New methods for assessing male germ line mutations in humans and genetic risks in their offspring (original) (raw)
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Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2002
A method to measure the germline mutations induced by cancer treatment in humans is needed. To establish such a method we used a transgenic mouse model consisting of a human DNA repeat locus that has a high spontaneous mutation frequency as a biomarker. Alterations in repeat number were measured in individual sperm from mice hemizygous for an expanded (CTG) 162 human myotonic dystrophy type 1 (DM1) microsatellite repeat using single genome-equivalent (g.e.) PCR and detection by a DNA fragment analyzer. Mutation frequencies were measured in DNA from sperm from controls and sperm derived from stem spermatogonia, differentiating spermatogonia, and spermatocytes exposed to radiation and from spermatocytes of mice treated with cyclophosphamide. There was no increase above control levels in mutations, scored as >1 repeat changes, in any of the treated groups. However, moderately large deletion mutants (between 9 and 20 repeat changes) were observed at frequencies of 2.2% when spermatocytes were treated with cyclophosphamide and, 1.8 and 2.5% when spermatocytes and stem cells, respectively, were treated with radiation, which were significantly higher than the frequency of 0.3% in controls. Thus, radiation and cyclophosphamide induced deletions in the expanded DM1 trinucleotide repeat. PCR artifacts were characterized in sperm DNA from controls and from mice treated with radiation; all artifacts involved losses of more than 20 DM1 repeats, and surprisingly the artifact frequency was higher in treated sperm than in control sperm. The radiation-induced increase in the frequency of PCR artifacts might reflect alterations in sperm DNA that destabilize the genome not only during PCR amplification but also during early embryonic development.
Fertility and Sterility, 2010
Objective: To report a more quantitative approach to study the influence of varying levels of sperm DNA damage on transgenerational changes in genomic instability in a mouse model. Design: Experimental prospective study. Setting: Embryology research laboratory. Animal(s): Swiss albino mice. Intervention(s): The sperm DNA damage was induced by different doses of g-irradiation to male mice followed by mating with healthy female mice. Main Outcome Measure(s): Genomic integrity in embryos, fetus, and spermatozoa of F1 mice derived from the DNA-damaged sperm. Result(s): The transgenerational changes in genetic integrity were attributed by a dose-dependent increase in the frequency of micronuclei in preimplantation embryos and a concomitant increase in genomic instability in fetal liver cells and sperm chromatin modifications in F1 males. A strong positive correlation was observed between the extent of sperm DNA damage and somatic and germ-line genomic instability. Conclusion(s): Sperm-mediated transgenerational genomic instability is dependent on the amount of DNA damage present in the sire's sperm at the time of fertilization. (Fertil Steril Ò 2010;93:2486-90. Ó2010 by American Society for Reproductive Medicine.
Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2016
Germline mutations are an important component of genetic toxicology; however, mutagenicity tests of germline cells are limited. Recent advances in sequencing technology can be used to detect mutations by direct sequencing of genomic DNA (gDNA). We previously reported induced de novo mutations detected using whole-exome sequencing in the offspring of N-ethyl-N-nitrosourea (ENU)-treated mice in a single-dose experiment (85 mg/kg, i.p., weekly on two occasions). In this study, two lower doses (10 and 30 mg/kg) were added, and dose-response of inherited germline mutations was analyzed. Male gpt delta transgenic mice treated with ENU in three dose groups were mated with untreated females 10 weeks after the last treatment, and offspring were obtained. The ENU-treated male mice showed dose-dependent increases in gpt mutant frequencies in their sperm, testis, and liver. gDNA of one family (parents and four offspring) from each dose group was used for whole-exome sequencing, and unique de novo mutations in the offspring were detected. Frequencies of inherited mutations increased with dosage more than 25-fold in the highest dose group. The mutation spectrum of the inherited mutations showed characteristics of ENU-induced mutations, such as A:T base substitutions. No confirmed mutations were observed in the control group. Filtering using the alternate reads ratio resulted in the mutation frequencies and spectra similar to those obtained by the Sanger sequencing confirmation. These results suggest that direct sequencing analysis may be a useful tool to investigate inherited germline mutations induced by environmental mutagens.
Transgenic Rodent Assay for Quantifying Male Germ Cell Mutant Frequency
Journal of Visualized Experiments, 2014
De novo mutations arise mostly in the male germline and may contribute to adverse health outcomes in subsequent generations. Traditional methods for assessing the induction of germ cell mutations require the use of large numbers of animals, making them impractical. As such, germ cell mutagenicity is rarely assessed during chemical testing and risk assessment. Herein, we describe an in vivo male germ cell mutation assay using a transgenic rodent model that is based on a recently approved Organisation for Economic Co-operation and Development (OECD) test guideline. This method uses an in vitro positive selection assay to measure in vivo mutations induced in a transgenic λgt10 vector bearing a reporter gene directly in the germ cells of exposed males. We further describe how the detection of mutations in the transgene recovered from germ cells can be used to characterize the stage-specific sensitivity of the various spermatogenic cell types to mutagen exposure by controlling three experimental parameters: the duration of exposure (administration time), the time between exposure and sample collection (sampling time), and the cell population collected for analysis. Because a large number of germ cells can be assayed from a single male, this method has superior sensitivity compared with traditional methods, requires fewer animals and therefore much less time and resources.
