Elevated germline mutation rate in teenage fathers

PubMed Central

Forster, Peter; Hohoff, Carsten; Dunkelmann, Bettina; Schürenkamp, Marianne; Pfeiffer, Heidi; Neuhuber, Franz; Brinkmann, Bernd

2015-01-01

Men age and die, while cells in their germline are programmed to be immortal. To elucidate how germ cells maintain viable DNA despite increasing parental age, we analysed DNA from 24 097 parents and their children, from Europe, the Middle East and Africa. We chose repetitive microsatellite DNA that mutates (unlike point mutations) only as a result of cellular replication, providing us with a natural ‘cell-cycle counter’. We observe, as expected, that the overall mutation rate for fathers is seven times higher than for mothers. Also as expected, mothers have a low and lifelong constant DNA mutation rate. Surprisingly, however, we discover that (i) teenage fathers already set out from a much higher mutation rate than teenage mothers (potentially equivalent to 77–196 male germline cell divisions by puberty); and (ii) ageing men maintain sperm DNA quality similar to that of teenagers, presumably by using fresh batches of stem cells known as ‘A-dark spermatogonia’. PMID:25694621

Direct estimate of the spontaneous germ line mutation rate in African green monkeys.

PubMed

Pfeifer, Susanne P

2017-12-01

Here, I provide the first direct estimate of the spontaneous mutation rate in an Old World monkey, using a seven individual, three-generation pedigree of African green monkeys. Eight de novo mutations were identified within ∼1.5 Gbp of accessible genome, corresponding to an estimated point mutation rate of 0.94 × 10 -8 per site per generation, suggesting an effective population size of ∼12000 for the species. This estimation represents a significant improvement in our knowledge of the population genetics of the African green monkey, one of the most important nonhuman primate models in biomedical research. Furthermore, by comparing mutation rates in Old World monkeys with the only other direct estimates in primates to date-humans and chimpanzees-it is possible to uniquely address how mutation rates have evolved over longer time scales. While the estimated spontaneous mutation rate for African green monkeys is slightly lower than the rate of 1.2 × 10 -8 per base pair per generation reported in chimpanzees, it is similar to the lower range of rates of 0.96 × 10 -8 -1.28 × 10 -8 per base pair per generation recently estimated from whole genome pedigrees in humans. This result suggests a long-term constraint on mutation rate that is quite different from similar evidence pertaining to recombination rate evolution in primates. © 2017 The Author(s). Evolution © 2017 The Society for the Study of Evolution.

Efficient Knock-in of a Point Mutation in Porcine Fibroblasts Using the CRISPR/Cas9-GMNN Fusion Gene.

PubMed

Gerlach, Max; Kraft, Theresia; Brenner, Bernhard; Petersen, Björn; Niemann, Heiner; Montag, Judith

2018-06-13

During CRISPR/Cas9 mediated genome editing, site-specific double strand breaks are introduced and repaired either unspecific by non-homologous end joining (NHEJ) or sequence dependent by homology directed repair (HDR). Whereas NHEJ-based generation of gene knock-out is widely performed, the HDR-based knock-in of specific mutations remains a bottleneck. Especially in primary cell lines that are essential for the generation of cell culture and animal models of inherited human diseases, knock-in efficacy is insufficient and needs significant improvement. Here, we tested two different approaches to increase the knock-in frequency of a specific point mutation into the MYH7 -gene in porcine fetal fibroblasts. We added a small molecule inhibitor of NHEJ, SCR7 (5,6-bis((E)-benzylideneamino)-2-mercaptopyrimidin-4-ol), during genome editing and screened cell cultures for the point mutation. However, this approach did not yield increased knock-in rates. In an alternative approach, we fused humanized Cas9 (hCas9) to the N-terminal peptide of the Geminin gene ( GMNN ). The fusion protein is degraded in NHEJ-dominated cell cycle phases, which should increase HDR-rates. Using hCas9- GMNN and point mutation-specific real time PCR screening, we found a two-fold increase in genome edited cell cultures. This increase of HDR by hCas9- GMNN provides a promising way to enrich specific knock-in in porcine fibroblast cultures for somatic cloning approaches.

Non-coding cancer driver candidates identified with a sample- and position-specific model of the somatic mutation rate

PubMed Central

Juul, Malene; Bertl, Johanna; Guo, Qianyun; Nielsen, Morten Muhlig; Świtnicki, Michał; Hornshøj, Henrik; Madsen, Tobias; Hobolth, Asger; Pedersen, Jakob Skou

2017-01-01

Non-coding mutations may drive cancer development. Statistical detection of non-coding driver regions is challenged by a varying mutation rate and uncertainty of functional impact. Here, we develop a statistically founded non-coding driver-detection method, ncdDetect, which includes sample-specific mutational signatures, long-range mutation rate variation, and position-specific impact measures. Using ncdDetect, we screened non-coding regulatory regions of protein-coding genes across a pan-cancer set of whole-genomes (n = 505), which top-ranked known drivers and identified new candidates. For individual candidates, presence of non-coding mutations associates with altered expression or decreased patient survival across an independent pan-cancer sample set (n = 5454). This includes an antigen-presenting gene (CD1A), where 5’UTR mutations correlate significantly with decreased survival in melanoma. Additionally, mutations in a base-excision-repair gene (SMUG1) correlate with a C-to-T mutational-signature. Overall, we find that a rich model of mutational heterogeneity facilitates non-coding driver identification and integrative analysis points to candidates of potential clinical relevance. DOI: http://dx.doi.org/10.7554/eLife.21778.001 PMID:28362259

New observations on maternal age effect on germline de novo mutations.

PubMed

Wong, Wendy S W; Solomon, Benjamin D; Bodian, Dale L; Kothiyal, Prachi; Eley, Greg; Huddleston, Kathi C; Baker, Robin; Thach, Dzung C; Iyer, Ramaswamy K; Vockley, Joseph G; Niederhuber, John E

2016-01-19

Germline mutations are the source of evolution and contribute substantially to many health-related processes. Here we use whole-genome deep sequencing data from 693 parents-offspring trios to examine the de novo point mutations (DNMs) in the offspring. Our estimate for the mutation rate per base pair per generation is 1.05 × 10(-8), well within the range of previous studies. We show that maternal age has a small but significant correlation with the total number of DNMs in the offspring after controlling for paternal age (0.51 additional mutations per year, 95% CI: 0.29, 0.73), which was not detectable in the smaller and younger parental cohorts of earlier studies. Furthermore, while the total number of DNMs increases at a constant rate for paternal age, the contribution from the mother increases at an accelerated rate with age.These observations have implications related to the incidence of de novo mutations relating to maternal age.

Estimating Divergence Dates and Substitution Rates in the Drosophila Phylogeny

PubMed Central

Obbard, Darren J.; Maclennan, John; Kim, Kang-Wook; Rambaut, Andrew; O’Grady, Patrick M.; Jiggins, Francis M.

2012-01-01

An absolute timescale for evolution is essential if we are to associate evolutionary phenomena, such as adaptation or speciation, with potential causes, such as geological activity or climatic change. Timescales in most phylogenetic studies use geologically dated fossils or phylogeographic events as calibration points, but more recently, it has also become possible to use experimentally derived estimates of the mutation rate as a proxy for substitution rates. The large radiation of drosophilid taxa endemic to the Hawaiian islands has provided multiple calibration points for the Drosophila phylogeny, thanks to the "conveyor belt" process by which this archipelago forms and is colonized by species. However, published date estimates for key nodes in the Drosophila phylogeny vary widely, and many are based on simplistic models of colonization and coalescence or on estimates of island age that are not current. In this study, we use new sequence data from seven species of Hawaiian Drosophila to examine a range of explicit coalescent models and estimate substitution rates. We use these rates, along with a published experimentally determined mutation rate, to date key events in drosophilid evolution. Surprisingly, our estimate for the date for the most recent common ancestor of the genus Drosophila based on mutation rate (25–40 Ma) is closer to being compatible with independent fossil-derived dates (20–50 Ma) than are most of the Hawaiian-calibration models and also has smaller uncertainty. We find that Hawaiian-calibrated dates are extremely sensitive to model choice and give rise to point estimates that range between 26 and 192 Ma, depending on the details of the model. Potential problems with the Hawaiian calibration may arise from systematic variation in the molecular clock due to the long generation time of Hawaiian Drosophila compared with other Drosophila and/or uncertainty in linking island formation dates with colonization dates. As either source of error will bias estimates of divergence time, we suggest mutation rate estimates be used until better models are available. PMID:22683811

Parkin dosage mutations have greater pathogenicity in familial PD than simple sequence mutations

PubMed Central

Pankratz, N; Kissell, D K.; Pauciulo, M W.; Halter, C A.; Rudolph, A; Pfeiffer, R F.; Marder, K S.; Foroud, T; Nichols, W C.

2009-01-01

Objective: Mutations in both alleles of parkin have been shown to result in Parkinson disease (PD). However, it is unclear whether haploinsufficiency (presence of a mutation in only 1 of the 2 parkin alleles) increases the risk for PD. Methods: We performed comprehensive dosage and sequence analysis of all 12 exons of parkin in a sample of 520 independent patients with familial PD and 263 controls. We evaluated whether presence of a single parkin mutation, either a sequence (point mutation or small insertion/deletion) or dosage (whole exon deletion or duplication) mutation, was found at increased frequency in cases as compared with controls. We then compared the clinical characteristics of cases with 0, 1, or 2 parkin mutations. Results: We identified 55 independent patients with PD with at least 1 parkin mutation and 9 controls with a single sequence mutation. Cases and controls had a similar frequency of single sequence mutations (3.1% vs 3.4%, p = 0.83); however, the cases had a significantly higher rate of dosage mutations (2.6% vs 0%, p = 0.009). Cases with a single dosage mutation were more likely to have an earlier age at onset (50% with onset at ≤45 years) compared with those with no parkin mutations (10%, p = 0.00002); this was not true for cases with only a single sequence mutation (25% with onset at ≤45 years, p = 0.06). Conclusions: Parkin haploinsufficiency, specifically for a dosage mutation rather than a point mutation or small insertion/deletion, is a risk factor for familial PD and may be associated with earlier age at onset. GLOSSARY ADL = Activities of Daily Living; GDS = Geriatric Depression Scale; MLPA = multiplex ligation-dependent probe amplification; MMSE = Mini-Mental State Examination; PD = Parkinson disease; UPDRS = Unified Parkinson’s Disease Rating Scale. PMID:19636047

Biochemical analysis of respiratory function in cybrid cell lines harbouring mitochondrial DNA mutations

PubMed Central

2004-01-01

We analysed key biochemical features that reflect the balance between glycolysis and glucose oxidation in cybrids (cytoplasmic hybrids) harbouring a representative sample of mitochondrial DNA point mutations and deletions. The cybrids analysed had the same 143B cell nuclear background and were isogenic for the mitochondrial background. The 143B cell line and its ρ0 counterpart were used as controls. All cells analysed were in a dynamic state, and cell number, time of plating, culture medium, extracellular volume and time of harvest and assay were strictly controlled. Intra- and extra-cellular lactate and pyruvate levels were measured in homoplasmic wild-type and mutant cells, and correlated with rates of ATP synthesis and O2 consumption. In all mutant cell lines, except those with the T8993C mutation in the ATPase 6 gene, glycolysis was increased even under conditions of low glucose, as demonstrated by increased levels of extracellular lactate and pyruvate. Extracellular lactate levels were strictly and inversely correlated with rates of ATP synthesis and O2 consumption. These results show increased glycolysis and defective oxidative phosphorylation, irrespective of the type or site of the point mutation or deletion in the mitochondrial genome. The different biochemical consequences of the T8993C mutation suggest a uniquely different pathogenic mechanism for this mutation. However, the distinct clinical features associated with some of these mutations still remain to be elucidated. PMID:15324306

The Impact of Mutation and Gene Conversion on the Local Diversification of Antigen Genes in African Trypanosomes

PubMed Central

Gjini, Erida; Haydon, Daniel T.; Barry, J. David; Cobbold, Christina A.

2012-01-01

Patterns of genetic diversity in parasite antigen gene families hold important information about their potential to generate antigenic variation within and between hosts. The evolution of such gene families is typically driven by gene duplication, followed by point mutation and gene conversion. There is great interest in estimating the rates of these processes from molecular sequences for understanding the evolution of the pathogen and its significance for infection processes. In this study, a series of models are constructed to investigate hypotheses about the nucleotide diversity patterns between closely related gene sequences from the antigen gene archive of the African trypanosome, the protozoan parasite causative of human sleeping sickness in Equatorial Africa. We use a hidden Markov model approach to identify two scales of diversification: clustering of sequence mismatches, a putative indicator of gene conversion events with other lower-identity donor genes in the archive, and at a sparser scale, isolated mismatches, likely arising from independent point mutations. In addition to quantifying the respective probabilities of occurrence of these two processes, our approach yields estimates for the gene conversion tract length distribution and the average diversity contributed locally by conversion events. Model fitting is conducted using a Bayesian framework. We find that diversifying gene conversion events with lower-identity partners occur at least five times less frequently than point mutations on variant surface glycoprotein (VSG) pairs, and the average imported conversion tract is between 14 and 25 nucleotides long. However, because of the high diversity introduced by gene conversion, the two processes have almost equal impact on the per-nucleotide rate of sequence diversification between VSG subfamily members. We are able to disentangle the most likely locations of point mutations and conversions on each aligned gene pair. PMID:22735079

Leveraging Distant Relatedness to Quantify Human Mutation and Gene-Conversion Rates

PubMed Central

Palamara, Pier Francesco; Francioli, Laurent C.; Wilton, Peter R.; Genovese, Giulio; Gusev, Alexander; Finucane, Hilary K.; Sankararaman, Sriram; Sunyaev, Shamil R.; de Bakker, Paul I.W.; Wakeley, John; Pe’er, Itsik; Price, Alkes L.

2015-01-01

The rate at which human genomes mutate is a central biological parameter that has many implications for our ability to understand demographic and evolutionary phenomena. We present a method for inferring mutation and gene-conversion rates by using the number of sequence differences observed in identical-by-descent (IBD) segments together with a reconstructed model of recent population-size history. This approach is robust to, and can quantify, the presence of substantial genotyping error, as validated in coalescent simulations. We applied the method to 498 trio-phased sequenced Dutch individuals and inferred a point mutation rate of 1.66 × 10−8 per base per generation and a rate of 1.26 × 10−9 for <20 bp indels. By quantifying how estimates varied as a function of allele frequency, we inferred the probability that a site is involved in non-crossover gene conversion as 5.99 × 10−6. We found that recombination does not have observable mutagenic effects after gene conversion is accounted for and that local gene-conversion rates reflect recombination rates. We detected a strong enrichment of recent deleterious variation among mismatching variants found within IBD regions and observed summary statistics of local sharing of IBD segments to closely match previously proposed metrics of background selection; however, we found no significant effects of selection on our mutation-rate estimates. We detected no evidence of strong variation of mutation rates in a number of genomic annotations obtained from several recent studies. Our analysis suggests that a mutation-rate estimate higher than that reported by recent pedigree-based studies should be adopted in the context of DNA-based demographic reconstruction. PMID:26581902

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

Perelson, Alan S; Gerrish, Philip J

The constructive creativity of natural selection originates from its paradoxical ability to foster cooperation through competition. Cooperating communities ranging from complex societies to somatic tissue are constantly under attack, however, by non-cooperating mutants or transformants, called 'cheaters'. Structure in these communities promotes the formation of cooperating clusters whose competitive superiority can alone be sufficient to thwart outgrowths of cheaters and thereby maintain cooperation. But we find that when cheaters appear too frequently -- exceeding a threshold mutation or transformation rate -- their scattered outgrowths infiltrate and break up cooperating clusters, resulting in a cascading loss of community integrity, a switchmore » to net positive selection for cheaters, and ultimately in the loss of cooperation. We find that this threshold mutation rate is directly proportional to the fitness support received from each cooperating neighbor minus the individual fitness benefit of cheating. When mutation rate also evolves, this threshold is crossed spontaneously after thousands of generations, at which point cheaters rapidly invade. In a structured community, cooperation can persist only if the mutation rate remains below a critical value.« less

Ntg1p, the base excision repair protein, generates mutagenic intermediates in yeast mitochondrial DNA.

PubMed

Phadnis, Naina; Mehta, Reema; Meednu, Nida; Sia, Elaine A

2006-07-13

Mitochondrial DNA is predicted to be highly prone to oxidative damage due to its proximity to free radicals generated by oxidative phosphorylation. Base excision repair (BER) is the primary repair pathway responsible for repairing oxidative damage in nuclear and mitochondrial genomes. In yeast mitochondria, three N-glycosylases have been identified so far, Ntg1p, Ogg1p and Ung1p. Ntg1p, a broad specificity N-glycosylase, takes part in catalyzing the first step of BER that involves the removal of the damaged base. In this study, we examined the role of Ntg1p in maintaining yeast mitochondrial genome integrity. Using genetic reporters and assays to assess mitochondrial mutations, we found that loss of Ntg1p suppresses mitochondrial point mutation rates, frameshifts and recombination rates. We also observed a suppression of respiration loss in the ntg1-Delta cells in response to ultraviolet light exposure implying an overlap between BER and UV-induced damage in the yeast mitochondrial compartment. Over-expression of the BER AP endonuclease, Apn1p, did not significantly affect the mitochondrial mutation rate in the presence of Ntg1p, whereas Apn1p over-expression in an ntg1-Delta background increased the frequency of mitochondrial mutations. In addition, loss of Apn1p also suppressed mitochondrial point mutations. Our work suggests that both Ntg1p and Apn1p generate mutagenic intermediates in the yeast mitochondrial genome.

The Cerebro-oculo-facio-skeletal Syndrome Point Mutation F231L in the ERCC1 DNA Repair Protein Causes Dissociation of the ERCC1-XPF Complex*

PubMed Central

Faridounnia, Maryam; Wienk, Hans; Kovačič, Lidija; Folkers, Gert E.; Jaspers, Nicolaas G. J.; Kaptein, Robert; Hoeijmakers, Jan H. J.; Boelens, Rolf

2015-01-01

The ERCC1-XPF heterodimer, a structure-specific DNA endonuclease, is best known for its function in the nucleotide excision repair (NER) pathway. The ERCC1 point mutation F231L, located at the hydrophobic interaction interface of ERCC1 (excision repair cross-complementation group 1) and XPF (xeroderma pigmentosum complementation group F), leads to severe NER pathway deficiencies. Here, we analyze biophysical properties and report the NMR structure of the complex of the C-terminal tandem helix-hairpin-helix domains of ERCC1-XPF that contains this mutation. The structures of wild type and the F231L mutant are very similar. The F231L mutation results in only a small disturbance of the ERCC1-XPF interface, where, in contrast to Phe231, Leu231 lacks interactions stabilizing the ERCC1-XPF complex. One of the two anchor points is severely distorted, and this results in a more dynamic complex, causing reduced stability and an increased dissociation rate of the mutant complex as compared with wild type. These data provide a biophysical explanation for the severe NER deficiencies caused by this mutation. PMID:26085086

A Mutation in PGM2 Causing Inefficient Galactose Metabolism in the Probiotic Yeast Saccharomyces boulardii.

PubMed

Liu, Jing-Jing; Zhang, Guo-Chang; Kong, In Iok; Yun, Eun Ju; Zheng, Jia-Qi; Kweon, Dae-Hyuk; Jin, Yong-Su

2018-05-15

The probiotic yeast Saccharomyces boulardii has been extensively studied for the prevention and treatment of diarrheal diseases, and it is now commercially available in some countries. S. boulardii displays notable phenotypic characteristics, such as a high optimal growth temperature, high tolerance against acidic conditions, and the inability to form ascospores, which differentiate S. boulardii from Saccharomyces cerevisiae The majority of prior studies stated that S. boulardii exhibits sluggish or halted galactose utilization. Nonetheless, the molecular mechanisms underlying inefficient galactose uptake have yet to be elucidated. When the galactose utilization of a widely used S. boulardii strain, ATCC MYA-796, was examined under various culture conditions, the S. boulardii strain could consume galactose, but at a much lower rate than that of S. cerevisiae While all GAL genes were present in the S. boulardii genome, according to analysis of genomic sequencing data in a previous study, a point mutation (G1278A) in PGM2 , which codes for phosphoglucomutase, was identified in the genome of the S. boulardii strain. As the point mutation resulted in the truncation of the Pgm2 protein, which is known to play a pivotal role in galactose utilization, we hypothesized that the truncated Pgm2 might be associated with inefficient galactose metabolism. Indeed, complementation of S. cerevisiae PGM2 in S. boulardii restored galactose utilization. After reverting the point mutation to a full-length PGM2 in S. boulardii by Cas9-based genome editing, the growth rates of wild-type (with a truncated PGM2 gene) and mutant (with a full-length PGM2 ) strains with glucose or galactose as the carbon source were examined. As expected, the mutant (with a full-length PGM2 ) was able to ferment galactose faster than the wild-type strain. Interestingly, the mutant showed a lower growth rate than that of the wild-type strain on glucose at 37°C. Also, the wild-type strain was enriched in the mixed culture of wild-type and mutant strains on glucose at 37°C, suggesting that the truncated PGM2 might offer better growth on glucose at a higher temperature in return for inefficient galactose utilization. Our results suggest that the point mutation in PGM2 might be involved in multiple phenotypes with different effects. IMPORTANCE Saccharomyces boulardii is a probiotic yeast strain capable of preventing and treating diarrheal diseases. However, the genetics and metabolism of this yeast are largely unexplored. In particular, molecular mechanisms underlying the inefficient galactose metabolism of S. boulardii remain unknown. Our study reports that a point mutation in PGM2 , which codes for phosphoglucomutase, is responsible for inferior galactose utilization by S. boulardii After correction of the mutated PGM2 via genome editing, the resulting strain was able to use galactose faster than a parental strain. While the PGM2 mutation made the yeast use galactose slowly, investigation of the genomic sequencing data of other S. boulardii strains revealed that the PGM2 mutation is evolutionarily conserved. Interestingly, the PGM2 mutation was beneficial for growth at a higher temperature on glucose. We speculate that the PGM2 mutation was enriched due to selection of S. boulardii in the natural habitat (sugar-rich fruits in tropical areas). Copyright © 2018 American Society for Microbiology.

Reduced Mutation Rate and Increased Transformability of Transposon-Free Acinetobacter baylyi ADP1-ISx

PubMed Central

Suárez, Gabriel A.; Renda, Brian A.; Dasgupta, Aurko

2017-01-01

ABSTRACT The genomes of most bacteria contain mobile DNA elements that can contribute to undesirable genetic instability in engineered cells. In particular, transposable insertion sequence (IS) elements can rapidly inactivate genes that are important for a designed function. We deleted all six copies of IS1236 from the genome of the naturally transformable bacterium Acinetobacter baylyi ADP1. The natural competence of ADP1 made it possible to rapidly repair deleterious point mutations that arose during strain construction. In the resulting ADP1-ISx strain, the rates of mutations inactivating a reporter gene were reduced by 7- to 21-fold. This reduction was higher than expected from the incidence of new IS1236 insertions found during a 300-day mutation accumulation experiment with wild-type ADP1 that was used to estimate spontaneous mutation rates in the strain. The extra improvement appears to be due in part to eliminating large deletions caused by IS1236 activity, as the point mutation rate was unchanged in ADP1-ISx. Deletion of an error-prone polymerase (dinP) and a DNA damage response regulator (umuDAb [the umuD gene of A. baylyi]) from the ADP1-ISx genome did not further reduce mutation rates. Surprisingly, ADP1-ISx exhibited increased transformability. This improvement may be due to less autolysis and aggregation of the engineered cells than of the wild type. Thus, deleting IS elements from the ADP1 genome led to a greater than expected increase in evolutionary reliability and unexpectedly enhanced other key strain properties, as has been observed for other clean-genome bacterial strains. ADP1-ISx is an improved chassis for metabolic engineering and other applications. IMPORTANCE Acinetobacter baylyi ADP1 has been proposed as a next-generation bacterial host for synthetic biology and genome engineering due to its ability to efficiently take up DNA from its environment during normal growth. We deleted transposable elements that are capable of copying themselves, inserting into other genes, and thereby inactivating them from the ADP1 genome. The resulting “clean-genome” ADP1-ISx strain exhibited larger reductions in the rates of inactivating mutations than expected from spontaneous mutation rates measured via whole-genome sequencing of lineages evolved under relaxed selection. Surprisingly, we also found that IS element activity reduces transformability and is a major cause of cell aggregation and death in wild-type ADP1 grown under normal laboratory conditions. More generally, our results demonstrate that domesticating a bacterial genome by removing mobile DNA elements that have accumulated during evolution in the wild can have unanticipated benefits. PMID:28667117

Reduced Mutation Rate and Increased Transformability of Transposon-Free Acinetobacter baylyi ADP1-ISx.

PubMed

Suárez, Gabriel A; Renda, Brian A; Dasgupta, Aurko; Barrick, Jeffrey E

2017-09-01

The genomes of most bacteria contain mobile DNA elements that can contribute to undesirable genetic instability in engineered cells. In particular, transposable insertion sequence (IS) elements can rapidly inactivate genes that are important for a designed function. We deleted all six copies of IS 1236 from the genome of the naturally transformable bacterium Acinetobacter baylyi ADP1. The natural competence of ADP1 made it possible to rapidly repair deleterious point mutations that arose during strain construction. In the resulting ADP1-ISx strain, the rates of mutations inactivating a reporter gene were reduced by 7- to 21-fold. This reduction was higher than expected from the incidence of new IS 1236 insertions found during a 300-day mutation accumulation experiment with wild-type ADP1 that was used to estimate spontaneous mutation rates in the strain. The extra improvement appears to be due in part to eliminating large deletions caused by IS 1236 activity, as the point mutation rate was unchanged in ADP1-ISx. Deletion of an error-prone polymerase ( dinP ) and a DNA damage response regulator ( umuD Ab [the umuD gene of A. baylyi ]) from the ADP1-ISx genome did not further reduce mutation rates. Surprisingly, ADP1-ISx exhibited increased transformability. This improvement may be due to less autolysis and aggregation of the engineered cells than of the wild type. Thus, deleting IS elements from the ADP1 genome led to a greater than expected increase in evolutionary reliability and unexpectedly enhanced other key strain properties, as has been observed for other clean-genome bacterial strains. ADP1-ISx is an improved chassis for metabolic engineering and other applications. IMPORTANCE Acinetobacter baylyi ADP1 has been proposed as a next-generation bacterial host for synthetic biology and genome engineering due to its ability to efficiently take up DNA from its environment during normal growth. We deleted transposable elements that are capable of copying themselves, inserting into other genes, and thereby inactivating them from the ADP1 genome. The resulting "clean-genome" ADP1-ISx strain exhibited larger reductions in the rates of inactivating mutations than expected from spontaneous mutation rates measured via whole-genome sequencing of lineages evolved under relaxed selection. Surprisingly, we also found that IS element activity reduces transformability and is a major cause of cell aggregation and death in wild-type ADP1 grown under normal laboratory conditions. More generally, our results demonstrate that domesticating a bacterial genome by removing mobile DNA elements that have accumulated during evolution in the wild can have unanticipated benefits. Copyright © 2017 American Society for Microbiology.

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.

Fast maximum likelihood estimation of mutation rates using a birth-death process.

PubMed

Wu, Xiaowei; Zhu, Hongxiao

2015-02-07

Since fluctuation analysis was first introduced by Luria and Delbrück in 1943, it has been widely used to make inference about spontaneous mutation rates in cultured cells. Under certain model assumptions, the probability distribution of the number of mutants that appear in a fluctuation experiment can be derived explicitly, which provides the basis of mutation rate estimation. It has been shown that, among various existing estimators, the maximum likelihood estimator usually demonstrates some desirable properties such as consistency and lower mean squared error. However, its application in real experimental data is often hindered by slow computation of likelihood due to the recursive form of the mutant-count distribution. We propose a fast maximum likelihood estimator of mutation rates, MLE-BD, based on a birth-death process model with non-differential growth assumption. Simulation studies demonstrate that, compared with the conventional maximum likelihood estimator derived from the Luria-Delbrück distribution, MLE-BD achieves substantial improvement on computational speed and is applicable to arbitrarily large number of mutants. In addition, it still retains good accuracy on point estimation. Published by Elsevier Ltd.

Molecular diagnosis of α-thalassemia in a multiethnic population.

PubMed

Gilad, Oded; Shemer, Orna Steinberg; Dgany, Orly; Krasnov, Tanya; Nevo, Michal; Noy-Lotan, Sharon; Rabinowicz, Ron; Amitai, Nofar; Ben-Dor, Shifra; Yaniv, Isaac; Yacobovich, Joanne; Tamary, Hannah

2017-06-01

α-Thalassemia, one of the most common genetic diseases, is caused by deletions or point mutations affecting one to four α-globin genes. Molecular diagnosis is important to prevent the most severe forms of the disease. However, the diagnosis of α-thalassemia is complex due to a high variability of the genetic defects involved, with over 250 described mutations. We summarize herein the findings of genetic analyses of DNA samples referred to our laboratory for the molecular diagnosis of α-thalassemia, along with a detailed clinical description. We utilized a diagnostic algorithm including Gap-PCR, to detect known deletions, followed by sequencing of the α-globin gene, to identify known and novel point mutations, and multiplex ligation-dependent probe amplification (MLPA) for the diagnosis of rare or novel deletions. α-Thalassemia was diagnosed in 662 of 975 samples referred to our laboratory. Most commonly found were deletions (75.3%, including two novel deletions previously described by us); point mutations comprised 25.4% of the cases, including five novel mutations. Our population included mostly Jews (of Ashkenazi and Sephardic origin) and Muslim Arabs, who presented with a higher rate of point mutations and hemoglobin H disease. Overall, we detected 53 different genotype combinations causing a spectrum of clinical phenotypes, from asymptomatic to severe anemia. Our work constitutes the largest group of patients with α-thalassemia originating in the Mediterranean whose clinical characteristics and molecular basis have been determined. We suggest a diagnostic algorithm that leads to an accurate molecular diagnosis in multiethnic populations. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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.

Prevalence and characterisation of plasmid-mediated quinolone resistance and mutations in the gyrase and topoisomerase IV genes among Shigella isolates from Henan, China, between 2001 and 2008.

PubMed

Yang, Haiyan; Duan, Guangcai; Zhu, Jingyuan; Zhang, Weidong; Xi, Yuanlin; Fan, Qingtang

2013-08-01

A total of 293 Shigella isolates were isolated from patients with diarrhoea in four villages of Henan, China. This study investigated the prevalence of the plasmid-mediated quinolone resistance (PMQR) genes qnrA, qnrB, qnrS, qepA and aac(6')-Ib-cr and compared the polymorphic quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC and parE. Of the isolates, 292 were found to be resistant to nalidixic acid and pipemidic acid, whereas 77 were resistant to ciprofloxacin (resistance rate of 26.3%). Resistance of the Shigella isolates to ciprofloxacin significantly increased from 2001 to 2008 (P<0.05). A mutation in gyrA was present in 277 (94.5%) of the isolates and a mutation in parC was present in 19 (6.5%) of the isolates. Moreover, 168 (57.3%) of the isolates contained only the gyrA (Ser83Leu) mutation. In addition, 107 isolates had two gyrA point mutations (Ser83Leu and either Asp87Gly, Asp87Asn or Asp113Tyr) and 13 isolates had two gyrA point mutations (Ser83Leu and Asp87Gly or Gly214Ala) and one parC mutation (Ser80Ile). In addition, qepA and aac(6')-Ib-cr were present in 6 (2.05%) and 19 (6.48%) of the isolates, respectively. All but one of the PMQR-positive isolates with a ciprofloxacin minimum inhibitory concentration in the range 4-32μg/mL had a mutation in the QRDR. It is known that PMQR-positive Shigella isolates are common in China. This study found that there was a significant increase in mutation rates of the QRDR and the resistant rates to ciprofloxacin. Other mechanisms may be present in the isolates that also contribute to their resistance to ciprofloxacin. Copyright © 2013 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Point Mutations in c-Myc Uncouple Neoplastic Transformation from Multiple Other Phenotypes in Rat Fibroblasts

PubMed Central

Graves, J. Anthony; Rothermund, Kristi; Wang, Tao; Qian, Wei; Van Houten, Bennett; Prochownik, Edward V.

2010-01-01

Deregulation of c-Myc (Myc) occurs in many cancers. In addition to transforming various cell types, Myc also influences additional transformation-associated cellular phenotypes including proliferation, survival, genomic instability, reactive oxygen species production, and metabolism. Although Myc is wild type in most cancers (wtMyc), it occasionally acquires point mutations in certain lymphomas. Some of these mutations confer a survival advantage despite partially attenuating proliferation and transformation. Here, we have evaluated four naturally-occurring or synthetic point mutations of Myc for their ability to affect these phenotypes, as well as to promote genomic instability, to generate reactive oxygen species and to up-regulate aerobic glycolysis and oxidative phosphorylation. Our findings indicate that many of these phenotypes are genetically and functionally independent of one another and are not necessary for transformation. Specifically, the higher rate of glucose metabolism known to be associated with wtMyc deregulation was found to be independent of transformation. One mutation (Q131R) was greatly impaired for nearly all of the studied Myc phenotypes, yet was able to retain some ability to transform. These findings indicate that, while the Myc phenotypes examined here make additive contributions to transformation, none, with the possible exception of increased reliance on extracellular glutamine for survival, are necessary for achieving this state. PMID:21060841

The Cerebro-oculo-facio-skeletal Syndrome Point Mutation F231L in the ERCC1 DNA Repair Protein Causes Dissociation of the ERCC1-XPF Complex.

PubMed

Faridounnia, Maryam; Wienk, Hans; Kovačič, Lidija; Folkers, Gert E; Jaspers, Nicolaas G J; Kaptein, Robert; Hoeijmakers, Jan H J; Boelens, Rolf

2015-08-14

The ERCC1-XPF heterodimer, a structure-specific DNA endonuclease, is best known for its function in the nucleotide excision repair (NER) pathway. The ERCC1 point mutation F231L, located at the hydrophobic interaction interface of ERCC1 (excision repair cross-complementation group 1) and XPF (xeroderma pigmentosum complementation group F), leads to severe NER pathway deficiencies. Here, we analyze biophysical properties and report the NMR structure of the complex of the C-terminal tandem helix-hairpin-helix domains of ERCC1-XPF that contains this mutation. The structures of wild type and the F231L mutant are very similar. The F231L mutation results in only a small disturbance of the ERCC1-XPF interface, where, in contrast to Phe(231), Leu(231) lacks interactions stabilizing the ERCC1-XPF complex. One of the two anchor points is severely distorted, and this results in a more dynamic complex, causing reduced stability and an increased dissociation rate of the mutant complex as compared with wild type. These data provide a biophysical explanation for the severe NER deficiencies caused by this mutation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Coalescent Inference Using Serially Sampled, High-Throughput Sequencing Data from Intrahost HIV Infection

PubMed Central

Dialdestoro, Kevin; Sibbesen, Jonas Andreas; Maretty, Lasse; Raghwani, Jayna; Gall, Astrid; Kellam, Paul; Pybus, Oliver G.; Hein, Jotun; Jenkins, Paul A.

2016-01-01

Human immunodeficiency virus (HIV) is a rapidly evolving pathogen that causes chronic infections, so genetic diversity within a single infection can be very high. High-throughput “deep” sequencing can now measure this diversity in unprecedented detail, particularly since it can be performed at different time points during an infection, and this offers a potentially powerful way to infer the evolutionary dynamics of the intrahost viral population. However, population genomic inference from HIV sequence data is challenging because of high rates of mutation and recombination, rapid demographic changes, and ongoing selective pressures. In this article we develop a new method for inference using HIV deep sequencing data, using an approach based on importance sampling of ancestral recombination graphs under a multilocus coalescent model. The approach further extends recent progress in the approximation of so-called conditional sampling distributions, a quantity of key interest when approximating coalescent likelihoods. The chief novelties of our method are that it is able to infer rates of recombination and mutation, as well as the effective population size, while handling sampling over different time points and missing data without extra computational difficulty. We apply our method to a data set of HIV-1, in which several hundred sequences were obtained from an infected individual at seven time points over 2 years. We find mutation rate and effective population size estimates to be comparable to those produced by the software BEAST. Additionally, our method is able to produce local recombination rate estimates. The software underlying our method, Coalescenator, is freely available. PMID:26857628

Genetic analysis of an Escherichia coli syndrome.

PubMed

Lennette, E T; Apirion, D

1971-12-01

A mutant strain of Escherichia coli that fails to recover from prolonged (72 hr) starvation also fails to grow at 43 C. Extracts of this mutant strain show an increased ribonuclease II activity as compared to extracts of the parental strain, and stable ribonucleic acid is degraded to a larger extent in this strain during starvation. Ts(+) transductants and revertants were tested for all the above-mentioned phenotypes. All the Ts(+) transductants and revertants tested behaved like the Ts(+) parental strain, which suggests that all the observed phenotypes are caused by a single sts (starvation-temperature sensitivity) mutation. The reversion rate from sts(-) to sts(+) is rather low but is within the range of reversion rates for other single-site mutations. Three-point transduction crosses located this sts mutation between the ilv and rbs genes. The properties of sts(+)/sts(-) merozygotes suggested that the Ts(-) phenotype of this mutation is recessive.

Point Mutations in Membrane Proteins Reshape Energy Landscape and Populate Different Unfolding Pathways

PubMed Central

Sapra, K. Tanuj; Balasubramanian, G. Prakash; Labudde, Dirk; Bowie, James U.; Muller, Daniel J.

2009-01-01

Using single-molecule force spectroscopy, we investigated the effect of single point mutations on the energy landscape and unfolding pathways of the transmembrane protein bacteriorhodopsin. We show that the unfolding energy barriers in the energy landscape of the membrane protein followed a simple two-state behavior and represent a manifestation of many converging unfolding pathways. Although the unfolding pathways of wild-type and mutant bacteriorhodopsin did not change, indicating the presence of same ensemble of structural unfolding intermediates, the free energies of the rate-limiting transition states of the bacteriorhodopsin mutants decreased as the distance of those transition states to the folded intermediate states decreased. Thus, all mutants exhibited Hammond behavior and a change in the free energies of the intermediates along the unfolding reaction coordinate and, consequently, their relative occupancies. This is the first experimental proof showing that point mutations can reshape the free energy landscape of a membrane protein and force single proteins to populate certain unfolding pathways over others. PMID:18191146

Mutation-selection equilibrium in games with mixed strategies.

PubMed

Tarnita, Corina E; Antal, Tibor; Nowak, Martin A

2009-11-07

We develop a new method for studying stochastic evolutionary game dynamics of mixed strategies. We consider the general situation: there are n pure strategies whose interactions are described by an nxn payoff matrix. Players can use mixed strategies, which are given by the vector (p(1),...,p(n)). Each entry specifies the probability to use the corresponding pure strategy. The sum over all entries is one. Therefore, a mixed strategy is a point in the simplex S(n). We study evolutionary dynamics in a well-mixed population of finite size. Individuals reproduce proportional to payoff. We consider the case of weak selection, which means the payoff from the game is only a small contribution to overall fitness. Reproduction can be subject to mutation; a mutant adopts a randomly chosen mixed strategy. We calculate the average abundance of every mixed strategy in the stationary distribution of the mutation-selection process. We find the crucial conditions that specify if a strategy is favored or opposed by selection. One condition holds for low mutation rate, another for high mutation rate. The result for any mutation rate is a linear combination of those two. As a specific example we study the Hawk-Dove game. We prove general statements about the relationship between games with pure and with mixed strategies.

Automatic extraction of protein point mutations using a graph bigram association.

PubMed

Lee, Lawrence C; Horn, Florence; Cohen, Fred E

2007-02-02

Protein point mutations are an essential component of the evolutionary and experimental analysis of protein structure and function. While many manually curated databases attempt to index point mutations, most experimentally generated point mutations and the biological impacts of the changes are described in the peer-reviewed published literature. We describe an application, Mutation GraB (Graph Bigram), that identifies, extracts, and verifies point mutations from biomedical literature. The principal problem of point mutation extraction is to link the point mutation with its associated protein and organism of origin. Our algorithm uses a graph-based bigram traversal to identify these relevant associations and exploits the Swiss-Prot protein database to verify this information. The graph bigram method is different from other models for point mutation extraction in that it incorporates frequency and positional data of all terms in an article to drive the point mutation-protein association. Our method was tested on 589 articles describing point mutations from the G protein-coupled receptor (GPCR), tyrosine kinase, and ion channel protein families. We evaluated our graph bigram metric against a word-proximity metric for term association on datasets of full-text literature in these three different protein families. Our testing shows that the graph bigram metric achieves a higher F-measure for the GPCRs (0.79 versus 0.76), protein tyrosine kinases (0.72 versus 0.69), and ion channel transporters (0.76 versus 0.74). Importantly, in situations where more than one protein can be assigned to a point mutation and disambiguation is required, the graph bigram metric achieves a precision of 0.84 compared with the word distance metric precision of 0.73. We believe the graph bigram search metric to be a significant improvement over previous search metrics for point mutation extraction and to be applicable to text-mining application requiring the association of words.

A mutation of the fission yeast EB1 overcomes negative regulation by phosphorylation and stabilizes microtubules

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

Iimori, Makoto; Ozaki, Kanako; Chikashige, Yuji

2012-02-01

Mal3 is a fission yeast homolog of EB1, a plus-end tracking protein (+ TIP). We have generated a mutation (89R) replacing glutamine with arginine in the calponin homology (CH) domain of Mal3. Analysis of the 89R mutant in vitro has revealed that the mutation confers a higher affinity to microtubules and enhances the intrinsic activity to promote the microtubule-assembly. The mutant Mal3 is no longer a + TIP, but binds strongly the microtubule lattice. Live cell imaging has revealed that while the wild type Mal3 proteins dissociate from the tip of the growing microtubules before the onset of shrinkage, themore » mutant Mal3 proteins persist on microtubules and reduces a rate of shrinkage after a longer pausing period. Consequently, the mutant Mal3 proteins cause abnormal elongation of microtubules composing the spindle and aster. Mal3 is phosphorylated at a cluster of serine/threonine residues in the linker connecting the CH and EB1-like C-terminal motif domains. The phosphorylation occurs in a microtubule-dependent manner and reduces the affinity of Mal3 to microtubules. We propose that because the 89R mutation is resistant to the effect of phosphorylation, it can associate persistently with microtubules and confers a stronger stability of microtubules likely by reinforcing the cylindrical structure. -- Highlights: Black-Right-Pointing-Pointer We characterize a mutation (mal3-89R) in fission yeast homolog of EB1. Black-Right-Pointing-Pointer The mutation enhances the activity to assemble microtubules. Black-Right-Pointing-Pointer Mal3 is phosphorylated in a microtubule-dependent manner. Black-Right-Pointing-Pointer The phosphorylation negatively regulates the Mal3 activity.« less

Vehicle Routing Problem Using Genetic Algorithm with Multi Compartment on Vegetable Distribution

NASA Astrophysics Data System (ADS)

Kurnia, Hari; Gustri Wahyuni, Elyza; Cergas Pembrani, Elang; Gardini, Syifa Tri; Kurnia Aditya, Silfa

2018-03-01

The problem that is often gained by the industries of managing and distributing vegetables is how to distribute vegetables so that the quality of the vegetables can be maintained properly. The problems encountered include optimal route selection and little travel time or so-called TSP (Traveling Salesman Problem). These problems can be modeled using the Vehicle Routing Problem (VRP) algorithm with rating ranking, a cross order based crossing, and also order based mutation mutations on selected chromosomes. This study uses limitations using only 20 market points, 2 point warehouse (multi compartment) and 5 vehicles. It is determined that for one distribution, one vehicle can only distribute to 4 market points only from 1 particular warehouse, and also one such vehicle can only accommodate 100 kg capacity.

Effect of mutation mechanisms on variant composition and distribution in Caenorhabditis elegans

PubMed Central

Wang, Jiou

2017-01-01

Genetic diversity is maintained by continuing generation and removal of variants. While examining over 800,000 DNA variants in wild isolates of Caenorhabditis elegans, we made a discovery that the proportions of variant types are not constant across the C. elegans genome. The variant proportion is defined as the fraction of a specific variant type (e.g. single nucleotide polymorphism (SNP) or indel) within a broader set of variants (e.g. all variants or all non-SNPs). The proportions of most variant types show a correlation with the recombination rate. These correlations can be explained as a result of a concerted action of two mutation mechanisms, which we named Morgan and Sanger mechanisms. The two proposed mechanisms act according to the distinct components of the recombination rate, specifically the genetic and physical distance. Regression analysis was used to explore the characteristics and contributions of the two mutation mechanisms. According to our model, ~20–40% of all mutations in C. elegans wild populations are derived from programmed meiotic double strand breaks, which precede chromosomal crossovers and thus may be the point of origin for the Morgan mechanism. A substantial part of the known correlation between the recombination rate and variant distribution appears to be caused by the mutations generated by the Morgan mechanism. Mathematically integrating the mutation model with background selection model gives a more complete depiction of how the variant landscape is shaped in C. elegans. Similar analysis should be possible in other species by examining the correlation between the recombination rate and variant landscape within the context of our mutation model. PMID:28135268

Dynamic evolution of plant mitochondrial genomes: Mobile genes and introns and highly variable mutation rates

PubMed Central

Palmer, Jeffrey D.; Adams, Keith L.; Cho, Yangrae; Parkinson, Christopher L.; Qiu, Yin-Long; Song, Keming

2000-01-01

We summarize our recent studies showing that angiosperm mitochondrial (mt) genomes have experienced remarkably high rates of gene loss and concomitant transfer to the nucleus and of intron acquisition by horizontal transfer. Moreover, we find substantial lineage-specific variation in rates of these structural mutations and also point mutations. These findings mostly arise from a Southern blot survey of gene and intron distribution in 281 diverse angiosperms. These blots reveal numerous losses of mt ribosomal protein genes but, with one exception, only rare loss of respiratory genes. Some lineages of angiosperms have kept all of their mt ribosomal protein genes whereas others have lost most of them. These many losses appear to reflect remarkably high (and variable) rates of functional transfer of mt ribosomal protein genes to the nucleus in angiosperms. The recent transfer of cox2 to the nucleus in legumes provides both an example of interorganellar gene transfer in action and a starting point for discussion of the roles of mechanistic and selective forces in determining the distribution of genetic labor between organellar and nuclear genomes. Plant mt genomes also acquire sequences by horizontal transfer. A striking example of this is a homing group I intron in the mt cox1 gene. This extraordinarily invasive mobile element has probably been acquired over 1,000 times separately during angiosperm evolution via a recent wave of cross-species horizontal transfers. Finally, whereas all previously examined angiosperm mtDNAs have low rates of synonymous substitutions, mtDNAs of two distantly related angiosperms have highly accelerated substitution rates. PMID:10860957

The Number of Point Mutations in Induced Pluripotent Stem Cells and Nuclear Transfer Embryonic Stem Cells Depends on the Method and Somatic Cell Type Used for Their Generation.

PubMed

Araki, Ryoko; Mizutani, Eiji; Hoki, Yuko; Sunayama, Misato; Wakayama, Sayaka; Nagatomo, Hiroaki; Kasama, Yasuji; Nakamura, Miki; Wakayama, Teruhiko; Abe, Masumi

2017-05-01

Induced pluripotent stem cells hold great promise for regenerative medicine but point mutations have been identified in these cells and have raised serious concerns about their safe use. We generated nuclear transfer embryonic stem cells (ntESCs) from both mouse embryonic fibroblasts (MEFs) and tail-tip fibroblasts (TTFs) and by whole genome sequencing found fewer mutations compared with iPSCs generated by retroviral gene transduction. Furthermore, TTF-derived ntESCs showed only a very small number of point mutations, approximately 80% less than the number observed in iPSCs generated using retrovirus. Base substitution profile analysis confirmed this greatly reduced number of point mutations. The point mutations in iPSCs are therefore not a Yamanaka factor-specific phenomenon but are intrinsic to genome reprogramming. Moreover, the dramatic reduction in point mutations in ntESCs suggests that most are not essential for genome reprogramming. Our results suggest that it is feasible to reduce the point mutation frequency in iPSCs by optimizing various genome reprogramming conditions. We conducted whole genome sequencing of ntES cells derived from MEFs or TTFs. We thereby succeeded in establishing TTF-derived ntES cell lines with far fewer point mutations. Base substitution profile analysis of these clones also indicated a reduced point mutation frequency, moving from a transversion-predominance to a transition-predominance. Stem Cells 2017;35:1189-1196. © 2017 AlphaMed Press.

Thiol peroxidase deficiency leads to increased mutational load and decreased fitness in Saccharomyces cerevisiae.

PubMed

Kaya, Alaattin; Lobanov, Alexei V; Gerashchenko, Maxim V; Koren, Amnon; Fomenko, Dmitri E; Koc, Ahmet; Gladyshev, Vadim N

2014-11-01

Thiol peroxidases are critical enzymes in the redox control of cellular processes that function by reducing low levels of hydroperoxides and regulating redox signaling. These proteins were also shown to regulate genome stability, but how their dysfunction affects the actual mutations in the genome is not known. Saccharomyces cerevisiae has eight thiol peroxidases of glutathione peroxidase and peroxiredoxin families, and the mutant lacking all these genes (∆8) is viable. In this study, we employed two independent ∆8 isolates to analyze the genome-wide mutation spectrum that results from deficiency in these enzymes. Deletion of these genes was accompanied by a dramatic increase in point mutations, many of which clustered in close proximity and scattered throughout the genome, suggesting strong mutational bias. We further subjected multiple lines of wild-type and ∆8 cells to long-term mutation accumulation, followed by genome sequencing and phenotypic characterization. ∆8 lines showed a significant increase in nonrecurrent point mutations and indels. The original ∆8 cells exhibited reduced growth rate and decreased life span, which were further reduced in all ∆8 mutation accumulation lines. Although the mutation spectrum of the two independent isolates was different, similar patterns of gene expression were observed, suggesting the direct contribution of thiol peroxidases to the observed phenotypes. Expression of a single thiol peroxidase could partially restore the growth phenotype of ∆8 cells. This study shows how deficiency in nonessential, yet critical and conserved oxidoreductase function, leads to increased mutational load and decreased fitness. Copyright © 2014 by the Genetics Society of America.

A new look at an old virus: patterns of mutation accumulation in the human H1N1 influenza virus since 1918

PubMed Central

2012-01-01

Background The H1N1 influenza A virus has been circulating in the human population for over 95 years, first manifesting itself in the pandemic of 1917–1918. Initial mortality was extremely high, but dropped exponentially over time. Influenza viruses have high mutation rates, and H1N1 has undergone significant genetic changes since 1918. The exact nature of H1N1 mutation accumulation over time has not been fully explored. Methods We have made a comprehensive historical analysis of mutational changes within H1N1 by examining over 4100 fully-sequenced H1N1 genomes. This has allowed us to examine the genetic changes arising within H1N1 from 1918 to the present. Results We document multiple extinction events, including the previously known extinction of the human H1N1 lineage in the 1950s, and an apparent second extinction of the human H1N1 lineage in 2009. These extinctions appear to be due to a continuous accumulation of mutations. At the time of its disappearance in 2009, the human H1N1 lineage had accumulated over 1400 point mutations (more than 10% of the genome), including approximately 330 non-synonymous changes (7.4% of all codons). The accumulation of both point mutations and non-synonymous amino acid changes occurred at constant rates (μ = 14.4 and 2.4 new mutations/year, respectively), and mutations accumulated uniformly across the entire influenza genome. We observed a continuous erosion over time of codon-specificity in H1N1, including a shift away from host (human, swine, and bird [duck]) codon preference patterns. Conclusions While there have been numerous adaptations within the H1N1 genome, most of the genetic changes we document here appear to be non-adaptive, and much of the change appears to be degenerative. We suggest H1N1 has been undergoing natural genetic attenuation, and that significant attenuation may even occur during a single pandemic. This process may play a role in natural pandemic cessation and has apparently contributed to the exponential decline in mortality rates over time, as seen in all major human influenza strains. These findings may be relevant to the development of strategies for managing influenza pandemics and strain evolution. PMID:23062055

Oscillatory dynamics in rock-paper-scissors games with mutations.

PubMed

Mobilia, Mauro

2010-05-07

We study the oscillatory dynamics in the generic three-species rock-paper-scissors games with mutations. In the mean-field limit, different behaviors are found: (a) for high mutation rate, there is a stable interior fixed point with coexistence of all species; (b) for low mutation rates, there is a region of the parameter space characterized by a limit cycle resulting from a Hopf bifurcation; (c) in the absence of mutations, there is a region where heteroclinic cycles yield oscillations of large amplitude (not robust against noise). After a discussion on the main properties of the mean-field dynamics, we investigate the stochastic version of the model within an individual-based formulation. Demographic fluctuations are therefore naturally accounted and their effects are studied using a diffusion theory complemented by numerical simulations. It is thus shown that persistent erratic oscillations (quasi-cycles) of large amplitude emerge from a noise-induced resonance phenomenon. We also analytically and numerically compute the average escape time necessary to reach a (quasi-)cycle on which the system oscillates at a given amplitude. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Polyploidy can drive rapid adaptation in yeast

NASA Astrophysics Data System (ADS)

Selmecki, Anna M.; Maruvka, Yosef E.; Richmond, Phillip A.; Guillet, Marie; Shoresh, Noam; Sorenson, Amber L.; de, Subhajyoti; Kishony, Roy; Michor, Franziska; Dowell, Robin; Pellman, David

2015-03-01

Polyploidy is observed across the tree of life, yet its influence on evolution remains incompletely understood. Polyploidy, usually whole-genome duplication, is proposed to alter the rate of evolutionary adaptation. This could occur through complex effects on the frequency or fitness of beneficial mutations. For example, in diverse cell types and organisms, immediately after a whole-genome duplication, newly formed polyploids missegregate chromosomes and undergo genetic instability. The instability following whole-genome duplications is thought to provide adaptive mutations in microorganisms and can promote tumorigenesis in mammalian cells. Polyploidy may also affect adaptation independently of beneficial mutations through ploidy-specific changes in cell physiology. Here we perform in vitro evolution experiments to test directly whether polyploidy can accelerate evolutionary adaptation. Compared with haploids and diploids, tetraploids undergo significantly faster adaptation. Mathematical modelling suggests that rapid adaptation of tetraploids is driven by higher rates of beneficial mutations with stronger fitness effects, which is supported by whole-genome sequencing and phenotypic analyses of evolved clones. Chromosome aneuploidy, concerted chromosome loss, and point mutations all provide large fitness gains. We identify several mutations whose beneficial effects are manifest specifically in the tetraploid strains. Together, these results provide direct quantitative evidence that in some environments polyploidy can accelerate evolutionary adaptation.

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.

An efficient method for the prediction of deleterious multiple-point mutations in the secondary structure of RNAs using suboptimal folding solutions

PubMed Central

Churkin, Alexander; Barash, Danny

2008-01-01

Background RNAmute is an interactive Java application which, given an RNA sequence, calculates the secondary structure of all single point mutations and organizes them into categories according to their similarity to the predicted structure of the wild type. The secondary structure predictions are performed using the Vienna RNA package. A more efficient implementation of RNAmute is needed, however, to extend from the case of single point mutations to the general case of multiple point mutations, which may often be desired for computational predictions alongside mutagenesis experiments. But analyzing multiple point mutations, a process that requires traversing all possible mutations, becomes highly expensive since the running time is O(nm) for a sequence of length n with m-point mutations. Using Vienna's RNAsubopt, we present a method that selects only those mutations, based on stability considerations, which are likely to be conformational rearranging. The approach is best examined using the dot plot representation for RNA secondary structure. Results Using RNAsubopt, the suboptimal solutions for a given wild-type sequence are calculated once. Then, specific mutations are selected that are most likely to cause a conformational rearrangement. For an RNA sequence of about 100 nts and 3-point mutations (n = 100, m = 3), for example, the proposed method reduces the running time from several hours or even days to several minutes, thus enabling the practical application of RNAmute to the analysis of multiple-point mutations. Conclusion A highly efficient addition to RNAmute that is as user friendly as the original application but that facilitates the practical analysis of multiple-point mutations is presented. Such an extension can now be exploited prior to site-directed mutagenesis experiments by virologists, for example, who investigate the change of function in an RNA virus via mutations that disrupt important motifs in its secondary structure. A complete explanation of the application, called MultiRNAmute, is available at [1]. PMID:18445289

Comparison of Epidermal Growth Factor Receptor Mutations between Metastatic Lymph Node Diagnosed by EBUS-TBNA and Primary Tumor in Non-Small Cell Lung Cancer

PubMed Central

Kang, Hyo Jae; Hwangbo, Bin; Lee, Jin Soo; Kim, Moon Soo; Lee, Jong Mog; Lee, Geon-Kook

2016-01-01

Introduction Although the use of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is increasing for epidermal growth factor receptor (EGFR) testing in lung cancer, the discordance rate in EGFR mutations between lymph node (LN) samples obtained by EBUS-TBNA and primary tumor (PT) is not well known. Thus, we compared the EGFR mutation status of LN samples obtained by EBUS-TBNA and PTs to estimate the efficacy of using EBUS-TBNA specimens for EGFR testing in advanced, non-squamous, non-small cell lung cancer (NSCLC). Materials and Methods Using data of patients from the EBUS-TBNA database (N = 1914) obtained between January 2009 and January 2013, we identified 100 treatment-naïve, advanced, non-squamous NSCLC patients (stage 3 and 4) with matched LN specimens obtained by EBUS-TBNA and PT specimens. Of these, 74 patients with paired specimens were feasible for EGFR mutation analysis, which we performed using a direct sequencing method. Results Of the 74 cases, at least one major [exon 19 deleted (19del) and L858R] or minor (T790M, exon 20 insertion, and other point mutations) EGFR mutation was detected in 31 cases (41.9%), which included PT (n = 31, 41.9%) and LN (n = 28, 37.8%) specimens. Major mutations were detected in 25 PT (33.8%, 19del = 13, L858R = 12) and 22 LN (29.8%, 19del = 11, L858R = 11) specimens. The discordance rate in major mutations between matched PT and LN specimens was 4.1% (3/74). Among minor mutations, T790M was detected in LN specimen only in 2 cases with L858R in PT and LN. The discordance rate major and minor EGFR mutations combined between matched PT and LN specimens was 12% (9/74). Conclusions We observed a high concordance rate of major EGFR mutations between matched LN specimens sampled by EBUS-TBNA and PTs, suggesting that LN samples obtained by EBUS-TBNA from advanced non-squamous NSCLC patients are effective for use in EGFR mutation testing. PMID:27685950

Long range dynamic effects of point-mutations trap a response regulator in an active conformation

PubMed Central

Bobay, Benjamin G.; Thompson, Richele J.; Hoch, James A.; Cavanagh, John

2010-01-01

When a point-mutation in a protein elicits a functional change, it is most common to assign this change to local structural perturbations. Here we show that point-mutations, distant from an essential highly dynamic kinase recognition loop in the response regulator Spo0F, lock this loop in an active conformation. This ‘conformational trapping’ results in functionally hyperactive Spo0F. Consequently, point-mutations are seen to affect functionally critical motions both close to and far from the mutational site. PMID:20828564

A Hearing-Loss Associated Myo1c Mutation (R156W) Decreases the Myosin Duty Ratio and Force Sensitivity†

PubMed Central

Lin, Tianming; Greenberg, Michael J.; Moore, Jeffrey R.; Ostap, E. Michael

2011-01-01

Myo1c is a member of the myosin superfamily that has been proposed to function as the adaptation motor in vestibular and auditory hair cells. A recent study identified a myo1c point mutation (R156W) in a person with bilateral sensorineural hearing loss. This mutated residue is located at the start of the highly conserved switch-1 region, which is a crucial element for the binding of nucleotide. We characterized the key steps on the ATPase pathway at 37 °C using recombinant wild-type (myo1c3IQ) and mutant myo1c (R156W-myo1c3IQ) constructs that consist of the motor domain and three IQ motifs. The R156W mutation only moderately affects the rates of ATP binding, ATP-induced actomyosin dissociation, and ADP release. The actin-activated ATPase rate of the mutant is inhibited > 4-fold, which is likely due to a decrease in the rate of phosphate release. The rate of actin gliding, as measured by the in vitro motility assay, is unaffected by the mutation at high myosin surface densities, but actin gliding is substantially reduced at low surface densities of R156W-myo1c3IQ. We used a frictional-loading assay to measure the affect of resisting forces on the rate of actin gliding and found that R156W-myo1c3IQ is less force sensitive than myo1c3IQ. Taken together, these results indicate that myo1c with the R156W mutation has a lower duty ratio than the wild-type protein and motile properties that are less sensitive to resisting forces. PMID:21265502

Revisiting the diffusion approximation to estimate evolutionary rates of gene family diversification.

PubMed

Gjini, Erida; Haydon, Daniel T; David Barry, J; Cobbold, Christina A

2014-01-21

Genetic diversity in multigene families is shaped by multiple processes, including gene conversion and point mutation. Because multi-gene families are involved in crucial traits of organisms, quantifying the rates of their genetic diversification is important. With increasing availability of genomic data, there is a growing need for quantitative approaches that integrate the molecular evolution of gene families with their higher-scale function. In this study, we integrate a stochastic simulation framework with population genetics theory, namely the diffusion approximation, to investigate the dynamics of genetic diversification in a gene family. Duplicated genes can diverge and encode new functions as a result of point mutation, and become more similar through gene conversion. To model the evolution of pairwise identity in a multigene family, we first consider all conversion and mutation events in a discrete manner, keeping track of their details and times of occurrence; second we consider only the infinitesimal effect of these processes on pairwise identity accounting for random sampling of genes and positions. The purely stochastic approach is closer to biological reality and is based on many explicit parameters, such as conversion tract length and family size, but is more challenging analytically. The population genetics approach is an approximation accounting implicitly for point mutation and gene conversion, only in terms of per-site average probabilities. Comparison of these two approaches across a range of parameter combinations reveals that they are not entirely equivalent, but that for certain relevant regimes they do match. As an application of this modelling framework, we consider the distribution of nucleotide identity among VSG genes of African trypanosomes, representing the most prominent example of a multi-gene family mediating parasite antigenic variation and within-host immune evasion. © 2013 Published by Elsevier Ltd. All rights reserved.

The correlations between alteration of p16 gene and clinicopathological factors and prognosis in squamous cell carcinomas of the buccal mucosa.

PubMed

Dong, Yuying; Wang, Jie; Dong, Fusheng; Wang, Xu; Zhang, Yinghuai

2012-07-01

To evaluate relationships between the alteration of p16 gene and the clinical status and prognosis of the patients with squamous cell carcinoma of the buccal mucosa. Thirty buccal cancers were included in the analysis. Deletion analysis was performed by PCR. Point mutation analysis was used by PCR-SSCP and direct sequencing. Methylation-specific PCR methods were adopted for the evaluation of p16 methylation. The correlation between alteration of p16 gene and clinicopathological factors buccal cancer was evaluated by Fisher's exact test. Kaplan-Meier and Cox regression were used to investigate the relationship between p16 alteration and survival time. The frequency of p16 alteration was 63.3% in buccal carcinomas. P16 deletion was associated significantly with tumor size (P = 0.01). P16 point mutation was associated significantly with differentiation (P = 0.006). P16 methylation was associated significantly with nodes metastasis (P = 0.027). The overall survival rate of 30 buccal carcinomas was 53.3%. The Log-rank test (P = 0.021) and univariate Cox regression analysis (P = 0.030) revealed that p16 methylation was significantly associated with the overall survival rate. Multivariate analysis showed that p16 deletion, p16 mutation, and p16 methylation were not statistically significant. The alterations of p16 gene may play a major role in malignancy and development and metastases of buccal carcinoma and may be an excellent marker of aggressive clinical behavior. P16 methylation has a prognostic value in buccal carcinoma but not an independent prognosis factor. P16 point mutation and p16 deletion have not prognostic significance in buccal carcinoma. © 2012 John Wiley & Sons A/S.

[MPLW515L point mutation in patients with myeloproliferative disease].

PubMed

Xia, Jun; Xu, Wei; Zhang, Su-Jiang; Fan, Lei; Qiao, Chun; Li, Jian-Yong

2008-12-01

In order to investigate the frequency of MPLW515L and JAK2V617F point mutations of the patients with myeloproliferative disease (MPD) in Nanjing area, MPLW515L and JAK2V617F point mutations were simultaneously detected by alleles specific polymerase chain reaction (AS-PCR) and sequencing in 190 MPD patients. The results showed that MPLW515L point mutation was detected in 1 out of 102 essential thrombocythemia (ET) patients (1.0%) and was not detected in 32 polycythemia vera (PV) patients, 13 idiopathic myelofibrosis (IMF) patients, 43 chronic myelogenous leukemia (CML) patients. JAK2V617F point mutation was detected in 20 out of 32 PV patients (62.5%), 43 out of 102 ET patients (42.2%), 5 out of 13 IMF patients (38.5%), and was not detected in 43 CML patients. It is concluded that MPLW515L point mutation exists in ET patient, but is not found in PV, IMF and CML. JAK2V617F point mutation exists in PV, ET and IMF, but not in CML.

A novel 'splice site' HCN4 Gene mutation, c.1737+1 G>T, causes familial bradycardia, reduced heart rate response, impaired chronotropic competence and increased short-term heart rate variability.

PubMed

Hategan, Lidia; Csányi, Beáta; Ördög, Balázs; Kákonyi, Kornél; Tringer, Annamária; Kiss, Orsolya; Orosz, Andrea; Sághy, László; Nagy, István; Hegedűs, Zoltán; Rudas, László; Széll, Márta; Varró, András; Forster, Tamás; Sepp, Róbert

2017-08-15

The most important molecular determinant of heart rate regulation in sino-atrial pacemaker cells includes hyperpolarization-activated, cyclic nucleotide-gated ion channels, the major isoform of which is encoded by the HCN4 gene. Mutations affecting the HCN4 gene are associated primarily with sick sinus syndrome. A novel c.1737+1 G>T 'splice-site' HCN4 mutation was identified in a large family with familial bradycardia which co-segregated with the disease providing a two-point LOD score of 4.87. Twelve out of the 22 investigated family members [4 males, 8 females average age 36 (SD 6) years] were considered as clinically affected (heart rate<60/min on resting ECG). Minimum [36 (SD 7) vs. 47 (SD 5) bpm, p=0.0087) and average heart rates [62 (SD 8) vs. 73 (SD 8) bpm, p=0.0168) were significantly lower in carriers on 24-hour Holter recordings. Under maximum exercise test carriers achieved significantly lower heart rates than non-carrier family members, and percent heart rate reserve and percent corrected heart rate reserve were significantly lower in carriers. Applying rigorous criteria for chronotropic incompetence a higher number of carriers exhibited chronotropic incompetence. Parameters, characterizing short-term variability of heart rate (i.e. rMSSD and pNN50%) were increased in carrier family members, even after normalization for heart rate, in the 24-hour ECG recordings with the same relative increase in 5-minute recordings. The identified novel 'splice site' HCN4 gene mutation, c.1737+1 G>T, causes familial bradycardia and leads to reduced heart rate response, impaired chronotropic competence and increased short-term heart rate variability in the mutation carriers. Copyright © 2017 Elsevier B.V. All rights reserved.

Improved survival with vemurafenib in melanoma with BRAF V600E mutation.

PubMed

Chapman, Paul B; Hauschild, Axel; Robert, Caroline; Haanen, John B; Ascierto, Paolo; Larkin, James; Dummer, Reinhard; Garbe, Claus; Testori, Alessandro; Maio, Michele; Hogg, David; Lorigan, Paul; Lebbe, Celeste; Jouary, Thomas; Schadendorf, Dirk; Ribas, Antoni; O'Day, Steven J; Sosman, Jeffrey A; Kirkwood, John M; Eggermont, Alexander M M; Dreno, Brigitte; Nolop, Keith; Li, Jiang; Nelson, Betty; Hou, Jeannie; Lee, Richard J; Flaherty, Keith T; McArthur, Grant A

2011-06-30

Phase 1 and 2 clinical trials of the BRAF kinase inhibitor vemurafenib (PLX4032) have shown response rates of more than 50% in patients with metastatic melanoma with the BRAF V600E mutation. We conducted a phase 3 randomized clinical trial comparing vemurafenib with dacarbazine in 675 patients with previously untreated, metastatic melanoma with the BRAF V600E mutation. Patients were randomly assigned to receive either vemurafenib (960 mg orally twice daily) or dacarbazine (1000 mg per square meter of body-surface area intravenously every 3 weeks). Coprimary end points were rates of overall and progression-free survival. Secondary end points included the response rate, response duration, and safety. A final analysis was planned after 196 deaths and an interim analysis after 98 deaths. At 6 months, overall survival was 84% (95% confidence interval [CI], 78 to 89) in the vemurafenib group and 64% (95% CI, 56 to 73) in the dacarbazine group. In the interim analysis for overall survival and final analysis for progression-free survival, vemurafenib was associated with a relative reduction of 63% in the risk of death and of 74% in the risk of either death or disease progression, as compared with dacarbazine (P<0.001 for both comparisons). After review of the interim analysis by an independent data and safety monitoring board, crossover from dacarbazine to vemurafenib was recommended. Response rates were 48% for vemurafenib and 5% for dacarbazine. Common adverse events associated with vemurafenib were arthralgia, rash, fatigue, alopecia, keratoacanthoma or squamous-cell carcinoma, photosensitivity, nausea, and diarrhea; 38% of patients required dose modification because of toxic effects. Vemurafenib produced improved rates of overall and progression-free survival in patients with previously untreated melanoma with the BRAF V600E mutation. (Funded by Hoffmann-La Roche; BRIM-3 ClinicalTrials.gov number, NCT01006980.).

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

Sequence analysis of the drug‑resistant rpoB gene in the Mycobacterium tuberculosis L‑form among patients with pneumoconiosis complicated by tuberculosis.

PubMed

Lu, Jun; Jiang, Shan; Ye, Song; Deng, Yun; Ma, Shuai; Li, Chao-Pin

2014-04-01

The aim of the present study was to investigate the mutational characteristics of the drug‑resistant Mycobacterium tuberculosis L‑form of the rpoB gene isolated from patients with pneumoconiosis complicated by tuberculosis, in order to reduce the occurrence of the drug resistance of patients and gain a more complete information on the resistance of the Mycobacterium tuberculosis L‑form. A total of 42 clinically isolated strains of Mycobacterium tuberculosis L‑form were collected, including 31 drug‑resistant strains. The genomic DNA was extracted, then the target genes were amplified by polymerase chain reaction and the hot mutational regions of the rpoB gene were analyzed by direct sequencing. The results revealed that no rpoB gene mutation was present in 11 rifampicin (RFP)‑sensitive strains, while conformational changes were identified in 31 RFP‑resistant strains. The mutation rate was 93.55% (29/31) in the resistant strains, and was frequently concentrated in codons 531 (51.61%; 16/31) and 526 (32.26%; 10/31), mainly occurring by case substitutions, including 27 unit point mutations and two two‑point mutations. The novel mutation identified in codon 516 had not been previously reported. The substitution of highly‑conserved amino acids encoded by the rpoB gene resulted in the molecular mechanism responsible for RFP resistance in the Mycobacterium tuberculosis L‑form. This also demonstrated that the rpoB gene is diversiform.

Distinct tumor protein p53 mutants in breast cancer subgroups.

PubMed

Dumay, Anne; Feugeas, Jean-Paul; Wittmer, Evelyne; Lehmann-Che, Jacqueline; Bertheau, Philippe; Espié, Marc; Plassa, Louis-François; Cottu, Paul; Marty, Michel; André, Fabrice; Sotiriou, Christos; Pusztai, Lajos; de Thé, Hugues

2013-03-01

Tumor protein p53 (TP53) is mutated in approximately 30% of breast cancers, but this frequency fluctuates widely between subclasses. We investigated the p53 mutation status in 572 breast tumors, classified into luminal, basal and molecular apocrine subgroups. As expected, the lowest mutation frequency was observed in luminal (26%), and the highest in basal (88%) tumors. Luminal tumors showed significantly higher frequency of substitutions (82 vs. 65%), notably A/T to G/C transitions (31 vs. 15%), whereas molecular apocrine and basal tumors presented much higher frequencies of complex mutations (deletions/insertions) (36 and 33%, respectively, vs. 18%). Accordingly, missense mutations were significantly more frequent in luminal tumors (75 vs. 54%), whereas basal tumors displayed significantly increased rates of TP53 truncations (43 vs. 25%), resulting in loss of function and/or expression. Interestingly, as basal tumors, molecular apocrine tumors presented with a high rate of complex mutations, but paradoxically, these were not associated with increased frequency of p53 truncation. As in luminal tumors, this could reflect a selective pressure for p53 gain of function, possibly through P63/P73 inactivation. Collectively, these observations point not only to different mechanisms of TP53 alterations, but also to different functional consequences in the different breast cancer subtypes. Copyright © 2012 UICC.

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.

Coevolution of CRISPR bacteria and phage in 2 dimensions

NASA Astrophysics Data System (ADS)

Han, Pu; Deem, Michael

2014-03-01

CRISPR (cluster regularly interspaced short palindromic repeats) is a newly discovered adaptive, heritable immune system of prokaryotes. It can prevent infection of prokaryotes by phage. Most bacteria and almost all archae have CRISPR. The CRISPR system incorporates short nucleotide sequences from viruses. These incorporated sequences provide a historical record of the host and predator coevolution. We simulate the coevolution of bacteria and phage in 2 dimensions. Each phage has multiple proto-spacers that the bacteria can incorporate. Each bacterium can store multiple spacers in its CRISPR. Phages can escape recognition by the CRISPR system via point mutation or recombination. We will discuss the different evolutionary consequences of point mutation or recombination on the coevolution of bacteria and phage. We will also discuss an intriguing ``dynamic phase transition'' in the number of phage as a function of time and mutation rate. We will show that due to the arm race between phages and bacteria, the frequency of spacers and proto-spacers in a population can oscillate quite rapidly.

Establishment of a nested-ASP-PCR method to determine the clarithromycin resistance of Helicobacter pylori.

PubMed

Luo, Xiao-Feng; Jiao, Jian-Hua; Zhang, Wen-Yue; Pu, Han-Ming; Qu, Bao-Jin; Yang, Bing-Ya; Hou, Min; Ji, Min-Jun

2016-07-07

To investigate clarithromycin resistance positions 2142, 2143 and 2144 of the 23SrRNA gene in Helicobacter pylori (H. pylori) by nested-allele specific primer-polymerase chain reaction (nested-ASP-PCR). The gastric tissue and saliva samples from 99 patients with positive results of the rapid urease test (RUT) were collected. The nested-ASP-PCR method was carried out with the external primers and inner allele-specific primers corresponding to the reference strain and clinical strains. Thirty gastric tissue and saliva samples were tested to determine the sensitivity of nested-ASP-PCR and ASP-PCR methods. Then, clarithromycin resistance was detected for 99 clinical samples by using different methods, including nested-ASP-PCR, bacterial culture and disk diffusion. The nested-ASP-PCR method was successfully established to test the resistance mutation points 2142, 2143 and 2144 of the 23SrRNA gene of H. pylori. Among 30 samples of gastric tissue and saliva, the H. pylori detection rate of nested-ASP-PCR was 90% and 83.33%, while the detection rate of ASP-PCR was just 63% and 56.67%. Especially in the saliva samples, nested-ASP-PCR showed much higher sensitivity in H. pylori detection and resistance mutation rates than ASP-PCR. In the 99 RUT-positive gastric tissue and saliva samples, the H. pylori-positive detection rate by nested-ASP-PCR was 87 (87.88%) and 67 (67.68%), in which there were 30 wild-type and 57 mutated strains in gastric tissue and 22 wild-type and 45 mutated strains in saliva. Genotype analysis showed that three-points mixed mutations were quite common, but different resistant strains were present in gastric mucosa and saliva. Compared to the high sensitivity shown by nested-ASP-PCR, the positive detection of bacterial culture with gastric tissue samples was 50 cases, in which only 26 drug-resistant strains were found through analyzing minimum inhibitory zone of clarithromycin. The nested-ASP-PCR assay showed higher detection sensitivity than ASP-PCR and drug sensitivity testing, which could be performed to evaluate clarithromycin resistance of H. pylori.

Establishment of a nested-ASP-PCR method to determine the clarithromycin resistance of Helicobacter pylori

PubMed Central

Luo, Xiao-Feng; Jiao, Jian-Hua; Zhang, Wen-Yue; Pu, Han-Ming; Qu, Bao-Jin; Yang, Bing-Ya; Hou, Min; Ji, Min-Jun

2016-01-01

AIM: To investigate clarithromycin resistance positions 2142, 2143 and 2144 of the 23SrRNA gene in Helicobacter pylori (H. pylori) by nested-allele specific primer-polymerase chain reaction (nested-ASP-PCR). METHODS: The gastric tissue and saliva samples from 99 patients with positive results of the rapid urease test (RUT) were collected. The nested-ASP-PCR method was carried out with the external primers and inner allele-specific primers corresponding to the reference strain and clinical strains. Thirty gastric tissue and saliva samples were tested to determine the sensitivity of nested-ASP-PCR and ASP-PCR methods. Then, clarithromycin resistance was detected for 99 clinical samples by using different methods, including nested-ASP-PCR, bacterial culture and disk diffusion. RESULTS: The nested-ASP-PCR method was successfully established to test the resistance mutation points 2142, 2143 and 2144 of the 23SrRNA gene of H. pylori. Among 30 samples of gastric tissue and saliva, the H. pylori detection rate of nested-ASP-PCR was 90% and 83.33%, while the detection rate of ASP-PCR was just 63% and 56.67%. Especially in the saliva samples, nested-ASP-PCR showed much higher sensitivity in H. pylori detection and resistance mutation rates than ASP-PCR. In the 99 RUT-positive gastric tissue and saliva samples, the H. pylori-positive detection rate by nested-ASP-PCR was 87 (87.88%) and 67 (67.68%), in which there were 30 wild-type and 57 mutated strains in gastric tissue and 22 wild-type and 45 mutated strains in saliva. Genotype analysis showed that three-points mixed mutations were quite common, but different resistant strains were present in gastric mucosa and saliva. Compared to the high sensitivity shown by nested-ASP-PCR, the positive detection of bacterial culture with gastric tissue samples was 50 cases, in which only 26 drug-resistant strains were found through analyzing minimum inhibitory zone of clarithromycin. CONCLUSION: The nested-ASP-PCR assay showed higher detection sensitivity than ASP-PCR and drug sensitivity testing, which could be performed to evaluate clarithromycin resistance of H. pylori. PMID:27433095

Mutation profile of KRAS and BRAF genes in patients with colorectal cancer: association with morphological and prognostic criteria.

PubMed

Samara, M; Kapatou, K; Ioannou, M; Kostopoulou, Ε; Papamichali, R; Papandreou, C; Athanasiadis, A; Koukoulis, G

2015-12-14

KRAS and BRAF mutations are well-recognized molecular alterations during colorectal carcinogenesis, but there is little agreement on their effect on tumor characteristics. Therefore, we aimed to evaluate the distribution of the most common KRAS and BRAF mutations in Greek patients with colorectal cancer and their possible associations with clinical histopathological parameters. In this study, 322 and 188 colorectal carcinomas were used for the mutation analysis of KRAS (exon 2) and BRAF (exon 15) genes, respectively. The mutational status of both genes was evaluated by polymerase chain reaction and sequencing analysis. Although the overall frequency of KRAS mutations (36.6%) seemed to be similar to those reported for other populations, the rate of point mutations at codon 13 was significantly lower (12%) in Greek patients with colorectal cancer and associated with male gender (P < 0.05). Tumors with G>T codon 12 transversions and G>C transitions showed more frequent lymph node metastasis (P < 0.05, P < 0.005, respectively). The rate of KRAS mutations gradually decreased with increasing histological grade (P < 0.05), as opposed to BRAF mutations, which were strongly associated with poorly differentiated tumors (P < 0.005). Additionally, we found that the histological features of preexisting adenoma were associated with the absence of BRAF mutations, in contrast to KRAS (P < 0.05). Our data suggested that there seems to be a correlation between morphological criteria and discrete genetic pathways in colorectal carcinogenesis. Moreover, ethnic or geographic factors may have an impact on genetic background of colorectal carcinomas, and specific types of KRAS mutations may influence the metastatic potential of colorectal tumors.

Rapid point-of-care testing for epidermal growth factor receptor gene mutations in patients with lung cancer using cell-free DNA from cytology specimen supernatants.

PubMed

Asaka, Shiho; Yoshizawa, Akihiko; Saito, Kazusa; Kobayashi, Yukihiro; Yamamoto, Hiroshi; Negishi, Tatsuya; Nakata, Rie; Matsuda, Kazuyuki; Yamaguchi, Akemi; Honda, Takayuki

2018-06-01

Epidermal growth factor receptor (EGFR) mutations are associated with responses to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in non-small-cell lung cancer (NSCLC). Our previous study revealed a rapid point-of-care system for detecting EGFR mutations. This system analyzes cell pellets from cytology specimens using droplet-polymerase chain reaction (d-PCR), and has a reaction time of 10 min. The present study aimed to validate the performance of the EGFR d-PCR assay using cell-free DNA (cfDNA) from supernatants obtained from cytology specimens. Assay results from cfDNA supernatant analyses were compared with those from cell pellets for 90 patients who were clinically diagnosed with, or suspected of having, lung cancer (80 bronchial lavage fluid samples, nine pleural effusion samples and one spinal fluid sample). EGFR mutations were identified in 12 and 15 cases using cfDNA supernatants and cell pellets, respectively. The concordance rates between cfDNA-supernatant and cell‑pellet assay results were 96.7% [kappa coefficient (K)=0.87], 98.9% (K=0.94), 98.9% (K=0.79) and 98.9% (K=0.79) for total EGFR mutations, L858R, E746_A750del and T790M, respectively. All 15 patients with EGFR mutation-positive results, as determined by EGFR d-PCR assay using cfDNA supernatants or cell pellets, also displayed positive results by conventional EGFR assays using tumor tissue or cytology specimens. Notably, EGFR mutations were even detected in five cfDNA supernatants for which the cytological diagnoses of the corresponding cell pellets were 'suspicious for malignancy', 'atypical' or 'negative for malignancy.' In conclusion, this rapid point-of-care system may be considered a promising novel screening method that may enable patients with NSCLC to receive EGFR-TKI therapy more rapidly, whilst also reserving cell pellets for additional morphological and molecular analyses.

Prevalence and Characterization of Somatic Mutations in Chinese Aldosterone-Producing Adenoma Patients

PubMed Central

Wang, Baojun; Li, Xintao; Zhang, Xu; Ma, Xin; Chen, Luyao; Zhang, Yu; Lyu, Xiangjun; Tang, Yuzhe; Huang, Qingbo; Gao, Yu; Fan, Yang; Ouyang, Jinzhi

2015-01-01

Abstract Recently somatic mutations of KCNJ5, ATP1A1, ATP2B3, and CACNA1D have been identified in patients with aldosterone-producing adenoma (APA). The present study sequenced the DNA in the tissues and blood samples from Chinese patients with APA for KCNJ5, ATP1A1, ATP2B3, and CACNA1D gene mutations. Among the 114 patients, 86 (75.4%) were identified with KCNJ5 somatic mutations, including 3 previously reported (G151R, L168R, T158A) and 2 other unreported mutations. One patient presented with both a point mutation (E147) and an insertion mutation, whereas another had a 36-base duplication, G153_G164dup. No mutation of ATP1A1 and ATP2B3 in the known hotspots was identified and only 1 male patient was detected with a novel CACNA1D mutation, V748I. Unlike other studies, male and female patients had similar KCNJ5 mutation rates (76.9% vs 74.2%). Mutation carriers were younger and had lower preoperative potassium level, whereas male (but not female) mutation carriers had higher preoperative plasma aldosterone concentration and preoperative blood pressures. Mutation carriers also had higher LV mass index (LVMI) than nonmutation carriers. After surgery, LVMI improved significantly in the KCNJ5 mutation group but not in the nonmutation group. The mRNA expression of KCNJ5, CYP11B2, and ATP2B3 was higher in the KCNJ5-mutated APA tissues. Functional characterization of the 2 novel KCNJ5 mutations showed that they were associated with decreased proliferation, membrane depolarization, elevated secretion of aldosterone, and increased expression of CYP11B1 and CYP11B2. In conclusion, Chinese APA patients appear to have a high frequency of somatic KCNJ5 mutation. Mutation prevalence rates are similar among men and women and 2 novel mutations are identified. KCNJ5-mutated patients benefit more from surgical resection of APA than nonmutated patients. PMID:25906099

RET/PTC and PAX8/PPARγ chromosomal rearrangements in post-Chernobyl thyroid cancer and their association with I-131 radiation dose and other characteristics

PubMed Central

Leeman-Neill, Rebecca J.; Brenner, Alina V.; Little, Mark P.; Bogdanova, Tetiana I.; Hatch, Maureen; Zurnadzy, Liudmyla Y.; Mabuchi, Kiyohiko; Tronko, Mykola D.; Nikiforov, Yuri E.

