Jediná tři lysosomální střádavé onemocnění s dědičností vázanou na X chromosom a provázená významným srdečním postižením jsou mukopolysacharidóza typu II (MPSII, deficit iduronát-2-sulfatázy), Danonova (DD, deficit LAMP2) a Fabryho nemoc (FD, deficit ?-galaktosidázy A). V projektu navrhujeme založení a charakterizaci kolekce buněčných modelů na bázi indukovaných pluripotentních buněk od dříve diagnostikovaných pacientů a pacientek. Modely plánujeme následně využít pro testování nových terapeutických a diagnostických postupů stejně jako pro další studium patogeneze zmíněných onemocnění. Jedním z našich hlavních cílů je, v liniích DD a FD pacientek, vývoj průtokově cytometrické metody pro separaci diferencovaných buněk (především kardiomyocytů) dle parentálního stavu jejich X-inaktivace. Separované buňky s příznivou X-inaktivací mutované alely lze, bez dalších genetických manipulací, považovat za vhodné pro další testování autologní buněčné terapie. V případě DD hodláme testovat terapeutický vliv kandidátních malých molekul interferujících s autofagií nebo lysosomální exocytózou.; The only lysosomal storage disorders with X-linked inheritance are mucopolysaccharidosis type II (MPSII, iduronate-2-sulfatase deficit), Danon (DD, LAMP2 deficit) and Fabry (FD, ?-galactosidase deficit) diseases. All express cardiac pathology of either primary cardiomyocytic (DD, FD) or secondary (due valvular dysfunction - MPSII) origin. In the project, we propose to establish and characterize induced pluripotent stem (iPS) cell based models from previously diagnosed male and female patients. The models will be used for testing novel diagnostic and therapeutic approaches as well as for further pathogenesis studies. One of our main goals, in cultures collected from DD and FD female patients, is to develop a protocol for flow cytometric sorting of the differentiated cells (mainly cardiomyocytes) based on their parental X-chromosome inactivation status. Supposedly, the favorably inactivated clones can be consequently utilized for autologous cell therapy testing. In DD, cellular impacts of candidate small molecules interfering with autophagy and/or lysosomal exocytosis will be tested.

• MeSH
• biologické modely MeSH
• Fabryho nemoc diagnóza   terapie   MeSH
• glykogenóza typu IIb diagnóza   terapie   MeSH
• inaktivace chromozomu X MeSH
• indukované pluripotentní kmenové buňky MeSH
• kardiomyopatie MeSH
• lyzozomální nemoci z ukládání diagnóza   terapie   MeSH
• mukopolysacharidóza II diagnóza   terapie   MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• genetika, lékařská genetika
• vnitřní lékařství
• cytologie, klinická cytologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

• Publikační typ
• abstrakt z konference MeSH

Mucopolysaccharidosis type II (MPS II) is an X-linked lysosomal storage disorder resulting from deficiency of iduronate-2-sulphatase activity. The disease manifests almost exclusively in males; only 16 symptomatic heterozygote girls have been reported so far. We describe the results of X-chromosome inactivation analysis in a 5-year-old girl with clinically severe disease and heterozygous mutation p.Arg468Gln in the IDS gene. X inactivation analysed at three X-chromosome loci showed extreme skewing (96/4 to 99/1) in two patient's cell types. This finding correlated with exclusive expression of the mutated allele. Induced pluripotent stem cells (iPSC) generated from the patient's peripheral blood demonstrated characteristic pluripotency markers, deficiency of enzyme activity, and mutation in the IDS gene. These cells were capable of differentiation into other cell types (cardiomyocytes, neurons). In MPS II iPSC clones, the X inactivation ratio remained highly skewed in culture conditions that led to partial X inactivation reset in Fabry disease iPSC clones. Our data, in accordance with the literature, suggest that extremely skewed X inactivation favouring the mutated allele is a crucial condition for manifestation of MPS II in females. This suggests that the X inactivation status and enzyme activity have a prognostic value and should be used to evaluate MPS II in females. For the first time, we show generation of iPSC from a symptomatic MPS II female patient that can serve as a cellular model for further research of the pathogenesis and treatment of this disease.

• MeSH
• iduronátsulfatasa genetika   metabolismus   MeSH
• inaktivace chromozomu X * MeSH
• indukované pluripotentní kmenové buňky * MeSH
• kultivované buňky MeSH
• lidé MeSH
• mukopolysacharidóza II diagnóza   enzymologie   genetika   MeSH
• mutace MeSH
• předškolní dítě MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH

Fabry disease is an X-linked lysosomal storage disease due to deficient α-galactosidase A (α-Gal A) activity and the resultant lysosomal accumulation of globotriaosylceramide (Gb3) and related lipids primarily in blood vessels, kidney, heart, and other organs. The renal distribution of stored glycolipid species in the α-Gal A knockout mouse model was compared to that in mice to assess relative distribution and absolute amounts of accumulated sphingolipid isoforms. Twenty isoforms of five sphingolipid groups were visualized by mass spectrometry imaging (MSI), and their distribution was compared with immunohistochemical (IHC) staining of Gb3, the major stored glycosphingolipid in consecutive tissue sections. Quantitative bulk lipid analysis of tissue sections was assessed by electrospray ionization with tandem mass spectrometry (ESI-MS/MS). In contrast to the findings in wild-type mice, all three analytical techniques (MSI, IHC, and ESI-MS/MS) revealed increases in Gb3 isoforms and ceramide dihexosides (composed mostly of galabiosylceramides), respectively. To our knowledge, this is the first report of the distribution of individual molecular species of Gb3 and galabiosylceramides in kidney sections in Fabry disease mouse. In addition, the spatial distribution of ceramides, ceramide monohexosides, and sphingomyelin forms in renal tissue is presented and discussed in the context of their biosynthesis.

• MeSH
• alfa-galaktosidasa genetika   metabolismus   MeSH
• Fabryho nemoc enzymologie   genetika   metabolismus   MeSH
• hmotnostní spektrometrie MeSH
• imunochemie MeSH
• ledviny chemie   metabolismus   MeSH
• lidé MeSH
• modely nemocí na zvířatech MeSH
• myši knockoutované MeSH
• myši MeSH
• sfingolipidy chemie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• Publikační typ
• abstrakt z konference MeSH

• Publikační typ
• abstrakt z konference MeSH

The function and intracellular delivery of enzyme therapeutics for Fabry disease were studied in cultured fibroblasts and in the biopsied tissues of two male patients to show diversity of affected cells in response to treatment. In the mutant fibroblasts cultures, the final cellular level of endocytosed recombinant alpha-galactosidases A (agalsidases, Fabrazyme, and Replagal) exceeded, by several fold, the amount in control fibroblasts and led to efficient direct intra-lysosomal hydrolysis of ((3)H)Gb3Cer. In contrast, in the samples from the heart and some other tissues biopsied after several months of enzyme replacement therapy (ERT) with Fabrazyme, only the endothelial cells were free of storage. Persistent Gb3Cer storage was found in cardiocytes (accompanied by increase of lipopigment), smooth muscle cells, fibroblasts, sweat glands, and skeletal muscle. Immunohistochemistry of cardiocytes demonstrated, for the first time, the presence of a considerable amount of the active enzyme in intimate contact with the storage compartment. Factors responsible for the limited ERT effectiveness are discussed, namely post-mitotic status of storage cells preventing their replacement by enzyme supplied precursors, modification of the lysosomal system by longstanding storage, and possible relative lack of Sap B. These observations support the strategy of early treatment for prevention of lysosomal storage.

• MeSH
• alfa-galaktosidasa metabolismus   terapeutické užití   MeSH
• biopsie MeSH
• Fabryho nemoc terapie   MeSH
• fibroblasty enzymologie   MeSH
• financování organizované MeSH
• genetická terapie metody   MeSH
• konfokální mikroskopie MeSH
• kultivované buňky MeSH
• lidé středního věku MeSH
• lidé MeSH
• myokard enzymologie   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH

PURPOSE: To confirm and define a molecular basis for a case of mucolipidosis type IV (ML IV) with an extremely atypical phenotype pattern. DESIGN: Observational case report of a patient with ML IV with disease progression restricted to ocular symptoms. METHODS: Complete ophthalmologic and neurologic examination. Ultrastructural examination of white blood cells, skin, conjunctiva, and corneal epithelium. The MCOLN1 gene was sequenced from cDNA and the proportion of splicing variants were assessed by quantitative allele-specific polymerase chain reaction. RESULTS: Absence of any neurological abnormalities. Retinal pathologic features were the main cause of visual disability: low visual acuity and cloudy corneas since 2 years of age, progressive decrease in visual acuity since the age of 9 years. Ultrastructural examination showed storage lysosomes filled with either concentric membranes or lucent precipitate in corneal and conjunctive epithelia and in vascular endothelium. Cultured fibroblasts were free of any autofluorescence. Sequencing of the MCOLN1 gene identified compound heterozygosity for D362Y and A-->T transition leading to the creation of a novel donor splicing site and a 4-bp deletion from exon 13 at the mRNA level. Both normal and pathologic splice forms were detected in skin fibroblasts and leukocytes, with the normal form being more abundant. CONCLUSIONS: The case of this patient with ML IV is unique and is characterized by a curious lack of generalized symptoms. In this patient, the disorder was limited to the eyes and appeared without the usual psychomotor deterioration. The resulting phenotype is the mildest seen to date.

