Minisatellite germline mutation rate in the Techa River population

Germline mutation at eight minisatellite loci has been studied among the irradiated families from the Techa River population and non-exposed families from the rural area of the Chelyabinsk and Kurgan Oblasts. The groups were matched by ethnicity, parental age, occupation and smoking habit. A statistically significant 1.7-fold increase in mutation rate was found in the germline of irradiated fathers, whereas maternal germline mutation rate in the exposed families was not elevated. Most of the minisatellite loci showed an elevated paternal mutation rate in the exposed group, indicating a generalised increase in minisatellite germline mutation rate in the Techa River population. These data suggest that the elevated minisatellite mutation rate can be attributed to radioactive exposure. The spectra of paternal mutation seen in the unexposed and exposed families were indistinguishable.     

Long-term genetic effects of radiation exposure

To date, there has been little experimental knowledge on the genetic risks of human exposure to ionising radiation for humans. Recent data suggest that hypervariable tandem repeat minisatellite loci provide a useful and sensitive experimental approach for monitoring radiation-induced germline mutation in humans. Here, I review the results of studies on minisatellite mutation rates in human populations exposed to radioactive fallout after the Chernobyl accident and nuclear weapon tests in Kazakhstan.     

Hereditary minisatellite mutations among the offspring of Estonian Chernobyl cleanup workers

A single accidental event such as the fallout released from the Chernobyl reactor in 1986 can expose millions of people to non-natural environmental radiation. Ionizing radiation increases the frequency of germline mutations in experimental studies, but the genetic effects of radiation in humans remain largely undefined. To evaluate the hereditary effects of low radiation doses, we compared the minisatellite mutation rates of 155 children born to Estonian Chernobyl cleanup workers after the accident with those of their siblings born prior to it. All together, 94 de novo paternal minisatellite mutations were found at eight tested loci (52 and 42 mutants among children born after and before the accident, respectively). The minisatellite mutation rate was nonsignificantly increased among children born after the accident (0.042 compared to 0.036, OR 1.33, 95% CI 0.80-2.20). Furthermore, there was some indication of an increased mutation rate among offspring born after the accident to workers who had received doses of 20 cSv or above compared with their siblings born before the accident (OR 3.0, 95% CI 0.97-9.30). The mutation rate was not associated with the father's age (OR 1.04, 95% CI 0.94-1.15) or the sex of the child (OR 0.95, 95% CI 0.50-1.79). Our results are consistent with both no effect of radiation on minisatellite mutations and a slight increase at dose levels exceeding 20 cSv.     

Mini-and microsatellite mutations in children from Chernobyl accident cleanup workers

Knowledge about possible genotoxic effects of low-dose radiation on the human germline is limited and relies primarily on extrapolations from high-dose exposures. To test whether ionizing radiation can cause paternal genetic mutations that are transmitted to offspring, we enrolled families of 88 Chernobyl cleanup workers exposed to ionizing radiation. We analyzed DNA isolated from lymphocytes for mutations via DNA blotting with the multi-locus minisatellite probes 33.6 and 33.15 and via PCR in a panel of six tetranucleotide repeats. Children conceived before and children conceived after their father's exposure showed no statistically significant differences in mutation frequencies. We saw an increase in germline microsatellite mutations after radiation exposure that was not statistically significant. We found no dependence of mutation rate on increasing exposure. A novel finding was that the tetranucleotide marker D7S1482 demonstrated germline hypermutability. In conclusion, our results do not support an increased level of germline minisatellite mutations but suggest a modest increase in germline mutations in tetranucleotide repeats. Small sample size, however, limited statistical power.     

Radiation and mutation induction in the human germline

This review describes the results of some publications on monitoring radiation-induced mutation in the human germline. Recent data have shown that minisatellite loci provide a useful and sensitive experimental approach for monitoring radiation-induced mutation in humans. The progress made in validating this approach and the results of recent publications on the analysis of minisatellite mutation rates in the irradiated families are presented.     

The use of synthetic tandem repeats to isolate new VNTR loci: cloning of a human hypermutable sequence.