2012-01-01

Background Childhood exposure to I-131 from the 1986 Chernobyl accident led to a sharp increase in papillary thyroid carcinoma (PTC) incidence in regions surrounding the reactor. Data concerning the association between genetic mutations in PTCs and individual radiation doses are limited. Methods We performed mutational analysis of 62 PTCs diagnosed in a Ukrainian cohort of patients who were <18 y.o. in 1986 and received 0.008-8.6 Gy of I-131 to the thyroid and explored associations between mutation types and I-131 dose and other characteristics. Results RET/PTC rearrangements were most common (35%), followed by BRAF (15%) and RAS (8%) point mutations. Two tumors carrying PAX8/PPARγ rearrangement were identified. We found a significant negative association with I-131 dose for BRAF and RAS point mutations and a significant concave association with I-131 dose, with an inflection point at 1.6 Gy and odds ratio 2.1, based on a linear-quadratic model for RET/PTC and PAX8/PPARγ rearrangements. The trends with dose were significantly different between tumors with point mutations and rearrangements. Compared to point mutations, rearrangements were associated with residence in the relatively iodine deficient Zhytomyr region, younger age at exposure or surgery, and male gender. Conclusions Our results provide the first demonstration of PAX8/PPARγ rearrangements in post-Chernobyl tumors and show different associations for point mutations and chromosomal rearrangements with I-131 dose and other factors. These data support the relationship between chromosomal rearrangements, but not point mutations, and I-131 exposure and point to a possible role of iodine deficiency in generation of RET/PTC rearrangements in these patients. PMID:23436219

JAK2V617F somatic mutation in the general population: myeloproliferative neoplasm development and progression rate

PubMed Central

Nielsen, Camilla; Bojesen, Stig E.; Nordestgaard, Børge G.; Kofoed, Klaus F.; Birgens, Henrik S.

2014-01-01

Clinical significance of the JAK2V617F mutation in patients with a myeloproliferative neoplasm has been the target of intensive research in recent years. However, there is considerably uncertainty about prognosis in JAK2V617F positive individuals without overt signs of myeloproliferative disease. In this study, we tested the hypothesis that increased JAK2V617F somatic mutation burden is associated with myeloproliferative neoplasm progression rate in the general population. Among 49,488 individuals from the Copenhagen General Population Study, 63 (0.1%) tested positive for the JAK2V617F mutation in the time period 2003–2008. Of these, 48 were available for re-examination in 2012. Level of JAK2V617F mutation burden was associated with myeloproliferative neoplasm progression rate, consistent with a biological continuum of increasing JAK2V617F mutation burden across increasing severity of myeloproliferative neoplasm from no disease (n=8 at re-examination) through essential thrombocythemia (n=20) and polycythemia vera (n=13) to primary myelofibrosis (n=7). Among those diagnosed with a myeloproliferative neoplasm only at re-examination in 2012, in the preceding years JAK2V617F mutation burden increased by 0.55% per year, erythrocyte volume fraction increased by 1.19% per year, and erythrocyte mean corpuscular volume increased by 1.25% per year, while there was no change in platelet count or erythropoietin levels. Furthermore, we established a JAK2V617F mutation burden cut-off point of 2% indicative of disease versus no disease; however, individuals with a mutation burden below 2% may suffer from a latent form of myeloproliferative disease revealed by a slightly larger spleen and/or slightly higher lactic acid dehydrogenase concentration compared to controls. Of all 63 JAK2V617F positive individuals, 48 were eventually diagnosed with a myeloproliferative neoplasm. PMID:24907356

The Frequency of EGFR Mutation in Lung Adenocarcinoma and the Efficacy of Tyrosine Kinase Inhibitor Therapy in a Hungarian Cohort of Patients.

PubMed

Sárosi, Veronika; Balikó, Zoltán; Smuk, Gábor; László, Terézia; Szabó, Mariann; Ruzsics, István; Mezősi, Emese

2016-10-01

In the last decades new therapeutic drugs have been developed for the treatment of non-small cell lung cancer (NSCLC) patients. Tyrosine kinase inhibitors (TKIs) significantly increase the progression free survival (PFS) of patients with NSCLC carrying epidermal growth factor receptor (EGFR) mutations. This type of lung cancer occurs mainly among non-smoking women and Asian origin. However, the new ESMO guideline recommends EGFR mutation analysis in every patient with NSCLC, because in patients with activating EGFR mutation, TKIs should be considered as first line therapy. In our recent work, we analyzed data of patients with EGFR-mutant adenocarcinoma from January 2009. The number of patients investigated was 446, among them 44 cases were positive for EGFR mutation. The ratio of positive cases was 9.86 % that is lower than the average mutation rate in Europe and much lower than that found in Asia. The exon 19 deletion was detected in 61.4 % of the patients, while L858R point mutation in exon 21 was observed in 34.1 % of them. In one subject, both exon 19 and 21 mutations were present simultaneously. A rare mutation located in exon 21 was found in another patient. TKI therapy was conducted in 38 patients. The disease control rate by TKI therapy was 85.7 %; primary resistance was documented in five subjects. Non-smoking patients with EGFR mutant adenocarcinoma had the highest benefit from TKI treatment. Our data support the recommendation that EGFR mutation status should be defined in all cases of locally advanced or metastatic lung adenocarcinoma.

Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer

PubMed Central

Chen, Qianqian; Chen, Xiaoxiang; Zhang, Sichao; Lan, Ke; Lu, Jian; Zhang, Chiyu

2015-01-01

The development of simple, accurate, rapid and cost-effective technologies for mutation detection is crucial to the early diagnosis and prevention of numerous genetic diseases, pharmacogenetics, and drug resistance. Proofreading PCR (PR-PCR) was developed for mutation detection in 1998 but is rarely applied due to its low efficiency in allele discrimination. Here we developed a modified PR-PCR method using a ddNTP-blocked primer and a mixture of DNA polymerases with and without the 3'-5' proofreading function. The ddNTP-blocked primer exhibited the best blocking efficiency to avoid nonspecific primer extension while the mixture of a tiny amount of high-fidelity DNA polymerase with a routine amount of Taq DNA polymerase provided the best discrimination and amplification effects. The modified PR-PCR method is quite capable of detecting various mutation types, including point mutations and insertions/deletions (indels), and allows discrimination amplification when the mismatch is located within the last eight nucleotides from the 3'-end of the ddNTP-blocked primer. The modified PR-PCR has a sensitivity of 1-5 × 102 copies and a selectivity of 5 × 10-5 mutant among 107 copies of wild-type DNA. It showed a 100% accuracy rate in the detection of P72R germ-line mutation in the TP53 gene among 60 clinical blood samples, and a high potential to detect rifampin-resistant mutations at low frequency in Mycobacterium tuberculosis using an adaptor and a fusion-blocked primer. These results suggest that the modified PR-PCR technique is effective in detection of various mutations or polymorphisms as a simple, sensitive and promising approach. PMID:25915410

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.

Label-free and high-sensitive detection for genetic point mutation based on hyperspectral interferometry

NASA Astrophysics Data System (ADS)

Fu, Rongxin; Li, Qi; Zhang, Junqi; Wang, Ruliang; Lin, Xue; Xue, Ning; Su, Ya; Jiang, Kai; Huang, Guoliang

2016-10-01

Label free point mutation detection is particularly momentous in the area of biomedical research and clinical diagnosis since gene mutations naturally occur and bring about highly fatal diseases. In this paper, a label free and high sensitive approach is proposed for point mutation detection based on hyperspectral interferometry. A hybridization strategy is designed to discriminate a single-base substitution with sequence-specific DNA ligase. Double-strand structures will take place only if added oligonucleotides are perfectly paired to the probe sequence. The proposed approach takes full use of the inherent conformation of double-strand DNA molecules on the substrate and a spectrum analysis method is established to point out the sub-nanoscale thickness variation, which benefits to high sensitive mutation detection. The limit of detection reach 4pg/mm2 according to the experimental result. A lung cancer gene point mutation was demonstrated, proving the high selectivity and multiplex analysis capability of the proposed biosensor.

Emerging Helicobacter pylori levofloxacin resistance and novel genetic mutation in Nepal.

PubMed

Miftahussurur, Muhammad; Shrestha, Pradeep Krishna; Subsomwong, Phawinee; Sharma, Rabi Prakash; Yamaoka, Yoshio

2016-11-04

The prevalence of Helicobacter pylori antibiotic susceptibility in the Nepalese strains is untracked. We determined the antibiotic susceptibility for H. pylori and analyzed the presence of genetic mutations associated with antibiotic resistance in Nepalese strains. This study included 146 consecutive patients who underwent gastroduodenal endoscopy in Kathmandu, Nepal. Among 42 isolated H. pylori, there was no resistance to amoxicillin and tetracycline. In contrast, similar with typical South Asian patterns; metronidazole resistance rate in Nepalese strains were extremely high (88.1 %, 37/42). Clarithromycin resistance rate in Nepalese strains were modestly high (21.4 %, 9/42). Most of metronidazole resistant strains had highly distributed rdxA and frxA mutations, but were relative coincidence without a synergistic effect to increase the minimum inhibitory concentration (MIC). Among strains with the high MIC, 63.6 % (7/11) were associated with frameshift mutation at position 18 of frxA with or without rdxA involvement. However, based on next generation sequencing data we found that one strain with the highest MIC value had a novel mutation in the form of amino acid substituted at Ala-212, Gln-382, Ile-485 of dppA and Leu-145, Thr-168, Glu-117, Val-121, Arg-221 in dapF aside from missense mutations in full-length rdxA. Mutations at Asn-87 and/or Asp-91 of the gyrA were predominantly in levofloxacin-resistant strains. The gyrB mutation had steady relationship with the gyrA 87-91 mutations. Although three (44.4 %) and two (22.2 %) of clarithromycin resistant strains had point mutation on A2143G and A2146G, we confirmed the involvement of rpl22 and infB in high MIC strains without an 23SrRNA mutation. The rates of resistance to clarithromycin, metronidazole and levofloxacin were high in Nepalese strains, indicating that these antibiotics-based triple therapies are not useful as first-line treatment in Nepal. Bismuth or non-bismuth-based quadruple regimens, furazolidone-based triple therapy or rifabutin-based triple therapy may become alternative strategy in Nepal.

A revised timescale for human evolution based on ancient mitochondrial genomes

PubMed Central

Johnson, Philip L.F.; Bos, Kirsten; Lari, Martina; Bollongino, Ruth; Sun, Chengkai; Giemsch, Liane; Schmitz, Ralf; Burger, Joachim; Ronchitelli, Anna Maria; Martini, Fabio; Cremonesi, Renata G.; Svoboda, Jiří; Bauer, Peter; Caramelli, David; Castellano, Sergi; Reich, David; Pääbo, Svante; Krause, Johannes

2016-01-01

Summary Background Recent analyses of de novo DNA mutations in modern humans have suggested a nuclear substitution rate that is approximately half that of previous estimates based on fossil calibration. This result has led to suggestions that major events in human evolution occurred far earlier than previously thought. Result Here we use mitochondrial genome sequences from 10 securely dated ancient modern humans spanning 40,000 years as calibration points for the mitochondrial clock, thus yielding a direct estimate of the mitochondrial substitution rate. Our clock yields mitochondrial divergence times that are in agreement with earlier estimates based on calibration points derived from either fossils or archaeological material. In particular, our results imply a separation of non-Africans from the most closely related sub-Saharan African mitochondrial DNAs (haplogroup L3) of less than 62,000-95,000 years ago. Conclusion Though single loci like mitochondrial DNA (mtDNA) can only provide biased estimates of population split times, they can provide valid upper bounds; our results exclude most of the older dates for African and non-African split times recently suggested by de novo mutation rate estimates in the nuclear genome. PMID:23523248

A revised timescale for human evolution based on ancient mitochondrial genomes.

PubMed

Fu, Qiaomei; Mittnik, Alissa; Johnson, Philip L F; Bos, Kirsten; Lari, Martina; Bollongino, Ruth; Sun, Chengkai; Giemsch, Liane; Schmitz, Ralf; Burger, Joachim; Ronchitelli, Anna Maria; Martini, Fabio; Cremonesi, Renata G; Svoboda, Jiří; Bauer, Peter; Caramelli, David; Castellano, Sergi; Reich, David; Pääbo, Svante; Krause, Johannes

2013-04-08

Recent analyses of de novo DNA mutations in modern humans have suggested a nuclear substitution rate that is approximately half that of previous estimates based on fossil calibration. This result has led to suggestions that major events in human evolution occurred far earlier than previously thought. Here, we use mitochondrial genome sequences from ten securely dated ancient modern humans spanning 40,000 years as calibration points for the mitochondrial clock, thus yielding a direct estimate of the mitochondrial substitution rate. Our clock yields mitochondrial divergence times that are in agreement with earlier estimates based on calibration points derived from either fossils or archaeological material. In particular, our results imply a separation of non-Africans from the most closely related sub-Saharan African mitochondrial DNAs (haplogroup L3) that occurred less than 62-95 kya. Though single loci like mitochondrial DNA (mtDNA) can only provide biased estimates of population divergence times, they can provide valid upper bounds. Our results exclude most of the older dates for African and non-African population divergences recently suggested by de novo mutation rate estimates in the nuclear genome. Copyright © 2013 Elsevier Ltd. All rights reserved.

Evaluation of a new commercial real-time PCR assay for diagnosis of Pneumocystis jirovecii pneumonia and identification of dihydropteroate synthase (DHPS) mutations.

PubMed

Montesinos, Isabel; Delforge, Marie-Luce; Ajjaham, Farida; Brancart, Françoise; Hites, Maya; Jacobs, Frederique; Denis, Olivier

2017-01-01

The PneumoGenius® real-time PCR assay is a new commercial multiplex real-time PCR method, which detects the Pneumocystis mitochondrial ribosomal large subunit (mtLSU) and two dihydropteroate synthase (DHPS) point mutations. To evaluate the clinical performance of this new real-time PCR assay we tested 120 extracted DNA samples from bronchoalveolar lavage specimens. These set of extracted DNA samples had already tested positive for Pneumocystis and patients had been classified in probable and unlikely PCP in a previous study. To evaluate de accuracy of the DHPS mutant's identification, an "in house" PCR and sequencing was performed. The sensitivity and specificity of PneumoGenius® PCR in discriminating between probable and unlikely Pneumocystis pneumonia (PCP) were 70% and 82% respectively. PneumoGenius® PCR was able to genotype more samples than "in house" DHPS PCR and sequencing. The same DHPS mutations were observed by both methods in four patients: two patients with a single mutation in position 171 (Pro57Ser) and two patients with a double mutation in position 165 (Thr55Ala) and in position 171 (Pro57Ser). A low rate of P. jirovecii (4.5%) harboring DHPS mutations was found, comparable to rates observed in other European countries. The PneumoGenius® real-time PCR is a suitable real-time PCR for PCP diagnosis and detection of DHPS mutants. The added value of DHPS mutation identification can assist in understanding the role of these mutations in prophylaxis failure or treatment outcome. Copyright © 2016 Elsevier Inc. All rights reserved.

Diverse point mutations in the human gene for polymorphic N-acetyltransferase

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

Vatsis, K.P.; Martell, K.J.; Weber, W.W.

1991-07-15

Classification of humans as rapid or slow acetylators is based on hereditary differences in rates of N-acetylation of therapeutic and carcinogenic agents, but N-acetylation of certain arylamine drugs displays no genetic variation. Two highly homologous human genes for N-acetyltransferase NAT1 and NAT2, presumably code for the genetically invariant and variant NAT proteins, respectively. In the present investigation, 1.9-kilobase human genomic EcoRI fragments encoding NAT2 were generated by the polymerase chain reaction with liver and leukocyte DNA from seven subjects phenotyped as homozygous and heterozygous acetylators. Direct sequencing revealed multiple point mutations in the coding region of two distinct NAT2 variants.more » One of these was derived from leukocytes of a slow acetylator and was distinguished by a silent mutation (coden 94) and a separate G {r arrow} A transition (position 590) leading to replacement of Arg-197 by Gln; the mutated guanine was part of a CpG dinucleotide and a Taq I site. The second NAT2 variant originated from liver with low N-acetylation activity. It was characterized by three nucleotide transitions giving rise to a silent mutation (codon 161), accompanied by obliteration of the sole Kpn I site, and two amino acid substitutions. The results show conclusively that the genetically variant NAT is encoded by NAT2.« less

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.

Evaluation of point mutations in dystrophin gene in Iranian Duchenne and Becker muscular dystrophy patients: introducing three novel variants.

PubMed

Haghshenas, Maryam; Akbari, Mohammad Taghi; Karizi, Shohreh Zare; Deilamani, Faravareh Khordadpoor; Nafissi, Shahriar; Salehi, Zivar

2016-06-01

Duchenne and Becker muscular dystrophies (DMD and BMD) are X-linked neuromuscular diseases characterized by progressive muscular weakness and degeneration of skeletal muscles. Approximately two-thirds of the patients have large deletions or duplications in the dystrophin gene and the remaining one-third have point mutations. This study was performed to evaluate point mutations in Iranian DMD/BMD male patients. A total of 29 DNA samples from patients who did not show any large deletion/duplication mutations following multiplex polymerase chain reaction (PCR) and multiplex ligation-dependent probe amplification (MLPA) screening were sequenced for detection of point mutations in exons 50-79. Also exon 44 was sequenced in one sample in which a false positive deletion was detected by MLPA method. Cycle sequencing revealed four nonsense, one frameshift and two splice site mutations as well as two missense variants.

Dynamic Harmony Search with Polynomial Mutation Algorithm for Valve-Point Economic Load Dispatch

PubMed Central

Karthikeyan, M.; Sree Ranga Raja, T.

2015-01-01

Economic load dispatch (ELD) problem is an important issue in the operation and control of modern control system. The ELD problem is complex and nonlinear with equality and inequality constraints which makes it hard to be efficiently solved. This paper presents a new modification of harmony search (HS) algorithm named as dynamic harmony search with polynomial mutation (DHSPM) algorithm to solve ORPD problem. In DHSPM algorithm the key parameters of HS algorithm like harmony memory considering rate (HMCR) and pitch adjusting rate (PAR) are changed dynamically and there is no need to predefine these parameters. Additionally polynomial mutation is inserted in the updating step of HS algorithm to favor exploration and exploitation of the search space. The DHSPM algorithm is tested with three power system cases consisting of 3, 13, and 40 thermal units. The computational results show that the DHSPM algorithm is more effective in finding better solutions than other computational intelligence based methods. PMID:26491710

Dynamic Harmony Search with Polynomial Mutation Algorithm for Valve-Point Economic Load Dispatch.

PubMed

Karthikeyan, M; Raja, T Sree Ranga

2015-01-01

Economic load dispatch (ELD) problem is an important issue in the operation and control of modern control system. The ELD problem is complex and nonlinear with equality and inequality constraints which makes it hard to be efficiently solved. This paper presents a new modification of harmony search (HS) algorithm named as dynamic harmony search with polynomial mutation (DHSPM) algorithm to solve ORPD problem. In DHSPM algorithm the key parameters of HS algorithm like harmony memory considering rate (HMCR) and pitch adjusting rate (PAR) are changed dynamically and there is no need to predefine these parameters. Additionally polynomial mutation is inserted in the updating step of HS algorithm to favor exploration and exploitation of the search space. The DHSPM algorithm is tested with three power system cases consisting of 3, 13, and 40 thermal units. The computational results show that the DHSPM algorithm is more effective in finding better solutions than other computational intelligence based methods.

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

Klesmith, Justin R.; Bacik, John -Paul; Michalczyk, Ryszard

Synthetic metabolic pathways often suffer from low specific productivity, and new methods that quickly assess pathway functionality for many thousands of variants are urgently needed. Here we present an approach that enables the rapid and parallel determination of sequence effects on flux for complete gene-encoding sequences. We show that this method can be used to determine the effects of over 8000 single point mutants of a pyrolysis oil catabolic pathway implanted in Escherichia coli. Experimental sequence-function data sets predicted whether fitness-enhancing mutations to the enzyme levoglucosan kinase resulted from enhanced catalytic efficiency or enzyme stability. A structure of one designmore » incorporating 38 mutations elucidated the structural basis of high fitness mutations. One design incorporating 15 beneficial mutations supported a 15-fold improvement in growth rate and greater than 24-fold improvement in enzyme activity relative to the starting pathway. Lastly, this technique can be extended to improve a wide variety of designed pathways.« less

Computational design of thermostabilizing point mutations for G protein-coupled receptors

PubMed Central

Popov, Petr; Peng, Yao; Shen, Ling; Stevens, Raymond C; Cherezov, Vadim; Liu, Zhi-Jie

2018-01-01

Engineering of GPCR constructs with improved thermostability is a key for successful structural and biochemical studies of this transmembrane protein family, targeted by 40% of all therapeutic drugs. Here we introduce a comprehensive computational approach to effective prediction of stabilizing mutations in GPCRs, named CompoMug, which employs sequence-based analysis, structural information, and a derived machine learning predictor. Tested experimentally on the serotonin 5-HT2C receptor target, CompoMug predictions resulted in 10 new stabilizing mutations, with an apparent thermostability gain ~8.8°C for the best single mutation and ~13°C for a triple mutant. Binding of antagonists confers further stabilization for the triple mutant receptor, with total gains of ~21°C as compared to wild type apo 5-HT2C. The predicted mutations enabled crystallization and structure determination for the 5-HT2C receptor complexes in inactive and active-like states. While CompoMug already shows high 25% hit rate and utility in GPCR structural studies, further improvements are expected with accumulation of structural and mutation data. PMID:29927385

RET/PTC and PAX8/PPARγ chromosomal rearrangements in post-Chernobyl thyroid cancer and their association with iodine-131 radiation dose and other characteristics.

PubMed

Leeman-Neill, Rebecca J; Brenner, Alina V; Little, Mark P; Bogdanova, Tetiana I; Hatch, Maureen; Zurnadzy, Liudmyla Y; Mabuchi, Kiyohiko; Tronko, Mykola D; Nikiforov, Yuri E

2013-05-15

Childhood exposure to iodine-131 from the 1986 nuclear accident in Chernobyl, Ukraine, led to a sharp increase in papillary thyroid carcinoma (PTC) incidence in regions surrounding the reactor. Data concerning the association between genetic mutations in PTCs and individual radiation doses are limited. Mutational analysis was performed on 62 PTCs diagnosed in a Ukrainian cohort of patients who were < 18 years old in 1986 and received 0.008 to 8.6 Gy of (131) I to the thyroid. Associations between mutation types and (131) I dose and other characteristics were explored. RET/PTC (ret proto-oncogene/papillary thyroid carcinoma) rearrangements were most common (35%), followed by BRAF (15%) and RAS (8%) point mutations. Two tumors carrying PAX8/PPARγ (paired box 8/peroxisome proliferator-activated receptor gamma) rearrangement were identified. A significant negative association with (131) I dose for BRAF and RAS point mutations and a significant concave association with (131) I dose, with an inflection point at 1.6 Gy and odds ratio of 2.1, based on a linear-quadratic model for RET/PTC and PAX8/PPARγ rearrangements were found. The trends with dose were significantly different between tumors with point mutations and rearrangements. Compared with point mutations, rearrangements were associated with residence in the relatively iodine-deficient Zhytomyr region, younger age at exposure or surgery, and male sex. These results provide the first demonstration of PAX8/PPARγ rearrangements in post-Chernobyl tumors and show different associations for point mutations and chromosomal rearrangements with (131) I dose and other factors. These data support the relationship between chromosomal rearrangements, but not point mutations, and (131) I exposure and point to a possible role of iodine deficiency in generation of RET/PTC rearrangements in these patients. Copyright © 2013 American Cancer Society.

Assessment of circulating copy number variant detection for cancer screening.

PubMed

Molparia, Bhuvan; Nichani, Eshaan; Torkamani, Ali

2017-01-01

Current high-sensitivity cancer screening methods, largely utilizing correlative biomarkers, suffer from false positive rates that lead to unnecessary medical procedures and debatable public health benefit overall. Detection of circulating tumor DNA (ctDNA), a causal biomarker, has the potential to revolutionize cancer screening. Thus far, the majority of ctDNA studies have focused on detection of tumor-specific point mutations after cancer diagnosis for the purpose of post-treatment surveillance. However, ctDNA point mutation detection methods developed to date likely lack either the scope or analytical sensitivity necessary to be useful for cancer screening, due to the low (<1%) ctDNA fraction derived from early stage tumors. On the other hand, tumor-derived copy number variant (CNV) detection is hypothetically a superior means of ctDNA-based cancer screening for many tumor types, given that, relative to point mutations, each individual tumor CNV contributes a much larger number of ctDNA fragments to the overall pool of circulating free DNA (cfDNA). A small number of studies have demonstrated the potential of ctDNA CNV-based screening in select cancer types. Here we perform an in silico assessment of the potential for ctDNA CNV-based cancer screening across many common cancers, and suggest ctDNA CNV detection shows promise as a broad cancer screening methodology.

Continuous and discontinuous phase transitions in the evolution of a polygenic trait under stabilizing selective pressure

NASA Astrophysics Data System (ADS)

Fierro, Annalisa; Cocozza, Sergio; Monticelli, Antonella; Scala, Giovanni; Miele, Gennaro

2017-06-01

The presence of phenomena analogous to phase transition in Statistical Mechanics has been suggested in the evolution of a polygenic trait under stabilizing selection, mutation and genetic drift. By using numerical simulations of a model system, we analyze the evolution of a population of N diploid hermaphrodites in random mating regime. The population evolves under the effect of drift, selective pressure in form of viability on an additive polygenic trait, and mutation. The analysis allows to determine a phase diagram in the plane of mutation rate and strength of selection. The involved pattern of phase transitions is characterized by a line of critical points for weak selective pressure (smaller than a threshold), whereas discontinuous phase transitions, characterized by metastable hysteresis, are observed for strong selective pressure. A finite-size scaling analysis suggests the analogy between our system and the mean-field Ising model for selective pressure approaching the threshold from weaker values. In this framework, the mutation rate, which allows the system to explore the accessible microscopic states, is the parameter controlling the transition from large heterozygosity ( disordered phase) to small heterozygosity ( ordered one).

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

PubMed

Ozaslan, Mehmet; Ozaslan, Ersan; Barsgan, Arzu; Koruk, Mehmet

2007-12-01

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

Influence of adaptive mutations, from thermal adaptation experiments, on the infection cycle of RNA bacteriophage Qβ.

PubMed

Kashiwagi, Akiko; Kadoya, Tamami; Kumasaka, Naoya; Kumagai, Tomofumi; Tsushima, Fumie Sano; Yomo, Tetsuya

2018-06-04

A population's growth rate is determined by multiple 'life history traits'. To quantitatively determine which life history traits should be improved to allow a living organism to adapt to an inhibitory environment is an important issue. Previously, we conducted thermal adaptation experiments on the RNA bacteriophage Qβ using three independent replicates and reported that all three end-point populations could grow at a temperature (43.6°C) that inhibited the growth of the ancestral strain. Even though the fitness values of the endpoint populations were almost the same, their genome sequence was not, indicating that the three thermally adapted populations may have different life history traits. In this study, we introduced each mutation observed in these three end-point populations into the cDNA of the Qβ genome and prepared three different mutants. Quantitative analysis showed that they tended to increase their fitness by increasing the adsorption rate to their host, shortening their latent period (i.e., the duration between phage infection and progeny release), and increasing the burst size (i.e., the number of progeny phages per infected cell), but all three mutants decreased their thermal stability. However, the degree to which these traits changed differed. The mutant with the least mutations showed a smaller decrease in thermal stability, the largest adsorption rate to the host, and the shortest latent period. These results indicated that several different adaptive routes exist by which Qβ can adapt to higher temperatures, even though Qβ is a simple RNA bacteriophage with a small genome size, encoding only four genes.

Adjuvant Dabrafenib plus Trametinib in Stage III BRAF-Mutated Melanoma.

PubMed

Long, Georgina V; Hauschild, Axel; Santinami, Mario; Atkinson, Victoria; Mandalà, Mario; Chiarion-Sileni, Vanna; Larkin, James; Nyakas, Marta; Dutriaux, Caroline; Haydon, Andrew; Robert, Caroline; Mortier, Laurent; Schachter, Jacob; Schadendorf, Dirk; Lesimple, Thierry; Plummer, Ruth; Ji, Ran; Zhang, Pingkuan; Mookerjee, Bijoyesh; Legos, Jeff; Kefford, Richard; Dummer, Reinhard; Kirkwood, John M

2017-11-09

Combination therapy with the BRAF inhibitor dabrafenib plus the MEK inhibitor trametinib improved survival in patients with advanced melanoma with BRAF V600 mutations. We sought to determine whether adjuvant dabrafenib plus trametinib would improve outcomes in patients with resected, stage III melanoma with BRAF V600 mutations. In this double-blind, placebo-controlled, phase 3 trial, we randomly assigned 870 patients with completely resected, stage III melanoma with BRAF V600E or V600K mutations to receive oral dabrafenib at a dose of 150 mg twice daily plus trametinib at a dose of 2 mg once daily (combination therapy, 438 patients) or two matched placebo tablets (432 patients) for 12 months. The primary end point was relapse-free survival. Secondary end points included overall survival, distant metastasis-free survival, freedom from relapse, and safety. At a median follow-up of 2.8 years, the estimated 3-year rate of relapse-free survival was 58% in the combination-therapy group and 39% in the placebo group (hazard ratio for relapse or death, 0.47; 95% confidence interval [CI], 0.39 to 0.58; P<0.001). The 3-year overall survival rate was 86% in the combination-therapy group and 77% in the placebo group (hazard ratio for death, 0.57; 95% CI, 0.42 to 0.79; P=0.0006), but this level of improvement did not cross the prespecified interim analysis boundary of P=0.000019. Rates of distant metastasis-free survival and freedom from relapse were also higher in the combination-therapy group than in the placebo group. The safety profile of dabrafenib plus trametinib was consistent with that observed with the combination in patients with metastatic melanoma. Adjuvant use of combination therapy with dabrafenib plus trametinib resulted in a significantly lower risk of recurrence in patients with stage III melanoma with BRAF V600E or V600K mutations than the adjuvant use of placebo and was not associated with new toxic effects. (Funded by GlaxoSmithKline and Novartis; COMBI-AD ClinicalTrials.gov, NCT01682083 ; EudraCT number, 2012-001266-15 .).

Transgenic mouse α- and β-cardiac myosins containing the R403Q mutation show isoform-dependent transient kinetic differences.

PubMed

Lowey, Susan; Bretton, Vera; Gulick, James; Robbins, Jeffrey; Trybus, Kathleen M

2013-05-24

Familial hypertrophic cardiomyopathy (FHC) is a major cause of sudden cardiac death in young athletes. The discovery in 1990 that a point mutation at residue 403 (R403Q) in the β-myosin heavy chain (MHC) caused a severe form of FHC was the first of many demonstrations linking FHC to mutations in muscle proteins. A mouse model for FHC has been widely used to study the mechanochemical properties of mutated cardiac myosin, but mouse hearts express α-MHC, whereas the ventricles of larger mammals express predominantly β-MHC. To address the role of the isoform backbone on function, we generated a transgenic mouse in which the endogenous α-MHC was partially replaced with transgenically encoded β-MHC or α-MHC. A His6 tag was cloned at the N terminus, along with R403Q, to facilitate isolation of myosin subfragment 1 (S1). Stopped flow kinetics were used to measure the equilibrium constants and rates of nucleotide binding and release for the mouse S1 isoforms bound to actin. For the wild-type isoforms, we found that the affinity of MgADP for α-S1 (100 μM) is ~ 4-fold weaker than for β-S1 (25 μM). Correspondingly, the MgADP release rate for α-S1 (350 s(-1)) is ~3-fold greater than for β-S1 (120 s(-1)). Introducing the R403Q mutation caused only a minor reduction in kinetics for β-S1, but R403Q in α-S1 caused the ADP release rate to increase by 20% (430 s(-1)). These transient kinetic studies on mouse cardiac myosins provide strong evidence that the functional impact of an FHC mutation on myosin depends on the isoform backbone.

A phase II trial of regorafenib in patients with metastatic and/or a unresectable gastrointestinal stromal tumor harboring secondary mutations of exon 17.

PubMed

Yeh, Chun-Nan; Chen, Ming-Huang; Chen, Yen-Yang; Yang, Ching-Yao; Yen, Chueh-Chuan; Tzen, Chin-Yuan; Chen, Li-Tzong; Chen, Jen-Shi

2017-07-04

Gastrointestinal stromal tumors (GISTs) are caused by the constitutive activation of KIT or platelet-derived growth factor receptor alpha (PDGFRA) mutations. Imatinib selectively inhibits KIT and PDGFR, leading to disease control for 80%-90% of patients with metastatic GIST. Imatinib resistance can occur within a median of 2-3 years due to secondary mutations in KIT. According to preclinical studies, both imatinib and sunitinib are ineffective against exon 17 mutations. However, the treatment efficacy of regorafenib for patients with GIST with exon 17 mutations is still unknown. Documented patients with GIST with exon 17 mutations were enrolled in this study. Patients received 160 mg of oral regorafenib daily on days 1-21 of a 28-day cycle. The primary end point of this trial was the clinical benefit rate (CBR; i.e., complete or partial response [PR], as well as stable disease [SD]) at 16 weeks. The secondary end points of this study included progression free survival (PFS), overall survival, and safety. Between June 2014 to May 2016, 18 patients were enrolled (15 of which were eligible for response evaluation). The CBR at 16 weeks was 93.3% (14 of 15; 6 PR and 8 SD). The median PFS was 22.1 months. The most common grade 3 toxicities were hand-and-foot skin reactions (10 of 18; 55.6%), followed by hypertension (5 of 18; 27.8%). Regorafenib significantly prolonged PFS in patients with advanced GIST harboring secondary mutations of exon 17. A phase III trial of regorafenib versus placebo is warranted. This trial is registered at ClinicalTrials.gov in November 2015, number NCT02606097.Key message: This phase II trial was conducted to assess the efficacy and safety of regorafenib in patients with GIST with exon 17 mutations. The results provide strong evidence that regorafenib significantly prolonged PFS in patients with advanced GIST harboring secondary mutations of exon 17.

MultiScheme: A Parallel Processing System Based on MIT (Massachusetts Institute of Technology) Scheme.

DTIC Science & Technology

1987-09-01

Later, when these alloca- :t)il t rate(-ies beconme a p)erfornmance concern, the schieduler can be inolded 4 toN fit the p~ articular appllicationi... distracts attention from the more important points that this example is intended to demonstrate. The implementation, therefore, is described separately in...for the benefit of outsiders. From the object’s point of view the pipeline is nothing but a list of messages that tell it how to mutate its own state

Molecular Profiling and Targeted Therapy for Advanced Thoracic Malignancies: A Biomarker-Derived, Multiarm, Multihistology Phase II Basket Trial

PubMed Central

Lopez-Chavez, Ariel; Thomas, Anish; Rajan, Arun; Raffeld, Mark; Morrow, Betsy; Kelly, Ronan; Carter, Corey Allan; Guha, Udayan; Killian, Keith; Lau, Christopher C.; Abdullaev, Zied; Xi, Liqiang; Pack, Svetlana; Meltzer, Paul S.; Corless, Christopher L.; Sandler, Alan; Beadling, Carol; Warrick, Andrea; Liewehr, David J.; Steinberg, Seth M.; Berman, Arlene; Doyle, Austin; Szabo, Eva; Wang, Yisong; Giaccone, Giuseppe

2015-01-01

Purpose We conducted a basket clinical trial to assess the feasibility of such a design strategy and to independently evaluate the effects of multiple targeted agents against specific molecular aberrations in multiple histologic subtypes concurrently. Patients and Methods We enrolled patients with advanced non–small-cell lung cancer (NSCLC), small-cell lung cancer, and thymic malignancies who underwent genomic characterization of oncogenic drivers. Patients were enrolled onto a not-otherwise-specified arm and treated with standard-of-care therapies or one of the following five biomarker-matched treatment groups: erlotinib for EGFR mutations; selumetinib for KRAS, NRAS, HRAS, or BRAF mutations; MK2206 for PIK3CA, AKT, or PTEN mutations; lapatinib for ERBB2 mutations or amplifications; and sunitinib for KIT or PDGFRA mutations or amplification. Results Six hundred forty-seven patients were enrolled, and 88% had their tumors tested for at least one gene. EGFR mutation frequency was 22.1% in NSCLC, and erlotinib achieved a response rate of 60% (95% CI, 32.3% to 83.7%). KRAS mutation frequency was 24.9% in NSCLC, and selumetinib failed to achieve its primary end point, with a response rate of 11% (95% CI, 0% to 48%). Completion of accrual to all other arms was not feasible. In NSCLC, patients with EGFR mutations had the longest median survival (3.51 years; 95% CI, 2.89 to 5.5 years), followed by those with ALK rearrangements (2.94 years; 95% CI, 1.66 to 4.61 years), those with KRAS mutations (2.3 years; 95% CI, 2.3 to 2.17 years), those with other genetic abnormalities (2.17 years; 95% CI, 1.3 to 2.74 years), and those without an actionable mutation (1.85 years; 95% CI, 1.61 to 2.13 years). Conclusion This basket trial design was not feasible for many of the arms with rare mutations, but it allowed the study of the genetics of less common malignancies. PMID:25667274

Pairwise contact energy statistical potentials can help to find probability of point mutations.

PubMed

Saravanan, K M; Suvaithenamudhan, S; Parthasarathy, S; Selvaraj, S

2017-01-01

To adopt a particular fold, a protein requires several interactions between its amino acid residues. The energetic contribution of these residue-residue interactions can be approximated by extracting statistical potentials from known high resolution structures. Several methods based on statistical potentials extracted from unrelated proteins are found to make a better prediction of probability of point mutations. We postulate that the statistical potentials extracted from known structures of similar folds with varying sequence identity can be a powerful tool to examine probability of point mutation. By keeping this in mind, we have derived pairwise residue and atomic contact energy potentials for the different functional families that adopt the (α/β) 8 TIM-Barrel fold. We carried out computational point mutations at various conserved residue positions in yeast Triose phosphate isomerase enzyme for which experimental results are already reported. We have also performed molecular dynamics simulations on a subset of point mutants to make a comparative study. The difference in pairwise residue and atomic contact energy of wildtype and various point mutations reveals probability of mutations at a particular position. Interestingly, we found that our computational prediction agrees with the experimental studies of Silverman et al. (Proc Natl Acad Sci 2001;98:3092-3097) and perform better prediction than i Mutant and Cologne University Protein Stability Analysis Tool. The present work thus suggests deriving pairwise contact energy potentials and molecular dynamics simulations of functionally important folds could help us to predict probability of point mutations which may ultimately reduce the time and cost of mutation experiments. Proteins 2016; 85:54-64. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

A pilot study of mitochondrial DNA point mutation A3243G in a sample of Croatian patients having type 2 diabetes mellitus associated with maternal inheritance.

PubMed

Martin-Kleiner, I; Pape-Medvidović, E; Pavlić-Renar, I; Metelko, Z; Kusec, R; Gabrilovac, J; Boranić, M

2004-12-01

In this work, patients having type 2 diabetes mellitus and diabetic mothers were tested for the presence of mitochondrial DNA point mutation A3243G. This mutation is associated with the MELAS syndrome (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes), diabetes and deafness. Twenty-two diabetic persons were screened. DNA was isolated from peripheral blood lymphocytes and from swabs of oral mucosa. The mitochondrial DNA point mutation A3243G was detected using PCR-RFLP test. The mutation was detected in oral mucosal DNA of two patients (but not from lymphocyte DNA). One patient was a man with hearing and visual impairments and proteinuria; the other was a woman having proteinuria but no hearing impairment. The mutation was not detectable in oral mucosal DNA from the control persons: 20 diabetic patients having diabetic fathers and 22 healthy, nondiabetic volunteers. The incidence of mitochondrial DNA point mutation A3243G in this study of Croatian diabetic patients is in line with data in the literature.

Reconstruction of thermotolerant yeast by one-point mutation identified through whole-genome analyses of adaptively-evolved strains.

PubMed

Satomura, Atsushi; Miura, Natsuko; Kuroda, Kouichi; Ueda, Mitsuyoshi

2016-03-17

Saccharomyces cerevisiae is used as a host strain in bioproduction, because of its rapid growth, ease of genetic manipulation, and high reducing capacity. However, the heat produced during the fermentation processes inhibits the biological activities and growth of the yeast cells. We performed whole-genome sequencing of 19 intermediate strains previously obtained during adaptation experiments under heat stress; 49 mutations were found in the adaptation steps. Phylogenetic tree revealed at least five events in which these strains had acquired mutations in the CDC25 gene. Reconstructed CDC25 point mutants based on a parental strain had acquired thermotolerance without any growth defects. These mutations led to the downregulation of the cAMP-dependent protein kinase (PKA) signaling pathway, which controls a variety of processes such as cell-cycle progression and stress tolerance. The one-point mutations in CDC25 were involved in the global transcriptional regulation through the cAMP/PKA pathway. Additionally, the mutations enabled efficient ethanol fermentation at 39 °C, suggesting that the one-point mutations in CDC25 may contribute to bioproduction.

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.

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.

Vascular Ehlers-Danlos syndrome mutations in type III collagen differently stall the triple helical folding.

PubMed

Mizuno, Kazunori; Boudko, Sergei; Engel, Jürgen; Bächinger, Hans Peter

2013-06-28

Vascular Ehlers-Danlos syndrome (EDS) type IV is the most severe form of EDS. In many cases the disease is caused by a point mutation of Gly in type III collagen. A slower folding of the collagen helix is a potential cause for over-modifications. However, little is known about the rate of folding of type III collagen in patients with EDS. To understand the molecular mechanism of the effect of mutations, a system was developed for bacterial production of homotrimeric model polypeptides. The C-terminal quarter, 252 residues, of the natural human type III collagen was attached to (GPP)7 with the type XIX collagen trimerization domain (NC2). The natural collagen domain forms a triple helical structure without 4-hydroxylation of proline at a low temperature. At 33 °C, the natural collagenous part is denatured, but the C-terminal (GPP)7-NC2 remains intact. Switching to a low temperature triggers the folding of the type III collagen domain in a zipper-like fashion that resembles the natural process. We used this system for the two known EDS mutations (Gly-to-Val) in the middle at Gly-910 and at the C terminus at Gly-1018. In addition, wild-type and Gly-to-Ala mutants were made. The mutations significantly slow down the overall rate of triple helix formation. The effect of the Gly-to-Val mutation is much more severe compared with Gly-to-Ala. This is the first report on the folding of collagen with EDS mutations, which demonstrates local delays in the triple helix propagation around the mutated residue.

KRAS and BRAF Mutations and PTEN Expression Do Not Predict Efficacy of Cetuximab-Based Chemoradiotherapy in Locally Advanced Rectal Cancer

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

Erben, Philipp, E-mail: philipp.erben@medma.uni-heidelberg.de; Stroebel, Philipp; Horisberger, Karoline

2011-11-15

Purpose: Mutations in KRAS and BRAF genes as well as the loss of expression of phosphatase and tensin homolog (PTEN) (deleted on chromosome 10) are associated with impaired activity of antibodies directed against epidermal growth factor receptor in patients with metastatic colorectal cancer. The predictive and prognostic value of the KRAS and BRAF point mutations as well as PTEN expression in patients with locally advanced rectal cancer (LARC) treated with cetuximab-based neoadjuvant chemoradiotherapy is unknown. Methods and Materials: We have conducted phase I and II trials of the combination of weekly administration of cetuximab and irinotecan and daily doses ofmore » capecitabine in conjunction with radiotherapy (45 Gy plus 5.4 Gy) in patients with LARC (stage uT3/4 or uN+). The status of KRAS and BRAF mutations was determined with direct sequencing, and PTEN expression status was determined with immunohistochemistry testing of diagnostic tumor biopsies. Tumor regression was evaluated by using standardized regression grading, and disease-free survival (DFS) was calculated according to the Kaplan-Meier method. Results: A total of 57 patients were available for analyses. A total of 31.6% of patients carried mutations in the KRAS genes. No BRAF mutations were found, while the loss of PTEN expression was observed in 9.6% of patients. Six patients achieved complete remission, and the 3-year DFS rate was 73%. No correlation was seen between tumor regression or DFS rate and a single marker or a combination of all markers. Conclusions: In the present series, no BRAF mutation was detected. The presence of KRAS mutations and loss of PTEN expression were not associated with impaired response to cetuximab-based chemoradiotherapy and 3-year DFS.« less

Comparison of the efficacy of icotinib in patients with non-small-cell lung cancer according to the type of epidermal growth factor receptor mutation.

PubMed

Xue, Zhang Xiao; Wen, Wang Xiu; Zhuang, Yu; Hua, Zang Jian; Xia, Yang Ni

2016-09-01

Icotinib hydrochloride is a novel epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) with preclinical and clinical activity in non-small-cell lung cancer (NSCLC). Exon 19 deletion and L858R point mutation are the most commonly encountered EGFR mutations in NSCLC, and they predict improved clinical outcomes following treatment with icotinib. The objective of this study was to evaluate the differential clinical efficacy of icotinib in patients with exon 19 deletion or L858R point mutation of the EGFR gene. A total of 104 patients with advanced NSCLC, who harbored exon 19 deletion or L858R point mutation of EGFR and were treated with icotinib, were enrolled in this study. The tumor response and progression-free survival were evaluated. There were no significant differences between patients with EGFR exon 19 deletion and those with L858R point mutation who received treatment with icotinib.

Mutation rate estimation for 15 autosomal STR loci in a large population from Mainland China.

PubMed

Zhao, Zhuo; Zhang, Jie; Wang, Hua; Liu, Zhi-Peng; Liu, Ming; Zhang, Yuan; Sun, Li; Zhang, Hui

2015-09-01

STR, short tandem repeats, are well known as a type of powerful genetic marker and widely used in studying human population genetics. Compared with the conventional genetic markers, the mutation rate of STR is higher. Additionally, the mutations of STR loci do not lead to genetic inconsistencies between the genotypes of parents and children; therefore, the analysis of STR mutation is more suited to assess the population mutation. In this study, we focused on 15 autosomal STR loci. DNA samples from a total of 42,416 unrelated healthy individuals (19,037 trios) from the population of Mainland China collected between Jan 2012 and May 2014 were successfully investigated. In our study, the allele frequencies, paternal mutation rates, maternal mutation rates and average mutation rates were detected. Furthermore, we also investigated the relationship between paternal ages, maternal ages, area, the time of pregnancy and average mutation rate. We found that the paternal mutation rate was higher than the maternal mutation rate and the paternal, maternal, and average mutation rates had a positive correlation with paternal age, maternal age and the time of pregnancy respectively. Additionally, the average mutation rate of coastal areas was higher than that of inland areas.

Frequency-dependent selection can lead to evolution of high mutation rates.

PubMed

Rosenbloom, Daniel I S; Allen, Benjamin

2014-05-01

Theoretical and experimental studies have shown that high mutation rates can be advantageous, especially in novel or fluctuating environments. Here we examine how frequency-dependent competition may lead to fluctuations in trait frequencies that exert upward selective pressure on mutation rates. We use a mathematical model to show that cyclical trait dynamics generated by "rock-paper-scissors" competition can cause the mutation rate in a population to converge to a high evolutionarily stable mutation rate, reflecting a trade-off between generating novelty and reproducing past success. Introducing recombination lowers the evolutionarily stable mutation rate but allows stable coexistence between mutation rates above and below the evolutionarily stable rate. Even considering strong mutational load and ignoring the costs of faithful replication, evolution favors positive mutation rates if the selective advantage of prevailing in competition exceeds the ratio of recombining to nonrecombining offspring. We discuss a number of genomic mechanisms that may meet our theoretical requirements for the adaptive evolution of mutation. Overall, our results suggest that local mutation rates may be higher on genes influencing cyclical competition and that global mutation rates in asexual species may be higher in populations subject to strong cyclical competition.

A comparative analysis of whole genome sequencing of esophageal adenocarcinoma pre- and post-chemotherapy

PubMed Central

Noorani, Ayesha; Lynch, Andy G.; Achilleos, Achilleas; Eldridge, Matthew; Bower, Lawrence; Weaver, Jamie M.J.; Crawte, Jason; Ong, Chin-Ann; Shannon, Nicholas; MacRae, Shona; Grehan, Nicola; Nutzinger, Barbara; O'Donovan, Maria; Hardwick, Richard; Tavaré, Simon; Fitzgerald, Rebecca C.

2017-01-01

The scientific community has avoided using tissue samples from patients that have been exposed to systemic chemotherapy to infer the genomic landscape of a given cancer. Esophageal adenocarcinoma is a heterogeneous, chemoresistant tumor for which the availability and size of pretreatment endoscopic samples are limiting. This study compares whole-genome sequencing data obtained from chemo-naive and chemo-treated samples. The quality of whole-genomic sequencing data is comparable across all samples regardless of chemotherapy status. Inclusion of samples collected post-chemotherapy increased the proportion of late-stage tumors. When comparing matched pre- and post-chemotherapy samples from 10 cases, the mutational signatures, copy number, and SNV mutational profiles reflect the expected heterogeneity in this disease. Analysis of SNVs in relation to allele-specific copy-number changes pinpoints the common ancestor to a point prior to chemotherapy. For cases in which pre- and post-chemotherapy samples do show substantial differences, the timing of the divergence is near-synchronous with endoreduplication. Comparison across a large prospective cohort (62 treatment-naive, 58 chemotherapy-treated samples) reveals no significant differences in the overall mutation rate, mutation signatures, specific recurrent point mutations, or copy-number events in respect to chemotherapy status. In conclusion, whole-genome sequencing of samples obtained following neoadjuvant chemotherapy is representative of the genomic landscape of esophageal adenocarcinoma. Excluding these samples reduces the material available for cataloging and introduces a bias toward the earlier stages of cancer. PMID:28465312

Insights into enzyme point mutation effect by molecular simulation: phenylethylamine oxidation catalyzed by monoamine oxidase A.

PubMed

Oanca, Gabriel; Purg, Miha; Mavri, Janez; Shih, Jean C; Stare, Jernej

2016-05-21

The I335Y point mutation effect on the kinetics of phenylethylamine decomposition catalyzed by monoamine oxidase A was elucidated by means of molecular simulation. The established empirical valence bond methodology was used in conjunction with the free energy perturbation sampling technique and a classical force field representing the state of reactants and products. The methodology allows for the simulation of chemical reactions, in the present case the breaking of the α-C-H bond in a phenylethylamine substrate and the subsequent hydrogen transfer to the flavin cofactor, resulting in the formation of the N-H bond on flavin. The empirical parameters were calibrated against the experimental data for the simulated reaction in a wild type protein and then used for the calculation of the reaction free energy profile in the I335Y mutant. In very good agreement with the measured kinetic data, mutation increases the free energy barrier for the rate limiting step by slightly more than 1 kcal mol(-1) and consequently decreases the rate constant by about an order of magnitude. The magnitude of the computed effect slightly varies with simulation settings, but always remains in reasonable agreement with the experiment. Analysis of trajectories reveals a major change in the interaction between phenyl rings of the substrate and the neighboring Phe352 residue upon the I335Y mutation due to the increased local polarity, leading to an attenuated quadrupole interaction between the rings and destabilization of the transition state. Additionally, the increased local polarity in the mutant allows for a larger number of water molecules to be present near the active site, effectively shielding the catalytic effect of the enzyme and contributing to the increased barrier.

Dabrafenib and Trametinib Treatment in Patients With Locally Advanced or Metastatic BRAF V600–Mutant Anaplastic Thyroid Cancer

PubMed Central

Subbiah, Vivek; Kreitman, Robert J.; Wainberg, Zev A.; Cho, Jae Yong; Schellens, Jan H.M.; Soria, Jean Charles; Wen, Patrick Y.; Zielinski, Christoph; Cabanillas, Maria E.; Urbanowitz, Gladys; Mookerjee, Bijoyesh; Wang, Dazhe; Rangwala, Fatima

2018-01-01

Purpose We report the efficacy and safety of dabrafenib (BRAF inhibitor) and trametinib (MEK inhibitor) combination therapy in BRAF V600E–mutated anaplastic thyroid cancer, a rare, aggressive, and highly lethal malignancy with poor patient outcomes and no systemic therapies with clinical benefit. Methods In this phase II, open-label trial, patients with predefined BRAF V600E–mutated malignancies received dabrafenib 150 mg twice daily and trametinib 2 mg once daily until unacceptable toxicity, disease progression, or death. The primary end point was investigator-assessed overall response rate. Secondary end points included duration of response, progression-free survival, overall survival, and safety. Results Sixteen patients with BRAF V600E–mutated anaplastic thyroid cancer were evaluable (median follow-up, 47 weeks; range, 4 to 120 weeks). All patients had received prior radiation treatment and/or surgery, and six had received prior systemic therapy. The confirmed overall response rate was 69% (11 of 16; 95% CI, 41% to 89%), with seven ongoing responses. Median duration of response, progression-free survival, and overall survival were not reached as a result of a lack of events, with 12-month estimates of 90%, 79%, and 80%, respectively. The safety population was composed of 100 patients who were enrolled with seven rare tumor histologies. Common adverse events were fatigue (38%), pyrexia (37%), and nausea (35%). No new safety signals were detected. Conclusion Dabrafenib plus trametinib is the first regimen demonstrated to have robust clinical activity in BRAF V600E–mutated anaplastic thyroid cancer and was well tolerated. These findings represent a meaningful therapeutic advance for this orphan disease. PMID:29072975

Dabrafenib and Trametinib Treatment in Patients With Locally Advanced or Metastatic BRAF V600-Mutant Anaplastic Thyroid Cancer.

PubMed

Subbiah, Vivek; Kreitman, Robert J; Wainberg, Zev A; Cho, Jae Yong; Schellens, Jan H M; Soria, Jean Charles; Wen, Patrick Y; Zielinski, Christoph; Cabanillas, Maria E; Urbanowitz, Gladys; Mookerjee, Bijoyesh; Wang, Dazhe; Rangwala, Fatima; Keam, Bhumsuk

2018-01-01

Purpose We report the efficacy and safety of dabrafenib (BRAF inhibitor) and trametinib (MEK inhibitor) combination therapy in BRAF V600E-mutated anaplastic thyroid cancer, a rare, aggressive, and highly lethal malignancy with poor patient outcomes and no systemic therapies with clinical benefit. Methods In this phase II, open-label trial, patients with predefined BRAF V600E-mutated malignancies received dabrafenib 150 mg twice daily and trametinib 2 mg once daily until unacceptable toxicity, disease progression, or death. The primary end point was investigator-assessed overall response rate. Secondary end points included duration of response, progression-free survival, overall survival, and safety. Results Sixteen patients with BRAF V600E-mutated anaplastic thyroid cancer were evaluable (median follow-up, 47 weeks; range, 4 to 120 weeks). All patients had received prior radiation treatment and/or surgery, and six had received prior systemic therapy. The confirmed overall response rate was 69% (11 of 16; 95% CI, 41% to 89%), with seven ongoing responses. Median duration of response, progression-free survival, and overall survival were not reached as a result of a lack of events, with 12-month estimates of 90%, 79%, and 80%, respectively. The safety population was composed of 100 patients who were enrolled with seven rare tumor histologies. Common adverse events were fatigue (38%), pyrexia (37%), and nausea (35%). No new safety signals were detected. Conclusion Dabrafenib plus trametinib is the first regimen demonstrated to have robust clinical activity in BRAF V600E-mutated anaplastic thyroid cancer and was well tolerated. These findings represent a meaningful therapeutic advance for this orphan disease.

Mutation rates among RNA viruses

PubMed Central

Drake, John W.; Holland, John J.

1999-01-01

The rate of spontaneous mutation is a key parameter in modeling the genetic structure and evolution of populations. The impact of the accumulated load of mutations and the consequences of increasing the mutation rate are important in assessing the genetic health of populations. Mutation frequencies are among the more directly measurable population parameters, although the information needed to convert them into mutation rates is often lacking. A previous analysis of mutation rates in RNA viruses (specifically in riboviruses rather than retroviruses) was constrained by the quality and quantity of available measurements and by the lack of a specific theoretical framework for converting mutation frequencies into mutation rates in this group of organisms. Here, we describe a simple relation between ribovirus mutation frequencies and mutation rates, apply it to the best (albeit far from satisfactory) available data, and observe a central value for the mutation rate per genome per replication of μg ≈ 0.76. (The rate per round of cell infection is twice this value or about 1.5.) This value is so large, and ribovirus genomes are so informationally dense, that even a modest increase extinguishes the population. PMID:10570172

Investigating the Impact of Asp181 Point Mutations on Interactions between PTP1B and Phosphotyrosine Substrate

NASA Astrophysics Data System (ADS)

Liu, Mengyuan; Wang, Lushan; Sun, Xun; Zhao, Xian

2014-05-01

Protein tyrosine phosphatase 1B (PTP1B) is a key negative regulator of insulin and leptin signaling, which suggests that it is an attractive therapeutic target in type II diabetes and obesity. The aim of this research is to explore residues which interact with phosphotyrosine substrate can be affected by D181 point mutations and lead to increased substrate binding. To achieve this goal, molecular dynamics simulations were performed on wild type (WT) and two mutated PTP1B/substrate complexes. The cross-correlation and principal component analyses show that point mutations can affect the motions of some residues in the active site of PTP1B. Moreover, the hydrogen bond and energy decomposition analyses indicate that apart from residue 181, point mutations have influence on the interactions of substrate with several residues in the active site of PTP1B.

Gcn4p and the Crabtree effect of yeast: drawing the causal model of the Crabtree effect in Saccharomyces cerevisiae and explaining evolutionary trade-offs of adaptation to galactose through systems biology.

PubMed

Martínez, José L; Bordel, Sergio; Hong, KuFk-Ki; Nielsen, Jens

2014-06-01

By performing an integrated comparative analysis on the physiology and transcriptome of four different S. cerevisiae strains growing on galactose and glucose, it was inferred that the transcription factors Bas1p, Pho2p, and Gcn4p play a central role in the regulatory events causing the Crabtree effect in S. cerevisiae. The analysis also revealed that a point mutation in the RAS2 observed in a galactose-adapted strain causes a lower Crabtree effect and growth rate on glucose by decreasing the activity of Gcn4p while at the same time is at the origin of higher growth rate on galactose due to a lower activity of the transcriptional repressor Sok2p. The role of Gcn4p on the trade-off effect observed on glucose was confirmed experimentally. This was done by showing that the point mutation in RAS2 does not result in a lower growth rate on glucose if it is introduced in a GCN4-negative background. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Experimental evolution and the dynamics of genomic mutation rate modifiers.

PubMed

Raynes, Y; Sniegowski, P D

2014-11-01

Because genes that affect mutation rates are themselves subject to mutation, mutation rates can be influenced by natural selection and other evolutionary forces. The population genetics of mutation rate modifier alleles has been a subject of theoretical interest for many decades. Here, we review experimental contributions to our understanding of mutation rate modifier dynamics. Numerous evolution experiments have shown that mutator alleles (modifiers that elevate the genomic mutation rate) can readily rise to high frequencies via genetic hitchhiking in non-recombining microbial populations. Whereas these results certainly provide an explanatory framework for observations of sporadically high mutation rates in pathogenic microbes and in cancer lineages, it is nonetheless true that most natural populations have very low mutation rates. This raises the interesting question of how mutator hitchhiking is suppressed or its phenotypic effect reversed in natural populations. Very little experimental work has addressed this question; with this in mind, we identify some promising areas for future experimental investigation.

Advances in radiation mutagenesis through studies on Drosophila

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

Muller, H. J.

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 confirmed for minute structural changes in spermatozoa. The dependence of gross structural changes, 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. However, these stages unlike spermatozoa are found to allow union of broken chromosomes. Therefore, the frequencies are lower for more dispersed exposures of these stages, and the precise dose relation variesmore » with the timing. Part of the dominant and even recessive lethals induced in late oocytes follow the same frequency pattern and therefore are multi-hit events. Yet there is a much lower chance after oocytic than spermatozoan irradiation that two broken ends derived from different hits will unite, hence most such unions are 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 the principle of marked reduction of radiation mutagenesis by anoxia, known for spermatozoa in adult males, to those in pupal males and in females to spermatids and to oocytes. In spermatids this reduction 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 mutagens, 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 mutations 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-mutations also showed similarity of spontaneous and radiation mutations. The doubling dose for back-mutations of forked induced in spermatozoa was several hundred roentgens, similar to that for direct point-mutations induced in gonia at diverse loci. Recent analyses of human mutational load lead to mutation-rate estimates like those earlier based on extrapolations from Drosophila, thus supporting the significance for man of the present studies. (auth)« less

Timing, rates and spectra of human germline mutation.

PubMed

Rahbari, Raheleh; Wuster, Arthur; Lindsay, Sarah J; Hardwick, Robert J; Alexandrov, Ludmil B; Turki, Saeed Al; Dominiczak, Anna; Morris, Andrew; Porteous, David; Smith, Blair; Stratton, Michael R; Hurles, Matthew E

2016-02-01

Germline mutations are a driving force behind genome evolution and genetic disease. We investigated genome-wide mutation rates and spectra in multi-sibling families. The mutation rate increased with paternal age in all families, but the number of additional mutations per year differed by more than twofold between families. Meta-analysis of 6,570 mutations showed that germline methylation influences mutation rates. In contrast to somatic mutations, we found remarkable consistency in germline mutation spectra between the sexes and at different paternal ages. In parental germ line, 3.8% of mutations were mosaic, resulting in 1.3% of mutations being shared by siblings. The number of these shared mutations varied significantly between families. Our data suggest that the mutation rate per cell division is higher during both early embryogenesis and differentiation of primordial germ cells but is reduced substantially during post-pubertal spermatogenesis. These findings have important consequences for the recurrence risks of disorders caused by de novo mutations.

Clock-like mutational processes in human somatic cells

PubMed Central

Alexandrov, Ludmil B.; Jones, Philip H.; Wedge, David C.; Sale, Julian E.; Campbell, Peter J.; Nik-Zainal, Serena; Stratton, Michael R.

2016-01-01

During the course of a lifetime somatic cells acquire mutations. Different mutational processes may contribute to the mutations accumulated in a cell, with each imprinting a mutational signature on the cell’s genome. Some processes generate mutations throughout life at a constant rate in all individuals and the number of mutations in a cell attributable to these processes will be proportional to the chronological age of the person. Using mutations from 10,250 cancer genomes across 36 cancer types, we investigated clock-like mutational processes that have been operating in normal human cells. Two mutational signatures show clock-like properties. Both exhibit different mutation rates in different tissues. However, their mutation rates are not correlated indicating that the underlying processes are subject to different biological influences. For one signature, the rate of cell division may influence its mutation rate. This study provides the first survey of clock-like mutational processes operative in human somatic cells. PMID:26551669

Teicoplanin resistance in Staphylococcus haemolyticus is associated with mutations in histidine kinases VraS and WalK.

PubMed

Vimberg, Vladimir; Cavanagh, Jorunn Pauline; Benada, Oldřich; Kofroňová, Olga; Hjerde, Erik; Zieglerová, Leona; Balíková Novotná, Gabriela

2018-03-01

We investigated the genetic basis of glycopeptide resistance in laboratory-derived strains of S. haemolyticus with emphasis on differences between vancomycin and teicoplanin. The genomes of two stable teicoplanin-resistant laboratory mutants selected on vancomycin or teicoplanin were sequenced and compared to parental S. haemolyticus strain W2/124. Only the two non-synonymous mutations, VraS Q289K and WalK V550L were identified. No other mutations or genome rearrangements were detected. Increased cell wall thickness, resistance to lysostaphin-induced lysis and adaptation of cell growth rates specifically to teicoplanin were phenotypes observed in a sequenced strain with the VraS Q289K mutation. Neither of the VraS Q289K and WalK V550L mutations was present in the genomes of 121S. haemolyticus clinical isolates. However, all but two of the teicoplanin resistant strains carried non-synonymous SNPs in vraSRTU and walKR-YycHIJ operons pointing to their importance for the glycopeptide resistance. Copyright © 2017 Elsevier Inc. All rights reserved.

Glutaric aciduria type I in the Arab and Jewish communities in Israel

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

Anikster, Y.; Shaag, A.; Elpeleg, O.N.

Mutation analysis was performed in eight families (16 patients) with glutaric aciduria type I (GA-I), which were all the families diagnosed in Israel in the years 1987 - 1994. Six families were of Moslem origin and two were non-Ashkenazi Jews. The entire coding region of the cDNA of the glutaryl-CoA dehydrogenase gene was sequenced in one patient of each family. Seven new mutations were identified in 15 of 16 mutated alleles, including six point mutations: T4161 (4 alleles), G39OR (1 allele), and S305L, A293T, L283P, and G101R (2 alleles each). In addition, a 1-bp deletion at position 1173 was identifiedmore » in two alleles. These findings do not provide a molecular basis for the clinical variability in GA-1 families. The occurrence of multiple novel mutations in a small geographic area may be explained by their recent onset in isolated communities with a high consanguinity rate. 22 refs., 3 figs., 3 tabs.« less

Adaptive evolutionary walks require neutral intermediates in RNA fitness landscapes.

PubMed

Rendel, Mark D

2011-01-01

In RNA fitness landscapes with interconnected networks of neutral mutations, neutral precursor mutations can play an important role in facilitating the accessibility of epistatic adaptive mutant combinations. I use an exhaustively surveyed fitness landscape model based on short sequence RNA genotypes (and their secondary structure phenotypes) to calculate the minimum rate at which mutants initially appearing as neutral are incorporated into an adaptive evolutionary walk. I show first, that incorporating neutral mutations significantly increases the number of point mutations in a given evolutionary walk when compared to estimates from previous adaptive walk models. Second, that incorporating neutral mutants into such a walk significantly increases the final fitness encountered on that walk - indeed evolutionary walks including neutral steps often reach the global optimum in this model. Third, and perhaps most importantly, evolutionary paths of this kind are often extremely winding in their nature and have the potential to undergo multiple mutations at a given sequence position within a single walk; the potential of these winding paths to mislead phylogenetic reconstruction is briefly considered. Copyright © 2010 Elsevier Inc. All rights reserved.

Scaling laws and universality for the strength of genetic interactions in yeast

NASA Astrophysics Data System (ADS)

Velenich, Andrea; Dai, Mingjie; Gore, Jeff

2012-02-01

Genetic interactions provide a window to the organization of the thousands of biochemical reactions in living cells. If two mutations affect unrelated cellular functions, the fitness effects of their combination can be easily predicted from the two separate fitness effects. However, because of interactions, for some pairs of mutations their combined fitness effect deviates from the naive prediction. We study genetic interactions in yeast cells by analyzing a publicly available database containing experimental growth rates of 5 million double mutants. We show that the characteristic strength of genetic interactions has a simple power law dependence on the fitness effects of the two interacting mutations and that the probability distribution of genetic interactions is a universal function. We further argue that the strength of genetic interactions depends only on the fitness effects of the interacting mutations and not on their biological origin in terms of single point mutations, entire gene knockouts or even more complicated physiological perturbations. Finally, we discuss the implications of the power law scaling of genetic interactions on the ruggedness of fitness landscapes and the consequent evolutionary dynamics.

How Crossover Speeds up Building Block Assembly in Genetic Algorithms.

PubMed

Sudholt, Dirk

2017-01-01

We reinvestigate a fundamental question: How effective is crossover in genetic algorithms in combining building blocks of good solutions? Although this has been discussed controversially for decades, we are still lacking a rigorous and intuitive answer. We provide such answers for royal road functions and OneMax, where every bit is a building block. For the latter, we show that using crossover makes every ([Formula: see text]+[Formula: see text]) genetic algorithm at least twice as fast as the fastest evolutionary algorithm using only standard bit mutation, up to small-order terms and for moderate [Formula: see text] and [Formula: see text]. Crossover is beneficial because it can capitalize on mutations that have both beneficial and disruptive effects on building blocks: crossover is able to repair the disruptive effects of mutation in later generations. Compared to mutation-based evolutionary algorithms, this makes multibit mutations more useful. Introducing crossover changes the optimal mutation rate on OneMax from [Formula: see text] to [Formula: see text]. This holds both for uniform crossover and k-point crossover. Experiments and statistical tests confirm that our findings apply to a broad class of building block functions.

Molecular basis for resistance to ACCase-inhibiting fluazifop in Eleusine indica from Malaysia.

PubMed

Cha, Thye San; Najihah, Mohamed Ghazani; Sahid, Ismail Bin; Chuah, Tse Seng

2014-05-01

Eleusine indica (goosegrass) populations resistant to fluazifop, an acetyl-CoA carboxylase (ACCase: EC6.4.1.2)-inhibiting herbicide, were found in several states in Malaysia. Dose-response assay indicated a resistance factor of 87.5, 62.5 and 150 for biotypes P2, P3 and P4, respectively. DNA sequencing and allele-specific PCR revealed that both biotypes P2 and P3 exhibit a single non-synonymous point mutation from TGG to TGC that leads to a well known Trp-2027-Cys mutation. Interestingly, the highly resistant biotype, P4, did not contain any of the known mutation except the newly discovered target point Asn-2097-Asp, which resulted from a nucleotide change in the codon AAT to GAT. ACCase gene expression was found differentially regulated in the susceptible biotype (P1) and highly resistant biotype P4 from 24 to 72h after treatment (HAT) when being treated with the recommended field rate (198gha(-1)) of fluazifop. However, the small and erratic differences of ACCase gene expression between biotype P1 and P4 does not support the 150-fold resistance in biotype P4. Therefore, the involvement of the target point Asn-2097-Asp and other non-target-site-based resistance mechanisms in the biotype P4 could not be ruled out. Copyright © 2014 Elsevier Inc. All rights reserved.

Large-Scale Discovery of Induced Point Mutations With High-Throughput TILLING

PubMed Central

Till, Bradley J.; Reynolds, Steven H.; Greene, Elizabeth A.; Codomo, Christine A.; Enns, Linda C.; Johnson, Jessica E.; Burtner, Chris; Odden, Anthony R.; Young, Kim; Taylor, Nicholas E.; Henikoff, Jorja G.; Comai, Luca; Henikoff, Steven

2003-01-01

TILLING (Targeting Induced Local Lesions in Genomes) is a general reverse-genetic strategy that provides an allelic series of induced point mutations in genes of interest. High-throughput TILLING allows the rapid and low-cost discovery of induced point mutations in populations of chemically mutagenized individuals. As chemical mutagenesis is widely applicable and mutation detection for TILLING is dependent only on sufficient yield of PCR products, TILLING can be applied to most organisms. We have developed TILLING as a service to the Arabidopsis community known as the Arabidopsis TILLING Project (ATP). Our goal is to rapidly deliver allelic series of ethylmethanesulfonate-induced mutations in target 1-kb loci requested by the international research community. In the first year of public operation, ATP has discovered, sequenced, and delivered >1000 mutations in >100 genes ordered by Arabidopsis researchers. The tools and methodologies described here can be adapted to create similar facilities for other organisms. PMID:12618384

Point mutations which should not be overlooked in Hb H disease.

PubMed

Farashi, Samaneh; Bayat, Nooshin; Vakili, Shadi; Faramarzi Garous, Negin; Ashki, Mehri; Imanian, Hashem; Najmabadi, Hossein; Azarkeivan, Azita

2016-01-01

Hb H disease is an alpha-thalassemia (α-thal) syndrome characterized by chronic hemolytic anemia that occurs when three of total four α-globin genes lost their function due to completely deletions or different kind of mutations. We here described 66 patients who have been diagnosed for Hb H disease during the last five years in our center. The genotypes involving point mutations present more severe phenotype than deletional forms that make them of primary important to health management. Hb H subjects carry different α-globin genotypes including deletional and non-deletional mutations showing heterogenous clinical manifestations. The Hb H patients presenting a wide range of phenotype carried different deletional, non-deletional mutations or compound heterozygosity of them. We emphasize the importance of some point mutations responsible for more severe form of Hb H disease in Iranian population and the necessity for consideration of prenatal diagnosis (PND) in high-risk couples.

Clock-like mutational processes in human somatic cells

DOE PAGES

Alexandrov, Ludmil B.; Jones, Philip H.; Wedge, David C.; ...

2015-11-09

During the course of a lifetime, somatic cells acquire mutations. Different mutational processes may contribute to the mutations accumulated in a cell, with each imprinting a mutational signature on the cell's genome. Some processes generate mutations throughout life at a constant rate in all individuals, and the number of mutations in a cell attributable to these processes will be proportional to the chronological age of the person. Using mutations from 10,250 cancer genomes across 36 cancer types, we investigated clock-like mutational processes that have been operating in normal human cells. Two mutational signatures show clock-like properties. Both exhibit different mutationmore » rates in different tissues. However, their mutation rates are not correlated, indicating that the underlying processes are subject to different biological influences. For one signature, the rate of cell division may influence its mutation rate. This paper provides the first survey of clock-like mutational processes operating in human somatic cells.« less

Identification of a mutation in CNNM4 by whole exome sequencing in an Amish family and functional link between CNNM4 and IQCB1.

PubMed

Li, Sisi; Xi, Quansheng; Zhang, Xiaoyu; Yu, Dong; Li, Lin; Jiang, Zhenyang; Chen, Qiuyun; Wang, Qing K; Traboulsi, Elias I

2018-06-01

We investigated an Amish family in which three siblings presented with an early-onset childhood retinal dystrophy inherited in an autosomal recessive fashion. Genome-wide linkage analysis identified significant linkage to marker D2S2216 on 2q11 with a two-point LOD score of 1.95 and a multi-point LOD score of 3.76. Whole exome sequencing was then performed for the three affected individuals and identified a homozygous nonsense mutation (c.C1813T, p.R605X) in the cyclin and CBS domain divalent metal cation transport mediator 4 (CNNM4) gene located within the 2p14-2q14 Jalili syndrome locus. The initial assessment and collection of the family were performed before the clinical delineation of Jalili syndrome. Another assessment was made after the discovery of the responsible gene and the dental abnormalities characteristic of Jalili syndrome were retrospectively identified. The p.R605X mutation represents the first probable founder mutation of Jalili syndrome identified in the Amish community. The molecular mechanism underlying Jalili syndrome is unknown. Here we show that CNNM4 interacts with IQCB1, which causes Leber congenital amaurosis (LCA) when mutated. A truncated CNNM4 protein starting at R605 significantly increased the rate of apoptosis, and significantly increased the interaction between CNNM4 and IQCB1. Mutation p.R605X may cause Jalili syndrome by a nonsense-mediated decay mechanism, affecting the function of IQCB1 and apoptosis, or both. Our data, for the first time, functionally link Jalili syndrome gene CNNM4 to LCA gene IQCB1, providing important insights into the molecular pathogenic mechanism of retinal dystrophy in Jalili syndrome.

Pituitary resistance to thyroid hormone associated with a base mutation in the hormone-binding domain of the human 3,5,3'-triiodothyronine receptor-beta.

PubMed

Sasaki, S; Nakamura, H; Tagami, T; Miyoshi, Y; Nogimori, T; Mitsuma, T; Imura, H

1993-05-01

Point mutations in the human T3 receptor-beta (TR beta) gene causing single amino acid substitutions have been identified in several different kindreds with generalized resistance to thyroid hormone. Until now, no study has been reported on the TR gene in cases of pituitary resistance (PRTH). In the present study, we analyzed the TR beta gene in a 30-yr-old Japanese female with PRTH. She exhibited clinical features of hyperthyroidism, elevated serum thyroid hormone levels accompanied by inappropriately increased secretion of TSH, mildly elevated basal metabolic rate, and increased urinary excretion of hydroxyproline. No pituitary tumor was detected. DNA fragments of exons 3-8 of the genomic TR beta gene were generated by the polymerase chain reaction and analyzed by a single stranded conformation polymorphism method. Exon 7 of the patient's TR beta gene showed an abnormal band, suggesting the existence of mutation(s). By subcloning and sequencing the DNA, a point mutation was identified in one allele at nucleotide 1297 (C to T), which altered the 333rd amino acid, arginine, to tryptophan. Neither of her apparently normal parents had any mutations of the TR beta gene. In vitro translation products of the mutant TR beta gene showed remarkably decreased T3-binding activity (Ka, 2.1 x 10(8) M-1; normal TR beta Ka, 1.1 x 10(10) M-1). Since the molecular defect detected in a patient with PRTH is similar to that seen in subjects with generalized resistance to thyroid hormone, both types of the syndrome may represent a continuous spectrum of the same etiological defect with variable tissue resistance to thyroid hormone.

Parent-progeny sequencing indicates higher mutation rates in heterozygotes.

PubMed

Yang, Sihai; Wang, Long; Huang, Ju; Zhang, Xiaohui; Yuan, Yang; Chen, Jian-Qun; Hurst, Laurence D; Tian, Dacheng

2015-07-23

Mutation rates vary within genomes, but the causes of this remain unclear. As many prior inferences rely on methods that assume an absence of selection, potentially leading to artefactual results, we call mutation events directly using a parent-offspring sequencing strategy focusing on Arabidopsis and using rice and honey bee for replication. Here we show that mutation rates are higher in heterozygotes and in proximity to crossover events. A correlation between recombination rate and intraspecific diversity is in part owing to a higher mutation rate in domains of high recombination/diversity. Implicating diversity per se as a cause, we find an ∼3.5-fold higher mutation rate in heterozygotes than in homozygotes, with mutations occurring in closer proximity to heterozygous sites than expected by chance. In a genome that is a patchwork of heterozygous and homozygous domains, mutations occur disproportionately more often in the heterozygous domains. If segregating mutations predispose to a higher local mutation rate, clusters of genes dominantly under purifying selection (more commonly homozygous) and under balancing selection (more commonly heterozygous), might have low and high mutation rates, respectively. Our results are consistent with this, there being a ten times higher mutation rate in pathogen resistance genes, expected to be under positive or balancing selection. Consequently, we do not necessarily need to evoke extremely weak selection on the mutation rate to explain why mutational hot and cold spots might correspond to regions under positive/balancing and purifying selection, respectively.

Mutations, mutation rates, and evolution at the hypervariable VNTR loci of Yersinia pestis.

PubMed

Vogler, Amy J; Keys, Christine E; Allender, Christopher; Bailey, Ira; Girard, Jessica; Pearson, Talima; Smith, Kimothy L; Wagner, David M; Keim, Paul

2007-03-01

VNTRs are able to discriminate among closely related isolates of recently emerged clonal pathogens, including Yersinia pestis the etiologic agent of plague, because of their great diversity. Diversity is driven largely by mutation but little is known about VNTR mutation rates, factors affecting mutation rates, or the mutational mechanisms. The molecular epidemiological utility of VNTRs will be greatly enhanced when this foundational knowledge is available. Here, we measure mutation rates for 43 VNTR loci in Y. pestis using an in vitro generated population encompassing approximately 96,000 generations. We estimate the combined 43-locus rate and individual rates for 14 loci. A comparison of Y. pestis and Escherichia coli O157:H7 VNTR mutation rates and products revealed a similar relationship between diversity and mutation rate in these two species. Likewise, the relationship between repeat copy number and mutation rate is nearly identical between these species, suggesting a generalized relationship that may be applicable to other species. The single- versus multiple-repeat mutation ratios and the insertion versus deletion mutation ratios were also similar, providing support for a general model for the mutations associated with VNTRs. Finally, we use two small sets of Y. pestis isolates to show how this general model and our estimated mutation rates can be used to compare alternate phylogenies, and to evaluate the significance of genotype matches, near-matches, and mismatches found in empirical comparisons with a reference database.

Molecular basis for the Kallmann syndrome-linked fibroblast growth factor receptor mutation

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

Thurman, Ryan D.; Kathir, Karuppanan Muthusamy; Rajalingam, Dakshinamurthy

Highlights: Black-Right-Pointing-Pointer The structural basis of the Kallmann syndrome is elucidated. Black-Right-Pointing-Pointer Kallmann syndrome mutation (A168S) induces a subtle conformational change(s). Black-Right-Pointing-Pointer Structural interactions mediated by beta-sheet G are most perturbed. Black-Right-Pointing-Pointer Ligand (FGF)-receptor interaction(s) is completely abolished by Kallmann mutation. Black-Right-Pointing-Pointer Kallmann mutation directly affects the FGF signaling process. -- Abstract: Kallmann syndrome (KS) is a developmental disease that expresses in patients as hypogonadotropic hypogonadism and anosmia. KS is commonly associated with mutations in the extracellular D2 domain of the fibroblast growth factor receptor (FGFR). In this study, for the first time, the molecular basis for the FGFR associatedmore » KS mutation (A168S) is elucidated using a variety of biophysical experiments, including multidimensional NMR spectroscopy. Secondary and tertiary structural analysis using far UV circular dichroism, fluorescence and limited trypsin digestion assays suggest that the KS mutation induces subtle tertiary structure change in the D2 domain of FGFR. Results of isothermal titration calorimetry experiments show the KS mutation causes a 10-fold decrease in heparin binding affinity and also a complete loss in ligand (FGF-1) binding. {sup 1}H-{sup 15}N chemical perturbation data suggest that complete loss in the ligand (FGF) binding affinity is triggered by a subtle conformational change that disrupts crucial structural interactions in both the heparin and the FGF binding sites in the D2 domain of FGFR. The novel findings reported in this study are expected to provide valuable clues toward a complete understanding of the other genetic diseases linked to mutations in the FGFR.« less

Spontaneous Mutation Rate in the Smallest Photosynthetic Eukaryotes

PubMed Central

Krasovec, Marc; Eyre-Walker, Adam; Sanchez-Ferandin, Sophie

2017-01-01

Abstract Mutation is the ultimate source of genetic variation, and knowledge of mutation rates is fundamental for our understanding of all evolutionary processes. High throughput sequencing of mutation accumulation lines has provided genome wide spontaneous mutation rates in a dozen model species, but estimates from nonmodel organisms from much of the diversity of life are very limited. Here, we report mutation rates in four haploid marine bacterial-sized photosynthetic eukaryotic algae; Bathycoccus prasinos, Ostreococcus tauri, Ostreococcus mediterraneus, and Micromonas pusilla. The spontaneous mutation rate between species varies from μ = 4.4 × 10−10 to 9.8 × 10−10 mutations per nucleotide per generation. Within genomes, there is a two-fold increase of the mutation rate in intergenic regions, consistent with an optimization of mismatch and transcription-coupled DNA repair in coding sequences. Additionally, we show that deviation from the equilibrium GC content increases the mutation rate by ∼2% to ∼12% because of a GC bias in coding sequences. More generally, the difference between the observed and equilibrium GC content of genomes explains some of the inter-specific variation in mutation rates. PMID:28379581

Mutation rate evolution in replicator dynamics.

PubMed

Allen, Benjamin; Rosenbloom, Daniel I Scholes

2012-11-01

The mutation rate of an organism is itself evolvable. In stable environments, if faithful replication is costless, theory predicts that mutation rates will evolve to zero. However, positive mutation rates can evolve in novel or fluctuating environments, as analytical and empirical studies have shown. Previous work on this question has focused on environments that fluctuate independently of the evolving population. Here we consider fluctuations that arise from frequency-dependent selection in the evolving population itself. We investigate how the dynamics of competing traits can induce selective pressure on the rates of mutation between these traits. To address this question, we introduce a theoretical framework combining replicator dynamics and adaptive dynamics. We suppose that changes in mutation rates are rare, compared to changes in the traits under direct selection, so that the expected evolutionary trajectories of mutation rates can be obtained from analysis of pairwise competition between strains of different rates. Depending on the nature of frequency-dependent trait dynamics, we demonstrate three possible outcomes of this competition. First, if trait frequencies are at a mutation-selection equilibrium, lower mutation rates can displace higher ones. Second, if trait dynamics converge to a heteroclinic cycle-arising, for example, from "rock-paper-scissors" interactions-mutator strains succeed against non-mutators. Third, in cases where selection alone maintains all traits at positive frequencies, zero and nonzero mutation rates can coexist indefinitely. Our second result suggests that relatively high mutation rates may be observed for traits subject to cyclical frequency-dependent dynamics.

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

Comprehensive sequence-flux mapping of a levoglucosan utilization pathway in E. coli

DOE PAGES

Klesmith, Justin R.; Bacik, John -Paul; Michalczyk, Ryszard; ...

