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.     

Elevated minisatellite mutation rate in the post-chernobyl families from ukraine

Germline mutation at eight human minisatellite loci has been studied among families from rural areas of the Kiev and Zhitomir regions of Ukraine, which were heavily contaminated by radionuclides after the Chernobyl accident. The control and exposed groups were composed of families containing children conceived before and after the Chernobyl accident, respectively. The groups were matched by ethnicity, maternal age, parental occupation, and smoking habits, and they differed only slightly by paternal age. A statistically significant 1.6-fold increase in mutation rate was found in the germline of exposed fathers, whereas the maternal germline mutation rate in the exposed families was not elevated. These data, together with the results of our previous analysis of the exposed families from Belarus, suggest that the elevated minisatellite mutation rate can be attributed to post-Chernobyl radioactive exposure. The mechanisms of mutation induction at human minisatellite loci are discussed.     

Differential genetic responses to ionizing irradiation in individual families of Japanese medaka, Oryzias latipes

Although no statistically significant hereditary effects have yet been detected in the children of survivors from the atomic bombings in Hiroshima and Nagasaki, recent animal studies have found that exposure to ionizing radiation can cause genomic and epigenomic instability in the exposed individuals, as well as their offspring, and therefore, may have much larger genetic effects than predicted by earlier studies. When individuals are exposed to various environmental insults, including radiation, individual sensitivity to the insults often varies. Variance in germ-line response to radiation among individuals has been widely recognized, but it is difficult to address due to the use of inbred strains and the limited number of offspring that can be produced by a pair of mice, the common model used to study genetic effects of radiation. Herein is the first study to examine individual family responses to ionizing radiation using a parent-pedigree approach in an outbred strain of a vertebrate model, the Japanese medaka fish. Changes in frequencies of radiation-induced germline mutations at nine microsatellite loci were examined in the same families before and after exposure to one of four acute doses of ionizing radiation (0.1, 0.5, 2.5, 5Gy, plus sham-exposed controls). Families varied significantly in pre-exposure mutation frequencies and responses to irradiation, but germline mutations were elevated in at least one family after 0.1, 0.5, and 5Gy exposures. Variance among individuals in sensitivity to radiation is well documented for many endpoints, and our work now extends these endpoints to include germ-line mutations. Further studies are needed to elucidate dose response, effects at varying stages of spermatogenesis, and the mechanisms underlying the variance in these individual responses to radiation.     

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.     

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.     

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.     

Factors affecting germline mutations in a hypervariable microsatellite: a comparative analysis of six species of swallows (Aves: Hirundinidae)

Microsatellites mutate frequently by replication slippage. Empirical evidence shows that the probability of such slippage mutations may increase with the length of the repeat region as well as exposure to environmental mutagens, but the mutation rate can also differ between the male and female germline. It has been hypothesized that more intense sexual selection or sperm competition can also lead to elevated mutation rates, but the empirical evidence is inconclusive. Here, we analyzed the occurrence of germline slippage mutations in the hypervariable pentanucleotide microsatellite locus HrU10 across six species of swallow (Aves: Hirundinidae). These species exhibit marked differences in the length range of the microsatellite, as well as differences in the intensity of sperm competition. We found a strong effect of microsatellite length on the probability of mutation, but no residual effect of species or their level of sperm competition when the length effect was accounted for. Neither could we detect any difference in mutation rate between tree swallows (Tachycineta bicolor) breeding in Hamilton Harbour, Ontario, an industrial site with previous documentation of elevated mutation rates for minisatellite DNA, and a rural reference population. However, our cross-species analysis revealed two significant patterns of sex differences in HrU10 germline mutations: (1) mutations in longer alleles occurred typically in the male germline, those in shorter alleles in the female germline, and (2) male germline mutations were more often expansions than contractions, whereas no directional bias was evident in the female germline. These results indicate some fundamental differences in male and female gametogenesis affecting the probability of slippage mutations. Our study also reflects the value of a comparative, multi-species approach for locus-specific mutation analyses, through which a wider range of influential factors can be assessed than in single-species studies.     

