Mitochondrial DNA mutation frequencies in experimentally irradiated compost worms, Eisenia fetida

The compost worm Eisenia fetida is routinely used in ecotoxicological studies. A standard assay to assess genetic damage in this species would be extremely valuable. Since mitochondrial DNA (mtDNA) is known to exhibit an increased mutation rate following exposure to ionising radiation we assessed the validity of a mtDNA-based assay for measuring increases in mutation rate in laboratory-irradiated compost worms. To this end the mutation frequency in the mtDNA of the compost worm E. fetida was quantified following in vivo gamma-irradiation of adult worms in three dose groups. Five adult worms exposed to 1.4 mGy/h for 55 days (total dose 1.85 Gy), five adult worms exposed to 8.5 mGy/h for 55 days (total dose 11.22 Gy) and five adult control worms were used to assess the effect of irradiation on mtDNA mutation induction. DNA samples extracted from irradiated adult worms were used in high-fidelity PCR of a 486 bp region of mtDNA spanning the ATPase 8 gene, chosen for its high spontaneous mutation rate. PCR products were cloned and sequenced to identify mutations, with 89-102 clones successfully sequenced per individual. A significant elevation in mtDNA mutation frequency (p=0.032) was seen in worms exposed at the higher dose rate (8.5 mGy/h, total dose 11.22 Gy; mutation frequency 27.98+/-4.85 x 10(-5)mutations/bp) in comparison to controls (mutation frequency 12.68+/-3.06 x 10(-5)mutations/bp), but no elevation in mutation frequency (p=0.764) was seen for the lower dose rate (1.4 mGy/h, total dose 1.85 Gy; mutation frequency 13.74+/-1.29 x 10(-5)mutations/bp) compared with controls. This indicates that although the technique has the potential to detect an elevation in mutation frequency, it does not have sufficient sensitivity at the doses likely to be encountered in environmental monitoring scenarios.     

The use of temporal temperature gradient gel electrophoresis to reveal mutations in peripheral blood mitochondrial DNA

The mutations in mitochondrial DNA (mtDNA) arise at a higher frequency than in nuclear DNA, and their appearance in peripheral blood can be considered as a sensitive marker to estimate the level of genotoxic load. For revealing the presence of mutations in mtDNA of peripheral blood, we used the method of temporal temperature gradient gel electrophoresis (TTGE). The samples of whole blood DNA from four donor groups were used. Group I contained 10 young (23-26 years) donors and Group II 12 elderly (65-74 years) donors. Group III was formed from patients with breast cancer (12 women) past sessions of radio-chemotherapies (RCHT). Group IV was made of professionals of a nucleus plant occupationally exposed to chronic gamma-irradiation. PCR was carried out on four coding sequences and on one hypervariable sequence of the D-loop (DloopI) of mtDNA. PCR products were tested with TTGE. Most mutations were revealed in the DloopI. Heteroplasmy in the region of DloopI was registered in the blood of each donor of Group III 7 days after the RCHT session. Also, mutations in mtDNA Dloop1 were found in 6 of 13 individuals of Group IV. The blood of this donor group was taken 16 to 28 years after prolonged irradiations in a dose range of 250-350 cGy. In the elderly donor group, the same results were observed in 3 of 12 individuals. The results show that the method of TTGE can be used in mass analyses to assess the effects of radiation and other genotoxic agents in man by detection of unknown mutations in peripheral blood mtDNA.     

