A candidate prostate cancer susceptibility gene at chromosome 17p.

It is difficult to identify genes that predispose to prostate cancer due to late age at diagnosis, presence of phenocopies within high-risk pedigrees and genetic complexity. A genome-wide scan of large, high-risk pedigrees from Utah has provided evidence for linkage to a locus on chromosome 17p. We carried out positional cloning and mutation screening within the refined interval, identifying a gene, ELAC2, harboring mutations (including a frameshift and a nonconservative missense change) that segregate with prostate cancer in two pedigrees. In addition, two common missense variants in the gene are associated with the occurrence of prostate cancer. ELAC2 is a member of an uncharacterized gene family predicted to encode a metal-dependent hydrolase domain that is conserved among eukaryotes, archaebacteria and eubacteria. The gene product bears amino acid sequence similarity to two better understood protein families, namely the PSO2 (SNM1) DNA interstrand crosslink repair proteins and the 73-kD subunit of mRNA 3' end cleavage and polyadenylation specificity factor (CPSF73).

BRG1, a component of the SWI-SNF complex, is mutated in multiple human tumor cell lines.

Human BRG1 is a component of the evolutionarily conserved SWI-SNF chromatin remodeling complex. BRG1 has been implicated in growth control through its interaction with the tumor suppressor pRb and may consequently serve as a negative regulator of proliferation. Postulating that BRG1 may itself be a tumor suppressor gene, we screened a panel of tumor cell lines to determine whether the gene is targeted for mutation. We report that the COOH-terminal region of BRG1 is homozygously deleted in two carcinoma cell lines, prostate TSU-Pr1 and lung A-427. In addition, biallelic inactivations of BRG1 were observed in four other cell lines derived from carcinomas of the breast, lung, pancreas, and prostate; their mutations in BRG1 included three frameshift lesions and one nonsense lesion. Point mutations were also discovered in a number of other cell lines, however in most cases any effect of these mutations on BRG1 function remains to be established. A variety of different mutations within BRG1, in several cell lines, suggest that BRG1 may be targeted for disruption in human tumors. Significantly, reintroduction of BRG1 into cells lacking BRG1 expression was sufficient to reverse their transformed phenotype inducing growth arrest and a flattened morphology. These data strongly support the model that BRG1 may function as a tumor suppressor and strengthen the hypothesis that the regulation of gene expression through chromatin remodeling is critical for cancer progression. It will be important to confirm these observations in primary tumors.

Genomic structure, chromosomal location, and mutation analysis of the human CDC14A gene.

Human CDC14A is a dual-specificity phosphatase that shares sequence similarity with the recently identified tumor suppressor, MMAC1/PTEN/TEP1. By radiation hybrid mapping, we localized CDC14A to chromosome band 1p21, a region that has been shown to exhibit loss of heterozygosity in highly differentiated breast carcinoma and malignant mesothelioma. We have mapped the exon-intron structure of CDC14A gene and found an in-frame ATG at 14 codons upstream of the previously reported start site (GenBank Accession No. AF000367). In screening a panel of 136 cDNAs from tumor cell lines for coding mutations, we have identified a 48-bp in-frame deletion in the cDNA of the breast carcinoma cell line, MDA-MB-436. This deletion is the result of an acceptor splice site mutation (AG to AT) in intron 12 that causes the skipping of exon 13 in the gene. Loss of expression of the wildtype allele in the same breast cell line supports the possibility that CDC14A may be a tumor suppressor gene that is targeted for inactivation during tumorigenesis.

BRCA1 sequence analysis in women at high risk for susceptibility mutations. Risk factor analysis and implications for genetic testing.

CONTEXT: A mutation in the BRCA1 gene may confer substantial risk for breast and/or ovarian cancer. However, knowledge regarding all possible mutations and the relationship between risk factors and mutations is incomplete. OBJECTIVES: To identify BRCA1 mutations and to determine factors that best predict presence of a deleterious BRCA1 mutation in patients with breast and/or ovarian cancer. DESIGN: A complete sequence analysis of the BRCA1 coding sequence and flanking intronic regions was performed in 798 women in a collaborative effort involving institutions from the United States, Italy, Germany, Finland, and Switzerland. PARTICIPANTS: Institutions selected 798 persons representing families (1 person for each family) thought to be at elevated a priori risk of BRCA1 mutation due to potential risk factors, such as multiple cases of breast cancer, early age of breast cancer diagnosis, and cases of ovarian cancer. No participant was from a family in which genetic markers showed linkage to the BRCA1 locus. MAJOR OUTCOME MEASURES: Sequence variants detected in this sample are presented along with analyses designed to determine predictive characteristics of those testing positive for BRCA1 mutations. RESULTS: In 102 women (12.8%), clearly deleterious mutations were detected. Fifty new genetic alterations were found including 24 deleterious mutations, 24 variants of unknown significance, and 2 rare polymorphisms. In a subset of 71 Ashkenazi Jewish women, only 2 distinct deleterious mutations were found: 185delAG in 17 cases and 5382insC in 7 cases. A bias in prior reports for mutations in exon 11 was revealed. Characteristics of a patient's specific diagnosis (unilateral or bilateral breast cancer, with or without ovarian cancer), early age at diagnosis, Ashkenazi Jewish ethnicity, and family history of cancer were positively associated with the probability of her carrying a deleterious BRCA1 mutation. CONCLUSIONS: Using logistic regression analysis, we provide a method for evaluating the probability of a woman's carrying a deleterious BRCA1 mutation for a wide range of cases, which can be an important tool for clinicians as they incorporate genetic susceptibility testing into their medical practice.

Mild radioiodination of insulin with dichloramine-T.

Iodination of insulin using N-dichloro-p-toluene sulphonamide(dichloramine-T) has been standardised. Dichloramine-T, a water insoluble derivative of chloramine-T showed excellent properties as an iodination reagent, for the preparation of radiolabeled insulin for use in radioimmunoassay. Iodination using dichloramine-T could be done at as low as 0.5-2 micrograms of the reagent, and at this concentration the molar ratio worked out to 1:3 to 1:12 (protein:dichloramine-T). Iodination yields of greater than 90% were obtained at pH 5-7. Evaluation of the iodinated tracer for suitability in radioimmunoassay was carried out by estimating radiochemical purity, immunological purity, non specific binding and stability on storage.