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




Estimation of the frequency of inherited germline mutations by whole exome sequencing in ethyl nitrosourea-treated and untreated gpt delta mice - Descarga este documento en PDF. Documentación en PDF para descargar gratis. Disponible también para leer online.

Genes and Environment

, 38:10

First Online: 01 April 2016Received: 23 December 2015Accepted: 11 February 2016

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 per base, which was 17-fold higher than that in the control family 11 × 10 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.

KeywordsExome sequencing Germline mutation Heritable mutation Mutation spectrum Transgenic rodent gene mutation assay Abbreviations6TG6-thioguanine

Cmchloramphenicol

ENUN-ethyl-N-nitrosourea

GbGigabase

GQgenotype quality

HPLChigh-performance liquid chromatography

MFmutation frequency

NGSnext-generation sequencing

SDSsodium dodecyl sulfate

SNVsingle nucleotide variant

SSCsaline sodium citrate

TGCEtemperature gradient capillary electrophoresis

TGRtransgenic rodent

Electronic supplementary materialThe online version of this article doi:10.1186-s41021-016-0035-y contains supplementary material, which is available to authorized users.

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Autor: Kenichi Masumura - Naomi Toyoda-Hokaiwado - Akiko Ukai - Yoichi Gondo - Masamitsu Honma - Takehiko Nohmi

Fuente: https://link.springer.com/



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