Microsatellite mutations show no increases in the children of the Chernobyl liquidators

We performed a study on Belarusian "liquidators", exploring whether increase in the frequencies of germline mutations at microsatellite loci could be found in their progeny. The liquidators, mostly young males, were those involved (during 1986 and 1987) in clean-up operations in the radioactively contaminated area following the Chernobyl nuclear power plant accident in 1986. Many liquidators fathered children during the clean-up period and after the work had been terminated. The numbers of families studied were 64 (liquidators) and 66 (controls). A total of 72 loci (31 autosomal, one X-linked and 40 Y-linked) were used. DNA was isolated from peripheral blood lymphocytes and the microsatellite loci were amplified by the polymerase chain reaction with fluorescence-labelled primers. Mutations were detected as variations in the length of the loci. At the Y-linked loci, the mutation rates (expressed as number of mutations among the total number of loci for the individuals included) are 2.9 x 10(-3) (4/1392) and 2.1 x 10(-3) (3/1458) in the children of exposed and control parents, respectively. This difference is not statistically significant. At the autosomal loci, the corresponding estimates are 5.9 x 10(-3) (11/1862; exposed group) and 8.5 x 10(-3) (18/2108; control). Again, the difference is not significant. The possibility that the Belarusian population might have been unexpectedly exposed to some chemical contaminants in the environment appears unlikely in view of the finding that the spontaneous mutation rates at the same set of loci in several non-Belarusian populations were similar to those in Belarus. The estimated mean radiation dose to the liquidators was small, being about 39 mSv, and this might be one reason why no increases in mutation rates due to radiation could be found.     

Analysis of heteroplasmy in the major noncoding region of mitochondrial DNA in the blood and atherosclerotic plaques of carotid arteries

For identification of somatic mitochondrial DNA (mtDNA) mutations, the mtDNA major noncoding region (D-loop) sequence in blood samples and carotid atherosclerosis plaques from patients with atherosclerosis was analyzed. Five point heteroplasmic positions were observed in 4 of 23 individuals (17%). Only in two cases could heteroplasmy have resulted from somatic mutation, whereas three heteroplasmic positions were found in both vascular tissue and blood. In addition, length heteroplasmy in a polycytosine stretches was registered at nucleotide positions 303–315 in 16 individuals, and also in the 16184–16193 region in four patients. The results suggest that somatic mtDNA mutations can occur during atherosclerosis, but some heteroplasmic mutations may appear in all tissues, possibly being inherited.     

Search for mutations altering protein charge and/or function in children of atomic bomb survivors: final report.

A sample of (1) children whose parents had been proximally exposed (i.e., less than 2,000 m from the hypocenter) at the time of the atomic bombings of Hiroshima and Nagasaki and (2) a suitable comparison group have been examined for the occurrence of mutations altering the electrophoretic mobility or activity of a series of 30 proteins. The examination of the equivalent of 667,404 locus products in the children of proximally exposed persons yielded three mutations altering electrophoretic mobility; the corresponding figure for the comparison group was three mutations in 466,881 tests. The examination of a subset of 60,529 locus products for loss of enzyme activity in the children of proximally exposed persons yielded one mutation; no mutations were encountered in 61,741 determinations on the children of the comparison group. When these two series are compared, the mutation rate observed in the children of proximally exposed persons is thus 0.60 x 10(-5)/locus/generation, with 95% confidence intervals between 0.2 and 1.5 x 10(-5), and that in the comparison children is 0.64 x 10(-5)/locus/generation, with 95% intervals between 0.1 and 1.9 x 10(-5). The average conjoint gonad doses for the proximally exposed parents are estimated to be 0.437 Gy of gamma radiation and 0.002 Gy of neutron radiation. If a relative biological effectiveness of 20 is assigned to the neutron radiation, the combined total gonad dose for the parents becomes 0.477 Sv. (Organ absorbed doses are expressed in gray [1 Gy = 100 rad]; where dose is a mixture of gamma and neutron radiation, it is necessary because of the differing relative biological effectiveness of gamma and neutron radiation to express the combined gamma-neutron gonad exposures in sieverts [1 Sv = 100 rem]).     

