Characteristics of induced mutations in offspring derived from irradiated mouse spermatogonia and mature oocytes.

The exposure of germ cells to radiation introduces mutations in the genomes of offspring, and a previous whole-genome sequencing study indicated that the irradiation of mouse sperm induces insertions/deletions (indels) and multisite mutations (clustered single nucleotide variants and indels). However, the current knowledge on the mutation spectra is limited, and the effects of radiation exposure on germ cells at stages other than the sperm stage remain unknown. Here, we performed whole-genome sequencing experiments to investigate the exposure of spermatogonia and mature oocytes. We compared de novo mutations in a total of 24 F1 mice conceived before and after the irradiation of their parents. The results indicated that radiation exposure, 4 Gy of gamma rays, induced 9.6 indels and 2.5 multisite mutations in spermatogonia and 4.7 indels and 3.1 multisite mutations in mature oocytes in the autosomal regions of each F1 individual. Notably, we found two types of deletions, namely, small deletions (mainly 1~12 nucleotides) in non-repeat sequences, many of which showed microhomology at the breakpoint junction, and single-nucleotide deletions in mononucleotide repeat sequences. The results suggest that these deletions and multisite mutations could be a typical signature of mutations induced by parental irradiation in mammals.

Radiation-Induced Deletions in Mouse Spermatogonia are Usually Large (over 200 kb) and Contain Little Sequence Similarity at the Junctions.

Ionizing radiation can induce mutations, and the majority of radiation-induced mutations in mammalian cells are deletions. The most critical types of radiation-induced DNA damage are DNA double-strand breaks, and these breaks are repaired by either the homologous recombination (HR) pathway or the non-homologous end joining (NHEJ) pathway. The HR pathway is not as mutagenic as the NHEJ pathway, and it is expected that radiation-induced deletions would usually have little sequence similarity around the deletion junction points. Here we report sequence data from the regions around the rejoined junctions of 33 de novo copy-number mutations (27 deletions and 6 duplications) obtained from offspring sired by male mice that were irradiated at the spermatogonia stage and from nonirradiated controls. The results indicate that deletions can be classified into three major groups. In group 1, nine deletions were found to share long blocks of similar sequences (200-6,000 bp) at the junctions and the deletion size varied extensively (1 kb to 2 Mb) (e.g., illegitimate recombination). In group 2, five deletions shared short identical sequences (0-7 bp) at the junctions, and the deletion sizes were shorter than 200 kb (e.g., micro-homology-mediated repair). Additional three-deletion candidates of this group were also found but turned out to be inherited from mosaic parents. They are therefore not included in germline mutations. In group 3, twelve deletions shared little sequence similarity (only 0-2 bp) at the junctions (likely due to NHEJ repair) and deletion sizes were longer than 200 kb. Group 1 consisted of deletions found in both spontaneous and irradiated genomes and thus, were probably caused by spontaneous events during meiosis or DNA replication. Group 2 consisted mainly of deletions found in nonexposed genomes. Group 3 consisted primarily of deletions that occurred in the irradiated genomes. Among the duplications, we found no indication of any association with radiation exposures. These results indicate that large size (>200 kb) and little sequence similarity around the rejoined sites are likely to be a hallmark of radiation-induced deletions in mice.

Genome-Wide Deletion Screening with the Array CGH Method in Mouse Offspring Derived from Irradiated Spermatogonia Indicates that Mutagenic Responses are Highly Variable among Genes.

Until the end of the 20th century, mouse germ cell data on induced mutation rates, which were collected using classical genetic methods at preselected specific loci, provided the principal basis for estimates of genetic risks from radiation in humans. The work reported on here is an extension of earlier efforts in this area using molecular methods. It focuses on validating the use of array comparative genomic hybridization (array CGH) methods for identifying radiation-induced copy number variants (CNVs) and specifically for DNA deletions. The emphasis on deletions stems from the view that it constitutes the predominant type of radiation-induced genetic damage, which is relevant for estimating genetic risks in humans. In the current study, deletion mutations were screened in the genomes of F1 mice born to unirradiated or 4 Gy irradiated sires at the spermatogonia stage (100 offspring each). The array CGH analysis was performed using a "2M array" with over 2 million probes with a mean interprobe distance of approximately 1 kb. The results provide evidence of five molecularly-confirmed paternally-derived deletions in the irradiated group (5/100) and one in the controls (1/100). These data support a calculation, which estimates that the mutation rate is 1 × 10-2/Gy per genome for induced deletions; this is much lower than would be expected if one assumes that the specific locus rate of 1 × 10-5/locus per Gy (at 34 loci) is applicable to other genes in the genome. The low observed rate of induced deletions suggests that the effective number of genes/genomic regions at which recoverable deletions could be induced would be only approximately 1,000. This estimate is far lower than expected from the size of the mouse genome (>20,000 genes). Such a discrepancy between observation and expectation can occur if the genome contains numerous genes that are far less sensitive to radiation-induced deletions, if many deletion-bearing offspring are not viable or if the current method is substandard for detecting small deletions.

