Analysis of N-acetylgalactosamine-4-sulfatase protein and kinetics in mucopolysaccharidosis type VI patients.

A sensitive and specific, monoclonal antibody-based immunoquantification assay has facilitated determination of the N-acetylgalactosamine-4-sulfatase (4-sulfatase) protein content in cultured fibroblasts from normal controls and mucopolysaccharidosis type VI (MPS VI) patients. The assay enabled the quantification of 4-sulfatase protein by using a panel of seven monoclonal antibodies and has shown that fibroblasts from 16 MPS VI patients contained less than or equal to 5% of the level determined for normal controls. Fibroblasts from the most severely affected patients contained the lowest levels of 4-sulfatase protein, usually with few epitopes detected, while fibroblasts from mildly affected patients had higher levels of 4-sulfatase protein, with all seven epitopes detected. The pattern of epitope expression is proposed to reflect the conformational changes in the 4-sulfatase protein that arise from different mutations in the 4-sulfatase gene. Immunoquantification in combination with a specific and highly sensitive 4-sulfated trisaccharide-based assay of enzyme activity in these MPS VI patient fibroblasts enabled the determination of residual 4-sulfatase catalytic efficiency (kcat/Km). The capacity of fibroblasts to degrade substrate (catalytic capacity) was calculated as the product of 4-sulfatase catalytic efficiency and the content of 4-sulfatase in fibroblasts. One patient, 2357, with no clinical signs of MPS VI but with reduced 4-sulfatase activity and protein (both 5% of normal) and dermatansulfaturia, had 5% of normal catalytic capacity. The other 15 MPS VI patient fibroblasts had 0%-1.4% of the catalytic capacity of fibroblasts from normal controls and were representative of the spectrum of MPS VI clinical phenotypes, from severe to mild.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification, expression, and biochemical characterization of N-acetylgalactosamine-4-sulfatase mutations and relationship with clinical phenotype in MPS-VI patients.

Maroteaux-Lamy syndrome, or mucopolysaccharidosis type VI (MPS-VI), is a lysosomal storage disorder characterized by the defective degradation of dermatan sulfate due to the deficiency of N-acetylgalactosamine-4-sulfatase (4S). The clinical severity of MPS-VI ranges in a continuum from mildly affected to severely affected patients. Mutations in MPS-VI patient samples were identified by chemical cleavage and direct DNA sequencing of PCR products derived from patient cDNA. Five amino acid substitutions were identified (T92M, R95Q, Y210C, H393P, and L498P), individually introduced into the wild-type 4S cDNA by site-directed in vitro mutagenesis, and transfected into Chinese hamster ovary cells. Three of the five mutations (R95Q, Y210C, and H393P) were observed in >1 of 25 unrelated MPS-VI patients; however, the mutations were not found in 20 control individuals. The residual 4S activity and protein (biochemical phenotype) were determined for each mutant in order to confirm their identity as mutations and to dissect the contribution of each mutant allele to the overall clinical phenotype of the patient. For each patient, the combined biochemical phenotypes of the two 4S mutant alleles demonstrated a good correspondence with the observed clinical phenotype (with the possible exception of a patient who was a compound heterozygote for T92M and L498P). This preliminary correspondence between genotype and the phenotype in MPS-VI may, with further refinement, contribute to the assessment of therapeutic approaches for MPS-VI patients.     

Mucopolysaccharidosis type VI: identification of three mutations in the arylsulfatase B gene of patients with the severe and mild phenotypes provides molecular evidence for genetic heterogeneity.