Germline mutation induction at tandem repeat DNA loci January 2002
2009
The ability to predict the genetic consequences for humans of exposure to ionising radiation has certainly been as a significant goal of human genetics in the past fifty years. However, despite numerous experimental studies, little is known about the effects of radiation exposure on germline mutation in humans. For example, data collected in Hiroshima and Nagasaki during the past 40 years on children of atomic bomb survivors using standard monitoring systems have not provided evidence of any statistically significant differences in mutation rate between exposed and control families (1). Similarly, a survey of survivors treated with radiotherapy showed that the occurrence of genetic diseases in their offspring was similar to that in control families (2). For this reason, germline mutation induction in mice still remains the main source of experimental data used to evaluate the genetic risk of human exposure to ionising radiation (3,4). Estimating the genetic hazards of radiation and other mutagens depends on extrapolation from experimental systems. The obvious shortcoming of current approaches for monitoring radiation-induced mutation is the necessity to use very large numbers of individuals (more than 100,000) to detect increases in mutation rate. Furthermore, the question of adequate scoring of radiation-induced mutations by these approaches remains uncertain (5). It is therefore clear that new experimental approaches for monitoring radiation-induced germline mutation in human populations need to be developed. We have proposed that hypervariable tandem repeat loci may provide a new experimental approach to the evaluation of germline mutation induction in mice and humans by low-dose exposure to ionising radiation (6, 7). These loci have a very high spontaneous mutation rate both in humans (8, 9) and mice (10, 11) and therefore capable of detecting changes in mutation rates in relatively small population samples. Here we present the summary of recent publications analysing mutation induction at mouse and human tandem repeat loci.
We aimed to estimate the rate of germline mutations in the offspring of individuals accidentally exposed to Cesium-137 ionizing radiation. The study included two distinct groups: one of cases, consisting of males and females accidentally exposed to low doses of ionizing radiation of Cs 137 , and a control group of non-exposed participants. The cases included 37 people representing 11 families and 15 children conceived after the accident. Exposed families incurred radiation absorbed doses in the range of 0.2 to 0.5 Gray. The control group included 15 families and 15 children also conceived after 1987 in Goiâ nia with no history of radiation exposure. DNA samples from peripheral blood were analyzed with the Affymetrix GeneChip ® CytoScanHD™ to estimate point mutations in autosomal SNPs. A set of scripts previously developed was used to detect de novo mutations by comparing parent and offspring genotypes at the level of each SNP marker. Overall numbers of observed Mendelian deviations were statistically significant between the exposed and control groups. Our retrospective transgenerational DNA analysis showed a 44.0% increase in the burden of SNP mutations in the offspring of cases when compared to controls, based on the average of MF MD for the two groups. Parent-of-origin and type of nucleotide substitution were also inferred. This proved useful in a retrospective estimation of the rate of de novo germline mutations in a human population accidentally exposed to low doses of radiation from Cesium-137. Our results suggested that observed burden of germline mutations identified in offspring was a potentially useful biomarker of effect to estimate parental exposure to low doses of IR and could become an important marker suitable for biomonitoring human population exposed to environmental mutagens.
Elevated mutation rates in the germ line of first-and second-generation offspring of …
Proceedings of the …, 2002
Mutation rates at two expanded simple tandem repeat loci were studied in the germ line of first-and second-generation offspring of inbred male CBA͞H, C57BL͞6, and BALB͞c mice exposed to either high linear energy transfer fission neutrons or low linear energy transfer x-rays. Paternal CBA͞H exposure to either x-rays or fission neutrons resulted in increased mutation rates in the germ line of two subsequent generations. Comparable transgenerational effects were observed also in neutron-irradiated C57BL͞6 and xirradiated BALB͞c mice. The levels of spontaneous mutation rates and radiation-induced transgenerational instability varied between strains (BALB͞c>CBA͞H>C57BL͞6). Pre-and postmeiotic paternal exposure resulted in similar increases in mutation rate in the germ line of both generations of CBA͞H mice, which together with our previous results suggests that radiation-induced expanded simple tandem repeat instability is manifested in diploid cells after fertilization. The remarkable finding that radiationinduced germ-line instability persists for at least two generations raises important issues of risk evaluation in humans.
Environmental and Molecular Mutagenesis, 2012
Large tandem repeat DNA loci such as expanded simple tandem repeats and minisatellites are efficient markers for detecting germline mutations; however, mutation detection using these loci can be imprecise and difficult to standardize across labs. Short-tandem repeats, such as microsatellites, offer more precise and high-throughput mutation detection, but germline mutation induction at these loci has not yet been studied in model organisms such as mice. In this study, we used microsatellite enrichment and large-scale DNA sequencing of several closely related inbred mouse lines to identify a panel of 19 polymorphic microsatellites with potentially high spontaneous mutation frequencies. We used this panel and four additional loci from other sources to quantify spontaneous mutation frequency in pedigrees of outbred Swiss-Webster mice. In addition, we also examined mutation induction in families in which sires were treated with acute doses of either 0.5 Gy or 1.0 Gy gamma-irradiation to spermatogonial stem cells. Per locus mutation frequencies ranged from 0 to 5.03 3 10 23 . Considering only the 11 loci with mutations, the mutation frequencies were: control 2.78 3 10 23 , 0.5 Gy 4.09 3 10 23 , and 1.0 Gy 1.82 3 10 23 . There were no statistically significant changes in mutation frequencies among treatment groups. Our study provides the first direct quantification of microsatellite mutation frequency in the mouse germline, but shows no evidence for mutation induction at pre-meiotic male germ cells following acute gamma-irradiation. Further work using the panel is needed to examine mutation induction at different doses of radiation, exposure durations, and stages during spermatogenesis. Environ. Mol. Mutagen. 00:000-000, 2012. V V C 2012 Wiley Periodicals, Inc.