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Congenital disorders of glycosylation and their impact on cellular and energetic metabolism
Rychtárová, Lucie ; Hansíková, Hana (advisor) ; Hodek, Petr (referee) ; Pecinová, Alena (referee)
Inherited disorders of glycosylation (CDG) are a large group of more than 160 types of metabolic disorders caused by genetic defects that lead to impaired glycan biosynthesis and modification. The lipid dolichol plays an essential role in glycan biosynthesis. Glycans play a key role in the function and structure of proteins and lipids and their deficiency leads to severe clinical symptoms. CDG usually manifests in childhood as a multisystem disorder. Families thus face a serious health problem due to the progressive and highly variable nature of the disease, the unfavorable prognosis and, with few exceptions, the unavailability of treatment. Currently, we still do not have a sufficient range of methods to recognize rare types of CDG and our knowledge of the pathophysiology of CDG is still limited. The first aim of this work was to optimize the method of determination of dolichol isoforms and to study them in physiology and pathology. The second aim of the work was to investigate the bioenergetic status and overall metabolism in the most common type of CDG - deficiency of phosphomannomutase 2 (PMM2-CDG), and in CDG caused by a defect in dolichol biosynthesis. The distribution of urinary dolichol isoforms in the population was characterized using an optimized method. The dolichol isoforms profile...
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Flow cytometry in the diagnostics and characterization of congenital disorders of glycosylation
Veselá, Šárka ; Hansíková, Hana (advisor) ; Hodek, Petr (referee)
Congenital disorders of glycosylation (CDG) are rare multisystem metabolic diseases and their number has rapidly grown in recent years. The clinical manifestation includes very broad spectrum of symptoms. In most of all cases CDG are caused by mutations in genes encoding the enzymes of glycosylation pathway. Based on the type of defect, CDG are divided into the following groups: disorders of N-glycosylation or O-glycosylation of proteins, defects in modification of proteins by GPI anchor, disorders of lipid glycosylation and defects that impact multiple glycosylation pathways. The aim of the thesis was to find new biochemical analyses suitable for diagnostics and characterization of CDG patients. The experimental conditions were optimized for selected markers (Sambucus Nigra (SNA) lectin, proaerolysin (FLAER), antibodies to proteins CD55 and CD59) and the staining was applied to cultivated skin fibroblasts from controls and patients diagnosed with CDG by whole-exome sequencing (ATP6AP1-CDG, PIGN-CDG, SLC10A7-CDG, PISD deficiency). The experiments were performed using flow cytometry (FACS) and fluorescent microscopy (FM). The detection of sialylation by SNA lectin and analysis of the mitochondrial membrane potential changes by a fluorescent labelled probe JC-1 with FCCP simulation of mitochondrial...
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Biochemical and molecular studies of the congenital disorders of glycosylation
Ondrušková, Nina ; Hansíková, Hana (advisor) ; Stiborová, Marie (referee) ; Hřebíček, Martin (referee)
Congenital disorders of glycosylation (CDG) represent a rapidly growing group of rare inherited metabolic diseases with estimated prevalence as high as 1:20 000, which are caused by genetic defects that impair the process of glycosylation, i.e. the enzymatic addition of a specific saccharide structure onto a protein or lipid backbone. Due to non-specificity and variability of clinical symptoms in the patients, the medical diagnosis of CDG remains extremely challenging and significantly relies on accurate biochemical and genetic analyses. The overall goal of the present dissertation thesis was to study CDG at the biochemical and molecular genetic level in the context of the Czech and Slovak Republic, which involved three specific aims: A.) to introduce and optimize laboratory screening methods for CDG detection in a group of clinically suspected patients, B.) to determine the corresponding genetic defect in the positive patients selected via CDG screening and to study the pathobiochemical aspects of specific CDG types at the cellular level, and C.) to analyze glycosylation disturbances of non- CDG etiology. Contributions of this work include optimization of isoelectric focusing of apolipoprotein C-III (ApoC-III) as a screening method for O-glycosylation abnormalities, as well as the description of...