Mucopolysaccharidosis type VI (MPS VI; Maroteaux-Lamy disease) results from the deficient activity of the lysosomal enzyme, arylsulfatase B (ASB; N-acetylgalactosamine-4-sulfatase E.C.3.1.6.1). The enzymatic defect leads to the accumulation of the glycosaminoglycan, dermatan sulfate, primarily in connective tissue and reticuloendothelial cell lysosomes. Although MPS VI patients have normal intelligence and no neurologic abnormalities, the disease is clinically heterogeneous: severely affected individuals expire in childhood or early adolescence while those with the mild or intermediate phenotypes have a slower, milder disease course and a longer life span. The recent isolation of the full-length cDNA-encoding human ASB permitted an investigation of the molecular lesions underlying the phenotypic heterogeneity in MPS VI. The ASB cDNA-coding sequences were determined from two unrelated MPS VI patients with the severe (proband 1) and mild (proband 2) phenotypes. These patients had about 2% and 7% of normal ASB activity in cultured fibroblasts, respectively. Proband 1 was homoallelic for a T-to-C transition in nucleotide (nt) 349, which predicted a cysteine-to-arginine substitution in the ASB polypeptide at residue 117 (C117R). Proband 2 was heteroallelic, having a T-to-C transition in nt 707, which predicted a leucine-to-proline replacement at ASB residue 236 (L236P), and having a G-to-A transition in nt 1214, which predicted a cysteine-to-tyrosine substitution at ASB residue 405 (C405Y). These mutations did not occur in three other unrelated MPS VI patients or in 120 ASB alleles from normal individuals, indicating that they were not polymorphisms. The identification of these three ASB mutations documents the first evidence of molecular heterogeneity in MPS VI and provides an initial basis for genotype/phenotype correlations in this lysosomal storage disease.     

Advantages of using same species enzyme for replacement therapy in a feline model of mucopolysaccharidosis type VI

In a feline model of mucopolysaccharidosis type VI (MPS VI), recombinant feline N-acetylgalactosamine-4-sulfatase (rf4S) administered at a dose of 1 mg/kg of body weight, altered the clinical course of the disease in two affected cats treated from birth. After 170 days of therapy, both cats were physically indistinguishable from normal cats with the exception of mild corneal clouding. Feline N-acetylgalactosamine-4-sulfatase was effective in reducing urinary glycosaminoglycan levels and lysosomal storage in all cell types examined except for corneal keratocytes and cartilage chondrocytes. In addition, skeletal pathology was nearly normalized as assessed by radiographic evidence and bone morphometric analysis. Comparison of results with a previous study in which recombinant human 4S (rh4S) was used at an equivalent dose and one 5 times higher indicated that rf4S had a more pronounced effect on reducing pathology than the same dose of rh4S, and in some instances such as bone pathology and lysosomal storage in aorta smooth muscle cells, it was as good as, or better than, the higher dose of rh4S. We conclude that in the feline MPS VI model the use of native or same species enzyme for enzyme replacement therapy has significant benefits.     

Hurler syndrome: a patient with abnormally high levels of alpha-L-iduronidase protein.

Mucopolysaccharidosis type I (MPS I: McKusick 25280) is a clinically heterogenous lysosomal storage disorder which is caused by a variable deficiency in alpha-L-iduronidase activity (alpha-L-iduronide iduronohydrolase, EC 3.2.1.76). Cultured fibroblasts from an MPS I patient (cell line 2827) with a severe clinical phenotype (Hurler syndrome) have been characterized using immunochemical and biochemical techniques. Using a specific immunoquantification assay, we have demonstrated that cell line 2827 had an alpha-L-iduronidase protein content (189 ng/mg of extracted cell protein) at least six times greater than the mean level found in normal control fibroblasts (30 ng/mg of extracted cell protein). This was the only MPS I cell line, from a group of 23 MPS I patients, that contained greater than 7% of the mean level of alpha-L-iduronidase protein detected in normal controls. Cell line 2827 had very low alpha-L-iduronidase activity toward the fluorogenic substrate 4-methylumbelliferyl-alpha-L-iduronide, and a radiolabeled disaccharide substrate derived from heparin. Maturation studies of alpha-L-iduronidase in cell line 2827 showed apparently normal levels of alpha-L-iduronidase synthesis with delayed processing to the mature form. Subcellular fractionation experiments demonstrated alpha-L-iduronidase protein in lysosomal-enriched fractions isolated from cell line 2827, suggesting a normal cell distribution and supporting the proposed delayed processing. It is proposed that the MPS I patient described has an alpha-L-iduronidase gene mutation which affects both the active site and post-translational processing of the enzyme. This mutation must be structurally conservative because it does not result in instability either during maturation or in the lysosome.     

