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ABSTRACT: It has been previously noted that the hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutant frequency of Russian subjects is significantly higher than age-matched Western counterparts. To further explore this difference, approximately 100 mutants collected from Russian twins reported in a previous study have been sequenced and compared to an aged-matched Western mutant dataset. The mutational spectrum of the Russian subjects was significantly different (Adams and Skopek Monte Carlo test, P = 0. 004). Curiously, this younger Russian spectrum resembles that recovered from older individuals in the West. Specifically, A:T-->C:G transversions are significantly over-represented (Fisher's Exact test, P = 0.003) in the twin spectrum as compared to the young (age </= 35) Western spectrum. Even more noteworthy is the observation that 42% (23/55) of the base substitutions, almost double the expected value, have not been previously reported. These observations lead to the conclusion that this group of young Russian subjects has a mutational pattern which is distinct from the pattern of mutation observed in Western counterparts. The origin of this difference, whether related to genotoxic challenge, repair, or avoidance, cannot be distinguished, but diet and other lifestyle factors clearly may be responsible.
Human Mutation 01/2000; 15(5):439-46. · 5.69 Impact Factor
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ABSTRACT: The molecular analysis of T-lymphocytes from experienced cosmonauts and seven pairs of unexposed twins was performed [M. Khaidakov, D. Young, H. Erfle, A. Mortimer, Y. Voronkov, B.W. Glickman, Molecular analysis of mutations in T-lymphocytes from experienced soviet cosmonauts, Environ. Mol. Mutagen, 30 (1997) 21-30; J. Curry, G. Bebb, J. Moffat, D. Young, M. Khaidakov, A. Mortimer, B.W. Glickman, Similar mutant frequencies observed between monozygotic twins, Human Mutation, 9 (1997) 445-451]. Hprt mutant frequencies (MF) in both datasets were considerably higher (38.0+/-14.6x10(-6) in cosmonauts, and 18.5+/-8.9x10(-6) in twins) than in the background Western control (8-12x10(-6)), [A.D. Tates, F.J. van Dam, H. van Mossel, H. Shoemaker, J.C.P. Thijssen, V.M. Woldring, A.H. Zwinderman, A.T. Natarajan, Use of the clonal assay for the measurement of frequencies of HPRT mutants in T-lymphocytes from five control populations, Mutation Res., 253 (1991) 199-213; R.F. Branda, L.M. Sullivan, J.P. O'Neill, M.T. Falta, J.A. Nicklas, B. Hirsch, P.M. Vacek, R.J. Albertini, Measurement of HPRT mutant frequencies in T-lymphocytes from healthy human populations, Mutation Res., 285 (1993) 267-279]. The distribution of mutations by class in the twin dataset was essentially similar to the background Western control, whereas cosmonaut samples demonstrated a significant excess of splice errors and complex mutations. The distribution of base substitutions showed similar trends in both the cosmonaut and twin samples, which are quite distinct compared to those seen in the Western control. The differences observed between cosmonaut and twin samples (a 2-fold higher MF and an excess of complex mutations in cosmonaut mutational spectra) could be an indication of possible effects of the space environment. However, these changes could also be age-related because the twin group was, on average, 17 years younger. Moreover, very similar patterns of base substitution distribution in both datasets suggest the involvement of certain region-specific factors reflected in mutational spectra. In order to discriminate between occupation and region-specific factors contributing to mutagenesis, an additional study involving trainees and cosmonauts with recent long-term flight experience is required.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 01/2000; 430(2):337-42. · 2.85 Impact Factor
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ABSTRACT: We discuss the relevance to space medicine of studies concerning human genetic variation and consequent variable disease susceptibility or sensitivity between individuals. The size of astronaut and cosmonaut populations is both presently and cumulatively small, and despite the launch of the International Space Station, unlikely to increase by orders of magnitude within the foreseeable future. In addition, astronauts-cosmonauts constitute unrepresentative samples of their national populations. While the context of exposure for the astronaut-cosmonaut group is one unlikely to be replicated elsewhere than in space, aspects of specific exposures may be simulated by events such as occupational radiation exposure or radiation therapy. Hence, population-based studies of genetic susceptibility or sensitivity to disease, especially where it is precipitated by events that may simulate consequences of the space environment, likely will prove of value in assessing long-term health risks.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 01/2000; 430(2):293-8. · 2.85 Impact Factor
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ABSTRACT: Examination of the literature for hprt mutant frequencies from peripheral T cells yielded data from 1194 human subjects. Relationships between mutant frequency, age, sex, and smoking were examined, and the kinetics were described. Mutant frequency increases rapidly with age until about age 15. Afterward, the rate of increase falls such that after age 53, the hprt mutant frequency is largely stabilized. Sex had no effect on mutant frequency. Cigarette smoking increased mean mutant frequency compared to nonsmokers, but did not alter age vs. mutant frequency relationships. An hprt in vivo mutant database containing 795 human hprt mutants from 342 individuals was prepared. No difference in mutational spectra was observed comparing smokers to nonsmokers, confirming previous reports. Sex affected the frequency of deletions (>1 bp) that are recovered more than twice as frequently in females (P = 0. 008) compared to males. There is no indication of a significant shift in mutational spectra with age for individuals older than 19 yr, with the exception of A:T --> C:G transversions. These events are recovered more frequently in older individuals.