2015-09-14

Synthetic metabolic pathways often suffer from low specific productivity, and new methods that quickly assess pathway functionality for many thousands of variants are urgently needed. Here we present an approach that enables the rapid and parallel determination of sequence effects on flux for complete gene-encoding sequences. We show that this method can be used to determine the effects of over 8000 single point mutants of a pyrolysis oil catabolic pathway implanted in Escherichia coli. Experimental sequence-function data sets predicted whether fitness-enhancing mutations to the enzyme levoglucosan kinase resulted from enhanced catalytic efficiency or enzyme stability. A structure of one designmore » incorporating 38 mutations elucidated the structural basis of high fitness mutations. One design incorporating 15 beneficial mutations supported a 15-fold improvement in growth rate and greater than 24-fold improvement in enzyme activity relative to the starting pathway. Lastly, this technique can be extended to improve a wide variety of designed pathways.« less

Periodontal Pathogens in the Etiology of Pancreatic Cancer.

PubMed

Öğrendik, Mesut

2017-03-01

Pancreatic cancer is the fourth leading cause of cancer-related deaths worldwide. Chronic pancreatitis is frequently observed in patients with pancreatic cancer, and a significant relationship between orodigestive cancer-related deaths and chronic periodontitis has been detected. Porphyromonas gingivalis , Tannerella forsythia , and Treponema denticola , collectively called the Red complex, are the major pathogens responsible for chronic periodontitis and secrete peptidylarginine deiminase. Anti- P. gingivalis antibodies titers are higher in pancreatic cancer patients than in healthy subjects. This review examines the association between oral bacteria and the etiology of pancreatic cancer. High rates of tumor suppressor gene p53 mutations, particularly p53 arginine mutations, were detected in pancreatic cancer patients. K-ras arginine mutations were detected in patients with pancreatic cancer. Oral bacteria peptidylarginine deiminases might lead to the p53 and K-ras point mutations by degrading arginine. Oral bacteria are likely to be responsible for the development of pancreatic cancer. If this hypothesis is true, it may reveal the real cause of pancreatic cancer, which is a fatal disease.

Differential effects on enzyme stability and kinetic parameters of mutants related to human triosephosphate isomerase deficiency.

PubMed

Cabrera, Nallely; Torres-Larios, Alfredo; García-Torres, Itzhel; Enríquez-Flores, Sergio; Perez-Montfort, Ruy

2018-06-01

Human triosephosphate isomerase (TIM) deficiency is a very rare disease, but there are several mutations reported to be causing the illness. In this work, we produced nine recombinant human triosephosphate isomerases which have the mutations reported to produce TIM deficiency. These enzymes were characterized biophysically and biochemically to determine their kinetic and stability parameters, and also to substitute TIM activity in supporting the growth of an Escherichia coli strain lacking the tim gene. Our results allowed us to rate the deleteriousness of the human TIM mutants based on the type and severity of the alterations observed, to classify four "unknown severity mutants" with altered residues in positions 62, 72, 122 and 154 and to explain in structural terms the mutation V231M, the most affected mutant from the kinetic point of view and the only homozygous mutation reported besides E104D. Copyright © 2018 Elsevier B.V. All rights reserved.

Meat and Livestock Association Plenary Lecture 2005. Oocyte signalling molecules and their effects on reproduction in ruminants.

PubMed

McNatty, Kenneth P; Lawrence, Stephen; Groome, Nigel P; Meerasahib, Mohammed F; Hudson, Norma L; Whiting, Lynda; Heath, Derek A; Juengel, Jennifer L

2006-01-01

Sheep (Ovis aries) are a highly diverse species, with more than 900 different breeds that vary significantly in their physiological characteristics, including ovulation rate and fecundity. From examination of inherited patterns of ovulation rate, several breeds have been identified with point mutations in two growth factor genes that are expressed in oocytes. Currently, five different point mutations have been identified in the BMP15 (GDF9b) gene and one in GDF9. Animals heterozygous for the GDF9 and/or the BMP15 mutations have higher ovulation rates than their wild-type counterparts. In contrast, those homozygous for any of the aforementioned BMP15 or GDF9 mutations are sterile owing to arrested follicular development. In bovine and ovine ovaries, GDF9 was expressed exclusively in oocytes throughout follicular growth from the primordial stage of development, whereas in sheep BMP15 was expressed exclusively in oocytes from the primary stage: no data for the ontogeny of BMP15 expression are currently available for cattle. In vitro, ovine growth differentiation factor 9 (oGDF9) has no effect on (3)H-thymidine incorporation by either bovine or ovine granulosa cells, whereas ovine bone morphogenetic protein 15 (oBMP15) has modest (1.2- to 1.6-fold; P < 0.05) stimulatory effects. Ovine GDF9 or oBMP15 alone inhibited progesterone production by bovine granulosa cells, whereas in ovine cells only oGDF9 was inhibitory. The effects of oGDF9 and oBMP15 together were often cooperative and not always the same as those observed for each factor alone. Active immunisation of ewes with BMP15 and/or GDF9 peptides affected ovarian follicular development and ovulation rate. Depending on the GDF9 and/or BMP15 vaccine formulation, ovulation rate was either increased or suppressed. A primary and single booster immunisation of ewes with a BMP15 peptide in a water-based adjuvant has led to 19-40% increases in lambs born per ewe lambing. Collectively, the evidence suggests that oocyte signalling molecules have profound effects on reproduction in mammals, including rodents, humans and ruminants. Moreover, in vivo manipulation of these oocyte signalling molecules provides new opportunities for the management of the fertility of ruminants.

Experimental Determination and Prediction of the Fitness Effects of Random Point Mutations in the Biosynthetic Enzyme HisA

PubMed Central

Lundin, Erik; Tang, Po-Cheng; Guy, Lionel; Näsvall, Joakim; Andersson, Dan I

2018-01-01

Abstract The distribution of fitness effects of mutations is a factor of fundamental importance in evolutionary biology. We determined the distribution of fitness effects of 510 mutants that each carried between 1 and 10 mutations (synonymous and nonsynonymous) in the hisA gene, encoding an essential enzyme in the l-histidine biosynthesis pathway of Salmonella enterica. For the full set of mutants, the distribution was bimodal with many apparently neutral mutations and many lethal mutations. For a subset of 81 single, nonsynonymous mutants most mutations appeared neutral at high expression levels, whereas at low expression levels only a few mutations were neutral. Furthermore, we examined how the magnitude of the observed fitness effects was correlated to several measures of biophysical properties and phylogenetic conservation.We conclude that for HisA: (i) The effect of mutations can be masked by high expression levels, such that mutations that are deleterious to the function of the protein can still be neutral with regard to organism fitness if the protein is expressed at a sufficiently high level; (ii) the shape of the fitness distribution is dependent on the extent to which the protein is rate-limiting for growth; (iii) negative epistatic interactions, on an average, amplified the combined effect of nonsynonymous mutations; and (iv) no single sequence-based predictor could confidently predict the fitness effects of mutations in HisA, but a combination of multiple predictors could predict the effect with a SD of 0.04 resulting in 80% of the mutations predicted within 12% of their observed selection coefficients. PMID:29294020

Evidence for a high mutation rate at rapidly evolving yeast centromeres.

PubMed

Bensasson, Douda

2011-07-18

Although their role in cell division is essential, centromeres evolve rapidly in animals, plants and yeasts. Unlike the complex centromeres of plants and aminals, the point centromeres of Saccharomcyes yeasts can be readily sequenced to distinguish amongst the possible explanations for fast centromere evolution. Using DNA sequences of all 16 centromeres from 34 strains of Saccharomyces cerevisiae and population genomic data from Saccharomyces paradoxus, I show that centromeres in both species evolve 3 times more rapidly even than selectively unconstrained DNA. Exceptionally high levels of polymorphism seen in multiple yeast populations suggest that rapid centromere evolution does not result from the repeated selective sweeps expected under meiotic drive. I further show that there is little evidence for crossing-over or gene conversion within centromeres, although there is clear evidence for recombination in their immediate vicinity. Finally I show that the mutation spectrum at centromeres is consistent with the pattern of spontaneous mutation elsewhere in the genome. These results indicate that rapid centromere evolution is a common phenomenon in yeast species. Furthermore, these results suggest that rapid centromere evolution does not result from the mutagenic effect of gene conversion, but from a generalised increase in the mutation rate, perhaps arising from the unusual chromatin structure at centromeres in yeast and other eukaryotes.

Evidence for a high mutation rate at rapidly evolving yeast centromeres

PubMed Central

2011-01-01

Background Although their role in cell division is essential, centromeres evolve rapidly in animals, plants and yeasts. Unlike the complex centromeres of plants and aminals, the point centromeres of Saccharomcyes yeasts can be readily sequenced to distinguish amongst the possible explanations for fast centromere evolution. Results Using DNA sequences of all 16 centromeres from 34 strains of Saccharomyces cerevisiae and population genomic data from Saccharomyces paradoxus, I show that centromeres in both species evolve 3 times more rapidly even than selectively unconstrained DNA. Exceptionally high levels of polymorphism seen in multiple yeast populations suggest that rapid centromere evolution does not result from the repeated selective sweeps expected under meiotic drive. I further show that there is little evidence for crossing-over or gene conversion within centromeres, although there is clear evidence for recombination in their immediate vicinity. Finally I show that the mutation spectrum at centromeres is consistent with the pattern of spontaneous mutation elsewhere in the genome. Conclusions These results indicate that rapid centromere evolution is a common phenomenon in yeast species. Furthermore, these results suggest that rapid centromere evolution does not result from the mutagenic effect of gene conversion, but from a generalised increase in the mutation rate, perhaps arising from the unusual chromatin structure at centromeres in yeast and other eukaryotes. PMID:21767380

Absence of ras-gene hot-spot mutations in canine fibrosarcomas and melanomas.

PubMed

Murua Escobar, Hugo; Günther, Kathrin; Richter, Andreas; Soller, Jan T; Winkler, Susanne; Nolte, Ingo; Bullerdiek, Jörn

2004-01-01

Point mutations within ras proto-oncogenes, particularly within the mutational hot-spot codons 12, 13 and 61, are frequently detected in human malignancies and in different types of experimentally-induced tumours in animals. So far little is known about ras mutations in naturally occurring canine fibrosarcomas or K-ras mutations in canine melanomas. To elucidate whether ras mutations exist in these naturally occurring tumours in dogs, in the present study we screened 13 canine fibrosarcomas, 2 feline fibrosarcomas and 11 canine melanomas for point mutations, particularly within the mutational hot-spots, making this the first study to investigate a large number of canine fibrosarcomas. None of the samples showed a K- or N-ras hot spot mutation. Thus, our data strongly suggest that ras mutations at the hot-spot loci are very rare and do not play a major role in the pathogenesis of the spontaneously occurring canine tumours investigated.

Impact of point mutation P29S in RAC1 on tumorigenesis.

PubMed

Rajendran, Vidya; Gopalakrishnan, Chandrasekhar; Purohit, Rituraj

2016-11-01

A point mutation (P29S) in the RAS-related C3 botulinum toxin substrate 1 (RAC1) was considered to be a trigger for melanoma, a form of skin cancer with highest mortality rate. In this study, we have investigated the pathogenic role of P29S based on the conformational behavior of RAC1 protein toward guanosine triphosphate (GTP). Molecular interaction, molecular dynamics trajectory analysis (RMSD, RMSF, Rg, SASA, DSSP, and PCA), and shape analysis of binding pocket were performed to analyze the interaction energy and the dynamic behavior of native and mutant RAC1 at the atomic level. Due to this mutation, the RAC1 switch I region acquired more flexibility and, to compensate it, the switch II region becomes rigid in their conformational space, as a result of which the interaction energy of the protein for GTP increased. The overall results strongly implied that the changes in atomic conformation of the switch I and II regions in mutant RAC1 protein were a significant reason for its malignant transformation and tumorigenesis. We raised the opportunity for researchers to design possible therapeutic molecule by considering our findings.

Anaerobically Grown Escherichia coli Has an Enhanced Mutation Rate and Distinct Mutational Spectra

PubMed Central

Shewaramani, Sonal; Finn, Thomas J.; Kassen, Rees; Rainey, Paul B.

2017-01-01

Oxidative stress is a major cause of mutation but little is known about how growth in the absence of oxygen impacts the rate and spectrum of mutations. We employed long-term mutation accumulation experiments to directly measure the rates and spectra of spontaneous mutation events in Escherichia coli populations propagated under aerobic and anaerobic conditions. To detect mutations, whole genome sequencing was coupled with methods of analysis sufficient to identify a broad range of mutational classes, including structural variants (SVs) generated by movement of repetitive elements. The anaerobically grown populations displayed a mutation rate nearly twice that of the aerobic populations, showed distinct asymmetric mutational strand biases, and greater insertion element activity. Consistent with mutation rate and spectra observations, genes for transposition and recombination repair associated with SVs were up-regulated during anaerobic growth. Together, these results define differences in mutational spectra affecting the evolution of facultative anaerobes. PMID:28103245

Effect of repeat copy number on variable-number tandem repeat mutations in Escherichia coli O157:H7.

PubMed

Vogler, Amy J; Keys, Christine; Nemoto, Yoshimi; Colman, Rebecca E; Jay, Zack; Keim, Paul

2006-06-01

Variable-number tandem repeat (VNTR) loci have shown a remarkable ability to discriminate among isolates of the recently emerged clonal pathogen Escherichia coli O157:H7, making them a very useful molecular epidemiological tool. However, little is known about the rates at which these sequences mutate, the factors that affect mutation rates, or the mechanisms by which mutations occur at these loci. Here, we measure mutation rates for 28 VNTR loci and investigate the effects of repeat copy number and mismatch repair on mutation rate using in vitro-generated populations for 10 E. coli O157:H7 strains. We find single-locus rates as high as 7.0 x 10(-4) mutations/generation and a combined 28-locus rate of 6.4 x 10(-4) mutations/generation. We observed single- and multirepeat mutations that were consistent with a slipped-strand mispairing mutation model, as well as a smaller number of large repeat copy number mutations that were consistent with recombination-mediated events. Repeat copy number within an array was strongly correlated with mutation rate both at the most mutable locus, O157-10 (r2= 0.565, P = 0.0196), and across all mutating loci. The combined locus model was significant whether locus O157-10 was included (r2= 0.833, P < 0.0001) or excluded (r2= 0.452, P < 0.0001) from the analysis. Deficient mismatch repair did not affect mutation rate at any of the 28 VNTRs with repeat unit sizes of >5 bp, although a poly(G) homomeric tract was destabilized in the mutS strain. Finally, we describe a general model for VNTR mutations that encompasses insertions and deletions, single- and multiple-repeat mutations, and their relative frequencies based upon our empirical mutation rate data.

Determining Y-STR mutation rates in deep-routing genealogies: Identification of haplogroup differences.

PubMed

Claerhout, Sofie; Vandenbosch, Michiel; Nivelle, Kelly; Gruyters, Leen; Peeters, Anke; Larmuseau, Maarten H D; Decorte, Ronny

2018-05-01

Knowledge of Y-chromosomal short tandem repeat (Y-STR) mutation rates is essential to determine the most recent common ancestor (MRCA) in familial searching or genealogy research. Up to now, locus-specific mutation rates have been extensively examined especially for commercially available forensic Y-STRs, while haplogroup specific mutation rates have not yet been investigated in detail. Through 450 patrilineally related namesakes distributed over 212 deep-rooting genealogies, the individual mutation rates of 42 Y-STR loci were determined, including 27 forensic Y-STR loci from the Yfiler ® Plus kit and 15 additional Y-STR loci (DYS388, DYS426, DYS442, DYS447, DYS454, DYS455, DYS459a/b, DYS549, DYS607, DYS643, DYS724a/b and YCAIIa/b). At least 726 mutations were observed over 148,596 meiosis and individual Y-STR mutation rates varied from 2.83 × 10 -4 to 1.86 × 10 -2 . The mutation rate was significantly correlated with the average allele size, the complexity of the repeat motif sequence and the age of the father. Significant differences in average Y-STR mutations rates were observed when haplogroup 'I & J' (4.03 × 10 -3 mutations/generation) was compared to 'R1b' (5.35 × 10 -3 mutations/generation) and to the overall mutation rate (5.03 × 10 -3 mutations/generation). A difference in allele size distribution was identified as the only cause for these haplogroup specific mutation rates. The haplogroup specific mutation rates were also present within the commercially available Y-STR kits (Yfiler ® , PowerPlex ® Y23 System and Yfiler ® Plus). This observation has consequences for applications where an average Y-STR mutation rate is used, e.g. tMRCA estimations in familial searching and genealogy research. Copyright © 2018 Elsevier B.V. All rights reserved.

Extensive de novo mutation rate variation between individuals and across the genome of Chlamydomonas reinhardtii

PubMed Central

Ness, Rob W.; Morgan, Andrew D.; Vasanthakrishnan, Radhakrishnan B.; Colegrave, Nick; Keightley, Peter D.

2015-01-01

Describing the process of spontaneous mutation is fundamental for understanding the genetic basis of disease, the threat posed by declining population size in conservation biology, and much of evolutionary biology. Directly studying spontaneous mutation has been difficult, however, because new mutations are rare. Mutation accumulation (MA) experiments overcome this by allowing mutations to build up over many generations in the near absence of natural selection. Here, we sequenced the genomes of 85 MA lines derived from six genetically diverse strains of the green alga Chlamydomonas reinhardtii. We identified 6843 new mutations, more than any other study of spontaneous mutation. We observed sevenfold variation in the mutation rate among strains and that mutator genotypes arose, increasing the mutation rate approximately eightfold in some replicates. We also found evidence for fine-scale heterogeneity in the mutation rate, with certain sequence motifs mutating at much higher rates, and clusters of multiple mutations occurring at closely linked sites. There was little evidence, however, for mutation rate heterogeneity between chromosomes or over large genomic regions of 200 kbp. We generated a predictive model of the mutability of sites based on their genomic properties, including local GC content, gene expression level, and local sequence context. Our model accurately predicted the average mutation rate and natural levels of genetic diversity of sites across the genome. Notably, trinucleotides vary 17-fold in rate between the most and least mutable sites. Our results uncover a rich heterogeneity in the process of spontaneous mutation both among individuals and across the genome. PMID:26260971

Role of the putative structural protein Sed1p in mitochondrial genome maintenance.

PubMed

Phadnis, Naina; Ayres Sia, Elaine

2004-09-24

The nuclear gene MIP1 encodes the mitochondrial DNA polymerase responsible for replicating the mitochondrial genome in Saccharomyces cerevisiae. A number of other factors involved in replicating and segregating the mitochondrial genome are yet to be identified. Here, we report that a bacterial two-hybrid screen using the mitochondrial polymerase, Mip1p, as bait identified the yeast protein Sed1p. Sed1p is a cell surface protein highly expressed in the stationary phase. We find that several modified forms of Sed1p are expressed and the largest of these forms interacts with the mitochondrial polymerase in vitro. Deletion of SED1 causes a 3.5-fold increase in the rate of mitochondrial DNA point mutations as well as a 4.3-fold increase in the rate of loss of respiration. In contrast, we see no change in the rate of nuclear point mutations indicating the specific role of Sed1p function in mitochondrial genome stability. Indirect immunofluorescence analysis of Sed1p localization shows that Sed1p is targeted to the mitochondria. Moreover, Sed1p is detected in purified mitochondrial fractions and the localization to the mitochondria of the largest modified form is insensitive to the action of proteinase K. Deletion of the sed1 gene results in a reduction in the quantity of Mip1p and also affects the levels of a mitochondrially-expressed protein, Cox3p. Our results point towards a role for Sed1p in mitochondrial genome maintenance.

Various mutations compensate for a deleterious lacZα insert in the replication enhancer of M13 bacteriophage

PubMed Central

Zygiel, Emily M.; Noren, Karen A.; Adamkiewicz, Marta A.; Aprile, Richard J.; Bowditch, Heather K.; Carroll, Christine L.; Cerezo, Maria Abigail S.; Dagher, Adelle M.; Hebert, Courtney R.; Hebert, Lauren E.; Mahame, Gloria M.; Milne, Stephanie C.; Silvestri, Kelly M.; Sutherland, Sara E.; Sylvia, Alexandria M.; Taveira, Caitlyn N.; VanValkenburgh, David J.; Noren, Christopher J.

2017-01-01

M13 and other members of the Ff class of filamentous bacteriophages have been extensively employed in myriad applications. The Ph.D. series of phage-displayed peptide libraries were constructed from the M13-based vector M13KE. As a direct descendent of M13mp19, M13KE contains the lacZα insert in the intergenic region between genes IV and II, where it interrupts the replication enhancer of the (+) strand origin. Phage carrying this 816-nucleotide insert are viable, but propagate in E. coli at a reduced rate compared to wild-type M13 phage, presumably due to a replication defect caused by the insert. We have previously reported thirteen compensatory mutations in the 5’-untranslated region of gene II, which encodes the replication initiator protein gIIp. Here we report several additional mutations in M13KE that restore a wild-type propagation rate. Several clones from constrained-loop variable peptide libraries were found to have ejected the majority of lacZα gene in order to reconstruct the replication enhancer, albeit with a small scar. In addition, new point mutations in the gene II 5’-untranslated region or the gene IV coding sequence have been spontaneously observed or synthetically engineered. Through phage propagation assays, we demonstrate that all these genetic modifications compensate for the replication defect in M13KE and restore the wild-type propagation rate. We discuss the mechanisms by which the insertion and ejection of the lacZα gene, as well as the mutations in the regulatory region of gene II, influence the efficiency of replication initiation at the (+) strand origin. We also examine the presence and relevance of fast-propagating mutants in phage-displayed peptide libraries. PMID:28445507

Various mutations compensate for a deleterious lacZα insert in the replication enhancer of M13 bacteriophage.

PubMed

Zygiel, Emily M; Noren, Karen A; Adamkiewicz, Marta A; Aprile, Richard J; Bowditch, Heather K; Carroll, Christine L; Cerezo, Maria Abigail S; Dagher, Adelle M; Hebert, Courtney R; Hebert, Lauren E; Mahame, Gloria M; Milne, Stephanie C; Silvestri, Kelly M; Sutherland, Sara E; Sylvia, Alexandria M; Taveira, Caitlyn N; VanValkenburgh, David J; Noren, Christopher J; Hall, Marilena Fitzsimons

2017-01-01

M13 and other members of the Ff class of filamentous bacteriophages have been extensively employed in myriad applications. The Ph.D. series of phage-displayed peptide libraries were constructed from the M13-based vector M13KE. As a direct descendent of M13mp19, M13KE contains the lacZα insert in the intergenic region between genes IV and II, where it interrupts the replication enhancer of the (+) strand origin. Phage carrying this 816-nucleotide insert are viable, but propagate in E. coli at a reduced rate compared to wild-type M13 phage, presumably due to a replication defect caused by the insert. We have previously reported thirteen compensatory mutations in the 5'-untranslated region of gene II, which encodes the replication initiator protein gIIp. Here we report several additional mutations in M13KE that restore a wild-type propagation rate. Several clones from constrained-loop variable peptide libraries were found to have ejected the majority of lacZα gene in order to reconstruct the replication enhancer, albeit with a small scar. In addition, new point mutations in the gene II 5'-untranslated region or the gene IV coding sequence have been spontaneously observed or synthetically engineered. Through phage propagation assays, we demonstrate that all these genetic modifications compensate for the replication defect in M13KE and restore the wild-type propagation rate. We discuss the mechanisms by which the insertion and ejection of the lacZα gene, as well as the mutations in the regulatory region of gene II, influence the efficiency of replication initiation at the (+) strand origin. We also examine the presence and relevance of fast-propagating mutants in phage-displayed peptide libraries.

Isolated growth hormone deficiency in two siblings because of paternal mosaicism for a mutation in the GH1 gene.

PubMed

Tsubahara, Mayuko; Hayashi, Yoshitaka; Niijima, Shin-ichi; Yamamoto, Michiyo; Kamijo, Takashi; Murata, Yoshiharu; Haruna, Hidenori; Okumura, Akihisa; Shimizu, Toshiaki

2012-03-01

  Mutations in the GH1 gene have been identified in patients with isolated growth hormone deficiency (IGHD). Mutations causing aberrant splicing of exon 3 of GH1 that have been identified in IGHD are inherited in an autosomal dominant manner, whereas other mutations in GH1 that have been identified in IGHD are inherited in an autosomal recessive manner.   Two siblings born from nonconsanguineous healthy parents exhibited IGHD. To elucidate the cause, GH1 in all family members was analysed.   Two novel mutations in GH1, a point mutation in intron 3 and a 16-bp deletion in exon 3, were identified by sequence analyses. The intronic mutation was present in both siblings and was predicted to cause aberrant splicing. The deletion was present in one of the siblings as well as the mother with normal stature and was predicted to cause rapid degradation of mRNA through nonsense-mediated mRNA decay. The point mutation was not identified in the parents' peripheral blood DNA; however, it was detected in the DNA extracted from the father's sperms. As a trace of the mutant allele was detected in the peripheral blood of the father using PCR-RFLP, the mutation is likely to have occurred de novo at an early developmental stage before differentiation of somatic cells and germline cells.   This is the first report of mosaicism for a mutation in GH1 in a family with IGHD. It is clear that the intronic mutation plays a dominant role in the pathogenesis of IGHD in this family, as one of the siblings who had only the point mutation was affected. On the other hand, the other sibling was a compound heterozygote for the point mutation and the 16-bp deletion and it may be arguable whether IGHD in this patient should be regarded as autosomal dominant or recessive. © 2012 Blackwell Publishing Ltd.

SHOX mutations in idiopathic short stature and Leri-Weill dyschondrosteosis: frequency and phenotypic variability.

PubMed

Jorge, Alexander A L; Souza, Silvia C; Nishi, Miriam Y; Billerbeck, Ana E; Libório, Débora C C; Kim, Chong A; Arnhold, Ivo J P; Mendonca, Berenice B

2007-01-01

The frequency of SHOX mutations in children with idiopathic short stature (ISS) has been found to be variable. We analysed the SHOX gene in children with ISS and Leri-Weill dyschondrosteosis (LWD) and evaluated the phenotypic variability in patients harbouring SHOX mutations. Sixty-three ISS, nine LWD children and 21 affected relatives. SHOX gene deletion was evaluated by fluorescence in situ hybridization (FISH), Southern blotting and segregation study of polymorphic marker. Point mutations were assessed by direct DNA sequencing. None of the ISS patients presented SHOX deletions, but two (3.2%) presented heterozygous point mutations, including the novel R147H mutation. However, when ISS patients were selected by sitting height : height ratio (SH/H) for age > 2 SD, mutation frequency detection increased to 22%. Eight (89%) LWD patients had SHOX deletions, but none had point mutations. Analysis of the other relatives in the families carrying SHOX mutations identified 14 children and 17 adult patients. A broad phenotypic variability was observed in all families regarding short stature severity and Madelung deformities. However, the presence of disproportional height, assessed by SH/H, was observed in all children and 82% of adult patients, being the most common feature in our patients with SHOX mutations. Patients with SHOX mutations present a broad phenotypic variability. SHOX mutations are very frequent in LWD (89%), in opposition to ISS (3.2%) in our cohort. The use of SH/H SDS as a selection criterion increases the frequency of SHOX mutation detection to 22% and should be used for selecting ISS children to undergo SHOX mutation molecular studies.

Determination of the DNA-binding kinetics of three related but heteroimmune bacteriophage repressors using EMSA and SPR analysis

PubMed Central

Henriksson-Peltola, Petri; Sehlén, Wilhelmina; Haggård-Ljungquist, Elisabeth

2007-01-01

Bacteriophages P2, P2 Hy dis and WΦ are very similar but heteroimmune Escherichia coli phages. The structural genes show over 96% identity, but the repressors show between 43 and 63% identities. Furthermore, the operators, which contain two directly repeated sequences, vary in sequence, length, location relative to the promoter and spacing between the direct repeats. We have compared the in vivo effects of the wild type and mutated operators on gene expression with the complexes formed between the repressors and their wild type or mutated operators using electrophoretic mobility shift assay (EMSA), and real-time kinetics of the protein–DNA interactions using surface plasmon resonance (SPR) analysis. Using EMSA, the repressors formed different protein–DNA complexes, and only WΦ was significantly affected by point mutations. However, SPR analysis showed a reduced association rate constant and an increased dissociation rate constant for P2 and WΦ operator mutants. The association rate constants of P2 Hy dis was too fast to be determined. The P2 Hy dis dissociation response curves were shown to be triphasic, while both P2 and WΦ C were biphasic. Thus, the kinetics of complex formation and the nature of the complexes formed differ extensively between these very closely related phages. PMID:17412705

Regulation of Catch Bonds by Rate of Force Application*

PubMed Central

Sarangapani, Krishna K.; Qian, Jin; Chen, Wei; Zarnitsyna, Veronika I.; Mehta, Padmaja; Yago, Tadayuki; McEver, Rodger P.; Zhu, Cheng

2011-01-01

The current paradigm for receptor-ligand dissociation kinetics assumes off-rates as functions of instantaneous force without impact from its prior history. This a priori assumption is the foundation for predicting dissociation from a given initial state using kinetic equations. Here we have invalidated this assumption by demonstrating the impact of force history with single-bond kinetic experiments involving selectins and their ligands that mediate leukocyte tethering and rolling on vascular surfaces during inflammation. Dissociation of bonds between L-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) loaded at a constant ramp rate to a constant hold force behaved as catch-slip bonds at low ramp rates that transformed to slip-only bonds at high ramp rates. Strikingly, bonds between L-selectin and 6-sulfo-sialyl Lewis X were impervious to ramp rate changes. This ligand-specific force history effect resembled the effect of a point mutation at the L-selectin surface (L-selectinA108H) predicted to contact the former but not the latter ligand, suggesting that the high ramp rate induced similar structural changes as the mutation. Although the A108H substitution in L-selectin eliminated the ramp rate responsiveness of its dissociation from PSGL-1, the inverse mutation H108A in P-selectin acquired the ramp rate responsiveness. Our data are well explained by the sliding-rebinding model for catch-slip bonds extended to incorporate the additional force history dependence, with Ala-108 playing a pivotal role in this structural mechanism. These results call for a paradigm shift in modeling the mechanical regulation of receptor-ligand bond dissociation, which includes conformational coupling between binding pocket and remote regions of the interacting molecules. PMID:21775439

Phase II and Biomarker Study of the Dual MET/VEGFR2 Inhibitor Foretinib in Patients With Papillary Renal Cell Carcinoma

PubMed Central

Choueiri, Toni K.; Vaishampayan, Ulka; Rosenberg, Jonathan E.; Logan, Theodore F.; Harzstark, Andrea L.; Bukowski, Ronald M.; Rini, Brian I.; Srinivas, Sandy; Stein, Mark N.; Adams, Laurel M.; Ottesen, Lone H.; Laubscher, Kevin H.; Sherman, Laurie; McDermott, David F.; Haas, Naomi B.; Flaherty, Keith T.; Ross, Robert; Eisenberg, Peter; Meltzer, Paul S.; Merino, Maria J.; Bottaro, Donald P.; Linehan, W. Marston; Srinivasan, Ramaprasad

2013-01-01

Purpose Foretinib is an oral multikinase inhibitor targeting MET, VEGF, RON, AXL, and TIE-2 receptors. Activating mutations or amplifications in MET have been described in patients with papillary renal cell carcinoma (PRCC). We aimed to evaluate the efficacy and safety of foretinib in patients with PRCC. Patients and Methods Patients were enrolled onto the study in two cohorts with different dosing schedules of foretinib: cohort A, 240 mg once per day on days 1 through 5 every 14 days (intermittent arm); cohort B, 80 mg daily (daily dosing arm). Patients were stratified on the basis of MET pathway activation (germline or somatic MET mutation, MET [7q31] amplification, or gain of chromosome 7). The primary end point was overall response rate (ORR). Results Overall, 74 patients were enrolled, with 37 in each dosing cohort. ORR by Response Evaluation Criteria in Solid Tumors (RECIST) 1.0 was 13.5%, median progression-free survival was 9.3 months, and median overall survival was not reached. The presence of a germline MET mutation was highly predictive of a response (five of 10 v five of 57 patients with and without germline MET mutations, respectively). The most frequent adverse events of any grade associated with foretinib were fatigue, hypertension, gastrointestinal toxicities, and nonfatal pulmonary emboli. Conclusion Foretinib demonstrated activity in patients with advanced PRCC with a manageable toxicity profile and a high response rate in patients with germline MET mutations. PMID:23213094

Evolution of Local Mutation Rate and Its Determinants.

PubMed

Terekhanova, Nadezhda V; Seplyarskiy, Vladimir B; Soldatov, Ruslan A; Bazykin, Georgii A

2017-05-01

Mutation rate varies along the human genome, and part of this variation is explainable by measurable local properties of the DNA molecule. Moreover, mutation rates differ between orthologous genomic regions of different species, but the drivers of this change are unclear. Here, we use data on human divergence from chimpanzee, human rare polymorphism, and human de novo mutations to predict the substitution rate at orthologous regions of non-human mammals. We show that the local mutation rates are very similar between human and apes, implying that their variation has a strong underlying cryptic component not explainable by the known genomic features. Mutation rates become progressively less similar in more distant species, and these changes are partially explainable by changes in the local genomic features of orthologous regions, most importantly, in the recombination rate. However, they are much more rapid, implying that the cryptic component underlying the mutation rate is more ephemeral than the known genomic features. These findings shed light on the determinants of mutation rate evolution. local mutation rate, molecular evolution, recombination rate. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

A Constant Rate of Spontaneous Mutation in DNA-Based Microbes

NASA Astrophysics Data System (ADS)

Drake, John W.

1991-08-01

In terms of evolution and fitness, the most significant spontaneous mutation rate is likely to be that for the entire genome (or its nonfrivolous fraction). Information is now available to calculate this rate for several DNA-based haploid microbes, including bacteriophages with single- or double-stranded DNA, a bacterium, a yeast, and a filamentous fungus. Their genome sizes vary by ≈6500-fold. Their average mutation rates per base pair vary by ≈16,000-fold, whereas their mutation rates per genome vary by only ≈2.5-fold, apparently randomly, around a mean value of 0.0033 per DNA replication. The average mutation rate per base pair is inversely proportional to genome size. Therefore, a nearly invariant microbial mutation rate appears to have evolved. Because this rate is uniform in such diverse organisms, it is likely to be determined by deep general forces, perhaps by a balance between the usually deleterious effects of mutation and the physiological costs of further reducing mutation rates.

Extensive Variation in the Mutation Rate Between and Within Human Genes Associated with Mendelian Disease.

PubMed

Smith, Thomas; Ho, Gladys; Christodoulou, John; Price, Elizabeth Ann; Onadim, Zerrin; Gauthier-Villars, Marion; Dehainault, Catherine; Houdayer, Claude; Parfait, Beatrice; van Minkelen, Rick; Lohman, Dietmar; Eyre-Walker, Adam

2016-05-01

We have investigated whether the mutation rate varies between genes and sites using de novo mutations (DNMs) from three genes associated with Mendelian diseases (RB1, NF1, and MECP2). We show that the relative frequency of mutations at CpG dinucleotides relative to non-CpG sites varies between genes and relative to the genomic average. In particular we show that the rate of transition mutation at CpG sites relative to the rate of non-CpG transversion is substantially higher in our disease genes than amongst DNMs in general; the rate of CpG transition can be several hundred-fold greater than the rate of non-CpG transversion. We also show that the mutation rate varies significantly between sites of a particular mutational type, such as non-CpG transversion, within a gene. We estimate that for all categories of sites, except CpG transitions, there is at least a 30-fold difference in the mutation rate between the 10% of sites with the highest and lowest mutation rates. However, our best estimate is that the mutation rate varies by several hundred-fold variation. We suggest that the presence of hypermutable sites may be one reason certain genes are associated with disease. © 2016 WILEY PERIODICALS, INC.

[Evaluation of performance of five bioinformatics software for the prediction of missense mutations].

PubMed

Chen, Qianting; Dai, Congling; Zhang, Qianjun; Du, Juan; Li, Wen

2016-10-01

To study the prediction performance evaluation with five kinds of bioinformatics software (SIFT, PolyPhen2, MutationTaster, Provean, MutationAssessor). From own database for genetic mutations collected over the past five years, Chinese literature database, Human Gene Mutation Database, and dbSNP, 121 missense mutations confirmed by functional studies, and 121 missense mutations suspected to be pathogenic by pedigree analysis were used as positive gold standard, while 242 missense mutations with minor allele frequency (MAF)>5% in dominant hereditary diseases were used as negative gold standard. The selected mutations were predicted with the five software. Based on the results, the performance of the five software was evaluated for their sensitivity, specificity, positive predict value, false positive rate, negative predict value, false negative rate, false discovery rate, accuracy, and receiver operating characteristic curve (ROC). In terms of sensitivity, negative predictive value and false negative rate, the rank was MutationTaster, PolyPhen2, Provean, SIFT, and MutationAssessor. For specificity and false positive rate, the rank was MutationTaster, Provean, MutationAssessor, SIFT, and PolyPhen2. For positive predict value and false discovery rate, the rank was MutationTaster, Provean, MutationAssessor, PolyPhen2, and SIFT. For area under the ROC curve (AUC) and accuracy, the rank was MutationTaster, Provean, PolyPhen2, MutationAssessor, and SIFT. The prediction performance of software may be different when using different parameters. Among the five software, MutationTaster has the best prediction performance.

Spontaneous mutation rate is a plastic trait associated with population density across domains of life.

PubMed

Krašovec, Rok; Richards, Huw; Gifford, Danna R; Hatcher, Charlie; Faulkner, Katy J; Belavkin, Roman V; Channon, Alastair; Aston, Elizabeth; McBain, Andrew J; Knight, Christopher G

2017-08-01

Rates of random, spontaneous mutation can vary plastically, dependent upon the environment. Such plasticity affects evolutionary trajectories and may be adaptive. We recently identified an inverse plastic association between mutation rate and population density at 1 locus in 1 species of bacterium. It is unknown how widespread this association is, whether it varies among organisms, and what molecular mechanisms of mutagenesis or repair are required for this mutation-rate plasticity. Here, we address all 3 questions. We identify a strong negative association between mutation rate and population density across 70 years of published literature, comprising hundreds of mutation rates estimated using phenotypic markers of mutation (fluctuation tests) from all domains of life and viruses. We test this relationship experimentally, determining that there is indeed density-associated mutation-rate plasticity (DAMP) at multiple loci in both eukaryotes and bacteria, with up to 23-fold lower mutation rates at higher population densities. We find that the degree of plasticity varies, even among closely related organisms. Nonetheless, in each domain tested, DAMP requires proteins scavenging the mutagenic oxidised nucleotide 8-oxo-dGTP. This implies that phenotypic markers give a more precise view of mutation rate than previously believed: having accounted for other known factors affecting mutation rate, controlling for population density can reduce variation in mutation-rate estimates by 93%. Widespread DAMP, which we manipulate genetically in disparate organisms, also provides a novel trait to use in the fight against the evolution of antimicrobial resistance. Such a prevalent environmental association and conserved mechanism suggest that mutation has varied plastically with population density since the early origins of life.

Spontaneous mutation rate is a plastic trait associated with population density across domains of life

PubMed Central

Gifford, Danna R.; Hatcher, Charlie; Faulkner, Katy J.; Belavkin, Roman V.; Channon, Alastair; Aston, Elizabeth; McBain, Andrew J.

2017-01-01

Rates of random, spontaneous mutation can vary plastically, dependent upon the environment. Such plasticity affects evolutionary trajectories and may be adaptive. We recently identified an inverse plastic association between mutation rate and population density at 1 locus in 1 species of bacterium. It is unknown how widespread this association is, whether it varies among organisms, and what molecular mechanisms of mutagenesis or repair are required for this mutation-rate plasticity. Here, we address all 3 questions. We identify a strong negative association between mutation rate and population density across 70 years of published literature, comprising hundreds of mutation rates estimated using phenotypic markers of mutation (fluctuation tests) from all domains of life and viruses. We test this relationship experimentally, determining that there is indeed density-associated mutation-rate plasticity (DAMP) at multiple loci in both eukaryotes and bacteria, with up to 23-fold lower mutation rates at higher population densities. We find that the degree of plasticity varies, even among closely related organisms. Nonetheless, in each domain tested, DAMP requires proteins scavenging the mutagenic oxidised nucleotide 8-oxo-dGTP. This implies that phenotypic markers give a more precise view of mutation rate than previously believed: having accounted for other known factors affecting mutation rate, controlling for population density can reduce variation in mutation-rate estimates by 93%. Widespread DAMP, which we manipulate genetically in disparate organisms, also provides a novel trait to use in the fight against the evolution of antimicrobial resistance. Such a prevalent environmental association and conserved mechanism suggest that mutation has varied plastically with population density since the early origins of life. PMID:28837573

Three novel PHEX gene mutations in four Chinese families with X-linked dominant hypophosphatemic rickets

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

Kang, Qing-lin; Xu, Jia; Metabolic Bone Disease and Genetic Research Unit, Department of Osteoporosis and Bone Diseases, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233

2012-07-13

Highlights: Black-Right-Pointing-Pointer In our study, all of the patients were of Han Chinese ethnicity, which were rarely reported. Black-Right-Pointing-Pointer We identified three novel PHEX gene mutations in four unrelated families with XLH. Black-Right-Pointing-Pointer We found that the relationship between the phenotype and genotype of the PHEX gene was not invariant. Black-Right-Pointing-Pointer We found that two PHEX gene sites, p.534 and p.731, were conserved. -- Abstract: Background: X-linked hypophosphatemia (XLH), the most common form of inherited rickets, is a dominant disorder that is characterized by renal phosphate wasting with hypophosphatemia, abnormal bone mineralization, short stature, and rachitic manifestations. The related genemore » with inactivating mutations associated with XLH has been identified as PHEX, which is a phosphate-regulating gene with homologies to endopeptidases on the X chromosome. In this study, a variety of PHEX mutations were identified in four Chinese families with XLH. Methods: We investigated four unrelated Chinese families who exhibited typical features of XLH by using PCR to analyze mutations that were then sequenced. The laboratory and radiological investigations were conducted simultaneously. Results: Three novel mutations were found in these four families: one frameshift mutation, c.2033dupT in exon 20, resulting in p.T679H; one nonsense mutation, c.1294A > T in exon 11, resulting in p.K432X; and one missense mutation, c.2192T > C in exon 22, resulting in p.F731S. Conclusions: We found that the PHEX gene mutations were responsible for XLH in these Chinese families. Our findings are useful for understanding the genetic basis of Chinese patients with XLH.« less

A novel OPA1 mutation in a Chinese family with autosomal dominant optic atrophy

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

Zhang, Juanjuan; Yuan, Yimin; Lin, Bing

2012-03-23

Highlights: Black-Right-Pointing-Pointer We report the characterization of a four-generation large Chinese family with ADOA. Black-Right-Pointing-Pointer We find a new heterozygous mutation c.C1198G in OPA1 gene which may be a novel pathogenic mutation in this pedigree. Black-Right-Pointing-Pointer We do not find any mitochondrial DNA mutations associated with optic atrophy. Black-Right-Pointing-Pointer Other factors may also contribute to the phenotypic variability of ADOA in this pedigree. -- Abstract: A large four-generation Chinese family with autosomal dominant optic atrophy (ADOA) was investigated in the present study. Eight of the family members were affected in this pedigree. The affected family members exhibited early-onset and progressivemore » visual impairment, resulting in mild to profound loss of visual acuity. The average age-at-onset was 15.9 years. A new heterozygous mutation c.C1198G was identified by sequence analysis of the 12th exon of the OPA1 gene. This mutation resulted in a proline to alanine substitution at codon 400, which was located in an evolutionarily conserved region. This missense mutation in the GTPase domain was supposed to result in a loss of function for the encoded protein and act through a dominant negative effect. No other mutations associated with optic atrophy were found in our present study. The c.C1198G heterozygous mutation in the OPA1 gene may be a novel key pathogenic mutation in this pedigree with ADOA. Furthermore, additional nuclear modifier genes, environmental factors, and psychological factors may also contribute to the phenotypic variability of ADOA in this pedigree.« less

A study of the mutational landscape of pediatric-type follicular lymphoma and pediatric nodal marginal zone lymphoma.

PubMed

Ozawa, Michael G; Bhaduri, Aparna; Chisholm, Karen M; Baker, Steven A; Ma, Lisa; Zehnder, James L; Luna-Fineman, Sandra; Link, Michael P; Merker, Jason D; Arber, Daniel A; Ohgami, Robert S

2016-10-01

Pediatric-type follicular lymphoma and pediatric marginal zone lymphoma are two of the rarest B-cell lymphomas. These lymphomas occur predominantly in the pediatric population and show features distinct from their more common counterparts in adults: adult-type follicular lymphoma and adult-type nodal marginal zone lymphoma. Here we report a detailed whole-exome deep sequencing analysis of a cohort of pediatric-type follicular lymphomas and pediatric marginal zone lymphomas. This analysis revealed a recurrent somatic variant encoding p.Lys66Arg in the transcription factor interferon regulatory factor 8 (IRF8) in 3 of 6 cases (50%) of pediatric-type follicular lymphoma. This specific point mutation was not detected in pediatric marginal zone lymphoma or in adult-type follicular lymphoma. Additional somatic point mutations in pediatric-type follicular lymphoma were observed in genes involved in transcription, intracellular signaling, and cell proliferation. In pediatric marginal zone lymphoma, no recurrent mutation was identified; however, somatic point mutations were observed in genes involved in cellular adhesion, cytokine regulatory elements, and cellular proliferation. A somatic variant in AMOTL1, a recurrently mutated gene in splenic marginal zone lymphoma, was also identified in a case of pediatric marginal zone lymphoma. The overall non-synonymous mutational burden was low in both pediatric-type follicular lymphoma and pediatric marginal zone lymphoma (4.6 mutations per exome). Altogether, these findings support a distinctive genetic basis for pediatric-type follicular lymphoma and pediatric marginal zone lymphoma when compared with adult subtypes and to one another. Moreover, identification of a recurrent point mutation in IRF8 provides insight into a potential driver mutation in the pathogenesis of pediatric-type follicular lymphoma with implications for novel diagnostic or therapeutic strategies.

A study of the mutational landscape of pediatric-type follicular lymphoma and pediatric nodal marginal zone lymphoma

PubMed Central

Ozawa, Michael G; Bhaduri, Aparna; Chisholm, Karen M; Baker, Steven A; Ma, Lisa; Zehnder, James L; Luna-Fineman, Sandra; Link, Michael P; Merker, Jason D; Arber, Daniel A; Ohgami, Robert S

2016-01-01

Pediatric-type follicular lymphoma and pediatric marginal zone lymphoma are two of the rarest B-cell lymphomas. These lymphomas occur predominantly in the pediatric population and show features distinct from their more common counterparts in adults: adult-type follicular lymphoma and adult-type nodal marginal zone lymphoma. Here we report a detailed whole-exome deep sequencing analysis of a cohort of pediatric-type follicular lymphomas and pediatric marginal zone lymphomas. This analysis revealed a recurrent somatic variant encoding p.Lys66Arg in the transcription factor interferon regulatory factor 8 (IRF8) in 3 of 6 cases (50%) of pediatric-type follicular lymphoma. This specific point mutation was not detected in pediatric marginal zone lymphoma or in adult-type follicular lymphoma. Additional somatic point mutations in pediatric-type follicular lymphoma were observed in genes involved in transcription, intracellular signaling, and cell proliferation. In pediatric marginal zone lymphoma, no recurrent mutation was identified; however, somatic point mutations were observed in genes involved in cellular adhesion, cytokine regulatory elements, and cellular proliferation. A somatic variant in AMOTL1, a recurrently mutated gene in splenic marginal zone lymphoma, was also identified in a case of pediatric marginal zone lymphoma. The overall non-synonymous mutational burden was low in both pediatric-type follicular lymphoma and pediatric marginal zone lymphoma (4.6 mutations per exome). Altogether, these findings support a distinctive genetic basis for pediatric-type follicular lymphoma and pediatric marginal zone lymphoma when compared with adult subtypes and to one another. Moreover, identification of a recurrent point mutation in IRF8 provides insight into a potential driver mutation in the pathogenesis of pediatric-type follicular lymphoma with implications for novel diagnostic or therapeutic strategies. PMID:27338637

Effect of Repeat Copy Number on Variable-Number Tandem Repeat Mutations in Escherichia coli O157:H7

PubMed Central

Vogler, Amy J.; Keys, Christine; Nemoto, Yoshimi; Colman, Rebecca E.; Jay, Zack; Keim, Paul

2006-01-01

Variable-number tandem repeat (VNTR) loci have shown a remarkable ability to discriminate among isolates of the recently emerged clonal pathogen Escherichia coli O157:H7, making them a very useful molecular epidemiological tool. However, little is known about the rates at which these sequences mutate, the factors that affect mutation rates, or the mechanisms by which mutations occur at these loci. Here, we measure mutation rates for 28 VNTR loci and investigate the effects of repeat copy number and mismatch repair on mutation rate using in vitro-generated populations for 10 E. coli O157:H7 strains. We find single-locus rates as high as 7.0 × 10−4 mutations/generation and a combined 28-locus rate of 6.4 × 10−4 mutations/generation. We observed single- and multirepeat mutations that were consistent with a slipped-strand mispairing mutation model, as well as a smaller number of large repeat copy number mutations that were consistent with recombination-mediated events. Repeat copy number within an array was strongly correlated with mutation rate both at the most mutable locus, O157-10 (r2 = 0.565, P = 0.0196), and across all mutating loci. The combined locus model was significant whether locus O157-10 was included (r2 = 0.833, P < 0.0001) or excluded (r2 = 0.452, P < 0.0001) from the analysis. Deficient mismatch repair did not affect mutation rate at any of the 28 VNTRs with repeat unit sizes of >5 bp, although a poly(G) homomeric tract was destabilized in the mutS strain. Finally, we describe a general model for VNTR mutations that encompasses insertions and deletions, single- and multiple-repeat mutations, and their relative frequencies based upon our empirical mutation rate data. PMID:16740932

Comparative lesion sequencing provides insights into tumor evolution.

PubMed

Jones, Siân; Chen, Wei-Dong; Parmigiani, Giovanni; Diehl, Frank; Beerenwinkel, Niko; Antal, Tibor; Traulsen, Arne; Nowak, Martin A; Siegel, Christopher; Velculescu, Victor E; Kinzler, Kenneth W; Vogelstein, Bert; Willis, Joseph; Markowitz, Sanford D

2008-03-18

We show that the times separating the birth of benign, invasive, and metastatic tumor cells can be determined by analysis of the mutations they have in common. When combined with prior clinical observations, these analyses suggest the following general conclusions about colorectal tumorigenesis: (i) It takes approximately 17 years for a large benign tumor to evolve into an advanced cancer but <2 years for cells within that cancer to acquire the ability to metastasize; (ii) it requires few, if any, selective events to transform a highly invasive cancer cell into one with the capacity to metastasize; (iii) the process of cell culture ex vivo does not introduce new clonal mutations into colorectal tumor cell populations; and (iv) the rates at which point mutations develop in advanced cancers are similar to those of normal cells. These results have important implications for understanding human tumor pathogenesis, particularly those associated with metastasis.

BRCA Mutation Frequency and Patterns of Treatment Response in BRCA Mutation–Positive Women With Ovarian Cancer: A Report From the Australian Ovarian Cancer Study Group

PubMed Central

Alsop, Kathryn; Fereday, Sian; Meldrum, Cliff; deFazio, Anna; Emmanuel, Catherine; George, Joshy; Dobrovic, Alexander; Birrer, Michael J.; Webb, Penelope M.; Stewart, Colin; Friedlander, Michael; Fox, Stephen; Bowtell, David; Mitchell, Gillian

2012-01-01

Purpose The frequency of BRCA1 and BRCA2 germ-line mutations in women with ovarian cancer is unclear; reports vary from 3% to 27%. The impact of germ-line mutation on response requires further investigation to understand its impact on treatment planning and clinical trial design. Patients and Methods Women with nonmucinous ovarian carcinoma (n = 1,001) enrolled onto a population-based, case-control study were screened for point mutations and large deletions in both genes. Survival outcomes and responses to multiple lines of chemotherapy were assessed. Results Germ-line mutations were found in 14.1% of patients overall, including 16.6% of serous cancer patients (high-grade serous, 22.6%); 44% had no reported family history of breast or ovarian cancer. Patients carrying germ-line mutations had improved rates of progression-free and overall survival. In the relapse setting, patients carrying mutations more frequently responded to both platin- and nonplatin-based regimens than mutation-negative patients, even in patients with early relapse after primary treatment. Mutation-negative patients who responded to multiple cycles of platin-based treatment were more likely to carry somatic BRCA1/2 mutations. Conclusion BRCA mutation status has a major influence on survival in ovarian cancer patients and should be an additional stratification factor in clinical trials. Treatment outcomes in BRCA1/2 carriers challenge conventional definitions of platin resistance, and mutation status may be able to contribute to decision making and systemic therapy selection in the relapse setting. Our data, together with the advent of poly(ADP-ribose) polymerase inhibitor trials, supports the recommendation that germ-line BRCA1/2 testing should be offered to all women diagnosed with nonmucinous, ovarian carcinoma, regardless of family history. PMID:22711857

The molecular basis of Canavan (Aspartoacylase deficiency) disease in European non-Jewish patients

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

Shaag, A.; Anikster, Y.; Glustein, J.Z.

Canavan disease is an infantile neurodegenerative disease that is due to aspartoacylase deficiency. The disease has been reported mainly in Ashkenazi Jews but also occurs in other ethnic groups. Determination of enzymatic activity for carrier detection and prenatal diagnosis is considered unreliable. In the present study, nine mutations were found in the aspartoacylase gene of 19 non-Jewish patients. These included four point mutations (A305E [39.5% of the mutated alleles], C218X [15.8%], F2955 [2.6%], and G274R [5.3%]); four deletion mutations (827delGT [5.3%], 870del4 [2.6%], 566del7 [2.6%], and 527del6 [2.6%]); and one exon skip (527del108 [5.3%]). The A305E mutation is pan-European andmore » probably the most ancient mutation, identified in patients of Greek, Polish, Danish, French, Spanish, Italian, and British origin. In contrast, the G274R and 527del108 mutations were found only in patients of Turkish origin, and the C218X mutation was identified only in patients of Gypsy origin. Homozygosity for the A305E mutation was identified in patients with both the severe and the mild forms of Canavan disease. Mutations were identified in 31 of the 38 alleles, resulting in an overall detection rate of 81.6%. All nine mutations identified in non-Jewish patients reside in exons 4-6 of the aspartoacylase gene. The results would enable accurate genetic counseling in the families of 13 (68.4%) of 19 patients, in whom two mutations were identified in the aspartoacylase cDNA. 19 refs., 9 figs., 3 tabs.« less

KIT D816V-mutated bone marrow mesenchymal stem cells in indolent systemic mastocytosis are associated with disease progression.

PubMed

Garcia-Montero, Andres C; Jara-Acevedo, Maria; Alvarez-Twose, Ivan; Teodosio, Cristina; Sanchez-Muñoz, Laura; Muñiz, Carmen; Muñoz-Gonzalez, Javier I; Mayado, Andrea; Matito, Almudena; Caldas, Carolina; Morgado, Jose M; Escribano, Luis; Orfao, Alberto

2016-02-11

Multilineage involvement of bone marrow (BM) hematopoiesis by the somatic KIT D816V mutation is present in a subset of adult indolent systemic mastocytosis (ISM) patients in association with a poorer prognosis. Here, we investigated the potential involvement of BM mesenchymal stem cells (MSCs) from ISM patients by the KIT D816V mutation and its potential impact on disease progression and outcome. This mutation was investigated in highly purified BM MSCs and other BM cell populations from 83 ISM patients followed for a median of 116 months. KIT D816V-mutated MSCs were detected in 22 of 83 cases. All MSC-mutated patients had multilineage KIT mutation (100% vs 30%, P = .0001) and they more frequently showed involvement of lymphoid plus myeloid BM cells (59% vs 22%; P = .03) and a polyclonal pattern of inactivation of the X-chromosome of KIT-mutated BM mast cells (64% vs 0%; P = .01) vs other multilineage ISM cases. Moreover, presence of KIT-mutated MSCs was associated with more advanced disease features, a greater rate of disease progression (50% vs 17%; P = .04), and a shorter progression-free survival (P ≤ .003). Overall, these results support the notion that ISM patients with mutated MSCs may have acquired the KIT mutation in a common pluripotent progenitor cell, prior to differentiation into MSCs and hematopoietic precursor cells, before the X-chromosome inactivation process occurs. From a clinical point of view, acquisition of the KIT mutation in an earlier BM precursor cell confers a significantly greater risk for disease progression and a poorer outcome. © 2016 by The American Society of Hematology.

Somatic diversification of chicken immunoglobulin light chains by point mutations.

PubMed

Parvari, R; Ziv, E; Lantner, F; Heller, D; Schechter, I

1990-04-01

The light-chain locus of chicken has 1 functional V lambda 1 gene, 1 J gene, and 25 pseudo-V lambda-genes (where V = variable and J = joining). A major problem is which somatic mechanisms expand this extremely limited germ-line information to generate many different antibodies. Weill's group [Reynaud, C. A., Anquez, V., Grimal, H. & Weill, J. C. (1987) Cell 48, 379-388] has shown that the pseudo-V lambda-genes diversify the rearranged V lambda 1 by gene conversion. Here we demonstrate that chicken light chains are further diversified by somatic point mutations and by V lambda 1-J flexible joining. Somatic point mutations were identified in the J and 3' noncoding DNA of rearranged light-chain genes of chicken. These regions were analyzed because point mutations in V lambda 1 are obscured by gene conversion; the J and 3' noncoding DNA are presented in one copy per haploid genome and are not subject to gene conversion. In rodents point mutations occur as frequently in the V-J coding regions as in the adjacent flanking DNA. Therefore, we conclude that somatic point mutations diversify the V lambda 1 of chicken. The frequency (0-1%) and distribution of the mutations (decreasing in number with increased distance from the V lambda 1 segment) in chicken were as observed in rodents. Sequence variability at the V lambda 1-J junctions could be attributed to imprecise joining of the V lambda 1 and J genes. The modification by gene conversion of rearranged V lambda 1 genes in the bursa was similar in chicken aged 3 months (9.5%) or 3 weeks (9.1%)--i.e., gene conversion that generates the preimmune repertoire in the bursa seems to level off around 3 weeks of age. This preimmune repertoire can be further diversified by somatic point mutations that presumably lead to the formation of antibodies with increased affinity. A segment with structural features of a matrix association region [(A + T)-rich and four topoisomerase II binding sites] was identified in the middle of the J-C lambda intron (where C = constant).

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.

Sensitive and reliable detection of Kit point mutation Asp 816 to Val in pathological material

PubMed Central

Kähler, Christian; Didlaukat, Sabine; Feller, Alfred C; Merz, Hartmut

2007-01-01

Background Human mastocytosis is a heterogenous disorder which is linked to a gain-of-function mutation in the kinase domain of the receptor tyrosine kinase Kit. This D816V mutation leads to constitutive activation and phosphorylation of Kit with proliferative disorders of mast cells in the peripheral blood, skin, and spleen. Most PCR applications used so far are labour-intensive and are not adopted to daily routine in pathological laboratories. The method has to be robust and working on such different materials like archival formalin-fixed, paraffin-embedded tissue (FFPE) and blood samples. Such a method is introduced in this publication. Methods The Kit point mutation Asp 816 to Val is heterozygous which means a problem in detection by PCR because the wild-type allele is also amplified and the number of cells which bear the point mutation is in most of the cases low. Most PCR protocols use probes to block the wild-type allele during amplification with more or less satisfying result. This is why point-mutated forward primers were designed and tested for efficiency in amplification of the mutated allele. Results One primer combination (A) fits the most for the introduced PCR assay. It was able just to amplify the mutated allele with high specificity from different patient's materials (FFPE or blood) of varying quality and quantity. Moreover, the sensitivity for this assay was convincing because 10 ng of DNA which bears the point mutation could be detected in a total volume of 200 ng of DNA. Conclusion The PCR assay is able to deal with different materials (blood and FFPE) this means quality and quantity of DNA and can be used for high-througput screening because of its robustness. Moreover, the method is easy-to-use, not labour-intensive, and easy to realise in a standard laboratory. PMID:17900365

Quantitative PCR high-resolution melting (qPCR-HRM) curve analysis, a new approach to simultaneously screen point mutations and large rearrangements: application to MLH1 germline mutations in Lynch syndrome.

PubMed

Rouleau, Etienne; Lefol, Cédrick; Bourdon, Violaine; Coulet, Florence; Noguchi, Tetsuro; Soubrier, Florent; Bièche, Ivan; Olschwang, Sylviane; Sobol, Hagay; Lidereau, Rosette

2009-06-01

Several techniques have been developed to screen mismatch repair (MMR) genes for deleterious mutations. Until now, two different techniques were required to screen for both point mutations and large rearrangements. For the first time, we propose a new approach, called "quantitative PCR (qPCR) high-resolution melting (HRM) curve analysis (qPCR-HRM)," which combines qPCR and HRM to obtain a rapid and cost-effective method suitable for testing a large series of samples. We designed PCR amplicons to scan the MLH1 gene using qPCR HRM. Seventy-six patients were fully scanned in replicate, including 14 wild-type patients and 62 patients with known mutations (57 point mutations and five rearrangements). To validate the detected mutations, we used sequencing and/or hybridization on a dedicated MLH1 array-comparative genomic hybridization (array-CGH). All point mutations and rearrangements detected by denaturing high-performance liquid chromatography (dHPLC)+multiplex ligation-dependent probe amplification (MLPA) were successfully detected by qPCR HRM. Three large rearrangements were characterized with the dedicated MLH1 array-CGH. One variant was detected with qPCR HRM in a wild-type patient and was located within the reverse primer. One variant was not detected with qPCR HRM or with dHPLC due to its proximity to a T-stretch. With qPCR HRM, prescreening for point mutations and large rearrangements are performed in one tube and in one step with a single machine, without the need for any automated sequencer in the prescreening process. In replicate, its reagent cost, sensitivity, and specificity are comparable to those of dHPLC+MLPA techniques. However, qPCR HRM outperformed the other techniques in terms of its rapidity and amount of data provided.

Cytotoxic chemotherapy and the evolution of cellular and viral resistance to antiretroviral therapy in HIV- infected individuals with lymphoma.

PubMed

McFaul, Katie; Liptrott, Neill; Cox, Alison; Martin, Phillip; Egan, Deirdre; Owen, Andrew; Kelly, Sarah; Karolia, Zeenat; Shaw, Kate; Bower, Mark; Boffito, Marta

2016-09-01

The use of combination antiretroviral therapy (cART) and cytotoxic chemotherapy for HIV-associated lymphoma runs the risks of inducing HIV drug resistance. This study examined two possible mechanisms: altered expression of membrane drug transporter protein (MTP) and acquisition of mutations in pro-viral DNA. Expression levels of MTP and pro-viral DNA resistance mutation analysis were performed on peripheral blood mononuclear cells (PBMC) before, during, and after chemotherapy. Twenty nine patients completed the three time point estimations. There were no significant variations before, during, and after chemotherapy in the expression of four MTPs: ABCB1, ABCC1, ABCC2, and SLCO3A1 (OATP3A1). Pro-viral DNA sequencing revealed that only one patient developed a new nucleos/tide reverse transcriptase inhibitor-associated mutation (184V) during the course of the study, giving a mutation rate of 0.0027 per person per year. In conclusion, concomitant administration of cytotoxic chemotherapy and cART does not induce expression of MTP. Furthermore, no significant changes in viral resistance were observed pre- and post-chemotherapy, suggesting mutagenic cytotoxic chemotherapy seems not to induce mutations in HIV pro-viral DNA.

Stochastic demography and the neutral substitution rate in class-structured populations.

PubMed

Lehmann, Laurent

2014-05-01

The neutral rate of allelic substitution is analyzed for a class-structured population subject to a stationary stochastic demographic process. The substitution rate is shown to be generally equal to the effective mutation rate, and under overlapping generations it can be expressed as the effective mutation rate in newborns when measured in units of average generation time. With uniform mutation rate across classes the substitution rate reduces to the mutation rate.

Engineering diverse changes in beta-turn propensities in the N-terminal beta-hairpin of ubiquitin reveals significant effects on stability and kinetics but a robust folding transition state.

PubMed

Simpson, Emma R; Meldrum, Jill K; Searle, Mark S

2006-04-04

Using the N-terminal 17-residue beta-hairpin of ubiquitin as a "host" for mutational studies, we have investigated the influence of the beta-turn sequence on protein stability and folding kinetics by replacing the native G-bulged turn (TLTGK) with more flexible analogues (TG3K and TG5K) and a series of four-residue type I' beta-turn sequences, commonly found in beta-hairpins. Although a statistical analysis of type I' turns demonstrates residue preferences at specific sites, the frequency of occurrence appears to only broadly correlate with experimentally determined protein stabilities. The subsequent engineering of context-dependent non-native tertiary contacts involving turn residues is shown to produce large changes in stability. Relatively few point mutations have been described that probe secondary structure formation in ubiquitin in a manner that is independent of tertiary contacts. To this end, we have used the more rigorous rate-equilibrium free energy relationship (Leffler analysis), rather than the two-point phi value analysis, to show for a family of engineered beta-turn mutants that stability (range of approximately 20 kJ/mol) and folding kinetics (190-fold variation in refolding rate) are linearly correlated (alpha(f) = 0.74 +/- 0.08). The data are consistent with a transition state that is robust with regard to a wide range of statistically favored and disfavored beta-turn mutations and implicate a loosely assembled beta-hairpin as a key template in transition state stabilization with the beta-turn playing a central role.

Rates of spontaneous mutation among RNA viruses.

PubMed Central

Drake, J W

1993-01-01

Simple methods are presented to estimate rates of spontaneous mutation from mutant frequencies and population parameters in RNA viruses. Published mutant frequencies yield a wide range of mutation rates per genome per replication, mainly because mutational targets have usually been small and, thus, poor samples of the mutability of the average base. Nevertheless, there is a clear central tendency for lytic RNA viruses (bacteriophage Q beta, poliomyelitis, vesicular stomatitis, and influenza A) to display rates of spontaneous mutation of approximately 1 per genome per replication. This rate is some 300-fold higher than previously reported for DNA-based microbes. Lytic RNA viruses thus mutate at a rate close to the maximum value compatible with viability. Retroviruses (spleen necrosis, murine leukemia, Rous sarcoma), however, mutate at an average rate about an order of magnitude lower than lytic RNA viruses. PMID:8387212

Rational evolutionary design: the theory of in vitro protein evolution.

PubMed

Voigt, C A; Kauffman, S; Wang, Z G

2000-01-01

Directed evolution uses a combination of powerful search techniques to generate proteins with improved properties. Part of the success is due to the stochastic element of random mutagenesis; improvements can be made without a detailed description of the complex interactions that constitute function or stability. However, optimization is not a conglomeration of random processes. Rather, it requires both knowledge of the system that is being optimized and a logical series of techniques that best explores the pathways of evolution (Eigen et al., 1988). The weighing of parameters associated with mutation, recombination, and screening to achieve the maximum fitness improvement is the beginning of rational evolutionary design. The optimal mutation rate is strongly influenced by the finite number of mutants that can be screened. A smooth fitness landscape implies that many mutations can be accumulated without disrupting the fitness. This has the effect of lowering the required library size to sample a higher mutation rate. As the sequence ascends the fitness landscape, the optimal mutation rate decreases as the probability of discovering improved mutations also decreases. Highly coupled regions require that many mutations be simultaneously made to generate a positive mutant. Therefore, positive mutations are discovered at uncoupled positions as the fitness of the parent increases. The benefit of recombination is twofold: it combines good mutations and searches more sequence space in a meaningful way. Recombination is most beneficial when the number of mutants that can be screened is limited and the landscape is of an intermediate ruggedness. The structure of schema in proteins leads to the conclusion that many cut points are required. The number of parents and their sequence identity are determined by the balance between exploration and exploitation. Many disparate parents can explore more space, but at the risk of losing information. The required screening effort is related to the number of uphill paths, which decreases more rapidly for rugged landscapes. Noise in the fitness measurements causes a dramatic increase in the required mutant library size, thus implying a smaller optimal mutation rate. Because of strict limitations on the number of mutants that can be screened, there is motivation to optimize the content of the mutant library. By restricting mutations to regions of the gene that are expected to show improvement, a greater return can be made with the same number of mutants. Initial studies with subtilisin E have shown that structurally tolerant positions tend to be where positive activity mutants are made during directed evolution. Mutant fitness information is produced by the screening step that has the potential to provide insight into the structure of the fitness landscape, thus aiding the setting of experimental parameters. By analyzing the mutant fitness distribution and targeting specific regions of the sequence, in vitro evolution can be accelerated. However, when expediting the search, there is a trade-off between rapid improvement and the quality of the long-term solution. The benefit of neutrality has yet to be captured with in vitro protein evolution. Neutral theory predicts the punctuated emergence of novel structure and function, however, with current methods, the required time scale is not feasible. Utilizing neutral evolution to accelerate the discovery of new functional and structural solutions requires a theory that predicts the behavior of mutational pathways between networks. Because the transition from neutral to adaptive evolution requires a multi-mutational switch, increasing the mutation rate decreases the time required for a punctuated change to occur. By limiting the search to the less coupled region of the sequence (smooth portion of the fitness landscape), the required larger mutation rate can be tolerated. Advances in directed evolution will be achieved when the driving forces behind such proce

Determining Mutation Rates in Bacterial Populations

PubMed Central

Rosche, William A.; Foster, Patricia L.

2010-01-01

When properly determined, spontaneous mutation rates are a more accurate and biologically meaningful reflection of the underlying mutagenic mechanism than are mutation frequencies. Because bacteria grow exponentially and mutations arise stochastically, methods to estimate mutation rates depend on theoretical models that describe the distribution of mutant numbers among parallel cultures, as in the original Luria-Delbrück fluctuation analysis. An accurate determination of mutation rate depends on understanding the strengths and limitations of these methods, and how to design fluctuation assays to optimize a given method. In this paper we describe a number of methods to estimate mutation rates, give brief accounts of their derivations, and discuss how they behave under various experimental conditions. PMID:10610800

Mutation spectrum of RB1 mutations in retinoblastoma cases from Singapore with implications for genetic management and counselling.

PubMed

Tomar, Swati; Sethi, Raman; Sundar, Gangadhara; Quah, Thuan Chong; Quah, Boon Long; Lai, Poh San

2017-01-01

Retinoblastoma (RB) is a rare childhood malignant disorder caused by the biallelic inactivation of RB1 gene. Early diagnosis and identification of carriers of heritable RB1 mutations can improve disease outcome and management. In this study, mutational analysis was conducted on fifty-nine matched tumor and peripheral blood samples from 18 bilateral and 41 unilateral unrelated RB cases by a combinatorial approach of Multiplex Ligation-dependent Probe Amplification (MLPA) assay, deletion screening, direct sequencing, copy number gene dosage analysis and methylation assays. Screening of both blood and tumor samples yielded a mutation detection rate of 94.9% (56/59) while only 42.4% (25/59) of mutations were detected if blood samples alone were analyzed. Biallelic mutations were observed in 43/59 (72.9%) of tumors screened. There were 3 cases (5.1%) in which no mutations could be detected and germline mutations were detected in 19.5% (8/41) of unilateral cases. A total of 61 point mutations were identified, of which 10 were novel. There was a high incidence of previously reported recurrent mutations, occurring at 38.98% (23/59) of all cases. Of interest were three cases of mosaic RB1 mutations detected in the blood from patients with unilateral retinoblastoma. Additionally, two germline mutations previously reported to be associated with low-penetrance phenotypes: missense-c.1981C>T and splice variant-c.607+1G>T, were observed in a bilateral and a unilateral proband, respectively. These findings have implications for genetic counselling and risk prediction for the affected families. This is the first published report on the spectrum of mutations in RB patients from Singapore and shows that further improved mutation screening strategies are required in order to provide a definitive molecular diagnosis for every case of RB. Our findings also underscore the importance of genetic testing in supporting individualized disease management plans for patients and asymptomatic family members carrying low-penetrance, germline mosaicism or heritable unilateral mutational phenotypes.

Efficacy of chemotherapy after first-line gefitinib therapy in EGFR mutation-positive advanced non-small cell lung cancer-data from a randomized Phase III study comparing gefitinib with carboplatin plus paclitaxel (NEJ002).

PubMed

Miyauchi, Eisaku; Inoue, Akira; Kobayashi, Kunihiko; Maemondo, Makoto; Sugawara, Shunichi; Oizumi, Satoshi; Isobe, Hiroshi; Gemma, Akihiko; Saijo, Yasuo; Yoshizawa, Hirohisa; Hagiwara, Koichi; Nukiwa, Toshihiro

2015-07-01

Epidermal growth factor receptor tyrosine kinase inhibitors are effective as first-line therapy for advanced non-small cell lung cancer patients harboring epidermal growth factor receptor mutations. However, it is unknown whether second-line platinum-based chemotherapy after epidermal growth factor receptor tyrosine kinase inhibitor therapy could lead to better outcomes. We evaluated the efficacy of second-line platinum-based chemotherapy after gefitinib for advanced non-small cell lung cancers harboring epidermal growth factor receptor mutations (the NEJ002 study). Seventy-one non-small cell lung cancers, treated with gefitinib as first-line therapy and then receiving platinum-based chemotherapy as second-line therapy were evaluated in NEJ002. Patients were evaluated for antitumor response to second-line chemotherapy by computed tomography according to the criteria of the Response Evaluation Criteria in Solid Tumors group (version 1.0). Of the 71 patients receiving platinum-based chemotherapy after first-line gefitinib, a partial response was documented in 25.4% (18/71), stable disease in 43.7% (31/71) and progression of disease in 21.1% (15/71). The objective response and disease control rates were 25.4% (18/71) and 69% (49/71), respectively. There was no significant difference between first- and second-line chemotherapy in objective response and disease control rates for advanced non-small cell lung cancer harboring activating epidermal growth factor receptor mutations. In the analysis of epidermal growth factor receptor mutation types, the objective responses of deletions in exon 19 and a point mutation in exon 21 (L858R) were 27.3% (9/33) and 28.1% (9/32), respectively, but these differences between objective response rates were not significant. The efficacy of second-line platinum-based chemotherapy followed at progression by gefitinib was similar to first-line platinum-based chemotherapy, and epidermal growth factor receptor mutation types did not influence the efficacy of second-line platinum-based chemotherapy. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Artemisinin resistance at the China-Myanmar border and association with mutations in the K13 propeller gene.

PubMed

Wang, Zenglei; Wang, Yingna; Cabrera, Mynthia; Zhang, Yanmei; Gupta, Bhavna; Wu, Yanrui; Kemirembe, Karen; Hu, Yue; Liang, Xiaoying; Brashear, Awtum; Shrestha, Sony; Li, Xiaolian; Miao, Jun; Sun, Xiaodong; Yang, Zhaoqing; Cui, Liwang

2015-11-01

Artemisinin resistance in Plasmodium falciparum parasites in Southeast Asia is a major concern for malaria control. Its emergence at the China-Myanmar border, where there have been more than 3 decades of artemisinin use, has yet to be investigated. Here, we comprehensively evaluated the potential emergence of artemisinin resistance and antimalarial drug resistance status in P. falciparum using data and parasites from three previous efficacy studies in this region. These efficacy studies of dihydroartemisinin-piperaquine combination and artesunate monotherapy of uncomplicated falciparum malaria in 248 P. falciparum patients showed an overall 28-day adequate clinical and parasitological response of >95% and day 3 parasite-positive rates of 6.3 to 23.1%. Comparison of the 57 K13 sequences (24 and 33 from day 3 parasite-positive and -negative cases, respectively) identified nine point mutations in 38 (66.7%) samples, of which F446I (49.1%) and an N-terminal NN insertion (86.0%) were predominant. K13 propeller mutations collectively, the F446I mutation alone, and the NN insertion all were significantly associated with day 3 parasite positivity. Increased ring-stage survival determined using the ring-stage survival assay (RSA) was highly associated with the K13 mutant genotype. Day 3 parasite-positive isolates had ∼10 times higher ring survival rates than day 3 parasite-negative isolates. Divergent K13 mutations suggested independent evolution of artemisinin resistance. Taken together, this study confirmed multidrug resistance and emergence of artemisinin resistance in P. falciparum at the China-Myanmar border. RSA and K13 mutations are useful phenotypic and molecular markers for monitoring artemisinin resistance. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Exome-wide Sequencing Shows Low Mutation Rates and Identifies Novel Mutated Genes in Seminomas.

PubMed

Cutcutache, Ioana; Suzuki, Yuka; Tan, Iain Beehuat; Ramgopal, Subhashini; Zhang, Shenli; Ramnarayanan, Kalpana; Gan, Anna; Lee, Heng Hong; Tay, Su Ting; Ooi, Aikseng; Ong, Choon Kiat; Bolthouse, Jonathan T; Lane, Brian R; Anema, John G; Kahnoski, Richard J; Tan, Patrick; Teh, Bin Tean; Rozen, Steven G

2015-07-01

Testicular germ cell tumors are the most common cancer diagnosed in young men, and seminomas are the most common type of these cancers. There have been no exome-wide examinations of genes mutated in seminomas or of overall rates of nonsilent somatic mutations in these tumors. The objective was to analyze somatic mutations in seminomas to determine which genes are affected and to determine rates of nonsilent mutations. Eight seminomas and matched normal samples were surgically obtained from eight patients. DNA was extracted from tissue samples and exome sequenced on massively parallel Illumina DNA sequencers. Single-nucleotide polymorphism chip-based copy number analysis was also performed to assess copy number alterations. The DNA sequencing read data were analyzed to detect somatic mutations including single-nucleotide substitutions and short insertions and deletions. The detected mutations were validated by independent sequencing and further checked for subclonality. The rate of nonsynonymous somatic mutations averaged 0.31 mutations/Mb. We detected nonsilent somatic mutations in 96 genes that were not previously known to be mutated in seminomas, of which some may be driver mutations. Many of the mutations appear to have been present in subclonal populations. In addition, two genes, KIT and KRAS, were affected in two tumors each with mutations that were previously observed in other cancers and are presumably oncogenic. Our study, the first report on exome sequencing of seminomas, detected somatic mutations in 96 new genes, several of which may be targetable drivers. Furthermore, our results show that seminoma mutation rates are five times higher than previously thought, but are nevertheless low compared to other common cancers. Similar low rates are seen in other cancers that also have excellent rates of remission achieved with chemotherapy. We examined the DNA sequences of seminomas, the most common type of testicular germ cell cancer. Our study identified 96 new genes in which mutations occurred during seminoma development, some of which might contribute to cancer development or progression. The study also showed that the rates of DNA mutations during seminoma development are higher than previously thought, but still lower than for other common solid-organ cancers. Such low rates are also observed among other cancers that, like seminomas, show excellent rates of disease remission after chemotherapy. Copyright © 2015 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Genome Sequencing and Comparative Genomics of the Broad Host-Range Pathogen Rhizoctonia solani AG8

PubMed Central

Hane, James K.; Anderson, Jonathan P.; Williams, Angela H.; Sperschneider, Jana; Singh, Karam B.

2014-01-01

Rhizoctonia solani is a soil-borne basidiomycete fungus with a necrotrophic lifestyle which is classified into fourteen reproductively incompatible anastomosis groups (AGs). One of these, AG8, is a devastating pathogen causing bare patch of cereals, brassicas and legumes. R. solani is a multinucleate heterokaryon containing significant heterozygosity within a single cell. This complexity posed significant challenges for the assembly of its genome. We present a high quality genome assembly of R. solani AG8 and a manually curated set of 13,964 genes supported by RNA-seq. The AG8 genome assembly used novel methods to produce a haploid representation of its heterokaryotic state. The whole-genomes of AG8, the rice pathogen AG1-IA and the potato pathogen AG3 were observed to be syntenic and co-linear. Genes and functions putatively relevant to pathogenicity were highlighted by comparing AG8 to known pathogenicity genes, orthology databases spanning 197 phytopathogenic taxa and AG1-IA. We also observed SNP-level “hypermutation” of CpG dinucleotides to TpG between AG8 nuclei, with similarities to repeat-induced point mutation (RIP). Interestingly, gene-coding regions were widely affected along with repetitive DNA, which has not been previously observed for RIP in mononuclear fungi of the Pezizomycotina. The rate of heterozygous SNP mutations within this single isolate of AG8 was observed to be higher than SNP mutation rates observed across populations of most fungal species compared. Comparative analyses were combined to predict biological processes relevant to AG8 and 308 proteins with effector-like characteristics, forming a valuable resource for further study of this pathosystem. Predicted effector-like proteins had elevated levels of non-synonymous point mutations relative to synonymous mutations (dN/dS), suggesting that they may be under diversifying selection pressures. In addition, the distant relationship to sequenced necrotrophs of the Ascomycota suggests the R. solani genome sequence may prove to be a useful resource in future comparative analysis of plant pathogens. PMID:24810276

The waiting time problem in a model hominin population.

PubMed

Sanford, John; Brewer, Wesley; Smith, Franzine; Baumgardner, John

2015-09-17

Functional information is normally communicated using specific, context-dependent strings of symbolic characters. This is true within the human realm (texts and computer programs), and also within the biological realm (nucleic acids and proteins). In biology, strings of nucleotides encode much of the information within living cells. How do such information-bearing nucleotide strings arise and become established? This paper uses comprehensive numerical simulation to understand what types of nucleotide strings can realistically be established via the mutation/selection process, given a reasonable timeframe. The program Mendel's Accountant realistically simulates the mutation/selection process, and was modified so that a starting string of nucleotides could be specified, and a corresponding target string of nucleotides could be specified. We simulated a classic pre-human hominin population of at least 10,000 individuals, with a generation time of 20 years, and with very strong selection (50% selective elimination). Random point mutations were generated within the starting string. Whenever an instance of the target string arose, all individuals carrying the target string were assigned a specified reproductive advantage. When natural selection had successfully amplified an instance of the target string to the point of fixation, the experiment was halted, and the waiting time statistics were tabulated. Using this methodology we tested the effect of mutation rate, string length, fitness benefit, and population size on waiting time to fixation. Biologically realistic numerical simulations revealed that a population of this type required inordinately long waiting times to establish even the shortest nucleotide strings. To establish a string of two nucleotides required on average 84 million years. To establish a string of five nucleotides required on average 2 billion years. We found that waiting times were reduced by higher mutation rates, stronger fitness benefits, and larger population sizes. However, even using the most generous feasible parameters settings, the waiting time required to establish any specific nucleotide string within this type of population was consistently prohibitive. We show that the waiting time problem is a significant constraint on the macroevolution of the classic hominin population. Routine establishment of specific beneficial strings of two or more nucleotides becomes very problematic.

Midostaurin and Decitabine in Treating Older Patients With Newly Diagnosed Acute Myeloid Leukemia and FLT3 Mutation

ClinicalTrials.gov

2017-11-29

Acute Myeloid Leukemia With FLT3/ITD Mutation; Acute Myeloid Leukemia With Gene Mutations; FLT3 Tyrosine Kinase Domain Point Mutation; Secondary Acute Myeloid Leukemia; Untreated Adult Acute Myeloid Leukemia

Mechanisms of viral mutation.

PubMed

Sanjuán, Rafael; Domingo-Calap, Pilar

2016-12-01

The remarkable capacity of some viruses to adapt to new hosts and environments is highly dependent on their ability to generate de novo diversity in a short period of time. Rates of spontaneous mutation vary amply among viruses. RNA viruses mutate faster than DNA viruses, single-stranded viruses mutate faster than double-strand virus, and genome size appears to correlate negatively with mutation rate. Viral mutation rates are modulated at different levels, including polymerase fidelity, sequence context, template secondary structure, cellular microenvironment, replication mechanisms, proofreading, and access to post-replicative repair. Additionally, massive numbers of mutations can be introduced by some virus-encoded diversity-generating elements, as well as by host-encoded cytidine/adenine deaminases. Our current knowledge of viral mutation rates indicates that viral genetic diversity is determined by multiple virus- and host-dependent processes, and that viral mutation rates can evolve in response to specific selective pressures.

A novel natural mutation AαPhe98Ile in the fibrinogen coiled-coil affects fibrinogen function.

PubMed

Riedelová-Reicheltová, Zuzana; Kotlín, Roman; Suttnar, Jiří; Geierová, Véra; Riedel, Tomáš; Májek, Pavel; Dyr, Jan Evangelista

2014-01-01

The aim of this study was to investigate the structure and function of fibrinogen obtained from a patient with normal coagulation times and idiopathic thrombophilia. This was done by SDS-PAGE and DNA sequence analyses, scanning electron microscopy, fibrinopeptide release, fibrin polymerisation initiated by thrombin and reptilase, fibrinolysis, and platelet aggregometry. A novel heterozygous point mutation in the fibrinogen Aα chain, Phe98 to Ile, was found and designated as fibrinogen Vizovice. The mutation, which is located in the RGDF sequence (Aα 95-98) of the fibrinogen coiled-coil region, significantly affected fibrin clot morphology. Namely, the clot formed by fibrinogen Vizovice contained thinner and curled fibrin fibers with reduced length. Lysis of the clots prepared from Vizovice plasma and isolated fibrinogen were found to be impaired. The lysis rate of Vizovice clots was almost four times slower than the lysis rate of control clots. In the presence of platelets agonists the mutant fibrinogen caused increased platelet aggregation. The data obtained show that natural mutation of Phe98 to Ile in the fibrinogen Aα chain influences lateral aggregation of fibrin protofibrils, fibrinolysis, and platelet aggregation. They also suggest that delayed fibrinolysis, together with the abnormal fibrin network morphology and increased platelet aggregation, may be the direct cause of thrombotic complications in the patient associated with pregnancy loss.

