ABSTRACT
BACKGROUND: The mucopolysaccharidoses (MPSs) are rare genetic disorders caused by mutations in lysosomal enzymes involved in the degradation of glycosaminoglycans (GAGs). In this study, we analyzed a total of 48 patients including MPSI (n=6), MPSII (n=18), MPSIIIA (n=11), MPSIVA (n=3), and MPSVI (n=10). METHODS: In MPS patients, urinary GAGs were colorimetrically assayed. Enzyme activity was quantified by colorimetric and fluorimetric assays. To find mutations, all IDUA, IDS, SGSH, GALNS, and ARSB exons and intronic flanks were sequenced. New mutations were functionally assessed by reconstructing mutant alleles with site-directed mutagenesis followed with expression of wild-type and mutant genetic variants in CHO cells, measuring enzymatic activity, and Western blot analysis of protein expression of normal and mutated enzymes in cell lysates. RESULTS: A total of five novel mutations were found including p.Asn348Lys (IDUA) in MPSI, p.Tyr240Cys (GALNS) in MPSIVA, and three ARSB mutations (p.Gln110*, p.Asn262Lysfs*14, and pArg315*) in MPSVI patients. In case of mutations p.Asn348Lys, p.Asn262Lysfs*14, and p.Gln110*, no mutant protein was detected while activity of the mutant protein was <1% of that of the normal enzyme. For p.Tyr240Cys, a trace of mutant protein was observed with a remnant activity of 3.6% of the wild-type GALNS activity. For pArg315*, a truncated 30-kDa protein that had 7.9% of activity of the normal ARSB was detected. CONCLUSIONS: These data further enrich our knowledge of the genetic background of MPSs.
Subject(s)
Glycosaminoglycans/metabolism , Mucopolysaccharidoses/enzymology , Mucopolysaccharidoses/genetics , Amino Acid Sequence , Animals , CHO Cells , Child , Cricetinae , Cricetulus , DNA Mutational Analysis , Female , Gene Expression Regulation, Enzymologic , Glycosaminoglycans/urine , Humans , Male , Molecular Sequence Data , Mucopolysaccharidoses/metabolism , Mucopolysaccharidoses/urine , RussiaABSTRACT
Mucopolysaccharidosis type II (MPS II) is a rare X-linked disorder caused by alterations in the iduronate-2-sulfatase (IDS) gene. In this study, IDS activity in peripheral mononuclear blood monocytes (PMBCs) was measured with a fluorimetric enzyme assay. Urinary glycosaminoglycans (GAGs) were quantified using a colorimetric assay. All IDS exons and intronic flanks were bidirectionally sequenced. A total of 15 mutations (all exonic region) were found in 17 MPS II patients. In this cohort of MPS II patients, all alterations in the IDS gene were caused by point nucleotide substitutions or small deletions. Mutations p.Arg88His and p.Arg172* occurred twice. All mutations were inherited except for p.Gly489Alafs*7, a germline mutation. We found four new mutations (p.Ser142Phe, p.Arg233Gly, p.Glu430*, and p.Ile360Tyrfs*31). In Epstein-Barr virus (EBV)-immortalized PMBCs derived from the MPS II patients, no IDS protein was detected in case of the p.Ser142Phe and p.Ile360Tyrfs*31 mutants. For p.Arg233Gly and p.Glu430*, we observed a residual expression of IDS. The p.Arg233Gly and p.Glu430* mutants had a residuary enzymatic activity that was lowered by 14.3 and 76-fold, respectively, compared with healthy controls. This observation may help explain the mild disease phenotype in MPS II patients who had these two mutations whereas the p.Ser142Phe and p.Ile360Tyrfs*31 mutations caused the severe disease manifestation.
