focusing on two evolutionarily distant organisms, human and yeast, we describe the first inter-species analysis of mutations of phenotypic relevance at the protein domain level. Results The results of this analysis reveal that phenotypic mutations from yeast cluster at specific positions on protein domains, a characteristic previously revealed to be displayed by human disease mutations. We found over one hundred domain hotspots in yeast with approximately 50% in the exact same domain position as known human disease mutations. Conclusions We describe an analysis using protein domains as a framework for transferring functional information by studying domain hotspots in human and yeast and relating phenotypic changes in yeast to diseases in human. This first-of-a-kind study of phenotypically relevant yeast mutations in relation to human disease mutations demonstrates the utility of a multi-species analysis for advancing the understanding of the relationship between genetic mutations and phenotypic changes at the organismal level. PMID:23819456

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

PubMed

Dong, Chongmei; Vincent, Kate; Sharp, Peter

2009-12-04

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

Mitrocomin from the jellyfish Mitrocoma cellularia with deleted C-terminal tyrosine reveals a higher bioluminescence activity compared to wild type photoprotein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Burakova, Ludmila P.; Natashin, Pavel V.; Markova, Svetlana V.

The full-length cDNA genes encoding five new isoforms of Ca2 +-regulated photoprotein mitrocomin from a small tissue sample of the outer bell margin containing photocytes of only one specimen of the luminous jellyfish Mitrocoma cellularia were cloned, sequenced, and characterized after their expression in Escherichia coli and subsequent purification. The analysis of cDNA nucleotide sequences encoding mitrocomin isoforms allowed suggestion that two isoforms might be the products of two allelic genes differing in one amino acid residue (64R/Q) whereas other isotypes appear as a result of transcriptional mutations. In addition, the crystal structure of mitrocomin was determined at 1.30 Åmore » resolution which expectedly revealed a high similarity with the structures of other hydromedusan photoproteins. Although mitrocomin isoforms reveal a high degree of identity of amino acid sequences, they vary in specific bioluminescence activities. At that, all isotypes displayed the identical bioluminescence spectra (473–474 nm with no shoulder at 400 nm). Fluorescence spectra of Ca2 +-discharged mitrocomins were almost identical to their light emission spectra similar to the case of Ca2 +-discharged aequorin, but different from Ca2 +-discharged obelins and clytin which fluorescence is red-shifted by 25–30 nm from bioluminescence spectra. The main distinction of mitrocomin from other hydromedusan photoproteins is an additional Tyr at the C-terminus. Using site-directed mutagenesis, we showed that this Tyr is not important for bioluminescence because its deletion even increases specific activity and efficiency of apo-mitrocomin conversion into active photoprotein, in contrast to C-terminal Pro of other photoproteins. Since genes in a population generally exist as different isoforms, it makes us anticipate the cloning of even more isoforms of mitrocomin and other hydromedusan photoproteins with different bioluminescence properties.« less

Genomic Context Analysis of de Novo STXBP1 Mutations Identifies Evidence of Splice Site DNA-Motif Associated Hotspots.

PubMed

Uddin, Mohammed; Woodbury-Smith, Marc; Chan, Ada J S; Albanna, Ammar; Minassian, Berge; Boelman, Cyrus; Scherer, Stephen W

2018-03-28

Mutations within STXBP1 have been associated with a range of neurodevelopmental disorders implicating the pleotropic impact of this gene. Although the frequency of de novo mutations within STXBP1 for selective cohorts with early onset epileptic encephalopathy is more than 1%, there is no evidence for a hotspot within the gene. In this study, we analyzed the genomic context of de novo STXBP1 mutations to examine whether certain motifs indicated a greater risk of mutation. Through a comprehensive context analysis of 136 de novo /rare mutation (SNV/Indels) sites in this gene, strikingly 26.92% of all SNV mutations occurred within 5bp upstream or downstream of a 'GTA' motif ( P < 0.0005). This implies a genomic context modulated mutagenesis. Moreover, 51.85% (14 out of 27) of the 'GTA' mutations are splicing compared to 14.70% (20 out of 136) of all reported mutations within STXBP1 We also noted that 11 of these 14 'GTA' associated mutations are de novo in origin. Our analysis provides strong evidence of DNA motif modulated mutagenesis for STXBP1 de novo splicing mutations. Copyright © 2018 Uddin et al.

The frequency and accuracy of replication past a thymine-thymine cyclobutane dimer are very different in Saccharomyces cerevisiae and Escherichia coli.

PubMed

Gibbs, P E; Kilbey, B J; Banerjee, S K; Lawrence, C W

1993-05-01

We have compared the mutagenic properties of a T-T cyclobutane dimer in baker's yeast, Saccharomyces cerevisiae, with those in Escherichia coli by transforming each of these species with the same single-stranded shuttle vector carrying either the cis-syn or the trans-syn isomer of this UV photoproduct at a unique site. The mutagenic properties investigated were the frequency of replicational bypass of the photoproduct, the error rate of bypass, and the mutation spectrum. In SOS-induced E. coli, the cis-syn dimer was bypassed in approximately 16% of the vector molecules, and 7.6% of the bypass products had targeted mutations. In S. cerevisiae, however, bypass occurred in about 80% of these molecules, and the bypass was at least 19-fold more accurate (approximately 0.4% targeted mutations). Each of these yeast mutations was a single unique event, and none were like those in E. coli, suggesting that in fact the difference in error rate is much greater. Bypass of the trans-syn dimer occurred in about 17% of the vector molecules in both species, but with this isomer the error rate was higher in S. cerevisiae (21 to 36% targeted mutations) than in E. coli (13%). However, the spectra of mutations induced by the latter photoproduct were virtually identical in the two organisms. We conclude that bypass and error frequencies are determined both by the structure of the photoproduct-containing template and by the particular replication proteins concerned but that the types of mutations induced depend predominantly on the structure of the template. Unlike E. coli, bypass in S. cerevisiae did not require UV-induced functions.

Analysis of ESR1 and PIK3CA mutations in plasma cell-free DNA from ER-positive breast cancer patients.

PubMed

Takeshita, Takashi; Yamamoto, Yutaka; Yamamoto-Ibusuki, Mutsuko; Tomiguchi, Mai; Sueta, Aiko; Murakami, Keiichi; Omoto, Yoko; Iwase, Hirotaka

2017-08-08

The measurement of ESR1 and PIK3CA mutations in plasma cell-free DNA (cfDNA) has been studied as a non-invasive method to quickly assess and monitor endocrine therapy (ET) resistant metastatic breast cancer (MBC) patients. The subjects of this retrospective study were a total of 185 plasma samples from 86 estrogen receptor-positive BC patients, of which 151 plasma samples were from 69 MBC patients and 34 plasma samples were from 17 primary BC (PBC) patients. We developed multiplex droplet digital PCR assays to verify the clinical significance of ESR1 and PIK3CA mutations both in a snapshot and serially in these patients. cfDNA ESR1 and PIK3CA mutations were found in 28.9% and 24.6 % of MBC patients, respectively. The relation between ESR1 or PIK3CA mutations and clinical features showed that ESR1 mutations occurred mostly in patients previously treated by ET, which was not the case for PIK3CA mutations. The analysis of the clinical impact of those mutations on subsequent lines of treatment for the 69 MBC patients revealed that both ESR1 and PIK3CA mutations detection were related to a shorter duration of ET effectiveness in univariate analysis but only for ESR1 mutations in multivariate analysis. The monitoring of cfDNA in a subset of 52 patients showed that loss of ESR1 mutations was related to a longer duration of response, which was not the case for PIK3CA mutations. We have demonstrated the clinical significance of on-treatment ESR1 mutations both in a snapshot and serially in comparison with PIK3CA mutations.

Analysis of ESR1 and PIK3CA mutations in plasma cell-free DNA from ER-positive breast cancer patients

PubMed Central

Takeshita, Takashi; Yamamoto, Yutaka; Yamamoto-Ibusuki, Mutsuko; Tomiguchi, Mai; Sueta, Aiko; Murakami, Keiichi; Omoto, Yoko; Iwase, Hirotaka

2017-01-01

Background The measurement of ESR1 and PIK3CA mutations in plasma cell-free DNA (cfDNA) has been studied as a non-invasive method to quickly assess and monitor endocrine therapy (ET) resistant metastatic breast cancer (MBC) patients. Methods The subjects of this retrospective study were a total of 185 plasma samples from 86 estrogen receptor-positive BC patients, of which 151 plasma samples were from 69 MBC patients and 34 plasma samples were from 17 primary BC (PBC) patients. We developed multiplex droplet digital PCR assays to verify the clinical significance of ESR1 and PIK3CA mutations both in a snapshot and serially in these patients. Results cfDNA ESR1 and PIK3CA mutations were found in 28.9% and 24.6 % of MBC patients, respectively. The relation between ESR1 or PIK3CA mutations and clinical features showed that ESR1 mutations occurred mostly in patients previously treated by ET, which was not the case for PIK3CA mutations. The analysis of the clinical impact of those mutations on subsequent lines of treatment for the 69 MBC patients revealed that both ESR1 and PIK3CA mutations detection were related to a shorter duration of ET effectiveness in univariate analysis but only for ESR1 mutations in multivariate analysis. The monitoring of cfDNA in a subset of 52 patients showed that loss of ESR1 mutations was related to a longer duration of response, which was not the case for PIK3CA mutations. Conclusions We have demonstrated the clinical significance of on-treatment ESR1 mutations both in a snapshot and serially in comparison with PIK3CA mutations. PMID:28881720

Novel USH2A compound heterozygous mutations cause RP/USH2 in a Chinese family.

PubMed

Liu, Xiaowen; Tang, Zhaohui; Li, Chang; Yang, Kangjuan; Gan, Guanqi; Zhang, Zibo; Liu, Jingyu; Jiang, Fagang; Wang, Qing; Liu, Mugen

2010-03-17

To identify the disease-causing gene in a four-generation Chinese family affected with retinitis pigmentosa (RP). Linkage analysis was performed with a panel of microsatellite markers flanking the candidate genetic loci of RP. These loci included 38 known RP genes. The complete coding region and exon-intron boundaries of Usher syndrome 2A (USH2A) were sequenced with the proband DNA to screen the disease-causing gene mutation. Restriction fragment length polymorphism (RFLP) analysis and direct DNA sequence analysis were done to demonstrate co-segregation of the USH2A mutations with the family disease. One hundred normal controls were used without the mutations. The disease-causing gene in this Chinese family was linked to the USH2A locus on chromosome 1q41. Direct DNA sequence analysis of USH2A identified two novel mutations in the patients: one missense mutation p.G1734R in exon 26 and a splice site mutation, IVS32+1G>A, which was found in the donor site of intron 32 of USH2A. Neither the p.G1734R nor the IVS32+1G>A mutation was found in the unaffected family members or the 100 normal controls. One patient with a homozygous mutation displayed only RP symptoms until now, while three patients with compound heterozygous mutations in the family of study showed both RP and hearing impairment. This study identified two novel mutations: p.G1734R and IVS32+1G>A of USH2A in a four-generation Chinese RP family. In this study, the heterozygous mutation and the homozygous mutation in USH2A may cause Usher syndrome Type II or RP, respectively. These two mutations expand the mutant spectrum of USH2A.

Null missense ABCR (ABCA4) mutations in a family with stargardt disease and retinitis pigmentosa.

PubMed

Shroyer, N F; Lewis, R A; Yatsenko, A N; Lupski, J R

2001-11-01

To determine the type of ABCR mutations that segregate in a family that manifests both Stargardt disease (STGD) and retinitis pigmentosa (RP), and the functional consequences of the underlying mutations. Direct sequencing of all 50 exons and flanking intronic regions of ABCR was performed for the STGD- and RP-affected relatives. RNA hybridization, Western blot analysis, and azido-adenosine triphosphate (ATP) labeling was used to determine the effect of disease-associated ABCR mutations in an in vitro assay system. Compound heterozygous missense mutations were identified in patients with STGD and RP. STGD-affected individual AR682-03 was compound heterozygous for the mutation 2588G-->C and a complex allele, [W1408R; R1640W]. RP-affected individuals AR682-04 and-05 were compound heterozygous for the complex allele [W1408R; R1640W] and the missense mutation V767D. Functional analysis of the mutation V767D by Western blot and ATP binding revealed a severe reduction in protein expression. In vitro analysis of ABCR protein with the mutations W1408R and R1640W showed a moderate effect of these individual mutations on expression and ATP-binding; the complex allele [W1408R; R1640W] caused a severe reduction in protein expression. These data reveal that missense ABCR mutations may be associated with RP. Functional analysis reveals that the RP-associated missense ABCR mutations are likely to be functionally null. These studies of the complex allele W1408R; R1640W suggest a synergistic effect of the individual mutations. These data are congruent with a model in which RP is associated with homozygous null mutations and with the notion that severity of retinal disease is inversely related to residual ABCR activity.

Mutation testing in Treacher Collins Syndrome.

PubMed

Ellis, P E; Dawson, M; Dixon, M J

2002-12-01

To report on a study where 97 subjects were screened for mutations in the Treacher Collins syndrome (TCS) gene TCOF1. Ninety-seven subjects with a clinical diagnosis of TCS were screened for potential mutations in TCOF1, by means of single strand conformation polymorphism (SSCP) analysis. In those subjects where potential mutations were detected, sequence analysis was performed to determine the site and type of mutation present. Thirty-six TCS-specific mutations are reported including 27 deletions, six point mutations, two splice junction mutations, and one insertion/deletion. This brings the total number of mutations reported to date to 105. The importance of detection of these mutations is mainly in postnatal diagnosis and genetic counselling. Knowledge of the family specific mutation may also be used in prenatal diagnosis to confirm whether the foetus is affected or not, and give the parents the choice of whether to continue with the pregnancy.

Genetic evolution of pancreatic cancer: lessons learnt from the pancreatic cancer genome sequencing project

PubMed Central

Iacobuzio-Donahue, Christine A

2012-01-01

Pancreatic cancer is a disease caused by the accumulation of genetic alterations in specific genes. Elucidation of the human genome sequence, in conjunction with technical advances in the ability to perform whole exome sequencing, have provided new insight into the mutational spectra characteristic of this lethal tumour type. Most recently, exomic sequencing has been used to clarify the clonal evolution of pancreatic cancer as well as provide time estimates of pancreatic carcinogenesis, indicating that a long window of opportunity may exist for early detection of this disease while in the curative stage. Moving forward, these mutational analyses indicate potential targets for personalised diagnostic and therapeutic intervention as well as the optimal timing for intervention based on the natural history of pancreatic carcinogenesis and progression. PMID:21749982

CCR4 frameshift mutation identifies a distinct group of adult T cell leukaemia/lymphoma with poor prognosis.

PubMed

Yoshida, Noriaki; Miyoshi, Hiroaki; Kato, Takeharu; Sakata-Yanagimoto, Mamiko; Niino, Daisuke; Taniguchi, Hiroaki; Moriuchi, Yukiyoshi; Miyahara, Masaharu; Kurita, Daisuke; Sasaki, Yuya; Shimono, Joji; Kawamoto, Keisuke; Utsunomiya, Atae; Imaizumi, Yoshitaka; Seto, Masao; Ohshima, Koichi

2016-04-01

Adult T cell leukaemia/lymphoma (ATLL) is an intractable T cell neoplasm caused by human T cell leukaemia virus type 1. Next-generation sequencing-based comprehensive mutation studies have revealed recurrent somatic CCR4 mutations in ATLL, although clinicopathological findings associated with CCR4 mutations remain to be delineated. In the current study, 184 cases of peripheral T cell lymphoma, including 113 cases of ATLL, were subjected to CCR4 mutation analysis. This sequence analysis identified mutations in 27% (30/113) of cases of ATLL and 9% (4/44) of cases of peripheral T cell lymphoma not otherwise specified. Identified mutations included nonsense (NS) and frameshift (FS) mutations. No significant differences in clinicopathological findings were observed between ATLL cases stratified by presence of CCR4 mutation. All ATLL cases with CCR4 mutations exhibited cell-surface CCR4 positivity. Semi-quantitative CCR4 protein analysis of immunohistochemical sections revealed higher CCR4 expression in cases with NS mutations of CCR4 than in cases with wild-type (WT) CCR4. Furthermore, among ATLL cases, FS mutation was significantly associated with a poor prognosis, compared with NS mutation and WT CCR4. These results suggest that CCR4 mutation is an important determinant of the clinical course in ATLL cases, and that NS and FS mutations of CCR4 behave differently with respect to ATLL pathophysiology. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Molecular diagnostics for hereditary hearing loss in children.

PubMed

Sommen, Manou; Wuyts, Wim; Van Camp, Guy

2017-08-01

Hearing loss (HL) is the most common birth defect in industrialized countries with far-reaching social, psychological and cognitive implications. It is an extremely heterogeneous disease, complicating molecular testing. The introduction of next-generation sequencing (NGS) has resulted in great progress in diagnostics allowing to study all known HL genes in a single assay. The diagnostic yield is currently still limited, but has the potential to increase substantially. Areas covered: In this review the utility of NGS and the problems for comprehensive molecular testing for HL are evaluated and discussed. Expert commentary: Different publications have proven the appropriateness of NGS for molecular testing of heterogeneous diseases such as HL. However, several problems still exist, such as pseudogenic background of some genes and problematic copy number variant analysis on targeted NGS data. Another main challenge for the future will be the establishment of population specific mutation-spectra to achieve accurate personalized comprehensive molecular testing for HL.

Mutational Signatures in Cancer (MuSiCa): a web application to implement mutational signatures analysis in cancer samples.

PubMed

Díaz-Gay, Marcos; Vila-Casadesús, Maria; Franch-Expósito, Sebastià; Hernández-Illán, Eva; Lozano, Juan José; Castellví-Bel, Sergi

2018-06-14

Mutational signatures have been proved as a valuable pattern in somatic genomics, mainly regarding cancer, with a potential application as a biomarker in clinical practice. Up to now, several bioinformatic packages to address this topic have been developed in different languages/platforms. MutationalPatterns has arisen as the most efficient tool for the comparison with the signatures currently reported in the Catalogue of Somatic Mutations in Cancer (COSMIC) database. However, the analysis of mutational signatures is nowadays restricted to a small community of bioinformatic experts. In this work we present Mutational Signatures in Cancer (MuSiCa), a new web tool based on MutationalPatterns and built using the Shiny framework in R language. By means of a simple interface suited to non-specialized researchers, it provides a comprehensive analysis of the somatic mutational status of the supplied cancer samples. It permits characterizing the profile and burden of mutations, as well as quantifying COSMIC-reported mutational signatures. It also allows classifying samples according to the above signature contributions. MuSiCa is a helpful web application to characterize mutational signatures in cancer samples. It is accessible online at http://bioinfo.ciberehd.org/GPtoCRC/en/tools.html and source code is freely available at https://github.com/marcos-diazg/musica .

Genetic Profiles of Korean Patients With Glucose-6-Phosphate Dehydrogenase Deficiency

PubMed Central

Lee, Jaewoong; Choi, Hayoung; Kim, Jiyeon; Kwon, Ahlm; Jang, Woori; Chae, Hyojin; Kim, Myungshin; Kim, Yonggoo; Lee, Jae Wook; Chung, Nack-Gyun

2017-01-01

Background We describe the genetic profiles of Korean patients with glucose-6-phosphate dehydrogenase (G6PD) deficiencies and the effects of G6PD mutations on protein stability and enzyme activity on the basis of in silico analysis. Methods In parallel with a genetic analysis, the pathogenicity of G6PD mutations detected in Korean patients was predicted in silico. The simulated effects of G6PD mutations were compared to the WHO classes based on G6PD enzyme activity. Four previously reported mutations and three newly diagnosed patients with missense mutations were estimated. Results One novel mutation (p.Cys385Gly, labeled G6PD Kangnam) and two known mutations [p.Ile220Met (G6PD São Paulo) and p.Glu416Lys (G6PD Tokyo)] were identified in this study. G6PD mutations identified in Koreans were also found in Brazil (G6PD São Paulo), Poland (G6PD Seoul), United States of America (G6PD Riley), Mexico (G6PD Guadalajara), and Japan (G6PD Tokyo). Several mutations occurred at the same nucleotide, but resulted in different amino acid residue changes in different ethnic populations (p.Ile380 variant, G6PD Calvo Mackenna; p.Cys385 variants, Tomah, Madrid, Lynwood; p.Arg387 variant, Beverly Hills; p.Pro396 variant, Bari; and p.Pro396Ala in India). On the basis of the in silico analysis, Class I or II mutations were predicted to be highly deleterious, and the effects of one Class IV mutation were equivocal. Conclusions The genetic profiles of Korean individuals with G6PD mutations indicated that the same mutations may have arisen by independent mutational events, and were not derived from shared ancestral mutations. The in silico analysis provided insight into the role of G6PD mutations in enzyme function and stability. PMID:28028996

Genetic Profiles of Korean Patients With Glucose-6-Phosphate Dehydrogenase Deficiency.

PubMed

Lee, Jaewoong; Park, Joonhong; Choi, Hayoung; Kim, Jiyeon; Kwon, Ahlm; Jang, Woori; Chae, Hyojin; Kim, Myungshin; Kim, Yonggoo; Lee, Jae Wook; Chung, Nack Gyun; Cho, Bin

2017-03-01

We describe the genetic profiles of Korean patients with glucose-6-phosphate dehydrogenase (G6PD) deficiencies and the effects of G6PD mutations on protein stability and enzyme activity on the basis of in silico analysis. In parallel with a genetic analysis, the pathogenicity of G6PD mutations detected in Korean patients was predicted in silico. The simulated effects of G6PD mutations were compared to the WHO classes based on G6PD enzyme activity. Four previously reported mutations and three newly diagnosed patients with missense mutations were estimated. One novel mutation (p.Cys385Gly, labeled G6PD Kangnam) and two known mutations [p.Ile220Met (G6PD São Paulo) and p.Glu416Lys (G6PD Tokyo)] were identified in this study. G6PD mutations identified in Koreans were also found in Brazil (G6PD São Paulo), Poland (G6PD Seoul), United States of America (G6PD Riley), Mexico (G6PD Guadalajara), and Japan (G6PD Tokyo). Several mutations occurred at the same nucleotide, but resulted in different amino acid residue changes in different ethnic populations (p.Ile380 variant, G6PD Calvo Mackenna; p.Cys385 variants, Tomah, Madrid, Lynwood; p.Arg387 variant, Beverly Hills; p.Pro396 variant, Bari; and p.Pro396Ala in India). On the basis of the in silico analysis, Class I or II mutations were predicted to be highly deleterious, and the effects of one Class IV mutation were equivocal. The genetic profiles of Korean individuals with G6PD mutations indicated that the same mutations may have arisen by independent mutational events, and were not derived from shared ancestral mutations. The in silico analysis provided insight into the role of G6PD mutations in enzyme function and stability.

Pyrosequencing analysis for detection of a BRAFV600E mutation in an FNAB specimen of thyroid nodules.

PubMed

Kim, Suk Kyeong; Kim, Dong-Lim; Han, Hye Seung; Kim, Wan Seop; Kim, Seung Ja; Moon, Won Jin; Oh, Seo Young; Hwang, Tae Sook

2008-06-01

Fine-needle aspiration biopsy (FNAB) is the primary means of distinguishing benign from malignant and of guiding therapeutic intervention in thyroid nodules. However, 10% to 30% of cases with indeterminate cytology in FNAB need other diagnostic tools to refine diagnosis. We compared the pyrosequencing method with the conventional direct DNA sequencing analysis and investigated the usefulness of preoperative BRAF mutation analysis as an adjunct diagnostic tool with routine FNAB. A total of 103 surgically confirmed patients' FNA slides were recruited and DNA was extracted after atypical cells were scraped from the slides. BRAF mutation was analyzed by pyrosequencing and direct DNA sequencing. Sixty-three (77.8%) of 81 histopathologically diagnosed malignant nodules revealed positive BRAF mutation on pyrosequencing analysis. In detail, 63 (84.0%) of 75 papillary thyroid carcinoma (PTC) samples showed positive BRAF mutation, whereas 3 follicular thyroid carcinomas, 1 anaplastic carcinoma, 1 medullary thyroid carcinoma, and 1 metastatic lung carcinoma did not show BRAF mutation. None of 22 benign nodules had BRAF mutation in both pyrosequencing and direct DNA sequencing. Out of 27 thyroid nodules classified as 'indeterminate' on cytologic examination preoperatively, 21 (77.8%) cases turned out to be malignant: 18 PTCs (including 2 follicular variant types) and 3 follicular thyroid carcinomas. Among these, 13 (61.9%) classic PTCs had BRAF mutation. None of 6 benign nodules, including 3 follicular adenomas and 3 nodular hyperplasias, had BRAF mutation. Among 63 PTCs with positive BRAF mutation detected by pyrosequencing analysis, 3 cases did not show BRAF mutation by direct DNA sequencing. Although it was not statistically significant, pyrosequencing was superior to direct DNA sequencing in detecting the BRAF mutation of thyroid nodules (P=0.25). Detecting BRAF mutation by pyrosequencing is more sensitive, faster, and less expensive than direct DNA sequencing and is proposed as an adjunct diagnostic tool in evaluating thyroid nodules of indeterminate cytology.

Database for Simulation of Electron Spectra for Surface Analysis (SESSA)Database for Simulation of Electron Spectra for Surface Analysis (SESSA)

National Institute of Standards and Technology Data Gateway

SRD 100 Database for Simulation of Electron Spectra for Surface Analysis (SESSA)Database for Simulation of Electron Spectra for Surface Analysis (SESSA) (PC database for purchase)   This database has been designed to facilitate quantitative interpretation of Auger-electron and X-ray photoelectron spectra and to improve the accuracy of quantitation in routine analysis. The database contains all physical data needed to perform quantitative interpretation of an electron spectrum for a thin-film specimen of given composition. A simulation module provides an estimate of peak intensities as well as the energy and angular distributions of the emitted electron flux.

Aristolochic acid-associated urothelial cancer in Taiwan

PubMed Central

Chen, Chung-Hsin; Dickman, Kathleen G.; Moriya, Masaaki; Zavadil, Jiri; Sidorenko, Viktoriya S.; Edwards, Karen L.; Gnatenko, Dmitri V.; Wu, Lin; Turesky, Robert J.; Wu, Xue-Ru; Pu, Yeong-Shiau; Grollman, Arthur P.

2012-01-01

Aristolochic acid, a potent human carcinogen produced by Aristolochia plants, is associated with urothelial carcinoma of the upper urinary tract (UUC). Following metabolic activation, aristolochic acid reacts with DNA to form aristolactam (AL)-DNA adducts. These lesions concentrate in the renal cortex, where they serve as a sensitive and specific biomarker of exposure, and are found also in the urothelium, where they give rise to a unique mutational signature in the TP53 tumor-suppressor gene. Using AL-DNA adducts and TP53 mutation spectra as biomarkers, we conducted a molecular epidemiologic study of UUC in Taiwan, where the incidence of UUC is the highest reported anywhere in the world and where Aristolochia herbal remedies have been used extensively for many years. Our study involves 151 UUC patients, with 25 patients with renal cell carcinomas serving as a control group. The TP53 mutational signature in patients with UUC, dominated by otherwise rare A:T to T:A transversions, is identical to that observed in UUC associated with Balkan endemic nephropathy, an environmental disease. Prominent TP53 mutational hotspots include the adenine bases of 5′AG (acceptor) splice sites located almost exclusively on the nontranscribed strand. A:T to T:A mutations also were detected at activating positions in the FGFR3 and HRAS oncogenes. AL-DNA adducts were present in the renal cortex of 83% of patients with A:T to T:A mutations in TP53, FGFR3, or HRAS. We conclude that exposure to aristolochic acid contributes significantly to the incidence of UUC in Taiwan, a finding with significant implications for global public health. PMID:22493262

Environmental Exposures, Genetic Polymorphisms and p53 Mutational Spectra in a Case-Control Study of Breast Cancer.

DTIC Science & Technology

1998-01-01

Further, preliminary clinical data suggest that serotonin reuptake inhibitors , such as fluoxetinc hydrochloride, may promote smoking cessation (15,16...benefits of serotonin reuptake inhibitors in smoking cessation (16) suggested that this gene may be a plausible candidate for predisposition to... used for random selection of women under age 65; women age 65 and over were randomly selected from the listing of the Health Care Finance

[Hereditary motor and sensory neuropathy with proximal dominant involvement (HMSN-P) is caused by a mutation in TFG].

PubMed

Ishiura, Hiroyuki; Tsuji, Shoji

2013-01-01

Hereditary motor and sensory neuropathy with proximal dominant involvement (HMSN-P) is an autosomal dominant neurodegenerative disease characterized by proximal predominant weakness and muscle atrophy accompanied by distal sensory disturbance. Linkage analysis using 4 families identified a region on chromosome 3 showing a LOD score exceeding 4. Further refinement of candidate region was performed by haplotype analysis using high-density SNP data, resulting in a minimum candidate region spanning 3.3 Mb. Exome analysis of an HMSN-P patient revealed a mutation (c.854C>T, p.Pro285Leu) in TRK-fused gene (TFG). The identical mutation was found in the four families, which cosegregated with the disease. The mutation was neither found in Japanese control subjects nor public databases. Detailed haplotype analysis suggested two independent origins of the mutation. These findings indicate that the mutation in TFG causes HMSN-P.

Pitfalls in molecular analysis for mismatch repair deficiency in a family with biallelic pms2 germline mutations.

PubMed

Leenen, C H M; Geurts-Giele, W R R; Dubbink, H J; Reddingius, R; van den Ouweland, A M; Tops, C M J; van de Klift, H M; Kuipers, E J; van Leerdam, M E; Dinjens, W N M; Wagner, A

2011-12-01

Heterozygous germline mutations in the mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2 cause Lynch syndrome. Biallelic mutations in the MMR genes are associated with a childhood cancer syndrome [constitutional mismatch repair deficiency (CMMR-D)]. This is predominantly characterized by hematological malignancies and tumors of the bowel and brain, often associated with signs of neurofibromatosis type 1 (NF1). Diagnostic strategies for selection of patients for MMR gene analysis include analysis of microsatellite instability (MSI) and immunohistochemical (IHC) analysis of MMR proteins in tumor tissue. We report the clinical characterization and molecular analyses of tumor specimens from a family with biallelic PMS2 germline mutations. This illustrates the pitfalls of present molecular screening strategies. Tumor tissues of five family members were analyzed for MSI and IHC. MSI was observed in only one of the analyzed tissues. However, IHC analysis of brain tumor tissue of the index patient and his sister showed absence of PMS2 expression, and germline mutation analyses showed biallelic mutations in PMS2: p.Ser46IIe and p.Pro246fs. The same heterozygous mutations were confirmed in the father and mother, respectively. These data support the conclusion that in case of a clinical phenotype of CMMR-D, it is advisable to routinely combine MSI analysis with IHC analysis for the expression of MMR proteins. With inconclusive or conflicting results, germline mutation analysis of the MMR genes should be considered after thorough counselling of the patients and/or their relatives. © 2011 John Wiley & Sons A/S.

Engineering a horseradish peroxidase C stable to radical attacks by mutating multiple radical coupling sites.

PubMed

Kim, Su Jin; Joo, Jeong Chan; Song, Bong Keun; Yoo, Young Je; Kim, Yong Hwan

2015-04-01

Peroxidases have great potential as industrial biocatalysts. In particular, the oxidative polymerization of phenolic compounds catalyzed by peroxidases has been extensively examined because of the advantage of this method over other conventional chemical methods. However, the industrial application of peroxidases is often limited because of their rapid inactivation by phenoxyl radicals during oxidative polymerization. In this work, we report a novel protein engineering approach to improve the radical stability of horseradish peroxidase isozyme C (HRPC). Phenylalanine residues that are vulnerable to modification by the phenoxyl radicals were identified using mass spectrometry analysis. UV-Vis and CD spectra showed that radical coupling did not change the secondary structure or the active site of HRPC. Four phenylalanine (Phe) residues (F68, F142, F143, and F179) were each mutated to alanine residues to generate single mutants to examine the role of these sites in radical coupling. Despite marginal improvement of radical stability, each single mutant still exhibited rapid radical inactivation. To further reduce inactivation by radical coupling, the four substitution mutations were combined in F68A/F142A/F143A/F179A. This mutant demonstrated dramatic enhancement of radical stability by retaining 41% of its initial activity compared to the wild-type, which was completely inactivated. Structure and sequence alignment revealed that radical-vulnerable Phe residues of HPRC are conserved in homologous peroxidases, which showed the same rapid inactivation tendency as HRPC. Based on our site-directed mutagenesis and biochemical characterization, we have shown that engineering radical-vulnerable residues to eliminate multiple radical coupling can be a good strategy to improve the stability of peroxidases against radical attack. © 2014 Wiley Periodicals, Inc.

Identification of a novel CLRN1 gene mutation in Usher syndrome type 3: two case reports.

PubMed

Yoshimura, Hidekane; Oshikawa, Chie; Nakayama, Jun; Moteki, Hideaki; Usami, Shin-Ichi

2015-05-01

This study examines the CLRN1 gene mutation analysis in Japanese patients who were diagnosed with Usher syndrome type 3 (USH3) on the basis of clinical findings. Genetic analysis using massively parallel DNA sequencing (MPS) was conducted to search for 9 causative USH genes in 2 USH3 patients. We identified the novel pathogenic mutation in the CLRN1 gene in 2 patients. The missense mutation was confirmed by functional prediction software and segregation analysis. Both patients were diagnosed as having USH3 caused by the CLRN1 gene mutation. This is the first report of USH3 with a CLRN1 gene mutation in Asian populations. Validating the presence of clinical findings is imperative for properly differentiating among USH subtypes. In addition, mutation screening using MPS enables the identification of causative mutations in USH. The clinical diagnosis of this phenotypically variable disease can then be confirmed. © The Author(s) 2015.

Mutation rates at 42 Y chromosomal short tandem repeats in Chinese Han population in Eastern China.

PubMed

Wu, Weiwei; Ren, Wenyan; Hao, Honglei; Nan, Hailun; He, Xin; Liu, Qiuling; Lu, Dejian

2018-01-31

Mutation analysis of 42 Y chromosomal short tandem repeats (Y-STRs) loci was performed using a sample of 1160 father-son pairs from the Chinese Han population in Eastern China. The results showed that the average mutation rate across the 42 Y-STR loci was 0.0041 (95% CI 0.0036-0.0047) per locus per generation. The locus-specific mutation rates varied from 0.000 to 0.0190. No mutation was found at DYS388, DYS437, DYS448, DYS531, and GATA_H4. DYS627, DYS570, DYS576, and DYS449 could be classified as rapidly mutating Y-STRs, with mutation rates higher than 1.0 × 10 -2 . DYS458, DYS630, and DYS518 were moderately mutating Y-STRs, with mutation rates ranging from 8 × 10 -3 to 1 × 10 -2 . Although the characteristics of the Y-STR mutations were consistent with those in previous studies, mutation rate differences between our data and previous published data were found at some rapidly mutating Y-STRs. The single-copy loci located on the short arm of the Y chromosome (Yp) showed relatively higher mutation rates more frequently than the multi-copy loci. These results will not only extend the data for Y-STR mutations but also be important for kinship analysis, paternal lineage identification, and family relationship reconstruction in forensic Y-STR analysis.

Norrie disease: first mutation report and prenatal diagnosis in an Indian family.

PubMed

Ghosh, Manju; Sharma, Shipra; Shastri, Shivaram; Arora, Sadhna; Shukla, Rashmi; Gupta, Neerja; Deka, Deepika; Kabra, Madhulika

2012-11-01

Norrie Disease (ND) is a rare X-linked recessive disorder characterised by congenital blindness due to severe retinal dysgenesis. Hearing loss and intellectual disability is present in 30-50 % cases. ND is caused by mutations in the NDP gene, located at Xp11.3. The authors describe mutation analysis of a proband with ND and subsequently prenatal diagnosis. Sequence analysis of the NDP gene revealed a hemizygous missense mutation arginine to serine in codon 41 (p.Arg41Ser) in the affected child. Mother was carrier for the mutation. In a subsequent di-chorionic di-amniotic pregnancy, the authors performed prenatal diagnosis by mutation analysis on chorionic villi sample at 11 wk of gestation. The fetuses were unaffected. This is a first mutation report and prenatal diagnosis of a familial case of Norrie disease from India. The importance of genetic testing of Norrie disease for confirmation, carrier testing, prenatal diagnosis and genetic counseling is emphasized.

Identification of mutations in Colombian patients affected with Fabry disease.

PubMed

Uribe, Alfredo; Mateus, Heidi Eliana; Prieto, Juan Carlos; Palacios, Maria Fernanda; Ospina, Sandra Yaneth; Pasqualim, Gabriela; da Silveira Matte, Ursula; Giugliani, Roberto

2015-12-15

Fabry Disease (FD) is an X-linked inborn error of glycosphingolipid catabolism, caused by a deficiency of the lisosomal α-galactosidase A (AGAL). The disorder leads to a vascular disease secondary to the involvement of kidney, heart and the central nervous system. The mutation analysis is a valuable tool for diagnosis and genetic counseling. Although more than 600 mutations have been identified, most mutations are private. Our objective was to describe the analysis of nine Colombian patients with Fabry disease by automated sequencing of the seven exons of the GLA gene. Two novel mutations were identified in two patients affected with the classical subtype of FD, in addition to other 6 mutations previously reported. The present study confirms the heterogeneity of mutations in Fabry disease and the importance of molecular analysis for genetic counseling, female heterozygotes detection as well as therapeutic decisions. Copyright © 2015 Elsevier B.V. All rights reserved.

Mutated form (G52E) of inactive diphtheria toxin CRM197: molecular simulations clearly display effect of the mutation to NAD binding.

PubMed

Salmas, Ramin Ekhteiari; Mestanoglu, Mert; Unlu, Ayhan; Yurtsever, Mine; Durdagi, Serdar

2016-11-01

Mutated form (G52E) of diphtheria toxin (DT) CRM197 is an inactive and nontoxic enzyme. Here, we provided a molecular insight using comparative molecular dynamics (MD) simulations to clarify the influence of a single point mutation on overall protein and active-site loop. Post-processing MD analysis (i.e. stability, principal component analysis, hydrogen-bond occupancy, etc.) is carried out on both wild and mutated targets to investigate and to better understand the mechanistic differences of structural and dynamical properties on an atomic scale especially at nicotinamide adenine dinucleotide (NAD) binding site when a single mutation (G52E) happens at the DT. In addition, a docking simulation is performed for wild and mutated forms. The docking scoring analysis and docking poses results revealed that mutant form is not able to properly accommodate the NAD molecule.

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

PubMed

Gonzalez-Bosquet, Jesus; Calcei, Jacob; Wei, Jun S; Garcia-Closas, Montserrat; Sherman, Mark E; Hewitt, Stephen; Vockley, Joseph; Lissowska, Jolanta; Yang, Hannah P; Khan, Javed; Chanock, Stephen

2011-01-17

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

Detection of Somatic Mutations by High-Resolution DNA Melting (HRM) Analysis in Multiple Cancers

PubMed Central

Gonzalez-Bosquet, Jesus; Calcei, Jacob; Wei, Jun S.; Garcia-Closas, Montserrat; Sherman, Mark E.; Hewitt, Stephen; Vockley, Joseph; Lissowska, Jolanta; Yang, Hannah P.; Khan, Javed; Chanock, Stephen

2011-01-01

Identification of somatic mutations in cancer is a major goal for understanding and monitoring the events related to cancer initiation and progression. High resolution melting (HRM) curve analysis represents a fast, post-PCR high-throughput method for scanning somatic sequence alterations in target genes. The aim of this study was to assess the sensitivity and specificity of HRM analysis for tumor mutation screening in a range of tumor samples, which included 216 frozen pediatric small rounded blue-cell tumors as well as 180 paraffin-embedded tumors from breast, endometrial and ovarian cancers (60 of each). HRM analysis was performed in exons of the following candidate genes known to harbor established commonly observed mutations: PIK3CA, ERBB2, KRAS, TP53, EGFR, BRAF, GATA3, and FGFR3. Bi-directional sequencing analysis was used to determine the accuracy of the HRM analysis. For the 39 mutations observed in frozen samples, the sensitivity and specificity of HRM analysis were 97% and 87%, respectively. There were 67 mutation/variants in the paraffin-embedded samples, and the sensitivity and specificity for the HRM analysis were 88% and 80%, respectively. Paraffin-embedded samples require higher quantity of purified DNA for high performance. In summary, HRM analysis is a promising moderate-throughput screening test for mutations among known candidate genomic regions. Although the overall accuracy appears to be better in frozen specimens, somatic alterations were detected in DNA extracted from paraffin-embedded samples. PMID:21264207

Digital PCR Improves Mutation Analysis in Pancreas Fine Needle Aspiration Biopsy Specimens.

PubMed

Sho, Shonan; Court, Colin M; Kim, Stephen; Braxton, David R; Hou, Shuang; Muthusamy, V Raman; Watson, Rabindra R; Sedarat, Alireza; Tseng, Hsian-Rong; Tomlinson, James S

2017-01-01

Applications of precision oncology strategies rely on accurate tumor genotyping from clinically available specimens. Fine needle aspirations (FNA) are frequently obtained in cancer management and often represent the only source of tumor tissues for patients with metastatic or locally advanced diseases. However, FNAs obtained from pancreas ductal adenocarcinoma (PDAC) are often limited in cellularity and/or tumor cell purity, precluding accurate tumor genotyping in many cases. Digital PCR (dPCR) is a technology with exceptional sensitivity and low DNA template requirement, characteristics that are necessary for analyzing PDAC FNA samples. In the current study, we sought to evaluate dPCR as a mutation analysis tool for pancreas FNA specimens. To this end, we analyzed alterations in the KRAS gene in pancreas FNAs using dPCR. The sensitivity of dPCR mutation analysis was first determined using serial dilution cell spiking studies. Single-cell laser-microdissection (LMD) was then utilized to identify the minimal number of tumor cells needed for mutation detection. Lastly, dPCR mutation analysis was performed on 44 pancreas FNAs (34 formalin-fixed paraffin-embedded (FFPE) and 10 fresh (non-fixed)), including samples highly limited in cellularity (100 cells) and tumor cell purity (1%). We found dPCR to detect mutations with allele frequencies as low as 0.17%. Additionally, a single tumor cell could be detected within an abundance of normal cells. Using clinical FNA samples, dPCR mutation analysis was successful in all preoperative FNA biopsies tested, and its accuracy was confirmed via comparison with resected tumor specimens. Moreover, dPCR revealed additional KRAS mutations representing minor subclones within a tumor that were not detected by the current clinical gold standard method of Sanger sequencing. In conclusion, dPCR performs sensitive and accurate mutation analysis in pancreas FNAs, detecting not only the dominant mutation subtype, but also the additional rare mutation subtypes representing tumor heterogeneity.

Digital PCR Improves Mutation Analysis in Pancreas Fine Needle Aspiration Biopsy Specimens

PubMed Central

Court, Colin M.; Kim, Stephen; Braxton, David R.; Hou, Shuang; Muthusamy, V. Raman; Watson, Rabindra R.; Sedarat, Alireza; Tseng, Hsian-Rong; Tomlinson, James S.

2017-01-01

Applications of precision oncology strategies rely on accurate tumor genotyping from clinically available specimens. Fine needle aspirations (FNA) are frequently obtained in cancer management and often represent the only source of tumor tissues for patients with metastatic or locally advanced diseases. However, FNAs obtained from pancreas ductal adenocarcinoma (PDAC) are often limited in cellularity and/or tumor cell purity, precluding accurate tumor genotyping in many cases. Digital PCR (dPCR) is a technology with exceptional sensitivity and low DNA template requirement, characteristics that are necessary for analyzing PDAC FNA samples. In the current study, we sought to evaluate dPCR as a mutation analysis tool for pancreas FNA specimens. To this end, we analyzed alterations in the KRAS gene in pancreas FNAs using dPCR. The sensitivity of dPCR mutation analysis was first determined using serial dilution cell spiking studies. Single-cell laser-microdissection (LMD) was then utilized to identify the minimal number of tumor cells needed for mutation detection. Lastly, dPCR mutation analysis was performed on 44 pancreas FNAs (34 formalin-fixed paraffin-embedded (FFPE) and 10 fresh (non-fixed)), including samples highly limited in cellularity (100 cells) and tumor cell purity (1%). We found dPCR to detect mutations with allele frequencies as low as 0.17%. Additionally, a single tumor cell could be detected within an abundance of normal cells. Using clinical FNA samples, dPCR mutation analysis was successful in all preoperative FNA biopsies tested, and its accuracy was confirmed via comparison with resected tumor specimens. Moreover, dPCR revealed additional KRAS mutations representing minor subclones within a tumor that were not detected by the current clinical gold standard method of Sanger sequencing. In conclusion, dPCR performs sensitive and accurate mutation analysis in pancreas FNAs, detecting not only the dominant mutation subtype, but also the additional rare mutation subtypes representing tumor heterogeneity. PMID:28125707

Altering the spectrum of immunoglobulin V gene somatic hypermutation by modifying the active site of AID.

PubMed

Wang, Meng; Rada, Cristina; Neuberger, Michael S

2010-01-18

High-affinity antibodies are generated by somatic hypermutation with nucleotide substitutions introduced into the IgV in a semirandom fashion, but with intrinsic mutational hotspots strategically located to optimize antibody affinity maturation. The process is dependent on activation-induced deaminase (AID), an enzyme that can deaminate deoxycytidine in DNA in vitro, where its activity is sensitive to the identity of the 5'-flanking nucleotide. As a critical test of whether such DNA deamination activity underpins antibody diversification and to gain insight into the extent to which the antibody mutation spectrum is dependent on the intrinsic substrate specificity of AID, we investigated whether it is possible to change the IgV mutation spectrum by altering AID's active site such that it prefers a pyrimidine (rather than a purine) flanking the targeted deoxycytidine. Consistent with the DNA deamination mechanism, B cells expressing the modified AID proteins yield altered IgV mutation spectra (exhibiting a purine-->pyrimidine shift in flanking nucleotide preference) and altered hotspots. However, AID-catalyzed deamination of IgV targets in vitro does not yield the same degree of hotspot dominance to that observed in vivo, indicating the importance of features beyond AID's active site and DNA local sequence environment in determining in vivo hotspot dominance.

Molecular epidemiology in environmental health: the potential of tumor suppressor gene p53 as a biomarker.

PubMed Central

Semenza, J C; Weasel, L H

1997-01-01

One of the challenges in environmental health is to attribute a certain health effect to a specific environmental exposure and to establish a cause-effect relationship. Molecular epidemiology offers a new approach to addressing these challenges. Mutations in the tumor suppressor gene p53 can shed light on past environmental exposure, and carcinogenic agents and doses can be distinguished on the basis of mutational spectra and frequency. Mutations in p53 have successfully been used to establish links between dietary aflatoxin exposure and liver cancer, exposure to ultraviolet light and skin cancer, smoking and cancers of the lung and bladder, and vinyl chloride exposure and liver cancer. In lung cancer, carcinogens from tobacco smoke have been shown to form adducts with DNA. The location of these adducts correlates with those positions in the p53 gene that are mutated in lung cancer, confirming a direct etiologic link between exposure and disease. Recent investigations have also explored the use of p53 as a susceptibility marker for cancer. Furthermore, studies in genetic toxicology have taken advantage of animals transgenic for p53 to screen for carcinogens in vivo. In this review, we summarize recent developments in p53 biomarker research and illustrate applications to environmental health. PMID:9114284

Mutational analysis of the HGO gene in Finnish alkaptonuria patients

PubMed Central

de Bernabe, D. B.-V.; Peterson, P.; Luopajarvi, K.; Matintalo, P.; Alho, A.; Konttinen, Y.; Krohn, K.; de Cordoba, S. R.; Ranki, A.

1999-01-01

Alkaptonuria (AKU), the prototypic inborn error of metabolism, has recently been shown to be caused by loss of function mutations in the homogentisate-1,2-dioxygenase gene (HGO). So far 17 mutations have been characterised in AKU patients of different ethnic origin. We describe three novel mutations (R58fs, R330S, and H371R) and one common AKU mutation (M368V), detected by mutational and polymorphism analysis of the HGO gene in five Finnish AKU pedigrees. The three novel AKU mutations are most likely specific for the Finnish population and have originated recently.


Keywords: alkaptonuria; homogentisate-1,2-dioxygenase; Finland PMID:10594001

Novel compound heterozygous mutations in MYO7A in a Chinese family with Usher syndrome type 1

PubMed Central

Liu, Fei; Li, Pengcheng; Liu, Ying; Li, Weirong; Wong, Fulton; Du, Rong; Wang, Lei; Li, Chang; Jiang, Fagang; Tang, Zhaohui

2013-01-01

Purpose To identify the disease-causing mutation(s) in a Chinese family with autosomal recessive Usher syndrome type 1 (USH1). Methods An ophthalmic examination and an audiometric test were conducted to ascertain the phenotype of two affected siblings. The microsatellite marker D11S937, which is close to the candidate gene MYO7A (USH1B locus), was selected for genotyping. From the DNA of the proband, all coding exons and exon-intron boundaries of MYO7A were sequenced to identify the disease-causing mutation(s). Restriction fragment length polymorphism (RFLP) analysis was performed to exclude the alternative conclusion that the mutations are non-pathogenic rare polymorphisms. Results Based on severe hearing impairment, unintelligible speech, and retinitis pigmentosa, a clinical diagnosis of Usher syndrome type 1 was made. The genotyping results did not exclude the USH1B locus, which suggested that the MYO7A gene was likely the gene associated with the disease-causing mutation(s) in the family. With direct DNA sequencing of MYO7A, two novel compound heterozygous mutations (c.3742G>A and c.6051+1G>A) of MYO7A were identified in the proband. DNA sequence analysis and RFLP analysis of other family members showed that the mutations cosegregated with the disease. Unaffected members, including the parents, uncle, and sister of the proband, carry only one of the two mutations. The mutations were not present in the controls (100 normal Chinese subjects=200 chromosomes) according to the RFLP analysis. Conclusions In this study, we identified two novel mutations, c.3742G>A (p.E1248K) and c.6051+1G>A (donor splice site mutation in intron 44), of MYO7A in a Chinese non-consanguineous family with USH1. The mutations cosegregated with the disease and most likely cause the phenotype in the two affected siblings who carry these mutations compound heterozygously. Our finding expands the mutational spectrum of MYO7A. PMID:23559863

Spectra-first feature analysis in clinical proteomics - A case study in renal cancer.

PubMed

Goh, Wilson Wen Bin; Wong, Limsoon

2016-10-01

In proteomics, useful signal may be unobserved or lost due to the lack of confident peptide-spectral matches. Selection of differential spectra, followed by associative peptide/protein mapping may be a complementary strategy for improving sensitivity and comprehensiveness of analysis (spectra-first paradigm). This approach is complementary to the standard approach where functional analysis is performed only on the finalized protein list assembled from identified peptides from the spectra (protein-first paradigm). Based on a case study of renal cancer, we introduce a simple spectra-binning approach, MZ-bin. We demonstrate that differential spectra feature selection using MZ-bin is class-discriminative and can trace relevant proteins via spectra associative mapping. Moreover, proteins identified in this manner are more biologically coherent than those selected directly from the finalized protein list. Analysis of constituent peptides per protein reveals high expression inconsistency, suggesting that the measured protein expressions are in fact, poor approximations of true protein levels. Moreover, analysis at the level of constituent peptides may provide higher resolution insight into the underlying biology: Via MZ-bin, we identified for the first time differential splice forms for the known renal cancer marker MAPT. We conclude that the spectra-first analysis paradigm is a complementary strategy to the traditional protein-first paradigm and can provide deeper level insight.

Catalysis of GTP Hydrolysis by Small GTPases at Atomic Detail by Integration of X-ray Crystallography, Experimental, and Theoretical IR Spectroscopy*

PubMed Central

Rudack, Till; Jenrich, Sarah; Brucker, Sven; Vetter, Ingrid R.; Gerwert, Klaus; Kötting, Carsten

2015-01-01

Small GTPases regulate key processes in cells. Malfunction of their GTPase reaction by mutations is involved in severe diseases. Here, we compare the GTPase reaction of the slower hydrolyzing GTPase Ran with Ras. By combination of time-resolved FTIR difference spectroscopy and QM/MM simulations we elucidate that the Mg2+ coordination by the phosphate groups, which varies largely among the x-ray structures, is the same for Ran and Ras. A new x-ray structure of a Ran·RanBD1 complex with improved resolution confirmed this finding and revealed a general problem with the refinement of Mg2+ in GTPases. The Mg2+ coordination is not responsible for the much slower GTPase reaction of Ran. Instead, the location of the Tyr-39 side chain of Ran between the γ-phosphate and Gln-69 prevents the optimal positioning of the attacking water molecule by the Gln-69 relative to the γ-phosphate. This is confirmed in the RanY39A·RanBD1 crystal structure. The QM/MM simulations provide IR spectra of the catalytic center, which agree very nicely with the experimental ones. The combination of both methods can correlate spectra with structure at atomic detail. For example the FTIR difference spectra of RasA18T and RanT25A mutants show that spectral differences are mainly due to the hydrogen bond of Thr-25 to the α-phosphate in Ran. By integration of x-ray structure analysis, experimental, and theoretical IR spectroscopy the catalytic center of the x-ray structural models are further refined to sub-Å resolution, allowing an improved understanding of catalysis. PMID:26272610

Catalysis of GTP hydrolysis by small GTPases at atomic detail by integration of X-ray crystallography, experimental, and theoretical IR spectroscopy.

PubMed

Rudack, Till; Jenrich, Sarah; Brucker, Sven; Vetter, Ingrid R; Gerwert, Klaus; Kötting, Carsten

2015-10-02

Small GTPases regulate key processes in cells. Malfunction of their GTPase reaction by mutations is involved in severe diseases. Here, we compare the GTPase reaction of the slower hydrolyzing GTPase Ran with Ras. By combination of time-resolved FTIR difference spectroscopy and QM/MM simulations we elucidate that the Mg(2+) coordination by the phosphate groups, which varies largely among the x-ray structures, is the same for Ran and Ras. A new x-ray structure of a Ran·RanBD1 complex with improved resolution confirmed this finding and revealed a general problem with the refinement of Mg(2+) in GTPases. The Mg(2+) coordination is not responsible for the much slower GTPase reaction of Ran. Instead, the location of the Tyr-39 side chain of Ran between the γ-phosphate and Gln-69 prevents the optimal positioning of the attacking water molecule by the Gln-69 relative to the γ-phosphate. This is confirmed in the RanY39A·RanBD1 crystal structure. The QM/MM simulations provide IR spectra of the catalytic center, which agree very nicely with the experimental ones. The combination of both methods can correlate spectra with structure at atomic detail. For example the FTIR difference spectra of RasA18T and RanT25A mutants show that spectral differences are mainly due to the hydrogen bond of Thr-25 to the α-phosphate in Ran. By integration of x-ray structure analysis, experimental, and theoretical IR spectroscopy the catalytic center of the x-ray structural models are further refined to sub-Å resolution, allowing an improved understanding of catalysis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Landscape of somatic mutations in 560 breast cancer whole-genome sequences

DOE PAGES

Nik-Zainal, Serena; Davies, Helen; Staaf, Johan; ...

2016-05-02

Here, we analysed whole-genome sequences of 560 breast cancers to advance understanding of the driver mutations conferring clonal advantage and the mutational processes generating somatic mutations. We found that 93 protein-coding cancer genes carried probable driver mutations. Some non-coding regions exhibited high mutation frequencies, but most have distinctive structural features probably causing elevated mutation rates and do not contain driver mutations. Mutational signature analysis was extended to genome rearrangements and revealed twelve base substitution and six rearrangement signatures. Three rearrangement signatures, characterized by tandem duplications or deletions, appear associated with defective homologous-recombination-based DNA repair: one with deficient BRCA1 function, anothermore » with deficient BRCA1 or BRCA2 function, the cause of the third is unknown. This analysis of all classes of somatic mutation across exons, introns and intergenic regions highlights the repertoire of cancer genes and mutational processes operating, and progresses towards a comprehensive account of the somatic genetic basis of breast cancer.« less

Landscape of somatic mutations in 560 breast cancer whole-genome sequences

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nik-Zainal, Serena; Davies, Helen; Staaf, Johan

Here, we analysed whole-genome sequences of 560 breast cancers to advance understanding of the driver mutations conferring clonal advantage and the mutational processes generating somatic mutations. We found that 93 protein-coding cancer genes carried probable driver mutations. Some non-coding regions exhibited high mutation frequencies, but most have distinctive structural features probably causing elevated mutation rates and do not contain driver mutations. Mutational signature analysis was extended to genome rearrangements and revealed twelve base substitution and six rearrangement signatures. Three rearrangement signatures, characterized by tandem duplications or deletions, appear associated with defective homologous-recombination-based DNA repair: one with deficient BRCA1 function, anothermore » with deficient BRCA1 or BRCA2 function, the cause of the third is unknown. This analysis of all classes of somatic mutation across exons, introns and intergenic regions highlights the repertoire of cancer genes and mutational processes operating, and progresses towards a comprehensive account of the somatic genetic basis of breast cancer.« less

Landscape of somatic mutations in 560 breast cancer whole genome sequences

PubMed Central

Nik-Zainal, Serena; Davies, Helen; Staaf, Johan; Ramakrishna, Manasa; Glodzik, Dominik; Zou, Xueqing; Martincorena, Inigo; Alexandrov, Ludmil B.; Martin, Sancha; Wedge, David C.; Van Loo, Peter; Ju, Young Seok; Smid, Marcel; Brinkman, Arie B; Morganella, Sandro; Aure, Miriam R.; Lingjærde, Ole Christian; Langerød, Anita; Ringnér, Markus; Ahn, Sung-Min; Boyault, Sandrine; Brock, Jane E.; Broeks, Annegien; Butler, Adam; Desmedt, Christine; Dirix, Luc; Dronov, Serge; Fatima, Aquila; Foekens, John A.; Gerstung, Moritz; Hooijer, Gerrit KJ; Jang, Se Jin; Jones, David R.; Kim, Hyung-Yong; King, Tari A.; Krishnamurthy, Savitri; Lee, Hee Jin; Lee, Jeong-Yeon; Li, Yilong; McLaren, Stuart; Menzies, Andrew; Mustonen, Ville; O’Meara, Sarah; Pauporté, Iris; Pivot, Xavier; Purdie, Colin A.; Raine, Keiran; Ramakrishnan, Kamna; Rodríguez-González, F. Germán; Romieu, Gilles; Sieuwerts, Anieta M.; Simpson, Peter T; Shepherd, Rebecca; Stebbings, Lucy; Stefansson, Olafur A; Teague, Jon; Tommasi, Stefania; Treilleux, Isabelle; Van den Eynden, Gert G.; Vermeulen, Peter; Vincent-Salomon, Anne; Yates, Lucy; Caldas, Carlos; van’t Veer, Laura; Tutt, Andrew; Knappskog, Stian; Tan, Benita Kiat Tee; Jonkers, Jos; Borg, Åke; Ueno, Naoto T; Sotiriou, Christos; Viari, Alain; Futreal, P. Andrew; Campbell, Peter J; Span, Paul N.; Van Laere, Steven; Lakhani, Sunil R; Eyfjord, Jorunn E.; Thompson, Alastair M.; Birney, Ewan; Stunnenberg, Hendrik G; van de Vijver, Marc J; Martens, John W.M.; Børresen-Dale, Anne-Lise; Richardson, Andrea L.; Kong, Gu; Thomas, Gilles; Stratton, Michael R.

2016-01-01

We analysed whole genome sequences of 560 breast cancers to advance understanding of the driver mutations conferring clonal advantage and the mutational processes generating somatic mutations. 93 protein-coding cancer genes carried likely driver mutations. Some non-coding regions exhibited high mutation frequencies but most have distinctive structural features probably causing elevated mutation rates and do not harbour driver mutations. Mutational signature analysis was extended to genome rearrangements and revealed 12 base substitution and six rearrangement signatures. Three rearrangement signatures, characterised by tandem duplications or deletions, appear associated with defective homologous recombination based DNA repair: one with deficient BRCA1 function; another with deficient BRCA1 or BRCA2 function; the cause of the third is unknown. This analysis of all classes of somatic mutation across exons, introns and intergenic regions highlights the repertoire of cancer genes and mutational processes operative, and progresses towards a comprehensive account of the somatic genetic basis of breast cancer. PMID:27135926

Genetic analysis of leukemic transformation of chronic myeloproliferative neoplasms

PubMed Central

Abdel-Wahab, Omar; Manshouri, Taghi; Patel, Jay; Harris, Kelly; Yao, JinJuan; Hedvat, Cyrus; Heguy, Adriana; Bueso-Ramos, Carlos; Kantarjian, Hagop; Levine, Ross L.; Verstovsek, Srdan

2009-01-01

The genetic events which contribute to transformation of myeloproliferative neoplasms (MPN) to acute myeloid leukemia (AML) are not well characterized. We investigated the role of JAK2, TET2, ASXL1, and IDH1 mutations in leukemic transformation of MPNs through mutational analysis of 63 patients with AML secondary to a preexisting MPN (sAML). We identified frequent TET2 (26.3%), ASXL1 (19.3%), IDH1 (9.5%), and JAK2 (36.8%) mutations in sAML; all possible mutational combinations of these genes were observed. Analysis of 14 patients for which paired samples from MPN and sAML were available demonstrated TET2 mutations were frequently acquired at leukemic transformation (6/14=43%). In contrast, ASXL1 mutations were almost always detected in both the MPN and AML clones from individual patients. A case was also observed where TET2 and ASXL1 mutations were found before the patient acquired a JAK2 mutation or developed clinical evidence of MPN. We conclude that mutations in TET2, ASXL1, and IDH1 are common in sAML derived from a pre-existing MPN. Although TET2/ASXL1 mutations may precede acquisition of JAK2 mutations by the MPN clone, mutations in TET2, but not ASXL1, are commonly acquired at the time of leukemic transformation. These data suggest the mutational order of events in MPN and sAML varies in different patients, and that TET2 and ASXL1 mutations have distinct roles in MPN pathogenesis and leukemic transformation. The presence of sAML with no pre-existing JAK2/TET2/ASXL1/IDH1 mutations indicates the existence of other mutations necessary for leukemic transformation. PMID:20068184

Multiplex picodroplet digital PCR to detect KRAS mutations in circulating DNA from the plasma of colorectal cancer patients.

