Our aim was to analyze the incidence of mutations in BRCA1 and BRCA2 genes in 54 families with breast/ovarian cancer. Families were selected from three Institutions following the standard criteria for hereditary breast/ovarian cancer. PCR amplification of all exons was performed, followed by SSCP, heteroduplex, PTT and sequencing analysis. We identified eight truncation mutations, three in the BRCA1 gene and five in the BRCA2 gene. Three of these mutations have not been reported previously by other groups: 308insA in one family, 3936 C>T in two families, for BRCA1, and 4970insTG in one family for BRCA2. In addition two families having Ashkenazi Jewish ancestors present the well known mutations 185delAG and 6174delT. Interestingly, 5 out of 11 families have mutations recurrent in Spanish families. Among the 54 families selected, seven have breast and ovary cancer cases, and only two presented a mutation in BRCA1 or BRCA2 genes. Other cancers as prostate and stomach are frequent among relatives carrying the mutation. Five cases of very early onset (<31 years old) breast cancer were detected. The frequencies of BRCA1 (0.074) and BRCA2 (0.13) mutations in our families is low but similar to the incidence found in other populations, like in Spain. Since is widely known that risk factors that modulate the development of breast cancer such as lifestyle risk factors, geographic location, country of origin and socioeconomic status, besides a familial history of breast cancer our findings suggest that the history of colonization and immigrations is very relevant when studying hereditary factors associated to breast cancer.

Germline mutations in BRCA1 account for a low proportion of hereditary cases in diverse populations. Several efforts have been made to find new genes involved in the inheritance of breast cancer with no success until today. The participation of BRCA1 in the development of breast cancer has been proposed in several studies where hypermethylation of its promoter and a decrease in expression has been reported for sporadic cases and one study on familial cases. To explore the participation of BRCA1 in hereditary carcinogenesis through a different mechanism than the inheritance of germline mutations, we studied the methylation status of its promoter in breast tumors, from patients previously screened for BRCA1/BRCA2 germline mutations. We also determined the presence of the BRCA1 protein in these tumors and correlated both events with tumor grade, hormone receptors and ERBB2 presence. Promoter hypermethylation of the BRCA1 gene was detected in 51% of our biopsies, among which 67% did not express the respective protein. This result leads us to suggest that hypermethylation could be considered as an inactivating mechanism for BRCA1 expression, either as a first or second hit. Moreover, a number of biopsies with absence of expression on BRCA1 showed negative detection of estrogen and progesterone receptors, a similar phenotype to BRCA1 mutated breast tumors.

Point mutations and small deletions and insertions in BRCA1 and BRCA2 genes are responsible of about 20% of hereditary breast cancer cases in Chilean population. Studies in other populations have identified the amplification and/or deletion of one or more exons in these genes as the cause of the disease. In this study the authors determined the presence of these types of alterations in BRCA1 and BRCA2, in 74 Chilean families with breast/ovarian cancer that were negative for germline mutations in these genes. Since these alterations are not detectable using the conventional PCR-based methods, the authors use MLPA (multiplex ligation-dependent probe amplification) to detect amplifications and/or deletions in BRCA1 and BRCA2 genes. The authors identified two different alterations in BRCA1: exon 10 duplication in one family and amplification of exons 3, 5, and 6 in two families. Duplication of exon 10 contains intronic adjacent sequences suggesting gene duplication. The second rearrangement consist of a 4 times amplification of a fragment containing exons 3, 5, and 6 joined together with no introns, suggesting the presence of a processed pseudogene. No alterations were detected in BRCA2. In order to validate the MLPA results and characterize the genomic alterations the authors performed qPCR, long range PCR, and sequencing.

Formation of reactive O2 species in biological systems can be accomplished by copper-(II) (Cu2+) catalysis, with the consequent cytotoxic response. We have evaluated the influence of Cu2+ on the respiratory activity of Kupffer cells in the perfused liver after colloidal carbon infusion. Studies were carried out in untreated rats and in animals pretreated with the Kupffer cell inactivator gadolinium chloride (GdCl3) or with the metallothionein (MT) inducing agent zinc sulphate, and results were correlated with changes in liver sinusoidal efflux of lactate dehydrogenase (LDH) as an index of hepatotoxicity. In the concentration range of 0.1-1 microM, Cu2+ did not modify carbon phagocytosis by Kupffer cells, whereas the carbon-induced liver O2 uptake showed a sigmoidal-type kinetics with a half-maximal concentration of 0.23 microM. Carbon-induced O2 uptake occurred concomitantly with an increased LDH efflux, effects that were significantly correlated and abolished by GdCl3 pretreatment or by MT induction. It is hypothesized that Cu2+ increases Kupffer cell-dependent O2 utilization by promotion of the free radical processes related to the respiratory burst of activated liver macrophages, which may contribute to the concomitant development of hepatocellular injury.

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