Estimation of the frequency of inherited germline mutations by whole exome sequencing in ethyl nitrosourea-treated and untreated gpt delta mice

Abstract

BackgroundGermline mutations are heritable and may cause health disadvantages in the next generation. To investigate trans-generational mutations, we treated male gpt delta mice with N-ethyl-N-nitrosourea (ENU) (85 mg/kg intraperitoneally, weekly on two occasions). The mice were mated with untreated female mice and offspring were obtained. Whole exome sequencing analyses were performed to identify de novo mutations in the offspring.ResultsAt 20 weeks after the treatment, the gpt mutant frequencies in the sperm of ENU-treated mice were 21-fold higher than those in the untreated control. Liver DNA was extracted from six mice, including the father, mother, and four offspring from each family of the ENU-treated or untreated mice. In total, 12 DNA samples were subjected to whole exome sequencing analyses. We identified de novo mutations in the offspring by comparing single nucleotide variations in the parents and offspring. In the ENU-treated group, we detected 148 mutation candidates in four offspring and 123 (82 %) were confirmed as true mutations by Sanger sequencing. In the control group, we detected 12 candidate mutations, of which, three (25 %) were confirmed. The frequency of inherited mutations in the offspring from the ENU-treated family was 184 × 10−8 per base, which was 17-fold higher than that in the control family (11 × 10−8 per base). The de novo mutation spectrum in the next generation exhibited characteristic ENU-induced somatic mutations, such as base substitutions at A:T bp.ConclusionsThese results suggest that direct sequencing analyses can be a useful tool for investigating inherited germline mutations and that the germ cells could be a good endpoint for evaluating germline mutations, which are transmitted to offspring as inherited mutations.Electronic supplementary materialThe online version of this article (doi:10.1186/s41021-016-0035-y) contains supplementary material, which is available to authorized users.

Effect of sampling time on somatic and germ cell mutations induced by acrylamide in gpt delta mice

“…Further study is necessary to elucidate genotoxic effects of chronically exposed AA in germlines and subsequent generations. Recent advances in sequencing technology has been able to detect de novo mutations induced in the offspring of mutagen-treated parents [ 35 , 45 , 46 ]. It is important to investigate whether dietary AA intake could induced DNA damage and results in germline mutations and heritable effects.…”

“…Briefly, high molecular weight genomic DNA was extracted from the testes and lung using RecoverEase DNA Isolation Kit (Agilent Technologies, Santa Clara, CA). Sperm DNA was extracted as previously described [ 35 ]. In brief, cauda epididymis was chopped in 1 mL of phosphate-buffered saline (pH 7.4), mesh-filtered, and pelleted by centrifugation.…”

Whole Genome Sequencing of the Mutamouse Model Reveals Strain- and Colony-Level Variation, and Genomic Features of the Transgene Integration Site

“…We attempted to identify the location of the breakpoints using a published method 55 . However, the aligned reads were located on many different chromosomes with no obvious pattern, suggesting that transgene integration took place in a genomic region containing repetitive elements.…”

Germline mutation: <i>de novo</i> mutation in reproductive lineage cells

“…Thus, confirmation by wet methods such as Sanger sequencing may be required to ascertain whether an identified mutation is true or false. Several parameters, such as sequencing depth and alternate/reference read ratio, could improve the efficiency of detecting DNMs (Masumura et al, 2016a(Masumura et al, , 2016b. Common standards regarding use of the same parameters and thresholds for the confirmation of mutations in addition to a better DNM detection pipeline would be helpful to compare mutation data among different studies.…”