Mark Plumb

Transgenerational mutation by radiation.

Parental exposure to ionizing radiation increases the frequency of germline mutations detectable in the next generation. Parental exposure can also increase the rate of mutation in somatic cells and confer a predisposition to cancer in offspring, suggesting that there could be an indirect effect of radiation on somatic genome stability that is transmissible through the germ line of the irradiated parents. We have found that this indirect effect extends to the germ line of unexposed first-generation offspring in mice, as revealed by an increased instability of repeat-DNA sequences in their descendants.

Genome stability: Transgenerational mutation by radiation

Parental exposure to ionizing radiation increases the frequency of germline mutations detectable in the next generation. Parental exposure can also increase the rate of mutation in somatic cells and confer a predisposition to cancer in offspring, suggesting that there could be an indirect effect of radiation on somatic genome stability that is transmissible through the germ line of the irradiated parents. We have found that this indirect effect extends to the germ line of unexposed first-generation offspring in mice, as revealed by an increased instability of repeat-DNA sequences in their descendants.

Radiation-induced germline instability at minisatellite loci

PURPOSE To review the results of recent studies on radiation-induced germline instability at mammalian minisatellite loci. RESULTS Evidence has been obtained recently that germline mutation at minisatellites is remarkably sensitive to ionizing radiation, in both mice and humans. In mice, an elevated mutation rate was found after acute irradiation of pre-meiotic spermatogonia, with a doubling dose of 0.33 Gy, a value close to those obtained in mice after acute spermatogonia irradiation using other systems for mutation detection. In humans, analysis of germline mutation rate at minisatellites among children born in areas of the Mogilev district of Belarus, which was heavily polluted after the Chernobyl accident, has shown a twofold higher mutation rate in exposed families compared with non-irradiated families from the United Kingdom. Within the Belarus cohort, the mutation rate was significantly greater in families exposed to a higher parental radiation dose, consistent with radiation induction of germline mutation. The data in this study also demonstrate the indirect nature of radiation-induced germline mutation at mammalian minisatellite loci suggesting a strong similarity with the phenomenon of genomic instability in somatic cells. CONCLUSIONS Minisatellite loci provide a powerful system for the efficient monitoring of germline mutation in humans and are capable of detecting induced mutations in relatively small population samples.

No correlation between germline mutation at repeat DNA and meiotic crossover in male mice exposed to X-rays or cisplatin

To test the hypothesis that mouse germline expanded simple tandem repeat (ESTR) mutations are associated with recombination events during spermatogenesis, crossover frequencies were compared with germline mutation rates at ESTR loci in male mice acutely exposed to 1Gy of X-rays or to 10mg/kg of the anticancer drug cisplatin. Ionising radiation resulted in a highly significant 2.7-3.6-fold increase in ESTR mutation rate in males mated 4, 5 and 6 weeks after exposure, but not 3 weeks after exposure. In contrast, irradiation had no effect on meiotic crossover frequencies assayed on six chromosomes using 25 polymorphic microsatellite loci spaced at approximately 20cM intervals and covering 421cM of the mouse genome. Paternal exposure to cisplatin did not affect either ESTR mutation rates or crossover frequencies, despite a report that cisplatin can increase crossover frequency in mice. Correlation analysis did not reveal any associations between the paternal ESTR mutation rate and crossover frequency in unexposed males and in those exposed to X-rays or cisplatin. This study does not, therefore, support the hypothesis that mutation induction at mouse ESTR loci results from a general genome-wide increase in meiotic recombination rate.

Ionising radiation and mutation induction at mouse minisatellite loci ☆: The story of the two generations

The analysis of the effects of ionising radiation on germline mutations is limited by the number of offspring that need to be analysed following exposure to a dose, which is relevant to risk assessment in humans. We have developed a new experimental approach using hypervariable mouse expanded simple tandem repeat (ESTR) loci (minisatellites) which are both highly sensitive to ionising radiation and which permit changes in mutation rates to be detected in relatively small samples. Here, we review the progress made in validating the model, and the unexpected features it has revealed, including a novel form of radiation-induced genetic instability that can be transmitted from one generation to the next.