Synthetic tandem repeats (STRs) of oligonucleotides have previously been shown to detect polymorphic loci in the human genome. Here, we report results from the use of three such probes to screen a human cosmid library. Nine of the 45 positive clones that were analyzed appear to contain highly polymorphic minisatellite or VNTR loci. The degree of enrichment for minisatellite sequences varied with the choice of STR: one provided a 15- to 20-fold enrichment (4 polymorphic loci among 10 clones), whereas 2 others gave a 3- to 5-fold enrichment (5 polymorphic probes in a total of 35 clones) compared to random screening. The 9 VNTR markers have been localized by linkage analysis in the CEPH panel and/or by in situ hybridization. Eight probes identify new loci, one of which maps to an interstitial region. One of the VNTR loci (identified by probe CEB1) was found to be hypermutable, with 52 mutation events identified among 310 children characterized in 40 CEPH families. The parental origin of the mutation could be identified in all instances, and only one mutation was found to be of maternal origin. The mutation rate in males was estimated to be approximately 15%. Segregation analysis of flanking markers suggests that mutations are not associated with crossing over. As the only previously described hypermutable minisatellite loci in humans have equal rates of male and female mutations, these observations establish that a second type of hypermutable minisatellite exists in the human genome. In neither case does the generation of new alleles appear to be associated with unequal crossing over.     

Somatic versus germline mutation processes at minisatellite CEB1 (D2S90) in humans and transgenic mice

The most variable human minisatellites show extreme germline instability dominated by complex intra-allelic rearrangements plus a lower frequency of inter-allelic transfers of repeat units. In contrast, little is known about somatic instability at such loci. We have therefore used single-molecule PCR to analyze mutation at minisatellite CEB1 (D2S90) in human blood DNA. Somatic mutants were rare and involved only relatively simple intra-allelic events, with no bias toward expansions, in sharp contrast to the complex gain-biased rearrangements seen in sperm. Somatic and germline mutation processes were further analyzed in mice transgenic for a cosmid insert containing CEB1. Mutant molecules in transgenic sperm and blood were detected but only at the low frequencies seen in human blood and arose mainly by simple duplications and deletions as seen for somatic mutations in human. These data suggest distinct pathways for germline and somatic CEB1 mutations with germline instability involving recombination-based repair of meiotic double-strand breaks and somatic mutation arising by replication slippage or mitotic recombination. The problem of transferring germline-specific features of minisatellite instability from human to mouse suggests, with other recent observations, that long-range chromatin conformation may be required for the recombination-based mode of germline instability at human minisatellites.     

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.     

Lack of effects of atomic bomb radiation on genetic instability of tandem-repetitive elements in human germ cells.

In a pilot study to detect the potential effects of atomic bomb radiation on germ-line instability, we screened 64 children from 50 exposed families and 60 from 50 control families for mutations at six minisatellite loci by using Southern blot analysis with Pc-1, lambda TM-18, ChdTC-15, p lambda 3, lambda MS-1, and CEB-1 probes. In the exposed families, one or both parents received a radiation dose > 0.01 Sv. Among the 64 children, only one child had parents who were both exposed. Thus, of a total of 128 gametes that produced the 64 children, 65 gametes were derived from exposed parents and 63 were from unexposed parents, the latter being included in a group of 183 unexposed gametes used for calculating mutation rates. The average parental gonadal dose for the 65 gametes was 1.9 Sv. We detected a total of 28 mutations at the p lambda g3, lambda MS-1, and CEB-1 loci, but no mutations at the Pc-1, lambda TM-18, and ChdTC-15 loci. We detected 6 mutations in 390 alleles of the 65 exposed gametes and 22 mutations in 1098 alleles of the 183 gametes from the unexposed parents. The mean mutation rate per locus per gamete in these six minisatellite loci was 1.5% in the exposed parents and 2.0% in the unexposed parents. We observed no significant difference in mutation rates in the children of the exposed and the unexposed parents (P = .37, Fisher's exact probability test).     

Mechanisms of human minisatellite mutation in yeast

Minisatellites are tandem repeat loci, with repeat units ranging in size from 5 bp to 100 bp. The total lengths of repeat arrays vary from about 0.5 kb to 30 kb, and excessive variability in allele length at human minisatellite loci is the result of germline-specific complex recombination events generating new length alleles. Minisatellite alleles also mutate to new lengths in somatic cells, but this occurs at a much lower rate than in the germline. Since recombination is involved in minisatellite mutation, the yeast Saccharomyces cerevisiae is a suitable model organism that has been employed to further dissect the molecular basis of mutation events at human minisatellites. These studies have shown that the mutational behaviour of a minisatellite in meiosis is not determined by the intrinsic properties of the repeat array, but are highly dependent on the position of the minisatellite in the genome. The processes for minisatellite mutation in yeast and humans are identical in the sense that mutation is indeed driven by meiotic recombination, but differ with regard to the types of structural changes that are generated by the recombination events. Tetrad analyses showed that inter-allelic transfers of repeats occur by conversion and not crossing over, and that several chromatids can be involved in successive recombination events in one meiosis, resulting in mutant alleles in several spores. It has been demonstrated that the genes SPO11 and RAD50, involved in the initiation of recombination events, are required for human minisatellite mutation in yeast meiosis. Intrinsic properties of the repeat array appear to determine the stability of human minisatellites in yeast mitosis, since mitotic mutation rates in yeast are highly variable between minisatellites. The repair genes RAD27 and DNA2 stabilise human minisatellites in yeast mitosis, while RAD5 has no effect on mitotic stability. MSH2 depresses human minisatellite frequency in meiotic cells of yeast.     

