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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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Involvement of Asthma-associated Protein ORMLDL3 in Mast Cell Signalling
Eitler, Jiří ; Dráber, Petr (advisor) ; Konvalinka, Jan (referee)
4 Abstract Mast cells are involved in variety of immunological processes, but they are mostly known for their role in allergy and asthma. As asthma and allergy are serious diseases with spreading tendency during last decades, mast cells are subject of intensive research. It is expected that studies of mast cell signalling pathways will contribute to our understanding of the nature of these diseases and help to design efficient treatment strategies. In an attempt to identify genes responsible for asthma disease, genome-wide screening methods have been currently applied. Using these methods, mutations in ORMDL3 (Orosomucoid1-like) protein were found out as a high risk asthma factor. ORMDL3 is a member of evolutionary conserved ORMDL family, comprising in mammals also of ORMDL1 and ORMDL2. Physiological function of these proteins is poorly understood and it has not been studied in mast cells. We decided to study the role of ORMDL proteins in mast cells. Lentiviral delivery system was optimised for generation of stable knock-downs (KD) of all three members of the ORMDL family in primary mast cells. The ORMDL gene expression was measured by improved qPCR (quantitative PCR) reaction buffers. We found that all ORMDL genes are expressed in mast cells in order ORMDL3 > ORMDL2 > ORMDL1. Next, we investigated the...
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