AbstractThe 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/Δexon) 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 BRCA2Δex11/Δex11 and BRCA2Δex11/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 BRCA2Δex11/Y3308Y, BRCA2Δex11/P3292L, and BRCA2Δex11/P3280H had wild-type function. A proposed phosphorylation site at codon 3291 affecting function was confirmed by substitution of an acidic residue (glutamate, BRCA2Δex11/S3291E) for the native serine, but in contrast to a prior report, phosphorylation was dispensable (alanine, BRCA2Δex11/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]