The effects of maternal irradiation during adulthood on mutation induction and transgenerational instability in mice

The long-term genetic effects of maternal irradiation remain poorly understood. To establish the effects of radiation exposure on mutation induction in the germline of directly exposed females and the possibility of transgenerational effects in their non-exposed offspring, adult female BALB/c and CBA/Ca mice were given 1Gy of acute X-rays and mated with control males. The frequency of mutation at expanded simple tandem repeat (ESTR) loci in the germline of directly exposed females did not differ from that of controls. Using a single-molecule PCR approach, ESTR mutation frequency was also established for both germline and somatic tissues in the first-generation offspring of irradiated parents. While the frequency of ESTR mutation in the offspring of irradiated males was significantly elevated, maternal irradiation did not affect stability in their F(1) offspring. Considering these data and the results of our previous study, we propose that, in sharp contrast to paternal exposure to ionising radiation, the transgenerational effects of maternal high-dose acute irradiation are likely to be negligible.     

Hypermutable minisatellites,a human affair?

Minisatellites are a class of highly polymorphic GC-rich tandem repeats. They include some of the most variable loci in the human genome, with mutation rates ranging from 0.5% to >20% per generation. Structurally, they consist of 10- to 100-bp intermingled variant repeats, making them ideal tools for dissecting mechanisms of instability at tandem repeats. Distinct mutation processes generate rare intra-allelic somatic events and frequent complex conversion-like germline mutations in these repeats. Furthermore, turnover of repeats at human minisatellites is controlled by intense recombinational activity in DNA flanking the repeat array. Surprisingly, whereas other mammalian genomes possess minisatellite-like sequences, hypermutable loci have not been identified that suggest human-specific turnover processes at minisatellite arrays. Attempts to transfer minisatellite germline instability to the mouse have failed. However, yeast models are now revealing valuable information regarding the mechanisms regulating instability at these tandem repeats. Finally, minisatellites and tandem repeats provide exquisitely sensitive molecular tools to detect genomic insults such as ionizing radiation exposure. Surprisingly, by a mechanism that remains elusive, there are transgenerational increases in minisatellite instability.     

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.     

Analysis of somatic mutations at human minisatellite loci in tumors and cell lines

Hypervariable human minisatellite loci show a substantial level of germline instability, and spontaneous mutation rates to new length alleles have been measured directly by pedigree analysis. We now show that mutation events altering the number of minisatellite repeat units are not restricted to the germline, but also arise in other tissues. Mutant alleles can be detected at a very low frequency in lymphoblastoid cell lines and at much higher frequencies in clonal tumor cell populations, most particularly in gastrointestinal adenocarcinomas. Mutant alleles in these tumors are usually present at a dosage equal to or greater than that of the progenitor allele, indicating that most or all of the tumor cells carry the same clonally derived mutant allele. As with germline mutation, the incidence of somatic mutations in tumors varies from locus to locus, with the same locus showing the highest level of germline and somatic instability. Most length changes, as those in the germline, are of only a few repeat units; however, very large changes are also observed, implying that such mutations can occur in the absence of meiosis.     

Induction of minisatellite mutations in the mouse germline by low-dose chronic exposure to gamma-radiation and fission neutrons

Germline mutation induction at mouse minisatellite loci by paternal low-dose (0.125-1 Gy) exposure to chronic (1.66 x 10(-4) Gy min(-1)) low-linear energy transfer (low-LET) gamma-irradiation and high-LET fission neutrons (0.003 Gy min(-1)) was studied at pre-meiotic stages of spermatogenesis. Both types of radiation produced linear dose-response curves for mutation of the paternal allele. In contrast to previous results using higher doses, the pattern of induction of minisatellite mutation after chronic gamma-irradiation was similar to acute (0.5 Gy min(-1)) exposure to X-rays, indicating that the elevated mutation rate was independent of the ability of the cell to repair damage induced immediately or over a period of up to 100 h. Chronic exposure to fission neutrons was more effective than acute or chronic low-LET exposure (relative biological effectiveness, RBE=3.36). The data also provide strong support for the previous conclusion that increases in minisatellite mutation rate are not caused by radiation-induced DNA damage at minisatellite loci themselves, but rather from damage induced by ionising radiation elsewhere in the genome/cell.     