Population-Scale Sequencing Data Enable Precise Estimates of Y-STR Mutation Rates

PubMed Central

Willems, Thomas; Gymrek, Melissa; Poznik, G. David; Tyler-Smith, Chris; Erlich, Yaniv

2016-01-01

Short tandem repeats (STRs) are mutation-prone loci that span nearly 1% of the human genome. Previous studies have estimated the mutation rates of highly polymorphic STRs by using capillary electrophoresis and pedigree-based designs. Although this work has provided insights into the mutational dynamics of highly mutable STRs, the mutation rates of most others remain unknown. Here, we harnessed whole-genome sequencing data to estimate the mutation rates of Y chromosome STRs (Y-STRs) with 2–6 bp repeat units that are accessible to Illumina sequencing. We genotyped 4,500 Y-STRs by using data from the 1000 Genomes Project and the Simons Genome Diversity Project. Next, we developed MUTEA, an algorithm that infers STR mutation rates from population-scale data by using a high-resolution SNP-based phylogeny. After extensive intrinsic and extrinsic validations, we harnessed MUTEA to derive mutation-rate estimates for 702 polymorphic STRs by tracing each locus over 222,000 meioses, resulting in the largest collection of Y-STR mutation rates to date. Using our estimates, we identified determinants of STR mutation rates and built a model to predict rates for STRs across the genome. These predictions indicate that the load of de novo STR mutations is at least 75 mutations per generation, rivaling the load of all other known variant types. Finally, we identified Y-STRs with potential applications in forensics and genetic genealogy, assessed the ability to differentiate between the Y chromosomes of father-son pairs, and imputed Y-STR genotypes. PMID:27126583

How the folding rates of two- and multistate proteins depend on the amino acid properties.

PubMed

Huang, Jitao T; Huang, Wei; Huang, Shanran R; Li, Xin

2014-10-01

Proteins fold by either two-state or multistate kinetic mechanism. We observe that amino acids play different roles in different mechanism. Many residues that are easy to form regular secondary structures (α helices, β sheets and turns) can promote the two-state folding reactions of small proteins. Most of hydrophilic residues can speed up the multistate folding reactions of large proteins. Folding rates of large proteins are equally responsive to the flexibility of partial amino acids. Other properties of amino acids (including volume, polarity, accessible surface, exposure degree, isoelectric point, and phase transfer energy) have contributed little to folding kinetics of the proteins. Cysteine is a special residue, it triggers two-state folding reaction and but inhibits multistate folding reaction. These findings not only provide a new insight into protein structure prediction, but also could be used to direct the point mutations that can change folding rate. © 2014 Wiley Periodicals, Inc.

Structural and functional studies of a 50 kDa antigenic protein from Salmonella enterica serovar Typhi.

PubMed

Choong, Yee Siew; Lim, Theam Soon; Chew, Ai Lan; Aziah, Ismail; Ismail, Asma

2011-04-01

The high typhoid incidence rate in developing and under-developed countries emphasizes the need for a rapid, affordable and accessible diagnostic test for effective therapy and disease management. TYPHIDOT®, a rapid dot enzyme immunoassay test for typhoid, was developed from the discovery of a ∼50 kDa protein specific for Salmonella enterica serovar Typhi. However, the structure of this antigen remains unknown till today. Studies on the structure of this antigen are important to elucidate its function, which will in turn increase the efficiency of the development and improvement of the typhoid detection test. This paper described the predictive structure and function of the antigenically specific protein. The homology modeling approach was employed to construct the three-dimensional structure of the antigen. The built structure possesses the features of TolC-like outer membrane protein. Molecular docking simulation was also performed to further probe the functionality of the antigen. Docking results showed that hexamminecobalt, Co(NH(3))(6)(3+), as an inhibitor of TolC protein, formed favorable hydrogen bonds with D368 and D371 of the antigen. The single point (D368A, D371A) and double point (D368A and D371A) mutations of the antigen showed a decrease (single point mutation) and loss (double point mutations) of binding affinity towards hexamminecobalt. The architecture features of the built model and the docking simulation reinforced and supported that this antigen is indeed the variant of outer membrane protein, TolC. As channel proteins are important for the virulence and survival of bacteria, therefore this ∼50 kDa channel protein is a good specific target for typhoid detection test. Copyright © 2011 Elsevier Inc. All rights reserved.

Peptide Nucleic Acid Array for Detection of Point Mutations in Hepatitis B Virus Associated with Antiviral Resistance ▿ †

PubMed Central

Jang, Hyunjung; Kim, Jihyun; Choi, Jae-jin; Son, Yeojin; Park, Heekyung

2010-01-01

The detection of antiviral-resistant hepatitis B virus (HBV) mutations is important for monitoring the response to treatment and for effective treatment decisions. We have developed an array using peptide nucleic acid (PNA) probes to detect point mutations in HBV associated with antiviral resistance. PNA probes were designed to detect mutations associated with resistance to lamivudine, adefovir, and entecavir. The PNA array assay was sensitive enough to detect 102 copies/ml. The PNA array assay was able to detect mutants present in more than 5% of the virus population when the total HBV DNA concentration was greater than 104 copies/ml. We analyzed a total of 68 clinical samples by this assay and validated its usefulness by comparing results to those of the sequencing method. The PNA array correctly identified viral mutants and has high concordance (98.3%) with direct sequencing in detecting antiviral-resistant mutations. Our results showed that the PNA array is a rapid, sensitive, and easily applicable assay for the detection of antiviral-resistant mutation in HBV. Thus, the PNA array is a useful and powerful diagnostic tool for the detection of point mutations or polymorphisms. PMID:20573874

Mutational activation of CheA, the protein kinase in the chemotaxis system of Escherichia coli.

PubMed Central

Tawa, P.; Stewart, R. C.

1994-01-01

In Escherichia coli and Salmonella typhimurium, appropriate changes of cell swimming patterns are mediated by CheA, an autophosphorylating histidine protein kinase whose activity is regulated by receptor/transducer proteins. The molecular mechanism underlying this regulation remains unelucidated but may involve CheA shifting between high-activity and low-activity conformations. We devised an in vivo screen to search for potential hyperkinase variants of CheA and used this screen to identify two cheA point mutations that cause the CheA protein to have elevated autokinase activity. Each point mutation resulted in alteration of proline 337. In vitro, CheA337PL and CheA337PS autophosphorylated significantly more rapidly than did wild-type CheA. This rate enhancement reflected the higher affinities of the mutant proteins for ATP and an increased rate constant for acquisition by CheA of the gamma-phosphoryl group of ATP within a kinetically defined CheA.ATP complex. In addition, the mutant proteins reacted with ADP more rapidly than did wild-type CheA. We considered the possibility that the mutations served to lock CheA into an activated signaling conformation; however, we found that both mutant proteins were regulated in a normal fashion by the transducer Tsr in the presence of CheW. We exploited the activated properties of one of these mutants to investigate whether the CheA subunits within a CheA dimer make equivalent contributions to the mechanism of trans phosphorylation. Our results indicate that CheA trans phosphorylation may involve active-site residues that are located both in cis and in trans to the autophosphorylation site and that the two protomers of a CheA dimer make nonequivalent contributions in determining the affinity of the ATP-binding site(s) of CheA. Images PMID:8021207

Molecular spectrum of c-KIT and PDGFRA gene mutations in gastro intestinal stromal tumor: determination of frequency, distribution pattern and identification of novel mutations in Indian patients.

PubMed

Ahmad, Firoz; Lad, Purnima; Bhatia, Simi; Das, Bibhu Ranjan

2015-01-01

KIT and PDGFRA gene mutations are the major genetic alterations seen in gastrointestinal stromal tumors (GISTs) and are being used clinically for predicting response to imatinib therapy. In the current study, we set out to explore the frequency and distribution pattern of c-KIT (exons 9, 11 and 13) and PDGFRA (exons 12 and 18) by direct sequencing in a series of 70 Indian GIST cases. Overall, 27 (38.5 %) and 4 (5.7 %) of the cases had c-KIT and PDGFRA mutations, respectively. Majority of KIT mutations involved exon 11 (85.7 %), followed by exon 9 (14.3 %), while none showed exon 13 mutation. Most exon 9 mutations showed Ala503-Tyr504 duplication, while one had novel point mutation at codon 476 (S476G). In contrast to exon 9 mutations, most exon 11 mutations were in-frame deletions (79 %, 19/24), predominantly at codons 550-560, while remaining exon 11 mutant cases were point mutations at codons 559, 560, 568, 573 and 575. Interestingly, P573T, Q556_V560delinsH, Q575H and Q575_P577 were novel variations observed in exon 11. The PDGFRA mutations were seen mostly in exon 18, which showed point mutation at codon 842 (D842V), while exon 12 showed a novel indel variation (V561_H570delinsT). No significant correlation between c-KIT/PDGFRA mutations and clinicopathological data was observed. In conclusion, this study highlights the frequency and distribution pattern of c-KIT/PDGFRA mutation in Indian cohort. The current study identified novel variations that added new insights into the genetic heterogeneity of GIST patients. Furthermore, this is the first study to report the presence of PDGFRA mutation from Indian subcontinent.

Clonal evolution in breast cancer revealed by single nucleus genome sequencing.

PubMed

Wang, Yong; Waters, Jill; Leung, Marco L; Unruh, Anna; Roh, Whijae; Shi, Xiuqing; Chen, Ken; Scheet, Paul; Vattathil, Selina; Liang, Han; Multani, Asha; Zhang, Hong; Zhao, Rui; Michor, Franziska; Meric-Bernstam, Funda; Navin, Nicholas E

2014-08-14

Sequencing studies of breast tumour cohorts have identified many prevalent mutations, but provide limited insight into the genomic diversity within tumours. Here we developed a whole-genome and exome single cell sequencing approach called nuc-seq that uses G2/M nuclei to achieve 91% mean coverage breadth. We applied this method to sequence single normal and tumour nuclei from an oestrogen-receptor-positive (ER(+)) breast cancer and a triple-negative ductal carcinoma. In parallel, we performed single nuclei copy number profiling. Our data show that aneuploid rearrangements occurred early in tumour evolution and remained highly stable as the tumour masses clonally expanded. In contrast, point mutations evolved gradually, generating extensive clonal diversity. Using targeted single-molecule sequencing, many of the diverse mutations were shown to occur at low frequencies (<10%) in the tumour mass. Using mathematical modelling we found that the triple-negative tumour cells had an increased mutation rate (13.3×), whereas the ER(+) tumour cells did not. These findings have important implications for the diagnosis, therapeutic treatment and evolution of chemoresistance in breast cancer.

Role of epistasis on the fixation probability of a non-mutator in an adapted asexual population.

PubMed

James, Ananthu

2016-10-21

The mutation rate of a well adapted population is prone to reduction so as to have a lower mutational load. We aim to understand the role of epistatic interactions between the fitness affecting mutations in this process. Using a multitype branching process, the fixation probability of a single non-mutator emerging in a large asexual mutator population is analytically calculated here. The mutator population undergoes deleterious mutations at constant, but at a much higher rate than that of the non-mutator. We find that antagonistic epistasis lowers the chances of mutation rate reduction, while synergistic epistasis enhances it. Below a critical value of epistasis, the fixation probability behaves non-monotonically with variation in the mutation rate of the background population. Moreover, the variation of this critical value of the epistasis parameter with the strength of the mutator is discussed in the appendix. For synergistic epistasis, when selection is varied, the fixation probability reduces overall, with damped oscillations. Copyright © 2016 Elsevier Ltd. All rights reserved.

A new nonsense mutation in the NF1 gene with neurofibromatosis-Noonan syndrome phenotype.

PubMed

Yimenicioğlu, Sevgi; Yakut, Ayten; Karaer, Kadri; Zenker, Martin; Ekici, Arzu; Carman, Kürşat Bora

2012-12-01

Neurofibromatosis-Noonan syndrome is a rare autosomal dominant disorder which combines neurofibromatosis type 1 (NF1) features with Noonan syndrome. NF1 gene mutations are reported in the majority of these patients. Sequence analysis of the established genes for Noonan syndrome revealed no mutation; a heterozygous NF1 point mutation c.7549C>T in exon 51, creating a premature stop codon (p.R2517X), had been demonstrated. Neurofibromatosis-Noonan syndrome recently has been considered a subtype of NF1 and caused by different NF1 mutations. We report the case of a 14-year-old boy with neurofibromatosis type 1 with Noonan-like features, who complained of headache with triventricular hydrocephaly and a heterozygous NF1 point mutation c.7549C>T in exon 51.

[Analysis of prevalence of point mutations in codon 12 of oncogene K-ras from non-cancerous samples of cervical cytology positive for type 16 or 18 PVH].

PubMed

Golijow, C D; Mourón, S A; Gómez, M A; Dulout, F N

1999-12-01

Ninety-one non cancerous samples from genital specimens positives for VPH 16 or 18 and 27 non-infected samples as controls were studied. Mutations at codon 12 in K-ras gene was analyzed using enriched alelic PCR technique. Among the samples studied 17.58% showed mutations in this codon. Significant differences were observed between the control group (negative DNA-HPV) and positives DNA-HPV samples (p < 0.01). No differences were found between both viral types in relation to the mutation frequency. The presence of mutations in the K-ras gene in non cancerous cytological samples point out new questions about the role of mutations in proto-oncogenes and the development of cervical cancer.

Genomic Landscape of Colorectal Mucosa and Adenomas in Familial Adenomatous Polyposis

PubMed Central

Borras, Ester; San Lucas, F. Anthony; Chang, Kyle; Zhou, Ruoji; Masand, Gita; Fowler, Jerry; Mork, Maureen E.; You, Y. Nancy; Taggart, Melissa W.; McAllister, Florencia; Jones, David A.; Davies, Gareth E.; Edelmann, Winfried; Ehli, Erik A.; Lynch, Patrick M.; Hawk, Ernest T.; Capella, Gabriel; Scheet, Paul; Vilar, Eduardo

2016-01-01

Purpose The molecular basis of the adenoma to carcinoma transition has been deduced using comparative analysis of genetic alterations observed through the sequential steps of intestinal carcinogenesis. However, comprehensive genomic analyses of adenomas and at-risk mucosa are still lacking. Therefore, our aim was to characterize the genomic landscape of colonic at-risk mucosa and adenomas. Experimental Design We analyzed the mutation profile and copy number changes of 25 adenomas and adjacent mucosa from 12 familial adenomatous polyposis patients using whole-exome sequencing and validated allelic imbalances in 37 adenomas using SNP arrays. We assessed for evidence of clonality and performed estimations on the proportions of driver and passenger mutations using a systems biology approach. Results Adenomas had lower mutational rates than did colorectal cancers and showed recurrent alterations in known cancer-driver genes (APC, KRAS, FBXW7, TCF7L2) and allelic imbalances in chromosomes 5, 7 and 13. Moreover, 80% of adenomas had somatic alterations in WNT pathway genes. Adenomas displayed evidence of multiclonality similar to stage I carcinomas. Strong correlations between mutational rate and patient age were observed in at-risk mucosa and adenomas. Our data indicate that at least 23% of somatic mutations are present in at-risk mucosa prior to adenoma initiation. Conclusions The genomic profiles of at-risk mucosa and adenomas illustrate the evolution from normal tissue to carcinoma via greater resolution of molecular changes at the inflection point of premalignant lesions. Furthermore, substantial genomic variation exists in at-risk mucosa before adenoma formation, and deregulation of the WNT pathway is required to foster carcinogenesis. PMID:27221540

Disease course in patients with autosomal recessive retinitis pigmentosa due to the USH2A gene.

PubMed

Sandberg, Michael A; Rosner, Bernard; Weigel-DiFranco, Carol; McGee, Terri L; Dryja, Thaddeus P; Berson, Eliot L

2008-12-01

To estimate the mean rates of ocular function loss in patients with autosomal recessive retinitis pigmentosa due to USH2A mutations. In 125 patients with USH2A mutations, longitudinal regression was used to estimate mean rates of change in Snellen visual acuity, Goldmann visual field area (V4e white test light), and 30-Hz (cone) full-field electroretinogram amplitude. These rates were compared with those of previously studied cohorts with dominant retinitis pigmentosa due to RHO mutations and with X-linked retinitis pigmentosa due to RPGR mutations. Rates of change in patients with the Cys759Phe mutation, the USH2A mutation associated with nonsyndromic disease, were compared with rates of change in patients with the Glu767fs mutation, the most common USH2A mutation associated with Usher syndrome type II (i.e., retinitis pigmentosa and hearing loss). Mean annual exponential rates of decline for the USH2A patients were 2.6% for visual acuity, 7.0% for visual field area, and 13.2% for electroretinogram amplitude. The rate of acuity loss fell between the corresponding rates for the RHO and RPGR patients, whereas the rates for field and ERG amplitude loss were faster than those for the RHO and RPGR patients. No significant differences were found for patients with the Cys759Phe mutation versus patients with the Glu767fs mutation. On average, USH2A patients lose visual acuity faster than RHO patients and slower than RPGR patients. USH2A patients lose visual field and cone electroretinogram amplitude faster than patients with RHO or RPGR mutations. Patients with a nonsyndromic USH2A mutation have the same retinal disease course as patients with syndromic USH2A disease.

COTIP: Cotton TILLING Platform, a Resource for Plant Improvement and Reverse Genetic Studies

PubMed Central

Aslam, Usman; Cheema, Hafiza M. N.; Ahmad, Sheraz; Khan, Iqrar A.; Malik, Waqas; Khan, Asif A.

2016-01-01

Cotton is cultivated worldwide for its white fiber, of which around 90% is tetraploid upland cotton (Gossypium hirsutum L.) carrying both A and D genome. Since centuries, yield increasing efforts for the cotton crop by conventional breeding approaches have caused an extensive erosion of natural genetic variability. Mutation based improvement strategies provide an effective way of creating new allelic variations. Targeting Induced Local Lesions IN Genomes (TILLING) provides a mutation based reverse genetic strategy to create and evaluate induced genetic variability at DNA level. Here, we report development and testing of TILLING populations of allotetraploid cotton (G. hirsutum) for functional genomic studies and mutation based enrichment of cotton genetic resources. Seed of two cotton cultivars “PB-899 and PB-900” were mutagenized with 0.3 and 0.2% (v/v) ethyl methanesulfonate, respectively. The phenotyping of M1 and M2 populations presented numerous mutants regarding the branching pattern, leaf morphology, disease resistance, photosynthetic lesions and flower sterility. Molecular screening for point mutations was performed by TILLING PCR aided CEL1 mismatch cleavage. To estimate the mutation frequency in the mutant genomes, five gene classes were TILLed in 8000 M2 plants of each var. “PB-899” and “PB-900.” These include actin (GhACT), Pectin Methyl Esterase (GhPME), sucrose synthase (GhSUS), resistance gene analog, and defense response gene (DRGs). The var. PB-899 was harboring 47% higher mutation induction rate than PB-900. The highest rate of mutation frequency was identified for NAC-TF5 (EU706348) of DRGs class, ranging from 1/58 kb in PB-899 to 1/105 kb in PB-900. The mutation screening assay revealed the presence of significant proportion of induced mutations in cotton TILLING populations such as 1/153 kb and 1/326 kb in var. “PB-899” and “PB-900,” respectively. The establishment of a cotton TILLING platform (COTIP) and data obtained from the resource TILLING population suggest its effectiveness in widening the genetic bases of cotton for improvement and utilizing it for subsequent reverse genetic studies of various genes. PMID:28082993

Selective gene amplification to detect the T790M mutation in plasma from patients with advanced non-small cell lung cancer (NSCLC) who have developed epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) resistance.

PubMed

Nishikawa, Shingo; Kimura, Hideharu; Koba, Hayato; Yoneda, Taro; Watanabe, Satoshi; Sakai, Tamami; Hara, Johsuke; Sone, Takashi; Kasahara, Kazuo; Nakao, Shinji

2018-03-01

The epidermal growth factor receptor (EGFR) T790M mutation is associated with resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in non-small cell lung cancer (NSCLC). However, tissues for the genotyping of the EGFR T790M mutation can be difficult to obtain in a clinical setting. The aims of this study were to evaluate a blood-based, non-invasive approach to detecting the EGFR T790M mutation in advanced NSCLC patients using the PointMan™ EGFR DNA enrichment kit, which is a novel method for the selective amplification of specific genotype sequences. Blood samples were collected from NSCLC patients who had activating EGFR mutations and who were resistant to EGFR-TKI treatment. Using cell-free DNA (cfDNA) from plasma, EGFR T790M mutations were amplified using the PointMan™ enrichment kit, and all the reaction products were confirmed using direct sequencing. The concentrations of plasma DNA were then determined using quantitative real-time PCR. Nineteen patients were enrolled, and 12 patients (63.2%) were found to contain EGFR T790M mutations in their cfDNA, as detected by the kit. T790M mutations were detected in tumor tissues in 12 cases, and 11 of these cases (91.7%) also exhibited the T790M mutation in cfDNA samples. The concentrations of cfDNA were similar between patients with the T790M mutation and those without the mutation. The PointMan™ kit provides a useful method for determining the EGFR T790M mutation status in cfDNA.

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.

An alternative derivation of the stationary distribution of the multivariate neutral Wright-Fisher model for low mutation rates with a view to mutation rate estimation from site frequency data.

PubMed

Schrempf, Dominik; Hobolth, Asger

2017-04-01

Recently, Burden and Tang (2016) provided an analytical expression for the stationary distribution of the multivariate neutral Wright-Fisher model with low mutation rates. In this paper we present a simple, alternative derivation that illustrates the approximation. Our proof is based on the discrete multivariate boundary mutation model which has three key ingredients. First, the decoupled Moran model is used to describe genetic drift. Second, low mutation rates are assumed by limiting mutations to monomorphic states. Third, the mutation rate matrix is separated into a time-reversible part and a flux part, as suggested by Burden and Tang (2016). An application of our result to data from several great apes reveals that the assumption of stationarity may be inadequate or that other evolutionary forces like selection or biased gene conversion are acting. Furthermore we find that the model with a reversible mutation rate matrix provides a reasonably good fit to the data compared to the one with a non-reversible mutation rate matrix. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Accumulation of Spontaneous Mutations in the Ciliate Tetrahymena thermophila

PubMed Central

Long, Hong-An; Paixão, Tiago; Azevedo, Ricardo B. R.; Zufall, Rebecca A.

2013-01-01

Knowledge of the rate and fitness effects of mutations is essential for understanding the process of evolution. Mutations are inherently difficult to study because they are rare and are frequently eliminated by natural selection. In the ciliate Tetrahymena thermophila, mutations can accumulate in the germline genome without being exposed to selection. We have conducted a mutation accumulation (MA) experiment in this species. Assuming that all mutations are deleterious and have the same effect, we estimate that the deleterious mutation rate per haploid germline genome per generation is U = 0.0047 (95% credible interval: 0.0015, 0.0125), and that germline mutations decrease fitness by s = 11% when expressed in a homozygous state (95% CI: 4.4%, 27%). We also estimate that deleterious mutations are partially recessive on average (h = 0.26; 95% CI: –0.022, 0.62) and that the rate of lethal mutations is <10% of the deleterious mutation rate. Comparisons between the observed evolutionary responses in the germline and somatic genomes and the results from individual-based simulations of MA suggest that the two genomes have similar mutational parameters. These are the first estimates of the deleterious mutation rate and fitness effects from the eukaryotic supergroup Chromalveolata and are within the range of those of other eukaryotes. PMID:23934880

Role of the mismatch repair gene, Msh6, in suppressing genome instability and radiation-induced mutations

PubMed Central

Barrera-Oro, Julio; Liu, Tzu-Yang; Gorden, Erin; Kucherlapati, Raju; Shao, Changshun; Tischfield, Jay A

2008-01-01

Mismatch repair (MMR) is critical for preserving genomic integrity. Failure of this system can accelerate somatic mutation and increase the risk of developing cancer. MSH6, in complex with MSH2, is the MMR protein that mediates DNA repair through the recognition of 1- and 2-bp mismatches. To evaluate the effects of MSH6 deficiency on genomic stability we compared the frequency of in vivo loss of heterozygosity (LOH) between MSH6-proficient and deficient, 129S2 x C57BL/6 F1 hybrid mice that were heterozygous for our reporter gene Aprt. We recovered mutant cells that had functionally lost APRT protein activity and categorized the spectrum of mutations responsible for the LOH events. We also measured the mutant frequency at the X-linked gene, Hprt, as a second reporter for point mutation. In Msh6−/−Aprt+/− mice, mutation frequency at Aprt was elevated in both T cells and fibroblasts by 2.5-fold and 5.7-fold, respectively, over Msh6+/+Aprt+/− littermate controls. While a modest increase in mitotic recombination (MR) was observed in MSH6-deficient fibroblasts compared to wild type controls, point mutation was the predominant mechanism leading to APRT deficiency in both cell types. Base substitution, consisting of multiple types of transitions, accounted for all of the point mutations identified within the Aprt coding region. We also assessed the role of MSH6 in preventing mutations caused by a common environmental mutagen, ionizing radiation (IR). In Msh6−/−Aprt+/− mice, 4 Gy of X-irradiation induced a significant increase in point mutations at both Aprt and Hprt in T cells, but not in fibroblasts. These findings indicate that MutSα reduces spontaneous and IR-induced mutation in a cell-type dependant manner. PMID:18538799

RNA Recombination Enhances Adaptability and Is Required for Virus Spread and Virulence.

PubMed

Xiao, Yinghong; Rouzine, Igor M; Bianco, Simone; Acevedo, Ashley; Goldstein, Elizabeth Faul; Farkov, Mikhail; Brodsky, Leonid; Andino, Raul

2016-04-13

Mutation and recombination are central processes driving microbial evolution. A high mutation rate fuels adaptation but also generates deleterious mutations. Recombination between two different genomes may resolve this paradox, alleviating effects of clonal interference and purging deleterious mutations. Here we demonstrate that recombination significantly accelerates adaptation and evolution during acute virus infection. We identified a poliovirus recombination determinant within the virus polymerase, mutation of which reduces recombination rates without altering replication fidelity. By generating a panel of variants with distinct mutation rates and recombination ability, we demonstrate that recombination is essential to enrich the population in beneficial mutations and purge it from deleterious mutations. The concerted activities of mutation and recombination are key to virus spread and virulence in infected animals. These findings inform a mathematical model to demonstrate that poliovirus adapts most rapidly at an optimal mutation rate determined by the trade-off between selection and accumulation of detrimental mutations. Copyright © 2016 Elsevier Inc. All rights reserved.

Discovery of Point Mutations in the Voltage-Gated Sodium Channel from African Aedes aegypti Populations: Potential Phylogenetic Reasons for Gene Introgression.

PubMed

Kawada, Hitoshi; Higa, Yukiko; Futami, Kyoko; Muranami, Yuto; Kawashima, Emiko; Osei, Joseph H N; Sakyi, Kojo Yirenkyi; Dadzie, Samuel; de Souza, Dziedzom K; Appawu, Maxwell; Ohta, Nobuo; Suzuki, Takashi; Minakawa, Noboru

2016-06-01

Yellow fever is endemic in some countries in Africa, and Aedes aegpyti is one of the most important vectors implicated in the outbreak. The mapping of the nation-wide distribution and the detection of insecticide resistance of vector mosquitoes will provide the beneficial information for forecasting of dengue and yellow fever outbreaks and effective control measures. High resistance to DDT was observed in all mosquito colonies collected in Ghana. The resistance and the possible existence of resistance or tolerance to permethrin were suspected in some colonies. High frequencies of point mutations at the voltage-gated sodium channel (F1534C) and one heterozygote of the other mutation (V1016I) were detected, and this is the first detection on the African continent. The frequency of F1534C allele and the ratio of F1534C homozygotes in Ae. aegypti aegypti (Aaa) were significantly higher than those in Ae. aegypti formosus (Aaf). We could detect the two types of introns between exon 20 and 21, and the F1534C mutations were strongly linked with one type of intron, which was commonly found in South East Asian and South and Central American countries, suggesting the possibility that this mutation was introduced from other continents or convergently selected after the introgression of Aaa genes from the above area. The worldwide eradication programs in 1940s and 1950s might have caused high selection pressure on the mosquito populations and expanded the distribution of insecticide-resistant Ae. aegypti populations. Selection of the F1534C point mutation could be hypothesized to have taken place during this period. The selection of the resistant population of Ae. aegypti with the point mutation of F1534C, and the worldwide transportation of vector mosquitoes in accordance with human activity such as trading of used tires, might result in the widespread distribution of F1534C point mutation in tropical countries.

Evolution of Escherichia coli to 42 °C and subsequent genetic engineering reveals adaptive mechanisms and novel mutations.

PubMed

Sandberg, Troy E; Pedersen, Margit; LaCroix, Ryan A; Ebrahim, Ali; Bonde, Mads; Herrgard, Markus J; Palsson, Bernhard O; Sommer, Morten; Feist, Adam M

2014-10-01

Adaptive laboratory evolution (ALE) has emerged as a valuable method by which to investigate microbial adaptation to a desired environment. Here, we performed ALE to 42 °C of ten parallel populations of Escherichia coli K-12 MG1655 grown in glucose minimal media. Tightly controlled experimental conditions allowed selection based on exponential-phase growth rate, yielding strains that uniformly converged toward a similar phenotype along distinct genetic paths. Adapted strains possessed as few as 6 and as many as 55 mutations, and of the 144 genes that mutated in total, 14 arose independently across two or more strains. This mutational recurrence pointed to the key genetic targets underlying the evolved fitness increase. Genome engineering was used to introduce the novel ALE-acquired alleles in random combinations into the ancestral strain, and competition between these engineered strains reaffirmed the impact of the key mutations on the growth rate at 42 °C. Interestingly, most of the identified key gene targets differed significantly from those found in similar temperature adaptation studies, highlighting the sensitivity of genetic evolution to experimental conditions and ancestral genotype. Additionally, transcriptomic analysis of the ancestral and evolved strains revealed a general trend for restoration of the global expression state back toward preheat stressed levels. This restorative effect was previously documented following evolution to metabolic perturbations, and thus may represent a general feature of ALE experiments. The widespread evolved expression shifts were enabled by a comparatively scant number of regulatory mutations, providing a net fitness benefit but causing suboptimal expression levels for certain genes, such as those governing flagellar formation, which then became targets for additional ameliorating mutations. Overall, the results of this study provide insight into the adaptation process and yield lessons important for the future implementation of ALE as a tool for scientific research and engineering. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Inefficient coupling between proton transport and ATP synthesis may be the pathogenic mechanism for NARP and Leigh syndrome resulting from the T8993G mutation in mtDNA.

PubMed

Sgarbi, Gianluca; Baracca, Alessandra; Lenaz, Giorgio; Valentino, Lucia M; Carelli, Valerio; Solaini, Giancarlo

2006-05-01

Mutations in the ATP6 gene of mtDNA (mitochondrial DNA) have been shown to cause several different neurological disorders. The product of this gene is ATPase 6, an essential component of the F1F0-ATPase. In the present study we show that the function of the F1F0-ATPase is impaired in lymphocytes from ten individuals harbouring the mtDNA T8993G point mutation associated with NARP (neuropathy, ataxia and retinitis pigmentosa) and Leigh syndrome. We show that the impaired function of both the ATP synthase and the proton transport activity of the enzyme correlates with the amount of the mtDNA that is mutated, ranging from 13-94%. The fluorescent dye RH-123 (Rhodamine-123) was used as a probe to determine whether or not passive proton flux (i.e. from the intermembrane space to the matrix) is affected by the mutation. Under state 3 respiratory conditions, a slight difference in RH-123 fluorescence quenching kinetics was observed between mutant and control mitochondria that suggests a marginally lower F0 proton flux capacity in cells from patients. Moreover, independent of the cellular mutant load the specific inhibitor oligomycin induced a marked enhancement of the RH-123 quenching rate, which is associated with a block in proton conductivity through F0 [Linnett and Beechey (1979) Inhibitors of the ATP synthethase system. Methods Enzymol. 55, 472-518]. Overall, the results rule out the previously proposed proton block as the basis of the pathogenicity of the mtDNA T8993G mutation. Since the ATP synthesis rate was decreased by 70% in NARP patients compared with controls, we suggest that the T8993G mutation affects the coupling between proton translocation through F0 and ATP synthesis on F1. We discuss our findings in view of the current knowledge regarding the rotary mechanism of catalysis of the enzyme.

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.

Sensitive detection of point mutation by electrochemiluminescence and DNA ligase-based assay

NASA Astrophysics Data System (ADS)

Zhou, Huijuan; Wu, Baoyan

2008-12-01

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

Confounders of mutation-rate estimators: selection and phenotypic lag in Thermus thermophilus

PubMed Central

Kissling, Grace E.; Grogan, Dennis W.; Drake, John W.

2015-01-01

In a recent description of the rate and character of spontaneous mutation in the hyperthermophilic bacterium Thermus thermophilus, the mutation rate was observed to be substantially lower than seen in several mesophiles. Subsequently, a report appeared indicating that this bacterium maintains an average of about 4.5 genomes per cell. This number of genomes might result in a segregation lag for the expression of a recessive mutation and might therefore lead to an underestimate of the rate of mutation. Here we describe some kinds of problems that may arise when estimating mutation rates and outline ways to adjust the rates accordingly. The emphasis is mainly on differential rates of growth of mutants versus their parents and on various kinds of phenotypic lag. We then apply these methods to the T. thermophilus data and conclude that there is as yet no reliable impact on a previously described rate. PMID:23916418

Looking back and to the future: Are we improving 'cure' in non-small cell lung cancer?

PubMed

Walder, David; O'Brien, Mary

2017-04-01

In surgical series, cancer-free survival at 5 years is often referred to as a cure. In recent years, attempts to improve cure rates in non-small cell lung cancer (NSCLC) have focussed on earlier diagnosis through cost-effective screening programs. Systemic therapies have historically added only a small benefit to overall survival in both the adjuvant and palliative setting. However, in the last two decades, the development of new treatment options has added incremental improvements in NSCLC survival rates. Patients with a targetable sensitising mutation including epidermal growth factor receptor gene mutations and anaplastic lymphoma kinase rearrangements have significantly better prognosis, and many will survive beyond 5 years. Immunotherapy is an effective treatment in selected patients with NSCLC and is set to cause another leap in 5 year survival rates. Although these patients are not free from disease, survival at 5 years may become the more important end-point as NSCLC becomes seen as a chronic oncological disease. Copyright © 2017 Elsevier Ltd. All rights reserved.

Translational autocontrol of the Escherichia coli ribosomal protein S15.

PubMed

Portier, C; Dondon, L; Grunberg-Manago, M

1990-01-20

When rpsO, the gene encoding the ribosomal protein S15 in Escherichia coli, is carried by a multicopy plasmid, the mRNA synthesis rate of S15 increases with the gene dosage but the rate of synthesis of S15 does not rise. A translational fusion between S15 and beta-galactosidase was introduced on the chromosome in a delta lac strain and the expression of beta-galactosidase studied under different conditions. The presence of S15 in trans represses the beta-galactosidase level five- to sixfold, while the synthesis rate of the S15-beta-galactosidase mRNA decreases by only 30 to 50%. These data indicate that S15 is subject to autogenous translational control. Derepressed mutants were isolated and sequenced. All the point mutations map in the second codon of S15, suggesting a location for the operator site that is very near to the translation initiation codon. However, the creation of deletion mutations shows that the operator extends into the 5' non-coding part of the message, thus overlapping the ribosome loading site.

Rates of spontaneous mutation.

PubMed Central

Drake, J W; Charlesworth, B; Charlesworth, D; Crow, J F

1998-01-01

Rates of spontaneous mutation per genome as measured in the laboratory are remarkably similar within broad groups of organisms but differ strikingly among groups. Mutation rates in RNA viruses, whose genomes contain ca. 10(4) bases, are roughly 1 per genome per replication for lytic viruses and roughly 0.1 per genome per replication for retroviruses and a retrotransposon. Mutation rates in microbes with DNA-based chromosomes are close to 1/300 per genome per replication; in this group, therefore, rates per base pair vary inversely and hugely as genome sizes vary from 6 x 10(3) to 4 x 10(7) bases or base pairs. Mutation rates in higher eukaryotes are roughly 0.1-100 per genome per sexual generation but are currently indistinguishable from 1/300 per cell division per effective genome (which excludes the fraction of the genome in which most mutations are neutral). It is now possible to specify some of the evolutionary forces that shape these diverse mutation rates. PMID:9560386

The impact of p53 protein core domain structural alteration on ovarian cancer survival.

PubMed

Rose, Stephen L; Robertson, Andrew D; Goodheart, Michael J; Smith, Brian J; DeYoung, Barry R; Buller, Richard E

2003-09-15

Although survival with a p53 missense mutation is highly variable, p53-null mutation is an independent adverse prognostic factor for advanced stage ovarian cancer. By evaluating ovarian cancer survival based upon a structure function analysis of the p53 protein, we tested the hypothesis that not all missense mutations are equivalent. The p53 gene was sequenced from 267 consecutive ovarian cancers. The effect of individual missense mutations on p53 structure was analyzed using the International Agency for Research on Cancer p53 Mutational Database, which specifies the effects of p53 mutations on p53 core domain structure. Mutations in the p53 core domain were classified as either explained or not explained in structural or functional terms by their predicted effects on protein folding, protein-DNA contacts, or mutation in highly conserved residues. Null mutations were classified by their mechanism of origin. Mutations were sequenced from 125 tumors. Effects of 62 of the 82 missense mutations (76%) could be explained by alterations in the p53 protein. Twenty-three (28%) of the explained mutations occurred in highly conserved regions of the p53 core protein. Twenty-two nonsense point mutations and 21 frameshift null mutations were sequenced. Survival was independent of missense mutation type and mechanism of null mutation. The hypothesis that not all missense mutations are equivalent is, therefore, rejected. Furthermore, p53 core domain structural alteration secondary to missense point mutation is not functionally equivalent to a p53-null mutation. The poor prognosis associated with p53-null mutation is independent of the mutation mechanism.

High prevalence of the point mutation in exon 6 of the xeroderma pigmentosum group A-complementing (XPAC) gene in xeroderma pigmentosum group A patients in Tunisia

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

Nishigori, Chikako; Imamura, Sadao; Yagi, Takashi

1993-11-01

Xeroderma pigmentosum (XP) patients in Tunisia who belong to the genetic complementation group A (XPA) have milder skin symptoms than do Japanese XPA patients. Such difference in the clinical features might be caused by the difference in the site of mutation in the XP A-complementing (XPAC) gene. The purpose of this study is to identify the genetic alterations in the XPAC gene in the Tunisian XPA patients and to investigate the relationship between the clinical symptoms and the genetic alterations. Three sites of mutation in the XPAC gene have been identified in the Japanese XPA patients, and about 85% ofmore » them have a G [yields] C point mutation at the splicing acceptor site of intron 3. The authors found that six (86%) of seven Tunisian XPA patients had a nonsense mutation in codon 228 in exon 6, because of a CGA [yields] TGA point mutation, which can be detected by the HphI RFLP. This type of mutation is the same as those found in two Japanese XPA patients with mild clinical RFLP. Milder skin symptoms in the XPA patients in Tunisia than in those in Japan, despite mostly sunny weather and the unsatisfactory sun protection in Tunisia, should be due to the difference in the mutation site. 11 refs., 2 figs., 2 tabs.« less

Erythrocytosis and Pulmonary Hypertension in a Mouse Model of Human HIF2A Gain of Function Mutation*

PubMed Central

Tan, Qiulin; Kerestes, Heddy; Percy, Melanie J.; Pietrofesa, Ralph; Chen, Li; Khurana, Tejvir S.; Christofidou-Solomidou, Melpo; Lappin, Terence R. J.; Lee, Frank S.

2013-01-01

The central pathway for oxygen-dependent control of red cell mass is the prolyl hydroxylase domain protein (PHD):hypoxia inducible factor (HIF) pathway. PHD site specifically prolyl hydroxylates the transcription factor HIF-α, thereby targeting the latter for degradation. Under hypoxia, this modification is attenuated, allowing stabilized HIF-α to activate target genes, including that for erythropoietin (EPO). Studies employing genetically modified mice point to Hif-2α, one of two main Hif-α isoforms, as being the critical regulator of Epo in the adult mouse. More recently, erythrocytosis patients with heterozygous point mutations in the HIF2A gene have been identified; whether these mutations were polymorphisms unrelated to the phenotype could not be ruled out. In the present report, we characterize a mouse line bearing a G536W missense mutation in the Hif2a gene that corresponds to the first such human mutation identified (G537W). We obtained mice bearing both heterozygous and homozygous mutations at this locus. We find that these mice display, in a mutation dose-dependent manner, erythrocytosis and pulmonary hypertension with a high degree of penetrance. These findings firmly establish missense mutations in HIF-2α as a cause of erythrocytosis, highlight the importance of this HIF-α isoform in erythropoiesis, and point to physiologic consequences of HIF-2α dysregulation. PMID:23640890

Rapid evolution of cis-regulatory sequences via local point mutations

NASA Technical Reports Server (NTRS)

Stone, J. R.; Wray, G. A.

2001-01-01

Although the evolution of protein-coding sequences within genomes is well understood, the same cannot be said of the cis-regulatory regions that control transcription. Yet, changes in gene expression are likely to constitute an important component of phenotypic evolution. We simulated the evolution of new transcription factor binding sites via local point mutations. The results indicate that new binding sites appear and become fixed within populations on microevolutionary timescales under an assumption of neutral evolution. Even combinations of two new binding sites evolve very quickly. We predict that local point mutations continually generate considerable genetic variation that is capable of altering gene expression.

CD79B and MYD88 Mutations in Splenic Marginal Zone Lymphoma

PubMed Central

Trøen, Gunhild; Warsame, Abdirashid; Delabie, Jan

2013-01-01

The mutation status of genes involved in the NF-κB signaling pathway in splenic marginal zone lymphoma was examined. DNA sequence analysis of four genes was performed: CD79A, CD79B, CARD11, and MYD88 that are activated through BCR signaling or Toll-like and interleukin signaling. A single point mutation was detected in the CD79B gene (Y196H) in one of ten SMZL cases. Additionally, one point mutation was identified in the MYD88 gene (L265P) in another SMZL case. No mutations were revealed in CD79A or CARD11 genes in these SMZL cases. Neither were mutations detected in these four genes studied in 13 control MZL samples. Interestingly, the two cases with mutations of CD79B and MYD88 showed increased numbers of immunoblasts spread among the smaller and typical marginal zone lymphoma cells. Although SMZL shows few mutations of NF-κB signaling genes, our results indicate that the presence of these mutations is associated with a higher histological grade. PMID:23378931

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

Effects of Darwinian Selection and Mutability on Rate of Broadly Neutralizing Antibody Evolution during HIV-1 Infection

PubMed Central

Sheng, Zizhang; Schramm, Chaim A.; Connors, Mark; Morris, Lynn; Mascola, John R.; Kwong, Peter D.; Shapiro, Lawrence

2016-01-01

Accumulation of somatic mutations in antibody variable regions is critical for antibody affinity maturation, with HIV-1 broadly neutralizing antibodies (bnAbs) generally requiring years to develop. We recently found that the rate at which mutations accumulate decreases over time, but the mechanism governing this slowing is unclear. In this study, we investigated whether natural selection and/or mutability of the antibody variable region contributed significantly to observed decrease in rate. We used longitudinally sampled sequences of immunoglobulin transcripts of single lineages from each of 3 donors, as determined by next generation sequencing. We estimated the evolutionary rates of the complementarity determining regions (CDRs), which are most significant for functional selection, and found they evolved about 1.5- to 2- fold faster than the framework regions. We also analyzed the presence of AID hotspots and coldspots at different points in lineage development and observed an average decrease in mutability of less than 10 percent over time. Altogether, the correlation between Darwinian selection strength and evolutionary rate trended toward significance, especially for CDRs, but cannot fully explain the observed changes in evolutionary rate. The mutability modulated by AID hotspots and coldspots changes correlated only weakly with evolutionary rates. The combined effects of Darwinian selection and mutability contribute substantially to, but do not fully explain, evolutionary rate change for HIV-1-targeting bnAb lineages. PMID:27191167

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

Coordinated Changes in Mutation and Growth Rates Induced by Genome Reduction.

PubMed

Nishimura, Issei; Kurokawa, Masaomi; Liu, Liu; Ying, Bei-Wen

2017-07-05

Genome size is determined during evolution, but it can also be altered by genetic engineering in laboratories. The systematic characterization of reduced genomes provides valuable insights into the cellular properties that are quantitatively described by the global parameters related to the dynamics of growth and mutation. In the present study, we analyzed a small collection of W3110 Escherichia coli derivatives containing either the wild-type genome or reduced genomes of various lengths to examine whether the mutation rate, a global parameter representing genomic plasticity, was affected by genome reduction. We found that the mutation rates of these cells increased with genome reduction. The correlation between genome length and mutation rate, which has been reported for the evolution of bacteria, was also identified, intriguingly, for genome reduction. Gene function enrichment analysis indicated that the deletion of many of the genes encoding membrane and transport proteins play a role in the mutation rate changes mediated by genome reduction. Furthermore, the increase in the mutation rate with genome reduction was highly associated with a decrease in the growth rate in a nutrition-dependent manner; thus, poorer media showed a larger change that was of higher significance. This negative correlation was strongly supported by experimental evidence that the serial transfer of the reduced genome improved the growth rate and reduced the mutation rate to a large extent. Taken together, the global parameters corresponding to the genome, growth, and mutation showed a coordinated relationship, which might be an essential working principle for balancing the cellular dynamics appropriate to the environment. IMPORTANCE Genome reduction is a powerful approach for investigating the fundamental rules for living systems. Whether genetically disturbed genomes have any specific properties that are different from or similar to those of natively evolved genomes has been under investigation. In the present study, we found that Escherichia coli cells with reduced genomes showed accelerated nucleotide substitution errors (mutation rates), although these cells retained the normal DNA mismatch repair systems. Intriguingly, this finding of correlation between reduced genome size and a higher mutation rate was consistent with the reported evolution of mutation rates. Furthermore, the increased mutation rate was quantitatively associated with a decreased growth rate, indicating that the global parameters related to the genome, growth, and mutation, which represent the amount of genetic information, the efficiency of propagation, and the fidelity of replication, respectively, are dynamically coordinated. Copyright © 2017 Nishimura et al.

PAX5 mutations occur frequently in adult B-cell progenitor acute lymphoblastic leukemia and PAX5 haploinsufficiency is associated with BCR-ABL1 and TCF3-PBX1 fusion genes: a GRAALL study.

PubMed

Familiades, J; Bousquet, M; Lafage-Pochitaloff, M; Béné, M-C; Beldjord, K; De Vos, J; Dastugue, N; Coyaud, E; Struski, S; Quelen, C; Prade-Houdellier, N; Dobbelstein, S; Cayuela, J-M; Soulier, J; Grardel, N; Preudhomme, C; Cavé, H; Blanchet, O; Lhéritier, V; Delannoy, A; Chalandon, Y; Ifrah, N; Pigneux, A; Brousset, P; Macintyre, E A; Huguet, F; Dombret, H; Broccardo, C; Delabesse, E

2009-11-01

Adult and child B-cell progenitor acute lymphoblastic leukemia (BCP-ALL) differ in terms of incidence and prognosis. These disparities are mainly due to the molecular abnormalities associated with these two clinical entities. A genome-wide analysis using oligo SNP arrays recently demonstrated that PAX5 (paired-box domain 5) is the main target of somatic mutations in childhood BCP-ALL being altered in 38.9% of the cases. We report here the most extensive analysis of alterations of PAX5 coding sequence in 117 adult BCP-ALL patients in the unique clinical protocol GRAALL-2003/GRAAPH-2003. Our study demonstrates that PAX5 is mutated in 34% of adult BCP-ALL, mutations being partial or complete deletion, partial or complete amplification, point mutation or fusion gene. PAX5 alterations are heterogeneous consisting in complete loss in 17%, focal deletions in 10%, point mutations in 7% and translocations in 1% of the cases. PAX5 complete loss and PAX5 point mutations differ. PAX5 complete loss seems to be a secondary event and is significantly associated with BCR-ABL1 or TCF3-PBX1 fusion genes and a lower white blood cell count.

Quartz crystal microbalance detection of DNA single-base mutation based on monobase-coded cadmium tellurium nanoprobe.

PubMed

Zhang, Yuqin; Lin, Fanbo; Zhang, Youyu; Li, Haitao; Zeng, Yue; Tang, Hao; Yao, Shouzhuo

2011-01-01

A new method for the detection of point mutation in DNA based on the monobase-coded cadmium tellurium nanoprobes and the quartz crystal microbalance (QCM) technique was reported. A point mutation (single-base, adenine, thymine, cytosine, and guanine, namely, A, T, C and G, mutation in DNA strand, respectively) DNA QCM sensor was fabricated by immobilizing single-base mutation DNA modified magnetic beads onto the electrode surface with an external magnetic field near the electrode. The DNA-modified magnetic beads were obtained from the biotin-avidin affinity reaction of biotinylated DNA and streptavidin-functionalized core/shell Fe(3)O(4)/Au magnetic nanoparticles, followed by a DNA hybridization reaction. Single-base coded CdTe nanoprobes (A-CdTe, T-CdTe, C-CdTe and G-CdTe, respectively) were used as the detection probes. The mutation site in DNA was distinguished by detecting the decreases of the resonance frequency of the piezoelectric quartz crystal when the coded nanoprobe was added to the test system. This proposed detection strategy for point mutation in DNA is proved to be sensitive, simple, repeatable and low-cost, consequently, it has a great potential for single nucleotide polymorphism (SNP) detection. 2011 © The Japan Society for Analytical Chemistry

Mutational spectrum of CDKL5 in early-onset encephalopathies: a study of a large collection of French patients and review of the literature.

PubMed

Nemos, C; Lambert, L; Giuliano, F; Doray, B; Roubertie, A; Goldenberg, A; Delobel, B; Layet, V; N'guyen, M A; Saunier, A; Verneau, F; Jonveaux, P; Philippe, C

2009-10-01

The CDKL5 gene has been implicated in the molecular etiology of early-onset intractable seizures with infantile spasms (IS), severe hypotonia and atypical Rett syndrome (RTT) features. So far, 48 deleterious alleles have been reported in the literature. We screened the CDKL5 gene in a cohort of 177 patients with early-onset seizures, including 30 men and 10 girls with Aicardi syndrome. The screening was negative for all men as well as for women with Aicardi syndrome, excluding the CDKL5 gene as a candidate for this neurodevelopmental disorder. We report 11 additional de novo mutations in CDKL5 in female patients. For the first time, the MLPA approach allowed the identification of a partial deletion encompassing the promoter and the first two exons of CDKL5. The 10-point mutations consist of five missenses (with recurrent amino acid changes at p.Ala40 and p.Arg178), four splicing variants and a 1-base pair duplication. We present a review of all mutated alleles published in the literature. In our study, the overall frequency of mutations in CDKL5 in women with early-onset seizures is around 8.6%, a result comparable with previous reports. Noteworthy, the CDKL5 mutation rate is high (28%) in women with early-onset seizures and IS.

Sleeping Beauty mutagenesis reveals cooperating mutations and pathways in pancreatic adenocarcinoma

PubMed Central

Mann, Karen M.; Ward, Jerrold M.; Yew, Christopher Chin Kuan; Kovochich, Anne; Dawson, David W.; Black, Michael A.; Brett, Benjamin T.; Sheetz, Todd E.; Dupuy, Adam J.; Chang, David K.; Biankin, Andrew V.; Waddell, Nicola; Kassahn, Karin S.; Grimmond, Sean M.; Rust, Alistair G.; Adams, David J.; Jenkins, Nancy A.; Copeland, Neal G.

2012-01-01

Pancreatic cancer is one of the most deadly cancers affecting the Western world. Because the disease is highly metastatic and difficult to diagnosis until late stages, the 5-y survival rate is around 5%. The identification of molecular cancer drivers is critical for furthering our understanding of the disease and development of improved diagnostic tools and therapeutics. We have conducted a mutagenic screen using Sleeping Beauty (SB) in mice to identify new candidate cancer genes in pancreatic cancer. By combining SB with an oncogenic Kras allele, we observed highly metastatic pancreatic adenocarcinomas. Using two independent statistical methods to identify loci commonly mutated by SB in these tumors, we identified 681 loci that comprise 543 candidate cancer genes (CCGs); 75 of these CCGs, including Mll3 and Ptk2, have known mutations in human pancreatic cancer. We identified point mutations in human pancreatic patient samples for another 11 CCGs, including Acvr2a and Map2k4. Importantly, 10% of the CCGs are involved in chromatin remodeling, including Arid4b, Kdm6a, and Nsd3, and all SB tumors have at least one mutated gene involved in this process; 20 CCGs, including Ctnnd1, Fbxo11, and Vgll4, are also significantly associated with poor patient survival. SB mutagenesis provides a rich resource of mutations in potential cancer drivers for cross-comparative analyses with ongoing sequencing efforts in human pancreatic adenocarcinoma. PMID:22421440

Identification of six novel mutations in Iranian patients with maple syrup urine disease and their in silico analysis.

PubMed

Abiri, Maryam; Karamzadeh, Razieh; Karimipoor, Morteza; Ghadami, Shirin; Alaei, Mohammad Reza; Bagheri, Samira Dabagh; Bagherian, Hamideh; Setoodeh, Aria; Noori-Daloii, Mohammad Reza; Sirous Zeinali

2016-04-01

Maple syrup urine disease (MSUD) is a rare inborn error of branched-chain amino acid metabolism. The disease prevalence is higher in populations with elevated rate of consanguineous marriages such as Iran. Different types of disease causing mutations have been previously reported in BCKDHA, BCKDHB, DBT and DLD genes known to be responsible for MSUD phenotype. In this study, two sets of multiplex polymorphic STR (Short Tandem Repeat) markers linked to the above genes were used to aid in homozygosity mapping in order to find probable pathogenic change(s) in the studied families. The families who showed homozygote haplotype for the BCKDHA gene were subsequently sequenced. Our findings showed that exons 2, 4 and 6 contain most of the mutations which are novel. The changes include two single nucleotide deletion (i.e. c. 143delT and c.702delT), one gross deletion covering the whole exon four c.(375+1_376-1)_(8849+1_885-1), two splice site changes (c.1167+1G>T, c. 288+1G>A), and one point mutation (c.731G>A). Computational approaches were used to analyze these two novel mutations in terms of their impact on protein structure. Computational structural modeling indicated that these mutations might affect structural stability and multimeric assembly of branched-chain α-keto acid dehydrogenase complex (BCKDC). Copyright © 2016. Published by Elsevier B.V.

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.

Subminimal Inhibitory Concentrations of the Disinfectant Benzalkonium Chloride Select for a Tolerant Subpopulation of Escherichia coli with Inheritable Characteristics

PubMed Central

Moen, Birgitte; Rudi, Knut; Bore, Erlend; Langsrud, Solveig

2012-01-01

Exposure of Escherichia coli to a subminimal inhibitory concentration (25% below MIC) of benzalkonium chloride (BC), an antimicrobial membrane-active agent commonly used in medical and food-processing environments, resulted in cell death and changes in cell morphology (filamentation). A small subpopulation (1–5% of the initial population) survived and regained similar morphology and growth rate as non-exposed cells. This subpopulation maintained tolerance to BC after serial transfers in medium without BC. To withstand BC during regrowth the cells up regulated a drug efflux associated gene (the acrB gene, member of the AcrAB-TolC efflux system) and changed expression of outer membrane porin genes (ompFW) and several genes involved in protecting the cell from the osmotic- and oxidative stress. Cells pre-exposed to osmotic- and oxidative stress (sodium chloride, salicylic acid and methyl viologen) showed higher tolerance to BC. A control and two selected isolates showing increased BC-tolerance after regrowth in BC was genome sequenced. No common point mutations were found in the BC- isolates but one point mutation in gene rpsA (Ribosomal protein S1) was observed in one of the isolates. The observed tolerance can therefore not solely be explained by the observed point mutation. The results indicate that there are several different mechanisms responsible for the regrowth of a tolerant subpopulation in BC, both BC-specific and general stress responses, and that sub-MIC of BC may select for phenotypic variants in a sensitive E. coli culture. PMID:22605968

Subminimal inhibitory concentrations of the disinfectant benzalkonium chloride select for a tolerant subpopulation of Escherichia coli with inheritable characteristics.

PubMed

Moen, Birgitte; Rudi, Knut; Bore, Erlend; Langsrud, Solveig

2012-01-01

Exposure of Escherichia coli to a subminimal inhibitory concentration (25% below MIC) of benzalkonium chloride (BC), an antimicrobial membrane-active agent commonly used in medical and food-processing environments, resulted in cell death and changes in cell morphology (filamentation). A small subpopulation (1-5% of the initial population) survived and regained similar morphology and growth rate as non-exposed cells. This subpopulation maintained tolerance to BC after serial transfers in medium without BC. To withstand BC during regrowth the cells up regulated a drug efflux associated gene (the acrB gene, member of the AcrAB-TolC efflux system) and changed expression of outer membrane porin genes (ompFW) and several genes involved in protecting the cell from the osmotic- and oxidative stress. Cells pre-exposed to osmotic- and oxidative stress (sodium chloride, salicylic acid and methyl viologen) showed higher tolerance to BC. A control and two selected isolates showing increased BC-tolerance after regrowth in BC was genome sequenced. No common point mutations were found in the BC- isolates but one point mutation in gene rpsA (Ribosomal protein S1) was observed in one of the isolates. The observed tolerance can therefore not solely be explained by the observed point mutation. The results indicate that there are several different mechanisms responsible for the regrowth of a tolerant subpopulation in BC, both BC-specific and general stress responses, and that sub-MIC of BC may select for phenotypic variants in a sensitive E. coli culture.

Methods for Determining Spontaneous Mutation Rates

PubMed Central

Foster, Patricia L.

2007-01-01

Spontaneous mutations arise as a result of cellular processes that act upon or damage DNA. Accurate determination of spontaneous mutation rates can contribute to our understanding of these processes and the enzymatic pathways that deal with them. The methods that are used to calculate mutation rates are based on the model for the expansion of mutant clones originally described by Luria and Delbrück and extended by Lea and Coulson. The accurate determination of mutation rates depends on understanding the strengths and limitations of these methods and how to optimize a fluctuation assay for a given method. This chapter describes the proper design of a fluctuation assay, several of the methods used to calculate mutation rates, and ways to evaluate the results statistically. PMID:16793403

Species abundance distributions in neutral models with immigration or mutation and general lifetimes.

PubMed

Lambert, Amaury

2011-07-01

We consider a general, neutral, dynamical model of biodiversity. Individuals have i.i.d. lifetime durations, which are not necessarily exponentially distributed, and each individual gives birth independently at constant rate λ. Thus, the population size is a homogeneous, binary Crump-Mode-Jagers process (which is not necessarily a Markov process). We assume that types are clonally inherited. We consider two classes of speciation models in this setting. In the immigration model, new individuals of an entirely new species singly enter the population at constant rate μ (e.g., from the mainland into the island). In the mutation model, each individual independently experiences point mutations in its germ line, at constant rate θ. We are interested in the species abundance distribution, i.e., in the numbers, denoted I(n)(k) in the immigration model and A(n)(k) in the mutation model, of species represented by k individuals, k = 1, 2, . . . , n, when there are n individuals in the total population. In the immigration model, we prove that the numbers (I(t)(k); k ≥ 1) of species represented by k individuals at time t, are independent Poisson variables with parameters as in Fisher's log-series. When conditioning on the total size of the population to equal n, this results in species abundance distributions given by Ewens' sampling formula. In particular, I(n)(k) converges as n → ∞ to a Poisson r.v. with mean γ/k, where γ : = μ/λ. In the mutation model, as n → ∞, we obtain the almost sure convergence of n (-1) A(n)(k) to a nonrandom explicit constant. In the case of a critical, linear birth-death process, this constant is given by Fisher's log-series, namely n(-1) A(n)(k) converges to α(k)/k, where α : = λ/(λ + θ). In both models, the abundances of the most abundant species are briefly discussed.

The Cardiomyopathy Mutation, R146G Troponin I, Stabilizes the Intermediate "C" State of Regulated Actin under High- and Low-Free Ca(2+) Conditions.

PubMed

Johnson, Dylan; Mathur, Mohit C; Kobayashi, Tomoyoshi; Chalovich, Joseph M

2016-08-16

The R146G mutation of troponin I (TnI) is associated with hypertrophic cardiomyopathy in humans. Earlier data pointed to stabilization of the intermediate, C state, of actin-tropomyosin-troponin by this mutant. Because cardiac disorders appear to be linked to changes in regulated actin distributions, we determined the extent to which the R146G TnI mutant alters the distribution of states at low and high Ca(2+) concentrations. We show, from measurements of the kcat for actin-activated ATPase activity at saturating Ca(2+) concentrations, that R146G TnI reduced the population of the active, M, state to 25% of the wild-type level. Together with acrylodan-tropomyosin fluorescence measurements of the B state, it appeared that the C state was populated at ∼91% of the total for the R146G TnI-containing actin filaments. The C state was also more heavily populated at low Ca(2+) concentrations. Acrylodan-tropomyosin fluorescence changes showed a large diminution in the inactive state value relative to the wild-type value without a comparable increase in the active state. Furthermore, the rate of binding of rigor S1 to pyrene-labeled actin filaments containing R146G TnI was faster than the rate of binding to wild-type filaments at low free Ca(2+) concentrations. These results indicate that the inhibitory region of TnI affects the B-C and M-C equilibria of actin-tropomyosin-troponin. The observation that a mutation in the inhibitory region affects the M-C equilibrium may point to a novel regulatory interaction.

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.

A novel mutation in the glucose-6-phosphate dehydrogenase gene in a subject with chronic nonspherocytic hemolytic anemia--characterization of enzyme using yeast expression system and molecular modeling.

PubMed

Grabowska, Dorota; Jablonska-Skwiecinska, Ewa; Plochocka, Danuta; Chelstowska, Anna; Lewandowska, Irmina; Witos, Iwona; Majewska, Zofia; Rokicka-Milewska, Roma; Burzynska, Beata

2004-01-01

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzymopathy. Human G6PD gene is highly polymorphic, with over 130 mutations identified, many of which cause hemolytic anemia. We studied a novel point mutation in the G6PD gene 1226 C-->G, predicting the proline 409 to arginine substitution (G6PD Suwalki). We expressed the human wild-type and mutated G6PD gene in yeast Saccharomyces cerevisiae which allowed the characterization of the Suwalki variant. We showed that human wild-type, as well as the mutated (1226 C-->G) G6PD gene, functionally complemented the phenotype displayed by the yeast strain with disruption of the ZWF1 gene (homologue of the human G6PD gene). Comparison of wild-type (wt) human G6PD purified from yeast and from blood shows no significant differences in the Km values for G6P and in the utilization rate for the substrate analogue, 2-deoxyG6P. The P409R substitution leads to drastic changes in G6PD kinetics. The specific activity as well as stability of mutated G6PD is also significantly reduced. Besides this, the effect of this mutation was analyzed using a model of the tertiary structure of the human enzyme. The localization of the P409R mutation suggests that it may influence the stability of the whole protein by changing tetramer interactions and disturbing the binding of structural NADP+.

Efficient gene-driven germ-line point mutagenesis of C57BL/6J mice

PubMed Central

Michaud, Edward J; Culiat, Cymbeline T; Klebig, Mitchell L; Barker, Paul E; Cain, KT; Carpenter, Debra J; Easter, Lori L; Foster, Carmen M; Gardner, Alysyn W; Guo, ZY; Houser, Kay J; Hughes, Lori A; Kerley, Marilyn K; Liu, Zhaowei; Olszewski, Robert E; Pinn, Irina; Shaw, Ginger D; Shinpock, Sarah G; Wymore, Ann M; Rinchik, Eugene M; Johnson, Dabney K

2005-01-01

Background Analysis of an allelic series of point mutations in a gene, generated by N-ethyl-N-nitrosourea (ENU) mutagenesis, is a valuable method for discovering the full scope of its biological function. Here we present an efficient gene-driven approach for identifying ENU-induced point mutations in any gene in C57BL/6J mice. The advantage of such an approach is that it allows one to select any gene of interest in the mouse genome and to go directly from DNA sequence to mutant mice. Results We produced the Cryopreserved Mutant Mouse Bank (CMMB), which is an archive of DNA, cDNA, tissues, and sperm from 4,000 G1 male offspring of ENU-treated C57BL/6J males mated to untreated C57BL/6J females. Each mouse in the CMMB carries a large number of random heterozygous point mutations throughout the genome. High-throughput Temperature Gradient Capillary Electrophoresis (TGCE) was employed to perform a 32-Mbp sequence-driven screen for mutations in 38 PCR amplicons from 11 genes in DNA and/or cDNA from the CMMB mice. DNA sequence analysis of heteroduplex-forming amplicons identified by TGCE revealed 22 mutations in 10 genes for an overall mutation frequency of 1 in 1.45 Mbp. All 22 mutations are single base pair substitutions, and nine of them (41%) result in nonconservative amino acid substitutions. Intracytoplasmic sperm injection (ICSI) of cryopreserved spermatozoa into B6D2F1 or C57BL/6J ova was used to recover mutant mice for nine of the mutations to date. Conclusions The inbred C57BL/6J CMMB, together with TGCE mutation screening and ICSI for the recovery of mutant mice, represents a valuable gene-driven approach for the functional annotation of the mammalian genome and for the generation of mouse models of human genetic diseases. The ability of ENU to induce mutations that cause various types of changes in proteins will provide additional insights into the functions of mammalian proteins that may not be detectable by knockout mutations. PMID:16300676

Decreased fracture rate, pharmacogenetics and BMD response in 79 Swedish children with osteogenesis imperfecta types I, III and IV treated with Pamidronate.

PubMed

Lindahl, K; Kindmark, A; Rubin, C-J; Malmgren, B; Grigelioniene, G; Söderhäll, S; Ljunggren, Ö; Åström, E

2016-06-01

Osteogenesis imperfecta (OI) is an inherited heterogeneous bone fragility disorder, usually caused by collagen I mutations. It is well established that bisphosphonate treatment increases lumbar spine (LS) bone mineral density (BMD), as well as improves vertebral geometry in severe OI; however, fracture reduction has been difficult to prove, pharmacogenetic studies are scarce, and it is not known at which age, or severity of disease, treatment should be initiated. COL1A1 and COL1A2 were analyzed in 79 children with OI (type I n=33, type III n=25 and type IV n=21) treated with Pamidronate. Data on LS BMD, height, and radiologically confirmed non-vertebral and vertebral fractures were collected prior to, and at several time points during treatment. An increase in LS BMD Z-score was observed for all types of OI, and a negative correlation to Δ LS BMD was observed for both age and LS BMD Z-score at treatment initiation. Supine height Z-scores were not affected by Pamidronate treatment, The fracture rate was reduced for all OI types at all time points during treatment (overall p<0.0003, <0.0001 and 0.0003 for all OI types I, III and IV respectively). The reduced fracture rate was maintained for types I and IV, while an additional decrease was observed over time for type III. The fracture rate was reduced also in individuals with continued low BMD after >4yrs Pamidronate. Twice as many boys as girls with OI type I were treated with Pamidronate, and the fracture rate the year prior treatment was 2.2 times higher for boys (p=0.0236). Greater Δ LS BMD, but smaller Δ fracture numbers were observed on Pamidronate for helical glycine mutations in COL1A1 vs. COL1A2. Vertebral compression fractures did not progress in any individual during treatment; however, they did not improve in 9%, and these individuals were all >11years of age at treatment initiation (p<0.0001). Pamidronate treatment in children with all types of OI increased LS BMD, decreased fracture rate, and improved vertebral compression fractures. Fracture reduction was prompt and maintained during treatment, irrespective of age at treatment initiation and collagen I mutation type. Copyright © 2016 Elsevier Inc. All rights reserved.

Effects of missense mutations in sortase A gene on enzyme activity in Streptococcus mutans.

PubMed

Zhuang, P L; Yu, L X; Tao, Y; Zhou, Y; Zhi, Q H; Lin, H C

2016-04-11

Streptococcus mutans (S. mutans) is the major aetiological agent of dental caries, and the transpeptidase Sortase A (SrtA) plays a major role in cariogenicity. The T168G and G470A missense mutations in the srtA gene may be linked to caries susceptibility, as demonstrated in our previous studies. This study aimed to investigate the effects of these missense mutations of the srtA gene on SrtA enzyme activity in S. mutans. The point mutated recombinant S.mutans T168G and G470A sortases were expressed in expression plasmid pET32a. S. mutans UA159 sortase coding gene srtA was used as the template for point mutation. Enzymatic activity was assessed by quantifying increases in the fluorescence intensity generated when a substrate Dabcyl-QALPNTGEE-Edans was cleaved by SrtA. The kinetic constants were calculated based on the curve fit for the Michaelis-Menten equation. SrtA△N40(UA159) and the mutant enzymes, SrtA△N40(D56E) and SrtA△N40(R157H), were expressed and purified. A kinetic analysis showed that the affinity of SrtA△N40(D56E) and SrtA△N40(R157H) remained approximately equal to the affinity of SrtA△N40(UA159), as determined by the Michaelis constant (K m ). However, the catalytic rate constant (k cat ) and catalytic efficiency (k cat /K m ) of SrtA△N40(D56E) were reduced compared with those of SrtA△N40(R157H) and SrtA△N40(UA159), whereas the k cat and k cat /K m values of SrtA△N40(R157H) were slightly lower than those of SrtA△N40(UA159). The findings of this study indicate that the T168G missense mutation of the srtA gene results in a significant reduction in enzymatic activity compared with S. mutans UA159, suggesting that the T168G missense mutation of the srtA gene may be related to low cariogenicity.

Rates of loss of heterozygosity and mitotic recombination in NF2 schwannomas, sporadic vestibular schwannomas and schwannomatosis schwannomas.

PubMed

Hadfield, K D; Smith, M J; Urquhart, J E; Wallace, A J; Bowers, N L; King, A T; Rutherford, S A; Trump, D; Newman, W G; Evans, D G

2010-11-25

Biallelic inactivation of the NF2 gene occurs in the majority of schwannomas. This usually involves a combination of a point mutation or multiexon deletion, in conjunction with either a second point mutation or loss of heterozygosity (LOH). We have performed DNA sequence and dosage analysis of the NF2 gene in a panel of 239 schwannoma tumours: 97 neurofibromatosis type 2 (NF2)-related schwannomas, 104 sporadic vestibular schwannomas (VS) and 38 schwannomatosis-related schwannomas. In total, we identified germline NF2 mutations in 86 out of 97 (89%) NF2 patients and a second mutational event in 77 out of 97 (79%). LOH was by far the most common form of second hit. A combination of microsatellite analysis with either conventional comparative genomic hybridization (CGH) or multiplex ligation-dependent probe amplification (MLPA) identified mitotic recombination (MR) as the cause of LOH in 14 out of 72 (19%) total evaluable tumours. Among sporadic VS, at least one NF2 mutation was identified by sequence analysis or MLPA in 65 out of 98 (66%) tumours. LOH occurred in 54 out of 96 (56%) evaluable tumours, but MR only accounted for 5 out of 77 (6%) tested. LOH was present in 28 out of 34 (82%) schwannomatosis-related schwannomas. In all eight patients who had previously tested positive for a germline SMARCB1 mutation, this involved loss of the whole, or part of the long arm, of chromosome 22. In contrast, 5 out of 22 (23%) tumours from patients with no germline SMARCB1 mutation exhibited MR. High-resolution Affymetrix SNP6 genotyping and copy number (CN) analysis (Affymetrix, Santa Clara, CA, USA) were used to determine the chromosomal breakpoint locations in tumours with MR. A range of unique recombination sites, spanning approximately 11.4 Mb, were identified. This study shows that MR is a mechanism of LOH in NF2 and SMARCB1-negative schwannomatosis-related schwannomas, occurring less frequently in sporadic VS. We found no evidence of MR in SMARCB1-positive schwannomatosis, suggesting that susceptibility to MR varies according to the disease context.

Insilico modeling and molecular dynamic simulation of claudin-1 point mutations in HCV infection.

PubMed

Vipperla, Bhavaniprasad; Dass, J Febin Prabhu; Jayanthi, S

2014-01-01

Claudin-1 (CLDN1) in association with envelope glycoprotein (CD81) mediates the fusion of HCV into the cytosol. Recent studies have indicated that point mutations in CLDN1 are important for the entry of hepatitis C virus (HCV). To validate these findings, we employed a computational platform to investigate the structural effect of two point mutations (I32M and E48K). Initially, three-dimensional co-ordinates for CLDN1 receptor sequence were generated. Then, three mutant models were built using the point mutation including a double mutant (I32M/E48K) model from the native model structure. Finally, all the four model structures including the native and three mutant models were subjected to molecular dynamics (MD) simulation for a period of 25 ns to appreciate their dynamic behavior. The MD trajectory files were analyzed using cluster and principal component method. The analysis suggested that either of the single mutation has negligible effect on the overall structure of CLDN1 compared to the double mutant form. However, the double mutant model of CLDN1 shows significant negative impact through the impairment of H-bonds and the simultaneous increase in solvent accessible surface area. Our simulation results are visibly consistent with the experimental report suggesting that the CLDN1 receptor distortion is prominent due to the double mutation with large surface accessibility. This increase in accessible surface area due to the coexistence of double mutation may be presumed as one of the key factor that results in permissive action of HCV attachment and infection.

Single point mutations distributed in 10 soluble and membrane regions of the Nicotiana plumbaginifolia plasma membrane PMA2 H+-ATPase activate the enzyme and modify the structure of the C-terminal region.

PubMed

Morsomme, P; Dambly, S; Maudoux, O; Boutry, M

1998-12-25

The Nicotiana plumbaginifolia pma2 (plasma membrane H+-ATPase) gene is capable of functionally replacing the H+-ATPase genes of the yeast Saccharomyces cerevisiae, provided that the external pH is kept above 5.0. Single point mutations within the pma2 gene were previously identified that improved H+-ATPase activity and allowed yeast growth at pH 4.0. The aim of the present study was to identify most of the PMA2 positions, the mutation of which would lead to improved growth and to determine whether all these mutations result in similar enzymatic and structural modifications. We selected additional mutants in total 42 distinct point mutations localized in 30 codons. They were distributed in 10 soluble and membrane regions of the enzyme. Most mutant PMA2 H+-ATPases were characterized by a higher specific activity, lower inhibition by ADP, and lower stimulation by lysophosphatidylcholine than wild-type PMA2. The mutants thus seem to be constitutively activated. Partial tryptic digestion and immunodetection showed that the PMA2 mutants had a conformational change making the C-terminal region more accessible. These data therefore support the hypothesis that point mutations in various H+-ATPase parts displace the inhibitory C-terminal region, resulting in enzyme activation. The high density of mutations within the first half of the C-terminal region suggests that this part is involved in the interaction between the inhibitory C-terminal region and the rest of the enzyme.

Rate of de novo mutations and the importance of father's age to disease risk.

PubMed

Kong, Augustine; Frigge, Michael L; Masson, Gisli; Besenbacher, Soren; Sulem, Patrick; Magnusson, Gisli; Gudjonsson, Sigurjon A; Sigurdsson, Asgeir; Jonasdottir, Aslaug; Jonasdottir, Adalbjorg; Wong, Wendy S W; Sigurdsson, Gunnar; Walters, G Bragi; Steinberg, Stacy; Helgason, Hannes; Thorleifsson, Gudmar; Gudbjartsson, Daniel F; Helgason, Agnar; Magnusson, Olafur Th; Thorsteinsdottir, Unnur; Stefansson, Kari

2012-08-23

Mutations generate sequence diversity and provide a substrate for selection. The rate of de novo mutations is therefore of major importance to evolution. Here we conduct a study of genome-wide mutation rates by sequencing the entire genomes of 78 Icelandic parent-offspring trios at high coverage. We show that in our samples, with an average father's age of 29.7, the average de novo mutation rate is 1.20 × 10(-8) per nucleotide per generation. Most notably, the diversity in mutation rate of single nucleotide polymorphisms is dominated by the age of the father at conception of the child. The effect is an increase of about two mutations per year. An exponential model estimates paternal mutations doubling every 16.5 years. After accounting for random Poisson variation, father's age is estimated to explain nearly all of the remaining variation in the de novo mutation counts. These observations shed light on the importance of the father's age on the risk of diseases such as schizophrenia and autism.

Adaptive tuning of mutation rates allows fast response to lethal stress in Escherichia coli

PubMed Central

Swings, Toon; Van den Bergh, Bram; Wuyts, Sander; Oeyen, Eline; Voordeckers, Karin; Verstrepen, Kevin J; Fauvart, Maarten; Verstraeten, Natalie; Michiels, Jan

2017-01-01

While specific mutations allow organisms to adapt to stressful environments, most changes in an organism's DNA negatively impact fitness. The mutation rate is therefore strictly regulated and often considered a slowly-evolving parameter. In contrast, we demonstrate an unexpected flexibility in cellular mutation rates as a response to changes in selective pressure. We show that hypermutation independently evolves when different Escherichia coli cultures adapt to high ethanol stress. Furthermore, hypermutator states are transitory and repeatedly alternate with decreases in mutation rate. Specifically, population mutation rates rise when cells experience higher stress and decline again once cells are adapted. Interestingly, we identified cellular mortality as the major force driving the quick evolution of mutation rates. Together, these findings show how organisms balance robustness and evolvability and help explain the prevalence of hypermutation in various settings, ranging from emergence of antibiotic resistance in microbes to cancer relapses upon chemotherapy. DOI: http://dx.doi.org/10.7554/eLife.22939.001 PMID:28460660

Frequent mutations in the p53 tumor suppressor gene in human leukemia T-cell lines.

PubMed Central

Cheng, J; Haas, M

1990-01-01

Human T-cell leukemia and T-cell acute lymphoblastic leukemia cell lines were studied for alterations in the p53 tumor suppressor gene. Southern blot analysis of 10 leukemic T-cell lines revealed no gross genomic deletions or rearrangements. Reverse transcription-polymerase chain reaction analysis of p53 mRNA indicated that all 10 lines produced p53 mRNA of normal size. By direct sequencing of polymerase chain reaction-amplified cDNA, we detected 11 missense and nonsense point mutations in 5 of the 10 leukemic T-cell lines studied. The mutations are primarily located in the evolutionarily highly conserved regions of the p53 gene. One of the five cell lines in which a mutation was detected possesses a homozygous point mutation in both p53 alleles, while the other four cell lines harbor from two to four different point mutations. An allelic study of two of the lines (CEM, A3/Kawa) shows that the two missense mutations found in each line are located on separate alleles, thus both alleles of the p53 gene may have been functionally inactivated by two different point mutations. Since cultured leukemic T-cell lines represent a late, fully tumorigenic stage of leukemic T cells, mutation of both (or more) alleles of the p53 gene may reflect the selection of cells possessing an increasingly tumorigenic phenotype, whether the selection took place in vivo or in vitro. Previously, we have shown that the HSB-2 T-cell acute lymphoblastic leukemia cell line had lost both alleles of the retinoblastoma tumor suppressor gene. Taken together, our data show that at least 6 of 10 leukemic T-cell lines examined may have lost the normal function of a known tumor suppressor gene, suggesting that this class of genes serves a critical role in the generation of fully tumorigenic leukemic T cells. Images PMID:2144611

Rapid polymerase chain reaction screening of Helicobacter pylori chromosomal point mutations.

PubMed

Ge, Z; Taylor, D E

1997-09-01

Microdiversity (within individual genes) in the genomes of different Helicobacter pylori strains has been demonstrated to be more frequent than that seen in other prokaryotes. Point mutations in some genes, such as the vacA and 23S ribosomal RNA genes could result in the alteration of pathogenicity or antibiotic susceptibility of individual H. pylori strains. Development of a simple, rapid, and reliable screening method would be useful in the molecular characterization of genetic variation among different H. pylori strains. The copP gene from H. pylori UA802 was used as a model for developing a mutation screening method. Four point mutations were introduced into the copP gene by in vitro site-directed mutagenesis and were verified by DNA sequencing. The mutated copP gene replaced the wild-type locus by natural transformation and homologous recombination. The site-specific mutants were screened by polymerase chain reaction (PCR) using 3'-end mismatched primers. The origins of the PCR fragments were demonstrated by Southern hybridization with the copP-derived DNA probe. Three of these four mutations were characterized by PCR with the specific primers that contained the 3'-terminal nucleotide complementary only to the mutated nucleotide on both plasmid and chromosomal DNA templates. One mutation was able to be identified with the foregoing primer containing an additional wild-type nucleotide at its 3'-end. Point mutant screening with these specific primers offers 100% sensitivity in the aforementioned conditions. To achieve optimal screening, the concentration of magnesium and the annealing temperature have to be adjusted. The procedure reported in this study is a simple, economical, rapid, and efficient approach in the identification of site-specific mutations on both plasmids and chromosomal DNA. Although the method was developed by using a specified H. pylori gene, it can be extended easily to other genes of interest in H. pylori or other organisms.

An evolutionary reduction principle for mutation rates at multiple Loci.

PubMed

Altenberg, Lee

2011-06-01

A model of mutation rate evolution for multiple loci under arbitrary selection is analyzed. Results are obtained using techniques from Karlin (Evolutionary Biology, vol. 14, pp. 61-204, 1982) that overcome the weak selection constraints needed for tractability in prior studies of multilocus event models.A multivariate form of the reduction principle is found: reduction results at individual loci combine topologically to produce a surface of mutation rate alterations that are neutral for a new modifier allele. New mutation rates survive if and only if they fall below this surface-a generalization of the hyperplane found by Zhivotovsky et al. (Proc. Natl. Acad. Sci. USA 91, 1079-1083, 1994) for a multilocus recombination modifier. Increases in mutation rates at some loci may evolve if compensated for by decreases at other loci. The strength of selection on the modifier scales in proportion to the number of germline cell divisions, and increases with the number of loci affected. Loci that do not make a difference to marginal fitnesses at equilibrium are not subject to the reduction principle, and under fine tuning of mutation rates would be expected to have higher mutation rates than loci in mutation-selection balance.Other results include the nonexistence of 'viability analogous, Hardy-Weinberg' modifier polymorphisms under multiplicative mutation, and the sufficiency of average transmission rates to encapsulate the effect of modifier polymorphisms on the transmission of loci under selection. A conjecture is offered regarding situations, like recombination in the presence of mutation, that exhibit departures from the reduction principle. Constraints for tractability are: tight linkage of all loci, initial fixation at the modifier locus, and mutation distributions comprising transition probabilities of reversible Markov chains.

Sample features associated with success rates in population-based EGFR mutation testing.

PubMed

Shiau, Carolyn J; Babwah, Jesse P; da Cunha Santos, Gilda; Sykes, Jenna R; Boerner, Scott L; Geddie, William R; Leighl, Natasha B; Wei, Cuihong; Kamel-Reid, Suzanne; Hwang, David M; Tsao, Ming-Sound

2014-07-01

Epidermal growth factor receptor (EGFR) mutation testing has become critical in the treatment of patients with advanced non-small-cell lung cancer. This study involves a large cohort and epidemiologically unselected series of EGFR mutation testing for patients with nonsquamous non-small-cell lung cancer in a North American population to determine sample-related factors that influence success in clinical EGFR testing. Data from consecutive cases of Canadian province-wide testing at a centralized diagnostic laboratory for a 24-month period were reviewed. Samples were tested for exon-19 deletion and exon-21 L858R mutations using a validated polymerase chain reaction method with 1% to 5% detection sensitivity. From 2651 samples submitted, 2404 samples were tested with 2293 samples eligible for analysis (1780 histology and 513 cytology specimens). The overall test-failure rate was 5.4% with overall mutation rate of 20.6%. No significant differences in the failure rate, mutation rate, or mutation type were found between histology and cytology samples. Although tumor cellularity was significantly associated with test-success or mutation rates in histology and cytology specimens, respectively, mutations could be detected in all specimen types. Significant rates of EGFR mutation were detected in cases with thyroid transcription factor (TTF)-1-negative immunohistochemistry (6.7%) and mucinous component (9.0%). EGFR mutation testing should be attempted in any specimen, whether histologic or cytologic. Samples should not be excluded from testing based on TTF-1 status or histologic features. Pathologists should report the amount of available tumor for testing. However, suboptimal samples with a negative EGFR mutation result should be considered for repeat testing with an alternate sample.