PubMed

Taly, Valerie; Pekin, Deniz; Benhaim, Leonor; Kotsopoulos, Steve K; Le Corre, Delphine; Li, Xinyu; Atochin, Ivan; Link, Darren R; Griffiths, Andrew D; Pallier, Karine; Blons, Hélène; Bouché, Olivier; Landi, Bruno; Hutchison, J Brian; Laurent-Puig, Pierre

2013-12-01

Multiplex digital PCR (dPCR) enables noninvasive and sensitive detection of circulating tumor DNA with performance unachievable by current molecular-detection approaches. Furthermore, picodroplet dPCR facilitates simultaneous screening for multiple mutations from the same sample. We investigated the utility of multiplex dPCR to screen for the 7 most common mutations in codons 12 and 13 of the KRAS (Kirsten rat sarcoma viral oncogene homolog) oncogene from plasma samples of patients with metastatic colorectal cancer. Fifty plasma samples were tested from patients for whom the primary tumor biopsy tissue DNA had been characterized by quantitative PCR. Tumor characterization revealed that 19 patient tumors had KRAS mutations. Multiplex dPCR analysis of the plasma DNA prepared from these samples identified 14 samples that matched the mutation identified in the tumor, 1 sample contained a different KRAS mutation, and 4 samples had no detectable mutation. Among the tumor samples that were wild type for KRAS, 2 KRAS mutations were identified in the corresponding plasma samples. Duplex dPCR (i.e., wild-type and single-mutation assay) was also used to analyze plasma samples from patients with KRAS-mutated tumors and 5 samples expected to contain the BRAF (v-raf murine sarcoma viral oncogene homolog B) V600E mutation. The results for the duplex analysis matched those for the multiplex analysis for KRAS-mutated samples and, owing to its higher sensitivity, enabled detection of 2 additional samples with low levels of KRAS-mutated DNA. All 5 samples with BRAF mutations were detected. This work demonstrates the clinical utility of multiplex dPCR to screen for multiple mutations simultaneously with a sensitivity sufficient to detect mutations in circulating DNA obtained by noninvasive blood collection.

Identification and analysis of mutational hotspots in oncogenes and tumour suppressors.

PubMed

Baeissa, Hanadi; Benstead-Hume, Graeme; Richardson, Christopher J; Pearl, Frances M G

2017-03-28

The key to interpreting the contribution of a disease-associated mutation in the development and progression of cancer is an understanding of the consequences of that mutation both on the function of the affected protein and on the pathways in which that protein is involved. Protein domains encapsulate function and position-specific domain based analysis of mutations have been shown to help elucidate their phenotypes. In this paper we examine the domain biases in oncogenes and tumour suppressors, and find that their domain compositions substantially differ. Using data from over 30 different cancers from whole-exome sequencing cancer genomic projects we mapped over one million mutations to their respective Pfam domains to identify which domains are enriched in any of three different classes of mutation; missense, indels or truncations. Next, we identified the mutational hotspots within domain families by mapping small mutations to equivalent positions in multiple sequence alignments of protein domainsWe find that gain of function mutations from oncogenes and loss of function mutations from tumour suppressors are normally found in different domain families and when observed in the same domain families, hotspot mutations are located at different positions within the multiple sequence alignment of the domain. By considering hotspots in tumour suppressors and oncogenes independently, we find that there are different specific positions within domain families that are particularly suited to accommodate either a loss or a gain of function mutation. The position is also dependent on the class of mutation.We find rare mutations co-located with well-known functional mutation hotspots, in members of homologous domain superfamilies, and we detect novel mutation hotspots in domain families previously unconnected with cancer. The results of this analysis can be accessed through the MOKCa database (http://strubiol.icr.ac.uk/extra/MOKCa).

Beta thalassemia in 31,734 cases with HBB gene mutations: Pathogenic and structural analysis of the common mutations; Iran as the crossroads of the Middle East.

PubMed

Mahdieh, Nejat; Rabbani, Bahareh

2016-11-01

Thalassemia is one of the most common single gene disorders worldwide. Nearly 80 to 90 million with minor beta thalassemia and 60-70 thousand affected infants are born annually worldwide. A comprehensive search on several databases including PubMed, InterScience, British Library Direct, and Science Direct was performed extracting papers about mutation detection and frequency of beta thalassemia. All papers reporting on the mutation frequency of beta thalassemia patients were selected to analyze the frequency of mutations in different regions and various ethnicities. Mutations of 31,734 individuals were identified. Twenty common mutations were selected for further analysis. Genotype-phenotype correlation, interactome, and in silico analyses of the mutations were performed using available bioinformatics tools. Secondary structure prediction was achieved for two common mutations with online tools. The mutations were also common among the countries neighboring Iran, which are responsible for 71% to 98% of mutations. Computational analyses could be used in addition to segregation and expression analysis to assess the extent of pathogenicity of the variant. The genetics of beta thalassemia in Iran is more extensively heterogeneous than in neighboring countries. Some common mutations have arisen historically from Iran and moved to other populations due to population migrations. Also, due to genetic drift, the frequencies of some mutations have increased in small populations. Copyright © 2016 Elsevier Ltd. All rights reserved.

Alpha-cardiac myosin heavy chain (MYH6) mutations affecting myofibril formation are associated with congenital heart defects.

PubMed

Granados-Riveron, Javier T; Ghosh, Tushar K; Pope, Mark; Bu'Lock, Frances; Thornborough, Christopher; Eason, Jacqueline; Kirk, Edwin P; Fatkin, Diane; Feneley, Michael P; Harvey, Richard P; Armour, John A L; David Brook, J

2010-10-15

Congenital heart defects (CHD) are collectively the most common form of congenital malformation. Studies of human cases and animal models have revealed that mutations in several genes are responsible for both familial and sporadic forms of CHD. We have previously shown that a mutation in MYH6 can cause an autosomal dominant form of atrial septal defect (ASD), whereas others have identified mutations of the same gene in patients with hypertrophic and dilated cardiomyopathy. In the present study, we report a mutation analysis of MYH6 in patients with a wide spectrum of sporadic CHD. The mutation analysis of MYH6 was performed in DNA samples from 470 cases of isolated CHD using denaturing high-performance liquid chromatography and sequence analysis to detect point mutations and small deletions or insertions, and multiplex amplifiable probe hybridization to detect partial or complete copy number variations. One non-sense mutation, one splicing site mutation and seven non-synonymous coding mutations were identified. Transfection of plasmids encoding mutant and non-mutant green fluorescent protein-MYH6 fusion proteins in mouse myoblasts revealed that the mutations A230P and A1366D significantly disrupt myofibril formation, whereas the H252Q mutation significantly enhances myofibril assembly in comparison with the non-mutant protein. Our data indicate that functional variants of MYH6 are associated with cardiac malformations in addition to ASD and provide a novel potential mechanism. Such phenotypic heterogeneity has been observed in other genes mutated in CHD.

GATA3 mutation in a family with hypoparathyroidism, deafness and renal dysplasia syndrome.

PubMed

Zhu, Zi-Yang; Zhou, Qiao-Li; Ni, Shi-Ning; Gu, Wei

2014-08-01

The hypoparathyroidism, deafness and renal dysplasia (HDR) syndrome is an autosomal dominant disorder primarily caused by GATA3 gene mutation. We report here a case that both of a Chinese boy and his father had HDR syndrome which caused by a novel mutation of GATA3. Polymerase chain reaction and DNA sequencing was performed to detect the exons of the GATA3 gene for mutation analysis. Sequence analysis of GATA3 revealed a heterozygous nonsense mutation in this family: a mutation of GATA3 at exon 2 (c.515C >A) that resulted in a premature stop at codon 172 (p.S172X) with a loss of two zinc finger domains. We identified a novel nonsense mutation which will expand the spectrum of HDR-associated GATA3 mutations.

Epidermal growth factor receptor mutations in adenocarcinoma in situ and minimally invasive adenocarcinoma detected using mutation-specific monoclonal antibodies.

PubMed

Nakamura, Haruhiko; Koizumi, Hirotaka; Kimura, Hiroyuki; Marushima, Hideki; Saji, Hisashi; Takagi, Masayuki

2016-09-01

Epidermal growth factor receptor (EGFR) mutation rates in adenocarcinoma in situ (AIS) and minimally invasive adenocarcinoma (MIA) were studied using both DNA analysis and mutation-specific immunohistochemistry. The peptide nucleic acid-locked nucleic acid polymerase chain reaction clamp method was used to detect mutations in exons 18, 19, 20, and 21 of the EGFR gene in DNA samples extracted from paraffin-embedded tissue sections. Simultaneously, immunohistochemical analysis with two EGFR mutation-specific monoclonal antibodies was used to identify proteins resulting from an in-frame deletion in exon 19 (E746_A750del) and a point mutation replacing leucine with arginine at codon 858 of exon 21 (L858R). Forty-three tumors (22 AIS and 21 MIA) were examined. The EGFR mutation rate in AIS detected by DNA analysis was 27.3% (L858R, 5/22; exon 19 deletion,1/22), whereas that detected in MIA was 42.9% (L858R,4/21; exon 19 deletion,5/21). Mutations detected by immunohistochemical analysis included 22.7% (L858R, 4/22; exon 19 deletion, 1/22) in AIS and 42.9% (L858R, 4/21; exon 19 deletion, 5/21) in MIA. Although some results were contradictory, concordant results were obtained using both assays in 38 of 43 cases (88.4%). DNA and immunohistochemical analyses revealed similar EGFR mutation rates in both MIA and AIS, suggesting that mutation-specific monoclonal antibodies are useful to confirm DNA assay results. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

A sensitive continuum analysis method for gamma ray spectra

NASA Technical Reports Server (NTRS)

Thakur, Alakh N.; Arnold, James R.

1993-01-01

In this work we examine ways to improve the sensitivity of the analysis procedure for gamma ray spectra with respect to small differences in the continuum (Compton) spectra. The method developed is applied to analyze gamma ray spectra obtained from planetary mapping by the Mars Observer spacecraft launched in September 1992. Calculated Mars simulation spectra and actual thick target bombardment spectra have been taken as test cases. The principle of the method rests on the extraction of continuum information from Fourier transforms of the spectra. We study how a better estimate of the spectrum from larger regions of the Mars surface will improve the analysis for smaller regions with poorer statistics. Estimation of signal within the continuum is done in the frequency domain which enables efficient and sensitive discrimination of subtle differences between two spectra. The process is compared to other methods for the extraction of information from the continuum. Finally we explore briefly the possible uses of this technique in other applications of continuum spectra.

DETECTION OF K-RAS AND P53 MUTATIONS IN SPUTUM SAMPLES OF LUNG CANCER PATIENTS USING LASER CAPTURE MICRODISSECTION MICROSCOPE AND MUTATION ANALYSIS

EPA Science Inventory

Detection of K-ras and p53 Mutations in Sputum Samples of Lung Cancer Patients Using Laser Capture Microdissection Microscope and Mutation Analysis

Phouthone Keohavong a,*, Wei-Min Gao a, Kui-Cheng Zheng a, Hussam Mady b, Qing Lan c, Mona Melhem b, and Judy Mumford d.
<...

Prognostic significance of SRSF2 mutations in myelodysplastic syndromes and chronic myelomonocytic leukemia: a meta-analysis.

PubMed

Arbab Jafari, Pourya; Ayatollahi, Hossein; Sadeghi, Ramin; Sheikhi, Maryam; Asghari, Amir

2018-05-14

Serine/arginine-rich splicing factor 2 (SRSF2) mutations were detected frequently in myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML) patients. However, its prognostic value has not yet been fully clarified. In this meta-analysis, Hazard Ratio (HR) and 95% confidence interval (CI) for overall-survival (OS) were chosen to evaluate the prognostic impact of SRSF2 mutations and to compare SRSF2 mutations to those with wild-type. A total of 2056 patients from 12 studies were obtained. The pooled HRs for OSsuggested that patients with MDS had a poorer prognosis (HR = 1.780, 95% CI (1.410-2.249)), while analysis on SRSF2 mutations revealed no significant effect on the prognosis of CMML patients (HR = 1.091, 95% CI (0.925-1.286)). The frequency of SRSF2 mutations was found to be 11.5% and 39.8% in patients with MDS and CMML, respectively. This meta-analysis suggests that SRSF2 has a poor prognosis in patients with MDS, but no prognosis impact on patients with CMML. In conclusion, SRSF2 mutations were significantly related to the shorter OS in patients with MDS which may consider as an adverse prognostic risk factor. Whereas, analysis did not show any prognostic effect on OS of CMML patients with SRSF2 mutations.

Increasing the yield in targeted next-generation sequencing by implicating CNV analysis, non-coding exons and the overall variant load: the example of retinal dystrophies.

PubMed

Eisenberger, Tobias; Neuhaus, Christine; Khan, Arif O; Decker, Christian; Preising, Markus N; Friedburg, Christoph; Bieg, Anika; Gliem, Martin; Charbel Issa, Peter; Holz, Frank G; Baig, Shahid M; Hellenbroich, Yorck; Galvez, Alberto; Platzer, Konrad; Wollnik, Bernd; Laddach, Nadja; Ghaffari, Saeed Reza; Rafati, Maryam; Botzenhart, Elke; Tinschert, Sigrid; Börger, Doris; Bohring, Axel; Schreml, Julia; Körtge-Jung, Stefani; Schell-Apacik, Chayim; Bakur, Khadijah; Al-Aama, Jumana Y; Neuhann, Teresa; Herkenrath, Peter; Nürnberg, Gudrun; Nürnberg, Peter; Davis, John S; Gal, Andreas; Bergmann, Carsten; Lorenz, Birgit; Bolz, Hanno J

2013-01-01

Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are major causes of blindness. They result from mutations in many genes which has long hampered comprehensive genetic analysis. Recently, targeted next-generation sequencing (NGS) has proven useful to overcome this limitation. To uncover "hidden mutations" such as copy number variations (CNVs) and mutations in non-coding regions, we extended the use of NGS data by quantitative readout for the exons of 55 RP and LCA genes in 126 patients, and by including non-coding 5' exons. We detected several causative CNVs which were key to the diagnosis in hitherto unsolved constellations, e.g. hemizygous point mutations in consanguineous families, and CNVs complemented apparently monoallelic recessive alleles. Mutations of non-coding exon 1 of EYS revealed its contribution to disease. In view of the high carrier frequency for retinal disease gene mutations in the general population, we considered the overall variant load in each patient to assess if a mutation was causative or reflected accidental carriership in patients with mutations in several genes or with single recessive alleles. For example, truncating mutations in RP1, a gene implicated in both recessive and dominant RP, were causative in biallelic constellations, unrelated to disease when heterozygous on a biallelic mutation background of another gene, or even non-pathogenic if close to the C-terminus. Patients with mutations in several loci were common, but without evidence for di- or oligogenic inheritance. Although the number of targeted genes was low compared to previous studies, the mutation detection rate was highest (70%) which likely results from completeness and depth of coverage, and quantitative data analysis. CNV analysis should routinely be applied in targeted NGS, and mutations in non-coding exons give reason to systematically include 5'-UTRs in disease gene or exome panels. Consideration of all variants is indispensable because even truncating mutations may be misleading.

Analysis of gene mutations among South Indian patients with maple syrup urine disease: identification of four novel mutations.

PubMed

Narayanan, M P; Menon, Krishnakumar N; Vasudevan, D M

2013-10-01

Maple syrup urine disease (MSUD) is predominantly caused by mutations in the BCKDHA, BCKDHB and DBT genes, which encode for the E1alpha, E1beta and E2 subunits of the branched-chain alpha-keto acid dehydrogenase complex, respectively. Because disease causing mutations play a major role in the development of the disease, prenatal diagnosis at gestational level may have significance in making decisions by parents. Thus, this study was aimed to screen South Indian MSUD patients for mutations and assess the genotype-phenotype correlation. Thirteen patients diagnosed with MSUD by conventional biochemical screening such as urine analysis by DNPH test, thin layer chromatography for amino acids and blood amino acid quantification by HPLC were selected for mutation analysis. The entire coding regions of the BCKDHA, BCKDHB and DBT genes were analyzed for mutations by PCR-based direct DNA sequencing. BCKDHA and BCKDHB mutations were seen in 43% of the total ten patients, while disease-causing DBT gene mutation was observed only in 14%. Three patients displayed no mutations. Novel mutations were c.130C>T in BCKDHA gene, c. 599C>T and c.121_122delAC in BCKDHB gene and c.190G>A in DBT gene. Notably, patients harbouring these mutations were non-responsive to thiamine supplementation and other treatment regimens and might have a worse prognosis as compared to the patients not having such mutations. Thus, identification of these mutations may have a crucial role in the treatment as well as understanding the molecular mechanisms in MSUD.

Somatic mutations in the transcriptional corepressor gene BCORL1 in adult acute myelogenous leukemia.

PubMed

Li, Meng; Collins, Roxane; Jiao, Yuchen; Ouillette, Peter; Bixby, Dale; Erba, Harry; Vogelstein, Bert; Kinzler, Kenneth W; Papadopoulos, Nickolas; Malek, Sami N

2011-11-24

To further our understanding of the genetic basis of acute myelogenous leukemia (AML), we determined the coding exon sequences of ∼ 18 000 protein-encoding genes in 8 patients with secondary AML. Here we report the discovery of novel somatic mutations in the transcriptional corepressor gene BCORL1 that is located on the X-chromosome. Analysis of BCORL1 in an unselected cohort of 173 AML patients identified a total of 10 mutated cases (6%) with BCORL1 mutations, whereas analysis of 19 AML cell lines uncovered 4 (21%) BCORL1 mutated cell lines. The majority (87%) of the mutations in BCORL1 were predicted to inactivate the gene product as a result of nonsense mutations, splice site mutation, or out-of-frame insertions or deletions. These results indicate that BCORL1 by genetic criteria is a novel candidate tumor suppressor gene, joining the growing list of genes recurrently mutated in AML.

MutationAligner: a resource of recurrent mutation hotspots in protein domains in cancer

PubMed Central

Gauthier, Nicholas Paul; Reznik, Ed; Gao, Jianjiong; Sumer, Selcuk Onur; Schultz, Nikolaus; Sander, Chris; Miller, Martin L.

2016-01-01

The MutationAligner web resource, available at http://www.mutationaligner.org, enables discovery and exploration of somatic mutation hotspots identified in protein domains in currently (mid-2015) more than 5000 cancer patient samples across 22 different tumor types. Using multiple sequence alignments of protein domains in the human genome, we extend the principle of recurrence analysis by aggregating mutations in homologous positions across sets of paralogous genes. Protein domain analysis enhances the statistical power to detect cancer-relevant mutations and links mutations to the specific biological functions encoded in domains. We illustrate how the MutationAligner database and interactive web tool can be used to explore, visualize and analyze mutation hotspots in protein domains across genes and tumor types. We believe that MutationAligner will be an important resource for the cancer research community by providing detailed clues for the functional importance of particular mutations, as well as for the design of functional genomics experiments and for decision support in precision medicine. MutationAligner is slated to be periodically updated to incorporate additional analyses and new data from cancer genomics projects. PMID:26590264

Potential energy distribution (PED) analysis of DFT calculated IR spectra of the most stable Li, Na, and Cu(I) diformate molecules

NASA Astrophysics Data System (ADS)

Jamróz, M. H.; Dobrowolski, J. Cz.

2001-05-01

For the most stable Li, Na, and Cu(I) diformates we present the vibrational spectra, supported by potential energy distribution (PED) analysis, and the interaction energies between formic acid and metal formate by the DFT (B3PW91) method. PED analysis of the theoretical spectra forms the basis for the elucidation of the future matrix isolation IR spectra.

Development and inter-laboratory validation of unlabeled probe melting curve analysis for detection of JAK2 V617F mutation in polycythemia vera.

PubMed

Wu, Zhiyuan; Yuan, Hong; Zhang, Xinju; Liu, Weiwei; Xu, Jinhua; Zhang, Wei; Guan, Ming

2011-01-01

JAK2 V617F, a somatic point mutation that leads to constitutive JAK2 phosphorylation and kinase activation, has been incorporated into the WHO classification and diagnostic criteria of myeloid neoplasms. Although various approaches such as restriction fragment length polymorphism, amplification refractory mutation system and real-time PCR have been developed for its detection, a generic rapid closed-tube method, which can be utilized on routine genetic testing instruments with stability and cost-efficiency, has not been described. Asymmetric PCR for detection of JAK2 V617F with a 3'-blocked unlabeled probe, saturate dye and subsequent melting curve analysis was performed on a Rotor-Gene® Q real-time cycler to establish the methodology. We compared this method to the existing amplification refractory mutation systems and direct sequencing. Hereafter, the broad applicability of this unlabeled probe melting method was also validated on three diverse real-time systems (Roche LightCycler® 480, Applied Biosystems ABI® 7500 and Eppendorf Mastercycler® ep realplex) in two different laboratories. The unlabeled probe melting analysis could genotype JAK2 V617F mutation explicitly with a 3% mutation load detecting sensitivity. At level of 5% mutation load, the intra- and inter-assay CVs of probe-DNA heteroduplex (mutation/wild type) covered 3.14%/3.55% and 1.72%/1.29% respectively. The method could equally discriminate mutant from wild type samples on the other three real-time instruments. With a high detecting sensitivity, unlabeled probe melting curve analysis is more applicable to disclose JAK2 V617F mutation than conventional methodologies. Verified with the favorable inter- and intra-assay reproducibility, unlabeled probe melting analysis provided a generic mutation detecting alternative for real-time instruments.

Differential effects of severe vs mild GBA mutations on Parkinson disease.

PubMed

Gan-Or, Ziv; Amshalom, Idan; Kilarski, Laura L; Bar-Shira, Anat; Gana-Weisz, Mali; Mirelman, Anat; Marder, Karen; Bressman, Susan; Giladi, Nir; Orr-Urtreger, Avi

2015-03-03

To better define the genotype-phenotype correlations between the type of GBA (glucosidase, beta, acid) mutation, severe or mild, and the risk and age at onset (AAO), and potential mechanism of Parkinson disease (PD). We analyzed 1,000 patients of Ashkenazi-Jewish descent with PD for 7 founder GBA mutations, and conducted a meta-analysis of risk and AAO according to GBA genotype (severe or mild mutation). The meta-analysis included 11,453 patients with PD and 14,565 controls from worldwide populations. The statistical analysis was done with and without continuity correction (constant or empirical), considering biases that could potentially affect the results. Among Ashkenazi-Jewish patients with PD, the odds ratios for PD were 2.2 and 10.3 for mild and severe GBA mutation carriers, respectively. The observed frequency of severe GBA mutation carriers among patients with PD was more than 4-fold than expected (4.4% vs 0.9%, respectively, p < 0.0001, Fisher exact test). In the different models of the meta-analysis, the odds ratios for PD ranged between 2.84 and 4.94 for mild GBA mutation carriers and 9.92 and 21.29 for severe GBA mutation carriers (p < 1 × 10(-6) for all analyses). Pooled analysis demonstrated AAO of 53.1 (±11.2) and 58.1 (±10.6) years for severe and mild GBA mutation carriers, respectively (p = 4.3 × 10(-5)). These data demonstrate that mild and severe heterozygous GBA mutations differentially affect the risk and the AAO of PD. Our results have important implications for genetic counseling and clinical follow-up. © 2015 American Academy of Neurology.

A single center analysis of nucleophosmin in acute myeloid leukemia: value of combining immunohistochemistry with molecular mutation analysis.

PubMed

Woolthuis, Carolien M; Mulder, André B; Verkaik-Schakel, Rikst Nynke; Rosati, Stefano; Diepstra, Arjan; van den Berg, Eva; Schuringa, Jan Jacob; Vellenga, Edo; Kluin, Philip M; Huls, Gerwin

2013-10-01

Mutations of nucleophosmin 1 are frequently found in acute myeloid leukemia and lead to aberrant cytoplasmic accumulation of nucleophosmin protein. Immunohistochemical staining is therefore recommended as the technique of choice in front-line screening. In this study, we assessed the sensitivity and specificity of immunohistochemistry on formalin-fixed bone marrow biopsies compared with gold standard molecular analysis to predict nucleophosmin 1 mutation status in 119 patients with acute myeloid leukemia. Discrepant cases were further characterized by gene expression analyses and fluorescence in situ hybridization. A large overlap between both methods was observed. Nevertheless, nine patients demonstrated discordant results at initial screening. Five cases demonstrated nuclear staining of nucleophosmin 1 by immunohistochemistry, but a nucleophosmin 1 mutation by molecular analysis. In two cases this could be attributed to technical issues and in three cases minor subpopulations of myeloblasts had not been discovered initially. All tested cases exhibited the characteristic nucleophosmin-mutated gene expression pattern. Four cases had cytoplasmic nucleophosmin 1 staining and a nucleophosmin-mutated gene expression pattern without a detectable nucleophosmin 1 mutation. In two of these cases we found the chromosomal translocation t(3;5)(q25;q35) encoding the NPM-MLF1 fusion protein. In the other discrepant cases the aberrant cytoplasmic nucleophosmin staining and gene expression could not be explained. In total six patients (5%) had true discordant results between immunohistochemistry and mutation analysis. We conclude that cytoplasmic nucleophosmin localization is not always caused by a conventional nucleophosmin 1 mutation and that in the screening for nucleophosmin 1 abnormalities, most information will be obtained by combining immunohistochemistry with molecular analysis.

A single center analysis of nucleophosmin in acute myeloid leukemia: value of combining immunohistochemistry with molecular mutation analysis

PubMed Central

Woolthuis, Carolien M.; Mulder, André B.; Verkaik-Schakel, Rikst Nynke; Rosati, Stefano; Diepstra, Arjan; van den Berg, Eva; Schuringa, Jan Jacob; Vellenga, Edo; Kluin, Philip M.; Huls, Gerwin

2013-01-01

Mutations of nucleophosmin 1 are frequently found in acute myeloid leukemia and lead to aberrant cytoplasmic accumulation of nucleophosmin protein. Immunohistochemical staining is therefore recommended as the technique of choice in front-line screening. In this study, we assessed the sensitivity and specificity of immunohistochemistry on formalin-fixed bone marrow biopsies compared with gold standard molecular analysis to predict nucleophosmin 1 mutation status in 119 patients with acute myeloid leukemia. Discrepant cases were further characterized by gene expression analyses and fluorescence in situ hybridization. A large overlap between both methods was observed. Nevertheless, nine patients demonstrated discordant results at initial screening. Five cases demonstrated nuclear staining of nucleophosmin 1 by immunohistochemistry, but a nucleophosmin 1 mutation by molecular analysis. In two cases this could be attributed to technical issues and in three cases minor subpopulations of myeloblasts had not been discovered initially. All tested cases exhibited the characteristic nucleophosmin-mutated gene expression pattern. Four cases had cytoplasmic nucleophosmin 1 staining and a nucleophosmin-mutated gene expression pattern without a detectable nucleophosmin 1 mutation. In two of these cases we found the chromosomal translocation t(3;5)(q25;q35) encoding the NPM-MLF1 fusion protein. In the other discrepant cases the aberrant cytoplasmic nucleophosmin staining and gene expression could not be explained. In total six patients (5%) had true discordant results between immunohistochemistry and mutation analysis. We conclude that cytoplasmic nucleophosmin localization is not always caused by a conventional nucleophosmin 1 mutation and that in the screening for nucleophosmin 1 abnormalities, most information will be obtained by combining immunohistochemistry with molecular analysis. PMID:23716555

Analysis of KRAS and BRAF genes mutation in the central nervous system metastases of non-small cell lung cancer.

PubMed

Nicoś, Marcin; Krawczyk, Paweł; Jarosz, Bożena; Sawicki, Marek; Szumiłło, Justyna; Trojanowski, Tomasz; Milanowski, Janusz

2016-05-01

KRAS mutations are associated with tumor resistance to EGFR TKIs (erlotinib, gefitinib) and to monoclonal antibody against EGFR (cetuximab). Targeted treatment of mutated RAS patients is still considered as a challenge. Inhibitors of c-Met (onartuzumab or tiwantinib) and MEK (selumetinib-a dual inhibitor of MEK1 and MEK2) signaling pathways showed activity in patients with mutations in KRAS that can became an effective approach in carriers of such disorders. BRAF mutation is very rare in patients with NSCLC, and its presence is associated with sensitivity of tumor cells to BRAF inhibitors (vemurafenib, dabrafenib). In the present study, the frequency and type of KRAS and BRAF mutation were assessed in 145 FFPE tissue samples from CNS metastases of NSCLC. In 30 patients, material from the primary tumor was simultaneously available. Real-time PCR technique with allele-specific molecular probe (KRAS/BRAF Mutation Analysis Kit, Entrogen, USA) was used for molecular tests. KRAS mutations were detected in 21.4 % of CNS metastatic lesions and in 23.3 % of corresponding primary tumors. Five mutations were identified both in primary and in metastatic lesions, while one mutation only in primary tumor and one mutation only in the metastatic tumor. Most of mutations were observed in codon 12 of KRAS; however, an individual patient had diagnosed a rare G13D and Q61R substitutions. KRAS mutations were significantly more frequent in adenocarcinoma patients and smokers. Additional analysis indicated one patient with rare coexistence of KRAS and DDR2 mutations. BRAF mutation was not detected in the examined materials. KRAS frequency appears to be similar in primary and CNS.

Environmental Exposures, Genetic Polymorphisms and p53 Mutational Spectra in a Case-Control Study of Breast Cancer

DTIC Science & Technology

1999-01-01

potential benefits of serotonin reuptake inhibitors in X1 tests were performed to evaluate associations of 5-HTT smoking cessation (15) suggested... dogs , sausages, bacon and cold diet assessment instrument used is a well-established tool for cuts was also assessed. A poultry index included...Controls were frequency-matched to cases on age and county. The listing of licensed New York State drivers was used for random selection of women

Environmental Exposures, Genetic Polymorphisms and p53 Mutational Spectra in a Case-Control Study of Breast Cancer.

DTIC Science & Technology

1997-01-01

frequently reported in premenopausal rather than postmenopausal women. It is currently unknown what might be the carcinogenic agents in alcoholic... agents in human main-that they could affect heterogeneity of Breast cancer and cigarette smoking. N Engl J mary epithelial cells. CancerRes. 1992;52:5617...trosamines, polycyclic aromatic hydrocarbons, aryl aro- Abbreviations: NMU, N-methyl-N- nitrosourea ; CYP2E1, cytochrome matic amines, and heterocyclic

Breast and ovarian cancer risks to carriers of the BRCA1 5382insC and 185delAG and BRCA2 6174delT mutations: a combined analysis of 22 population based studies

PubMed Central

Antoniou, A; Pharoah, P; Narod, S; Risch, H; Eyfjord, J; Hopper, J; Olsson, H; Johannsson, O; Borg, A; Pasini, B; Radice, P; Manoukian, S; Eccles, D; Tang, N; Olah, E; Anton-Culver, H; Warner, E; Lubinski, J; Gronwald, J; Gorski, B; Tulinius, H; Thorlacius, S; Eerola, H; Nevanlinna, H; Syrjakoski, K; Kallioniemi, O; Thompson, D; Evans, C; Peto, J; Lalloo, F; Evans, D; Easton, D

2005-01-01

A recent report estimated the breast cancer risks in carriers of the three Ashkenazi founder mutations to be higher than previously published estimates derived from population based studies. In an attempt to confirm this, the breast and ovarian cancer risks associated with the three Ashkenazi founder mutations were estimated using families included in a previous meta-analysis of populatrion based studies. The estimated breast cancer risks for each of the founder BRCA1 and BRCA2 mutations were similar to the corresponding estimates based on all BRCA1 or BRCA2 mutations in the meta-analysis. These estimates appear to be consistent with the observed prevalence of the mutations in the Ashkenazi Jewish population. PMID:15994883

Man-made mineral fiber hazardous properties assessment using transgenic rodents: example of glass fiber testing.

PubMed

Bottin, M C; Vigneron, J C; Rousseau, R; Micillino, J C; Eypert-Blaison, C; Kauffer, E; Martin, P; Binet, S; Rihn, B H

2003-09-01

Transgenic BigBlue rats were exposed to CM 44 glass fibers (6.3 mg/m3) by nose only, 6 h/day for 5 days. Two endpoints were examined 1, 3, 14, 28, and 90 days following exposure: fiber biopersistence and mutations in lung DNA. The half-time of the fibers >20 microm was 12.8 days, and mutant frequencies of control and exposed rats were similar across all time points. The mutation spectra of both series were similar after 28 days of fixation time. These results showed that a glass fiber with a high clearance in the lung seems to not present any significant effect on mutagenesis on lung DNA and are in marked contrast to results for asbestos, which caused a twofold mutant frequency increase as described in a previous study.

Structural Analysis of Single-Point Mutations Given an RNA Sequence: A Case Study with RNAMute

NASA Astrophysics Data System (ADS)

Churkin, Alexander; Barash, Danny

2006-12-01

We introduce here for the first time the RNAMute package, a pattern-recognition-based utility to perform mutational analysis and detect vulnerable spots within an RNA sequence that affect structure. Mutations in these spots may lead to a structural change that directly relates to a change in functionality. Previously, the concept was tried on RNA genetic control elements called "riboswitches" and other known RNA switches, without an organized utility that analyzes all single-point mutations and can be further expanded. The RNAMute package allows a comprehensive categorization, given an RNA sequence that has functional relevance, by exploring the patterns of all single-point mutants. For illustration, we apply the RNAMute package on an RNA transcript for which individual point mutations were shown experimentally to inactivate spectinomycin resistance in Escherichia coli. Functional analysis of mutations on this case study was performed experimentally by creating a library of point mutations using PCR and screening to locate those mutations. With the availability of RNAMute, preanalysis can be performed computationally before conducting an experiment.

Python Spectral Analysis Tool (PySAT) for Preprocessing, Multivariate Analysis, and Machine Learning with Point Spectra

NASA Astrophysics Data System (ADS)

Anderson, R. B.; Finch, N.; Clegg, S.; Graff, T.; Morris, R. V.; Laura, J.

2017-06-01

We present a Python-based library and graphical interface for the analysis of point spectra. The tool is being developed with a focus on methods used for ChemCam data, but is flexible enough to handle spectra from other instruments.

Hereditary cancer genes are highly susceptible to splicing mutations

PubMed Central

Soemedi, Rachel; Maguire, Samantha; Murray, Michael F.; Monaghan, Sean F.

2018-01-01

Substitutions that disrupt pre-mRNA splicing are a common cause of genetic disease. On average, 13.4% of all hereditary disease alleles are classified as splicing mutations mapping to the canonical 5′ and 3′ splice sites. However, splicing mutations present in exons and deeper intronic positions are vastly underreported. A recent re-analysis of coding mutations in exon 10 of the Lynch Syndrome gene, MLH1, revealed an extremely high rate (77%) of mutations that lead to defective splicing. This finding is confirmed by extending the sampling to five other exons in the MLH1 gene. Further analysis suggests a more general phenomenon of defective splicing driving Lynch Syndrome. Of the 36 mutations tested, 11 disrupted splicing. Furthermore, analyzing past reports suggest that MLH1 mutations in canonical splice sites also occupy a much higher fraction (36%) of total mutations than expected. When performing a comprehensive analysis of splicing mutations in human disease genes, we found that three main causal genes of Lynch Syndrome, MLH1, MSH2, and PMS2, belonged to a class of 86 disease genes which are enriched for splicing mutations. Other cancer genes were also enriched in the 86 susceptible genes. The enrichment of splicing mutations in hereditary cancers strongly argues for additional priority in interpreting clinical sequencing data in relation to cancer and splicing. PMID:29505604

A multidisciplinary approach for the diagnosis of hypocalcified amelogenesis imperfecta in two Chilean families.

PubMed

Urzúa, Blanca; Ortega-Pinto, Ana; Farias, Daniela Adorno; Franco, Eugenia; Morales-Bozo, Irene; Moncada, Gustavo; Escobar-Pezoa, Nicolás; Scholz, Ursula; Cifuentes, Victor

2012-01-01

The purpose of this study was to conduct a multidisciplinary analysis of a specific type of tooth enamel disturbance (amelogenesis imperfecta) affecting two Chilean families to obtain a precise diagnosis and to investigate possible underlying mutations. Two non-related families affected with amelogenesis imperfecta were evaluated with clinical, radiographic and histopathological methods. Furthermore, pedigrees of both families were constructed and the presence of eight mutations in the enamelin gene (ENAM) and three mutations in the enamelysin gene (MMP-20) were investigated by PCR and direct sequencing. In the two affected patients, the dental malformation presented as soft and easily disintegrated enamel and exposed dark dentin. Neither of the affected individuals presented with a dental and skeletal open bite. Histologically, a high level of an organic matrix with prismatic organization was found. Genetic analysis indicated that the condition is autosomal recessive in one family and either autosomal recessive or due to a new mutation in the other family. Molecular mutational analysis revealed that none of the eight mutations previously described in the ENAM gene or the three mutations in the MMP-20 gene were present in the probands. A multidisciplinary analysis allowed for a diagnosis of hypocalcified amelogenesis imperfecta, Witkop type III, which was unrelated to previously described mutations in the ENAM or MMP-20 genes.

An Usher syndrome type 1 patient diagnosed before the appearance of visual symptoms by MYO7A mutation analysis.

PubMed

Yoshimura, Hidekane; Iwasaki, Satoshi; Kanda, Yukihiko; Nakanishi, Hiroshi; Murata, Toshinori; Iwasa, Yoh-ichiro; Nishio, Shin-ya; Takumi, Yutaka; Usami, Shin-ichi

2013-02-01

Usher syndrome type 1 (USH1) appears to have only profound non-syndromic hearing loss in childhood and retinitis pigmentosa develops in later years. This study examined the frequency of USH1 before the appearance of visual symptoms in Japanese deaf children by MYO7A mutation analysis. We report the case of 6-year-old male with profound hearing loss, who did not have visual symptoms. The frequency of MYO7A mutations in profound hearing loss children is also discussed. We sequenced all exons of the MYO7A gene in 80 Japanese children with severe to profound non-syndromic HL not due to mutations of the GJB2 gene (ages 0-14 years). A total of nine DNA variants were found and six of them were presumed to be non-pathogenic variants. In addition, three variants of them were found in two patients (2.5%) with deafness and were classified as possible pathogenic variants. Among them, at least one nonsense mutation and one missense mutation from the patient were confirmed to be responsible for deafness. After MYO7A mutation analysis, the patient was diagnosed with RP, and therefore, also diagnosed with USH1. This is the first case report to show the advantage of MYO7A mutation analysis to diagnose USH1 before the appearance of visual symptoms. We believed that MYO7A mutation analysis is valid for the early diagnosis of USH1. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

The application of a linear algebra to the analysis of mutation rates.

PubMed

Jones, M E; Thomas, S M; Clarke, K

1999-07-07

Cells and bacteria growing in culture are subject to mutation, and as this mutation is the ultimate substrate for selection and evolution, the factors controlling the mutation rate are of some interest. The mutational event is not observed directly, but is inferred from the phenotype of the original mutant or of its descendants; the rate of mutation is inferred from the number of such mutant phenotypes. Such inference presumes a knowledge of the probability distribution for the size of a clone arising from a single mutation. We develop a mathematical formulation that assists in the design and analysis of experiments which investigate mutation rates and mutant clone size distribution, and we use it to analyse data for which the classical Luria-Delbrück clone-size distribution must be rejected. Copyright 1999 Academic Press.

Cost-effective PKHD1 genetic testing for autosomal recessive polycystic kidney disease.

PubMed

Krall, Paola; Pineda, Cristina; Ruiz, Patricia; Ejarque, Laia; Vendrell, Teresa; Camacho, Juan Antonio; Mendizábal, Santiago; Oliver, Artur; Ballarín, José; Torra, Roser; Ars, Elisabet

2014-02-01

Genetic diagnosis of autosomal recessive polycystic kidney disease (ARPKD) is challenging due to the length and allelic heterogeneity of the PKHD1 gene. Mutations appear to be clustered at specific exons, depending on the geographic origin of the patient. We aimed to identify the PKHD1 exons most likely mutated in Spanish ARPKD patients. Mutation analysis was performed in 50 ARPKD probands and nine ARPKD-suspicious patients by sequencing PKHD1 exons arranged by their reported mutation frequency. Haplotypes containing the most frequent mutations were analyzed. Other PKD genes (HNF1B, PKD1, PKD2) were sequenced in PKHD1-negative cases. Thirty-six different mutations (concentrated in 24 PKHD1 exons) were detected, giving a mutation detection rate of 86%. The screening of five exons (58, 32, 34, 36, 37) yielded a 54% chance of detecting one mutation; the screening of nine additional exons (3, 9, 39, 61, 5, 22, 26, 41, 57) increased the chance to 76%. The c.9689delA mutation was present in 17 (34%) patients, all of whom shared the same haplotype. Two HNF1B mutations and one PKD1 variant were detected in negative cases. Establishing a PKHD1 exon mutation profile in a specific population and starting the analysis with the most likely mutated exons might significantly enhance the efficacy of genetic testing in ARPKD. Analysis of other PKD genes might be considered, especially in suspicious cases.

Mutational analysis of genes coding for cell surface proteins in colorectal cancer cell lines reveal novel altered pathways, druggable mutations and mutated epitopes for targeted therapy

PubMed Central

Correa, Bruna R.; Bettoni, Fabiana; Koyama, Fernanda C.; Navarro, Fabio C.P.; Perez, Rodrigo O.; Mariadason, John; Sieber, Oliver M.; Strausberg, Robert L.; Simpson, Andrew J.G.; Jardim, Denis L.F.; Reis, Luiz Fernando L.; Parmigiani, Raphael B.; Galante, Pedro A.F.; Camargo, Anamaria A.

2014-01-01

We carried out a mutational analysis of 3,594 genes coding for cell surface proteins (Surfaceome) in 23 colorectal cancer cell lines, searching for new altered pathways, druggable mutations and mutated epitopes for targeted therapy in colorectal cancer. A total of 3,944 somatic non-synonymous substitutions and 595 InDels, occurring in 2,061 (57%) Surfaceome genes were catalogued. We identified 48 genes not previously described as mutated in colorectal tumors in the TCGA database, including genes that are mutated and expressed in >10% of the cell lines (SEMA4C, FGFRL1, PKD1, FAM38A, WDR81, TMEM136, SLC36A1, SLC26A6, IGFLR1). Analysis of these genes uncovered important roles for FGF and SEMA4 signaling in colorectal cancer with possible therapeutic implications. We also found that cell lines express on average 11 druggable mutations, including frequent mutations (>20%) in the receptor tyrosine kinases AXL and EPHA2, which have not been previously considered as potential targets for colorectal cancer. Finally, we identified 82 cell surface mutated epitopes, however expression of only 30% of these epitopes was detected in our cell lines. Notwithstanding, 92% of these epitopes were expressed in cell lines with the mutator phenotype, opening new venues for the use of “general” immune checkpoint drugs in this subset of patients. PMID:25193853

Comprehensive Mutation Analysis of PMS2 in a Large Cohort of Probands Suspected of Lynch Syndrome or Constitutional Mismatch Repair Deficiency Syndrome.

PubMed

van der Klift, Heleen M; Mensenkamp, Arjen R; Drost, Mark; Bik, Elsa C; Vos, Yvonne J; Gille, Hans J J P; Redeker, Bert E J W; Tiersma, Yvonne; Zonneveld, José B M; García, Encarna Gómez; Letteboer, Tom G W; Olderode-Berends, Maran J W; van Hest, Liselotte P; van Os, Theo A; Verhoef, Senno; Wagner, Anja; van Asperen, Christi J; Ten Broeke, Sanne W; Hes, Frederik J; de Wind, Niels; Nielsen, Maartje; Devilee, Peter; Ligtenberg, Marjolijn J L; Wijnen, Juul T; Tops, Carli M J

2016-11-01

Monoallelic PMS2 germline mutations cause 5%-15% of Lynch syndrome, a midlife cancer predisposition, whereas biallelic PMS2 mutations cause approximately 60% of constitutional mismatch repair deficiency (CMMRD), a rare childhood cancer syndrome. Recently improved DNA- and RNA-based strategies are applied to overcome problematic PMS2 mutation analysis due to the presence of pseudogenes and frequent gene conversion events. Here, we determined PMS2 mutation detection yield and mutation spectrum in a nationwide cohort of 396 probands. Furthermore, we studied concordance between tumor IHC/MSI (immunohistochemistry/microsatellite instability) profile and mutation carrier state. Overall, we found 52 different pathogenic PMS2 variants explaining 121 Lynch syndrome and nine CMMRD patients. In vitro mismatch repair assays suggested pathogenicity for three missense variants. Ninety-one PMS2 mutation carriers (70%) showed isolated loss of PMS2 in their tumors, for 31 (24%) no or inconclusive IHC was available, and eight carriers (6%) showed discordant IHC (presence of PMS2 or loss of both MLH1 and PMS2). Ten cases with isolated PMS2 loss (10%; 10/97) harbored MLH1 mutations. We confirmed that recently improved mutation analysis provides a high yield of PMS2 mutations in patients with isolated loss of PMS2 expression. Application of universal tumor prescreening methods will however miss some PMS2 germline mutation carriers. © 2016 WILEY PERIODICALS, INC.

Development of a practical NF1 genetic testing method through the pilot analysis of five Japanese families with neurofibromatosis type 1.

PubMed

Okumura, Akiko; Ozaki, Mamoru; Niida, Yo

2015-08-01

Mutation analysis of NF1, the responsible gene for neurofibromatosis type 1 (NF1), is still difficult due to its large size, lack of mutational hotspots, the presence of many pseudogenes, and its wide spectrum of mutations. To develop a simple and inexpensive NF1 genetic testing for clinical use, we analyzed five Japanese families with NF1 as a pilot study. Our original method, CEL endonuclease mediated heteroduplex incision with polyacrylamide gel electrophoresis and silver staining (CHIPS) was optimized for NF1 mutation screening, and reverse transcription polymerase chain reaction (RT-PCR) was performed to determine the effect of transcription. Also, we employed DNA microarray analysis to evaluate the break points of the large deletion. A new nonsense mutation, p.Gln209(∗), was detected in family 1 and the splicing donor site mutation, c.2850+1G>T, was detected in family 2. In family 3, c.4402A>G was detected in exon 34 and the p.Ser1468Gly missense mutation was predicted. However mRNA analysis revealed that this substitution created an aberrant splicing acceptor site, thereby causing the p.Phe1457(∗) nonsense mutation. In the other two families, type-1 and unique NF1 microdeletions were detected by DNA microarray analysis. Our results show that the combination of CHIPS and RT-PCR effectively screen and characterize NF1 point mutations, and both DNA and RNA level analysis are required to understand the nature of the NF1 mutation. Our results also suggest the possibility of a higher incidence and unique profile of NF1 large deletions in the Japanese population as compared to previous studies performed in Europe. Copyright © 2014 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

High resolution melting analysis for epidermal growth factor receptor mutations in formalin-fixed paraffin-embedded tissue and plasma free DNA from non-small cell lung cancer patients.

PubMed

Jing, Chang-Wen; Wang, Zhuo; Cao, Hai-Xia; Ma, Rong; Wu, Jian-Zhong

2014-01-01

The aim of the research was to explore a cost effective, fast, easy to perform, and sensitive method for epidermal growth factor receptor (EGFR) mutation testing. High resolution melting analysis (HRM) was introduced to evaluate the efficacy of the analysis for dectecting EGFR mutations in exons 18 to 21 using formalin-fixed paraffin-embedded (FFPE) tissues and plasma free DNA from 120 patients. The total EGFR mutation rate was 37.5% (45/120) detected by direct sequencing. There were 48 mutations in 120 FFPE tissues assessed by HRM. For plasma free DNA, the EGFR mutation rate was 25.8% (31/120). The sensitivity of HRM assays in FFPE samples was 100% by HRM. There was a low false-positive mutation rate but a high false-negative rate in plasma free DNA detected by HRM. Our results show that HRM analysis has the advantage of small tumor sample need. HRM applied with plasma free DNA showed a high false-negative rate but a low false-positive rate. Further research into appropriate methods and analysis needs to be performed before HRM for plasma free DNA could be accepted as an option in diagnostic or screening settings.

Deep sequencing reveals double mutations in cis of MPL exon 10 in myeloproliferative neoplasms.

PubMed

Pietra, Daniela; Brisci, Angela; Rumi, Elisa; Boggi, Sabrina; Elena, Chiara; Pietrelli, Alessandro; Bordoni, Roberta; Ferrari, Maurizio; Passamonti, Francesco; De Bellis, Gianluca; Cremonesi, Laura; Cazzola, Mario

2011-04-01

Somatic mutations of MPL exon 10, mainly involving a W515 substitution, have been described in JAK2 (V617F)-negative patients with essential thrombocythemia and primary myelofibrosis. We used direct sequencing and high-resolution melt analysis to identify mutations of MPL exon 10 in 570 patients with myeloproliferative neoplasms, and allele specific PCR and deep sequencing to further characterize a subset of mutated patients. Somatic mutations were detected in 33 of 221 patients (15%) with JAK2 (V617F)-negative essential thrombocythemia or primary myelofibrosis. Only one patient with essential thrombocythemia carried both JAK2 (V617F) and MPL (W515L). High-resolution melt analysis identified abnormal patterns in all the MPL mutated cases, while direct sequencing did not detect the mutant MPL in one fifth of them. In 3 cases carrying double MPL mutations, deep sequencing analysis showed identical load and location in cis of the paired lesions, indicating their simultaneous occurrence on the same chromosome.

Prenatal diagnosis in a cystic fibrosis family: a combined molecular strategy for a precise diagnosis.

PubMed

Chávez-Saldaña, Margarita; García-Cavazos, Ricardo; Vigueras, Rosa María; Orozco, Lorena

2011-01-01

The high genetic heterogeneity in populations with a wide spectrum of mutations in the CF transmembrane conductance regulator gene (CFTR), makes the detection of mutations a very hard and difficult task, thereby limiting the accurate diagnosis of the disease, mainly in patients with uncharacterized mutations. Molecular strategies, like targeted identification of the most frequent CFTR mutations in Mexican population combined with linkage analysis using markers, is very useful for carrier detection and for prenatal diagnosis in affected families with CF. In this paper we show that the combination of methodologies was a crucial alternative to reach a precise prenatal CF diagnosis. We documented CF diagnosis in a 14th-week fetus combining the screening of the most common mutations in Mexican population with linkage analysis of two extragenic polymorphisms (XV2C/TaqI and KM19/PstI). We determined that the fetus inherited the PG542X mutation from its mother and an unknown mutation from its father through the chromosomal phases analysis.

Prenatal diagnosis of fetal glutaric aciduria type 1 with rare compound heterozygous mutations in GCDH gene.

PubMed

Peng, Hsiu-Huei; Shaw, Sheng-Wen; Huang, Kuan-Gen

2018-02-01

Glutaric aciduria type 1 is a rare disease, with the estimated prevalence about 1 in 100,000 newborns. GCDH gene mutation can lead to glutaric acid and 3- OH glutaric acid accumulation, with clinical manifestation of neuronal damage, brain atrophy, microencephalic macrocephaly, decreased coordination of swallowing, poor muscle coordination, spasticity, and severe dystonic movement disorder. A 22-year-old female, Gravida 4 Para 2, is pregnancy at 13 weeks of gestational age. Her first child is normal, however, the second child was diagnosed as glutaric aciduria type I after birth. She came to our hospital for prenatal genetic counselling of her fetus at 13 weeks of gestational age. We performed GCDH gene mutation analysis of maternal blood showed IVS 3 + 1 G > A heterozygous mutation, GCDH gene mutation analysis of paternal blood showed c. 1240 G > A heterozygous mutation, and the second child has compound heterozygous IVS 3 + 1 G > A and c. 1240 G > A mutations. Later, we performed amniocentesis at 16 weeks of gestational age for chromosome study and GCDH gene mutation analysis for the fetus. The fetal chromosome study showed normal karyotype, however, GCDH gene mutation analysis showed compound heterozygous IVS 3 + 1 G > A and c. 1240 G > A mutations. The couple decided to termination of pregnancy thereafter. Glutaric acidemia type 1 is an autosomal recessive disorder because of pathogenic mutations in the GCDH gene. Early diagnosis and therapy of glutaric acidemia type 1 can reduce the risk of neuronal damage and acute dystonia. We report a case of prenatal diagnosis of fetal glutaric aciduria type 1 with rare compound heterozygous GCDH gene mutation at IVS 3 + 1 G > A and c. 1240 G > A mutations, which provide better genetic counselling for the couples. Copyright © 2018. Published by Elsevier B.V.

Increasing the Yield in Targeted Next-Generation Sequencing by Implicating CNV Analysis, Non-Coding Exons and the Overall Variant Load: The Example of Retinal Dystrophies

PubMed Central

Eisenberger, Tobias; Neuhaus, Christine; Khan, Arif O.; Decker, Christian; Preising, Markus N.; Friedburg, Christoph; Bieg, Anika; Gliem, Martin; Issa, Peter Charbel; Holz, Frank G.; Baig, Shahid M.; Hellenbroich, Yorck; Galvez, Alberto; Platzer, Konrad; Wollnik, Bernd; Laddach, Nadja; Ghaffari, Saeed Reza; Rafati, Maryam; Botzenhart, Elke; Tinschert, Sigrid; Börger, Doris; Bohring, Axel; Schreml, Julia; Körtge-Jung, Stefani; Schell-Apacik, Chayim; Bakur, Khadijah; Al-Aama, Jumana Y.; Neuhann, Teresa; Herkenrath, Peter; Nürnberg, Gudrun; Nürnberg, Peter; Davis, John S.; Gal, Andreas; Bergmann, Carsten; Lorenz, Birgit; Bolz, Hanno J.

2013-01-01

Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are major causes of blindness. They result from mutations in many genes which has long hampered comprehensive genetic analysis. Recently, targeted next-generation sequencing (NGS) has proven useful to overcome this limitation. To uncover “hidden mutations” such as copy number variations (CNVs) and mutations in non-coding regions, we extended the use of NGS data by quantitative readout for the exons of 55 RP and LCA genes in 126 patients, and by including non-coding 5′ exons. We detected several causative CNVs which were key to the diagnosis in hitherto unsolved constellations, e.g. hemizygous point mutations in consanguineous families, and CNVs complemented apparently monoallelic recessive alleles. Mutations of non-coding exon 1 of EYS revealed its contribution to disease. In view of the high carrier frequency for retinal disease gene mutations in the general population, we considered the overall variant load in each patient to assess if a mutation was causative or reflected accidental carriership in patients with mutations in several genes or with single recessive alleles. For example, truncating mutations in RP1, a gene implicated in both recessive and dominant RP, were causative in biallelic constellations, unrelated to disease when heterozygous on a biallelic mutation background of another gene, or even non-pathogenic if close to the C-terminus. Patients with mutations in several loci were common, but without evidence for di- or oligogenic inheritance. Although the number of targeted genes was low compared to previous studies, the mutation detection rate was highest (70%) which likely results from completeness and depth of coverage, and quantitative data analysis. CNV analysis should routinely be applied in targeted NGS, and mutations in non-coding exons give reason to systematically include 5′-UTRs in disease gene or exome panels. Consideration of all variants is indispensable because even truncating mutations may be misleading. PMID:24265693

Cost-effectiveness analysis of EGFR mutation testing in patients with non-small cell lung cancer (NSCLC) with gefitinib or carboplatin-paclitaxel.

PubMed

Arrieta, Oscar; Anaya, Pablo; Morales-Oyarvide, Vicente; Ramírez-Tirado, Laura Alejandra; Polanco, Ana C

2016-09-01

Assess the cost-effectiveness of an EGFR-mutation testing strategy for advanced NSCLC in first-line therapy with either gefitinib or carboplatin-paclitaxel in Mexican institutions. Cost-effectiveness analysis using a discrete event simulation (DES) model to simulate two therapeutic strategies in patients with advanced NSCLC. Strategy one included patients tested for EGFR-mutation and therapy given accordingly. Strategy two included chemotherapy for all patients without testing. All results are presented in 2014 US dollars. The analysis was made with data from the Mexican frequency of EGFR-mutation. A univariate sensitivity analysis was conducted on EGFR prevalence. Progression-free survival (PFS) transition probabilities were estimated on data from the IPASS and simulated with a Weibull distribution, run with parallel trials to calculate a probabilistic sensitivity analysis. PFS of patients in the testing strategy was 6.76 months (95 % CI 6.10-7.44) vs 5.85 months (95 % CI 5.43-6.29) in the non-testing group. The one-way sensitivity analysis showed that PFS has a direct relationship with EGFR-mutation prevalence, while the ICER and testing cost have an inverse relationship with EGFR-mutation prevalence. The probabilistic sensitivity analysis showed that all iterations had incremental costs and incremental PFS for strategy 1 in comparison with strategy 2. There is a direct relationship between the ICER and the cost of EGFR testing, with an inverse relationship with the prevalence of EGFR-mutation. When prevalence is >10 % ICER remains constant. This study could impact Mexican and Latin American health policies regarding mutation detection testing and treatment for advanced NSCLC.

Genetic epidemiology of amyotrophic lateral sclerosis: a systematic review and meta-analysis.

PubMed

Zou, Zhang-Yu; Zhou, Zhi-Rui; Che, Chun-Hui; Liu, Chang-Yun; He, Rao-Li; Huang, Hua-Pin

2017-07-01

Genetic studies have shown that C9orf72 , SOD1 , TARDBP and FUS are the most common mutated genes in amyotrophic lateral sclerosis (ALS). Here, we performed a meta-analysis to determine the mutation frequencies of these major ALS-related genes in patients with ALS. We performed an extensive literature research to identify all original articles reporting frequencies of C9orf72 , SOD1 , TARDBP and FUS mutations in ALS. The mutation frequency and effect size of each study were combined. Possible sources of heterogeneity across studies were determined by meta-regression, sensitivity analysis and subgroup analysis. 111 studies were included in the meta-analysis. The overall pooled mutation frequencies of these major ALS-related genes were 47.7% in familial amyotrophic lateral sclerosis (FALS) and 5.2% in sporadic ALS (SALS). A significant difference was identified regarding the frequencies of mutations in major ALS genes between European and Asian patients. In European populations, the most common mutations were the C9orf72 repeat expansions (FALS 33.7%, SALS 5.1%), followed by SOD1 (FALS 14.8%, SALS 1.2%), TARDBP (FALS 4.2%, SALS 0.8%) and FUS mutations (FALS 2.8%, SALS 0.3%), while in Asian populations the most common mutations were SOD1 mutations (FALS 30.0%, SALS 1.5%), followed by FUS (FALS 6.4%, SALS 0.9%), C9orf72 (FALS 2.3%, SALS 0.3%) and TARDBP (FALS 1.5%, SALS 0.2%) mutations. These findings demonstrated that the genetic architecture of ALS in Asian populations is distinct from that in European populations, which need to be given appropriate consideration when performing genetic testing of patients with ALS. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

The p16INK4alpha/p19ARF gene mutations are infrequent and are mutually exclusive to p53 mutations in Indian oral squamous cell carcinomas.

PubMed

Kannan, K; Munirajan, A K; Krishnamurthy, J; Bhuvarahamurthy, V; Mohanprasad, B K; Panishankar, K H; Tsuchida, N; Shanmugam, G

2000-03-01

Eighty-seven untreated primary oral squamous cell carcinomas (SCCs) associated with betel quid and tobacco chewing from Indian patients were analysed for the presence of mutations in the commonly shared exon 2 of p16INK4alpha/p19ARF genes. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and sequencing analysis were used to detect mutations. SSCP analysis indicated that only 9% (8/87) of the tumours had mutation in p16INK4alpha/p19ARF genes. Seventy-two tumours studied here were previously analysed for p53 mutations and 21% (15/72) of them were found to have mutations in p53 gene. Only one tumour was found to have mutation at both p53 and p16INK4alpha/p19ARF genes. Thus, the mutation rates observed were 21% for p53, 9% for p16INK4alpha/p19ARF, and 1% for both. Sequencing analysis revealed two types of mutations; i) G to C (GCAG to CCAG) transversion type mutation at intron 1-exon 2 splice junction and ii) another C to T transition type mutation resulting in CGA to TGA changing arginine to a termination codon at p16INK4alpha gene codon 80 and the same mutation will alter codon 94 of p19ARF gene from CCG to CTG (proline to leucine). These results suggest that p16INK4alpha/p19ARF mutations are less frequent than p53 mutations in Indian oral SCCs. The p53 and p16INK4alpha/p19ARF mutational events are independent and are mutually exclusive suggesting that mutational inactivation of either p53 or p16INK4alpha/p19ARF may alleviate the need for the inactivation of the other gene.

Presence of a consensus DNA motif at nearby DNA sequence of the mutation susceptible CG nucleotides.

