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Molecular causes and mechanisms of hereditary cholestasis and statin-induced myopathy
Neřoldová, Magdaléna ; Jirsa, Milan (advisor) ; Dvořáková, Lenka (referee) ; Bronský, Jiří (referee)
The discovery of the molecular basis of Rotor syndrome consisting in biallelic inactivating mutations in both SLCO1B1 and SLCO1B3 genes encoding hepatic transporters OATP1B1 and OATP1B3, together with the previously described association of the rs4149056 variant in OATP1B1 with statin-induced myopathy (SM), led us to hypothesis that private variants in OATP1B1 and OATP1B3 confer predisposition to SM. This hypothesis was not supported by our study of 88 patients in whom exome sequencing was performed. Interestingly, we detected candidate variants in several genes mutated in recessively inherited muscle disorders, namely CLCN1, whose carriage may predispose to SM. Pathogenic variants in several dozens of causative genes underlie hereditary cholestasis. We performed exome sequencing in 51 unexplained cases and revealed several unexpected diagnoses such as autosomal recessive polycystic polyposis, cutaneous porphyria or nephronophthisis. The most remarkable finding was that of yet unreported F11R deficiency due to a homozygous splice mutation found simultaneously in an index patient suffering from liver cirrhosis and her healthy sister. The F11R protein is involved in formation of tight intercellular junctions and mutations in F11r predispose to liver failure in mice. The finding of bialellic variants...
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Metabolic effects of bile lipids
Žížalová, Kateřina ; Leníček, Martin (advisor) ; Jirsa, Milan (referee) ; Dvořák, Karel (referee)
Bile acids act as important fat emulsifiers in the human body. However, it is increasingly becoming apparent that they also play a role as important signalling molecules. Bile acids are a relatively heterogeneous group differing also in their metabolic effects. Their individual proportion in the body is highly variable and strongly influenced by the composition of the microbiome. The importance of individual bile acids is not yet well enough described to be fully exploited in clinical practice. For this reason, the determination of bile acids by enzymatic methods seems to be considered sufficient in most departments. Another and more reliable option is the increasingly widespread use of liquid chromatography-tandem mass spectrometry (LC-MS/MS). The aim of my work was to test the reliability of the enzymatic method and to introduce a method for the determination of bile acids, including the measurement of such atypical ones for example microbial conjugates and keto- and isoderivatives. Moreover, I aimed to test sample preparation for the measurement of 7α-hydroxy-4-cholesten-3-one, the bile acid biosynthesis marker. We used these methods to investigate the changes in bile acid composition changes in iron overload rats. In this rat model of genetically determined disorders of iron metabolism, bile...
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Role of host-dependent factors in prediction of antiviral treatment response in chronic hepatitis C
Fraňková, Soňa ; Jirsa, Milan (advisor) ; Brůha, Radan (referee) ; Plíšek, Stanislav (referee)
Soňa Fraňková: Role of host-dependent factors in prediction of antiviral treatment response in chronic hepatitis C Abstract Hepatitis C virus infection represents a leading cause of liver disease in western countries. The primary goal of HCV therapy is elimination of the virus, i.e. sustained virological response (SVR) achievement. Genetic factors have long been suspected of playing a crucial role in determining response to IFN-α-based therapies, but pretreatment predictors of response were only poorly defined and did not allow personalization of therapy. The aim of the thesis is to describe the role of host-dependent factors in prediction of antiviral treatment response in chronic hepatitis C in specific groups of patients. First, we focused on the role of the IFNG -764G/C promoter variant in SVR achievement. We did not prove that this variant predicted SVR in Czech HCV-infected individuals. Next, we focused on the role of IL28B and IFNL4 in HCV-infected patients: we confirmed that the IL28B rs12979860 CC genotype slows down the progression of liver fibrosis in chronic HCV infection and that IFNL4 ss469415590 TT|ΔG genotyping does not bring a better prediction of treatment success than IL28B rs12979860 in the Czech population. Third, we assessed prediction of treatment response in HCV positive liver...
