Functional analysis of BRCA1 C-terminal missense mutations identified in breast and ovarian cancer families

Abstract

Germline mutations in the breast and ovarian cancer susceptibility gene BRCA1 are responsible for the majority of cases involving hereditary breast and ovarian cancer. Whereas all truncating mutations are considered as functionally deleterious, most of the missense variants identified to date cannot be readily distinguished as either disease-associated mutations or benign polymorphisms. The C-terminal domain of BRCA1 displays an intrinsic transactivation activity, and mutations linked to disease predisposition have been shown to confer loss of such activity in yeast and mammalian cells. In an attempt to clarify the functional importance of the BRCA1 C-terminus as a transcription activator in cancer predisposition, we have characterized the effect of C-terminal germline variants identified in Scandinavian breast and ovarian cancer families. Missense variants A1669S, C1697R, R1699W, R1699Q, A1708E, S1715R and G1738E and a truncating mutation, W1837X, were characterized using yeast- and mammalian-based transcription assays. In addition, four additional missense variants (V1665M, D1692N, S1715N and D1733G) and one in-frame deletion (V1688del) were included in the study. Our findings demonstrate that transactivation activity may reflect a tumor-suppressing function of BRCA1 and further support the role of BRCA1 missense mutations in disease predisposition. We also report a discrepancy between results from yeast- and mammalian-based assays, indicating that it may not be possible to unambiguously characterize variants with the yeast assay alone. We show that transcription-based assays can aid in the characterization of deleterious mutations in the C-terminal part of BRCA1 and may form the basis of a functional assay.

Figure 1. Domain structure of BRCA1

Top panel. Schematic representation of full length BRCA1 protein featuring the RING domain in the N-terminal region and the BRCT domains in the C-terminal region. The region analyzed in this study (aa 1560–1863) is contained in the box, which is enlarged and represented in the bottom panel. Gray circles represent the two BRCT domains, BRCT-N (aa 1649–1736) and BRCT-C (aa 1756–1855). NLS, nuclear localization signals. Bottom panel. GAL4- and LexA- DNA binding domain (DBD) fusions to BRCA1 C-terminal region (aa 1560–1863). Mutations analyzed in this study are depicted as black (missense) or open (nonsense and in-frame deletion) triangles.

Figure 2. Scandinavian breast and breast-ovarian cancer families with germline BRCA1 C-terminal missense or truncating mutations

Shown are cancer types, age at diagnosis, and mutation status (*mutation; °confirmed from blood sample not to carry mutation; ^determined from paraffin embedded tumor tissue not to carry mutation). Cancer type abbreviations: Br (breast), Ov (ovary), Ut (uterus), Gyn (gynecological), Pr (prostate), Co (colon), Rec (rectal), Leuk (leukemia), Lu (lung), Bn (brain), Bl (bladder), Pa (pancreas), Ga (gastric), Pen (Penile), Abd (Abdominal) Ca (cancer of unknown type), Ca? (possibly affected).

Figure 3. Transcriptional activity of BRCA1 variants

A. Activity in yeast cells. Structure of the reporter plasmid is depicted on top of the graph. Variants (black bars) are in order of location in the structure of BRCA1 with the exception of last three constructs (gray bars), which correspond to negative (M1775R and Y1853X) and positive (wild type) controls. Shaded area represents range of activity equal or higher than wild type. B. Activity in human cells. Structure of the reporter plasmid is depicted on top of the graph. Variants (black bars) are in order of location in the structure of BRCA1 with the exception of last three constructs (gray bars), which correspond to negative (M1775R and Y1853X) and positive (wild type) controls. Shaded area represents range of activity equal or higher than wild type. C. Mutant R1699W is expressed at the same level as wild type (gray arrow). White arrow indicates expression of the GAL4 DBD moiety in the absence of any fusion fragment.