PubMed

Chowdhury, Kaushik; Kumar, Suresh; Sharma, Tanu; Sharma, Ankit; Bhagat, Meenakshi; Kamai, Asangla; Ford, Bridget M; Asthana, Shailendra; Mandal, Chandi C

2018-01-10

Complexity in tissues affected by cancer arises from somatic mutations and epigenetic modifications in the genome. The mutation susceptible hotspots present within the genome indicate a non-random nature and/or a position specific selection of mutation. An association exists between the occurrence of mutations and epigenetic DNA methylation. This study is primarily aimed at determining mutation status, and identifying a signature for predicting mutation prone zones of tumor suppressor (TS) genes. Nearby sequences from the top five positions having a higher mutation frequency in each gene of 42 TS genes were selected from a cosmic database and were considered as mutation prone zones. The conserved motifs present in the mutation prone DNA fragments were identified. Molecular docking studies were done to determine putative interactions between the identified conserved motifs and enzyme methyltransferase DNMT1. Collective analysis of 42 TS genes found GC as the most commonly replaced and AT as the most commonly formed residues after mutation. Analysis of the top 5 mutated positions of each gene (210 DNA segments for 42 TS genes) identified that CG nucleotides of the amino acid codons (e.g., Arginine) are most susceptible to mutation, and found a consensus DNA "T/AGC/GAGGA/TG" sequence present in these mutation prone DNA segments. Similar to TS genes, analysis of 54 oncogenes not only found CG nucleotides of the amino acid Arg as the most susceptible to mutation, but also identified the presence of similar consensus DNA motifs in the mutation prone DNA fragments (270 DNA segments for 54 oncogenes) of oncogenes. Docking studies depicted that, upon binding of DNMT1 methylates to this consensus DNA motif (C residues of CpG islands), mutation was likely to occur. Thus, this study proposes that DNMT1 mediated methylation in chromosomal DNA may decrease if a foreign DNA segment containing this consensus sequence along with CG nucleotides is exogenously introduced to dividing cancer cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Impact of tissue type and content of neoplastic cells of samples on the quality of epidermal growth factor receptor mutation analysis among patients with lung adenocarcinoma

PubMed Central

PALIOGIANNIS, PANAGIOTIS; ATTENE, FEDERICO; COSSU, ANTONIO; DEFRAIA, EFISIO; PORCU, GIUSEPPE; CARTA, ANNAMARIA; SOTGIU, MARIA IGNAZIA; PAZZOLA, ANTONIO; CORDERO, LORENZO; CAPELLI, FRANCESCA; FADDA, GIOVANNI MARIA; ORTU, SALVATORE; SOTGIU, GIOVANNI; PALOMBA, GRAZIA; SINI, MARIA CRISTINA; PALMIERI, GIUSEPPE; COLOMBINO, MARIA

2015-01-01

Assessment of the epidermal growth factor receptor (EGFR) mutational status has become crucial in recent years in the molecular classification of patients with lung cancer. The impact of the type and quantity of malignant cells of the neoplastic specimen on the quality of mutation analysis remains to be elucidated, and only empirical and sporadic data are available. The aim of the present study was to investigate the impact of tissue type and content of neoplastic cells in the specimen on the quality of EGFR mutation analysis among patients with lung adenocarcinoma. A total of 515 patients with histologically-confirmed disease were included in the present study. Formalin-fixed paraffin embedded tissue samples were used for the mutation analysis and the content of the neoplastic cells was evaluated using light microscopy. Genomic DNA was isolated using a standard protocol. The coding sequences and splice junctions of exons 18, 19 and 21 in the EGFR gene were then screened for mutations by direct automated sequencing. The mean age of the patients examined was 64.9 years and 357 (69.3%) were male. A total of 429 tissue samples (83.3%) were obtained by biopsy and the remaining samples were obtained by surgery. A total of 456 samples (88.5%) were observed from primary lung adenocarcinomas, while 59 (11.5%) were from metastatic lesions. EGFR mutations occurred in 59 cases (11.5%); exon 18 mutations were detected in one case (1.7%), whereas exon 19 and 21 mutations were detected in 30 (51%) and 28 (47.3%) cases, respectively. EGFR mutations were more frequent in females and patients that had never smoked. The distribution of the mutations among primary and metastatic tissues exhibited no significant differences in the proportions of EGFR mutations detected. However, a statistically significant difference in the number of mutations detected was found between samples with at least 50% of neoplastic cells (450 cases-57 mutations; 12.7%) and those with <50% of neoplastic cells (65 cases-2 mutations; 3.1%). PMID:25683726

KRAS, NRAS and BRAF mutations in Greek and Romanian patients with colorectal cancer: a cohort study

PubMed Central

Negru, Serban; Papadopoulou, Eirini; Apessos, Angela; Stanculeanu, Dana Lucia; Ciuleanu, Eliade; Volovat, Constantin; Croitoru, Adina; Kakolyris, Stylianos; Aravantinos, Gerasimos; Ziras, Nikolaos; Athanasiadis, Elias; Touroutoglou, Nikolaos; Pavlidis, Nikolaos; Kalofonos, Haralabos P; Nasioulas, George

2014-01-01

Objectives Treatment decision-making in colorectal cancer is often guided by tumour tissue molecular analysis. The aim of this study was the development and validation of a high-resolution melting (HRM) method for the detection of KRAS, NRAS and BRAF mutations in Greek and Romanian patients with colorectal cancer and determination of the frequency of these mutations in the respective populations. Setting Diagnostic molecular laboratory located in Athens, Greece. Participants 2425 patients with colorectal cancer participated in the study. Primary and secondary outcome measures 2071 patients with colorectal cancer (1699 of Greek and 372 of Romanian origin) were analysed for KRAS exon 2 mutations. In addition, 354 tumours from consecutive patients (196 Greek and 161 Romanian) were subjected to full KRAS (exons 2, 3 and 4), NRAS (exons 2, 3 and 4) and BRAF (exon 15) analysis. KRAS, NRAS and BRAF mutation detection was performed by a newly designed HRM analysis protocol, followed by Sanger sequencing. Results KRAS exon 2 mutations (codons 12/13) were detected in 702 of the 1699 Greek patients with colorectal carcinoma analysed (41.3%) and in 39.2% (146/372) of the Romanian patients. Among the 354 patients who were subjected to full KRAS, NRAS and BRAF analysis, 40.96% had KRAS exon 2 mutations (codons 12/13). Among the KRAS exon 2 wild-type patients 15.31% harboured additional RAS mutations and 12.44% BRAF mutations. The newly designed HRM method used showed a higher sensitivity compared with the sequencing method. Conclusions The HRM method developed was shown to be a reliable method for KRAS, NRAS and BRAF mutation detection. Furthermore, no difference in the mutation frequency of KRAS, NRAS and BRAF was observed between Greek and Romanian patients with colorectal cancer. PMID:24859998

Multi-institutional oncogenic driver mutation analysis in lung adenocarcinoma: The Lung Cancer Mutation Consortium experience

PubMed Central

Dias-Santagata, Dora; Wistuba, Ignacio I.; Chen, Heidi; Fujimoto, Junya; Kugler, Kelly; Franklin, Wilbur A.; Iafrate, A. John; Ladanyi, Marc; Kris, Mark G.; Johnson, Bruce E.; Bunn, Paul A.; Minna, John D.; Kwiatkowski, David J.

2015-01-01

Introduction Molecular genetic analyses of lung adenocarcinoma have recently become standard of care for treatment selection. The Lung Cancer Mutation Consortium was formed to enable collaborative multi-institutional analyses of 10 potential oncogenic driver mutations. Technical aspects of testing, and clinicopathologic correlations are presented. Methods Mutation testing in at least one of 8 genes (EGFR, KRAS, ERBB2, AKT1, BRAF, MEK1, NRAS, PIK3CA) using SNaPshot, mass spectrometry, Sanger sequencing +/− PNA and/or sizing assays, along with ALK and/or MET FISH were performed in 6 labs on 1007 patients from 14 institutions. Results 1007 specimens had mutation analysis performed, and 733 specimens had all 10 genes analyzed. Mutation identification rates did not vary by analytic method. Biopsy and cytology specimens were inadequate for testing in 26% and 35% of cases compared to 5% of surgical specimens. Among the 1007 cases with mutation analysis performed, EGFR, KRAS, ALK, and ERBB2 alterations were detected in 22, 25, 8.5, and 2.4% of cases, respectively. EGFR mutations were highly associated with female sex, Asian race, and never smoking status; and less strongly associated with stage IV disease, presence of bone metastases, and absence of adrenal metastases. ALK rearrangements were strongly associated with never smoking status, and more weakly associated with presence of liver metastases. ERBB2 mutations were strongly associated with Asian race and never smoking status. Two mutations were seen in 2.7% of samples, all but one of which involved one or more of PIK3CA, ALK or MET. Conclusion Multi-institutional molecular analysis across multiple platforms, sample types, and institutions can yield consistent results and novel clinicopathological observations. PMID:25738220

Further screening of the rhodopsin gene in patients with autosomal dominant retinitis pigmentosa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vaithinathan, R.; Berson, E.L.; Dryja, T.P.

Here the authors report 8 novel mutations and 8 previously reported mutations found from further analysis of the rhodopsin gene in a large set of additional patients with autosomal dominant retinitis pigmentosa. Leukocyte DNA was purified from 122 unrelated patients with autosomal dominant retinitis pigmentosa who were not included in previous analyses. The coding region and splice donor and acceptor sites of the rhodopsin gene were screened for mutations using single-strand conformation polymorphism analysis and direct genomic sequencing. They found 29 patients with varient bands that were due to mutations. Sequence analysis showed that 20 cases each had 1 ofmore » 9 previously published mutations: Pro23His, Thr58Arg, Gly89Asp, Pro171Leu, Glu181Lys, Pro347Leu, Phe45Leu, Arg135Trp, and Lys296Glu. In 9 other cases, they found 8 novel mutations. One was a 3-bp deletion (Cys264-del), and the rest were point mutations resulting in an altered amino acid: Gly51Arg (GGC [yields] CGC), Cys110Tyr (TCG [yields] TAC), Gly114Asp (GGC [yields] GAC), Ala164Glu (GCG [yields] GAG), Pro171Ser (CCA [yields] TCA), Val345Leu (GTG [yields] CTG), and Pro347Gln (CCG [yields] CAG). Each of these novel mutations was found in only one family except for Gly51Arg, which was found in two. In every family tested, the mutation cosegregated with the disease. However, in pedigree D865 only one affected member was available for analysis. About two-thirds of the mutations affect amino acids in transmembrane domains, yet only one-half of opsin's residues are in these regions. One-third of the mutations alter residues in the extracellular/intradiscal space, which includes only 25% of the protein.« less

Fragment length analysis screening for detection of CEBPA mutations in intermediate-risk karyotype acute myeloid leukemia.

PubMed

Fuster, Oscar; Barragán, Eva; Bolufer, Pascual; Such, Esperanza; Valencia, Ana; Ibáñez, Mariam; Dolz, Sandra; de Juan, Inmaculada; Jiménez, Antonio; Gómez, Maria Teresa; Buño, Ismael; Martínez, Joaquín; Cervera, José; Montesinos, Pau; Moscardó, Federico; Sanz, Miguel Ángel

2012-01-01

During last years, molecular markers have been increased as prognostic factors routinely screened in acute myeloid leukemia (AML). Recently, an increasing interest has been reported in introducing to clinical practice screening for mutations in the CCAAT/enhancer-binding protein α (CEBPA) gene in AML, as it seems to be a good prognostic factor. However, there is no reliable established method for assessing CEBPA mutations during the diagnostic work-up of AMLs. We describe here a straightforward and reliable fragment analysis method based in PCR capillary electrophoresis (PCR-CE) for screening of CEBPA mutations; moreover, we present the results obtained in 151 intermediate-risk karyotype AML patients (aged 16-80 years). The method gave a specificity of 100% and sensitivity of 93% with a lower detection limit of 1-5% for CEBPA mutations. The series found 19 mutations and four polymorphisms in 12 patients, seven of whom (58%) presented two mutations. The overall frequency of CEBPA mutations in AML was 8% (n = 12). CEBPA mutations showed no coincidence with FLT3-ITD or NPM1 mutations. CEBPA mutation predicted better disease-free survival in the group of patients without FLT3-ITD, NPM, or both genes mutated (HR 3.6, IC 95%; 1.0-13.2, p = 0.05) and better overall survival in patients younger than 65 of this group without molecular markers (HR 4.0, IC 95%; 1.0-17.4, p = 0.05). In conclusion, the fragment analysis method based in PCR-CE is a rapid, specific, and sensitive method for CEBPA mutation screening and our results confirm that CEBPA mutations can identify a subgroup of patients with favorable prognosis in AML with intermediate-risk karyotype.

Occult HBV among Anti-HBc Alone: Mutation Analysis of an HBV Surface Gene and Pre-S Gene.

PubMed

Kim, Myeong Hee; Kang, So Young; Lee, Woo In

2017-05-01

The aim of this study is to investigate the molecular characteristics of occult hepatitis B virus (HBV) infection in 'anti-HBc alone' subjects. Twenty-four patients with 'anti-HBc alone' and 20 control patients diagnosed with HBV were analyzed regarding S and pre-S gene mutations. All specimens were analyzed for HBs Ag, anti-HBc, and anti-HBs. For specimens with an anti-HBc alone, quantitative analysis of HBV DNA, as well as sequencing and mutation analysis of S and pre-S genes, were performed. A total 24 were analyzed for the S gene, and 14 were analyzed for the pre-S gene through sequencing. A total of 20 control patients were analyzed for S and pre-S gene simultaneously. Nineteen point mutations of the major hydrophilic region were found in six of 24 patients. Among them, three mutations, S114T, P127S/T, M133T, were detected in common. Only one mutation was found in five subjects of the control group; this mutation was not found in the occult HBV infection group, however. Pre-S mutations were detected in 10 patients, and mutations of site aa58-aa100 were detected in 9 patients. A mutation on D114E was simultaneously detected. Although five mutations from the control group were found at the same location (aa58-aa100), no mutations of occult HBV infection were detected. The prevalence of occult HBV infection is not low among 'anti-HBc alone' subjects. Variable mutations in the S gene and pre-S gene were associated with the occurrence of occult HBV infection. Further larger scale studies are required to determine the significance of newly detected mutations. © Copyright: Yonsei University College of Medicine 2017

EMPCA and Cluster Analysis of Quasar Spectra: Construction and Application to Simulated Spectra

NASA Astrophysics Data System (ADS)

Marrs, Adam; Leighly, Karen; Wagner, Cassidy; Macinnis, Francis

2017-01-01

Quasars have complex spectra with emission lines influenced by many factors. Therefore, to fully describe the spectrum requires specification of a large number of parameters, such as line equivalent width, blueshift, and ratios. Principal Component Analysis (PCA) aims to construct eigenvectors-or principal components-from the data with the goal of finding a few key parameters that can be used to predict the rest of the spectrum fairly well. Analysis of simulated quasar spectra was used to verify and justify our modified application of PCA.We used a variant of PCA called Weighted Expectation Maximization PCA (EMPCA; Bailey 2012) along with k-means cluster analysis to analyze simulated quasar spectra. Our approach combines both analytical methods to address two known problems with classical PCA. EMPCA uses weights to account for uncertainty and missing points in the spectra. K-means groups similar spectra together to address the nonlinearity of quasar spectra, specifically variance in blueshifts and widths of the emission lines.In producing and analyzing simulations, we first tested the effects of varying equivalent widths and blueshifts on the derived principal components, and explored the differences between standard PCA and EMPCA. We also tested the effects of varying signal-to-noise ratio. Next we used the results of fits to composite quasar spectra (see accompanying poster by Wagner et al.) to construct a set of realistic simulated spectra, and subjected those spectra to the EMPCA /k-means analysis. We concluded that our approach was validated when we found that the mean spectra from our k-means clusters derived from PCA projection coefficients reproduced the trends observed in the composite spectra.Furthermore, our method needed only two eigenvectors to identify both sets of correlations used to construct the simulations, as well as indicating the linear and nonlinear segments. Comparing this to regular PCA, which can require a dozen or more components, or to direct spectral analysis that may need measurement of 20 fit parameters, shows why the dual application of these two techniques is such a powerful tool.

Impact of proto-oncogene mutation detection in cytological specimens from thyroid nodules improves the diagnostic accuracy of cytology.

PubMed

Cantara, Silvia; Capezzone, Marco; Marchisotta, Stefania; Capuano, Serena; Busonero, Giulia; Toti, Paolo; Di Santo, Andrea; Caruso, Giuseppe; Carli, Anton Ferdinando; Brilli, Lucia; Montanaro, Annalisa; Pacini, Furio

2010-03-01

Fine-needle aspiration cytology (FNAC) is the gold standard for the differential diagnosis of thyroid nodules but has the limitation of inadequate sampling or indeterminate lesions. We aimed to verify whether search of thyroid cancer-associated protooncogene mutations in cytological samples may improve the diagnostic accuracy of FNAC. One hundred seventy-four consecutive patients undergoing thyroid surgery were submitted to FNAC (on 235 thyroid nodules) that was used for cytology and molecular analysis of BRAF, RAS, RET, TRK, and PPRgamma mutations. At surgery these nodules were sampled to perform the same molecular testing. Mutations were found in 67 of 235 (28.5%) cytological samples. Of the 67 mutated samples, 23 (34.3%) were mutated by RAS, 33 (49.3%) by BRAF, and 11 (16.4%) by RET/PTC. In 88.2% of the cases, the mutation was confirmed in tissue sample. The presence of mutations at cytology was associated with cancer 91.1% of the times and follicular adenoma 8.9% of the time. BRAF or RET/PTC mutations were always associated with cancer, whereas RAS mutations were mainly associated with cancer (74%) but also follicular adenoma (26%). The diagnostic performance of molecular analysis was superior to that of traditional cytology, with better sensitivity and specificity, and the combination of the two techniques further contributed to improve the total accuracy (93.2%), compared with molecular analysis (90.2%) or traditional cytology (83.0%). Our findings demonstrate that molecular analysis of cytological specimens is feasible and that its results in combination with cytology improves the diagnostic performance of traditional cytology.

Genetic analysis of hispanic individuals with cystic fibrosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grebe, T.A.; Doane, W.W.; Norman, R.A.

1994-03-01

The authors have performed molecular genetic analysis of Hispanic individuals with cystic fibrosis (CF) in the southwestern United States. Of 129 CF chromosomes analyzed, oly 46% (59/129) carry [Delta]F508. The G542X mutation was found on 5% (7/129) of CF chromosomes. The 3849+10kbC[yields]T mutation, detected primarily in Ashkenazi Jews, was present on 2% (3/129). R1162X and R334W, mutations identified in Spain and Italy, each occurred on 1.6% (2/129) of CF chromosomes. W1282X and R553X were each detected once. G551D and N1303K were not found. Overall, screening for 22 or more mutations resulted in detection of only 58% of CF transmembrane conductancemore » regulator gene mutations among Hispanic individuals. Analysis of KM19/XV2c haplotypes revealed an unusual distribution. Although the majority of [Delta]508 mutations are on chromosomes of B haplotypes, the other CF mutations are on A and C haplotypes at higher-than-expected frequencies. These genetic analysis demonstrate significant differences between Hispanic individuals with CF and those of the general North American population. Assessment of carrier/affected risk in Hispanic CF individuals cannot, therefore, be based on the mutation frequencies found through studies of the general population but must be adjusted to better reflect the genetic makeup of this ethnic group. Further studies are necessary to identify the causative mutation(s) in this population and to better delineate genotype/phenotype correlations. These will enable counselors to provide more accurate genetic counseling. 22 refs., 2 tabs.« less

High-resolution Melting Analysis for Gene Scanning of Adenomatous Polyposis Coli (APC) Gene With Oral Squamous Cell Carcinoma Samples.

PubMed

Chang, Ya-Sian; Lin, Chien-Yu; Yang, Shu-Fen; Ho, Cheng Mao; Chang, Jan-Gowth

2016-02-01

There have been many different mutations reported for the large adenomatous polyposis coli (APC) tumor suppressor gene. APC mutations result in inactivation of APC tumor suppressor action, allowing the progression of tumorigenesis. The present study utilized a highly efficient method to identify APC mutations and investigated the association between the APC genetic variants Y486Y, A545A, T1493T, and D1822V and susceptibility to oral squamous cell carcinoma (OSCC). High-resolution melting (HRM) analysis was used to characterize APC mutations. Genomic DNA was extracted from 83 patient specimens of OSCC and 50 blood samples from healthy control subjects. The 14 exons and mutation cluster region of exon 15 were screened by HRM analysis. All mutations were confirmed by direct DNA sequencing. Three mutations and 4 single nucleotide polymorphisms (SNPs) were found in this study. The mutations were c.573T>C (Y191Y) in exon 5, c.1005A>G (L335L) in exon 9, and c.1488A>T (T496T) in exon 11. Two SNPs, c.4479G>A (T1493T) and c.5465A>T (D1822V), were located in exon 15, whereas c.1458T>C (Y486Y) and c.1635G>A (A545A) were located in exon 11 and 13, respectively. There was no observed association between OSCC risk and genotype for any of the 4 APC SNPs. The mutation of APC is rare in Taiwanese patients with OSCC. HRM analysis is a reliable, accurate, and fast screening method for APC mutations.

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

PubMed

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

2012-01-01

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

Detection of sdhB Gene Mutations in SDHI-Resistant Isolates of Botrytis cinerea Using High Resolution Melting (HRM) Analysis.

PubMed

Samaras, Anastasios; Madesis, Panagiotis; Karaoglanidis, George S

2016-01-01

Botrytis cinerea , is a high risk pathogen for fungicide resistance development. Pathogen' resistance to SDHIs is associated with several mutations in sdh gene. The diversity of mutations and their differential effect on cross-resistance patterns among SDHIs and the fitness of resistant strains necessitate the availability of a tool for their rapid identification. This study was initiated to develop and validate a high-resolution melting (HRM) analysis for the identification of P225H/F/L//T, N230I, and H272L/R/Y mutations. Based on the sequence of sdh B subunit of resistant and sensitive isolates, a universal primer pair was designed. The specificity of the HRM analysis primers was verified to ensure against the cross-reaction with other fungal species and its sensitivity was evaluated using concentrations of known amounts of mutant's DNA. The melting curve analysis generated nine distinct curve profiles, enabling the discrimination of all the four mutations located at codon 225, the N230I mutation, the three mutations located in codon 272, and the non-mutated isolates (isolates of wild-type sensitivity). Similar results were obtained when DNA was extracted directly from artificially inoculated strawberry fruit. The method was validated by monitoring the presence of sdh B mutations in samples of naturally infected strawberry fruits and stone fruit rootstock seedling plants showing damping-off symptoms. HRM analysis data were compared with a standard PIRA-PCR technique and an absolute agreement was observed suggesting that in both populations the H272R mutation was the predominant one, while H272Y, N230I, and P225H were detected in lower frequencies. The results of the study suggest that HRM analysis can be a useful tool for sensate, accurate, and rapid identification of several sdh B mutations in B. cinerea and it is expected to contribute in routine fungicide resistance monitoring or assessments of the effectiveness of anti-resistance strategies implemented in crops heavily treated with botryticides.

Detection of sdhB Gene Mutations in SDHI-Resistant Isolates of Botrytis cinerea Using High Resolution Melting (HRM) Analysis

PubMed Central

Samaras, Anastasios; Madesis, Panagiotis; Karaoglanidis, George S.

2016-01-01

Botrytis cinerea, is a high risk pathogen for fungicide resistance development. Pathogen’ resistance to SDHIs is associated with several mutations in sdh gene. The diversity of mutations and their differential effect on cross-resistance patterns among SDHIs and the fitness of resistant strains necessitate the availability of a tool for their rapid identification. This study was initiated to develop and validate a high-resolution melting (HRM) analysis for the identification of P225H/F/L//T, N230I, and H272L/R/Y mutations. Based on the sequence of sdhB subunit of resistant and sensitive isolates, a universal primer pair was designed. The specificity of the HRM analysis primers was verified to ensure against the cross-reaction with other fungal species and its sensitivity was evaluated using concentrations of known amounts of mutant’s DNA. The melting curve analysis generated nine distinct curve profiles, enabling the discrimination of all the four mutations located at codon 225, the N230I mutation, the three mutations located in codon 272, and the non-mutated isolates (isolates of wild-type sensitivity). Similar results were obtained when DNA was extracted directly from artificially inoculated strawberry fruit. The method was validated by monitoring the presence of sdhB mutations in samples of naturally infected strawberry fruits and stone fruit rootstock seedling plants showing damping-off symptoms. HRM analysis data were compared with a standard PIRA–PCR technique and an absolute agreement was observed suggesting that in both populations the H272R mutation was the predominant one, while H272Y, N230I, and P225H were detected in lower frequencies. The results of the study suggest that HRM analysis can be a useful tool for sensate, accurate, and rapid identification of several sdhB mutations in B. cinerea and it is expected to contribute in routine fungicide resistance monitoring or assessments of the effectiveness of anti-resistance strategies implemented in crops heavily treated with botryticides. PMID:27895633

Role of damage-specific DNA polymerases in M13 phage mutagenesis induced by a major lipid peroxidation product trans-4-hydroxy-2-nonenal.

PubMed

Janowska, Beata; Kurpios-Piec, Dagmara; Prorok, Paulina; Szparecki, Grzegorz; Komisarski, Marek; Kowalczyk, Paweł; Janion, Celina; Tudek, Barbara

2012-01-03

One of the major lipid peroxidation products trans-4-hydroxy-2-nonenal (HNE), forms cyclic propano- or ethenoadducts bearing six- or seven-carbon atom side chains to G>C≫A>T. To specify the role of SOS DNA polymerases in HNE-induced mutations, we tested survival and mutation spectra in the lacZα gene of M13mp18 phage, whose DNA was treated in vitro with HNE, and which was grown in uvrA(-)Escherichia coli strains, carrying one, two or all three SOS DNA polymerases. When Pol IV was the only DNA SOS polymerase in the bacterial host, survival of HNE-treated M13 DNA was similar to, but mutation frequency was lower than in the strain containing all SOS DNA polymerases. When only Pol II or Pol V were present in host bacteria, phage survival decreased dramatically. Simultaneously, mutation frequency was substantially increased, but exclusively in the strain carrying only Pol V, suggesting that induction of mutations by HNE is mainly dependent on Pol V. To determine the role of Pol II and Pol IV in HNE induced mutagenesis, Pol II or Pol IV were expressed together with Pol V. This resulted in decrease of mutation frequency, suggesting that both enzymes can compete with Pol V, and bypass HNE-DNA adducts in an error-free manner. However, HNE-DNA adducts were easily bypassed by Pol IV and only infrequently by Pol II. Mutation spectrum established for strains expressing only Pol V, showed that in uvrA(-) bacteria the frequency of base substitutions and recombination increased in relation to NER proficient strains, particularly mutations at adenine sites. Among base substitutions A:T→C:G, A:T→G:C, G:C→A:T and G:C→T:A prevailed. The results suggest that Pol V can infrequently bypass HNE-DNA adducts inducing mutations at G, C and A sites, while bypass by Pol IV and Pol II is error-free, but for Pol II infrequent. Copyright © 2011 Elsevier B.V. All rights reserved.

BCOR and BCORL1 mutations in myelodysplastic syndromes and related disorders.

PubMed

Damm, Frederik; Chesnais, Virginie; Nagata, Yasunobu; Yoshida, Kenichi; Scourzic, Laurianne; Okuno, Yusuke; Itzykson, Raphael; Sanada, Masashi; Shiraishi, Yuichi; Gelsi-Boyer, Véronique; Renneville, Aline; Miyano, Satoru; Mori, Hiraku; Shih, Lee-Yung; Park, Sophie; Dreyfus, François; Guerci-Bresler, Agnes; Solary, Eric; Rose, Christian; Cheze, Stéphane; Prébet, Thomas; Vey, Norbert; Legentil, Marion; Duffourd, Yannis; de Botton, Stéphane; Preudhomme, Claude; Birnbaum, Daniel; Bernard, Olivier A; Ogawa, Seishi; Fontenay, Michaela; Kosmider, Olivier

2013-10-31

Patients with low-risk myelodysplastic syndromes (MDS) that rapidly progress to acute myeloid leukemia (AML) remain a challenge in disease management. Using whole-exome sequencing of an MDS patient, we identified a somatic mutation in the BCOR gene also mutated in AML. Sequencing of BCOR and related BCORL1 genes in a cohort of 354 MDS patients identified 4.2% and 0.8% of mutations respectively. BCOR mutations were associated with RUNX1 (P = .002) and DNMT3A mutations (P = .015). BCOR is also mutated in chronic myelomonocytic leukemia patients (7.4%) and BCORL1 in AML patients with myelodysplasia-related changes (9.1%). Using deep sequencing, we show that BCOR mutations arise after mutations affecting genes involved in splicing machinery or epigenetic regulation. In univariate analysis, BCOR mutations were associated with poor prognosis in MDS (overall survival [OS]: P = .013; cumulative incidence of AML transformation: P = .005). Multivariate analysis including age, International Prognostic Scoring System, transfusion dependency, and mutational status confirmed a significant inferior OS to patients with a BCOR mutation (hazard ratio, 3.3; 95% confidence interval, 1.4-8.1; P = .008). These data suggest that BCOR mutations define the clinical course rather than disease initiation. Despite infrequent mutations, BCOR analyses should be considered in risk stratification.

Simultaneous screening for JAK2 and calreticulin gene mutations in myeloproliferative neoplasms with high resolution melting.

PubMed

Matsumoto, Nariyoshi; Mori, Sayaka; Hasegawa, Hiroo; Sasaki, Daisuke; Mori, Hayato; Tsuruda, Kazuto; Imanishi, Daisuke; Imaizumi, Yoshitaka; Hata, Tomoko; Kaku, Norihito; Kosai, Kousuke; Uno, Naoki; Miyazaki, Yasushi; Yanagihara, Katsunori

2016-11-01

Recently, novel calreticulin (CALR) mutations were discovered in Janus kinase 2 (JAK2) non-mutated myelofibrosis (PMF) and essential thrombocythemia (ET) cases, with a frequency of 60-80%. We examined clinical correlations and CALR mutation frequency in our myeloproliferative neoplasms (MPN) cases, and introduce an effective test method for use in clinical practice. We examined 177 samples previously investigated for the JAK2 mutation for differential diagnosis of MPN. JAK2 and CALR mutations were analyzed using melting curve analysis and microchip electrophoresis, respectively. Next, we constructed a test for simultaneous screening of the JAK2 and CALR mutations utilizing high resolution melting (HRM). Among 99 MPN cases, 60 possessed the JAK2 mutation alone. Of the 39 MPN cases without the JAK2 mutation, 14 were positive for the CALR mutation, all of which were ET. Using our novel screening test for the JAK2 and CALR mutations by HRM, the concordance rate of conventional analysis with HRM was 96% for the JAK2 mutation and 95% for the CALR mutation. Our novel simultaneous screening test for the JAK2 and CALR gene mutations with HRM is useful for diagnosis of MPN. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Loss of second and sixth conserved cysteine residues from trypsin inhibitor-like cysteine-rich domain-type protease inhibitors in Bombyx mori may induce activity against microbial proteases.

PubMed

Li, Youshan; Liu, Huawei; Zhu, Rui; Xia, Qingyou; Zhao, Ping

2016-12-01

Previous studies have indicated that most trypsin inhibitor-like cysteine-rich domain (TIL)-type protease inhibitors, which contain a single TIL domain with ten conserved cysteines, inhibit cathepsin, trypsin, chymotrypsin, or elastase. Our recent findings suggest that Cys 2nd and Cys 6th were lost from the TIL domain of the fungal-resistance factors in Bombyx mori, BmSPI38 and BmSPI39, which inhibit microbial proteases and the germination of Beauveria bassiana conidia. To reveal the significance of these two missing cysteines in relation to the structure and function of TIL-type protease inhibitors in B. mori, cysteines were introduced at these two positions (D36 and L56 in BmSPI38, D38 and L58 in BmSPI39) by site-directed mutagenesis. The homology structure model of TIL domain of the wild-type and mutated form of BmSPI39 showed that two cysteine mutations may cause incorrect disulfide bond formation of B. mori TIL-type protease inhibitors. The results of Far-UV circular dichroism (CD) spectra indicated that both the wild-type and mutated form of BmSPI39 harbored predominantly random coil structures, and had slightly different secondary structure compositions. SDS-PAGE and Western blotting analysis showed that cysteine mutations affected the multimerization states and electrophoretic mobility of BmSPI38 and BmSPI39. Activity staining and protease inhibition assays showed that the introduction of cysteine mutations dramaticly reduced the activity of inhibitors against microbial proteases, such as subtilisin A from Bacillus licheniformis, protease K from Engyodontium album, protease from Aspergillus melleus. We also systematically analyzed the key residue sites, which may greatly influence the specificity and potency of TIL-type protease inhibitors. We found that the two missing cysteines in B. mori TIL-type protease inhibitors might be crucial for their inhibitory activities against microbial proteases. The genetic engineering of TIL-type protease inhibitors may be applied in both health care and agricultural industries, and could lead to new methods for breeding fungus-resistant transgenic crops and antifungal transgenic silkworm strains. Copyright © 2016 Elsevier Inc. All rights reserved.

A universal method for the mutational analysis of K-ras and p53 gene in non-small-cell lung cancer using formalin-fixed paraffin-embedded tissue.

PubMed

Sarkar, F H; Valdivieso, M; Borders, J; Yao, K L; Raval, M M; Madan, S K; Sreepathi, P; Shimoyama, R; Steiger, Z; Visscher, D W

1995-12-01

The p53 tumor suppressor gene has been found to be altered in almost all human solid tumors, whereas K-ras gene mutations have been observed in a limited number of human cancers (adenocarcinoma of colon, pancreas, and lung). Studies of mutational inactivation for both genes in the same patient's sample on non-small-cell lung cancer have been limited. In an effort to perform such an analysis, we developed and compared methods (for the mutational detection of p53 and K-ras gene) that represent a modified and universal protocol, in terms of DNA extraction, polymerase chain reaction (PCR) amplification, and nonradioisotopic PCR-single-strand conformation polymorphism (PCR-SSCP) analysis, which is readily applicable to either formalin-fixed, paraffin-embedded tissues or frozen tumor specimens. We applied this method to the evaluation of p53 (exons 5-8) and K-ras (codon 12 and 13) gene mutations in 55 cases of non-small-cell lung cancer. The mutational status in the p53 gene was evaluated by radioisotopic PCR-SSCP and compared with PCR-SSCP utilizing our standardized nonradioisotopic detection system using a single 6-microns tissue section. The mutational patterns observed by PCR-SSCP were subsequently confirmed by PCR-DNA sequencing. The mutational status in the K-ras gene was similarly evaluated by PCR-SSCP, and the specific mutation was confirmed by Southern slot-blot hybridization using 32P-labeled sequence-specific oligonucleotide probes for codons 12 and 13. Mutational changes in K-ras (codon 12) were found in 10 of 55 (18%) of non-small-cell lung cancers. Whereas adenocarcinoma showed K-ras mutation in 33% of the cases at codon 12, only one mutation was found at codon 13. As expected, squamous cell carcinoma samples (25 cases) did not show K-ras mutations. Mutations at exons 5-8 of the p53 gene were documented in 19 of 55 (34.5%) cases. Ten of the 19 mutations were single nucleotide point mutations, leading to amino acid substitution. Six showed insertional mutation, and three showed deletion mutations. Only three samples showed mutations of both K-ras and p53 genes. We conclude that although K-ras and p53 gene mutations are frequent in non-small-cell lung cancer, mutations of both genes in the same patient's samples are not common. We also conclude that this universal nonradioisotopic method is superior to other similar methods and is readily applicable to the rapid screening of large numbers of formalin-fixed, paraffin-embedded or frozen samples for the mutational analysis of multiple genes.

Analysis of AIS data of the Bonanza Creek Experimental Forest, Alaska

NASA Technical Reports Server (NTRS)

Spanner, M. A.; Peterson, D. L.

1986-01-01

Airborne Imaging Spectrometer (AIS) data were acquired in 1985 over the Bonanza Creek Experimental Forest, Alaska for the analysis of canopy characteristics including biochemistry. Concurrent with AIS overflights, foliage from fifteen coniferous and deciduous forest stands were analyzed for a variety of biochemical constituents including nitrogen, lignin, protein, and chlorophyll. Preliminary analysis of AIS spectra indicates that the wavelength region between 1450 to 1800 namometers (nm) displays distinct differences in spectral response for some of the forest stands. A flat field subtraction (forest stand spectra - flat field spectra) of the AIS spectra assisted in the interpretation of features of the spectra that are related to biology.

Molecular analysis of Fanconi anemia: the experience of the Bone Marrow Failure Study Group of the Italian Association of Pediatric Onco-Hematology

PubMed Central

De Rocco, Daniela; Bottega, Roberta; Cappelli, Enrico; Cavani, Simona; Criscuolo, Maria; Nicchia, Elena; Corsolini, Fabio; Greco, Chiara; Borriello, Adriana; Svahn, Johanna; Pillon, Marta; Mecucci, Cristina; Casazza, Gabriella; Verzegnassi, Federico; Cugno, Chiara; Locasciulli, Anna; Farruggia, Piero; Longoni, Daniela; Ramenghi, Ugo; Barberi, Walter; Tucci, Fabio; Perrotta, Silverio; Grammatico, Paola; Hanenberg, Helmut; Della Ragione, Fulvio; Dufour, Carlo; Savoia, Anna

2014-01-01

Fanconi anemia is an inherited disease characterized by congenital malformations, pancytopenia, cancer predisposition, and sensitivity to cross-linking agents. The molecular diagnosis of Fanconi anemia is relatively complex for several aspects including genetic heterogeneity with mutations in at least 16 different genes. In this paper, we report the mutations identified in 100 unrelated probands enrolled into the National Network of the Italian Association of Pediatric Hematoly and Oncology. In approximately half of these cases, mutational screening was carried out after retroviral complementation analyses or protein analysis. In the other half, the analysis was performed on the most frequently mutated genes or using a next generation sequencing approach. We identified 108 distinct variants of the FANCA, FANCG, FANCC, FANCD2, and FANCB genes in 85, 9, 3, 2, and 1 families, respectively. Despite the relatively high number of private mutations, 45 of which are novel Fanconi anemia alleles, 26% of the FANCA alleles are due to 5 distinct mutations. Most of the mutations are large genomic deletions and nonsense or frameshift mutations, although we identified a series of missense mutations, whose pathogenetic role was not always certain. The molecular diagnosis of Fanconi anemia is still a tiered procedure that requires identifying candidate genes to avoid useless sequencing. Introduction of next generation sequencing strategies will greatly improve the diagnostic process, allowing a rapid analysis of all the genes. PMID:24584348

MutationAligner: a resource of recurrent mutation hotspots in protein domains in cancer.

PubMed

Gauthier, Nicholas Paul; Reznik, Ed; Gao, Jianjiong; Sumer, Selcuk Onur; Schultz, Nikolaus; Sander, Chris; Miller, Martin L

2016-01-04

The MutationAligner web resource, available at http://www.mutationaligner.org, enables discovery and exploration of somatic mutation hotspots identified in protein domains in currently (mid-2015) more than 5000 cancer patient samples across 22 different tumor types. Using multiple sequence alignments of protein domains in the human genome, we extend the principle of recurrence analysis by aggregating mutations in homologous positions across sets of paralogous genes. Protein domain analysis enhances the statistical power to detect cancer-relevant mutations and links mutations to the specific biological functions encoded in domains. We illustrate how the MutationAligner database and interactive web tool can be used to explore, visualize and analyze mutation hotspots in protein domains across genes and tumor types. We believe that MutationAligner will be an important resource for the cancer research community by providing detailed clues for the functional importance of particular mutations, as well as for the design of functional genomics experiments and for decision support in precision medicine. MutationAligner is slated to be periodically updated to incorporate additional analyses and new data from cancer genomics projects. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

[Two novel pathogenic mutations of GAN gene identified in a patient with giant axonal neuropathy].

PubMed

Wang, Juan; Ma, Qingwen; Cai, Qin; Liu, Yanna; Wang, Wei; Ren, Zhaorui

2016-06-01

To explore the disease-causing mutations in a patient suspected for giant axonal neuropathy(GAN). Target sequence capture sequencing was used to screen potential mutations in genomic DNA extracted from peripheral blood sample of the patient. Sanger sequencing was applied to confirm the detected mutation. The mutation was verified among 400 GAN alleles from 200 healthy individuals by Sanger sequencing. The function of the mutations was predicted by bioinformatics analysis. The patient was identified as a compound heterozygote carrying two novel pathogenic GAN mutations, i.e., c.778G>T (p.Glu260Ter) and c.277G>A (p.Gly93Arg). Sanger sequencing confirmed that the c.778G>T (p.Glu260Ter) mutation was inherited from his father, while c.277G>A (p.Gly93Arg) was inherited from his mother. The same mutations was not found in the 200 healthy individuals. Bioinformatics analysis predicted that the two mutations probably caused functional abnormality of gigaxonin. Two novel GAN mutations were detected in a patient with GAN. Both mutations are pathogenic and can cause abnormalities of gigaxonin structure and function, leading to pathogenesis of GAN. The results may also offer valuable information for similar diseases.

Discriminant analysis of fused positive and negative ion mobility spectra using multivariate self-modeling mixture analysis and neural networks.

PubMed

Chen, Ping; Harrington, Peter B

2008-02-01

A new method coupling multivariate self-modeling mixture analysis and pattern recognition has been developed to identify toxic industrial chemicals using fused positive and negative ion mobility spectra (dual scan spectra). A Smiths lightweight chemical detector (LCD), which can measure positive and negative ion mobility spectra simultaneously, was used to acquire the data. Simple-to-use interactive self-modeling mixture analysis (SIMPLISMA) was used to separate the analytical peaks in the ion mobility spectra from the background reactant ion peaks (RIP). The SIMPLSIMA analytical components of the positive and negative ion peaks were combined together in a butterfly representation (i.e., negative spectra are reported with negative drift times and reflected with respect to the ordinate and juxtaposed with the positive ion mobility spectra). Temperature constrained cascade-correlation neural network (TCCCN) models were built to classify the toxic industrial chemicals. Seven common toxic industrial chemicals were used in this project to evaluate the performance of the algorithm. Ten bootstrapped Latin partitions demonstrated that the classification of neural networks using the SIMPLISMA components was statistically better than neural network models trained with fused ion mobility spectra (IMS).

Molecular genetic analysis of the F11 gene in 14 Turkish patients with factor XI deficiency: identification of novel and recurrent mutations and their inheritance within families.

PubMed

Colakoglu, Seyma; Bayhan, Turan; Tavil, Betül; Keskin, Ebru Yılmaz; Cakir, Volkan; Gümrük, Fatma; Çetin, Mualla; Aytaç, Selin; Berber, Ergul

2018-01-01

Factor XI (FXI) deficiency is an autosomal bleeding disease associated with genetic defects in the F11 gene which cause decreased FXI levels or impaired FXI function. An increasing number of mutations has been reported in the FXI mutation database, most of which affect the serine protease domain of the protein. FXI is a heterogeneous disorder associated with a variable bleeding tendency and a variety of causative F11 gene mutations. The molecular basis of FXI deficiency in 14 patients from ten unrelated families in Turkey was analysed to establish genotype-phenotype correlations and inheritance of the mutations in the patients' families. Fourteen index cases with a diagnosis of FXI deficiency and family members of these patients were enrolled into the study. The patients' F11 genes were amplified by polymerase chain reaction and subjected to direct DNA sequencing analysis. The findings were analysed statistically using bivariate correlations, Pearson's correlation coefficient and the nonparametric Mann-Whitney test. Direct DNA sequencing analysis of the F11 genes revealed that all of the 14 patients had a F11 gene mutation. Eight different mutations were identified in the apple 1, apple 2 or serine protease domains, except one which was a splice site mutation. Six of the mutations were recurrent. Two of the mutations were novel missense mutations, p.Val522Gly and p.Cys581Arg, within the catalytic domain. The p.Trp519Stop mutation was observed in two families whereas all the other mutations were specific to a single family. Identification of mutations confirmed the genetic heterogeneity of FXI deficiency. Most of the patients with mutations did not have any bleeding complications, whereas some had severe bleeding symptoms. Genetic screening for F11 gene mutations is important to decrease the mortality and morbidity rate associated with FXI deficiency, which can be life-threatening if bleeding occurs in tissues with high fibrinolytic activity.

Mutations in Splicing Factor Genes Are a Major Cause of Autosomal Dominant Retinitis Pigmentosa in Belgian Families

PubMed Central

Coppieters, Frauke; Roels, Dimitri; De Jaegere, Sarah; Flipts, Helena; De Zaeytijd, Julie; Walraedt, Sophie; Claes, Charlotte; Fransen, Erik; Van Camp, Guy; Depasse, Fanny; Casteels, Ingele; de Ravel, Thomy

2017-01-01

Purpose Autosomal dominant retinitis pigmentosa (adRP) is characterized by an extensive genetic heterogeneity, implicating 27 genes, which account for 50 to 70% of cases. Here 86 Belgian probands with possible adRP underwent genetic testing to unravel the molecular basis and to assess the contribution of the genes underlying their condition. Methods Mutation detection methods evolved over the past ten years, including mutation specific methods (APEX chip analysis), linkage analysis, gene panel analysis (Sanger sequencing, targeted next-generation sequencing or whole exome sequencing), high-resolution copy number screening (customized microarray-based comparative genomic hybridization). Identified variants were classified following American College of Medical Genetics and Genomics (ACMG) recommendations. Results Molecular genetic screening revealed mutations in 48/86 cases (56%). In total, 17 novel pathogenic mutations were identified: four missense mutations in RHO, five frameshift mutations in RP1, six mutations in genes encoding spliceosome components (SNRNP200, PRPF8, and PRPF31), one frameshift mutation in PRPH2, and one frameshift mutation in TOPORS. The proportion of RHO mutations in our cohort (14%) is higher than reported in a French adRP population (10.3%), but lower than reported elsewhere (16.5–30%). The prevalence of RP1 mutations (10.5%) is comparable to other populations (3.5%-10%). The mutation frequency in genes encoding splicing factors is unexpectedly high (altogether 19.8%), with PRPF31 the second most prevalent mutated gene (10.5%). PRPH2 mutations were found in 4.7% of the Belgian cohort. Two families (2.3%) have the recurrent NR2E3 mutation p.(Gly56Arg). The prevalence of the recurrent PROM1 mutation p.(Arg373Cys) was higher than anticipated (3.5%). Conclusions Overall, we identified mutations in 48 of 86 Belgian adRP cases (56%), with the highest prevalence in RHO (14%), RP1 (10.5%) and PRPF31 (10.5%). Finally, we expanded the molecular spectrum of PRPH2, PRPF8, RHO, RP1, SNRNP200, and TOPORS-associated adRP by the identification of 17 novel mutations. PMID:28076437

Clinical and mutation analysis of 51 probands with anophthalmia and/or severe microphthalmia from a single center

PubMed Central

Gerth-Kahlert, Christina; Williamson, Kathleen; Ansari, Morad; Rainger, Jacqueline K; Hingst, Volker; Zimmermann, Theodor; Tech, Stefani; Guthoff, Rudolf F; van Heyningen, Veronica; FitzPatrick, David R

2013-01-01

Clinical evaluation and mutation analysis was performed in 51 consecutive probands with severe eye malformations – anophthalmia and/or severe microphthalmia – seen in a single specialist ophthalmology center. The mutation analysis consisted of bidirectional sequencing of the coding regions of SOX2, OTX2, PAX6 (paired domain), STRA6, BMP4, SMOC1, FOXE3, and RAX, and genome-wide array-based copy number assessment. Fifteen (29.4%) of the 51 probands had likely causative mutations affecting SOX2 (9/51), OTX2 (5/51), and STRA6 (1/51). Of the cases with bilateral anophthalmia, 9/12 (75%) were found to be mutation positive. Three of these mutations were large genomic deletions encompassing SOX2 (one case) or OTX2 (two cases). Familial inheritance of three intragenic, plausibly pathogenic, and heterozygous mutations was observed. An unaffected carrier parent of an affected child with an identified OTX2 mutation confirmed the previously reported nonpenetrance for this disorder. Two families with SOX2 mutations demonstrated a parent and child both with significant but highly variable eye malformations. Heterozygous loss-of-function mutations in SOX2 and OTX2 are the most common genetic pathology associated with severe eye malformations and bi-allelic loss-of-function in STRA6 is confirmed as an emerging cause of nonsyndromal eye malformations. PMID:24498598

Diagnosis of becker muscular dystrophy: Results of Re-analysis of DNA samples.

PubMed

Straathof, Chiara S M; Van Heusden, Dave; Ippel, Pieternella F; Post, Jan G; Voermans, Nicol C; De Visser, Marianne; Brusse, Esther; Van Den Bergen, Janneke C; Van Der Kooi, Anneke J; Verschuuren, Jan J G M; Ginjaar, Hendrika B

2016-01-01

The phenotype of Becker muscular dystrophy (BMD) is highly variable, and the disease may be underdiagnosed. We searched for new mutations in the DMD gene in a cohort of previously undiagnosed patients who had been referred in the period 1985-1995. All requests for DNA analysis of the DMD gene in probands with suspected BMD were re-evaluated. If the phenotype was compatible with BMD, and no deletions or duplications were detected, DNA samples were screened for small mutations. In 79 of 185 referrals, no mutation was found. Analysis could be performed on 31 DNA samples. Seven different mutations, including 3 novel ones, were found. Long-term clinical follow-up is described. Refining DNA analysis in previously undiagnosed cases can identify mutations in the DMD gene and provide genetic diagnosis of BMD. A delayed diagnosis can still be valuable for the proband or the relatives of BMD patients. © 2015 Wiley Periodicals, Inc.

Cytology smears as diagnostic material for EGFR gene testing in non-small cell lung cancer.

PubMed

Powrózek, Tomasz; Krawczyk, Paweł; Pankowski, Juliusz; Reszka, Katarzyna; Jakubiak, Magdalena; Obrochta, Anna; Wojas-Krawczyk, Kamila; Buczkowski, Jarosław; Milanowski, Janusz

2015-11-14

Cytology smears can be effectively used for EGFR mutation testing in the qualification of NSCLC patients for EGFR tyrosine kinase inhibitor therapy. However, tissue specimens are preferred for EGFR mutation analysis. The aim of this study was to estimate the effectiveness of the real-time PCR method for EGFR testing in histology and cytology materials obtained simultaneously from NSCLC patients. Fourteen adenocarcinoma patients with EGFR-mutation-positive primary tumor tissues were included in the study. Corresponding cytological smears of metastatic lymph nodes obtained by EBUS-TBNA were examined. EGFR Mutation Analysis Kit (EntroGen, USA) and real-time PCR (m2000rt system, Abbott, USA) were used for EGFR mutation analysis in both types of material. In primary tumor tissues, 12 deletions in exon 19 and 2 substitutions in exon 21 (L858R mutation) of the EGFR gene were found. Except for 1 deletion in exon 19, the same EGFR gene mutations were detected in all corresponding cytology samples. The percentage of tumor cells, DNA concentration, percentage of mutated DNA as well as ΔCt values were similar in cytology slides and histology material. In both types of materials, no significant correlations were found between the percentage of tumor cells and the percentage of mutated DNA nor between the DNA concentration and the percentage of mutated DNA. We demonstrated the high effectiveness of a sensitive real-time PCR method in EGFR gene mutation detection in cytology smears.

[Study of a family with epidermolysis bullosa simplex resulting from a novel mutation of KRT14 gene].

PubMed

Meng, Lanlan; Du, Juan; Li, Wen; Lu, Guangxiu; Tan, Yueqiu

2017-08-10

To determine the molecular etiology for a Chinese pedigree affected with epidermolysis bullosa simplex (EBS). Target region sequencing using a hereditary epidermolysis bullosa capture array combined with Sanger sequencing and bioinformatics analysis were used. Mutation taster, PolyPhen-2, Provean, and SIFT software and NCBI online were employed to assess the pathogenicity and conservation of detected mutations. One hundred healthy unrelated individuals were used as controls. Target region sequencing showed that the proband has carried a unreported heterozygous c.1234A>G (p.Ile412Val) mutation of the KRT14 gene, which was confirmed by Sanger sequencing in other 8 affected individuals but not among healthy members of the pedigree. Bioinformatics analysis indicated that the mutation is highly pathogenic. Remarkably, 3 members of the family (2 affected and 1 unaffected) have carried a heterozygous c.1237G>A (p.Ala413Thr) mutation of the KRT14 gene, which was collected in Human Gene Mutation Database (HGMD). Bioinformatics analysis indicated that the mutation may not be pathogenic. Both mutations were not detected among the 100 healthy controls. The novel c.1234A>G(p.Ile412Val) mutation of the KRT14 gene is probably responsible for the disease, while c.1237G>A (p.Ala413Thr) mutation of KRT14 gene may be a polymorphism. Compared with Sanger sequencing, target region capture sequencing is more efficient and can significantly reduce the cost of genetic testing for EBS.

Precise Detection of IDH1/2 and BRAF Hotspot Mutations in Clinical Glioma Tissues by a Differential Calculus Analysis of High-Resolution Melting Data

PubMed Central

Hatae, Ryusuke; Yoshimoto, Koji; Kuga, Daisuke; Akagi, Yojiro; Murata, Hideki; Suzuki, Satoshi O.; Mizoguchi, Masahiro; Iihara, Koji

2016-01-01

High resolution melting (HRM) is a simple and rapid method for screening mutations. It offers various advantages for clinical diagnostic applications. Conventional HRM analysis often yields equivocal results, especially for surgically obtained tissues. We attempted to improve HRM analyses for more effective applications to clinical diagnostics. HRM analyses were performed for IDH1R132 and IDH2R172 mutations in 192 clinical glioma samples in duplicate and these results were compared with sequencing results. BRAFV600E mutations were analyzed in 52 additional brain tumor samples. The melting profiles were used for differential calculus analyses. Negative second derivative plots revealed additional peaks derived from heteroduplexes in PCR products that contained mutations; this enabled unequivocal visual discrimination of the mutations. We further developed a numerical expression, the HRM-mutation index (MI), to quantify the heteroduplex-derived peak of the mutational curves. Using this expression, all IDH1 mutation statuses matched those ascertained by sequencing, with the exception of three samples. These discordant results were all derived from the misinterpretation of sequencing data. The effectiveness of our approach was further validated by analyses of IDH2R172 and BRAFV600E mutations. The present analytical method enabled an unequivocal and objective HRM analysis and is suitable for reliable mutation scanning in surgically obtained glioma tissues. This approach could facilitate molecular diagnostics in clinical environments. PMID:27529619

Software and database for the analysis of mutations in the human FBN1 gene.

PubMed Central

Collod, G; Béroud, C; Soussi, T; Junien, C; Boileau, C

1996-01-01

Fibrillin is the major component of extracellular microfibrils. Mutations in the fibrillin gene on chromosome 15 (FBN1) were described at first in the heritable connective tissue disorder, Marfan syndrome (MFS). More recently, FBN1 has also been shown to harbor mutations related to a spectrum of conditions phenotypically related to MFS and many mutations will have to be accumulated before genotype/phenotype relationships emerge. To facilitate mutational analysis of the FBN1 gene, a software package along with a computerized database (currently listing 63 entries) have been created. PMID:8594563

Monoallelic mutation analysis (MAMA) for identifying germline mutations.

PubMed

Papadopoulos, N; Leach, F S; Kinzler, K W; Vogelstein, B

1995-09-01

Dissection of germline mutations in a sensitive and specific manner presents a continuing challenge. In dominantly inherited diseases, mutations occur in only one allele and are often masked by the normal allele. Here we report the development of a sensitive and specific diagnostic strategy based on somatic cell hybridization termed MAMA (monoallelic mutation analysis). We have demonstrated the utility of this strategy in two different hereditary colorectal cancer syndromes, one caused by a defective tumour suppressor gene on chromosome 5 (familial adenomatous polyposis, FAP) and the other caused by a defective mismatch repair gene on chromosome 2 (hereditary non-polyposis colorectal cancer, HNPCC).

Somatic mutations in the transcriptional corepressor gene BCORL1 in adult acute myelogenous leukemia

PubMed Central

Li, Meng; Collins, Roxane; Jiao, Yuchen; Ouillette, Peter; Bixby, Dale; Erba, Harry; Vogelstein, Bert; Kinzler, Kenneth W.

2011-01-01

To further our understanding of the genetic basis of acute myelogenous leukemia (AML), we determined the coding exon sequences of ∼ 18 000 protein-encoding genes in 8 patients with secondary AML. Here we report the discovery of novel somatic mutations in the transcriptional corepressor gene BCORL1 that is located on the X-chromosome. Analysis of BCORL1 in an unselected cohort of 173 AML patients identified a total of 10 mutated cases (6%) with BCORL1 mutations, whereas analysis of 19 AML cell lines uncovered 4 (21%) BCORL1 mutated cell lines. The majority (87%) of the mutations in BCORL1 were predicted to inactivate the gene product as a result of nonsense mutations, splice site mutation, or out-of-frame insertions or deletions. These results indicate that BCORL1 by genetic criteria is a novel candidate tumor suppressor gene, joining the growing list of genes recurrently mutated in AML. PMID:21989985

[Rapid detection of hot spot mutations of FGFR3 gene with PCR-high resolution melting assay].

PubMed

Li, Shan; Wang, Han; Su, Hua; Gao, Jinsong; Zhao, Xiuli

2017-08-10

To identify the causative mutations in five individuals affected with dyschondroplasia and develop an efficient procedure for detecting hot spot mutations of the FGFR3 gene. Genomic DNA was extracted from peripheral blood samples with a standard phenol/chloroform method. PCR-Sanger sequencing was used to analyze the causative mutations in the five probands. PCR-high resolution melting (HRM) was developed to detect the identified mutations. A c.1138G>A mutation in exon 8 was found in 4 probands, while a c.1620C>G mutation was found in exon 11 of proband 5 whom had a mild phenotype. All patients were successfully distinguished from healthy controls with the PCR-HRM method. The results of HRM analysis were highly consistent with that of Sanger sequencing. The Gly380Arg and Asn540Lys are hot spot mutations of the FGFR3 gene among patients with ACH/HCH. PCR-HRM analysis is more efficient for detecting hot spot mutations of the FGFR3 gene.

[Mutation analysis for a pedigree affected with keratitis-ichthyosis-deafness syndrome].

PubMed

Li, Lulu; Li, Yuan; Lin, Wei; Zhao, Xiuli

2017-10-10

To identify mutation of GJB2 gene and provide genetic counseling for a family affected with keratitis-ichthyosis-deafness (KID) syndrome. Genomic DNA was extracted from peripheral blood samples with a standard phenol-chloroform method. PCR and Sanger sequencing were used to analyze potential mutation in the proband. Suspected mutation was verified with a PCR-high-resolution melting (PCR-HRM) method. T-clone sequencing was applied to determine the parental origin of the mutation. A heterozygous mutation, c.148G>A (p.Asp50Asn), which is located in the exon 1 of the GJB2 gene, was found in the proband. The results was confirmed by HRM analysis. Cloning sequencing suggested that the mutation was derived from the father's germline. The hot-spot mutation c.148G>A (p.Asp50Asn) in the GJB2 gene probably underlies the KID syndrome in this Chinese family. A PCR-HRM method has been established to rapidly detect common mutations associated with this disease.

RHO Mutations (p.W126L and p.A346P) in Two Japanese Families with Autosomal Dominant Retinitis Pigmentosa

PubMed Central

Akahori, Masakazu; Itabashi, Takeshi; Nishino, Jo; Yoshitake, Kazutoshi; Ikeo, Kazuho; Tsuneoka, Hiroshi

2014-01-01

Purpose. To investigate genetic and clinical features of patients with rhodopsin (RHO) mutations in two Japanese families with autosomal dominant retinitis pigmentosa (adRP). Methods. Whole-exome sequence analysis was performed in ten adRP families. Identified RHO mutations for the cosegregation analysis were confirmed by Sanger sequencing. Ophthalmic examinations were performed to evaluate the RP phenotypes. The impact of the RHO mutation on the rhodopsin conformation was examined by molecular modeling analysis. Results. In two adRP families, we identified two RHO mutations (c.377G>T (p.W126L) and c.1036G>C (p.A346P)), one of which was novel. Complete cosegregation was confirmed for each mutation exhibiting the RP phenotype in both families. Molecular modeling predicted that the novel mutation (p.W126L) might impair rhodopsin function by affecting its conformational transition in the light-adapted form. Clinical phenotypes showed that patients with p.W126L exhibited sector RP, whereas patients with p.A346P exhibited classic RP. Conclusions. Our findings demonstrated that the novel mutation (p.W126L) may be associated with the phenotype of sector RP. Identification of RHO mutations is a very useful tool for predicting disease severity and providing precise genetic counseling. PMID:25485142

A frame-shift mutation of PMS2 is a widespread cause of Lynch syndrome.

PubMed

Clendenning, M; Senter, L; Hampel, H; Robinson, K Lagerstedt; Sun, S; Buchanan, D; Walsh, M D; Nilbert, M; Green, J; Potter, J; Lindblom, A; de la Chapelle, A

2008-06-01

When compared to the other mismatch repair genes involved in Lynch syndrome, the identification of mutations within PMS2 has been limited (<2% of all identified mutations), yet the immunohistochemical analysis of tumour samples indicates that approximately 5% of Lynch syndrome cases are caused by PMS2. This disparity is primarily due to complications in the study of this gene caused by interference from pseudogene sequences. Using a recently developed method for detecting PMS2 specific mutations, we have screened 99 patients who are likely candidates for PMS2 mutations based on immunohistochemical analysis. We have identified a frequently occurring frame-shift mutation (c.736_741del6ins11) in 12 ostensibly unrelated Lynch syndrome patients (20% of patients we have identified with a deleterious mutation in PMS2, n = 61). These individuals all display the rare allele (population frequency <0.05) at a single nucleotide polymorphism (SNP) in exon 11, and have been shown to possess a short common haplotype, allowing us to calculate that the mutation arose around 1625 years ago (65 generations; 95% confidence interval 22 to 120). Ancestral analysis indicates that this mutation is enriched in individuals with British and Swedish ancestry. We estimate that there are >10 000 carriers of this mutation in the USA alone. The identification of both the mutation and the common haplotype in one Swedish control sample (n = 225), along with evidence that Lynch syndrome associated cancers are rarer than expected in the probands' families, would suggest that this is a prevalent mutation with reduced penetrance.

