Abstract. The Hedgehog/Patched signaling pathway plays a prominent role during mammalian development but it is also involved in oncogenic transformation. We investigated the methylation status of the Patched promotor in a set of basocellular carcinomas of the skin and ovarian tumors as an alternative to mutational causes of the pathway deregulation. Our aim was to define a possible role of genetic and/or epigenetic mechanisms of Hedgehog/Patched signal transduction in the development of these tumors. Bisulfite-converted DNA from tumors and from matched healthy tissue was amplified by a specific PCR and the CpG-rich regions of the Patched promoter were sequenced. Two promoter regions showed statistically significant hypermethylation compared to healthy controls in ovarian tumors; more significantly in the region in the vicinity of Gli1-binding sites and less significantly in the region containing the ATG codon. But, in basocellular carcinomas of the skin we observed no difference in methylation, suggesting different mechanisms of neoplasia in these tumors.

Background: Mutations in BRCA1 and BRCA2 genes are associated with family predisposition to breast and ovarian cancer. Novel screening methods are required for efficient and rapid detection of sequence variants in cancer patients and their family members. Methods: The screening for variants in the breast and ovarian cancer susceptibility genes BRCA1 and BRCA2 in Croatia was performed by a high-resolution melting approach, which is based on differences in melting curves caused by variations in nucleotide sequence. This is the first screening in Croatia on elderly healthy women with no family history of cancer. BRCA1 screening was performed on 220 and BRCA2 screening on 115 samples.

A new approach for the evaluation of the human papillomavirus type 16 variability with high resolution melting analysis. Journal of Virological Methods 162, 142-147. doi:10.1016Methods 162, 142-147. doi:10. /j.jviromet.2009 This study showed that HRM analysis can be useful for the identification of HPV 16 variants. The HRM method will be useful in low resource settings as it saves considerable time and resources compared to sequencing.Key words: human papillomavirus 16; variant; high resolution melting; sequencing Abbreviations: HPV -human papillomavirus; PCR -polymerase chain reaction; HRM -high resolution melting; SNP -single nucleotide polymorphisms Sabol, I., Čretnik, M., Hadžisejdić, I., Si-Mohamed, A., Matovina, M., Grahovac, B., Levanat, S., Grce, M., 2009. A new approach for the evaluation of the human papillomavirus type 16 variability with high resolution melting analysis.

Background: Gorlin syndrome is a rare autosomal-dominant disorder characterized by a wide range of developmental abnormalities and various tumors. The syndrome is caused by mutations in PTCH1, a tumor suppressor gene located at 9q22.32. We describe a Gorlin syndrome case with typical features of the syndrome and no mutations in PTCH1, but with a large deletion of the 9q22 region that has rarely been described. Objective: To fully characterize the large deletion in the patient. Methods: In order to map the size and position of the deletion, we developed quantitative multiplex fluorescent PCR with polymorphic markers surrounding the PTCH1 gene, followed by long-range PCR and sequencing. Results: The deleted segment of 4.5 Mb in the 9q22.32–q22.33 region was determined, and included the entire PTCH1, its promoter and 22 OMIM genes. Conclusion: We suggest that screening for large deletions should be included in standard mutation screening for Gorlin syndrome patients.

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Abstract. In the Milan area (Northern Italy), we identified a family characterized by a high prevalence of ovarian and breast cancer cases (5 out of 6 subjects, over 3 generations), and a predominant prevalence of ovarian lesions (4 out of 5 patients). Analysis of BRCA1 and BRCA2 genes allowed the identification of the missense c.190T>C mutation in codon 64 (Cys64Arg) of BRCA1. The aims of the present investigation were to characterize the functional implications of the c.190T>C mutation at the molecular level, and to search whether additional polymorphisms might be linked to the peculiar phenotypic features observed in the Italian pedigree. Molecular modelling studies suggested that substitution of the cysteine 64 with an arginine likely disrupts the architecture of the BRCA1 RING finger domain, responsible for the interaction with BARD1, essential for the tumor-suppressor activity of the BRCA1-BARD1 complex. By splicing site information analysis, exonic splicing enhancer site characterization, and analysis of transcript fragment length and sequence, we showed that the c.190T>C mutation was able to modulate the splicing of exon 5 in a fashion opposite to the c.190T>G transversion, responsible for the functionally-related Cys64Gly amino acid substitution. Genotyping of BRCA1 and BRCA2 in the Italian family revealed the presence of two significant polymorphisms: the cancer-associated c.2612C>T SNP in BRCA1, and the c.-26G>A SNP in the BRCA2 gene, acting as an ovarian cancer risk modifier in carriers of deleterious BRCA1 mutations. Analysis of these SNPs in a genotypically-unrelated Polish family, characterized by prevalent breast neoplasms in carriers of the c.190T>C mutation, revealed a genetic profile consistent with the hypothetic role of both polymorphisms. IntroductionHereditary breast cancer accounts for 5-10% of all breast cancers worldwide. Prevalence of BRCA1 and BRCA2 mutations among high-risk cancer patients may vary by ethnicity, study inclusion criteria and mutation detection techniques. Since their early characterization, inherited BRCA1 mutations were found to be responsible for ~45% of inherited early-onset breast cancers, and for >80% of inherited breast and ovarian cancers (1). In Europe, BRCA1 mutation frequencies in breast cancer populations show considerable variability and range between 0.4% in Finland and 7% in Sweden (2). Meta-analytic mean cumulative cancer risks for mutation carriers at age 70 years are 57% for BRCA1 and 49% for BRCA2 mutation carriers; ovarian cancer risk is 40% for BRCA1 and 18% for BRCA2 mutation carriers (3). In Italy, breast cancer penetrance, estimated in a series of 568 families, was found to be 27% at age 50 and 39% at age 70 in BRCA1 mutation carriers, and 26% at age 50 and 44% at age 70 in BRCA2 mutation carriers (4). Ovarian cancer penetrances in Italy were estimated to be 14% at age 50 and 43% at age 70 in BRCA1 mutation carriers, and 3% at age 50 and 15% at age 70 in BRCA2 mutation carriers (4).BRCA1 is a gene with a coding sequence spanning over 23 exons, i...

Abstract. Transition from malignant schwannoma to malignant triton tumor is analyzed in a case report on a patient with recurring cancers and suspected familial predisposition. It is hypothesized that rhabdomyoblastic differentiation, which distinguishes triton from schwannoma, might be attributable to Hedgehog-Patched pathway malfunctioning. Loss of one Patched gene allele was found in the tissue of advanced triton, but the retained allele had no exon or promoter mutations. Protein levels at early cancer stages indicated possible Patched response to the pathway activation in the first occurrence of triton tumor. Later, in the recurring triton, Patched expression was several times lower than in the control tissue, suggesting that haploinsufficiency was aided by silencing of the remaining allele, although its promoter was not hypermethylated. These findings may justify further investigation of the HedgehogPatched pathway role in triton malignancies, especially because of the recent research on the therapeutical potential of the pathway.

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