Identification of BRCA1-IRIS, a BRCA1 locus product

Breast cancer is the most common malignancy among women, and mutations in the BRCA genes produce increased susceptibility to these malignancies in certain families. Here we identify BRCA1-IRIS as a 1,399-amino-acid BRCA1 gene product encoded by an uninterrupted open reading frame that extends from codon 1 of the known BRCA1 open reading frame to a termination point 34 triplets into intron 11. Unlike full-length BRCA1 (p220), BRCA1-IRIS is exclusively chromatin-associated, fails to interact with BARD1 in vivo or in vitro and exhibits unique nuclear immunostaining. Unlike BRCA1FL (or p220), BRCA1-IRIS also co-immunoprecipitated with DNA-replication-licensing proteins and with known replication initiation sites. Suppression of BRCA1-IRIS expression hindered the normal departure of geminin from pre-replication complexes, and depressed the rate of cellular DNA replication and possibly initiation-related synthesis. In contrast, BRCA1-IRIS overexpression stimulated DNA replication. These data imply that endogenous BRCA1-IRIS positively influences the DNA replication initiation machinery.     

Characterization of common BRCA1 and BRCA2 variants

Many missense variants identified in BRCA1 and BRCA2, two genes responsible for the majority of hereditary breast and ovarian cancer, are of unclear clinical significance. Characterizing the significance of such variants is important for medical management of patients in whom they are identified. The aim of this study was to characterize eight of the most common reported missense mutations in BRCA1 and BRCA2 occurring in patients tested for hereditary risk of breast and ovarian cancers. The prevalence of each variant in a control population, co-segregation of the variant with cancer within families, location of the variant within the gene, the nature of the amino acid substitution and conservation of the wild-type amino acid among species were considered. In a control population, the BRCA1 variants M1652I, R1347G, and S1512I, were each observed at a frequency of 4.08%, 2.04%, and 2.04%, respectively, and the BRCA2 variants A2951T, V2728I, and D1420Y, were seen at 1.02%, 0.68%, and 0.34%, respectively. Although the BRCA2 variants T598A and R2034C were not seen in this group of controls, other clinical and published observations indicate that these variants are not deleterious. Based on epidemiological and biological criteria, we therefore conclude that the BRCA1 missense mutations R1347G, S1512I and M1652I, and the BRCA2 missense mutations T598A, D1420Y, R2034C, V2728I, and A2951T, are not deleterious mutations.     

Genomic rearrangements in BRCA1 and BRCA2: A literature review

Women with mutations in the breast cancer genes BRCA1 or BRCA2 have an increased lifetime risk of developing breast, ovarian and other BRCA-associated cancers. However, the number of detected germline mutations in families with hereditary breast and ovarian cancer (HBOC) syndrome is lower than expected based upon genetic linkage data. Undetected deleterious mutations in the BRCA genes in some high-risk families are due to the presence of intragenic rearrangements such as deletions, duplications or insertions that span whole exons. This article reviews the molecular aspects of BRCA1 and BRCA2 rearrangements and their frequency among different populations. An overview of the techniques used to screen for large rearrangements in BRCA1 and BRCA2 is also presented. The detection of rearrangements in BRCA genes, especially BRCA1, offers a promising outlook for mutation screening in clinical practice, particularly in HBOC families that test negative for a germline mutation assessed by traditional methods.     

Loss of nuclear BRCA1 protein staining in normal tissue cells derived from BRCA1 and BRCA2 mutation carriers

Enhanced genomic instability has been recently reported in normal cells derived from BRCA1/2 mutation carriers when placed in vitro in non-physiological stress conditions. We present here original data which help to explain the observed genomic instability. Leucocytes from BRCA1/2 mutation carriers, sporadic breast cancer patients and controls were prepared for BRCA1 immunocytochemistry. We show that BRCA1 containing nuclear dot like structures are detectable in about 80% of the leucocytes from controls and sporadic breast cancer patients, but are absent in the majority of normal cells from BRCA1 as well as BRCA2 mutation carriers (also in their normal breast cells). Our results thus indicate that the genomic instability observed in normal cells from BRCA1 and BRCA2 mutation carriers is associated with a down-regulation of nuclear BRCA1 protein accumulation in the dot like structures. These results suggest in addition that immunocytochemical or alternative molecular screening strategies might help to identify women with a high risk for breast (ovarian) cancer even when the underlying genetic defect remains undetectable.     

