Genetic analysis of familial breast and ovarian cancer indi cates that BRCA1 is a tumor suppressor gene. The BRCA1 gene encodes a 190 kDa protein with Sequence homology and biochemical analogy to the granin family of proteins. Granins are Secreted from endocrine cells via the regulated Secretory pathway and are proteolytically cleaved to yield biologically active peptides. BRCA1 protein localizes to Secretory vesicles, and was demonstrated to be Secreted. Gene transfer of BRCA1 inhibits growth and tumorigenesis of breast and ovarian cancer cells, but not colon or lung cancer cells or fibroblasts, Suggesting that BRCA1 encodes a tissue-specific growth inhibitor. Thus, BRCA1 is a Secreted growth inhibitor and functions by a mechanism not previously described for tumor Suppressor genes. The BRCA2 breast and ovarian cancer gene encodes a protein that also includes a granin region, indicating that the BRCA2 protein is also a Secreted tumor Suppressor. Therapeutic methods using the BRCA1 and BRCA proteins and genes are also described. A method of Screening for the receptors of the BRCA1 protein and BRCA2 proteins is also described.

Summary: Bisulfite sequencing allows cytosine methylation, an important epigenetic marker, to be detected via nucleotide substitutions. Since the Applied Biosystems SOLiD System uses a unique di-base encoding that increases confidence in the detection of nucleotide substitutions, it is a potentially advantageous platform for this application. However, the di-base encoding also makes reads with many nucleotide substitutions difficult to align to a reference sequence with existing tools, preventing the platform's potential utility for bisulfite sequencing from being realized. Here, we present SOCS-B, a reference-based, un-gapped alignment algorithm for the SOLiD System that is tolerant of both bisulfite-induced nucleotide substitutions and a parametric number of sequencing errors, facilitating bisulfite sequencing on this platform. An implementation of the algorithm has been integrated with the previously reported SOCS alignment tool, and was used to align CpG methylation-enriched Arabidopsis thaliana bisulfite sequence data, exhibiting a 2-fold increase in sensitivity compared to existing methods for aligning SOLiD bisulfite data. Availability: Executables, source code, and sample data are available at http://solidsoftwaretools.com/gf/project/socs/ Contact: bergmann@nbacc.net Supplementary information: Supplementary data are available at Bioinformatics online.