• MeSH
• alternativní sestřih genetika   MeSH
• degenerace retiny genetika   patologie   MeSH
• dítě MeSH
• elektroretinografie MeSH
• epitelové buňky ultrastruktura   MeSH
• fenotyp MeSH
• fibroblasty ultrastruktura   MeSH
• financování organizované MeSH
• kationtové kanály TRPM genetika   MeSH
• kůže ultrastruktura   MeSH
• leukocyty ultrastruktura   MeSH
• lidé MeSH
• lyzozomy genetika   ultrastruktura   MeSH
• messenger RNA genetika   MeSH
• mukolipidózy genetika   patologie   MeSH
• mutace MeSH
• mutační analýza DNA MeSH
• nemoci rohovky genetika   patologie   MeSH
• nemoci spojivky genetika   patologie   MeSH
• polymerázová řetězová reakce MeSH
• rohovkový epitel ultrastruktura   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• kazuistiky MeSH

BACKGROUND: Anderson-Fabry disease (AFD) is an X-linked disorder caused by deficient activity of enzyme alpha-galactosidase A, resulting in the accumulation of glycosphingolipids within lysosomes. Pulmonary involvement in AFD has previously been documented, but until now has been studied only in a few series of patients without any longitudinal follow-up. The aim of this study was to compare spirometric changes in AFD patients with a matched control population and to follow the subsequent progression of the disease. MATERIALS AND METHODS: Fifty individuals (27 women, 23 men, mean age 40 +/- 14 years) with AFD from 14 families underwent a static spirometric examination under standard conditions. A set of indices was compared with that of the control population. Out of this cohort, 39 individuals not receiving enzyme replacement therapy were longitudinally evaluated (median follow-up time 24 months). RESULTS: A clinically significant reduction in spirometric parameters, corresponding to mild to severe airway obstruction, was observed in 26% of women and 61% of men. During the serial follow-up, a significant (p < 0.05) age-dependent reduction of predicted %FVC and %FEV1 values was observed in male patients, while the influence of age was not seen in female patients. The %FEF(25-75) values decreased by similar degrees in men and women and in older and younger patients, indicating that progressive bronchial disease affects the small airways first. CONCLUSIONS: We have demonstrated a clinically relevant age- and sex-dependent progressive pulmonary involvement in AFD patients. The effects of enzyme replacement therapy on pulmonary involvement remain to be demonstrated.

• MeSH
• alfa-galaktosidasa genetika   metabolismus   MeSH
• časové faktory MeSH
• dospělí MeSH
• dýchání MeSH
• Fabryho nemoc enzymologie   genetika   patofyziologie   MeSH
• fenotyp MeSH
• financování organizované MeSH
• genetická predispozice k nemoci MeSH
• genotyp MeSH
• lidé středního věku MeSH
• lidé MeSH
• mutace MeSH
• následné studie MeSH
• obstrukce dýchacích cest MeSH
• prognóza MeSH
• progrese nemoci MeSH
• sexuální faktory MeSH
• spirometrie MeSH
• studie případů a kontrol MeSH
• stupeň závažnosti nemoci MeSH
• usilovný výdechový objem MeSH
• věkové faktory MeSH
• vitální kapacita MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• multicentrická studie MeSH
• Geografické názvy
• Česká republika MeSH

BACKGROUND: Fabry disease (FD) is a genetic disorder characterized by accumulation of trihexosylceramide in lysosomes of various tissues leading to multiorgan manifestations, including progressive renal disease. Previous screening studies have shown that a non-neglectable proportion of haemodialysis(HD) patients have unsuspected FD. An extensive FD screening study, the largest to date, has been conducted in HD patients in Czech Republic. We aimed to uncover previously undiagnosed FD patients, to enable them to benefit from cause-specific therapeutic intervention with enzyme replacement therapy (ERT). METHODS: Large-scale screening was executed using a convenient automated enzymatic (alpha-galactosidose A, alpha-Gal A) dried blood spot on filter paper fluorescence method. RESULTS: In total, 3370 (45.1% males, 54.9% females) out of 4058 HD patients (83%) in Czech Republic participated in this blood spot screening (BSS) study. Abnormal low fluorescence readings were obtained in 117 patients (3.5%). Subsequent determination of plasma alpha-Gal A activity identified four males and three females with deficient plasma enzyme activity. Determination of alpha-Gal A activity in peripheral blood leucocytes and confirmatory molecular analysis resulted in four newly diagnosed Fabry males and one female. Subsequent family screening identified 10 family members with genotypically proven FD. Based on these screening results, ERT could be offered to five male FD patients. CONCLUSIONS: BSS represents a promising screening tool that has proven to be convenient and effective in uncovering unrecognized FD patients among the chronic HD population in Czech Republic.

• MeSH
• alfa-galaktosidasa chemie   MeSH
• dialýza ledvin metody   MeSH
• dospělí MeSH
• enzymoterapie MeSH
• enzymy MeSH
• Fabryho nemoc diagnóza   krev   terapie   MeSH
• financování organizované MeSH
• fluorescenční spektrometrie MeSH
• konformace proteinů MeSH
• lidé středního věku MeSH
• lidé MeSH
• lyzozomy metabolismus   MeSH
• rodokmen MeSH
• senioři MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Geografické názvy
• Česká republika MeSH