Active site mutation in DNA polymerase gamma associated with progressive external ophthalmoplegia causes error-prone DNA synthesis

Progressive external ophthalmoplegia (PEO) is a heritable mitochondrial disorder characterized by the accumulation of multiple point mutations and large deletions in mtDNA. Autosomal dominant PEO was recently shown to co-segregate with a heterozygous Y955C mutation in the human gene encoding the sole mitochondrial DNA polymerase, DNA polymerase gamma (pol gamma). Since Tyr-955 is a highly conserved residue critical for nucleotide recognition among family A DNA polymerases, we analyzed the effects of the Y955C mutation on the kinetics and fidelity of DNA synthesis by the purified human mutant polymerase in complex with its accessory subunit. The Y955C enzyme retains a wild-type catalytic rate (k(cat)) but suffers a 45-fold decrease in apparent binding affinity for the incoming nucleoside triphosphate (K(m)). The Y955C derivative is 2-fold less accurate for base pair substitutions than wild-type pol gamma despite the action of intrinsic exonucleolytic proofreading. The full mutator effect of the Y955C substitution was revealed by genetic inactivation of the exonuclease, and error rates for certain mismatches were elevated by 10-100-fold. The error-prone DNA synthesis observed for the Y955C pol gamma is consistent with the accumulation of mtDNA mutations in patients with PEO.     

Radiation-induced germline mutations detected by a direct comparison of parents and first-generation offspring DNA sequences containing SNPs

Germline mutation induction has been detected in mice but not in humans. To estimate the genetic risk of germline mutation induction in humans, new techniques for extrapolating from animal data to humans or directly detecting radiation-induced mutations in man are expected to be developed. We have developed a new method to detect germline mutations by directly comparing the DNA sequences of parents and first-generation offspring. C3H male mice were irradiated with gamma-rays of 3, 2 and 1 Gy and 3 weeks later were mated with C57BL female mice of the same age. The nucleotide sequences of 160 UniSTS markers containing 300-900 bp and SNPs of the DNA of parent and offspring mice were determined by direct sequencing. At each dose of radiation, a total of 5 Mb DNA sequences were examined for radiation-induced mutations. We found 7 deletions in 3 Gy-irradiated mice, 1 deletion in 2 Gy-irradiated mice, 1 deletion in 1 Gy-irradiated mice and no mutations in control mice. The maximum mutation frequency was 2.0 x 10(-4)/locus/Gy at 3 Gy, and these results suggested that a non-linear increase of mutations with dose.     

The use of next generation sequencing technology to study the effect of radiation therapy on mitochondrial DNA mutation

The human mitochondrial genome has an exclusively maternal mode of inheritance. Mitochondrial DNA (mtDNA) is particularly vulnerable to environmental insults due in part to an underdeveloped DNA repair system, limited to base excision and homologous recombination repair. Radiation exposure to the ovaries may cause mtDNA mutations in oocytes, which may in turn be transmitted to offspring. We hypothesized that the children of female cancer survivors who received radiation therapy may have an increased rate of mtDNA heteroplasmy mutations, which conceivably could increase their risk of developing cancer and other diseases. We evaluated 44 DNA blood samples from 17 Danish and 1 Finnish families (18 mothers and 26 children). All mothers had been treated for cancer as children and radiation doses to their ovaries were determined based on medical records and computational models. DNA samples were sequenced for the entire mitochondrial genome using the Illumina GAII system. Mother's age at sample collection was positively correlated with mtDNA heteroplasmy mutations. There was evidence of heteroplasmy inheritance in that 9 of the 18 families had at least one child who inherited at least one heteroplasmy site from his or her mother. No significant difference in single nucleotide polymorphisms between mother and offspring, however, was observed. Radiation therapy dose to ovaries also was not significantly associated with the heteroplasmy mutation rate among mothers and children. No evidence was found that radiotherapy for pediatric cancer is associated with the mitochondrial genome mutation rate in female cancer survivors and their children.     