Advances in the application of germline tandem repeat instability for in situ monitoring

Alterations in tandem repetitive DNA sequences such as minisatellite DNA and expanded simple tandem repeats (ESTRs) may provide useful biomarkers of induced germline effects. In this review, I describe the differences between ESTRs and minisatellites with respect to their structure and mutational mechanisms, and discuss field applications measuring induced germline instability. It is evident that both types of loci have high rates of mutation that facilitate the measurement of induced mutation measured in relatively small numbers of samples following environmentally relevant exposures. Several research groups have used these loci to demonstrate a significant increase in germline mutation in humans and animals exposed to radioactive or chemical pollutants in their natural environment. Mutations are manifested as gains or losses in repeat units and are detected either by pedigree screening or by PCR amplification of sperm DNA. Mutations at both ESTRs and minisatellites appear to arise via indirect mechanisms rather than by direct damage to the repeat locus itself. Most interestingly, ESTR instability following radiation has been shown to be heritable and transmitted to subsequent generations. An understanding of the mechanisms involved in induced instability is required in order to begin to decipher the potential biological implications of increased germline tandem repeat mutation. Furthermore, relatively few studies have investigated the ability of different genotoxins to induce tandem repeat instability. Such laboratory-based experiments will be crucial in clarifying the particular environmental or occupational exposures that should be targeted for future studies and for isolating and subsequently identifying the putative mutagens in complex environmental matrices.     

Germline mutations of tetranucleotide DNA repeats in families with normal children and reproductive pathology

We have previously reported a high rate of tetranucleotide DNA repeat mutations, including mutations of both germline and somatic origin, in spontaneous human abortuses. To analyze in more detail mutational microsatellite (MS) variability in meiosis and its possible association with disturbed embryonic development, we have conducted a comparative study of mutation rates of a complex of 15 autosomal tetranucleotide MSs in 55 families with healthy children and in 103 families that have had spontaneous abortuses with normal karyotypes. In the families with miscarriage, the gametic MS mutation rate was higher than in the families with normal reproductive function (4.36 x 10(-3) versus 2.32 x 10(-3) per locus per gamete per generation), but this difference was statistically nonsignificant (P = 0.25). No association of MS mutations with familiar miscarriage was found. Mutations at the MS loci studied were recorded almost 3 times as often in spermatogenesis as in oogenesis, which is likely to result from a greater number of DNA replication cycles in male germline cell precursors than in female ones. Mutations increasing and reducing the MS sequence length appeared at virtually the same rate. Changes in MS DNA sequence length per one repeated element, i.e., single-step mutations (93% of cases) exceeded all other events of allele length change. The highest number of mutations (81.2%) was found in longer alleles. This distribution of mutations by size, direction, and parental origin corresponds to the multistep mutation model of their emergence via mechanism of DNA strand slippage during replication.     

Inhibition of human cytochrome P450 1B1, 1A1 and 1A2 by antigenotoxic compounds,purpurin and alizarin

Expanded simple tandem repeat (ESTR) loci include some of the most unstable DNA in the mouse genome and have been extensively used in pedigree studies of germline mutation. We now show that repeat DNA instability at the mouse ESTR locus Ms6-hm can also be monitored by single molecule PCR analysis of genomic DNA. Unlike unstable human minisatellites which mutate almost exclusively in the germline by a meiotic recombination-based process, mouse Ms6-hm shows repeat instability both in germinal (sperm) DNA and in somatic (spleen, brain) DNA. There is no significant variation in mutation frequency between mice of the same inbred strain. However, significant variation occurs between tissues, with mice showing the highest mutation frequency in sperm. The size spectra of somatic and sperm mutants are indistinguishable and heavily biased towards gains and losses of only a few repeat units, suggesting repeat turnover by a mitotic replication slippage process operating both in the soma and in the germline. Analysis of male mice following acute pre-meiotic exposure to X-rays showed a significant increase in sperm but not somatic mutation frequency, though no change in the size spectrum of mutants. The level of radiation-induced mutation at Ms6-hm was indistinguishable from that established by conventional pedigree analysis following paternal irradiation. This confirms that mouse ESTR loci are very sensitive to ionizing radiation and establishes that induced germline mutation results from radiation-induced mutant alleles being present in sperm, rather than from unrepaired sperm DNA lesions that subsequently lead to the appearance of mutants in the early embryo. This single molecule monitoring system has the potential to substantially reduce the number of mice needed for germline mutation monitoring, and can be used to study not only germline mutation but also somatic mutation in vivo and in cell culture.     