A single point mutation in the low-density lipoprotein receptor switches the degradation of its mature protein from the proteasome to the lysosome

Pathogenic mutations in the low-density lipoprotein receptor prevent cholesterol uptake and cause familial hypercholesterolemia. In comparison to the biogenesis and endocytic trafficking of this receptor and some of its mutants, their degradation mechanisms are not well understood. Therefore, to gain some insights into this aspect, we analyzed the effects of proteasomal and lysosomal inhibitors on the levels of the wild type low-density lipoprotein receptor and a mutant form, C358Y, which was prevalent in a sample of Spanish familial hypercholesterolemia patients. In transfected cells, the mutant C358Y exhibited lower activity than the wild type receptor, as well as retarded post-translational processing of its precursor to the mature form. Interestingly, about 30% of the mutant precursor was degraded by a lysosomal pathway. Moreover, its mature form was more rapidly degraded than the wild type receptor (half lives of 5.3 and 10.9 h, respectively) and its degradation was exclusively dependent on a lysosomal pathway. In contrast, the mature form of the wild type receptor was mainly degraded by proteasomes and, to a minor extent (30%), by lysosomes. We conclude that a single mutation in the low-density lipoprotein receptor switches the degradation of the mature receptor from a proteasomal to a lysosomal pathway which degrades the protein at a faster rate. This suggests cooperation of proteasomes and lysosomes in the degradation of the low-density lipoprotein receptor and adds an intriguing new aspect to our understanding of receptor-mediated endocytosis.     

Human N-acetylgalactosamine-4-sulphatase biosynthesis and maturation in normal, Maroteaux-Lamy and multiple-sulphatase-deficient fibroblasts.

The biosynthesis and maturation of N-acetylgalactosamine-4-sulphatase (4-sulphatase) was studied in normal fibroblasts and in fibroblasts from patients with either mucopolysaccharidosis type VI (MPS VI; Maroteaux-Lamy syndrome) or multiple sulphatase deficiency (MSD). Fibroblasts were incubated in culture medium containing [3H]leucine or [35S]methionine, and radiolabelled 4-sulphatase was isolated by immunoaffinity chromatography using 4-sulphatase-specific monoclonal antibodies. In normal fibroblasts a precursor of 66 kDa, detected intracellularly after 3 h and in NH4Cl-induced secretions, was processed intracellularly, within an additional 3 h, to a polypeptide of 57 kDa composed of disulphide-linked polypeptides of 43 kDa and 8 kDa. All fibroblast lines obtained from MPS VI patients, exhibiting clinical characteristics ranging from no clearly recognized symptoms to the severe classical phenotype, incorporated radioactivity into immune-purified 4-sulphatase at a rate less than 10% of that seen in normal fibroblasts. Maturation of the residual 4-sulphatase showed, variously, features which may be indicative of delayed intracellular transport, decreased intracellular stability, failure of lysosomal targetting or resistance to enzyme processing. Although some features of the residual enzyme synthesis and maturation were consistent with the patient's clinical phenotype, this was infrequent. The maturation of 4-sulphatase in fibroblasts from MSD patients was indistinguishable from that in normal fibroblasts, and the half-life of 4-sulphatase in these fibroblasts, determined after a 24 h pulse and prolonged chase, was only slightly less than that in normal fibroblasts.     

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.     

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.     

Glycosidase active site mutations in human alpha-L-iduronidase

Mucopolysaccharidosis type I (MPS I; McKusick 25280) results from a deficiency in alpha-L-iduronidase activity. Using a bioinformatics approach, we have previously predicted the putative acid/base catalyst and nucleophile residues in the active site of this human lysosomal glycosidase to be Glu182 and Glu299, respectively. To obtain experimental evidence supporting these predictions, wild-type alpha-L-iduronidase and site-directed mutants E182A and E299A were individually expressed in Chinese hamster ovary-K1 cell lines. We have compared the synthesis, processing, and catalytic properties of the two mutant proteins with wild-type human alpha-L-iduronidase. Both E182A and E299A transfected cells produced catalytically inactive human alpha-L-iduronidase protein at levels comparable to the wild-type control. The E182A protein was synthesized, processed, targeted to the lysosome, and secreted in a similar fashion to wild-type alpha-L-iduronidase. The E299A mutant protein was also synthesized and secreted similarly to the wild-type enzyme, but there were alterations in its rate of traffic and proteolytic processing. These data indicate that the enzymatic inactivity of the E182A and E299A mutants is not due to problems of synthesis/folding, but to the removal of key catalytic residues. In addition, we have identified a MPS I patient with an E182K mutant allele. The E182K mutant protein was expressed in CHO-K1 cells and also found to be enzymatically inactive. Together, these results support the predicted role of E182 and E299 in the catalytic mechanism of alpha-L-iduronidase and we propose that the mutation of either of these residues would contribute to a very severe clinical phenotype in a MPS I patient.     