Genetics 07/1999; 152(3):1065-77. · 4.01 Impact Factor
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ABSTRACT: We treated transformed human fibroblasts with diphtheria toxin (DT) and isolated 40 single cells that were toxin resistant but unable to propagate. In 13 of them toxin resistance was associated with the presence of one or more aberrant transcripts of the structural gene for elongation factor 2 (EF-2). cDNA obtained from these transcripts had 164-447 bp-long deletions. Each of these deletions was associated with 2-8 base pairs-long repeats at its breakpoints. Only 10 out of 16 cDNA deletions were associated with presumed exon junctions. A role is suggested for errors in transcription in producing the aberrant transcripts which gave rise to the deletion-bearing cDNA species.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 11/1997; 379(2):109-15. · 2.85 Impact Factor
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ABSTRACT: Our laboratory has characterized several hundred mutant hprt cDNAs produced using Moloney murine leukemia reverse transcriptase to convert mRNA to cDNA. During the characterization of these mutants we have detected six T-lymphocyte mutants that demonstrate multiple G:C --> A:T transitions along the hprt cDNA coding sequence. Attempts to repeat the mRNA to cDNA conversion and subsequent characterization have demonstrated that the multiple transitions are likely artifacts. We suggest that reverse transcriptase is directly responsible for these multiple base substitutions and as such, that multiple mutations be viewed as suspect requiring confirmation at the genomic level.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 04/1997; 374(1):145-8. · 2.85 Impact Factor
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ABSTRACT: The development of radiological and nuclear technologies and the deployment of nuclear weapons have made ionizing radiation one of the most studied human mutagens. Exposure to ionizing radiation produces DNA damage which can result in mutation and cancer, making the risk associated with human exposure a critical issue. In this paper we estimate the risk associated with radiation exposure for individuals exposed to 137Cs during the 1987 Goiânia radiological accident. Using combined regression slopes from both the in vivo hprt mutant frequency and micronucleus frequency data we estimated a doubling dose of 173 (+/-47) cGy for these two endpoints. This is in close agreement with the published estimates for low dose rate and chronic exposure to low-LET radiation. We obtained risk estimates of about 24-fold increase in dominant disorders in the post-exposure generation of the directly exposed population. No detectable increase was found in the population at large. The risk of carcinogenesis in the directly exposed population was found to be increased by a factor in the range of 1.4 to 1.5. The small sample size in this study requires a large element of caution with respect to risk estimates interpretation. Moreover, the doubling dose estimates prepared here are derived from lymphocytes. This somatic data may require additional considerations for both cancer and certainly germ-line events. Nevertheless, the risk of carcinogenesis and genetic harm for this population are good indicators of the potential genetic damage imposed by ionizing radiation to the Goiânia population.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 03/1997; 373(2):207-14. · 2.85 Impact Factor
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ABSTRACT: The relative contribution of both genetic and environmental factors to spontaneous mutation frequency in humans is unknown. We have investigated the contribution of genetic factors to this phenomenon by determining the in vivo mutant frequency at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in circulating T-lymphocytes obtained from pairs of monozygotic twins. hprt mutant frequencies were determined three times over fourteen days in six sets of monozygotic male twins (mean age 30) taking part in a Russian Space Program inclined bed rest experiment. Blood samples were obtained prior to, during, and immediately following the experiment. Mononuclear cells were separated, frozen, and flown to Canada for analysis using the hprt T-lymphocyte clonal assay. There is no evidence within this data set to demonstrate that the period of inclined bed rest to simulate the effects of weightlessness had any effect on the observed mutant frequency. However, the average mutant frequency for the six sets of Russian twins was found to be three times higher than that of Western counterparts. More surprisingly, the spontaneous mutant frequency of monozygotic twins was found to be much more similar within pairs than between pairs of twins. These data suggest that the contribution of genetics in the determination of mutation frequency is substantial. However, whether high concordance within twin pairs reflects shared environmental experience as well as common genetic factors is not entirely clear. More data will be required to distinguish genetic from environmental factors and to determine the degree to which mutant frequency is genetically determined.
