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Mechanisms of immune dysregulation leading to inflammatory bowel disease
Horáčková, Klára ; Froňková, Eva (advisor) ; Filipp, Dominik (referee)
Bc. Klára Horáčková DIPLOMA THESIS Mechanisms of immune dysregulation leading to inflammatory bowel disease Abstract Inflammatory bowel disease (IBD) is a complex disorder characterized by chronic inflammation of the gastrointestinal tract. Classical IBD is a multifactorial disease with adulthood or later-childhood onset. However, children with very early onset IBD (VEO-IBD, before 6 years of age) are a specific cohort, whose pathology can be caused by severe genetic defects in genes connected to immune homeostasis in the gut. We aimed to identify the causal genetic variants in 20 pediatric patients diagnosed with IBD (age of onset from 3 to 154 months) using whole exome sequencing (WES). We evaluated several bioinformatical approaches for WES data analysis. This included a comparison of two methods of variant identification using VarScan2 or GATK4-based tools. Furthermore, we compared 4 gene lists ("virtual panels") for variant filtering, one of which was compiled purposefully for this thesis. We identified and validated via segregation analysis 5 causal variants in 4 genes (DUOX2 compound heterozygote, FOXP3, NLRP3 and NOD2) accounting for 20 % of the cohort. NOD2 (p.A755V) variant has already been reported in IBD cases, while DUOX2 (p.R1216W + p.A1131T), FOXP3 (p.H400L) and NLRP3 (p.V200M) were newly...
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Cell signaling aberrations in primary immunodeficiencies
Fejtková, Martina ; Kanderová, Veronika (advisor) ; Vlková, Marcela (referee)
Primary immunodeficiencies (PID) are genetic disorders characterized by increased susceptibility to infections and various degrees of immune dysregulation. With the expansion of massive parallel sequencing, an increasing number of defects in immune-related genes is being identified in PID. However, the biological impact of the found mutations is often unknown. It is necessary to devise methods to clarify their causality for disease development, which may also aid therapeutic decisions. One of the novel discoveries are gain-of-function mutations in STAT1 gene, resulting in chronic mucocutaneous candidiasis. Candidiasis may be ameliorated with antimycotics or with targeted JAK-STAT inhibitor, ruxolitinib. For our patient with a novel mutation in STAT1, we developed a simple test for the detection of phospho-STAT molecules in peripheral blood lymphocytes. The test confirmed the gain-of-function character of the identified mutation and was used to monitor ruxolitinib treatment efficacy. In the second patient, who presented with lymphadenopathy and immunodeficiency, the as yet undescribed mutation in CASP8 was found. We proved its loss-of-function property expressed as reduced caspase-8 and caspase-3 cleavage, impaired cellular apoptosis, and decreased NFB-related signaling. The third patient who...
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Mechanisms of immune dysregulation leading to inflammatory bowel disease
Horáčková, Klára ; Froňková, Eva (advisor) ; Filipp, Dominik (referee)
Bc. Klára Horáčková DIPLOMA THESIS Mechanisms of immune dysregulation leading to inflammatory bowel disease Abstract Inflammatory bowel disease (IBD) is a complex disorder characterized by chronic inflammation of the gastrointestinal tract. Classical IBD is a multifactorial disease with adulthood or later-childhood onset. However, children with very early onset IBD (VEO-IBD, before 6 years of age) are a specific cohort, whose pathology can be caused by severe genetic defects in genes connected to immune homeostasis in the gut. We aimed to identify the causal genetic variants in 20 pediatric patients diagnosed with IBD (age of onset from 3 to 154 months) using whole exome sequencing (WES). We evaluated several bioinformatical approaches for WES data analysis. This included a comparison of two methods of variant identification using VarScan2 or GATK4-based tools. Furthermore, we compared 4 gene lists ("virtual panels") for variant filtering, one of which was compiled purposefully for this thesis. We identified and validated via segregation analysis 5 causal variants in 4 genes (DUOX2 compound heterozygote, FOXP3, NLRP3 and NOD2) accounting for 20 % of the cohort. NOD2 (p.A755V) variant has already been reported in IBD cases, while DUOX2 (p.R1216W + p.A1131T), FOXP3 (p.H400L) and NLRP3 (p.V200M) were newly...
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Cell signaling aberrations in primary immunodeficiencies
Fejtková, Martina ; Kanderová, Veronika (advisor) ; Vlková, Marcela (referee)
Primary immunodeficiencies (PID) are genetic disorders characterized by increased susceptibility to infections and various degrees of immune dysregulation. With the expansion of massive parallel sequencing, an increasing number of defects in immune-related genes is being identified in PID. However, the biological impact of the found mutations is often unknown. It is necessary to devise methods to clarify their causality for disease development, which may also aid therapeutic decisions. One of the novel discoveries are gain-of-function mutations in STAT1 gene, resulting in chronic mucocutaneous candidiasis. Candidiasis may be ameliorated with antimycotics or with targeted JAK-STAT inhibitor, ruxolitinib. For our patient with a novel mutation in STAT1, we developed a simple test for the detection of phospho-STAT molecules in peripheral blood lymphocytes. The test confirmed the gain-of-function character of the identified mutation and was used to monitor ruxolitinib treatment efficacy. In the second patient, who presented with lymphadenopathy and immunodeficiency, the as yet undescribed mutation in CASP8 was found. We proved its loss-of-function property expressed as reduced caspase-8 and caspase-3 cleavage, impaired cellular apoptosis, and decreased NFB-related signaling. The third patient who...
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Eight color flow cytometry test development for primary imunodeficiency patients
Šinkorová, Vendula ; Kalina, Tomáš (advisor) ; Brdička, Tomáš (referee)
Primary immunodeficiencies represent a heterogeneous group of hereditary immune system malfunctions with very variable causes and symptoms. Multiparametric flow cytometry has become an important tool in primary immunodeficiency diagnostics and research because it provides detailed information on the phenotype of individual immune cells and their proportions in circulation. We have developed a complex monoclonal antibody panel composed of five eight-color tubes which is designed for immunophenotyping of basic lymphocyte subsets and further analysis of B and T cell subpopulations. We have optimized and standardized the panels so they will identify any changes originating from primary immunodeficiencies and provide comparable data on the level of cooperation between more laboratories. This was achieved by cooperation of six European research facilities which are all parts of the Euroflow consortium. The panels have been validated both on peripheral blood samples from healthy donors and patients with either gentically defined primary immunodeficiency or common variable immunodeficiency. Keywords: T lymphocyte, B lymphocyte, primary immunodeficiency, flow cytometry, immunophenotyping, Euroflow, optimization, standardization
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