Identification of a novel missense mutation in Brazilian patient with a severe form of mucopolysaccharidosis type IVA

Mucopolysaccharidosis type IVA (MPS IVA) or Morquio syndrome type A is an autosomal recessive disease caused by deficiency of the lysosomal enzyme N-acetylgalactosamine-6-sulfatase (GALNS). We report molecular characterization of a patient who presents the new missense mutation p.C165Y in homozygosis. Bioinformatics analysis predicted this mutation as being probably pathogenic. To evaluate the possibility that this alteration was a polymorphism we tested 100 alleles and all the results were negative. These findings together with the observation that this alteration is not present in controls, suggest that it is a disease-causing mutation, which was correlated with the severe phenotype observed in our patient. We conclude that molecular analysis of the GALNS gene, in addition to enzyme assays, is important for diagnosis and contributes to the better understanding of the relationship between genotype and phenotype, which is important as enzyme replacement therapy (ERT) will soon become available and treatment decisions will have to be take in such cases.     

ARSB gene variants causing Mucopolysaccharidosis VI in Miniature Pinscher and Miniature Schnauzer dogs

Mucopolysaccharidosis (MPS) VI is a lysosomal storage disease caused by a deficiency of N-acetylgalactosamine-4-sulfatase, also called arylsulfatase B (ARSB, EC 3.1.6.12). Dogs with MPS VI show progressive predominantly oculoskeletal signs homologous to those in human and feline patients. We report herein two pathogenic ARSB gene variants in Miniature Pinscher and Miniature Schnauzer dogs with MPS VI and a genotyping survey in these breeds. All exons and adjacent regions of the ARSB gene were sequenced from three affected Miniature Pinschers and three affected Miniature Schnauzers. Allelic discrimination assays were used for genotyping. A missense variant (NM_001048133.1:c.910G>A) was found in exon 5 of MPS VI-affected Miniature Pinschers that is predicted to result in a deleterious amino acid substitution of a highly conserved glycine to arginine (NP_001041598.1:p.Gly304Arg). In MPS VI-affected Miniature Schnauzers, a 56 bp deletion (NM_001048133.1:c.-24_32del) was found at the junction of exon 1 and its upstream region, predicting no enzyme synthesis. All clinically affected Miniature Pinschers and Miniature Schnauzers were homozygous for the respective variants, and screened healthy dogs in each breed were either heterozygous or homozygous for the wt allele. Whereas the Miniature Pinscher variant seemed to occur commonly (0.133 allele frequency), the Miniature Schnauzer variant was presumed to be rare. In conclusion, two breed-specific pathogenic ARSB gene variants were identified in Miniature Pinscher and Miniature Schnauzer dogs with MPS VI, allowing for genotyping and informed breeding to prevent the production of affected offspring.     

Mucopolysaccharidosis VI (Maroteaux-Lamy syndrome): six unique arylsulfatase B gene alleles causing variable disease phenotypes.

Mucopolysaccharidosis type VI, or Maroteaux-Lamy syndrome, is a lysosomal storage disorder caused by a deficiency of the enzyme arylsulfatase B (ASB), also known as N-acetylgalactosamine-4-sulfatase. Multiple clinical phenotypes of this autosomal recessively inherited disease have been described. Recent isolation and characterization of the human ASB gene facilitated the analysis of molecular defects underlying the different phenotypes. Conditions for PCR amplification of the entire open reading frame from genomic DNA and for subsequent direct automated DNA sequencing of the resulting DNA fragments were established. Besides two polymorphisms described elsewhere that cause methionine-for-valine substitutions in the arylsulfatase B gene, six new mutations in six patients were detected: four point mutations resulting in amino acid substitutions, a 1-bp deletion, and a 1-bp insertion. The point mutations were two G-to-A and two T-to-C transitions. The G-to-A transitions cause an arginine-for-glycine substitution at residue 144 in a homoallelic patient with a severe disease phenotype and a tyrosine-for-cysteine substitution at residue 521 in a potentially heteroallelic patient with the severe form of the disease. The T-to-C transitions cause an arginine-for-cysteine substitution at amino acid residue 192 in a homoallelic patient with mild symptoms and a proline-for-leucine substitution at amino acid 321 in a homoallelic patient with the intermediate form. The insertion between nucleotides T1284 and G1285 resulted in a loss of the 100 C-terminal amino acids of the wild-type protein and in the deletion of nucleotide C1577 in a 39-amino-acid C-terminal extension of the ASB polypeptide. Both mutations were detected in homoallelic patients with the severe form of the disease. Expression of mutant cDNAs encoding the four amino acid substitutions and the deletion resulted in severe reduction of both ASB protein levels and arylsulfatase enzyme activity in comparison with a wild-type control. The six mutations described in the present study were unique among 25 unrelated mucopolysaccharidosis VI patients, suggesting a broad molecular heterogeneity of the Maroteaux-Lamy syndrome.     