BARD1 induces BRCA1 intranuclear foci formation by increasing RING-dependent BRCA1 nuclear import and inhibiting BRCA1 nuclear export

BRCA1 is a tumor suppressor with several important nuclear functions. BRCA1 has no known cytoplasmic functions. We show here that the two previously identified nuclear localization signals (NLSs) are insufficient for nuclear localization of BRCA1 due to the opposing action of an NH2-terminal nuclear export signal. In transfected breast cancer cells, BRCA1 nuclear localization requires both the NLSs and NH2-terminal RING domain region; mutating either of these sequences shifts BRCA1 to the cytoplasm. The BRCA1 RING element mediates nuclear import via association with BARD1, and this is not affected by cancer-associated RING mutations. Moreover, BARD1 directly masks the BRCA1 nuclear export signal, and the resulting block to nuclear export is requisite for efficient import and nuclear localization of ectopic and endogenous BRCA1. Our results explain why BRCA1 exon 11 splice variants, which lack the NLSs but retain the RING domain, are frequently detected in the nucleus and in nuclear foci in vivo. In fact, co-expression of BARD1 promoted formation of DNA damage-induced nuclear foci comprising ectopic wild-type or NLS-deficient BRCA1, implicating BARD1 in nuclear targeting of BRCA1 for DNA repair. Our identification of BARD1 as a BRCA1 nuclear chaperone has regulatory implications for its reported effects on BRCA1 protein stability, ubiquitin ligase activity, and DNA repair.     

Genetic heterogeneity in hereditary breast cancer: role of BRCA1 and BRCA2.

The common hereditary forms of breast cancer have been largely attributed to the inheritance of mutations in the BRCA1 or BRCA2 genes. However, it is not yet clear what proportion of hereditary breast cancer is explained by BRCA1 and BRCA2 or by some other unidentified susceptibility gene(s). We describe the proportion of hereditary breast cancer explained by BRCA1 or BRCA2 in a sample of North American hereditary breast cancers and assess the evidence for additional susceptibility genes that may confer hereditary breast or ovarian cancer risk. Twenty-three families were identified through two high-risk breast cancer research programs. Genetic analysis was undertaken to establish linkage between the breast or ovarian cancer cases and markers on chromosomes 17q (BRCA1) and 13q (BRCA2). Mutation analysis in the BRCA1 and BRCA2 genes was also undertaken in all families. The pattern of hereditary cancer in 14 (61%) of the 23 families studied was attributed to BRCA1 by a combination of linkage and mutation analyses. No families were attributed to BRCA2. Five families (22%) provided evidence against linkage to both BRCA1 and BRCA2. No BRCA1 or BRCA2 mutations were detected in these five families. The BRCA1 or BRCA2 status of four families (17%) could not be determined. BRCA1 and BRCA2 probably explain the majority of hereditary breast cancer that exists in the North American population. However, one or more additional genes may yet be found that explain some proportion of hereditary breast cancer.     

The predictive value of BRCA1 and BRCA2 mutation testing

Genetic testing for mutations in BRCA1 and BRCA2, two genes predisposing to breast and ovarian cancers, is available to women with a relevant family history. The aim of this study was to estimate the positive and negative predictive value of clinical sequence analysis of these genes. A reference graph showing positive and negative predictive values over a range of pre-test risk was derived, taking into account the sensitivity and specificity of a full-sequence analysis test. High positive and negative predictive values were found for women with pre-test risk between 4% and 40%, a range of risk commonly seen in clinical testing. The predictive value of full sequence and single-site analysis of BRCA1 and BRCA2, therefore, compares favorably with other diagnostic medical tests. Our results provide a numerical estimate of the predictive value of BRCA testing, and as such, provide a valuable tool to healthcare providers and families as they interpret BRCA1 and BRCA2 test results.     

