Assuntos
Deleção Cromossômica , Mecanismo Genético de Compensação de Dose , Iduronato Sulfatase/genética , Mucopolissacaridose II/genética , Mutação/genética , Cromossomo X/genética , Alelos , Animais , Southern Blotting , Linhagem Celular , Criança , Pré-Escolar , Metilação de DNA , Evolução Fatal , Feminino , Fibroblastos , França , Genes Recessivos/genética , Humanos , Células Híbridas/efeitos dos fármacos , Células Híbridas/enzimologia , Células Híbridas/metabolismo , Iduronato Sulfatase/metabolismo , Hibridização in Situ Fluorescente , Íntrons/genética , Masculino , Mucopolissacaridose II/enzimologia , Ouabaína/farmacologia , Fenótipo , Polimorfismo Genético/genética , RNA Mensageiro/análise , RNA Mensageiro/genéticaRESUMO
Deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS) is responsible for mucopolysaccharidosis type II (OMIM 309900). The IDS gene (Xq28) has been completely sequenced (accession number L35485). Northern blot analysis of poly(A(+)) RNA from different tissues, hybridized with the total IDS cDNA, has revealed three major species of 2.1, 5.4 and 5.7 kb and one minor of 1.4 kb. The 1.4-kb mRNA has been previously described and we show that the three major IDS mRNA are the result of alternative polyadenylation site selection: a non-canonical ATTAAA signal at genomic position 23631 for the 2.1-kb mRNA, a AATAAA signal at position 27156 for the 5.4-kb mRNA and a AATAAA signal at position 27399 for the 5.7-kb mRNA. The different IDS mRNA encode for the same polypeptide and the most abundant transcripts have a long 3'-untranslated region (3'-UTR). The absence of obvious correlation between transcripts content and size, IDS protein amount and IDS activity in the four human fetal tissues tested suggests that it is IDS protein processing that may be regulated rather than IDS gene transcription.
Assuntos
Iduronato Sulfatase/genética , Proteínas de Ligação a RNA/metabolismo , Sequência de Bases , Northern Blotting , Éxons , Fibroblastos/metabolismo , Humanos , Iduronato Sulfatase/metabolismo , Dados de Sequência Molecular , Mucopolissacaridose II/enzimologia , Mucopolissacaridose II/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Poliadenilação e Clivagem de mRNARESUMO
We studied 70 unrelated Hunter patients and found a gene alteration in every patient. The molecular heterogeneity was very important. Large gene rearrangements were identified in 14 patients. Forty-three different mutations were identified in the 56 other patients and 31 were not previously described. Deletions and insertions, splice site mutations were associated with a severe phenotype as nonsense mutations except Q531X. Only a few mutations were present in several patients making difficult genotype-phenotype correlations. Mutation identification allows accurate carrier detection improving prenatal diagnosis. The mother was not found to be a carrier in five cases among the 44 sporadic cases. Haplotype analysis demonstrated a higher frequency of mutations in male meiosis.
Assuntos
Genes/genética , Iduronato Sulfatase/genética , Mucopolissacaridose II/genética , Southern Blotting , DNA/análise , DNA/genética , Análise Mutacional de DNA , Família , Saúde da Família , Feminino , Deleção de Genes , Genótipo , Humanos , Masculino , Mucopolissacaridose II/enzimologia , Mutação/genética , FenótipoRESUMO
Three missense mutations identified in the IDS gene of our Hunter's disease patients (P86L, P480L and P480Q) and the previously described P86R mutation were expressed in COS cells to evaluate their functional consequence on iduronate-2-sulfatase (IDS) activity and processing. The 86-proline residue belongs to the highly conserved pentapeptide C-X-P-S-R in which cysteine modification to a formylglycine is required for sulfatase activity. The substitution of the 86-proline residue led to a severe mutation as no mature form was targeted to the lysosome in agreement with the severe phenotype observed in patients carrying P86L and P86R mutations. Expression studies with P480L and P480Q mutant cDNAs showed the presence of a small amount of 55 kDa mature form in the lysosomes of transfected COS cells. IDS activity of the P480L and P480Q mutants in cell extracts represents 16.6% and 5.4% of the wild-type, respectively.