The Structure of Human GALNS Reveals the Molecular Basis for Mucopolysaccharidosis IV A

Lysosomal enzymes catalyze the breakdown of macromolecules in the cell. In humans, loss of activity of a lysosomal enzyme leads to an inherited metabolic defect known as a lysosomal storage disorder. The human lysosomal enzyme galactosamine-6-sulfatase (GALNS, also known as N-acetylgalactosamine-6-sulfatase and GalN6S; E.C. 3.1.6.4) is deficient in patients with the lysosomal storage disease mucopolysaccharidosis IV A (also known as MPS IV A and Morquio A). Here, we report the three-dimensional structure of human GALNS, determined by X-ray crystallography at 2.2 Å resolution. The structure reveals a catalytic gem diol nucleophile derived from modification of a cysteine side chain. The active site of GALNS is a large, positively charged trench suitable for binding polyanionic substrates such as keratan sulfate and chondroitin-6-sulfate. Enzymatic assays on the insect‐cell-expressed human GALNS indicate activity against synthetic substrates and inhibition by both substrate and product. Mapping 120 MPS IV A missense mutations onto the structure reveals that a majority of mutations affect the hydrophobic core of the structure, indicating that most MPS IV A cases result from misfolding of GALNS. Comparison of the structure of GALNS to paralogous sulfatases shows a wide variety of active‐site geometries in the family but strict conservation of the catalytic machinery. Overall, the structure and the known mutations establish the molecular basis for MPS IV A and for the larger MPS family of diseases.     

Niemann-Pick type C disease: NPC1 mutations associated with severe and mild cellular cholesterol trafficking alterations

Niemann-Pick type C disease (NPC) is a rare neurodegenerative disorder characterised by lysosomal/late endosomal accumulation of endocytosed unesterified cholesterol and delayed induction of cholesterol homeostatic reactions. The large majority of mutations in the NPC1 gene described thus far have been associated with severe cellular cholesterol trafficking impairment (classic biochemical phenotype, present in about 85% of NPC patients). In our population of 13 unrelated NP-C1 patients, among which 12 were of Portuguese extraction, we observed an unusually large proportion of families presenting mild alterations of intracellular cholesterol transport (variant biochemical phenotype), without strict correlation between the biochemical phenotype and the clinical expression of the disease. Mutational studies were carried out to compare molecular lesions associated with severe and mild cholesterol traffic impairment. Levels of NPC1 protein were studied by Western blot in cultured fibroblasts of four patients with homozygous mutant alleles. Ten novel mutations were identified (Q92R, C177Y, R518W, W942C, R978C, A1035V, 2129delA, 3662delT, IVS23+1 G>A and IVS16-82 G>A). The mutational profile appeared to be correlated with the biochemical phenotype. Splicing mutations, I1061T and A1035V, corresponded to "classic" alleles, while three missense mutations, C177Y, R978C and P1007A, could be defined as "variant" alleles. All "variant" mutations described so far appear to be clustered within the cysteine-rich luminal loop between TM 8 and 9, with the remarkable exception of C177Y. The latter mutant allele, at variance with P1007A, was correlated to a decreased level of NPC1 protein and a severe course of the disease, and disclosed a new location for "variant" mutations, the luminal loop located at the N-terminal end of the protein.     