Denaturing high-performance liquid chromatography detects reliably BRCA1 and BRCA2 mutations

Denaturing high-performance liquid chromatography (DHPLC) is a recently developed method of comparative sequencing based upon heteroduplex detection. To assess the reliability of this method, 180 different mutations (54 deletions, 12 insertions, and 117 single base substitutions) in BRCA1 and BRCA2 were tested. Second, 25 index individuals with complete DHPLC analysis of BRCA1 were reanalyzed by dye-terminator sequencing. Third, 41 index individuals were analyzed concomitantly by both DGGE and DHPLC. Of the 180 different BRCA1 and BRCA2 mutations, 179 showed heterozygous DHPLC elution profiles. Dye-terminator sequencing of the entire BRCA1 gene, including 5592 bp of coding sequence and 5206 bp of flanking noncoding sequence, in 25 index individuals did not reveal additional variants missed by DHPLC. The concomitant analysis of 41 index cases showed that 4 probably disease-associated mutations were identified by DHPLC while only 3 of those 4 sites were detected by denaturing gradient gel electrophoresis. We conclude that DHPLC is a sensitive and cost-effective method for the screening of BRCA1 and BRCA2.     

Management updates for women with a BRCA1 or BRCA2 mutation

In most cases of families with breast and ovarian cancer, the pattern of cancers in the family can be attributed to mutations in the BRCA1 and BRCA2 genes. Genetic testing for these cancer susceptibility genes typically takes place in the context of comprehensive genetic counseling. Strategies have been developed for the medical management of women at high risk of developing breast cancer, including options for screening and prophylactic surgery. BRCA1 and BRCA2 carriers are recommended to undergo prophylactic bilateral salpingo-oophorectomy by age 35-40 years or when childbearing is complete. This surgery significantly reduces the risk of ovarian cancer and also reduces the risk of breast cancer when performed in premenopausal mutation carriers. For breast cancer management, BRCA1 and BRCA2 carriers are offered the options of increased surveillance, with or without chemoprevention, or prophylactic surgery. Currently, BRCA carrier status is not used as an independent prognostic factor regarding systemic treatment options.     

Presence of BRCA1 and BRCA2 proteins in human milk fat globules after delivery.

We evaluated BRCA1 and BRCA2 oncosuppressor protein expression in 26 milk samples in women just after delivery. The quantification of BRCA1 and BRCA2 proteins was performed in isolated milk fat globules using an affinity chromatography strategy. The amounts of BRCA1 and BRCA2 proteins were found to be similar. We explained the presence of BRCA1 and BRCA2 proteins in human milk fat globules by the fact that they are formed by exocytosis of lipids from epithelial cells of the mammary gland and are enveloped by plasma membrane from the apical part of the milk-secreting cells. This raises the possibility that BRCA1 and BRCA2 proteins are a protective response to proliferation and play a possible role in newborn nutrition.     

Three novel BRCA1/BRCA2 mutations in breast/ovarian cancer families in Croatia

BRCA1 and BRCA2 genes from 167 candidates (145 families) were scanned for mutations. We identified 14 pathogenic point mutations in 17 candidates, 9 in BRCA1 and 5 in BRCA2. Of those, 11 have been previously described and 3 were novel (c.5335C>T in BRCA1 and c.4139_4140dupTT and c.8175G>A in BRCA2). No large deletions or duplications involving BRCA1 and BRCA2 genes were identified. No founder mutations were detected for the Croatian population. Croatia shares most of the mutations with neighboring Slovenia and also with Germany, Austria and Poland. Two common sequence variants in BRCA1, c.2077G>A and c.4956G>A, were found more frequently in mutation carriers compared to healthy controls. No difference in BRCA2 variants was detected between the groups. Haplotype inference showed no difference in haplotype distributions between deleterious mutation carriers and non-carriers in neither BRCA1 nor BRCA2. In silico analyses identified one BRCA1 sequence variant (c.4039A>G) and two BRCA2 variants (c.5986G>A and c.6884G>C) as harmful with high probability, and inconclusive results were obtained for our novel BRCA2 variant c.3864_3866delTAA. Combination of QMPSF and HRMA methods provides high detection rate and complete coverage of BRCA1/2 genes. Benefit of BRCA1/2 mutation testing is clear, since we detected mutations in young unaffected women, who will be closely monitored for breast and ovarian cancer.     

