Next generation sequencing of benzo(a)pyrene-induced lacZ mutants identifies a germ cell-specific mutation spectrum.

Jason M O’Brien,Carole L Yauk,Marc A Beal,Francesco Marchetti

doi:10.1038/srep36743

Abstract

De novo mutations are implicated in a variety of genetic diseases and arise primarily in the male germline. We investigated whether male germ cells have unique mechanisms for spontaneous or chemically-induced mutation relative to somatic cells using the MutaMouse model. We recovered lacZ transgenes from sperm 42 days after a 28-day exposure to benzo(a)pyrene (BaP, 100 mg/kg/day) to assess mutations arising in dividing spermatogonia. BaP caused a 3.4-fold increase in lacZ mutant frequency over controls which increased to 4.1-fold after clonal correction. We then used next generation sequencing to compare the spontaneous and BaP-induced mutation spectra in sperm and bone marrow. The spontaneous spectrum in sperm had significantly more G:C to A:T transitions and fewer mutations at A:T basepairs than bone marrow. BaP predominantly induced G:C to T:A transversions in both cell types, and both were enriched for mutations at CpG dinucleotides. However, BaP induced significantly more deletions in sperm, but more G:C to A:T transitions and G:C to C:G transversions in bone marrow. Differences in error-prone translesion DNA synthesis polymerases may underlie the observed spectrum differences between sperm and bone marrow. These findings suggest that mutations in sperm can arise via mechanisms that are unique to male germ cells.

Highlights

Over long periods of time, germline mutation is one of the primary driving forces of evolution
There are several mechanistic factors, some of which are unique to males, that may contribute to germline specific mutation[5]: (a) male germ cells progressively lose DNA repair capacity as they advance through spermatogenesis[6,7]; (b) the chromatin remodeling required for the replacement of histone proteins with protamines during spermiogenesis[8] may promote additional opportunities for misrepair of endogenous strand breaks[9]; (c) the process of meiosis provides opportunities to affect nucleotide composition that are unique to germ cells; and, (d) post-meiotic haploid germ cells cannot rely on sister chromatids as templates for homology-based repair mechanisms[10]
The animals used in the present study are a subset from a previously reported dose-response study on the induction of lacZ mutations in dividing spermatogonia[26]

Summary

Introduction

Over long periods of time, germline mutation is one of the primary driving forces of evolution. In addition to influencing mutation rate, these factors may contribute to the generation of different types of mutation (ie, mutation spectrum) in male germ cells relative to other cell types It stands to reason, that mutagenic chemicals may induce mutations via germ cell-specific mechanisms, resulting in a unique spectrum of mutations relative to other cell types. We use the TGR mutation assay coupled with NGS to produce high-resolution spectra of mutation types in the germ cells of untreated and BaP-exposed male mice. We compare these to the spontaneous and BaP-induced mutation spectra in bone marrow[16] and speculate on the possible mechanisms responsible for any spectral discrepancies between these tissues. This study provides important evidence for elucidating whether or not there are germ cell-specific mechanisms for spontaneous and BaP-induced mutations

Methods

Results

Conclusion