Functional characterization of BRCA1 sequence variants using a yeast small colony phenotype assay.
Germline mutations that inactivate the tumor suppressor gene BRCA1 are associated with an increased risk of cancers of the breast and other tissues, but the functional consequence of many missense variants found in the human population is uncertain. Several predictive methods have been proposed to distinguish cancer-predisposing missense mutations from harmless polymorphisms, including a small colony phenotype (SCP) assay performed in the model organism, yeast (Saccharomyces cerevisiae). The goal of this study was to further evaluate this colony size assay. We constructed 28 missense mutations throughout the C-terminal 305 amino acid residues of BRCA1. Mutated proteins were expressed in yeast and evaluated using the SCP assay. We conclude there is as yet no evidence the assay can identify inactivating mutations upstream of the BRCT repeats. However, within and between the BRCT repeats, results of the assay are in general agreement with predictions based on structural modeling, other in vitro and in vivo assays, and cross-species sequence conservation. Thus, the yeast assay appears to provide confirmatory in vivo evidence to aid in characterizing some BRCA1 missense variants.
Coyne
Robert S
RS
Department of Biochemistry and Molecular Genetics, University of Virginia Health Sciences Center, Charlottesville, Virginia, USA. rcoyne@nsf.gov
McDonald
Heather B
HB
Edgemon
Keith
K
Brody
Lawrence C
LC
D019313:BRCA1 Protein / Q000032:analysis*; D001943:Breast Neoplasms / Q000145:classification / Q000175:diagnosis / Q000235:genetics; D005260:Female; D014644:Genetic Variation; D006801:Humans; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D014411:Neoplastic Stem Cells; D010641:Phenotype; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development*; D015533:Transcriptional Activation / Q000235:genetics*
D019313:BRCA1 Protein