A Syngeneic Variance Library for Functional Annotation of Human Variation: Application toBRCA2
Open Access
- 1 July 2008
- journal article
- Published by American Association for Cancer Research (AACR) in Cancer Research
- Vol. 68 (13), 5023-5030
- https://doi.org/10.1158/0008-5472.can-07-6189
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/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]This publication has 39 references indexed in Scilit:
- Knock-in of Mutant K-ras in Nontumorigenic Human Epithelial Cells as a New Model for Studying K-ras–Mediated TransformationCancer Research, 2007
- Distinguishing Rational from Irrational Applications of Pharmacogenetic Synergies from the Bench to Clinical TrialsCell Cycle, 2007
- Targeting Fanconi Anemia/BRCA2 Pathway Defects in Cancer: The Significance of Preclinical Pharmacogenomic ModelsClinical Cancer Research, 2007
- Mutation analysis of 24 known cancer genes in the NCI-60 cell line setMolecular Cancer Therapeutics, 2006
- The Consensus Coding Sequences of Human Breast and Colorectal CancersScience, 2006
- Cellular characterization of cells from the Fanconi anemia complementation group, FA-D1/BRCA2Mutation Research, 2006
- Chinese hamster cell mutant, V-C8, a model for analysis of Brca2 functionMutation Research - Reviews in Mutation Research, 2006
- Statistical Analysis of Pathogenicity of Somatic Mutations in CancerGenetics, 2006
- Targeted Disruption of FANCC and FANCG in Human Cancer Provides a Preclinical Model for Specific Therapeutic OptionsGastroenterology, 2006
- Breast and Ovarian Cancer Risks Due to Inherited Mutations in BRCA1 and BRCA2Science, 2003