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. 2008 Jul 1;68(13):5023-30.
doi: 10.1158/0008-5472.CAN-07-6189.

A syngeneic variance library for functional annotation of human variation: application to BRCA2

Tomas Hucl  1 , Carlo Rago, Eike Gallmeier, Jonathan R Brody, Myriam Gorospe, Scott E Kern
Affiliations

A syngeneic variance library for functional annotation of human variation: application to BRCA2

Tomas Hucl et al. Cancer Res. .

Abstract

The enormous scope of natural human genetic variation is now becoming defined. To accurately annotate these variants, and to identify those with clinical importance, is often difficult to assess through functional assays. We explored systematic annotation by using homologous recombination to modify a native gene in hemizygous (wt/Deltaexon) human cancer cells, generating a novel syngeneic variance library (SyVaL). We created a SyVaL of BRCA2 variants: nondeleterious, proposed deleterious, deleterious, and of uncertain significance. We found that the null states BRCA2(Deltaex11/Deltaex11) and BRCA2(Deltaex11/Y3308X) were deleterious as assessed by a loss of RAD51 focus formation on genotoxic damage and by acquisition of toxic hypersensitivity to mitomycin C and etoposide, whereas BRCA2(Deltaex11/Y3308Y), BRCA2(Deltaex11/P3292L), and BRCA2(Deltaex11/P3280H) had wild-type function. A proposed phosphorylation site at codon 3291 affecting function was confirmed by substitution of an acidic residue (glutamate, BRCA2(Deltaex11/S3291E)) for the native serine, but in contrast to a prior report, phosphorylation was dispensable (alanine, BRCA2(Deltaex11/S3291A)) for BRCA2-governed cellular phenotypes. These results show that SyVaLs offer a means to comprehensively annotate gene function, facilitating numerical and unambiguous readouts. SyVaLs may be especially useful for genes in which functional assays using exogenous expression are toxic or otherwise unreliable. They also offer a stable, distributable cellular resource for further research.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest: The Johns Hopkins University School of Medicine has property rights in the materials (cell lines) reported in this article. T. Hucl and S.E. Kern are entitled as inventors to a share of the royalty received by the University for licensed uses of the materials by commercial entities. The terms of licensing agreement(s) are being managed by the Johns Hopkins University School of Medicine Committee on Conflict of Interest in accordance with its conflict of interest policy. (Licensing agreements covering the materials in this article total and inventors' interest of less than $10,000 in total.) The other authors disclosed no potential conflicts of interest.

Figures

Figure 1
Figure 1
Structural and functional demonstration of BRCA2 gene disruption. A, targeting scheme. OF and OR, primers outside the deleted sequence; IF and IR, primers inside the deleted sequence; SF, SR, SF2, and SR2, screening primer pairs; LHA and RHA, left and right homology arms. Numbers on light background indicate exons, and numbers on dark background indicate BRC repeats. B, PCR detecting the wt and targeted alleles using genomic DNA as template. C, Northern blot detecting the targeted exon 11 mRNA transcript.
Figure 2
Figure 2
Characterization of BRCA2-null cells. A, immunofluorescence of RAD51 nuclear foci in untreated and treated cells (2.4 μg/mL MMC). BRCA2 +/−, BRCA2wt/Δex11; BRCA2−/−, BRCA2Δex11/Δex11. B, analysis of 50 metaphases for chromosomal aberrations in untreated and treated (equitoxic doses of MMC) heterozygous and knockout clones. C.1, colony formation assays on irradiation. BRCA2 par, parental BRCA2wt/mut × 2. Points, mean of three independent experiments; bars, SE. C.2, cell cycle analysis 48 h after MMC treatment. Representative cell cycle profiles obtained after treatment with indicated concentrations of MMC. D, cell proliferation following treatment with selected drugs. Two BRCA2 −/− (BRCA2Δex11/Δex11) subclones were analyzed. Points, mean of three independent experiments; bars, SE.
Figure 3
Figure 3
Drug sensitivity studies. Cell proliferation on treatment with various drugs at the indicated concentrations compared with untreated cells. Points, mean of three independent experiments; bars, SE.
Figure 4
Figure 4
Structural demonstration of BRCA2 exon 27 targeting. Targeting scheme. OF, primer outside the deleted sequence; IR, primer inside the deleted sequence; SF, SR, SF2, and SR2, screening primer pairs; INF and INR, intron 25 spanning primers; LHA and RHA, left and right homology arms.
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