Phenotypic rescue after adeno-associated virus-mediated delivery of 4-sulfatase to the retinal pigment epithelium of feline mucopolysaccharidosis VI

Background

Mucopolysaccharidosis VI (MPS VI), due to recessively inherited 4‐sulfatase (4S) deficiency, results in lysosomal storage of dermatan sulfate in numerous tissues. Retinal involvement is limited to the retinal pigment epithelium (RPE). This study aimed to determine whether recombinant adeno‐associated virus (AAV)‐mediated delivery of 4S would reverse the RPE pathology seen in MPS VI cats.

Methods

AAV.f4S, containing the feline 4S cDNA, was delivered unilaterally to eyes of affected cats by subretinal or intravitreal injection. Contralateral eyes received AAV with the green fluorescent protein (GFP) reporter gene as control. At 2–11 months post‐injection, the cats were sacrificed and the treatment effects were evaluated histologically.

Results

By ophthalmoscopy and histological analyses, GFP was evident as early as 4 weeks and persisted through the latest time point (11 months). Untreated and AAV.GFP‐treated diseased retinas contained massively hypertrophied RPE cells secondary to accumulation of dilated lysosomal inclusions containing dermatan sulfate. MPS VI eyes treated subretinally with AAV.f4S had minimal RPE cell inclusions and, consequently, were not hypertrophied.

Conclusions

AAV‐mediated subretinal delivery of f4S provided correction of the disease phenotype in RPE cells of feline MPS VI, supporting the utility of AAV as a vector for the treatment of RPE‐specific as well as lysosomal storage diseases. Copyright © 2002 John Wiley & Sons, Ltd.

     

Generation of a novel disease model mouse for mucopolysaccharidosis type VI via c. 252T>C human ARSB mutation knock-in

Mucopolysaccharidosis type VI (MPS VI) is an autosomal recessive lysosomal disorder caused by a mutation in the ARSB gene, which encodes arylsulfatase B (ARSB), and is characterized by glycosaminoglycan accumulation. Some pathogenic mutations have been identified in or near the substrate-binding pocket of ARSB, whereas many missense mutations present far from the substrate-binding pocket. Each MPS VI patient shows different severity of clinical symptoms. To understand the relationship between mutation patterns and the severity of MPS VI clinical symptoms, mutations located far from the substrate-binding pocket must be investigated using mutation knock-in mice. Here, I generated a knock-in mouse model of human ARSB Y85H mutation identified in Japanese MPS VI patients using a CRISPR-Cas9-mediated approach. The generated mouse model exhibited phenotypes similar to those of MPS VI patients, including facial features, mucopolysaccharide accumulation, and smaller body size, suggesting that this mouse will be a valuable model for understanding MPS VI pathology.     

alpha-L-iduronidase premature stop codons and potential read-through in mucopolysaccharidosis type I patients

alpha-L-Iduronidase is a glycosyl hydrolase involved in the sequential degradation of the glycosaminoglycans heparan sulphate and dermatan sulphate. A deficiency in alpha-L-iduronidase results in the lysosomal accumulation and urinary secretion of partially degraded glycosaminoglycans and is the cause of the lysosomal storage disorder mucopolysaccharidosis type I (MPS I; Hurler and Scheie syndromes; McKusick 25280). The premature stop codons Q70X and W402X are two of the most common alpha-l-iduronidase gene (IDUA) mutations accounting for up to 70% of MPS I disease alleles in some populations. Here, we have reported a new mutation, making a total of 15 different mutations that can cause premature IDUA stop codons and have investigated the biochemistry of these mutations. Natural stop codon read-through was dependent on the fidelity of the codon when evaluated at Q70X and W402X in CHO-K1 cells, but the three possible stop codons TAA, TAG and TGA, had different effects on mRNA stability and this effect was context dependent. In CHO-K1 cells expressing the Q70X and W402X mutations, the level of gentamicin-enhanced stop codon read-through was slightly less than the increment in activity caused by a lower fidelity stop codon. In this system, gentamicin had more effect on read-through for the TAA and TGA stop codons when compared to the TAG stop codon. In an MPS I patient study, premature TGA stop codons were associated with a slightly attenuated clinical phenotype, when compared to classical Hurler syndrome (e.g. W402X/W402X and Q70X/Q70X genotypes with TAG stop codons). Natural read-through of premature stop codons is a potential explanation for variable clinical phenotype in MPS I patients. Enhanced stop codon read-through is a potential treatment strategy for a large sub-group of MPS I patients.