The AMBRE Project: Stellar parameterisation of the ESO:FEROS archived spectra

NASA Astrophysics Data System (ADS)

Worley, C. C.; de Laverny, P.; Recio-Blanco, A.; Hill, V.; Bijaoui, A.; Ordenovic, C.

2012-06-01

Context. The AMBRE Project is a collaboration between the European Southern Observatory (ESO) and the Observatoire de la Côte d'Azur (OCA) that has been established in order to carry out the determination of stellar atmospheric parameters for the archived spectra of four ESO spectrographs. Aims: The analysis of the FEROS archived spectra for their stellar parameters (effective temperatures, surface gravities, global metallicities, alpha element to iron ratios and radial velocities) has been completed in the first phase of the AMBRE Project. From the complete ESO:FEROS archive dataset that was received, a total of 21 551 scientific spectra have been identified, covering the period 2005 to 2010. These spectra correspond to 6285 stars. Methods: The determination of the stellar parameters was carried out using the stellar parameterisation algorithm, MATISSE (MATrix Inversion for Spectral SynthEsis), which has been developed at OCA to be used in the analysis of large scale spectroscopic studies in galactic archaeology. An analysis pipeline has been constructed that integrates spectral normalisation, cleaning and radial velocity correction procedures in order that the FEROS spectra could be analysed automatically with MATISSE to obtain the stellar parameters. The synthetic grid against which the MATISSE analysis is carried out is currently constrained to parameters of FGKM stars only. Results: Stellar atmospheric parameters, effective temperature, surface gravity, metallicity and alpha element abundances, were determined for 6508 (30.2%) of the FEROS archived spectra (~3087 stars). Radial velocities were determined for 11 963 (56%) of the archived spectra. 2370 (11%) spectra could not be analysed within the pipeline due to very low signal-to-noise ratios or missing spectral orders. 12 673 spectra (58.8%) were analysed in the pipeline but their parameters were discarded based on quality criteria and error analysis determined within the automated process. The majority of these rejected spectra were found to have broad spectral features, as probed both by the direct measurement of the features and cross-correlation function breadths, indicating that they may be hot and/or fast rotating stars, which are not considered within the adopted reference synthetic spectra grid. The current configuration of the synthetic spectra grid is devoted to slow-rotating FGKM stars. Hence non-standard spectra (binaries, chemically peculiar stars etc.) that could not be identified may pollute the analysis.

Biochemical and genetic diagnosis of the primary hyperoxalurias: a review.

PubMed

Rumsby, G

2000-01-01

The primary hyperoxalurias are a group of inherited disorders of endogenous oxalate overproduction. Diagnosis of the two best-characterized disorders, primary hyperoxaluria (PH) Types 1 and 2, is achieved by sequential measurement of alanine:glyoxylate aminotransferase and glyoxylate reductase enzyme activity in a single needle liver biopsy. While genetic analysis of PH2 is still at a relatively early stage, the AGXT gene defective in the Type 1 disorder is well characterized, and a number of mutations have been identified. To determine whether mutation analysis could replace enzymatic analysis for the diagnosis of PH1, DNA samples from 127 consecutive unrelated patients in whom there was a high clinical suspicion of primary hyperoxaluria were analyzed for the presence of the G630A and T853C mutations, which together account for approximately 34% of the mutant alleles in our patient cohort. The sensitivity of mutation detection was 47% in those patients with enzymologically confirmed Type 1 disease, showing that mutation analysis cannot effectively replace enzymology at the present time. However, there is little doubt of the value of genetic methods (mutation and linkage analysis) for diagnosing PH1 (and eventually PH2) in other family members and for prenatal diagnosis and carrier testing.

Radiation risk predictions for Space Station Freedom orbits

NASA Technical Reports Server (NTRS)

Cucinotta, Francis A.; Atwell, William; Weyland, Mark; Hardy, Alva C.; Wilson, John W.; Townsend, Lawrence W.; Shinn, Judy L.; Katz, Robert

1991-01-01

Risk assessment calculations are presented for the preliminary proposed solar minimum and solar maximum orbits for Space Station Freedom (SSF). Integral linear energy transfer (LET) fluence spectra are calculated for the trapped proton and GCR environments. Organ dose calculations are discussed using the computerized anatomical man model. The cellular track model of Katz is applied to calculate cell survival, transformation, and mutation rates for various aluminum shields. Comparisons between relative biological effectiveness (RBE) and quality factor (QF) values for SSF orbits are made.

Molecular subgroups of medulloblastoma

PubMed Central

Northcott, Paul A; Dubuc, Adrian M; Pfister, Stefan; Taylor, Michael D

2014-01-01

Recent efforts at stratifying medulloblastomas based on their molecular features have revolutionized our understanding of this morbidity. Collective efforts by multiple independent groups have subdivided medulloblastoma from a single disease into four distinct molecular subgroups characterized by disparate transcriptional signatures, mutational spectra, copy number profiles and, most importantly, clinical features. We present a summary of recent studies that have contributed to our understanding of the core medulloblastoma subgroups, focusing largely on clinically relevant discoveries that have already, and will continue to, shape research. PMID:22853794

Role of genetic mutations in folate-related enzyme genes on Male Infertility

PubMed Central

Liu, Kang; Zhao, Ruizhe; Shen, Min; Ye, Jiaxin; Li, Xiao; Huang, Yuan; Hua, Lixin; Wang, Zengjun; Li, Jie

2015-01-01

Several studies showed that the genetic mutations in the folate-related enzyme genes might be associated with male infertility; however, the results were still inconsistent. We performed a meta-analysis with trial sequential analysis to investigate the associations between the MTHFR C677T, MTHFR A1298C, MTR A2756G, MTRR A66G mutations and the MTHFR haplotype with the risk of male infertility. Overall, a total of 37 studies were selected. Our meta-analysis showed that the MTHFR C677T mutation was a risk factor for male infertility in both azoospermia and oligoasthenoteratozoospermia patients, especially in Asian population. Men carrying the MTHFR TC haplotype were most liable to suffer infertility while those with CC haplotype had lowest risk. On the other hand, the MTHFR A1298C mutation was not related to male infertility. MTR A2756G and MTRR A66G were potential candidates in the pathogenesis of male infertility, but more case-control studies were required to avoid false-positive outcomes. All of these results were confirmed by the trial sequential analysis. Finally, our meta-analysis with trial sequential analysis proved that the genetic mutations in the folate-related enzyme genes played a significant role in male infertility. PMID:26549413

Mutational Analysis of the Adaptor Protein 2 Sigma Subunit (AP2S1) Gene: Search for Autosomal Dominant Hypocalcemia Type 3 (ADH3)

PubMed Central

Rogers, Angela; Nesbit, M. Andrew; Hannan, Fadil M.; Howles, Sarah A.; Gorvin, Caroline M.; Cranston, Treena; Allgrove, Jeremy; Bevan, John S.; Bano, Gul; Brain, Caroline; Datta, Vipan; Grossman, Ashley B.; Hodgson, Shirley V.; Izatt, Louise; Millar-Jones, Lynne; Pearce, Simon H.; Robertson, Lisa; Selby, Peter L.; Shine, Brian; Snape, Katie; Warner, Justin

2014-01-01

Context: Autosomal dominant hypocalcemia (ADH) types 1 and 2 are due to calcium-sensing receptor (CASR) and G-protein subunit-α11 (GNA11) gain-of-function mutations, respectively, whereas CASR and GNA11 loss-of-function mutations result in familial hypocalciuric hypercalcemia (FHH) types 1 and 2, respectively. Loss-of-function mutations of adaptor protein-2 sigma subunit (AP2σ 2), encoded by AP2S1, cause FHH3, and we therefore sought for gain-of-function AP2S1 mutations that may cause an additional form of ADH, which we designated ADH3. Objective: The objective of the study was to investigate the hypothesis that gain-of-function AP2S1 mutations may cause ADH3. Design: The sample size required for the detection of at least one mutation with a greater than 95% likelihood was determined by binomial probability analysis. Nineteen patients (including six familial cases) with hypocalcemia in association with low or normal serum PTH concentrations, consistent with ADH, but who did not have CASR or GNA11 mutations, were ascertained. Leukocyte DNA was used for sequence and copy number variation analysis of AP2S1. Results: Binomial probability analysis, using the assumption that AP2S1 mutations would occur in hypocalcemic patients at a prevalence of 20%, which is observed in FHH patients without CASR or GNA11 mutations, indicated that the likelihood of detecting at least one AP2S1 mutation was greater than 95% and greater than 98% in sample sizes of 14 and 19 hypocalcemic patients, respectively. AP2S1 mutations and copy number variations were not detected in the 19 hypocalcemic patients. Conclusion: The absence of AP2S1 abnormalities in hypocalcemic patients, suggests that ADH3 may not occur or otherwise represents a rare hypocalcemic disorder. PMID:24708097

Cancerouspdomains: comprehensive analysis of cancer type-specific recurrent somatic mutations in proteins and domains.

PubMed

Hashemi, Seirana; Nowzari Dalini, Abbas; Jalali, Adrin; Banaei-Moghaddam, Ali Mohammad; Razaghi-Moghadam, Zahra

2017-08-16

Discriminating driver mutations from the ones that play no role in cancer is a severe bottleneck in elucidating molecular mechanisms underlying cancer development. Since protein domains are representatives of functional regions within proteins, mutations on them may disturb the protein functionality. Therefore, studying mutations at domain level may point researchers to more accurate assessment of the functional impact of the mutations. This article presents a comprehensive study to map mutations from 29 cancer types to both sequence- and structure-based domains. Statistical analysis was performed to identify candidate domains in which mutations occur with high statistical significance. For each cancer type, the corresponding type-specific domains were distinguished among all candidate domains. Subsequently, cancer type-specific domains facilitated the identification of specific proteins for each cancer type. Besides, performing interactome analysis on specific proteins of each cancer type showed high levels of interconnectivity among them, which implies their functional relationship. To evaluate the role of mitochondrial genes, stem cell-specific genes and DNA repair genes in cancer development, their mutation frequency was determined via further analysis. This study has provided researchers with a publicly available data repository for studying both CATH and Pfam domain regions on protein-coding genes. Moreover, the associations between different groups of genes/domains and various cancer types have been clarified. The work is available at http://www.cancerouspdomains.ir .

Mutation analysis of the MYO7A and CDH23 genes in Japanese patients with Usher syndrome type 1.

PubMed

Nakanishi, Hiroshi; Ohtsubo, Masafumi; Iwasaki, Satoshi; Hotta, Yoshihiro; Takizawa, Yoshinori; Hosono, Katsuhiro; Mizuta, Kunihiro; Mineta, Hiroyuki; Minoshima, Shinsei

2010-12-01

Usher syndrome (USH) is an autosomal recessive disorder characterized by retinitis pigmentosa and hearing loss. USH type 1 (USH1), the second common type of USH, is frequently caused by MYO7A and CDH23 mutations, accounting for 70-80% of the cases among various ethnicities, including Caucasians, Africans and Asians. However, there have been no reports of mutation analysis for any responsible genes for USH1 in Japanese patients. This study describes the first mutation analysis of MYO7A and CDH23 in Japanese USH1 patients. Five mutations (three in MYO7A and two in CDH23) were identified in four of five unrelated patients. Of these mutations, two were novel. One of them, p.Tyr1942SerfsX23 in CDH23, was a large deletion causing the loss of 3 exons. This is the first large deletion to be found in CDH23. The incidence of the MYO7A and CDH23 mutations in the study population was 80%, which is consistent with previous findings. Therefore, mutation screening for these genes is expected to be a highly sensitive method for diagnosing USH1 among the Japanese.

Ocular findings associated with a Cys39Arg mutation in the Norrie disease gene.

PubMed

Joos, K M; Kimura, A E; Vandenburgh, K; Bartley, J A; Stone, E M

1994-12-01

To diagnose the carriers and noncarriers in a family affected with Norrie disease based on molecular analysis. Family members from three generations, including one affected patient, two obligate carriers, one carrier identified with linkage analysis, one noncarrier identified with linkage analysis, and one female family member with indeterminate carrier status, were examined clinically and electrophysiologically. Linkage analysis had previously failed to determine the carrier status of one female family member in the third generation. Blood samples were screened for mutations in the Norrie disease gene with single-strand conformation polymorphism analysis. The mutation was characterized by dideoxy-termination sequencing. Ophthalmoscopy and electroretinographic examination failed to detect the carrier state. The affected individuals and carriers in this family were found to have a transition from thymidine to cytosine in the first nucleotide of codon 39 of the Norrie disease gene, causing a cysteine-to-arginine mutation. Single-strand conformation polymorphism analysis identified a patient of indeterminate status (by linkage) to be a noncarrier of Norrie disease. Ophthalmoscopy and electroretinography could not identify carriers of this Norrie disease mutation. Single-strand conformation polymorphism analysis was more sensitive and specific than linkage analysis in identifying carriers in this family.

Comparison of Thoracic Radiotherapy Efficacy Between Patients With and Without EGFR-mutated Lung Adenocarcinoma.

PubMed

Li, Ming-Hsien; Tsai, Jo-Ting; Ting, Lai-Lei; Lin, Jang-Chun; Liu, Yu-Chang

2018-01-01

To investigate the association between tumor response to thoracic radiotherapy and epidermal growth factor receptor (EGFR) mutation status in patients with lung adenocarcinoma, we collected 48 patients treated between January 2010 and December 2013. Of the 18 patients with EGFR mutation, 15 (83.3%) had a single mutation, and three (16.7%) had double mutation. Different EGFR mutation subtypes exhibited different responses to radiotherapy. The identified double EGFR mutations were associated with reduction of residual tumor burden (RTB) after radiotherapy. In univariate analysis, EGFR mutations in exon 18, 20, and 21 and double EGFR mutation were significant factors predicting RTB. In multivariate analysis, exon 20 mutation was the only significant factor. Patients with EGFR mutation seemed to have longer mean overall survival (OS) compared to the group with wild-type EGFR (31.1 vs. 26.6 months, p=0.49). The median and mean OS in patients with double EGFR mutation vs. wild-type EGFR were 20.1 vs. 16.9 months and 28.9 vs. 26.6 months, respectively. Further studies with larger sample size are warranted to clarify the association of EGFR mutation status with the lung tumor response after radiotherapy. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Identification of Mutations Underlying 20 Inborn Errors of Metabolism in the United Arab Emirates Population

PubMed Central

Ben-Rebeh, Imen; Hertecant, Jozef L.; Al-Jasmi, Fatma A.; Aburawi, Hanan E.; Al-Yahyaee, Said A.; Al-Gazali, Lihadh

2012-01-01

Inborn errors of metabolism (IEM) are frequently encountered by physicians in the United Arab Emirates (UAE). However, the mutations underlying a large number of these disorders have not yet been determined. Therefore, the objective of this study was to identify the mutations underlying a number of IEM disorders among UAE residents from both national and expatriate families. A case series of patients from 34 families attending the metabolic clinic at Tawam Hospital were clinically evaluated, and molecular testing was carried out to determine their causative mutations. The mutation analysis was carried out at molecular genetics diagnostic laboratories. Thirty-eight mutations have been identified as responsible for twenty IEM disorders, including in the metabolism of amino acids, lipids, steroids, metal transport and mitochondrial energy metabolism, and lysosomal storage disorders. Nine of the identified mutations are novel, including two missense mutations, three premature stop codons and four splice site mutations. Mutation analysis of IEM disorders in the UAE population has an important impact on molecular diagnosis and genetic counseling for families affected by these disorders. PMID:22106832

Comparison of cross-platform technologies for EGFR T790M testing in patients with non-small cell lung cancer

PubMed Central

Li, Xuefei; Zhou, Caicun

2017-01-01

Somatic mutations in the gene encoding epidermal growth factor receptor (EGFR) play an important role in determining targeted treatment modalities in non-small cell lung cancer (NSCLC). The EGFR T790M mutation emerges in approximately 50% of cases who acquire resistance to tyrosine kinase inhibitors. Detecting EGFR T790M mutation in tumor tissue is challenging due to heterogeneity of the tumor, low abundance of the mutation and difficulty for re-biopsy in patients with advanced disease. Alternatively, circulating tumor DNA (ctDNA) has been proposed as a non-invasive method for mutational analysis. The presence of EGFR mutations in ctDNA predicts response to the EGFR TKIs in the first-line setting. Molecular testing is now considered a standard care for NSCLC. The advent of standard commercially available kits and targeted mutational analysis has revolutionized the accuracy of mutation detection platforms for detection of EGFR mutations. Our review provides an overview of various commonly used platforms for detecting EGFR T790M mutation in tumor tissue and plasma. PMID:29246024

Whole-exome analysis to detect congenital hemolytic anemia mimicking congenital dyserythropoietic anemia.

PubMed

Hamada, Motoharu; Doisaki, Sayoko; Okuno, Yusuke; Muramatsu, Hideki; Hama, Asahito; Kawashima, Nozomu; Narita, Atsushi; Nishio, Nobuhiro; Yoshida, Kenichi; Kanno, Hitoshi; Manabe, Atsushi; Taga, Takashi; Takahashi, Yoshiyuki; Miyano, Satoru; Ogawa, Seishi; Kojima, Seiji

2018-06-23

Congenital dyserythropoietic anemia (CDA) is a heterogeneous group of rare congenital disorders characterized by ineffective erythropoiesis and dysplastic changes in erythroblasts. Diagnosis of CDA is based primarily on the morphology of bone marrow erythroblasts; however, genetic tests have recently become more important. Here, we performed genetic analysis of 10 Japanese patients who had been diagnosed with CDA based on laboratory findings and morphological characteristics. We examined 10 CDA patients via central review of bone marrow morphology and genetic analysis for congenital bone marrow failure syndromes. Sanger sequencing for CDAN1, SEC23B, and KLF1 was performed for all patients. We performed whole-exome sequencing in patients without mutation in these genes. Three patients carried pathogenic CDAN1 mutations, whereas no SEC23B mutations were identified in our cohort. WES unexpectedly identified gene mutations known to cause congenital hemolytic anemia in two patients: canonical G6PD p.Val394Leu mutation and SPTA1 p.Arg28His mutation. Comprehensive genetic analysis is warranted for more effective diagnosis of patients with suspected CDA.

Congenital combined pituitary hormone deficiency attributable to a novel PROP1 mutation (467insT).

PubMed

Nose, Osamu; Tatsumi, Keita; Nakano, Yukiko; Amino, Nobuyuki

2006-04-01

Combined pituitary hormone deficiency (CPHD) is an anterior pituitary disorder, commonly resulting in growth retardation. PROP1 gene mutations appear to be frequently responsible for CPHD, particularly in Middle and Eastern Europe and the Americas, but few cases have been reported in Japan. Two sisters (aged 8.4 and 4.3 years at presentation) exhibited proportional short stature from about 2 years of age. Genetic analysis determined the nature and location of mutations. Pituitary size by magnetic resonance imaging (MRI) indicated only slight hypoplasia, while hormone analysis revealed deficiencies in secretion of growth hormone (GH), thyroid stimulating hormone, prolactin and gonadotropins; adrenocortinotropin secretion appeared adequate. Genetic analysis revealed a novel familial inherited PROP1 mutation. A unique insertion mutation was found in codon 156 (467insT) located in the transcription-activating region of the PROP1 gene. The resulting PROP1 protein (191 amino acids) would lack the transcription activation domain and consequently be non-functional. Gene analysis suggested that the siblings had inherited a unique autosomal recessive PROP1 gene mutation resulting in severe GH deficiency and subsequent growth retardation.

Identification of a Comprehensive Spectrum of Genetic Factors for Hereditary Breast Cancer in a Chinese Population by Next-Generation Sequencing

PubMed Central

Yang, Xiaochen; Wu, Jiong; Lu, Jingsong; Liu, Guangyu; Di, Genhong; Chen, Canming; Hou, Yifeng; Sun, Menghong; Yang, Wentao; Xu, Xiaojing; Zhao, Ying; Hu, Xin; Li, Daqiang; Cao, Zhigang; Zhou, Xiaoyan; Huang, Xiaoyan; Liu, Zhebin; Chen, Huan; Gu, Yanzi; Chi, Yayun; Yan, Xia; Han, Qixia; Shen, Zhenzhou; Shao, Zhimin; Hu, Zhen

2015-01-01

The genetic etiology of hereditary breast cancer has not been fully elucidated. Although germline mutations of high-penetrance genes such as BRCA1/2 are implicated in development of hereditary breast cancers, at least half of all breast cancer families are not linked to these genes. To identify a comprehensive spectrum of genetic factors for hereditary breast cancer in a Chinese population, we performed an analysis of germline mutations in 2,165 coding exons of 152 genes associated with hereditary cancer using next-generation sequencing (NGS) in 99 breast cancer patients from families of cancer patients regardless of cancer types. Forty-two deleterious germline mutations were identified in 21 genes of 34 patients, including 18 (18.2%) BRCA1 or BRCA2 mutations, 3 (3%) TP53 mutations, 5 (5.1%) DNA mismatch repair gene mutations, 1 (1%) CDH1 mutation, 6 (6.1%) Fanconi anemia pathway gene mutations, and 9 (9.1%) mutations in other genes. Of seven patients who carried mutations in more than one gene, 4 were BRCA1/2 mutation carriers, and their average onset age was much younger than patients with only BRCA1/2 mutations. Almost all identified high-penetrance gene mutations in those families fulfill the typical phenotypes of hereditary cancer syndromes listed in the National Comprehensive Cancer Network (NCCN) guidelines, except two TP53 and three mismatch repair gene mutations. Furthermore, functional studies of MSH3 germline mutations confirmed the association between MSH3 mutation and tumorigenesis, and segregation analysis suggested antagonism between BRCA1 and MSH3. We also identified a lot of low-penetrance gene mutations. Although the clinical significance of those newly identified low-penetrance gene mutations has not been fully appreciated yet, these new findings do provide valuable epidemiological information for the future studies. Together, these findings highlight the importance of genetic testing based on NCCN guidelines and a multi-gene analysis using NGS may be a supplement to traditional genetic counseling. PMID:25927356

The MATISSE analysis of large spectral datasets from the ESO Archive

NASA Astrophysics Data System (ADS)

Worley, C.; de Laverny, P.; Recio-Blanco, A.; Hill, V.; Vernisse, Y.; Ordenovic, C.; Bijaoui, A.

2010-12-01

The automated stellar classification algorithm, MATISSE, has been developed at the Observatoire de la Côte d'Azur (OCA) in order to determine stellar temperatures, gravities and chemical abundances for large datasets of stellar spectra. The Gaia Data Processing and Analysis Consortium (DPAC) has selected MATISSE as one of the key programmes to be used in the analysis of the Gaia Radial Velocity Spectrometer (RVS) spectra. MATISSE is currently being used to analyse large datasets of spectra from the ESO archive with the primary goal of producing advanced data products to be made available in the ESO database via the Virtual Observatory. This is also an invaluable opportunity to identify and address issues that can be encountered with the analysis large samples of real spectra prior to the launch of Gaia in 2012. The analysis of the archived spectra of the FEROS spectrograph is currently underway and preliminary results are presented.

Roles of Rev1, Pol ζ, Pol32 and Pol η in the bypass of chromosomal abasic sites in Saccharomyces cerevisiae

PubMed Central

Auerbach, Paul A.; Demple, Bruce

2010-01-01

Translesion synthesis (TLS) on DNA is a process by which potentially cytotoxic replication-blocking lesions are bypassed, but at the risk of increased mutagenesis. The exact in vivo role of the individual TLS enzymes in Saccharomyces cerevisiae has been difficult to determine from previous studies due to differing results from the variety of systems used. We have generated a series of S.cerevisiae strains in which each of the TLS-related genes REV1, REV3, REV7, RAD30 and POL32 was deleted, and in which chromosomal apyrimidinic sites were generated during normal cell growth by the activity of altered forms of human uracil-DNA glycosylase that remove undamaged cytosines or thymines. Deletion of REV1, REV3 or REV7 resulted in slower growth dependent on (rev3Δ and rev7Δ) or enhanced by (rev1Δ) expression of the mutator glycosylases and a nearly complete abolition of glycosylase-induced mutagenesis. Deletion of POL32 resulted in cell death when the mutator glycosylases were expressed and, in their absence, diminished spontaneous mutagenesis. RAD30 appeared to be unnecessary for mutagenesis in response to abasic sites, as deleting this gene caused no significant change in either the mutation rates or the mutational spectra due to glycosylase expression. PMID:19901007

On the molecular mechanism of GC content variation among eubacterial genomes.

PubMed

Wu, Hao; Zhang, Zhang; Hu, Songnian; Yu, Jun

2012-01-10

As a key parameter of genome sequence variation, the GC content of bacterial genomes has been investigated for over half a century, and many hypotheses have been put forward to explain this GC content variation and its relationship to other fundamental processes. Previously, we classified eubacteria into dnaE-based groups (the dimeric combination of DNA polymerase III alpha subunits), according to a hypothesis where GC content variation is essentially governed by genome replication and DNA repair mechanisms. Further investigation led to the discovery that two major mutator genes, polC and dnaE2, may be responsible for genomic GC content variation. Consequently, an in-depth analysis was conducted to evaluate various potential intrinsic and extrinsic factors in association with GC content variation among eubacterial genomes. Mutator genes, especially those with dominant effects on the mutation spectra, are biased towards either GC or AT richness, and they alter genomic GC content in the two opposite directions. Increased bacterial genome size (or gene number) appears to rely on increased genomic GC content; however, it is unclear whether the changes are directly related to certain environmental pressures. Certain environmental and bacteriological features are related to GC content variation, but their trends are more obvious when analyzed under the dnaE-based grouping scheme. Most terrestrial, plant-associated, and nitrogen-fixing bacteria are members of the dnaE1|dnaE2 group, whereas most pathogenic or symbiotic bacteria in insects, and those dwelling in aquatic environments, are largely members of the dnaE1|polV group. Our studies provide several lines of evidence indicating that DNA polymerase III α subunit and its isoforms participating in either replication (such as polC) or SOS mutagenesis/translesion synthesis (such as dnaE2), play dominant roles in determining GC variability. Other environmental or bacteriological factors, such as genome size, temperature, oxygen requirement, and habitat, either play subsidiary roles or rely indirectly on different mutator genes to fine-tune the GC content. These results provide a comprehensive insight into mechanisms of GC content variation and the robustness of eubacterial genomes in adapting their ever-changing environments over billions of years.

Novel types of COMP mutations and genotype-phenotype association in pseudoachondroplasia and multiple epiphyseal dysplasia.

PubMed

Mabuchi, Akihiko; Manabe, Noriyo; Haga, Nobuhiko; Kitoh, Hiroshi; Ikeda, Toshiyuki; Kawaji, Hiroyuki; Tamai, Kazuya; Hamada, Junichiro; Nakamura, Shigeru; Brunetti-Pierri, Nicola; Kimizuka, Mamori; Takatori, Yoshio; Nakamura, Kozo; Nishimura, Gen; Ohashi, Hirofumi; Ikegawa, Shiro

2003-01-01

Mutations in the gene encoding cartilage oligomeric matrix protein ( COMP) cause two skeletal dysplasias, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED). More than 40 mutations have been identified; however, genotype-phenotype relationships are not well delineated. Further, mutations other than in-frame insertion/deletions and substitutions have not been found, and currently known mutations are clustered within relatively small regions. Here we report the identification of nine novel and three recurrent COMP mutations in PSACH and MED patients. These include two novel types of mutations; the first, a gross deletion spanning an exon-intron junction, causes an exon deletion. The second, a frameshift mutation that results in a truncation of the C-terminal domain, is the first known truncating mutation in the COMP gene. The remaining mutations, other than a novel exon 18 mutation, affected highly conserved aspartate or cysteine residues in the calmodulin-like repeat (CLR) region. Genotype-phenotype analysis revealed a correlation between the position and type of mutations and the severity of short stature. Mutations in the seventh CLR produced more severe short stature compared with mutations elsewhere in the CLRs ( P=0.0003) and elsewhere in the COMP gene ( P=0.0007). Patients carrying mutations within the five-aspartates repeat (aa 469-473) in the seventh CLR were extremely short (below -6 SD). Patients with deletion mutations were significantly shorter than those with substitution mutations ( P=0.0024). These findings expand the mutation spectrum of the COMP gene and highlight genotype-phenotype relationships, facilitating improved genetic diagnosis and analysis of COMP function in humans.

Transcription factor mutations in myelodysplastic/myeloproliferative neoplasms

PubMed Central

Ernst, Thomas; Chase, Andrew; Zoi, Katerina; Waghorn, Katherine; Hidalgo-Curtis, Claire; Score, Joannah; Jones, Amy; Grand, Francis; Reiter, Andreas; Hochhaus, Andreas; Cross, Nicholas C.P.

2010-01-01

Background Aberrant activation of tyrosine kinases, caused by either mutation or gene fusion, is of major importance for the development of many hematologic malignancies, particularly myeloproliferative neoplasms. We hypothesized that hitherto unrecognized, cytogenetically cryptic tyrosine kinase fusions may be common in non-classical or atypical myeloproliferative neoplasms and related myelodysplastic/myeloproliferative neoplasms. Design and Methods To detect genomic copy number changes associated with such fusions, we performed a systematic search in 68 patients using custom designed, targeted, high-resolution array comparative genomic hybridization. Arrays contained 44,000 oligonucleotide probes that targeted 500 genes including all 90 tyrosine kinases plus downstream tyrosine kinase signaling components, other translocation targets, transcription factors, and other factors known to be important for myelopoiesis. Results No abnormalities involving tyrosine kinases were detected; however, nine cytogenetically cryptic copy number imbalances were detected in seven patients, including hemizygous deletions of RUNX1 or CEBPA in two cases with atypical chronic myeloid leukemia. Mutation analysis of the remaining alleles revealed non-mutated RUNX1 and a frameshift insertion within CEBPA. A further mutation screen of 187 patients with myelodysplastic/myeloproliferative neoplasms identified RUNX1 mutations in 27 (14%) and CEBPA mutations in seven (4%) patients. Analysis of other transcription factors known to be frequently mutated in acute myeloid leukemia revealed NPM1 mutations in six (3%) and WT1 mutations in two (1%) patients with myelodysplastic/myeloproliferative neoplasms. Univariate analysis indicated that patients with mutations had a shorter overall survival (28 versus 44 months, P=0.019) compared with patients without mutations, with the prognosis for cases with CEBPA, NPM1 or WT1 mutations being particularly poor. Conclusions We conclude that mutations of transcription and other nuclear factors are frequent in myelodysplastic/myeloproliferative neoplasms and are generally mutually exclusive. CEBPA, NPM1 or WT1 mutations may be associated with a poor prognosis, an observation that will need to be confirmed by detailed prospective studies. PMID:20421268

Clinical implications of genomic profiles in metastatic breast cancer with a focus on TP53 and PIK3CA, the most frequently mutated genes.

PubMed

Kim, Ji-Yeon; Lee, Eunjin; Park, Kyunghee; Park, Woong-Yang; Jung, Hae Hyun; Ahn, Jin Seok; Im, Young-Hyuck; Park, Yeon Hee

2017-04-25

Breast cancer (BC) has been genetically profiled through large-scale genome analyses. However, the role and clinical implications of genetic alterations in metastatic BC (MBC) have not been evaluated. Therefore, we conducted whole-exome sequencing (WES) and RNA-Seq of 37 MBC samples and targeted deep sequencing of another 29 MBCs. We evaluated somatic mutations from WES and targeted sequencing and assessed gene expression and performed pathway analysis from RNA-Seq. In this analysis, PIK3CA was the most commonly mutated gene in estrogen receptor (ER)-positive BC, while in ER-negative BC, TP53 was the most commonly mutated gene (p = 0.018 and p < 0.001, respectively). TP53 stopgain/loss and frameshift mutation was related to low expression of TP53 in contrast nonsynonymous mutation was related to high expression. The impact of TP53 mutation on clinical outcome varied with regard to ER status. In ER-positive BCs, wild type TP53 had a better prognosis than mutated TP53 (median overall survival (OS) (wild type vs. mutated): 88.5 ± 54.4 vs. 32.6 ± 10.7 (months), p = 0.002). In contrast, mutated TP53 had a protective effect in ER-negative BCs (median OS: 0.10 vs. 32.6 ± 8.2, p = 0.026). However, PIK3CA mutation did not affect patient survival. In gene expression analysis, CALM1, a potential regulator of AKT, was highly expressed in PIK3CA-mutated BCs. In conclusion, mutation of TP53 was associated with expression status and affect clinical outcome according to ER status in MBC. Although mutation of PIK3CA was not related to survival in this study, mutation of PIK3CA altered the expression of other genes and pathways including CALM1 and may be a potential predictive marker of PI3K inhibitor effectiveness.

Altered Pre-mRNA Splicing Caused by a Novel Intronic Mutation c.1443+5G>A in the Dihydropyrimidinase (DPYS) Gene

PubMed Central

Nakajima, Yoko; Meijer, Judith; Zhang, Chunhua; Wang, Xu; Kondo, Tomomi; Ito, Tetsuya; Dobritzsch, Doreen; Van Kuilenburg, André B. P.

2016-01-01

Dihydropyrimidinase (DHP) deficiency is an autosomal recessive disease caused by mutations in the DPYS gene. Patients present with highly elevated levels of dihydrouracil and dihydrothymine in their urine, blood and cerebrospinal fluid. The analysis of the effect of mutations in DPYS on pre-mRNA splicing is hampered by the fact that DHP is primarily expressed in liver and kidney cells. The minigene approach can detect mRNA splicing aberrations using cells that do not express the endogenous mRNA. We have used a minigene-based approach to analyze the effects of a presumptive pre-mRNA splicing mutation in two newly identified Chinese pediatric patients with DHP deficiency. Mutation analysis of DPYS showed that both patients were compound heterozygous for a novel intronic mutation c.1443+5G>A in intron 8 and a previously described missense mutation c.1001A>G (p.Q334R) in exon 6. Wild-type and the mutated minigene constructs, containing exons 7, 8 and 9 of DPYS, yielded different splicing products after expression in HEK293 cells. The c.1443+5G>A mutation resulted in altered pre-mRNA splicing of the DPYS minigene construct with full skipping of exon 8. Analysis of the DHP crystal structure showed that the deletion of exon 8 severely affects folding, stability and homooligomerization of the enzyme as well as disruption of the catalytic site. Thus, the analysis suggests that the c.1443+5G>A mutation results in aberrant splicing of the pre-mRNA encoding DHP, underlying the DHP deficiency in two unrelated Chinese patients. PMID:26771602

Altered Pre-mRNA Splicing Caused by a Novel Intronic Mutation c.1443+5G>A in the Dihydropyrimidinase (DPYS) Gene.

PubMed

Nakajima, Yoko; Meijer, Judith; Zhang, Chunhua; Wang, Xu; Kondo, Tomomi; Ito, Tetsuya; Dobritzsch, Doreen; Van Kuilenburg, André B P

2016-01-12

Dihydropyrimidinase (DHP) deficiency is an autosomal recessive disease caused by mutations in the DPYS gene. Patients present with highly elevated levels of dihydrouracil and dihydrothymine in their urine, blood and cerebrospinal fluid. The analysis of the effect of mutations in DPYS on pre-mRNA splicing is hampered by the fact that DHP is primarily expressed in liver and kidney cells. The minigene approach can detect mRNA splicing aberrations using cells that do not express the endogenous mRNA. We have used a minigene-based approach to analyze the effects of a presumptive pre-mRNA splicing mutation in two newly identified Chinese pediatric patients with DHP deficiency. Mutation analysis of DPYS showed that both patients were compound heterozygous for a novel intronic mutation c.1443+5G>A in intron 8 and a previously described missense mutation c.1001A>G (p.Q334R) in exon 6. Wild-type and the mutated minigene constructs, containing exons 7, 8 and 9 of DPYS, yielded different splicing products after expression in HEK293 cells. The c.1443+5G>A mutation resulted in altered pre-mRNA splicing of the DPYS minigene construct with full skipping of exon 8. Analysis of the DHP crystal structure showed that the deletion of exon 8 severely affects folding, stability and homooligomerization of the enzyme as well as disruption of the catalytic site. Thus, the analysis suggests that the c.1443+5G>A mutation results in aberrant splicing of the pre-mRNA encoding DHP, underlying the DHP deficiency in two unrelated Chinese patients.

[Clinical-based study of ovarian cancer patients with and without BRCA1/2 genes mutation: clinical features and pedigree analysis].

PubMed

Tao, T; Yang, J X; Shen, K; Cao, D Y

2017-01-25

Objective: To compare the clinical and histological features and prognosis of patients with ovarian cancer from different genetic background, and to make further understanding of the genetic model of BRCA genes used pedigree analysis. Methods: There were 71 patients from 67 independent families enrolled in our study from Apr. 2000 to Jun. 2009 in Peking Union Medical College Hospital. All exons of BRCA1/2 genes were analyzed using denaturing high-performance liquid chromatography(DHPLC) followed by direct sequencing, and clinical features of patients were compared by statistical analysis. Pedigree analysis of two families with BRCA genes mutation were performed. Results: The mutation rate of BRCA genes was 28% (20/71). The frequency of BRCA1 and BRCA2 gene mutation was 23% (16/71) and 6% (4/71), respectively ( P= 0.004). Histology types of patients with and without BRCA genes mutation were different. The onset age between patients with and without BRCA genes mutation was similar (52.6 versus 54.6 years old, P= 0.393), and tend to be early-onset breast or ovarian cancer in high-risk group. There was no significant difference of platinum-resistant rate, disease free survival and overall survival rate between patients with and without BRCA genes mutation (all P> 0.05). According to the pedigree analysis, up to 100% of female offspring inherited pathogenic mutations, and male offspring could be a mutation carrier. Conclusions: The genetic screening and clinical intervention should be performed as early as possible for the members from families at risk of hereditary ovarian cancer. Genetic consulting is important for patients with high-grade papillary serous adenocarcinoma of ovary. It is still unknown that whether the patients with BRCA gene mutations have better prognosis than sporadic ones, and further perspective, randomized controlled trial is still needed.

Characterisation of ATM mutations in Slavic Ataxia telangiectasia patients.

PubMed

Soukupova, Jana; Pohlreich, Petr; Seemanova, Eva

2011-09-01

Ataxia telangiectasia (AT) is a genomic instability syndrome characterised, among others, by progressive cerebellar degeneration, oculocutaneous telangiectases, immunodeficiency, elevated serum alpha-phetoprotein level, chromosomal breakage, hypersensitivity to ionising radiation and increased cancer risk. This autosomal recessive disorder is caused by mutations in the ataxia telangiectasia mutated (ATM) gene coding for serine/threonine protein kinase with a crucial role in response to DNA double-strand breaks. We characterised genotype and phenotype of 12 Slavic AT patients from 11 families. Mutation analysis included sequencing of the entire coding sequence, adjacent intron regions, 3'UTR and 5'UTR of the ATM gene and multiplex ligation-dependent probe amplification (MLPA) for the detection of large deletions/duplications at the ATM locus. The high incidence of new and individual mutations demonstrates a marked mutational heterogeneity of AT in the Czech Republic. Our data indicate that sequence analysis of the entire coding region of ATM is sufficient for a high detection rate of mutations in ATM and that MLPA analysis for the detection of deletions/duplications seems to be redundant in the Slavic population.

Large-scale whole-genome sequencing of the Icelandic population.

PubMed

Gudbjartsson, Daniel F; Helgason, Hannes; Gudjonsson, Sigurjon A; Zink, Florian; Oddson, Asmundur; Gylfason, Arnaldur; Besenbacher, Soren; Magnusson, Gisli; Halldorsson, Bjarni V; Hjartarson, Eirikur; Sigurdsson, Gunnar Th; Stacey, Simon N; Frigge, Michael L; Holm, Hilma; Saemundsdottir, Jona; Helgadottir, Hafdis Th; Johannsdottir, Hrefna; Sigfusson, Gunnlaugur; Thorgeirsson, Gudmundur; Sverrisson, Jon Th; Gretarsdottir, Solveig; Walters, G Bragi; Rafnar, Thorunn; Thjodleifsson, Bjarni; Bjornsson, Einar S; Olafsson, Sigurdur; Thorarinsdottir, Hildur; Steingrimsdottir, Thora; Gudmundsdottir, Thora S; Theodors, Asgeir; Jonasson, Jon G; Sigurdsson, Asgeir; Bjornsdottir, Gyda; Jonsson, Jon J; Thorarensen, Olafur; Ludvigsson, Petur; Gudbjartsson, Hakon; Eyjolfsson, Gudmundur I; Sigurdardottir, Olof; Olafsson, Isleifur; Arnar, David O; Magnusson, Olafur Th; Kong, Augustine; Masson, Gisli; Thorsteinsdottir, Unnur; Helgason, Agnar; Sulem, Patrick; Stefansson, Kari

2015-05-01

Here we describe the insights gained from sequencing the whole genomes of 2,636 Icelanders to a median depth of 20×. We found 20 million SNPs and 1.5 million insertions-deletions (indels). We describe the density and frequency spectra of sequence variants in relation to their functional annotation, gene position, pathway and conservation score. We demonstrate an excess of homozygosity and rare protein-coding variants in Iceland. We imputed these variants into 104,220 individuals down to a minor allele frequency of 0.1% and found a recessive frameshift mutation in MYL4 that causes early-onset atrial fibrillation, several mutations in ABCB4 that increase risk of liver diseases and an intronic variant in GNAS associating with increased thyroid-stimulating hormone levels when maternally inherited. These data provide a study design that can be used to determine how variation in the sequence of the human genome gives rise to human diversity.

Quantification of GC-biased gene conversion in the human genome

PubMed Central

Glémin, Sylvain; Arndt, Peter F.; Messer, Philipp W.; Petrov, Dmitri; Galtier, Nicolas; Duret, Laurent

2015-01-01

Much evidence indicates that GC-biased gene conversion (gBGC) has a major impact on the evolution of mammalian genomes. However, a detailed quantification of the process is still lacking. The strength of gBGC can be measured from the analysis of derived allele frequency spectra (DAF), but this approach is sensitive to a number of confounding factors. In particular, we show by simulations that the inference is pervasively affected by polymorphism polarization errors and by spatial heterogeneity in gBGC strength. We propose a new general method to quantify gBGC from DAF spectra, incorporating polarization errors, taking spatial heterogeneity into account, and jointly estimating mutation bias. Applying it to human polymorphism data from the 1000 Genomes Project, we show that the strength of gBGC does not differ between hypermutable CpG sites and non-CpG sites, suggesting that in humans gBGC is not caused by the base-excision repair machinery. Genome-wide, the intensity of gBGC is in the nearly neutral area. However, given that recombination occurs primarily within recombination hotspots, 1%–2% of the human genome is subject to strong gBGC. On average, gBGC is stronger in African than in non-African populations, reflecting differences in effective population sizes. However, due to more heterogeneous recombination landscapes, the fraction of the genome affected by strong gBGC is larger in non-African than in African populations. Given that the location of recombination hotspots evolves very rapidly, our analysis predicts that, in the long term, a large fraction of the genome is affected by short episodes of strong gBGC. PMID:25995268

Linkage Study Revealed Complex Haplotypes in a Multifamily due to Different Mutations in CAPN3 Gene in an Iranian Ethnic Group.

PubMed

Mojbafan, Marzieh; Tonekaboni, Seyed Hassan; Abiri, Maryam; Kianfar, Soudeh; Sarhadi, Ameneh; Nilipour, Yalda; Tavakkoly-Bazzaz, Javad; Zeinali, Sirous

2016-07-01

Calpainopathy is an autosomal recessive form of limb girdle muscular dystrophies which is caused by mutation in CAPN3 gene. In the present study, co-segregation of this disorder was analyzed with four short tandem repeat markers linked to the CAPN3 gene. Three apparently unrelated Iranian families with same ethnicity were investigated. Haplotype analysis and sequencing of the CAPN3 gene were performed. DNA sample from one of the patients was simultaneously sent for next-generation sequencing. DNA sequencing identified two mutations. It was seen as a homozygous c.2105C>T in exon 19 in one family, a homozygous novel mutation c.380G>A in exon 3 in another family, and a compound heterozygote form of these two mutations in the third family. Next-generation sequencing also confirmed our results. It was expected that, due to the rare nature of limb girdle muscular dystrophies, affected individuals from the same ethnic group share similar mutations. Haplotype analysis showed two different homozygote patterns in two families, yet a compound heterozygote pattern in the third family as seen in the mutation analysis. This study shows that haplotype analysis would help in determining presence of different founders.

A Streamlined Protocol for Molecular Testing of the DMD Gene within a Diagnostic Laboratory: A Combination of Array Comparative Genomic Hybridization and Bidirectional Sequence Analysis

PubMed Central

Marquis-Nicholson, Renate; Lai, Daniel; Love, Jennifer M.; Love, Donald R.

2013-01-01

Purpose. The aim of this study was to develop a streamlined mutation screening protocol for the DMD gene in order to confirm a clinical diagnosis of Duchenne or Becker muscular dystrophy in affected males and to clarify the carrier status of female family members. Methods. Sequence analysis and array comparative genomic hybridization (aCGH) were used to identify mutations in the dystrophin DMD gene. We analysed genomic DNA from six individuals with a range of previously characterised mutations and from eight individuals who had not previously undergone any form of molecular analysis. Results. We successfully identified the known mutations in all six patients. A molecular diagnosis was also made in three of the four patients with a clinical diagnosis who had not undergone prior genetic screening, and testing for familial mutations was successfully completed for the remaining four patients. Conclusion. The mutation screening protocol described here meets best practice guidelines for molecular testing of the DMD gene in a diagnostic laboratory. The aCGH method is a superior alternative to more conventional assays such as multiplex ligation-dependent probe amplification (MLPA). The combination of aCGH and sequence analysis will detect mutations in 98% of patients with the Duchenne or Becker muscular dystrophy. PMID:23476807

Advances in Radiation Mutagenesis through Studies on Drosophila

DOE R&D Accomplishments Database

Muller, H. J.

1958-06-01

The approximately linear relation between radiation dose and induced lethals known for Drosophila spermatozoa, is now extended to spermatids. Data are included regarding oogonia. The linearity principle has been confined for minute structural changes in sperm as multi-hit events, on about the 1.5 power of the dose, long known for spermatozoa, is now extended to spermatids and late oocytes, for relatively short exposures. are found to allow union of broken chromosomes. Therefore, the frequencies are lower for more dispersed exposures of varies with lethals induced in late oocytes follow the same frequency pattern and there fore are multi-hit events. Yet han spermatozoan irradiation that two broken ends derived from nonreciprocal. The following is the order of decreasing radiation mutability of different stages found by ourselves and others: spermatids, spermatozoa in females, spermatozoa 0 to 1 day before ejaculation, earlier spermatozoa, late oocytes, gonia of either sex. Lethal frequencies for these stages range over approximately an order of magnitude, gross structural changes far more widely. Of potential usefulness is our extension of genesis by anoxia, known for spermatozoa in adult males, to those in pupal males and in females, to sperion is especially marked but the increase caused by substituting oxygen for air is less marked, perhaps because of enzymatic differences. In contrast, the induction of gross structural changes in oocytes, but not in spermatids, is markedly reduced by oxygen post-treatment; it is increased by dehydration. The efficacy of induction of structural changes by treatment of spermatozoa, whether with radiation or chemical mutagen, is correlated with the conditions of sperm utilization and egg production. Improving our perspective on radiation effects, some 800,000 offspring have been scored for spontaneous visible mutations of 13 specific loci. The average point-mutation rate was 0.5 to 1.0 per locus among 10/sup 5/ germ cells. Most mutation occurred in peri- fertilization stages. All loci studied mutated from one to nine times. Loci mutating oftener spontaneously also gave more radiation mutation, in other studies, Spectra of individual loci prove similar for spontaneous and induced mutation. Studies on back-mutation also showed similarity of spontaneous and radiation mutations. The doubling dose for back-mutations of forked induced in spermatozoa was several hundred roentgens, gonia at diverse loci. Recent analyses of human mutational load lead to mutation-rate estimated like those earlier based on extrapolations from Drosophila, thus supporting the significance for man of the present studies. (auth)

Using Molecular Modeling in Teaching Group Theory Analysis of the Infrared Spectra of Organometallic Compounds

ERIC Educational Resources Information Center

Wang, Lihua

2012-01-01

A new method is introduced for teaching group theory analysis of the infrared spectra of organometallic compounds using molecular modeling. The main focus of this method is to enhance student understanding of the symmetry properties of vibrational modes and of the group theory analysis of infrared (IR) spectra by using visual aids provided by…

Spectral analysis of the structure of ultradispersed diamonds

NASA Astrophysics Data System (ADS)

Uglov, V. V.; Shimanski, V. I.; Rusalsky, D. P.; Samtsov, M. P.

2008-07-01

The structure of ultradispersed diamonds (UDD) is studied by spectral methods. The presence of diamond crystal phase in the UDD is found based on x-ray analysis and Raman spectra. The Raman spectra also show sp2-and sp3-hybridized carbon. Analysis of IR absorption spectra suggests that the composition of functional groups present in the particles changes during the treatment.

Frequent germline deleterious mutations in DNA repair genes in familial prostate cancer cases are associated with advanced disease.

PubMed

Leongamornlert, D; Saunders, E; Dadaev, T; Tymrakiewicz, M; Goh, C; Jugurnauth-Little, S; Kozarewa, I; Fenwick, K; Assiotis, I; Barrowdale, D; Govindasami, K; Guy, M; Sawyer, E; Wilkinson, R; Antoniou, A C; Eeles, R; Kote-Jarai, Z

2014-03-18

Prostate cancer (PrCa) is one of the most common diseases to affect men worldwide and among the leading causes of cancer-related death. The purpose of this study was to use second-generation sequencing technology to assess the frequency of deleterious mutations in 22 tumour suppressor genes in familial PrCa and estimate the relative risk of PrCa if these genes are mutated. Germline DNA samples from 191 men with 3 or more cases of PrCa in their family were sequenced for 22 tumour suppressor genes using Agilent target enrichment and Illumina technology. Analysis for genetic variation was carried out by using a pipeline consisting of BWA, Genome Analysis Toolkit (GATK) and ANNOVAR. Clinical features were correlated with mutation status using standard statistical tests. Modified segregation analysis was used to determine the relative risk of PrCa conferred by the putative loss-of-function (LoF) mutations identified. We discovered 14 putative LoF mutations in 191 samples (7.3%) and these mutations were more frequently associated with nodal involvement, metastasis or T4 tumour stage (P=0.00164). Segregation analysis of probands with European ancestry estimated that LoF mutations in any of the studied genes confer a relative risk of PrCa of 1.94 (95% CI: 1.56-2.42). These findings show that LoF mutations in DNA repair pathway genes predispose to familial PrCa and advanced disease and therefore warrants further investigation. The clinical utility of these findings will become increasingly important as targeted screening and therapies become more widespread.

Molecular genetics of Leber congenital amaurosis in Chinese: New data from 66 probands and mutation overview of 159 probands.

PubMed

Xu, Yan; Xiao, Xueshan; Li, Shiqiang; Jia, Xiaoyun; Xin, Wei; Wang, Panfeng; Sun, Wenmin; Huang, Li; Guo, Xiangming; Zhang, Qingjiong

2016-08-01

Leber congenital amaurosis (LCA) is the most severe form of inherited retinal dystrophy. We have previously performed a mutational analysis of the known LCA-associated genes in probands with LCA by both Sanger and whole exome sequencing. In this study, whole exome sequencing was carried out on 66 new probabds with LCA. In conjunction with these data, the present study provides a comprehensive analysis of the spectrum and frequency of all known genes associated with retinal dystrophy in a total of 159 Chinese probands with LCA. The known genes responsible for all forms hereditary retinal dystrophy were included based on information from RetNet. The candidate variants were filtered by bioinformatics analysis and confirmed by Sanger sequencing. Potentially causative mutations were further validated in available family members. Overall, a total of 118 putative pathogenic mutations from 23 genes were identified in 56.6% (90/159) of probands. These mutations were harbored in 13 LCA-associated genes and in ten genes related to other forms of retinal dystrophy. The most frequently mutated gene in probands with LCA was GUCY2D (10.7%, 17/159). A series of mutational analyses suggests that all known genes associated with retinal dystrophy account for 56.6% of Chinese patients with LCA. A comprehensive molecular genetic analysis of Chinese patients with LCA provides an overview of the spectrum and frequency of ethno-specific mutations of all known genes, as well as indications about other unknown genes in the remaining probands who lacked identified mutations. Copyright © 2016 Elsevier Ltd. All rights reserved.

Principal component analysis and analysis of variance on the effects of Entellan New on the Raman spectra of fibers.

PubMed

Yu, Marcia M L; Sandercock, P Mark L

2012-01-01

During the forensic examination of textile fibers, fibers are usually mounted on glass slides for visual inspection and identification under the microscope. One method that has the capability to accurately identify single textile fibers without subsequent demounting is Raman microspectroscopy. The effect of the mountant Entellan New on the Raman spectra of fibers was investigated to determine if it is suitable for fiber analysis. Raman spectra of synthetic fibers mounted in three different ways were collected and subjected to multivariate analysis. Principal component analysis score plots revealed that while spectra from different fiber classes formed distinct groups, fibers of the same class formed a single group regardless of the mounting method. The spectra of bare fibers and those mounted in Entellan New were found to be statistically indistinguishable by analysis of variance calculations. These results demonstrate that fibers mounted in Entellan New may be identified directly by Raman microspectroscopy without further sample preparation. © 2011 American Academy of Forensic Sciences.

Amino-Acid Network Clique Analysis of Protein Mutation Non-Additive Effects: A Case Study of Lysozme.

PubMed

Ming, Dengming; Chen, Rui; Huang, He

2018-05-10

Optimizing amino-acid mutations in enzyme design has been a very challenging task in modern bio-industrial applications. It is well known that many successful designs often hinge on extensive correlations among mutations at different sites within the enzyme, however, the underpinning mechanism for these correlations is far from clear. Here, we present a topology-based model to quantitively characterize non-additive effects between mutations. The method is based on the molecular dynamic simulations and the amino-acid network clique analysis. It examines if the two mutation sites of a double-site mutation fall into to a 3-clique structure, and associates such topological property of mutational site spatial distribution with mutation additivity features. We analyzed 13 dual mutations of T4 phage lysozyme and found that the clique-based model successfully distinguishes highly correlated or non-additive double-site mutations from those additive ones whose component mutations have less correlation. We also applied the model to protein Eglin c whose structural topology is significantly different from that of T4 phage lysozyme, and found that the model can, to some extension, still identify non-additive mutations from additive ones. Our calculations showed that mutation non-additive effects may heavily depend on a structural topology relationship between mutation sites, which can be quantitatively determined using amino-acid network k -cliques. We also showed that double-site mutation correlations can be significantly altered by exerting a third mutation, indicating that more detailed physicochemical interactions should be considered along with the network clique-based model for better understanding of this elusive mutation-correlation principle.

Detection of Ultra-Rare Mitochondrial Mutations in Breast Stem Cells by Duplex Sequencing.

PubMed

Ahn, Eun Hyun; Hirohata, Kensen; Kohrn, Brendan F; Fox, Edward J; Chang, Chia-Cheng; Loeb, Lawrence A

2015-01-01

Long-lived adult stem cells could accumulate non-repaired DNA damage or mutations that increase the risk of tumor formation. To date, studies on mutations in stem cells have concentrated on clonal (homoplasmic) mutations and have not focused on rarely occurring stochastic mutations that may accumulate during stem cell dormancy. A major challenge in investigating these rare mutations is that conventional next generation sequencing (NGS) methods have high error rates. We have established a new method termed Duplex Sequencing (DS), which detects mutations with unprecedented accuracy. We present a comprehensive analysis of mitochondrial DNA mutations in human breast normal stem cells and non-stem cells using DS. The vast majority of mutations occur at low frequency and are not detectable by NGS. The most prevalent point mutation types are the C>T/G>A and A>G/T>C transitions. The mutations exhibit a strand bias with higher prevalence of G>A, T>C, and A>C mutations on the light strand of the mitochondrial genome. The overall rare mutation frequency is significantly lower in stem cells than in the corresponding non-stem cells. We have identified common and unique non-homoplasmic mutations between non-stem and stem cells that include new mutations which have not been reported previously. Four mutations found within the MT-ND5 gene (m.12684G>A, m.12705C>T, m.13095T>C, m.13105A>G) are present in all groups of stem and non-stem cells. Two mutations (m.8567T>C, m.10547C>G) are found only in non-stem cells. This first genome-wide analysis of mitochondrial DNA mutations may aid in characterizing human breast normal epithelial cells and serve as a reference for cancer stem cell mutation profiles.

ImatraNMR: Novel software for batch integration and analysis of quantitative NMR spectra

NASA Astrophysics Data System (ADS)

Mäkelä, A. V.; Heikkilä, O.; Kilpeläinen, I.; Heikkinen, S.

2011-08-01

Quantitative NMR spectroscopy is a useful and important tool for analysis of various mixtures. Recently, in addition of traditional quantitative 1D 1H and 13C NMR methods, a variety of pulse sequences aimed for quantitative or semiquantitative analysis have been developed. To obtain actual usable results from quantitative spectra, they must be processed and analyzed with suitable software. Currently, there are many processing packages available from spectrometer manufacturers and third party developers, and most of them are capable of analyzing and integration of quantitative spectra. However, they are mainly aimed for processing single or few spectra, and are slow and difficult to use when large numbers of spectra and signals are being analyzed, even when using pre-saved integration areas or custom scripting features. In this article, we present a novel software, ImatraNMR, designed for batch analysis of quantitative spectra. In addition to capability of analyzing large number of spectra, it provides results in text and CSV formats, allowing further data-analysis using spreadsheet programs or general analysis programs, such as Matlab. The software is written with Java, and thus it should run in any platform capable of providing Java Runtime Environment version 1.6 or newer, however, currently it has only been tested with Windows and Linux (Ubuntu 10.04). The software is free for non-commercial use, and is provided with source code upon request.

[FANCA gene mutation analysis in Fanconi anemia patients].

PubMed

Chen, Fei; Peng, Guang-Jie; Zhang, Kejian; Hu, Qun; Zhang, Liu-Qing; Liu, Ai-Guo

2005-10-01

To screen the FANCA gene mutation and explore the FANCA protein function in Fanconi anemia (FA) patients. FANCA protein expression and its interaction with FANCF were analyzed using Western blot and immunoprecipitation in 3 cases of FA-A. Genomic DNA was used for MLPA analysis followed by sequencing. FANCA protein was undetectable and FANCA and FANCF protein interaction was impaired in these 3 cases of FA-A. Each case of FA-A contained biallelic pathogenic mutations in FANCA gene. No functional FANCA protein was found in these 3 cases of FA-A, and intragenic deletion, frame shift and splice site mutation were the major pathogenic mutations found in FANCA gene.

An N-terminal glycine to cysteine mutation in the collagen COL1A1 gene produces moderately severe osteogenesis imperfecta

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wilcox, W.; Scott, L.; Cohn, D.

Osteogenesis imperfecta (OI) is usually due to mutations in the type I procollagen genes COL1A1 and COL1A2. Point mutations close to the N-terminus are generally milder than those near the C-terminus of the molecule (the gradient hypothesis of collagen mutations). We describe a patient with moderately severe OI due to a mutation in the N-terminal portion of the triple helical domain of the {alpha}1(I) chain. Electrophoretic analysis of collagen isolated from fibroblast cultures suggested the abnormal presence of a cysteine in the N-terminal portion of the {alpha}1(I) chain. Five overlapping DNA fragments amplified from fibroblast RNA were screened for mutationsmore » using single strand conformational polymorphism (SSCP) and heteroduplex analyses. Direct DNA sequence analysis of the single positive fragment demonstrated a G to T transversion, corresponding to a glycine to cysteine substitution at position 226 of the triple helical domain of the {alpha}1(I) chain. The mutation was confirmed by restriction enzyme analysis of amplified genomic DNA. The mutation was not present in fibroblasts from either phenotypically normal parent. Combining this mutation with other reported mutations, glycine to cysteine substitutions at positions 205, 211, 223, and 226 produce a moderately severe phenotype whereas flanking mutations at positions 175 and 382 produce a mild phenotype. This data supports a regional rather than a gradient model of the relationship between the nature and location of type I collagen mutations and OI phenotype.« less

Evolutionary Analysis Predicts Sensitive Positions of MMP20 and Validates Newly- and Previously-Identified MMP20 Mutations Causing Amelogenesis Imperfecta

PubMed Central

Gasse, Barbara; Prasad, Megana; Delgado, Sidney; Huckert, Mathilde; Kawczynski, Marzena; Garret-Bernardin, Annelyse; Lopez-Cazaux, Serena; Bailleul-Forestier, Isabelle; Manière, Marie-Cécile; Stoetzel, Corinne; Bloch-Zupan, Agnès; Sire, Jean-Yves

2017-01-01

Amelogenesis imperfecta (AI) designates a group of genetic diseases characterized by a large range of enamel disorders causing important social and health problems. These defects can result from mutations in enamel matrix proteins or protease encoding genes. A range of mutations in the enamel cleavage enzyme matrix metalloproteinase-20 gene (MMP20) produce enamel defects of varying severity. To address how various alterations produce a range of AI phenotypes, we performed a targeted analysis to find MMP20 mutations in French patients diagnosed with non-syndromic AI. Genomic DNA was isolated from saliva and MMP20 exons and exon-intron boundaries sequenced. We identified several homozygous or heterozygous mutations, putatively involved in the AI phenotypes. To validate missense mutations and predict sensitive positions in the MMP20 sequence, we evolutionarily compared 75 sequences extracted from the public databases using the Datamonkey webserver. These sequences were representative of mammalian lineages, covering more than 150 million years of evolution. This analysis allowed us to find 324 sensitive positions (out of the 483 MMP20 residues), pinpoint functionally important domains, and build an evolutionary chart of important conserved MMP20 regions. This is an efficient tool to identify new- and previously-identified mutations. We thus identified six functional MMP20 mutations in unrelated families, finding two novel mutated sites. The genotypes and phenotypes of these six mutations are described and compared. To date, 13 MMP20 mutations causing AI have been reported, making these genotypes and associated hypomature enamel phenotypes the most frequent in AI. PMID:28659819

Evolutionary Analysis Predicts Sensitive Positions of MMP20 and Validates Newly- and Previously-Identified MMP20 Mutations Causing Amelogenesis Imperfecta.