Mutation spectrum of RB1 mutations in retinoblastoma cases from Singapore with implications for genetic management and counselling

PubMed Central

Tomar, Swati; Sethi, Raman; Sundar, Gangadhara; Quah, Thuan Chong; Quah, Boon Long; Lai, Poh San

2017-01-01

Retinoblastoma (RB) is a rare childhood malignant disorder caused by the biallelic inactivation of RB1 gene. Early diagnosis and identification of carriers of heritable RB1 mutations can improve disease outcome and management. In this study, mutational analysis was conducted on fifty-nine matched tumor and peripheral blood samples from 18 bilateral and 41 unilateral unrelated RB cases by a combinatorial approach of Multiplex Ligation-dependent Probe Amplification (MLPA) assay, deletion screening, direct sequencing, copy number gene dosage analysis and methylation assays. Screening of both blood and tumor samples yielded a mutation detection rate of 94.9% (56/59) while only 42.4% (25/59) of mutations were detected if blood samples alone were analyzed. Biallelic mutations were observed in 43/59 (72.9%) of tumors screened. There were 3 cases (5.1%) in which no mutations could be detected and germline mutations were detected in 19.5% (8/41) of unilateral cases. A total of 61 point mutations were identified, of which 10 were novel. There was a high incidence of previously reported recurrent mutations, occurring at 38.98% (23/59) of all cases. Of interest were three cases of mosaic RB1 mutations detected in the blood from patients with unilateral retinoblastoma. Additionally, two germline mutations previously reported to be associated with low-penetrance phenotypes: missense-c.1981C>T and splice variant-c.607+1G>T, were observed in a bilateral and a unilateral proband, respectively. These findings have implications for genetic counselling and risk prediction for the affected families. This is the first published report on the spectrum of mutations in RB patients from Singapore and shows that further improved mutation screening strategies are required in order to provide a definitive molecular diagnosis for every case of RB. Our findings also underscore the importance of genetic testing in supporting individualized disease management plans for patients and asymptomatic family members carrying low-penetrance, germline mosaicism or heritable unilateral mutational phenotypes. PMID:28575107

Rates of Spontaneous Mutation in Bacteriophage T4 Are Independent of Host Fidelity Determinants

PubMed Central

Santos, M. E.; Drake, J. W.

1994-01-01

Bacteriophage T4 encodes most of the genes whose products are required for its DNA metabolism, and host (Escherichia coli) genes can only infrequently complement mutationally inactivated T4 genes. We screened the following host mutator mutations for effects on spontaneous mutation rates in T4: mutT (destruction of aberrant dGTPs), polA, polB and polC (DNA polymerases), dnaQ (exonucleolytic proofreading), mutH, mutS, mutL and uvrD (methyl-directed DNA mismatch repair), mutM and mutY (excision repair of oxygen-damaged DNA), mutA (function unknown), and topB and osmZ (affecting DNA topology). None increased T4 spontaneous mutation rates within a resolving power of about twofold (nor did optA, which is not a mutator but overexpresses a host dGTPase). Previous screens in T4 have revealed strong mutator mutations only in the gene encoding the viral DNA polymerase and proofreading 3'-exonuclease, plus weak mutators in several polymerase accessory proteins or determinants of dNTP pool sizes. T4 maintains a spontaneous mutation rate per base pair about 30-fold greater than that of its host. Thus, the joint high fidelity of insertion by T4 DNA polymerase and proofreading by its associated 3'-exonuclease appear to determine the T4 spontaneous mutation rate, whereas the host requires numerous additional systems to achieve high replication fidelity. PMID:7851754

Point mutations in the tumor suppressor Smad4/DPC4 enhance its phosphorylation by GSK3 and reversibly inactivate TGF-β signaling

PubMed Central

Demagny, Hadrien; De Robertis, Edward M

2016-01-01

The tumor suppressor Smad4/DPC4 is an essential transcription factor in the TGF-β pathway and is frequently mutated or deleted in prostate, colorectal, and pancreatic carcinomas. We recently discovered that Smad4 activity and stability are regulated by the FGF/EGF and Wnt signaling pathways through a series of MAPK and GSK3 phosphorylation sites located in its linker region. In the present study, we report that loss-of-function associated with 2 point mutations commonly found in colorectal and pancreatic cancers results from enhanced Smad4 phosphorylation by GSK3, generating a phosphodegron that leads to subsequent β-TrCP–mediated polyubiquitination and proteasomal degradation. Using chemical GSK3 inhibitors, we show that Smad4 point mutant proteins can be stabilized and TGF-β signaling restored in cancer cells harboring such mutations. PMID:27308538

Low Base-Substitution Mutation Rate in the Germline Genome of the Ciliate Tetrahymena thermophila

DTIC Science & Technology

2016-09-15

generations of mutation accumulation (MA). We applied an existing mutation-calling pipeline and developed a new probabilistic mutation detection approach...noise introduced by mismapped reads. We used both our new method and an existing mutation-calling pipeline (Sung, Tucker, et al. 2012) to analyse the...and larger MA experiments will be required to confidently estimate the mutational spectrum of a species with such a low mutation rate. Materials and

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

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)

Evidence and age-related distribution of mtDNA D-loop point mutations in skeletal muscle from healthy subjects and mitochondrial patients.

PubMed

Del Bo, Roberto; Bordoni, Andreina; Martinelli Boneschi, Filippo; Crimi, Marco; Sciacco, Monica; Bresolin, Nereo; Scarlato, Guglielmo; Comi, Giacomo Pietri

2002-10-15

The progressive accumulation of mitochondrial DNA (mtDNA) alterations, ranging from single mutations to large-scale deletions, in both the normal ageing process and pathological conditions is a relevant phenomenon in terms of frequency and heteroplasmic degree. Recently, two point mutations (A189G and T408A) within the Displacement loop (D-loop) region, the control region for mtDNA replication, were shown to occur in skeletal muscles from aged individuals. We evaluated the presence and the heteroplasmy levels of these two mutations in muscle biopsies from 91 unrelated individuals of different ages (21 healthy subjects and 70 patients affected by mitochondrial encephalomyopathies). Overall, both mutations significantly accumulate with age. However, a different relationship was discovered among the different subgroups of patients: a higher number of A189G positive subjects younger than 53 years was detected in the subgroup of multiple-deleted patients; furthermore, a trend towards an increased risk for the mutations was evidenced among patients carrying multiple deletions when compared to healthy controls. These findings support the idea that a common biological mechanism determines the accumulation of somatic point mutations in the D-loop region, both in healthy subjects and in mitochondrial myopathy patients. At the same time, it appears that disorders caused by mutations of nuclear genes controlling mtDNA replication (the "mtDNA multiple deletions" syndromes) present a temporal advantage to mutate in the D-loop region. This observation may be relevant to the definition of the molecular pathogenesis of these latter syndromes. Copyright 2002 Elsevier Science B.V.

Destabilization of the metal site as a hub for the pathogenic mechanism of five ALS-linked mutants of copper, zinc superoxide dismutase.

PubMed

Mera-Adasme, Raúl; Erdmann, Hannes; Bereźniak, Tomasz; Ochsenfeld, Christian

2016-10-01

Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease, with no effective pharmacological treatment. Its pathogenesis is unknown, although a subset of the cases is linked to genetic mutations. A significant fraction of the mutations occur in one protein, copper, zinc superoxide dismutase (SOD1). The toxic function of mutant SOD1 has not been elucidated, but damage to the metal site of the protein is believed to play a major role. In this work, we study the electrostatic loop of SOD1, which we had previously proposed to work as a "solvent seal" isolating the metal site from water molecules. Out of the five contact points identified between the electrostatic loop and its dock in the rest of the protein, three points were found to be affected by ALS-linked mutations, with a total of five mutations identified. The effect of the five mutations was studied using methods of computational chemistry. We found that four of the mutations destabilize the proposed solvent seal, while the fifth mutation directly affects the metal-site stability. In the two contact points unaffected by ALS-linked mutations, the side chains of the residues were not found to play a stabilizing role. Our results show that the docking of the electrostatic loop to the rest of SOD1 plays a role in ALS pathogenesis, in support of that structure acting as a solvent barrier for the metal site. The results provide a unified pathogenic mechanism for five different ALS-linked mutations of SOD1.

Congenital heart defect causing mutation in Nkx2.5 displays in vivo functional deficit.

PubMed

Zakariyah, Abeer F; Rajgara, Rashida F; Veinot, John P; Skerjanc, Ilona S; Burgon, Patrick G

2017-04-01

The Nkx2.5 gene encodes a transcription factor that plays a critical role in heart development. In humans, heterozygous mutations in NKX2.5 result in congenital heart defects (CHDs). However, the molecular mechanisms by which these mutations cause the disease remain unknown. NKX2.5-R142C is a mutation that was reported to be associated with atrial septal defect (ASD) and atrioventricular (AV) block in 13-patients from one family. The R142C mutation is located within both the DNA-binding domain and the nuclear localization sequence of NKX2.5 protein. The pathogenesis of CHDs in humans with R142C point mutation is not well understood. To examine the functional deficit associated with this mutation in vivo, we generated and characterized a knock-in mouse that harbours the human mutation R142C. Systematic structural and functional examination of the embryonic, newborn, and adult mice revealed that the homozygous embryos Nkx2.5 R141C/R141C are developmentally arrested around E10.5 with delayed heart morphogenesis and downregulation of Nkx2.5 target genes, Anf, Mlc2v, Actc1 and Cx40. Histological examination of Nkx2.5 R141C/+ newborn hearts showed that 36% displayed ASD, with at least 80% 0f adult heterozygotes displaying a septal defect. Moreover, heterozygous Nkx2.5 R141C/+ newborn mice have downregulation of ion channel genes with 11/12 adult mice manifesting a prolonged PR interval that is indicative of 1st degree AV block. Collectively, the present study demonstrates that mice with the R141C point mutation in the Nkx2.5 allele phenocopies humans with the NKX2.5 R142C point mutation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mutations in the Norrie disease gene.

PubMed

Schuback, D E; Chen, Z Y; Craig, I W; Breakefield, X O; Sims, K B

1995-01-01

We report our experience to date in mutation identification in the Norrie disease (ND) gene. We carried out mutational analysis in 26 kindreds in an attempt to identify regions presumed critical to protein function and potentially correlated with generation of the disease phenotype. All coding exons, as well as noncoding regions of exons 1 and 2, 636 nucleotides in the noncoding region of exon 3, and 197 nucleotides of 5' flanking sequence, were analyzed for single-strand conformation polymorphisms (SSCP) by polymerase chain reaction (PCR) amplification of genomic DNA. DNA fragments that showed altered SSCP band mobilities were sequenced to locate the specific mutations. In addition to three previously described submicroscopic deletions encompassing the entire ND gene, we have now identified 6 intragenic deletions, 8 missense (seven point mutations, one 9-bp deletion), 6 nonsense (three point mutations, three single bp deletions/frameshift) and one 10-bp insertion, creating an expanded repeat in the 5' noncoding region of exon 1. Thus, mutations have been identified in a total of 24 of 26 (92%) of the kindreds we have studied to date. With the exception of two different mutations, each found in two apparently unrelated kindreds, these mutations are unique and expand the genotype database. Localization of the majority of point mutations at or near cysteine residues, potentially critical in protein tertiary structure, supports a previous protein model for norrin as member of a cystine knot growth factor family (Meitinger et al., 1993). Genotype-phenotype correlations were not evident with the limited clinical data available, except in the cases of larger submicroscopic deletions associated with a more severe neurologic syndrome.(ABSTRACT TRUNCATED AT 250 WORDS)

Hybrid Capture-Based Comprehensive Genomic Profiling Identifies Lung Cancer Patients with Well-Characterized Sensitizing Epidermal Growth Factor Receptor Point Mutations That Were Not Detected by Standard of Care Testing.

PubMed

Suh, James H; Schrock, Alexa B; Johnson, Adrienne; Lipson, Doron; Gay, Laurie M; Ramkissoon, Shakti; Vergilio, Jo-Anne; Elvin, Julia A; Shakir, Abdur; Ruehlman, Peter; Reckamp, Karen L; Ou, Sai-Hong Ignatius; Ross, Jeffrey S; Stephens, Philip J; Miller, Vincent A; Ali, Siraj M

2018-03-14

In our recent study, of cases positive for epidermal growth factor receptor ( EGFR ) exon 19 deletions using comprehensive genomic profiling (CGP), 17/77 (22%) patients with prior standard of care (SOC) EGFR testing results available were previously negative for exon 19 deletion. Our aim was to compare the detection rates of CGP versus SOC testing for well-characterized sensitizing EGFR point mutations (pm) in our 6,832-patient cohort. DNA was extracted from 40 microns of formalin-fixed paraffin-embedded sections from 6,832 consecutive cases of non-small cell lung cancer (NSCLC) of various histologies (2012-2015). CGP was performed using a hybrid capture, adaptor ligation-based next-generation sequencing assay to a mean coverage depth of 576×. Genomic alterations (pm, small indels, copy number changes and rearrangements) involving EGFR were recorded for each case and compared with prior testing results if available. Overall, there were 482 instances of EGFR exon 21 L858R (359) and L861Q (20), exon 18 G719X (73) and exon 20 S768I (30) pm, of which 103 unique cases had prior EGFR testing results that were available for review. Of these 103 cases, CGP identified 22 patients (21%) with sensitizing EGFR pm that were not detected by SOC testing, including 9/75 (12%) patients with L858R, 4/7 (57%) patients with L861Q, 8/20 (40%) patients with G719X, and 4/7 (57%) patients with S768I pm (some patients had multiple EGFR pm). In cases with available clinical data, benefit from small molecule inhibitor therapy was observed. CGP, even when applied to low tumor purity clinical-grade specimens, can detect well-known EGFR pm in NSCLC patients that would otherwise not be detected by SOC testing. Taken together with EGFR exon 19 deletions, over 20% of patients who are positive for EGFR -activating mutations using CGP are previously negative by SOC EGFR mutation testing, suggesting that thousands of such patients per year in the U.S. alone could experience improved clinical outcomes when hybrid capture-based CGP is used to inform therapeutic decisions. This study points out that genomic profiling, as based on hybrid capture next-generation sequencing, can identify lung cancer patients with point mutation in epidermal growth factor receptor (EGFR) missed by standard molecular testing who can likely benefit from anti-EGFR targeted therapy. Beyond the specific findings regarding false-negative point mutation testing for EGFR, this study highlights the need for oncologists and pathologists to be cognizant of the performance characteristics of testing deployed and the importance of clinical intuition in questioning the results of laboratory testing. © AlphaMed Press 2018.

Distribution of fixed beneficial mutations and the rate of adaptation in asexual populations

PubMed Central

Good, Benjamin H.; Rouzine, Igor M.; Balick, Daniel J.; Hallatschek, Oskar; Desai, Michael M.

2012-01-01

When large asexual populations adapt, competition between simultaneously segregating mutations slows the rate of adaptation and restricts the set of mutations that eventually fix. This phenomenon of interference arises from competition between mutations of different strengths as well as competition between mutations that arise on different fitness backgrounds. Previous work has explored each of these effects in isolation, but the way they combine to influence the dynamics of adaptation remains largely unknown. Here, we describe a theoretical model to treat both aspects of interference in large populations. We calculate the rate of adaptation and the distribution of fixed mutational effects accumulated by the population. We focus particular attention on the case when the effects of beneficial mutations are exponentially distributed, as well as on a more general class of exponential-like distributions. In both cases, we show that the rate of adaptation and the influence of genetic background on the fixation of new mutants is equivalent to an effective model with a single selection coefficient and rescaled mutation rate, and we explicitly calculate these effective parameters. We find that the effective selection coefficient exactly coincides with the most common fixed mutational effect. This equivalence leads to an intuitive picture of the relative importance of different types of interference effects, which can shift dramatically as a function of the population size, mutation rate, and the underlying distribution of fitness effects. PMID:22371564

Genome-Wide Mutation Rate Response to pH Change in the Coral Reef Pathogen Vibrio shilonii AK1.

PubMed

Strauss, Chloe; Long, Hongan; Patterson, Caitlyn E; Te, Ronald; Lynch, Michael

2017-08-22

Recent application of mutation accumulation techniques combined with whole-genome sequencing (MA/WGS) has greatly promoted studies of spontaneous mutation. However, such explorations have rarely been conducted on marine organisms, and it is unclear how marine habitats have influenced genome stability. This report resolves the mutation rate and spectrum of the coral reef pathogen Vibrio shilonii , which causes coral bleaching and endangers the biodiversity maintained by coral reefs. We found that its mutation rate and spectrum are highly similar to those of other studied bacteria from various habitats, despite the saline environment. The mutational properties of this marine bacterium are thus controlled by other general evolutionary forces such as natural selection and genetic drift. We also found that as pH drops, the mutation rate decreases and the mutation spectrum is biased in the direction of generating G/C nucleotides. This implies that evolutionary features of this organism and perhaps other marine microbes might be altered by the increasingly acidic ocean water caused by excess CO 2 emission. Nonetheless, further exploration is needed as the pH range tested in this study was rather narrow and many other possible mutation determinants, such as carbonate increase, are associated with ocean acidification. IMPORTANCE This study explored the pH dependence of a bacterial genome-wide mutation rate. We discovered that the genome-wide rates of appearance of most mutation types decrease linearly and that the mutation spectrum is biased in generating more G/C nucleotides with pH drop in the coral reef pathogen V. shilonii . Copyright © 2017 Strauss et al.

A protein-targeting strategy used to develop a selective inhibitor of the E17K point mutation in the PH domain of Akt1

NASA Astrophysics Data System (ADS)

Deyle, Kaycie M.; Farrow, Blake; Qiao Hee, Ying; Work, Jeremy; Wong, Michelle; Lai, Bert; Umeda, Aiko; Millward, Steven W.; Nag, Arundhati; Das, Samir; Heath, James R.

2015-05-01

Ligands that can bind selectively to proteins with single amino-acid point mutations offer the potential to detect or treat an abnormal protein in the presence of the wild type (WT). However, it is difficult to develop a selective ligand if the point mutation is not associated with an addressable location, such as a binding pocket. Here we report an all-chemical synthetic epitope-targeting strategy that we used to discover a 5-mer peptide with selectivity for the E17K-transforming point mutation in the pleckstrin homology domain of the Akt1 oncoprotein. A fragment of Akt1 that contained the E17K mutation and an I19[propargylglycine] substitution was synthesized to form an addressable synthetic epitope. Azide-presenting peptides that clicked covalently onto this alkyne-presenting epitope were selected from a library using in situ screening. One peptide exhibits a 10:1 in vitro selectivity for the oncoprotein relative to the WT, with a similar selectivity in cells. This 5-mer peptide was expanded into a larger ligand that selectively blocks the E17K Akt1 interaction with its PIP3 (phosphatidylinositol (3,4,5)-trisphosphate) substrate.

Different rates of spontaneous mutation of chloroplastic and nuclear viroids as determined by high-fidelity ultra-deep sequencing.

PubMed

López-Carrasco, Amparo; Ballesteros, Cristina; Sentandreu, Vicente; Delgado, Sonia; Gago-Zachert, Selma; Flores, Ricardo; Sanjuán, Rafael

2017-09-01

Mutation rates vary by orders of magnitude across biological systems, being higher for simpler genomes. The simplest known genomes correspond to viroids, subviral plant replicons constituted by circular non-coding RNAs of few hundred bases. Previous work has revealed an extremely high mutation rate for chrysanthemum chlorotic mottle viroid, a chloroplast-replicating viroid. However, whether this is a general feature of viroids remains unclear. Here, we have used high-fidelity ultra-deep sequencing to determine the mutation rate in a common host (eggplant) of two viroids, each representative of one family: the chloroplastic eggplant latent viroid (ELVd, Avsunviroidae) and the nuclear potato spindle tuber viroid (PSTVd, Pospiviroidae). This revealed higher mutation frequencies in ELVd than in PSTVd, as well as marked differences in the types of mutations produced. Rates of spontaneous mutation, quantified in vivo using the lethal mutation method, ranged from 1/1000 to 1/800 for ELVd and from 1/7000 to 1/3800 for PSTVd depending on sequencing run. These results suggest that extremely high mutability is a common feature of chloroplastic viroids, whereas the mutation rates of PSTVd and potentially other nuclear viroids appear significantly lower and closer to those of some RNA viruses.

Different rates of spontaneous mutation of chloroplastic and nuclear viroids as determined by high-fidelity ultra-deep sequencing

PubMed Central

Ballesteros, Cristina; Sentandreu, Vicente; Gago-Zachert, Selma

2017-01-01

Mutation rates vary by orders of magnitude across biological systems, being higher for simpler genomes. The simplest known genomes correspond to viroids, subviral plant replicons constituted by circular non-coding RNAs of few hundred bases. Previous work has revealed an extremely high mutation rate for chrysanthemum chlorotic mottle viroid, a chloroplast-replicating viroid. However, whether this is a general feature of viroids remains unclear. Here, we have used high-fidelity ultra-deep sequencing to determine the mutation rate in a common host (eggplant) of two viroids, each representative of one family: the chloroplastic eggplant latent viroid (ELVd, Avsunviroidae) and the nuclear potato spindle tuber viroid (PSTVd, Pospiviroidae). This revealed higher mutation frequencies in ELVd than in PSTVd, as well as marked differences in the types of mutations produced. Rates of spontaneous mutation, quantified in vivo using the lethal mutation method, ranged from 1/1000 to 1/800 for ELVd and from 1/7000 to 1/3800 for PSTVd depending on sequencing run. These results suggest that extremely high mutability is a common feature of chloroplastic viroids, whereas the mutation rates of PSTVd and potentially other nuclear viroids appear significantly lower and closer to those of some RNA viruses. PMID:28910391

Quantitative evaluation of DNA damage and mutation rate by atmospheric and room-temperature plasma (ARTP) and conventional mutagenesis.

PubMed

Zhang, Xue; Zhang, Chong; Zhou, Qian-Qian; Zhang, Xiao-Fei; Wang, Li-Yan; Chang, Hai-Bo; Li, He-Ping; Oda, Yoshimitsu; Xing, Xin-Hui

2015-07-01

DNA damage is the dominant source of mutation, which is the driving force of evolution. Therefore, it is important to quantitatively analyze the DNA damage caused by different mutagenesis methods, the subsequent mutation rates, and their relationship. Atmospheric and room temperature plasma (ARTP) mutagenesis has been used for the mutation breeding of more than 40 microorganisms. However, ARTP mutagenesis has not been quantitatively compared with conventional mutation methods. In this study, the umu test using a flow-cytometric analysis was developed to quantify the DNA damage in individual viable cells using Salmonella typhimurium NM2009 as the model strain and to determine the mutation rate. The newly developed method was used to evaluate four different mutagenesis systems: a new ARTP tool, ultraviolet radiation, 4-nitroquinoline-1-oxide (4-NQO), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenesis. The mutation rate was proportional to the corresponding SOS response induced by DNA damage. ARTP caused greater DNA damage to individual living cells than the other conventional mutagenesis methods, and the mutation rate was also higher. By quantitatively comparing the DNA damage and consequent mutation rate after different types of mutagenesis, we have shown that ARTP is a potentially powerful mutagenesis tool with which to improve the characteristics of microbial cell factories.

The rate and character of spontaneous mutation in an RNA virus.

PubMed Central

Malpica, José M; Fraile, Aurora; Moreno, Ignacio; Obies, Clara I; Drake, John W; García-Arenal, Fernando

2002-01-01

Estimates of spontaneous mutation rates for RNA viruses are few and uncertain, most notably due to their dependence on tiny mutation reporter sequences that may not well represent the whole genome. We report here an estimate of the spontaneous mutation rate of tobacco mosaic virus using an 804-base cognate mutational target, the viral MP gene that encodes the movement protein (MP). Selection against newly arising mutants was countered by providing MP function from a transgene. The estimated genomic mutation rate was on the lower side of the range previously estimated for lytic animal riboviruses. We also present the first unbiased riboviral mutational spectrum. The proportion of base substitutions is the same as that in a retrovirus but is lower than that in most DNA-based organisms. Although the MP mutant frequency was 0.02-0.05, 35% of the sequenced mutants contained two or more mutations. Therefore, the mutation process in populations of TMV and perhaps of riboviruses generally differs profoundly from that in populations of DNA-based microbes and may be strongly influenced by a subpopulation of mutator polymerases. PMID:12524327

DeepGene: an advanced cancer type classifier based on deep learning and somatic point mutations.

PubMed

Yuan, Yuchen; Shi, Yi; Li, Changyang; Kim, Jinman; Cai, Weidong; Han, Zeguang; Feng, David Dagan

2016-12-23

With the developments of DNA sequencing technology, large amounts of sequencing data have become available in recent years and provide unprecedented opportunities for advanced association studies between somatic point mutations and cancer types/subtypes, which may contribute to more accurate somatic point mutation based cancer classification (SMCC). However in existing SMCC methods, issues like high data sparsity, small volume of sample size, and the application of simple linear classifiers, are major obstacles in improving the classification performance. To address the obstacles in existing SMCC studies, we propose DeepGene, an advanced deep neural network (DNN) based classifier, that consists of three steps: firstly, the clustered gene filtering (CGF) concentrates the gene data by mutation occurrence frequency, filtering out the majority of irrelevant genes; secondly, the indexed sparsity reduction (ISR) converts the gene data into indexes of its non-zero elements, thereby significantly suppressing the impact of data sparsity; finally, the data after CGF and ISR is fed into a DNN classifier, which extracts high-level features for accurate classification. Experimental results on our curated TCGA-DeepGene dataset, which is a reformulated subset of the TCGA dataset containing 12 selected types of cancer, show that CGF, ISR and DNN all contribute in improving the overall classification performance. We further compare DeepGene with three widely adopted classifiers and demonstrate that DeepGene has at least 24% performance improvement in terms of testing accuracy. Based on deep learning and somatic point mutation data, we devise DeepGene, an advanced cancer type classifier, which addresses the obstacles in existing SMCC studies. Experiments indicate that DeepGene outperforms three widely adopted existing classifiers, which is mainly attributed to its deep learning module that is able to extract the high level features between combinatorial somatic point mutations and cancer types.

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

NASA Astrophysics Data System (ADS)

Su, Qiang; Zhou, Xiaoming

2008-12-01

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

X-linked Charcot-Marie-Tooth disease predominates in a cohort of multiethnic Malaysian patients.

PubMed

Shahrizaila, Nortina; Samulong, Sarimah; Tey, Shelisa; Suan, Liaw Chiew; Meng, Lao Kah; Goh, Khean Jin; Ahmad-Annuar, Azlina

2014-02-01

Data regarding Charcot-Marie-Tooth disease is lacking in Southeast Asian populations. We investigated the frequency of the common genetic mutations in a multiethnic Malaysian cohort. Patients with features of Charcot-Marie-Tooth disease or hereditary liability to pressure palsies were investigated for PMP22 duplication, deletion, and point mutations and GJB1, MPZ, and MFN2 point mutations. Over a period of 3 years, we identified 25 index patients. A genetic diagnosis was reached in 60%. The most common were point mutations in GJB1, accounting for X-linked Charcot-Marie-Tooth disease (24% of the total patient population), followed by PMP22 duplication causing Charcot-Marie-Tooth disease type 1A (20%). We also discovered 2 novel GJB1 mutations, c.521C>T (Proline174Leucine) and c.220G>A (Valine74Methionine). X-linked Charcot-Marie-Tooth disease was found to predominate in our patient cohort. We also found a better phenotype/genotype correlation when applying a more recently recommended genetic approach to Charcot-Marie-Tooth disease. Copyright © 2013 Wiley Periodicals, Inc.

Coordinated Changes in Mutation and Growth Rates Induced by Genome Reduction

PubMed Central

Nishimura, Issei; Kurokawa, Masaomi; Liu, Liu

2017-01-01

ABSTRACT Genome size is determined during evolution, but it can also be altered by genetic engineering in laboratories. The systematic characterization of reduced genomes provides valuable insights into the cellular properties that are quantitatively described by the global parameters related to the dynamics of growth and mutation. In the present study, we analyzed a small collection of W3110 Escherichia coli derivatives containing either the wild-type genome or reduced genomes of various lengths to examine whether the mutation rate, a global parameter representing genomic plasticity, was affected by genome reduction. We found that the mutation rates of these cells increased with genome reduction. The correlation between genome length and mutation rate, which has been reported for the evolution of bacteria, was also identified, intriguingly, for genome reduction. Gene function enrichment analysis indicated that the deletion of many of the genes encoding membrane and transport proteins play a role in the mutation rate changes mediated by genome reduction. Furthermore, the increase in the mutation rate with genome reduction was highly associated with a decrease in the growth rate in a nutrition-dependent manner; thus, poorer media showed a larger change that was of higher significance. This negative correlation was strongly supported by experimental evidence that the serial transfer of the reduced genome improved the growth rate and reduced the mutation rate to a large extent. Taken together, the global parameters corresponding to the genome, growth, and mutation showed a coordinated relationship, which might be an essential working principle for balancing the cellular dynamics appropriate to the environment. PMID:28679744

Detection of KRAS G12D in colorectal cancer stool by droplet digital PCR

PubMed Central

Olmedillas-López, Susana; Lévano-Linares, Dennis César; Alexandre, Carmen Laura Aúz; Vega-Clemente, Luz; Sánchez, Edurne León; Villagrasa, Alejandro; Ruíz-Tovar, Jaime; García-Arranz, Mariano; García-Olmo, Damián

2017-01-01

AIM To assess KRAS G12D mutation detection by droplet digital PCR (ddPCR) in stool-derived DNA from colorectal cancer (CRC) patients. METHODS In this study, tumor tissue and stool samples were collected from 70 patients with stage I-IV CRC diagnosed by preoperative biopsy. KRAS mutational status was determined by pyrosequencing analysis of DNA obtained from formalin-fixed paraffin-embedded (FFPE) tumor tissues. The KRAS G12D mutation was then analyzed by ddPCR in FFPE tumors and stool-derived DNA from patients with this point mutation. Wild-type (WT) tumors, as determined by pyrosequencing, were included as controls; analysis of FFPE tissue and stool-derived DNA by ddPCR was performed for these patients as well. RESULTS Among the total 70 patients included, KRAS mutations were detected by pyrosequencing in 32 (45.71%), whereas 38 (54.29%) had WT tumors. The frequency of KRAS mutations was higher in left-sided tumors (11 located in the right colon, 15 in the left, and 6 in the rectum). The predominant point mutation was KRAS G12D (14.29%, n = 10), which was more frequent in early-stage tumors (I-IIA, n = 7). In agreement with pyrosequencing results, the KRAS G12D mutation was detected by ddPCR in FFPE tumor-derived DNA, and only a residual number of mutated copies was found in WT controls. The KRAS G12D mutation was also detected in stool-derived DNA in 80% of all fecal samples from CRC patients with this point mutation. CONCLUSION ddPCR is a reliable and sensitive method to analyze KRAS G12D mutation in stool-derived DNA from CRC patients, especially at early stages. This non-invasive approach is potentially applicable to other relevant biomarkers for CRC management. PMID:29093617

Mapping mitochondrial heteroplasmy in a Leydig tumor by laser capture micro-dissection and cycling temperature capillary electrophoresis.

PubMed

Refinetti, Paulo; Arstad, Christian; Thilly, William G; Morgenthaler, Stephan; Ekstrøm, Per Olaf

2017-01-01

The growth of tumor cells is accompanied by mutations in nuclear and mitochondrial genomes creating marked genetic heterogeneity. Tumors also contain non-tumor cells of various origins. An observed somatic mitochondrial mutation would have occurred in a founding cell and spread through cell division. Micro-anatomical dissection of a tumor coupled with assays for mitochondrial point mutations permits new insights into this growth process. More generally, the ability to detect and trace, at a histological level, somatic mitochondrial mutations in human tissues and tumors, makes these mutations into markers for lineage tracing. A tumor was first sampled by a large punch biopsy and scanned for any significant degree of heteroplasmy in a set of sequences containing known mutational hotspots of the mitochondrial genome. A heteroplasmic tumor was sliced at a 12 μm thickness and placed on membranes. Laser capture micro-dissection was used to take 25000 μm 2 subsamples or spots. After DNA amplification, cycling temperature capillary electrophoresis (CTCE) was used on the laser captured samples to quantify mitochondrial mutant fractions. Of six testicular tumors studied, one, a Leydig tumor, was discovered to carry a detectable degree of heteroplasmy for two separate point mutations: a C → T mutation at bp 64 and a T → C mutation found at bp 152. From this tumor, 381 spots were sampled with laser capture micro-dissection. The ordered distribution of spots exhibited a wide range of fractions of the mutant sequences from 0 to 100% mutant copies. The two mutations co-distributed in the growing tumor indicating they were present on the same genome copies in the founding cell. Laser capture microdissection of sliced tumor samples coupled with CTCE-based point mutation assays provides an effective and practical means to obtain maps of mitochondrial mutational heteroplasmy within human tumors.

Lumacaftor-Ivacaftor in Patients with Cystic Fibrosis Homozygous for Phe508del CFTR.

PubMed

Wainwright, Claire E; Elborn, J Stuart; Ramsey, Bonnie W; Marigowda, Gautham; Huang, Xiaohong; Cipolli, Marco; Colombo, Carla; Davies, Jane C; De Boeck, Kris; Flume, Patrick A; Konstan, Michael W; McColley, Susanna A; McCoy, Karen; McKone, Edward F; Munck, Anne; Ratjen, Felix; Rowe, Steven M; Waltz, David; Boyle, Michael P

2015-07-16

Cystic fibrosis is a life-limiting disease that is caused by defective or deficient cystic fibrosis transmembrane conductance regulator (CFTR) protein activity. Phe508del is the most common CFTR mutation. We conducted two phase 3, randomized, double-blind, placebo-controlled studies that were designed to assess the effects of lumacaftor (VX-809), a CFTR corrector, in combination with ivacaftor (VX-770), a CFTR potentiator, in patients 12 years of age or older who had cystic fibrosis and were homozygous for the Phe508del CFTR mutation. In both studies, patients were randomly assigned to receive either lumacaftor (600 mg once daily or 400 mg every 12 hours) in combination with ivacaftor (250 mg every 12 hours) or matched placebo for 24 weeks. The primary end point was the absolute change from baseline in the percentage of predicted forced expiratory volume in 1 second (FEV1) at week 24. A total of 1108 patients underwent randomization and received study drug. The mean baseline FEV1 was 61% of the predicted value. In both studies, there were significant improvements in the primary end point in both lumacaftor-ivacaftor dose groups; the difference between active treatment and placebo with respect to the mean absolute improvement in the percentage of predicted FEV1 ranged from 2.6 to 4.0 percentage points (P<0.001), which corresponded to a mean relative treatment difference of 4.3 to 6.7% (P<0.001). Pooled analyses showed that the rate of pulmonary exacerbations was 30 to 39% lower in the lumacaftor-ivacaftor groups than in the placebo group; the rate of events leading to hospitalization or the use of intravenous antibiotics was lower in the lumacaftor-ivacaftor groups as well. The incidence of adverse events was generally similar in the lumacaftor-ivacaftor and placebo groups. The rate of discontinuation due to an adverse event was 4.2% among patients who received lumacaftor-ivacaftor versus 1.6% among those who received placebo. These data show that lumacaftor in combination with ivacaftor provided a benefit for patients with cystic fibrosis homozygous for the Phe508del CFTR mutation. (Funded by Vertex Pharmaceuticals and others; TRAFFIC and TRANSPORT ClinicalTrials.gov numbers, NCT01807923 and NCT01807949.).

Restriction digest screening facilitates efficient detection of site-directed mutations introduced by CRISPR in C. albicans UME6.

PubMed

Evans, Ben A; Smith, Olivia L; Pickerill, Ethan S; York, Mary K; Buenconsejo, Kristen J P; Chambers, Antonio E; Bernstein, Douglas A

2018-01-01

Introduction of point mutations to a gene of interest is a powerful tool when determining protein function. CRISPR-mediated genome editing allows for more efficient transfer of a desired mutation into a wide range of model organisms. Traditionally, PCR amplification and DNA sequencing is used to determine if isolates contain the intended mutation. However, mutation efficiency is highly variable, potentially making sequencing costly and time consuming. To more efficiently screen for correct transformants, we have identified restriction enzymes sites that encode for two identical amino acids or one or two stop codons. We used CRISPR to introduce these restriction sites directly upstream of the Candida albicans UME6 Zn 2+ -binding domain, a known regulator of C. albicans filamentation. While repair templates coding for different restriction sites were not equally successful at introducing mutations, restriction digest screening enabled us to rapidly identify isolates with the intended mutation in a cost-efficient manner. In addition, mutated isolates have clear defects in filamentation and virulence compared to wild type C. albicans . Our data suggest restriction digestion screening efficiently identifies point mutations introduced by CRISPR and streamlines the process of identifying residues important for a phenotype of interest.

Critical Mutation Rate Has an Exponential Dependence on Population Size in Haploid and Diploid Populations

PubMed Central

Aston, Elizabeth; Channon, Alastair; Day, Charles; Knight, Christopher G.

2013-01-01

Understanding the effect of population size on the key parameters of evolution is particularly important for populations nearing extinction. There are evolutionary pressures to evolve sequences that are both fit and robust. At high mutation rates, individuals with greater mutational robustness can outcompete those with higher fitness. This is survival-of-the-flattest, and has been observed in digital organisms, theoretically, in simulated RNA evolution, and in RNA viruses. We introduce an algorithmic method capable of determining the relationship between population size, the critical mutation rate at which individuals with greater robustness to mutation are favoured over individuals with greater fitness, and the error threshold. Verification for this method is provided against analytical models for the error threshold. We show that the critical mutation rate for increasing haploid population sizes can be approximated by an exponential function, with much lower mutation rates tolerated by small populations. This is in contrast to previous studies which identified that critical mutation rate was independent of population size. The algorithm is extended to diploid populations in a system modelled on the biological process of meiosis. The results confirm that the relationship remains exponential, but show that both the critical mutation rate and error threshold are lower for diploids, rather than higher as might have been expected. Analyzing the transition from critical mutation rate to error threshold provides an improved definition of critical mutation rate. Natural populations with their numbers in decline can be expected to lose genetic material in line with the exponential model, accelerating and potentially irreversibly advancing their decline, and this could potentially affect extinction, recovery and population management strategy. The effect of population size is particularly strong in small populations with 100 individuals or less; the exponential model has significant potential in aiding population management to prevent local (and global) extinction events. PMID:24386200

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.

Discovery of Point Mutations in the Voltage-Gated Sodium Channel from African Aedes aegypti Populations: Potential Phylogenetic Reasons for Gene Introgression

PubMed Central

Muranami, Yuto; Kawashima, Emiko; Osei, Joseph H. N.; Sakyi, Kojo Yirenkyi; Dadzie, Samuel; de Souza, Dziedzom K.; Appawu, Maxwell; Ohta, Nobuo; Minakawa, Noboru

2016-01-01

Background Yellow fever is endemic in some countries in Africa, and Aedes aegpyti is one of the most important vectors implicated in the outbreak. The mapping of the nation-wide distribution and the detection of insecticide resistance of vector mosquitoes will provide the beneficial information for forecasting of dengue and yellow fever outbreaks and effective control measures. Methodology/Principal Findings High resistance to DDT was observed in all mosquito colonies collected in Ghana. The resistance and the possible existence of resistance or tolerance to permethrin were suspected in some colonies. High frequencies of point mutations at the voltage-gated sodium channel (F1534C) and one heterozygote of the other mutation (V1016I) were detected, and this is the first detection on the African continent. The frequency of F1534C allele and the ratio of F1534C homozygotes in Ae. aegypti aegypti (Aaa) were significantly higher than those in Ae. aegypti formosus (Aaf). We could detect the two types of introns between exon 20 and 21, and the F1534C mutations were strongly linked with one type of intron, which was commonly found in South East Asian and South and Central American countries, suggesting the possibility that this mutation was introduced from other continents or convergently selected after the introgression of Aaa genes from the above area. Conclusions/Significance The worldwide eradication programs in 1940s and 1950s might have caused high selection pressure on the mosquito populations and expanded the distribution of insecticide-resistant Ae. aegypti populations. Selection of the F1534C point mutation could be hypothesized to have taken place during this period. The selection of the resistant population of Ae. aegypti with the point mutation of F1534C, and the worldwide transportation of vector mosquitoes in accordance with human activity such as trading of used tires, might result in the widespread distribution of F1534C point mutation in tropical countries. PMID:27304430

A novel natural killer cell line (KHYG-1) from a patient with aggressive natural killer cell leukemia carrying a p53 point mutation.

PubMed

Yagita, M; Huang, C L; Umehara, H; Matsuo, Y; Tabata, R; Miyake, M; Konaka, Y; Takatsuki, K

2000-05-01

We present the establishment of a natural killer (NK) leukemia cell line, designated KHYG-1, from the blood of a patient with aggressive NK leukemia, which both possessed the same p53 point mutation. The immunophenotype of the primary leukemia cells was CD2+, surface CD3-, cytoplasmic CD3epsilon+, CD7+, CD8alphaalpha+, CD16+, CD56+, CD57+ and HLA-DR+. A new cell line (KHYG-1) was established by culturing peripheral leukemia cells with 100 units of recombinant interleukin (IL)-2. The KHYG-1 cells showed LGL morphology with a large nucleus, coarse chromatin, conspicuous nucleoli, and abundant basophilic cytoplasm with many azurophilic granules. The immunophenotype of KHYG-1 cells was CD1-, CD2+, surface CD3-, cytoplasmic CD3epsilon+, CD7+, CD8alphaalpha+, CD16-, CD25-, CD33+, CD34-, CD56+, CD57-, CD122+, CD132+, and TdT-. Southern blot analysis of these cells revealed a normal germline configuration for the beta, delta, and gamma chains of the T cell receptor and the immunoglobulin heavy-chain genes. Moreover, the KHYG-1 cells displayed NK cell activity and IL-2-dependent proliferation in vitro, suggesting that they are of NK cell origin. Epstein-Barr virus (EBV) DNA was not detected in KHYG-1 cells by Southern blot analysis with a terminal repeat probe from an EBV genome. A point mutation in exon 7 of the p53 gene was detected in the KHYG-1 cells by PCR/SSCP analysis, and direct sequencing revealed the conversion of C to T at nucleotide 877 in codon 248. The primary leukemia cells also carried the same point mutation. Although the precise role of the p53 point mutation in leukemogenesis remains to be clarified, the establishment of an NK leukemia cell line with a p53 point mutation could be valuable in the study of leukemogenesis.

Novel MSH2 splice-site mutation in a young patient with Lynch syndrome

PubMed Central

Liccardo, Raffaella; De Rosa, Marina; Izzo, Paola; Duraturo, Francesca

2018-01-01

Lynch Syndrome (LS) is associated with germline mutations in one of the mismatch repair (MMR) genes, including MutL homolog 1 (MLH1), MutS homolog 2 (MSH2), MSH6, PMS1 homolog 2, mismatch repair system component (PMS2), MLH3 and MSH3. The mutations identified in MMR genes are point mutations or large rearrangements. The point mutations are certainly pathogenetic whether they determine formation of truncated protein. The mutations that arise in splice sites are classified as ‘likely pathogenic’ variants. In the present study, a novel splicing mutation was identified, (named c.212-1g>a), in the MSH2 gene. This novel mutation in the consensus splice site of MSH2 exon 2 leads to the loss of the canonical splice site, without skipping in-frame of exon 2; also with the formation of 2 aberrant transcripts, due to the activation of novel splice sites in exon 2. This mutation was identified in a young patient who developed colon cancer at the age of 26 years and their belongs to family that met the ‘Revised Amsterdam Criteria’. The present study provided insight into the molecular mechanism determining the pathogenicity of this novel MSH2 mutation and it reaffirms the importance of genetic testing in LS. PMID:29568967

Death and population dynamics affect mutation rate estimates and evolvability under stress in bacteria

PubMed Central

Bonhoeffer, Sebastian

2018-01-01

The stress-induced mutagenesis hypothesis postulates that in response to stress, bacteria increase their genome-wide mutation rate, in turn increasing the chances that a descendant is able to better withstand the stress. This has implications for antibiotic treatment: exposure to subinhibitory doses of antibiotics has been reported to increase bacterial mutation rates and thus probably the rate at which resistance mutations appear and lead to treatment failure. More generally, the hypothesis posits that stress increases evolvability (the ability of a population to generate adaptive genetic diversity) and thus accelerates evolution. Measuring mutation rates under stress, however, is problematic, because existing methods assume there is no death. Yet subinhibitory stress levels may induce a substantial death rate. Death events need to be compensated by extra replication to reach a given population size, thus providing more opportunities to acquire mutations. We show that ignoring death leads to a systematic overestimation of mutation rates under stress. We developed a system based on plasmid segregation that allows us to measure death and division rates simultaneously in bacterial populations. Using this system, we found that a substantial death rate occurs at the tested subinhibitory concentrations previously reported to increase mutation rate. Taking this death rate into account lowers and sometimes removes the signal for stress-induced mutagenesis. Moreover, even when antibiotics increase mutation rate, we show that subinhibitory treatments do not increase genetic diversity and evolvability, again because of effects of the antibiotics on population dynamics. We conclude that antibiotic-induced mutagenesis is overestimated because of death and that understanding evolvability under stress requires accounting for the effects of stress on population dynamics as much as on mutation rate. Our goal here is dual: we show that population dynamics and, in particular, the numbers of cell divisions are crucial but neglected parameters in the evolvability of a population, and we provide experimental and computational tools and methods to study evolvability under stress, leading to a reassessment of the magnitude and significance of the stress-induced mutagenesis paradigm. PMID:29750784

Death and population dynamics affect mutation rate estimates and evolvability under stress in bacteria.

PubMed

Frenoy, Antoine; Bonhoeffer, Sebastian

2018-05-01

The stress-induced mutagenesis hypothesis postulates that in response to stress, bacteria increase their genome-wide mutation rate, in turn increasing the chances that a descendant is able to better withstand the stress. This has implications for antibiotic treatment: exposure to subinhibitory doses of antibiotics has been reported to increase bacterial mutation rates and thus probably the rate at which resistance mutations appear and lead to treatment failure. More generally, the hypothesis posits that stress increases evolvability (the ability of a population to generate adaptive genetic diversity) and thus accelerates evolution. Measuring mutation rates under stress, however, is problematic, because existing methods assume there is no death. Yet subinhibitory stress levels may induce a substantial death rate. Death events need to be compensated by extra replication to reach a given population size, thus providing more opportunities to acquire mutations. We show that ignoring death leads to a systematic overestimation of mutation rates under stress. We developed a system based on plasmid segregation that allows us to measure death and division rates simultaneously in bacterial populations. Using this system, we found that a substantial death rate occurs at the tested subinhibitory concentrations previously reported to increase mutation rate. Taking this death rate into account lowers and sometimes removes the signal for stress-induced mutagenesis. Moreover, even when antibiotics increase mutation rate, we show that subinhibitory treatments do not increase genetic diversity and evolvability, again because of effects of the antibiotics on population dynamics. We conclude that antibiotic-induced mutagenesis is overestimated because of death and that understanding evolvability under stress requires accounting for the effects of stress on population dynamics as much as on mutation rate. Our goal here is dual: we show that population dynamics and, in particular, the numbers of cell divisions are crucial but neglected parameters in the evolvability of a population, and we provide experimental and computational tools and methods to study evolvability under stress, leading to a reassessment of the magnitude and significance of the stress-induced mutagenesis paradigm.

Retroviral mutation rates and A-to-G hypermutations during different stages of retroviral replication.

PubMed Central

Kim, T; Mudry, R A; Rexrode, C A; Pathak, V K

1996-01-01

Retroviruses mutate at a high rate in vivo during viral replication. Mutations may occur during proviral transcription by RNA polymerase II, during minus-strand DNA synthesis (RNA template) by viral reverse transcriptase, or during plus-strand DNA synthesis (DNA template) by reverse transcriptase. To determine the contributions of different stages of replication to the retroviral mutation rates, we developed a spleen necrosis virus-based in vivo system to selectively identify mutations occurring during the early stage (RNA transcription plus minus-strand synthesis) and the late stage (plus-strand synthesis plus DNA repair). A lacZalpha reporter gene was inserted into the long terminal repeat (LTR) of a spleen necrosis virus shuttle vector, and proviruses were recovered from infected cells as plasmids containing either one or both LTRs. Plasmids containing both LTRs generated a mutant phenotype only if the lacZalpha genes in both LTRs were mutated, which is most likely to occur during the early stage. Mutant phenotypes were identified from plasmids containing one LTR regardless of the stage at which the mutations occurred. Thus, mutant frequencies obtained after recovery of plasmids containing both LTRs or one LTR provided early-stage and total mutation rates, respectively. Analysis of 56,409 proviruses suggested that the retroviral mutation rates during the early and late stages of replication were equal or within twofold of each other. In addition, two mutants with A-to-G hypermutations were discovered, suggesting a role for mammalian double-stranded RNA adenosine deaminase enzyme in retroviral mutations. These experiments provide a system to selectively identify mutations in the early stage of retroviral replication and to provide upper and lower limits to the in vivo mutation rates during minus-strand and plus-strand synthesis, respectively. PMID:8892879

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

DTIC Science & Technology

2016-10-15

Mutation rates may be nonuniform because of greater rates of damage, asymmetric nucleotide pools, structural differences affecting polymerase fidelity...strains of these two significant bacterial species. In the MMR-deficient strains, mutation rates were nonuniform among genome regions and varied in pat

Mutation at a distance caused by homopolymeric guanine repeats in Saccharomyces cerevisiae

PubMed Central

McDonald, Michael J.; Yu, Yen-Hsin; Guo, Jheng-Fen; Chong, Shin Yen; Kao, Cheng-Fu; Leu, Jun-Yi

2016-01-01

Mutation provides the raw material from which natural selection shapes adaptations. The rate at which new mutations arise is therefore a key factor that determines the tempo and mode of evolution. However, an accurate assessment of the mutation rate of a given organism is difficult because mutation rate varies on a fine scale within a genome. A central challenge of evolutionary genetics is to determine the underlying causes of this variation. In earlier work, we had shown that repeat sequences not only are prone to a high rate of expansion and contraction but also can cause an increase in mutation rate (on the order of kilobases) of the sequence surrounding the repeat. We perform experiments that show that simple guanine repeats 13 bp (base pairs) in length or longer (G13+) increase the substitution rate 4- to 18-fold in the downstream DNA sequence, and this correlates with DNA replication timing (R = 0.89). We show that G13+ mutagenicity results from the interplay of both error-prone translesion synthesis and homologous recombination repair pathways. The mutagenic repeats that we study have the potential to be exploited for the artificial elevation of mutation rate in systems biology and synthetic biology applications. PMID:27386516

Osimertinib - effective treatment of NSCLC with activating EGFR mutations after progression on EGFR tyrosine kinase inhibitors.

PubMed

Skrzypski, Marcin; Szymanowska-Narloch, Amelia; Dziadziuszko, Rafał

2017-01-01

Non-small cell lung cancer (NSCLC) driven by activating mutations in epidermal growth factor receptor (EGFR) constitutes up to 10% of NSCLC cases. According to the NCCN recommendations, all patients (with the exception of smoking patients with squamous cell lung cancer) should be screened for the presence of activating EGFR mutations, i.e. deletion in exon 19 or point mutation L858R in exon 21, in order to select the group that benefits from EGFR tyrosine kinase inhibitors (EGFR TKIs) treatment. Among approved agents there are the 1 st generation reversible EGFR TKIs, erlotinib and gefitinib, and the 2 nd generation irreversible EGFR TKI, afatinib. The objective response rates to these drugs in randomised clinical trials were in the range of 56-74%, and median time to progression 9-13 months. The most common determinant of resistance to these drugs is the clonal expansion of cancer cells with T790M mutation (Thr790Met) in exon 20 of EGFR. Osimertinib (Tagrisso™), a 3 rd generation, irreversible EGFR tyrosine kinase inhibitor, constitutes a novel, highly efficacious treatment for NSCLC patients progressing on EGFR TKIs with T790M mutation confirmed as the resistance mechanism. Resistance mutation can be determined in tissue or liquid biopsy obtained after progression on EGFR TKIs. Osimertinib has a favourable toxicity profile, with mild rash and diarrhoea being the most common. In this article, we present three cases that were successfully treated with osimertinib after progression on 1st and 2nd generation EGFR TKIs.

Genotyping of K-ras codons 12 and 13 mutations in colorectal cancer by capillary electrophoresis.

PubMed

Chen, Yen-Ling; Chang, Ya-Sian; Chang, Jan-Gowth; Wu, Shou-Mei

2009-06-26

Point mutations of the K-ras gene located in codons 12 and 13 cause poor responses to the anti-epidermal growth factor receptor (anti-EGFR) therapy of colorectal cancer (CRC) patients. Besides, mutations of K-ras gene have also been proven to play an important role in human tumor progression. We established a simple and effective capillary electrophoresis (CE) method for simultaneous point mutation detection in codons 12 and 13 of K-ras gene. We combined one universal fluorescence-based nonhuman-sequence primer and two fragment-oriented primers in one tube, and performed this two-in-one polymerase chain reaction (PCR). PCR fragments included wild type and seven point mutations at codons 12 and 13 of K-ras gene. The amplicons were analyzed by single-strand conformation polymorphism (SSCP)-CE method. The CE analysis was performed by using a 1x Tris-borate-EDTA (TBE) buffer containing 1.5% (w/v) hydroxyethylcellulose (HEC) (MW 250,000) under reverse polarity with 15 degrees C and 30 degrees C. Ninety colorectal cancer patients were blindly genotyped using this developed method. The results showed good agreement with those of DNA sequencing method. The SSCP-CE was feasible for mutation screening of K-ras gene in populations.

Development and validation of a whole-exome sequencing test for simultaneous detection of point mutations, indels and copy-number alterations for precision cancer care

PubMed Central

Rennert, Hanna; Eng, Kenneth; Zhang, Tuo; Tan, Adrian; Xiang, Jenny; Romanel, Alessandro; Kim, Robert; Tam, Wayne; Liu, Yen-Chun; Bhinder, Bhavneet; Cyrta, Joanna; Beltran, Himisha; Robinson, Brian; Mosquera, Juan Miguel; Fernandes, Helen; Demichelis, Francesca; Sboner, Andrea; Kluk, Michael; Rubin, Mark A; Elemento, Olivier

2016-01-01

We describe Exome Cancer Test v1.0 (EXaCT-1), the first New York State-Department of Health-approved whole-exome sequencing (WES)-based test for precision cancer care. EXaCT-1 uses HaloPlex (Agilent) target enrichment followed by next-generation sequencing (Illumina) of tumour and matched constitutional control DNA. We present a detailed clinical development and validation pipeline suitable for simultaneous detection of somatic point/indel mutations and copy-number alterations (CNAs). A computational framework for data analysis, reporting and sign-out is also presented. For the validation, we tested EXaCT-1 on 57 tumours covering five distinct clinically relevant mutations. Results demonstrated elevated and uniform coverage compatible with clinical testing as well as complete concordance in variant quality metrics between formalin-fixed paraffin embedded and fresh-frozen tumours. Extensive sensitivity studies identified limits of detection threshold for point/indel mutations and CNAs. Prospective analysis of 337 cancer cases revealed mutations in clinically relevant genes in 82% of tumours, demonstrating that EXaCT-1 is an accurate and sensitive method for identifying actionable mutations, with reasonable costs and time, greatly expanding its utility for advanced cancer care. PMID:28781886

The androgen receptor gene mutations database.

PubMed

Patterson, M N; Hughes, I A; Gottlieb, B; Pinsky, L

1994-09-01

The androgen receptor gene mutations database is a comprehensive listing of mutations published in journals and meetings proceedings. The majority of mutations are point mutations identified in patients with androgen insensitivity syndrome. Information is included regarding the phenotype, the nature and location of the mutations, as well as the effects of the mutations on the androgen binding activity of the receptor. The current version of the database contains 149 entries, of which 114 are unique mutations. The database is available from EMBL (NetServ@EMBL-Heidelberg.DE) or as a Macintosh Filemaker file (mc33001@musica.mcgill.ca).

Identification of TCT, a novel knockdown resistance allele mutation and analysis of resistance detection methods in the voltage-gated Na⁺ channel of Culex pipiens pallens from Shandong Province, China.

PubMed

Liu, Hong-Mei; Cheng, Peng; Huang, Xiaodan; Dai, Yu-Hua; Wang, Hai-Fang; Liu, Li-Juan; Zhao, Yu-Qiang; Wang, Huai-Wei; Gong, Mao-Qing

2013-02-01

The present study aimed to investigate deltamethrin resistance in Culex pipiens pallens (C. pipiens pallens) mosquitoes and its correlation with knockdown resistance (kdr) mutations. In addition, mosquito‑resistance testing methods were analyzed. Using specific primers in polymerase chain reaction (PCR) and allele-specific (AS)-PCR, kdr gene sequences isolated from wild C. pipiens pallens mosquitoes were sequenced. Linear regression analysis was used to determine the correlation between the mutations and deltamethrin resistance. A kdr allelic gene was cloned and sequenced. Analysis of the DNA sequences revealed the presence of two point mutations at the L1014 residue in the IIS6 transmembrane segment of the voltage‑gated sodium channel (VGSC): L1014F, TTA→TTT, replacing a leucine (L) with a phenylalanine (F); L1014S, TTA→TCA, replacing leucine (L) with serine (S). Two alternative kdr-like mutations, L1014F and L1014S, were identified to be positively correlated with the deltamethrin-resistant phenotype. In addition a novel mutation, TCT, was identified in the VGSC of C. pipiens pallens. PCR and AS-PCR yielded consistent results with respect to mosquito resistance. However, the detection rate of PCR was higher than that of AS-PCR. Further studies are required to determine the specific resistance mechanism. PCR and AS-PCR demonstrated suitability for mosquito resistance field tests, however, the former method may be superior to the latter.

Electroclinical presentation and genotype-phenotype relationships in patients with Unverricht-Lundborg disease carrying compound heterozygous CSTB point and indel mutations.

PubMed

Canafoglia, Laura; Gennaro, Elena; Capovilla, Giuseppe; Gobbi, Giuseppe; Boni, Antonella; Beccaria, Francesca; Viri, Maurizio; Michelucci, Roberto; Agazzi, Pamela; Assereto, Stefania; Coviello, Domenico A; Di Stefano, Maria; Rossi Sebastiano, Davide; Franceschetti, Silvana; Zara, Federico

2012-12-01

Unverricht-Lundborg disease (EPM1A) is frequently due to an unstable expansion of a dodecamer repeat in the CSTB gene, whereas other types of mutations are rare. EPM1A due to homozygous expansion has a rather stereotyped presentation with prominent action myoclonus. We describe eight patients with five different compound heterozygous CSTB point or indel mutations in order to highlight their particular phenotypical presentations and evaluate their genotype-phenotype relationships. We screened CSTB mutations by means of Southern blotting and the sequencing of the genomic DNA of each proband. CSTB messenger RNA (mRNA) aberrations were characterized by sequencing the complementary DNA (cDNA) of lymphoblastoid cells, and assessing the protein concentrations in the lymphoblasts. The patient evaluations included the use of a simplified myoclonus severity rating scale, multiple neurophysiologic tests, and electroencephalography (EEG)-polygraphic recordings. To highlight the particular clinical features and disease time-course in compound heterozygous patients, we compared some of their characteristics with those observed in a series of 40 patients carrying the common homozygous expansion mutation observed at the C. Besta Foundation, Milan, Italy. The eight compound heterozygous patients belong to six EPM1A families (out of 52; 11.5%) diagnosed at the Laboratory of Genetics of the Galliera Hospitals in Genoa, Italy. They segregated five different heterozygous point or indel mutations in association with the common dodecamer expansion. Four patients from three families had previously reported CSTB mutations (c.67-1G>C and c.168+1_18del); one had a novel nonsense mutation at the first exon (c.133C>T) leading to a premature stop codon predicting a short peptide; the other three patients from two families had a complex novel indel mutation involving the donor splice site of intron 2 (c.168+2_169+21delinsAA) and leading to an aberrant transcript with a partially retained intron. The protein dose (cystatin B/β-actin) in our heterozygous patients was 0.24 ± 0.02, which is not different from that assessed in patients bearing the homozygous dodecamer expansion. The compound heterozygous patients had a significantly earlier disease onset (7.4 ± 1.7 years) than the homozygous patients, and their disease presentations included frequent myoclonic seizures and absences, often occurring in clusters throughout the course of the disease. The seizures were resistant to the pharmacologic treatments that usually lead to complete seizure control in homozygous patients. EEG-polygraphy allowed repeated seizures to be recorded. Action myoclonus progressively worsened and all of the heterozygous patients older than 30 years were in wheelchairs. Most of the patients showed moderate to severe cognitive impairment, and six had psychiatric symptoms. EPM1A due to compound heterozygous CSTB mutations presents with variable but often markedly severe and particular phenotypes. Most of our patients presented with the electroclinical features of severe epilepsy, which is unexpected in homozygous patients, and showed frequent seizures resistant to pharmacologic treatment. The presence of variable phenotypes (even in siblings) suggests interactions with other genetic factors influencing the final disease presentation. Wiley Periodicals, Inc. © 2012 International League Against Epilepsy.

Evolutionary rescue of a parasite population by mutation rate evolution.

PubMed

Greenspoon, Philip B; Mideo, Nicole

2017-10-01

The risk of antibiotic resistance evolution in parasites is a major problem for public health. Identifying factors which promote antibiotic resistance evolution is thus a priority in evolutionary medicine. The rate at which new mutations enter the parasite population is one important predictor; however, mutation rate is not necessarily a fixed quantity, as is often assumed, but can itself evolve. Here we explore the possible impacts of mutation rate evolution on the fate of a disease circulating in a host population, which is being treated with drugs, the use of which varies over time. Using an evolutionary rescue framework, we find that mutation rate evolution provides a dramatic increase in the probability that a parasite population survives treatment in only a limited region, while providing little or no advantage in other regions. Both epidemiological features, such as the virulence of infection, and population genetic parameters, such as recombination rate, play important roles in determining the probability of evolutionary rescue and whether mutation rate evolution enhances the probability of evolutionary rescue or not. While efforts to curtail mutation rate evolution in parasites may be worthwhile under some circumstances, our results suggest that this need not always be the case. Copyright © 2017 Elsevier Inc. All rights reserved.

The induction of mutation and recombination following UV irradiation during meiosis in Saccharomyces cerevisiae.

PubMed

Kelly, S L; Parry, J M

1983-03-01

Irradiation of yeast cultures with ultraviolet light at discrete stages during meiosis produces cyclic variations in sensitivity, i.e. cells are more sensitive to the lethal effects of UV light prior to entry into the meiotic DNA synthesis, and this corresponds to a peak of induction of point mutation. Cells become more resistant to both induced point mutation and lethality as they enter meiotic DNA synthesis, but become more sensitive again during spore formation. The induced level of intragenic recombination rises during the period of commitment to recombination to a level indistinguishable from the full meiotic level of spontaneous intragenic recombination. Induced reciprocal recombination remains above the spontaneous level up to the point of commitment to sporulation.

Comparison of Detection Rate and Mutational Pattern of Drug-Resistant Mutations Between a Large Cohort of Genotype B and Genotype C Hepatitis B Virus-Infected Patients in North China.

PubMed

Li, Xiaodong; Liu, Yan; Xin, Shaojie; Ji, Dong; You, Shaoli; Hu, Jinhua; Zhao, Jun; Wu, Jingjing; Liao, Hao; Zhang, Xin-Xin; Xu, Dongping

2017-06-01

The study aimed to investigate the association of prevalent genotypes in China (HBV/C and HBV/B) with HBV drug-resistant mutations. A total of 13,847 nucleos(t)ide analogue (NA)-treated patients with chronic HBV infection from North China were enrolled. HBV genotypes and resistant mutations were determined by direct sequencing and confirmed by clonal sequencing if necessary. HBV/B, HBV/C, and HBV/D occupied 14.3%, 84.9%, and 0.8% across the study population, respectively. NA usage had no significant difference between HBV/B- and HBV/C-infected patients. Lamivudine-resistant mutations were more frequently detected in HBV/C-infected patients, compared with HBV/B-infected patients (31.67% vs. 25.26%, p < 0.01). Adefovir- and entecavir-resistant mutation detection rates were similar, but the mutational pattern was different between the two genotypes. For adefovir-resistant mutations, HBV/C-infected patients had a higher detection rate of rtA181 V (HBV/C 5.29% vs. HBV/B 1.36%, p < 0.01) and a lower detection rate of rtN236T (2.70% vs. 6.54%, p < 0.01). For entecavir-resistant mutations, HBV/C-infected patients had a higher detection rate of rtM204 V/I+T184 substitution or S202G/C (3.66% vs. 2.16%, p < 0.01) and a lower detection rate of rtM204 V/I+M250 V/I/L substitution (0.67% vs. 1.46%, p < 0.01). Multidrug-resistant mutations (defined as coexistence of mutation to nucleoside and nucleotide analogues) were detected in 104 patients. HBV/C-infected patients had a higher detection rate of multidrug-resistant mutation than HBV/B-infected patients (0.83% vs. 0.35%, p < 0.05). The study for the first time clarified that HBV/C-infected patients had a higher risk to develop multidrug-resistant mutations, compared with HBV/B-infected patients; and HBV/C- and HBV/B-infected patients had different inclinations in the ETV-resistant mutational pattern.

Investigating the Consequences of Interference between Multiple CD8+ T Cell Escape Mutations in Early HIV Infection

PubMed Central

Garcia, Victor; Feldman, Marcus W.; Regoes, Roland R.

2016-01-01

During early human immunodeficiency virus (HIV) infection multiple CD8+ T cell responses are elicited almost simultaneously. These responses exert strong selective pressures on different parts of HIV’s genome, and select for mutations that escape recognition and are thus beneficial to the virus. Some studies reveal that the later these escape mutations emerge, the more slowly they go to fixation. This pattern of escape rate decrease(ERD) can arise by distinct mechanisms. In particular, in large populations with high beneficial mutation rates interference among different escape strains –an effect that can emerge in evolution with asexual reproduction and results in delayed fixation times of beneficial mutations compared to sexual reproduction– could significantly impact the escape rates of mutations. In this paper, we investigated how interference between these concurrent escape mutations affects their escape rates in systems with multiple epitopes, and whether it could be a source of the ERD pattern. To address these issues, we developed a multilocus Wright-Fisher model of HIV dynamics with selection, mutation and recombination, serving as a null-model for interference. We also derived an interference-free null model assuming initial neutral evolution before immune response elicitation. We found that interference between several equally selectively advantageous mutations can generate the observed ERD pattern. We also found that the number of loci, as well as recombination rates substantially affect ERD. These effects can be explained by the underexponential decline of escape rates over time. Lastly, we found that the observed ERD pattern in HIV infected individuals is consistent with both independent, interference-free mutations as well as interference effects. Our results confirm that interference effects should be considered when analyzing HIV escape mutations. The challenge in estimating escape rates and mutation-associated selective coefficients posed by interference effects cannot simply be overcome by improved sampling frequencies or sizes. This problem is a consequence of the fundamental shortcomings of current estimation techniques under interference regimes. Hence, accounting for the stochastic nature of competition between mutations demands novel estimation methodologies based on the analysis of HIV strains, rather than mutation frequencies. PMID:26829720

Genetic interaction analysis of point mutations enables interrogation of gene function at a residue-level resolution

PubMed Central

Braberg, Hannes; Moehle, Erica A.; Shales, Michael; Guthrie, Christine; Krogan, Nevan J.

2014-01-01

We have achieved a residue-level resolution of genetic interaction mapping – a technique that measures how the function of one gene is affected by the alteration of a second gene – by analyzing point mutations. Here, we describe how to interpret point mutant genetic interactions, and outline key applications for the approach, including interrogation of protein interaction interfaces and active sites, and examination of post-translational modifications. Genetic interaction analysis has proven effective for characterizing cellular processes; however, to date, systematic high-throughput genetic interaction screens have relied on gene deletions or knockdowns, which limits the resolution of gene function analysis and poses problems for multifunctional genes. Our point mutant approach addresses these issues, and further provides a tool for in vivo structure-function analysis that complements traditional biophysical methods. We also discuss the potential for genetic interaction mapping of point mutations in human cells and its application to personalized medicine. PMID:24842270

Tissue-specific mutation accumulation in human adult stem cells during life

NASA Astrophysics Data System (ADS)

Blokzijl, Francis; de Ligt, Joep; Jager, Myrthe; Sasselli, Valentina; Roerink, Sophie; Sasaki, Nobuo; Huch, Meritxell; Boymans, Sander; Kuijk, Ewart; Prins, Pjotr; Nijman, Isaac J.; Martincorena, Inigo; Mokry, Michal; Wiegerinck, Caroline L.; Middendorp, Sabine; Sato, Toshiro; Schwank, Gerald; Nieuwenhuis, Edward E. S.; Verstegen, Monique M. A.; van der Laan, Luc J. W.; de Jonge, Jeroen; Ijzermans, Jan N. M.; Vries, Robert G.; van de Wetering, Marc; Stratton, Michael R.; Clevers, Hans; Cuppen, Edwin; van Boxtel, Ruben

2016-10-01

The gradual accumulation of genetic mutations in human adult stem cells (ASCs) during life is associated with various age-related diseases, including cancer. Extreme variation in cancer risk across tissues was recently proposed to depend on the lifetime number of ASC divisions, owing to unavoidable random mutations that arise during DNA replication. However, the rates and patterns of mutations in normal ASCs remain unknown. Here we determine genome-wide mutation patterns in ASCs of the small intestine, colon and liver of human donors with ages ranging from 3 to 87 years by sequencing clonal organoid cultures derived from primary multipotent cells. Our results show that mutations accumulate steadily over time in all of the assessed tissue types, at a rate of approximately 40 novel mutations per year, despite the large variation in cancer incidence among these tissues. Liver ASCs, however, have different mutation spectra compared to those of the colon and small intestine. Mutational signature analysis reveals that this difference can be attributed to spontaneous deamination of methylated cytosine residues in the colon and small intestine, probably reflecting their high ASC division rate. In liver, a signature with an as-yet-unknown underlying mechanism is predominant. Mutation spectra of driver genes in cancer show high similarity to the tissue-specific ASC mutation spectra, suggesting that intrinsic mutational processes in ASCs can initiate tumorigenesis. Notably, the inter-individual variation in mutation rate and spectra are low, suggesting tissue-specific activity of common mutational processes throughout life.

Mutation rates for 20 STR loci in a population from São Paulo state, Southeast, Brazil.

PubMed

Martinez, Juliana; Braganholi, Danilo Faustino; Ambrósio, Isabela Brunelli; Polverari, Fernanda Silva; Cicarelli, Regina Maria Barretto

2017-11-01

Short tandem repeats (STRs) are genetic markers largely employed in forensic analysis and paternity investigation cases. When an inconsistency between the parent and child is considered as a possible mutation, the mutation rate should be incorporated into paternity index calculations to give a robust result and to reduce the chance of misinterpretation. The aim of this study was to estimate the mutation rates of 20 autosomal STRs loci used for paternity tests. In these loci we analysed 29,831 parent-child allelic transfers from 929 duo or trio paternity tests carried out during 2012?2016 from São Paulo State, Brazil. We identified 35 mutations in 16 loci, and they were more frequent in the paternal germline compared to the maternal germline. The loci with the highest rate were vWA and FGA and the ones with the lowest rate were PENTA E, PENTA D, D21S11, D7S820 and D6S1043. We did not identified any mutation in D2S1338, TH01, TPOX and D16S539 loci. All mutations consisted of losses or gains of one repeat unit. Mutation rates found in the São Paulo population have peculiarities, which justifies the use of regional databases in laboratories.

Impact of Mutations on the Midpoint Potential of the [4Fe-4S]+1,+2 Cluster and on Catalytic Activity in Electron Transfer Flavoprotein-ubiquinone Oxidoreductase (ETF-QO)†

PubMed Central

Usselman, Robert J.; Fielding, Alistair J.; Frerman, Frank E.; Watmough, Nicholas J.; Eaton, Gareth R.; Eaton, Sandra S.

2011-01-01

Electron transfer flavoprotein - ubiquinone oxidoreductase (ETF-QO) is an iron-sulfur flavoprotein that accepts electrons from electron-transfer flavoprotein (ETF) and reduces ubiquinone from the Q-pool. ETF-QO contains a single [4Fe-4S]2+,1+ cluster and one equivalent of FAD, which are diamagnetic in the isolated oxidized enzyme and can be reduced to paramagnetic forms by enzymatic donors or dithionite. Mutations were introduced by site-directed mutagenesis of amino acids in the vicinity of the iron-sulfur cluster of Rhodobacter sphaeroides ETF-QO. Y501 and T525 are equivalent to Y533 and T558 in the porcine ETF-QO. In the porcine protein, these residues are within hydrogen bonding distance of the Sγ of the cysteine ligands to the iron-sulfur cluster. Y501F, T525A, and Y501F/T525A substitutions were made to determine the effects on midpoint potential, activity, and EPR spectral properties of the cluster. The integrity of the mutated proteins was confirmed by optical spectra, EPR g-values, and spin-lattice relaxation rates, and the cluster to flavin point-dipole distance was determined by relaxation enhancement. Potentiometric titrations were monitored by changes in the CW EPR signals of the cluster and semiquinone. Single mutations decreased the mid-point potentials of the iron-sulfur cluster from +37 mV for wild type to −60 mV for Y501F and T525A and to −128 mV for Y501F/T525A. Lowering the midpoint potential resulted in a decrease in steady-state ubiquinone reductase activity and in ETF semiquinone disproportionation. The decrease in activity demonstrates that reduction of the iron-sulfur cluster is required for activity. There was no detectable effect of the mutations on the flavin midpoint potentials. PMID:18069858

Adenylosuccinate lyase (ADSL) and infantile autism: Absence of previously reported point mutation

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

Fon, E.A.; Sarrazin, J.; Rouleau, G.A.

Autism is a heterogeneous neuropsychiatric syndrome of unknown etiology. There is evidence that a deficiency in the enzyme adenylosuccinate lyase (ADSL), essential for de novo purine biosynthesis, could be involved in the pathogenesis of certain cases. A point mutation in the ADSL gene, resulting in a predicted serine-to-proline substitution and conferring structural instability to the mutant enzyme, has been reported previously in 3 affected siblings. In order to determine the prevalence of the mutation, we PCR-amplified the exon spanning the site of this mutation from the genomic DNA of patients fulfilling DSM-III-R criteria for autistic disorder. None of the 119more » patients tested were found to have this mutation. Furthermore, on preliminary screening using single-strand conformation polymorphism (SSCP), no novel mutations were detected in the coding sequence of four ADSL exons, spanning approximately 50% of the cDNA. In light of these findings, it appears that mutations in the ADSL gene represent a distinctly uncommon cause of autism. 12 refs., 2 figs.« less

Third trimester fetal heart rate predicts phenotype and mutation burden in the type 1 long QT syndrome.

PubMed

Winbo, Annika; Fosdal, Inger; Lindh, Maria; Diamant, Ulla-Britt; Persson, Johan; Wettrell, Göran; Rydberg, Annika

2015-08-01

Early diagnosis and risk stratification is of clinical importance in the long QT syndrome (LQTS), however, little genotype-specific data are available regarding fetal LQTS. We investigate third trimester fetal heart rate, routinely recorded within public maternal health care, as a possible marker for LQT1 genotype and phenotype. This retrospective study includes 184 fetuses from 2 LQT1 founder populations segregating p.Y111C and p.R518X (74 noncarriers and 110 KCNQ1 mutation carriers, whereof 13 double mutation carriers). Pedigree-based measured genotype analysis revealed significant associations between fetal heart rate, genotype, and phenotype; mean third trimester prelabor fetal heart rates obtained from obstetric records (gestational week 29-41) were lower per added mutation (no mutation, 143±5 beats per minute; single mutation, 134±8 beats per minute; double mutations, 111±6 beats per minute; P<0.0001), and lower in symptomatic versus asymptomatic mutation carriers (122±10 versus 137±9 beats per minute; P<0.0001). Strong correlations between fetal heart rate and neonatal heart rate (r=0.700; P<0.001), and postnatal QTc (r=-0.762; P<0.001) were found. In a multivariable model, fetal genotype explained the majority of variance in fetal heart rate (-10 beats per minute per added mutation; P<1.0×10(-23)). Arrhythmia symptoms and intrauterine β-blocker exposure each predicted -7 beats per minute, P<0.0001. In this study including 184 fetuses from 2 LQT1 founder populations, third trimester fetal heart rate discriminated between fetal genotypes and correlated with severity of postnatal cardiac phenotype. This finding strengthens the role of fetal heart rate in the early detection and risk stratification of LQTS, particularly for fetuses with double mutations, at high risk of early life-threatening arrhythmias. © 2015 American Heart Association, Inc.

Hepatitis B virus pre-S/S variants in liver diseases.

PubMed

Chen, Bing-Fang

2018-04-14

Chronic hepatitis B is a global health problem. The clinical outcomes of chronic hepatitis B infection include asymptomatic carrier state, chronic hepatitis (CH), liver cirrhosis (LC), and hepatocellular carcinoma (HCC). Because of the spontaneous error rate inherent to viral reverse transcriptase, the hepatitis B virus (HBV) genome evolves during the course of infection under the antiviral pressure of host immunity. The clinical significance of pre-S/S variants has become increasingly recognized in patients with chronic HBV infection. Pre-S/S variants are often identified in hepatitis B carriers with CH, LC, and HCC, which suggests that these naturally occurring pre-S/S variants may contribute to the development of progressive liver damage and hepatocarcinogenesis. This paper reviews the function of the pre-S/S region along with recent findings related to the role of pre-S/S variants in liver diseases. According to the mutation type, five pre-S/S variants have been identified: pre-S deletion, pre-S point mutation, pre-S1 splice variant, C-terminus S point mutation, and pre-S/S nonsense mutation. Their associations with HBV genotype and the possible pathogenesis of pre-S/S variants are discussed. Different pre-S/S variants cause liver diseases through different mechanisms. Most cause the intracellular retention of HBV envelope proteins and induction of endoplasmic reticulum stress, which results in liver diseases. Pre-S/S variants should be routinely determined in HBV carriers to help identify individuals who may be at a high risk of less favorable liver disease progression. Additional investigations are required to explore the molecular mechanisms of the pre-S/S variants involved in the pathogenesis of each stage of liver disease.

High terbinafine resistance in Trichophyton interdigitale isolates in Delhi, India harbouring mutations in the squalene epoxidase gene.

PubMed

Singh, Ashutosh; Masih, Aradhana; Khurana, Ananta; Singh, Pradeep Kumar; Gupta, Meenakshi; Hagen, Ferry; Meis, Jacques F; Chowdhary, Anuradha

2018-03-25

In the last few years, infections caused by dermatophytes along with a concomitant increase in the number of difficult to treat cases have increasingly been recognised, indicating that dermatophytosis remains a challenging public health problem. The majority of infections are caused by Trichophyton rubrum and Trichophyton mentagrophytes complex. Terbinafine, an allylamine antifungal used orally and topically is considered to be a first-line drug in the therapy of dermatophyte infections. Terbinafine resistance has been predominately attributed to point mutations in the squalene epoxidase (SQLE) target gene a key enzyme in the ergosterol biosynthetic pathway leading to single amino acid substitutions. Here, we report the largest series of 20 terbinafine-resistant Trichophyton interdigitale isolates obtained predominately from cases of tinea corporis/cruris in three hospitals in Delhi, India exhibiting elevated MICs (4 to ≥32 μg/mL) to terbinafine and all harbouring single-point mutations Leu393Phe or Phe397Leu in the SQLE gene. In 12 (60%) T. interdigitale isolates, the Phe397Leu substitution was observed, whereas in the remaining 8 (40%) isolates the substitution Leu393Phe was reported for the first time in T. interdigitale. Furthermore, 10 susceptible T. interdigitale isolates (0.125-2 μg/mL) had a wild-type genotype. Remarkably, considerably high terbinafine resistance rate of 32% was observed among 63 T. interdigitale isolates identified by sequencing of the internal transcribed spacer region. This high level of terbinafine resistance of Indian dermatophyte isolates is worrisome warranting antifungal susceptibility testing and mutation analysis for monitoring this emerging resistance. © 2018 Blackwell Verlag GmbH.

Zinc finger point mutations within the WT1 gene in Wilms tumor patients.

PubMed Central

Little, M H; Prosser, J; Condie, A; Smith, P J; Van Heyningen, V; Hastie, N D

1992-01-01

A proposed Wilms tumor gene, WT1, which encodes a zinc finger protein, has previously been isolated from human chromosome 11p13. Chemical mismatch cleavage analysis was used to identify point mutations in the zinc finger region of this gene in a series of 32 Wilms tumors. Two exonic single base changes were detected. In zinc finger 3 of a bilateral Wilms tumor patient, a constitutional de novo C----T base change was found changing an arginine to a stop codon. One tumor from this patient showed allele loss leading to 11p hemizygosity of the abnormal allele. In zinc finger 2 of a sporadic Wilms tumor patient, a C----T base change resulted in an arginine to cysteine amino acid change. To our knowledge, a WT1 gene missense mutation has not been detected previously in a Wilms tumor. By comparison with a recent NMR and x-ray crystallographic analysis of an analogous zinc finger gene, early growth response gene 1 (EGR1), this amino acid change in WT1 occurs at a residue predicted to be critical for DNA binding capacity and site specificity. The detection of one nonsense point mutation and one missense WT1 gene point mutation adds to the accumulating evidence implicating this gene in a proportion of Wilms tumor patients. Images PMID:1317572

Method for detecting point mutations in DNA utilizing fluorescence energy transfer

DOEpatents

Parkhurst, Lawrence J.; Parkhurst, Kay M.; Middendorf, Lyle

2001-01-01

A method for detecting point mutations in DNA using a fluorescently labeled oligomeric probe and Forster resonance energy transfer (FRET) is disclosed. The selected probe is initially labeled at each end with a fluorescence dye, which act together as a donor/acceptor pair for FRET. The fluorescence emission from the dyes changes dramatically from the duplex stage, wherein the probe is hybridized to the complementary strand of DNA, to the single strand stage, when the probe is melted to become detached from the DNA. The change in fluorescence is caused by the dyes coming into closer proximity after melting occurs and the probe becomes detached from the DNA strand. The change in fluorescence emission as a function of temperature is used to calculate the melting temperature of the complex or T.sub.m. In the case where there is a base mismatch between the probe and the DNA strand, indicating a point mutation, the T.sub.m has been found to be significantly lower than the T.sub.m for a perfectly match probelstand duplex. The present invention allows for the detection of the existence and magnitude of T.sub.m, which allows for the quick and accurate detection of a point mutation in the DNA strand and, in some applications, the determination of the approximate location of the mutation within the sequence.

Population data and mutation rates of 20 autosomal STR loci in a Chinese Han population from Yunnan Province, Southwest China.

PubMed

Zhang, Xiufeng; Liu, Linlin; Xie, Runfang; Wang, Guiyi; Shi, Yuan; Gu, Tao; Hu, Liping; Nie, Shengjie

2018-07-01

The genetic polymorphisms of 20 autosomal short tandem repeat (STR) loci included in the PowerPlex® 21 kit were evaluated from 2068 unrelated, healthy individuals from the Chinese Han population of Yunnan Province in southwest China. All of the loci reached Hardy-Weinberg equilibrium. These loci were examined to determine allele frequencies and forensic statistical parameters. The genetic relationships among the Yunnan Han and other Chinese populations were also estimated. The combined discrimination power and probability of excluding paternity of the 20 STR loci were 0.99999999999999999999999126 and 0.999999975, respectively. In addition, mutation rates from 4363 parentage cases (2215 trios and 2148 duos) were investigated in this study. A total of 164 mutations were observed in 6578 meioses from the 20 loci. The highest mutation rate was observed in D12S391 (0.30%), and the lowest mutation rates were observed in D13S317 (0.03%) and TPOX (0.03%). The average mutation rate for the 20 loci was estimated to be 1.246 × 10 -3 per meiosis. The mutations were primarily single-step and paternal mutations.

An evaluation of germline mutations and reproductive impacts in fathead minnow (Pimephales promelas) exposed to contaminated sediment.