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Molecular genetic diagnostics of cystic fibrosis, hyperhomocysteinemia-related disorders and male infertility: validation and application of high resolution melting
Norambuena Baraquet, Patricia Alejandra Del Pilar ; Macek, Milan (advisor) ; Jirsa, Milan (referee) ; Vrtěl, Radek (referee)
Diagnostic test results are crucial for treatment management and family planning of an individual. Considering that around 80% of medical decisions are based on diagnostic tests and that genotyping is usually concluded only once in a lifetime, it is of a great importance to assure highly accurate test results and provided under high quality standards. Cystic fibrosis (CF) is one of the most common and life-threatening autosomal recessive genetic disease affecting mainly Caucasian populations. CF is caused by mutations in the CFTR gene and until this date, more than 1900 mutations have been detected, while only few of them have frequencies higher than 1% worldwide. Thus, to confirm the diagnosis of cystic fibrosis in patients where only one disease-causing mutation has been found, it is necessary to apply a sensitive test to search for uncommon CFTR gene mutations/variants. In this work, we have successfully used HRM for gene scanning of certain exons of the CFTR gene. We have confirmed the numerous advantages of the HRM method for gene scanning and also detect some limitations that must be considered through an implementation process in a DNA diagnostic laboratory. Hyperhomocysteinemia has been proposed as a risk factor for several diseases such as recurrent pregnancy loss and inherited thrombophilia and...
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Molecular Basis of Hereditary Hyperuricaemic Nephropathies
Vyleťal, Petr ; Kmoch, Stanislav (advisor) ; Viklický, Ondřej (referee) ; Jirsa, Milan (referee)
Familial juvenile hyperuricemic nephropathy (FJHN) and medullary cystic kidney disease type 1 (MCKD1) and type 2 (MCKD2) are autosomal dominant tubulointerstitial nephropathies characterized by combinations of hyperuricaemia, gouty arthritis, progressive renal insufficiency, and in some but not all families, medullary cysts. The phenotypic expression of these diseases is inconsistent, overlaps and indicates broader genetic and allelic heterogeneity. Their pathophysiology was mainly unknown. Previous studies localized FJHN/MCKD genes to chromosomes 16p11 and 1q21. This thesis was primarily aimed at identification of molecular bases and mechanisms underlying FJHN/MCKD. To follow this aim, we focused on collection and characterization of FJHN/MCKD patients and families, identification of disease causing genes in affected families, characterisation of identified proteins and their mutated forms and the isolation and characterisation of interacting partners of newly identified proteins. We employed and established numerous molecular genetic, molecular biological and biochemical methods. We gathered one of the largest sets of families with FJHN/MCKD in the world. In about 26% of families we identified UMOD (uromodulin encoding) gene mutations and characterised by various approaches 6 uromodulin mutant proteins....
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The role of toll-like receptors in the pathogenesis of liver diseases
Petrášek, Jan ; Jirsa, Milan (advisor) ; Tlaskalová - Hogenová, Helena (referee) ; Červinková, Zuzana (referee)
Identifikační záznam: PETRÁŠEK, Jan. ÚLOHA TOLL-LIKE RECEPTORŮ V PATOGENEZI JATERNÍCH ONEMOCNĚNÍ. [The role of toll-like receptors in the pathogenesis of liver diseases]. Praha, 2010. 198s., Disertační práce. Univerzita Karlova v Praze, 1. lékařská fakulta, Laboratoř Experimentální Hepatologie IKEM. Vedoucí práce: Milan Jirsa. Abstrakt Společným jmenovatelem nejčastějších onemocnění jater je aktivace mechanismů vrozené imunity, které přispívají k rozvoji zánětu a poškození jaterního parenchymu. Klíčovou úlohu v rozvoji jaterního poškození hrají Toll-like receptory, jejichž charakterizace v posledním desetiletí vedla přehodnocení patofyziologie některých jaterních onemocnění. Předkládaná práce studuje význam alelických variant v genech kódujících proteiny Toll-like receptorové signální kaskády a mezibuněčné signalizace v patogenezi alkoholické nemoci jater, přináší nový pohled na probiotika v léčbě nealkoholické steatohepatitidy a nové poznatky o protizánětlivém působení interferonů I. typu u některých jaterních chorob. Abstract Recent reports suggest that majority of chronic and acute liver diseases share a significant degree of liver inflammation and injury attributable to innate immunity, activated through Toll-like receptors. Detailed characterization of Toll-like receptor sigaling cascades in the last...