PubMed

Gasse, Barbara; Prasad, Megana; Delgado, Sidney; Huckert, Mathilde; Kawczynski, Marzena; Garret-Bernardin, Annelyse; Lopez-Cazaux, Serena; Bailleul-Forestier, Isabelle; Manière, Marie-Cécile; Stoetzel, Corinne; Bloch-Zupan, Agnès; Sire, Jean-Yves

2017-01-01

Amelogenesis imperfecta (AI) designates a group of genetic diseases characterized by a large range of enamel disorders causing important social and health problems. These defects can result from mutations in enamel matrix proteins or protease encoding genes. A range of mutations in the enamel cleavage enzyme matrix metalloproteinase-20 gene ( MMP20 ) produce enamel defects of varying severity. To address how various alterations produce a range of AI phenotypes, we performed a targeted analysis to find MMP20 mutations in French patients diagnosed with non-syndromic AI. Genomic DNA was isolated from saliva and MMP20 exons and exon-intron boundaries sequenced. We identified several homozygous or heterozygous mutations, putatively involved in the AI phenotypes. To validate missense mutations and predict sensitive positions in the MMP20 sequence, we evolutionarily compared 75 sequences extracted from the public databases using the Datamonkey webserver. These sequences were representative of mammalian lineages, covering more than 150 million years of evolution. This analysis allowed us to find 324 sensitive positions (out of the 483 MMP20 residues), pinpoint functionally important domains, and build an evolutionary chart of important conserved MMP20 regions. This is an efficient tool to identify new- and previously-identified mutations. We thus identified six functional MMP20 mutations in unrelated families, finding two novel mutated sites. The genotypes and phenotypes of these six mutations are described and compared. To date, 13 MMP20 mutations causing AI have been reported, making these genotypes and associated hypomature enamel phenotypes the most frequent in AI.

Mutational analysis of GALT gene in Greek patients with galactosaemia: identification of two novel mutations and clinical evaluation.

PubMed

Schulpis, Kleopatra H; Thodi, Georgia; Iakovou, Konstantinos; Chatzidaki, Maria; Dotsikas, Yannis; Molou, Elina; Triantafylli, Olga; Loukas, Yannis L

2017-10-01

Classical galactosaemia is an inborn error of metabolism due to the deficiency of the enzyme galactose-1-phosphate uridylyltransferase (GALT). The aim of the study was to identify the underlying mutations in Greek patients with GALT deficiency and evaluate their psychomotor and speech development. Patients with GALT deficiency (n = 17) were picked up through neonatal screening. Mutational analysis was conducted via Sanger sequencing, while in silico analysis was used in the cases of novel missense mutations. Psychomotor speech development tests were utilized for the clinical evaluation of the patients. Eleven different mutations in the GALT gene were detected in the patient cohort, including two novel ones. The most frequent mutation was p.Q188R (c.563 A > G). As for the novel mutations, p.M298I (c.894 G > A) was identified in four out of 32 independent alleles, while p.P115S (c.343 C > T) was identified once. Psychomotor evaluation revealed that most of the patients were found in the borderline area (Peabody test), while only two had speech delay problems. The WISK test revealed three patients at borderline limits and two were at lower than normal limits. The mutational spectrum of the GALT gene in Greek patients is presented for the first time. The mutation p.Q188R is the most frequent among Greek patients. Two novel mutations were identified and their potential pathogenicity was estimated. Regarding the phenotypic characteristics, psychomotor disturbances and speech delay were mainly observed among GALT-deficient patients.

Correlational study on mitochondrial DNA mutations as potential risk factors in breast cancer.

PubMed

Li, Linhai; Chen, Lidan; Li, Jun; Zhang, Weiyun; Liao, Yang; Chen, Jianyun; Sun, Zhaohui

2016-05-24

The presented study performed an mtDNA genome-wide association analysis to screen the peripheral blood of breast cancer patients for high-risk germline mutations. Unlike previous studies, which have used breast tissue in analyzing somatic mutations, we looked for germline mutations in our study, since they are better predictors of breast cancer in high-risk groups, facilitate early, non-invasive diagnoses of breast cancer and may provide a broader spectrum of therapeutic options. The data comprised 22 samples of healthy group and 83 samples from breast cancer patients. The sequencing data showed 170 mtDNA mutations in the healthy group and 393 mtDNA mutations in the disease group. Of these, 283 mtDNA mutations (88 in the healthy group and 232 in the disease group) had never been reported in the literature. Moreover, correlation analysis indicated there was a significant difference in 32 mtDNA mutations. According to our relative risk analysis of these 32 mtDNA mutations, 27 of the total had odds ratio values (ORs) of less than 1, meaning that these mutations have a potentially protective role to play in breast cancer. The remaining 5 mtDNA mutations, RNR2-2463 indelA, COX1-6296 C>A, COX1-6298 indelT, ATP6-8860 A>G, and ND5-13327 indelA, whose ORs were 8.050, 4.464, 4.464, 5.254 and 4.853, respectively, were regarded as risk factors of increased breast cancer. The five mutations identified here may serve as novel indicators of breast cancer and may have future therapeutic applications. In addition, the use of peripheral blood samples was procedurally simple and could be applied as a non-invasive diagnostic technique.

Multiple Origins of a Mitochondrial Mutation Conferring Deafness

PubMed Central

Hutchin, T. P.; Cortopassi, G. A.

1997-01-01

A point mutation (1555G) in the smaller ribosomal subunit of the mitochondrial DNA (mtDNA) has been associated with maternally inherited traits of hypersensitivity to streptomycin and sensorineural deafness in a number of families from China, Japan, Israel, and Africa. To determine whether this distribution was the result of a single or multiple mutational events, we carried out genetic distance analysis and phylogenetic analysis of 10 independent mtDNA D-loop sequences from Africa and Asia. The mtDNA sequence diversity was high (2.21%). Phylogenetic analysis assigned 1555G-bearing haplotypes at very divergent points in the human mtDNA evolutionary tree, and the 1555G mutations occur in many cases on race-specific mtDNA haplotypes, both facts are inconsistent with a recent introgression of the mutation into these races. The simplest interpretation of the available data is that there have been multiple origins of the 1555G mutation. The genetic distance among mtDNAs bearing the pathogenic 1555G mutation is much larger than among mtDNAs bearing either evolutionarily neutral or weakly deleterious nucleotide substitutions (such as the 4336G mutation). These results are consistent with the view that pathogenic mtDNA haplotypes such as 1555G arise on disparate mtDNA lineages which because of negative natural selection leave relatively few related descendants. The co-existence of the same mutation with deafness in individuals with very different nuclear and mitochondrial genetic backgrounds confirms the pathogenicity of the 1555G mutation. PMID:9055086

The Application of Next-Generation Sequencing for Mutation Detection in Autosomal-Dominant Hereditary Hearing Impairment.

PubMed

Gürtler, Nicolas; Röthlisberger, Benno; Ludin, Katja; Schlegel, Christoph; Lalwani, Anil K

2017-07-01

Identification of the causative mutation using next-generation sequencing in autosomal-dominant hereditary hearing impairment, as mutation analysis in hereditary hearing impairment by classic genetic methods, is hindered by the high heterogeneity of the disease. Two Swiss families with autosomal-dominant hereditary hearing impairment. Amplified DNA libraries for next-generation sequencing were constructed from extracted genomic DNA, derived from peripheral blood, and enriched by a custom-made sequence capture library. Validated, pooled libraries were sequenced on an Illumina MiSeq instrument, 300 cycles and paired-end sequencing. Technical data analysis was performed with SeqMonk, variant analysis with GeneTalk or VariantStudio. The detection of mutations in genes related to hearing loss by next-generation sequencing was subsequently confirmed using specific polymerase-chain-reaction and Sanger sequencing. Mutation detection in hearing-loss-related genes. The first family harbored the mutation c.5383+5delGTGA in the TECTA-gene. In the second family, a novel mutation c.2614-2625delCATGGCGCCGTG in the WFS1-gene and a second mutation TCOF1-c.1028G>A were identified. Next-generation sequencing successfully identified the causative mutation in families with autosomal-dominant hereditary hearing impairment. The results helped to clarify the pathogenic role of a known mutation and led to the detection of a novel one. NGS represents a feasible approach with great potential future in the diagnostics of hereditary hearing impairment, even in smaller labs.

[Analysis of genotype and phenotype correlation of MYH7-V878A mutation among ethnic Han Chinese pedigrees affected with hypertrophic cardiomyopathy].

PubMed

Wang, Bo; Guo, Ruiqi; Zuo, Lei; Shao, Hong; Liu, Ying; Wang, Yu; Ju, Yan; Sun, Chao; Wang, Lifeng; Zhang, Yanmin; Liu, Liwen

2017-08-10

To analyze the phenotype-genotype correlation of MYH7-V878A mutation. Exonic amplification and high-throughput sequencing of 96-cardiovascular disease-related genes were carried out on probands from 210 pedigrees affected with hypertrophic cardiomyopathy (HCM). For the probands, their family members, and 300 healthy volunteers, the identified MYH7-V878A mutation was verified by Sanger sequencing. Information of the HCM patients and their family members, including clinical data, physical examination, echocardiography (UCG), electrocardiography (ECG), and conserved sequence of the mutation among various species were analyzed. A MYH7-V878A mutation was detected in five HCM pedigrees containing 31 family members. Fourteen members have carried the mutation, among whom 11 were diagnosed with HCM, while 3 did not meet the diagnostic criteria. Some of the fourteen members also carried other mutations. Family members not carrying the mutation had normal UCG and ECG. No MYH7-V878A mutation was found among the 300 healthy volunteers. Analysis of sequence conservation showed that the amino acid is located in highly conserved regions among various species. MYH7-V878A is a hot spot among ethnic Han Chinese with a high penetrance. Functional analysis of the conserved sequences suggested that the mutation may cause significant alteration of the function. MYH7-V878A has a significant value for the early diagnosis of HCM.

A Novel Missense Mutation of Doublecortin: Mutation Analysis of Korean Patients with Subcortical Band Heterotopia

PubMed Central

Kim, Myeong-Kyu; Park, Man-Seok; Kim, Byeong-Chae; Cho, Ki-Hyun; Kim, Young-Seon; Kim, Jin-Hee; Heo, Tag; Kim, Eun-Young

2005-01-01

The neuronal migration disorders, X-linked lissencephaly syndrome (XLIS) and subcortical band heterotopia (SBH), also called "double cortex", have been linked to missense, nonsense, aberrant splicing, deletion, and insertion mutations in doublecortin (DCX) in families and sporadic cases. Most DCX mutations identified to date are located in two evolutionarily conserved domains. We performed mutation analysis of DCX in two Korean patients with SBH. The SBH patients had mild to moderate developmental delays, drug-resistant generalized seizures, and diffuse thick SBH upon brain MRI. Sequence analysis of the DCX coding region in Patient 1 revealed a c.386 C>T change in exon 3. The sequence variation results in a serine to leucine amino acid change at position 129 (S129L), which has not been found in other family members of Patient 1 or in a large panel of 120 control X-chromosomes. We report here a novel c.386 C>T mutation of DCX that is responsible for SBH. PMID:16100463

Novel HSF4 mutation causes congenital total white cataract in a Chinese family.

PubMed

Ke, Tie; Wang, Qing K; Ji, Binchu; Wang, Xu; Liu, Ping; Zhang, Xianqin; Tang, Zhaohui; Ren, Xiang; Liu, Mugen

2006-08-01

To identify the disease-causing gene (mutation) in a Chinese family affected with autosomal dominant congenital total white cataract. Observational case series. Genotyping and linkage analyses were used to identify the linkage of the disease-causing gene in the Chinese family to the HSF4 gene encoding a member of the family of heat shock transcription factors (HSFs). Direct DNA sequence analysis was used to identify the disease-causing mutation. Polymerase chain reaction/restriction fragment length polymorphism analysis was used to demonstrate cosegregation of the HSF4 mutation with the cataract and the absence of the mutation in the normal controls. The cataract gene in the Chinese family was linked to marker D16S3043, and further haplotype analysis defined the causative gene between D16S515 and D16S415 within which HSF4 is located. A novel mutation c.221G>A was identified in HSF4, which results in substitution of a highly conserved arginine residue by histidine at codon 74 (p.R74H). The R74H mutation cosegregated with the affected individuals in the family and did not exist in unaffected family members and 150 unrelated normal controls. These results identified a novel missense mutation R74H in the transcription factor gene HSF4 in a Chinese cataract family and expand the spectrum of HSF4 mutations causing cataract.

Exome Sequencing Identifies Potentially Druggable Mutations in Nasopharyngeal Carcinoma.

PubMed

Chow, Yock Ping; Tan, Lu Ping; Chai, San Jiun; Abdul Aziz, Norazlin; Choo, Siew Woh; Lim, Paul Vey Hong; Pathmanathan, Rajadurai; Mohd Kornain, Noor Kaslina; Lum, Chee Lun; Pua, Kin Choo; Yap, Yoke Yeow; Tan, Tee Yong; Teo, Soo Hwang; Khoo, Alan Soo-Beng; Patel, Vyomesh

2017-03-03

In this study, we first performed whole exome sequencing of DNA from 10 untreated and clinically annotated fresh frozen nasopharyngeal carcinoma (NPC) biopsies and matched bloods to identify somatically mutated genes that may be amenable to targeted therapeutic strategies. We identified a total of 323 mutations which were either non-synonymous (n = 238) or synonymous (n = 85). Furthermore, our analysis revealed genes in key cancer pathways (DNA repair, cell cycle regulation, apoptosis, immune response, lipid signaling) were mutated, of which those in the lipid-signaling pathway were the most enriched. We next extended our analysis on a prioritized sub-set of 37 mutated genes plus top 5 mutated cancer genes listed in COSMIC using a custom designed HaloPlex target enrichment panel with an additional 88 NPC samples. Our analysis identified 160 additional non-synonymous mutations in 37/42 genes in 66/88 samples. Of these, 99/160 mutations within potentially druggable pathways were further selected for validation. Sanger sequencing revealed that 77/99 variants were true positives, giving an accuracy of 78%. Taken together, our study indicated that ~72% (n = 71/98) of NPC samples harbored mutations in one of the four cancer pathways (EGFR-PI3K-Akt-mTOR, NOTCH, NF-κB, DNA repair) which may be potentially useful as predictive biomarkers of response to matched targeted therapies.

Novel EDA mutation in X-linked hypohidrotic ectodermal dysplasia and genotype-phenotype correlation.

PubMed

Zeng, B; Lu, H; Xiao, X; Zhou, L; Lu, J; Zhu, L; Yu, D; Zhao, W

2015-11-01

X-linked hypohidrotic ectodermal dysplasia (XLHED) is characterized by abnormalities of hair, teeth, and sweat glands, while non-syndromic hypodontia (NSH) affects only teeth. Mutations in Ectodysplasin A (EDA) underlie both XLHED and NSH. This study investigated the genetic causes of six hypohidrotic ectodermal dysplasia (HED) patients and genotype-phenotype correlation. The EDA gene of six patients with HED was sequenced. Bioinformatics analysis and structural modeling for the mutations were performed. The records of 134 patients with XLHED and EDA-related NSH regarding numbers of missing permanent teeth from this study and 20 articles were reviewed. Nonparametric tests were used to analyze genotype-phenotype correlations. In four of the six patients, we identified a novel mutation c.852T>G (p.Phe284Leu) and three reported mutations: c.467G>A (p.Arg156His), c.776C>A (p.Ala259Glu), and c.871G>A (p.Gly291Arg). They were predicted to be pathogenic by bioinformatics analysis and structural modeling. Genotype-phenotype correlation analysis revealed that truncating mutations were associated with more missing teeth. Missense mutations and the mutations affecting the TNF homology domain were correlated with fewer missing teeth. This study extended the mutation spectrum of XLHED and revealed the relationship between genotype and the number of missing permanent teeth. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Genetic Epidemiology of Glucose-6-Dehydrogenase Deficiency in the Arab World.

PubMed

Doss, C George Priya; Alasmar, Dima R; Bux, Reem I; Sneha, P; Bakhsh, Fadheela Dad; Al-Azwani, Iman; Bekay, Rajaa El; Zayed, Hatem

2016-11-17

A systematic search was implemented using four literature databases (PubMed, Embase, Science Direct and Web of Science) to capture all the causative mutations of Glucose-6-phosphate dehydrogenase (G6PD) deficiency (G6PDD) in the 22 Arab countries. Our search yielded 43 studies that captured 33 mutations (23 missense, one silent, two deletions, and seven intronic mutations), in 3,430 Arab patients with G6PDD. The 23 missense mutations were then subjected to phenotypic classification using in silico prediction tools, which were compared to the WHO pathogenicity scale as a reference. These in silico tools were tested for their predicting efficiency using rigorous statistical analyses. Of the 23 missense mutations, p.S188F, p.I48T, p.N126D, and p.V68M, were identified as the most common mutations among Arab populations, but were not unique to the Arab world, interestingly, our search strategy found four other mutations (p.N135T, p.S179N, p.R246L, and p.Q307P) that are unique to Arabs. These mutations were exposed to structural analysis and molecular dynamics simulation analysis (MDSA), which predicting these mutant forms as potentially affect the enzyme function. The combination of the MDSA, structural analysis, and in silico predictions and statistical tools we used will provide a platform for future prediction accuracy for the pathogenicity of genetic mutations.

Exome Sequencing Identifies Potentially Druggable Mutations in Nasopharyngeal Carcinoma

PubMed Central

Chow, Yock Ping; Tan, Lu Ping; Chai, San Jiun; Abdul Aziz, Norazlin; Choo, Siew Woh; Lim, Paul Vey Hong; Pathmanathan, Rajadurai; Mohd Kornain, Noor Kaslina; Lum, Chee Lun; Pua, Kin Choo; Yap, Yoke Yeow; Tan, Tee Yong; Teo, Soo Hwang; Khoo, Alan Soo-Beng; Patel, Vyomesh

2017-01-01

In this study, we first performed whole exome sequencing of DNA from 10 untreated and clinically annotated fresh frozen nasopharyngeal carcinoma (NPC) biopsies and matched bloods to identify somatically mutated genes that may be amenable to targeted therapeutic strategies. We identified a total of 323 mutations which were either non-synonymous (n = 238) or synonymous (n = 85). Furthermore, our analysis revealed genes in key cancer pathways (DNA repair, cell cycle regulation, apoptosis, immune response, lipid signaling) were mutated, of which those in the lipid-signaling pathway were the most enriched. We next extended our analysis on a prioritized sub-set of 37 mutated genes plus top 5 mutated cancer genes listed in COSMIC using a custom designed HaloPlex target enrichment panel with an additional 88 NPC samples. Our analysis identified 160 additional non-synonymous mutations in 37/42 genes in 66/88 samples. Of these, 99/160 mutations within potentially druggable pathways were further selected for validation. Sanger sequencing revealed that 77/99 variants were true positives, giving an accuracy of 78%. Taken together, our study indicated that ~72% (n = 71/98) of NPC samples harbored mutations in one of the four cancer pathways (EGFR-PI3K-Akt-mTOR, NOTCH, NF-κB, DNA repair) which may be potentially useful as predictive biomarkers of response to matched targeted therapies. PMID:28256603

Genetic analysis in Bartter syndrome from India.

PubMed

Sharma, Pradeep Kumar; Saikia, Bhaskar; Sharma, Rachna; Ankur, Kumar; Khilnani, Praveen; Aggarwal, Vinay Kumar; Cheong, Hae

2014-10-01

Bartter syndrome is a group of inherited, salt-losing tubulopathies presenting as hypokalemic metabolic alkalosis with normotensive hyperreninemia and hyperaldosteronism. Around 150 cases have been reported in literature till now. Mutations leading to salt losing tubulopathies are not routinely tested in Indian population. The authors have done the genetic analysis for the first time in the Bartter syndrome on two cases from India. First case was antenatal Bartter syndrome presenting with massive polyuria and hyperkalemia. Mutational analysis revealed compound heterozygous mutations in KCNJ1(ROMK) gene [p(Leu220Phe), p(Thr191Pro)]. Second case had a phenotypic presentation of classical Bartter syndrome however, genetic analysis revealed only heterozygous novel mutation in SLC12A gene p(Ala232Thr). Bartter syndrome is a clinical diagnosis and genetic analysis is recommended for prognostication and genetic counseling.

Structure-Based Analysis Reveals Cancer Missense Mutations Target Protein Interaction Interfaces.

PubMed

Engin, H Billur; Kreisberg, Jason F; Carter, Hannah

2016-01-01

Recently it has been shown that cancer mutations selectively target protein-protein interactions. We hypothesized that mutations affecting distinct protein interactions involving established cancer genes could contribute to tumor heterogeneity, and that novel mechanistic insights might be gained into tumorigenesis by investigating protein interactions under positive selection in cancer. To identify protein interactions under positive selection in cancer, we mapped over 1.2 million nonsynonymous somatic cancer mutations onto 4,896 experimentally determined protein structures and analyzed their spatial distribution. In total, 20% of mutations on the surface of known cancer genes perturbed protein-protein interactions (PPIs), and this enrichment for PPI interfaces was observed for both tumor suppressors (Odds Ratio 1.28, P-value < 10(-4)) and oncogenes (Odds Ratio 1.17, P-value < 10(-3)). To study this further, we constructed a bipartite network representing structurally resolved PPIs from all available human complexes in the Protein Data Bank (2,864 proteins, 3,072 PPIs). Analysis of frequently mutated cancer genes within this network revealed that tumor-suppressors, but not oncogenes, are significantly enriched with functional mutations in homo-oligomerization regions (Odds Ratio 3.68, P-Value < 10(-8)). We present two important examples, TP53 and beta-2-microglobulin, for which the patterns of somatic mutations at interfaces provide insights into specifically perturbed biological circuits. In patients with TP53 mutations, patient survival correlated with the specific interactions that were perturbed. Moreover, we investigated mutations at the interface of protein-nucleotide interactions and observed an unexpected number of missense mutations but not silent mutations occurring within DNA and RNA binding sites. Finally, we provide a resource of 3,072 PPI interfaces ranked according to their mutation rates. Analysis of this list highlights 282 novel candidate cancer genes that encode proteins participating in interactions that are perturbed recurrently across tumors. In summary, mutation of specific protein interactions is an important contributor to tumor heterogeneity and may have important implications for clinical outcomes.

The 253-kb inversion and deep intronic mutations in UNC13D are present in North American patients with familial hemophagocytic lymphohistiocytosis 3.

PubMed

Qian, Yaping; Johnson, Judith A; Connor, Jessica A; Valencia, C Alexander; Barasa, Nathaniel; Schubert, Jeffery; Husami, Ammar; Kissell, Diane; Zhang, Ge; Weirauch, Matthew T; Filipovich, Alexandra H; Zhang, Kejian

2014-06-01

The mutations in UNC13D are responsible for familial hemophagocytic lymphohistiocytosis (FHL) type 3. A 253-kb inversion and two deep intronic mutations, c.118-308C > T and c.118-307G > A, in UNC13D were recently reported in European and Asian FHL3 patients. We sought to determine the prevalence of these three non-coding mutations in North American FHL patients and evaluate the significance of examining these new mutations in genetic testing. We performed DNA sequencing of UNC13D and targeted analysis of these three mutations in 1,709 North American patients with a suspected clinical diagnosis of hemophagocytic lymphohistiocytosis (HLH). The 253-kb inversion, intronic mutations c.118-308C > T and c.118-307G > A were found in 11, 15, and 4 patients, respectively, in which the genetic basis (bi-allelic mutations) explained 25 additional patients. Taken together with previously diagnosed FHL3 patients in our HLH patient registry, these three non-coding mutations were found in 31.6% (25/79) of the FHL3 patients. The 253-kb inversion, c.118-308C > T and c.118-307G > A accounted for 7.0%, 8.9%, and 1.3% of mutant alleles, respectively. Significantly, eight novel mutations in UNC13D are being reported in this study. To further evaluate the expression level of the newly reported intronic mutation c.118-307G > A, reverse transcription PCR and Western blot analysis revealed a significant reduction of both RNA and protein levels suggesting that the c.118-307G > A mutation affects transcription. These specified non-coding mutations were found in a significant number of North American patients and inclusion of them in mutation analysis will improve the molecular diagnosis of FHL3. © 2014 Wiley Periodicals, Inc.

Mutation profiles in early-stage lung squamous cell carcinoma with clinical follow-up and correlation with markers of immune function.

PubMed

Choi, M; Kadara, H; Zhang, J; Parra, E R; Rodriguez-Canales, J; Gaffney, S G; Zhao, Z; Behrens, C; Fujimoto, J; Chow, C; Kim, K; Kalhor, N; Moran, C; Rimm, D; Swisher, S; Gibbons, D L; Heymach, J; Kaftan, E; Townsend, J P; Lynch, T J; Schlessinger, J; Lee, J; Lifton, R P; Herbst, R S; Wistuba, I I

2017-01-01

Lung squamous cell carcinoma (LUSC) accounts for 20–30% of non-small cell lung cancers (NSCLCs). There are limited treatment strategies for LUSC in part due to our inadequate understanding of the molecular underpinnings of the disease. We performed whole-exome sequencing (WES) and comprehensive immune profiling of a unique set of clinically annotated early-stage LUSCs to increase our understanding of the pathobiology of this malignancy. Matched pairs of surgically resected stage I-III LUSCs and normal lung tissues (n = 108) were analyzed by WES. Immunohistochemistry and image analysis-based profiling of 10 immune markers were done on a subset of LUSCs (n = 91). Associations among mutations, immune markers and clinicopathological variables were statistically examined using analysis of variance and Fisher’s exact test. Cox proportional hazards regression models were used for statistical analysis of clinical outcome. This early-stage LUSC cohort displayed an average of 209 exonic mutations per tumor. Fourteen genes exhibited significant enrichment for somatic mutation: TP53, MLL2, PIK3CA, NFE2L2, CDH8, KEAP1, PTEN, ADCY8, PTPRT, CALCR, GRM8, FBXW7, RB1 and CDKN2A. Among mutated genes associated with poor recurrence-free survival, MLL2 mutations predicted poor prognosis in both TP53 mutant and wild-type LUSCs. We also found that in treated patients, FBXW7 and KEAP1 mutations were associated with poor response to adjuvant therapy, particularly in TP53-mutant tumors. Analysis of mutations with immune markers revealed that ADCY8 and PIK3CA mutations were associated with markedly decreased tumoral PD-L1 expression, LUSCs with PIK3CA mutations exhibited elevated CD45ro levels and CDKN2A-mutant tumors displayed an up-regulated immune response. Our findings pinpoint mutated genes that may impact clinical outcome as well as personalized strategies for targeted immunotherapies in early-stage LUSC.

Cell lineage analysis in human brain using endogenous retroelements

PubMed Central

Evrony, Gilad D.; Lee, Eunjung; Mehta, Bhaven K.; Benjamini, Yuval; Johnson, Robert M.; Cai, Xuyu; Yang, Lixing; Haseley, Psalm; Lehmann, Hillel S.; Park, Peter J.; Walsh, Christopher A.

2015-01-01

Summary Somatic mutations occur during brain development and are increasingly implicated as a cause of neurogenetic disease. However, the patterns in which somatic mutations distribute in the human brain are unknown. We used high-coverage whole-genome sequencing of single neurons from a normal individual to identify spontaneous somatic mutations as clonal marks to track cell lineages in human brain. Somatic mutation analyses in >30 locations throughout the nervous system identified multiple lineages and sub-lineages of cells marked by different LINE-1 (L1) retrotransposition events and subsequent mutation of poly-A microsatellites within L1. One clone contained thousands of cells limited to the left middle frontal gyrus, whereas a second distinct clone contained millions of cells distributed over the entire left hemisphere. These patterns mirror known somatic mutation disorders of brain development, and suggest that focally distributed mutations are also prevalent in normal brains. Single-cell analysis of somatic mutation enables tracing of cell lineage clones in human brain. PMID:25569347

Kit receptor tyrosine kinase dysregulations in feline splenic mast cell tumours.

PubMed

Sabattini, S; Barzon, G; Giantin, M; Lopparelli, R M; Dacasto, M; Prata, D; Bettini, G

2017-09-01

This study investigated Kit receptor dysregulations (cytoplasmic immunohistochemical expression and/or c-KIT mutations) in cats affected with splenic mast cell tumours. Twenty-two cats were included. Median survival time was 780 days (range: 1-1219). An exclusive splenic involvement was significantly (P = 0.042) associated with longer survival (807 versus 120 days). Eighteen tumours (85.7%) showed Kit cytoplasmic expression (Kit pattern 2, 3). Mutation analysis was successful in 20 cases. Fourteen missense mutations were detected in 13 out of 20 tumours (65%). Eleven (78.6%) were located in exon 8, and three (21.6%) in exon 9. No mutations were detected in exons 11 and 17. Seven mutations corresponded to the same internal tandem duplication in exon 8 (c.1245_1256dup). Although the association between Kit cytoplasmic expression and mutations was significant, immunohistochemistry cannot be considered a surrogate marker for mutation analysis. No correlation was observed between c-Kit mutations and tumour differentiation, mitotic activity or survival. © 2016 John Wiley & Sons Ltd.

Laboratory practice guidelines for detecting and reporting JAK2 and MPL mutations in myeloproliferative neoplasms: a report of the Association for Molecular Pathology.

PubMed

Gong, Jerald Z; Cook, James R; Greiner, Timothy C; Hedvat, Cyrus; Hill, Charles E; Lim, Megan S; Longtine, Janina A; Sabath, Daniel; Wang, Y Lynn

2013-11-01

Recurrent mutations in JAK2 and MPL genes are genetic hallmarks of BCR-ABL1-negative myeloproliferative neoplasms. Detection of JAK2 and MPL mutations has been incorporated into routine diagnostic algorithms for these diseases. This Special Article summarizes results from a nationwide laboratory survey of JAK2 and MPL mutation analysis. Based on the current practice pattern and the literature, this Special Article provides recommendations and guidelines for laboratory practice for detection of mutations in the JAK2 and MPL genes, including clinical manifestations for prompting the mutation analysis, current and recommended methodologies for testing the mutations, and standardization for reporting the test results. This Special Article also points to future directions for genomic testing in BCR-ABL1-negative myeloproliferative neoplasms. Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

DMD mutation spectrum analysis in 613 Chinese patients with dystrophinopathy.

PubMed

Guo, Ruolan; Zhu, Guosheng; Zhu, Huimin; Ma, Ruiyu; Peng, Ying; Liang, Desheng; Wu, Lingqian

2015-08-01

Dystrophinopathy is a group of inherited diseases caused by mutations in the DMD gene. Within the dystrophinopathy spectrum, Duchenne and Becker muscular dystrophies are common X-linked recessive disorders that mainly feature striated muscle necrosis. We combined multiplex ligation-dependent probe amplification with Sanger sequencing to detect large deletions/duplications and point mutations in the DMD gene in 613 Chinese patients. A total of 571 (93.1%) patients were diagnosed, including 428 (69.8%) with large deletions/duplications and 143 (23.3%) with point mutations. Deletion/duplication breakpoints gathered mostly in introns 44-55. Reading frame rules could explain 88.6% of deletion mutations. We identified seventy novel point mutations that had not been previously reported. Spectrum expansion and genotype-phenotype analysis of DMD mutations on such a large sample size in Han Chinese population would provide new insights into the pathogenic mechanism underlying dystrophinopathies.

Rapid identification of mutations in the IDS gene of Hunter patients: Analysis of mRNA by the protein truncation test

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hogervorst, F.B.L.; Tuijn, A.C. van der; Ommen, G.J.B. van

Hunter syndrome is an X-linked recessive disorder constituting phenotypes ranging from mild to severe. The gene affected in Hunter syndrome is iduronate-2-sulfatase (IDS). The identification of mutations leading to a defective enzyme could be of benefit for the diagnosis and prognosis of patients. At this moment a variety of mutations have been found, including large deletions and base substitutions. We have previously described a method, designated the protein truncation test (PTT), for the detection of mutations leading to premature translation termination. The method combines reverse transcription and PCR (RT-PCR) with in vitro transcript/translation of the products generated. To facilitate amore » PTT analysis, the forward primer is modified by addition of a T7 promoter sequence and an in-frame protein translation initiation sequence. In our department the method has been successfully applied for DMD and FAP. Here we report on the PTT analysis of 8 Hunter patients, all of them without major gene alterations as determined by Southern analysis. Total RNA was isolated from cultured skin fibroblasts or peripheral blood lymphocytes. PTT analysis revealed 4 novel mutations in the IDS gene: two missense mutations and two frameshift mutations (splice donor site alteration in intron 6 and a 13 bp deletion in exon 9). Furthermore, PTT proved to be a simple method to identify carriers. Currently, we use the generated RT-PCR products of the remaining patients for automated sequence analysis. PTT may be of great value in screening disorders in which affected genes give rise to truncated protein products.« less

The Effect of Chain Length on Mid-Infrared and Near-Infrared Spectra of Aliphatic 1-Alcohols.

PubMed

Kwaśniewicz, Michał; Czarnecki, Mirosław A

2018-02-01

Effect of the chain length on mid-infrared (MIR) and near-infrared (NIR) spectra of aliphatic 1-alcohols from methanol to 1-decanol was examined in detail. Of particular interest were the spectra-structure correlations in the NIR region and the correlation between MIR and NIR spectra of 1-alcohols. An application of two-dimensional correlation analysis (2D-COS) and chemometric methods provided comprehensive information on spectral changes in the data set. Principal component analysis (PCA) and cluster analysis evidenced that the spectra of methanol, ethanol, and 1-propanol are noticeably different from the spectra of higher 1-alcohols. The similarity between the spectra increases with an increase in the chain length. Hence, the most similar are the spectra of 1-nonanol and 1-decanol. Two-dimensional hetero-correlation analysis is very helpful for identification of the origin of bands and may guide selection of the best spectral ranges for the chemometric analysis. As shown, normalization of the spectra pronounces the intensity changes in various spectral regions and provides information not accessible from the raw data. The spectra of alcohols cannot be represented as a sum of the CH 3 , CH 2 , and OH group spectra since the OH group is involved in the hydrogen bonding. As a result, the spectral changes of this group are nonlinear and its spectral profile cannot be properly resolved. Finally, this work provides a lot of evidence that the degree of self-association of 1-alcohols decreases with the increase in chain length because of the growing meaning of the hydrophobic interactions. For butyl alcohol and higher 1-alcohols the hydrophobic interactions are more important than the OH OH interactions. Therefore, methanol, ethanol, and 1-propanol have unlimited miscibility with water, whereas 1-butanol and higher 1-alcohols have limited miscibility with water.

Pitfalls in genetic analysis of pheochromocytomas/paragangliomas-case report.

PubMed

Canu, Letizia; Rapizzi, Elena; Zampetti, Benedetta; Fucci, Rossella; Nesi, Gabriella; Richter, Susan; Qin, Nan; Giachè, Valentino; Bergamini, Carlo; Parenti, Gabriele; Valeri, Andrea; Ercolino, Tonino; Eisenhofer, Graeme; Mannelli, Massimo

2014-07-01

About 35% of patients with pheochromocytoma/paraganglioma carry a germline mutation in one of the 10 main susceptibility genes. The recent introduction of next-generation sequencing will allow the analysis of all these genes in one run. When positive, the analysis is generally unequivocal due to the association between a germline mutation and a concordant clinical presentation or positive family history. When genetic analysis reveals a novel mutation with no clinical correlates, particularly in the presence of a missense variant, the question arises whether the mutation is pathogenic or a rare polymorphism. We report the case of a 35-year-old patient operated for a pheochromocytoma who turned out to be a carrier of a novel SDHD (succinate dehydrogenase subunit D) missense mutation. With no positive family history or clinical correlates, we decided to perform additional analyses to test the clinical significance of the mutation. We performed in silico analysis, tissue loss of heterozygosity analysis, immunohistochemistry, Western blot analysis, SDH enzymatic assay, and measurement of the succinate/fumarate concentration ratio in the tumor tissue by tandem mass spectrometry. Although the in silico analysis gave contradictory results according to the different methods, all the other tests demonstrated that the SDH complex was conserved and normally active. We therefore came to the conclusion that the variant was a nonpathogenic polymorphism. Advancements in technology facilitate genetic analysis of patients with pheochromocytoma but also offer new challenges to the clinician who, in some cases, needs clinical correlates and/or functional tests to give significance to the results of the genetic assay.

Non-invasive prenatal diagnosis of multiple endocrine neoplasia type 2A using COLD-PCR combined with HRM genotyping analysis from maternal serum.

PubMed

Macher, Hada C; Martinez-Broca, Maria A; Rubio-Calvo, Amalia; Leon-Garcia, Cristina; Conde-Sanchez, Manuel; Costa, Alzenira; Navarro, Elena; Guerrero, Juan M

2012-01-01

The multiple endocrine neoplasia type 2A (MEN2A) is a monogenic disorder characterized by an autosomal dominant pattern of inheritance which is characterized by high risk of medullary thyroid carcinoma in all mutation carriers. Although this disorder is classified as a rare disease, the patients affected have a low life quality and a very expensive and continuous treatment. At present, MEN2A is diagnosed by gene sequencing after birth, thus trying to start an early treatment and by reduction of morbidity and mortality. We first evaluated the presence of MEN2A mutation (C634Y) in serum of 25 patients, previously diagnosed by sequencing in peripheral blood leucocytes, using HRM genotyping analysis. In a second step, we used a COLD-PCR approach followed by HRM genotyping analysis for non-invasive prenatal diagnosis of a pregnant woman carrying a fetus with a C634Y mutation. HRM analysis revealed differences in melting curve shapes that correlated with patients diagnosed for MEN2A by gene sequencing analysis with 100% accuracy. Moreover, the pregnant woman carrying the fetus with the C634Y mutation revealed a melting curve shape in agreement with the positive controls in the COLD-PCR study. The mutation was confirmed by sequencing of the COLD-PCR amplification product. In conclusion, we have established a HRM analysis in serum samples as a new primary diagnosis method suitable for the detection of C634Y mutations in MEN2A patients. Simultaneously, we have applied the increase of sensitivity of COLD-PCR assay approach combined with HRM analysis for the non-invasive prenatal diagnosis of C634Y fetal mutations using pregnant women serum.

Overrepresentation of missense mutations in mild hemophilia A patients from Belgium: founder effect or independent occurrence?

PubMed

Lannoy, N; Lambert, C; Vikkula, M; Hermans, C

2015-06-01

Roughly 40% of observed mutations responsible for hemophilia A (HA) are novel and present in either a single family or a limited number of unrelated families. During routine diagnostic analysis of 73 unrelated Belgian patients with mild HA, 4 out of 43 different mutations (p.Ser2030Asn, p.Arg2178Cys, p.Arg2178His, and p.Pro2311His) were detected in more than one family, representing 35% of total identified mutations. To discriminate between an independent recurrence or a founder effect, an analysis of intra- and -extragenic single nucleotide polymorphisms (SNPs) and short tandem repeats (STRs) flanking the F8 gene was conducted. SNP haplotype and microsatellite analysis revealed strong evidence that p.Ser2030Asn and p.Pro2311His mutations were probably associated with a founder effect. The two other mutations localized in an F8 cytosine-phosphate-guanine (CpG) site likely resulted from recurrent de novo events. This study suggests that missense mutations producing C-to-T or G-to-A substitutions in CpG dinucleotide can occur de novo with more repetition than other causal substitutions that do not affect the CpG site. Analysis of F8 database implied that CpG sites throughout the F8 gene are not all mutated with the same frequency. Causes are still unknown and remain to be identified. Copyright © 2015 Elsevier Ltd. All rights reserved.

Mutation analysis of the STRA6 gene in isolated and non-isolated anophthalmia/microphthalmia.

PubMed

Chassaing, N; Ragge, N; Kariminejad, A; Buffet, A; Ghaderi-Sohi, S; Martinovic, J; Calvas, P

2013-03-01

PDAC syndrome [Pulmonary hypoplasia/agenesis, Diaphragmatic hernia/eventration, Anophthalmia/microphthalmia (A/M) and Cardiac Defect] is a condition associated with recessive mutations in the STRA6 gene in some of these patients. Recently, cases with isolated anophthalmia have been associated with STRA6 mutations. To determine the minimal findings associated with STRA6 mutations, we performed mutation analysis of the STRA6 gene in 28 cases with anophthalmia. In 7 of the cases the anophthalmia was isolated, in 14 cases it was associated with one of the major features included in PDAC and 7 had other abnormalities. Mutations were identified in two individuals: one with bilateral anophthalmia and some features included in PDAC, who was a compound heterozygote for a missense mutation and a large intragenic deletion, and the second case with all the major features of PDAC and who had a homozygous splicing mutation. This study suggests that STRA6 mutations are more likely to be identified in individuals with A/M and other abnormalities included in the PDAC spectrum, rather than in isolated A/M cases. © 2012 John Wiley & Sons A/S.

Mutational analysis of the TCOF1 gene in 11 Japanese patients with Treacher Collins Syndrome and mechanism of mutagenesis.

PubMed

Horiuchi, Katsumi; Ariga, Tadashi; Fujioka, Hirotaka; Kawashima, Kunihiro; Yamamoto, Yuhei; Igawa, Hiroharu; Sugihara, Tsuneki; Sakiyama, Yukio

2005-05-01

Treacher Collins Syndrome (TCS) (OMIM 154500) is a congenital, craniofacial disorder inherited as an autosomal dominant trait. The responsible gene for TCS, TCOF1, was mapped to 5q32-33.1 and identified in 1996. Since then, TCOF1 mutations in patients with TCS have been reported from Europe, North and South America, however, no TCS cases from an Asian country have been molecularly characterized. Here we report mutational analysis for 11 Japanese patients with TCS for the first time, and have identified TCOF1 mutations in 9 of them. The mutations detected were various, but most likely all the mutations are predicted to result in a truncated gene product, known as treacle. One mutation frequently reported was included in our cases, but no missense mutations were detected. These findings are similar to those for the previous studies for TCS in other races. We have speculated about the molecular mechanisms of the mutations in most cases. Collectively, we have defined some of the characteristic molecular features commonly observed in TCS patients, irrespective of racial difference. 2005 Wiley-Liss, Inc.

A de novo deletion mutation in SOX10 in a Chinese family with Waardenburg syndrome type 4.

PubMed

Wang, Xiong; Zhu, Yaowu; Shen, Na; Peng, Jing; Wang, Chunyu; Liu, Haiyi; Lu, Yanjun

2017-01-27

Waardenburg syndrome type 4 (WS4) or Waardenburg-Shah syndrome is a rare genetic disorder with a prevalence of <1/1,000,000 and characterized by the association of congenital sensorineural hearing loss, pigmentary abnormalities, and intestinal aganglionosis. There are three types of WS4 (WS4A-C) caused by mutations in endothelin receptor type B, endothelin 3, and SRY-box 10 (SOX10), respectively. This study investigated a genetic mutation in a Chinese family with one WS4 patient in order to improve genetic counselling. Genomic DNA was extracted, and mutation analysis of the three WS4 related genes was performed using Sanger sequencing. We detected a de novo heterozygous deletion mutation [c.1333delT (p.Ser445Glnfs*57)] in SOX10 in the patient; however, this mutation was absent in the unaffected parents and 40 ethnicity matched healthy controls. Subsequent phylogenetic analysis and three-dimensional modelling of the SOX10 protein confirmed that the c.1333delT heterozygous mutation was pathogenic, indicating that this mutation might constitute a candidate disease-causing mutation.

A de novo deletion mutation in SOX10 in a Chinese family with Waardenburg syndrome type 4

PubMed Central

Wang, Xiong; Zhu, Yaowu; Shen, Na; Peng, Jing; Wang, Chunyu; Liu, Haiyi; Lu, Yanjun

2017-01-01

Waardenburg syndrome type 4 (WS4) or Waardenburg-Shah syndrome is a rare genetic disorder with a prevalence of <1/1,000,000 and characterized by the association of congenital sensorineural hearing loss, pigmentary abnormalities, and intestinal aganglionosis. There are three types of WS4 (WS4A–C) caused by mutations in endothelin receptor type B, endothelin 3, and SRY-box 10 (SOX10), respectively. This study investigated a genetic mutation in a Chinese family with one WS4 patient in order to improve genetic counselling. Genomic DNA was extracted, and mutation analysis of the three WS4 related genes was performed using Sanger sequencing. We detected a de novo heterozygous deletion mutation [c.1333delT (p.Ser445Glnfs*57)] in SOX10 in the patient; however, this mutation was absent in the unaffected parents and 40 ethnicity matched healthy controls. Subsequent phylogenetic analysis and three-dimensional modelling of the SOX10 protein confirmed that the c.1333delT heterozygous mutation was pathogenic, indicating that this mutation might constitute a candidate disease-causing mutation. PMID:28128317

Intronic splicing mutations in PTCH1 cause Gorlin syndrome.

PubMed

Bholah, Zaynab; Smith, Miriam J; Byers, Helen J; Miles, Emma K; Evans, D Gareth; Newman, William G

2014-09-01

Gorlin syndrome is an autosomal dominant disorder characterized by multiple early-onset basal cell carcinoma, odontogenic keratocysts and skeletal abnormalities. It is caused by heterozygous mutations in the tumour suppressor PTCH1. Routine clinical genetic testing, by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA) to confirm a clinical diagnosis of Gorlin syndrome, identifies a mutation in 60-90 % of cases. We undertook RNA analysis on lymphocytes from ten individuals diagnosed with Gorlin syndrome, but without known PTCH1 mutations by exonic sequencing or MLPA. Two altered PTCH1 transcripts were identified. Genomic DNA sequence analysis identified an intron 7 mutation c.1068-10T>A, which created a strong cryptic splice acceptor site, leading to an intronic insertion of eight bases; this is predicted to create a frameshift p.(His358Alafs*12). Secondly, a deep intronic mutation c.2561-2057A>G caused an inframe insertion of 78 intronic bases in the cDNA transcript, leading to a premature stop codon p.(Gly854fs*3). The mutations are predicted to cause loss of function of PTCH1, consistent with its tumour suppressor function. The findings indicate the importance of RNA analysis to detect intronic mutations in PTCH1 not identified by routine screening techniques.

Noninvasive prenatal diagnosis of hemophilia by microfluidics digital PCR analysis of maternal plasma DNA.

PubMed

Tsui, Nancy B Y; Kadir, Rezan A; Chan, K C Allen; Chi, Claudia; Mellars, Gillian; Tuddenham, Edward G; Leung, Tak Y; Lau, Tze K; Chiu, Rossa W K; Lo, Y M Dennis

2011-03-31

Hemophilia is a bleeding disorder with X-linked inheritance. Current prenatal diagnostic methods for hemophilia are invasive and pose a risk to the fetus. Cell-free fetal DNA analysis in maternal plasma provides a noninvasive mean of assessing fetal sex in such pregnancies. However, the disease status of male fetuses remains unknown if mutation-specific confirmatory analysis is not performed. Here we have developed a noninvasive test to diagnose whether the fetus has inherited a causative mutation for hemophilia from its mother. The strategy is based on a relative mutation dosage approach, which we have previously established for determining the mutational status of fetuses for autosomal disease mutations. In this study, the relative mutation dosage method is used to deduce whether a fetus has inherited a hemophilia mutation on chromosome X by detecting whether the concentration of the mutant or wild-type allele is overrepresented in the plasma of heterozygous women carrying male fetuses. We correctly detected fetal genotypes for hemophilia mutations in all of the 12 studied maternal plasma samples obtained from at-risk pregnancies from as early as the 11th week of gestation. This development would make the decision to undertake prenatal testing less traumatic and safer for at-risk families.

Mutation analysis of 272 Spanish families affected by autosomal recessive retinitis pigmentosa using a genotyping microarray.

PubMed

Ávila-Fernández, Almudena; Cantalapiedra, Diego; Aller, Elena; Vallespín, Elena; Aguirre-Lambán, Jana; Blanco-Kelly, Fiona; Corton, M; Riveiro-Álvarez, Rosa; Allikmets, Rando; Trujillo-Tiebas, María José; Millán, José M; Cremers, Frans P M; Ayuso, Carmen

2010-12-03

Retinitis pigmentosa (RP) is a genetically heterogeneous disorder characterized by progressive loss of vision. The aim of this study was to identify the causative mutations in 272 Spanish families using a genotyping microarray. 272 unrelated Spanish families, 107 with autosomal recessive RP (arRP) and 165 with sporadic RP (sRP), were studied using the APEX genotyping microarray. The families were also classified by clinical criteria: 86 juveniles and 186 typical RP families. Haplotype and sequence analysis were performed to identify the second mutated allele. At least one-gene variant was found in 14% and 16% of the juvenile and typical RP groups respectively. Further study identified four new mutations, providing both causative changes in 11% of the families. Retinol Dehydrogenase 12 (RDH12) was the most frequently mutated gene in the juvenile RP group, and Usher Syndrome 2A (USH2A) and Ceramide Kinase-Like (CERKL) were the most frequently mutated genes in the typical RP group. The only variant found in CERKL was p.Arg257Stop, the most frequent mutation. The genotyping microarray combined with segregation and sequence analysis allowed us to identify the causative mutations in 11% of the families. Due to the low number of characterized families, this approach should be used in tandem with other techniques.

The use of high resolution melting analysis to detect Fabry mutations in heterozygous females via dry bloodspots.

PubMed

Tai, Chang-Long; Liu, Mei-Ying; Yu, Hsiao-Chi; Chiang, Chiang-Chuan; Chiang, Hung; Suen, Jeng-Hung; Kao, Shu-Min; Huang, Yu-Hsiu; Wu, Tina Jui-Ting; Yang, Chia-Feng; Tsai, Fang-Chih; Lin, Ching-Yuang; Chang, Jan-Gowth; Chen, Hong-Duo; Niu, Dau-Ming

2012-02-18

As an X-linked genetic disorder, Fabry disease was first thought to affect males only, and females were generally considered to be asymptomatic carriers. However, recent research suggests that female carriers of Fabry disease may still develop vital organ damage causing severe morbidity and mortality. In the previous newborn screening, from 299,007 newborns, we identified a total of 20 different Fabry mutations and 121 newborns with Fabry mutations. However, we found that most female carriers are not detected by enzyme assays. A streamlined method for high resolution melting (HRM) analysis was designed to screen for GLA gene mutations using a same PCR and melting program. Primer sets were designed to cover the 7 exons and the Chinese common intronic mutation, IVS4+919G>A of GLA gene. The HRM analysis was successful in identifying heterozygous and hemizygous patients with the 20 surveyed mutations. We were also successful in using this method to test dry blood spots of newborns afflicted with Fabry mutations without having to determine DNA concentration before PCR amplification. The results of this study show that HRM could be a reliable and sensitive method for use in the rapid screening of females for GLA mutations. Copyright © 2011 Elsevier B.V. All rights reserved.

Rational design of a monomeric and photostable far-red fluorescent protein for fluorescence imaging in vivo.

PubMed

Yu, Dan; Dong, Zhiqiang; Gustafson, William Clay; Ruiz-González, Rubén; Signor, Luca; Marzocca, Fanny; Borel, Franck; Klassen, Matthew P; Makhijani, Kalpana; Royant, Antoine; Jan, Yuh-Nung; Weiss, William A; Guo, Su; Shu, Xiaokun

2016-02-01

Fluorescent proteins (FPs) are powerful tools for cell and molecular biology. Here based on structural analysis, a blue-shifted mutant of a recently engineered monomeric infrared fluorescent protein (mIFP) has been rationally designed. This variant, named iBlueberry, bears a single mutation that shifts both excitation and emission spectra by approximately 40 nm. Furthermore, iBlueberry is four times more photostable than mIFP, rendering it more advantageous for imaging protein dynamics. By tagging iBlueberry to centrin, it has been demonstrated that the fusion protein labels the centrosome in the developing zebrafish embryo. Together with GFP-labeled nucleus and tdTomato-labeled plasma membrane, time-lapse imaging to visualize the dynamics of centrosomes in radial glia neural progenitors in the intact zebrafish brain has been demonstrated. It is further shown that iBlueberry can be used together with mIFP in two-color protein labeling in living cells and in two-color tumor labeling in mice. © 2015 The Protein Society.

Fraction of boroxol rings in vitreous boron oxide from a first-principles analysis of Raman and NMR spectra.

PubMed

Umari, P; Pasquarello, Alfredo

2005-09-23

We determine the fraction f of B atoms belonging to boroxol rings in vitreous boron oxide through a first-principles analysis. After generating a model structure of vitreous B2O3 by first-principles molecular dynamics, we address a large set of properties, including the neutron structure factor, the neutron density of vibrational states, the infrared spectra, the Raman spectra, and the 11B NMR spectra, and find overall good agreement with corresponding experimental data. From the analysis of Raman and 11B NMR spectra, we yield consistently for both probes a fraction f of approximately 0.75. This result indicates that the structure of vitreous boron oxide is largely dominated by boroxol rings.

Excitation wavelength selection for quantitative analysis of carotenoids in tomatoes using Raman spectroscopy.

PubMed

Hara, Risa; Ishigaki, Mika; Kitahama, Yasutaka; Ozaki, Yukihiro; Genkawa, Takuma

2018-08-30

The difference in Raman spectra for different excitation wavelengths (532 nm, 785 nm, and 1064 nm) was investigated to identify an appropriate wavelength for the quantitative analysis of carotenoids in tomatoes. For the 532 nm-excited Raman spectra, the intensity of the peak assigned to the carotenoid has no correlation with carotenoid concentration, and the peak shift reflects carotenoid composition changing from lycopene to β-carotene and lutein. Thus, 532 nm-excited Raman spectra are useful for the qualitative analysis of carotenoids. For the 785 nm- and 1064 nm-excited Raman spectra, the peak intensity of the carotenoid showed good correlation with carotenoid concentration; thus, regression models for carotenoid concentration were developed using these Raman spectra and partial least squares regression. A regression model designed using the 785 nm-excited Raman spectra showed a better result than the 532 nm- and 1064 nm-excited Raman spectra. Therefore, it can be concluded that 785 nm is the most suitable excitation wavelength for the quantitative analysis of carotenoid concentration in tomatoes. Copyright © 2018 Elsevier Ltd. All rights reserved.

Molecular genetic analysis of the F11 gene in 14 Turkish patients with factor XI deficiency: identification of novel and recurrent mutations and their inheritance within families

PubMed Central

Colakoglu, Seyma; Bayhan, Turan; Tavil, Betül; Keskin, Ebru Yılmaz; Cakir, Volkan; Gümrük, Fatma; Çetin, Mualla; Aytaç, Selin; Berber, Ergul

2018-01-01

Background Factor XI (FXI) deficiency is an autosomal bleeding disease associated with genetic defects in the F11 gene which cause decreased FXI levels or impaired FXI function. An increasing number of mutations has been reported in the FXI mutation database, most of which affect the serine protease domain of the protein. FXI is a heterogeneous disorder associated with a variable bleeding tendency and a variety of causative F11 gene mutations. The molecular basis of FXI deficiency in 14 patients from ten unrelated families in Turkey was analysed to establish genotype-phenotype correlations and inheritance of the mutations in the patients’ families. Material and methods Fourteen index cases with a diagnosis of FXI deficiency and family members of these patients were enrolled into the study. The patients’ F11 genes were amplified by polymerase chain reaction and subjected to direct DNA sequencing analysis. The findings were analysed statistically using bivariate correlations, Pearson’s correlation coefficient and the nonparametric Mann-Whitney test. Results Direct DNA sequencing analysis of the F11 genes revealed that all of the 14 patients had a F11 gene mutation. Eight different mutations were identified in the apple 1, apple 2 or serine protease domains, except one which was a splice site mutation. Six of the mutations were recurrent. Two of the mutations were novel missense mutations, p.Val522Gly and p.Cys581Arg, within the catalytic domain. The p.Trp519Stop mutation was observed in two families whereas all the other mutations were specific to a single family. Discussion Identification of mutations confirmed the genetic heterogeneity of FXI deficiency. Most of the patients with mutations did not have any bleeding complications, whereas some had severe bleeding symptoms. Genetic screening for F11 gene mutations is important to decrease the mortality and morbidity rate associated with FXI deficiency, which can be life-threatening if bleeding occurs in tissues with high fibrinolytic activity. PMID:27723456

Significance of KRAS, NRAS, BRAF and PIK3CA mutations in metastatic colorectal cancer patients receiving Bevacizumab: a single institution experience

PubMed Central

Baltruškevičienė, Edita; Mickys, Ugnius; Žvirblis, Tadas; Stulpinas, Rokas; Pipirienė Želvienė, Teresė; Aleknavičius, Eduardas

2016-01-01

Background. KRAS mutation is an important predictive and prognostic factor for patients receiving anti-EGFR therapy. An expanded KRAS, NRAS, BRAF, PIK3CA mutation analysis provides additional prognostic information, but its role in predicting bevacizumab efficacy is unclear. The aim of our study was to evaluate the incidence of KRAS, NRAS, BRAF and PIK3CA mutations in metastatic colorectal cancer patients receiving first line oxaliplatin based chemotherapy with or without bevacizumab and to evaluate their prognostic and predictive significance. Methods. 55 patients with the first-time diagnosed CRC receiving FOLFOX ± bevacizumab were involved in the study. Tumour blocks were tested for KRAS mutations in exons 2, 3 and 4, NRAS mutations in exons 2, 3 and 4, BRAF mutation in exon 15 and PIK3CA mutations in exons 9 and 20. The association between mutations and clinico-pathological factors, treatment outcomes and survival was analyzed. Results. KRAS mutations were detected in 67.3% of the patients, BRAF in 1.8%, PIK3CA in 5.5% and there were no NRAS mutations. A significant association between the high CA 19–9 level and KRAS mutation was detected (mean CA 19–9 levels were 276 and 87 kIU/l, respectively, p = 0.019). There was a significantly higher response rate in the KRAS, NRAS, BRAF and PIK3CA wild type cohort receiving bevacizumab compared to any gene mutant type (100 and 60%, respectively, p = 0.030). The univariate Cox regression analysis did not confirm KRAS and other tested mutations as prognostic factors for PFS or OS. Conclusions. Our study revealed higher KRAS and lower NRAS, BRAF and PIK3CA mutation rates in the Lithuanian population than those reported in the literature. KRAS mutation was associated with the high CA 19–9 level and mucinous histology type, but did not show any predictive or prognostic significance. The expanded KRAS, NRAS, BRAF and PIK3CA mutation analysis provided additional significant predictive information. PMID:28356789

Determination of EGFR and KRAS mutational status in Greek non-small-cell lung cancer patients

PubMed Central

PAPADOPOULOU, EIRINI; TSOULOS, NIKOLAOS; TSIRIGOTI, ANGELIKI; APESSOS, ANGELA; AGIANNITOPOULOS, KONSTANTINOS; METAXA-MARIATOU, VASILIKI; ZAROGOULIDIS, KONSTANTINOS; ZAROGOULIDIS, PAVLOS; KASARAKIS, DIMITRIOS; KAKOLYRIS, STYLIANOS; DAHABREH, JUBRAIL; VLASTOS, FOTIS; ZOUBLIOS, CHARALAMPOS; RAPTI, AGGELIKI; PAPAGEORGIOU, NIKI GEORGATOU; VELDEKIS, DIMITRIOS; GAGA, MINA; ARAVANTINOS, GERASIMOS; KARAVASILIS, VASILEIOS; KARAGIANNIDIS, NAPOLEON; NASIOULAS, GEORGE

2015-01-01

It has been reported that certain patients with non-small-cell lung cancer (NSCLC) that harbor activating somatic mutations within the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene may be effectively treated using targeted therapy. The use of EGFR inhibitors in patient therapy has been demonstrated to improve response and survival rates; therefore, it was suggested that clinical screening for EGFR mutations should be performed for all patients. Numerous clinicopathological factors have been associated with EGFR and Kirsten-rat sarcoma oncogene homolog (KRAS) mutational status including gender, smoking history and histology. In addition, it was reported that EGFR mutation frequency in NSCLC patients was ethnicity-dependent, with an incidence rate of ~30% in Asian populations and ~15% in Caucasian populations. However, limited data has been reported on intra-ethnic differences throughout Europe. The present study aimed to investigate the frequency and spectrum of EGFR mutations in 1,472 Greek NSCLC patients. In addition, KRAS mutation analysis was performed in patients with known smoking history in order to determine the correlation of type and mutation frequency with smoking. High-resolution melting curve (HRM) analysis followed by Sanger sequencing was used to identify mutations in exons 18–21 of the EGFR gene and in exon 2 of the KRAS gene. A sensitive next-generation sequencing (NGS) technology was also employed to classify samples with equivocal results. The use of sensitive mutation detection techniques in a large study population of Greek NSCLC patients in routine diagnostic practice revealed an overall EGFR mutation frequency of 15.83%. This mutation frequency was comparable to that previously reported in other European populations. Of note, there was a 99.8% concordance between the HRM method and Sanger sequencing. NGS was found to be the most sensitive method. In addition, female non-smokers demonstrated a high prevalence of EGFR mutations. Furthermore, KRAS mutation analysis in patients with a known smoking history revealed no difference in mutation frequency according to smoking status; however, a different mutation spectrum was observed. PMID:26622815

Genetic analysis of a four generation Indian family with Usher syndrome: a novel insertion mutation in MYO7A.