Mutagenic effects of ionizing radiation on immature rat oocytes.

Estimates of genetic risks from radiation delivered to humans are derived largely from mouse studies. In males, the target is spermatogonia and a large amount of information is available. In contrast, in females, immature oocytes are the target, but extrapolations from mice to humans are not very definitive because immature mouse oocytes are highly sensitive to radiation and die by apoptosis, which is not the case in humans. Since mouse offspring derived from surviving immature oocytes have to date not shown any signs of mutation induction, two alternative hypotheses are proposed: 1. Apoptotic death effectively eliminates damaged oocytes in mice and therefore human immature oocytes may be highly mutable; and 2. Immature oocytes are inherently resistant to mutation induction and apoptotic death is not relevant to mutagenesis. To test these hypotheses, rat immature oocytes, which are not as sensitive as those in mice to radiation-induced apoptosis were exposed to 2.5 Gy of gamma rays and the offspring were examined using a two-dimensional DNA analysis method. Screening of a total of 2.26 million DNA fragments, we identified 32 and 18 mutations in the control and exposed groups, respectively. Of these, in the two groups, 29 and 14 mutations were microsatellite mutations, two and one were base changes, and one and three were deletions. Among the four deletions most relevant to radiation exposure, only one was possibly derived from the irradiated dam (but not determined) and three were paternal in origin. Although the number of mutations was small, the results appear to support the second hypothesis and indicate that immature oocytes are generally less sensitive than mature oocytes to mutation induction.

The genetic risk in mice from radiation: an estimate of the mutation induction rate per genome.

Restriction Landmark Genome Scanning (RLGS) is a method that uses end-labeled (32)P NotI sites that are mostly associated with coding genes to visualizes thousands of DNA fragments as spots in two-dimensional autoradiograms. This approach allows direct detection of autosomal deletions as spots with half normal intensity. The method was applied to mouse offspring derived from spermatogonia exposed to 4 Gy of X rays. A genome-wide assessment of the mutation induction rate was estimated from the detected deletions. Examinations were made of 1,007 progeny (502 derived from control males and 505 from irradiated males) and 1,190 paternal and 1,240 maternal spots for each mouse. The results showed one deletion mutation in the unirradiated paternal genomes of 502 offspring (0.2%) and 5 deletions in the irradiated paternal genomes of 505 offspring (1%). The difference was marginally significant, with the deletion sizes ranged from 2-13 Mb. If the frequencies are taken at face value, the net increase was 0.8% after an exposure of 4 Gy, or 0.2% per Gy per individual if a linear dose response is assumed. Since the present RLGS analysis examined 1,190 NotI sites, while the mouse genome contains ∼25,000 genes, the genomic probability of any gene undergoing a deletion mutation would be 25× 0.2%, or 5% per Gy. Furthermore, since the present RLGS screened about 0.2% of the total genome, the probability of detecting a deletion anywhere in the total genome would be estimated to be 500 times 0.2% or 100% (i.e., 1 deletion per Gy). These results are discussed with reference to copy number variation in the human genome.

Restriction landmark genome scanning for the detection of mutations.

There is a continuing need for more efficient methods to examine mutations in humans and other species resulting from exposures to environmental toxins and radiation. Environmental genomic studies, which often utilize nonmodel system species and as a result, there is a particular need for a method that does not rely on the availability of genome sequence information. Restriction landmark genome scanning (RLGS) is a two-dimensional electrophoresis (2-DE) of end-labeled DNA fragments. A vertical giant gel 2-DE system has been developed and applied to the RLGS. On a single RLGS pattern of mouse or human DNA, approx 2000 DNA fragments (spots) varying in size from 1.0 to 5.0 kb in the first dimension and 0.2 to 3.0 kb in the second dimension are visualized. In principle, this system will detect genomic alterations of two types: (1) that due to gain or loss of a cut site for the three restriction fragment enzymes employed in the study and (2) that due to insertion/deletion/rearrangement (I/D/R) events. After optimization of the sample preparation and electrophoresis conditions, the gel quality reached a level such that the electrophoresis patterns derived from a single DNA sample gave distribution patterns of spots able to be superimposed. This technology can visualize up to 3000 DNA fragments per gel without using any probes, and thus should be highly efficient in monitoring for mutations resulting in I/D/R events in DNA fragments distributed throughout the genome. This method relies on direct labeling of DNA fragments rather than hybridization and therefore precise information on genome sequences is not required. As a result this method is applicable to any species.

