Correction of CNS defects in the MPSII mouse model via systemic enzyme replacement therapy.

Mucopolysaccharidosis type II (MPSII), or Hunter syndrome, is a devastating disorder associated with a shortened life expectancy. Patients affected by MPSII have a variety of symptoms that affect all organs of the body and may include progressive cognitive impairment. MPSII is due to inactivity of the enzyme iduronate-2-sulfatase (IDS), which results in the accumulation of storage material in the lysosomes, such as dermatan and heparan sulfates, with consequent cell degeneration in all tissues including, in the severe phenotype, neurodegeneration in the central nervous system (CNS). To date, the only treatment available is systemic infusion of IDS, which ameliorates exclusively certain visceral defects. Therefore, it is important to simultaneously treat the visceral and CNS defects of the MPSII patients. Here, we have developed enzyme replacement therapy (ERT) protocols in a mouse model that allow the IDS to reach the brain, with the substantial correction of the CNS phenotype and of the neurobehavioral features. Treatments were beneficial even in adult and old MPSII mice, using relatively low doses of infused IDS over long intervals. This study demonstrates that CNS defects of MPSII mice can be treated by systemic ERT, providing the potential for development of an effective treatment for MPSII patients.

IDS crossing of the blood-brain barrier corrects CNS defects in MPSII mice.

Mucopolysaccharidosis type II (MPSII), or Hunter syndrome, arises from a deficiency in iduronate 2-sulfatase (IDS), and it is characterized by progressive somatic and neurological involvement. The MPSII mouse model reproduces the features of MPSII patients. Systemic administration of the AAV2/5CMV-hIDS vector in MPSII mouse pups results in the full correction of glycosaminoglycan (GAG) accumulation in visceral organs and in the rescue of the defects and GAG accumulation in the central nervous system (CNS). Remarkably, in treated MPSII animals, this CNS correction arises from the crossing of the blood-brain barrier by the IDS enzyme itself, not from the brain transduction. Thus, we show here that early treatment of MPSII mice with one systemic injection of AAV2/5CMV-hIDS results in prolonged and high levels of circulating IDS that can efficiently and simultaneously rescue both visceral and CNS defects for up to 18 months after therapy.

A novel fluorometric enzyme analysis method for Hunter syndrome using dried blood spots.

Mucopolysaccharidosis type II (MPS II) or Hunter syndrome is a lysosomal storage disease caused by deficiency of iduronate-2-sulfatase (IDS). A convenient single-step fluorometric microplate enzyme assay has been developed and validated for clinical diagnosis of MPS II using dried blood spots (DBS). The assay compared well with a recently reported digital microfluidic method, from which it was adapted. Results show that this DBS assay is robust and reproducible using both technologies.

Screening patients referred to a metabolic clinic for lysosomal storage disorders.

BACKGROUND: Lysosomal protein profiling is being developed as a high throughput method to screen populations for lysosomal storage disorders (LSD). DESIGN: 1415 blood spots from patients referred to a metabolic clinic for LSD were screened using a single multiplex assay for 14 proteins in a dried blood spot. RESULTS: All patients with Pompe disease, metachromatic leukodystrophy, and mucopolysaccharidosis (MPS) type I, IIIA, IIIB and VI were identified by reduced lysosomal protein. Five samples were identified as possible pseudo-arylsulfatase A deficiency; four were confirmed. One multiple sulfatase deficiency patient was identified with multiple reduced sulfatase proteins. There were 10 MPS II patients identified with reduced iduronate 2-sulfatase, and one MPS II patient with iduronate 2-sulfatase in the unaffected range. For Fabry disease, 10 male patients were identified with reduced α-galactosidase and 2/6 female Fabry heterozygotes returned α-galactosidase concentrations in the male Fabry range. All 10 mucolipidosis II/III patients were identified with multiple raised proteins. For 79 blood spots with chitotriosidase >3.4mg/l, a follow-up one-plex chitotriosidase assay enabled identification of all nine Gaucher patients. CONCLUSION: This study demonstrates the sensitivity and specificity of this technology to accurately identify 99% of LSD patients, with the exception of one MPS II false negative.