Radiation-induced untargeted germline mutations in Japanese medaka

Radiation has been shown to increase mutation frequencies at tandem repeat loci by indirect interactions of radiation with DNA. We studied germline mutations in chronically exposed Japanese medaka (Oryzias latipes) using microsatellite loci. After screening 26 randomly selected loci among unirradiated parents and their 200 offspring, we selected seven highly mutable loci (0.5-1.0 x 10(-2) mutants per locus per gamete) and two bonus loci for further study. To determine if radiation exposure increases mutation frequencies in these loci, medaka were chronically irradiated from subadults through maturation at relatively low dose rates of 68 mGy/d. Total doses for males and females were 10.4 and 3 Gy, respectively. The mean number of mutations for the offspring of exposed families (0.149+/-0.044) was significantly higher (P=0.018) than for control families (0.080+/-0.028), indicating induction of germline mutations from chronic irradiation. This increase in the microsatellite mutation rate is greater than expected from direct interaction of radiation with DNA, suggesting indirect, untargeted mechanism(s) for mutations. This study identified microsatellite loci with a high mutational background in medaka, variation among loci and families as important variables, and demonstrated the usefulness of this fish model for studying radiation-induced germline mutations.     

The pedigree rate of sequence divergence in the human mitochondrial genome: there is a difference between phylogenetic and pedigree rates

We have extended our previous analysis of the pedigree rate of control-region divergence in the human mitochondrial genome. One new germline mutation in the mitochondrial DNA (mtDNA) control region was detected among 185 transmission events (generations) from five Leber hereditary optic neuropathy (LHON) pedigrees. Pooling the LHON pedigree analyses yields a control-region divergence rate of 1.0 mutation/bp/10(6) years (Myr). When the results from eight published studies that used a similar approach were pooled with the LHON pedigree studies, totaling >2,600 transmission events, a pedigree divergence rate of 0.95 mutations/bp/Myr for the control region was obtained with a 99.5% confidence interval of 0.53-1.57. Taken together, the cumulative results support the original conclusion that the pedigree divergence rate for the control region is approximately 10-fold higher than that obtained with phylogenetic analyses. There is no evidence that any one factor explains this discrepancy, and the possible roles of mutational hotspots (rate heterogeneity), selection, and random genetic drift and the limitations of phylogenetic approaches to deal with high levels of homoplasy are discussed. In addition, we have extended our pedigree analysis of divergence in the mtDNA coding region. Finally, divergence of complete mtDNA sequences was analyzed in two tissues, white blood cells and skeletal muscle, from each of 17 individuals. In three of these individuals, there were four instances in which an mtDNA mutation was found in one tissue but not in the other. These results are discussed in terms of the occurrence of somatic mtDNA mutations.     

Extracellular mutant mitochondrial DNA content is dramatically elevated in the blood plasma of irradiated mice

Nucleic acids circulating in blood plasma and other biological fluids are of interest as potential markers for the diagnosis of various pathologies and the monitoring of stresses. Mitochondrial DNA (mtDNA) is a more vulnerable target for many genotoxic agents than nuclear DNA, and mutations in the mitochondrial genome can serve as markers for many diseases. In the present study, extracellular mtDNA with mutations was assayed in the blood plasma of mice exposed to X radiation at a dose of 5 Gy. For this purpose, heteroduplexes obtained by the hybridization of mtDNA PCR amplicons (ND3 gene and D loop region) from the blood plasma of irradiated and control mice were cleaved with CEL endonuclease, a mismatch-specific enzyme. The total amount of mtDNA (ND4 gene) copies vs. nuclear DNA (GAPDH gene) was measured by real-time PCR. The content of mtDNA with mutations in murine blood plasma remained high within one month after irradiation but varied with time. The measurements were performed on days 1, 4, 8, 14, and 28 after irradiation, and the maximum level was detected on day 14. The elevated content of extracellular mutant mtDNA in blood plasma of X-irradiated mice is a sensitive candidate biomarker for the assessment of radiation injury and effects of other genotoxic agents.     