PubMed

Kumar, Arun; Babu, Mohan; Kimberling, William J; Venkatesh, Conjeevaram P

2004-11-24

Usher syndrome (USH) is a rare autosomal recessive disorder characterized by deafness and retinitis pigmentosa. The purpose of this study was to determine the genetic cause of USH in a four generation Indian family. Peripheral blood samples were collected from individuals for genomic DNA isolation. To determine the linkage of this family to known USH loci, microsatellite markers were selected from the candidate regions of known loci and used to genotype the family. Exon specific intronic primers for the MYO7A gene were used to amplify DNA samples from one affected individual from the family. PCR products were subsequently sequenced to detect mutation. PCR-SSCP analysis was used to determine if the mutation segregated with the disease in the family and was not present in 50 control individuals. All affected individuals had a classic USH type I (USH1) phenotype which included deafness, vestibular dysfunction and retinitis pigmentosa. Pedigree analysis suggested an autosomal recessive mode of inheritance of USH in the family. Haplotype analysis suggested linkage of this family to the USH1B locus on chromosome 11q. DNA sequence analysis of the entire coding region of the MYO7A gene showed a novel insertion mutation c.2663_2664insA in a homozygous state in all affected individuals, resulting in truncation of MYO7A protein. This is the first study from India which reports a novel MYO7A insertion mutation in a four generation USH family. The mutation is predicted to produce a truncated MYO7A protein. With the novel mutation reported here, the total number of USH causing mutations in the MYO7A gene described to date reaches to 75.

[Pigment-protein complexes nd the number of the reaction photosystem centers in pea chlorophyll mutants].

PubMed

Ladygin, V G

2004-01-01

We studied fluorescent and absorption properties of the chloroplasts and pigment-protein complexes isolated by gel electrophoresis from the leaves of pea, the initial cultivar Torsdag and mutants chlorotica 2004 and 2014. Specific maxima of fluorescence and chlorophyll forms in individual complexes have been determined from the absorption and fluorescence spectra of the chloroplast chlorophyll and their secondary derivatives at 23 and -196 degrees C. Chlorotica 2004 mutant proved to have an increased intensity of a long-wave band at both 23 degrees C (745 nm) and -196 degrees C (728 nm) of the light-harvesting complex I. At the same time, this mutant featured a decreased accumulation of chlorophyll forms at 690, 697, and 708 nm forming the nearest-neighbor antenna of PSI reaction center. No spectral differences have been revealed between chlorotica 2014 mutant and the initial cultivar. Gel electrophoresis demonstrated synthesis of all chlorophyll-protein complexes in both mutants. At the same time, analysis of photochemical activity of PSI and PSII reaction centers and evaluation of the light-harvesting antenna as well as the number of reaction centers of the photosystems suggest that chlorotica 2004 mutant has 1.7 times less PSI reaction centers due to a mutation-disturbed chlorophyll a-protein complex of PSI. The primary effect of chlorotica 2014 mutation remains unclear. The proportional changes in the photosystem complexes in this mutant suggest that they are secondary and result from a 50% decrease in chlorophyll content.

Massively Parallel DNA Sequencing Facilitates Diagnosis of Patients with Usher Syndrome Type 1

PubMed Central

Yoshimura, Hidekane; Iwasaki, Satoshi; Nishio, Shin-ya; Kumakawa, Kozo; Tono, Tetsuya; Kobayashi, Yumiko; Sato, Hiroaki; Nagai, Kyoko; Ishikawa, Kotaro; Ikezono, Tetsuo; Naito, Yasushi; Fukushima, Kunihiro; Oshikawa, Chie; Kimitsuki, Takashi; Nakanishi, Hiroshi; Usami, Shin-ichi

2014-01-01

Usher syndrome is an autosomal recessive disorder manifesting hearing loss, retinitis pigmentosa and vestibular dysfunction, and having three clinical subtypes. Usher syndrome type 1 is the most severe subtype due to its profound hearing loss, lack of vestibular responses, and retinitis pigmentosa that appears in prepuberty. Six of the corresponding genes have been identified, making early diagnosis through DNA testing possible, with many immediate and several long-term advantages for patients and their families. However, the conventional genetic techniques, such as direct sequence analysis, are both time-consuming and expensive. Targeted exon sequencing of selected genes using the massively parallel DNA sequencing technology will potentially enable us to systematically tackle previously intractable monogenic disorders and improve molecular diagnosis. Using this technique combined with direct sequence analysis, we screened 17 unrelated Usher syndrome type 1 patients and detected probable pathogenic variants in the 16 of them (94.1%) who carried at least one mutation. Seven patients had the MYO7A mutation (41.2%), which is the most common type in Japanese. Most of the mutations were detected by only the massively parallel DNA sequencing. We report here four patients, who had probable pathogenic mutations in two different Usher syndrome type 1 genes, and one case of MYO7A/PCDH15 digenic inheritance. This is the first report of Usher syndrome mutation analysis using massively parallel DNA sequencing and the frequency of Usher syndrome type 1 genes in Japanese. Mutation screening using this technique has the power to quickly identify mutations of many causative genes while maintaining cost-benefit performance. In addition, the simultaneous mutation analysis of large numbers of genes is useful for detecting mutations in different genes that are possibly disease modifiers or of digenic inheritance. PMID:24618850

Massively parallel DNA sequencing facilitates diagnosis of patients with Usher syndrome type 1.

PubMed

Yoshimura, Hidekane; Iwasaki, Satoshi; Nishio, Shin-Ya; Kumakawa, Kozo; Tono, Tetsuya; Kobayashi, Yumiko; Sato, Hiroaki; Nagai, Kyoko; Ishikawa, Kotaro; Ikezono, Tetsuo; Naito, Yasushi; Fukushima, Kunihiro; Oshikawa, Chie; Kimitsuki, Takashi; Nakanishi, Hiroshi; Usami, Shin-Ichi

2014-01-01

Usher syndrome is an autosomal recessive disorder manifesting hearing loss, retinitis pigmentosa and vestibular dysfunction, and having three clinical subtypes. Usher syndrome type 1 is the most severe subtype due to its profound hearing loss, lack of vestibular responses, and retinitis pigmentosa that appears in prepuberty. Six of the corresponding genes have been identified, making early diagnosis through DNA testing possible, with many immediate and several long-term advantages for patients and their families. However, the conventional genetic techniques, such as direct sequence analysis, are both time-consuming and expensive. Targeted exon sequencing of selected genes using the massively parallel DNA sequencing technology will potentially enable us to systematically tackle previously intractable monogenic disorders and improve molecular diagnosis. Using this technique combined with direct sequence analysis, we screened 17 unrelated Usher syndrome type 1 patients and detected probable pathogenic variants in the 16 of them (94.1%) who carried at least one mutation. Seven patients had the MYO7A mutation (41.2%), which is the most common type in Japanese. Most of the mutations were detected by only the massively parallel DNA sequencing. We report here four patients, who had probable pathogenic mutations in two different Usher syndrome type 1 genes, and one case of MYO7A/PCDH15 digenic inheritance. This is the first report of Usher syndrome mutation analysis using massively parallel DNA sequencing and the frequency of Usher syndrome type 1 genes in Japanese. Mutation screening using this technique has the power to quickly identify mutations of many causative genes while maintaining cost-benefit performance. In addition, the simultaneous mutation analysis of large numbers of genes is useful for detecting mutations in different genes that are possibly disease modifiers or of digenic inheritance.

New VMD2 gene mutations identified in patients affected by Best vitelliform macular dystrophy

PubMed Central

Marchant, D; Yu, K; Bigot, K; Roche, O; Germain, A; Bonneau, D; Drouin‐Garraud, V; Schorderet, D F; Munier, F; Schmidt, D; Neindre, P Le; Marsac, C; Menasche, M; Dufier, J L; Fischmeister, R; Hartzell, C; Abitbol, M

2007-01-01

Purpose The mutations responsible for Best vitelliform macular dystrophy (BVMD) are found in a gene called VMD2. The VMD2 gene encodes a transmembrane protein named bestrophin‐1 (hBest1) which is a Ca2+‐sensitive chloride channel. This study was performed to identify disease‐specific mutations in 27 patients with BVMD. Because this disease is characterised by an alteration in Cl− channel function, patch clamp analysis was used to test the hypothesis that one of the VMD2 mutated variants causes the disease. Methods Direct sequencing analysis of the 11 VMD2 exons was performed to detect new abnormal sequences. The mutant of hBest1 was expressed in HEK‐293 cells and the associated Cl− current was examined using whole‐cell patch clamp analysis. Results Six new VMD2 mutations were identified, located exclusively in exons four, six and eight. One of these mutations (Q293H) was particularly severe. Patch clamp analysis of human embryonic kidney cells expressing the Q293H mutant showed that this mutant channel is non‐functional. Furthermore, the Q293H mutant inhibited the function of wild‐type bestrophin‐1 channels in a dominant negative manner. Conclusions This study provides further support for the idea that mutations in VMD2 are a necessary factor for Best disease. However, because variable expressivity of VMD2 was observed in a family with the Q293H mutation, it is also clear that a disease‐linked mutation in VMD2 is not sufficient to produce BVMD. The finding that the Q293H mutant does not form functional channels in the membrane could be explained either by disruption of channel conductance or gating mechanisms or by improper trafficking of the protein to the plasma membrane. PMID:17287362

Mutation analysis in 129 genes associated with other forms of retinal dystrophy in 157 families with retinitis pigmentosa based on exome sequencing.

PubMed

Xu, Yan; Guan, Liping; Xiao, Xueshan; Zhang, Jianguo; Li, Shiqiang; Jiang, Hui; Jia, Xiaoyun; Yang, Jianhua; Guo, Xiangming; Yin, Ye; Wang, Jun; Zhang, Qingjiong

2015-01-01

Mutations in 60 known genes were previously identified by exome sequencing in 79 of 157 families with retinitis pigmentosa (RP). This study analyzed variants in 129 genes associated with other forms of hereditary retinal dystrophy in the same cohort. Apart from the 73 genes previously analyzed, a further 129 genes responsible for other forms of hereditary retinal dystrophy were selected based on RetNet. Variants in the 129 genes determined by whole exome sequencing were selected and filtered by bioinformatics analysis. Candidate variants were confirmed by Sanger sequencing and validated by analysis of available family members and controls. A total of 90 candidate variants were present in the 129 genes. Sanger sequencing confirmed 83 of the 90 variants. Analysis of family members and controls excluded 76 of these 83 variants. The remaining seven variants were considered to be potential pathogenic mutations; these were c.899A>G, c.1814C>G, and c.2107C>T in BBS2; c.1073C>T and c.1669C>T in INPP5E; and c.3582C>G and c.5704-5C>G in CACNA1F. Six of these seven mutations were novel. The mutations were detected in five unrelated patients without a family history, including three patients with homozygous or compound heterozygous mutations in BBS2 and INPP5E, and two patients with hemizygous mutations in CACNA1F. None of the patients had mutations in the genes associated with autosome dominant retinal dystrophy. Only a small portion of patients with RP, about 3% (5/157), had causative mutations in the 129 genes associated with other forms of hereditary retinal dystrophy.

Combined Microsatellite Instability, MLH1 Methylation Analysis, and Immunohistochemistry for Lynch Syndrome Screening in Endometrial Cancers From GOG210: An NRG Oncology and Gynecologic Oncology Group Study.

PubMed

Goodfellow, Paul J; Billingsley, Caroline C; Lankes, Heather A; Ali, Shamshad; Cohn, David E; Broaddus, Russell J; Ramirez, Nilsa; Pritchard, Colin C; Hampel, Heather; Chassen, Alexis S; Simmons, Luke V; Schmidt, Amy P; Gao, Feng; Brinton, Louise A; Backes, Floor; Landrum, Lisa M; Geller, Melissa A; DiSilvestro, Paul A; Pearl, Michael L; Lele, Shashikant B; Powell, Matthew A; Zaino, Richard J; Mutch, David

2015-12-20

The best screening practice for Lynch syndrome (LS) in endometrial cancer (EC) remains unknown. We sought to determine whether tumor microsatellite instability (MSI) typing along with immunohistochemistry (IHC) and MLH1 methylation analysis can help identify women with LS. ECs from GOG210 patients were assessed for MSI, MLH1 methylation, and mismatch repair (MMR) protein expression. Each tumor was classified as having normal MMR, defective MMR associated with MLH1 methylation, or probable MMR mutation (ie, defective MMR but no methylation). Cancer family history and demographic and clinical features were compared for the three groups. Lynch mutation testing was performed for a subset of women. Analysis of 1,002 ECs suggested possible MMR mutation in 11.8% of tumors. The number of patients with a family history suggestive of LS was highest among women whose tumors were classified as probable MMR mutation (P = .001). Lynch mutations were identified in 41% of patient cases classified as probable mutation (21 of 51 tested). One of the MSH6 Lynch mutations was identified in a patient whose tumor had intact MSH6 expression. Age at diagnosis was younger for mutation carriers than noncarriers (54.3 v 62.3 years; P < .01), with five carriers diagnosed at age > 60 years. Combined MSI, methylation, and IHC analysis may prove useful in Lynch screening in EC. Twenty-four percent of mutation carriers presented with ECs at age > 60 years, and one carrier had an MSI-positive tumor with no IHC defect. Restricting Lynch testing to women diagnosed at age < 60 years or to women with IHC defects could result in missing a substantial fraction of genetic disease. © 2015 by American Society of Clinical Oncology.

Combined Microsatellite Instability, MLH1 Methylation Analysis, and Immunohistochemistry for Lynch Syndrome Screening in Endometrial Cancers From GOG210: An NRG Oncology and Gynecologic Oncology Group Study

PubMed Central

Goodfellow, Paul J.; Billingsley, Caroline C.; Lankes, Heather A.; Ali, Shamshad; Cohn, David E.; Broaddus, Russell J.; Ramirez, Nilsa; Pritchard, Colin C.; Hampel, Heather; Chassen, Alexis S.; Simmons, Luke V.; Schmidt, Amy P.; Gao, Feng; Brinton, Louise A.; Backes, Floor; Landrum, Lisa M.; Geller, Melissa A.; DiSilvestro, Paul A.; Pearl, Michael L.; Lele, Shashikant B.; Powell, Matthew A.; Zaino, Richard J.; Mutch, David

2015-01-01

Purpose The best screening practice for Lynch syndrome (LS) in endometrial cancer (EC) remains unknown. We sought to determine whether tumor microsatellite instability (MSI) typing along with immunohistochemistry (IHC) and MLH1 methylation analysis can help identify women with LS. Patients and Methods ECs from GOG210 patients were assessed for MSI, MLH1 methylation, and mismatch repair (MMR) protein expression. Each tumor was classified as having normal MMR, defective MMR associated with MLH1 methylation, or probable MMR mutation (ie, defective MMR but no methylation). Cancer family history and demographic and clinical features were compared for the three groups. Lynch mutation testing was performed for a subset of women. Results Analysis of 1,002 ECs suggested possible MMR mutation in 11.8% of tumors. The number of patients with a family history suggestive of LS was highest among women whose tumors were classified as probable MMR mutation (P = .001). Lynch mutations were identified in 41% of patient cases classified as probable mutation (21 of 51 tested). One of the MSH6 Lynch mutations was identified in a patient whose tumor had intact MSH6 expression. Age at diagnosis was younger for mutation carriers than noncarriers (54.3 v 62.3 years; P < .01), with five carriers diagnosed at age > 60 years. Conclusion Combined MSI, methylation, and IHC analysis may prove useful in Lynch screening in EC. Twenty-four percent of mutation carriers presented with ECs at age > 60 years, and one carrier had an MSI-positive tumor with no IHC defect. Restricting Lynch testing to women diagnosed at age < 60 years or to women with IHC defects could result in missing a substantial fraction of genetic disease. PMID:26552419

Nanofluidic Digital PCR and Extended Genotyping of RAS and BRAF for Improved Selection of Metastatic Colorectal Cancer Patients for Anti-EGFR Therapies.

PubMed

Azuara, Daniel; Santos, Cristina; Lopez-Doriga, Adriana; Grasselli, Julieta; Nadal, Marga; Sanjuan, Xavier; Marin, Fátima; Vidal, Joana; Montal, Robert; Moreno, Victor; Bellosillo, Beatriz; Argiles, Guillem; Elez, Elena; Dienstmann, Rodrigo; Montagut, Clara; Tabernero, Josep; Capellá, Gabriel; Salazar, Ramon

2016-05-01

The clinical significance of low-frequent RAS pathway-mutated alleles and the optimal sensitivity cutoff value in the prediction of response to anti-EGFR therapy in metastatic colorectal cancer (mCRC) patients remains controversial. We aimed to evaluate the added value of genotyping an extended RAS panel using a robust nanofluidic digital PCR (dPCR) approach. A panel of 34 hotspots, including RAS (KRAS and NRAS exons 2/3/4) and BRAF (V600E), was analyzed in tumor FFPE samples from 102 mCRC patients treated with anti-EGFR therapy. dPCR was compared with conventional quantitative PCR (qPCR). Response rates, progression-free survival (PFS), and overall survival (OS) were correlated to the mutational status and the mutated allele fraction. Tumor response evaluations were not available in 9 patients and were excluded for response rate analysis. Twenty-two percent of patients were positive for one mutation with qPCR (mutated alleles ranged from 2.1% to 66.6%). Analysis by dPCR increased the number of positive patients to 47%. Mutated alleles for patients only detected by dPCR ranged from 0.04% to 10.8%. An inverse correlation between the fraction of mutated alleles and radiologic response was observed. ROC analysis showed that a fraction of 1% or higher of any mutated alleles offered the best predictive value for all combinations of RAS and BRAF analysis. In addition, this threshold also optimized prediction both PFS and OS. We conclude that mutation testing using an extended gene panel, including RAS and BRAF with a threshold of 1% improved prediction of response to anti-EGFR therapy. Mol Cancer Ther; 15(5); 1106-12. ©2016 AACR. ©2016 American Association for Cancer Research.

A9 region in EPHB2 mutation is frequent in tumors with microsatellite instability. Analysis of prognosis.

PubMed

Rafael, Sara; Vidaurreta, Marta; Veganzones, Silvia; De La Orden, Virgnia; Mediero, Beatriz; Gutierrez, Maria Luisa; Maestro, Maria Luisa

2013-11-01

The aim of the present study was to determine the relation of EPH tyrosine kinase receptor B2 (EPHB2) A9 region mutation and microsatellite instability (MSI); and to analyze their influence in prognosis of patients with sporadic colorectal cancer (CRC). A total of 481 patients with CRC were examined. MSI (NCI criteria) and EPHB2 were analyzed using PCR and fragment analysis software. EPHB2 mutation was detected in 3.1% of patients. Mutation of EPHB2 was associated with location and with MSI status. We considered low instability (L-MSI) when only one marker showed instability, high instability (H-MSI) when two or more markers were positive and microsatelllite stable (MSS) when no instability was detected. The stratified analysis of overall survival (OS) and disease-free survival (DFS) in MSI according to EPHB2 status revealed no statistically significant differences. However, the risk of recurrence of H-MSI tumors with EPHB2 mutation carriers was 3.6-times higher than in non-mutation carriers. The frequency of EPHB2 mutation is higher in patients with H-MSI than MSS tumors. Promising results were found regarding the prognostic influence of EPHB2 in H-MSI.

A novel loss-of-function mutation in OTX2 in a patient with anophthalmia and isolated growth hormone deficiency.

PubMed

Ashkenazi-Hoffnung, Liat; Lebenthal, Yael; Wyatt, Alexander W; Ragge, Nicola K; Dateki, Sumito; Fukami, Maki; Ogata, Tsutomu; Phillip, Moshe; Gat-Yablonski, Galia

2010-06-01

Heterozygous mutations of the gene encoding transcription factor OTX2 were recently shown to be responsible for ocular as well as pituitary abnormalities. Here, we describe a patient with unilateral anophthalmia and short stature. Endocrine evaluation of the hypothalamic-pituitary axis revealed isolated growth hormone deficiency (IGHD) with small anterior pituitary gland, invisible stalk, ectopic posterior lobe, and right anophthalmia on brain magnetic resonance imaging. DNA was analyzed for mutations in the HESX1, SOX2, and OTX2 genes. Molecular analysis yielded a novel heterozygous OTX2 mutation (c.270A>T, p.R90S) within the homeodomain. Functional analysis revealed that the mutation inhibited both the DNA binding and transactivation activities of the protein. This novel loss-of-function mutation is associated with anophthalmia and IGHD in a patient of Sephardic Jewish descent. We recommend that patients with GH deficiency and ocular malformation in whom genetic analysis for classic transcription factor genes (PROP1, POU1F1, HESX1, and LHX4) failed to identify alterations should be checked for the presence of mutations in the OTX2 gene.

KRAS Mutation as a Potential Prognostic Biomarker of Biliary Tract Cancers

PubMed Central

Yokoyama, Masaaki; Ohnishi, Hiroaki; Ohtsuka, Kouki; Matsushima, Satsuki; Ohkura, Yasuo; Furuse, Junji; Watanabe, Takashi; Mori, Toshiyuki; Sugiyama, Masanori

2016-01-01

BACKGROUND The aim of this study was to identify the unique molecular characteristics of biliary tract cancer (BTC) for the development of novel molecular-targeted therapies. MATERIALS AND METHODS We performed mutational analysis of KRAS, BRAF, PIK3CA, and FBXW7 and immunohistochemical analysis of EGFR and TP53 in 63 Japanese patients with BTC and retrospectively evaluated the association between the molecular characteristics and clinicopathological features of BTC. RESULTS KRAS mutations were identified in 9 (14%) of the 63 BTC patients; no mutations were detected within the analyzed regions of BRAF, PIK3CA, and FBXW7. EGFR overexpression was observed in 5 (8%) of the 63 tumors, while TP53 overexpression was observed in 48% (30/63) of the patients. Overall survival of patients with KRAS mutation was significantly shorter than that of patients with the wild-type KRAS gene (P = 0.005). By multivariate analysis incorporating molecular and clinicopathological features, KRAS mutations and lymph node metastasis were identified to be independently associated with shorter overall survival (KRAS, P = 0.004; lymph node metastasis, P = 0.015). CONCLUSIONS Our data suggest that KRAS mutation is a poor prognosis predictive biomarker for the survival in BTC patients. PMID:28008299

A de novo mutation in the AGXT gene causing primary hyperoxaluria type 1.

PubMed

Williams, Emma L; Kemper, Markus J; Rumsby, Gill

2006-09-01

Primary hyperoxaluria type 1 is caused by mutations in the alanine-glyoxylate aminotransferase (AGXT) gene. In cases in which no mutation was identified, linkage analysis can be used to confirm or exclude the diagnosis in other siblings. We present a family in which a sibling of the index case predicted to have primary hyperoxaluria type 1 by means of linkage analysis failed to show hyperoxaluria during the following 7 years, putting the diagnosis into question. Whole-gene sequence analysis identified 2 causative mutations in the index case, of which only 1, c.646A (Gly216Arg), was inherited. The other sequence change, c.33_34insC, was a de novo mutation occurring on the paternal allele. This particular mutation is a relatively common cause of primary hyperoxaluria type 1. It occurs in a run of 8 cytosines and therefore potentially is susceptible to polymerase slippage. This case illustrates 2 important points. First, biochemical confirmation of a genetic diagnosis should always be made in siblings diagnosed by using genetic tests. Second, de novo mutations should be considered as a potential, albeit rare, cause of primary hyperoxaluria type 1.

PIMMS (Pragmatic Insertional Mutation Mapping System) Laboratory Methodology a Readily Accessible Tool for Identification of Essential Genes in Streptococcus

PubMed Central

Blanchard, Adam M.; Egan, Sharon A.; Emes, Richard D.; Warry, Andrew; Leigh, James A.

2016-01-01

The Pragmatic Insertional Mutation Mapping (PIMMS) laboratory protocol was developed alongside various bioinformatics packages (Blanchard et al., 2015) to enable detection of essential and conditionally essential genes in Streptococcus and related bacteria. This extended the methodology commonly used to locate insertional mutations in individual mutants to the analysis of mutations in populations of bacteria. In Streptococcus uberis, a pyogenic Streptococcus associated with intramammary infection and mastitis in ruminants, the mutagen pGhost9:ISS1 was shown to integrate across the entire genome. Analysis of >80,000 mutations revealed 196 coding sequences, which were not be mutated and a further 67 where mutation only occurred beyond the 90th percentile of the coding sequence. These sequences showed good concordance with sequences within the database of essential genes and typically matched sequences known to be associated with basic cellular functions. Due to the broad utility of this mutagen and the simplicity of the methodology it is anticipated that PIMMS will be of value to a wide range of laboratories in functional genomic analysis of a wide range of Gram positive bacteria (Streptococcus, Enterococcus, and Lactococcus) of medical, veterinary, and industrial significance. PMID:27826289

On the construction of a new stellar classification template library for the LAMOST spectral analysis pipeline

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wei, Peng; Luo, Ali; Li, Yinbi

2014-05-01

The LAMOST spectral analysis pipeline, called the 1D pipeline, aims to classify and measure the spectra observed in the LAMOST survey. Through this pipeline, the observed stellar spectra are classified into different subclasses by matching with template spectra. Consequently, the performance of the stellar classification greatly depends on the quality of the template spectra. In this paper, we construct a new LAMOST stellar spectral classification template library, which is supposed to improve the precision and credibility of the present LAMOST stellar classification. About one million spectra are selected from LAMOST Data Release One to construct the new stellar templates, andmore » they are gathered in 233 groups by two criteria: (1) pseudo g – r colors obtained by convolving the LAMOST spectra with the Sloan Digital Sky Survey ugriz filter response curve, and (2) the stellar subclass given by the LAMOST pipeline. In each group, the template spectra are constructed using three steps. (1) Outliers are excluded using the Local Outlier Probabilities algorithm, and then the principal component analysis method is applied to the remaining spectra of each group. About 5% of the one million spectra are ruled out as outliers. (2) All remaining spectra are reconstructed using the first principal components of each group. (3) The weighted average spectrum is used as the template spectrum in each group. Using the previous 3 steps, we initially obtain 216 stellar template spectra. We visually inspect all template spectra, and 29 spectra are abandoned due to low spectral quality. Furthermore, the MK classification for the remaining 187 template spectra is manually determined by comparing with 3 template libraries. Meanwhile, 10 template spectra whose subclass is difficult to determine are abandoned. Finally, we obtain a new template library containing 183 LAMOST template spectra with 61 different MK classes by combining it with the current library.« less

Frequency of TERT promoter mutations in primary tumors of the liver.

PubMed

Quaas, Alexander; Oldopp, Theresa; Tharun, Lars; Klingenfeld, Catina; Krech, Till; Sauter, Guido; Grob, Tobias J

2014-12-01

Transcriptional regulation of the TERT gene is a major cause of the cancer-specific increase in telomerase activity. Recently, frequent somatic mutations in the TERT promoter have been described in several tumor entities such as melanoma, glioblastoma, bladder cancer, and hepatocellular carcinoma. By generating a putative consensus binding site for ETS transcription factors within the TERT promoter, these mutations are predicted to increase promoter activity and TERT transcription. In order to improve the understanding of the role of TERT promoter mutation in liver tumorigenesis, the mutational status of the TERT promoter was analyzed in 78 hepatocellular carcinomas, 15 hepatocellular adenomas, and 52 intrahepatic cholangiocarciomas. The promoter region of TERT was screened for the two hotspot mutations using PCR and restriction fragment length analysis, utilizing the introduction of novel restriction sites by the somatic mutations. TERT promoter mutation was found in 37 of 78 hepatocellular carcinomas (47 %) and was restricted to the -124C>T mutation. Frequency of mutations was associated with grade of differentiation ranging from 39 % in well-differentiated tumors to 73 % in high-grade hepatocellular carcinomas. TERT promoter mutations were not found in 15 hepatocellular adenomas and 52 intrahepatic cholangiocarcinomas. These data show that TERT promoter mutation is the most frequent genetic alteration in hepatocellular carcinoma known at this time. The striking predominance of the -124C>T mutation compared with other tumor entities suggest a biological difference of the two hotspot mutations. Analysis of TERT promoter mutation might become a diagnostic tool distinguishing hepatocellular adenoma from well-differentiated hepatocellular carcinoma.

The prognostic value of KRAS mutation by cell-free DNA in cancer patients: A systematic review and meta-analysis.

PubMed

Zhuang, Rongyuan; Li, Song; Li, Qian; Guo, Xi; Shen, Feng; Sun, Hong; Liu, Tianshu

2017-01-01

KRAS mutation has been found in various types of cancer. However, the prognostic value of KRAS mutation in cell-free DNA (cfDNA) in cancer patients was conflicting. In the present study, a meta-analysis was conducted to clarify its prognostic significance. Literature searches of Cochrane Library, EMBASE, PubMed and Web of Science were performed to identify studies related to KRAS mutation detected by cfDNA and survival in cancer patients. Two evaluators reviewed and extracted the information independently. Review Manager 5.3 software was used to perform the statistical analysis. Thirty studies were included in the present meta-analysis. Our analysis showed that KRAS mutation in cfDNA was associated with a poorer survival in cancer patients for overall survival (OS, HR 2.02, 95% CI 1.63-2.51, P<0.01) and progression-free survival (PFS, HR 1.64, 95% CI 1.27-2.13, P<0.01). In subgroup analyses, KRAS mutation in pancreatic cancer, colorectal cancer, non-small cell lung cancer and ovarian epithelial cancer had HRs of 2.81 (95% CI 1.83-4.30, P<0.01), 1.67 (95% CI 1.25-2.42, P<0.01), 1.64 (95% CI 1.13-2.39, P = 0.01) and 2.17 (95% 1.12-4.21, p = 0.02) for OS, respectively. In addition, the ethnicity didn't influence the prognostic value of KRAS mutation in cfDNA in cancer patients (p = 0.39). Prognostic value of KRAS mutation was slightly higher in plasma than in serum (HR 2.13 vs 1.65), but no difference was observed (p = 0.37). Briefly, KRAS mutation in cfDNA was a survival prognostic biomarker in cancer patients. Its prognostic value was different in various types of cancer.

Mutation analysis of the COL1A1 and COL1A2 genes in Vietnamese patients with osteogenesis imperfecta.

PubMed

Ho Duy, Binh; Zhytnik, Lidiia; Maasalu, Katre; Kändla, Ivo; Prans, Ele; Reimann, Ene; Märtson, Aare; Kõks, Sulev

2016-08-12

The genetics of osteogenesis imperfecta (OI) have not been studied in a Vietnamese population before. We performed mutational analysis of the COL1A1 and COL1A2 genes in 91 unrelated OI patients of Vietnamese origin. We then systematically characterized the mutation profiles of these two genes which are most commonly related to OI. Genomic DNA was extracted from EDTA-preserved blood according to standard high-salt extraction methods. Sequence analysis and pathogenic variant identification was performed with Mutation Surveyor DNA variant analysis software. Prediction of the pathogenicity of mutations was conducted using Alamut Visual software. The presence of variants was checked against Dalgleish's osteogenesis imperfecta mutation database. The sample consisted of 91 unrelated osteogenesis imperfecta patients. We identified 54 patients with COL1A1/2 pathogenic variants; 33 with COL1A1 and 21 with COL1A2. Two patients had multiple pathogenic variants. Seventeen novel COL1A1 and 10 novel COL1A2 variants were identified. The majority of identified COL1A1/2 pathogenic variants occurred in a glycine substitution (36/56, 64.3 %), usually serine (23/36, 63.9 %). We found two pathogenic variants of the COL1A1 gene c.2461G > A (p.Gly821Ser) in four unrelated patients and one, c.2005G > A (p.Ala669Thr), in two unrelated patients. Our data showed a lower number of collagen OI pathogenic variants in Vietnamese patients compared to reported rates for Asian populations. The OI mutational profile of the Vietnamese population is unique and related to the presence of a high number of recessive mutations in non-collagenous OI genes. Further analysis of OI patients negative for collagen mutations, is required.

Retrieval of complex χ(2) parts for quantitative analysis of sum-frequency generation intensity spectra

PubMed Central

Hofmann, Matthias J.; Koelsch, Patrick

2015-01-01

Vibrational sum-frequency generation (SFG) spectroscopy has become an established technique for in situ surface analysis. While spectral recording procedures and hardware have been optimized, unique data analysis routines have yet to be established. The SFG intensity is related to probing geometries and properties of the system under investigation such as the absolute square of the second-order susceptibility χ(2)2. A conventional SFG intensity measurement does not grant access to the complex parts of χ(2) unless further assumptions have been made. It is therefore difficult, sometimes impossible, to establish a unique fitting solution for SFG intensity spectra. Recently, interferometric phase-sensitive SFG or heterodyne detection methods have been introduced to measure real and imaginary parts of χ(2) experimentally. Here, we demonstrate that iterative phase-matching between complex spectra retrieved from maximum entropy method analysis and fitting of intensity SFG spectra (iMEMfit) leads to a unique solution for the complex parts of χ(2) and enables quantitative analysis of SFG intensity spectra. A comparison between complex parts retrieved by iMEMfit applied to intensity spectra and phase sensitive experimental data shows excellent agreement between the two methods. PMID:26450297

X-linked Alport syndrome caused by splicing mutations in COL4A5.

PubMed

Nozu, Kandai; Vorechovsky, Igor; Kaito, Hiroshi; Fu, Xue Jun; Nakanishi, Koichi; Hashimura, Yuya; Hashimoto, Fusako; Kamei, Koichi; Ito, Shuichi; Kaku, Yoshitsugu; Imasawa, Toshiyuki; Ushijima, Katsumi; Shimizu, Junya; Makita, Yoshio; Konomoto, Takao; Yoshikawa, Norishige; Iijima, Kazumoto

2014-11-07

X-linked Alport syndrome is caused by mutations in the COL4A5 gene. Although many COL4A5 mutations have been detected, the mutation detection rate has been unsatisfactory. Some men with X-linked Alport syndrome show a relatively mild phenotype, but molecular basis investigations have rarely been conducted to clarify the underlying mechanism. In total, 152 patients with X-linked Alport syndrome who were suspected of having Alport syndrome through clinical and pathologic investigations and referred to the hospital for mutational analysis between January of 2006 and January of 2013 were genetically diagnosed. Among those patients, 22 patients had suspected splice site mutations. Transcripts are routinely examined when suspected splice site mutations for abnormal transcripts are detected; 11 of them showed expected exon skipping, but others showed aberrant splicing patterns. The mutation detection strategy had two steps: (1) genomic DNA analysis using PCR and direct sequencing and (2) mRNA analysis using RT-PCR to detect RNA processing abnormalities. Six splicing consensus site mutations resulting in aberrant splicing patterns, one exonic mutation leading to exon skipping, and four deep intronic mutations producing cryptic splice site activation were identified. Interestingly, one case produced a cryptic splice site with a single nucleotide substitution in the deep intron that led to intronic exonization containing a stop codon; however, the patient showed a clearly milder phenotype for X-linked Alport syndrome in men with a truncating mutation. mRNA extracted from the kidney showed both normal and abnormal transcripts, with the normal transcript resulting in the milder phenotype. This novel mechanism leads to mild clinical characteristics. This report highlights the importance of analyzing transcripts to enhance the mutation detection rate and provides insight into genotype-phenotype correlations. This approach can clarify the cause of atypically mild phenotypes in X-linked Alport syndrome. Copyright © 2014 by the American Society of Nephrology.

A comprehensive functional analysis of PTEN mutations: implications in tumor- and autism-related syndromes.

PubMed

Rodríguez-Escudero, Isabel; Oliver, María D; Andrés-Pons, Amparo; Molina, María; Cid, Víctor J; Pulido, Rafael

2011-11-01

The PTEN (phosphatase and tensin homolog) phosphatase is unique in mammals in terms of its tumor suppressor activity, exerted by dephosphorylation of the lipid second messenger PIP(3) (phosphatidylinositol 3,4,5-trisphosphate), which activates the phosphoinositide 3-kinase/Akt/mTOR (mammalian target of rapamycin) oncogenic pathway. Loss-of-function mutations in the PTEN gene are frequent in human cancer and in the germline of patients with PTEN hamartoma tumor-related syndromes (PHTSs). In addition, PTEN is mutated in patients with autism spectrum disorders (ASDs), although no functional information on these mutations is available. Here, we report a comprehensive in vivo functional analysis of human PTEN using a heterologous yeast reconstitution system. Ala-scanning mutagenesis at the catalytic loops of PTEN outlined the critical role of residues within the P-catalytic loop for PIP(3) phosphatase activity in vivo. PTEN mutations that mimic the P-catalytic loop of mammalian PTEN-like proteins (TPTE, TPIP, tensins and auxilins) affected PTEN function variably, whereas tumor- or PHTS-associated mutations targeting the PTEN P-loop produced complete loss of function. Conversely, Ala-substitutions, as well as tumor-related mutations at the WPD- and TI-catalytic loops, displayed partial activity in many cases. Interestingly, a tumor-related D92N mutation was partially active, supporting the notion that the PTEN Asp92 residue might not function as the catalytic general acid. The analysis of a panel of ASD-associated hereditary PTEN mutations revealed that most of them did not substantially abrogate PTEN activity in vivo, whereas most of PHTS-associated mutations did. Our findings reveal distinctive functional patterns among PTEN mutations found in tumors and in the germline of PHTS and ASD patients, which could be relevant for therapy.

Mutations in POGLUT1, Encoding Protein O-Glucosyltransferase 1, Cause Autosomal-Dominant Dowling-Degos Disease

PubMed Central

Basmanav, F. Buket; Oprisoreanu, Ana-Maria; Pasternack, Sandra M.; Thiele, Holger; Fritz, Günter; Wenzel, Jörg; Größer, Leopold; Wehner, Maria; Wolf, Sabrina; Fagerberg, Christina; Bygum, Anette; Altmüller, Janine; Rütten, Arno; Parmentier, Laurent; El Shabrawi-Caelen, Laila; Hafner, Christian; Nürnberg, Peter; Kruse, Roland; Schoch, Susanne; Hanneken, Sandra; Betz, Regina C.

2014-01-01

Dowling-Degos disease (DDD) is an autosomal-dominant genodermatosis characterized by progressive and disfiguring reticulate hyperpigmentation. We previously identified loss-of-function mutations in KRT5 but were only able to detect pathogenic mutations in fewer than half of our subjects. To identify additional causes of DDD, we performed exome sequencing in five unrelated affected individuals without mutations in KRT5. Data analysis identified three heterozygous mutations from these individuals, all within the same gene. These mutations, namely c.11G>A (p.Trp4∗), c.652C>T (p.Arg218∗), and c.798-2A>C, are within POGLUT1, which encodes protein O-glucosyltransferase 1. Further screening of unexplained cases for POGLUT1 identified six additional mutations, as well as two of the above described mutations. Immunohistochemistry of skin biopsies of affected individuals with POGLUT1 mutations showed significantly weaker POGLUT1 staining in comparison to healthy controls with strong localization of POGLUT1 in the upper parts of the epidermis. Immunoblot analysis revealed that translation of either wild-type (WT) POGLUT1 or of the protein carrying the p.Arg279Trp substitution led to the expected size of about 50 kDa, whereas the c.652C>T (p.Arg218∗) mutation led to translation of a truncated protein of about 30 kDa. Immunofluorescence analysis identified a colocalization of the WT protein with the endoplasmic reticulum and a notable aggregating pattern for the truncated protein. Recently, mutations in POFUT1, which encodes protein O-fucosyltransferase 1, were also reported to be responsible for DDD. Interestingly, both POGLUT1 and POFUT1 are essential regulators of Notch activity. Our results furthermore emphasize the important role of the Notch pathway in pigmentation and keratinocyte morphology. PMID:24387993

Functional and splicing defect analysis of 23 ACVRL1 mutations in a cohort of patients affected by Hereditary Hemorrhagic Telangiectasia

PubMed Central

Alaa el Din, Ferdos; Patri, Sylvie; Thoreau, Vincent; Rodriguez-Ballesteros, Montserrat; Hamade, Eva; Bailly, Sabine; Gilbert-Dussardier, Brigitte; Abou Merhi, Raghida; Kitzis, Alain

2015-01-01

Hereditary Hemorrhagic Telangiectasia syndrome (HHT) or Rendu-Osler-Weber (ROW) syndrome is an autosomal dominant vascular disorder. Two most common forms of HHT, HHT1 and HHT2, have been linked to mutations in the endoglin (ENG) and activin receptor-like kinase 1 (ACVRL1or ALK1) genes respectively. This work was designed to examine the pathogenicity of 23 nucleotide variations in ACVRL1 gene detected in more than 400 patients. Among them, 14 missense mutations and one intronic variant were novels, and 8 missense mutations were previously identified with questionable implication in HHT2. The functionality of missense mutations was analyzed in response to BMP9 (specific ligand of ALK1), the maturation of the protein products and their localization were analyzed by western blot and fluorescence microscopy. The splicing impairment of the intronic and of two missense mutations was examined by minigene assay. Functional analysis showed that 18 out of 22 missense mutations were defective. Splicing analysis revealed that one missense mutation (c.733A>G, p.Ile245Val) affects the splicing of the harboring exon 6. Similarly, the intronic mutation outside the consensus splicing sites (c.1048+5G>A in intron 7) was seen pathogenic by splicing study. Both mutations induce a frame shift creating a premature stop codon likely resulting in mRNA degradation by NMD surveillance mechanism. Our results confirm the haploinsufficiency model proposed for HHT2. The affected allele of ACVRL1 induces mRNA degradation or the synthesis of a protein lacking the receptor activity. Furthermore, our data demonstrate that functional and splicing analyses together, represent two robust diagnostic tools to be used by geneticists confronted with novel or conflicted ACVRL1 mutations. PMID:26176610

TERT promoter mutation and its interaction with IDH mutations in glioma: Combined TERT promoter and IDH mutations stratifies lower-grade glioma into distinct survival subgroups-A meta-analysis of aggregate data.

PubMed

Vuong, Huy Gia; Altibi, Ahmed M A; Duong, Uyen N P; Ngo, Hanh T T; Pham, Thong Quang; Chan, Aden Ka-Yin; Park, Chul-Kee; Fung, Kar-Ming; Hassell, Lewis

2017-12-01

The clinical significance of telomerase reverse transcriptase (TERT) promoter mutation in glioma remains unclear. The aim of our meta-analysis is to investigate the prognostic impact TERT promoter mutation in glioma patients and its interaction with other molecular markers, particularly Isocitrate Dehydrogenase (IDH) mutation from aggregate level data. Relevant articles were searched in four electronic databases including PubMed, Scopus, Web of Science and Virtual Health Library. Pooled HRs were calculated using random effect model weighted by inverse variance method. From 1010 studies, we finally included 28 studies with 11519 patients for meta-analyses. TERT mutation is significantly associated with compromised overall survival (OS) (HR=1.38; 95% CI=1.15-1.67) and progression-free survival (PFS) (HR=1.31; 95% CI=1.06-1.63) in glioma patients. In studying its reaction with IDH, TERT promoter mutation was associated with reduced OS in both IDH-mutant (IDH-mut) and IDH-wild type (IDH-wt) glioblastomas but shown to have inverse effects on IDH-mut and IDH-wt grade II/III tumors. Our analysis categorized WHO grade II/III glioma patients into four distinct survival subgroups with descending survival as follow: TERT-mut/IDH-mut≫TERT-wt/IDH-mut≫TERT-wt/IDH-wt≫TERT-mut/IDH-wt. Prognostic value of TERT promoter mutations in gliomas is dependent on tumor grade and the IDH mutational status. With the same tumor grade in WHO grade II and III tumors and the same IDH mutation status, TERT-mut is a prognostic factor. Copyright © 2017 Elsevier B.V. All rights reserved.

Integrated Analysis of Mutation Data from Various Sources Identifies Key Genes and Signaling Pathways in Hepatocellular Carcinoma

PubMed Central

Wei, Lin; Tang, Ruqi; Lian, Baofeng; Zhao, Yingjun; He, Xianghuo; Xie, Lu

2014-01-01

Background Recently, a number of studies have performed genome or exome sequencing of hepatocellular carcinoma (HCC) and identified hundreds or even thousands of mutations in protein-coding genes. However, these studies have only focused on a limited number of candidate genes, and many important mutation resources remain to be explored. Principal Findings In this study, we integrated mutation data obtained from various sources and performed pathway and network analysis. We identified 113 pathways that were significantly mutated in HCC samples and found that the mutated genes included in these pathways contained high percentages of known cancer genes, and damaging genes and also demonstrated high conservation scores, indicating their important roles in liver tumorigenesis. Five classes of pathways that were mutated most frequently included (a) proliferation and apoptosis related pathways, (b) tumor microenvironment related pathways, (c) neural signaling related pathways, (d) metabolic related pathways, and (e) circadian related pathways. Network analysis further revealed that the mutated genes with the highest betweenness coefficients, such as the well-known cancer genes TP53, CTNNB1 and recently identified novel mutated genes GNAL and the ADCY family, may play key roles in these significantly mutated pathways. Finally, we highlight several key genes (e.g., RPS6KA3 and PCLO) and pathways (e.g., axon guidance) in which the mutations were associated with clinical features. Conclusions Our workflow illustrates the increased statistical power of integrating multiple studies of the same subject, which can provide biological insights that would otherwise be masked under individual sample sets. This type of bioinformatics approach is consistent with the necessity of making the best use of the ever increasing data provided in valuable databases, such as TCGA, to enhance the speed of deciphering human cancers. PMID:24988079

Integrated analysis of mutation data from various sources identifies key genes and signaling pathways in hepatocellular carcinoma.

PubMed

Zhang, Yuannv; Qiu, Zhaoping; Wei, Lin; Tang, Ruqi; Lian, Baofeng; Zhao, Yingjun; He, Xianghuo; Xie, Lu

2014-01-01

Recently, a number of studies have performed genome or exome sequencing of hepatocellular carcinoma (HCC) and identified hundreds or even thousands of mutations in protein-coding genes. However, these studies have only focused on a limited number of candidate genes, and many important mutation resources remain to be explored. In this study, we integrated mutation data obtained from various sources and performed pathway and network analysis. We identified 113 pathways that were significantly mutated in HCC samples and found that the mutated genes included in these pathways contained high percentages of known cancer genes, and damaging genes and also demonstrated high conservation scores, indicating their important roles in liver tumorigenesis. Five classes of pathways that were mutated most frequently included (a) proliferation and apoptosis related pathways, (b) tumor microenvironment related pathways, (c) neural signaling related pathways, (d) metabolic related pathways, and (e) circadian related pathways. Network analysis further revealed that the mutated genes with the highest betweenness coefficients, such as the well-known cancer genes TP53, CTNNB1 and recently identified novel mutated genes GNAL and the ADCY family, may play key roles in these significantly mutated pathways. Finally, we highlight several key genes (e.g., RPS6KA3 and PCLO) and pathways (e.g., axon guidance) in which the mutations were associated with clinical features. Our workflow illustrates the increased statistical power of integrating multiple studies of the same subject, which can provide biological insights that would otherwise be masked under individual sample sets. This type of bioinformatics approach is consistent with the necessity of making the best use of the ever increasing data provided in valuable databases, such as TCGA, to enhance the speed of deciphering human cancers.

Stickler syndrome caused by COL2A1 mutations: genotype–phenotype correlation in a series of 100 patients

PubMed Central

Hoornaert, Kristien P; Vereecke, Inge; Dewinter, Chantal; Rosenberg, Thomas; Beemer, Frits A; Leroy, Jules G; Bendix, Laila; Björck, Erik; Bonduelle, Maryse; Boute, Odile; Cormier-Daire, Valerie; De Die-Smulders, Christine; Dieux-Coeslier, Anne; Dollfus, Hélène; Elting, Mariet; Green, Andrew; Guerci, Veronica I; Hennekam, Raoul C M; Hilhorts-Hofstee, Yvonne; Holder, Muriel; Hoyng, Carel; Jones, Kristi J; Josifova, Dragana; Kaitila, Ilkka; Kjaergaard, Suzanne; Kroes, Yolande H; Lagerstedt, Kristina; Lees, Melissa; LeMerrer, Martine; Magnani, Cinzia; Marcelis, Carlo; Martorell, Loreto; Mathieu, Michèle; McEntagart, Meriel; Mendicino, Angela; Morton, Jenny; Orazio, Gabrielli; Paquis, Véronique; Reish, Orit; Simola, Kalle O J; Smithson, Sarah F; Temple, Karen I; Van Aken, Elisabeth; Van Bever, Yolande; van den Ende, Jenneke; Van Hagen, Johanna M; Zelante, Leopoldo; Zordania, Riina; De Paepe, Anne; Leroy, Bart P; De Buyzere, Marc; Coucke, Paul J; Mortier, Geert R

2010-01-01

Stickler syndrome is an autosomal dominant connective tissue disorder caused by mutations in different collagen genes. The aim of our study was to define more precisely the phenotype and genotype of Stickler syndrome type 1 by investigating a large series of patients with a heterozygous mutation in COL2A1. In 188 probands with the clinical diagnosis of Stickler syndrome, the COL2A1 gene was analyzed by either a mutation scanning technique or bidirectional fluorescent DNA sequencing. The effect of splice site alterations was investigated by analyzing mRNA. Multiplex ligation-dependent amplification analysis was used for the detection of intragenic deletions. We identified 77 different COL2A1 mutations in 100 affected individuals. Analysis of the splice site mutations showed unusual RNA isoforms, most of which contained a premature stop codon. Vitreous anomalies and retinal detachments were found more frequently in patients with a COL2A1 mutation compared with the mutation-negative group (P<0.01). Overall, 20 of 23 sporadic patients with a COL2A1 mutation had either a cleft palate or retinal detachment with vitreous anomalies. The presence of vitreous anomalies, retinal tears or detachments, cleft palate and a positive family history were shown to be good indicators for a COL2A1 defect. In conclusion, we confirm that Stickler syndrome type 1 is predominantly caused by loss-of-function mutations in the COL2A1 gene as >90% of the mutations were predicted to result in nonsense-mediated decay. On the basis of binary regression analysis, we developed a scoring system that may be useful when evaluating patients with Stickler syndrome. PMID:20179744

Autosomal dominant tubulointerstitial kidney disease caused by uromodulin mutations: seek and you will find.

PubMed

Raffler, Gabriele; Zitt, Emanuel; Sprenger-Mähr, Hannelore; Nagel, Mato; Lhotta, Karl

2016-04-01

Uromodulin (UMOD)-associated kidney disease belongs to the group of autosomal dominant interstitial kidney diseases and is caused by mutations in the UMOD gene. Affected patients present with hyperuricemia, gout, and progressive renal failure. The disease is thought to be very rare but is probably underdiagnosed. Two index patients from two families with tubulointerstitial nephropathy and hyperuricemia were examined, including blood and urine chemistry, ultrasound, and mutation analysis of the UMOD gene. In addition, other available family members were studied. In a 46-year-old female patient with a fractional excretion of uric acid of 3 %, analysis of the UMOD gene revealed a p.W202S missense mutation. The same mutation was found in her 72-year-old father, who suffers from gout and end-stage renal disease. The second index patient was a 47-year-old female with chronic kidney disease and gout for more than 10 years. Her fractional uric acid excretion was 3.5 %. Genetic analysis identified a novel p.H250Q UMOD mutation that was also present in her 12-year-old son, who had normal renal function and uric acid levels. In patients suffering from chronic tubulointerstitial nephropathy, hyperuricemia, and a low fractional excretion of uric acid mutation, analysis of the UMOD gene should be performed to diagnose UMOD-associated kidney disease.

A high proportion of ADA point mutations associated with a specific alanine-to-valine substitution.

PubMed

Markert, M L; Norby-Slycord, C; Ward, F E

1989-09-01

In 15%-20% of children with severe combined immunodeficiency (SCID), the underlying defect is adenosine deaminase (ADA) deficiency. The overall goal of our research has been to identify the precise molecular defects in patients with ADA-deficient SCID. In this study, we focused on a patient whom we found to have normal sized ADA mRNA by Northern analysis and an intact ADA structural gene by Southern analysis. By cloning and sequencing this patient's ADA cDNA, we found a C-to-T point mutation in exon 11. This resulted in the amino acid substitution of a valine for an alanine at position 329 of the ADA protein. Sequence analysis revealed that this mutation created a new BalI restriction site. Using Southern analyses, we were able to directly screen individuals to determine the frequency of this mutation. By combining data on eight families followed at our institution with data on five other families reported in the literature, we established that five of 13 patients (seven of 22 alleles) with known or suspected point mutations have this defect. This mutation was found to be associated with three different ADA haplotypes. This argues against a founder effect and suggests that the mutation is very old. In summary, a conservative amino acid substitution is found in a high proportion of patients with ADA deficiency; this can easily be detected by Southern analysis.

Patterns of haplotypes for 92 cystic fibrosis mutations: Variability, association and recurrence

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morral, N.; Llevadot, R.; Estivill, X.

1994-09-01

Most CFTR mutations are very uncommon among the cystic fibrosis population, with frequencies of less than 1%, and many are found only in specific areas. We have analyzed 92 CF mutations for several markers (4 microsatellites and 3 other polymorphisms) scattered in the CFTR gene. Haplotypes associated with these mutations can be used as a framework in the screening of chromosomes carrying unknown mutations. The association between mutation and haplotype reduces the number of mutations it is necessary to search for to a maximum of 16 for the same haplotype. Only mutations {triangle}F508, G542X and N1303K are associated with moremore » than one haplotype as a result of slippage at more than one microsatellite loci, suggesting that these three are the most ancient CF mutations. Recurrence has been found for at least 7 mutations: H199Y, R347P, L558S, R553X, 2184insA, 3272-26A{r_arrow}G, 3849+10kbC{r_arrow}T and R1162X. Also microsatellite analysis of chromosomes of several ethnic origins (Czech, Italian, Russian, Slovac and Spanish) suggested that possibility of three or more independent origins for mutations R334W, R347P, R1162X, and 3849+10kbC{r_arrow}T, which was confirmed by analysis of markers flanking these mutations.« less

The first USH2A mutation analysis of Japanese autosomal recessive retinitis pigmentosa patients: a totally different mutation profile with the lack of frequent mutations found in Caucasian patients.

PubMed

Zhao, Yang; Hosono, Katsuhiro; Suto, Kimiko; Ishigami, Chie; Arai, Yuuki; Hikoya, Akiko; Hirami, Yasuhiko; Ohtsubo, Masafumi; Ueno, Shinji; Terasaki, Hiroko; Sato, Miho; Nakanishi, Hiroshi; Endo, Shiori; Mizuta, Kunihiro; Mineta, Hiroyuki; Kondo, Mineo; Takahashi, Masayo; Minoshima, Shinsei; Hotta, Yoshihiro

2014-09-01

Retinitis pigmentosa (RP) is a highly heterogeneous genetic disease. The USH2A gene, which accounts for approximately 74-90% of Usher syndrome type 2 (USH2) cases, is also one of the major autosomal recessive RP (arRP) causative genes among Caucasian populations. To identify disease-causing USH2A gene mutations in Japanese RP patients, all 73 exons were screened for mutations by direct sequencing. In total, 100 unrelated Japanese RP patients with no systemic manifestations were identified, excluding families with obvious autosomal dominant inheritance. Of these 100 patients, 82 were included in this present study after 18 RP patients with very likely pathogenic EYS (eyes shut homolog) mutations were excluded. The mutation analysis of the USH2A revealed five very likely pathogenic mutations in four patients. A patient had only one very likely pathogenic mutation and the others had two of them. Caucasian frequent mutations p.C759F in arRP and p.E767fs in USH2 were not found. All the four patients exhibited typical clinical features of RP. The observed prevalence of USH2A gene mutations was approximately 4% among Japanese arRP patients, and the profile of the USH2A gene mutations differed largely between Japanese patients and previously reported Caucasian populations.

ImatraNMR: novel software for batch integration and analysis of quantitative NMR spectra.

PubMed

Mäkelä, A V; Heikkilä, O; Kilpeläinen, I; Heikkinen, S

2011-08-01

Quantitative NMR spectroscopy is a useful and important tool for analysis of various mixtures. Recently, in addition of traditional quantitative 1D (1)H and (13)C NMR methods, a variety of pulse sequences aimed for quantitative or semiquantitative analysis have been developed. To obtain actual usable results from quantitative spectra, they must be processed and analyzed with suitable software. Currently, there are many processing packages available from spectrometer manufacturers and third party developers, and most of them are capable of analyzing and integration of quantitative spectra. However, they are mainly aimed for processing single or few spectra, and are slow and difficult to use when large numbers of spectra and signals are being analyzed, even when using pre-saved integration areas or custom scripting features. In this article, we present a novel software, ImatraNMR, designed for batch analysis of quantitative spectra. In addition to capability of analyzing large number of spectra, it provides results in text and CSV formats, allowing further data-analysis using spreadsheet programs or general analysis programs, such as Matlab. The software is written with Java, and thus it should run in any platform capable of providing Java Runtime Environment version 1.6 or newer, however, currently it has only been tested with Windows and Linux (Ubuntu 10.04). The software is free for non-commercial use, and is provided with source code upon request. Copyright © 2011 Elsevier Inc. All rights reserved.

ssaD1, a suppressor of secA51(Ts) that renders growth of Escherichia coli cold sensitive, is an early amber mutation in the transcription factor gene nusB.

PubMed Central

Rajapandi, T.; Oliver, D.

1994-01-01

Complementation analysis of the ssaD1 mutation, isolated as a suppressor of the secA51(Ts) mutation that renders growth of Escherichia coli cold sensitive, was used to show that ssaD corresponds to nusB, a gene known to be important in transcription antitermination. DNA sequence analysis of the ssaD1 allele showed that it creates an amber mutation in the 15th codon of nusB. Analysis of the effect of different levels of NusB protein on secA transcription and translation suggested that NusB plays little or no role in the control of secA expression. Accordingly, mechanisms by which nusB inactivation can lead to suppression of secA51(Ts) and secY24(Ts) mutations without affecting secA expression need to be considered. PMID:8021230

Molecular and Clinical Characterization of Albinism in a Large Cohort of Italian Patients

PubMed Central

Gargiulo, Annagiusi; Testa, Francesco; Rossi, Settimio; Di Iorio, Valentina; Fecarotta, Simona; de Berardinis, Teresa; Iovine, Antonello; Magli, Adriano; Signorini, Sabrina; Fazzi, Elisa; Galantuomo, Maria Silvana; Fossarello, Maurizio; Montefusco, Sandro; Ciccodicola, Alfredo; Neri, Alberto; Macaluso, Claudio; Simonelli, Francesca; Surace, Enrico Maria

2011-01-01

Purpose. The purpose of this study was to identify the molecular basis of albinism in a large cohort of Italian patients showing typical ocular landmarks of the disease and to provide a full characterization of the clinical ophthalmic manifestations. Methods. DNA samples from 45 patients with ocular manifestations of albinism were analyzed by direct sequencing analysis of five genes responsible for albinism: TYR, P, TYRP1, SLC45A2 (MATP), and OA1. All patients studied showed a variable degree of skin and hair hypopigmentation. Eighteen patients with distinct mutations in each gene associated with OCA were evaluated by detailed ophthalmic analysis, optical coherence tomography (OCT), and fundus autofluorescence. Results. Disease-causing mutations were identified in more than 95% of analyzed patients with OCA (28/45 [62.2%] cases with two or more mutations; 15/45 [33.3%] cases with one mutation). Thirty-five different mutant alleles were identified of which 15 were novel. Mutations in TYR were the most frequent (73.3%), whereas mutations in P occurred more rarely (13.3%) than previously reported. Novel mutations were also identified in rare loci such as TYRP1 and MATP. Mutations in the OA1 gene were not detected. Clinical assessment revealed that patients with iris and macular pigmentation had significantly higher visual acuity than did severe hypopigmented phenotypes. Conclusions. TYR gene mutations represent a relevant cause of oculocutaneous albinism in Italy, whereas mutations in P present a lower frequency than that found in other populations. Clinical analysis revealed that the severity of the ocular manifestations depends on the degree of retinal pigmentation. PMID:20861488

Muscle RAS oncogene homolog (MRAS) recurrent mutation in Borrmann type IV gastric cancer.

