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, 3 (3), 255-60

Disruption of Brca2 Increases the Spontaneous Mutation Rate in Vivo: Synergism With Ionizing Radiation

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Disruption of Brca2 Increases the Spontaneous Mutation Rate in Vivo: Synergism With Ionizing Radiation

Andrew N J Tutt et al. EMBO Rep.

Abstract

The breast cancer predisposition gene BRCA2 encodes a protein involved in the repair of DNA double-strand breaks, which arise spontaneously and following exposure to ionizing radiation (IR). To develop a mouse model that examines the effect of BRCA2 mutation and IR exposure on in vivo somatic mutation acquisition, we crossed mice with targeted disruption of Brca2 with a LacZ transgenic mutation reporter strain. Loss of both wild-type Brca2 alleles caused a 2.3-fold increase, equivalent to an extra 100 mutations per cell, in the in vivo acquisition of spontaneous somatic mutation by 2 weeks gestation. IR (4 Gy) had a disproportionate effect on animals homozygous for Brca2 disruption, inducing 3.4-fold more mutations compared with wild-type animals. These data provide the first evidence that loss of Brca2 increases in vivo somatic mutation acquisition and synergizes with IR exposure, with potential attendant implications for mammographic screening and therapeutic IR in mutation carriers.

Figures

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Fig. 1. Schematic diagram of a LacZ plasmid transgenic mouse model for analysis of the effect of Brca2 loss of function on in vivo somatic mutation frequency. Mice are shown of the three potential Brca2 genotypes either treated or untreated with IR. These mice are also homozygous for two pUR288 LacZ plasmid concatamer chromosome integration sites on chromosomes 3 and 4 (marked as black chromosomes). Each concatamer contains ∼10 copies of pUR288. The ampicillinr gene is shown as a blue box, the bacterial origin of replication as a yellow box and the LacZ reporter gene in red. HindIII sites between the plasmids are marked (H). Flanking regions of genomic DNA are represented by the green line. Individual linear plasmids are shown after release from genomic DNA by HindIII digestion. The central plasmid has a point mutation within LacZ represented by a green dot. The plasmid on the right-hand side has suffered a deletion or genomic rearrangement removing the 3′ LacZ sequence with a break point extending into flanking genomic DNA. This removes the original HindIII site and thus HindIII digestion recovers a LacZ mutant plasmid of different size to the wild type. Single reporter plasmids are recovered from genomic DNA, transformed into E. coli and mutation frequency determined by comparing colony numbers on mutant selection (P-gal) and non-selective rescue efficiency (X-gal) plates.
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Fig. 2. Analysis of the effect of loss of Brca2 on in vivo somatic mutation frequency and mutation type. (A) Scatter plot showing the somatic LacZ mutation frequency in the pUR288 reporter in Brca2Wt/Wt (n = 4), Brca2Wt/Tr (n = 5) and Brca2Tr/Tr (n = 3) day E14.5 embryos. Data points represent individual animals each based on >105 recovered reporter plasmids. Horizontal bars represent the mean. (B) AvaI–PstI restriction fragments from recovered LacZ mutant pUR288 plasmids run as an example of Size-change and No-change mutants compared with the wild-type pUR288 restriction digest pattern. (C) The bar chart shows the frequency of no-change, size-change and mouse genome rearrangement LacZ mutants in Brca2Wt/Wt (n = 4), Brca2Wt/Tr (n = 5) and Brca2Tr/Tr (n = 3) day E14.5 embryos. Error bars represent the standard error of the mean (SEM).
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Fig. 3. Analysis of the effect of IR on in vivo somatic mutation frequency and mutation type. (A) The bar chart shows the somatic mutation frequency in the LacZ pUR288 reporter in 4 Gy irradiated Brca2Wt/Wt (n = 8), Brca2Wt/Tr (n = 9) and Brca2Tr/Tr (n = 6) day E14.5 embryos compared with the unirradiated embryos of the same genotype. Error bars represent SEM. (B) Scatter plot showing the frequency of somatic LacZ mutation in the pUR288 reporter induced over the spontaneous background in 4 Gy-irradiated Brca2Wt/Wt (n = 8), Brca2Wt/Tr (n = 9) and Brca2Tr/Tr (n = 6) day E14.5 embryos. (C) The bar chart shows the frequency of size-change and no-change LacZ mutants in 4 Gy-irradiated Brca2Wt/Wt (n = 8), Brca2Wt/Tr (n = 9) and Brca2Tr/Tr (n = 6) day E14.5 embryos. Error bars represent the SEM.

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