PubMed

Miller, Jason L; Sherry, Jim; Parrott, Joanne; Quinn, James S

2018-06-18

Polycyclic aromatic hydrocarbons (PAHs) have become ubiquitous in the aquatic environment. Some PAHs are mutagenic, potentially causing germline mutations in fish that inhabit PAH contaminated waters. We evaluated the effect of exposure to sediment-borne PAHs on reproduction and germline mutation rates in fathead minnows (Pimephales promelas). Exposure to the contaminated sediment had no significant impact on the reproductive endpoints measured in this study. Germline mutations rates at three microsatellite DNA loci were 1.69 × 10 -3 in fish exposed to PAH-contaminated sediment and 0.55 × 10 -3 in control fish, with zero mutations being observed in fish exposed to sediment from a reference site. While the difference in mutation rates between treatments was not statistically significant for the sample size used (15-19 families per treatment), the observed mutations rates enabled us to estimate the sample size required to detect a significant effect. To our knowledge, this is the first report of germline mutation rates in fathead minnow exposed to an environmental contaminant, providing baseline data for use in the design of future experiments. Crown Copyright © 2018. Published by Elsevier Inc. All rights reserved.

Destabilizing Mutations Alter the Hydrogen Exchange Mechanism in Ribonuclease A

PubMed Central

Bruix, Marta; Ribó, Marc; Benito, Antoni; Laurents, Douglas V.; Rico, Manuel; Vilanova, Maria

2008-01-01

The effect of strongly destabilizing mutations, I106A and V108G of Ribonuclease A (RNase A), on its structure and stability has been determined by NMR. The solution structures of these variants are essentially equivalent to RNase A. The exchange rates of the most protected amide protons in RNase A (35°C), the I106A variant (35°C), and the V108G variant (10°C) yield stability values of 9.9, 6.0, and 6.8 kcal/mol, respectively, when analyzed assuming an EX2 exchange mechanism. Thus, the destabilization induced by these mutations is propagated throughout the protein. Simulation of RNase A hydrogen exchange indicates that the most protected protons in RNase A and the V108G variant exchange via the EX2 regime, whereas those of I106A exchange through a mixed EX1 + EX2 process. It is striking that a single point mutation can alter the overall exchange mechanism. Thus, destabilizing mutations joins high temperatures, high pH and the presence of denaturating agents as a factor that induces EX1 exchange in proteins. The calculations also indicate a shift from the EX2 to the EX1 mechanism for less protected groups within the same protein. This should be borne in mind when interpreting exchange data as a measure of local stability in less protected regions. PMID:18192347

Single-cell paired-end genome sequencing reveals structural variation per cell cycle

PubMed Central

Voet, Thierry; Kumar, Parveen; Van Loo, Peter; Cooke, Susanna L.; Marshall, John; Lin, Meng-Lay; Zamani Esteki, Masoud; Van der Aa, Niels; Mateiu, Ligia; McBride, David J.; Bignell, Graham R.; McLaren, Stuart; Teague, Jon; Butler, Adam; Raine, Keiran; Stebbings, Lucy A.; Quail, Michael A.; D’Hooghe, Thomas; Moreau, Yves; Futreal, P. Andrew; Stratton, Michael R.; Vermeesch, Joris R.; Campbell, Peter J.

2013-01-01

The nature and pace of genome mutation is largely unknown. Because standard methods sequence DNA from populations of cells, the genetic composition of individual cells is lost, de novo mutations in cells are concealed within the bulk signal and per cell cycle mutation rates and mechanisms remain elusive. Although single-cell genome analyses could resolve these problems, such analyses are error-prone because of whole-genome amplification (WGA) artefacts and are limited in the types of DNA mutation that can be discerned. We developed methods for paired-end sequence analysis of single-cell WGA products that enable (i) detecting multiple classes of DNA mutation, (ii) distinguishing DNA copy number changes from allelic WGA-amplification artefacts by the discovery of matching aberrantly mapping read pairs among the surfeit of paired-end WGA and mapping artefacts and (iii) delineating the break points and architecture of structural variants. By applying the methods, we capture DNA copy number changes acquired over one cell cycle in breast cancer cells and in blastomeres derived from a human zygote after in vitro fertilization. Furthermore, we were able to discover and fine-map a heritable inter-chromosomal rearrangement t(1;16)(p36;p12) by sequencing a single blastomere. The methods will expedite applications in basic genome research and provide a stepping stone to novel approaches for clinical genetic diagnosis. PMID:23630320

Genetic epidemiology of Charcot-Marie-Tooth disease.

PubMed

Braathen, G J

2012-01-01

Charcot-Marie-Tooth disease (CMT) is the most common inherited disorder of the peripheral nervous system. The frequency of different CMT genotypes has been estimated in clinic populations, but prevalence data from the general population is lacking. Point mutations in the mitofusin 2 (MFN2) gene has been identified exclusively in Charcot-Marie-Tooth disease type 2 (CMT2), and in a single family with intermediate CMT. MFN2 point mutations are probably the most common cause of CMT2. The CMT phenotype caused by mutation in the myelin protein zero (MPZ) gene varies considerably, from early onset and severe forms to late onset and milder forms. The mechanism is not well understood. The myelin protein zero (P(0) ) mediates adhesion in the spiral wraps of the Schwann cell's myelin sheath. X-linked Charcot-Marie Tooth disease (CMTX) is caused by mutations in the connexin32 (cx32) gene that encodes a polypeptide which is arranged in hexameric array and form gap junctions. Estimate prevalence of CMT. Estimate frequency of Peripheral Myelin Protein 22 (PMP22) duplication and point mutations, insertions and deletions in Cx32, Early growth response 2 (EGR2), MFN2, MPZ, PMP22 and Small integral membrane protein of lysosome/late endosome (SIMPLE) genes. Description of novel mutations in Cx32, MFN2 and MPZ. Description of de novo mutations in MFN2. Our population based genetic epidemiological survey included persons with CMT residing in eastern Akershus County, Norway. The participants were interviewed and examined by one geneticist/neurologist, and classified clinically, neurophysiologically and genetically. Two-hundred and thirty-two consecutive unselected and unrelated CMT families with available DNA from all regions in Norway were included in the MFN2 study. We screened for point mutations in the MFN2 gene. We describe four novel mutations, two in the connexin32 gene and two in the MPZ gene. A total of 245 affected from 116 CMT families from the general population of eastern Akershus county were included in the genetic epidemiological survey. In the general population 1 per 1214 persons (95% CI 1062-1366) has CMT. Charcot-Marie-Tooth disease type 1 (CMT1), CMT2 and intermediate CMT were found in 48.2%, 49.4% and 2.4% of the families, respectively. A mutation in the investigated genes was found in 27.2% of the CMT families and in 28.6% of the affected. The prevalence of the PMP22 duplication and mutations in the Cx32, MPZ and MFN2 genes was found in 13.6%, 6.2%, 1.2%, 6.2% of the families, and in 19.6%, 4.8%, 1.1%, 3.2% of the affected, respectively. None of the families had point mutations, insertions or deletions in the EGR2, PMP22 or SIMPLE genes. Four known and three novel mitofusin 2 (MFN2) point mutations in 8 unrelated Norwegian CMT families were identified. The novel point mutations were not found in 100 healthy controls. This corresponds to 3.4% (8/232) of CMT families having point mutations in MFN2. The phenotypes were compatible with CMT1 in two families, CMT2 in four families, intermediate CMT in one family and distal hereditary motor neuronopathy (dHMN) in one family. A point mutation in the MFN2 gene was found in 2.3% of CMT1, 5.5% of CMT2, 12.5% of intermediate CMT and 6.7% of dHMN families. Two novel missense mutations in the MPZ gene were identified. Family 1 had a c.368G>A (Gly123Asp) transition while family 2 and 3 had a c.103G>A (Asp35Asn) transition. The affected in family 1 had early onset and severe symptoms compatible with Dejerine-Sottas syndrome (DSS), while affected in family 2 and 3 had late onset, milder symptoms and axonal neuropathy compatible with CMT2. Two novel connexin32 mutations that cause early onset X-linked CMT were identified. Family 1 had a deletion c.225delG (R75fsX83) which causes a frameshift and premature stop codon at position 247 while family 2 had a c.536G>A (Cys179Tyr) transition which causes a change of the highly conserved cysteine residue, i.e. disruption of at least one of three disulfide bridges. The mean age at onset was in the first decade and the nerve conduction velocities were in the intermediate range. Charcot-Marie-Tooth disease is the most common inherited neuropathy. At present 47 hereditary neuropathy genes are known, and an examination of all known genes would probably only identify mutations in approximately 50% of those with CMT. Thus, it is likely that at least 30-50 CMT genes are yet to be identified. The identified known and novel point mutations in the MFN2 gene expand the clinical spectrum from CMT2 and intermediate CMT to also include possibly CMT1 and the dHMN phenotypes. Thus, genetic analyses of the MFN2 gene should not be restricted to persons with CMT2. The phenotypic variation caused by different missense mutations in the MPZ gene is likely caused by different conformational changes of the MPZ protein which affects the functional tetramers. Severe changes of the MPZ protein cause dysfunctional tetramers and predominantly uncompacted myelin, i.e. the severe phenotypes congenital hypomyelinating neuropathy and DSS, while milder changes cause the phenotypes CMT1 and CMT2. The two novel mutations in the connexin32 gene are more severe than the majority of previously described mutations possibly due to the severe structural change of the gap junction they encode. Charcot-Marie-Tooth disease is the most common inherited disorder of the peripheral nervous system with an estimated prevalence of 1 in 1214. CMT1 and CMT2 are equally frequent in the general population. The prevalence of PMP22 duplication and of mutations in Cx32, MPZ and MFN2 is 19.6%, 4.8%, 1.1% and 3.2%, respectively. The ratio of probable de novo mutations in CMT families was estimated to be 22.7%. Genotype- phenotype correlations for seven novel mutations in the genes Cx32 (2), MFN2 (3) and MPZ (2) are described. Two novel phenotypes were ascribed to the MFN2 gene, however further studies are needed to confirm that MFN2 mutations can cause CMT1 and dHMN. © 2012 John Wiley & Sons A/S.

Efficient Mutagenesis Independent of Ligation (EMILI).

PubMed

Füzik, Tibor; Ulbrich, Pavel; Ruml, Tomáš

2014-11-01

Site-directed mutagenesis is one of the most widely used techniques in life sciences. Here we describe an improved and simplified method for introducing mutations at desired sites. It consists of an inverse PCR using a plasmid template and two partially complementary primers. The synthesis step is followed by annealing of the PCR product's sticky ends, which are generated by exonuclease digestion. This method is fast, extremely efficient and cost-effective. It can be used to introduce large insertions and deletions, but also for multiple point mutations in a single step. To show the principle and to prove the efficiency of the method, we present a series of basic mutations (insertions, deletions, point mutations) on pUC19 plasmid DNA. Copyright © 2014 Elsevier B.V. All rights reserved.

Analysis of mutational spectra by denaturant capillary electrophoresis

PubMed Central

Ekstrøm, Per O.; Khrapko, Konstantin; Li-Sucholeiki, Xiao-Cheng; Hunter, Ian W.; Thilly, William G.

2009-01-01

Numbers and kinds of point mutant within DNA from cells, tissues and human population may be discovered for nearly any 75–250bp DNA sequence. High fidelity DNA amplification incorporating a thermally stable DNA “clamp” is followed by separation by denaturing capillary electrophoresis (DCE). DCE allows for peak collection and verification sequencing. DCE in a mode of cycling temperature, e.g.+/− 5°C, CyDCE, permits high resolution of mutant sequences using computer defined analytes without preliminary optimization experiments. DNA sequencers have been modified to permit higher throughput CyDCE and a massively parallel,~25,000 capillary system, has been designed for pangenomic scans in large human populations. DCE has been used to define quantitative point mutational spectra for study a wide variety of genetic phenomena: errors of DNA polymerases, mutations induced in human cells by chemicals and irradiation, testing of human gene-common disease associations and the discovery of origins of point mutations in human development and carcinogenesis. PMID:18600220

Gene Amplification and Point Mutations in Pyrimidine Metabolic Genes in 5-Fluorouracil Resistant Leishmania infantum

PubMed Central

Ritt, Jean-François; Raymond, Frédéric; Leprohon, Philippe; Légaré, Danielle; Corbeil, Jacques; Ouellette, Marc

2013-01-01

Background The human protozoan parasites Leishmania are prototrophic for pyrimidines with the ability of both de novo biosynthesis and uptake of pyrimidines. Methodology/Principal Findings Five independent L. infantum mutants were selected for resistance to the pyrimidine analogue 5-fluorouracil (5-FU) in the hope to better understand the metabolism of pyrimidine in Leishmania. Analysis of the 5-FU mutants by comparative genomic hybridization and whole genome sequencing revealed in selected mutants the amplification of DHFR-TS and a deletion of part of chromosome 10. Point mutations in uracil phosphorybosyl transferase (UPRT), thymidine kinase (TK) and uridine phosphorylase (UP) were also observed in three individual resistant mutants. Transfection experiments confirmed that these point mutations were responsible for 5-FU resistance. Transport studies revealed that one resistant mutant was defective for uracil and 5-FU import. Conclusion/Significance This study provided further insights in pyrimidine metabolism in Leishmania and confirmed that multiple mutations can co-exist and lead to resistance in Leishmania. PMID:24278495

Restriction digest screening facilitates efficient detection of site-directed mutations introduced by CRISPR in C. albicans UME6

PubMed Central

Evans, Ben A.; Smith, Olivia L.; Pickerill, Ethan S.; York, Mary K.; Buenconsejo, Kristen J.P.; Chambers, Antonio E.

2018-01-01

Introduction of point mutations to a gene of interest is a powerful tool when determining protein function. CRISPR-mediated genome editing allows for more efficient transfer of a desired mutation into a wide range of model organisms. Traditionally, PCR amplification and DNA sequencing is used to determine if isolates contain the intended mutation. However, mutation efficiency is highly variable, potentially making sequencing costly and time consuming. To more efficiently screen for correct transformants, we have identified restriction enzymes sites that encode for two identical amino acids or one or two stop codons. We used CRISPR to introduce these restriction sites directly upstream of the Candida albicans UME6 Zn2+-binding domain, a known regulator of C. albicans filamentation. While repair templates coding for different restriction sites were not equally successful at introducing mutations, restriction digest screening enabled us to rapidly identify isolates with the intended mutation in a cost-efficient manner. In addition, mutated isolates have clear defects in filamentation and virulence compared to wild type C. albicans. Our data suggest restriction digestion screening efficiently identifies point mutations introduced by CRISPR and streamlines the process of identifying residues important for a phenotype of interest. PMID:29892505

Thermal Stability of Rhodopsin and Progression of Retinitis Pigmentosa

PubMed Central

Liu, Monica Yun; Liu, Jian; Mehrotra, Devi; Liu, Yuting; Guo, Ying; Baldera-Aguayo, Pedro A.; Mooney, Victoria L.; Nour, Adel M.; Yan, Elsa C. Y.

2013-01-01

Over 100 point mutations in the rhodopsin gene have been associated with retinitis pigmentosa (RP), a family of inherited visual disorders. Among these, we focused on characterizing the S186W mutation. We compared the thermal properties of the S186W mutant with another RP-causing mutant, D190N, and with WT rhodopsin. To assess thermal stability, we measured the rate of two thermal reactions contributing to the thermal decay of rhodopsin as follows: thermal isomerization of 11-cis-retinal and hydrolysis of the protonated Schiff base linkage between the 11-cis-retinal chromophore and opsin protein. We used UV-visible spectroscopy and HPLC to examine the kinetics of these reactions at 37 and 55 °C for WT and mutant rhodopsin purified from HEK293 cells. Compared with WT rhodopsin and the D190N mutant, the S186W mutation dramatically increases the rates of both thermal isomerization and dark state hydrolysis of the Schiff base by 1–2 orders of magnitude. The results suggest that the S186W mutant thermally destabilizes rhodopsin by disrupting a hydrogen bond network at the receptor's active site. The decrease in the thermal stability of dark state rhodopsin is likely to be associated with higher levels of dark noise that undermine the sensitivity of rhodopsin, potentially accounting for night blindness in the early stages of RP. Further studies of the thermal stability of additional pathogenic rhodopsin mutations in conjunction with clinical studies are expected to provide insight into the molecular mechanism of RP and test the correlation between rhodopsin's thermal stability and RP progression in patients. PMID:23625926

Importance of DNA repair in tumor suppression

NASA Astrophysics Data System (ADS)

Brumer, Yisroel; Shakhnovich, Eugene I.

2004-12-01

The transition from a normal to cancerous cell requires a number of highly specific mutations that affect cell cycle regulation, apoptosis, differentiation, and many other cell functions. One hallmark of cancerous genomes is genomic instability, with mutation rates far greater than those of normal cells. In microsatellite instability (MIN tumors), these are often caused by damage to mismatch repair genes, allowing further mutation of the genome and tumor progression. These mutation rates may lie near the error catastrophe found in the quasispecies model of adaptive RNA genomes, suggesting that further increasing mutation rates will destroy cancerous genomes. However, recent results have demonstrated that DNA genomes exhibit an error threshold at mutation rates far lower than their conservative counterparts. Furthermore, while the maximum viable mutation rate in conservative systems increases indefinitely with increasing master sequence fitness, the semiconservative threshold plateaus at a relatively low value. This implies a paradox, wherein inaccessible mutation rates are found in viable tumor cells. In this paper, we address this paradox, demonstrating an isomorphism between the conservatively replicating (RNA) quasispecies model and the semiconservative (DNA) model with post-methylation DNA repair mechanisms impaired. Thus, as DNA repair becomes inactivated, the maximum viable mutation rate increases smoothly to that of a conservatively replicating system on a transformed landscape, with an upper bound that is dependent on replication rates. On a specific single fitness peak landscape, the repair-free semiconservative system is shown to mimic a conservative system exactly. We postulate that inactivation of post-methylation repair mechanisms is fundamental to the progression of a tumor cell and hence these mechanisms act as a method for the prevention and destruction of cancerous genomes.

Mutation and Evolutionary Rates in Adélie Penguins from the Antarctic

PubMed Central

Millar, Craig D.; Dodd, Andrew; Anderson, Jennifer; Gibb, Gillian C.; Ritchie, Peter A.; Baroni, Carlo; Woodhams, Michael D.; Hendy, Michael D.; Lambert, David M.

2008-01-01

Precise estimations of molecular rates are fundamental to our understanding of the processes of evolution. In principle, mutation and evolutionary rates for neutral regions of the same species are expected to be equal. However, a number of recent studies have shown that mutation rates estimated from pedigree material are much faster than evolutionary rates measured over longer time periods. To resolve this apparent contradiction, we have examined the hypervariable region (HVR I) of the mitochondrial genome using families of Adélie penguins (Pygoscelis adeliae) from the Antarctic. We sequenced 344 bps of the HVR I from penguins comprising 508 families with 915 chicks, together with both their parents. All of the 62 germline heteroplasmies that we detected in mothers were also detected in their offspring, consistent with maternal inheritance. These data give an estimated mutation rate (μ) of 0.55 mutations/site/Myrs (HPD 95% confidence interval of 0.29–0.88 mutations/site/Myrs) after accounting for the persistence of these heteroplasmies and the sensitivity of current detection methods. In comparison, the rate of evolution (k) of the same HVR I region, determined using DNA sequences from 162 known age sub-fossil bones spanning a 37,000-year period, was 0.86 substitutions/site/Myrs (HPD 95% confidence interval of 0.53 and 1.17). Importantly, the latter rate is not statistically different from our estimate of the mutation rate. These results are in contrast to the view that molecular rates are time dependent. PMID:18833304

IFITM5 mutations and osteogenesis imperfecta.

PubMed

Hanagata, Nobutaka

2016-03-01

Interferon-induced transmembrane protein 5 (IFITM5) is an osteoblast-specific membrane protein that has been shown to be a positive regulatory factor for mineralization in vitro. However, Ifitm5 knockout mice do not exhibit serious bone abnormalities, and thus the function of IFITM5 in vivo remains unclear. Recently, a single point mutation (c.-14C>T) in the 5' untranslated region of IFITM5 was identified in patients with osteogenesis imperfecta type V (OI-V). Furthermore, a single point mutation (c.119C>T) in the coding region of IFITM5 was identified in OI patients with more severe symptoms than patients with OI-V. Although IFITM5 is not directly involved in the formation of bone in vivo, the reason why IFITM5 mutations cause OI remains a major mystery. In this review, the current state of knowledge of OI pathological mechanisms due to IFITM5 mutations will be reviewed.

Epistatic Interactions within the Influenza A Virus Polymerase Complex Mediate Mutagen Resistance and Replication Fidelity

PubMed Central

Pauly, Matthew D.; Lyons, Daniel M.; Fitzsimmons, William J.

2017-01-01

ABSTRACT Lethal mutagenesis is a broad-spectrum antiviral strategy that employs mutagenic nucleoside analogs to exploit the high mutation rate and low mutational tolerance of many RNA viruses. Studies of mutagen-resistant viruses have identified determinants of replicative fidelity and the importance of mutation rate to viral population dynamics. We have previously demonstrated the effective lethal mutagenesis of influenza A virus using three nucleoside analogs as well as the virus’s high genetic barrier to mutagen resistance. Here, we investigate the mutagen-resistant phenotypes of mutations that were enriched in drug-treated populations. We find that PB1 T123A has higher replicative fitness than the wild type, PR8, and maintains its level of genome production during 5-fluorouracil (2,4-dihydroxy-5-fluoropyrimidine) treatment. Surprisingly, this mutagen-resistant variant also has an increased baseline rate of C-to-U and G-to-A mutations. A second drug-selected mutation, PA T97I, interacts epistatically with PB1 T123A to mediate high-level mutagen resistance, predominantly by limiting the inhibitory effect of nucleosides on polymerase activity. Consistent with the importance of epistatic interactions in the influenza virus polymerase, our data suggest that nucleoside analog resistance and replication fidelity are strain dependent. Two previously identified ribavirin {1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1H-1,2,4-triazole-3-carboxamide} resistance mutations, PB1 V43I and PB1 D27N, do not confer drug resistance in the PR8 background, and the PR8-PB1 V43I polymerase exhibits a normal baseline mutation rate. Our results highlight the genetic complexity of the influenza A virus polymerase and demonstrate that increased replicative capacity is a mechanism by which an RNA virus can counter the negative effects of elevated mutation rates. IMPORTANCE RNA viruses exist as genetically diverse populations. This standing genetic diversity gives them the potential to adapt rapidly, evolve resistance to antiviral therapeutics, and evade immune responses. Viral mutants with altered mutation rates or mutational tolerance have provided insights into how genetic diversity arises and how it affects the behavior of RNA viruses. To this end, we identified variants within the polymerase complex of influenza virus that are able to tolerate drug-mediated increases in viral mutation rates. We find that drug resistance is highly dependent on interactions among mutations in the polymerase complex. In contrast to other viruses, influenza virus counters the effect of higher mutation rates primarily by maintaining high levels of genome replication. These findings suggest the importance of maintaining large population sizes for viruses with high mutation rates and show that multiple proteins can affect both mutation rate and genome synthesis. PMID:28815216

Computer-guided design, synthesis, and biological evaluation of quinoxalinebisarylureas as FLT3 inhibitors.

PubMed

Göring, Stefan; Bensinger, Dennis; Naumann, Eva C; Schmidt, Boris

2015-03-01

Activating mutations of FMS-like tyrosine kinase 3 (FLT3) are present in ∼30 % of patients with acute myeloid leukemia (AML) and are associated with poor prognosis. Point mutations in the tyrosine kinase domain (TKD) are observed as primary mutations or are acquired as secondary mutations in FLT3 with internal tandem duplications (ITDs) after treatment with tyrosine kinase inhibitors (TKIs). Although dozens of potent inhibitors against FLT3 ITD have been reported, activating TKD point mutations, especially at residues F691 and D835, remain the leading cause for therapy resistance, highlighting the consistent need for new potent inhibitors. Herein we report the identification and characterization of novel quinoxaline-based FLT3 inhibitors. We used the pharmacophore features of diverse known inhibitors as a starting point for a new optimization algorithm for type II TKIs, starting from an in silico library pharmacophore search and induced-fit docking in the known FLT3 structure. This led to the design of a set of diverse quinoxalinebisarylureas, which were profiled in an FLT3 kinase activity assay. The most promising compounds were further evaluated in a zebrafish embryo phenotype assay. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Deep sequencing of natural and experimental populations of Drosophila melanogaster reveals biases in the spectrum of new mutations.

PubMed

Assaf, Zoe June; Tilk, Susanne; Park, Jane; Siegal, Mark L; Petrov, Dmitri A

2017-12-01

Mutations provide the raw material of evolution, and thus our ability to study evolution depends fundamentally on having precise measurements of mutational rates and patterns. We generate a data set for this purpose using (1) de novo mutations from mutation accumulation experiments and (2) extremely rare polymorphisms from natural populations. The first, mutation accumulation (MA) lines are the product of maintaining flies in tiny populations for many generations, therefore rendering natural selection ineffective and allowing new mutations to accrue in the genome. The second, rare genetic variation from natural populations allows the study of mutation because extremely rare polymorphisms are relatively unaffected by the filter of natural selection. We use both methods in Drosophila melanogaster , first generating our own novel data set of sequenced MA lines and performing a meta-analysis of all published MA mutations (∼2000 events) and then identifying a high quality set of ∼70,000 extremely rare (≤0.1%) polymorphisms that are fully validated with resequencing. We use these data sets to precisely measure mutational rates and patterns. Highlights of our results include: a high rate of multinucleotide mutation events at both short (∼5 bp) and long (∼1 kb) genomic distances, showing that mutation drives GC content lower in already GC-poor regions, and using our precise context-dependent mutation rates to predict long-term evolutionary patterns at synonymous sites. We also show that de novo mutations from independent MA experiments display similar patterns of single nucleotide mutation and well match the patterns of mutation found in natural populations. © 2017 Assaf et al.; Published by Cold Spring Harbor Laboratory Press.

Genetic evolution of HIV in patients remaining on a stable HAART regimen despite insufficient viral suppression.

PubMed

Kristiansen, Thomas B; Pedersen, Anders G; Eugen-Olsen, Jesper; Katzenstein, Terese L; Lundgren, Jens D

2005-01-01

Our objective was to investigate whether steadily increasing resistance levels are inevitable in the course of a failing but unchanged Highly Active Antiretroviral Therapy (HAART) regimen. Patients having an unchanged HAART regimen and a good CD4 response (100 cells/microl above nadir) despite consistent HIV-RNA levels above 200 copies/ml were included in the study. The study period spanned at least 12 months and included 47 plasma samples from 17 patients that were sequenced and analysed with respect to evolutionary changes. At inclusion, the median CD4 count was 300 cells/ml (inter-quartile range (IQR): 231-380) and the median HIV-RNA was 2000 copies/ml (IQR: 1301-6090). Reverse transcription inhibitor (RTI) mutations increased 0.5 mutations per y (STD = 0.8 mutations per y), while major protease inhibitor (PI) resistance mutations increased at a rate of 0.2 mutations per y (STD = 0.8 mutations per y) and minor PI resistance mutations increased at a rate of 0.3 mutations per y (STD = 0.7 mutations per y). The rate at which RTI mutations accumulated decreased during the study period (p = 0.035). Interestingly, the rate of mutation accumulation was not associated with HIV-RNA level. The majority of patients kept accumulating new resistance mutations. However, 3 out of 17 patients with viral failure were caught in an apparent mutational deadlock, thus the development of additional resistance during a failing HAART is not inevitable. We hypothesize that certain patterns of mutations can cause a mutational deadlock where the evolutionary benefit of further resistance mutation is limited if the patient is kept on a stable HAART regimen.

The Role of MC1R in Speciation & Phylogeny

ERIC Educational Resources Information Center

Offner, Susan

2013-01-01

A point mutation in the MC1R gene, a G-protein-coupled receptor, has been found that could have led to the formation of two subspecies of Solomon Island flycatcher from a single ancestral population. I discuss the many roles that G-protein-coupled receptors play in vertebrate physiology and how one particular point mutation can have enormous…

Glassy Dynamics in the Adaptive Immune Response Prevents Autoimmune Disease

NASA Astrophysics Data System (ADS)

Sun, Jun; Deem, Michael

2006-03-01

The immune system normally protects the human host against death by infection. However, when an immune response is mistakenly directed at self antigens, autoimmune disease can occur. We describe a model of protein evolution to simulate the dynamics of the adaptive immune response to antigens. Computer simulations of the dynamics of antibody evolution show that different evolutionary mechanisms, namely gene segment swapping and point mutation, lead to different evolved antibody binding affinities. Although a combination of gene segment swapping and point mutation can yield a greater affinity to a specific antigen than point mutation alone, the antibodies so evolved are highly cross-reactive and would cause autoimmune disease, and this is not the chosen dynamics of the immune system. We suggest that in the immune system a balance has evolved between binding affinity and specificity in the mechanism for searching the amino acid sequence space of antibodies. Our model predicts that chronic infection may lead to autoimmune disease as well due to cross-reactivity and suggests a broad distribution for the time of onset of autoimmune disease due to chronic exposure. The slow search of antibody sequence space by point mutation leads to the broad of distribution times.

Multiplex sequence analysis demonstrates the competitive growth advantage of the A-to-G mutants of clarithromycin-resistant Helicobacter pylori.

PubMed

Wang, G; Rahman, M S; Humayun, M Z; Taylor, D E

1999-03-01

Clarithromycin resistance in Helicobacter pylori is due to point mutation within the 23S rRNA. We examined the growth rates of different types of site-directed mutants and demonstrated quantitatively the competitive growth advantage of A-to-G mutants over other types of mutants by a multiplex sequencing assay. The results provide a rational explanation of why A-to-G mutants are predominantly observed among clarithromycin-resistant clinical isolates.

Multiplex Sequence Analysis Demonstrates the Competitive Growth Advantage of the A-to-G Mutants of Clarithromycin-Resistant Helicobacter pylori

PubMed Central

Wang, Ge; Rahman, M. Sayeedur; Humayun, M. Zafri; Taylor, Diane E.

1999-01-01

Clarithromycin resistance in Helicobacter pylori is due to point mutation within the 23S rRNA. We examined the growth rates of different types of site-directed mutants and demonstrated quantitatively the competitive growth advantage of A-to-G mutants over other types of mutants by a multiplex sequencing assay. The results provide a rational explanation of why A-to-G mutants are predominantly observed among clarithromycin-resistant clinical isolates. PMID:10049289

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

Role and Mechanism of Structural Variation in Progression of Breast Cancer

DTIC Science & Technology

2013-09-01

mutations that occurred throughout tumor evolution, we identified 9 early nonsynonymous point mutations that occurred in cancer genes . Only five of...identified, are mutations in the TP53 gene suggesting its role as a driver mutation   5   • Our data also suggests that in the case of this one patient...generated by breakage-fusion- bridge cycles that promote repeated rounds of mutation within a chromosome arm, or from progressive amplification of genes that

Identification of point mutations in clinical Staphylococcus aureus strains that produce small-colony variants auxotrophic for menadione.

PubMed

Dean, Melissa A; Olsen, Randall J; Long, S Wesley; Rosato, Adriana E; Musser, James M

2014-04-01

Staphylococcus aureus small-colony variants (SCVs) are implicated in chronic and relapsing infections that are difficult to diagnose and treat. Despite many years of study, the underlying molecular mechanisms and virulence effect of the small-colony phenotype remain incompletely understood. We sequenced the genomes of five S. aureus SCV strains recovered from human patients and discovered previously unidentified nonsynonymous point mutations in three genes encoding proteins in the menadione biosynthesis pathway. Analysis of genetic revertants and complementation with wild-type alleles confirmed that these mutations caused the SCV phenotype and decreased virulence for mice.

A Fluorescence Quenching Assay Based on Molecular Beacon Formation through a Ligase Detection Reaction for Facile and Rapid Detection of Point Mutations.

PubMed

Sawamura, Kensuke; Hashimoto, Masahiko

2017-01-01

A fluorescence quenching assay based on a ligase detection reaction was developed for facile and rapid detection of point mutations present in a mixed population of non-variant DNA. If the test DNA carried a targeted mutation, then the two allele-specific primers were ligated to form a molecular beacon resulting in the expected fluorescence quenching signatures. Using this method, we successfully detected as low as 5% mutant DNA in a mixture of wild-type DNA (t test at 99% confidence level).

Chromobacterium violaceum ω-transaminase variant Trp60Cys shows increased specificity for (S)-1-phenylethylamine and 4'-substituted acetophenones, and follows Swain-Lupton parameterisation.

PubMed

Cassimjee, Karim Engelmark; Humble, Maria Svedendahl; Land, Henrik; Abedi, Vahak; Berglund, Per

2012-07-28

For biocatalytic production of pharmaceutically important chiral amines the ω-transaminase enzymes have proven useful. Engineering of these enzymes has to some extent been accomplished by rational design, but mostly by directed evolution. By use of a homology model a key point mutation in Chromobacterium violaceum ω-transaminase was found upon comparison with engineered variants from homologous enzymes. The variant Trp60Cys gave increased specificity for (S)-1-phenylethylamine (29-fold) and 4'-substituted acetophenones (∼5-fold). To further study the effect of the mutation the reaction rates were Swain-Lupton parameterised. On comparison with the wild type, reactions of the variant showed increased resonance dependence; this observation together with changed pH optimum and cofactor dependence suggests an altered reaction mechanism.

Contextualizing Genetics for Regional Heart Failure Care

PubMed Central

Iyngkaran, Pupalan; Thomas, Merlin C.; Johnson, Renee; French, John; Ilton, Marcus; McDonald, Peter; Hare, David L.; Fatkin, Diane

2016-01-01

Congestive heart failure (CHF) is a chronic and often devastating cardiovascular disorder with no cure. There has been much advancement in the last two decades that has seen improvements in morbidity and mortality. Clinicians have also noted variations in the responses to therapies. More detailed observations also point to clusters of diseases, phenotypic groupings, unusual severity and the rates at which CHF occurs. Medical genetics is playing an increasingly important role in answering some of these observations. This developing field in many respects provides more information than is currently clinically applicable. This includes making sense of the established single gene mutations or uncommon private mutations. In this thematic series which discusses the many factors that could be relevant for CHF care, once established treatments are available in the communities; this section addresses a contextual role for medical genetics. PMID:27280306

Faster-X evolution: Theory and evidence from Drosophila.

PubMed

Charlesworth, Brian; Campos, José L; Jackson, Benjamin C

2018-02-12

A faster rate of adaptive evolution of X-linked genes compared with autosomal genes can be caused by the fixation of recessive or partially recessive advantageous mutations, due to the full expression of X-linked mutations in hemizygous males. Other processes, including recombination rate and mutation rate differences between X chromosomes and autosomes, may also cause faster evolution of X-linked genes. We review population genetics theory concerning the expected relative values of variability and rates of evolution of X-linked and autosomal DNA sequences. The theoretical predictions are compared with data from population genomic studies of several species of Drosophila. We conclude that there is evidence for adaptive faster-X evolution of several classes of functionally significant nucleotides. We also find evidence for potential differences in mutation rates between X-linked and autosomal genes, due to differences in mutational bias towards GC to AT mutations. Many aspects of the data are consistent with the male hemizygosity model, although not all possible confounding factors can be excluded. © 2018 John Wiley & Sons Ltd.

High-Throughput Identification of Loss-of-Function Mutations for Anti-Interferon Activity in the Influenza A Virus NS Segment

PubMed Central

Wu, Nicholas C.; Young, Arthur P.; Al-Mawsawi, Laith Q.; Olson, C. Anders; Feng, Jun; Qi, Hangfei; Luan, Harding H.; Li, Xinmin; Wu, Ting-Ting

2014-01-01

ABSTRACT Viral proteins often display several functions which require multiple assays to dissect their genetic basis. Here, we describe a systematic approach to screen for loss-of-function mutations that confer a fitness disadvantage under a specified growth condition. Our methodology was achieved by genetically monitoring a mutant library under two growth conditions, with and without interferon, by deep sequencing. We employed a molecular tagging technique to distinguish true mutations from sequencing error. This approach enabled us to identify mutations that were negatively selected against, in addition to those that were positively selected for. Using this technique, we identified loss-of-function mutations in the influenza A virus NS segment that were sensitive to type I interferon in a high-throughput fashion. Mechanistic characterization further showed that a single substitution, D92Y, resulted in the inability of NS to inhibit RIG-I ubiquitination. The approach described in this study can be applied under any specified condition for any virus that can be genetically manipulated. IMPORTANCE Traditional genetics focuses on a single genotype-phenotype relationship, whereas high-throughput genetics permits phenotypic characterization of numerous mutants in parallel. High-throughput genetics often involves monitoring of a mutant library with deep sequencing. However, deep sequencing suffers from a high error rate (∼0.1 to 1%), which is usually higher than the occurrence frequency for individual point mutations within a mutant library. Therefore, only mutations that confer a fitness advantage can be identified with confidence due to an enrichment in the occurrence frequency. In contrast, it is impossible to identify deleterious mutations using most next-generation sequencing techniques. In this study, we have applied a molecular tagging technique to distinguish true mutations from sequencing errors. It enabled us to identify mutations that underwent negative selection, in addition to mutations that experienced positive selection. This study provides a proof of concept by screening for loss-of-function mutations on the influenza A virus NS segment that are involved in its anti-interferon activity. PMID:24965464

A site specific model and analysis of the neutral somatic mutation rate in whole-genome cancer data.

PubMed

Bertl, Johanna; Guo, Qianyun; Juul, Malene; Besenbacher, Søren; Nielsen, Morten Muhlig; Hornshøj, Henrik; Pedersen, Jakob Skou; Hobolth, Asger

2018-04-19

Detailed modelling of the neutral mutational process in cancer cells is crucial for identifying driver mutations and understanding the mutational mechanisms that act during cancer development. The neutral mutational process is very complex: whole-genome analyses have revealed that the mutation rate differs between cancer types, between patients and along the genome depending on the genetic and epigenetic context. Therefore, methods that predict the number of different types of mutations in regions or specific genomic elements must consider local genomic explanatory variables. A major drawback of most methods is the need to average the explanatory variables across the entire region or genomic element. This procedure is particularly problematic if the explanatory variable varies dramatically in the element under consideration. To take into account the fine scale of the explanatory variables, we model the probabilities of different types of mutations for each position in the genome by multinomial logistic regression. We analyse 505 cancer genomes from 14 different cancer types and compare the performance in predicting mutation rate for both regional based models and site-specific models. We show that for 1000 randomly selected genomic positions, the site-specific model predicts the mutation rate much better than regional based models. We use a forward selection procedure to identify the most important explanatory variables. The procedure identifies site-specific conservation (phyloP), replication timing, and expression level as the best predictors for the mutation rate. Finally, our model confirms and quantifies certain well-known mutational signatures. We find that our site-specific multinomial regression model outperforms the regional based models. The possibility of including genomic variables on different scales and patient specific variables makes it a versatile framework for studying different mutational mechanisms. Our model can serve as the neutral null model for the mutational process; regions that deviate from the null model are candidates for elements that drive cancer development.

Deletion Mutagenesis Downstream of the 5′ Long Terminal Repeat of Human Immunodeficiency Virus Type 1 Is Compensated for by Point Mutations in both the U5 Region and gag Gene

PubMed Central

Liang, Chen; Rong, Liwei; Russell, Rodney S.; Wainberg, Mark A.

2000-01-01

We have studied the role of an RNA region at nucleotides (nt) +200 to +233, just downstream of the 5′ long terminal repeat, in encapsidation of human immunodeficiency virus type 1 genomic RNA. Three deletion mutations, namely, BH-D0, BH-D1, and BH-D2, were generated to eliminate sequences at positions nt +200 to +219, +200 to +226, and +200 to +233. The result in each case was decreased levels of packaging of viral RNA into the mutated viruses, with the BH-D2 virus being the most severely affected. Consistently, all three deletions resulted in impaired viral infectiousness and the BH-D2 mutation showed the most dramatic impact in this regard. Further analysis revealed additional defects in Gag precursor processing and in the extension efficiency of the tRNA3Lys primer in reverse transcription reactions performed with these mutated viruses. To shed further light on the function of these deleted sequences in viral replication, the mutated viruses were cultured in MT-2 cells over prolonged periods to enable them to reacquire wild-type replication kinetics. Sequencing of the reverted viruses revealed point mutations in both the noncoding region and the gag gene. In the case of the BH-D0 revertant, two mutations were observed at positions G112A in the U5 region, termed M1, and T24I in the nucleocapsid protein, termed MNC, respectively. Either of these two mutations was able to confer wild-type replication capacity on BH-D0. In the case of BH-D1, each of the M1 mutations, a mutation termed M2, i.e., C227T, just downstream of the primer binding site, a mutation termed MP2 (T12I) in the p2 protein, and the MNC mutation were observed. A combination of either M1 and M2 or MP2 and MNC was able to rescue BH-D1. In the case of the BH-D2 deletion-containing viruses, three point mutations, i.e., M1, MP2, and MNC, were observed and the presence of all three was required to restore viral replication to wild-type levels. PMID:10864634

Evaluating the evaluation of cancer driver genes

PubMed Central

Tokheim, Collin J.; Papadopoulos, Nickolas; Kinzler, Kenneth W.; Vogelstein, Bert; Karchin, Rachel

2016-01-01

Sequencing has identified millions of somatic mutations in human cancers, but distinguishing cancer driver genes remains a major challenge. Numerous methods have been developed to identify driver genes, but evaluation of the performance of these methods is hindered by the lack of a gold standard, that is, bona fide driver gene mutations. Here, we establish an evaluation framework that can be applied to driver gene prediction methods. We used this framework to compare the performance of eight such methods. One of these methods, described here, incorporated a machine-learning–based ratiometric approach. We show that the driver genes predicted by each of the eight methods vary widely. Moreover, the P values reported by several of the methods were inconsistent with the uniform values expected, thus calling into question the assumptions that were used to generate them. Finally, we evaluated the potential effects of unexplained variability in mutation rates on false-positive driver gene predictions. Our analysis points to the strengths and weaknesses of each of the currently available methods and offers guidance for improving them in the future. PMID:27911828

Analysis of Clinical Isolates of Helicobacter pylori in Pakistan Reveals High Degrees of Pathogenicity and High Frequencies of Antibiotic Resistance

PubMed Central

Rasheed, Faisal; Campbell, Barry James; Alfizah, Hanafiah; Varro, Andrea; Zahra, Rabaab; Yamaoka, Yoshio; Pritchard, David Mark

2014-01-01

Background Antibiotic resistance in Helicobacter pylori contributes to failure in eradicating the infection and is most often due to point and missense mutations in a few key genes. Methods The antibiotic susceptibility profiles of H. pylori isolates from 46 Pakistani patients were determined by Etest. Resistance and pathogenicity genes were amplified, and sequences were analyzed to determine the presence of mutations. Results A high percentage of isolates (73.9%) were resistant to metronidazole (MTZ), with considerable resistance to clarithromycin (CLR; 47.8%) and amoxicillin (AML; 54.3%) also observed. Relatively few isolates were resistant to tetracycline (TET; 4.3%) or to ciprofloxacin (CIP; 13%). However, most isolates (n = 43) exhibited resistance to one or more antibiotics. MTZ-resistant isolates contained missense mutations in oxygen-independent NADPH nitroreductase (RdxA; 8 mutations found) and NADH flavin oxidoreductase (FrxA; 4 mutations found). In the 23S rRNA gene, responsible for CLR resistance, a new point mutation (A2181G) and 4 previously reported mutations were identified. Pathogenicity genes cagA, dupA, and vacA s1a/m1 were detected frequently in isolates which were also found to be resistant to MTZ, CLR, and AML. A high percentage of CagA and VacA seropositivity was also observed in these patients. Phylogenetic analysis of partial sequences showed uniform distribution of the 3′ region of cagA throughout the tree. Conclusions We have identified H. pylori isolates in Pakistan which harbor pathogenicity genes and worrying antibiotic resistance profiles as a result of having acquired multiple point and missense mutations. H. pylori eradication regimens should therefore be reevaluated in this setting. PMID:24827414

Analysis of clinical isolates of Helicobacter pylori in Pakistan reveals high degrees of pathogenicity and high frequencies of antibiotic resistance.

PubMed

Rasheed, Faisal; Campbell, Barry James; Alfizah, Hanafiah; Varro, Andrea; Zahra, Rabaab; Yamaoka, Yoshio; Pritchard, David Mark

2014-10-01

Antibiotic resistance in Helicobacter pylori contributes to failure in eradicating the infection and is most often due to point and missense mutations in a few key genes. The antibiotic susceptibility profiles of H. pylori isolates from 46 Pakistani patients were determined by Etest. Resistance and pathogenicity genes were amplified, and sequences were analyzed to determine the presence of mutations. A high percentage of isolates (73.9%) were resistant to metronidazole (MTZ), with considerable resistance to clarithromycin (CLR; 47.8%) and amoxicillin (AML; 54.3%) also observed. Relatively few isolates were resistant to tetracycline (TET; 4.3%) or to ciprofloxacin (CIP; 13%). However, most isolates (n = 43) exhibited resistance to one or more antibiotics. MTZ-resistant isolates contained missense mutations in oxygen-independent NADPH nitroreductase (RdxA; 8 mutations found) and NADH flavin oxidoreductase (FrxA; 4 mutations found). In the 23S rRNA gene, responsible for CLR resistance, a new point mutation (A2181G) and 4 previously reported mutations were identified. Pathogenicity genes cagA, dupA, and vacA s1a/m1 were detected frequently in isolates which were also found to be resistant to MTZ, CLR, and AML. A high percentage of CagA and VacA seropositivity was also observed in these patients. Phylogenetic analysis of partial sequences showed uniform distribution of the 3' region of cagA throughout the tree. We have identified H. pylori isolates in Pakistan which harbor pathogenicity genes and worrying antibiotic resistance profiles as a result of having acquired multiple point and missense mutations. H. pylori eradication regimens should therefore be reevaluated in this setting. © 2014 John Wiley & Sons Ltd.

Radiobiological studies of plants orbited in Biosatellite II.

PubMed

Schairer, L A; Sparrow, A H; Marimuthu, K M

1970-01-01

The Biosatellite II Tradescantia experiment probed the effects of the space environment on spontaneous and radiation-induced mutation rates and on cytological changes in Tradescantia clone 02. Thirty two young flowering plants arranged in a plastic housing with the roots immersed in nutrient solution were exposed to gamma radiation from an on-board 85 Strontium source during the two-day orbital flight. Unirradiated plants were flown in a package in the spacecraft behind a tungsten radiation shield and identical non-flight control packages (with and without irradiation) were maintained at the launch site. After retrieval of the spacecraft near Hawaii, samples of root tip, ovary and stamen tissues were collected. These and the intact plants were flown to the Brookhaven National Laboratory for observations on the following end points: somatic mutation, cell size, loss of reproductive integrity resulting in stunted stamen hairs, pollen grain mortality, frequency of micronuclei in pollen, disturbed mitotic spindle function and chromosome aberrations. Analysis of data on somatic mutation, cell size and chromosome aberration end points showed no significant differences between flight and non-flight samples. However, pollen abortion, frequency of micronuclei in pollen and loss of reproductive integrity (stamen hair stunting) showed increases associated with weightlessness in irradiated material. Root tip and microspore cells showed effects of disturbed mitotic spindle function in orbited plants both with and without irradiation. Clearly differences exist between flight and non-flight material and the significance and possible mechanisms for these effects are being studied in continuing non-flight tests.

Osimertinib in Untreated EGFR-Mutated Advanced Non-Small-Cell Lung Cancer.

PubMed

Soria, Jean-Charles; Ohe, Yuichiro; Vansteenkiste, Johan; Reungwetwattana, Thanyanan; Chewaskulyong, Busyamas; Lee, Ki Hyeong; Dechaphunkul, Arunee; Imamura, Fumio; Nogami, Naoyuki; Kurata, Takayasu; Okamoto, Isamu; Zhou, Caicun; Cho, Byoung Chul; Cheng, Ying; Cho, Eun Kyung; Voon, Pei Jye; Planchard, David; Su, Wu-Chou; Gray, Jhanelle E; Lee, Siow-Ming; Hodge, Rachel; Marotti, Marcelo; Rukazenkov, Yuri; Ramalingam, Suresh S

2018-01-11

Osimertinib is an oral, third-generation, irreversible epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) that selectively inhibits both EGFR-TKI-sensitizing and EGFR T790M resistance mutations. We compared osimertinib with standard EGFR-TKIs in patients with previously untreated, EGFR mutation-positive advanced non-small-cell lung cancer (NSCLC). In this double-blind, phase 3 trial, we randomly assigned 556 patients with previously untreated, EGFR mutation-positive (exon 19 deletion or L858R) advanced NSCLC in a 1:1 ratio to receive either osimertinib (at a dose of 80 mg once daily) or a standard EGFR-TKI (gefitinib at a dose of 250 mg once daily or erlotinib at a dose of 150 mg once daily). The primary end point was investigator-assessed progression-free survival. The median progression-free survival was significantly longer with osimertinib than with standard EGFR-TKIs (18.9 months vs. 10.2 months; hazard ratio for disease progression or death, 0.46; 95% confidence interval [CI], 0.37 to 0.57; P<0.001). The objective response rate was similar in the two groups: 80% with osimertinib and 76% with standard EGFR-TKIs (odds ratio, 1.27; 95% CI, 0.85 to 1.90; P=0.24). The median duration of response was 17.2 months (95% CI, 13.8 to 22.0) with osimertinib versus 8.5 months (95% CI, 7.3 to 9.8) with standard EGFR-TKIs. Data on overall survival were immature at the interim analysis (25% maturity). The survival rate at 18 months was 83% (95% CI, 78 to 87) with osimertinib and 71% (95% CI, 65 to 76) with standard EGFR-TKIs (hazard ratio for death, 0.63; 95% CI, 0.45 to 0.88; P=0.007 [nonsignificant in the interim analysis]). Adverse events of grade 3 or higher were less frequent with osimertinib than with standard EGFR-TKIs (34% vs. 45%). Osimertinib showed efficacy superior to that of standard EGFR-TKIs in the first-line treatment of EGFR mutation-positive advanced NSCLC, with a similar safety profile and lower rates of serious adverse events. (Funded by AstraZeneca; FLAURA ClinicalTrials.gov number, NCT02296125 .).

Nearing saturation of cancer driver gene discovery.

PubMed

Hsiehchen, David; Hsieh, Antony

2018-06-15

Extensive sequencing efforts of cancer genomes such as The Cancer Genome Atlas (TCGA) have been undertaken to uncover bona fide cancer driver genes which has enhanced our understanding of cancer and revealed therapeutic targets. However, the number of driver gene mutations is bounded, indicating that there must be a point when further sequencing efforts will be excessive. We found that there was a significant positive correlation between sample size and identified driver gene mutations across 33 cancers sequenced by the TCGA, which is expected if additional sequencing is still leading to the identification of more driver genes. However, the rate of new cancer driver genes being discovered with larger samples is declining rapidly. Our analysis provides a general guide for determining which cancer types would likely benefit from additional sequencing efforts, particularly those with relatively high rates of cancer driver gene discovery. Our results argue that past strategies of indiscriminately sequencing as many specimens as possible for all cancer types is becoming inefficient. In addition, without significant investments into applying our knowledge of cancer genomes, we risk sequencing more cancer genomes for the sake of sequencing rather than meaningful patient benefit.

Mutation-selection equilibrium in games with multiple strategies.

PubMed

Antal, Tibor; Traulsen, Arne; Ohtsuki, Hisashi; Tarnita, Corina E; Nowak, Martin A

2009-06-21

In evolutionary games the fitness of individuals is not constant but depends on the relative abundance of the various strategies in the population. Here we study general games among n strategies in populations of large but finite size. We explore stochastic evolutionary dynamics under weak selection, but for any mutation rate. We analyze the frequency dependent Moran process in well-mixed populations, but almost identical results are found for the Wright-Fisher and Pairwise Comparison processes. Surprisingly simple conditions specify whether a strategy is more abundant on average than 1/n, or than another strategy, in the mutation-selection equilibrium. We find one condition that holds for low mutation rate and another condition that holds for high mutation rate. A linear combination of these two conditions holds for any mutation rate. Our results allow a complete characterization of nxn games in the limit of weak selection.

How mutation affects evolutionary games on graphs

PubMed Central

Allen, Benjamin; Traulsen, Arne; Tarnita, Corina E.; Nowak, Martin A.

2011-01-01

Evolutionary dynamics are affected by population structure, mutation rates and update rules. Spatial or network structure facilitates the clustering of strategies, which represents a mechanism for the evolution of cooperation. Mutation dilutes this effect. Here we analyze how mutation influences evolutionary clustering on graphs. We introduce new mathematical methods to evolutionary game theory, specifically the analysis of coalescing random walks via generating functions. These techniques allow us to derive exact identity-by-descent (IBD) probabilities, which characterize spatial assortment on lattices and Cayley trees. From these IBD probabilities we obtain exact conditions for the evolution of cooperation and other game strategies, showing the dual effects of graph topology and mutation rate. High mutation rates diminish the clustering of cooperators, hindering their evolutionary success. Our model can represent either genetic evolution with mutation, or social imitation processes with random strategy exploration. PMID:21473871

Evaluation of gefitinib efficacy according to body mass index, body surface area, and body weight in patients with EGFR-mutated advanced non-small cell lung cancer.

PubMed

Imai, Hisao; Kuwako, Tomohito; Kaira, Kyoichi; Masuda, Tomomi; Miura, Yosuke; Seki, Kaori; Sakurai, Reiko; Utsugi, Mitsuyoshi; Shimizu, Kimihiro; Sunaga, Noriaki; Tomizawa, Yoshio; Ishihara, Shinichi; Ishizuka, Takao; Mogi, Akira; Hisada, Takeshi; Minato, Koichi; Takise, Atsushi; Saito, Ryusei; Yamada, Masanobu

2017-03-01

In patients with epidermal growth factor receptor (EGFR)-mutated, advanced, non-small cell lung cancer (NSCLC), common gefitinib-sensitive EGFR mutations that predict a greater response to therapy include the exon 19 deletion and L858R point mutation. The objective of this study was to evaluate whether body surface area (BSA), body weight (BW), and body mass index (BMI) affect gefitinib efficacy in such patients. The medical charts of 138 consecutive patients with advanced NSCLC harboring sensitive EGFR mutations, who underwent gefitinib treatment, were reviewed. The median BSA and BW were used as cutoff values to evaluate their impact on gefitinib efficacy. BMI was categorized as underweight (<18.5 kg/m 2 ), normal (18.5-25 kg/m 2 ), and overweight (≥25 kg/m 2 ). The median BSA and BW were 1.48 m 2 and 53 kg, respectively. The overall response rate, progression-free survival (PFS), and overall survival (OS) were 65.2%, 12.2, and 24.2 months, respectively. There were no significant differences in clinical outcomes according to BSA, BW, or BMI alone. Subgroup analysis based on the mutation type and BSA revealed no significant differences in PFS between the groups; however, the median OS in those with exon 19 deletion combined with low BSA was significantly favorable compared with the other groups. Gefitinib efficacy in patients with NSCLC harboring sensitive EGFR mutations did not differ according to BSA, BW, and BMI. However, OS was superior in patients with both the exon 19 deletion and low BSA.

Molecular Determinants of Mutant Phenotypes, Inferred from Saturation Mutagenesis Data.

PubMed

Tripathi, Arti; Gupta, Kritika; Khare, Shruti; Jain, Pankaj C; Patel, Siddharth; Kumar, Prasanth; Pulianmackal, Ajai J; Aghera, Nilesh; Varadarajan, Raghavan

2016-11-01

Understanding how mutations affect protein activity and organismal fitness is a major challenge. We used saturation mutagenesis combined with deep sequencing to determine mutational sensitivity scores for 1,664 single-site mutants of the 101 residue Escherichia coli cytotoxin, CcdB at seven different expression levels. Active-site residues could be distinguished from buried ones, based on their differential tolerance to aliphatic and charged amino acid substitutions. At nonactive-site positions, the average mutational tolerance correlated better with depth from the protein surface than with accessibility. Remarkably, similar results were observed for two other small proteins, PDZ domain (PSD95 pdz3 ) and IgG-binding domain of protein G (GB1). Mutational sensitivity data obtained with CcdB were used to derive a procedure for predicting functional effects of mutations. Results compared favorably with those of two widely used computational predictors. In vitro characterization of 80 single, nonactive-site mutants of CcdB showed that activity in vivo correlates moderately with thermal stability and solubility. The inability to refold reversibly, as well as a decreased folding rate in vitro, is associated with decreased activity in vivo. Upon probing the effect of modulating expression of various proteases and chaperones on mutant phenotypes, most deleterious mutants showed an increased in vivo activity and solubility only upon over-expression of either Trigger factor or SecB ATP-independent chaperones. Collectively, these data suggest that folding kinetics rather than protein stability is the primary determinant of activity in vivo This study enhances our understanding of how mutations affect phenotype, as well as the ability to predict fitness effects of point mutations. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Association of a novel point mutation in MSH2 gene with familial multiple primary cancers.

PubMed

Hu, Hai; Li, Hong; Jiao, Feng; Han, Ting; Zhuo, Meng; Cui, Jiujie; Li, Yixue; Wang, Liwei

2017-10-03

Multiple primary cancers (MPC) have been identified as two or more cancers without any subordinate relationship that occur either simultaneously or metachronously in the same or different organs of an individual. Lynch syndrome is an autosomal dominant genetic disorder that increases the risk of many types of cancers. Lynch syndrome patients who suffer more than two cancers can also be considered as MPC; patients of this kind provide unique resources to learn how genetic mutation causes MPC in different tissues. We performed a whole genome sequencing on blood cells and two tumor samples of a Lynch syndrome patient who was diagnosed with five primary cancers. The mutational landscape of the tumors, including somatic point mutations and copy number alternations, was characterized. We also compared Lynch syndrome with sporadic cancers and proposed a model to illustrate the mutational process by which Lynch syndrome progresses to MPC. We revealed a novel pathologic mutation on the MSH2 gene (G504 splicing) that associates with Lynch syndrome. Systematical comparison of the mutation landscape revealed that multiple cancers in the proband were evolutionarily independent. Integrative analysis showed that truncating mutations of DNA mismatch repair (MMR) genes were significantly enriched in the patient. A mutation progress model that included germline mutations of MMR genes, double hits of MMR system, mutations in tissue-specific driver genes, and rapid accumulation of additional passenger mutations was proposed to illustrate how MPC occurs in Lynch syndrome patients. Our findings demonstrate that both germline and somatic alterations are driving forces of carcinogenesis, which may resolve the carcinogenic theory of Lynch syndrome.

Functional connectivity in autosomal dominant and late-onset Alzheimer disease.

PubMed

Thomas, Jewell B; Brier, Matthew R; Bateman, Randall J; Snyder, Abraham Z; Benzinger, Tammie L; Xiong, Chengjie; Raichle, Marcus; Holtzman, David M; Sperling, Reisa A; Mayeux, Richard; Ghetti, Bernardino; Ringman, John M; Salloway, Stephen; McDade, Eric; Rossor, Martin N; Ourselin, Sebastien; Schofield, Peter R; Masters, Colin L; Martins, Ralph N; Weiner, Michael W; Thompson, Paul M; Fox, Nick C; Koeppe, Robert A; Jack, Clifford R; Mathis, Chester A; Oliver, Angela; Blazey, Tyler M; Moulder, Krista; Buckles, Virginia; Hornbeck, Russ; Chhatwal, Jasmeer; Schultz, Aaron P; Goate, Alison M; Fagan, Anne M; Cairns, Nigel J; Marcus, Daniel S; Morris, John C; Ances, Beau M

2014-09-01

Autosomal dominant Alzheimer disease (ADAD) is caused by rare genetic mutations in 3 specific genes in contrast to late-onset Alzheimer disease (LOAD), which has a more polygenetic risk profile. To assess the similarities and differences in functional connectivity changes owing to ADAD and LOAD. We analyzed functional connectivity in multiple brain resting state networks (RSNs) in a cross-sectional cohort of participants with ADAD (n = 79) and LOAD (n = 444), using resting-state functional connectivity magnetic resonance imaging at multiple international academic sites. For both types of AD, we quantified and compared functional connectivity changes in RSNs as a function of dementia severity measured by the Clinical Dementia Rating Scale. In ADAD, we qualitatively investigated functional connectivity changes with respect to estimated years from onset of symptoms within 5 RSNs. A decrease in functional connectivity with increasing Clinical Dementia Rating scores were similar for both LOAD and ADAD in multiple RSNs. Ordinal logistic regression models constructed in one type of Alzheimer disease accurately predicted clinical dementia rating scores in the other, further demonstrating the similarity of functional connectivity loss in each disease type. Among participants with ADAD, functional connectivity in multiple RSNs appeared qualitatively lower in asymptomatic mutation carriers near their anticipated age of symptom onset compared with asymptomatic mutation noncarriers. Resting-state functional connectivity magnetic resonance imaging changes with progressing AD severity are similar between ADAD and LOAD. Resting-state functional connectivity magnetic resonance imaging may be a useful end point for LOAD and ADAD therapy trials. Moreover, the disease process of ADAD may be an effective model for the LOAD disease process.

Accelerated telomere shortening and replicative senescence in human fibroblasts overexpressing mutant and wild-type lamin A

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

Huang Shurong; Risques, Rosa Ana; Martin, George M.

2008-01-01

LMNA mutations are responsible for a variety of genetic disorders, including muscular dystrophy, lipodystrophy, and certain progeroid syndromes, notably Hutchinson-Gilford Progeria. Although a number of clinical features of these disorders are suggestive of accelerated aging, it is not known whether cells derived from these patients exhibit cellular phenotypes associated with accelerated aging. We examined a series of isogenic skin fibroblast lines transfected with LMNA constructs bearing known pathogenic point mutations or deletion mutations found in progeroid syndromes. Fibroblasts overexpressing mutant lamin A exhibited accelerated rates of loss of telomeres and shortened replicative lifespans, in addition to abnormal nuclear morphology. Tomore » our surprise, these abnormalities were also observed in lines overexpressing wild-type lamin A. Copy number variants are common in human populations; those involving LMNA, whether arising meiotically or mitotically, might lead to progeroid phenotypes. In an initial pilot study of 23 progeroid cases without detectable WRN or LMNA mutations, however, no cases of altered LMNA copy number were detected. Nevertheless, our findings raise a hypothesis that changes in lamina organization may cause accelerated telomere attrition, with different kinetics for overexpession of wild-type and mutant lamin A, which leads to rapid replicative senescence and progroid phenotypes.« less

Epistasis increases the rate of conditionally neutral substitution in an adapting population.

PubMed

Draghi, Jeremy A; Parsons, Todd L; Plotkin, Joshua B

2011-04-01

Kimura observed that the rate of neutral substitution should equal the neutral mutation rate. This classic result is central to our understanding of molecular evolution, and it continues to influence phylogenetics, genomics, and the interpretation of evolution experiments. By demonstrating that neutral mutations substitute at a rate independent of population size and selection at linked sites, Kimura provided an influential justification for the idea of a molecular clock and emphasized the importance of genetic drift in shaping molecular evolution. But when epistasis among sites is common, as numerous empirical studies suggest, do neutral mutations substitute according to Kimura's expectation? Here we study simulated, asexual populations of RNA molecules, and we observe that conditionally neutral mutations--i.e., mutations that do not alter the fitness of the individual in which they arise, but that may alter the fitness effects of subsequent mutations--substitute much more often than expected while a population is adapting. We quantify these effects using a simple population-genetic model that elucidates how the substitution rate at conditionally neutral sites depends on the population size, mutation rate, strength of selection, and prevalence of epistasis. We discuss the implications of these results for our understanding of the molecular clock, and for the interpretation of molecular variation in laboratory and natural populations.

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.

[Mutations of EGFR gene and EML4-ALK fusion gene in superficial lymph node of non-small cell lung cancer].

PubMed

Wei, Lili; Li, Xingzhou; Yu, Zhonghe

2015-07-14

To explore the mutation status of epidermal growth factor receptor (EGFR) fusion gene and microtubule associated protein like 4-anaplastic lymphoma kinase (EML4-ALK) fusion gene in superficial lymph nodes of non-small cell lung cancer (NSCLC). The technique of fluorescent quantitative polymerase chain reaction (FQ-PCR) was employed for detecting the mutation rate of EGFR gene and EML4-ALK fusion gene for 40 cases of superficial lymph node tissue of NSCLC inpatients at General Military Hospital of Beijing PLA Command from February 2013 to November 2014. And then the correlations were analyzed between EMIA-ALK fusion gene and EGFR gene with clinical features and the clinical efficacies of targeted therapy. The mutation rate of EGFR gene was 35% (14/40) and 50% (10/20) in non-smokers and 46.7% (14/30) in adenocarcinoma patients. The mutation distribution was as follows: exon 18 (n = 1), exon 19 (n =8) and exon 21 (n =5). The mutation rate of EML4-ALK fusion gene was 2. 5% (1/40). EGFR gene mutation was predominantly present in non-smokers (P < 0. 05) and adenocarcinoma (P <0. 01) while no significant difference existed between gender, age or stage (P >0. 05). Those on a targeted therapy had a disease control rate of 93. 3%. Both EGFR gene and EMI4-ALK fusion gene may be detected in superficial lymph nodes of NSCLC patients. The mutation rate of EGFR gene is high in adenocarcinoma and non-smokers while EML4-ALK fusion gene has a low mutation rate.

Comparison of droplet digital PCR and conventional quantitative PCR for measuring EGFR gene mutation

PubMed Central

ZHANG, BO; XU, CHUN-WEI; SHAO, YUN; WANG, HUAI-TAO; WU, YONG-FANG; SONG, YE-YING; LI, XIAO-BING; ZHANG, ZHE; WANG, WEN-JING; LI, LI-QIONG; CAI, CONG-LI

2015-01-01

Early detection of epidermal growth factor receptor (EGFR) mutation, particularly EGFR T790M mutation, is of clinical significance. The aim of the present study was to compare the performances of amplification refractory mutation system-based quantitative polymerase chain reaction (ARMS-qPCR) and droplet digital polymerase chain reaction (ddPCR) approaches in the detection of EGFR mutation and explore the feasibility of using ddPCR in the detection of samples with low mutation rates. EGFR gene mutations in plasmid samples with different T790M mutation rates (0.1–5%) and 10 clinical samples were detected using the ARMS-qPCR and ddPCR approaches. The results demonstrated that the ARMS-qPCR method stably detected the plasmid samples (6,000 copies) with 5 and 1% mutation rates, while the ddPCR approach reliably detected those with 5% (398 copies), 1% (57 copies), 0.5% (24 copies) and 0.1% (average 6 copies) mutation rates. For the 10 clinical samples, the results for nine samples by the ARMS-qPCR and ddPCR methods were consistent; however, the sample N006, indicated to be EGFR wild-type by ARMS-qPCR, was revealed to have a clear EGFR T790M mutation with seven copies of mutant alleles in a background of 6,000 wild-type copies using ddPCR technology. This study demonstrates the feasibility of applying the ddPCR system to detect EGFR mutation and identified the advantage of ddPCR in the detection of samples with a low EGFR mutation abundance, particularly the secondary EGFR T790M resistance mutation, which enables early diagnosis before acquired resistance to tyrosine kinase inhibitors becomes clinically detectable. PMID:25780439

Long-term follow-up of chronic pancreatitis patients with K-ras mutation in the pancreatic juice.

PubMed

Kamisawa, Terumi; Takuma, Kensuke; Tabata, Taku; Egawa, Naoto; Yamaguchi, Toshikazu

2011-01-01

Pancreatic cancer is known to occur during the course of chronic pancreatitis in some patients. This study aimed to identify a high risk group for developing pancreatic cancer associated with chronic pancreatitis, particularly the presence of K-ras mutations in the pancreatic juice. K-ras mutation was analyzed by enriched polymerase chain reaction-enzyme linked mini-sequence assay in endoscopically-collected pancreatic juice of 21 patients with chronic pancreatitis between 1995 and 2000. All of them were followed-up for 6.0 +/- 3.8 (mean +/- SD) years (range, 2.1-14.2 years). K-ras point mutation was observed in the pancreatic juice of 11 patients with chronic pancreatitis (2+, n=2; 1+, n=6; +/-, n=3). Of these, 2 chronic pancreatitis patients with 2+K-ras point mutation developed pancreatic cancer 4.5 and 10.8 years, respectively, after the examination. Two chronic pancreatitis patients with K-ras mutation developed pancreatic cancer 4.5 and 10.8 years later. Semiquantitative analysis of K-ras mutation in endoscopically-collected pancreatic juice appears to be a useful tool for identifying chronic pancreatitis patients at high risk for developing pancreatic cancer.

High frequency of AML1/RUNX1 point mutations in radiation-associated myelodysplastic syndrome around Semipalatinsk nuclear test site.

PubMed

Zharlyganova, Dinara; Harada, Hironori; Harada, Yuka; Shinkarev, Sergey; Zhumadilov, Zhaxybay; Zhunusova, Aigul; Tchaizhunusova, Naylya J; Apsalikov, Kazbek N; Kemaikin, Vadim; Zhumadilov, Kassym; Kawano, Noriyuki; Kimura, Akiro; Hoshi, Masaharu

2008-09-01

It is known that bone marrow is a sensitive organ to ionizing radiation, and many patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) have been diagnosed in radiation-treated cases and atomic bomb survivors in Hiroshima and Nagasaki. The AML1/RUNX1 gene has been known to be frequently mutated in MDS/AML patients among atomic bomb survivors and radiation therapy-related MDS/AML patients. In this study, we investigated the AML1 mutations in radiation-exposed patients with MDS/AML among the residents near the Semipalatinsk Nuclear Test Site (SNTS), where the risk of solid cancers and leukemias was increased due to the radiation effects. AML1 mutations were identified in 7 (39%) of 18 radiation-exposed MDS/AML patients. In contrast, no AML1 mutation was found in 13 unexposed MDS/AML cases. The frequency of AML1 mutations in radiation-exposed patients with MDS/AML was significantly higher compared with unexposed patients (p < 0.05).We also found a significant correlation between individual estimated doses and AML1 mutations (p < 0.05). Considering these results, AML1 point mutations might be a useful biomarker that differentiates radio-induced MDS/AML from spontaneous MDS/AML.

Genetic Correction and Hepatic Differentiation of Hemophilia B-specific Human Induced Pluripotent Stem Cells.

PubMed

He, Qiong; Wang, Hui-Hui; Cheng, Tao; Yuan, Wei-Ping; Ma, Yu-Po; Jiang, Yong-Ping; Ren, Zhi-Hua

2017-09-27

Objective To genetically correct a disease-causing point mutation in human induced pluripotent stem cells (iPSCs) derived from a hemophilia B patient. Methods First, the disease-causing mutation was detected by sequencing the encoding area of human coagulation factor IX (F IX) gene. Genomic DNA was extracted from the iPSCs, and the primers were designed to amplify the eight exons of F IX. Next, the point mutation in those iPSCs was genetically corrected using CRISPR/Cas9 technology in the presence of a 129-nucleotide homologous repair template that contained two synonymous mutations. Then, top 8 potential off-target sites were subsequently analyzed using Sanger sequencing. Finally, the corrected clones were differentiated into hepatocyte-like cells, and the secretion of F IX was validated by immunocytochemistry and ELISA assay. Results The cell line bore a missense mutation in the 6 th coding exon (c.676 C>T) of F IX gene. Correction of the point mutation was achieved via CRISPR/Cas9 technology in situ with a high efficacy at about 22% (10/45) and no off-target effects detected in the corrected iPSC clones. F IX secretion, which was further visualized by immunocytochemistry and quantified by ELISA in vitro, reached about 6 ng/ml on day 21 of differentiation procedure. Conclusions Mutations in human disease-specific iPSCs could be precisely corrected by CRISPR/Cas9 technology, and corrected cells still maintained hepatic differentiation capability. Our findings might throw a light on iPSC-based personalized therapies in the clinical application, especially for hemophilia B.

Identification of a splicing enhancer in MLH1 using COMPARE a new assay for determination of relative RNA splicing efficiencies

PubMed Central

Xu, Dong-Qing; Mattox, William

2006-01-01

Exonic splicing enhancers (ESEs) are sequences that facilitate recognition of splice sites and prevent exon-skipping. Because ESEs are often embedded within proteincoding sequences, alterations in them can also often be interpreted as nonsense, missense or silent mutations. To correctly interpret exonic mutations and their roles in disease, it is important to develop strategies that identify ESE mutations. Potential ESEs can be found computationally in many exons but it has proven difficult to predict if a given mutation will have effects on splicing based on sequence alone. Here we describe a flexible in vitro method that can be used to functionally compare the effects of multiple sequence variants on ESE activity in a single in vitro splicing reaction. We have applied this method in parallel with conventional splicing assays to test for a splicing enhancer in exon 17 of the human MLH1 gene. Point mutations associated with hereditary nonpolyposis colorectal cancer (HNPCC) have previously been found to correlate with exon-skipping in both lymphocytes and tumors from patients. We show that sequences from this exon can replace an ESE from the mouse IgM gene to support RNA splicing in HeLa nuclear extracts. ESE activity was reduced by HNPCC point mutations in codon 659 indicating that their primary effect is on splicing. Surprisingly the strongest enhancer function mapped to a different region of the exon upstream of this codon. Together our results indicate that HNPCC point mutations in codon 659 affect an auxillary element that augments the enhancer function to ensure exon inclusion. PMID:16357104

Nonlinear dynamics of the rock-paper-scissors game with mutations.

PubMed

Toupo, Danielle F P; Strogatz, Steven H

2015-05-01

We analyze the replicator-mutator equations for the rock-paper-scissors game. Various graph-theoretic patterns of mutation are considered, ranging from a single unidirectional mutation pathway between two of the species, to global bidirectional mutation among all the species. Our main result is that the coexistence state, in which all three species exist in equilibrium, can be destabilized by arbitrarily small mutation rates. After it loses stability, the coexistence state gives birth to a stable limit cycle solution created in a supercritical Hopf bifurcation. This attracting periodic solution exists for all the mutation patterns considered, and persists arbitrarily close to the limit of zero mutation rate and a zero-sum game.

The allele-frequency spectrum in a decoupled Moran model with mutation, drift, and directional selection, assuming small mutation rates.

PubMed

Vogl, Claus; Clemente, Florian

2012-05-01

We analyze a decoupled Moran model with haploid population size N, a biallelic locus under mutation and drift with scaled forward and backward mutation rates θ(1)=μ(1)N and θ(0)=μ(0)N, and directional selection with scaled strength γ=sN. With small scaled mutation rates θ(0) and θ(1), which is appropriate for single nucleotide polymorphism data in highly recombining regions, we derive a simple approximate equilibrium distribution for polymorphic alleles with a constant of proportionality. We also put forth an even simpler model, where all mutations originate from monomorphic states. Using this model we derive the sojourn times, conditional on the ancestral and fixed allele, and under equilibrium the distributions of fixed and polymorphic alleles and fixation rates. Furthermore, we also derive the distribution of small samples in the diffusion limit and provide convenient recurrence relations for calculating this distribution. This enables us to give formulas analogous to the Ewens-Watterson estimator of θ for biased mutation rates and selection. We apply this theory to a polymorphism dataset of fourfold degenerate sites in Drosophila melanogaster. Copyright © 2012 Elsevier Inc. All rights reserved.

Mitochondrial DNA sequence characteristics modulate the size of the genetic bottleneck.

PubMed

Wilson, Ian J; Carling, Phillipa J; Alston, Charlotte L; Floros, Vasileios I; Pyle, Angela; Hudson, Gavin; Sallevelt, Suzanne C E H; Lamperti, Costanza; Carelli, Valerio; Bindoff, Laurence A; Samuels, David C; Wonnapinij, Passorn; Zeviani, Massimo; Taylor, Robert W; Smeets, Hubert J M; Horvath, Rita; Chinnery, Patrick F

2016-03-01

With a combined carrier frequency of 1:200, heteroplasmic mitochondrial DNA (mtDNA) mutations cause human disease in ∼1:5000 of the population. Rapid shifts in the level of heteroplasmy seen within a single generation contribute to the wide range in the severity of clinical phenotypes seen in families transmitting mtDNA disease, consistent with a genetic bottleneck during transmission. Although preliminary evidence from human pedigrees points towards a random drift process underlying the shifting heteroplasmy, some reports describe differences in segregation pattern between different mtDNA mutations. However, based on limited observations and with no direct comparisons, it is not clear whether these observations simply reflect pedigree ascertainment and publication bias. To address this issue, we studied 577 mother-child pairs transmitting the m.11778G>A, m.3460G>A, m.8344A>G, m.8993T>G/C and m.3243A>G mtDNA mutations. Our analysis controlled for inter-assay differences, inter-laboratory variation and ascertainment bias. We found no evidence of selection during transmission but show that different mtDNA mutations segregate at different rates in human pedigrees. m.8993T>G/C segregated significantly faster than m.11778G>A, m.8344A>G and m.3243A>G, consistent with a tighter mtDNA genetic bottleneck in m.8993T>G/C pedigrees. Our observations support the existence of different genetic bottlenecks primarily determined by the underlying mtDNA mutation, explaining the different inheritance patterns observed in human pedigrees transmitting pathogenic mtDNA mutations. © The Author 2016. Published by Oxford University Press.

Targeted mutations and MD simulations of a methanol-stable lipase YLIP9 from Yarrowia lipolytica MSR80 to develop a biodiesel enzyme.

PubMed

Syal, Poonam; Verma, Ved Vrat; Gupta, Rani

2017-11-01

Biodiesel, an environment friendly alternative for fuels, contains methyl esters of long-chain fatty acids. Our group has reported a methanol-stable YLIP9 from Yarrowia lipolytica MSR80 that shows poor catalysis of long-chain fatty acids. To shift its substrate specificity, residues within lid and binding pocket were identified for sequential mutations using YLIP2 as the template. Of the two point mutations (Glu116Leu and Ser119Val) introduced in the lid, the former mutation (YLIP9L1) increased the catalytic rate by ∼2-fold without any change in substrate specificity. In this mutant, six binding pocket residues (Bp2-Bp7) were further mutated to obtain six double mutants. YLIP9L1Bp3 showed significant shift in substrate specificity towards long-chain pNPesters with 11-fold increase in catalytic efficiency than YLIP9. Double mutations also led to increased thermostability and lowered activation energy of YLIP9L1Bp3 thereby shifting its optimum temperature from 60°C to 50°C. In silico molecular dynamics simulations revealed improved lid flexibility and increased catalytic triad volume in YLIP9L1Bp3. The enzyme YLIP9L1Bp3 was methanol-stable having selectivity for long-chain fatty acids with improved catalytic efficiency. Its application as a biodiesel enzyme was validated by transesterification of palm oil in presence of methanol, where it showed 8-fold increase in conversion of oil to methyl esters. Copyright © 2017 Elsevier B.V. All rights reserved.

An automated microfluidic DNA microarray platform for genetic variant detection in inherited arrhythmic diseases.

PubMed

Huang, Shu-Hong; Chang, Yu-Shin; Juang, Jyh-Ming Jimmy; Chang, Kai-Wei; Tsai, Mong-Hsun; Lu, Tzu-Pin; Lai, Liang-Chuan; Chuang, Eric Y; Huang, Nien-Tsu

2018-03-12

In this study, we developed an automated microfluidic DNA microarray (AMDM) platform for point mutation detection of genetic variants in inherited arrhythmic diseases. The platform allows for automated and programmable reagent sequencing under precise conditions of hybridization flow and temperature control. It is composed of a commercial microfluidic control system, a microfluidic microarray device, and a temperature control unit. The automated and rapid hybridization process can be performed in the AMDM platform using Cy3 labeled oligonucleotide exons of SCN5A genetic DNA, which produces proteins associated with sodium channels abundant in the heart (cardiac) muscle cells. We then introduce a graphene oxide (GO)-assisted DNA microarray hybridization protocol to enable point mutation detection. In this protocol, a GO solution is added after the staining step to quench dyes bound to single-stranded DNA or non-perfectly matched DNA, which can improve point mutation specificity. As proof-of-concept we extracted the wild-type and mutant of exon 12 and exon 17 of SCN5A genetic DNA from patients with long QT syndrome or Brugada syndrome by touchdown PCR and performed a successful point mutation discrimination in the AMDM platform. Overall, the AMDM platform can greatly reduce laborious and time-consuming hybridization steps and prevent potential contamination. Furthermore, by introducing the reciprocating flow into the microchannel during the hybridization process, the total assay time can be reduced to 3 hours, which is 6 times faster than the conventional DNA microarray. Given the automatic assay operation, shorter assay time, and high point mutation discrimination, we believe that the AMDM platform has potential for low-cost, rapid and sensitive genetic testing in a simple and user-friendly manner, which may benefit gene screening in medical practice.

Human Germline Mutation and the Erratic Evolutionary Clock

PubMed Central

Przeworski, Molly

2016-01-01

Our understanding of the chronology of human evolution relies on the “molecular clock” provided by the steady accumulation of substitutions on an evolutionary lineage. Recent analyses of human pedigrees have called this understanding into question by revealing unexpectedly low germline mutation rates, which imply that substitutions accrue more slowly than previously believed. Translating mutation rates estimated from pedigrees into substitution rates is not as straightforward as it may seem, however. We dissect the steps involved, emphasizing that dating evolutionary events requires not “a mutation rate” but a precise characterization of how mutations accumulate in development in males and females—knowledge that remains elusive. PMID:27760127

[Clinical significance of JAK2、CALR and MPL gene mutations in 1 648 Philadelphia chromosome negative myeloproliferative neoplasms patients from a single center].

PubMed

Li, M Y; Chao, H Y; Sun, A N; Qiu, H Y; Jin, Z M; Tang, X W; Han, Y; Fu, C C; Chen, S N; Wu, D P

2017-04-14

Objective: To explore the prevalences of JAK2, CALR and MPL gene mutations and the mutation types in patients with Philadelphia chromosome negative myeloproliferative neoplasms (MPNs) , and to compare their clinical characteristics of different mutation types with each other and mutation negative group. Methods: The mutations of JAK2 V617F, JAK2 gene at exon 12, CALR gene at exon 9 and MPL gene at exon 10 in 1 648 Ph negative MPNs patients were detected by direct sequencing. Results: ① The JAK2V617F mutation was found in 471 (92.7%) of 508 PV patients, 819 (78.1%) of 1 049 ET patients and 74 (81.3%) of 91 PMF patients respectively, with the total mutation rate as 82.8% (1 364/1 648) . The JAK2 exon12 mutation was found in 9 (1.7%) of 508 PV patients, none was found in ET or PMF patients, with the total mutation rate as 0.5% (9/1 648) . The CALR mutation was found in 132 (12.6%) of 1 049 ET patients and 11 (12.1%) of 91 PMF patients respectively, with the total mutation rate as 8.7% (143/1 648) ; the MPL mutation was found in 9 (0.9%) of 1 049 ET patients and 1 (1.1%) of 91 PMF patients respectively, with the total mutation rate as 0.6% (10/1 648) . The co-occurrence of any two types of driver gene mutations was not detected by direct sequencing. ②The median onset age of patients with JAK2V617F[61 (15-95) y] was significant higher than of with JAK2 exon12 mutation[49 (33-62) y] or without mutations[42 (3-78) y] ( P <0.001) , but not for patients with CALR[57 (17-89) y] or MPL mutation[59 (22-71) y] ( P >0.05) . Patients with JAK2V617F had higher white blood cell count and hemoglobin level ( P <0.05) when compared with patients with CALR mutation or without mutations, or only significantly higher white blood cell count when compared with patients with MPL mutation ( P =0.013) . The platelet count of patients with CALR mutation was significantly higher than of with JAK2V617F[966 (400-2 069) ×10(9)/L vs 800 (198-3 730) ×10(9)/L, P <0.001]. ③Karyotype analysis was conducted in 1 160 patients with MPNs, the rates of karyotype abnormality of patients with and without CALR mutation were 9.8% (8/82) and 7.4% (80/1 078) ( P =0.441) respectively; The rates of karyotype abnormality of patients with and without JAK2V617F mutation were 7.7% (75/971) and 6.9% (13/189) ( P =0.688) respectively. The incidence of karyotype abnormality of patients with CALR mutation was higher than of with JAK2V617F[9.8% (8/82) vs 7.7% (75/971) ] without statistically significant difference ( P =0.512) . The karyotype analysis of 7 cases of JAK2 exon12 mutation and 6 ones with MPL gene mutation revealed normal karyotype. Conclusions: Driver gene mutations detection could ensure the diagnosis and prognosis judgment of MPN more reliable, different subtypes of MPNs had different profiles of driver gene mutations, the latter lead to unique clinical phenotype.