PubMed

Yasumoto, Makiko; Sakamoto, Etsuko; Ogasawara, Sachiko; Isobe, Taro; Kizaki, Junya; Sumi, Akiko; Kusano, Hironori; Akiba, Jun; Torimura, Takuji; Akagi, Yoshito; Itadani, Hiraku; Kobayashi, Tsutomu; Hasako, Shinichi; Kumazaki, Masafumi; Mizuarai, Shinji; Oie, Shinji; Yano, Hirohisa

2017-01-01

The prognosis of patients with Borrmann type IV gastric cancer (Type IV) is extremely poor. Thus, there is an urgent need to elucidate the molecular mechanisms underlying the oncogenesis of Type IV and to identify new therapeutic targets. Although previous studies using whole-exome and whole-genome sequencing have elucidated genomic alterations in gastric cancer, none has focused on comprehensive genetic analysis of Type IV. To discover cancer-relevant genes in Type IV, we performed whole-exome sequencing and genome-wide copy number analysis on 13 patients with Type IV. Exome sequencing identified 178 somatic mutations in protein-coding sequences or at splice sites. Among the mutations, we found a mutation in muscle RAS oncogene homolog (MRAS), which is predicted to cause molecular dysfunction. MRAS belongs to the Ras subgroup of small G proteins, which includes the prototypic RAS oncogenes. We analyzed an additional 46 Type IV samples to investigate the frequency of MRAS mutation. There were eight nonsynonymous mutations (mutation frequency, 17%), showing that MRAS is recurrently mutated in Type IV. Copy number analysis identified six focal amplifications and one homozygous deletion, including insulin-like growth factor 1 receptor (IGF1R) amplification. The samples with IGF1R amplification had remarkably higher IGF1R mRNA and protein expression levels compared with the other samples. This is the first report of MRAS recurrent mutation in human tumor samples. Our results suggest that MRAS mutation and IGF1R amplification could drive tumorigenesis of Type IV and could be new therapeutic targets. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Network Analysis of Protein Adaptation: Modeling the Functional Impact of Multiple Mutations

PubMed Central

Beleva Guthrie, Violeta; Masica, David L; Fraser, Andrew; Federico, Joseph; Fan, Yunfan; Camps, Manel; Karchin, Rachel

2018-01-01

Abstract The evolution of new biochemical activities frequently involves complex dependencies between mutations and rapid evolutionary radiation. Mutation co-occurrence and covariation have previously been used to identify compensating mutations that are the result of physical contacts and preserve protein function and fold. Here, we model pairwise functional dependencies and higher order interactions that enable evolution of new protein functions. We use a network model to find complex dependencies between mutations resulting from evolutionary trade-offs and pleiotropic effects. We present a method to construct these networks and to identify functionally interacting mutations in both extant and reconstructed ancestral sequences (Network Analysis of Protein Adaptation). The time ordering of mutations can be incorporated into the networks through phylogenetic reconstruction. We apply NAPA to three distantly homologous β-lactamase protein clusters (TEM, CTX-M-3, and OXA-51), each of which has experienced recent evolutionary radiation under substantially different selective pressures. By analyzing the network properties of each protein cluster, we identify key adaptive mutations, positive pairwise interactions, different adaptive solutions to the same selective pressure, and complex evolutionary trajectories likely to increase protein fitness. We also present evidence that incorporating information from phylogenetic reconstruction and ancestral sequence inference can reduce the number of spurious links in the network, whereas preserving overall network community structure. The analysis does not require structural or biochemical data. In contrast to function-preserving mutation dependencies, which are frequently from structural contacts, gain-of-function mutation dependencies are most commonly between residues distal in protein structure. PMID:29522102

Functional Analysis of Somatic Mutations in Lung Cancer

DTIC Science & Technology

2015-10-01

antibody cetuximab [11]. Finally, we have developed novel single cell sequencing approaches to uncover EGFR mutational variants in glioblastoma and their...assessed which mutations are epistatic to EGFR or capable of initiating xenograft tumor formation in vivo. Using eVIP, we identified 69% of mutations...analyzed as impactful whereas 31% appear functionally neutral. A subset of the impactful mutations induce xenograft tumor formation in mice and/or

A simple approach to quantitative analysis using three-dimensional spectra based on selected Zernike moments.

PubMed

Zhai, Hong Lin; Zhai, Yue Yuan; Li, Pei Zhen; Tian, Yue Li

2013-01-21

A very simple approach to quantitative analysis is proposed based on the technology of digital image processing using three-dimensional (3D) spectra obtained by high-performance liquid chromatography coupled with a diode array detector (HPLC-DAD). As the region-based shape features of a grayscale image, Zernike moments with inherently invariance property were employed to establish the linear quantitative models. This approach was applied to the quantitative analysis of three compounds in mixed samples using 3D HPLC-DAD spectra, and three linear models were obtained, respectively. The correlation coefficients (R(2)) for training and test sets were more than 0.999, and the statistical parameters and strict validation supported the reliability of established models. The analytical results suggest that the Zernike moment selected by stepwise regression can be used in the quantitative analysis of target compounds. Our study provides a new idea for quantitative analysis using 3D spectra, which can be extended to the analysis of other 3D spectra obtained by different methods or instruments.

Metastatic site location influences the diagnostic accuracy of ctDNA EGFR- mutation testing in NSCLC patients: a pooled analysis.

PubMed

Passiglia, Francesco; Rizzo, Sergio; Rolfo, Christian; Galvano, Antonio; Bronte, Enrico; Incorvaia, Lorena; Listi, Angela; Barraco, Nadia; Castiglia, Marta; Calo, Valentina; Bazan, Viviana; Russo, Antonio

2018-03-08

Recent studies evaluated the diagnostic accuracy of circulating tumor DNA (ctDNA) in the detection of epidermal growth factor receptor (EGFR) mutations from plasma of NSCLC patients, overall showing a high concordance as compared to standard tissue genotyping. However it is less clear if the location of metastatic site may influence the ability to identify EGFR mutations in plasma. This pooled analysis aims to evaluate the association between the metastatic site location and the sensitivity of ctDNA analysis in detecting EGFR mutations in NSCLC patients. Data from all published studies, evaluating the sensitivity of plasma-based EGFR-mutation testing, stratified by metastatic site location (extrathoracic (M1b) vs intrathoracic (M1a)) were collected by searching in PubMed, Cochrane Library, American Society of Clinical Oncology, and World Conference of Lung Cancer, meeting proceedings. Pooled Odds ratio (OR) and 95% confidence intervals (95% CIs) were calculated for the ctDNA analysis sensitivity, according to metastatic site location. A total of ten studies, with 1425 patients, were eligible. Pooled analysis showed that the sensitivity of ctDNA-based EGFR-mutation testing is significantly higher in patients with M1b vs M1a disease (OR: 5.09; 95% CIs: 2.93 - 8.84). A significant association was observed for both EGFR-activating (OR: 4.30, 95% CI: 2.35-7.88) and resistant T790M mutations (OR: 11.89, 95% CI: 1.45-97.22), regardless of the use of digital-PCR (OR: 5.85, 95% CI: 3.56-9.60) or non-digital PCR technologies (OR: 2.96, 95% CI: 2.24-3.91). These data suggest that the location of metastatic sites significantly influences the diagnostic accuracy of ctDNA analysis in detecting EGFR mutations in NSCLC patients. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Effects of point mutations on the thermostability of B. subtilis lipase: investigating nonadditivity

NASA Astrophysics Data System (ADS)

Singh, Bipin; Bulusu, Gopalakrishnan; Mitra, Abhijit

2016-10-01

Molecular level understanding of mutational effects on stability and activity of enzymes is challenging particularly when several point mutations are incorporated during the directed evolution experiments. In our earlier study, we have suggested the lack of consistency in the effect of point mutations incorporated during the initial generations of directed evolution experiments, towards conformational stabilization of B. subtilis lipase mutants of later generations. Here, we report that the cumulative point mutations incorporated in mutants 2M (with two point mutations) to 6M (with six point mutations) possibly do not retain their original stabilizing nature in the most thermostable 12M mutant (with 12 point mutations). We have carried out MD simulations using structures incorporating reversal of different sets of point mutations to assess their effect on the conformational stability and activity of 12M. Our analysis has revealed that reversal of certain point mutations in 12M had little effect on its conformational stability, suggesting that these mutations were probably inconsequential towards the thermostability of the 12M mutant. Interestingly these mutations involved evolutionarily conserved residues. On the other hand, some of the other point mutations incorporated in nonconserved regions, appeared to contribute significantly towards the conformational stability and/or activity of 12M. Based on the analysis of dynamics of in silico mutants generated using the consensus sequence, we identified experimentally verifiable residue positions to further increase the conformational stability and activity of the 12M mutant.

Effects of point mutations on the thermostability of B. subtilis lipase: investigating nonadditivity.

PubMed

Singh, Bipin; Bulusu, Gopalakrishnan; Mitra, Abhijit

2016-10-01

Molecular level understanding of mutational effects on stability and activity of enzymes is challenging particularly when several point mutations are incorporated during the directed evolution experiments. In our earlier study, we have suggested the lack of consistency in the effect of point mutations incorporated during the initial generations of directed evolution experiments, towards conformational stabilization of B. subtilis lipase mutants of later generations. Here, we report that the cumulative point mutations incorporated in mutants 2M (with two point mutations) to 6M (with six point mutations) possibly do not retain their original stabilizing nature in the most thermostable 12M mutant (with 12 point mutations). We have carried out MD simulations using structures incorporating reversal of different sets of point mutations to assess their effect on the conformational stability and activity of 12M. Our analysis has revealed that reversal of certain point mutations in 12M had little effect on its conformational stability, suggesting that these mutations were probably inconsequential towards the thermostability of the 12M mutant. Interestingly these mutations involved evolutionarily conserved residues. On the other hand, some of the other point mutations incorporated in nonconserved regions, appeared to contribute significantly towards the conformational stability and/or activity of 12M. Based on the analysis of dynamics of in silico mutants generated using the consensus sequence, we identified experimentally verifiable residue positions to further increase the conformational stability and activity of the 12M mutant.

Clinical utility of routine MPL exon 10 analysis in the diagnosis of essential thrombocythaemia and primary myelofibrosis.

PubMed

Boyd, Elaine M; Bench, Anthony J; Goday-Fernández, Andrea; Anand, Shubha; Vaghela, Krishna J; Beer, Phillip; Scott, Mike A; Bareford, David; Green, Anthony R; Huntly, Brian; Erber, Wendy N

2010-04-01

Approximately 50% of essential thrombocythaemia and primary myelo-fibrosis patients do not have a JAK2 V617F mutation. Up to 5% of these are reported to have a MPL exon 10 mutation but testing for MPL is not routine as there are multiple mutation types. The ability to routinely assess both JAK2 and MPL mutations would be beneficial in the differential diagnosis of unexplained thrombocytosis or myelofibrosis. We developed and applied a high resolution melt (HRM) assay, capable of detecting all known MPL mutations in a single analysis, for the detection of MPL exon 10 mutations. We assessed 175 ET and PMF patients, including 67 that were JAK2 V617F-negative by real time polymerase chain reaction (PCR). Overall, 19/175 (11%) patients had a MPL exon 10 mutation, of whom 16 were JAK2 V617F-negative (16/67; 24%). MPL mutation types were W515L (11), W515K (4), W515R (2) and W515A (1). One patient had both W515L and S505N MPL mutations and these were present in the same haemopoietic colonies. Real time PCR for JAK2 V617F analysis and HRM for MPL exon 10 status identified one or more clonal marker in 71% of patients. This combined genetic approach increases the sensitivity of meeting the World Health Organization diagnostic criteria for these myeloproliferative neoplasms.

Molecular basis of cystic fibrosis in Lithuania: incomplete CFTR mutation detection by PCR-based screening protocols.

PubMed

Giannattasio, S; Bobba, A; Jurgelevicius, V; Vacca, R A; Lattanzio, P; Merafina, R S; Utkus, A; Kucinskas, V; Marra, E

2006-01-01

Mutational analysis of the cystic fibrosis transmembrane regulator (CFTR) gene was performed in 98 unrelated CF chromosomes from 49 Lithuanian CF patients through a combined approach in which the p.F508del mutation was first screened by allele-specific PCR while CFTR mutations in nonp.F508del chromosomes have been screened for by denaturing gradient gel electrophoresis analysis. A CFTR mutation was characterized in 62.2% of CF chromosomes, two of which (2.0%) have been previously shown to carry a large gene deletion CFTRdele2,3(21 kb). The most frequent Lithuanian CF mutation is p.F508del (52.0%). Seven CFTR mutations, p.N1303K (2.0%), p.R75Q (1.0%), p.G314R (1.0%), p.R553X (4.2%), p.W1282X (1.0%), and g.3944delGT (1.0%), accounted for 10.1% of Lithuanian CF chromosomes. It was not possible to characterize 35.8% of the CF Lithuanian chromosomes. Analysis of intron 8 (TG)mTn and M470V polymorphic loci did not permit the characterization of the CFTR dysfunction underlying the CF phenotype in the patients for which no CFTR mutation was identified. Thus, screening of the eight CFTR mutations identified in this study and of the large deletion CFTRdele2,3(21 kb) allows the implementation of an early molecular or confirmatory CF diagnosis for 65% of Lithuanian CF chromosomes.

[Mutation Analysis of 19 STR Loci in 20 723 Cases of Paternity Testing].

PubMed

Bi, J; Chang, J J; Li, M X; Yu, C Y

2017-06-01

To observe and analyze the confirmed cases of paternity testing, and to explore the mutation rules of STR loci. The mutant STR loci were screened from 20 723 confirmed cases of paternity testing by Goldeneye 20A system．The mutation rates, and the sources, fragment length, steps and increased or decreased repeat sequences of mutant alleles were counted for the analysis of the characteristics of mutation-related factors. A total of 548 mutations were found on 19 STR loci, and 557 mutation events were observed. The loci mutation rate was 0.07‰-2.23‰. The ratio of paternal to maternal mutant events was 3.06:1. One step mutation was the main mutation, and the number of the increased repeat sequences was almost the same as the decreased repeat sequences. The repeat sequences were more likely to decrease in two steps mutation and above. Mutation mainly occurred in the medium allele, and the number of the increased repeat sequences was almost the same as the decreased repeat sequences. In long allele mutations, the decreased repeat sequences were significantly more than the increased repeat sequences. The number of the increased repeat sequences was almost the same as the decreased repeat sequences in paternal mutation, while the decreased repeat sequences were more than the increased in maternal mutation. There are significant differences in the mutation rate of each locus. When one or two loci do not conform to the genetic law, other detection system should be added, and PI value should be calculated combined with the information of the mutate STR loci in order to further clarify the identification opinions. Copyright© by the Editorial Department of Journal of Forensic Medicine

Mutation analysis of the APC gene in Taiwanese FAP families: low incidence of APC germline mutation in a distinct subgroup of FAP families.

PubMed

Chiang, J M; Chen, H W; Tang, R P; Chen, J S; Changchien, C R; Hsieh, P S; Wang, J Y

2010-06-01

Familial adenomatous polyposis (FAP) is an autosomal-dominant disease caused by germline mutations in the adenomatous polyposis coli (APC) gene. The affected individuals develop colorectal polyposis and show various extra-colonic manifestations. In this study, we aimed to investigate the genetic and clinical characteristics of FAP in Taiwanese families and analyze the genotype-phenotype correlations. Blood samples were obtained from 66 FAP patients registered in the hereditary colorectal cancer database. Then, germline mutations in the APC genes of these 66 polyposis patients from 47 unrelated FAP families were analyzed. The germline-mutation-negative cases were analyzed by performing multiplex ligation-dependent probe amplification (MLPA) and single-strand conformation polymorphism (SSCP) analysis of the MUTYH gene. Among the analyzed families, 79% (37/47) of the families showed 28 APC mutations, including 19 frameshift mutations, 4 nonsense mutations, 3 genomic deletion mutations, 1 missense mutation, and 1 splice-site mutation. In addition, we identified 15 novel mutations in 32% (15/47) of the families. The cases in which APC mutations were not identified showed significantly lower incidence of profuse polyposis (P = 0.034) and gastroduodenal polyps (P = 0.027). Furthermore, FAP families in which some affected individuals had less than 100 polyps showed significant association with low incidence of APC germline mutations (P = 0.002). We have added the APC germline-mutation data for Taiwanese FAP patients and indicated the presence of an FAP subgroup comprising affected individuals with nonadenomatous polyps or less than 100 adenomatous polyps; this form of FAP is less frequently caused by germline mutations of the APC gene.

Comparison of neutron spectra measured with three sizes of organic liquid scintillators using differentiation analysis

NASA Technical Reports Server (NTRS)

Shook, D. F.; Pierce, C. R.

1972-01-01

Proton recoil distributions were obtained by using organic liquid scintillators of different size. The measured distributions are converted to neutron spectra by differentiation analysis for comparison to the unfolded spectra of the largest scintillator. The approximations involved in the differentiation analysis are indicated to have small effects on the precision of neutron spectra measured with the smaller scintillators but introduce significant error for the largest scintillator. In the case of the smallest cylindrical scintillator, nominally 1.2 by 1.3 cm, the efficiency is shown to be insensitive to multiple scattering and to the angular distribution to the incident flux. These characteristics of the smaller scintillator make possible its use to measure scalar flux spectra within media high efficiency is not required.

Is High Resolution Melting Analysis (HRMA) Accurate for Detection of Human Disease-Associated Mutations? A Meta Analysis

PubMed Central

Ma, Feng-Li; Jiang, Bo; Song, Xiao-Xiao; Xu, An-Gao

2011-01-01

Background High Resolution Melting Analysis (HRMA) is becoming the preferred method for mutation detection. However, its accuracy in the individual clinical diagnostic setting is variable. To assess the diagnostic accuracy of HRMA for human mutations in comparison to DNA sequencing in different routine clinical settings, we have conducted a meta-analysis of published reports. Methodology/Principal Findings Out of 195 publications obtained from the initial search criteria, thirty-four studies assessing the accuracy of HRMA were included in the meta-analysis. We found that HRMA was a highly sensitive test for detecting disease-associated mutations in humans. Overall, the summary sensitivity was 97.5% (95% confidence interval (CI): 96.8–98.5; I2 = 27.0%). Subgroup analysis showed even higher sensitivity for non-HR-1 instruments (sensitivity 98.7% (95%CI: 97.7–99.3; I2 = 0.0%)) and an eligible sample size subgroup (sensitivity 99.3% (95%CI: 98.1–99.8; I2 = 0.0%)). HRMA specificity showed considerable heterogeneity between studies. Sensitivity of the techniques was influenced by sample size and instrument type but by not sample source or dye type. Conclusions/Significance These findings show that HRMA is a highly sensitive, simple and low-cost test to detect human disease-associated mutations, especially for samples with mutations of low incidence. The burden on DNA sequencing could be significantly reduced by the implementation of HRMA, but it should be recognized that its sensitivity varies according to the number of samples with/without mutations, and positive results require DNA sequencing for confirmation. PMID:22194806

Establishing high resolution melting analysis: method validation and evaluation for c-RET proto-oncogene mutation screening.

PubMed

Benej, Martin; Bendlova, Bela; Vaclavikova, Eliska; Poturnajova, Martina

2011-10-06

Reliable and effective primary screening of mutation carriers is the key condition for common diagnostic use. The objective of this study is to validate the method high resolution melting (HRM) analysis for routine primary mutation screening and accomplish its optimization, evaluation and validation. Due to their heterozygous nature, germline point mutations of c-RET proto-oncogene, associated to multiple endocrine neoplasia type 2 (MEN2), are suitable for HRM analysis. Early identification of mutation carriers has a major impact on patients' survival due to early onset of medullary thyroid carcinoma (MTC) and resistance to conventional therapy. The authors performed a series of validation assays according to International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines for validation of analytical procedures, along with appropriate design and optimization experiments. After validated evaluation of HRM, the method was utilized for primary screening of 28 pathogenic c-RET mutations distributed among nine exons of c-RET gene. Validation experiments confirm the repeatability, robustness, accuracy and reproducibility of HRM. All c-RET gene pathogenic variants were detected with no occurrence of false-positive/false-negative results. The data provide basic information about design, establishment and validation of HRM for primary screening of genetic variants in order to distinguish heterozygous point mutation carriers among the wild-type sequence carriers. HRM analysis is a powerful and reliable tool for rapid and cost-effective primary screening, e.g., of c-RET gene germline and/or sporadic mutations and can be used as a first line potential diagnostic tool.

Novel and recurrent NDP gene mutations in familial cases of Norrie disease and X-linked exudative vitreoretinopathy.

PubMed

Pelcastre, Erika L; Villanueva-Mendoza, Cristina; Zenteno, Juan C

2010-05-01

To present the results of molecular analysis of the NDP gene in Mexican families with Norrie disease (ND) and X-linked familial exudative vitreoretinopathy (XL-FEVR). Two unrelated families with ND and two with XL-FEVR were studied. Clinical diagnosis was suspected on the basis of a complete ophthalmologic examination. Molecular methods included DNA isolation from peripheral blood leucocytes, polymerase chain reaction amplification and direct nucleotide sequencing analysis of the complete coding region and exon-intron junctions of NDP. Haplotype analysis using NDP-linked microsatellites markers was performed in both ND families. A novel Norrin missense mutation, p.Arg41Thr, was identified in two apparently unrelated families with ND. Haplotype analysis demonstrated that affected males in these two families shared the same ND-linked haplotype, suggesting a common origin for this novel mutation. The previously reported p.Arg121Trp and p.Arg121Gln Norrin mutations were identified in the two families with XL-FEVR. Our results expand the mutational spectrum in ND. This is the first report of ND resulting from mutation at arginine position 41 of Norrin. Interestingly, mutations at the same residue but resulting in a different missense change were previously described in subjects with XL-FEVR (p.Arg41Lys) or persistent fetal vasculature syndrome (p.Arg41Ser), indicating that the novel p.Arg41Thr change causes a more severe retinal phenotype. Preliminary data suggest a founder effect for the ND p.Arg41Thr mutation in these two Mexican families.

Identification of novel mutations in the α-galactosidase A gene in patients with Fabry disease: pitfalls of mutation analyses in patients with low α-galactosidase A activity.

PubMed

Yoshimitsu, Makoto; Higuchi, Koji; Miyata, Masaaki; Devine, Sean; Mattman, Andre; Sirrs, Sandra; Medin, Jeffrey A; Tei, Chuwa; Takenaka, Toshihiro

2011-05-01

Fabry disease is an X-linked lysosomal storage disorder caused by mutations of the α-galactosidase A (GLA) gene, and the disease is a relatively prevalent cause of left ventricular hypertrophy followed by conduction abnormalities and arrhythmias. Mutation analysis of the GLA gene is a valuable tool for accurate diagnosis of affected families. In this study, we carried out molecular studies of 10 unrelated families diagnosed with Fabry disease. Genetic analysis of the GLA gene using conventional genomic sequencing was performed in 9 hemizygous males and 6 heterozygous females. In patients with no mutations in coding DNA sequence, multiplex ligation-dependent probe amplification (MLPA) and/or cDNA sequencing were performed. We identified a novel exon 2 deletion (IVS1_IVS2) in a heterozygous female by MLPA, which was undetectable by conventional sequencing methods. In addition, the g.9331G>A mutation that has previously been found only in patients with cardiac Fabry disease was found in 3 unrelated, newly-diagnosed, cardiac Fabry patients by sequencing GLA genomic DNA and cDNA. Two other novel mutations, g.8319A>G and 832delA were also found in addition to 4 previously reported mutations (R112C, C142Y, M296I, and G373D) in 6 other families. We could identify GLA gene mutations in all hemizygotes and heterozygotes from 10 families with Fabry disease. Mutations in 4 out of 10 families could not be identified by classical genomic analysis, which focuses on exons and the flanking region. Instead, these data suggest that MLPA analysis and cDNA sequence should be considered in genetic testing surveys of patients with Fabry disease. Copyright © 2011 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.

CHEK2 mutation and risk of prostate cancer: a systematic review and meta-analysis

PubMed Central

Wang, Yue; Dai, Bo; Ye, Dingwei

2015-01-01

Background: CHEK2 encodes for a G2 checkpoint kinase which plays a critical role in DNA repair. Its mutation confers an increased risk of breast cancer. It has also been suggested to increase risks of prostate cancer, but its involvement with this type of cancer has not been confirmed. Methods: We performed a systematic review and meta-analysis to clarify the association between CHEK2 1100delC, IVS2+1G>A, I157T mutation and risk of Prostate Cancer. A comprehensive, computerized literature search of PubMed until December 27, 2014 was carried out. Eligible studies were included according to specific inclusion criteria. Pooled hazard ratio was estimated using the fixed effects model or random effects model according to heterogeneity between studies. Results: Eight eligible studies were included in the analysis, all were retrospective studies. The overall meta-analysis demonstrated that the CHEK2 1100delC mutation (OR 3.29; 95% confidence interval: 1.85-5.85; P = 0.00) and I157T missense mutation (OR 1.80; 95% confidence interval: 1.51-2.14; P = 0.00) was associated with higher risk of Prostate Cancer, and CHEK2 1100delC mutation is irrelevant to familial aggregation phenomenon of prostate cancer (OR 1.59; 95% confidence interval: 0.79-3.20; P = 0.20). The IVS2+1G>A mutation is also irrelevant to Prostate Cancer (OR = 1.59, 95% CI = 0.93-2.71, P = 0.09). None of the single studies materially altered the original results and no evidence of publication bias was found. Conclusion: CHEK2 1100delC mutation and I157T missense mutation in males indicates higher risk of Prostate Cancer, but there’s no evidence to prove the CHEK2 1100delC mutation was associated with Familial prostate cancer. PMID:26629066

CHEK2 mutation and risk of prostate cancer: a systematic review and meta-analysis.

PubMed

Wang, Yue; Dai, Bo; Ye, Dingwei

2015-01-01

CHEK2 encodes for a G2 checkpoint kinase which plays a critical role in DNA repair. Its mutation confers an increased risk of breast cancer. It has also been suggested to increase risks of prostate cancer, but its involvement with this type of cancer has not been confirmed. We performed a systematic review and meta-analysis to clarify the association between CHEK2 1100delC, IVS2+1G>A, I157T mutation and risk of Prostate Cancer. A comprehensive, computerized literature search of PubMed until December 27, 2014 was carried out. Eligible studies were included according to specific inclusion criteria. Pooled hazard ratio was estimated using the fixed effects model or random effects model according to heterogeneity between studies. Eight eligible studies were included in the analysis, all were retrospective studies. The overall meta-analysis demonstrated that the CHEK2 1100delC mutation (OR 3.29; 95% confidence interval: 1.85-5.85; P = 0.00) and I157T missense mutation (OR 1.80; 95% confidence interval: 1.51-2.14; P = 0.00) was associated with higher risk of Prostate Cancer, and CHEK2 1100delC mutation is irrelevant to familial aggregation phenomenon of prostate cancer (OR 1.59; 95% confidence interval: 0.79-3.20; P = 0.20). The IVS2+1G>A mutation is also irrelevant to Prostate Cancer (OR = 1.59, 95% CI = 0.93-2.71, P = 0.09). None of the single studies materially altered the original results and no evidence of publication bias was found. CHEK2 1100delC mutation and I157T missense mutation in males indicates higher risk of Prostate Cancer, but there's no evidence to prove the CHEK2 1100delC mutation was associated with Familial prostate cancer.

Fixation probability of a nonmutator in a large population of asexual mutators.

PubMed

Jain, Kavita; James, Ananthu

2017-11-21

In an adapted population of mutators in which most mutations are deleterious, a nonmutator that lowers the mutation rate is under indirect selection and can sweep to fixation. Using a multitype branching process, we calculate the fixation probability of a rare nonmutator in a large population of asexual mutators. We show that when beneficial mutations are absent, the fixation probability is a nonmonotonic function of the mutation rate of the mutator: it first increases sublinearly and then decreases exponentially. We also find that beneficial mutations can enhance the fixation probability of a nonmutator. Our analysis is relevant to an understanding of recent experiments in which a reduction in the mutation rates has been observed. Copyright © 2017 Elsevier Ltd. All rights reserved.

Identification of a Novel HADHB Gene Mutation in an Iranian Patient with Mitochondrial Trifunctional Protein Deficiency.

PubMed

Shahrokhi, Mahdiyeh; Shafiei, Mohammad; Galehdari, Hamid; Shariati, Gholamreza

2017-01-01

Mitochondrial trifunctional protein (MTP) is a hetero-octamer composed of eight parts (subunits): four α-subunits containing LCEH (long-chain 2,3-enoyl-CoA  hydratase) and LCHAD (long-chain 3-hydroxyacyl CoA dehydrogenase) activity, and four β-subunits that possess LCKT (long-chain  3-ketoacyl-CoA thiolase) activity which catalyzes three out of four steps in β-oxidation spiral of long-chain fatty acid. Its deficiency is an autosomal recessive disorder that causes a clinical spectrum of diseases. A blood spot was collected from the patient's original newborn screening card with parental informed consent. A newborn screening test and quantity plasma acylcarnitine profile analysis by MS/MS were performed. After isolation of DNA and Amplification of all exons of the HADHA and HADHB, directly Sequence analyses of all exons and the flanking introns both of genes were performed. Here, we report a novel mutation in a patient with MTP deficiency diagnosed with newborn screening test and quantity plasma acylcarnitine profile analysis by MS/MS and then confirmed by enzyme analysis in cultured fibroblasts and direct sequencing of the HADHA and HADHB genes. Molecular analysis of causative genes showed a missense mutation (p.Q385P) c.1154A > C in exon 14 of HADHB gene. Since this mutation was not found in 50 normal control cases; so it was concluded that c.1154A > C mutation was a causative mutation. Phenotype analysis of this mutation predicted pathogenesis which reduces the stability of the MTP protein complex.

Analysis of atmospheric trace constituents from high resolution infrared balloon-borne and ground-based solar absorption spectra

NASA Technical Reports Server (NTRS)

Goldman, A.; Murcray, F. J.; Rinsland, C. P.; Blatherwick, R. D.; Murcray, F. H.; Murcray, D. G.

1991-01-01

Results of ongoing studies of high-resolution solar absorption spectra aimed at the identification and quantification of trace constituents of importance in the chemistry of the stratosphere and upper troposphere are presented. An analysis of balloon-borne and ground-based spectra obtained at 0.0025/cm covering the 700-2200/cm interval is presented. The 0.0025/cm spectra, along with corresponding laboratory spectra, improves the spectral line parameters, and thus the accuracy of quantifying trace constituents. Results for COF2, F22, SF6, and other species are presented. The retrieval methods used for total column density and altitude distribution for both ground-based and balloon-borne spectra are also discussed.

Comprehensive Molecular Characterization of Muscle-Invasive Bladder Cancer. | Office of Cancer Genomics

Cancer.gov

We report a comprehensive analysis of 412 muscle-invasive bladder cancers characterized by multiple TCGA analytical platforms. Fifty-eight genes were significantly mutated, and the overall mutational load was associated with APOBEC-signature mutagenesis. Clustering by mutation signature identified a high-mutation subset with 75% 5-year survival.

KRAS mutation testing in metastatic colorectal cancer

PubMed Central

Tan, Cong; Du, Xiang

2012-01-01

The KRAS oncogene is mutated in approximately 35%-45% of colorectal cancers, and KRAS mutational status testing has been highlighted in recent years. The most frequent mutations in this gene, point substitutions in codons 12 and 13, were validated as negative predictors of response to anti-epidermal growth factor receptor antibodies. Therefore, determining the KRAS mutational status of tumor samples has become an essential tool for managing patients with colorectal cancers. Currently, a variety of detection methods have been established to analyze the mutation status in the key regions of the KRAS gene; however, several challenges remain related to standardized and uniform testing, including the selection of tumor samples, tumor sample processing and optimal testing methods. Moreover, new testing strategies, in combination with the mutation analysis of BRAF, PIK3CA and loss of PTEN proposed by many researchers and pathologists, should be promoted. In addition, we recommend that microsatellite instability, a prognostic factor, be added to the abovementioned concomitant analysis. This review provides an overview of KRAS biology and the recent advances in KRAS mutation testing. This review also addresses other aspects of status testing for determining the appropriate treatment and offers insight into the potential drawbacks of mutational testing. PMID:23066310

Interplay between DMD Point Mutations and Splicing Signals in Dystrophinopathy Phenotypes

PubMed Central

Juan-Mateu, Jonàs; González-Quereda, Lidia; Rodríguez, Maria José; Verdura, Edgard; Lázaro, Kira; Jou, Cristina; Nascimento, Andrés; Jiménez-Mallebrera, Cecilia; Colomer, Jaume; Monges, Soledad; Lubieniecki, Fabiana; Foncuberta, Maria Eugenia; Pascual-Pascual, Samuel Ignacio; Molano, Jesús; Baiget, Montserrat; Gallano, Pia

2013-01-01

DMD nonsense and frameshift mutations lead to severe Duchenne muscular dystrophy while in-frame mutations lead to milder Becker muscular dystrophy. Exceptions are found in 10% of cases and the production of alternatively spliced transcripts is considered a key modifier of disease severity. Several exonic mutations have been shown to induce exon-skipping, while splice site mutations result in exon-skipping or activation of cryptic splice sites. However, factors determining the splicing pathway are still unclear. Point mutations provide valuable information regarding the regulation of pre-mRNA splicing and elements defining exon identity in the DMD gene. Here we provide a comprehensive analysis of 98 point mutations related to clinical phenotype and their effect on muscle mRNA and dystrophin expression. Aberrant splicing was found in 27 mutations due to alteration of splice sites or splicing regulatory elements. Bioinformatics analysis was performed to test the ability of the available algorithms to predict consequences on mRNA and to investigate the major factors that determine the splicing pathway in mutations affecting splicing signals. Our findings suggest that the splicing pathway is highly dependent on the interplay between splice site strength and density of regulatory elements. PMID:23536893

Idiopathic restrictive cardiomyopathy is part of the clinical expression of cardiac troponin I mutations

PubMed Central

Mogensen, Jens; Kubo, Toru; Duque, Mauricio; Uribe, William; Shaw, Anthony; Murphy, Ross; Gimeno, Juan R.; Elliott, Perry; McKenna, William J.

2003-01-01

Restrictive cardiomyopathy (RCM) is an uncommon heart muscle disorder characterized by impaired filling of the ventricles with reduced volume in the presence of normal or near normal wall thickness and systolic function. The disease may be associated with systemic disease but is most often idiopathic. We recognized a large family in which individuals were affected by either idiopathic RCM or hypertrophic cardiomyopathy (HCM). Linkage analysis to selected sarcomeric contractile protein genes identified cardiac troponin I (TNNI3) as the likely disease gene. Subsequent mutation analysis revealed a novel missense mutation, which cosegregated with the disease in the family (lod score: 4.8). To determine if idiopathic RCM is part of the clinical expression of TNNI3 mutations, genetic investigations of the gene were performed in an additional nine unrelated RCM patients with restrictive filling patterns, bi-atrial dilatation, normal systolic function, and normal wall thickness. TNNI3 mutations were identified in six of these nine RCM patients. Two of the mutations identified in young individuals were de novo mutations. All mutations appeared in conserved and functionally important domains of the gene. PMID:12531876

A multisite blinded study for the detection of BRAF mutations in formalin-fixed, paraffin-embedded malignant melanoma

PubMed Central

Richter, Anna; Grieu, Fabienne; Carrello, Amerigo; Amanuel, Benhur; Namdarian, Kateh; Rynska, Aleksandra; Lucas, Amanda; Michael, Victoria; Bell, Anthony; Fox, Stephen B.; Hewitt, Chelsee A.; Do, Hongdo; McArthur, Grant A.; Wong, Stephen Q.; Dobrovic, Alexander; Iacopetta, Barry

2013-01-01

Melanoma patients with BRAF mutations respond to treatment with vemurafenib, thus creating a need for accurate testing of BRAF mutation status. We carried out a blinded study to evaluate various BRAF mutation testing methodologies in the clinical setting. Formalin-fixed, paraffin-embedded melanoma samples were macrodissected before screening for mutations using Sanger sequencing, single-strand conformation analysis (SSCA), high resolution melting analysis (HRM) and competitive allele-specific TaqMan® PCR (CAST-PCR). Concordance of 100% was observed between the Sanger sequencing, SSCA and HRM techniques. CAST-PCR gave rapid and accurate results for the common V600E and V600K mutations, however additional assays are required to detect rarer BRAF mutation types found in 3–4% of melanomas. HRM and SSCA followed by Sanger sequencing are effective two-step strategies for the detection of BRAF mutations in the clinical setting. CAST-PCR was useful for samples with low tumour purity and may also be a cost-effective and robust method for routine diagnostics. PMID:23584600

Tissue or blood: which is more suitable for detection of EGFR mutations in non-small cell lung cancer?

PubMed

Biaoxue, Rong; Shuanying, Yang

2018-01-01

Many studies have evaluated the accuracy of EGFR mutation status in blood against that in tumor tissues as the reference. We conducted this systematic review and meta-analysis to assess whether blood can be used as a substitute for tumor tissue in detecting EGFR mutations. Investigations that provided data on EGFR mutation status in blood were searched in the databases of Medline, Embase, Ovid Technologies and Web of Science. The detect efficiency of EGFR mutations in paired blood and tissues was compared using a random-effects model of meta-analysis. Pooled sensitivity and specificity and diagnostic accuracy were calculated by receiver operating characteristic curve. A total of 19 studies with 2,922 individuals were involved in this meta-analysis. The pooled results showed the positive detection rate of EGFR mutations in lung cancer tissues was remarkably higher than that of paired blood samples (odds ratio [OR] = 1.47, p<0.001). The pooled sensitivity and specificity of blood were 0.65 and 0.91, respectively, and the area under the receiver operating characteristic curve was 0.89. Although blood had a better specificity for detecting EGFR mutations, the absence of blood positivity should not necessarily be construed as confirmed negativity. Patients with negative results for blood should decidedly undergo further biopsies to ascertain EGFR mutations.

Single-cell genetic analysis validates cytopathological identification of circulating cancer cells in patients with clear cell renal cell carcinoma.

PubMed

Broncy, Lucile; Njima, Basma Ben; Méjean, Arnaud; Béroud, Christophe; Romdhane, Khaled Ben; Ilie, Marius; Hofman, Veronique; Muret, Jane; Hofman, Paul; Bouhamed, Habiba Chaabouni; Paterlini-Bréchot, And Patrizia

2018-04-13

Circulating Rare Cells (CRC) are non-haematological cells circulating in blood. They include Circulating Cancer Cells (CCC) and cells with uncertain malignant features (CRC-UMF) according to cytomorphology. Clear cell renal cell carcinomas frequently bear a mutated Von Hippel-Lindau (VHL) gene. To match blind genetic analysis of CRC and tumor samples with CRC cytopathological diagnosis. 29/30 patients harboured CRC (20 harboured CCC, 29 CRC-UMF) and 25/29 patients carried VHL mutations in their tumour. 205 single CRC (64 CCC, 141 CRC-UMF) provided genetic data. 57/57 CCC and 104/125 CRC-UMF from the 25 patients with VHL-mutated tumor carried the same VHL mutation detected in the tumor. Seven CCC and 16 CRC-UMF did not carry VHL mutations but were found in patients with wild-type VHL tumor tissue. All the CCC and 83,2% (104/125) of the CRC-UMF were found to carry the same VHL mutation identified in the corresponding tumorous tissue, validating cytopathological identification of CCC in patients with clear cell renal cell carcinoma. The blood of 30 patients with clear cell renal cell carcinoma was treated by ISET ® for CRC isolation, cytopathology and single-cell VHL mutations analysis, performed blindly and compared to VHL mutations of corresponding tumor tissues and leukocytes.

Analysis of MSH3 in endometrial cancers with defective DNA mismatch repair.

PubMed

Swisher, E M; Mutch, D G; Herzog, T J; Rader, J S; Kowalski, L D; Elbendary, A; Goodfellow, P J

1998-01-01

To clarify the origin of defective mismatch repair (MMR) in sporadic endometrial cancers with microsatellite instability (MSI), a thorough mutation analysis was performed on the human mismatch repair gene MSH3. Twenty-eight MSI-positive endometrial cancers were investigated for mutations in the human mismatch repair gene MSH3 using single-strand conformation variant (SSCV) analysis of all 24 exons. All variants were sequenced. Loss of heterozygosity was investigated at all MSH3 polymorphisms discovered. A subset of tumors were investigated for methylation of the 5' promoter region of MSH3 using Southern blot hybridization. An identical single-base deletion (delta A) predicted to result in a truncated proteins was discovered in six tumors (21.4%). This deletion occurs in a string of eight consecutive adenosine residues (A8). Because simple repeat sequences are unstable in cells with defective MMR, the observed mutation may be an effect, rather than a cause, of MSI. Evidence of inactivation of the second MSH3 allele in tumors with the delta A mutation would strongly support a causal role for these MSH3 mutations. However, there was no evidence of a second mutation, loss of sequences, or methylation of the promoter region in any of the tumors with the delta A mutation. Although the delta A mutation is a frequent event in sporadic MSI-positive endometrial cancers, it may not be causally associated with defective DNA MMR.

Surgical perspective of T1799A BRAF mutation diagnostic value in papillary thyroid carcinoma.

PubMed

Brahma, Bayu; Yulian, Erwin Danil; Ramli, Muchlis; Setianingsih, Iswari; Gautama, Walta; Brahma, Putri; Sastroasmoro, Sudigdo; Harimurti, Kuntjoro

2013-01-01

Throughout Indonesia, thyroid cancer is one of the ten commonest malignancies, with papillary thyroid carcinoma (PTC) in our hospital accounting for about 60% of all thyroid nodules. Although fine needle aspiration biopsy (FNAB) is the most reliable diagnostic tool, some nodules are diagnosed as indeterminate and second surgery is common for PTC. The aim of this study was to establish the diagnostic value and feasibility of testing the BRAF T1799A mutation on FNA specimens for improving PTC diagnosis. This prospective study enrolled 95 patients with thyroid nodules and future surgery planned. Results of mutational status were compared with surgical pathology diagnosis. Of the 70 cases included in the final analysis, 62.8% were PTC and the prevalence of BRAF mutation was 38.6%. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for BRAF mutation analysis were 36%, 100%, 100% and 48%, respectively. With other data findings, nodules with "onset less than 5 year" and "hard consistency" were proven as diagnostic determinants for BRAF mutation with a probability of 62.5%. This mutation was also a significant risk factor for extra-capsular extension. Molecular analysis of the BRAF T1799A mutation in FNAB specimens has high specificity and positive predictive value for PTC. It could be used in the selective patients with clinical characteristics to facilitate PTC diagnosis and for guidance regarding extent of thyroidectomy.

Developments for Personalized Medicine of Lung Cancer Subtypes: Mass Spectrometry-Based Clinical Proteogenomic Analysis of Oncogenic Mutations.

PubMed

Nishimura, Toshihide; Nakamura, Haruhiko

2016-01-01

Molecular therapies targeting lung cancers with mutated epidermal growth factor receptor (EGFR) by EGFR-tyrosin kinase inhibitors (EGFR-TKIs), gefitinib and erlotinib, changed the treatment system of lung cancer. It was revealed that drug efficacy differs by race (e.g., Caucasians vs. Asians) due to oncogenic driver mutations specific to each race, exemplified by gefitinib / erlotinib. The molecular target drugs for lung cancer with anaplastic lymphoma kinase (ALK) gene translocation (the fusion gene, EML4-ALK) was approved, and those targeting lung cancers addicted ROS1, RET, and HER2 have been under development. Both identification and quantification of gatekeeper mutations need to be performed using lung cancer tissue specimens obtained from patients to improve the treatment for lung cancer patients: (1) identification and quantitation data of targeted mutated proteins, including investigation of mutation heterogeneity within a tissue; (2) exploratory mass spectrometry (MS)-based clinical proteogenomic analysis of mutated proteins; and also importantly (3) analysis of dynamic protein-protein interaction (PPI) networks of proteins significantly related to a subgroup of patients with lung cancer not only with good efficacy but also with acquired resistance. MS-based proteogenomics is a promising approach to directly capture mutated and fusion proteins expressed in a clinical sample. Technological developments are further expected, which will provide a powerful solution for the stratification of patients and drug discovery (Precision Medicine).

Colorectal cancer cell lines are representative models of the main molecular subtypes of primary cancer.

PubMed

Mouradov, Dmitri; Sloggett, Clare; Jorissen, Robert N; Love, Christopher G; Li, Shan; Burgess, Antony W; Arango, Diego; Strausberg, Robert L; Buchanan, Daniel; Wormald, Samuel; O'Connor, Liam; Wilding, Jennifer L; Bicknell, David; Tomlinson, Ian P M; Bodmer, Walter F; Mariadason, John M; Sieber, Oliver M

2014-06-15

Human colorectal cancer cell lines are used widely to investigate tumor biology, experimental therapy, and biomarkers. However, to what extent these established cell lines represent and maintain the genetic diversity of primary cancers is uncertain. In this study, we profiled 70 colorectal cancer cell lines for mutations and DNA copy number by whole-exome sequencing and SNP microarray analyses, respectively. Gene expression was defined using RNA-Seq. Cell line data were compared with those published for primary colorectal cancers in The Cancer Genome Atlas. Notably, we found that exome mutation and DNA copy-number spectra in colorectal cancer cell lines closely resembled those seen in primary colorectal tumors. Similarities included the presence of two hypermutation phenotypes, as defined by signatures for defective DNA mismatch repair and DNA polymerase ε proofreading deficiency, along with concordant mutation profiles in the broadly altered WNT, MAPK, PI3K, TGFβ, and p53 pathways. Furthermore, we documented mutations enriched in genes involved in chromatin remodeling (ARID1A, CHD6, and SRCAP) and histone methylation or acetylation (ASH1L, EP300, EP400, MLL2, MLL3, PRDM2, and TRRAP). Chromosomal instability was prevalent in nonhypermutated cases, with similar patterns of chromosomal gains and losses. Although paired cell lines derived from the same tumor exhibited considerable mutation and DNA copy-number differences, in silico simulations suggest that these differences mainly reflected a preexisting heterogeneity in the tumor cells. In conclusion, our results establish that human colorectal cancer lines are representative of the main subtypes of primary tumors at the genomic level, further validating their utility as tools to investigate colorectal cancer biology and drug responses. ©2014 American Association for Cancer Research.

Proteogenomic strategies for identification of aberrant cancer peptides using large-scale Next Generation Sequencing data

DOE PAGES

Woo, Sunghee; Cha, Seong Won; Na, Seungjin; ...

2014-11-17

Cancer is driven by the acquisition of somatic DNA lesions. Distinguishing the early driver mutations from subsequent passenger mutations is key to molecular sub-typing of cancers, and the discovery of novel biomarkers. The availability of genomics technologies (mainly wholegenome and exome sequencing, and transcript sampling via RNA-seq, collectively referred to as NGS) have fueled recent studies on somatic mutation discovery. However, the vision is challenged by the complexity, redundancy, and errors in genomic data, and the difficulty of investigating the proteome using only genomic approaches. Recently, combination of proteomic and genomic technologies are increasingly employed. However, the complexity and redundancymore » of NGS data remains a challenge for proteogenomics, and various trade-offs must be made to allow for the searches to take place. This paperprovides a discussion of two such trade-offs, relating to large database search, and FDR calculations, and their implication to cancer proteogenomics. Moreover, it extends and develops the idea of a unified genomic variant database that can be searched by any mass spectrometry sample. A total of 879 BAM files downloaded from TCGA repository were used to create a 4.34 GB unified FASTA database which contained 2,787,062 novel splice junctions, 38,464 deletions, 1105 insertions, and 182,302 substitutions. Proteomic data from a single ovarian carcinoma sample (439,858 spectra) was searched against the database. By applying the most conservative FDR measure, we have identified 524 novel peptides and 65,578 known peptides at 1% FDR threshold. The novel peptides include interesting examples of doubly mutated peptides, frame-shifts, and non-sample-recruited mutations, which emphasize the strength of our approach.« less

Accumulation of the Cyclobutane Thymine Dimer in Defined Sequences of Free and Nucleosomal DNA

DTIC Science & Technology

2013-08-01

cyclobutane dimer in a single-stranded vector , Proc. Natl. Acad. Sci. U. S. A., 1988, 85, 8141–8145. 11 C. A. Smith, M. Wang, N. Jiang, L. Che, X. Zhao and...J.-S. Taylor, Mutation spectra of M13 vectors containing site-specific cis–syn, trans–syn-I, (6-4), and Dewar pyrimi- done photoproducts of thymidylyl...Bypass of a site-specific cis–syn thymine dimer in a SV40 vector during in vitro replication by HeLa and XPV cell-free extracts, Biochemistry, 1998

Toxicity, Mutagenicity, and Mutational Spectra of Vinyl Chloride, 2- Chloroethylene Oxide, and Chloracetaldehyde in a Human Lymphoblastoid Line Expressing Cytochrome P450IIE1.

DTIC Science & Technology

1992-01-01

concluded that CEO was the alkylating agent involved in conversion of adenosine to l,N 6 -ethenoadenosine. 1,N6 - Ethenoadenosine was not produced by CEO...guanines as nearest neighbors upon the alkylation of a guanine residue in DNA. N-methyl-N- nitrosourea (MNU) was reacted with a synthetic polynucleotide...the alkylating agent MNNG or the intercalating agent ICR-191. In the study they determined that mutants comprising at least one percent of the total

A meta-analysis of prognostic value of KIT mutation status in gastrointestinal stromal tumors

PubMed Central

Jiang, Zhiqiang; Zhang, Jian; Li, Zhi; Liu, Yingjun; Wang, Daohai; Han, Guangsen

2016-01-01

Numerous types of KIT mutations have been reported in gastrointestinal stromal tumors (GISTs); however, controversy still exists regarding their clinicopathological significance. In this study, we reviewed the publicly available literature to assess the data by a meta-analysis to characterize KIT mutations and different types of KIT mutations in prognostic prediction in patients with GISTs. Twenty-eight studies that included 4,449 patients were identified and analyzed. We found that KIT mutation status was closely correlated with size of tumors and different mitosis indexes, but not with tumor location. KIT mutation was also observed to be significantly correlated with tumor recurrence, metastasis, as well as the overall survival of patients. Interestingly, there was higher risk of progression in KIT exon 9-mutated patients than in exon 11-mutated patients. Five-year relapse-free survival (RFS) rate was significantly higher in KIT exon 11-deleted patients than in those with other types of KIT exon 11 mutations. In addition, RFS for 5 years was significantly worse in patients bearing KIT codon 557–558 deletions than in those bearing other KIT exon 11 deletions. Our results strongly support the hypothesis that KIT mutation status is another evaluable factor for prognosis prediction in GISTs. PMID:27350754

Systematic analysis of mutation distribution in three dimensional protein structures identifies cancer driver genes.

PubMed

Fujimoto, Akihiro; Okada, Yukinori; Boroevich, Keith A; Tsunoda, Tatsuhiko; Taniguchi, Hiroaki; Nakagawa, Hidewaki

2016-05-26

Protein tertiary structure determines molecular function, interaction, and stability of the protein, therefore distribution of mutation in the tertiary structure can facilitate the identification of new driver genes in cancer. To analyze mutation distribution in protein tertiary structures, we applied a novel three dimensional permutation test to the mutation positions. We analyzed somatic mutation datasets of 21 types of cancers obtained from exome sequencing conducted by the TCGA project. Of the 3,622 genes that had ≥3 mutations in the regions with tertiary structure data, 106 genes showed significant skew in mutation distribution. Known tumor suppressors and oncogenes were significantly enriched in these identified cancer gene sets. Physical distances between mutations in known oncogenes were significantly smaller than those of tumor suppressors. Twenty-three genes were detected in multiple cancers. Candidate genes with significant skew of the 3D mutation distribution included kinases (MAPK1, EPHA5, ERBB3, and ERBB4), an apoptosis related gene (APP), an RNA splicing factor (SF1), a miRNA processing factor (DICER1), an E3 ubiquitin ligase (CUL1) and transcription factors (KLF5 and EEF1B2). Our study suggests that systematic analysis of mutation distribution in the tertiary protein structure can help identify cancer driver genes.

Systematic analysis of mutation distribution in three dimensional protein structures identifies cancer driver genes

PubMed Central

Fujimoto, Akihiro; Okada, Yukinori; Boroevich, Keith A.; Tsunoda, Tatsuhiko; Taniguchi, Hiroaki; Nakagawa, Hidewaki

2016-01-01

Protein tertiary structure determines molecular function, interaction, and stability of the protein, therefore distribution of mutation in the tertiary structure can facilitate the identification of new driver genes in cancer. To analyze mutation distribution in protein tertiary structures, we applied a novel three dimensional permutation test to the mutation positions. We analyzed somatic mutation datasets of 21 types of cancers obtained from exome sequencing conducted by the TCGA project. Of the 3,622 genes that had ≥3 mutations in the regions with tertiary structure data, 106 genes showed significant skew in mutation distribution. Known tumor suppressors and oncogenes were significantly enriched in these identified cancer gene sets. Physical distances between mutations in known oncogenes were significantly smaller than those of tumor suppressors. Twenty-three genes were detected in multiple cancers. Candidate genes with significant skew of the 3D mutation distribution included kinases (MAPK1, EPHA5, ERBB3, and ERBB4), an apoptosis related gene (APP), an RNA splicing factor (SF1), a miRNA processing factor (DICER1), an E3 ubiquitin ligase (CUL1) and transcription factors (KLF5 and EEF1B2). Our study suggests that systematic analysis of mutation distribution in the tertiary protein structure can help identify cancer driver genes. PMID:27225414

Calreticulin mutation-specific immunostaining in myeloproliferative neoplasms: pathogenetic insight and diagnostic value

PubMed Central

Vannucchi, A M; Rotunno, G; Bartalucci, N; Raugei, G; Carrai, V; Balliu, M; Mannarelli, C; Pacilli, A; Calabresi, L; Fjerza, R; Pieri, L; Bosi, A; Manfredini, R; Guglielmelli, P

2014-01-01

Mutations in the gene calreticulin (CALR) occur in the majority of JAK2- and MPL-unmutated patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF); identifying CALR mutations contributes to the diagnostic pathway of ET and PMF. CALR mutations are heterogeneous spanning over the exon 9, but all result in a novel common protein C terminus. We developed a polyclonal antibody against a 17-amino-acid peptide derived from mutated calreticulin that was used for immunostaining of bone marrow biopsies. We show that this antibody specifically recognized patients harboring different types of CALR mutation with no staining in healthy controls and JAK2- or MPL-mutated ET and PMF. The labeling was mostly localized in megakaryocytes, whereas myeloid and erythroid cells showed faint staining, suggesting a preferential expression of calreticulin in megakaryocytes. Megakaryocytic-restricted expression of calreticulin was also demonstrated using an antibody against wild-type calreticulin and by measuring the levels of calreticulin RNA by gene expression analysis. Immunostaining using an antibody specific for mutated calreticulin may become a rapid, simple and cost-effective method for identifying CALR-mutated patients complementing molecular analysis; furthermore, the labeling pattern supports the preferential expansion of megakaryocytic cell lineage as a result of CALR mutation in an immature hematopoietic stem cell. PMID:24618731

Targeted mutation analysis of endometrial clear cell carcinoma.

PubMed

Hoang, Lien N; McConechy, Melissa K; Meng, Bo; McIntyre, John B; Ewanowich, Carol; Gilks, Cyril Blake; Huntsman, David G; Köbel, Martin; Lee, Cheng-Han

2015-04-01

Endometrial clear cell carcinomas (CCC) constitute fewer than 5% of all carcinomas of the endometrium. Currently, little is known regarding the genetic basis of endometrial CCC. We performed genomic and immunohistochemical analyses on 14 rigorously reviewed pure endometrial CCC. The genomic analysis consisted of sequencing the coding regions of 26 genes implicated previously in endometrial carcinoma. Twelve of 14 tumours displayed a prototypical CCC immunophenotype [napsin A+, hepatocyte nuclear factor-1β (HNF1β(+) ) and oestrogen receptor(-) ] and all showed intact mismatch repair protein expression. We detected mutations in 11 of 14 tumours, and there was a predominance of mutations involving genes that are mutated more frequently in endometrial serous carcinomas than in endometrioid carcinomas. Two tumours displayed a prototypical serous carcinoma mutation profile (concurrent TP53 and PPP2R1A mutations, without PTEN, CTNNB1 or ARID1A mutation). No mutations in PTEN, CTNNB1 or POLE were identified. The overall mutation profile of this cohort of endometrial CCC appears to be more serous-like than endometrioid-like, with a minor subset in the TP53-mutated CCC showing serous carcinoma profile. These findings provide new insights into the molecular features of morphologically prototypical endometrial CCC, and underscore the need for further investigations into the oncogenesis of endometrial CCC. © 2014 John Wiley & Sons Ltd.

[Ultrastructure and Raman Spectral Characteristics of Two Kinds of Acute Myeloid Leukemia Cells].

PubMed

Liang, Hao-Yue; Cheng, Xue-Lian; Dong, Shu-Xu; Zhao, Shi-Xuan; Wang, Ying; Ru, Yong-Xin

2018-02-01

To investigate the Raman spectral characteristics of leukemia cells from 4 patients with acute promyelocytic leukemia (M 3 ) and 3 patients with acute monoblastic leukemia (M 5 ), establish a novel Raman label-free method to distinguish 2 kinds of acute myeloid leukemia cells so as to provide basis for clinical research. Leukemia cells were collected from bone marrow of above-mentioned patients. Raman spectra were acquired by Horiba Xplora Raman spectrometer and Raman spectra of 30-50 cells from each patient were recorded. The diagnostic model was established according to principle component analysis (PCA), discriminant function analysis (DFA) and cluster analysis, and the spectra of leukemia cells from 7 patients were analyzed and classified. Characteristics of Raman spectra were analyzed combining with ultrastructure of leukemia cells. There were significant differences between Raman spectra of 2 kinds of leukemia cells. Compared with acute monoblastic leukemia cells, the spectra of acute promyelocytic leukemia cells showed stronger peaks in 622, 643, 757, 852, 1003, 1033, 1117, 1157, 1173, 1208, 1340, 1551, 1581 cm -1 . The diagnostic models established by PCA-DFA and cluster analysis could successfully classify these Raman spectra of different samples with a high accuracy of 100% (233/233). The model was evaluated by "Leave-one-out" cross-validation and reached a high accuracy of 97% (226/233). The level of macromolecules of M 3 cells is higher than that of M 5 . The diagnostic models established by PCA-DFA can classify these Raman spectra of different cells with a high accuracy. Raman spectra shows consistent result with ultrastructure by TEM.

Alport Syndrome: De Novo Mutation in the COL4A5 Gene Converting Glycine 1205 to Valine.

PubMed

Antón-Martín, Pilar; Aparicio López, Cristina; Ramiro-León, Soraya; Santillán Garzón, Sonia; Santos-Simarro, Fernando; Gil-Fournier, Belén

2012-01-01

Alport syndrome is a primary basement membrane disorder arising from mutations in genes encoding the type IV collagen protein family. It is a genetically heterogeneous disease with different mutations and forms of inheritance that presents with renal affection, hearing loss and eye defects. Several new mutations related to X-linked forms have been previously determined. We report the case of a 12 years old male and his family diagnosed with Alport syndrome after genetic analysis was performed. A new mutation determining a nucleotide change c.3614G > T (p.Gly1205Val) in hemizygosis in the COL4A5 gene was found. This molecular defect has not been previously described. Molecular biology has helped us to comprehend the mechanisms of pathophysiology in Alport syndrome. Genetic analysis provides the only conclusive diagnosis of the disorder at the moment. Our contribution with a new mutation further supports the need of more sophisticated molecular methods to increase the mutation detection rates with lower costs and less time.

Single quantum dot analysis enables multiplexed point mutation detection by gap ligase chain reaction.

PubMed

Song, Yunke; Zhang, Yi; Wang, Tza-Huei

2013-04-08

Gene point mutations present important biomarkers for genetic diseases. However, existing point mutation detection methods suffer from low sensitivity, specificity, and a tedious assay processes. In this report, an assay technology is proposed which combines the outstanding specificity of gap ligase chain reaction (Gap-LCR), the high sensitivity of single-molecule coincidence detection, and the superior optical properties of quantum dots (QDs) for multiplexed detection of point mutations in genomic DNA. Mutant-specific ligation products are generated by Gap-LCR and subsequently captured by QDs to form DNA-QD nanocomplexes that are detected by single-molecule spectroscopy (SMS) through multi-color fluorescence burst coincidence analysis, allowing for multiplexed mutation detection in a separation-free format. The proposed assay is capable of detecting zeptomoles of KRAS codon 12 mutation variants with near 100% specificity. Its high sensitivity allows direct detection of KRAS mutation in crude genomic DNA without PCR pre-amplification. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rosatelli, M.C.; Faa, V.; Sardu, R.

This study reports the molecular characterization of [beta]-thalassemia in the Sardinian population. Three thousand [beta]-thalassemia chromosomes from prospective parents presenting at the genetic service were initially analyzed by dot blot analysis with oligonucleotide probes complementary to the most common [beta]-thalassemia mutations in the Mediterranean at-risk populations. The mutation which remained uncharacterized by this approach were defined by denaturing gradient gel electrophoresis (DGGE) followed by direct sequence analysis on amplified DNA. The authors reconfirmed that the predominant mutation in the Sardinian population is the codon 39 nonsense mutation, which accounts for 95.7% of the [beta]-thalassemia chromosomes. The other two relatively commonmore » mutations are frameshifts at codon 6 (2.1%) and at codon 76 (0.7%), relatively uncommon in other Mediterranean-origin populations. In this study they have detected a novel [beta]-thalassemia mutation, i.e., a frameshift at codon 1, in three [beta]-thalassemia chromosomes. The DGGE procedure followed by direct sequencing on amplified DNA is a powerful approach for the characterization of unknown mutations in this genetic system.« less

Mutations in myosin VIIA (MYO7A) and usherin (USH2A) in Spanish patients with Usher syndrome types I and II, respectively.

PubMed

Nájera, Carmen; Beneyto, Magdalena; Blanca, José; Aller, Elena; Fontcuberta, Ana; Millán, José María; Ayuso, Carmen

2002-07-01

Usher syndrome is an autosomal recessive disorder characterized by congenital hearing impairment and retinitis pigmentosa. Three clinical types are known (USH1, USH2 and USH3), and there is an extensive genetic heterogeneity, with at least ten genes implicated. The most frequently mutated genes are MYO7A, which causes USH1B, and usherin, which causes USH2A. We carried out a mutation analysis of these two genes in the Spanish population. Analysis of the MYO7A gene in patients from 30 USH1 families and sporadic cases identified 32% of disease alleles, with mutation Q821X being the most frequent. Most of the remaining variants are private mutations. With regard to USH2, mutation 2299delG was detected in 25% of the Spanish patients. Altogether the mutations detected in USH2A families account for 23% of the disease alleles. Copyright 2002 Wiley-Liss, Inc.