Human Mutation 02/1997; 9(5):445-51. · 5.69 Impact Factor
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ABSTRACT: We have characterized 54 HPRT- point mutations in T-lymphocytes from 17 individuals exposed to ionizing radiation of 137Cs in Goiania, Brazil and compared this spectrum to that of 30 HPRT- mutants from 9 unexposed Brazilian controls. The average internal exposure of the exposed group was 205 mCi, and the average external exposure was 1.7 Gy. The average HPRT- mutant frequency for the exposed group was 13.3 x 10(-5), approximately a 10-fold increase over the mutant frequency of the unexposed controls, which was 1.56 x 10(-5). The types of point mutations characterized included base substitutions, small deletions, frameshifts, insertions, complex mutations, and losses of exon sequences from the mRNA. The relative frequency of the different mutation types was similar in the two studied groups. However, in our study the distribution of events within the hprt coding sequence seemed to cluster at the same regions of the gene. These observations imply that the hprt gene does not present a homogeneous target to radiation mutagenesis, and perhaps this class of information may be used to detect radiation exposure in human populations.
Environmental and Molecular Mutagenesis 02/1997; 29(2):107-16. · 3.71 Impact Factor
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ABSTRACT: Mutational specificity describes the composite of all of the genetic alterations in a collection of mutations arising from a specific treatment. The information includes not only the nature of the genetic change (e.g., a base substitution or a frameshift), but also information about nucleotide position and hence the DNA context. As both the type of DNA damage and its position can be expected to reflect the nature of the chemical and physical mutagen, mutational specificity can be expected to provide insights into mechanisms of mutation. Conversely, mutational spectra should also provide insights into the identity of the mutagen. Indeed, the pioneering work on mutational specificity in Escherichia coli indicates that each physical or chemical treatment produces a unique spectrum of mutations. With the application of biotechnology to the field of genotoxicology, the database of sequenced mutations has become quite substantial. Both in vitro and in vivo data has been obtained following exposure to a variety of agents. In this communication we will critically assess whether the reality of mutational specificity has fulfilled the expectations and to examine what potential remains to be explored, especially in the area of monitoring human populations. The usefulness of both mutational spectra analysis and population monitoring with regards to chemoprevention are discussed.
Journal of cellular biochemistry. Supplement 02/1996; 25:99-107.
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ABSTRACT: We have examined the effects of ionizing radiation on somatic mutations in vivo, using the hprt clonal assay. The study was performed on blood samples obtained from children exposed during a radiological accident that happened in 1987, in Goiânia, Brazil. The group of children exposed to ionizing radiation includes six males and four females ranging in age from 6 to 14 years at the time of exposure. The radiation doses ranged from 15 to 70 cGy. A Brazilian control group, not exposed to ionizing radiation, was also analyzed under similar conditions. the mean hprt mutant frequency for the exposed group was 4.6 times higher than the control group, although the cloning efficiency from the exposed group was significantly reduced. Linear regression analysis of the mutant frequency and ionizing radiation dose did not show a significant relationship between these two parameters. However, a reliable inverse relationship was demonstrated when the regression analysis was performed with nonselective cloning efficiency and ionizing radiation dose. It was demonstrated that nonselective cloning efficiency diminishes as ionizing radiation dose increases. To correct mutant frequencies for clonal events, the clonal relationship between the hprt mutant clones was examined by T-cell receptor analysis. The majority of the mutants analyzed represented individual clones, thus validating the observed mutant frequencies.
Environmental and Molecular Mutagenesis 02/1996; 28(3):267-75. · 3.71 Impact Factor
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ABSTRACT: Modern technologies have provided the opportunity to monitor mutations in people in vivo. The subjects of this study were accidentally exposed to 137Cesium in a radiological accident that occurred in September 1987 in Goiânia, Brazil, during which more than 150 people received doses greater than 0.1 Gy and as high as 7 Gy. The objective of this study was to determine how long the hprt mutant T-cells in the peripheral blood contribute to mutant frequency by examining the time-course of the T-lymphocyte response to ionizing radiation. This report describes the results obtained over a period of 2.3 to 4.5 years subsequent to the accident, from 11 subjects with doses ranging from 1 to 7 Gy, and from nine control subjects selected from the same population. The mean In MF (+/- SE) of the control group was 2.5 (+/- 0.2) + In10(-6). The exposed group had a significantly increased mutant frequency; the mean In MF (+/- SE) were 3.3 (+/- 0.3) + In10(-6), 2.8 (+/- 0.2) + In10(-6), and 2.3 (+/- 0.2) + In10(-6), in the years 1990-1992 respectively. Based on the decline of mutant frequency and using Buckton's models [Buckton et al. (1967): Nature 214:470-473], we demonstrated that mutant T-cells have a short-term memory with a half-life of 2.1 years. This relatively short half-life limits the effective use of the hprt assay as the method of choice to monitor past exposure. The data also demonstrate a positive correlation with age, and an inverse correlation with plating efficiency.