Prevalence and characterization of cardiac involvement in Hunter syndrome.

OBJECTIVES: To assess the prevalence of cardiovascular signs and symptoms in a large group of patients with Hunter syndrome, an X-linked metabolic disorder caused by a deficiency of the lysosomal enzyme iduronate-2-sulfatase. STUDY DESIGN: The Hunter Outcome Survey was established to characterize the natural history of Hunter syndrome and to assess the response to enzyme replacement therapy. Echocardiographic and electrocardiographic examination results were available for 102 patients who were enzyme replacement therapy-naïve in the Hunter Outcome Survey (median age at examination, approximately 8 years) as of Jan 23, 2009. RESULTS: The most common cardiovascular finding was valve disease, which was present in 63% of patients. Left ventricular hypertrophy (defined as left ventricular mass indexed to height(2.7) ≥50 g/m(2.7)) was found in 48% of patients <18 years old. Elevated blood pressure (defined as a Z score ≥2 for systolic blood pressure or diastolic blood pressure) was present in 25% of patients <18 years old. Other findings included abnormal heart frequency (7%), arrhythmia (5%), and congestive heart failure (6%). CONCLUSIONS: Treating physicians should be aware of the early emergence of cardiovascular manifestations in patients with Hunter syndrome so that appropriate treatment can be initiated.

Safety evaluation of chronic intrathecal administration of idursulfase-IT in cynomolgus monkeys.

Recombinant human idursulfase, an intravenous enzyme replacement therapy indicated for treatment of somatic symptoms of mucopolysaccharidosis II (Hunter syndrome), is anticipated to have minimal benefit for the cognitive impairment associated with the severe phenotype. Because intrathecal (IT) administration of enzyme replacement therapy for other lysosomal enzyme disorders has shown efficacy in animal models, an IT formulation of idursulfase (idursulfase-IT) and a drug-delivery device (subcutaneous port connected to a lumbar IT catheter) were developed for treating central nervous system (CNS) involvement. In this chronic safety study, cynomolgus monkeys were dosed weekly with IV idursulfase (0.5 mg/kg) and every four weeks with idursulfase-IT (3, 30, and 100 mg) for six months, with device and vehicle controls treated similarly (n = 6, all groups). Necropsies were performed twenty-four hours post-final IT dose or after a recovery period (four weeks post-final dose in vehicle-control, 3 mg, and 100 mg IT groups: n = 6). No clinical signs or gross central nervous system lesions were observed. Compared to controls, more pronounced cellular infiltrates in brain and spinal cord meninges were noted, which largely resolved after the recovery period. Central nervous sytem levels of idursulfase-IT were dose dependent, as determined by enzyme activity and immunohistochemistry. The no-observed-adverse-effect level of idursulfase-IT was 100 mg.

Enzyme replacement therapy by fibroblast transplantation: long-term biochemical study in three cases of Hunter's syndrome.

We have assessed the effectiveness of transplanted histocompatible fibroblasts as a long-lived source of lysosomal enzymes for replacement therapy in three patients with Hunter's syndrome, over periods ranging from 2.5 to 3.75 yr. The level of Hunter corrective factor excreted by all three patients increased after transplantation, as did the activity of alpha-L-idurono-2-sulfate sulfatase in serum, when measured directly with a radioactive disulfated disaccharide substrate. Sulfatase activity was also raised in leukocyte homogenates from the two patients that we were able to assess. These increases in enzyme activity were accompanied by corresponding increases in catabolism of heparan and dermatan sulfates, as shown by (a) a decrease in sulfate:uronic ratios of urinary oligosaccharides, (b) an increase in iduronic acid monosaccharide, and (c) a normalization of Bio-Gel P-2 gel filtration profiles. Both the increase in enzyme activity and increased catabolism were maintained during the period of study and were not affected by either a gradual decrease or total withdrawal of immunosuppressive therapy.

Prevention of Neurocognitive Deficiency in Mucopolysaccharidosis Type II Mice by Central Nervous System-Directed, AAV9-Mediated Iduronate Sulfatase Gene Transfer.

