engleski

High-Resolution Melting Analysis of BRCA1 and BRCA2 Sequence Variants in Healthy Women in Croatia and Computational Analysis of New Variants of Unknown Clinical Significance

BRCA1 and BRCA2 are the major hereditary breast and/or ovarian cancer predisposing genes and their mutations increase the risk of developing cancer, both sporadic and hereditary forms. At least 10% of cases are attributable to familial inheritance. In Croatia, more than 2200 new cases of breast cancer are diagnosed yearly, and about 900 women die of this malignancy. We analyzed distribution and occurrence of sequence variants in BRCA1 and BRCA2 genes in a healthy population of women from Croatia in an attempt to distinguish non-tumorigenic from tumorigenic changes in genomic sequences of BRCA1 and BRCA2 genes. The screening was performed by high resolution melting analysis, which is based on differences in melting curves caused by variations in nucleotide sequence ; detected variants were confirmed by direct sequencing. In total, we analyzed 230 samples for BRCA1 and 140 samples for BRCA2 gene. We found 21 different sequence variants in BRCA1 (one novel) and 36 variants in BRCA2 gene (7 novel). At present, almost half of all BRCA1 and BRCA2 sequence variants found are unclassified variants (UVs) so their clinical significance is unknown or uncertain. That represents problem for risk assessment in genetic counseling. We used different web-based tools to assess preliminary functional significance of newly found UVs and found that two non-synonymous amino acid changes and one amino acid deletion could have no impact on structure and function of BRCA2 protein. Two synonymous substitutions could have impact on splicing by disrupting and creating exonic splicing enhancers. Our only intronic UV shows no potential impact on normal splicing and all exonic UVs do not lead to creation of potential cryptic splice sites.

BRCA1; BRCA2; polymorphism; high-resolution melting

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