Heparan N-sulfatase gene: two novel mutations and transient expression of 15 defects

Sanfilippo syndrome type A or mucopolysaccharidosis IIIA (MPS IIIA) results from the deficiency of the enzyme heparan N-sulfatase (NS, EC 3.10.1.1), r equired for the degradation of heparan sulfate. Molecular defects of 24 Ita lian MPS IIIA patients were recently reported by our group. We report here two novel mutations: 1040insT and Q365X and the expression studies on 15 of the identified defects. Transient expression of COS cells by cDNA mutageni zed to correspond to heparan N-sulfatase mutations Y40N, A44T, 166delG, G12 2R, P128L, L146P, R150Q, D179N, R182C, R206P, P227R, 1040insT, 1093insG, E3 69K, R377C did not yield active enzyme, demonstrating the deleterious natur e of the mutations. Western blot analysis and metabolic labeling experiment s revealed, for cells transfected with wild-type enzyme, a precursor 62-kDa form and a mature 56-kDa form. Western blot resulted, for 11 mutations, in the presence of both forms, indicating a normal maturation of the mutant e nzyme. Western blot, metabolic labeling and immunofluorescence experiments suggested, for mutations 166delG, L146P, 1040insT and 1093insG, an increase d degradation of the mutant enzymes. (C) 2000 Published by Elsevier Science B.V. All rights reserved.