Expression of five iduronate-2-sulfatase site-directed mutations.

Five point mutations (R88H, R88P, T118I, 959delT, R468Q) previously identified in the iduronate-2-sulfatase (IDS) gene of Italian Hunter patients were expressed in COS cells to evaluate their functional consequence on enzyme activity, processing and intracellular localization. The 88 arginine residue belongs to the CXPSR pentapeptide conserved in all human sulfatases, where cysteine modification to formylglycine is required for enzyme activity. Substitution of arginine with histidine residue resulted in 13.7% residual enzyme activity, with an apparent K(m) value (133 microM) lower than that found for the normal enzyme (327 microM), indicating a higher affinity for the substrate; substitution of arginine with proline resulted in total absence of residual activity, in agreement with the phenotypes observed in patients carrying R88H and R88P mutations. For the four missense mutations, pulse-chase labelling experiments showed an apparently normal maturation; however, subcellular fractionation demonstrated poor transport to lysosomes. Therefore, residues 88, 118 and 468 appear to be not essential for processing but important for IDS conformation.

Maroteaux-lamy syndrome: five novel mutations and their structural localization.

Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI, MPS VI) is an autosomal recessive disorder due to the deficiency of the lysosomal enzyme N-acetylgalactosamine-4-sulfatase (arylsulfatase B, ASB). Mutation analysis in Maroteaux-Lamy syndrome resulted in the identification of approximately 40 molecular defects underlying a great genetic heterogeneity. Here we report five novel mutations in Italian subjects: S65F, P116H, R315Q, Q503X, P531R; each defect was confirmed by restriction enzyme or amplification refractory mutation system (ARMS) analysis. We also performed a three-dimensional (3-D) structure analysis of the alterations identified by us, and of an additional 22 point mutations reported by other groups, in an attempt to draw helpful information about their possible effects on protein conformation.

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), 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.

[Mutation analysis in Hunter patients]. / Analisi di mutazioni in pazienti Hunter.

Mutations of the iduronate-2-sulfatase gene have been identified as responsible of Hunter syndrome or mucopolysaccharidosis type II. About 20% of the patients have deletions of the whole gene or other major structural alterations. The mutations found so far include: 34 missense, 8 nonsense, 11 small deletions from 1 to 3 bp, 2 deletions of 8 pb, 2 insertions of 1 bp and 2 insertions of 14 bp, with most leading to a frameshift and premature chain termination. Also 8 different splice-site mutations leading to insertions or deletions in the mRNA have been tabulated. Knowledge of the primary genetic defect allows insight into genotype-phenotype correlation and allows a better understanding of the structure and function of iduronate-2-sulfatase.

[Mucopolysaccharidosis II (Hunter syndrome): identification of the carrier state of the disease by means of mutation analysis]. / Mucopolisaccaridosi tipo II (sindrome di Hunter): identificazione dello stato di portatrice della malattia mediante analisi delle mutazioni.

Identification of iduronate-2-sulfatase (IDS) gene mutations in patients with mucopolysaccharidosis type II (MPS II, Hunter syndrome) allows fast and reliable carrier detection in females, relatives of the patients. We describe here a study on a Hunter family where, after the identification of the primary genetic defect in the patients, we identified carriers unambiguously among at-risk female relatives and excluded such status in other subjects. This is an example of direct, DNA based diagnosis.

[Atopy, environmental pollution and respiratory function. Study of 1000 children from 2 areas of the city of Naples]. / Atopia, inquinamento ambientale e funzionalità respiratoria. Studio su 1000 bambini di due zone della città di Napoli.

The aim of this study was to observe the chronic respiratory deficiency in childhood caused by two possible motives: allergy and ambiental pollution. We executed aerial samples in two zones of Naples, one (USL 45) with heavy pollution because many factories present and other with slight pollution (USL 40). The authors calculated the PEF (Peak Expiratory Flow at the first second) and in a group of subject also the spirometric values, in two groups of subjects, one of atopic children and in the other of non atopic children. The study showed a greater number of atopic subjects with abnormal PEF in the zone with heavy pollution; in non atopic children the abnormal PEF was greater in the zone with heavy pollution (35.2%) in comparison with slight pollution zone (3%).

Two novel mutations of the arylsulfatase B gene in two Italian patients with severe form of mucopolysaccharidosis. Mutations in brief no. 127. Online.