Mucopolysaccharidosis type II (MPS II; Hunter syndrome) is a rare X-linked recessive lysosomal disorder caused by defective iduronate-2-sulfatase (IDS), resulting in accumulation of heparan sulfate and dermatan sulfate glycosaminoglycans (GAGs). Enzyme replacement is the only Food and Drug Administration-approved therapy available for MPS II, but it is expensive and does not improve neurologic outcomes in MPS II patients. This study evaluated the effectiveness of adeno-associated virus (AAV) vector encoding human IDS delivered intracerebroventricularly in a murine model of MPS II. Supraphysiological levels of IDS were observed in the circulation (160-fold higher than wild type) for at least 28 weeks post injection and in most tested peripheral organs (up to 270-fold) at 10 months post injection. In contrast, only low levels of IDS were observed (7-40% of wild type) in all areas of the brain. Sustained IDS expression had a profound effect on normalization of GAG in all tested tissues and on prevention of hepatomegaly. Additionally, sustained IDS expression in the central nervous system (CNS) had a prominent effect in preventing neurocognitive deficit in MPS II mice treated at 2 months of age. This study demonstrates that CNS-directed, AAV9 mediated gene transfer is a potentially effective treatment for Hunter syndrome, as well as other monogenic disorders with neurologic involvement.

Analysis of normal and mutant iduronate-2-sulphatase conformation.

Mammalian sulphatases (EC 3.1.6) are a family of enzymes that have a high degree of similarity in amino acid sequence, structure and catalytic mechanism. IDS (iduronate-2-sulphatase; EC 3.1.6.13) is a lysosomal exo-sulphatase that belongs to this protein family and is involved in the degradation of the glycosaminoglycans heparan sulphate and dermatan sulphate. An IDS deficiency causes the lysosomal storage disorder MPS II (mucopolysaccharidosis type II). To examine the structural alterations in heat-denatured and mutant IDS, a panel of four monoclonal antibodies was raised to the denatured protein and used as probes of protein conformation. The linear sequence epitope reactivity of a polyclonal antibody raised against the native protein and the monoclonal antibodies were defined and mapped to distinct regions on the IDS protein. The antigenicity of native IDS was higher in regions without glycosylation, but reactivity was not restricted to protein surface epitopes. One monoclonal epitope was relatively surface accessible and in close proximity to an N-linked glycosylation site, while three others required additional thermal energy to expose the epitopes. The monoclonal antibodies demonstrated the capacity to differentiate progressive structural changes in IDS and could be used to characterize the severity of MPS type II in patients based on variable denatured microstates.

Fragile X mental retardation and the iduronate sulphatase locus: testing Laird's model of fra(X) inheritance.

Fragile X [fra(X)] mental retardation syndrome is the most frequent familial cause of mental handicap. The clinical phenotype is associated with a rare fragile site at Xq27.3. The mutation underlying the disorder, an insertion into the FMR-1 gene, has been characterized, but the pathogenesis of the condition is obscure and the pattern of inheritance is still not fully understood. One model of fra(X) pathogenesis was proposed by Laird in 1987, suggesting that the fra(X) mutation acts as a cis-acting, local block to the pre-oogenesis reactivation of the inactivated X chromosome. To test this model, we examined the activity of the F8, F9 and iduronate sulphatase (IDS) loci. The level of IDS in the serum of fra(X) males was found to be very significantly reduced in the fra(X) group when compared to that of control males: this lends support to Laird's model of fra(X) pathogenesis. However, we detected no methylation differences between fra(X) and control samples at the IDS locus, although such changes are known in fra(X) males at sites closer to the fragile site. Thus the mechanism of the reduction in IDS activity has not been identified.

Heterozygote detection in Hunter syndrome.

Iduronate sulfate sulfatase activity was determined in 36 women, relatives of Hunter syndrome patients. The use of serum and lymphocyte extracts for the determination of enzyme levels enabled the detection of 13 out of 15 (86%) obligate heterozygotes and identification of 10 of 21 other relatives as carriers. These methods are relatively simple and can easily be applied for routine examinations of all women at risk of being a Hunter heterozygote. These results permit for the first time meaningful genetic counseling for the families of Hunter patients.