Environmental and Molecular Mutagenesis 02/1996; 27(3):165-75. · 3.71 Impact Factor
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ABSTRACT: The mutagenic impact of various environmental and therapeutic agents can now be directly assayed in humans by the T-lymphocyte cloning assay. We have previously reported that following radioimmunoglobulin therapy, cancer patients exhibited increased mutant frequency of the hprt locus and an increased yield of large intergenic deletions compared to unexposed controls. Here we report the results of the analysis of 26 independent hprt mutations in nine cancer patients who underwent radioimmunoglobulin therapy. The majority of mutations (52%) had lost exon sequences from the mRNA. The remaining mutations were 20% small deletions and frameshifts and 28% base substitutions. The type of mutations observed were similar to those seen in unexposed controls. The site distribution of the mutations, however, indicates that some sequence contexts may be more sensitive to radiation mutagenesis than others.
Environmental and Molecular Mutagenesis 02/1995; 26(3):213-7. · 3.71 Impact Factor
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ABSTRACT: Using a T-lymphocyte clonal assay, 73 6-thioguanine resistant T-lymphocytes were isolated from two blood samples obtained 4 months apart from a 50-year-old male subject. Sixty-six of these mutants were characterized at the DNA sequence level using cDNA. One particular single base substitution was recovered a total of 23 times. The majority of T-cell receptors (TCR) of these mutants all share a common gamma-TCR rearrangement, and thus likely represent a single mutational event that underwent clonal expansion in vivo. Siblings of this clone were recovered in both collections. Three other single base substitutions were also recovered more than once. In two of the three cases, the mutants were also found to be clonally related, while in one case they were not. A number of identical exon loss events were also recovered, yet none of these were clonally related. This probably reflects the multiple pathways by which these mutations can arise. The TCR data was used to correct the observed mutant frequency to produce an estimate of the actual mutation frequency. The two mutant frequencies, 18 x 10(-6) and 19 x 10(-6), obtained from the first and second sampling periods, respectively, can thus be corrected to yield true mutation frequency's of 12 x 10(-6) each.
Environmental and Molecular Mutagenesis 02/1995; 25(3):167-79. · 3.71 Impact Factor
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ABSTRACT: Spontaneous or background mutation in mammals plays an important role in both medical and evolutionary contexts. However, establishing mutation frequencies or rates has not always been easy. When the field of mammalian mutagenesis was in its infancy, the word "variant" rather than "mutant" was often used because the genetic nature of the observed phenotypic alterations could not be adequately proven. Nowadays numerous target genes have been identified in which mutant frequencies can be measured, and occasionally even rates can be estimated. Indeed, the genetic basis for 'variants' now often comes from direct DNA sequencing. This review describes the most often used and best understood genetic markers for mutation research and examines their usefulness. In addition, mutational specificity is compared for several loci and the use of DNA-sequence data in determining the origins of spontaneous mutation is also discussed. An important observation is that spontaneous mutation frequencies of similarly sized genes can vary by more than an order of magnitude. Chromosomal location, the nature of the gene product and mutational specificity may offer a partial explanation.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 02/1994; 304(1):19-32. · 2.85 Impact Factor
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ABSTRACT: The nature of mutation at the HPRT locus in human T-lymphocytes in vivo is currently a subject of considerable interest. Determination of clonality in individual mutant T-lymphocytes is essential for the proper interpretation. This requires the molecular analysis of their respective T-cell receptors (TCR). We have developed a polymerase chain reaction (PCR)-based method for coamplification of hprt cDNA and the rearranged gamma T-cell receptor genes from crude cell lysates of individual 6-thioguanine resistant human T-lymphocytes. Following reverse transcription to produce hprt cDNA, the crude cell lysate is treated with proteinase K and subjected to a primary PCR with two sets of amplification primers, one specific for the hprt cDNA and the other for the rearranged gamma TCR gene. A secondary round of PCR, employing appropriate sets of nested amplification primers, are then used to produce sufficient quantities of DNA for both the sequencing and restriction fragment length analysis, of the hprt cDNA and gamma TCR gene respectively.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 09/1993; 288(2):269-75. · 2.85 Impact Factor