Ubc9 mediates nuclear localization and growth suppression of BRCA1 and BRCA1a proteins

BRCA1 gene mutations are responsible for hereditary breast and ovarian cancers. In sporadic breast tumors, BRCA1 dysfunction or aberrant subcellular localization is thought to be common. BRCA1 is a nuclear-cytoplasm shuttling protein and the reason for cytoplasmic localization of BRCA1 in young breast cancer patients is not yet known. We have previously reported BRCA1 proteins unlike K109R and cancer-predisposing mutant C61G to bind Ubc9 and modulate ER-α turnover. In the present study, we have examined the consequences of altered Ubc9 binding and knockdown on the subcellular localization and growth inhibitory function of BRCA1 proteins. Our results using live imaging of YFP, GFP, RFP-tagged BRCA1, BRCA1a and BRCA1b proteins show enhanced cytoplasmic localization of K109 R and C61G mutant BRCA1 proteins in normal and cancer cells. Furthermore, down-regulation of Ubc9 in MCF-7 cells using Ubc9 siRNA resulted in enhanced cytoplasmic localization of BRCA1 protein and exclusive cytoplasmic retention of BRCA1a and BRCA1b proteins. These mutant BRCA1 proteins were transforming and impaired in their capacity to inhibit growth of MCF-7 and CAL51 breast cancer cells. Interestingly, cytoplasmic BRCA1a mutants showed more clonogenicity in soft agar and higher levels of expression of Ubc9 than parental MCF7 cells. This is the first report demonstrating the physiological link between cytoplasmic mislocalization of mutant BRCA1 proteins, loss of ER-α repression, loss of ubiquitin ligase activity and loss of growth suppression of BRCA1 proteins. Thus, binding of BRCA1 proteins to nuclear chaperone Ubc9 provides a novel mechanism for nuclear import and control of tumor growth.     

Functional assays for BRCA1 and BRCA2

Genetic testing for the two major breast cancer susceptibility genes has now been available for several years with more than 70,000 people tested in the USA alone. While the current genetic testing identifies many sequence alterations there are problems with both sensitivity and specificity of the assay. In particular, the genetic testing is limited in its ability to determine which of the many missense mutations identified in BRCA1 and BRCA2 actually predispose to cancer and which are simply neutral alterations. Here we will focus on the limitations in test result interpretation and we will explore how biochemistry and cell biology can help to clarify these issues. Although we limit our discussion to genetic testing of BRCA1 and BRCA2, the problem is common to an expanding group of genes, including ATM and MSH2, in which germ-line missense mutations may also confer increased risk of cancer. Here we advocate the use of functional assays to complement genetic data in the analysis of unclassified missense mutations and propose a set of standards to conduct and interpret these assays.     

Moderate frequency of BRCA1 and BRCA2 germ-line mutations in Scandinavian familial breast cancer.

Previous studies of high-risk breast cancer families have proposed that two major breast cancer-susceptibility genes, BRCA1 and BRCA2, may account for at least two-thirds of all hereditary breast cancer. We have screened index cases from 106 Scandinavian (mainly southern Swedish) breast cancer and breast-ovarian cancer families for germ-line mutations in all coding exons of the BRCA1 and BRCA2 genes, using the protein-truncation test, SSCP analysis, or direct sequencing. A total of 24 families exhibited 11 different BRCA1 mutations, whereas 11 different BRCA2 mutations were detected in 12 families, of which 3 contained cases of male breast cancer. One BRCA2 mutation, 4486delG, was found in two families of the present study and, in a separate study, also in breast tumors from three unrelated males with unknown family history, suggesting that at least one BRCA2 founder mutation exists in the Scandinavian population. We report 1 novel BRCA1 mutation, eight additional cases of 4 BRCA1 mutations described elsewhere, and 11 novel BRCA2 mutations (9 frameshift deletions and 2 nonsense mutations), of which all are predicted to cause premature truncation of the translated products. The relatively low frequency of BRCA1 and BRCA2 mutations in the present study could be explained by insufficient screening sensitivity to the location of mutations in uncharacterized regulatory regions, the analysis of phenocopies, or, most likely, within predisposed families, additional uncharacterized BRCA genes.     

Extracellular matrix enhances heregulin-dependent BRCA1 phosphorylation and suppresses BRCA1 expression through its C terminus

