title: Dose-dependent de novo germline mutations detected by whole-exome sequencing in progeny of ENU-treated male gpt delta mice.
abstract: 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 (85mg/kg, i.p., weekly on two occasions). In this study, two lower doses (10 and 30mg/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.
authors: Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan. Electronic address: masumura@nihs.go.jp.
authors: Kenichi
authors: K
authors: Masumura
authors: Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
authors: Naomi
authors: N
authors: Toyoda-Hokaiwado
authors: Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
authors: Akiko
authors: A
authors: Ukai
authors: RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan.
authors: Yoichi
authors: Y
authors: Gondo
authors: Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
authors: Masamitsu
authors: M
authors: Honma
authors: Biological Safety Research Center, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
authors: Takehiko
authors: T
authors: Nohmi
mesh_terms: D000818:Animals; D029968:Escherichia coli Proteins / Q000235:genetics*; D005038:Ethylnitrosourea / Q000633:toxicity*; D059472:Exome; D005260:Female; D018095:Germ-Line Mutation; D059014:High-Throughput Nucleotide Sequencing; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D010430:Pentosyltransferases / Q000235:genetics*
chemical_list: D029968:Escherichia coli Proteins; D010430:Pentosyltransferases; C071962:Gpt protein, E coli; D005038:Ethylnitrosourea
keywords: Germline mutation; Inherited mutation; Mutation frequency; Transgenic rodent gene mutation assay; Whole-exome sequencing; gpt delta mouse