Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 6 (4), R284-90

The Icelandic Founder Mutation BRCA2 999del5: Analysis of Expression

Affiliations

The Icelandic Founder Mutation BRCA2 999del5: Analysis of Expression

Evgenia K Mikaelsdottir et al. Breast Cancer Res.

Abstract

Introduction: A founder mutation in the BRCA2 gene (BRCA2 999del5) accounts for 7-8% of female breast cancers and for 40% of male breast cancers in Iceland. If expressed, the mutant gene would encode a protein consisting of the first 256 amino acids of the BRCA2 protein. The purpose of this study was to determine whether this mutant protein is produced in heterozygous individuals and, if so, what might be the functional consequences of mutant protein production.

Methods: The presence of BRCA2 999del5 transcripts in fibroblasts from heterozygous individuals was assayed by cDNA synthesis and sequencing. The potential protein-coding portion of BRCA2 999del5 was cloned into the pIND(SP1)/V5-His vector and expressed in COS7 cells. The presence of the mutant protein in cell lysates from heterozygous fibroblasts and from COS7 cells was tested by a number of methods including immunoprecipitation, affinity purification with nickel-coated agarose beads, Western blotting and ELISA, using antibodies to the N-terminal end of BRCA2, antiserum specific for the 16 nonrelevant amino acids at the carboxyl end and antibodies to fusion partners of recombinant proteins.

Results: The frequency of the BRCA2 999del5 transcript in heterozygous fibroblasts was about one-fifth of the wild-type transcript; however, no mutant protein could be detected. Overexpression of BRCA2 999del5 mRNA in COS7 cells failed to produce a mutant protein unless degradation by proteasomes was blocked.

Conclusion: Our results show that the protein product of BRCA2 999del5 is extremely unstable. Therefore, an increase in breast cancer risk in BRCA2 999del5 carriers is due to haploinsufficiency at the BRCA2 locus.

Figures

Figure 1
Figure 1
Overexpression of BRCA2 999del5 mRNA in COS7 cells. The potential coding sequence of BRCA2 999del5 was cloned into the pIND(SP1)/V5-His expression vector, transfected into COS7 cells and induced with ponasterone A: 1, mock transfection; 2, cells transfected with pIND-BRCA2 999del5; and 3, untransfected COS7 cells. (a) Expression of BRCA2 999del5 was confirmed by northern blotting with a labeled BRCA2 999del5 probe. (b) RNA samples that were used in northern blotting stained with ethidium bromide and visualized with UV light.
Figure 2
Figure 2
No mutant BRCA2 protein was detected in overexpression experiments. (a) Lysates of transfected COS7 cells were subjected to electrophoresis through 6–18% gradient gel and blotted with an antibody against the V5 tag (α-V5): 1, untransfected cells; 2, pIND-LacZ transfected cells; 3, cells cotransfected with both pIND-LacZ and pIND-BRCA2 999del5; and 4, cells transfected with pIND-BRCA2 999del5. (b) Nickel-coated agarose beads were used to adsorb recombinant proteins under native conditions. The proteins were subjected to electrophoresis through 6–18% gradient gel and blotted with α-V5: 1, untransfected COS7 cells; 2, cells overexpressing pIND-BRCA2 999del5; and 3, cells overexpressing pIND-LacZ. (c) α-V5 was used to precipitate recombinant proteins and for immunoblotting: 1, untransfected COS7 cells; 2, cells transfected with pIND-LacZ; 3, cells transfected with pIND-BRCA2 999del5 without induction; and 4, cells transfected with pIND-BRCA2 999del5 with induction.
Figure 3
Figure 3
No mutant protein could be detected by ELISA. (a) Lysates of fibroblasts with wild-type BRCA2 (wt) and fibroblasts heterozygous for BRCA2 999del5 (Mut) were tested for the presence of BRCA2 999del5 protein: bar pairs 3 and 4, lysates diluted 1:10; bar pairs 5 and 6, lysates diluted 1:100; bar pairs 7 and 8, lysates diluted 1:1000. (b) The same experiment was performed using lysates from untransfected COS7 cells (COS7), and COS7 cells overexpressing pIND-LacZ (Mock) or pIND-BRCA2 999del5 (Mut): bar pairs 3, 4 and 5, lysates diluted 1:10; bar pairs 6, 7 and 8, lysates diluted 1:100; bar pairs 9, 10 and 11, lysates diluted 1:1000. The synthetic peptide, representing the 16 out-of-frame amino acids (a.a.) of BRCA2 999del5 and used to raise the antiserum, was used as a positive control.
Figure 4
Figure 4
Detection of BRCA2 999del5 protein under denaturing conditions and after proteasome inhibition. (a) COS7 cells were transfected with pIND-BRCA2 999del5 or pIND-LacZ without proteasome inhibition. Samples were treated under denaturing conditions. (b) COS7 cells were transfected with pIND-BRCA2 999del5 or pIND-LacZ with proteasome inhibition. Samples were purified with nickel-coated agarose beads under denaturing conditions. Samples in both (a) and (b) were blotted with an antibody against the V5 tag (α-V5).

Similar articles

See all similar articles

Cited by 14 articles

See all "Cited by" articles

References

    1. Welcsh PL, King MC. BRCA1 and BRCA2 and the genetics of breast and ovarian cancer. Hum Mol Genet. 2001;10:705–713. doi: 10.1093/hmg/10.7.705. - DOI - PubMed
    1. Spain BH, Larson CJ, Shihabuddin LS, Gage FH, Verma IM. Truncated BRCA2 is cytoplasmic: implications for cancer-linked mutations. Proc Natl Acad Sci USA. 1999;96:13920–13925. doi: 10.1073/pnas.96.24.13920. - DOI - PMC - PubMed
    1. Fan S, Yuan R, Ma YX, Meng Q, Goldberg ID, Rosen EM. Mutant BRCA1 genes antagonize phenotype of wild-type BRCA1. Oncogene. 2001;20:8215–8235. doi: 10.1038/sj.onc.1205033. - DOI - PubMed
    1. Brown MA, Nicolai H, Howe K, Katagiri T, Lalani el-N, Simpson KJ, Manning NW, Deans A, Chen P, Khanna KK, Wati MR, Griffiths BL, Xu CF, Stamp GW, Solomon E. Expression of a truncated Brca1 protein delays lactational mammary development in transgenic mice. Transgenic Res. 2002;11:467–478. doi: 10.1023/A:1020348025139. - DOI - PubMed
    1. Sylvain V, Lafarge S, Bignon YJ. Dominant-negative activity of a Brca1 truncation mutant: effects on proliferation, tumorigenicity in vivo, and chemosensitivity in a mouse ovarian cancer cell line. Int J Oncol. 2002;20:845–853. - PubMed

Publication types

MeSH terms

Feedback