Multimodal assessment of protein functional deficiency supports pathogenicity of BRCA1 p.V1688del

Unequivocal discrimination between neutral variants and deleterious mutations is crucial for appropriate counseling of individuals with a BRCA1 or BRCA2 sequence change. An increasing number of variants of uncertain significance (VUS) are being identified, the unclassified biological effect of which poses clinical concerns. A multifactorial likelihood-based approach recently suggested disease causality for BRCA1 p.V1688del, a VUS recurrent in Italian breast/ovarian cancer families. Whether and how this single amino acid deletion in the BRCA1 COOH terminus (BRCT) domain affects the function of the mutant protein (Delta ValBRCA1) has not been elucidated. We undertook comprehensive functional characterization of AValBRCA1, comprising comparative structural modeling, analysis of protein stability and associations, and analysis of DNA repair function. Our model predicted BRCT domain destabilization and folding disruption caused by BRCA1 p.V1688del. Consistently, the recombinant Delta ValBRCA1 was less stable than wild-type BRCA1 and, unlike the latter, failed to associate with BRIP1, CtIP, and Rap80 and to relocalize to sites of DNA damage. Yeast two-hybrid analysis revealed a compromised interaction with FHL2 and KPNA2, which is likely responsible for improper subcellular localization of Delta ValBRCA1. In addition, we found four new breast/ovarian cancer families of Italian ancestry who carried this sequence alteration. These results provide the first evidence of the effect of BRCA1 p.V1688del on protein stability and function, supporting the view that it is a deleterious mutation. Multimodal analyses like ours could advance understanding of tumor suppression by BRCA1 and ultimately contribute to developing efficient strategies for screening and characterization of VUS. [Cancer Res 2009;69(17):7030-7

Establishment and characterization of xenografts and cancer cell cultures derived from BRCA1 -/- epithelial ovarian cancers

The BRCA1 gene is responsible for a high number of hereditary breast and ovarian cancers that cluster in families with a strong genetic predisposition. Despite intense investigation, the accumulating findings on BRCA1 biological functions have not yet been translated into specific therapeutic approaches, also due to the lack of suitable experimental models. The purpose of this study was to establish and characterize cell cultures and xenografts from patients with BRCA1 -/- ovarian cancers. We derived two ovarian cancer cell lines, termed PD-OVCA1 and PD-OVCA2, both from patients previously treated with chemotherapy, that propagate in SCID mice as well as in vitro for a limited number of passages. Both cell lines expressed cytokeratins and the CA125 tumour marker. A detailed molecular characterization highlighted both constitutive and somatic genetic events that abrogate BRCA1 gene function. Both cell lines were shown to lose the wild type BRCA1 allele; intriguingly, these deletions were apparently accompanied by gain of one or more copies of the mutant alleles. Finally, a genomic profile of major chromosomal aberrations was obtained by the Multiplex Ligation-dependent Probe Amplification (MLPA) technique, which disclosed chromosomal imbalances targeting specific genes in each cell line. The PD-OVCA1 and PD-OVCA2 ovarian cancer cell lines will provide a valuable tool for new experimental models for the study of BRCA1-associated tumour biology. © 2006 Elsevier Ltd. All rights reserved