Somatic mutations in the D-loop and decrease in the copy number of mitochondrial DNA in human hepatocellular carcinoma

Somatic mutations in mitochondrial DNA (mtDNA) have been detected in many human cancers, including hepatocellular carcinoma (HCC). The D-loop region was found to be a "hot spot" for mutation in mtDNA of the tumors. However, effects of the D-loop mutations on the copy number of mtDNA in tumor tissues are poorly understood. Using direct sequencing, we examined mutations in the D-loop region of mtDNA in 61 HCCs and the corresponding non-tumor liver tissues. The results revealed that 39.3% of the HCCs carried somatic mutation(s) in the D-loop of mtDNA, and most of these mutations were homoplasmic. Moreover, 37.0% (10/27) of these mutations were T-to-C and G-to-A transitions and 40.7% (11/27) of them were located in the polycytidine stretch between nucleotide position (np) 303 and 309 of mtDNA. In addition, we found that mtDNA copy number of HCC was significantly decreased in 60.5% of the patients with hepatoma, especially in those with somatic mutation(s) in the D-loop of mtDNA (17/24). This decrease in mtDNA copy number was highly associated with the occurrence of point mutations near the replication origin of the heavy-strand of mtDNA. Interestingly, we found that 42.9% (6/14) of the HCCs without mutation in the D-loop had a reduced copy number of mtDNA, indicating that other unidentified factors involved in mitochondrial biogenesis might be defective in the tumor. The results obtained in this study strongly suggest that somatic mutations in the D-loop together with the decrease in the copy number of mtDNA may be an important event during the early phase of liver carcinogenesis.     

TT virus is distributed in various leukocyte subpopulations at distinct levels, with the highest viral load in granulocytes.

When TT virus (TTV) DNA was quantitated in whole blood and plasma aliquots from 27 viremic individuals by real-time detection PCR that can detect essentially all TTV genotypes, the TTV load was 6.9 +/- 3.5 (mean +/- standard deviation)-fold higher in the whole blood than in the plasma samples [P < 0.002 (paired t test)]. To clarify the reason for this difference, peripheral blood cells of various types including red blood cells, granulocytes (CD15+), B cells (CD19+), T cells (CD3+), monocytes (CD14+), and NK cells (CD3-/CD56+) were separated at a purity of 95.4-99.5% from each of three infected individuals with relatively high TTV viremia, and their TTV viral loads were determined. Red blood cells were uniformly negative, but the other cell types were positive for TTV DNA at various titers. In all three patients, the highest TTV load was found in granulocytes (4.2 x 10(4)-3.1 x 10(5) copies/10(6) cells), followed by monocytes (1.4-2.2 x 10(4) copies/10(6) cells) and NK cells (5.4-6.5 x 10(3) copies/10(6) cells); B and T cells were positive, with a low viral load (6.7 x 10(1)-2.7 x 10(3) copies/10(6) cells). These results indicate that TTV is distributed in various peripheral blood cell types at distinct levels, with the highest viral load in granulocytes, and that a significant proportion of the TTV DNA in peripheral blood is not identified by the standard plasma/serum DNA detection methods.     

Highly efficient random mutagenesis in transcription-reverse-transcription cycles by a hydrogen bond ambivalent nucleoside 5'-triphosphate analogue: potential candidates for a selective anti-retroviral therapy

The nucleoside P can base pair with both A and G. We evaluated the mutation frequency induced by the 5'-triphospbate of the ribonucleoside P (PTP) in an in vitro retroviral replication model. After 4 cycles of replication in the presence of PTP, the mutation frequency was raised to 3.8 x 10(-2) per nucleotide and C-to-U and U-to-C mutations were dominantly observed. These results suggest that ambivalent NTP analogues, like PTP, could induce mutations beyond the error threshold of retroviruses.     

The mutation rate of the human mtDNA deletion mtDNA4977.