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.     

Analysis of genome instability in offspring of Mayak workers’ families: Minisatellite CEB

Genome instability transmission in offspring was analyzed in order to evaluate the risk of delayed genetic effects of exposure in 95 family triplets in which only fathers experienced prolonged occupational radiation exposure. The mean total preconceptive absorbed dose (TPAD) of external gamma radiation in the paternal gonads was 1.65 ± 0.080 Gy (dose range of 0.57–5.70 Gy), and the mean TPAD of internal alpha radiation from incorporated plutonium-239 in the gonads was 0.0015 ± 0.0003 Gy (dose range 0.000–0.015 Gy). The control group consisted of 50 family triplets in which parents were not occupationally exposed. The mutation process was studied using PCR based on hypervariable minisatellite marker CEB1 (chromosome 2, 2q37.3). The paternal type of inheritance of mutations for minisatellite CEB1 was found in 80% of cases. The analysis revealed a statistically significant increase in minisatellite CEB1 mutations in the common group of families in which fathers experienced prolonged occupational radiation exposure and in the group of families in which fathers were exposed to radiation in a dosage range of 0.5–1.0 Gy as compared to the control, reaching a significance level of p = 0.109 and p = 0.058, respectively. The dose threshold of mutation detection in the offspring of Mayak PA workers was estimated.     

Induced minisatellite germline mutations in herring gulls (Larus argentatus) living near steel mills

Despite widespread industrial release of genotoxic contaminants, little is understood of their role in inducing germline mutations in natural populations. We used multilocus DNA fingerprinting to quantify germline minisatellite mutations in families of herring gulls (Larus argentatus) in three nesting categories: (a) near cities with large steel mills operating coking ovens; (b) near cities without steel mills; and (c) in rural locations removed from point sources of contamination. Gulls nesting near integrated steel mills showed significantly higher mutation rates than gulls from rural locations (Fisher's exact, P=0.0004); urban sites without steel mills fell midway between steel and rural sites (difference from rural; Fisher's exact, P=0.19). Distance of the nesting location of herring gulls from the steel industries' coking ovens was negatively correlated with minisatellite mutation rate demonstrating significant risk for induced germline mutations in cities with steel operations (Kendall Tau; tau=0.119; P<0.0001).     

Germline mutation rates in mice following in utero exposure to diesel exhaust particles by maternal inhalation

The induction of inherited DNA sequence mutations arising in the germline (i.e., sperm or egg) of mice exposed in utero to diesel exhaust particles (DEPs) via maternal inhalation compared to unexposed controls was investigated in this study. Previous work has shown that particulate air pollutants (PAPs) from industrial environments cause DNA damage and mutations in the sperm of adult male mice. Effects on the female and male germline during critical stages of development (in utero) are unknown. In mice, previous studies have shown that expanded simple tandem repeat (ESTR) loci exhibit high rates of spontaneous mutation, making this endpoint a valuable tool for studying inherited mutation and genomic instability. In the present study, pregnant C57Bl/6 mice were exposed to 19mg/m(3) DEP from gestational day 7 through 19, alongside air exposed controls. Male and female F1 offspring were raised to maturity and mated with control CBA mice. The F2 descendents were collected and ESTR germline mutation rates were derived from full pedigrees (mother, father, offspring) of F1 male and female mice. We found no evidence for increased ESTR mutation rates in females exposed in utero to DEP relative to control females. In contrast, a statistically significant increase in the mutation frequency of male mice exposed in utero to DEP was observed (2-fold; Fisher's exact p<0.05). Thus, maternal exposure to DEP results in increased mutation in sperm during development.     

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.     

