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8-oxoguanine Causes Spontaneous De Novo Germline Mutations in Mice

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8-oxoguanine Causes Spontaneous De Novo Germline Mutations in Mice

Mizuki Ohno et al. Sci Rep.

Abstract

Spontaneous germline mutations generate genetic diversity in populations of sexually reproductive organisms, and are thus regarded as a driving force of evolution. However, the cause and mechanism remain unclear. 8-oxoguanine (8-oxoG) is a candidate molecule that causes germline mutations, because it makes DNA more prone to mutation and is constantly generated by reactive oxygen species in vivo. We show here that endogenous 8-oxoG caused de novo spontaneous and heritable G to T mutations in mice, which occurred at different stages in the germ cell lineage and were distributed throughout the chromosomes. Using exome analyses covering 40.9 Mb of mouse transcribed regions, we found increased frequencies of G to T mutations at a rate of 2 × 10(-7) mutations/base/generation in offspring of Mth1/Ogg1/Mutyh triple knockout (TOY-KO) mice, which accumulate 8-oxoG in the nuclear DNA of gonadal cells. The roles of MTH1, OGG1, and MUTYH are specific for the prevention of 8-oxoG-induced mutation, and 99% of the mutations observed in TOY-KO mice were G to T transversions caused by 8-oxoG; therefore, we concluded that 8-oxoG is a causative molecule for spontaneous and inheritable mutations of the germ lineage cells.

Figures

Figure 1
Figure 1. Phenotype of TOY-KO mice.
(a) Accumulation of 8-oxodG in TOY-KO mouse tissues. LC-MS/MS was used to determine the amount of 8-oxodG. Data are presented as the means ± SD. Wilcoxon tests were used to analyze differences between TOY-KO (gray) and C57BL/6J:Jcl (open) mouse tissues (* P < 0.05; ** P < 0.001). (b) Survival of TOY-KO mice. The survival curve of TOY-KO mice (n = 56, indicated in red) was compared with that of Mth1+/−/Ogg1+/−/Mutyh+/− (TOY-hetero) mice (n = 14, indicated in black). (c) A Harderian gland tumor (left) and a trichoepithelioma (right) observed in a TOY-KO mice (indicated by arrows). Hematoxylin and eosin staining of each tumor is shown. Scale bars, 200 μm. (d) Numbers of newborn and weaned mice. Gray and red bars indicate the numbers of newborn and weaned mice in each generation of TOY-KO mice, respectively.
Figure 2
Figure 2. Phenotypic variations observed in the progeny of TOY-KO mice.
(a) The hydrocephalus trait was transmitted to the next generation in the TOY-KO pedigree. A hematoxylin/eosin-stained section showing the typical features of the hydrocephalus trait. Blue indicates a mouse with hydrocephalus, and green indicates a mouse carrying the causative mutation without the hydrocephalus phenotype (also shown in Supplementary Fig. S2 online). (b) Hydrocephalus. MRI, hematoxylin/eosin staining and X-ray images of normal (C57BL/6J) and hydrocephalus TOY-KO mice are shown in the upper panel. MRI images were obtained using an MRI mini SA (DS Pharma Biomedical Co. Ltd., Suita, Japan). X-ray images were obtained using a μFX-1000 (Fuji Photo File Co. Ltd.). (c) Pedigrees of the TOY-KO mouse mated with C57BL/6J (shown as B6) and 129Sv mice are shown in the lower panel. Blue indicates a mouse with hydrocephalus, and green indicates a mouse carrying the causative mutation without the hydrocephalus phenotype.
Figure 3
Figure 3. Identification of de novo germline mutations in TOY-KO mice.
(a) Scheme for screening of germline mutations. (b) Pedigree of TOY-KO mice used for germline mutation analysis. TOY365F, TOY609F and TOY450F were used to identify de novo germline mutations. Blue numbers, 84, 98, and 114, indicate the number of mutations detected in TOY365F, TOY609F and TOY450F, respectively. Numbers in parentheses indicate the number of original mutations in each generation, which were found in tail DNA for the first time in the pedigree. The DNA of TOY110F was unavailable; therefore, the mouse was excluded from the analysis. (c) The numbers of base substitution mutations found in TOY365F, TOY609F and TOY450F.
Figure 4
Figure 4. Heritable mutations mapped in the pedigree of TOY-KO mice.
The number in each box indicates the mutation ID number shown in Supplementary Data S1 online, and the color indicates the mutation category.
Figure 5
Figure 5. Genome-wide distribution of mutations and site preferences of G to T mutations in di- and trinucleotide sequences.
(a) Mutations detected in G2–G8 were mapped on a mouse G-band ideogram using Ideographica (http://www.ncrna.org/idiographica/). Each black transverse line on the right side of the chromosome represents a mutation site. (b) Site preferences of G to T mutations in di-nucleotide sequences. The plots represent the relative ratio of the actual value of detected mutations (G to T mutations in G2–G8) in each di-nucleotide to its occurrence level in the analyzed exome sequences. ‘g' indicates the position of a mutated guanine. (c) Site preferences of G to T mutations in tri-nucleotides. For each nucleotide sequence, a chi square test (detected vs. expected) was performed, and the colored sequences indicate a significant difference: P < 0.001 (pink), P < 0.01 (blue), and P < 0.05 (orange).
Figure 6
Figure 6. Fate of a germline mutation.
Mutation #187 (Ch. 15) was chosen to show the fate of a mutation generated in TOY-KO mice through the generations. This mutation initially appeared in TOY108M (G3) as a heterozygous allele. It was transmitted to progeny TOY-114M, TOY-115F, and TOY-132F. At G5, mutation #187 became homozygous in TOY138M and TOY131F, and thus were fixed in the progeny. Conversely, in another branch, the mutation was not transmitted from TOY-234M and TOY-236F (G6) to their offspring and eventually disappeared. The mutated locus is indicated in red.
Figure 7
Figure 7. Germ line mutations occur at different stages of the germ cell lineage.
Mutations detected in the tail DNA of the first mutant mouse had occurred either in the germ lineage cells of the previous generation or during the very early developmental stage of the mutant mouse. Mutations start to accumulate from the first replication of fertilized egg DNA; however, each mutation is diluted out in the tissue DNA. Therefore, we used the tail DNA sequence as a reference sequence of fertilized egg DNA. In contrast to tail tissue, differentiated gametes can transmit their sequence information monoclonally to offspring. If the original mutated allele was mapped in multiple mice of the same generation, such as mutation #54 (in Fig. 4, Supplementary Data S1 online), the mutation probably occurred in the germ lineage cells of the parents (indicated as A). For mutations in the X chromosome (such as mutation #261), which began in the male with a heterozygous status (see Supplementary Fig. S4 online), the mutation probably occurred in a cell during the early stage of embryonic development (shown as B), resulting in mosaicism of tail tissue. These results indicate that germline mutations occur at different developmental stages of the germ cell lineage.

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