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A Syngeneic Variance Library for Functional Annotation of Human Variation: Application to BRCA2

¹ The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University; ² Howard Hughes Medical Institute and the Ludwig Center for Cancer Genetics and Therapeutics, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University; and ³ Laboratory of Cellular and Molecular Biology, National Institute on Aging-Intramural Research Program, NIH, Baltimore, Maryland

Requests for reprints: Scott E. Kern, Department of Oncology, Johns Hopkins University, 1650 Orleans Street, Baltimore, MD 21231. Phone: 410-614-3314; Fax: 443-287-4653; E-mail: sk{at}jhmi.edu.

Key Words: BRCA2 • knockout • syngeneic library

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/exon) 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^ex11/ex11 and BRCA2^ex11/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^ex11/Y3308Y, BRCA2^ex11/P3292L, and BRCA2^ex11/P3280H had wild-type function. A proposed phosphorylation site at codon 3291 affecting function was confirmed by substitution of an acidic residue (glutamate, BRCA2^ex11/S3291E) for the native serine, but in contrast to a prior report, phosphorylation was dispensable (alanine, BRCA2^ex11/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. [Cancer Res 2008;68(13):5023–30]