J. J. Hopwood

Long-term clinical progress in bone marrow transplanted mucopolysaccharidosis type I patients with a defined genotype

SummaryTwo mucopolysaccharidosis type I (MPS-I) patients, subjected to bone marrow transplantation (BMT) more than 10 years ago, have recently had their α-L-iduronidase genotypes defined. Both patients, homozygous for the relatively common W402X mutation, received BMT when they were 14 and 11 months of age, and are now 12 and 14 years old, respectively. Untreated MPS-I patients, homozygous for W402X, have an extremely severe clinical phenotype with rapid clinical deterioration and death before 6 years of age. The 12-year-old patient, with limited mobility, is coping well at school, while the other patient is wheelchair-bound with severe disability in his lower limbs, and attends a school for the physically handicapped. Both patients have less than normal intelligence with slowly continuing losses. A third MPS-I patients, diagnosed at the age of 6 months, was felt, prior to BMT at 14 months, to have a severe phenotype. Twelve years post-BMT, he is ambulatory, albeit with restricted movement, and has normal intelligence. This patient did not have a defined MPS-I genotype and had α-L-iduronidase protein and activity consistent with a less severe outcome than the first two patients. We conclude that BMT has significantly slowed down the clinical regression of the W402X phenotype. We propose that if further gains are to be made, BMT should be performed within the first few months of life. Early diagnosis is therefore essential.

Enzyme replacement therapy for mucopolysaccharidosis VI: long-term cardiac effects of galsulfase (Naglazyme ® ) therapy

Characteristic cardiac valve abnormalities and left ventricular hypertrophy are present in untreated patients with mucopolysaccharidosis type VI (MPS VI). Cardiac ultrasound was performed to investigate these findings in subjects during long-term enzyme replacement therapy (ERT) with recombinant human arylsulfatase B (rhASB, rhN-acetylgalactosamine 4-sulfatase, galsulfase, Naglazyme®). Studies were conducted in 54 subjects before ERT was begun and at specific intervals for up to 96xa0weeks of weekly infusions of rhASB at 1xa0mg/kg during phase 1/2, phase 2, and phase 3 trials of rhASB. At baseline, mitral and aortic valve obstruction was present and was significantly greater in those ≥12xa0years of age. Mild mitral and trace aortic regurgitation were present, the former being significantly greater in those <12xa0years. Left ventricular hypertrophy, with averaged z-scores ranging from 1.6–1.9 SD greater than normal, was present for ages both <12 and ≥12xa0years. After 96xa0weeks of ERT, ventricular septal hypertrophy regressed in those <12xa0years. For those ≥12xa0years, septal hypertrophy was unchanged, and aortic regurgitation increased statistically but not physiologically. Obstructive gradients across mitral and aortic valves remained unchanged. The results suggest that long-term ERT is effective in reducing intraventricular septal hypertrophy and preventing progression of cardiac valve abnormalities when administered to those <12xa0years of age.

Mutations among Italian mucopolysaccharidosis type I patients

A group of 27 Italian patients was screened for α-L-iduronidase mucopolysaccharidosis type I mutations. Mutations were found in 18 patients, with 28 alleles identified. The two most common mutations in northern Europeans (W402X and Q70X) accounted for 11% and 13% of the alleles, respectively. The R89Q mutation, uncommon in Europeans, was found only in one patient, accounting for 1 of 54 alleles (1.9%). The other mutations, P533R, A327P and G51D, accounted for 11%, 5.6% and 9.3% of the total alleles, respectively. Interestingly, the high frequency of the P533R mutation seems to be confined to Sicily and is higher than the 3% reported in a British/Australian study.

Huntington disease-linked locus D4S111 exposed as the alpha-L-iduronidase gene.