Carrier detection in Hunter syndrome.

We have studied the carrier state of the Hunter syndrome using a series of obligate carriers, females at high genetic risk, and normal control women. Specific odds of a female being a carrier of Hunter syndrome were based on serum levels of iduronate 2-sulphate sulphatase activity. These, together with the prior genetic odds, may be used in calculating the overall odds of a woman being a carrier. Iduronate 2-sulphate sulphatase levels were found to increase significantly with age. Obligate carriers from families of severe cases had significantly lower enzyme levels compared with those from families of mild cases. In contrast, enzyme levels in sera of mild and severe cases were not significantly different. With the accumulation of more data the effect of age of the potential carrier and the severity of the disease may have to be taken into consideration in the risk calculation. Hair-root analysis was more reliable in the detection of carriers than estimation of serum enzyme levels, but some individuals could not be classified with confidence by hair-root analysis alone. Carrier detection was most reliable when hair-root analysis and serum enzyme levels were taken together.

Donor bone marrow from a sibling with inborn error of metabolism for treatment of acute leukaemia - clinical and biochemical consequences in the non-affected recipient.

Bone marrow transplantation (BMT) is increasingly used in an attempt to correct inborn errors of metabolism (IEM). However, little is known about effects of BMT from patients with IEM donating for non-affected recipients. We present data from a 8.5-year-old girl who underwent BMT in second remission for relapsed acute lymphoblastic leukaemia (ALL) at the age of 7 years from her HLA-identical brother who was severely affected by Hunter syndrome (Mucopolysaccharidosis type II, iduronate-2-sulphatase (IDS) deficiency). After BMT not only leukocyte but also plasma activity of IDS was absent. Mixing experiments and immunoadsorption suggest antibody-mediated enzyme inhibition. However, her urinary glycosaminoglycan excretion has not increased post BMT and clinical signs of mucopolysaccharidosis are absent 20 months after BMT. We conclude that patients with white cell enzyme deficiencies and other IEMs do not have to be excluded from bone marrow donation. Antibody production by the graft may occur and be reflected by a marked reduction in plasma enzyme levels but not tissue activity. Similar antibody responses resulting in enzyme inactivation might also affect other enzyme replacement strategies for individuals with IEM.

An improved assay for iduronate 2-sulphate sulphatase in serum and its use in the detection of carriers of the Hunter syndrome.

A more sensitive assay procedure has been developed for the enzyme iduronate 2-sulphate sulphatase which is deficient in the Hunter syndrome. The substrate is the same as previously described by Lim et al. [1], O-(alpha-L-idopyranosyluronic acid 2-sulphate)-(1leads to 4)-2,5 anhydro-D-[3H-1]mannitol 6-sulphate, but, after incubation, it is separated from the product by ion-exchange chromatography on a micro-column of Dowex 1 x 2 (Cl-1) instead of high voltage electrophoresis or ECTEOLA cellulose chromatography. Since the blank correction is then much smaller, a shorter incubation time can be used and conversion of the substrate reduced from approximately 50% down to levels where complications resulting from substrate depletion and product inhibition are minimal. Using whole serum the apparent Km for the substrate is 0.2 mmol/l. With an incubation time of 20 min, sera from heterozygotes exhibited approximately 35% of the normal levels of iduronate 2-sulphate sulphatase (0.11-0.61, mean 0.34 nmol.h-1.mg-1 protein for carriers; 0.24-2.35, mean 0.94 nmol.h-1.mg-1 protein for 37 normal females). Serum analyses can thus be used to supplement those on hair roots in the detection of carriers of the Hunter syndrome.

Mucopolysaccharidoses type II: enzymatic activity and quantitative and qualitative studies of urinary glycosaminoglycans in five patients.

