Germline Minisatellite Mutations Research Articles

Germline minisatellite mutations in workers occupationally exposed to radiation at the Sellafield nuclear facility

Germline minisatellite mutation rates were investigated in male workers occupationally exposed to radiation at the Sellafield nuclear facility. DNA samples from 160 families with 255 offspring were analysed for mutations at eight hypervariable minisatellite loci (B6.7, CEB1, CEB15, CEB25, CEB36, MS1, MS31, MS32) by Southern hybridisation. No significant difference was observed between the paternal mutation rate of 5.0% (37 mutations in 736 alleles) for control fathers with a mean preconceptional testicular dose of 9 mSv and that of 5.8% (66 in 1137 alleles) for exposed fathers with a mean preconceptional testicular dose of 194 mSv. Subgrouping the exposed fathers into two dose groups with means of 111 mSv and 274 mSv revealed paternal mutation rates of 6.0% (32 mutations in 536 alleles) and 5.7% (34 mutations in 601 alleles), respectively, neither of which was significantly different in comparisons with the rate for the control fathers. Maternal mutation rates of 1.6% (12 mutations in 742 alleles) for the partners of control fathers and 1.7% (19 mutations in 1133 alleles) for partners of exposed fathers were not significantly different. This study provides evidence that paternal preconceptional occupational radiation exposure does not increase the germline minisatellite mutation rate and therefore refutes suggestions that such exposure could result in a destabilisation of the germline that can be passed on to future generations.

Paternal lifestyle as a potential source of germline mutations transmitted to offspring

Paternal exposure to high levels of radioactivity causes heritable germline minisatellite mutations. However, the effect of more general paternal exposures, such as cigarette smoking, on germline mutations remains unexplored. We analyzed two of the most commonly used minisatellite loci (CEB1 and B6.7) to identify germline mutations in blood samples of complete mother-father-child triads from the Norwegian Mother and Child Cohort Study (MoBa). The presence of mutations was subsequently related to general lifestyle factors, including paternal smoking before the partner became pregnant. Paternally derived mutations at the B6.7 locus (mutation frequency 0.07) were not affected by lifestyle. In contrast, high gross yearly income as a general measure of a healthy lifestyle coincided with low-mutation frequencies at the CEB1 locus (P=0.047). Income was inversely related to smoking behavior, and paternally derived CEB1 mutations were dose dependently increased when the father smoked in the 6 mo before pregnancy, 0.21 vs. 0.05 in smoking and nonsmoking fathers, respectively (P=0.061). These results suggest that paternal lifestyle can affect the chance of heritable mutations in unstable repetitive DNA sequences. To our knowledge, this is the first study reporting an effect of lifestyle on germline minisatellite mutation frequencies in a human population with moderate paternal exposures.

Does Cancer Treatment in Childhood Induce Transgenerational Genetic Damage?

Does Cancer Treatment in Childhood Induce Transgenerational Genetic Damage?

Germline minisatellite mutations in survivors of childhood and young adult cancer treated with radiation

Purpose: To investigate minisatellite germline mutation rates in survivors of childhood and young adult cancer who received radiotherapy.Materials and methods: DNA samples from 100 families, where one parent was a cancer survivor, were analysed for mutations at eight hypervariable minisatellite loci (B6.7, CEB1, CEB15, CEB25, CEB36, MS1, MS31, MS32) by Southern hybridisation.Results: No significant difference was observed between the paternal mutation rate of 5.6% in exposed fathers with a mean preconceptional testicular dose of 1.23 Gy (56 mutations in 998 informative alleles) and that of 5.8% in unexposed fathers (17 in 295 informative alleles). Subgrouping the exposed fathers into dose groups of <0.10 Gy, 0.10–0.99 Gy, 1.00–1.99 Gy, ≥2.00 Gy revealed no significant differences in paternal mutation rate in comparison with the unexposed fathers. Maternal mutation rates of 1.6% in cancer survivor mothers with a mean preconceptional ovarian dose of 0.58 Gy (five mutations in 304 informative alleles) and 2.1% in unexposed mothers (21 in 987 informative alleles) were not significantly different. There were no differences in minisatellite mutation rates associated with treatment with chemotherapeutic agents.Conclusions: This study provides evidence that preconception radiotherapy for childhood or early adulthood cancer does not increase the germline minisatellite mutation rate.

Comparison of germ line minisatellite mutation detection at the CEB1 locus by Southern blotting and PCR amplification

Identification of de novo minisatellite mutations in the offspring of parents exposed to mutagenic agents offers a potentially sensitive measure of germ line genetic events induced by ionizing radiation and genotoxic chemicals. Germ line minisatellite mutations (GMM) are usually detected by hybridizing Southern blots of unamplified size-fractionated genomic DNA with minisatellite probes. However, this consumes a relatively large amount of DNA, requires several steps and may lack sensitivity. We have developed a polymerase chain reaction (PCR)-based GMM assay, which we applied to the hypermutable minisatellite, CEB1. Here, we compare the sensitivity and specificity of this assay with the conventional Southern hybridization method using DNA from 10 spouse pairs, one parent of each pair being a survivor of cancer in childhood, and their 20 offspring. We report that both methods have similar specificity but that the PCR method uses 250 times less DNA, has fewer steps and is better at detecting GMM with single repeats provided that specific guidelines for allele sizing are followed. The PCR GMM method is easier to apply to families where the amount of offspring DNA sample is limited.