The human mitochondrial mutation mtDNA4977 is a 4,977-bp deletion that originates between two 13-bp direct repeats. We grew 220 colonies of cells, each from a single human cell. For each colony, we counted the number of cells and amplified the DNA by PCR to test for the presence of a deletion. To estimate the mutation fate, we used a model that describes the relationship between the mutation rate and the probability that a colony of a given size will contain no mutants, taking into account such factors as possible mitochondrial turnover and mistyping due to PCR error. We estimate that the mutation rate for mtDNA4977 in cultured human cells is 5.95 x 10(-8) per mitochondrial genome replication. This method can be applied to specific chromosomal, as well as mitochondrial, mutations.     

Mitochondrial DNA mutation in a Chinese family with myoclonic epilepsy and ragged-red fiber disease

We analyzed the mitochondrial DNA of blood cells of 5 patients from a Chinese family with myoclonic epilepsy and ragged-red fiber disease. The results showed that in all the affected individuals there was a point mutation from A to G at the 8344th nucleotide pair, which was located in the tRNA(Lys) gene. No such a mutation was found in mtDNA of either unaffected members of that family or other healthy Chinese subjects. These findings are consistent with the recent report of Shoffner et al. (Cell 1990, 61: 931-937), and confirm that the point mutation is indeed the cause of this disease.     

The Effect of DNA Sequence Polymorphisms on Intragenic Recombination in the Rosy Locus of Drosophila Melanogaster

The effect of simple DNA sequence polymorphisms on intragenic recombination in the rosy locus of Drosophila melanogaster was assayed. Two crosses were performed involving nearly identical molecular distances between selective ry null mutations (3778 nucleotides and 3972 nucleotides). In one heterozygote (ry606/ry531), in addition to the nucleotide substitution ry- mutations, there were 11 simple nucleotide polymorphisms between the selective markers as well as additional flanking simple nucleotide polymorphisms within the rosy locus. In the other heterozygote (ry606/ry609), there were no additional polymorphisms because the two rosy nucleotide substitution mutations were induced on the same rosy isoallele (ry+6). From ry606/ry531 heterozygous females, 27 intragenic crossovers and five marker conversions were seen among 4.53 x 10(5) progeny. From ry606/ry609 heterozygous females, 23 intragenic crossovers and eight marker conversions were seen among 4.18 x 10(5) progeny. The intragenic crossover frequencies per kilobase of DNA were very similar, 1.6 x 10(-5) for ry606/ry531 and 1.4 x 10(-5) for ry606/ry609. Thus, simple DNA sequence polymorphisms neither inhibit nor promote intragenic recombination in D. melanogaster.     

High mutation rates in the mitochondrial genomes of Daphnia pulex

Despite the great utility of mitochondrial DNA (mtDNA) sequence data in population genetics and phylogenetics, key parameters describing the process of mitochondrial mutation (e.g., the rate and spectrum of mutational change) are based on few direct estimates. Furthermore, the variation in the mtDNA mutation process within species or between lineages with contrasting reproductive strategies remains poorly understood. In this study, we directly estimate the mtDNA mutation rate and spectrum using Daphnia pulex mutation-accumulation (MA) lines derived from sexual (cyclically parthenogenetic) and asexual (obligately parthenogenetic) lineages. The nearly complete mitochondrial genome sequences of 82 sexual and 47 asexual MA lines reveal high mtDNA mutation rate of 1.37 × 10(-7) and 1.73 × 10(-7) per nucleotide per generation, respectively. The Daphnia mtDNA mutation rate is among the highest in eukaryotes, and its spectrum is dominated by insertions and deletions (70%), largely due to the presence of mutational hotspots at homopolymeric nucleotide stretches. Maximum likelihood estimates of the Daphnia mitochondrial effective population size reveal that between five and ten copies of mitochondrial genomes are transmitted per female per generation. Comparison between sexual and asexual lineages reveals no statistically different mutation rates and highly similar mutation spectra.