Five patients presenting Hunter's syndrome were biochemically studied. Quantification of urinary glycosaminoglycans (GAGs), electrophoretic characterization and correlation with enzymatic activity in leucocytes were carried out. In all cases, urinary GAGs/creatinine ratio was increased. Electrophoresis revealed the presence of heparan sulfate (HS) and dermatan sulfate (DS) in four cases (80%), but in the remaining patient, only DS was present. In all patients, deficient enzymatic activity was demonstrated. These results show evidences of biochemical differences in this syndrome.

Genetic analysis of 17 children with Hunter syndrome: identification and functional characterization of four novel mutations in the iduronate-2-sulfatase gene.

Mucopolysaccharidosis type II (MPS II) is a rare X-linked disorder caused by alterations in the iduronate-2-sulfatase (IDS) gene. In this study, IDS activity in peripheral mononuclear blood monocytes (PMBCs) was measured with a fluorimetric enzyme assay. Urinary glycosaminoglycans (GAGs) were quantified using a colorimetric assay. All IDS exons and intronic flanks were bidirectionally sequenced. A total of 15 mutations (all exonic region) were found in 17 MPS II patients. In this cohort of MPS II patients, all alterations in the IDS gene were caused by point nucleotide substitutions or small deletions. Mutations p.Arg88His and p.Arg172* occurred twice. All mutations were inherited except for p.Gly489Alafs*7, a germline mutation. We found four new mutations (p.Ser142Phe, p.Arg233Gly, p.Glu430*, and p.Ile360Tyrfs*31). In Epstein-Barr virus (EBV)-immortalized PMBCs derived from the MPS II patients, no IDS protein was detected in case of the p.Ser142Phe and p.Ile360Tyrfs*31 mutants. For p.Arg233Gly and p.Glu430*, we observed a residual expression of IDS. The p.Arg233Gly and p.Glu430* mutants had a residuary enzymatic activity that was lowered by 14.3 and 76-fold, respectively, compared with healthy controls. This observation may help explain the mild disease phenotype in MPS II patients who had these two mutations whereas the p.Ser142Phe and p.Ile360Tyrfs*31 mutations caused the severe disease manifestation.

Hunter syndrome follow-up after 1 year of enzyme-replacement therapy.

Mucopolysaccharidosis II (Hunter syndrome) is a rare x-linked disorder caused by a deficiency in the lysosomal enzyme iduronate-2-sulphatase, leading to an accumulation of the glycosaminoglycans (GAGs) dermatansulphate and heparan sulphate. The consequence of GAGs accumulation is progressive, multiorgan disease. Enzyme-replacement therapy is hypothesised to result in disease stabilisation and improved prognosis. We present a severe case of Hunter syndrome diagnosed at age 2 years and 4 months, who started enzyme-replacement therapy at the age of 3 years and 3 months. We report his evolution after 1 year of treatment. The treatment response was good and there was significant improvement in the quality of life. Owing to the rarity of Hunter syndrome, the multisystem nature and the heterogeneity of disease progression, patient care implies interdisciplinary consultations with a wide range of specialists. The best management can be provided in reference centres for metabolic diseases.

Extension of the molecular analysis to the promoter region of the iduronate 2-sulfatase gene reveals genomic alterations in mucopolysaccharidosis type II patients with normal coding sequence.

Hunter disease or mucopolysaccharidosis type II (MPS II) is an X-linked recessive lysosomal disorder caused by the deficit of the enzyme iduronate-2-sulfatase (IDS), involved in the catabolism of the glycosaminoglycans heparan and dermatan sulfate. Our aim was to search for molecular defects in the promoter region of the IDS gene in patients with previous biochemical diagnosis of MPS II and after we sequenced the whole IDS coding region and the exon/intron boundaries without detecting any pathogenic mutations. Screening of the promoter region of four patients detected in two of them a 178 bp deletion and in the other two a single nucleotide substitution 818 bp upstream of the coding region. The latter had never been described before in MPS II patients and it turned out to be a polymorphism. Our experience suggests that MPS II patients with no mutations detected in the IDS coding region should be screened in the promoter region of the gene. Findings will hopefully help to clarify the relationship between genotype and phenotype and will be useful for the correct molecular diagnosis of Hunter patients and the identification of female carriers, the latter particularly important for genetic counseling.