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Molecular Basis of Familial Hyperuricemic Nephropathies
Živná, Martina ; Kmoch, Stanislav (advisor) ; Jirsa, Milan (referee) ; Sedláček, Zdeněk (referee)
In 1960 Duncan and Dixon described family whth chronic tubulointerstitial kidney disease associated with juvenile onset of hyperuricemia and gout. Based on combination of these clinical symptoms they named the disease familial juvenile hyperuricemic nephropathy (FJHN) [1]. Disease with very similar clinical presentation but different age of onset and kidney histology was described as a medullary cystic kidney disease (MCKD) in 1977 [2]. Until recently the molecular basis and pathogenesis of this syndrome remained unknown. The long term aim of our research group is to elucidate the genetic basis of the disease and to solve pathogenetic mechanisms leading to the individual clinical and biochemical symptoms (e.g. hyperuricemia) and kidney damage in general. We systematically identify patients with this disease and healthy family members and collect relevant clinical information and samples for classification (urine, blood, tissue biopsies) and subsequent clinical, biochemical, molecular biology and cell pathology correlations. We [3, 4] and others [5-7] proved genetic heterogeneity of FJHN and defined four FJHN loci on chromosomes 1q21, 1q41, 16p11.2. and 17q21.3. Further research defined disease causing mutations in three genes - uromodulin (UMOD) [8], hepatonuclear factor 1-beta (HNF-1) [9] and renin (REN)...
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Molecular analysis in cases of inherited diseas
Mrázová, Lenka ; Kmoch, Stanislav (advisor) ; Jirsa, Milan (referee) ; Macek, Milan (referee)
Urcení vztahu mezi onemocnením a jeho molekulární podstatou je jedním z hlavních cílu lékarské genetiky. V rámci studie: "Molekulární analýza vybraných dedicne podmínených onemocnení" byly popsány postupy, které vedou k objasnení molekulární podstaty u onemocnení s již známými odpovednými geny i u onemocnení, u kterého byl odpovedný gen teprve identifikován. Studie prispela k rozšírení znalostí zkoumaných dedicných onemocnení. Zavedené postupy molekulárních analýz jsou soucástí diagnostiky na Ústavu dedicných metabolických poruch v Praze. Výsledky techto analýz slouží k prevenci, prognóze i lécbe onemocnení a u postižených rodin byla umožnena prenatální diagnostika. Výsledky byly publikovány v nekolika domácích i zahranicních odborných casopisech a prezentovány na mezinárodních konferencích. Nekteré z publikací byly dále citovány.
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Genetic factors in aetiology and phatogenesisof low gamma-glutamyltransferase cholestasis and hereditary jaundice
Cebecauerová, Dita ; Jirsa, Milan (advisor) ; Brdička, Radim (referee) ; Macek, Milan (referee)
(English) Recent progress in understanding the molecular mechanism of hepatobiliary disorders enabled the improvement of diagnostic accuracy and promoted the study of the regulation of gene expression and its potential modifying factors. Current achievement in the field of genetically determined cholestatic disorders is well illustrated in this thesis, focused on low gamma-glutamyltransferase (γGT) cholestasis and hereditary jaundice. The study describes several distinct defects of hepatocyte transport system, characterises underlying mutations and their phenotypic consequences and, finally, extends these studies for detailed characterisation of ATP8B1 gene regulatory regions. Chapters related to low γGT cholestasis - characterise rare type of mutation associated with benign course of PFIC type I (formerly BRIC1) and explain the putative mechanisms of mutation origin. - provide extensive study of severe forms of ABCB11 deficiency (PFIC2) including genotype-phenotype correlations in 109 affected families, evaluation of the specific ABCB11 genotypes' impact on BSEP immunostaining and risk of hepatobiliary malignancy. - identify and characterise yet unknown regulatory regions of ATP8B1, a gene mutated in Progressive Familial Intrahepatic Cholestasis type I. The studies demonstrate the complex structure...
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Bilirubin secretory pathway and its disorders.
Sticová, Eva ; Jirsa, Milan (advisor) ; Bronský, Jiří (referee) ; Jirásek, Tomáš (referee)
Identification and functional characterization of numerous transport systems at the sinusoidal and canalicular membrane of hepatocytes have significantly expanded our understanding of bilirubin metabolism and contributed to elucidation of molecular basis of hereditary jaundice. Moreover, dysregulation of hepatobiliary transport systems could explain jaundice in many acquired liver disorders. This thesis is focused on the new aspects of bilirubin handling in hepatocytes based on elucidation of the molecular basis of Rotor syndrome. The first study is focused on the antioxidative properties of bilirubin in liver tissue in a model of obstructive cholestasis. In the second part of the thesis we present several novel mutations in ABCC2, the gene associated with Dubin-Johnson syndrome, identified in patients selected for the Rotor locus mapping study. In the key third study concerned with Rotor syndrome we demonstrated that biallelic inactivating mutations causing complete absence of transport proteins OATP1B1 and OATP1B3 result in disruption of hepatic reuptake of bilirubin, which is the molecular basis of Rotor-type jaundice. These results indicate that apart from secretion of conjugated bilirubin into bile, a significant fraction of bilirubin glucuronide is secreted via MRP3 into sinusoidal blood and...
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