Genotype-phenotype correlation in 44 Czech, Slovak, Croatian and Serbian patients with mucopolysaccharidosis type II.

Mucopolysaccharidosis type II (Hunter syndrome, MPS II, OMIM 309900) is an X-linked lysosomal storage disorder caused by deficiency of iduronate-2-sulfatase (IDS). We analyzed clinical and laboratory data from 44 Slavic patients with this disease. In total, 21 Czech, 7 Slovak, 9 Croatian and 7 Serbian patients (43 M/1 F) were included in the study (median age 11.0 years, range 1.2-43 years). Birth prevalence ranged from 1:69,223 (Serbia) to 1:192,626 (Czech Rep.). In the majority of patients (71%), the disease manifested in infancy. Cognitive functions were normal in 10 patients. Four, six and 24 patients had mild, moderate, and severe developmental delay, respectively, typically subsequent to developmental regression (59%). Residual enzyme activity showed no predictive value, and estimation of glycosaminoglycans (GAGs) had only limited importance for prognosis. Mutation analysis performed in 36 families led to the identification of 12 novel mutations, eight of which were small deletions/insertions. Large deletions/rearrangements and all but one small deletion/insertion led to a severe phenotype. This genotype-phenotype correlation was also identified in six cases with recurrent missense mutations. Based on patient genotype, the severity of the disease may be predicted with high probability in approximately half of MPS II patients.

X-Chromosome Inactivation Analysis in Different Cell Types and Induced Pluripotent Stem Cells Elucidates the Disease Mechanism in a Rare Case of Mucopolysaccharidosis Type II in a Female.

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.

Ornithine carbamoyltransferase deficiency: molecular characterization of 29 families.

Ornithine carbamoyltransferase deficiency is the most common inherited defect of the urea cycle. We examined 28 male and 9 female patients from 29 families and identified 25 distinct mutations in OTC, 14 of which were novel. Three novel missense mutations (p.Ala102Pro, p.Pro158Ser, p.Lys210Glu) and a novel deletion of the Leu43 are not directly involved either in the enzyme active site or in the intersubunit interactions; however, the mutations include conserved residues involved in intramolecular interaction network essential for the function of the enzyme. Three novel large deletions - a 444 kb deletion affecting RPGR, OTC and TSPAN7, a 10 kb-deletion encompassing OTC exons 5 and 6 and a 24.5 kb-deletion encompassing OTC exons 9 and 10 - have probably been initiated by double strand breaks at recombination-promoting motifs with subsequent non-homologous end joining repair. Finally, we present a manifesting heterozygote carrying a hypomorphic mutation p.Arg129His in combination with unfavorably skewed X-inactivation in three peripheral tissues.

Novel mutations in a boy with dihydrolipoamide dehydrogenase deficiency.

BACKGROUND: Dihydrolipoamide dehydrogenase (DLD) deficiency is a rare cause of primary lactic acidosis in infancy. MATERIAL AND METHODS: This article presents the results of biochemical and molecular analyses and metabolic response to treatment procedures in a 10-week old boy presenting with vomiting, progressive hypotonia, lactic acidosis (pH 7.04; BE - 20; B-lactate 6.6 mmol/l, controls <2.1; CSF-lactate 4.8 mmol/l, controls <2.0), increased levels of branched chain amino acids in blood, and increased urinary excretion of branched chain oxo-acids due to DLD deficiency. RESULTS: DLD activity was less than 5% of control values in lymphocytes, muscle mitochondria and fibroblasts. Western blot analysis in muscle tissue showed a decrease in the quantity of DLD protein to 40% in comparison to control. A high-fat, low-protein diet supplemented with MCT oils and sodium dichloroacetate resulted in normalization of lactate, amino acids and organic acids in body fluids, but there was no improvement in psychomotor development. Novel heterozygous mutations were found in the DLD gene: A1081G and G1123A. Both mutations affect the same region of the binding site for FAD. The G1123A mutation, resulting in the substitution of Glu 375 > Lys, breaks down the possible interaction of glutamic acid with neighboring lysine and causes electrostatic and steric repulsion, which is likely to destabilize structure in this part of the protein. In case of the A1081G mutation, resulting in substitution of Met 361 > Val, no important intermolecular interactions are broken and the reason for destabilization of the protein is not as clear. CONCLUSIONS: The prognosis for children with DLD deficiency is unfavorable, although long-term normalization of most metabolites in body fluids may be achieved with the proper diet and the administration of sodium dichloroacetate.

Identification of mutations in the glucose-6-phosphatase gene in Czech and Slovak patients with glycogen storage disease type ia, including novel mutations K76N, V166A and 540del5.

Mutations in the glucose-6-phosphatase (G6Pase) gene are responsible for glycogen storage disease type Ia (GSD Ia). A study of the molecular basis of GSD Ia was carried out in 12 Czech and Slovak GSD Ia patients from 10 unrelated families. Mutation analysis was performed for the entire coding region of G6Pase gene using DGGE, sequencing and PCR/digestion. With the strategy used, all mutant alleles were identified in this study. Three novel mutations (K76N, V166A and 540del5), six previously described mutations (W77R, R83C, G188R, R295C, Q347X and 158delC) and one known polymorphism (1176T-->C) were detected. The most common mutation identified was R83C, accounting for 8 out of 20 (40%) mutant alleles. The K76N mutation was found in a Gypsy family: two siblings with GSD Ia were homozygous for this mutation. These findings expand our knowledge of mutations responsible for glycogen storage disease type Ia.