Analysis of pre-mRNA alternative splicing products and their importance in breast cancer oncogenesis

Breast cancer is the most common tumor disease diagnosed in women worldwide. The hereditary character of this disease is observed in 5-10 % of all cases, and it is usually caused by a pathogenic mutation in one of the predisposition genes. Although a variety of pathogenic mutations in the coding sequences of these genes was described, the cause of the disease is still unknown in many familial cases (> 50%). A great number of identified pathogenic mutations were localized in the consensus splicing sites, which results in the formation of aberrant mRNA splicing variants and their damaged protein isoforms. However, little is known about mutations affecting regulatory splicing sites, which can result in the translation of similarly affected mRNAs. In this work, we proposed a method for indirect detection of mutations affecting the natural splicing pattern of any gene of our interest based on multiplex PCR and NGS with high sensitivity. Verification of this method on the BRCA1 model gene revealed the presence of the total of 94 splicing variants in peripheral leucocytes and healthy breast and adjacent fat tissues. This is the most detailed catalogue of physically occurring BRCA1 mRNA variants thus far. The most commonly occurring variants, maintaining open reading frame, were quantified by RT-qPCR which..