Molecular Clock of Neutral Mutations in a Fitness-Increasing Evolutionary Process

PubMed Central

Iijima, Leo; Suzuki, Shingo; Hashimoto, Tomomi; Oyake, Ayana; Kobayashi, Hisaka; Someya, Yuki; Narisawa, Dai; Yomo, Tetsuya

2015-01-01

The molecular clock of neutral mutations, which represents linear mutation fixation over generations, is theoretically explained by genetic drift in fitness-steady evolution or hitchhiking in adaptive evolution. The present study is the first experimental demonstration for the molecular clock of neutral mutations in a fitness-increasing evolutionary process. The dynamics of genome mutation fixation in the thermal adaptive evolution of Escherichia coli were evaluated in a prolonged evolution experiment in duplicated lineages. The cells from the continuously fitness-increasing evolutionary process were subjected to genome sequencing and analyzed at both the population and single-colony levels. Although the dynamics of genome mutation fixation were complicated by the combination of the stochastic appearance of adaptive mutations and clonal interference, the mutation fixation in the population was simply linear over generations. Each genome in the population accumulated 1.6 synonymous and 3.1 non-synonymous neutral mutations, on average, by the spontaneous mutation accumulation rate, while only a single genome in the population occasionally acquired an adaptive mutation. The neutral mutations that preexisted on the single genome hitchhiked on the domination of the adaptive mutation. The successive fixation processes of the 128 mutations demonstrated that hitchhiking and not genetic drift were responsible for the coincidence of the spontaneous mutation accumulation rate in the genome with the fixation rate of neutral mutations in the population. The molecular clock of neutral mutations to the fitness-increasing evolution suggests that the numerous neutral mutations observed in molecular phylogenetic trees may not always have been fixed in fitness-steady evolution but in adaptive evolution. PMID:26177190

Molecular Clock of Neutral Mutations in a Fitness-Increasing Evolutionary Process.

PubMed

Kishimoto, Toshihiko; Ying, Bei-Wen; Tsuru, Saburo; Iijima, Leo; Suzuki, Shingo; Hashimoto, Tomomi; Oyake, Ayana; Kobayashi, Hisaka; Someya, Yuki; Narisawa, Dai; Yomo, Tetsuya

2015-07-01

The molecular clock of neutral mutations, which represents linear mutation fixation over generations, is theoretically explained by genetic drift in fitness-steady evolution or hitchhiking in adaptive evolution. The present study is the first experimental demonstration for the molecular clock of neutral mutations in a fitness-increasing evolutionary process. The dynamics of genome mutation fixation in the thermal adaptive evolution of Escherichia coli were evaluated in a prolonged evolution experiment in duplicated lineages. The cells from the continuously fitness-increasing evolutionary process were subjected to genome sequencing and analyzed at both the population and single-colony levels. Although the dynamics of genome mutation fixation were complicated by the combination of the stochastic appearance of adaptive mutations and clonal interference, the mutation fixation in the population was simply linear over generations. Each genome in the population accumulated 1.6 synonymous and 3.1 non-synonymous neutral mutations, on average, by the spontaneous mutation accumulation rate, while only a single genome in the population occasionally acquired an adaptive mutation. The neutral mutations that preexisted on the single genome hitchhiked on the domination of the adaptive mutation. The successive fixation processes of the 128 mutations demonstrated that hitchhiking and not genetic drift were responsible for the coincidence of the spontaneous mutation accumulation rate in the genome with the fixation rate of neutral mutations in the population. The molecular clock of neutral mutations to the fitness-increasing evolution suggests that the numerous neutral mutations observed in molecular phylogenetic trees may not always have been fixed in fitness-steady evolution but in adaptive evolution.

[Analysis of the parental origin of MECP2 mutations in patients with Rett syndrome].

PubMed

Zhang, Jing-jing; Bao, Xin-hua; Cao, Guang-na; Jiang, Sheng-ling; Zhu, Xing-wang; Lu, Hong-mei; Jia, Li-fang; Pan, Hong; Wu, Xi-ru

2010-04-01

To identify the parental origin of methyl-CpG-binding protein 2 (MECP2) gene mutations in Chinese patients with Rett syndrome. Single nucleotide polymorphisms (SNPs) in intron 3 of the MECP2 gene were analyzed by PCR and sequencing in 115 patients with Rett syndrome. Then sequencing of the SNP region was performed for the fathers of the patients who had at least one SNP, to determine which allele was from the father. Then allele-specific PCR was performed and the products were sequenced to see whether the allele from father or mother harbored the mutation. Seventy-six of the 115 patients had at least one SNP. Three hot SNPs were found in these patients. They were: IVS3+22C >G, IVS3+266C >T and IVS3+683C>T. Among the 76 cases, 73 had a paternal origin of MECP2 mutations, and the other 3 had a maternal origin. There were multiple types of MECP2 mutation of the paternal origin, including 4 frame shift, 2 deletion and 67 point (56C >T, 6C >G, 2A >G, 2G >T and 1A >T) mutations. The mutation types of the 3 patients with maternal origin included 2 frame shift and 1 point (C >T) mutation. In Chinese RTT patients, the MECP2 mutations are mostly of paternal origin.

Elevated mutation rate during meiosis in Saccharomyces cerevisiae.

PubMed

Rattray, Alison; Santoyo, Gustavo; Shafer, Brenda; Strathern, Jeffrey N

2015-01-01

Mutations accumulate during all stages of growth, but only germ line mutations contribute to evolution. While meiosis contributes to evolution by reassortment of parental alleles, we show here that the process itself is inherently mutagenic. We have previously shown that the DNA synthesis associated with repair of a double-strand break is about 1000-fold less accurate than S-phase synthesis. Since the process of meiosis involves many programmed DSBs, we reasoned that this repair might also be mutagenic. Indeed, in the early 1960's Magni and Von Borstel observed elevated reversion of recessive alleles during meiosis, and found that the revertants were more likely to be associated with a crossover than non-revertants, a process that they called "the meiotic effect." Here we use a forward mutation reporter (CAN1 HIS3) placed at either a meiotic recombination coldspot or hotspot near the MAT locus on Chromosome III. We find that the increased mutation rate at CAN1 (6 to 21 -fold) correlates with the underlying recombination rate at the locus. Importantly, we show that the elevated mutation rate is fully dependent upon Spo11, the protein that introduces the meiosis specific DSBs. To examine associated recombination we selected for random spores with or without a mutation in CAN1. We find that the mutations isolated this way show an increased association with recombination (crossovers, loss of crossover interference and/or increased gene conversion tracts). Polζ appears to contribute about half of the mutations induced during meiosis, but is not the only source of mutations for the meiotic effect. We see no difference in either the spectrum or distribution of mutations between mitosis and meiosis. The correlation of hotspots with elevated mutagenesis provides a mechanism for organisms to control evolution rates in a gene specific manner.

Mitochondrial Mutation Rate, Spectrum and Heteroplasmy in Caenorhabditis elegans Spontaneous Mutation Accumulation Lines of Differing Population Size.

PubMed

Konrad, Anke; Thompson, Owen; Waterston, Robert H; Moerman, Donald G; Keightley, Peter D; Bergthorsson, Ulfar; Katju, Vaishali

2017-06-01

Mitochondrial genomes of metazoans, given their elevated rates of evolution, have served as pivotal markers for phylogeographic studies and recent phylogenetic events. In order to determine the dynamics of spontaneous mitochondrial mutations in small populations in the absence and presence of selection, we evolved mutation accumulation (MA) lines of Caenorhabditis elegans in parallel over 409 consecutive generations at three varying population sizes of N = 1, 10, and 100 hermaphrodites. The N =1 populations should have a minimal influence of natural selection to provide the spontaneous mutation rate and the expected rate of neutral evolution, whereas larger population sizes should experience increasing intensity of selection. New mutations were identified by Illumina paired-end sequencing of 86 mtDNA genomes across 35 experimental lines and compared with published genomes of natural isolates. The spontaneous mitochondrial mutation rate was estimated at 1.05 × 10-7/site/generation. A strong G/C→A/T mutational bias was observed in both the MA lines and the natural isolates. This suggests that the low G + C content at synonymous sites is the product of mutation bias rather than selection as previously proposed. The mitochondrial effective population size per worm generation was estimated to be 62. Although it was previously concluded that heteroplasmy was rare in C. elegans, the vast majority of mutations in this study were heteroplasmic despite an experimental regime exceeding 400 generations. The frequencies of frameshift and nonsynonymous mutations were negatively correlated with population size, which suggests their deleterious effects on fitness and a potent role for selection in their eradication. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Monotonicity of fitness landscapes and mutation rate control.

PubMed

Belavkin, Roman V; Channon, Alastair; Aston, Elizabeth; Aston, John; Krašovec, Rok; Knight, Christopher G

2016-12-01

A common view in evolutionary biology is that mutation rates are minimised. However, studies in combinatorial optimisation and search have shown a clear advantage of using variable mutation rates as a control parameter to optimise the performance of evolutionary algorithms. Much biological theory in this area is based on Ronald Fisher's work, who used Euclidean geometry to study the relation between mutation size and expected fitness of the offspring in infinite phenotypic spaces. Here we reconsider this theory based on the alternative geometry of discrete and finite spaces of DNA sequences. First, we consider the geometric case of fitness being isomorphic to distance from an optimum, and show how problems of optimal mutation rate control can be solved exactly or approximately depending on additional constraints of the problem. Then we consider the general case of fitness communicating only partial information about the distance. We define weak monotonicity of fitness landscapes and prove that this property holds in all landscapes that are continuous and open at the optimum. This theoretical result motivates our hypothesis that optimal mutation rate functions in such landscapes will increase when fitness decreases in some neighbourhood of an optimum, resembling the control functions derived in the geometric case. We test this hypothesis experimentally by analysing approximately optimal mutation rate control functions in 115 complete landscapes of binding scores between DNA sequences and transcription factors. Our findings support the hypothesis and find that the increase of mutation rate is more rapid in landscapes that are less monotonic (more rugged). We discuss the relevance of these findings to living organisms.

The molecular defect of ferrochelatase in a patient with erythropoietic protoporphyria.

PubMed Central

Nakahashi, Y; Fujita, H; Taketani, S; Ishida, N; Kappas, A; Sassa, S

1992-01-01

The molecular basis of an inherited defect of ferrochelatase in a patient with erythropoietic protoporphyria (EPP) was investigated. Ferrochelatase is the terminal enzyme in the heme biosynthetic pathway and catalyzes the insertion of ferrous iron into protoporphyrin IX to form heme. In Epstein-Barr virus-transformed lymphoblastoid cells from a proband with EPP, enzyme activity, an immunochemically quantifiable protein, and mRNA content of ferrochelatase were about one-half the normal level. In contrast, the rate of transcription of ferrochelatase mRNA in the proband's cells was normal, suggesting that decreased ferrochelatase mRNA is due to an unstable transcript. cDNA clones encoding ferrochelatase in the proband, isolated by amplification using the polymerase chain reaction, were found to be classified either into those encoding the normal protein or into those encoding an abnormal protein that lacked exon 2 of the ferrochelatase gene, indicating that the proband is heterozygous for the ferrochelatase defect. Genomic DNA analysis revealed that the abnormal allele had a point mutation, C----T, near the acceptor site of intron 1. This point mutation appears to be responsible for the post-transcriptional splicing abnormality resulting in an aberrant transcript of ferrochelatase in this patient. Images PMID:1729699

Roles for the Rad27 Flap Endonuclease in Mitochondrial Mutagenesis and Double-Strand Break Repair in Saccharomyces cerevisiae.

PubMed

Nagarajan, Prabha; Prevost, Christopher T; Stein, Alexis; Kasimer, Rachel; Kalifa, Lidza; Sia, Elaine A

2017-06-01

The structure-specific nuclease, Rad27p/FEN1, plays a crucial role in DNA repair and replication mechanisms in the nucleus. Genetic assays using the rad27-∆ mutant have shown altered rates of DNA recombination, microsatellite instability, and point mutation in mitochondria. In this study, we examined the role of Rad27p in mitochondrial mutagenesis and double-strand break (DSB) repair in Saccharomyces cerevisiae Our findings show that Rad27p is essential for efficient mitochondrial DSB repair by a pathway that generates deletions at a region flanked by direct repeat sequences. Mutant analysis suggests that both exonuclease and endonuclease activities of Rad27p are required for its role in mitochondrial DSB repair. In addition, we found that the nuclease activities of Rad27p are required for the prevention of mitochondrial DNA (mtDNA) point mutations, and in the generation of spontaneous mtDNA rearrangements. Overall, our findings underscore the importance of Rad27p in the maintenance of mtDNA, and demonstrate that it participates in multiple DNA repair pathways in mitochondria, unlinked to nuclear phenotypes. Copyright © 2017 by the Genetics Society of America.

STRUM: structure-based prediction of protein stability changes upon single-point mutation.

PubMed

Quan, Lijun; Lv, Qiang; Zhang, Yang

2016-10-01

Mutations in human genome are mainly through single nucleotide polymorphism, some of which can affect stability and function of proteins, causing human diseases. Several methods have been proposed to predict the effect of mutations on protein stability; but most require features from experimental structure. Given the fast progress in protein structure prediction, this work explores the possibility to improve the mutation-induced stability change prediction using low-resolution structure modeling. We developed a new method (STRUM) for predicting stability change caused by single-point mutations. Starting from wild-type sequences, 3D models are constructed by the iterative threading assembly refinement (I-TASSER) simulations, where physics- and knowledge-based energy functions are derived on the I-TASSER models and used to train STRUM models through gradient boosting regression. STRUM was assessed by 5-fold cross validation on 3421 experimentally determined mutations from 150 proteins. The Pearson correlation coefficient (PCC) between predicted and measured changes of Gibbs free-energy gap, ΔΔG, upon mutation reaches 0.79 with a root-mean-square error 1.2 kcal/mol in the mutation-based cross-validations. The PCC reduces if separating training and test mutations from non-homologous proteins, which reflects inherent correlations in the current mutation sample. Nevertheless, the results significantly outperform other state-of-the-art methods, including those built on experimental protein structures. Detailed analyses show that the most sensitive features in STRUM are the physics-based energy terms on I-TASSER models and the conservation scores from multiple-threading template alignments. However, the ΔΔG prediction accuracy has only a marginal dependence on the accuracy of protein structure models as long as the global fold is correct. These data demonstrate the feasibility to use low-resolution structure modeling for high-accuracy stability change prediction upon point mutations. http://zhanglab.ccmb.med.umich.edu/STRUM/ CONTACT: qiang@suda.edu.cn and zhng@umich.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

STRUM: structure-based prediction of protein stability changes upon single-point mutation

PubMed Central

Quan, Lijun; Lv, Qiang; Zhang, Yang

2016-01-01

Motivation: Mutations in human genome are mainly through single nucleotide polymorphism, some of which can affect stability and function of proteins, causing human diseases. Several methods have been proposed to predict the effect of mutations on protein stability; but most require features from experimental structure. Given the fast progress in protein structure prediction, this work explores the possibility to improve the mutation-induced stability change prediction using low-resolution structure modeling. Results: We developed a new method (STRUM) for predicting stability change caused by single-point mutations. Starting from wild-type sequences, 3D models are constructed by the iterative threading assembly refinement (I-TASSER) simulations, where physics- and knowledge-based energy functions are derived on the I-TASSER models and used to train STRUM models through gradient boosting regression. STRUM was assessed by 5-fold cross validation on 3421 experimentally determined mutations from 150 proteins. The Pearson correlation coefficient (PCC) between predicted and measured changes of Gibbs free-energy gap, ΔΔG, upon mutation reaches 0.79 with a root-mean-square error 1.2 kcal/mol in the mutation-based cross-validations. The PCC reduces if separating training and test mutations from non-homologous proteins, which reflects inherent correlations in the current mutation sample. Nevertheless, the results significantly outperform other state-of-the-art methods, including those built on experimental protein structures. Detailed analyses show that the most sensitive features in STRUM are the physics-based energy terms on I-TASSER models and the conservation scores from multiple-threading template alignments. However, the ΔΔG prediction accuracy has only a marginal dependence on the accuracy of protein structure models as long as the global fold is correct. These data demonstrate the feasibility to use low-resolution structure modeling for high-accuracy stability change prediction upon point mutations. Availability and Implementation: http://zhanglab.ccmb.med.umich.edu/STRUM/ Contact: qiang@suda.edu.cn and zhng@umich.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27318206

Pathway level alterations rather than mutations in single genes predict response to HER2-targeted therapies in the neo-ALTTO trial.

PubMed

Shi, W; Jiang, T; Nuciforo, P; Hatzis, C; Holmes, E; Harbeck, N; Sotiriou, C; Peña, L; Loi, S; Rosa, D D; Chia, S; Wardley, A; Ueno, T; Rossari, J; Eidtmann, H; Armour, A; Piccart-Gebhart, M; Rimm, D L; Baselga, J; Pusztai, L

2017-01-01

We performed whole-exome sequencing of pretreatment biopsies and examined whether genome-wide metrics of overall mutational load, clonal heterogeneity or alterations at variant, gene, and pathway levels are associated with treatment response and survival. Two hundred and three biopsies from the NeoALTTO trial were analyzed. Mutations were called with MuTect, and Strelka, using pooled normal DNA. Associations between DNA alterations and outcome were evaluated by logistic and Cox-proportional hazards regression. There were no recurrent single gene mutations significantly associated with pathologic complete response (pCR), except PIK3CA [odds ratio (OR) = 0.42, P = 0.0185]. Mutations in 33 of 714 pathways were significantly associated with response, but different genes were affected in different individuals. PIK3CA was present in 23 of these pathways defining a ‘trastuzumab resistance-network’ of 459 genes. Cases with mutations in this network had low pCR rates to trastuzumab (2/50, 4%) compared with cases with no mutations (9/16, 56%), OR = 0.035; P < 0.001. Mutations in the ‘Regulation of RhoA activity’ pathway were associated with higher pCR rate to lapatinib (OR = 14.8, adjusted P = 0.001), lapatinib + trastuzumab (OR = 3.0, adjusted P = 0.09), and all arms combined (OR = 3.77, adjusted P = 0.02). Patients (n = 124) with mutations in the trastuzumab resistance network but intact RhoA pathway had 2% (1/41) pCR rate with trastuzumab alone (OR = 0.026, P = 0.001) but adding lapatinib increased pCR rate to 45% (17/38, OR = 1.68, P = 0.3). Patients (n = 46) who had no mutations in either gene set had 6% pCR rate (1/15) with lapatinib, but had the highest pCR rate, 52% (8/15) with trastuzumab alone. Mutations in the RhoA pathway are associated with pCR to lapatinib and mutations in a PIK3CA-related network are associated with resistance to trastuzumab. The combined mutation status of these two pathways could define patients with very low response rate to trastuzumab alone that can be augmented by adding lapatinib or substituting trastuzumab with lapatinib.

Muver, a computational framework for accurately calling accumulated mutations.

PubMed

Burkholder, Adam B; Lujan, Scott A; Lavender, Christopher A; Grimm, Sara A; Kunkel, Thomas A; Fargo, David C

2018-05-09

Identification of mutations from next-generation sequencing data typically requires a balance between sensitivity and accuracy. This is particularly true of DNA insertions and deletions (indels), that can impart significant phenotypic consequences on cells but are harder to call than substitution mutations from whole genome mutation accumulation experiments. To overcome these difficulties, we present muver, a computational framework that integrates established bioinformatics tools with novel analytical methods to generate mutation calls with the extremely low false positive rates and high sensitivity required for accurate mutation rate determination and comparison. Muver uses statistical comparison of ancestral and descendant allelic frequencies to identify variant loci and assigns genotypes with models that include per-sample assessments of sequencing errors by mutation type and repeat context. Muver identifies maximally parsimonious mutation pathways that connect these genotypes, differentiating potential allelic conversion events and delineating ambiguities in mutation location, type, and size. Benchmarking with a human gold standard father-son pair demonstrates muver's sensitivity and low false positive rates. In DNA mismatch repair (MMR) deficient Saccharomyces cerevisiae, muver detects multi-base deletions in homopolymers longer than the replicative polymerase footprint at rates greater than predicted for sequential single-base deletions, implying a novel multi-repeat-unit slippage mechanism. Benchmarking results demonstrate the high accuracy and sensitivity achieved with muver, particularly for indels, relative to available tools. Applied to an MMR-deficient Saccharomyces cerevisiae system, muver mutation calls facilitate mechanistic insights into DNA replication fidelity.

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.

Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase that increases the production rate of D-tagatose.

PubMed

Kim, H-J; Kim, J-H; Oh, H-J; Oh, D-K

2006-07-01

Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase used to increase the production rate of D-tagatose. A mutated gene was obtained by an error-prone polymerase chain reaction using L-arabinose isomerase gene from G. stearothermophilus as a template and the gene was expressed in Escherichia coli. The expressed mutated L-arabinose isomerase exhibited the change of three amino acids (Met322-->Val, Ser393-->Thr, and Val408-->Ala), compared with the wild-type enzyme and was then purified to homogeneity. The mutated enzyme had a maximum galactose isomerization activity at pH 8.0, 65 degrees C, and 1.0 mM Co2+, while the wild-type enzyme had a maximum activity at pH 8.0, 60 degrees C, and 1.0-mM Mn2+. The mutated L-arabinose isomerase exhibited increases in D-galactose isomerization activity, optimum temperature, catalytic efficiency (kcat/Km) for D-galactose, and the production rate of D-tagatose from D-galactose. The mutated L-arabinose isomerase from G. stearothermophilus is valuable for the commercial production of D-tagatose. This work contributes knowledge on the characterization of a mutated L-arabinose isomerase, and allows an increased production rate for D-tagatose from D-galactose using the mutated enzyme.

A novel twelve class fluctuation test reveals higher than expected mutation rates for influenza A viruses

PubMed Central

Pauly, Matthew D; Procario, Megan C; Lauring, Adam S

2017-01-01

Influenza virus’ low replicative fidelity contributes to its capacity for rapid evolution. Clonal sequencing and fluctuation tests have suggested that the influenza virus mutation rate is 2.7 × 10–6 - 3.0 × 10–5 substitutions per nucleotide per strand copied (s/n/r). However, sequencing assays are biased toward mutations with minimal fitness impacts and fluctuation tests typically investigate only a subset of all possible single nucleotide mutations. We developed a fluctuation test based on reversion to fluorescence in a set of virally encoded mutant green fluorescent proteins, which allowed us to measure the rates of selectively neutral mutations representative of the twelve different mutation types. We measured an overall mutation rate of 1.8 × 10–4 s/n/r for PR8 (H1N1) and 2.5 × 10–4 s/n/r for Hong Kong 2014 (H3N2) and a transitional bias of 2.7–3.6. Our data suggest that each replicated genome will have an average of 2–3 mutations and highlight the importance of mutational load in influenza virus evolution. DOI: http://dx.doi.org/10.7554/eLife.26437.001 PMID:28598328

A point mutation of valine-311 to methionine in Bacillus subtilis protoporphyrinogen oxidase does not greatly increase resistance to the diphenyl ether herbicide oxyfluorfen.

PubMed

Jeong, Eunjoo; Houn, Thavrak; Kuk, Yongin; Kim, Eun-Seon; Chandru, Hema Kumar; Baik, Myunggi; Back, Kyoungwhan; Guh, Ja-Ock; Han, Oksoo

2003-10-01

In an effort to asses the effect of Val311Met point mutation of Bacillus subtilis protoporphyrinogen oxidase on the resistance to diphenyl ether herbicides, a Val311Met point mutant of B. subtilis protoporphyrinogen oxidase was prepared, heterologously expressed in Escherichia coli, and the purified recombinant Val311Met mutant protoporphyrinogen oxidase was kinetically characterized. The mutant protoporphyrinogen oxidase showed very similar kinetic patterns to wild type protoporphyrinogen oxidase, with slightly decreased activity dependent on pH and the concentrations of NaCl, Tween 20, and imidazole. When oxyfluorfen was used as a competitive inhibitor, the Val311Met mutant protoporphyrinogen oxidase showed an increased inhibition constant about 1.5 times that of wild type protoporphyrinogen oxidase. The marginal increase of the inhibition constant indicates that the Val311Met point mutation in B. subtilis protoporphyrinogen oxidase may not be an important determinant in the mechanism that protects protoporphyrinogen oxidase against diphenyl ether herbicides.

THE ACTION OF RADIATION AND OTHER MUTAGENIC AGENTS (1) IN INDUCING MUTATION IN DROSOPHILA FEMALES, AND (2) IN CONTROLLING THE ACTION OF SPECIFIC GENES RESPONSIBLE FOR SUPPRESSING UNCONTROLLED GROWTH. Report Covering 9-Year Period, May 1, 1953-April 30, 1962

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

Glass, H.B.

1962-02-01

Studies of the comparative mutagenic effects of ionizing radiations on males and females of Drosophila melanogaster are described. Sex-linked recessive lethal mutations were induced in nitrogen, air, and oxygen at doses of obtained in spermatozoa were uniformly about one-third higher than the frequencies obtained for the same dose and condition of atmosphere in mature oocytes. The relative frequencies of recessive autosomal lethals in mature male and female germ cells were identical with the relative fre quencies of sex-linked recessive lethals. In studies of point mutations and deficiencies involving specific loci, the rates in the male germ cells exceeded those inmore » the female germ cells by a proportion equal to that found to apply to autosomal and sex-linked recessive lethals. Spontaneous mutation rates were determined for a number of specific loci marked by recessive genes used in the tested stocks. Fertility was lost in both males and females when they were x-rayed as 80-hr-old larvae and bred upon emerging as adults. Females recovered their fertility rapidly but the males did so at a much slower rate. The brown; scarlet'' stock was found to carry two mutants each suppressed by a particular suppressor gene. It was concluded that the two suppressors act along different metabolic pathways departing from tryplophan, but both involving an x-ray-sensitive step. A study was made of the effects on the life span of two different mating regimens: immediate and deferred. It was found that the lines previously subjected to immediate mating significantly outlived the lines previously subjected to deferred mating when the mating regimen in the test was immediate mating. Exactly the opposite happened when the mating regimen in the test was deferred mating. (M.C.G.)« less

Application of oligonucleotide array CGH to the simultaneous detection of a deletion in the nuclear TK2 gene and mtDNA depletion.

PubMed

Zhang, Shulin; Li, Fang-Yuan; Bass, Harold N; Pursley, Amber; Schmitt, Eric S; Brown, Blaire L; Brundage, Ellen K; Mardach, Rebecca; Wong, Lee-Jun

2010-01-01

Thymidine kinase 2 (TK2), encoded by the TK2 gene on chromosome 16q22, is one of the deoxyribonucleoside kinases responsible for the maintenance of mitochondrial deoxyribonucleotide pools. Defects in TK2 mainly cause a myopathic form of the mitochondrial DNA depletion syndrome (MDDS). Currently, only point mutations and small insertions and deletions have been reported in TK2 gene; gross rearrangements of TK2 gene and possible hepatic involvement in patients with TK2 mutations have not been described. We report a non-consanguineous Jordanian family with three deceased siblings due to mtDNA depletion. Sequence analysis of the father detected a heterozygous c.761T>A (p.I254N) mutation in his TK2 gene; however, point mutations in the mother were not detected. Subsequent gene dosage analysis using oligonucleotide array CGH identified an intragenic approximately 5.8-kb deletion encompassing the 5'UTR to intron 2 of her TK2 gene. Sequence analysis confirmed that the deletion spans c.1-495 to c.283-2899 of the TK2 gene (nucleotide 65,136,256-65,142,086 of chromosome 16). Analysis of liver and muscle specimens from one of the deceased infants in this family revealed compound heterozygosity for the paternal point mutation and maternal intragenic deletion. In addition, a significant reduction of the mtDNA content in liver and muscle was detected (10% and 20% of age- and tissue-matched controls, respectively). Prenatal diagnosis was performed in the third pregnancy. The fetus was found to carry both the point mutation and the deletion. This child died 6months after birth due to myopathy. A serum specimen demonstrated elevated liver transaminases in two of the infants from whom results were available. This report expands the mutation spectrum associated with TK2 deficiency. While the myopathic form of MDDS appears to be the main phenotype of TK2 mutations, liver dysfunction may also be a part of the mitochondrial depletion syndrome caused by TK2 gene defects.

Widespread Distribution of a Newly Found Point Mutation in Voltage-Gated Sodium Channel in Pyrethroid-Resistant Aedes aegypti Populations in Vietnam

PubMed Central

Kawada, Hitoshi; Higa, Yukiko; Komagata, Osamu; Kasai, Shinji; Tomita, Takashi; Thi Yen, Nguyen; Loan, Luu Lee; Sánchez, Rodrigo A. P.; Takagi, Masahiro

2009-01-01

Background Resistance of Aedes aegypti to photostable pyrethroid insecticides is a major problem for disease-vector control programs. Pyrethroids target the voltage-gated sodium channel on the insects' neurons. Single amino acid substitutions in this channel associated with pyrethroid resistance are one of the main factors that cause knockdown resistance in insects. Although kdr has been observed in several mosquito species, point mutations in the para gene have not been fully characterized in Ae. aegypti populations in Vietnam. The aim of this study was to determine the types and frequencies of mutations in the para gene in Ae. aegypti collected from used tires in Vietnam. Methods and Findings Several point mutations were examined that cause insensitivity of the voltage-gated sodium channel in the insect nervous system due to the replacement of the amino acids L1014F, the most commonly found point mutation in several mosquitoes; I1011M (or V) and V1016G (or I), which have been reported to be associated to knockdown resistance in Ae. aegypti located in segment 6, domain II; and a recently found amino acid replacement in F1269 in Ae. aegypti, located in segment 6, domain III. Among 756 larvae from 70 locations, no I1011M or I1011V nor L1014F mutations were found, and only two heterozygous V1016G mosquitoes were detected. However, F1269C mutations on domain III were distributed widely and with high frequency in 269 individuals among 757 larvae (53 collection sites among 70 locations surveyed). F1269C frequencies were low in the middle to north part of Vietnam but were high in the areas neighboring big cities and in the south of Vietnam, with the exception of the southern mountainous areas located at an elevation of 500–1000 m. Conclusions The overall percentage of homozygous F1269C seems to remain low (7.4%) in the present situation. However, extensive and uncontrolled frequent use of photostable pyrethroids might be a strong selection pressure for this mutation to cause serious problems in the control of dengue fever in Vietnam. PMID:19806205

[Analysis of EML4-ALK gene fusion mutation in patients 
with non-small cell lung cancer].

PubMed

Wang, Xuzhou; Chen, Weisheng; Yu, Yinghao

2015-02-01

Non-small cell lung cancer (NSCLC) is the main type of lung cancer, and the related locus mutation detection research has become a hot direction of molecular targeted therapy, studying on gene mutation status of echinodem microtubule associated protein like 4-Anaplastic lymphoma kinase (EML4-ALK) and epidermal growth factor receptor (EGFR), detecting the sensitivity of EML4-ALK gene fusion and gene mutation of EGFR. EML4-ALK gene fusion in 85 cases of paraffin embedded tumor tissue and adjacent lung tissue was detected with the application of immunohistochemistry (IHC), Scorpions amplification refractory mutation system (Scorpions ARMS) fluorescence quantitative PCR and fluorescence in situ hybridization (FISH) technology, and EGFR gene in 18, 19, 20 and 21 exon mutation status was detected with the application of ARMS method. In 115 cases of NSCLC, IHC showed 32 cases with ALK (D5F3) expression, the expression rate was 27.8%; ARMS showed 27 cases with EML4-ALK fusion gene mutation, the mutation detection rate was 23.5%; 53 cases were detected with EGFR mutation, the mutation rate was 46%. While FISH showed 23 cases with EML4-ALK fusion gene mutation, the detection rate was 20%, slightly lower than the ARMS detection results, suggesting that ARMS more sensitive. The application of IHC, ARMS fluorescence quantitative PCR and FISH technology can make a rapid and accurate evaluation of EML4-ALK gene fusion.

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

Mutation-induced protein interaction kinetics changes affect apoptotic network dynamic properties and facilitate oncogenesis

PubMed Central

Zhao, Linjie; Sun, Tanlin; Pei, Jianfeng; Ouyang, Qi

2015-01-01

It has been a consensus in cancer research that cancer is a disease caused primarily by genomic alterations, especially somatic mutations. However, the mechanism of mutation-induced oncogenesis is not fully understood. Here, we used the mitochondrial apoptotic pathway as a case study and performed a systematic analysis of integrating pathway dynamics with protein interaction kinetics to quantitatively investigate the causal molecular mechanism of mutation-induced oncogenesis. A mathematical model of the regulatory network was constructed to establish the functional role of dynamic bifurcation in the apoptotic process. The oncogenic mutation enrichment of each of the protein functional domains involved was found strongly correlated with the parameter sensitivity of the bifurcation point. We further dissected the causal mechanism underlying this correlation by evaluating the mutational influence on protein interaction kinetics using molecular dynamics simulation. We analyzed 29 matched mutant–wild-type and 16 matched SNP—wild-type protein systems. We found that the binding kinetics changes reflected by the changes of free energy changes induced by protein interaction mutations, which induce variations in the sensitive parameters of the bifurcation point, were a major cause of apoptosis pathway dysfunction, and mutations involved in sensitive interaction domains show high oncogenic potential. Our analysis provided a molecular basis for connecting protein mutations, protein interaction kinetics, network dynamics properties, and physiological function of a regulatory network. These insights provide a framework for coupling mutation genotype to tumorigenesis phenotype and help elucidate the logic of cancer initiation. PMID:26170328

[Observation and analysis on mutation of routine STR locus].

PubMed

Li, Qiu-yang; Feng, Wei-jun; Yang, Qin-gen

2005-05-01

To observe and analyze the characteristic of mutation at STR locus. 27 mutant genes observed in 1211 paternity testing cases were checked by PAGE-silver stained and PowerPlex 16 System Kit and validated by sequencing. Mutant genes locate on 15 loci. The pattern of mutation was accord with stepwise mutation model. The mutation ratio of male-to-female was 8:1 and correlated to the age of father. Mutation rate is correlated to the geometric mean of the number of homogeneous repeats of locus. The higher the mean, the higher the mutation rate. These loci are not so appropriate for use in paternity testing.

Phase II trial of veliparib in patients with previously treated BRCA-mutated pancreas ductal adenocarcinoma.

PubMed

Lowery, Maeve A; Kelsen, David P; Capanu, Marinela; Smith, Sloane C; Lee, Jonathan W; Stadler, Zsofia K; Moore, Malcolm J; Kindler, Hedy L; Golan, Talia; Segal, Amiel; Maynard, Hannah; Hollywood, Ellen; Moynahan, MaryEllen; Salo-Mullen, Erin E; Do, Richard Kinh Gian; Chen, Alice P; Yu, Kenneth H; Tang, Laura H; O'Reilly, Eileen M

2018-01-01

BRCA-associated cancers have increased sensitivity to poly(ADP-ribose) polymerase inhibitors (PARPis). This single arm, non-randomised, multicentre phase II trial evaluated the response rate of veliparib in patients with previously treated BRCA1/2- or PALB2-mutant pancreatic adenocarcinoma (PDAC). Patients with stage III/IV PDAC and known germline BRCA1/2 or PALB2 mutation, 1-2 lines of treatment, Eastern Cooperative Oncology Group 0-2, were enrolled. Veliparib was dosed at a volume of 300 mg twice-daily (N = 3), then 400 mg twice-daily (N = 15) days 1-28. The primary end-point was to determine the response rate of veliparib; secondary end-points included progression-free survival (PFS), duration of response, overall survival (OS) and safety. Sixteen patients were enrolled; male N = 8 (50%). Median age was 52 years (range 43-77). Five (31%) had a BRCA1 and 11 (69%) had a BRCA2 mutation. Fourteen (88%) patients had received prior platinum-based therapy. No confirmed partial responses (PRs) were seen: one (6%) unconfirmed PR was observed at 4 months with disease progression (PD) at 6 months; four (25%) had stable disease (SD), whereas 11 (69%) had PD as best response including one with clinical PD. Median PFS was 1.7 months (95% confidence interval [CI] 1.57-1.83) and median OS was 3.1 months (95% CI 1.9-4.1). Six (38%) patients had grade III toxicity, including fatigue (N = 3), haematology (N = 2) and nausea (N = 1). Veliparib was well tolerated, but no confirmed response was observed although four (25%) patients remained on study with SD for ≥ 4 months. Additional strategies in this population are needed, and ongoing trials are evaluating PARPis combined with chemotherapy (NCT01585805) and as a maintenance strategy (NCT02184195). Copyright © 2017 Elsevier Ltd. All rights reserved.

Meiotic recombination counteracts male-biased mutation (male-driven evolution).

PubMed

Mawaribuchi, Shuuji; Ito, Michihiko; Ogata, Mitsuaki; Oota, Hiroki; Katsumura, Takafumi; Takamatsu, Nobuhiko; Miura, Ikuo

2016-01-27

Meiotic recombination is believed to produce greater genetic variation despite the fact that deoxyribonucleic acid (DNA)-replication errors are a major source of mutations. In some vertebrates, mutation rates are higher in males than in females, which developed the theory of male-driven evolution (male-biased mutation). However, there is little molecular evidence regarding the relationships between meiotic recombination and male-biased mutation. Here we tested the theory using the frog Rana rugosa, which has both XX/XY- and ZZ/ZW-type sex-determining systems within the species. The male-to-female mutation-rate ratio (α) was calculated from homologous sequences on the X/Y or Z/W sex chromosomes, which supported male-driven evolution. Surprisingly, each α value was notably higher in the XX/XY-type group than in the ZZ/ZW-type group, although α should have similar values within a species. Interestingly, meiotic recombination between homologous chromosomes did not occur except at terminal regions in males of this species. Then, by subdividing α into two new factors, a replication-based male-to-female mutation-rate ratio (β) and a meiotic recombination-based XX-to-XY/ZZ-to-ZW mutation-rate ratio (γ), we constructed a formula describing the relationship among a nucleotide-substitution rate and the two factors, β and γ. Intriguingly, the β- and γ-values were larger and smaller than 1, respectively, indicating that meiotic recombination might reduce male-biased mutations. © 2016 The Author(s).

Identification of Point Mutations in Clinical Staphylococcus aureus Strains That Produce Small-Colony Variants Auxotrophic for Menadione

PubMed Central

Dean, Melissa A.; Olsen, Randall J.; Long, S. Wesley; Rosato, Adriana E.

2014-01-01

Staphylococcus aureus small-colony variants (SCVs) are implicated in chronic and relapsing infections that are difficult to diagnose and treat. Despite many years of study, the underlying molecular mechanisms and virulence effect of the small-colony phenotype remain incompletely understood. We sequenced the genomes of five S. aureus SCV strains recovered from human patients and discovered previously unidentified nonsynonymous point mutations in three genes encoding proteins in the menadione biosynthesis pathway. Analysis of genetic revertants and complementation with wild-type alleles confirmed that these mutations caused the SCV phenotype and decreased virulence for mice. PMID:24452687

Studies on congenital hereditary cataract and microphthalmia of the miniature schnauzer dog.

PubMed

Shastry, B S; Reddy, V N

1994-09-30

Hereditary cataract in dogs occurs as an autosomal recessive trait. The opacity is primarily in the lens nucleus and posterior cortex. The affected animals also have other ocular abnormalities such as microphthalmia. To understand the genetic basis of this disorder, we have analyzed leukocyte DNA from affected and normal dogs for possible mutations in the homeobox containing gene and myotonic dystrophy locus. The results show that there are no signs of microdeletion, insertion, point mutation and rearrangements in these loci. Although these observations cannot completely rule out the possibility of point mutations, they suggest that the above loci are unlikely to be associated with the disease.

Stress-induced mutagenesis: Stress diversity facilitates the persistence of mutator genes

PubMed Central

2017-01-01

Mutator strains are expected to evolve when the availability and effect of beneficial mutations are high enough to counteract the disadvantage from deleterious mutations that will inevitably accumulate. As the population becomes more adapted to its environment, both availability and effect of beneficial mutations necessarily decrease and mutation rates are predicted to decrease. It has been shown that certain molecular mechanisms can lead to increased mutation rates when the organism finds itself in a stressful environment. While this may be a correlated response to other functions, it could also be an adaptive mechanism, raising mutation rates only when it is most advantageous. Here, we use a mathematical model to investigate the plausibility of the adaptive hypothesis. We show that such a mechanism can be mantained if the population is subjected to diverse stresses. By simulating various antibiotic treatment schemes, we find that combination treatments can reduce the effectiveness of second-order selection on stress-induced mutagenesis. We discuss the implications of our results to strategies of antibiotic therapy. PMID:28719607

Single-Molecule Counting of Point Mutations by Transient DNA Binding

NASA Astrophysics Data System (ADS)

Su, Xin; Li, Lidan; Wang, Shanshan; Hao, Dandan; Wang, Lei; Yu, Changyuan

2017-03-01

High-confidence detection of point mutations is important for disease diagnosis and clinical practice. Hybridization probes are extensively used, but are hindered by their poor single-nucleotide selectivity. Shortening the length of DNA hybridization probes weakens the stability of the probe-target duplex, leading to transient binding between complementary sequences. The kinetics of probe-target binding events are highly dependent on the number of complementary base pairs. Here, we present a single-molecule assay for point mutation detection based on transient DNA binding and use of total internal reflection fluorescence microscopy. Statistical analysis of single-molecule kinetics enabled us to effectively discriminate between wild type DNA sequences and single-nucleotide variants at the single-molecule level. A higher single-nucleotide discrimination is achieved than in our previous work by optimizing the assay conditions, which is guided by statistical modeling of kinetics with a gamma distribution. The KRAS c.34 A mutation can be clearly differentiated from the wild type sequence (KRAS c.34 G) at a relative abundance as low as 0.01% mutant to WT. To demonstrate the feasibility of this method for analysis of clinically relevant biological samples, we used this technology to detect mutations in single-stranded DNA generated from asymmetric RT-PCR of mRNA from two cancer cell lines.

A valveless rotary microfluidic device for multiplex point mutation identification based on ligation-rolling circle amplification.

PubMed

Heo, Hyun Young; Chung, Soyi; Kim, Yong Tae; Kim, Do Hyun; Seo, Tae Seok

2016-04-15

Genetic variations such as single nucleotide polymorphism (SNP) and point mutations are important biomarkers to monitor disease prognosis and diagnosis. In this study, we developed a novel rotary microfluidic device which can perform multiplex SNP typing on the mutation sites of TP53 genes. The microdevice consists of three glass layers: a channel wafer, a Ti/Pt electrode-patterned resistance temperature detector (RTD) wafer, and a rotary plate in which twelve reaction chambers were fabricated. A series of sample injection, ligation-rolling circle amplification (L-RCA) reaction, and fluorescence detection of the resultant amplicons could be executed by rotating the top rotary plate, identifying five mutation points related with cancer prognosis. The use of the rotary plate eliminates the necessity of microvalves and micropumps to control the microfluidic flow in the channel, simplifying the chip design and chip operation for multiplex SNP detection. The proposed microdevice provides an advanced genetic analysis platform in terms of multiplexity, simplicity, and portability in the fields of biomedical diagnostics. Copyright © 2015 Elsevier B.V. All rights reserved.

Increasing thermal stability and catalytic activity of glutamate decarboxylase in E. coli: An in silico study.

PubMed

Tavakoli, Yasaman; Esmaeili, Abolghasem; Saber, Hossein

2016-10-01

Glutamate decarboxylase (GAD) is an enzyme that converts l-glutamate to gamma amino butyric acid (GABA) that is a widely used drug to treat mental disorders like Alzheimer's disease. In this study for the first time point mutation was performed virtually in the active site of the E. coli GAD in order to increase thermal stability and catalytic activity of the enzyme. Energy minimization and addition of water box were performed using GROMACS 5.4.6 package. PoPMuSiC 2.1 web server was used to predict potential spots for point mutation and Modeller software was used to perform point mutation on three dimensional model. Molegro virtual docker software was used for cavity detection and stimulated docking study. Results indicate that performing mutation separately at positions 164, 302, 304, 393, 396, 398 and 410 increase binding affinity to substrate. The enzyme is predicted to be more thermo- stable in all 7 mutants based on ΔΔG value. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mutation screening of the PCDH15 gene in Spanish patients with Usher syndrome type I.

PubMed

Jaijo, Teresa; Oshima, Aki; Aller, Elena; Carney, Carol; Usami, Shin-ichi; Millán, José M; Kimberling, William J

2012-01-01

PCDH15 codes for protocadherin-15, a cell-cell adhesion protein essential in the morphogenesis and cohesion of stereocilia bundles and in the function or preservation of photoreceptor cells. Mutations in the PCDH15 gene are responsible for Usher syndrome type I (USH1F) and non-syndromic hearing loss (DFNB23). The purpose of this work was to perform PCDH15 mutation screening to identify the genetic cause of the disease in a cohort of Spanish patients with Usher syndrome type I and establish phenotype-genotype correlation. Mutation analysis of PCDH15 included additional exons recently identified and was performed by direct sequencing. The screening was performed in 19 probands with USH already screened for mutations in the most prevalent USH1 genes, myosin VIIA (MYO7A) and cadherin-23 (CDH23), and for copy number variants in PCDH15. Seven different point mutations, five novel, were detected. Including the large PCDH15 rearrangements previously reported in our cohort of patients, a total of seven of 19 patients (36.8%) were carriers of at least one pathogenic allele. Thirteen out of the 38 screened alleles carried pathogenic PCDH15 variants (34.2%). Five out of the seven point mutations reported in the present study are novel, supporting the idea that most PCDH15 mutations are private. Furthermore, no mutational hotspots have been identified. In most patients, detected mutations led to a truncated protein, reinforcing the hypothesis that severe mutations cause the Usher I phenotype and that missense variants are mainly responsible for non-syndromic hearing impairment.

Mutation screening of the PCDH15 gene in Spanish patients with Usher syndrome type I

PubMed Central

Jaijo, Teresa; Oshima, Aki; Aller, Elena; Carney, Carol; Usami, Shin-ichi; Kimberling, William J.

2012-01-01

Purpose PCDH15 codes for protocadherin-15, a cell-cell adhesion protein essential in the morphogenesis and cohesion of stereocilia bundles and in the function or preservation of photoreceptor cells. Mutations in the PCDH15 gene are responsible for Usher syndrome type I (USH1F) and non-syndromic hearing loss (DFNB23). The purpose of this work was to perform PCDH15 mutation screening to identify the genetic cause of the disease in a cohort of Spanish patients with Usher syndrome type I and establish phenotype-genotype correlation. Methods Mutation analysis of PCDH15 included additional exons recently identified and was performed by direct sequencing. The screening was performed in 19 probands with USH already screened for mutations in the most prevalent USH1 genes, myosin VIIA (MYO7A) and cadherin-23 (CDH23), and for copy number variants in PCDH15. Results Seven different point mutations, five novel, were detected. Including the large PCDH15 rearrangements previously reported in our cohort of patients, a total of seven of 19 patients (36.8%) were carriers of at least one pathogenic allele. Thirteen out of the 38 screened alleles carried pathogenic PCDH15 variants (34.2%). Conclusions Five out of the seven point mutations reported in the present study are novel, supporting the idea that most PCDH15 mutations are private. Furthermore, no mutational hotspots have been identified. In most patients, detected mutations led to a truncated protein, reinforcing the hypothesis that severe mutations cause the Usher I phenotype and that missense variants are mainly responsible for non-syndromic hearing impairment. PMID:22815625

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

How long ago did smallpox virus emerge?

PubMed

Shchelkunov, Sergei N

2009-01-01

Unlike vertebrates, for which paleontological data are available, and RNA viruses, which display a high rate of genetic variation, an objective estimate of time parameters for the molecular evolution of DNA viruses, which display a low rate of accumulation of mutations, is a complex problem. Genomic studies of a set of smallpox (variola) virus (VARV) isolates demonstrated the patterns of phylogenetic relationships between geographic variants of this virus. Using archival data on smallpox outbreaks and the results of phylogenetic analyses of poxvirus genomes, different research teams have obtained contradictory data on the possible time point of VARV origin. I discuss the approaches used for dating of VARV evolution and adduce the arguments favoring its historically recent origin.

Clinical outcomes of EGFR-TKI treatment and genetic heterogeneity in lung adenocarcinoma patients with EGFR mutations on exons 19 and 21.

PubMed

Yu, Jiang-Yong; Yu, Si-Fan; Wang, Shu-Hang; Bai, Hua; Zhao, Jun; An, Tong-Tong; Duan, Jian-Chun; Wang, Jie

2016-03-21

Epidermal growth factor receptor (EGFR) mutations, including a known exon 19 deletion (19 del) and exon 21 L858R point mutation (L858R mutation), are strong predictors of the response to EGFR tyrosine kinase inhibitor (EGFR-TKI) treatment in lung adenocarcinoma. However, whether patients carrying EGFR 19 del and L858R mutations exhibit different responsiveness to EGFR-TKIs and what are the potential mechanism for this difference remain controversial. This study aimed to investigate the clinical outcomes of EGFR-TKI treatment in patients with EGFR 19 del and L858R mutations and explore the genetic heterogeneity of tumors with the two mutation subtypes. Of 1127 patients with advanced lung adenocarcinoma harboring EGFR 19 del or L858R mutations, 532 received EGFR-TKI treatment and were included in this study. EGFR 19 del and L858R mutations were detected by using denaturing high-performance liquid chromatography (DHPLC). T790M mutation, which is a common resistant mutation on exon 20 of EGFR, was detected by amplification refractory mutation system (ARMS). Next-generation sequencing (NGS) was used to explore the genetic heterogeneity of tumors with EGFR 19 del and L858R mutations. Of the 532 patients, 319 (60.0%) had EGFR 19 del, and 213 (40.0%) had L858R mutations. The patients with EGFR 19 del presented a significantly higher overall response rate (ORR) for EGFR-TKI treatment (55.2% vs. 43.7%, P = 0.017) and had a longer progression-free survival (PFS) after first-line EGFR-TKI treatment (14.4 vs. 11.4 months, P = 0.034) compared with those with L858R mutations. However, no statistically significant difference in overall survival (OS) was observed between the two groups of patients. T790M mutation status was analyzed in 88 patients before EGFR-TKI treatment and 134 after EGFR-TKI treatment, and there was no significant difference in the co-existence of T790M mutation with EGFR 19 del and L858R mutations before EGFR-TKI treatment (5.6% vs. 8.8%, P = 0.554) or after treatment (24.4% vs. 35.4%, P = 0.176). In addition, 24 patients with EGFR 19 del and 19 with L858R mutations were analyzed by NGS, and no significant difference in the presence of multiple somatic mutations was observed between the two genotypes. Patients with EGFR 19 del exhibit longer PFS and higher ORR compared with those with L858R mutations. Whether the heterogeneity of tumors with EGFR 19 del and L858R mutations contribute to a therapeutic response difference needs further investigation.

Mutation analysis of Australasian Gaucher disease patients

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

Nelson, P.V.; Carey, W.F.; Morris, C.P.

1995-09-25

We have previously reported phenotype and genotype analyses in 28 Australasian Gaucher patients who were screened for several of the common Gaucher mutations: N370S, L444P, 84GG, and R463C. Horowitz and Zimran have reported that the complex alleles recNciI and recTL, which contain several point mutations including L444P, are relatively common, especially in non-Jewish Gaucher patients. Zimran and Horowitz have also stated that these recombinant alleles could easily be missed by laboratories testing only for the common Gaucher point mutations. Failure to correctly identify these mutations would influence any attempt to correlate genotype with phenotype. We have therefore retested our Gauchermore » patients for recNciI (L444P, A456P, and V46OV) and recTL (D409H, L444P, A456P, and V46OV) by PCR amplification, followed by hybridization with allele-specific oligonucleotides. 4 refs.« less

A variant c-KIT mutation, D816H, fundamental to the sequential development of an ovarian mixed germ cell tumor and systemic mastocytosis with chronic myelomonocytic leukemia.

PubMed

Mitchell, Sarah G; Bunting, Silvia T; Saxe, Debra; Olson, Thomas; Keller, Frank G

2017-04-01

An activating point mutation of the c-KIT tyrosine kinase receptor gene, D816H, has been described in germ cell tumors (GCTs). We report an adolescent diagnosed with an ovarian mixed GCT and systemic mastocytosis with chronic myelomonocytic leukemia (SM-CMML). The teratoma and dysgerminoma differed by copy number aberrations via single nucleotide polymorphism (SNP) microarray, but were inclusive of the same c-KIT D816H point mutation (c.2446G>C) also identified in blood and bone marrow mast cells. These findings indicate not only a clonal origin of the GCT and hematologic malignancy, but also suggest a rare KIT mutation may be playing a fundamental role in malignancy development. © 2016 Wiley Periodicals, Inc.

A molecular dynamics investigation on the crizotinib resistance mechanism of C1156Y mutation in ALK

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

Sun, Hui-Yong; Ji, Feng-Qin, E-mail: fengqinji@mail.hzau.edu.cn; Center for Bioinformatics, Huazhong Agricultural University, Wuhan 430070

2012-06-29

Highlights: Black-Right-Pointing-Pointer The study revealed the detailed resistance mechanism of the non-active mutation C1156Y in ALK. Black-Right-Pointing-Pointer C1156Y leads to crizotinib displacement and conformational changes in the binding cavity. Black-Right-Pointing-Pointer The conformations cause a decline in the vdW and electrostatic energy between crizotinib and ALK. -- Abstract: Crizotinib is an anaplastic lymphoma kinase (ALK) inhibitor that has recently been approved in the US for the treatment of non-small cell lung carcinoma (NSCLC). Despite its outstanding safety and efficacy, several resistant mutations against crizotinib have been detected in the treatment of NSCLC. However, in contrast to the widely accepted mechanism ofmore » steric hindrance by mutations at the active site, the mechanism by which the C1156Y non-active site mutation confers resistance against crizotinib remains unclear. In the present study, the resistance mechanism of C1156Y in ALK was investigated using molecular dynamics simulations. The results suggest that despite the non-active site mutation, C1156Y causes the dislocation of crizotinib as well as the indirect conformational changes in the binding cavity, which results in a marked decrease in the van der Waals and electrostatic interactions between crizotinib and ALK. The obtained results provide a detailed explanation of the resistance caused by C1156Y and may give a vital clue for the design of drugs to combat crizotinib resistance.« less

Impact of whole brain radiation therapy on CSF penetration ability of Icotinib in EGFR-mutated non-small cell lung cancer patients with brain metastases: Results of phase I dose-escalation study.

PubMed

Zhou, Lin; He, Jiazhuo; Xiong, Weijie; Liu, Yongmei; Xiang, Jing; Yu, Qin; Liang, Maozhi; Zhou, Xiaojuan; Ding, Zhenyu; Huang, Meijuan; Ren, Li; Zhu, Jiang; Li, Lu; Hou, Mei; Ding, Lieming; Tan, Fenlai; Lu, You

2016-06-01

Whole-brain radiation therapy (WBRT) and epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are both treatment options for EGFR-mutated non-small cell lung cancer (NSCLC) patients with brain metastases. However, the dose-escalation toxicity and efficacy of combination therapy, and the effect of WBRT on cerebrospinal fluid (CSF) penetration of EGFR-TKIs are still unclear. EGFR-mutated NSCLC patients with brain metastases were enrolled in this study, and the cohorts were constructed with a 3+3 design. The patients received icotinib with escalating doses (125-625mg, tid), and the concurrent WBRT (37.5Gy/15f/3weeks) started a week later. The CSF penetration rates of icotinib were tested before, immediately after, and 4 weeks after WBRT, respectively. Potential toxicities and benefits from dose-escalation treatment were analyzed. Fifteen patients were included in this study, 3 at each dose level from 125mg-375mg and 6 at 500mg with 3 occurred dose-limiting toxicities. The maximal tolerated dose of icotinib was 375mg tid in this combination therapy. There was a significant correlation between icotinib concentration in the CSF and plasma (R(2)=0.599, P<0.001). The CSF penetration rate of icotinib, from 1.2% to 9.7%, reached a maximum at 375mg (median, 6.1%). There was no significant difference for CSF penetration rates among the three test points (median, 4.1% vs. 2.8% vs. 2.8%, P=0.16). The intracranial objective response rate and median intracranial progression free survival are 80% and 18.9 months. WBRT plus concurrent icotinib is well tolerated in EGFR-mutated NSCLC patients with brain metastases, up to an icotinib dose of 375mg tid. The icotinib CSF concentration seemed to have a potential ceiling effect with the dose escalation, and WBRT seemed to have no significant impact on CSF penetration of icotinib till 4 weeks after the treatment. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

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.

Increasing Nucleosome Occupancy Is Correlated with an Increasing Mutation Rate so Long as DNA Repair Machinery Is Intact

PubMed Central

Taylor, Jared F.; Khattab, Omar S.; Chen, Yu-Han; Chen, Yumay; Jacobsen, Steven E.; Wang, Ping H.

2015-01-01

Deciphering the multitude of epigenomic and genomic factors that influence the mutation rate is an area of great interest in modern biology. Recently, chromatin has been shown to play a part in this process. To elucidate this relationship further, we integrated our own ultra-deep sequenced human nucleosomal DNA data set with a host of published human genomic and cancer genomic data sets. Our results revealed, that differences in nucleosome occupancy are associated with changes in base-specific mutation rates. Increasing nucleosome occupancy is associated with an increasing transition to transversion ratio and an increased germline mutation rate within the human genome. Additionally, cancer single nucleotide variants and microindels are enriched within nucleosomes and both the coding and non-coding cancer mutation rate increases with increasing nucleosome occupancy. There is an enrichment of cancer indels at the theoretical start (74 bp) and end (115 bp) of linker DNA between two nucleosomes. We then hypothesized that increasing nucleosome occupancy decreases access to DNA by DNA repair machinery and could account for the increasing mutation rate. Such a relationship should not exist in DNA repair knockouts, and we thus repeated our analysis in DNA repair machinery knockouts to test our hypothesis. Indeed, our results revealed no correlation between increasing nucleosome occupancy and increasing mutation rate in DNA repair knockouts. Our findings emphasize the linkage of the genome and epigenome through the nucleosome whose properties can affect genome evolution and genetic aberrations such as cancer. PMID:26308346

[Clinical significance of drug resistance-associated mutations in treatment of hepatitis C with direct-acting antiviral agents].

PubMed

Li, Z; Chen, Z W; Ren, H; Hu, P

2017-03-20

Direct-acting antiviral agents (DAAs) achieve a high sustained virologic response rate in the treatment of chronic hepatitis C virus infection. However, drug resistance-associated mutations play an important role in treatment failure and have attracted more and more attention. This article elaborates on the clinical significance of drug resistance-associated mutations from the aspects of their definition, association with genotype, known drug resistance-associated mutations and their prevalence rates, the impact of drug resistance-associated mutations on treatment naive and treatment-experienced patients, and the role of clinical detection, in order to provide a reference for clinical regimens with DAAs and help to achieve higher sustained virologic response rates.

Reduced mutation rate in exons due to differential mismatch repair

PubMed Central

Mularoni, Loris; Muiños, Ferran; Gonzalez-Perez, Abel; López-Bigas, Núria

2017-01-01

While recent studies have revealed higher than anticipated heterogeneity of mutation rate across genomic regions, mutations in exons and introns are assumed to be generated at the same rate. Here we find fewer somatic mutations in exons than expected based on their sequence content, and demonstrate that this is not due to purifying selection. Moreover, we show that it is caused by higher mismatch repair activity in exonic than in intronic regions. Our findings have important implications for our understanding of mutational and DNA repair processes, our knowledge of the evolution of eukaryotic genes, and practical ramifications for the study of the evolution of both tumors and species. PMID:29106418

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 ...

AMPA receptor flip/flop mutants affecting deactivation, desensitization, and modulation by cyclothiazide, aniracetam, and thiocyanate.

PubMed

Partin, K M; Fleck, M W; Mayer, M L

1996-11-01

AMPA receptor GluRA subunits with mutations at position 750, a residue shown previously to control allosteric regulation by cyclothiazide, were analyzed for modulation of deactivation and desensitization by cyclothiazide, aniracetam, and thiocyanate. Point mutations from Ser to Asn, Ala, Asp, Gly, Gln, Met, Cys, Thr, Leu, Val, and Tyr were constructed in GluRAflip. The last four of these mutants were not functional; S750D was active only in the presence of cyclothiazide, and the remaining mutants exhibited altered rates of deactivation and desensitization for control responses to glutamate, and showed differential modulation by cyclothiazide and aniracetam. Results from kinetic analysis are consistent with aniracetam and cyclothiazide acting via distinct mechanisms. Our experiments demonstrate for the first time the functional importance of residue 750 in regulating intrinsic channel-gating kinetics and emphasize the biological significance of alternative splicing in the M3-M4 extracellular loop.

Inducible transgenics. New lessons on events governing the induction and commitment in mammary tumorigenesis.

PubMed

Hulit, J; Di Vizio, D; Pestell, R G

2001-01-01

Breast cancer arises from multiple genetic events that together contribute to the established, irreversible malignant phenotype. The development of inducible tissue-specific transgenics has allowed a careful dissection of the events required for induction and subsequent maintenance of tumorigenesis. Mammary gland targeted expression of oncogenic Ras or c-Myc is sufficient for the induction of mammary gland tumorigenesis in the rodent, and when overexpressed together the rate of tumor onset is substantially enhanced. In an exciting recent finding, D'Cruz et al discovered tetracycline-regulated c-Myc overexpression in the mammary gland induced invasive mammary tumors that regressed upon withdrawal of c-Myc expression. Almost one-half of the c-Myc-induced tumors harbored K-ras or N-ras gene point mutations, correlating with tumor persistence on withdrawal of c-Myc transgene expression. These findings suggest maintenance of tumorigenesis may involve a second mutation within the Ras pathway.

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

PubMed Central

Pritchard, Jonathan K.

2016-01-01

The site frequency spectrum (SFS) has long been used to study demographic history and natural selection. Here, we extend this summary by examining the SFS conditional on the alleles found at the same site in other species. We refer to this extension as the “phylogenetically-conditioned SFS” or cSFS. Using recent large-sample data from the Exome Aggregation Consortium (ExAC), combined with primate genome sequences, we find that human variants that occurred independently in closely related primate lineages are at higher frequencies in humans than variants with parallel substitutions in more distant primates. We show that this effect is largely due to sites with elevated mutation rates causing significant departures from the widely-used infinite sites mutation model. Our analysis also suggests substantial variation in mutation rates even among mutations involving the same nucleotide changes. In summary, we show that variable mutation rates are key determinants of the SFS in humans. PMID:27977673

Germline stem cell competition, mutation hot spots, genetic disorders and older dads

PubMed Central

Arnheim, Norman; Calabrese, Peter

2016-01-01

Some de novo human mutations arise at frequencies far exceeding the genome average mutation rate. Examples are the common mutations at one or a few sites in the genes causing achondroplasia, Noonan syndrome, multiple endocrine neoplasia 2B and Apert syndrome. These mutations are recurrent, provide a gain of function, are paternally derived and are more likely transmitted as the father ages. Recent experiments tested whether the high mutation frequencies are due to an elevated mutation rate per cell division, as expected, or an advantage of the mutant spermatogonial stem cells over wild-type stem cells. The evidence, which includes the surprising discovery of testis mutation clusters, rejects the former model but not the latter. We propose how the mutations might alter spermatogonial stem cell function and discuss how germline selection contributes to the paternal age effect, the human mutational load and adaptive evolution. PMID:27070266

Biomarker Detection Using NAPPA Tumor Antigen Arrays: EDRN Supplement — EDRN Public Portal

Cancer.gov

The overall goal of this project application for the EDRN set-aside funds is to focus our collaborative efforts to identify p53 mutation-specific antibody biomarkers in breast, prostate, and ovarian cancer. P53-specific gene mutations are frequent in multiple cancer types. Of the common solid tumors, p53 mutations have been identified in 50% of lung and ovarian cancers, 45% of colon cancers, 20% of breast cancers, and 10-30% of prostate cancers (The p53 Mutation Handbook, T. Soussi, http://p53/free/fr). The most common mutations vary from cancer to cancer, with 50 point mutations covering the 10 most common mutations for all major solid tumors

High mitochondrial mutation rates estimated from deep-rooting Costa Rican pedigrees

PubMed Central

Madrigal, Lorena; Melendez-Obando, Mauricio; Villegas-Palma, Ramon; Barrantes, Ramiro; Raventos, Henrieta; Pereira, Reynaldo; Luiselli, Donata; Pettener, Davide; Barbujani, Guido

2012-01-01

Estimates of mutation rates for the noncoding hypervariable Region I (HVR-I) of mitochondrial DNA (mtDNA) vary widely, depending on whether they are inferred from phylogenies (assuming that molecular evolution is clock-like) or directly from pedigrees. All pedigree-based studies so far were conducted on populations of European origin. In this paper we analyzed 19 deep-rooting pedigrees in a population of mixed origin in Costa Rica. We calculated two estimates of the HVR-I mutation rate, one considering all apparent mutations, and one disregarding changes at sites known to be mutational hot spots and eliminating genealogy branches which might be suspected to include errors, or unrecognized adoptions along the female lines. At the end of this procedure, we still observed a mutation rate equal to 1.24 × 10−6, per site per year, i.e., at least three-fold as high as estimates derived from phylogenies. Our results confirm that mutation rates observed in pedigrees are much higher than estimated assuming a neutral model of long-term HVRI evolution. We argue that, until the cause of these discrepancies will be fully understood, both lower estimates (i.e., those derived from phylogenetic comparisons) and higher, direct estimates such as those obtained in this study, should be considered when modeling evolutionary and demographic processes. PMID:22460349

No correlation between germline mutation at repeat DNA and meiotic crossover in male mice exposed to X-rays or cisplatin.

PubMed

Barber, R; Plumb, M; Smith, A G; Cesar, C E; Boulton, E; Jeffreys, A J; Dubrova, Y E

2000-12-20

To test the hypothesis that mouse germline expanded simple tandem repeat (ESTR) mutations are associated with recombination events during spermatogenesis, crossover frequencies were compared with germline mutation rates at ESTR loci in male mice acutely exposed to 1Gy of X-rays or to 10mg/kg of the anticancer drug cisplatin. Ionising radiation resulted in a highly significant 2.7-3.6-fold increase in ESTR mutation rate in males mated 4, 5 and 6 weeks after exposure, but not 3 weeks after exposure. In contrast, irradiation had no effect on meiotic crossover frequencies assayed on six chromosomes using 25 polymorphic microsatellite loci spaced at approximately 20cM intervals and covering 421cM of the mouse genome. Paternal exposure to cisplatin did not affect either ESTR mutation rates or crossover frequencies, despite a report that cisplatin can increase crossover frequency in mice. Correlation analysis did not reveal any associations between the paternal ESTR mutation rate and crossover frequency in unexposed males and in those exposed to X-rays or cisplatin. This study does not, therefore, support the hypothesis that mutation induction at mouse ESTR loci results from a general genome-wide increase in meiotic recombination rate.

Comparison of small biopsy specimens and surgical specimens for the detection of EGFR mutations and EML4-ALK in non-small-cell lung cancer

PubMed Central

Xiao, DeSheng; Lu, Can; Zhu, Wei; He, QiuYan; Li, Yong; Fu, ChunYan; Zhou, JianHua; Liu, Shuang; Tao, YongGuang

2016-01-01

Epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) fusion genes represent novel oncogenes that are associated with non–small-cell lung cancers (NSCLC). The feasibility of detecting EGFR mutations and ALK fusion genes in small biopsy specimens or surgical specimens was determined. Of the 721 NSCLC patients, a total of 305 cases were positive for EGFR mutations (42.3%). The rate of EGFR mutations in women was significantly higher than that in men. Histologically, the EGFR mutation rate in adenocarcinomas was significantly higher than that in squamous cell carcinomas. No difference in the EGFR mutation rate was observed between surgical specimens (42.1%) and small biopsy specimens (42.4%), which indicated that the EGFR mutation ratios in surgical specimens and small biopsy specimens were not different. In 385 NSCLC patients, 26 cases were positive for EML4-ALK (6.8%). However, 11.7% of the surgical specimens were EML4-ALK-positive, whereas the positive proportion in the small biopsy specimens was only 4.7%, which indicated that EML4-ALK-positive rate in the surgical specimens was significantly higher than that in the small biopsy specimens. Detection of EGFR gene mutations was feasible in small biopsy specimens, and screening for EML4-ALK expression in small biopsy specimens can be used to guide clinical treatments. PMID:27322143

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

Comparison of small biopsy specimens and surgical specimens for the detection of EGFR mutations and EML4-ALK in non-small-cell lung cancer.

PubMed

Xiao, DeSheng; Lu, Can; Zhu, Wei; He, QiuYan; Li, Yong; Fu, ChunYan; Zhou, JianHua; Liu, Shuang; Tao, YongGuang

2016-09-13

Epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) fusion genes represent novel oncogenes that are associated with non-small-cell lung cancers (NSCLC). The feasibility of detecting EGFR mutations and ALK fusion genes in small biopsy specimens or surgical specimens was determined. Of the 721 NSCLC patients, a total of 305 cases were positive for EGFR mutations (42.3%). The rate of EGFR mutations in women was significantly higher than that in men. Histologically, the EGFR mutation rate in adenocarcinomas was significantly higher than that in squamous cell carcinomas. No difference in the EGFR mutation rate was observed between surgical specimens (42.1%) and small biopsy specimens (42.4%), which indicated that the EGFR mutation ratios in surgical specimens and small biopsy specimens were not different. In 385 NSCLC patients, 26 cases were positive for EML4-ALK (6.8%). However, 11.7% of the surgical specimens were EML4-ALK-positive, whereas the positive proportion in the small biopsy specimens was only 4.7%, which indicated that EML4-ALK-positive rate in the surgical specimens was significantly higher than that in the small biopsy specimens. Detection of EGFR gene mutations was feasible in small biopsy specimens, and screening for EML4-ALK expression in small biopsy specimens can be used to guide clinical treatments.

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.

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.

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

Yagishita, Shigehiro; Horinouchi, Hidehito, E-mail: hhorinou@ncc.go.jp; Katsui Taniyama, Tomoko

Purpose: To determine the frequency and clinical significance of epidermal growth factor receptor (EGFR) mutations in patients with potentially curable stage III non–small-cell lung cancer (NSCLC) who are eligible for definitive chemoradiotherapy (CRT). Patients and Methods: Between January 2001 and December 2010, we analyzed the EGFR mutational status in consecutive NSCLC patients who were treated by CRT. The response rate, relapse-free survival, 2-year relapse-free rate, initial relapse sites, and overall survival of the patients were investigated. Results: A total of 528 patients received CRT at our hospital during the study period. Of these, 274 were diagnosed as having nonsquamous NSCLC. Sufficientmore » specimens for mutational analyses could be obtained from 198 of these patients. The proportion of patients with EGFR activating mutations was 17%. In addition to the well-known characteristics of patients carrying EGFR mutations (female, adenocarcinoma, and never/light smoker), the proportion of cases with smaller primary lesions (T1/2) was found to be higher in patients with EGFR mutations than in those with wild-type EGFR. Patients with EGFR mutations showed similar response rate, relapse-free survival, and 2-year relapse-free rates as compared to patients with wild-type EGFR. Local relapses as the site of initial relapse occurred significantly less frequently in patients with EGFR mutation (4% vs 21%; P=.045). Patients with EGFR mutations showed longer local control (adjusted hazard ratio 0.49; P=.043). After disease progression, a majority of the patients with EGFR mutations received EGFR tyrosine kinase inhibitors (62%), and these patients showed longer postprogression survival than those with wild-type EGFR. Conclusions: Our study is the first to show radiosensitive biology of EGFR-mutated tumors in definitive CRT with curative intent. This finding could serve as a credible baseline estimate of EGFR-mutated population in stage III nonsquamous NSCLC.« less

Uncommon EGFR mutations in cytological specimens of 1,874 newly diagnosed Indonesian lung cancer patients.

PubMed

Syahruddin, Elisna; Wulandari, Laksmi; Sri Muktiati, Nunuk; Rima, Ana; Soeroso, Noni; Ermayanti, Sabrina; Levi, Michael; Hidajat, Heriawaty; Widjajahakim, Grace; Utomo, Ahmad Rusdan Handoyo

2018-01-01

We aimed to evaluate the distribution of individual epidermal growth factor receptor ( EGFR ) mutation subtypes found in routine cytological specimens. A retrospective audit was performed on EGFR testing results of 1,874 consecutive cytological samples of newly diagnosed or treatment-naïve Indonesian lung cancer patients (years 2015-2016). Testing was performed by ISO15189 accredited central laboratory. Overall test failure rate was 5.1%, with the highest failure (7.1%) observed in pleural effusion and lowest (1.6%) in needle aspiration samples. EGFR mutation frequency was 44.4%. Tyrosine kinase inhibitor (TKI)-sensitive common EGFR mutations (ins/dels exon 19, L858R) and uncommon mutations (G719X, T790M, L861Q) contributed 57.1% and 29%, respectively. Approximately 13.9% of mutation-positive patients carried a mixture of common and uncommon mutations. Women had higher EGFR mutation rate (52.9%) vs men (39.1%; p <0.05). In contrast, uncommon mutations conferring either TKI responsive (G719X, L861Q) or TKI resistance (T790M, exon 20 insertions) were consistently more frequent in men than in women (67.3% vs 32.7% or 69.4% vs 30.6%; p <0.05). Up to 10% EGFR mutation-positive patients had baseline single mutation T790M, exon 20 insertion, or in coexistence with TKI-sensitive mutations. Up to 9% patients had complex or multiple EGFR mutations, whereby 48.7% patients harbored TKI-resistant mutations. One patient presented third-generation TKI-resistant mutation L792F simultaneously with T790M. Routine diagnostic cytological techniques yielded similar success rate to detect EGFR mutations. Uncommon EGFR mutations were frequent events in Indonesian lung cancer patients.

Personalized Oncology Through Integrative High-Throughput Sequencing: A Pilot Study

PubMed Central

Roychowdhury, Sameek; Iyer, Matthew K.; Robinson, Dan R.; Lonigro, Robert J.; Wu, Yi-Mi; Cao, Xuhong; Kalyana-Sundaram, Shanker; Sam, Lee; Balbin, O. Alejandro; Quist, Michael J.; Barrette, Terrence; Everett, Jessica; Siddiqui, Javed; Kunju, Lakshmi P.; Navone, Nora; Araujo, John C.; Troncoso, Patricia; Logothetis, Christopher J.; Innis, Jeffrey W.; Smith, David C.; Lao, Christopher D.; Kim, Scott Y.; Roberts, J. Scott; Gruber, Stephen B.; Pienta, Kenneth J.; Talpaz, Moshe; Chinnaiyan, Arul M.

2012-01-01

Individual cancers harbor a set of genetic aberrations that can be informative for identifying rational therapies currently available or in clinical trials. We implemented a pilot study to explore the practical challenges of applying high-throughput sequencing in clinical oncology. We enrolled patients with advanced or refractory cancer who were eligible for clinical trials. For each patient, we performed whole-genome sequencing of the tumor, targeted whole-exome sequencing of tumor and normal DNA, and transcriptome sequencing (RNA-Seq) of the tumor to identify potentially informative mutations in a clinically relevant time frame of 3 to 4 weeks. With this approach, we detected several classes of cancer mutations including structural rearrangements, copy number alterations, point mutations, and gene expression alterations. A multidisciplinary Sequencing Tumor Board (STB) deliberated on the clinical interpretation of the sequencing results obtained. We tested our sequencing strategy on human prostate cancer xenografts. Next, we enrolled two patients into the clinical protocol and were able to review the results at our STB within 24 days of biopsy. The first patient had metastatic colorectal cancer in which we identified somatic point mutations in NRAS, TP53, AURKA, FAS, and MYH11, plus amplification and overexpression of cyclin-dependent kinase 8 (CDK8). The second patient had malignant melanoma, in which we identified a somatic point mutation in HRAS and a structural rearrangement affecting CDKN2C. The STB identified the CDK8 amplification and Ras mutation as providing a rationale for clinical trials with CDK inhibitors or MEK (mitogenactivated or extracellular signal–regulated protein kinase kinase) and PI3K (phosphatidylinositol 3-kinase) inhibitors, respectively. Integrative high-throughput sequencing of patients with advanced cancer generates a comprehensive, individual mutational landscape to facilitate biomarker-driven clinical trials in oncology. PMID:22133722

Epidermodysplasia verruciformis in lipoid proteinosis: case report and discussion of pathophysiology.

PubMed

O'Blenes, Catherine; Pasternak, Sylvia; Issekutz, Andrew; Gillis, Jane; Chowdhury, Dhiman; Finlayson, Laura

2015-01-01

Lipoid proteinosis (LP) is a rare autosomal recessive genodermatosis caused by mutations in extracellular matrix protein 1 (ECM1) that involves deposition of basement membrane-like material in the skin and other organs. Epidermodysplasia verruciformis (EV) is also a rare autosomal recessive genodermatosis involving susceptibility to human papillomavirus (HPV) infections and squamous cell carcinoma, caused in most cases by homozygous mutations in EVER1 or EVER2. We describe a case of EV in a patient with LP and discuss the pathophysiology. A 3-year-old Lebanese girl presented with hoarseness, beaded papules along the eyelid margins, waxy papules and plaques on her head and neck, and lichenoid verrucous papules on the forearms and hands. Histopathology of the waxy papules exhibited deposition of periodic acid Schiff-positive basement membrane-like material in the superficial dermis, characteristic of LP. The verruca plana-like lesions exhibited acanthosis and enlarged keratinocytes with pale blue-grey cytoplasm and a perinuclear halo, consistent with verrucae and EV. Polymerase chain reaction amplification and sequencing of ECM1, EVER1, and EVER2 demonstrated a homozygous point mutation, c.389C>T (p.Thr130Met), in exon 6 of ECM1 and a heterozygous point mutation, c.917 A>T (p.Asn306Ile), in exon 8 in EVER2, known to cause EV in homozygous patients. The homozygous point mutation c.389C>T in ECM1 may be a novel mutation causing LP. Verruca plana-like lesions seen in LP appear to represent a form of acquired EV. In this patient, a heterozygous mutation in EVER2 at c.917 A>T may also have conferred susceptibility to HPV infection. © 2013 Wiley Periodicals, Inc.

Structural Basis of Glyphosate Resistance Resulting from the Double Mutation Thr97 → Ile and Pro101 → Ser in 5-Enolpyruvylshikimate-3-phosphate Synthase from Escherichia coli*S⃞

PubMed Central

Funke, Todd; Yang, Yan; Han, Huijong; Healy-Fried, Martha; Olesen, Sanne; Becker, Andreas; Schönbrunn, Ernst

2009-01-01

The shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is the target of the broad spectrum herbicide glyphosate. The genetic engineering of EPSPS led to the introduction of glyphosate-resistant crops worldwide. The genetically engineered corn lines NK603 and GA21 carry distinct EPSPS enzymes. CP4 EPSPS, expressed in NK603 corn and transgenic soybean, cotton, and canola, belongs to class II EPSPS, glyphosate-insensitive variants of this enzyme isolated from certain Gram-positive bacteria. GA21 corn, on the other hand, was created by point mutations of class I EPSPS, such as the enzymes from Zea mays or Escherichia coli, which are sensitive to low glyphosate concentrations. The structural basis of the glyphosate resistance resulting from these point mutations has remained obscure. We studied the kinetic and structural effects of the T97I/P101S double mutation, the molecular basis for GA21 corn, using EPSPS from E. coli. The T97I/P101S enzyme is essentially insensitive to glyphosate (Ki = 2.4 mm) but maintains high affinity for the substrate phosphoenolpyruvate (PEP) (Km = 0.1 mm). The crystal structure at 1.7-Å resolution revealed that the dual mutation causes a shift of residue Gly96 toward the glyphosate binding site, impairing efficient binding of glyphosate, while the side chain of Ile97 points away from the substrate binding site, facilitating PEP utilization. The single site T97I mutation renders the enzyme sensitive to glyphosate and causes a substantial decrease in the affinity for PEP. Thus, only the concomitant mutations of Thr97 and Pro101 induce the conformational changes necessary to produce catalytically efficient, glyphosate-resistant class I EPSPS. PMID:19211556

Structural basis of glyphosate resistance resulting from the double mutation Thr97 -> Ile and Pro101 -> Ser in 5-enolpyruvylshikimate-3-phosphate synthase from Escherichia coli.

PubMed

Funke, Todd; Yang, Yan; Han, Huijong; Healy-Fried, Martha; Olesen, Sanne; Becker, Andreas; Schönbrunn, Ernst

2009-04-10

The shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is the target of the broad spectrum herbicide glyphosate. The genetic engineering of EPSPS led to the introduction of glyphosate-resistant crops worldwide. The genetically engineered corn lines NK603 and GA21 carry distinct EPSPS enzymes. CP4 EPSPS, expressed in NK603 corn and transgenic soybean, cotton, and canola, belongs to class II EPSPS, glyphosate-insensitive variants of this enzyme isolated from certain Gram-positive bacteria. GA21 corn, on the other hand, was created by point mutations of class I EPSPS, such as the enzymes from Zea mays or Escherichia coli, which are sensitive to low glyphosate concentrations. The structural basis of the glyphosate resistance resulting from these point mutations has remained obscure. We studied the kinetic and structural effects of the T97I/P101S double mutation, the molecular basis for GA21 corn, using EPSPS from E. coli. The T97I/P101S enzyme is essentially insensitive to glyphosate (K(i) = 2.4 mm) but maintains high affinity for the substrate phosphoenolpyruvate (PEP) (K(m) = 0.1 mm). The crystal structure at 1.7-A resolution revealed that the dual mutation causes a shift of residue Gly(96) toward the glyphosate binding site, impairing efficient binding of glyphosate, while the side chain of Ile(97) points away from the substrate binding site, facilitating PEP utilization. The single site T97I mutation renders the enzyme sensitive to glyphosate and causes a substantial decrease in the affinity for PEP. Thus, only the concomitant mutations of Thr(97) and Pro(101) induce the conformational changes necessary to produce catalytically efficient, glyphosate-resistant class I EPSPS.

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.

A tale of two subunits: how the neomorphic R132H IDH1 mutation enhances production of αHG.

PubMed

Pietrak, Beth; Zhao, Huizhen; Qi, Hongwei; Quinn, Chad; Gao, Enoch; Boyer, Joseph G; Concha, Nestor; Brown, Kristin; Duraiswami, Chaya; Wooster, Richard; Sweitzer, Sharon; Schwartz, Benjamin

2011-05-31

Heterozygously expressed single-point mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2, respectively) render these dimeric enzymes capable of producing the novel metabolite α-hydroxyglutarate (αHG). Accumulation of αHG is used as a biomarker for a number of cancer types, helping to identify tumors with similar IDH mutations. With IDH1, it has been shown that one role of the mutation is to increase the rate of conversion from αKG to αHG. To improve our understanding of the function of this mutation, we have detailed the kinetics of the normal (isocitrate to αKG) and neomorphic (αKG to αHG) reactions, as well as the coupled conversion of isocitrate to αHG. We find that the mutant IDH1 is very efficient in this coupled reaction, with the ability to form αHG from isocitrate and NADP(+). The wild type/wild type IDH1 is also able to catalyze this conversion, though it is much more sensitive to concentrations of isocitrate. This difference in behavior can be attributed to the competitive binding between isocitrate and αKG, which is made more favorable for αKG by the neomorphic mutation at arginine 132. Thus, each partial reaction in the heterodimer is functionally isolated from the other. To test whether there is a cooperative effect resulting from the two subunits being in a dimer, we selectively inactivated each subunit with a secondary mutation in the NADP/H binding site. We observed that the remaining, active subunit was unaffected in its associated activity, reinforcing the notion of each subunit being functionally independent. This was further demonstrated using a monomeric form of IDH from Azotobacter vinelandii, which can be shown to gain the same neomorphic reaction when a homologous mutation is introduced into that protein.

Dew inspired breathing-based detection of genetic point mutation visualized by naked eye

PubMed Central

Xie, Liping; Wang, Tongzhou; Huang, Tianqi; Hou, Wei; Huang, Guoliang; Du, Yanan

2014-01-01

A novel label-free method based on breathing-induced vapor condensation was developed for detection of genetic point mutation. The dew-inspired detection was realized by integration of target-induced DNA ligation with rolling circle amplification (RCA). The vapor condensation induced by breathing transduced the RCA-amplified variances in DNA contents into visible contrast. The image could be recorded by a cell phone for further or even remote analysis. This green assay offers a naked-eye-reading method potentially applied for point-of-care liver cancer diagnosis in resource-limited regions. PMID:25199907

Dew inspired breathing-based detection of genetic point mutation visualized by naked eye

NASA Astrophysics Data System (ADS)

Xie, Liping; Wang, Tongzhou; Huang, Tianqi; Hou, Wei; Huang, Guoliang; Du, Yanan

2014-09-01

A novel label-free method based on breathing-induced vapor condensation was developed for detection of genetic point mutation. The dew-inspired detection was realized by integration of target-induced DNA ligation with rolling circle amplification (RCA). The vapor condensation induced by breathing transduced the RCA-amplified variances in DNA contents into visible contrast. The image could be recorded by a cell phone for further or even remote analysis. This green assay offers a naked-eye-reading method potentially applied for point-of-care liver cancer diagnosis in resource-limited regions.

