Fabry disease (FD) is an X-linked lysosomal storage disorder caused by mutations in the GLA gene encoding alpha-galactosidase A (AGAL). The impact of X-chromosome inactivation (XCI) on the phenotype of female FD patients remains unclear. In this study we aimed to determine pitfalls of XCI testing in a cohort of 35 female FD patients. XCI was assessed by two methylation-based and two allele-specific expression assays. The results correlated, although some variance among the four assays was observed. GLA transcript analyses identified crossing-over in three patients and detected mRNA instability in three out of four analyzed null alleles. AGAL activity correlated with XCI pattern and was not influenced by the mutation type or by reduced mRNA stability. Therefore, AGAL activity may help to detect crossing-over in patients with unstable GLA alleles. Tissue-specific XCI patterns in six patients, and age-related changes in two patients were observed. To avoid misinterpretation of XCI results in female FD patients we show that (i) a combination of several XCI assays generates more reliable results and minimizes possible biases; (ii) correlating XCI to GLA expression and AGAL activity facilitates identification of cross-over events; (iii) age- and tissue-related XCI specificities of XCI patterning should be considered.
- MeSH
- alfa-galaktosidasa genetika MeSH
- chromozomy MeSH
- Fabryho nemoc * diagnóza genetika MeSH
- fenotyp MeSH
- inaktivace chromozomu X genetika MeSH
- lidé MeSH
- mutace MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Niemann-Pick type C (NP-C) is a rare neurovisceral genetic disorder caused by mutations in the NPC1 or the NPC2 gene. NPC1 is a multipass-transmembrane protein essential for egress of cholesterol from late endosomes/lysosomes. To evaluate impacts of NPC1 mutations, we examined fibroblast cultures from 26 NP-C1 patients with clinical phenotypes ranging from infantile to adult neurologic onset forms. The cells were tested with multiple assays including NPC1 mRNA expression levels and allele expression ratios, assessment of NPC1 promoter haplotypes, NPC1 protein levels, cellular cholesterol staining, localization of the mutant NPC1 proteins to lysosomes, and cholesterol/cholesteryl ester ratios. These results were correlated with phenotypes of the individual patients. RESULTS: Overall we identified 5 variant promoter haplotypes. Three of them showed reporter activity decreased down to 70% of the control sequence. None of the haplotypes were consistently associated with more severe clinical presentation of NP-C. Levels of transcripts carrying null NPC1 alleles were profoundly lower than levels of the missense variants. Low levels of the mutant NPC1 protein were identified in most samples. The protein localised to lysosomes in cultures expressing medium to normal NPC1 levels. Fibroblasts from patients with severe infantile phenotypes had higher cholesterol levels and higher cholesterol/cholesteryl ester ratios. On the contrary, cell lines from patients with juvenile and adolescent/adult phenotypes showed values comparable to controls. CONCLUSION: No single assay fully correlated with the disease severity. However, low residual levels of NPC1 protein and high cholesterol/cholesteryl ester ratios associated with severe disease. The results suggest not only low NPC1 expression due to non-sense mediated decay or low mutant protein stability, but also dysfunction of the stable mutant NPC1 as contributors to the intracellular lipid transport defect.
- MeSH
- fibroblasty metabolismus MeSH
- intracelulární signální peptidy a proteiny MeSH
- lidé MeSH
- membránové glykoproteiny * genetika metabolismus MeSH
- mladiství MeSH
- mutace genetika MeSH
- transportní proteiny * genetika MeSH
- Check Tag
- lidé MeSH
- mladiství MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- ABC transportéry genetika metabolismus MeSH
- dna (nemoc) * genetika metabolismus patofyziologie MeSH
- lidé MeSH
- molekulární biologie MeSH
- nádorové proteiny genetika metabolismus MeSH
- pohybová aktivita fyziologie MeSH
- polymorfismus genetický * MeSH
- stupeň závažnosti nemoci MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- dopisy MeSH
- práce podpořená grantem 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
- Publikační typ
- abstrakt z konference MeSH
The HUMARA assay, the most common method for evaluation of X-inactivation skewing in blood cells, has been reported to be usable in only about 80% of females, emphasizing the need for alternative methods for testing of HUMARA-uninformative individuals. We conducted an in silico search for potentially polymorphic tri-to-hexanucleotide repeats in the proximity of CpG islands located in 5' regions of X-chromosome genes to design five candidate assays (numbered I, II, III, IV, and V) combining methylation-specific restriction digest with PCR amplification in a manner similar to the HUMARA assay. The results obtained by these assays in 100 healthy females were compared to X-inactivation skewing measured by the AR-MSP method which is based on methylation-specific PCR amplification of the first exon of the AR gene. On the basis of statistical evidence, three of the novel assays (II, IV, and V), which were informative in 18%, 61%, and 55% of females in the cohort, respectively, may be used as alternatives or conjointly with the HUMARA assay to improve its reliability. The three new assays were combined with the HUMARA assay into a novel X-inactivation test leading to the increase of informative females in the cohort from 67% to 96%.
- MeSH
- androgenní receptory genetika MeSH
- biotest * MeSH
- CpG ostrůvky MeSH
- dítě MeSH
- DNA primery chemická syntéza MeSH
- dospělí MeSH
- exony MeSH
- inaktivace chromozomu X * MeSH
- lidé středního věku MeSH
- lidé MeSH
- lidské chromozomy X chemie MeSH
- metylace DNA MeSH
- mikrosatelitní repetice MeSH
- mladiství MeSH
- modely genetické MeSH
- počítačová simulace MeSH
- polymerázová řetězová reakce metody MeSH
- polymorfismus genetický MeSH
- senioři MeSH
- senzitivita a specificita MeSH
- studie případů a kontrol MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In a female patient with signs of ornithine carbamoyltransferase deficiency (OTCD), the only variation found was a heterozygous single nucleotide substitution c.-366A>G. Determination of transcription start sites of human OTC 95, 119 and 169 bp upstream of the initiation codon located the variation upstream of the 5'-untranslated region. We predicted the human promoter and enhancer elements from homology with rat and mouse, performed function analysis of both regulatory regions and assessed the impact of the promoter variation in functional studies using dual luciferase reporter assay. Our data indicate that: (i) Full transcriptional activity of human OTC promoter depends on an upstream enhancer, as do the rodent promoters. (ii) The promoter variation c.-366A>G does not affect the function of the promoter alone but it disrupts the interaction of the promoter with the enhancer. (iii) The promoter-enhancer interaction contributes to tissue specific expression of OTC in the liver. We conclude that mutations in the regulatory regions of OTC can lead to OTCD and should be included in genetic testing.
- MeSH
- buněčné linie MeSH
- kojenec MeSH
- lidé MeSH
- luciferasy metabolismus MeSH
- molekulární sekvence - údaje MeSH
- mutace genetika MeSH
- mutační analýza DNA MeSH
- nemoc z nedostatku ornithinkarbamoyltransferázy enzymologie genetika MeSH
- novorozenec MeSH
- ornithinkarbamoyltransferasa genetika MeSH
- počátek transkripce MeSH
- předškolní dítě MeSH
- promotorové oblasti (genetika) MeSH
- reportérové geny MeSH
- sekvence nukleotidů MeSH
- těhotenství MeSH
- zesilovače transkripce MeSH
- Check Tag
- kojenec MeSH
- lidé MeSH
- mužské pohlaví MeSH
- novorozenec MeSH
- předškolní dítě MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
104 stran ; 30 cm
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