New germline mutations in the hypervariable minisatellite CEB1 in the parents of children with leukaemia

Gardner and co-workers advanced the hypothesis that the Seascale leukaemia cluster could have been caused by new mutations in germ cells, induced by paternal preconceptional irradiation (PPI) exposure at the Sellafield nuclear installation. Since evidence has shown that PPI can increase the de novo germline mutation rate in hypervariable minisatellite loci, we investigated the hypothesis that sporadic childhood leukaemia might be associated with an increased parental germline minisatellite mutation rate. To test this hypothesis, we compared de novo germline mutation rates in the hypervariable minisatellite locus, CEB1, in family trios (both parents and their child) of children with leukaemia (n=135) compared with unaffected control families (n=124). The majority of case and control germline mutations were paternal (94%); the mean paternal germline mutation rates of children with leukaemia (0.083) and control children (0.156) were not significantly different (odds ratio, 95% confidence interval: 0.50, 0.23–1.08; P=0.11). There were no significant differences in case and control parental allele sizes, case and control germline mutation progenitor allele sizes (2.74 vs 2.54 kb; P=0.56), case and control mutant allele sizes (2.71 vs 2.67 kb; P=0.90), mutant allele size changes (0.13 vs 0.26 kb; P=0.10), or mutational spectra. Within the limitation of the number of families available for study, we conclude that childhood leukaemia is unlikely to be associated with increased germline minisatellite instability.

A pilot study examining germline minisatellite mutations in the offspring of Danish childhood and adolescent cancer survivors treated with radiotherapy

Purpose:To investigate germline mutation rate at eight minisatellite loci in 24 Danish families, where one parent is the survivor of childhood or adolescent cancer treated with radiotherapy.Materials and methods:Parents and offspring were profiled for eight hypervariable minisatellite loci (B6.7, CEB1, CEB15, CEB25, CEB36, MS1, MS31, MS32) by Southern blotting.Results:Seven paternal mutations were observed for 130 informative alleles in 18 offspring from 11 radiation-exposed fathers (mean preconceptional dose for offspring 0.29 Gy, range <0.01 – 1.2 Gy), compared to six mutations for 146 informative alleles in 21 offspring from 13 unexposed fathers. No statistically significant difference between the total paternal mutation rates was observed (5.4% for exposed fathers and 4.1% for unexposed fathers). Three maternal mutations were observed for 148 informative alleles in 21 offspring from 13 radiation-exposed mothers (mean preconceptional dose for offspring 0.71 Gy, range <0.01 – 9.2 Gy), compared to one mutation for 130 informative alleles in 18 offspring from 11 unexposed mothers. Again, no statistically significant difference was observed between the total maternal mutation rates (2.0% for exposed mothers and 0.8% for unexposed mothers).Conclusions:The data from this pilot study demonstrate no statistically significant increase in germline minisatellite mutation rate associated with radiotherapy for childhood and adolescent cancer.

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.

Evolution and population genetics of the H-ras minisatellite and cancer predisposition.

Case-control studies have implicated rare length H-ras minisatellite alleles in cancer risk. In Europeans, this locus has four common alleles, and a larger number of rare alleles; possession of a rare allele has been identified as a risk factor responsible for 5-10% of some cancers. This unusual model of predisposition has been controversial in case-control studies, but also makes characteristic predictions about the population genetics of the locus, which we examine in this study. Using minisatellite variant repeat ("MVR") mapping, and compound haplotypes composed of the minisatellite and surrounding substitutional polymorphisms, we have reconstructed the main steps in the evolution of this locus in human populations. MVR-calibrated measurements of allele length yield rare allele frequencies significantly higher than most previous studies, and show that most other analyses have not distinguished two common alleles of 84 and 85 repeat units. Alleles classified as "rare" in European populations predominate (70%) in the African sample studied. Small-pool PCR (SP-PCR) analysis on common alleles in sperm DNA gives an estimate for the germline minisatellite mutation rate of about 0.05% (95% confidence upper limit 0.15%). Overall, our results do not reflect a locus subject to frequent mutation and strong selection, and are difficult to reconcile with the proposed cancer predisposition. Restricted variation at this locus is most simply explained by low mutation rate, and although definitive case-control studies can only be performed using methods capable of reproducibly distinguishing rare and common alleles, our work suggests that most studies to date have not resolved alleles adequately.

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; τ=0.119; P<0.0001).