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

Abstract

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), required for the degradation of heparan sulfate. Molecular defects of 24 Italian 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 mutagenized to correspond to heparan N-sulfatase mutations Y40N, A44T, 166delG, G122R, P128L, L146P, R150Q, D179N, R182C, R206P, P227R, 1040insT, 1093insG, E369K, R377C did not yield active enzyme, demonstrating the deleterious nature of the mutations. Western blot analysis and metabolic labeling experiments 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 enzyme. Western blot, metabolic labeling and immunofluorescence experiments suggested, for mutations 166delG, L146P, 1040insT and 1093insG, an increased degradation of the mutant enzymes.

The Mutation p.Ser298Pro in the sulphamidase gene (SGSH) is associated with a slowly progressive clinical phenotype in mucopolysaccharidosis type IIIA (Sanfilippo A Syndrome)

“…Additionally, there are some other reports describing mutations identified in the sulphamidase gene of patients exhibiting milder clinical symptoms (Table 2). Residual sulphamidase activity between 3 and 11% of normal has been reported in these patients or after expression of mutant sulphamidase cDNAs in vitro [Weber et al, 1998;Beesley et al, 2000;Esposito et al, 2000, Perkins et al, 2001Miyazaki et al, 2002;Di Natale et al, 2003;Muschol et al, 2004;Gabrielli et al, 2005]. In contrast to the individuals described in Table 2, who have seemingly unique genotypes, the p.Ser298Pro mutation appears to be more common (accounting for 9.5% of the patient alleles in this study).…”

“…At present we have identified 13 non-related MPS IIIA patients heterozygous for p.Ser298Pro in Germany, seven in the UK and two in France [Muschol et al; manuscript in [Muschol et al, 2004] 7 p.Arg206Pro p.Arg206Pro n.r. >20 [Gabrielli et al, 2005;Esposito et al, 2000] n.i., not identified; n.r., not reported preparation]. In the MPS IIIA population studied here, the p.Ser298Pro mutation was found in combination with p.Arg74Cys (3/10), p.Arg245His (3/10), p.Gly251Ala (2/10), p.Asp32Glu (1/10), and c. 184-190del (1/10) on the other allele.…”

“…Substitution of Arg74 by a cysteine residue (c.220C>T (p.Arg74Cys)) affects the active site of the enzyme and is assumed to be associated with a severe clinical phenotype. By contrast, the mutation c.318C>A (p.Ser106Arg) is associated with residual enzyme activity and a slowly progressive phenotype [Perkins et al, 1999;Esposito et al, 2000;Muschol et al, 2004]. Recently we described the natural course of MPS IIIA in 71 MPS IIIA patients examining the age of onset, symptoms, diagnosis and progression of the disease [Meyer et al, 2007].…”

A novel missense mutation in lysosomal sulfamidase is the basis of MPS III A in a spontaneous mouse mutant

“…3. Clustal W analysis of human and mouse (mainly CD1/129 SvJ background) sulfamidase sequences with the location of the inactivating point mutation identified here in the mouse (*) and those identified previously in humans (Scott et al, 1995;Blanch et al, 1997;Bunge et al, 1997;Weber et al, 1997;Di Natale et al, 1998;Montfort et al, 1998;Esposito et al, 2000) (Mutant AA). The amino acids proposed as critical for sulfatase activity in N-acetylgalactosamine-4-sulfatase (Bond et al, 1997) are identified by a dot.…”

“…The prevalence of a particular subtype varies with geographical region, MPS III A being the most common subtype in the United Kingdom (Whiteman and Young, 1977) and in the Netherlands (Poorthuis et al, 1999). To date approximately 42 mutations have been described in the sulfamidase gene of MPS III A patients from different geographical regions (Scott et al, 1995;Bhaumik et al, 1999;Bunge et al, 1997;Weber et al, 1997;Di Natale et al, 1998;Montfort et al, 1998;Esposito et al, 2000), and about 50% are missense point mutations.…”