Repeat instability at human minisatellites arising from meiotic recombination.

Little is known about the role of meiotic recombination processes such as unequal crossover in driving instability at tandem repeat DNA. Methods have therefore been developed to detect meiotic crossovers within two different GC-rich minisatellite repeat arrays in humans, both in families and in sperm DNA. Both loci normally mutate in the germline by complex conversion-like transfer of repeats between alleles. Analysis shows that inter-allelic unequal crossovers also occur at both loci, although at low frequency, to yield simple recombinant repeat arrays with exchange of flanking markers. Equal crossovers between aligned alleles, resulting in recombinant alleles but without change in repeat copy number, also occur in sperm at a similar frequency to unequal crossovers. Both crossover and conversion show polarity in the repeat array and are co-suppressed in an allele showing unusual germline stability. This provides evidence that minisatellite conversion and crossover arise by a common mechanism, perhaps by alternative processing of a meiotic recombination initiation complex, and implies that minisatellite instability is a by-product of meiotic recombination in repeat DNA. While minisatellite recombination is infrequent, crossover rates indicate that the unstable end of a human minisatellite can act as a recombination warm-spot, even between sequence-heterologous alleles.     

Meiotic interallelic conversion at the human minisatellite MS32 in yeast triggers recombination in several chromatids

Tandem repetitive DNA sequences such as minisatellites include the most polymorphic loci yet identified in the human genome. The high mutation rates at many of these loci are driven by incompletely understood recombination-based mechanisms that operate in the germline. To analyse aspects of minisatellite mutation processes and general eukaryotic recombination in meiosis that cannot be studied in humans or other mammals, including crosstalk and interplay between all four chromatids, we have previously constructed a eukaryotic model system, enabling the analysis of all four products of meiosis. In this system we have integrated alleles of the human minisatellite MS32, flanked by synthetic markers, in the vicinity of a meiotic recombination hot spot in chromosome III of Saccharomyces cerevisiae. In the present study, tetrad analysis showed that gene conversion is the predominant and possibly the universal pathway leading to interallelic transfer of repeats, with or without exchange of flanking regions. The data also suggest a hyper-recombinogenic state, triggered by interallelic mutation processes which generate a cascade of mutant alleles in the same meiosis. A number of tetrads contained identical mutant alleles of meiotic origin. Several tetrads could not be explained by the current models for minisatellite mutation. Accordingly, we here present a modified model based on the successive repair of multiple double-strand breaks.     

Very high mutation rate in offspring of Chernobyl accident liquidators

Exposure to ionizing radiation has long been suspected to increase mutation load in humans. Nevertheless, such events as atomic bombing seem not to have yielded significant genetic defects. The Chernobyl accident created a different, long-term exposure to radiation. Clean-up teams (or 'liquidators') of the Chernobyl reactor are among those who received the highest doses, presumably in some combination of acute and chronic forms. In this study, children born to liquidator families (currently either in the Ukraine or Israel) conceived after (CA) parental exposure to radiation were screened for the appearance of new fragments using multi-site DNA fingerprinting. Their sibs conceived before (CB) exposure served as critical internal controls, in addition to external controls (non-exposed families). An unexpectedly high (sevenfold) increase in the number of new bands in CA individuals compared with the level seen in controls was recorded. A strong tendency for the number of new bands to decrease with elapsed time between exposure and offspring conception was established for the Ukrainian families. These results indicate that low doses of radiation can induce multiple changes in human germline DNA.     

Microsatellite mutations in the offspring of irradiated parents 19 years after the Cesium-137 accident

In September of 1987, a radiotherapy unit containing 50.9 TBq of Cs¹³⁷Cl was removed from an abandoned radiotherapy clinic. This unit was subsequently disassembled leading to the most serious radiological accident yet to occur in the Western hemisphere. This event provides an opportunity to assess the genetic effects of ionizing radiation. We surveyed genetic variation of 12 microsatellite loci in 10 families of exposed individuals and their offspring and also in non-exposed families from the same area of Goias state. We found an increase in the number of new alleles in the offspring of the exposed individuals. The mutation rate was found to be higher in the exposed families compared to the control group. These results indicated that exposure to ionizing radiation can be detected in offspring of exposed individuals and also suggest that the elevated microsatellite mutation rate can be attributed to radioactive exposure.