Susceptibility to radiation-induced leukaemia/lymphoma is genetically separable from sensitivity to radiation-induced genomic instability.

PURPOSE To determine whether there is a relationship between the genetics underlying the susceptibility to radiation-induced leukaemia in CBA/H (acute myeloid leukaemia, AML) and C57BL/6 (thymic lymphoma, TL) mice, and the genetics underlying the sensitivity of CBA/H (sensitive) and C57BL/6 (resistant) mice to radiation-induced chromosomal instability. MATERIALS AND METHODS CBA/H, (CBA/H x C57BL/6)F1, F1 x CBA/H, F1 x C57BL/6 and F1 x F1 mice were exposed to a single acute dose of 3.0 Gy X-rays. AML and TL were diagnosed over the subsequent 30 months. RESULTS There was no statistically significant difference in the incidence of AML in F1, F1 x F1, F1 x CBA/H and F1 x C57BL/6 mice, which was approximately 50% that in CBA/H mice. AML susceptibility is therefore a dominant polygenic trait, and both susceptibility and resistance (variable penetrance) CBA/H and C57BL/6 loci are involved. The incidence of TL in the FM and F1 x CBA/H mice was negligible, indicating that TL susceptibility is a recessive trait. As the TL incidence in the F1 x C57BL/6 mice was about half that in C57BL/6 mice, one recessive locus is probably involved. CONCLUSIONS AML susceptibility in CBA/H mice is a dominant trait in contrast to the recessive inheritance of CBA/H sensitivity to radiation-induced chromosomal instability. TL-susceptibility in C57BL/6 is a recessive trait in contrast to the dominant inheritance of C57BL/6 resistance to radiation-induced chromosomal instability.Purpose : To determine whether there is a relationship between the genetics underlying the susceptibility to radiation-induced leukaemia in CBA/H (acute myeloid leukaemia, AML) and C57BL/6 (thymic lymphoma, TL) mice, and the genetics underlying the sensitivity of CBA/H (sensitive) and C57BL/6 (resistant) mice to radiation-induced chromosomal instability. Materials and methods : CBA/H, (CBA/H ×C57BL/6)F 1, F 1 ×CBA/H, F 1 ×C57BL/6 and F 1 ×F 1 mice were exposed to a single acute dose of 3.0 Gy X-rays. AML and TL were diagnosed over the subsequent 30 months. Results : There was no statistically significant difference in the incidence of AML in F 1, F 1 ×F 1, F 1 ×CBA/H and F 1 ×C57BL/6 mice, which was ~50% that in CBA/H mice. AML susceptibility is therefore a dominant polygenic trait, and both susceptibility and resistance (variable penetrance) CBA/H and C57BL/6 loci are involved. The incidence of TL in the F 1 and F 1 ×CBA/H mice was negligible, indicating that TL susceptibility is a recessive trait. As the TL incidence in the F 1 ×C57BL/6 mice was about half that in C57BL/6 mice, one recessive locus is probably involved. Conclusions : AML susceptibility in CBA/H mice is a dominant trait in contrast to the recessive inheritance of CBA/H sensitivity to radiation-induced chromosomal instability. TL-susceptibility in C57BL/6 is a recessive trait in contrast to the dominant inheritance of C57BL/6 resistance to radiation-induced chromosomal instability.

Comments on the paper: Microsatellite instability in acute myelocytic leukaemia developed from A-bomb survivors--a biological perspective.

