Browsing by Subject "RNA sekvensointi"

The RNA splicing process is an important part of gene expression in which the introns are removed from the pre-mRNA so that the mature mRNA only contains protein coding exons. The splicing process is executed by the spliceosome in two subsequent transesterification reactions that occur partly co-transcriptionally. In the first step an intron lariat is formed between the exons. This is followed by splicing of the intron lariat and ligating the exons together. Genetic variants that affect the splicing of a particular gene are called splicing variants and they may disrupt the normal splicing process. Splicing variants can be both exonic or intronic and have effects on splice site recognition, activate cryptic splice sites or create new splice sites. These changes can lead to for example exon skipping or intron retention in the transcript. Diagnosing splicing variants is challenging because of the unknown functional effects of the variants. Splicing prediction tools can help predict the possible effects of variants. Different sequencing methods enable the detection of aberrant splicing transcripts and thus may help in variant interpretation. The aim of this master’s thesis was to develop a detection method suitable for the diagnostic laboratory for RNA splicing variants in congenital disorders. The methods that were tested included RNA and Sanger sequencing. First, the patient selection was performed using splicing predictions and previous research on the variants. Secondly, after receiving patient samples, RNA was extracted, and its integrity measured. The laboratory work was then divided into two parts, the other leading to the RNA sequencing and the other to Sanger sequencing. Before Sanger sequencing primer design, RT-PCR, PCR and analysis of the PCR fragment sizes was performed. RNA sequencing was preceded by RNA library preparation. The studied variants in this thesis were BRCA2 c.476-3C>A, MSH2 c.2005+3A>T and CYLD c.2350+5G>A. The PCR fragment analysis and Sanger sequencing was able to detect an aberrant splicing transcript with exon skipping on two patients caused by a variant in the CYLD gene. The RNA sequencing results confirmed the aberrant splicing transcript. In addition, fragment analysis showed evidence of a possible splicing isoform with skipping of two exons caused by a variant in the BRCA2 gene that was not expressed enough to show on the Sanger sequencing results. The RNA sequencing detected a splicing transcript with exon skipping in two BRCA2 patients. However, this was not the same transcript as interpreted from the fragment analysis results and no results in the RNA sequencing indicated a transcript with skipping of two exons.