Mucopolysaccharidosis type VI (MPS VI) or Maroteaux-Lamy syndrome, is a autosomal recessive disorder, due to the deficiency of the lysosomal enzyme N-acetylgalactosamine-4-sulfatase (arylsufatase B, ASB: EC 3.1.6.12). Three classical forms of the disease have been differentiated: severe, intermediate, mild. Mutational analysis of the ASB gene resulted in the identification of 30 ASB mutant alleles, each of which was found to be unique among unrelated patients, demonstrating a broad molecular heterogeneity of the disease. In this communication we present two novel mutant alleles in two severely affected subjects. Both alterations, the missense mutation G302R and the nonsense Q456X, were found in homozygosity and were confirmed by amplification refractory mutation system (ARMS) or restriction analysis. The missense G302R mutation concerns an amino acid which may be of special importance to the polypeptide, since 302 position is completely conserved in all the eukaryotic sulfatases aligned so far; the nonsense mutation Q456X leads to the translation of a putative mutant ASB protein lacking the last 78 amino acids with a loss of the 8 kD mature polypeptide, one of the two peptides generated by intralysosomal proteolytic processing of the 64kD precursor.

Identification of molecular defects in Italian Sanfilippo A patients including 13 novel mutations.

Sanfilippo syndrome type A or mucopolysaccharidosis IIIA (MPS IIIA) is a lysosomal storage disorder caused by the deficiency of the enzyme heparin sulfamidase (EC 3.10.1.1), required for the degradation of the mucopolysaccharide heparan sulfate. Patients develop central nervous system degeneration resulting in progressive dementia, developmental delay, hyperactivity, and aggressive behaviour; subjects may present a wide spectrum of clinical severity. Here, we report the results from molecular analysis of 24 Italian MPS IIIA patients diagnosed over the last 15 years in our laboratory. Altogether, we were able to characterize 38 out of the 48 (79%) pathogenic alleles. We identified 16 molecular defects, 13 novel. The majority of alterations were missense mutations: on exon two (Y40N; A44T; S66W; R74C), on exon four (G122R; P128L; L146P; R150Q), on exon five (D179N; R182C), on exon six (P227R) and on exon eight (E369K; R377C). Single base pair deletions: on exon two (A52nt-1) and on exon eight (T360nt-1) and one base pair insertion on exon eight (V361nt+1) were also identified. Restriction enzyme or ARMS analyses were used to confirm each alteration. S66W represents the most common alteration in our patients population accounting for 33% of the total alleles. Interestingly, all six patients from Sardinia present this mutation, and five of them are homozygous for this change, suggesting that these subjects may have been derived from a common founder.

Detection of four novel mutations in the iduronate-2-sulfatase gene. Mutations in brief no. 123. Online.

Hunter disease (mucopolysaccharidosis type II or MPS II) is an X-linked recessive disorder caused by the deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS) (E.C.3.1.6.13.) involved in the catabolism of mucopolysaccharides dermatan sulfate and heparan sulfate. A large variety of alterations have been detected at the IDS locus. We report here the identification, in 7 unrelated Italian patients, of IDS gene mutations, four of which are novel and have been confirmed by amplification refractory system (ARMS) or restriction analysis. Our findings include: the missense mutation P86L found in a severe phenotype, the splicing mutation G374G and the nonsense mutation W475X, both associated with mild phenotypes. The four novel mutations were: the missense mutations R88P and R88H, associated with severe phenotypes, concerning a position found to be a mutational "hot-spot" for the IDS gene due to a mutation-prone CpG dinucleotide; mutations T1181 and P266H, both in mild patients. Interestingly, four of our mutations are located on exon III of IDS gene, confirming the high mutation frequency of this exon. After this manuscript was submitted, Rathman et al (Am. J.Hum.Genet.59,1202,1996) reported a total of 101 mutations including one R88H which is one of the novel mutations in this report.

Molecular genetic characterization and prenatal diagnosis in a family with Hunter disease.

Hunter disease, Mucopolysaccharidosis type II, is an X-linked recessive lysosomal storage disorder caused by a deficiency in iduronate sulfatase activity. We studied at molecular level a Neapolitan family with the disease. We report, in patient, the delta 139 mutation on the third exon of the gene, on female family members, the DNA analysis that allowed to assess or exclude their carrier status and on fetal DNA from a pregnancy of patient's mother, a prenatal diagnosis that resulted negative.