Merkel Cell Polyomavirus Exhibits Dominant Control of the Tumor Genome and Transcriptome in Virus-Associated Merkel Cell Carcinoma.

PubMed

Starrett, Gabriel J; Marcelus, Christina; Cantalupo, Paul G; Katz, Joshua P; Cheng, Jingwei; Akagi, Keiko; Thakuria, Manisha; Rabinowits, Guilherme; Wang, Linda C; Symer, David E; Pipas, James M; Harris, Reuben S; DeCaprio, James A

2017-01-03

Merkel cell polyomavirus is the primary etiological agent of the aggressive skin cancer Merkel cell carcinoma (MCC). Recent studies have revealed that UV radiation is the primary mechanism for somatic mutagenesis in nonviral forms of MCC. Here, we analyze the whole transcriptomes and genomes of primary MCC tumors. Our study reveals that virus-associated tumors have minimally altered genomes compared to non-virus-associated tumors, which are dominated by UV-mediated mutations. Although virus-associated tumors contain relatively small mutation burdens, they exhibit a distinct mutation signature with observable transcriptionally biased kataegic events. In addition, viral integration sites overlap focal genome amplifications in virus-associated tumors, suggesting a potential mechanism for these events. Collectively, our studies indicate that Merkel cell polyomavirus is capable of hijacking cellular processes and driving tumorigenesis to the same severity as tens of thousands of somatic genome alterations. A variety of mutagenic processes that shape the evolution of tumors are critical determinants of disease outcome. Here, we sequenced the entire genome of virus-positive and virus-negative primary Merkel cell carcinomas (MCCs), revealing distinct mutation spectra and corresponding expression profiles. Our studies highlight the strong effect that Merkel cell polyomavirus has on the divergent development of viral MCC compared to the somatic alterations that typically drive nonviral tumorigenesis. A more comprehensive understanding of the distinct mutagenic processes operative in viral and nonviral MCCs has implications for the effective treatment of these tumors. Copyright © 2017 Starrett et al.

Overexpressed DNA polymerase iota regulated by JNK/c-Jun contributes to hypermutagenesis in bladder cancer.

PubMed

Yuan, Fang; Xu, Zhigang; Yang, Mingzhen; Wei, Quanfang; Zhang, Yi; Yu, Jin; Zhi, Yi; Liu, Yang; Chen, Zhiwen; Yang, Jin

2013-01-01

Human DNA polymerase iota (pol ι) possesses high error-prone DNA replication features and performs translesion DNA synthesis. It may be specialized and strictly regulated in normal mammalian cells. Dysregulation of pol ι may contribute to the acquisition of a mutator phenotype. However, there are few reports describing the transcription regulatory mechanism of pol ι, and there is controversy regarding its role in carcinogenesis. In this study, we performed the deletion and point-mutation experiment, EMSA, ChIP, RNA interference and western blot assay to prove that c-Jun activated by c-Jun N-terminal kinase (JNK) regulates the transcription of pol ι in normal and cancer cells. Xeroderma pigmentosum group C protein (XPC) and ataxia-telangiectasia mutated related protein (ATR) promote early JNK activation in response to DNA damage and consequently enhance the expression of pol ι, indicating that the novel role of JNK signal pathway is involved in DNA damage response. Furthermore, associated with elevated c-Jun activity, the overexpression of pol ι is positively correlated with the clinical tumor grade in 97 bladder cancer samples and may contribute to the hypermutagenesis. The overexpressed pol ι-involved mutagenesis is dependent on JNK/c-Jun pathway in bladder cancer cells identifying by the special mutation spectra. Our results support the conclusion that dysregulation of pol ι by JNK/c-Jun is involved in carcinogenesis and offer a novel understanding of the role of pol ι or c-Jun in mutagenesis.

Overexpressed DNA Polymerase Iota Regulated by JNK/c-Jun Contributes to Hypermutagenesis in Bladder Cancer

PubMed Central

Yuan, Fang; Xu, Zhigang; Yang, Mingzhen; Wei, Quanfang; Zhang, Yi; Yu, Jin; Zhi, Yi; Liu, Yang; Chen, Zhiwen; Yang, Jin

2013-01-01

Human DNA polymerase iota (pol ι) possesses high error-prone DNA replication features and performs translesion DNA synthesis. It may be specialized and strictly regulated in normal mammalian cells. Dysregulation of pol ι may contribute to the acquisition of a mutator phenotype. However, there are few reports describing the transcription regulatory mechanism of pol ι, and there is controversy regarding its role in carcinogenesis. In this study, we performed the deletion and point-mutation experiment, EMSA, ChIP, RNA interference and western blot assay to prove that c-Jun activated by c-Jun N-terminal kinase (JNK) regulates the transcription of pol ι in normal and cancer cells. Xeroderma pigmentosum group C protein (XPC) and ataxia-telangiectasia mutated related protein (ATR) promote early JNK activation in response to DNA damage and consequently enhance the expression of pol ι, indicating that the novel role of JNK signal pathway is involved in DNA damage response. Furthermore, associated with elevated c-Jun activity, the overexpression of pol ι is positively correlated with the clinical tumor grade in 97 bladder cancer samples and may contribute to the hypermutagenesis. The overexpressed pol ι-involved mutagenesis is dependent on JNK/c-Jun pathway in bladder cancer cells identifying by the special mutation spectra. Our results support the conclusion that dysregulation of pol ι by JNK/c-Jun is involved in carcinogenesis and offer a novel understanding of the role of pol ι or c-Jun in mutagenesis. PMID:23922701

Detection of PIK3CA gene mutations with HRM analysis and association with IGFBP-5 expression levels in breast cancer.

PubMed

Dirican, Ebubekir; Kaya, Zehra; Gullu, Gokce; Peker, Irem; Ozmen, Tolga; Gulluoglu, Bahadir M; Kaya, Handan; Ozer, Ayse; Akkiprik, Mustafa

2014-01-01

Breast cancer is the second most common cancer and second leading cause of cancer deaths in women. Phosphatidylinositol-3-kinase (PI3K)/AKT pathway mutations are associated with cancer and phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) gene mutations have been observed in 25-45% of breast cancer samples. Insulin growth factor binding protein-5 (IGFBP-5) can show different effects on apoptosis, cell motility and survival in breast cancer. We here aimed to determine the association between PIK3CA gene mutations and IGFBP-5 expressions for the first time in breast cancer patients. Frozen tumor samples from 101 Turkish breast cancer patients were analyzed with high resolution melting (HRM) for PIK3CA mutations (exon 9 and exon 20) and 37 HRM positive tumor samples were analyzed by DNA sequencing, mutations being found in 31. PIK3CA exon 9 mutations (Q546R, E542Q, E545K, E542K and E545D) were found in 10 tumor samples, exon 20 mutations (H1047L, H1047R, T1025T and G1049R) in 21, where only 1 tumor sample had two exon 20 mutations (T1025T and H1047R). Moreover, we detected one sample with both exon 9 (E542Q) and exon 20 (H1047R) mutations. 35% of the tumor samples with high IGFBP-5 mRNA expression and 29.4% of the tumor samples with low IGFBP-5 mRNA expression had PIK3CA mutations (p=0.9924). This is the first study of PIK3CA mutation screening results in Turkish breast cancer population using HRM analysis. This approach appears to be a very effective and reliable screening method for the PIK3CA exon 9 and 20 mutation detection. Further analysis with a greater number of samples is needed to clarify association between PIK3CA gene mutations and IGFBP-5 mRNA expression, and also clinical outcome in breast cancer patients.

BRCA1 and BRCA2 founder mutations account for 78% of germline carriers among hereditary breast cancer families in Chile

PubMed Central

Alvarez, Carolina; Tapia, Teresa; Perez-Moreno, Elisa; Gajardo-Meneses, Patricia; Ruiz, Catalina; Rios, Mabel; Missarelli, Claudio; Silva, Mariela; Cruz, Adolfo; Matamala, Luis; Carvajal-Carmona, Luis; Camus, Mauricio; Carvallo, Pilar

2017-01-01

Identifying founder mutations in BRCA1 and BRCA2 in specific populations constitute a valuable opportunity for genetic screening. Several studies from different populations have reported recurrent and/or founder mutations representing a relevant proportion of BRCA mutation carriers. In Latin America, only few founder mutations have been described. We screened 453 Chilean patients with hereditary breast cancer for mutations in BRCA1 and BRCA2. For recurrent mutations, we genotyped 11 microsatellite markers in BRCA1 and BRCA2 in order to determine a founder effect through haplotype analysis. We found a total of 25 mutations (6 novel) in 71 index patients among which, nine are present exclusively in Chilean patients. Our analysis revealed the presence of nine founder mutations, 4 in BRCA1 and 5 in BRCA2, shared by 2 to 10 unrelated families and spread in different regions of Chile. Our panel contains the highest amount of founder mutations until today and represents the highest percentage (78%) of BRCA1 and BRCA2 mutation carriers. We suggest that the dramatic reduction of Amerindian population due to smallpox and wars with Spanish conquerors, a scarce population increase during 300 years, and the geographic position of Chile constituted a favorable scenario to establish founder genetic markers in our population. PMID:29088781

Mutation spectrum of primary hyperoxaluria type 1 in Tunisia: implication for diagnosis in North Africa.

PubMed

Nagara, Majdi; Tiar, Afaf; Ben Halim, Nizar; Ben Rhouma, Faten; Messaoud, Olfa; Bouyacoub, Yosra; Kefi, Rym; Hassayoun, Saida; Zouari, Noura; Ben Ammar, Mohamed Slim; Abdelhak, Sonia; Chemli, Jalel

2013-09-15

Primary hyperoxaluria type 1 (PH1) is an autosomal recessive inherited metabolic disease, characterized by progressive kidney failure due to renal deposition of calcium oxalate. Mutations in the AGXT gene, encoding the liver-specific enzyme alanine glyoxylate aminotransferase, are responsible for the disease. We aimed to determine the mutational spectrum causing PH1 and to provide an accurate tool for diagnosis as well as for prenatal diagnosis in the affected families. Direct sequencing was used to detect mutations in the AGXT gene in DNA samples from 13 patients belonging to 12 Tunisian families. Molecular analysis revealed five mutations causing PH1 in Tunisia. The mutations were identified along exons 1, 2, 4, 5 and 7. The most predominant mutations were the Maghrebian "p.I244T" and the Arabic "p.G190R". Furthermore, three other mutations characteristic of different ethnic groups were found in our study population. These results confirm the mutational heterogeneity related to PH1 in Tunisian population. All the mutations are in a homozygous state, reflecting the high impact of endogamy in our population. Mutation analysis through DNA sequencing can provide a useful first line investigation for PH1. This identification could provide an accurate tool for prenatal diagnosis, genetic counseling and screen for potential presymptomatic individuals. © 2013 Elsevier B.V. All rights reserved.

A meta-analysis of the relationship between FGFR3 and TP53 mutations in bladder cancer.

PubMed

Neuzillet, Yann; Paoletti, Xavier; Ouerhani, Slah; Mongiat-Artus, Pierre; Soliman, Hany; de The, Hugues; Sibony, Mathilde; Denoux, Yves; Molinie, Vincent; Herault, Aurélie; Lepage, May-Linda; Maille, Pascale; Renou, Audrey; Vordos, Dimitri; Abbou, Claude-Clément; Bakkar, Ashraf; Asselain, Bernard; Kourda, Nadia; El Gaaied, Amel; Leroy, Karen; Laplanche, Agnès; Benhamou, Simone; Lebret, Thierry; Allory, Yves; Radvanyi, François

2012-01-01

TP53 and FGFR3 mutations are the most common mutations in bladder cancers. FGFR3 mutations are most frequent in low-grade low-stage tumours, whereas TP53 mutations are most frequent in high-grade high-stage tumours. Several studies have reported FGFR3 and TP53 mutations to be mutually exclusive events, whereas others have reported them to be independent. We carried out a meta-analysis of published findings for FGFR3 and TP53 mutations in bladder cancer (535 tumours, 6 publications) and additional unpublished data for 382 tumours. TP53 and FGFR3 mutations were not independent events for all tumours considered together (OR = 0.25 [0.18-0.37], p = 0.0001) or for pT1 tumours alone (OR = 0.47 [0.28-0.79], p = 0.0009). However, if the analysis was restricted to pTa tumours or to muscle-invasive tumours alone, FGFR3 and TP53 mutations were independent events (OR = 0.56 [0.23-1.36] (p = 0.12) and OR = 0.99 [0.37-2.7] (p = 0.35), respectively). After stratification of the tumours by stage and grade, no dependence was detected in the five tumour groups considered (pTaG1 and pTaG2 together, pTaG3, pT1G2, pT1G3, pT2-4). These differences in findings can be attributed to the putative existence of two different pathways of tumour progression in bladder cancer: the CIS pathway, in which FGFR3 mutations are rare, and the Ta pathway, in which FGFR3 mutations are frequent. TP53 mutations occur at the earliest stage of the CIS pathway, whereas they occur would much later in the Ta pathway, at the T1G3 or muscle-invasive stage.

A Meta-Analysis of the Relationship between FGFR3 and TP53 Mutations in Bladder Cancer

PubMed Central

Ouerhani, Slah; Mongiat-Artus, Pierre; Soliman, Hany; de The, Hugues; Sibony, Mathilde; Denoux, Yves; Molinie, Vincent; Herault, Aurélie; Lepage, May-Linda; Maille, Pascale; Renou, Audrey; Vordos, Dimitri; Abbou, Claude-Clément; Bakkar, Ashraf; Asselain, Bernard; Kourda, Nadia; El Gaaied, Amel; Leroy, Karen; Laplanche, Agnès; Benhamou, Simone; Lebret, Thierry; Allory, Yves; Radvanyi, François

2012-01-01

TP53 and FGFR3 mutations are the most common mutations in bladder cancers. FGFR3 mutations are most frequent in low-grade low-stage tumours, whereas TP53 mutations are most frequent in high-grade high-stage tumours. Several studies have reported FGFR3 and TP53 mutations to be mutually exclusive events, whereas others have reported them to be independent. We carried out a meta-analysis of published findings for FGFR3 and TP53 mutations in bladder cancer (535 tumours, 6 publications) and additional unpublished data for 382 tumours. TP53 and FGFR3 mutations were not independent events for all tumours considered together (OR = 0.25 [0.18–0.37], p = 0.0001) or for pT1 tumours alone (OR = 0.47 [0.28–0.79], p = 0.0009). However, if the analysis was restricted to pTa tumours or to muscle-invasive tumours alone, FGFR3 and TP53 mutations were independent events (OR = 0.56 [0.23–1.36] (p = 0.12) and OR = 0.99 [0.37–2.7] (p = 0.35), respectively). After stratification of the tumours by stage and grade, no dependence was detected in the five tumour groups considered (pTaG1 and pTaG2 together, pTaG3, pT1G2, pT1G3, pT2-4). These differences in findings can be attributed to the putative existence of two different pathways of tumour progression in bladder cancer: the CIS pathway, in which FGFR3 mutations are rare, and the Ta pathway, in which FGFR3 mutations are frequent. TP53 mutations occur at the earliest stage of the CIS pathway, whereas they occur would much later in the Ta pathway, at the T1G3 or muscle-invasive stage. PMID:23272046

Molecular analysis of mucopolysaccharidosis IVA: Common mutations and racial difference

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tomatsu, S.; Hori, T.; Nakashima, Y.

1994-09-01

Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disorder caused by a deficiency in N-acetylgalactosamine -6-sulfate sulfatase (GALNS). Studies on the molecular basis of MPS IVA have been facilitated following cloning of the full-length cDNA and genomic DNA. In this study we detected mutations from 20 Caucasian and 19 Japanese MPS IVA patients using SSCP system and compared mutations of Caucasian origin with those of Japanese origin. The results showed the presence of 16 various mutations (3 small, deletions, 2 nonsense and 11 missense mutations) for Caucasian patients and 15 (1 deletion, 1 large alteration and 13 missense mutations) formore » Japanese. Moreover, two common mutations existed; one is double gene deletion characteristic for Japanese (6 alleles; 15%) and the other is a point mutation (1113F A{yields}T transition) characteristic for Caucasian (9 alleles; 22.5%). And the clear genotype/phenotype relationship among 1342delCA, IVS1(-2), P151S, Q148X, R386C, I113F, Q473X, W220G, P151L, A291T, R90W, and P77R, for a severe type, G96B N204K and V138A for a milder type, was observed. Only R386 mutation was seen in both of the populations. Further, the precise DNA analysis for double gene deletion of a common double gene deletion has been performed by defining the breakpoints and the results showed that one deletion was caused by homologous recombination due to Alu repetitive sequences and the other was due to nonhomologous recombination of short direct repeat. Haplotype analysis for six alleles with double deletion were different, indicating the different origin of this mutation or the frequent recombination events before a mutational event. Thus the mutations in GALNS gene are very heterogeneous and the racial difference is characteristic.« less

Drug Resistance Missense Mutations in Cancer Are Subject to Evolutionary Constraints

PubMed Central

Friedman, Ran

2013-01-01

Several tumour types are sensitive to deactivation of just one or very few genes that are constantly active in the cancer cells, a phenomenon that is termed ‘oncogene addiction’. Drugs that target the products of those oncogenes can yield a temporary relief, and even complete remission. Unfortunately, many patients receiving oncogene-targeted therapies relapse on treatment. This often happens due to somatic mutations in the oncogene (‘resistance mutations’). ‘Compound mutations’, which in the context of cancer drug resistance are defined as two or more mutations of the drug target in the same clone may lead to enhanced resistance against the most selective inhibitors. Here, it is shown that the vast majority of the resistance mutations occurring in cancer patients treated with tyrosin kinase inhibitors aimed at three different proteins follow an evolutionary pathway. Using bioinformatic analysis tools, it is found that the drug-resistance mutations in the tyrosine kinase domains of Abl1, ALK and exons 20 and 21 of EGFR favour transformations to residues that can be identified in similar positions in evolutionary related proteins. The results demonstrate that evolutionary pressure shapes the mutational landscape in the case of drug-resistance somatic mutations. The constraints on the mutational landscape suggest that it may be possible to counter single drug-resistance point mutations. The observation of relatively many resistance mutations in Abl1, but not in the other genes, is explained by the fact that mutations in Abl1 tend to be biochemically conservative, whereas mutations in EGFR and ALK tend to be radical. Analysis of Abl1 compound mutations suggests that such mutations are more prevalent than hitherto reported and may be more difficult to counter. This supports the notion that such mutations may provide an escape route for targeted cancer drug resistance. PMID:24376513

Four Novel p.N385K, p.V36A, c.1033–1034insT and c.1417–1418delCT Mutations in the Sphingomyelin Phosphodiesterase 1 (SMPD1) Gene in Patients with Types A and B Niemann-Pick Disease (NPD)

PubMed Central

Manshadi, Masoumeh Dehghan; Kamalidehghan, Behnam; Keshavarzi, Fatemeh; Aryani, Omid; Dadgar, Sepideh; Arastehkani, Ahoora; Tondar, Mahdi; Ahmadipour, Fatemeh; Meng, Goh Yong; Houshmand, Massoud

2015-01-01

Background: Types A and B Niemann-Pick disease (NPD) are autosomal-recessive lysosomal storage disorders caused by the deficient activity of acid sphingomyelinase due to mutations in the sphingomyelin phosphodiesterase 1 (SMPD1) gene. Methods: In order to determine the prevalence and distribution of SMPD1 gene mutations, the genomic DNA of 15 unrelated Iranian patients with types A and B NPD was examined using PCR, DNA sequencing and bioinformatics analysis. Results: Of 8 patients with the p.G508R mutation, 5 patients were homozygous, while the other 3 were heterozygous. One patient was heterozygous for both the p.N385K and p.G508R mutations. Another patient was heterozygous for both the p.A487V and p.G508R mutations. Two patients (one homozygous and one heterozygous) showed the p.V36A mutation. One patient was homozygous for the c.1033–1034insT mutation. One patient was homozygous for the c.573delT mutation, and 1 patient was homozygous for the c.1417–1418delCT mutation. Additionally, bioinformatics analysis indicated that two new p.V36A and p.N385K mutations decreased the acid sphingomyelinase (ASM) protein stability, which might be evidence to suggest the pathogenicity of these mutations. Conclusion: with detection of these new mutations, the genotypic spectrum of types A and B NPD is extended, facilitating the definition of disease-related mutations. However, more research is essential to confirm the pathogenic effect of these mutations. PMID:25811928

The clinical phenotype of Lynch syndrome due to germline PMS2 mutations

PubMed Central

Senter, Leigha; Clendenning, Mark; Sotamaa, Kaisa; Hampel, Heather; Green, Jane; Potter, John D.; Lindblom, Annika; Lagerstedt, Kristina; Thibodeau, Stephen N.; Lindor, Noralane M.; Young, Joanne; Winship, Ingrid; Dowty, James G.; White, Darren M.; Hopper, John L.; Baglietto, Laura; Jenkins, Mark A.; de la Chapelle, Albert

2009-01-01

Background and Aims Although the clinical phenotype of Lynch syndrome (also known as Hereditary Nonpolyposis Colorectal Cancer) has been well described, little is known about disease in PMS2 mutation carriers. Now that mutation detection methods can discern mutations in PMS2 from mutations in its pseudogenes, more mutation carriers have been identified. Information about the clinical significance of PMS2 mutations is crucial for appropriate counseling. Here, we report the clinical characteristics of a large series of PMS2 mutation carriers. Methods We performed PMS2 mutation analysis using long range PCR and MLPA for 99 probands diagnosed with Lynch syndrome-associated tumors showing isolated loss of PMS2 by immunohistochemistry. Penetrance was calculated using a modified segregation analysis adjusting for ascertainment. Results Germline PMS2 mutations were detected in 62% of probands (n = 55 monoallelic; 6 biallelic). Among families with monoallelic PMS2 mutations, 65.5% met revised Bethesda guidelines. Compared with the general population, in mutation carriers, the incidence of colorectal cancer was 5.2 fold higher and the incidence of endometrial cancer was 7.5 fold higher. In North America, this translates to a cumulative cancer risk to age 70 of 15–20% for colorectal cancer, 15% for endometrial cancer, and 25–32% for any Lynch syndrome-associated cancer. No elevated risk for non-Lynch syndrome-associated cancers was observed. Conclusions PMS2 mutations contribute significantly to Lynch syndrome but the penetrance for monoallelic mutation carriers appears to be lower than that for the other mismatch repair genes. Modified counseling and cancer surveillance guidelines for PMS2 mutation carriers are proposed. PMID:18602922

The clinical phenotype of Lynch syndrome due to germ-line PMS2 mutations.

PubMed

Senter, Leigha; Clendenning, Mark; Sotamaa, Kaisa; Hampel, Heather; Green, Jane; Potter, John D; Lindblom, Annika; Lagerstedt, Kristina; Thibodeau, Stephen N; Lindor, Noralane M; Young, Joanne; Winship, Ingrid; Dowty, James G; White, Darren M; Hopper, John L; Baglietto, Laura; Jenkins, Mark A; de la Chapelle, Albert

2008-08-01

Although the clinical phenotype of Lynch syndrome (also known as hereditary nonpolyposis colorectal cancer) has been well described, little is known about disease in PMS2 mutation carriers. Now that mutation detection methods can discern mutations in PMS2 from mutations in its pseudogenes, more mutation carriers have been identified. Information about the clinical significance of PMS2 mutations is crucial for appropriate counseling. Here, we report the clinical characteristics of a large series of PMS2 mutation carriers. We performed PMS2 mutation analysis using long-range polymerase chain reaction and multiplex ligation-dependent probe amplification for 99 probands diagnosed with Lynch syndrome-associated tumors showing isolated loss of PMS2 by immunohistochemistry. Penetrance was calculated using a modified segregation analysis adjusting for ascertainment. Germ-line PMS2 mutations were detected in 62% of probands (n = 55 monoallelic; 6 biallelic). Among families with monoallelic PMS2 mutations, 65.5% met revised Bethesda guidelines. Compared with the general population, in mutation carriers, the incidence of colorectal cancer was 5.2-fold higher, and the incidence of endometrial cancer was 7.5-fold higher. In North America, this translates to a cumulative cancer risk to age 70 years of 15%-20% for colorectal cancer, 15% for endometrial cancer, and 25%-32% for any Lynch syndrome-associated cancer. No elevated risk for non-Lynch syndrome-associated cancers was observed. PMS2 mutations contribute significantly to Lynch syndrome, but the penetrance for monoallelic mutation carriers appears to be lower than that for the other mismatch repair genes. Modified counseling and cancer surveillance guidelines for PMS2 mutation carriers are proposed.

The AMBRE project: Parameterisation of FGK-type stars from the ESO:HARPS archived spectra

NASA Astrophysics Data System (ADS)

De Pascale, M.; Worley, C. C.; de Laverny, P.; Recio-Blanco, A.; Hill, V.; Bijaoui, A.

2014-10-01

Context. The AMBRE project is a collaboration between the European Southern Observatory (ESO) and the Observatoire de la Côte d'Azur (OCA). It has been established to determine the stellar atmospheric parameters of the archived spectra of four ESO spectrographs. Aims: The analysis of the ESO:HARPS archived spectra for the determination of their atmospheric parameters (effective temperature, surface gravity, global metallicities, and abundance of α-elements over iron) is presented. The sample being analysed (AMBRE:HARPS) covers the period from 2003 to 2010 and is comprised of 126 688 scientific spectra corresponding to ~17 218 different stars. Methods: For the analysis of the AMBRE:HARPS spectral sample, the automated pipeline developed for the analysis of the AMBRE:FEROS archived spectra has been adapted to the characteristics of the HARPS spectra. Within the pipeline, the stellar parameters are determined by the MATISSE algorithm, which has been developed at OCA for the analysis of large samples of stellar spectra in the framework of galactic archaeology. In the present application, MATISSE uses the AMBRE grid of synthetic spectra, which covers FGKM-type stars for a range of gravities and metallicities. Results: We first determined the radial velocity and its associated error for the ~15% of the AMBRE:HARPS spectra, for which this velocity had not been derived by the ESO:HARPS reduction pipeline. The stellar atmospheric parameters and the associated chemical index [α/Fe] with their associated errors have then been estimated for all the spectra of the AMBRE:HARPS archived sample. Based on key quality criteria, we accepted and delivered the parameterisation of 93 116 (74% of the total sample) spectra to ESO. These spectra correspond to ~10 706 stars; each are observed between one and several hundred times. This automatic parameterisation of the AMBRE:HARPS spectra shows that the large majority of these stars are cool main-sequence dwarfs with metallicities greater than -0.5 dex (as expected, given that HARPS has been extensively used for planet searches around GK-stars).

Gene analysis of PROP1 in dwarfism with combined pituitary hormone deficiency.

PubMed

Takamura, N; Fofanova, O V; Kinoshita, E; Yamashita, S

1999-06-01

The prophet of Pit-1 gene (PROP1), a novel pituitary-specific homeodomain factor, has been proved to be one of the causative genes for combined pituitary hormone deficiency (CPHD). Recently, PROP1 mutations have been identified in CPHD families, including our Russian cohort. The 2-bp deletion, 296delGA (A301G302del), is the most common mutational hot spot. Furthermore, in our cohort, PROP1 mutations are more common in comparison with human POU1F1 gene mutations. Here we review the gene analysis of PROP1 in patients with CPHD.

Statistical properties of Fermi GBM GRBs' spectra

NASA Astrophysics Data System (ADS)

Rácz, István I.; Balázs, Lajos G.; Horvath, Istvan; Tóth, L. Viktor; Bagoly, Zsolt

2018-03-01

Statistical studies of gamma-ray burst (GRB) spectra may result in important information on the physics of GRBs. The Fermi GBM catalogue contains GRB parameters (peak energy, spectral indices, and intensity) estimated fitting the gamma-ray spectral energy distribution of the total emission (fluence, flnc), and during the time of the peak flux (pflx). Using contingency tables, we studied the relationship of the models best-fitting pflx and flnc time intervals. Our analysis revealed an ordering of the spectra into a power law - Comptonized - smoothly broken power law - Band series. This result was further supported by a correspondence analysis of the pflx and flnc spectra categorical variables. We performed a linear discriminant analysis (LDA) to find a relationship between categorical (spectral) and model independent physical data. LDA resulted in highly significant physical differences among the spectral types, that is more pronounced in the case of the pflx spectra, than for the flnc spectra. We interpreted this difference as caused by the temporal variation of the spectrum during the outburst. This spectral variability is confirmed by the differences in the low-energy spectral index and peak energy, between the pflx and flnc spectra. We found that the synchrotron radiation is significant in GBM spectra. The mean low-energy spectral index is close to the canonical value of α = -2/3 during the peak flux. However, α is ˜ -0.9 for the spectra of the fluences. We interpret this difference as showing that the effect of cooling is important only for the fluence spectra.

Clinical follow up of mexican women with early onset of breast cancer and mutations in the BRCA1 and BRCA2 genes.

PubMed

Calderón-Garcidueñas, Ana Laura; Ruiz-Flores, Pablo; Cerda-Flores, Ricardo M; Barrera-Saldaña, Hugo A

2005-01-01

This study describes the presence of mutations in BRCA1 and BRCA2 genes in a group of Mexican women and the clinical evolution of early onset breast cancer (EOBC). A prospective hospital-based study was performed in a sample of 22 women with EOBC (7 in clinical stage IIA, 8 in IIB, and 7 in IIIA). The patients attended a tertiary care hospital in northeastern Mexico in 1997 and were followed up over a 5-year period. Molecular analysis included: 1) a mutation screening by heteroduplex analysis (HA) of BRCA1 and BRCA2 genes and 2) a sequence analysis. Of 22 patients, 14 (63.6%) showed a variant band detected by heteroduplex analysis of the BRCA1 and BRCA2 genes: 8 polymorphisms, 4 mutations of uncertain significance, and 2 novel truncated protein mutations, one in BRCAI (exon 11, 3587delT) and the other in the BRCA2 gene (exon 11, 2664InsA). These findings support future studies to determine the significance and impact of the genetic factor in this Mexican women population.

PRISM: Processing routines in IDL for spectroscopic measurements (installation manual and user's guide, version 1.0)

USGS Publications Warehouse

Kokaly, Raymond F.

2011-01-01

This report describes procedures for installing and using the U.S. Geological Survey Processing Routines in IDL for Spectroscopic Measurements (PRISM) software. PRISM provides a framework to conduct spectroscopic analysis of measurements made using laboratory, field, airborne, and space-based spectrometers. Using PRISM functions, the user can compare the spectra of materials of unknown composition with reference spectra of known materials. This spectroscopic analysis allows the composition of the material to be identified and characterized. Among its other functions, PRISM contains routines for the storage of spectra in database files, import/export of ENVI spectral libraries, importation of field spectra, correction of spectra to absolute reflectance, arithmetic operations on spectra, interactive continuum removal and comparison of spectral features, correction of imaging spectrometer data to ground-calibrated reflectance, and identification and mapping of materials using spectral feature-based analysis of reflectance data. This report provides step-by-step instructions for installing the PRISM software and running its functions.

Emergence of DNA Polymerase ε Antimutators That Escape Error-Induced Extinction in Yeast

PubMed Central

Williams, Lindsey N.; Herr, Alan J.; Preston, Bradley D.

2013-01-01

DNA polymerases (Pols) ε and δ perform the bulk of yeast leading- and lagging-strand DNA synthesis. Both Pols possess intrinsic proofreading exonucleases that edit errors during polymerization. Rare errors that elude proofreading are extended into duplex DNA and excised by the mismatch repair (MMR) system. Strains that lack Pol proofreading or MMR exhibit a 10- to 100-fold increase in spontaneous mutation rate (mutator phenotype), and inactivation of both Pol δ proofreading (pol3-01) and MMR is lethal due to replication error-induced extinction (EEX). It is unclear whether a similar synthetic lethal relationship exists between defects in Pol ε proofreading (pol2-4) and MMR. Using a plasmid-shuffling strategy in haploid Saccharomyces cerevisiae, we observed synthetic lethality of pol2-4 with alleles that completely abrogate MMR (msh2Δ, mlh1Δ, msh3Δ msh6Δ, or pms1Δ mlh3Δ) but not with partial MMR loss (msh3Δ, msh6Δ, pms1Δ, or mlh3Δ), indicating that high levels of unrepaired Pol ε errors drive extinction. However, variants that escape this error-induced extinction (eex mutants) frequently emerged. Five percent of pol2-4 msh2Δ eex mutants encoded second-site changes in Pol ε that reduced the pol2-4 mutator phenotype between 3- and 23-fold. The remaining eex alleles were extragenic to pol2-4. The locations of antimutator amino-acid changes in Pol ε and their effects on mutation spectra suggest multiple mechanisms of mutator suppression. Our data indicate that unrepaired leading- and lagging-strand polymerase errors drive extinction within a few cell divisions and suggest that there are polymerase-specific pathways of mutator suppression. The prevalence of suppressors extragenic to the Pol ε gene suggests that factors in addition to proofreading and MMR influence leading-strand DNA replication fidelity. PMID:23307893

Mutation analysis of 28 gaucher disease patients: The Australasian experience

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lewis, B.D.; Nelson, P.V.; Robertson, E.F.

1994-01-15

Gaucher disease is the most common lysomal storage disease. It is an autosomal recessive disorder that results from a deficiency of {beta}-glucocerrebrosidase. Three clinical phenotypes have been described: non-neuronopathic, acute neuronopathic, and subacuteneuronopathic. Genomic DNA from 28 Australasian patients of diverse ethnic origin with Gaucher disease was screened for 3 common mutations (1226G, 1448C and 84GG) using the amplification refractory mutation system (ARMS), and one uncommon mutation (1504T) by restriction enzyme digestion. Thirty-eight of the 56 independent alleles in these patients were characterized, with 1448C present in 42% and 1226G in 28% of the alleles. The 1226G mutation was associatedmore » only with the nonneuronopathic phenotype and 7 of the 15 patients who carried the 1448C mutation developed neuronopathic disease. Three infants who died in the neonatal period following a rapidly progressive neurodegenerative course carried no identifiable mutations. The 84GG mutation was carried by 2 Jewish patients and 1504T was present in one patient. It is now possible to rapidly identify the common Gaucher mutations using ARMS and restriction enzyme digestion, and our findings confirm the heterogeneity of mutations in Gaucher disease. It is also possible to predict in part the phenotypic outcome when screening patients for these mutations. The authors consider mutation analysis to be of most use in prenatal diagnosis and for carrier detection within affected families. 27 refs., 2 figs., 2 tabs.« less

Identification of novel mutations in Mexican patients with Aarskog-Scott syndrome.

PubMed

Pérez-Coria, Mariana; Lugo-Trampe, José J; Zamudio-Osuna, Michell; Rodríguez-Sánchez, Iram P; Lugo-Trampe, Angel; de la Fuente-Cortez, Beatriz; Campos-Acevedo, Luis D; Martínez-de-Villarreal, Laura E

2015-05-01

Aarskog-Scott syndrome (AAS), also known as faciogenital dysplasia (FGD, OMIM # 305400), is an X-linked disorder of recessive inheritance, characterized by short stature and facial, skeletal, and urogenital abnormalities. AAS is caused by mutations in the FGD1 gene (Xp11.22), with over 56 different mutations identified to date. We present the clinical and molecular analysis of four unrelated families of Mexican origin with an AAS phenotype, in whom FGD1 sequencing was performed. This analysis identified two stop mutations not previously reported in the literature: p.Gln664* and p.Glu380*. Phenotypically, every male patient met the clinical criteria of the syndrome, whereas discrepancies were found between phenotypes in female patients. Our results identify two novel mutations in FGD1, broadening the spectrum of reported mutations; and provide further delineation of the phenotypic variability previously described in AAS.

Identification of novel mutations in Mexican patients with Aarskog–Scott syndrome

PubMed Central

Pérez-Coria, Mariana; Lugo-Trampe, José J; Zamudio-Osuna, Michell; Rodríguez-Sánchez, Iram P; Lugo-Trampe, Angel; de la Fuente-Cortez, Beatriz; Campos-Acevedo, Luis D; Martínez-de-Villarreal, Laura E

2015-01-01

Aarskog–Scott syndrome (AAS), also known as faciogenital dysplasia (FGD, OMIM # 305400), is an X-linked disorder of recessive inheritance, characterized by short stature and facial, skeletal, and urogenital abnormalities. AAS is caused by mutations in the FGD1 gene (Xp11.22), with over 56 different mutations identified to date. We present the clinical and molecular analysis of four unrelated families of Mexican origin with an AAS phenotype, in whom FGD1 sequencing was performed. This analysis identified two stop mutations not previously reported in the literature: p.Gln664* and p.Glu380*. Phenotypically, every male patient met the clinical criteria of the syndrome, whereas discrepancies were found between phenotypes in female patients. Our results identify two novel mutations in FGD1, broadening the spectrum of reported mutations; and provide further delineation of the phenotypic variability previously described in AAS. PMID:26029706

[Analysis of H63D mutation in hemochromatosis (HFE) gene in populations of central Eurasia].

PubMed

Khusainova, R I; Khusnutdinova, N N; Litvinov, S S; Khusnutdinova, E K

2013-02-01

An analysis of the frequency of H63D (c. 187C>G) mutations in the HFEgene in 19 populations from Central Eurasia demonstrated that the distribution of the mutation in the region of interest was not uniform and that there were the areas of H63D accumulation. The investigation of three polymorphic variants, c.340+4T>C (rs2071303, IVS2(+4)T>C), c.893-44T>C (rs1800708, IVS4(-44)T>C), and c.1007-47G>A (rs1572982, IVS5(-47)A>G), in the HFE gene in individuals homozygous for H63D mutations in the HFE gene revealed the linkage of H63D with three haplotypes, *CTA, *TG, and *TTA. These findings indicated the partial spread of the mutation in Central Eurasia from Western Europe, as well as the possible repeated appearance of the mutation on the territory on interest.

Impact of the number of mutations in survival and response outcomes to hypomethylating agents in patients with myelodysplastic syndromes or myelodysplastic/myeloproliferative neoplasms

PubMed Central

Montalban-Bravo, Guillermo; Takahashi, Koichi; Patel, Keyur; Wang, Feng; Xingzhi, Song; Nogueras, Graciela M.; Huang, Xuelin; Pierola, Ana Alfonso; Jabbour, Elias; Colla, Simona; Gañan-Gomez, Irene; Borthakur, Gautham; Daver, Naval; Estrov, Zeev; Kadia, Tapan; Pemmaraju, Naveen; Ravandi, Farhad; Bueso-Ramos, Carlos; Chamseddine, Ali; Konopleva, Marina; Zhang, Jianhua; Kantarjian, Hagop; Futreal, Andrew; Garcia-Manero, Guillermo

2018-01-01

The prognostic and predictive value of sequencing analysis in myelodysplastic syndromes (MDS) has not been fully integrated into clinical practice. We performed whole exome sequencing (WES) of bone marrow samples from 83 patients with MDS and 31 with MDS/MPN identifying 218 driver mutations in 31 genes in 98 (86%) patients. A total of 65 (57%) patients received therapy with hypomethylating agents. By univariate analysis, mutations in BCOR, STAG2, TP53 and SF3B1 significantly influenced survival. Increased number of mutations (≥ 3), but not clonal heterogeneity, predicted for shorter survival and LFS. Presence of 3 or more mutations also predicted for lower likelihood of response (26 vs 50%, p = 0.055), and shorter response duration (3.6 vs 26.5 months, p = 0.022). By multivariate analysis, TP53 mutations (HR 3.1, CI 1.3–7.5, p = 0.011) and number of mutations (≥ 3) (HR 2.5, CI 1.3–4.8, p = 0.005) predicted for shorter survival. A novel prognostic model integrating this mutation data with IPSS-R separated patients into three categories with median survival of not reached, 29 months and 12 months respectively (p < 0.001) and increased stratification potential, compared to IPSS-R, in patients with high/very-high IPSS-R. This model was validated in a separate cohort of 413 patients with untreated MDS. Although the use of WES did not provide significant more information than that obtained with targeted sequencing, our findings indicate that increased number of mutations is an independent prognostic factor in MDS and that mutation data can add value to clinical prognostic models. PMID:29515765

Molecular analysis of congenital goitres with hypothyroidism caused by defective thyroglobulin synthesis. Identification of a novel c.7006C>T [p.R2317X] mutation and expression of minigenes containing nonsense mutations in exon 7.

PubMed

Machiavelli, Gloria A; Caputo, Mariela; Rivolta, Carina M; Olcese, María C; Gruñeiro-Papendieck, Laura; Chiesa, Ana; González-Sarmiento, Rogelio; Targovnik, Héctor M

2010-01-01

Thyroglobulin (TG) deficiency is an autosomal-recessive disorder that results in thyroid dyshormonogenesis. A number of distinct mutations have been identified as causing human hypothyroid goitre. The purpose of this study was to identify and characterize new mutations in the TG gene in an attempt to increase the understanding of the genetic mechanism responsible for this disorder. A total of six patients from four nonconsanguineous families with marked impairment of TG synthesis were studied. Single-strand conformation polymorphism (SSCP) analysis, sequencing of DNA, genotyping, expression of chimeric minigenes and bioinformatic analysis were performed. Four different inactivating TG mutations were identified: one novel mutation (c.7006C>T [p.R2317X]) and three previously reported (c.886C>T [p.R277X], c.6701C>A [p.A2215D] and c.6725G>A [p.R2223H]). Consequently, one patient carried a compound heterozygous for p.R2223H/p.R2317X mutations; two brothers showed a homozygous p.A2215D substitution and the remaining three patients, from two families with typical phenotype, had a single p.R277X mutated allele. We also showed functional evidences that premature stop codons inserted at different positions in exon 7, which disrupt exonic splicing enhancer (ESE) sequences, do not interfere with exon definition and processing. In this study, we have identified a novel nonsense mutation p.R2317X in the acetylcholinesterase homology domain of TG. We have also observed that nonsense mutations do not interfere with the pre-mRNA splicing of exon 7. The results are in accordance with previous observations confirming the genetic heterogeneity of TG defects.

Longitudinal monitoring of EGFR mutations in plasma predicts outcomes of NSCLC patients treated with EGFR TKIs: Korean Lung Cancer Consortium (KLCC-12-02).

PubMed

Lee, Ji Yun; Qing, Xu; Xiumin, Wei; Yali, Bai; Chi, Sangah; Bak, So Hyeon; Lee, Ho Yun; Sun, Jong-Mu; Lee, Se-Hoon; Ahn, Jin Seok; Cho, Eun Kyung; Kim, Dong-Wan; Kim, Hye Ryun; Min, Young Joo; Jung, Sin-Ho; Park, Keunchil; Mao, Mao; Ahn, Myung-Ju

2016-02-09

We hypothesized that plasma-based EGFR mutation analysis for NSCLC may be feasible for monitoring treatment response to EGFR TKIs and also predict drug resistance.Clinically relevant mutations including exon 19 deletion (ex19del), L858R and T790M were analyzed using droplet digital PCR (ddPCR) in longitudinally collected plasma samples (n = 367) from 81 NSCLC patients treated with EGFR TKI. Of a total 58 baseline cell-free DNA (cfDNA) samples available for ddPCR analysis, 43 (74.1%) had the same mutation in the matched tumors (clinical sensitivity: 70.8% [17/24] for L858R and 76.5% [26/34] for ex19del). The concordance rates of plasma with tissue-based results of EGFR mutations were 87.9% for L858R and 86.2% for ex19del. All 40 patients who were detected EGFR mutations at baseline showed a dramatic decrease of mutant copies (>50%) in plasma during the first two months after treatment. Median progression-free survival (PFS) was 10.1 months for patients with undetectable EGFR v 6.3 months for detectable EGFR mutations in blood after two-month treatment (HR 3.88, 95% CI 1.48-10.19, P = 0.006). We observed emerging resistance with early detection of T790M as a secondary mutation in 14 (28.6%) of 49 patients. Plasma-based EGFR mutation analysis using ddPCR can monitor treatment response to EGFR TKIs and can lead to early detection of EGFR TKIs resistance. Further studies confirming clinical implications of EGFR mutation in plasma are warranted to guide optimal therapeutic strategies upon knowledge of treatment response and resistance.

SDHB-related pheochromocytoma and paraganglioma penetrance and genotype-phenotype correlations.

PubMed

Jochmanova, Ivana; Wolf, Katherine I; King, Kathryn S; Nambuba, Joan; Wesley, Robert; Martucci, Victoria; Raygada, Margarita; Adams, Karen T; Prodanov, Tamara; Fojo, Antonio Tito; Lazurova, Ivica; Pacak, Karel

2017-08-01

Succinate dehydrogenase subunit B (SDHB) gene mutations are associated with an aggressive clinical disease course of pheochromocytoma/paraganglioma (PHEO/PGL). Limited information is available concerning PHEO/PGL penetrance among SDHB mutation carriers with regards to primary tumor location, specific mutation type, and gender. We assessed PHEO/PGL penetrance in SDHB mutation carriers and described the clinical presentation and disease course. Asymptomatic relatives (N = 611) of 103 index patients were tested for SDHB mutations. Mutation carriers (N = 328) were offered PHEO/PGL screening, of which 241 participated and were included in penetrance analysis. For additional disease outcome analysis, the 103 index patients and 40 screened individuals who developed PHEO/PGL were included. Clinical data were collected between October 2004 and June 2016. Forty (16.60%) of the 241 screened individuals developed PHEO/PGL during the study. The penetrance estimate in this population was 49.80% (95% CI 29-74.9) at 85 years. A significantly higher age-related penetrance of disease was observed in males compared to females, with 50% penetrance achieved at age 74 vs. not reached. Age-related penetrance analysis demonstrated 4 mutations (Ile127Ser, IVS1+1G>T, Exon 1 deletion, Arg90X) presenting with a slower rate of disease development (50% penetrance ages, respectively: not achieved, 70, 63, 61 years) compared to Arg46X and Val140Phe mutations (50% penetrance at 38 years). Here, we found a higher estimated penetrance compared to several other studies, and a striking difference in age-related penetrance between male and female SDHB mutation carriers with no association between mutation and gender or tumor location.

Mucopolysaccharidosis type I in 21 Czech and Slovak patients: Mutation analysis suggests a functional importance of C-terminus of the IDUA protein

PubMed Central

Vazna, Alzbeta; Beesley, Clare; Berna, Linda; Stolnaja, Larisa; Myskova, Helena; Bouckova, Michaela; Vlaskova, Hana; Poupetova, Helena; Zeman, Jiri; Magner, Martin; Hlavata, Anna; Winchester, Bryan; Hrebicek, Martin; Dvorakova, Lenka

2009-01-01

Abstract Mucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disorder that is caused by a deficiency of the enzyme α-l-iduronidase (IDUA). Of the 21 Czech and Slovak patients who have been diagnosed with MPS I in the last 30 years, 16 have a severe clinical presentation (Hurler syndrome), 2 less severe manifestations (Scheie syndrome), and 3 an intermediate severity (Hurler/Scheie phenotype). Mutation analysis was performed in 20 MPS I patients and 39 mutant alleles were identified. There was a high prevalence of the null mutations p.W402X (12 alleles) and p.Q70X (7 alleles) in this cohort. Four of the 13 different mutations were novel: p.V620F (3 alleles), p.W626X (1 allele), c.1727 + 2T > G (1 allele) and c.1918_1927del (2 alleles). The pathogenicity of the novel mutations was verified by transient expression studies in Chinese hamster ovary cells. Seven haplotypes were observed in the patient alleles using 13 intragenic polymorphisms. One of the two haplotypes associated with the mutation p.Q70X was not found in any of the controls. Haplotype analysis showed, that mutations p.Q70X, p.V620F, and p.D315Y probably have more than one ancestor. Missense mutations localized predominantly in the hydrophobic core of the enzyme are associated with the severe phenotype, whereas missense mutations localized to the surface of the enzyme are usually associated with the attenuated phenotypes. Mutations in the 130 C-terminal amino acids lead to clinical manifestations, which indicates a functional importance of the C-terminus of the IDUA protein. © 2009 Wiley-Liss, Inc. PMID:19396826

Clinical Utility of Genetic Testing in Children and Adults with Steroid-Resistant Nephrotic Syndrome

PubMed Central

Santín, Sheila; Bullich, Gemma; Tazón-Vega, Bárbara; García-Maset, Rafael; Giménez, Isabel; Silva, Irene; Ruíz, Patricia; Ballarín, José

2011-01-01

Summary Background and objectives The increasing number of podocyte-expressed genes implicated in steroid-resistant nephrotic syndrome (SRNS), the phenotypic variability, and the uncharacterized relative frequency of mutations in these genes in pediatric and adult patients with SRNS complicate their routine genetic analysis. Our aim was to compile the clinical and genetic data of eight podocyte genes analyzed in 110 cases (125 patients) with SRNS (ranging from congenital to adult onset) to provide a genetic testing approach. Design, setting, participants, & measurements Mutation analysis was performed by sequencing the NPHS1, NPHS2, TRPC6, CD2AP, PLCE1, INF2, WT1 (exons 8 and 9), and ACTN4 (exons 1 to 10) genes. Results We identified causing mutations in 34% (37/110) of SRNS patients, representing 67% (16/24) familial and 25% (21/86) sporadic cases. Mutations were detected in 100% of congenital-onset, 57% of infantile-onset, 24 and 36% of early and late childhood-onset, 25% of adolescent-onset, and 14% of adult-onset patients. The most frequently mutated gene was NPHS1 in congenital onset and NPHS2 in the other groups. A partial remission was observed in 7 of 26 mutation carriers treated with immunosuppressive agents and/or angiotensin-converting enzyme inhibitors. Patients with NPHS1 mutations showed a faster progression to ESRD than patients with NPHS2 mutations. None of these mutation carriers relapsed after kidney transplantation. Conclusions We propose a genetic testing algorithm for SRNS based on the age at onset and the familial/sporadic status. Mutation analysis of specific podocyte-genes has a clinical value in all age groups, especially in children. PMID:21415313

A novel COLD-PCR/FMCA assay enhances the detection of low-abundance IDH1 mutations in gliomas.

PubMed

Pang, Brendan; Durso, Mary B; Hamilton, Ronald L; Nikiforova, Marina N

2013-03-01

Point mutations in isocitrate dehydrogenase 1 (IDH1) have been identified in many gliomas. The detection of IDH1 mutations becomes challenging on suboptimal glioma biopsies when a limited number of tumor cells is available for analysis. Coamplification at lower denaturing-polymerase chain reaction (COLD-PCR) is a PCR technique that deliberately lowers the denaturing cycle temperature to selectively favor amplification of mutant alleles, allowing for the sensitive detection of low-abundance mutations. We developed a novel COLD-PCR assay on the LightCycler platform (Roche, Applied Science, Indianapolis, IN), using post-PCR fluorescent melting curve analysis (FMCA) for the detection of mutant IDH1 with a detection limit of 1%. Thirty-five WHO grade I to IV gliomas and 9 non-neoplastic brain and spinal cord biopsies were analyzed with this technique and the results were compared with the conventional real-time PCR and the Sanger sequencing analysis. COLD-PCR/FMCA was able to detect the most common IDH1 R132H mutation and rare mutation types including R132H, R132C, R132L, R132S, and R132G mutations. Twenty-five glioma cases were positive for IDH1 mutations by COLD-PCR/FMCA, and 23 gliomas were positive by the conventional real-time PCR and Sanger sequencing. A pilocytic astrocytoma (PA I) and a glioblastoma multiforme (GBM IV) showed low-abundance IDH1 mutations detected by COLD-PCR/FMCA. The remaining 10 glioma and 9 non-neoplastic samples were negative by all the 3 methods. In summary, we report a novel COLD-PCR/FMCA method that provides rapid and sensitive detection of IDH1 mutations in formalin-fixed paraffin-embedded tissue and can be used in the clinical setting to assess the small brain biopsies.

A novel WFS1 mutation in a family with dominant low frequency sensorineural hearing loss with normal VEMP and EcochG findings

PubMed Central

Bramhall, Naomi F; Kallman, Jeremy C; Verrall, Aimee M; Street, Valerie A

2008-01-01

Background Low frequency sensorineural hearing loss (LFSNHL) is an uncommon clinical finding. Mutations within three different identified genes (DIAPH1, MYO7A, and WFS1) are known to cause LFSNHL. The majority of hereditary LFSNHL is associated with heterozygous mutations in the WFS1 gene (wolframin protein). The goal of this study was to use genetic analysis to determine if a small American family's hereditary LFSNHL is linked to a mutation in the WFS1 gene and to use VEMP and EcochG testing to further characterize the family's audiovestibular phenotype. Methods The clinical phenotype of the American family was characterized by audiologic testing, vestibular evoked myogenic potentials (VEMP), and electrocochleography (EcochG) evaluation. Genetic characterization was performed by microsatellite analysis and direct sequencing of WFS1 for mutation detection. Results Sequence analysis of the WFS1 gene revealed a novel heterozygous mutation at c.2054G>C predicting a p.R685P amino acid substitution in wolframin. The c.2054G>C mutation segregates faithfully with hearing loss in the family and is absent in 230 control chromosomes. The p.R685 residue is located within the hydrophilic C-terminus of wolframin and is conserved across species. The VEMP and EcochG findings were normal in individuals segregating the WFS1 c.2054G>C mutation. Conclusion We discovered a novel heterozygous missense mutation in exon 8 of WFS1 predicting a p.R685P amino acid substitution that is likely to underlie the LFSNHL phenotype in the American family. For the first time, we describe VEMP and EcochG findings for individuals segregating a heterozygous WFS1 mutation. PMID:18518985

A novel WFS1 mutation in a family with dominant low frequency sensorineural hearing loss with normal VEMP and EcochG findings.

PubMed

Bramhall, Naomi F; Kallman, Jeremy C; Verrall, Aimee M; Street, Valerie A

2008-06-02

Low frequency sensorineural hearing loss (LFSNHL) is an uncommon clinical finding. Mutations within three different identified genes (DIAPH1, MYO7A, and WFS1) are known to cause LFSNHL. The majority of hereditary LFSNHL is associated with heterozygous mutations in the WFS1 gene (wolframin protein). The goal of this study was to use genetic analysis to determine if a small American family's hereditary LFSNHL is linked to a mutation in the WFS1 gene and to use VEMP and EcochG testing to further characterize the family's audiovestibular phenotype. The clinical phenotype of the American family was characterized by audiologic testing, vestibular evoked myogenic potentials (VEMP), and electrocochleography (EcochG) evaluation. Genetic characterization was performed by microsatellite analysis and direct sequencing of WFS1 for mutation detection. Sequence analysis of the WFS1 gene revealed a novel heterozygous mutation at c.2054G>C predicting a p.R685P amino acid substitution in wolframin. The c.2054G>C mutation segregates faithfully with hearing loss in the family and is absent in 230 control chromosomes. The p.R685 residue is located within the hydrophilic C-terminus of wolframin and is conserved across species. The VEMP and EcochG findings were normal in individuals segregating the WFS1 c.2054G>C mutation. We discovered a novel heterozygous missense mutation in exon 8 of WFS1 predicting a p.R685P amino acid substitution that is likely to underlie the LFSNHL phenotype in the American family. For the first time, we describe VEMP and EcochG findings for individuals segregating a heterozygous WFS1 mutation.

BRAFV600E mutation and its association with clinicopathological features of colorectal cancer: a systematic review and meta-analysis.

PubMed

Chen, Dong; Huang, Jun-Fu; Liu, Kai; Zhang, Li-Qun; Yang, Zhao; Chuai, Zheng-Ran; Wang, Yun-Xia; Shi, Da-Chuan; Huang, Qing; Fu, Wei-Ling

2014-01-01

Colorectal cancer (CRC) is a heterogeneous disease with multiple underlying causative genetic mutations. The B-type Raf proto-oncogene (BRAF) plays an important role in the mitogen-activated protein kinase (MAPK) signaling cascade during CRC. The presence of BRAFV600E mutation can determine the response of a tumor to chemotherapy. However, the association between the BRAFV600E mutation and the clinicopathological features of CRC remains controversial. We performed a systematic review and meta-analysis to estimate the effect of BRAFV600E mutation on the clinicopathological characteristics of CRC. We identified studies that examined the effect of BRAFV600E mutation on CRC within the PubMed, ISI Science Citation Index, and Embase databases. The effect of BRAFV600E on outcome parameters was estimated by odds ratios (ORs) with 95% confidence intervals (CIs) for each study using a fixed effects or random effects model. 25 studies with a total of 11,955 CRC patients met inclusion criteria. The rate of BRAFV600 was 10.8% (1288/11955). The BRAFV600E mutation in CRC was associated with advanced TNM stage, poor differentiation, mucinous histology, microsatellite instability (MSI), CpG island methylator phenotype (CIMP). This mutation was also associated with female gender, older age, proximal colon, and mutL homolog 1 (MLH1) methylation. This meta-analysis demonstrated that BRAFV600E mutation was significantly correlated with adverse pathological features of CRC and distinct clinical characteristics. These data suggest that BRAFV600E mutation could be used to supplement standard clinical and pathological staging for the better management of individual CRC patients, and could be considered as a poor prognostic marker for CRC.

Meta-analysis of neuroblastomas reveals a skewed ALK mutation spectrum in tumors with MYCN amplification.

PubMed

De Brouwer, Sara; De Preter, Katleen; Kumps, Candy; Zabrocki, Piotr; Porcu, Michaël; Westerhout, Ellen M; Lakeman, Arjan; Vandesompele, Jo; Hoebeeck, Jasmien; Van Maerken, Tom; De Paepe, Anne; Laureys, Geneviève; Schulte, Johannes H; Schramm, Alexander; Van Den Broecke, Caroline; Vermeulen, Joëlle; Van Roy, Nadine; Beiske, Klaus; Renard, Marleen; Noguera, Rosa; Delattre, Olivier; Janoueix-Lerosey, Isabelle; Kogner, Per; Martinsson, Tommy; Nakagawara, Akira; Ohira, Miki; Caron, Huib; Eggert, Angelika; Cools, Jan; Versteeg, Rogier; Speleman, Frank

2010-09-01

Activating mutations of the anaplastic lymphoma kinase (ALK) were recently described in neuroblastoma. We carried out a meta-analysis of 709 neuroblastoma tumors to determine their frequency and mutation spectrum in relation to genomic and clinical parameters, and studied the prognostic significance of ALK copy number and expression. The frequency and type of ALK mutations, copy number gain, and expression were analyzed in a new series of 254 neuroblastoma tumors. Data from 455 published cases were used for further in-depth analysis. ALK mutations were present in 6.9% of 709 investigated tumors, and mutations were found in similar frequencies in favorable [International Neuroblastoma Staging System (INSS) 1, 2, and 4S; 5.7%] and unfavorable (INSS 3 and 4; 7.5%) neuroblastomas (P = 0.087). Two hotspot mutations, at positions R1275 and F1174, were observed (49% and 34.7% of the mutated cases, respectively). Interestingly, the F1174 mutations occurred in a high proportion of MYCN-amplified cases (P = 0.001), and this combined occurrence was associated with a particular poor outcome, suggesting a positive cooperative effect between both aberrations. Furthermore, the F1174L mutant was characterized by a higher degree of autophosphorylation and a more potent transforming capacity as compared with the R1275Q mutant. Chromosome 2p gains, including the ALK locus (91.8%), were associated with a significantly increased ALK expression, which was also correlated with poor survival. ALK mutations occur in equal frequencies across all genomic subtypes, but F1174L mutants are observed in a higher frequency of MYCN-amplified tumors and show increased transforming capacity as compared with the R1275Q mutants.

Impact of the number of mutations in survival and response outcomes to hypomethylating agents in patients with myelodysplastic syndromes or myelodysplastic/myeloproliferative neoplasms.

PubMed

Montalban-Bravo, Guillermo; Takahashi, Koichi; Patel, Keyur; Wang, Feng; Xingzhi, Song; Nogueras, Graciela M; Huang, Xuelin; Pierola, Ana Alfonso; Jabbour, Elias; Colla, Simona; Gañan-Gomez, Irene; Borthakur, Gautham; Daver, Naval; Estrov, Zeev; Kadia, Tapan; Pemmaraju, Naveen; Ravandi, Farhad; Bueso-Ramos, Carlos; Chamseddine, Ali; Konopleva, Marina; Zhang, Jianhua; Kantarjian, Hagop; Futreal, Andrew; Garcia-Manero, Guillermo

2018-02-09

The prognostic and predictive value of sequencing analysis in myelodysplastic syndromes (MDS) has not been fully integrated into clinical practice. We performed whole exome sequencing (WES) of bone marrow samples from 83 patients with MDS and 31 with MDS/MPN identifying 218 driver mutations in 31 genes in 98 (86%) patients. A total of 65 (57%) patients received therapy with hypomethylating agents. By univariate analysis, mutations in BCOR, STAG2, TP53 and SF3B1 significantly influenced survival. Increased number of mutations (≥ 3), but not clonal heterogeneity, predicted for shorter survival and LFS. Presence of 3 or more mutations also predicted for lower likelihood of response (26 vs 50%, p = 0.055), and shorter response duration (3.6 vs 26.5 months, p = 0.022). By multivariate analysis, TP53 mutations (HR 3.1, CI 1.3-7.5, p = 0.011) and number of mutations (≥ 3) (HR 2.5, CI 1.3-4.8, p = 0.005) predicted for shorter survival. A novel prognostic model integrating this mutation data with IPSS-R separated patients into three categories with median survival of not reached, 29 months and 12 months respectively ( p < 0.001) and increased stratification potential, compared to IPSS-R, in patients with high/very-high IPSS-R. This model was validated in a separate cohort of 413 patients with untreated MDS. Although the use of WES did not provide significant more information than that obtained with targeted sequencing, our findings indicate that increased number of mutations is an independent prognostic factor in MDS and that mutation data can add value to clinical prognostic models.