Effects of hydroxyurea on CNV induction in the mouse germline

Abstract

Copy number variants (CNVs) are important in genome variation and genetic disease, with new mutations arising frequently in the germline and somatic cells. Replication stress caused by aphidicolin and hydroxyurea (HU) is a potent inducer of de novo CNVs in cultured mammalian cells. HU is used extensively for long-term management of sickle cell disease. Here, we examined the effects of HU treatment on germline CNVs in vivo in male mice to explore whether replication stress can act as a CNV mutagen in germline mitotic divisions as in cultured cells and whether this would support a concern for increased CNV mutations in offspring of men treated with HU. Several trials of HU administration were performed by oral gavage and subcutaneous pump, with CNVs characterized in C57BL/6 x C3H/HeJ hybrid mouse offspring by microarray and mate-pair sequencing. HU had a short half-life of ~14 min and a narrow dose window over which studies could be performed while maintaining fertility. Tissue histopathology and reticulocyte micronucleus assays verified that doses had a substantial tissue and genetic toxicity. CNVs were readily detected in offspring that originated in both paternal and maternal mouse strains, as de novo and inherited events. However, HU did not increase CNV formation above baseline levels. These results reveal a high rate of CNV mutagenesis in the mouse germline but do not support the hypothesis that HU would increase CNV formation during mammalian spermatogenesis, perhaps due to highly toxic effects on sperm development or experimental variables related to HU pharmacology in mice. Environ. Mol. Mutagen. 59:698-714, 2018. © 2018 Wiley Periodicals, Inc.

Paternal exposure to benzo(a)pyrene induces genome-wide mutations in mouse offspring

“…This resulted in a final set of ten confirmed de novo and two germline mosaic CNVs (Supplementary Data 6). The spontaneous de novo deletion CNV frequency observed in our control mice (1.8%; 3/171) was comparable with previous estimates (1.5%; 2/136) using combined data from two published mouse studies 11,35 . The frequency of deletion CNVs in the treated group was 0.5–1% (1/83, 0/98 for the two time points, respectively; 1/181 total); hence, there was no evidence for induction of deletions (Table 1).…”

“…All of the de novo CNVs with detectable breakpoints appear to have been mediated by overlaps consisting of short microhomologies of 1–6 bp between sequence breakpoints (Supplementary Data 6). These events are similar to the CNVs detected in irradiated human fibroblasts 36 and in the germline of mice exposed to hydroxyurea 35 where 1–8 -bp homologies were observed at most breakpoint junctions. The CNVs with microhomology preclude involvement of homologous recombination repair (HRR) because the sequence homology required for HRR is >200 bp 37 .…”

“…Using sires as the biological unit, and considering the number of offspring analyzed for each sire (Supplementary Data 7), yields a statistically significant increase in the frequency of duplication CNVs (0.0278 vs. 0; Fisher’s exact test, adjusted P < 0.001) demonstrating that this result is not due to clustering within a litter or within a BaP-exposed male. Indeed, the spontaneous frequency of duplications appears to be rare compared with deletions, as only a single duplication was detected from 93 control animals from Adewoye et al 11 , 43 control animals from Arlt et al 35 , and 171 animals in this study. Thus, although we have recovered few duplications overall, this increase in the exposed mice is unlikely to be due to chance alone.

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DNA Copy Number Variations as Markers of Mutagenic Impact

“…The effects of HU on germline CNVs was studied in mice [16] to test the idea that HU-induced replication stress will act as a CNV-inducing mutagen in germline mitotic divisions in the same manner as in cultured somatic cells [11]. However, HU did not increase the control level of CNV, perhaps due to highly toxic effects on sperm development or experimental variables related to HU pharmacology in mice.…”

“…CNV formation, like all classes of mutation, will likely include exposure to environmental mutagens and inherited genetic predispositions. The spectrum of DNA damaging agents known to lead to increased rates of CNV formation is still quite limited [10,16]. In this review, we will focus on radiation- and chemical mutagen-induced CNVs in cells, organisms and germline.…”