Inherited mutations in the BRCA1 breast-ovarian cancer gene, isolated in 1994 (/), have been estimated to be responsible for approximately 5% of ovarian cancer diagnosed in women under the age of 50 years (2). Recently, the BRCA2 gene was identified (3), and it appears to confer a much lower risk of ovarian cancers (2,4). BRCA1 and BRCA2 are believed to be tumor suppressor genes, since both alleles appear to be inactivated during neoplastic development (5,6). Despite extensive BRCA1-screening efforts, only five somatic and 11 germline mutations have been identified among 267 sporadic (i.e., nonfamilial) ovarian cancers studied to date (7-11). As BRCA1 is a large gene with widely distributed alterations, mutation screening is labor intensive. Approximately 90% of the reported BRCA1 alterations are either frameshift or nonsense mutations, which result in the premature termination of protein synthesis (12-15). Therefore, we developed the protein truncation test as an efficient strategy to determine the role of BRCA1 mutations in patients with early-onset ovarian cancer. Epithelial ovarian tumor tissue and matched blood lymphocytes were obtained, with institutional review boardapproved, written informed consent, from 16 patients treated at the Division of Gynecologic Oncology at Duke University Medical Center. The mean age of the patients at disease onset was 48 years. Exon 11 (61% of coding sequence) was screened by the protein truncation test using primers and protocols that are available on-line in the Breast Cancer Information Core database (http://www. nchgr.nih.gov/intramural_research/lab_transfer/BIC/) (16). In addition, the entire BRCA1 -coding region of these tumor DNAs was analyzed by single-strand conformation analysis, as previously described (7,16). Analysis using the protein truncation test revealed alterations in BRCA1 protein size in patients EOO473 and EOO1906 (Fig. 1, A). Direct DNA sequencing identified an identical, previously unreported, single base-pair deletion, 2575delC, in exon 11, causing a frameshift and subsequent premature stop codon (Table 1). This deletion was present in the germline of both patients (data not shown) who were apparently unrelated. Patient EOO473 had no known family history of cancer, but patient EOO1906 reported a strong family history (Table 1). In patient EOO23, single-strand conformation analysis of tumor DNA revealed a mobility shift that was shown by DNA sequencing to result from a T—>G transversion 11 base pairs prior to the 3' splice site of intron 5 (Table 1). This previously described alteration (14,15) activates a cryptic splice site that includes 59 nucleotides of intron 5 in the BRCA1 messenger RNA (mRNA) of this patient. This mutation was also detectable by the protein truncation test using tumor complementary DNA (Fig. 1, B). Direct sequencing of lymphocyte DNA indicated that this was a germline alteration (data not shown), and this patient reported a sister with dual primary breast and ovarian cancers (Table 1). In this study, we have identified germline BRCA1 mutations in three of 16 women with early-onset ovarian cancer. Despite extensive searches by several groups (7-11), somatic mutations in BRCA1 appear to be quite rare. It is interesting that the sporadic ovarian cancer patients with germline mutations described previously (10,11) and in this brief communication (average age at disease onset = 45 years) developed cancer approximately 18 years earlier than patients with somatic mutations (average age at disease onset = 63 years) (8,9). This significant age difference at disease diagnosis (Student's t test, P<.003) is consistent with the need for two somatic events to inactivate BRCA1. Although mutation detection in BRCA1 remains technically challenging, the results of the current study and those obtained by other groups (17,18) suggest that the protein truncation test is an efficient screening tool. This test does have certain shortcomings, however. In this test, missense alterations are undetectable, and sensitivity is limited for mutations that yield either short or very large products. Adapting gel concentrations and electrophoresis times as well as substituting [H]leucine for [S]methionine may help to circumvent these problems (17,19). Screening the entire coding region of the gene by the protein truncation test requires the availability of cellular RNA, whereas only exon 11 can be screened using genomic DNA. Finally, in specific cases, the instability of mRNA-containing, premature stop codons may also reduce detection by this technique (20). In summary, although somatic BRCA1 mutations are responsible for only a

The second hereditary breast cancer gene, BRCA2, was recently isolated. Germline mutations of this gene predispose carriers to breast cancer, and, to a lesser extent, ovarian cancer. Loss of heterozygosity (LOH) at the BRCA2 locus has been observed in 30-40% of sporadic breast and ovarian tumours, implying that BRCA2 may act as a tumour suppressor gene in a proportion of sporadic cases. To define the role of BRCA2 in sporadic breast and ovarian cancer, we screened the entire gene for mutations using a combination of techniques in 70 primary breast carcinomas and in 55 primary epithelial ovarian carcinomas. Our analysis revealed alterations in 2/70 breast tumours and none of the ovarian carcinomas. One alteration found in the breast cancers was a 2-basepair (bp) deletion (4710delAG) which was subsequently shown to be a germline mutation, the other was a somatic missense mutation (Asp3095Glu) of unknown significance. Our results suggest that BRCA2 is a very infrequent target for somatic inactivation in breast and ovarian carcinomas, similar to the results obtained for BRCA1.

Analyses of losses of heterozygosity and linkage studies have implicated a gene(s) on chromosome 17q in the genesis of sporadic and early-onset familial breast carcinomas, respectively. To define the critical region of 17q, we examined DNAs from a series of 20 sporadic breast carcinomas and corresponding blood samples for allelic losses of chromosome 17q using microsatellite length polymorphisms. With these highly informative markers (average heterozygosity, 0.73), we observed frequent deletions of 17q at several loci. We found that D17S250 was deleted in 50% (7 of 14), THRA1 in 79% (11 of 14), D17S579 in 59% (11 of 19), NME1 in 29% (5 of 17), MPO in 36% (4 of 11), and GH in 25% (4 of 16) in the tumor set examined. A common region of deletion was found that was flanked by D17S250 to D15S579. These markers have recently been localized to a 6-cM interval of proximal chromosome 17q in bands 17q11.2-q21 and map within the region of the early-onset familial breast cancer locus, implying that the same gene or genes may be involved in both sporadic and familial breast tumors. Thyroid hormone receptor alpha and retinoic acid receptor alpha are two potential candidate genes in this region.