Dew inspired breathing-based detection of genetic point mutation visualized by naked eye.

PubMed

Xie, Liping; Wang, Tongzhou; Huang, Tianqi; Hou, Wei; Huang, Guoliang; Du, Yanan

2014-09-09

A novel label-free method based on breathing-induced vapor condensation was developed for detection of genetic point mutation. The dew-inspired detection was realized by integration of target-induced DNA ligation with rolling circle amplification (RCA). The vapor condensation induced by breathing transduced the RCA-amplified variances in DNA contents into visible contrast. The image could be recorded by a cell phone for further or even remote analysis. This green assay offers a naked-eye-reading method potentially applied for point-of-care liver cancer diagnosis in resource-limited regions.

Darwinism for the Genomic Age: Connecting Mutation to Diversification

PubMed Central

Hua, Xia; Bromham, Lindell

2017-01-01

A growing body of evidence suggests that rates of diversification of biological lineages are correlated with differences in genome-wide mutation rate. Given that most research into differential patterns of diversification rate have focused on species traits or ecological parameters, a connection to the biochemical processes of genome change is an unexpected observation. While the empirical evidence for a significant association between mutation rate and diversification rate is mounting, there has been less effort in explaining the factors that mediate this connection between genetic change and species richness. Here we draw together empirical studies and theoretical concepts that may help to build links in the explanatory chain that connects mutation to diversification. First we consider the way that mutation rates vary between species. We then explore how differences in mutation rates have flow-through effects to the rate at which populations acquire substitutions, which in turn influences the speed at which populations become reproductively isolated from each other due to the acquisition of genomic incompatibilities. Since diversification rate is commonly measured from phylogenetic analyses, we propose a conceptual approach for relating events of reproductive isolation to bifurcations on molecular phylogenies. As we examine each of these relationships, we consider theoretical models that might shine a light on the observed association between rate of molecular evolution and diversification rate, and critically evaluate the empirical evidence for these links, focusing on phylogenetic comparative studies. Finally, we ask whether we are getting closer to a real understanding of the way that the processes of molecular evolution connect to the observable patterns of diversification. PMID:28224003

Darwinism for the Genomic Age: Connecting Mutation to Diversification.

PubMed

Hua, Xia; Bromham, Lindell

2017-01-01

A growing body of evidence suggests that rates of diversification of biological lineages are correlated with differences in genome-wide mutation rate. Given that most research into differential patterns of diversification rate have focused on species traits or ecological parameters, a connection to the biochemical processes of genome change is an unexpected observation. While the empirical evidence for a significant association between mutation rate and diversification rate is mounting, there has been less effort in explaining the factors that mediate this connection between genetic change and species richness. Here we draw together empirical studies and theoretical concepts that may help to build links in the explanatory chain that connects mutation to diversification. First we consider the way that mutation rates vary between species. We then explore how differences in mutation rates have flow-through effects to the rate at which populations acquire substitutions, which in turn influences the speed at which populations become reproductively isolated from each other due to the acquisition of genomic incompatibilities. Since diversification rate is commonly measured from phylogenetic analyses, we propose a conceptual approach for relating events of reproductive isolation to bifurcations on molecular phylogenies. As we examine each of these relationships, we consider theoretical models that might shine a light on the observed association between rate of molecular evolution and diversification rate, and critically evaluate the empirical evidence for these links, focusing on phylogenetic comparative studies. Finally, we ask whether we are getting closer to a real understanding of the way that the processes of molecular evolution connect to the observable patterns of diversification.

The SHOX region and its mutations.

PubMed

Capone, L; Iughetti, L; Sabatini, S; Bacciaglia, A; Forabosco, A

2010-06-01

The short stature homeobox-containing (SHOX) gene lies in the pseudoautosomal region 1 (PAR1) that comprises 2.6 Mb of the short-arm tips of both the X and Y chromosomes. It is known that its heterozygous mutations cause Leri-Weill dyschondrosteosis (LWD) (OMIM #127300), while its homozygous mutations cause a severe form of dwarfism known as Langer mesomelic dysplasia (LMD) (OMIM #249700). The analysis of 238 LWD patients between 1998 and 2007 by multiple authors shows a prevalence of deletions (46.4%) compared to point mutations (21.2%). On the whole, deletions and point mutations account for about 67% of LWD patients. SHOX is located within a 1000 kb desert region without genes. The comparative genomic analysis of this region between genomes of different vertebrates has led to the identification of evolutionarily conserved non-coding DNA elements (CNE). Further functional studies have shown that one of these CNE downstream of the SHOX gene is necessary for the expression of SHOX; this is considered to be typical "enhancer" activity. Including the enhancer, the overall mutation of the SHOX region in LWD patients does not hold in 100% of cases. Various authors have demonstrated the existence of other CNE both downstream and upstream of SHOX regions. The resulting conclusion is that it is necessary to reanalyze all LWD/LMD patients without SHOX mutations for the presence of mutations in the 5'- and 3'-flanking SHOX regions.

Development of a PCR/LDR/capillary electrophoresis assay with potential for the detection of a beta-thalassemia fetal mutation in maternal plasma.

PubMed

Yi, Ping; Chen, Zhuqin; Yu, Lili; Zheng, Yingru; Liu, Guodong; Xie, Haichang; Zhou, Yuanguo; Zheng, Xiuhui; Han, Jian; Li, Li

2010-08-01

Analysis of fetal DNA in maternal plasma has recently been introduced for non-invasive prenatal diagnosis. We have now investigated the feasibility of polymerase chain reaction (PCR)/ligase detection reaction (LDR)/capillary electrophoresis for the detection of fetal point mutations, such as the beta-thalassemia mutation, IVS2 654(C --> T), in maternal plasma DNA. The sensitivity of LDR/capillary electrophoresis was examined by quantifying the mutant PCR products in the presence of a vast excess of non-mutant competitor template, a situation that mimics the detection of rare fetal mutations in the presence of excess maternal DNA. PCR/LDR/capillary electrophoresis was applied to detect the mutation, IVS2 654(C --> T), in an experimental model at different sensitivity levels and from 10 maternal plasma samples. Our results demonstrated that this approach to detect a low abundance IVS2 654(C --> T) mutation achieved a sensitivity of approximately 1:10,000. The approach was applied to maternal plasma DNA to detect the paternally inherited fetal IVS2 654(C --> T) mutation, and the results were equivalent to those obtained by PCR/reverse dot blot of amniotic fluid cell DNA. PCR/LDR/capillary electrophoresis has a very high sensitivity that can distinguish low abundance single nucleotide differences and can detect paternally inherited fetal point mutations in maternal plasma.

[A preliminary study on p53 gene in lung cancer tissues of workers exposed to silica and welding fumes].

PubMed

Liu, B; Zhou, P; Miao, Q

1997-05-01

Mutations of suppressor gene p53 was studied in 36 cases of silica related lung cancer and 6 cases of welding fume related lung cancer with immunohistochemical and PCR-SSCP methods. Cancer tissues were embedded in paraffin and stored for 13.4 years in average. Results revealed that there was abnormal mobility shift of electrophoresis in 18 cases with 20 point mutations of 42 specimens tested, accounted for 42.9%, and 50% (10/20) of the mutations were clustered in exon 8. This finding differed from mutational spectrum of gene in non-occupational lung cancer, in which mutation frequency of exon 8 ranged from 17.5% to 23.5%. Gene mutation frequency in varied pathological categories of pneumoconiosis related lung cancer also differed from that in common lung cancer. In the latter, the highest one was in small cell lung cancer (70%) and the lowest in adenocarcinoma (33%), but in the former, the highest in adenocarcinoma (53.9%) and the lowest in small cell lung cancer (30.8%). Immunohistochemical observations also showed a very high prevalence of p53 gene mutation expression (46.9%). Sequencing, which was determined in two cases of this study, revealed that two point mutations all occurred in non-hotspot codon 144 of p53 gene. Difference in gene mutation spectrum suggests that there exist specific carcinogens and carcinogenesis in silica and welding fume related lung cancer.

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

Koivisto, U.M.; Viikari, J.S.; Kontula, K.

Two deletions of the low-density lipoprotein (LDL) receptor gene were previously shown to account for about two thirds of all mutations causing familial hypercholesterolemia (FH) in Finland. We screened the DNA samples from a cohort representing the remaining 30% of Finnish heterozygous FH patients by amplifying all the 18 exons of the receptor gene by PCR and searching for DNA variations with the SSCP technique. Ten novel mutations were identified, comprising two nonsense and seven missense mutations as well as one frameshift mutation caused by a 13-bp deletion. A single nucleotide change, substituting adenine for guanidine at position 2533 andmore » resulting in an amino acid change of glycine to aspartic acid at codon 823, was found in DNA samples from 14 unrelated FH probands. This mutation (FH-Turku) affects the sequence encoding the putative basolateral sorting signal of the LDL receptor protein; however, the exact functional consequences of this mutation are yet to be examined. The FH-Turku gene and another point mutation (Leu380{r_arrow}His or FH-Pori) together account for {approximately}8% of the FH-causing genes in Finland and are particularly common among FH patients from the southwestern part of the country (combined, 30%). Primer-introduced restriction analysis was applied for convenient assay of the FH-Turku and FH-Pori point mutations. In conclusion, this paper demonstrates the unique genetic background of FH in Finland and describes a commonly occurring FH gene with a missense mutation closest to the C terminus thus far reported. 32 refs., 5 figs., 2 tabs.« less

Next-generation sequencing reveals the mutational landscape of clinically diagnosed Usher syndrome: copy number variations, phenocopies, a predominant target for translational read-through, and PEX26 mutated in Heimler syndrome.

PubMed

Neuhaus, Christine; Eisenberger, Tobias; Decker, Christian; Nagl, Sandra; Blank, Cornelia; Pfister, Markus; Kennerknecht, Ingo; Müller-Hofstede, Cornelie; Charbel Issa, Peter; Heller, Raoul; Beck, Bodo; Rüther, Klaus; Mitter, Diana; Rohrschneider, Klaus; Steinhauer, Ute; Korbmacher, Heike M; Huhle, Dagmar; Elsayed, Solaf M; Taha, Hesham M; Baig, Shahid M; Stöhr, Heidi; Preising, Markus; Markus, Susanne; Moeller, Fabian; Lorenz, Birgit; Nagel-Wolfrum, Kerstin; Khan, Arif O; Bolz, Hanno J

2017-09-01

Combined retinal degeneration and sensorineural hearing impairment is mostly due to autosomal recessive Usher syndrome (USH1: congenital deafness, early retinitis pigmentosa (RP); USH2: progressive hearing impairment, RP). Sanger sequencing and NGS of 112 genes (Usher syndrome, nonsyndromic deafness, overlapping conditions), MLPA, and array-CGH were conducted in 138 patients clinically diagnosed with Usher syndrome. A molecular diagnosis was achieved in 97% of both USH1 and USH2 patients, with biallelic mutations in 97% (USH1) and 90% (USH2), respectively. Quantitative readout reliably detected CNVs (confirmed by MLPA or array-CGH), qualifying targeted NGS as one tool for detecting point mutations and CNVs. CNVs accounted for 10% of identified USH2A alleles, often in trans to seemingly monoallelic point mutations. We demonstrate PTC124-induced read-through of the common p.Trp3955* nonsense mutation (13% of detected USH2A alleles), a potential therapy target. Usher gene mutations were found in most patients with atypical Usher syndrome, but the diagnosis was adjusted in case of double homozygosity for mutations in OTOA and NR2E3 , genes implicated in isolated deafness and RP. Two patients with additional enamel dysplasia had biallelic PEX26 mutations, for the first time linking this gene to Heimler syndrome. Targeted NGS not restricted to Usher genes proved beneficial in uncovering conditions mimicking Usher syndrome.

Exploring environmental causes of altered ras effects: fragmentation plus integration?

PubMed

Porta, Miquel; Ayude, Daniel; Alguacil, Juan; Jariod, Manuel

2003-02-01

Mutations in ras genes are the most common abnormality of oncogenes in human cancer and a major example of activation by point mutation. Experimental and epidemiological studies support the notion that Ki-ras activation and expression may be chemically related. We discuss the potential role of several environmental compounds in the induction or promotion of ras mutations in humans, with a focus on exocrine pancreatic cancer, the human tumor with the highest prevalence at diagnosis of Ki-ras mutations. Organochlorine compounds, organic solvents, and coffee compounds may play an indirect role in causing Ki-ras mutations, rather than as direct inducers of the mutations. Although for some organochlorine compounds the induction of point mutations in ras oncogenes cannot be excluded, it seems more likely that the effects of these compounds are mediated through nongenomic or indirectly genotoxic mechanisms of action. Organic solvents also may act via enzymatic induction of ras mutagens or by providing a proliferation advantage to ras-mutated cell clones. In exocrine pancreatic cancer, caffeine, other coffee compounds, or other factors with which coffee drinking is associated could modulate Ki-ras activation by interfering with DNA repair, cell-cycle checkpoints, and apoptosis. Asbestos, cigarette smoking, and some dietary factors also may be involved in the initiation or the promotion of Ki-ras mutations in lung and colon cancers. Further development of the mechanistic scenarios proposed here could contribute to a meaningful integration of biological, clinical, and environmental knowledge on the causes of altered ras effects. Copyright 2003 Wiley-Liss, Inc.

The proto-oncogene KRAS and BRAF profiles and some clinical characteristics in colorectal cancer in the Turkish population.

PubMed

Ozen, Filiz; Ozdemir, Semra; Zemheri, Ebru; Hacimuto, Gizem; Silan, Fatma; Ozdemir, Ozturk

2013-02-01

The aim of the current study was to investigate the prevalence and predictive significance of the KRAS and BRAF mutations in Turkish patients with colorectal cancer (CRC). Totally, 53 fresh tumoral tissue specimens were investigated in patients with CRC. All specimens were obtained during routine surgery of patients who were histopathologically diagnosed and genotyped for common KRAS and BRAF point mutations. After DNA extraction, the target mutations were analyzed using the AutoGenomics INFINITI(®) assay, and some samples were confirmed by quantitative real-time polymerase chain reaction fluorescence melting curve analyses. KRAS mutations were found in 26 (49.05%) CRC samples. Twenty-seven samples (50.95%) had wild-type profiles for KRAS codon 12, 13, and 61 in the current cohort. In 17 (65.38%) samples, codon 12; in 7 (26.93%) samples, codon 13; and in 2 (7.69%) samples, codon 61 were found to be mutated, particularly in grade 2 of tumoral tissues. No point mutation was detected in BRAF codon Val600Glu for the studied CRC patients. Our study, based on a representative collection of human CRC tumors, indicates that KRAS gene mutations were detected in 49.05% of the samples, and the most frequent mutation was in the G12D codon. Results also showed that codons 12 and 13 of KRAS are relatively frequently without BRAF mutation in a CRC cohort from the Turkish population.

Relationship of body mass index with BRAF (V600E) mutation in papillary thyroid cancer.

PubMed

Shi, Rong-Liang; Qu, Ning; Liao, Tian; Wei, Wen-Jun; Lu, Zhong-Wu; Ma, Ben; Wang, Yu-Long; Ji, Qing-Hai

2016-06-01

Current evidences suggest an influence of overweight body mass index (BMI) on the carcinogenesis in malignancies. However, the role of BMI is unclear in papillary thyroid cancer (PTC). The aim of the present study is to investigate the relationship between BMI and BRAF (V600E) mutation status in PTC. BRAF (V600E) mutation in 108 patients with PTC was analyzed by Sanger sequencing. The cutoff point of BMI was identified by X-tile for predicting mutation by overweight. Odds ratios (OR) and 95 % confidence interval (CI) of BRAF (V600E) mutation according to BMI and clinicopathologic variables were calculated using logistic regression models. Fifty-one patients were positive for BRAF (V600E) mutation. A positive relationship existed between BRAF (V600E) mutation and BMI (p = 0.039). A 24.3 kg/m(2) was identified as cutoff point for differentiating greater than 52.0 % observed probability of mutation for BRAF (V600E) in entire cohort, which was similar to the midpoint between the upper limit of normal BMI and overweight defined by WHO (≥24 kg/m(2)). Multivariate analysis confirmed the association between BRAF (V600E) mutation with overweight BMI range (OR 7.645, 95 % CI 1.275-45.831, p = 0.026). This study suggests an influence of overweight BMI on the status of BRAF (V600E) in patients with PTC, whereas the underlying mechanism need to be further investigated.

Computational Analysis of Epidermal Growth Factor Receptor Mutations Predicts Differential Drug Sensitivity Profiles toward Kinase Inhibitors.

PubMed

Akula, Sravani; Kamasani, Swapna; Sivan, Sree Kanth; Manga, Vijjulatha; Vudem, Dashavantha Reddy; Kancha, Rama Krishna

2018-05-01

A significant proportion of patients with lung cancer carry mutations in the EGFR kinase domain. The presence of a deletion mutation in exon 19 or L858R point mutation in the EGFR kinase domain has been shown to cause enhanced efficacy of inhibitor treatment in patients with NSCLC. Several less frequent (uncommon) mutations in the EGFR kinase domain with potential implications in treatment response have also been reported. The role of a limited number of uncommon mutations in drug sensitivity was experimentally verified. However, a huge number of these mutations remain uncharacterized for inhibitor sensitivity or resistance. A large-scale computational analysis of clinically reported 298 point mutants of EGFR kinase domain has been performed, and drug sensitivity profiles for each mutant toward seven kinase inhibitors has been determined by molecular docking. In addition, the relative inhibitor binding affinity toward each drug as compared with that of adenosine triphosphate was calculated for each mutant. The inhibitor sensitivity profiles predicted in this study for a set of previously characterized mutants correlated well with the published clinical, experimental, and computational data. Both the single and compound mutations displayed differential inhibitor sensitivity toward first- and next-generation kinase inhibitors. The present study provides predicted drug sensitivity profiles for a large panel of uncommon EGFR mutations toward multiple inhibitors, which may help clinicians in deciding mutant-specific treatment strategies. Copyright © 2018 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

HMG CoA lyase deficiency: identification of five causal point mutations in codons 41 and 42, including a frequent Saudi Arabian mutation, R41Q.

PubMed Central

Mitchell, G A; Ozand, P T; Robert, M F; Ashmarina, L; Roberts, J; Gibson, K M; Wanders, R J; Wang, S; Chevalier, I; Plöchl, E; Miziorko, H

1998-01-01

The hereditary deficiency of 3-hydroxy-3-methylglutaryl (HMG) CoA lyase (HL; OMIM 246450 [http://www3.ncbi.nlm.nih. gov:80/htbin-post/Omim/dispmim?246450]) results in episodes of hypoketotic hypoglycemia and coma and is reported to be frequent and clinically severe in Saudi Arabia. We found genetic diversity among nine Saudi HL-deficient probands: six were homozygous for the missense mutation R41Q, and two were homozygous for the frameshift mutation F305fs(-2). In 32 non-Saudi HL-deficient probands, we found three R41Q alleles and also discovered four other deleterious point mutations in codons 41 and 42: R41X, D42E, D42G, and D42H. In purified mutant recombinant HL, all four missense mutations in codons 41 and 42 cause a marked decrease in HL activity. We developed a screening procedure for HL missense mutations that yields residual activity at levels comparable to those obtained using purified HL peptides. Codons 41 and 42 are important for normal HL catalysis and account for a disproportionate 21 (26%) of 82 of mutant alleles in our group of HL-deficient probands. PMID:9463337

Mutation Rates across Budding Yeast Chromosome VI Are Correlated with Replication Timing

PubMed Central

Lang, Gregory I.; Murray, Andrew W.

2011-01-01

Previous experimental studies suggest that the mutation rate is nonuniform across the yeast genome. To characterize this variation across the genome more precisely, we measured the mutation rate of the URA3 gene integrated at 43 different locations tiled across Chromosome VI. We show that mutation rate varies 6-fold across a single chromosome, that this variation is correlated with replication timing, and we propose a model to explain this variation that relies on the temporal separation of two processes for replicating past damaged DNA: error-free DNA damage tolerance and translesion synthesis. This model is supported by the observation that eliminating translesion synthesis decreases this variation. PMID:21666225

A KCNH2 branch point mutation causing aberrant splicing contributes to an explanation of genotype-negative long QT syndrome.

PubMed

Crotti, Lia; Lewandowska, Marzena A; Schwartz, Peter J; Insolia, Roberto; Pedrazzini, Matteo; Bussani, Erica; Dagradi, Federica; George, Alfred L; Pagani, Franco

2009-02-01

Genetic screening of long QT syndrome (LQTS) fails to identify disease-causing mutations in about 30% of patients. So far, molecular screening has focused mainly on coding sequence mutations or on substitutions at canonical splice sites. The purpose of this study was to explore the possibility that intronic variants not at canonical splice sites might affect splicing regulatory elements, lead to aberrant transcripts, and cause LQTS. Molecular screening was performed through DHPLC and sequence analysis. The role of the intronic mutation identified was assessed with a hybrid minigene splicing assay. A three-generation LQTS family was investigated. Molecular screening failed to identify an obvious disease-causing mutation in the coding sequences of the major LQTS genes but revealed an intronic A-to-G substitution in KCNH2 (IVS9-28A/G) cosegregating with the clinical phenotype in family members. In vitro analysis proved that the mutation disrupts the acceptor splice site definition by affecting the branch point (BP) sequence and promoting intron retention. We further demonstrated a tight functional relationship between the BP and the polypyrimidine tract, whose weakness is responsible for the pathological effect of the IVS9-28A/G mutation. We identified a novel BP mutation in KCNH2 that disrupts the intron 9 acceptor splice site definition and causes LQT2. The present finding demonstrates that intronic mutations affecting pre-mRNA processing may contribute to the failure of traditional molecular screening in identifying disease-causing mutations in LQTS subjects and offers a rationale strategy for the reduction of genotype-negative cases.

Discovery and prioritization of somatic mutations in diffuse large B-cell lymphoma (DLBCL) by whole-exome sequencing

PubMed Central

Lohr, Jens G.; Stojanov, Petar; Lawrence, Michael S.; Auclair, Daniel; Chapuy, Bjoern; Sougnez, Carrie; Cruz-Gordillo, Peter; Knoechel, Birgit; Asmann, Yan W.; Slager, Susan L.; Novak, Anne J.; Dogan, Ahmet; Ansell, Stephen M.; Zou, Lihua; Gould, Joshua; Saksena, Gordon; Stransky, Nicolas; Rangel-Escareño, Claudia; Fernandez-Lopez, Juan Carlos; Hidalgo-Miranda, Alfredo; Melendez-Zajgla, Jorge; Hernández-Lemus, Enrique; Schwarz-Cruz y Celis, Angela; Imaz-Rosshandler, Ivan; Ojesina, Akinyemi I.; Jung, Joonil; Pedamallu, Chandra S.; Lander, Eric S.; Habermann, Thomas M.; Cerhan, James R.; Shipp, Margaret A.; Getz, Gad; Golub, Todd R.

2012-01-01

To gain insight into the genomic basis of diffuse large B-cell lymphoma (DLBCL), we performed massively parallel whole-exome sequencing of 55 primary tumor samples from patients with DLBCL and matched normal tissue. We identified recurrent mutations in genes that are well known to be functionally relevant in DLBCL, including MYD88, CARD11, EZH2, and CREBBP. We also identified somatic mutations in genes for which a functional role in DLBCL has not been previously suspected. These genes include MEF2B, MLL2, BTG1, GNA13, ACTB, P2RY8, PCLO, and TNFRSF14. Further, we show that BCL2 mutations commonly occur in patients with BCL2/IgH rearrangements as a result of somatic hypermutation normally occurring at the IgH locus. The BCL2 point mutations are primarily synonymous, and likely caused by activation-induced cytidine deaminase–mediated somatic hypermutation, as shown by comprehensive analysis of enrichment of mutations in WRCY target motifs. Those nonsynonymous mutations that are observed tend to be found outside of the functionally important BH domains of the protein, suggesting that strong negative selection against BCL2 loss-of-function mutations is at play. Last, by using an algorithm designed to identify likely functionally relevant but infrequent mutations, we identify KRAS, BRAF, and NOTCH1 as likely drivers of DLBCL pathogenesis in some patients. Our data provide an unbiased view of the landscape of mutations in DLBCL, and this in turn may point toward new therapeutic strategies for the disease. PMID:22343534

FireProt: Energy- and Evolution-Based Computational Design of Thermostable Multiple-Point Mutants.

PubMed

Bednar, David; Beerens, Koen; Sebestova, Eva; Bendl, Jaroslav; Khare, Sagar; Chaloupkova, Radka; Prokop, Zbynek; Brezovsky, Jan; Baker, David; Damborsky, Jiri

2015-11-01

There is great interest in increasing proteins' stability to enhance their utility as biocatalysts, therapeutics, diagnostics and nanomaterials. Directed evolution is a powerful, but experimentally strenuous approach. Computational methods offer attractive alternatives. However, due to the limited reliability of predictions and potentially antagonistic effects of substitutions, only single-point mutations are usually predicted in silico, experimentally verified and then recombined in multiple-point mutants. Thus, substantial screening is still required. Here we present FireProt, a robust computational strategy for predicting highly stable multiple-point mutants that combines energy- and evolution-based approaches with smart filtering to identify additive stabilizing mutations. FireProt's reliability and applicability was demonstrated by validating its predictions against 656 mutations from the ProTherm database. We demonstrate that thermostability of the model enzymes haloalkane dehalogenase DhaA and γ-hexachlorocyclohexane dehydrochlorinase LinA can be substantially increased (ΔTm = 24°C and 21°C) by constructing and characterizing only a handful of multiple-point mutants. FireProt can be applied to any protein for which a tertiary structure and homologous sequences are available, and will facilitate the rapid development of robust proteins for biomedical and biotechnological applications.

Ras mutations are rare in solitary cold and toxic thyroid nodules.

PubMed

Krohn, K; Reske, A; Ackermann, F; Müller, A; Paschke, R

2001-08-01

Activation of ras proto-oncogenes as a result of point mutations is detectable in a significant percentage of most types of tumour. Similar to neoplasms of other organs, mutations of all three ras genes can be found in thyroid tumours. H-, K- and N-ras mutations have been detected in up to 20% of follicular adenomas and adenomatous nodules which were not functionally characterized. This raises the question as to whether ras mutations are specific for hypofunctional nodules and TSH receptor mutations for hyperfunctioning nodules. To investigate ras and TSH receptor mutations with respect to functional differentiation we studied 41 scintigraphically cold nodules and 47 toxic thyroid nodules. To address the likelihood of a somatic mutation we also studied the clonal origin of these tumours. Genomic DNA was extracted from nodular and surrounding tissue. Mutational hot spots in exons 1 and 2 of the H- and K-ras gene were PCR amplified and sequenced using big dye terminator chemistry. Denaturing gradient gel electrophoresis (DGGE) was used to verify sequencing results for the H-ras gene and to analyse the N-ras gene because its greater sensitivity in detecting somatic mutations. Clonality of nodular thyroid tissue was evaluated using X-Chromosome inactivation based on PCR amplification of the human androgen receptor locus. Monoclonal origin was detectable in 14 of 23 informative samples from cold thyroid nodules. In toxic thyroid nodules the frequency of clonal tissue was 20 in 30 informative cases. Only one point mutation could be found in the N-ras gene codon 61 (Gly to Arg) in a cold adenomatous nodule which was monoclonal. In toxic thyroid nodules no ras mutation was detectable. Our study suggests that ras mutations are rare in solitary cold and toxic thyroid nodules and that the frequent monoclonal origin of these tumours implies somatic mutations in genes other than H-, K- and N-ras.

Novel pathogenic mutations and skin biopsy analysis in Knobloch syndrome

PubMed Central

Suzuki, Oscar; Kague, Erika; Bagatini, Kelly; Tu, Hongmin; Heljasvaara, Ritva; Carvalhaes, Lorenza; Gava, Elisandra; de Oliveira, Gisele; Godoi, Paulo; Oliva, Glaucius; Kitten, Gregory; Pihlajaniemi, Taina; Passos-Bueno, Maria-Rita

2009-01-01

Purpose To facilitate future diagnosis of Knobloch syndrome (KS) and better understand its etiology, we sought to identify not yet described COL18A1 mutations in KS patients. In addition, we tested whether mutations in this gene lead to absence of the COL18A1 gene product and attempted to better characterize the functional effect of a previously reported missense mutation. Methods Direct sequencing of COL18A1 exons was performed in KS patients from four unrelated pedigrees. We used immunofluorescent histochemistry in skin biopsies to evaluate the presence of type XVIII collagen in four KS patients carrying two already described mutations: c.3277C>T, a nonsense mutation, and c.3601G>A, a missense mutation. Furthermore, we determined the binding properties of the mutated endostatin domain p.A1381T (c.3601G>A) to extracellular matrix proteins using ELISA and surface plasmon resonance assays. Results We identified four novel mutations in COL18A1, including a large deletion involving exon 41. Skin biopsies from KS patients revealed lack of type XVIII collagen in epithelial basement membranes and blood vessels. We also found a reduced affinity of p.A1381T endostatin to some extracellular matrix components. Conclusions COL18A1 mutations involved in Knobloch syndrome have a distribution bias toward the coding exons of the C-terminal end. Large deletions must also be considered when point mutations are not identified in patients with characteristic KS phenotype. We report, for the first time, lack of type XVIII collagen in KS patients by immunofluorescent histochemistry in skin biopsy samples. As a final point, we suggest the employment of this technique as a preliminary and complementary test for diagnosis of KS in cases when mutation screening either does not detect mutations or reveals mutations of uncertain effect, such as the p.A1381T change. PMID:19390655

Novel pathogenic mutations and skin biopsy analysis in Knobloch syndrome.

PubMed

Suzuki, Oscar; Kague, Erika; Bagatini, Kelly; Tu, Hongmin; Heljasvaara, Ritva; Carvalhaes, Lorenza; Gava, Elisandra; de Oliveira, Gisele; Godoi, Paulo; Oliva, Glaucius; Kitten, Gregory; Pihlajaniemi, Taina; Passos-Bueno, Maria-Rita

2009-01-01

To facilitate future diagnosis of Knobloch syndrome (KS) and better understand its etiology, we sought to identify not yet described COL18A1 mutations in KS patients. In addition, we tested whether mutations in this gene lead to absence of the COL18A1 gene product and attempted to better characterize the functional effect of a previously reported missense mutation. Direct sequencing of COL18A1 exons was performed in KS patients from four unrelated pedigrees. We used immunofluorescent histochemistry in skin biopsies to evaluate the presence of type XVIII collagen in four KS patients carrying two already described mutations: c.3277C>T, a nonsense mutation, and c.3601G>A, a missense mutation. Furthermore, we determined the binding properties of the mutated endostatin domain p.A1381T (c.3601G>A) to extracellular matrix proteins using ELISA and surface plasmon resonance assays. We identified four novel mutations in COL18A1, including a large deletion involving exon 41. Skin biopsies from KS patients revealed lack of type XVIII collagen in epithelial basement membranes and blood vessels. We also found a reduced affinity of p.A1381T endostatin to some extracellular matrix components. COL18A1 mutations involved in Knobloch syndrome have a distribution bias toward the coding exons of the C-terminal end. Large deletions must also be considered when point mutations are not identified in patients with characteristic KS phenotype. We report, for the first time, lack of type XVIII collagen in KS patients by immunofluorescent histochemistry in skin biopsy samples. As a final point, we suggest the employment of this technique as a preliminary and complementary test for diagnosis of KS in cases when mutation screening either does not detect mutations or reveals mutations of uncertain effect, such as the p.A1381T change.

Knockdown resistance (kdr)-like mutations in the voltage-gated sodium channel of a malaria vector Anopheles stephensi and PCR assays for their detection.

PubMed

Singh, Om P; Dykes, Cherry L; Lather, Manila; Agrawal, Om P; Adak, Tridibes

2011-03-14

Knockdown resistance (kdr) in insects, resulting from mutation(s) in the voltage-gated sodium channel (vgsc) gene is one of the mechanisms of resistance against DDT and pyrethroid-group of insecticides. The most common mutation(s) associated with knockdown resistance in insects, including anophelines, has been reported to be present at residue Leu1014 in the IIS6 transmembrane segment of the vgsc gene. This study reports the presence of two alternative kdr-like mutations, L1014S and L1014F, at this residue in a major malaria vector Anopheles stephensi and describes new PCR assays for their detection. Part of the vgsc (IIS4-S5 linker-to-IIS6 transmembrane segment) of An. stephensi collected from Alwar (Rajasthan, India) was PCR-amplified from genomic DNA, sequenced and analysed for the presence of deduced amino acid substitution(s). Analysis of DNA sequences revealed the presence of two alternative non-synonymous point mutations at L1014 residue in the IIS6 transmembrane segment of vgsc, i.e., T>C mutation on the second position and A>T mutation on the third position of the codon, leading to Leu (TTA)-to-Ser (TCA) and -Phe (TTT) amino acid substitutions, respectively. Polymerase chain reaction (PCR) assays were developed for identification of each of these two point mutations. Genotyping of An. stephensi mosquitoes from Alwar by PCR assays revealed the presence of both mutations, with a high frequency of L1014S. The PCR assays developed for detection of the kdr mutations were specific as confirmed by DNA sequencing of PCR-genotyped samples. Two alternative kdr-like mutations, L1014S and L1014F, were detected in An. stephensi with a high allelic frequency of L1014S. The occurrence of L1014S is being reported for the first time in An. stephensi. Two specific PCR assays were developed for detection of two kdr-like mutations in An. stephensi.

High mutation detection rate in TCOF1 among Treacher Collins syndrome patients reveals clustering of mutations and 16 novel pathogenic changes.

PubMed

Splendore, A; Silva, E O; Alonso, L G; Richieri-Costa, A; Alonso, N; Rosa, A; Carakushanky, G; Cavalcanti, D P; Brunoni, D; Passos-Bueno, M R

2000-10-01

Twenty-eight families with a clinical diagnosis of Treacher Collins syndrome were screened for mutations in the 25 coding exons of TCOF1 and their adjacent splice junctions through SSCP and direct sequencing. Pathogenic mutations were detected in 26 patients, yielding the highest detection rate reported so far for this disease (93%) and bringing the number of known disease-causing mutations from 35 to 51. This is the first report to describe clustering of pathogenic mutations. Thirteen novel polymorphic alterations were characterized, confirming previous reports that TCOF1 has an unusually high rate of single-nucleotide polymorphisms (SNPs) within its coding region. We suggest a possible different mechanism leading to TCS or genetic heterogeneity for this condition, as we identified two families with no apparent pathogenic mutation in the gene. Furthermore, our data confirm the absence of genotype-phenotype correlation and reinforce that the apparent anticipation often observed in TCS families is due to ascertainment bias. Copyright 2000 Wiley-Liss, Inc.

Uncommon EGFR mutations in cytological specimens of 1,874 newly diagnosed Indonesian lung cancer patients

PubMed Central

Syahruddin, Elisna; Wulandari, Laksmi; Sri Muktiati, Nunuk; Rima, Ana; Soeroso, Noni; Ermayanti, Sabrina; Levi, Michael; Hidajat, Heriawaty; Widjajahakim, Grace; Utomo, Ahmad Rusdan Handoyo

2018-01-01

Purpose We aimed to evaluate the distribution of individual epidermal growth factor receptor (EGFR) mutation subtypes found in routine cytological specimens. Patients and methods A retrospective audit was performed on EGFR testing results of 1,874 consecutive cytological samples of newly diagnosed or treatment-naïve Indonesian lung cancer patients (years 2015–2016). Testing was performed by ISO15189 accredited central laboratory. Results Overall test failure rate was 5.1%, with the highest failure (7.1%) observed in pleural effusion and lowest (1.6%) in needle aspiration samples. EGFR mutation frequency was 44.4%. Tyrosine kinase inhibitor (TKI)-sensitive common EGFR mutations (ins/dels exon 19, L858R) and uncommon mutations (G719X, T790M, L861Q) contributed 57.1% and 29%, respectively. Approximately 13.9% of mutation-positive patients carried a mixture of common and uncommon mutations. Women had higher EGFR mutation rate (52.9%) vs men (39.1%; p<0.05). In contrast, uncommon mutations conferring either TKI responsive (G719X, L861Q) or TKI resistance (T790M, exon 20 insertions) were consistently more frequent in men than in women (67.3% vs 32.7% or 69.4% vs 30.6%; p<0.05). Up to 10% EGFR mutation–positive patients had baseline single mutation T790M, exon 20 insertion, or in coexistence with TKI-sensitive mutations. Up to 9% patients had complex or multiple EGFR mutations, whereby 48.7% patients harbored TKI-resistant mutations. One patient presented third-generation TKI-resistant mutation L792F simultaneously with T790M. Conclusion Routine diagnostic cytological techniques yielded similar success rate to detect EGFR mutations. Uncommon EGFR mutations were frequent events in Indonesian lung cancer patients. PMID:29615847

[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

Genetic analysis of children of atomic bomb survivors.

PubMed Central

Satoh, C; Takahashi, N; Asakawa, J; Kodaira, M; Kuick, R; Hanash, S M; Neel, J V

1996-01-01

Studies are under way for the detection of potential genetic effects of atomic bomb radiation at the DNA level in the children of survivors. In a pilot study, we have examined six minisatellites and five microsatellites in DNA derived from 100 families including 124 children. We detected a total of 28 mutations in three minisatellite loci. The mean mutation rates per locus per gamete in the six minisatellite loci were 1.5% for 65 exposed gametes for which mean parental gonadal dose was 1.9 Sv and 2.0% for 183 unexposed gametes. We detected four mutations in two tetranucleotide repeat sequences but no mutations in three trinucleotide repeat sequences. The mean mutation rate per locus per gamete was o% for the exposed gametes and 0.5% for the unexposed gametes in the five microsatellite loci. No significant differences in the mutation rates between the exposed and the unexposed gametes were detected in these repetitive sequences. Additional loci are being analyzed to increase the power of our study to observe a significant difference in the mutation rates at the 0.05 level of significance. Images Figure 1. Figure 2. Figure 2. Figure 2. Figure 2. Figure 2. Figure 2. PMID:8781374

PCR-RFLP to Detect Codon 248 Mutation in Exon 7 of "p53" Tumor Suppressor Gene

ERIC Educational Resources Information Center

Ouyang, Liming; Ge, Chongtao; Wu, Haizhen; Li, Suxia; Zhang, Huizhan

2009-01-01

Individual genome DNA was extracted fast from oral swab and followed up with PCR specific for codon 248 of "p53" tumor suppressor gene. "Msp"I restriction mapping showed the G-C mutation in codon 248, which closely relates to cancer susceptibility. Students learn the concepts, detection techniques, and research significance of point mutations or…

Mutational and structural analysis of diffuse large B-cell lymphoma using whole genome sequencing | Office of Cancer Genomics

Cancer.gov

Abstract: Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous cancer comprising at least two molecular subtypes that differ in gene expression and distribution of mutations. Recently, application of genome/exome sequencing and RNA-seq to DLBCL has revealed numerous genes that are recurrent targets of somatic point mutation in this disease.

Point mutation in the MITF gene causing Waardenburg syndrome type II in a three-generation Indian family.

PubMed

Lalwani, A K; Attaie, A; Randolph, F T; Deshmukh, D; Wang, C; Mhatre, A; Wilcox, E

1998-12-04

Waardenburg syndrome (WS) is an autosomal-dominant neural crest cell disorder phenotypically characterized by hearing impairment and disturbance of pigmentation. A presence of dystopia canthorum is indicative of WS type 1, caused by loss of function mutation in the PAX3 gene. In contrast, type 2 WS (WS2) is characterized by normally placed medial canthi and is genetically heterogeneous; mutations in MITF (microphthalmia associated transcription factor) associated with WS2 have been identified in some but not all affected families. Here, we report on a three-generation Indian family with a point mutation in the MITF gene causing WS2. This mutation, initially reported in a Northern European family, creates a stop codon in exon 7 and is predicted to result in a truncated protein lacking the HLH-Zip or Zip structure necessary for normal interaction with its target DNA motif. Comparison of the phenotype between the two families demonstrates a significant difference in pigmentary disturbance of the eye. This family, with the first documented case of two unrelated WS2 families harboring identical mutations, provides additional evidence for the importance of genetic background on the clinical phenotype.

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

PubMed

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

2006-08-28

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

Antimalarial drug susceptibility and point mutations associated with drug resistance in 248 Plasmodium falciparum isolates imported from Comoros to Marseille, France in 2004 2006.

PubMed

Parola, Philippe; Pradines, Bruno; Simon, Fabrice; Carlotti, Marie-Paule; Minodier, Philippe; Ranjeva, Marie-Pierre; Badiaga, Sékéné; Bertaux, Lionel; Delmont, Jean; Morillon, Marc; Silai, Ramatou; Brouqui, Philippe; Parzy, Daniel

2007-09-01

A total of 248 Plasmodium falciparum isolates were sampled in travelers with malaria who came to Marseille, France from Comoros to investigate in vitro activities of antimalarial drugs and molecular markers of drug resistance. Of the 248 isolates, 126 were maintained in culture. Of these, 53% were resistant to chloroquine, and 3% had reduced susceptibility to quinine, mefloquine, and atovaquone; 1% had reduced susceptibility to halofantrine and dihydroartemisinin; 7% had reduced susceptibility to monodesethylamodiaquine; 37% had reduced susceptibility to cycloguanil; and none had reduced susceptibility to lumefantrine. Resistance-associated point mutations were screened in 207 isolates. No mutations in the cytochrome b gene were found. Of the 207 isolates, 119 (58%) had a mutation in the P. falciparum dihydrofolate reductase (Pfdhfr) gene at codon 108, 6 (5%) had mutations in both Pfdhfr codon 108 and the P. falciparum dihydropteroate synthase codon 437, and 115 (56%) had the chloroquine resistance-associated K76T mutation in the P. falciparum chloroquine resistance transporter gene. This study represents a unique opportunity to improve surveillance of P. falciparum drug resistance in Comoros with consequences for treatment and chemoprophylaxis guidelines.

De novo point mutations in patients diagnosed with ataxic cerebral palsy

PubMed Central

Parolin Schnekenberg, Ricardo; Perkins, Emma M.; Miller, Jack W.; Davies, Wayne I. L.; D’Adamo, Maria Cristina; Pessia, Mauro; Fawcett, Katherine A.; Sims, David; Gillard, Elodie; Hudspith, Karl; Skehel, Paul; Williams, Jonathan; O’Regan, Mary; Jayawant, Sandeep; Jefferson, Rosalind; Hughes, Sarah; Lustenberger, Andrea; Ragoussis, Jiannis

2015-01-01

Cerebral palsy is a sporadic disorder with multiple likely aetiologies, but frequently considered to be caused by birth asphyxia. Genetic investigations are rarely performed in patients with cerebral palsy and there is little proven evidence of genetic causes. As part of a large project investigating children with ataxia, we identified four patients in our cohort with a diagnosis of ataxic cerebral palsy. They were investigated using either targeted next generation sequencing or trio-based exome sequencing and were found to have mutations in three different genes, KCNC3, ITPR1 and SPTBN2. All the mutations were de novo and associated with increased paternal age. The mutations were shown to be pathogenic using a combination of bioinformatics analysis and in vitro model systems. This work is the first to report that the ataxic subtype of cerebral palsy can be caused by de novo dominant point mutations, which explains the sporadic nature of these cases. We conclude that at least some subtypes of cerebral palsy may be caused by de novo genetic mutations and patients with a clinical diagnosis of cerebral palsy should be genetically investigated before causation is ascribed to perinatal asphyxia or other aetiologies. PMID:25981959

A mechanism for exon skipping caused by nonsense or missense mutations in BRCA1 and other genes.

PubMed

Liu, H X; Cartegni, L; Zhang, M Q; Krainer, A R

2001-01-01

Point mutations can generate defective and sometimes harmful proteins. The nonsense-mediated mRNA decay (NMD) pathway minimizes the potential damage caused by nonsense mutations. In-frame nonsense codons located at a minimum distance upstream of the last exon-exon junction are recognized as premature termination codons (PTCs), targeting the mRNA for degradation. Some nonsense mutations cause skipping of one or more exons, presumably during pre-mRNA splicing in the nucleus; this phenomenon is termed nonsense-mediated altered splicing (NAS), and its underlying mechanism is unclear. By analyzing NAS in BRCA1, we show here that inappropriate exon skipping can be reproduced in vitro, and results from disruption of a splicing enhancer in the coding sequence. Enhancers can be disrupted by single nonsense, missense and translationally silent point mutations, without recognition of an open reading frame as such. These results argue against a nuclear reading-frame scanning mechanism for NAS. Coding-region single-nucleotide polymorphisms (cSNPs) within exonic splicing enhancers or silencers may affect the patterns or efficiency of mRNA splicing, which may in turn cause phenotypic variability and variable penetrance of mutations elsewhere in a gene.

Fukutin-related protein localizes to the Golgi apparatus and mutations lead to mislocalization in muscle in vivo.

PubMed

Keramaris-Vrantsis, Elizabeth; Lu, Pei J; Doran, Timothy; Zillmer, Allen; Ashar, Jignya; Esapa, Christopher T; Benson, Matthew A; Blake, Derek J; Rosenfeld, Jeffrey; Lu, Qi L

2007-10-01

Mutations in the fukutin-related protein gene (FKRP) are associated with a spectrum of diseases from mild limb-girdle muscular dystrophy type 2I to severe congenital muscular dystrophy type 1C, muscle-eye-brain disease (MEB), and Walker-Warburg syndrome (WWS). The effect of mutations on the transportation of the mutant proteins may constitute the underlying mechanisms for the pathogenesis of these diseases. Here we examined the subcellular localization of mouse and human normal and mutant FKRP proteins in cells and in muscle in vivo. Both normal human and mouse FKRPs localize in part of the Golgi apparatus in muscle fibers. Mutations in the FKRP gene invariably altered the localization of the protein, leading to endoplasmic reticulum retention within cells and diminished Golgi localization in muscle fibers. Our results therefore suggest that an individual missense point mutation can confer at least two independent effects on the protein, causing (1) reduction or loss of the presumed glycosyltransferase activity directly and (2) mislocalization that could further alter the function of the protein. The complexity of the effect of individual missense point mutations may partly explain the wide variation of the FKRP-related myopathies.

Myelin protein zero gene mutated in Charcot-Marie-Tooth type 1B patients

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

Su, Ying; Li, Lanying; Lepercq, J.

1993-11-15

The autosomal dominant of Charcot-Marie-Tooth disease (CMT), whose gene is type 1B (CMT1B), has slow nerve conduction with demyelinated Schwann cells. In this study the abundant peripheral myelin protein zero (MPZ) gene, MPZ, was mapped 130 kb centromeric to the Fc receptor immunoglobulin gene cluster in band 1q22, and a major MPZ point mutation was found to cosegregate with CMT1B in one large CMT1B family. The MPZ point mutation in 18 of 18 related CMT1B pedigree 1 patients converts a positively charged lysine in codon 96 to a negatively charged glutamate. The same MPZ locus cosegregates with the CMT1B diseasemore » gene in a second CMT1B family [total multipoint logarithm of odds (lod) = 11.4 at [theta] = 0.00] with a splice junction mutation. Both mutations occur in MPZ protein regions otherwise conserved identically in human, rat, and cow since these species diverged 100 million years ago. MPZ protein, expressed exclusively in myelinated peripheral nerve Schwann cells, constitutes >50% of myelin protein. These mutations are anticipated to disrupt homophilic MPZ binding and result in CMT1B peripheral nerve demyelination.« less

Development and application of loop-mediated isothermal amplification for detection of the F167Y mutation of carbendazim-resistant isolates in Fusarium graminearum

PubMed Central

Duan, Yabing; Zhang, Xiaoke; Ge, Changyan; Wang, Yong; Cao, Junhong; Jia, Xiaojing; Wang, Jianxin; Zhou, Mingguo

2014-01-01

Resistance of Fusarium graminearum to carbendazim is caused by point mutations in the β2-tubulin gene. The point mutation at codon 167 (TTT → TAT, F167Y) occurs in more than 90% of field resistant isolates in China. To establish a suitable method for rapid detection of the F167Y mutation in F. graminearum, an efficient and simple method with high specificity was developed based on loop-mediated isothermal amplification (LAMP). A set of four primers was designed and optimized to specially distinguish the F167Y mutation genotype. The LAMP reaction was optimal at 63°C for 60 min. When hydroxynaphthol blue dye (HNB) was added prior to amplification, samples with DNA of the F167Y mutation developed a characteristic sky blue color after the reaction but those without DNA or with different DNA did not. Results of HNB staining method were reconfirmed by gel electrophoresis. The developed LAMP had good specificity, stability and repeatability and was suitable for monitoring carbendazim-resistance populations of F. graminearum in agricultural production. PMID:25403277

The Rate of Beneficial Mutations Surfing on the Wave of a Range Expansion

PubMed Central

Lehe, Rémi; Hallatschek, Oskar; Peliti, Luca

2012-01-01

Many theoretical and experimental studies suggest that range expansions can have severe consequences for the gene pool of the expanding population. Due to strongly enhanced genetic drift at the advancing frontier, neutral and weakly deleterious mutations can reach large frequencies in the newly colonized regions, as if they were surfing the front of the range expansion. These findings raise the question of how frequently beneficial mutations successfully surf at shifting range margins, thereby promoting adaptation towards a range-expansion phenotype. Here, we use individual-based simulations to study the surfing statistics of recurrent beneficial mutations on wave-like range expansions in linear habitats. We show that the rate of surfing depends on two strongly antagonistic factors, the probability of surfing given the spatial location of a novel mutation and the rate of occurrence of mutations at that location. The surfing probability strongly increases towards the tip of the wave. Novel mutations are unlikely to surf unless they enjoy a spatial head start compared to the bulk of the population. The needed head start is shown to be proportional to the inverse fitness of the mutant type, and only weakly dependent on the carrying capacity. The precise location dependence of surfing probabilities is derived from the non-extinction probability of a branching process within a moving field of growth rates. The second factor is the mutation occurrence which strongly decreases towards the tip of the wave. Thus, most successful mutations arise at an intermediate position in the front of the wave. We present an analytic theory for the tradeoff between these factors that allows to predict how frequently substitutions by beneficial mutations occur at invasion fronts. We find that small amounts of genetic drift increase the fixation rate of beneficial mutations at the advancing front, and thus could be important for adaptation during species invasions. PMID:22479175

An Ethyl-Nitrosourea-Induced Point Mutation in Phex Causes Exon Skipping, X-Linked Hypophosphatemia, and Rickets

PubMed Central

Carpinelli, Marina R.; Wicks, Ian P.; Sims, Natalie A.; O’Donnell, Kristy; Hanzinikolas, Katherine; Burt, Rachel; Foote, Simon J.; Bahlo, Melanie; Alexander, Warren S.; Hilton, Douglas J.

2002-01-01

We describe the clinical, genetic, biochemical, and molecular characterization of a mouse that arose in the first generation (G1) of a random mutagenesis screen with the chemical mutagen ethyl-nitrosourea. The mouse was observed to have skeletal abnormalities inherited with an X-linked dominant pattern of inheritance. The causative mutation, named Skeletal abnormality 1 (Ska1), was shown to be a single base pair mutation in a splice donor site immediately following exon 8 of the Phex (phosphate-regulating gene with homologies to endopeptidases located on the X-chromosome) gene. This point mutation caused skipping of exon 8 from Phex mRNA, hypophosphatemia, and features of rickets. This experimentally induced phenotype mirrors the human condition X-linked hypophosphatemia; directly confirms the role of Phex in phosphate homeostasis, normal skeletal development, and rickets; and illustrates the power of mutagenesis in exploring animal models of human disease. PMID:12414538

An ethyl-nitrosourea-induced point mutation in phex causes exon skipping, x-linked hypophosphatemia, and rickets.

PubMed

Carpinelli, Marina R; Wicks, Ian P; Sims, Natalie A; O'Donnell, Kristy; Hanzinikolas, Katherine; Burt, Rachel; Foote, Simon J; Bahlo, Melanie; Alexander, Warren S; Hilton, Douglas J

2002-11-01

We describe the clinical, genetic, biochemical, and molecular characterization of a mouse that arose in the first generation (G(1)) of a random mutagenesis screen with the chemical mutagen ethyl-nitrosourea. The mouse was observed to have skeletal abnormalities inherited with an X-linked dominant pattern of inheritance. The causative mutation, named Skeletal abnormality 1 (Ska1), was shown to be a single base pair mutation in a splice donor site immediately following exon 8 of the Phex (phosphate-regulating gene with homologies to endopeptidases located on the X-chromosome) gene. This point mutation caused skipping of exon 8 from Phex mRNA, hypophosphatemia, and features of rickets. This experimentally induced phenotype mirrors the human condition X-linked hypophosphatemia; directly confirms the role of Phex in phosphate homeostasis, normal skeletal development, and rickets; and illustrates the power of mutagenesis in exploring animal models of human disease.

Transthyretin Amyloidosis: Chaperone Concentration Changes and Increased Proteolysis in the Pathway to Disease

PubMed Central

Ribeiro, Raquel; Gilberto, Samuel; Gomes, Ricardo A.; Ferreira, António; Mateus, Élia; Barroso, Eduardo; Coelho, Ana V.; Freire, Ana Ponces; Cordeiro, Carlos

2015-01-01

Transthyretin amyloidosis is a conformational pathology characterized by the extracellular formation of amyloid deposits and the progressive impairment of the peripheral nervous system. Point mutations in this tetrameric plasma protein decrease its stability and are linked to disease onset and progression. Since non-mutated transthyretin also forms amyloid in systemic senile amyloidosis and some mutation bearers are asymptomatic throughout their lives, non-genetic factors must also be involved in transthyretin amyloidosis. We discovered, using a differential proteomics approach, that extracellular chaperones such as fibrinogen, clusterin, haptoglobin, alpha-1-anti-trypsin and 2-macroglobulin are overrepresented in transthyretin amyloidosis. Our data shows that a complex network of extracellular chaperones are over represented in human plasma and we speculate that they act synergistically to cope with amyloid prone proteins. Proteostasis may thus be as important as point mutations in transthyretin amyloidosis. PMID:26147092

Late-onset nonketotic hyperglycinemia with a heterozygous novel point mutation of the GLDC gene.

PubMed

Brenton, J Nicholas; Rust, Robert S

2014-05-01

Atypical nonketotic hyperglycinemia is characterized by heterogeneous phenotypes that often include nonspecific behavioral problems, cognitive deficits, and developmental delays. We describe a girl with late-onset nonketotic hyperglycinemia presenting at 5 years of age with hypotonia, chorea, ataxia, and alterations in consciousness in the setting of febrile illness. Serum amino acid analysis was mildly elevated; however, urine amino acid analysis was instrumental in demonstrating marked hyperglycinuria. Mutation testing showed a heterozygous novel sequence change/point mutation in the glycine decarboxylase gene. This patient illustrates the importance of obtaining urine amino acids in individuals whose clinical manifestations are suspicious for any form of nonketotic hyperglycinemia, because this testing may provide more prominent evidence of elevations in glycine. She also illustrates the potential for a heterozygous mutation to result in manifestations of an atypical form of nonketotic hyperglycinemia. Copyright © 2014 Elsevier Inc. All rights reserved.

Variation in genome-wide mutation rates within and between human families.

PubMed

Conrad, Donald F; Keebler, Jonathan E M; DePristo, Mark A; Lindsay, Sarah J; Zhang, Yujun; Casals, Ferran; Idaghdour, Youssef; Hartl, Chris L; Torroja, Carlos; Garimella, Kiran V; Zilversmit, Martine; Cartwright, Reed; Rouleau, Guy A; Daly, Mark; Stone, Eric A; Hurles, Matthew E; Awadalla, Philip

2011-06-12

J.B.S. Haldane proposed in 1947 that the male germline may be more mutagenic than the female germline. Diverse studies have supported Haldane's contention of a higher average mutation rate in the male germline in a variety of mammals, including humans. Here we present, to our knowledge, the first direct comparative analysis of male and female germline mutation rates from the complete genome sequences of two parent-offspring trios. Through extensive validation, we identified 49 and 35 germline de novo mutations (DNMs) in two trio offspring, as well as 1,586 non-germline DNMs arising either somatically or in the cell lines from which the DNA was derived. Most strikingly, in one family, we observed that 92% of germline DNMs were from the paternal germline, whereas, in contrast, in the other family, 64% of DNMs were from the maternal germline. These observations suggest considerable variation in mutation rates within and between families.

Computational analysis of the human sinus node action potential: model development and effects of mutations

PubMed Central

Fabbri, Alan; Fantini, Matteo; Wilders, Ronald

2017-01-01

Key points We constructed a comprehensive mathematical model of the spontaneous electrical activity of a human sinoatrial node (SAN) pacemaker cell, starting from the recent Severi–DiFrancesco model of rabbit SAN cells.Our model is based on electrophysiological data from isolated human SAN pacemaker cells and closely matches the action potentials and calcium transient that were recorded experimentally.Simulated ion channelopathies explain the clinically observed changes in heart rate in corresponding mutation carriers, providing an independent qualitative validation of the model.The model shows that the modulatory role of the ‘funny current’ (I f) in the pacing rate of human SAN pacemaker cells is highly similar to that of rabbit SAN cells, despite its considerably lower amplitude.The model may prove useful in the design of experiments and the development of heart‐rate modulating drugs. Abstract The sinoatrial node (SAN) is the normal pacemaker of the mammalian heart.  Over several decades, a large amount of data on the ionic mechanisms underlying the spontaneous electrical activity of SAN pacemaker cells has been obtained, mostly in experiments on single cells isolated from rabbit SAN. This wealth of data has allowed the development of mathematical models of the electrical activity of rabbit SAN pacemaker cells. The present study aimed to construct a comprehensive model of the electrical activity of a human SAN pacemaker cell using recently obtained electrophysiological data from human SAN pacemaker cells.  We based our model on the recent Severi–DiFrancesco model of a rabbit SAN pacemaker cell. The action potential and calcium transient of the resulting model are close to the experimentally recorded values. The model has a much smaller ‘funny current’ (I f) than do rabbit cells, although its modulatory role is highly similar. Changes in pacing rate upon the implementation of mutations associated with sinus node dysfunction agree with the clinical observations. This agreement holds for both loss‐of‐function and gain‐of‐function mutations in the HCN4, SCN5A and KCNQ1 genes, underlying ion channelopathies in I f, fast sodium current and slow delayed rectifier potassium current, respectively. We conclude that our human SAN cell model can be a useful tool in the design of experiments and the development of drugs that aim to modulate heart rate. PMID:28185290

Molecular identification of sulfadoxine-pyrimethamine resistance in malaria infected women who received intermittent preventive treatment in the Democratic Republic of Congo.

PubMed

Ruh, Emrah; Bateko, Jean Paul; Imir, Turgut; Taylan-Ozkan, Aysegul

2018-01-09

Point mutations in Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes which confer resistance to sulfadoxine-pyrimethamine (SP) occur at increasing rates. The present study aimed to identify Pfdhfr and Pfdhps mutations in P. falciparum isolates recovered from women who received two doses of SP during pregnancy in Bandundu, the Democratic Republic of Congo (DRC). A total of 48 women with confirmed P. falciparum infection were enrolled in the study. Finger-prick blood samples that were collected on filter paper at the time of delivery were used for DNA isolation. Pfdhfr and Pfdhps genes were amplified by a nested PCR protocol. DNA sequencing was performed on both strands, and the point mutations were analysed. All of the 48 (100.0%) P. falciparum isolates carried at least one polymorphism in both genes. The wild-type haplotypes of Pfdhfr (CNCSI [C50, N51, C59, S108, I164]) and Pfdhps (SAKAA [S436, A437, K540, A581, A613]) were not observed in the study. In Pfdhfr, N51I (85.4%), C59R (60.4%), and S108N (100.0%) polymorphisms were detected. Triple mutation (CIRNI) (mutant amino acids are underlined) was the most prevalent (47.9%) Pfdhfr haplotype. In the study, all P. falciparum isolates (100.0%) harboured the A437G allele in Pfdhps gene. Also, K540E and A581G polymorphisms were observed in one (2.1%) isolate. Single mutant haplotype (SGKAA) was detected in 97.9% of the isolates. Mutant Pfdhfr and Pfdhps allele combinations revealed quintuple (CICNI-SGEGA; 2.1%), quadruple (CIRNI-SGKAA; 47.9%), triple (CICNI-SGKAA; 35.4%, CNRNI-SGKAA; 12.5%), and double (CNCNI-SGKAA; 2.1%) haplotypes. In the study, the rate of SGEGA haplotype was low (2.1%). Although K540E and A581G alleles are more common in Eastern Africa, a distinct lineage of SGEGA is also present in the DRC, which is located in Central Africa. This haplotype is associated with decreased efficacy of SP in pregnant women and infants, therefore, it should be carefully considered in the DRC and SP resistance should be routinely monitored.

A pivot mutation impedes reverse evolution across an adaptive landscape for drug resistance in Plasmodium vivax.

PubMed

Ogbunugafor, C Brandon; Hartl, Daniel

2016-01-25

The study of reverse evolution from resistant to susceptible phenotypes can reveal constraints on biological evolution, a topic for which evolutionary theory has relatively few general principles. The public health catastrophe of antimicrobial resistance in malaria has brought these constraints on evolution into a practical realm, with one proposed solution: withdrawing anti-malarial medication use in high resistance settings, built on the assumption that reverse evolution occurs readily enough that populations of pathogens may revert to their susceptible states. While past studies have suggested limits to reverse evolution, there have been few attempts to properly dissect its mechanistic constraints. Growth rates were determined from empirical data on the growth and resistance from a set of combinatorially complete set of mutants of a resistance protein (dihydrofolate reductase) in Plasmodium vivax, to construct reverse evolution trajectories. The fitness effects of individual mutations were calculated as a function of drug environment, revealing the magnitude of epistatic interactions between mutations and genetic backgrounds. Evolution across the landscape was simulated in two settings: starting from the population fixed for the quadruple mutant, and from a polymorphic population evenly distributed between double mutants. A single mutation of large effect (S117N) serves as a pivot point for evolution to high resistance regions of the landscape. Through epistatic interactions with other mutations, this pivot creates an epistatic ratchet against reverse evolution towards the wild type ancestor, even in environments where the wild type is the most fit of all genotypes. This pivot mutation underlies the directional bias in evolution across the landscape, where evolution towards the ancestor is precluded across all examined drug concentrations from various starting points in the landscape. The presence of pivot mutations can dictate dynamics of evolution across adaptive landscape through epistatic interactions within a protein, leaving a population trapped on local fitness peaks in an adaptive landscape, unable to locate ancestral genotypes. This irreversibility suggests that the structure of an adaptive landscape for a resistance protein should be understood before considering resistance management strategies. This proposed mechanism for constraints on reverse evolution corroborates evidence from the field indicating that phenotypic reversal often occurs via compensatory mutation at sites independent of those associated with the forward evolution of resistance. Because of this, molecular methods that identify resistance patterns via single SNPs in resistance-associated markers might be missing signals for resistance and compensatory mutation throughout the genome. In these settings, whole genome sequencing efforts should be used to identify resistance patterns, and will likely reveal a more complicated genomic signature for resistance and susceptibility, especially in settings where anti-malarial medications have been used intermittently. Lastly, the findings suggest that, given their role in dictating the dynamics of evolution across the landscape, pivot mutations might serve as future targets for therapy.

Clinical Impact of Additional Cytogenetic Aberrations, cKIT and RAS Mutations, and Treatment Elements in Pediatric t(8;21)-AML: Results From an International Retrospective Study by the International Berlin-Frankfurt-Münster Study Group

PubMed Central

Klein, Kim; Kaspers, Gertjan; Harrison, Christine J.; Beverloo, H. Berna; Reedijk, Ardine; Bongers, Mathilda; Cloos, Jacqueline; Pession, Andrea; Reinhardt, Dirk; Zimmerman, Martin; Creutzig, Ursula; Dworzak, Michael; Alonzo, Todd; Johnston, Donna; Hirsch, Betsy; Zapotocky, Michal; De Moerloose, Barbara; Fynn, Alcira; Lee, Vincent; Taga, Takashi; Tawa, Akio; Auvrignon, Anne; Zeller, Bernward; Forestier, Erik; Salgado, Carmen; Balwierz, Walentyna; Popa, Alexander; Rubnitz, Jeffrey; Raimondi, Susana; Gibson, Brenda

2015-01-01

Purpose This retrospective cohort study aimed to determine the predictive relevance of clinical characteristics, additional cytogenetic aberrations, and cKIT and RAS mutations, as well as to evaluate whether specific treatment elements were associated with outcomes in pediatric t(8;21)-positive patients with acute myeloid leukemia (AML). Patients and Methods Karyotypes of 916 pediatric patients with t(8;21)-AML were reviewed for the presence of additional cytogenetic aberrations, and 228 samples were screened for presence of cKIT and RAS mutations. Multivariable regression models were used to assess the relevance of anthracyclines, cytarabine, and etoposide during induction and overall treatment. End points were the probability of achieving complete remission, cumulative incidence of relapse (CIR), probability of event-free survival, and probability of overall survival. Results Of 838 patients included in final analyses, 92% achieved complete remission. The 5-year overall survival, event-free survival, and CIR were 74%, 58%, and 26%, respectively. cKIT mutations and RAS mutations were not significantly associated with outcome. Patients with deletions of chromosome arm 9q [del(9q); n = 104] had a lower probability of complete remission (P = .01). Gain of chromosome 4 (+4; n = 21) was associated with inferior CIR and survival (P < .01). Anthracycline doses greater than 150 mg/m2 and etoposide doses greater than 500 mg/m2 in the first induction course and high-dose cytarabine 3 g/m2 during induction were associated with better outcomes on various end points. Cumulative doses of cytarabine greater than 30 g/m2 and etoposide greater than 1,500 mg/m2 were associated with lower CIR rates and better probability of event-free survival. Conclusion Pediatric patients with t(8;21)-AML and additional del(9q) or additional +4 might not be considered at good risk. Patients with t(8;21)-AML likely benefit from protocols that have high doses of anthracyclines, etoposide, and cytarabine during induction, as well as from protocols comprising cumulative high doses of cytarabine and etoposide. PMID:26573082

Circulating mutational portrait of cancer: manifestation of aggressive clonal events in both early and late stages.

PubMed

Yang, Meng; Topaloglu, Umit; Petty, W Jeffrey; Pagni, Matthew; Foley, Kristie L; Grant, Stefan C; Robinson, Mac; Bitting, Rhonda L; Thomas, Alexandra; Alistar, Angela T; Desnoyers, Rodwige J; Goodman, Michael; Albright, Carol; Porosnicu, Mercedes; Vatca, Mihaela; Qasem, Shadi A; DeYoung, Barry; Kytola, Ville; Nykter, Matti; Chen, Kexin; Levine, Edward A; Staren, Edgar D; D'Agostino, Ralph B; Petro, Robin M; Blackstock, William; Powell, Bayard L; Abraham, Edward; Pasche, Boris; Zhang, Wei

2017-05-04

Solid tumors residing in tissues and organs leave footprints in circulation through circulating tumor cells (CTCs) and circulating tumor DNAs (ctDNA). Characterization of the ctDNA portraits and comparison with tumor DNA mutational portraits may reveal clinically actionable information on solid tumors that is traditionally achieved through more invasive approaches. We isolated ctDNAs from plasma of patients of 103 lung cancer and 74 other solid tumors of different tissue origins. Deep sequencing using the Guardant360 test was performed to identify mutations in 73 clinically actionable genes, and the results were associated with clinical characteristics of the patient. The mutation profiles of 37 lung cancer cases with paired ctDNA and tumor genomic DNA sequencing were used to evaluate clonal representation of tumor in circulation. Five lung cancer cases with longitudinal ctDNA sampling were monitored for cancer progression or response to treatments. Mutations in TP53, EGFR, and KRAS genes are most prevalent in our cohort. Mutation rates of ctDNA are similar in early (I and II) and late stage (III and IV) cancers. Mutation in DNA repair genes BRCA1, BRCA2, and ATM are found in 18.1% (32/177) of cases. Patients with higher mutation rates had significantly higher mortality rates. Lung cancer of never smokers exhibited significantly higher ctDNA mutation rates as well as higher EGFR and ERBB2 mutations than ever smokers. Comparative analysis of ctDNA and tumor DNA mutation data from the same patients showed that key driver mutations could be detected in plasma even when they were present at a minor clonal population in the tumor. Mutations of key genes found in the tumor tissue could remain in circulation even after frontline radiotherapy and chemotherapy suggesting these mutations represented resistance mechanisms. Longitudinal sampling of five lung cancer cases showed distinct changes in ctDNA mutation portraits that are consistent with cancer progression or response to EGFR drug treatment. This study demonstrates that ctDNA mutation rates in the key tumor-associated genes are clinical parameters relevant to smoking status and mortality. Mutations in ctDNA may serve as an early detection tool for cancer. This study quantitatively confirms the hypothesis that ctDNAs in circulation is the result of dissemination of aggressive tumor clones and survival of resistant clones. This study supports the use of ctDNA profiling as a less-invasive approach to monitor cancer progression and selection of appropriate drugs during cancer evolution.

Invasive advance of an advantageous mutation: nucleation theory.

PubMed

O'Malley, Lauren; Basham, James; Yasi, Joseph A; Korniss, G; Allstadt, Andrew; Caraco, Thomas

2006-12-01

For sedentary organisms with localized reproduction, spatially clustered growth drives the invasive advance of a favorable mutation. We model competition between two alleles where recurrent mutation introduces a genotype with a rate of local propagation exceeding the resident's rate. We capture ecologically important properties of the rare invader's stochastic dynamics by assuming discrete individuals and local neighborhood interactions. To understand how individual-level processes may govern population patterns, we invoke the physical theory for nucleation of spatial systems. Nucleation theory discriminates between single-cluster and multi-cluster dynamics. A sufficiently low mutation rate, or a sufficiently small environment, generates single-cluster dynamics, an inherently stochastic process; a favorable mutation advances only if the invader cluster reaches a critical radius. For this mode of invasion, we identify the probability distribution of waiting times until the favored allele advances to competitive dominance, and we ask how the critical cluster size varies as propagation or mortality rates vary. Increasing the mutation rate or system size generates multi-cluster invasion, where spatial averaging produces nearly deterministic global dynamics. For this process, an analytical approximation from nucleation theory, called Avrami's Law, describes the time-dependent behavior of the genotype densities with remarkable accuracy.

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

PubMed

Sahakyan, Aleksandr B; Balasubramanian, Shankar

2017-01-13

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

RNA2DMut: a web tool for the design and analysis of RNA structure mutations.

PubMed

Moss, Walter N

2018-03-01

With the widespread application of high-throughput sequencing, novel RNA sequences are being discovered at an astonishing rate. The analysis of function, however, lags behind. In both the cis - and trans -regulatory functions of RNA, secondary structure (2D base-pairing) plays essential regulatory roles. In order to test RNA function, it is essential to be able to design and analyze mutations that can affect structure. This was the motivation for the creation of the RNA2DMut web tool. With RNA2DMut, users can enter in RNA sequences to analyze, constrain mutations to specific residues, or limit changes to purines/pyrimidines. The sequence is analyzed at each base to determine the effect of every possible point mutation on 2D structure. The metrics used in RNA2DMut rely on the calculation of the Boltzmann structure ensemble and do not require a robust 2D model of RNA structure for designing mutations. This tool can facilitate a wide array of uses involving RNA: for example, in designing and evaluating mutants for biological assays, interrogating RNA-protein interactions, identifying key regions to alter in SELEX experiments, and improving RNA folding and crystallization properties for structural biology. Additional tools are available to help users introduce other mutations (e.g., indels and substitutions) and evaluate their effects on RNA structure. Example calculations are shown for five RNAs that require 2D structure for their function: the MALAT1 mascRNA, an influenza virus splicing regulatory motif, the EBER2 viral noncoding RNA, the Xist lncRNA repA region, and human Y RNA 5. RNA2DMut can be accessed at https://rna2dmut.bb.iastate.edu/. © 2018 Moss; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Crystal Structure and Biochemical Characterization of Chlamydomonas FDX2 Reveal Two Residues that, When Mutated, Partially Confer FDX2 the Redox Potential and Catalytic Properties of FDX1

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

Boehm, Marko; Alahuhta, Markus; Mulder, David W.

2015-11-03

The green alga Chlamydomonas reinhardtii contains six plastidic [2Fe2S]-cluster ferredoxins (FDXs), with FDX1 as the predominant isoform under photoautotrophic growth. FDX2 is highly similar to FDX1 and has been shown to interact with specific enzymes (such as nitrite reductase), as well as to share interactors with FDX1, such as the hydrogenases (HYDA), ferredoxin:NAD(P) reductase I (FNR1), and pyruvate:ferredoxin oxidoreductase (PFR1), albeit performing at low catalytic rates. Here we report the FDX2 crystal structure solved at 1.18 Å resolution. Based on differences between the Chlorella fusca FDX1 and C. reinhardtii FDX2 structures, we generated and purified point-mutated versions of the FDX2more » protein and assayed them in vitro for their ability to catalyze hydrogen and NADPH photo-production. The data show that structural differences at two amino acid positions contribute to functional differences between FDX1 and FDX2, suggesting that FDX2 might have evolved from FDX1 toward a different physiological role in the cell. Moreover, we demonstrate that the mutations affect both the midpoint potentials of the FDX and kinetics of the FNR reaction, possibly due to altered binding between FDX and FNR. An effect on H 2 photo-production rates was also observed, although the kinetics of the reaction were not further characterized.« less

Mutations in Prickle Orthologs Cause Seizures in Flies, Mice, and Humans

PubMed Central

Tao, Hirotaka; Manak, J. Robert; Sowers, Levi; Mei, Xue; Kiyonari, Hiroshi; Abe, Takaya; Dahdaleh, Nader S.; Yang, Tian; Wu, Shu; Chen, Shan; Fox, Mark H.; Gurnett, Christina; Montine, Thomas; Bird, Thomas; Shaffer, Lisa G.; Rosenfeld, Jill A.; McConnell, Juliann; Madan-Khetarpal, Suneeta; Berry-Kravis, Elizabeth; Griesbach, Hilary; Saneto, Russell P.; Scott, Matthew P.; Antic, Dragana; Reed, Jordan; Boland, Riley; Ehaideb, Salleh N.; El-Shanti, Hatem; Mahajan, Vinit B.; Ferguson, Polly J.; Axelrod, Jeffrey D.; Lehesjoki, Anna-Elina; Fritzsch, Bernd; Slusarski, Diane C.; Wemmie, John; Ueno, Naoto; Bassuk, Alexander G.

2011-01-01

Epilepsy is heritable, yet few causative gene mutations have been identified, and thus far no human epilepsy gene mutations have been found to produce seizures in invertebrates. Here we show that mutations in prickle genes are associated with seizures in humans, mice, and flies. We identified human epilepsy patients with heterozygous mutations in either PRICKLE1 or PRICKLE2. In overexpression assays in zebrafish, prickle mutations resulted in aberrant prickle function. A seizure phenotype was present in the Prickle1-null mutant mouse, two Prickle1 point mutant (missense and nonsense) mice, and a Prickle2-null mutant mouse. Drosophila with prickle mutations displayed seizures that were responsive to anti-epileptic medication, and homozygous mutant embryos showed neuronal defects. These results suggest that prickle mutations have caused seizures throughout evolution. PMID:21276947

Tether mutations that restore function and suppress pleiotropic phenotypes of the C. elegans isp-1(qm150) Rieske iron–sulfur protein

PubMed Central

Jafari, Gholamali; Wasko, Brian M.; Tonge, Ashley; Schurman, Nathan; Dong, Cindy; Li, Zhongyu; Peters, Rebecca; Kayser, Ernst-Bernhard; Pitt, Jason N.; Morgan, Phil G.; Sedensky, Margaret M.; Crofts, Antony R.; Kaeberlein, Matt

2015-01-01

Mitochondria play an important role in numerous diseases as well as normative aging. Severe reduction in mitochondrial function contributes to childhood disorders such as Leigh Syndrome, whereas mild disruption can extend the lifespan of model organisms. The Caenorhabditis elegans isp-1 gene encodes the Rieske iron–sulfur protein subunit of cytochrome c oxidoreductase (complex III of the electron transport chain). The partial loss of function allele, isp-1(qm150), leads to several pleiotropic phenotypes. To better understand the molecular mechanisms of ISP-1 function, we sought to identify genetic suppressors of the delayed development of isp-1(qm150) animals. Here we report a series of intragenic suppressors, all located within a highly conserved six amino acid tether region of ISP-1. These intragenic mutations suppress all of the evaluated isp-1(qm150) phenotypes, including developmental rate, pharyngeal pumping rate, brood size, body movement, activation of the mitochondrial unfolded protein response reporter, CO2 production, mitochondrial oxidative phosphorylation, and lifespan extension. Furthermore, analogous mutations show a similar effect when engineered into the budding yeast Rieske iron–sulfur protein Rip1, revealing remarkable conservation of the structure–function relationship of these residues across highly divergent species. The focus on a single subunit as causal both in generation and in suppression of diverse pleiotropic phenotypes points to a common underlying molecular mechanism, for which we propose a “spring-loaded” model. These observations provide insights into how gating and control processes influence the function of ISP-1 in mediating pleiotropic phenotypes including developmental rate, movement, sensitivity to stress, and longevity. PMID:26504246

The Effect of Coexistence of a Pair of Mutated Oncogenes on the Survival Rate of Invasive Breast Carcinoma Patients

NASA Astrophysics Data System (ADS)

Nair, D. R.

2017-12-01

The purpose of this project was to determine the effect of two mutated oncogenes on the survival rate from invasive breast carcinoma when in comparison to the mutation of a single oncogene on the survival rate. An oncogene is defined as a gene, that when mutated, can lead to cancer. The two oncogenes used in this project were human epidermal growth factor receptor 2 (HER2) and c-myc (MYC). HER2 and MYC are both oncogenes that contribute to the formation of cancer. HER2 proteins are receptors on breast cells, and when the HER2 gene is mutated, there is an overexpression of HER2 protein on the breast cell. This makes the breast cells proliferate uncontrollably. MYC is a gene that codes for a transcription factor that plays a role in cell cycle progression. The overexpression of MYC also leads to the proliferation of cells. I hypothesized that if there is a mutation in both the MYC and HER2 genes, then the survival rate of invasive breast carcinoma patients will be lower compared to patients with the mutations of only MYC or HER2. To test this hypothesis, we conducted individual gene searches in CBioPortal for HER2 in the datasets from the studies titled TCGA Nature 2012, TCGA Cell 2015, and TCGA Provisional. We conducted individual gene searches in CBioPortal for MYC in the same datasets. The survival rate data was then exported and analyzed for patients with mutations of either HER2 or MYC and with mutations of both genes. To determine the cases that had both HER2 and MYC mutations, we found the overlapping cases in both HER2 and MYC groups for all three datasets. We calculated the median of the survival data for cases where either HER2 or MYC was mutated and cases where both MYC and HER2 were mutated. From the first dataset, the median of MYC data was 95.53, HER2 data was 95.83, and both HER2 and MYC data was 91.24. In the second dataset, the median of MYC data was 92.17 , HER2 data was 93.5, and both HER2 and MYC data was 87.95 . In the third dataset, the median of MYC data was 92.18, HER2 data was 94.22, and both HER2 and MYC data was 89.45. The median survival rates all showed that cases with mutations in both genes had a lower survival rate than those with single mutations. My hypothesis was supported. Some sources of error are the fewer number of cases in the TCGA Nature 2012 dataset, making this data statistically insignificant.

Fitness of RNA virus decreased by Muller's ratchet

NASA Astrophysics Data System (ADS)

Chao, Lin

1990-11-01

WHY sex exists remains an unsolved problem in biology1-3. If mutations are on the average deleterious, a high mutation rate can account for the evolution of sex4. One form of this mutational hypothesis is Muller's ratchet5,6. If the mutation rate is high, mutation-free individuals become rare and they can be lost by genetic drift in small populations. In asexual populations, as Muller5 noted, the loss is irreversible and the load of deleterious mutations increases in a ratchet-like manner with the successive loss of the least-mutated individuals. Sex can be advantageous because it increases the fitness of sexual populations by re-creating mutation-free individuals from mutated individuals and stops (or slows) Muller's ratchet. Although Muller's ratchet is an appealing hypothesis, it has been investigated and documented experimentally in only one group of organisms-ciliated protozoa2. I initiated a study to examine the role of Muller's ratchet on the evolution of sex in RNA viruses and report here a significant decrease in fitness due to Muller's ratchet in 20 lineages of the RNA bacteriophage Φ6. These results show that deleterious mutations are generated at a sufficiently high rate to advance Muller's ratchet in an RNA virus and that beneficial, backward and compensatory mutations cannot stop the ratchet in the observed range of fitness decrease.

The mutation-drift balance in spatially structured populations.

PubMed

Schneider, David M; Martins, Ayana B; de Aguiar, Marcus A M

2016-08-07

In finite populations the action of neutral mutations is balanced by genetic drift, leading to a stationary distribution of alleles that displays a transition between two different behaviors. For small mutation rates most individuals will carry the same allele at equilibrium, whereas for high mutation rates of the alleles will be randomly distributed with frequencies close to one half for a biallelic gene. For well-mixed haploid populations the mutation threshold is μc=1/2N, where N is the population size. In this paper we study how spatial structure affects this mutation threshold. Specifically, we study the stationary allele distribution for populations placed on regular networks where connected nodes represent potential mating partners. We show that the mutation threshold is sensitive to spatial structure only if the number of potential mates is very small. In this limit, the mutation threshold decreases substantially, increasing the diversity of the population at considerably low mutation rates. Defining kc as the degree of the network for which the mutation threshold drops to half of its value in well-mixed populations we show that kc grows slowly as a function of the population size, following a power law. Our calculations and simulations are based on the Moran model and on a mapping between the Moran model with mutations and the voter model with opinion makers. Copyright © 2016 Elsevier Ltd. All rights reserved.

Regulation of MDM2 Activity by Nucleolin

DTIC Science & Technology

2005-06-01

tumorigenesis with -50% of human cancers showing mutation of the TP53 gene , often a loss of one gene copy and a point mutation within the second. p53...Sordat B, Gillet M, Schorderet D, Bosman FT, Chaubert P (2001) Methylation silencing and mutations of the p14ARF and pl6INK4a genes in colon cancer. Lab...for the first machinery (for example, see reference 53 and references step of pre-rRNA processing (22). Mutation of the genes en- therein). It is

First Report of the 23S rRNA Gene A2058G Point Mutation Associated With Macrolide Resistance in Treponema pallidum From Syphilis Patients in Cuba.

PubMed

Noda, Angel A; Matos, Nelvis; Blanco, Orestes; Rodríguez, Islay; Stamm, Lola Virginia

2016-05-01

This study aimed to assess the presence of macrolide-resistant Treponema pallidum subtypes in Havana, Cuba. Samples from 41 syphilis patients were tested for T. pallidum 23S rRNA gene mutations. Twenty-five patients (61%) harbored T. pallidum with the A2058G mutation, which was present in all 8 subtypes that were identified. The A2059G mutation was not detected.

Detection of JAK2 V617F mutation increases the diagnosis of myeloproliferative neoplasms

PubMed Central

ZHANG, SHU-PENG; LI, HUI; LAI, REN-SHENG

2015-01-01

The Janus kinase (JAK)2 gene, which is located on chromosome 9p24, is involved in the signaling transduction pathways of the hematopoietic and immune system. Mutations in the JAK2 gene have served as disease markers for myeloproliferative neoplasms (MPNs). The aim of the present study was to investigate the occurrence of the JAK2 gene mutation in 140 clinical samples, and to evaluate its clinical significance in MPNs and other hematological diseases. Genomic DNA was extracted from the peripheral blood leukocytes or bone marrow karyocytes of 140 clinical samples, which included 130 patients with various types of hematological disease and 10 control patients. In addition, exons 12 and 14 of the JAK2 gene were analyzed by direct sequencing and the mutation rates of various MPN subtypes were evaluated. Of the 140 samples, exons 12 and 14 were tested in 74 samples, however, exon 14 only was tested in 66 samples. No mutations were identified in exon 12. The V617F mutation rate in polycythemia vera was 82.1% (23/28), and the mutation rates in essential thrombocythemia histiocytosis, primary myelofibrosis and other MPNs were 53.1% (17/32), 40.0% (4/10) and 60.0% (6/10), respectively. Therefore, the total mutation rate of the JAK2 gene in MPN was 62.5% (50/80). For non-MPN hematological diseases, four V617F mutations were detected in samples of leukocytosis of unknown origin (4/12), however, no JAK2 V617F mutations were identified in the 10 controls. Therefore, JAK2 V617F mutations may present a novel marker for diagnosis of MPNs. Furthermore, the direct sequencing method appeared to be satisfactory for the clinical gene testing of hematological samples. PMID:25624900