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Sequencing of the ORMDL3 gene
KRUPILOVÁ, Kristýna
The main topic of the presented bachelor thesis is a sequence analysis of the ORMDL3 gene, which is identified as a potential risk factor for many diseases, the most discussed of which is childhood asthma. This gene is a member of the ORMDL gene family, which encodes transmembrane proteins anchored in the endoplasmic reticulum. In humans, ORMDL3 is located on chromosome 17, in region 17q21.1. In the theoretical part of the thesis the autoimmune diseases, on which origin and development the mutations in the ORMDL3 gene participate to a certain extent, are specified. These diseases include the aforementioned asthma, Crohn's disease, rheumatoid arthritis, insulin-dependent diabetes mellitus and primary biliary cirrhosis. Also, the Sanger sequencing method by which the samples were sequenced, and the modern NGS method are described here. The practical part of the thesis is focused on preparation of samples for Sanger sequencing, and evaluation of sequencing data. To amplify the desired region for the sequencing reaction, the PCR method was used. 20 anonymized DNA samples were prepared this way. For sequencing by Genseq s.r.o. 15 samples were sent. Furthermore, 20 provided samples were evaluated, 10 of which were from children from the České Budějovice region and 10 from children from the Karviná region. These samples were also anonymized. To analyze the sequences of individual DNA samples, freely available BioEdit program and the NCBI database was used. The sequences were read from both sides (forward and reverse), the found mutations were identified and the resulting data were plotted in graphs. After the sequences were read, a heterozygous mutation was present in 5 of the 15 samples. The same mutation was also present in the provided sequences, specifically in 8 out of 10 from České Budějovice and 3 out of 10 from Karviná. This mutation has not yet been described in the NCBI database.
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