Mutation rates in scots pine (Pinus sylvestris L.) from the Chernobyl exclusion zone evaluated with amplified fragment-length polymorphisms (AFLPs) and microsatellite markers

Ionizing radiation is a strong mutagenic factor and, accordingly, elevated mutation rates would be expected in plants exposed to high chronic or acute radiation after the Chernobyl accident in 1986. Somatic mutations were analyzed in pines (Pinus sylvestris L.) planted before and after the Chernobyl accident and in control material of the same origin planted in sites with natural radiation. Microsatellites (SSRs) and amplified fragment-length polymorphisms (AFLPs) were investigated. The mutation rates for microsatellites were estimated as 2.8 × 10(-4)-7.1 × 10(-4) per locus for different irradiated tree populations; no mutations were detected in the controls. In the case of AFLPs, the observed mutation rates were 3.74 × 10(-3) -3.99 × 10(-3) and 1.06 × 10(-3) per locus for contaminated and control areas, respectively. Thus a statistically highly significant three-fold increase in number of mutations was found by the use of AFLP markers, indicating that ionizing radiation causes strong DNA damage across the entire genome and that AFLPs may be the appropriate marker system for this kind of analysis.     

Germline Mutations of Tetranucleotide DNA Repeats in Families with Normal Children and Reproductive Pathology

We have previously reported a high rate of tetranucleotide DNA repeat mutations, including mutations of both germline and somatic origin, in spontaneous human abortions. To analyze in more detail mutational microsatellite (MS) variability in meiosis and its possible association with disturbed embryonic development, we have conducted a comparative study of mutation rates of a panel of 15 autosomal tetranucleotide MSs in 55 families with healthy children and in 103 families that have had spontaneous abortions with normal karyotypes. In the families with miscarriage, the gametic MS mutation rate was higher than in the families with normal reproductive function (4.36 × 10⁻³ versus 2.32 × 10⁻³ per locus per gamete per generation), but this difference was statistically nonsignificant (P = 0.25). No association of MS mutations with familiar miscarriage was found. Mutations at the MS loci studied were recorded almost 3 times as often in spermatogenesis as in oogenesis, which is likely to result from a greater number of DNA replication cycles in male germline cell precursors than in female ones. Mutations increasing and reducing the MS sequence length appeared at virtually the same rate. Changes in MS DNA sequence length per one repeated element, i.e., single-step mutations (93% of cases) exceeded all other events of allele length change. The highest number of mutations (81.2%) was found in longer alleles. This distribution of mutations by size, direction, and parental origin corresponds to the multistep mutation model of their emergence via mechanism of DNA strand slippage during replication.__________Translated from Genetika, Vol. 41, No. 7, 2005, pp. 943–953.Original Russian Text Copyright © 2005 by Nikitina, Lebedev, Sukhanova, Nazarenko.     

Variable germline and embryonic instability of the human minisatellite MS32 (D1S8) in transgenic mice.

Tandem repeat loci such as minisatellites and trinucleotide repeats frequently show instability. We have investigated mutation at human minisatellite MS32 (locus D1S8) transferred to transgenic mice. Three lines of hemizygous transgenic mice were studied. A single-copy line (110D) was seen to be relatively stable, whilst two multicopy lines showed structural instability of the transgene in pedigrees (lines 109 and 110A). For both these lines, mutant structures were detected as a result of mutation events having occurred in the germline or early embryo. Structural changes seen included gain or loss of minisatellite repeat units (110A and 109), alteration of DNA flanking the minisatellite repeat array (109 only) or deletion of the entire transgene (109 only). This work demonstrates that tandem repeat transgenes can show instability and thus provide additional systems for the analysis of repetitive DNA structural change in mice.     

Minisatellite mutation frequency in human sperm following radiotherapy

Screening pedigrees for inherited minisatellite length changes provides an efficient means of monitoring repeat DNA instability but has given rise to apparently contradictory results regarding the effects of radiation on the human germline. To explore this further in individuals with known radiation doses and to potentially gain information on the timing of mutation induction, we have used an extremely sensitive single molecule approach to quantify the frequencies of mutation at the hypervariable minisatellites B6.7 and CEB1 in the sperm of three seminoma patients following hemipelvic radiotherapy. Scattered radiation doses to the testicles were monitored and pre-treatment sperm DNA was compared with sperm derived from irradiated pre-meiotic, meiotic and post-meiotic cells. We show no evidence for mutation induction in any of the patients and discuss this finding in the context of previous population studies using minisatellites as reporter systems, one of which provided evidence for radiation-induced germline mutation.