Genotype–phenotype correlations in mucopolysaccharidosis type I using enzyme kinetics, immunoquantification and in vitro turnover studies

Abstract

Fibroblasts from 16 patients with known alpha-L-iduronidase gene mutations and different clinical phenotypes of mucopolysaccharidosis type I (MPS I) were investigated in order to establish genotype/phenotype correlations. Enzyme kinetic studies were performed using the specific alpha-L-iduronidase substrate iduronosyl anhydro[1-3H]mannitol-6-sulfate. Specific residual enzyme activities were estimated using the kinetic parameters and an immunoquantification assay which determines levels of alpha-L-iduronidase protein. Cells were cultured in the presence of [35S]sulfate and the in vivo degradation of accumulated labelled glycosaminoglycans measured after different chase times. Residual enzyme activity and different amounts of residual enzyme protein were present in extracts from 9 of 16 cell lines covering a wide spectrum of clinical severity. Catalytic capacity, calculated as the product of kcat/Km and ng iduronidase protein per mg cell protein, was shown in most cases to be directly related to the severity of clinical phenotype, with up to 7% of normal values for patients with the attenuated form of MPS I (Scheie) and less than 0.13% for severely affected patients (Hurler) In vitro turnover studies allowed further refinement of correlations between genotype and phenotype. Scheie disease compared to Hurler disease patients were shown to accumulate smaller amounts of glycosaminoglycans that were also turned over faster. A combination of turnover and residual enzyme data established a correlation between the genotype, the biochemical phenotype and the clinical course of this lysosomal storage disorder.

Functional abnormalities of heparan sulfate in mucopolysaccharidosis-I are associated with defective biologic activity of FGF-2 on human multipotent progenitor cells

“…[16][17][18]33,[38][39][40] Overall, our findings are consistent with these studies. A unique aspect of our study is that we examined metabolically radiolabeled GAGs obtained from the progeny of primary, multipotent stem-cell populations from the BM of patients with Hurler syndrome and healthy volunteers.…”

“…Sera used for all studies in this paper were heat-inactivated (2 hours at 55°C) to inactivate bovine ␣-L-iduronidase. 33 …”

Inhibition of Glycosaminoglycan Synthesis Using Rhodamine B in a Mouse Model of Mucopolysaccharidosis Type IIIA

“…Both methods will result in an overall reduction in the lysosomal burden of gag. Pathology in MPS has been shown to correlate to the level of gag storage (29); thus, any treatment that reduces lysosomal storage is likely to have a positive patient outcome.…”

Lysosomal storage diseases: Diagnostic confirmation and management of presymptomatic individuals

“…Evidently, as little as 0.4% of normal enzyme activity is sufficient to produce a mild phenotype. 224 Enzymatic activity alone is unreliable for prediction of phenotype because some MPS I-H patient fibroblasts had more enzyme activity than those from MPS I-HS patients; similarly, there were MPS I-HS cell lines with more activity than MPS I-S cells. 224,225 Enzymatic analysis is also insufficient for carrier testing because of overlap in activity between normal individuals and heterozygotes.…”