Germ line mutations in the breast cancer susceptibility gene BRCA1 account for the increased risk of early onset of familial breast cancer, whereas overexpression of the ErbB family of receptor tyrosine kinases has been linked to the development of nonfamilial or sporadic breast cancer. To analyze whether there is a link between these two regulatory molecules, we studied the effects of ErbB-2 activation by heregulin (HRG) on BRCA1 function. It was previously demonstrated that HRG induced the phosphorylation of BRCA1, which was mediated by the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Since altered interaction between cells and the surrounding extracellular matrix (ECM) is a common feature in a variety of tumors and since ECM modulates intracellular signaling, we hypothesized that ECM may affect the expression and HRG-dependent phosphorylation of BRCA1. Following stimulation by HRG, a strong increase in [(3)H]thymidine incorporation was observed in human T47D breast cancer cells seeded on plastic (PL). When T47D cells were seeded on laminin (LAM) or Matrigel, HRG induced a significantly higher proliferation than it did in cells seeded on PL. T47D cells seeded on poly-L-lysine had an abrogated mitogenic response, indicating the involvement of integrins in this process. HRG treatment induced a transient phosphorylation of BRCA1 that was enhanced in T47D cells grown on LAM. LAM-enhanced BRCA1 phosphorylation was mediated through alpha(6) integrin upon HRG stimulation. Accordingly, T47D cells grown on LAM had the greatest increase in ErbB-2 activation, PI3K activity, and phosphorylation of Akt. A similar pattern of BRCA1 mRNA expression was observed when T47D cells were seeded on PL, LAM, or COL4. There was a significant decrease in the steady state of the BRCA1 mRNA level on both the LAM and COL4 matrices compared to that for cells seeded on PL. In addition, HRG stimulation caused a significant decrease in BRCA1 mRNA expression that was dependent on protein synthesis. Pretreatment with both the calpain inhibitor ALLN (N-acetyl-Leu-Leu-norleucinal) and the proteosome inhibitor lactacystin inhibited the HRG-induced down-regulation of BRCA1 mRNA expression. Likewise, there was a strong decrease in the protein level of BRCA1 in T47D cells 4 h after treatment with HRG compared to its level in control nontreated T47D cells. Pretreatment with the proteosome inhibitors ALLN, lactacystin, and PSI [N-benzyloxycarbonyl-Ile-Glu-(O-t-butyl)-Ala-leucinal] inhibited also the HRG-induced down-regulation of BRCA1 protein in breast cancer cells. Interestingly, BRCA1 mRNA expression in HCC-1937 breast cancer cells, which express C-terminally truncated BRCA1, was not affected by either LAM or CL4. No phosphorylation of BRCA1 from HCC-1937 cells was observed in response to HRG. While Cdk4 phosphorylated wild-type BRCA1 in response to HRG in T47D cells, Cdk4 failed to phosphorylate the truncated form of BRCA1 in HCC-1937 cells. Furthermore, overexpression of wild-type BRCA1 in HCC-1937 cells resulted in the phosphorylation of BRCA1 and decreased BRCA1 expression upon HRG stimulation while overexpression of truncated BRCA1 in T47D cells resulted in a lack of BRCA1 phosphorylation and restoration of BRCA1 expression. These findings suggest that ECM enhances HRG-dependent BRCA1 phosphorylation and that ECM and HRG down-regulate BRCA1 expression in breast cancer cells. Furthermore, ECM suppresses BRCA1 expression through the C terminus of BRCA1.     

Haplotype structure in Ashkenazi Jewish BRCA1 and BRCA2 mutation carriers

Three founder mutations in BRCA1 and BRCA2 contribute to the risk of hereditary breast and ovarian cancer in Ashkenazi Jews (AJ). They are observed at increased frequency in the AJ compared to other BRCA mutations in Caucasian non-Jews (CNJ). Several authors have proposed that elevated allele frequencies in the surrounding genomic regions reflect adaptive or balancing selection. Such proposals predict long-range linkage disequilibrium (LD) resulting from a selective sweep, although genetic drift in a founder population may also act to create long-distance LD. To date, few studies have used the tools of statistical genomics to examine the likelihood of long-range LD at a deleterious locus in a population that faced a genetic bottleneck. We studied the genotypes of hundreds of women from a large international consortium of BRCA1 and BRCA2 mutation carriers and found that AJ women exhibited long-range haplotypes compared to CNJ women. More than 50% of the AJ chromosomes with the BRCA1 185delAG mutation share an identical 2.1?Mb haplotype and nearly 16% of AJ chromosomes carrying the BRCA2 6174delT mutation share a 1.4?Mb haplotype. Simulations based on the best inference of Ashkenazi population demography indicate that long-range haplotypes are expected in the context of a genome-wide survey. Our results are consistent with the hypothesis that a local bottleneck effect from population size constriction events could by chance have resulted in the large haplotype blocks observed at high frequency in the BRCA1 and BRCA2 regions of Ashkenazi Jews.