Estimation of mutation induction rates in AT-rich sequences using a genome scanning approach after X irradiation of mouse spermatogonia.

We have previously used NotI as the marker enzyme (recognizing GCGGCCGC) in a genome scanning approach for detection of mutations induced in mouse spermatogonia and estimated the mutation induction rate as about 0.7 x 10(-5) per locus per Gy. To see whether different parts of the genome have different sensitivities for mutation induction, we used AflII (recognizing CTTAAG) as the marker enzyme in the present study. After the screening of 1,120 spots in each mouse offspring, we found five mutations among 92,655 spots from the unirradiated paternal genome, five mutations among 218,411 spots from the unirradiated maternal genome, and 13 mutations among 92,789 spots from 5 Gy-exposed paternal genome. Among the 23 mutations, 11 involved mouse satellite DNA sequences (AT-rich), and the remaining 12 mutations also involved AT-rich but non-satellite sequences. Both types of sequences were found as multiple, similar-sequence blocks in the genome. Counting each member of cluster mutations separately and excluding results on one hypermutable spot, the spontaneous mutation rates were estimated as 3.2 (+/- 1.9) x 10(-5) and 2.3 (+/- 1.0) x 10(-5) per locus per generation in the male and female genomes, respectively, and the mutation induction rate as 1.1 (+/- 1.2) x 10(-5) per locus per Gy. The induction rate would be reduced to 0.9 x 10(-5) per locus per Gy if satellite sequence mutations were excluded from this analysis. The results indicate that mutation induction rates do not largely differ between GC-rich and AT-rich regions: 1 x 10(-5) per locus per Gy or less, which is close to 1.08 x 10(-5) per locus per Gy, the current estimate for the mean mutation induction rate in mice.

A genome scanning approach to assess the genetic effects of radiation in mice and humans.

We used Restriction Landmark Genome Scanning (RLGS) to assess, on a genome-wide basis, the mutation induction rate in mouse germ cells after radiation exposure. Analyses of 1,115 autosomal NotI DNA fragments per mouse for reduced spot intensity, indicative of loss of one copy, in 506 progeny derived from X-irradiated spermatogonia (190, 237 and 79 mice in 0-, 3-, and 5-Gy groups, respectively), permitted us to identify 16 mutations affecting 23 fragments in 20 mice. The 16 mutations were composed of eight small changes (1-9 bp) at microsatellite sequences, five large deletions (more than 25 kb), and three insertions of SINE B2 or LINE1 transposable elements. The maximum induction rate of deletion mutations was estimated as (0.17 +/- 0.09) x 10(-5)/locus Gy(-1). The estimate is considerably lower than 1 x 10(-5)/locus Gy(-1), the mean induction rate of deletion mutations at Russell's 7 loci, which assumed that deletion mutations comprise 50% of all mutations. We interpret the results as indicating that the mean induction rate of mutations in the whole genome may be substantially lower than that at the 7 loci. We also demonstrate the applicability of RLGS for detection of human mutations, which allows direct comparisons between the two species.

Two-dimensional complementary deoxyribonucleic acid electrophoresis revealing up-regulated human epididymal protein-1 and down-regulated CL-100 in thyroid papillary carcinoma.

Two-dimensional cDNA electrophoresis was used to analyze gene expressions in papillary carcinoma and normal tissue of thyroid glands. Pooled thyroid tissues were used to extract mRNA. Complementary DNAs, synthesized with NotI anchor primers, were digested with three restriction enzymes, NotI, EcoRV, and PvuII. The protruding NotI ends were filled in with (32)P deoxynucleotide triphosphates, and the radiolabeled cDNA fragments were separated in two dimensions. Approximately 500 cDNA fragments were visualized as discrete spots without probes. A total of 20 spots, 9 up-regulated and 11 down-regulated cDNAs in papillary carcinoma, were selected and cloned for sequencing. This experiment lent itself to a novel discovery of up-regulated human epididymal protein 1 (HE-1) and down-regulated CL-100 genes in thyroid papillary carcinomas confirmed by Northern blot analysis. Immunohistochemical stains showed abundant HE-1 protein in the papillary carcinoma, whereas little or no HE-1 protein was detected in other types of thyroid cancers and normal thyroid tissues. The restricted localization of HE-1 protein to the portions of papillary projections suggests an involvement of HE-1 protein for forming papillary shape. Our study showed that two-dimensional cDNA electrophoresis is a useful method of detecting differentially expressed genes in human diseases as demonstrated for HE-1 and CL-100 in papillary carcinoma.