During RNA splicing, genetic variants can alter the identification of splice sites recognized by the spliceosome. Altered recognition of splice sites can cause aberrant mRNA molecule, which can be the underlying cause of genetic syndrome development. One of the approaches to detect variants that cause misrecognition of splice sites is to determine the effect of the variant directly on the patient’s mRNA molecule. In this doctoral thesis, we developed and validated a new cDNA sequencing approach to identify splicing aberrations resulting from germline variants. By using new approach, we discovered 18, 49, and 7 new alternative transcripts in the STK11, NBN, and BRIP1 genes, respectively. We annotated all the discovered alternative transcripts and published the first comprehensive catalog of annotated alternative transcripts for these genes. In the second part of the study, we performed a bioinformatics assessment of variants' impact on splicing. We functionally assessed 32 variants of unknown clinical significance (VUS) in genes associated with hereditary cancer syndromes. Using the novel cDNA sequencing approach, we successfully reclassified 65.6 % of all VUSs (46.9 % were reclassified as a likely benign variant and 18.8 % as a likely pathogenic variant). Among 2808 patients included in the study, we found that 2 % of them carried a VUS with a potential impact on splicing. We were able to successfully reclassify VUS in 1.4 % of patients through mRNA analysis. The causative variant was identified through mRNA analysis in 0.2 % of patients.
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