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Biochemical and molecular studies of the congenital disorders of glycosylation
Ondrušková, Nina
Congenital disorders of glycosylation (CDG) represent a rapidly growing group of rare inherited metabolic diseases with estimated prevalence as high as 1:20 000, which are caused by genetic defects that impair the process of glycosylation, i.e. the enzymatic addition of a specific saccharide structure onto a protein or lipid backbone. Due to non-specificity and variability of clinical symptoms in the patients, the medical diagnosis of CDG remains extremely challenging and significantly relies on accurate biochemical and genetic analyses. The overall goal of the present dissertation thesis was to study CDG at the biochemical and molecular genetic level in the context of the Czech and Slovak Republic, which involved three specific aims: A.) to introduce and optimize laboratory screening methods for CDG detection in a group of clinically suspected patients, B.) to determine the corresponding genetic defect in the positive patients selected via CDG screening and to study the pathobiochemical aspects of specific CDG types at the cellular level, and C.) to analyze glycosylation disturbances of non- CDG etiology. Contributions of this work include optimization of isoelectric focusing of apolipoprotein C-III (ApoC-III) as a screening method for O-glycosylation abnormalities, as well as the description of...
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Dolichol content analysis by mass spectrometry in urine from patients with congenital disorders of glycosylation
Zdražilová, Lucie ; Hansíková, Hana (advisor) ; Kohoutová, Michaela (referee)
Dolichol is a membrane lipid, which carries monnosaccharides and glycans for N-linked protein glycosylation and glycosylphosphatidylinositol-anchor biosynthesis occuring in endoplasmic reticulum. Its structure is composed of isoprenoid units. Dolichol is present in all tissues and in most of the membrane organelles of eukaryotic cells. Recently some types of congenital disorders of glycosylation have been described as a consequence of dolichol biosynthesis and metabolism defects, which are not detectable by standard methods. The aim of this diploma thesis was to analyze dolichol content in urine and in different tissues from patients with deficiency in dolichol biosynthesis by mass spectrometry and to study the impact of these defects on energetic metabolism. Biological material for this study consisted of urine samples from 76 controls with age ranging from 1 months to 81 years, 6 patients with congenital disorders of glycosylation and 43 patients with suspicion of congenital disorder of glycosylation; samples of frontal cortex, liver, muscle and heart tissues from 2 patients with mutation in NUS1 gene and controls. Urine samples were stored at -20 řC and tissue homogenates were stored in -80 řC until analysis. Lipid fraction after extraction was separated by liquid chromatography. Dolichols were...
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Biochemical and molecular studies of the congenital disorders of glycosylation
Ondrušková, Nina
Congenital disorders of glycosylation (CDG) represent a rapidly growing group of rare inherited metabolic diseases with estimated prevalence as high as 1:20 000, which are caused by genetic defects that impair the process of glycosylation, i.e. the enzymatic addition of a specific saccharide structure onto a protein or lipid backbone. Due to non-specificity and variability of clinical symptoms in the patients, the medical diagnosis of CDG remains extremely challenging and significantly relies on accurate biochemical and genetic analyses. The overall goal of the present dissertation thesis was to study CDG at the biochemical and molecular genetic level in the context of the Czech and Slovak Republic, which involved three specific aims: A.) to introduce and optimize laboratory screening methods for CDG detection in a group of clinically suspected patients, B.) to determine the corresponding genetic defect in the positive patients selected via CDG screening and to study the pathobiochemical aspects of specific CDG types at the cellular level, and C.) to analyze glycosylation disturbances of non- CDG etiology. Contributions of this work include optimization of isoelectric focusing of apolipoprotein C-III (ApoC-III) as a screening method for O-glycosylation abnormalities, as well as the description of...
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Biochemical and molecular studies of the congenital disorders of glycosylation
Ondrušková, Nina ; Hansíková, Hana (advisor) ; Stiborová, Marie (referee) ; Hřebíček, Martin (referee)
Congenital disorders of glycosylation (CDG) represent a rapidly growing group of rare inherited metabolic diseases with estimated prevalence as high as 1:20 000, which are caused by genetic defects that impair the process of glycosylation, i.e. the enzymatic addition of a specific saccharide structure onto a protein or lipid backbone. Due to non-specificity and variability of clinical symptoms in the patients, the medical diagnosis of CDG remains extremely challenging and significantly relies on accurate biochemical and genetic analyses. The overall goal of the present dissertation thesis was to study CDG at the biochemical and molecular genetic level in the context of the Czech and Slovak Republic, which involved three specific aims: A.) to introduce and optimize laboratory screening methods for CDG detection in a group of clinically suspected patients, B.) to determine the corresponding genetic defect in the positive patients selected via CDG screening and to study the pathobiochemical aspects of specific CDG types at the cellular level, and C.) to analyze glycosylation disturbances of non- CDG etiology. Contributions of this work include optimization of isoelectric focusing of apolipoprotein C-III (ApoC-III) as a screening method for O-glycosylation abnormalities, as well as the description of...
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