Sir, I read with interest the recent correspondences by Little (2002) and Cox and Edwards (2002) discussing the papers by Nakanishi et al. (1999, 2001) on microsatellite (MSI) and genetic instability in the acute myeloid leukaemias (AML) that arose in A-bomb survivors. Several suggestions were made in the correspondence that require further consideration. Specifically, it was suggested that the MSI rates in A-bomb survivor AML cases should be related to the radiation dose received (Little 2002); if causal, then radiation-induced leukaemogenic development should be specifically driven by the induction of persistently expressed genomic instability (Cox and Edwards 2002); and, that there is a dose-related association between the expression of genomic instability in AML cases and the probability of radiation causation (Cox and Edwards 2002). These issues deserve some comment, in particular with reference to therapy-related AML/MDS (t-AML) and the underlying assumption that the 86 572 A-bomb survivors (Pierce et al. 1996) represent a genetically homogenous population.

Evidence for clustered tumour suppressor gene loci on mouse chromosomes 2 and 4 in radiation-induced Acute Myeloid Leukaemia

Purpose: To investigate the influence of genetic and epigenetic factors on allelic loss on chromosomes 2 and 4 in mouse radiation-induced acute myeloid leukaemia (r-AML). Methods: r-AML that arose in (CBA/H×C57BL/6)F1×CBA/H and F1×C57BL/6 mice were screened for transcription factor PU1 (also known as SPI-1) gene mutations and methylation of the paired box gene 5 (Pax5) gene promoter. We have increased the statistical significance of a genetic linkage analysis of affected F1×CBA/H mice to test for linkage to loci implicated directly or indirectly with r-AML-susceptibility. Results: There was a statistically significant difference ( p < 10−4) in the frequency of PU1 gene mutations in F1×CBA/H and F1×C57BL/6 r-AML, implicating a second linked but genotype-dependent myeloid leukaemia suppressor gene on chromosome 2. A suggestive CBA/H r-AML-resistance locus maps within 10 cM of the minimally deleted region on chromosome 4. The Pax5 gene promoter is subject to ongoing subclonal promoter methylation in the r-AML, evidence that Pax5 gene silencing confers a selective advantage during clonal expansion in vivo. Conclusions: Allelic loss in mouse r-AML and subsequent tumour suppressor gene mutation (PU1) or silencing (Pax5) is strongly influenced by genetic background and/or epigenetic factors, and driven by in vivo clonal selection.

QTL analyses of lineage-negative mouse bone marrow cells labeled with Sca-1 and c-Kit

Differences in the number of functionally and/or phenotypically defined bone marrow cells in inbred mouse strains have been exploited to map quantitative trait loci (QTL) that determine the variation in cell frequency. To extend this approach to the differences in the stem/progenitor cell compartment in CBA/H and C57BL/6 mice, we have exploited the resolution of flow cytometry and the power of QTL analyses in 124 F2 mice to analyze lineage-negative (Lin−) bone marrow cells according to the intensity of labeling with Sca-1 and c-Kit. In the Lin− Sca-1+ c-Kit+ enriched population, six QTL were identified: one significant and five suggestive. Whereas previous in vitro clonogenic, LTC-IC, day 35 CAFC, and flow cytometry each identified different QTL, our approach identified the same or very similar QTL at all three loci (chromosomes 1, 17, and 18) as well as QTL on chromosomes 6 and 10. In silico analyses implicate hematopoietic stem cell homing involving Cxcr4 and Cxcl12 as being the determining pathway. The mapping of the same or very similar QTL in independent studies using different assay(s) suggests a common genetic determinant, and thus reinforces the biological and genetic significance of the QTL. These data also suggest that mouse bone marrow cell subpopulations can be functionally, phenotypically, and genetically defined.

Transgenerational mutation by radiation: Genome stability

Parental exposure to ionizing radiation increases the frequency of germline mutations detectable in the next generation. Parental exposure can also increase the rate of mutation in somatic cells and confer a predisposition to cancer in offspring, suggesting that there could be an indirect effect of radiation on somatic genome stability that is transmissible through the germ line of the irradiated parents. We have found that this indirect effect extends to the germ line of unexposed first-generation offspring in mice, as revealed by an increased instability of repeat-DNA sequences in their descendants.