Ταυτοποίηση νέων αλληλομόρφων που σχετίζονται με προδιάθεση για καρκίνο μαστού-ωοθηκών

Abstract

Nowadays, genetic analysis of hereditary breast/ovarian cancer plays an important role in understanding the molecular mechanisms leading to carcinogenesis and it significantly contributes to the clinical management of patients with breast/ovarian cancer. More than 12 genes have been implicated in hereditary breast/ovarian cancer, while in the present work BRCA1, BRCA2, CHEK2, PALB2 and RAD51C genes were analyzed in patients with early onset of cancer (17-40 years) or/and more than 3 cases of breast/ovarian cancer in the same family. The goal of this study was the identification of new pathogenic mutations in these genes, as well as the characterization of recurrent mutations of high frequency in the Greek population. The total number of families tested for pathogenic mutations in the BRCA1 and BRCA2 genes is 132 and 114 respectively. 26 of these families were found to carry a pathogenic mutation in the BRCA1 gene (19.7%), while pathogenic mutations in the BRCA2 gene were found in 4 women (3.8%) and 2 men (22.2%). In addition, a total of 20 unclassified variants were identified, 9 of them in the BRCA1 gene (7.8%) and 11 in the BRCA2 gene (8.3%). Some of the patients with a severe family history of breast/ovarian cancer that had been tested negative for mutations in the BRCA1 and BRCA2 genes, were tested for mutations in the CHEK2 (12 families), PALB2 (16 families) and RAD51C (26 families). No pathogenic mutations were identified in these genes. Furthermore, patient samples were tested for large genomic rearrangements in the BRCA1 and BRCA2 genes using 2 different techniques (QMPSF and MLPA). QMPSF technique was applied in 49 patients and MLPA was applied in 25 patients. In 2 of these patients, a deletion of exons 23 and 24 in the BRCA1 gene was identified, whereas no genomic rearrangements were identified in the BRCA2 gene. In addition, 4 recurrent mutations in the BRCA1 gene were characterised (p.Gly1738Arg, 3.2kB deletion of exon 20, 4.2kB deletion of exon 24 and a 11kB deletion of exons 23 and 24) as Greek founder mutations. More than 1000 breast/ovarian cancer patients were screened for these mutations in order to estimate their frequency in the Greek population. In order to confirm our hypothesis that all carriers of these mutations shared a common, but distinct in each case, ancestor, the haplotype for Single Nucleotide Polymorphisms and for Microsatellite Repeats was analyzed. In each case the disease-associated haplotype was identified and proven to be shared by all mutation carriers. These mutations were also identified as Greek founder mutations The study performed at the International Agency for Research on Cancer (Lyon-France) concentrated on the characterization of missense mutations of unknown pathogenicity, using the in silico prediction program Align-GVGD. 15 unclassified variants, 8 in the BRCA1 gene and 7 in the BRCA2 gene were tested using this in silico program. The analysis showed that all but two are considered neutral. Finally, the effect of 7 missense and 1 intronic variants in the BRCA1 and BRCA2 genes on mRNA splicing was studied by establishing Lymphoblastoid Cell Lines from blood samples of patients carrying these variants. mRNA extracted from these cell lines that have previously been treated with or without puromycin was then tested for the effect of these variants on mRNA splicing. Results showed no negative effect on mRNA splicing for any of these variants.