Abstractα-l-Iduronidase (IDUA) has been intensively studied due to its causative role in mucopolysaccharidosis type I (Hurler, Scheie and Hurler/Scheie syndromes). The recent cloning of a human IDUA cDNA has resulted in a reevaluation of the chromosomal location of this gene. Previously assigned to chromosome 22, IDUA now has been localized to 4p16.3, the region of chromosome 4 associated with Huntingtons disease (HD). The existence of a battery of cloned DNA, physical map information, and genetic polymorphism data for this region has allowed the rapid fine mapping of IDUA within the terminal cytogenetic band of 4p. IDUA was found to be coincident with D4S111, an anonymous locus displaying a highly informative multiallele DNA polymorphism. This map location, 1.1×106 bp from the telomere, makes IDUA the most distal cloned gene assigned to 4p. However, it falls within a segment of 4p16.3 that has been eliminated from the HD candidate region, excluding a role for IDUA in this disorder.

Hurler syndrome: A patient with abnormally high levels of α-l-iduronidase protein

Abstract Mucopolysaccharidosis type I (MPS I: McKusick 25280) is a clinically heterogenous lysosomal storage disorder which is caused by a variable deficiency in α- l -iduronidase activity (α- 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 α- 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 α- l -iduronidase protein detected in normal controls. Cell line 2827 had very low α- l -iduronidase activity toward the fluorogenic substrate 4-methylumbelliferyl-α- l -iduronide, and a radiolabeled disaccharide substrate derived from heparin. Maturation studies of α- l -iduronidase in cell line 2827 showed apparently normal levels of α- l -iduronidase synthesis with delayed processing to the mature form. Subcellular fractionation experiments demonstrated α- 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 α- 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.

Biochemical characterization of patients and prenatal diagnosis of sialic acid storage disease for three families.

SummaryModifications of the assay method of Aminoff (1961) for the determination of sialic acid levels in urine, amniotic fluid, cultured cell homogenates and tissue homogenates, which reduce the interference from proteins by precipitation and from interfering chromogens by second derivative spectroscopy are described.Biochemical profiles of patients from three families with different clinical forms of sialic acid storage disease were made using data obtained with the new method. A family with two patients with the clinically severe, early-onset form is described, in which a 9-fold elevation of sialic acid can be detected in the skin fibroblasts and a 12-fold elevation in the urine. A patient from the second family is described with very severe clinical features and with 160-fold and 16-fold elevations of sialic acid in the urine and skin fibroblasts respectively. A patient from a third family is described with mild clinical features but with a 160-fold and 6-fold elevation of sialic acid in urine and skin fibroblasts respectively. The data obtained in this study are compared with data obtained in other laboratories on other patients.The method was used to assess the levels of sialic acid present in amniotic cells and chorionic villus cells obtained prenatally from pregnancies in each of the three families. In one case, in which amniotic cells were elevated in sialic acid and were vacuolated, the pregnancy was terminated. Follow-up studies confirmed the diagnosis. Pregnancies from the other two families were assessed to be not affected.

Biochemical profiling to predict disease severity in metachromatic leukodystrophy

Metachromatic leukodystrophy is a neurodegenerative disease that is characterized by a deficiency of arylsulfatase A, resulting in the accumulation of sulfatide and other lipids in the lysosomal network of affected cells. Accumulation of sulfatide in the nervous system leads to severe impairment of neurological function with a fatal outcome. Prognosis is often poor unless treatment is carried out before the onset of clinical symptoms. Pre-symptomatic detection of affected individuals may be possible with the introduction of newborn screening programs. The ability to accurately predict clinical phenotype and rate of disease progression in asymptomatic individuals will be essential to assist selection of the most appropriate treatment strategy. Biochemical profiling, incorporating the determination of residual enzyme protein/activity using immune-based assays, and metabolite profiling using electrospray ionization-tandem mass spectrometry, was performed on urine and cultured skin fibroblasts from a cohort of patients representing the clinical spectrum of metachromatic leukodystrophy and on unaffected controls. Residual enzyme protein/activity in fibroblasts was able to differentiate unaffected controls, arylsulfatase A pseudo-deficient individuals, pseudo-deficient compound heterozygotes and affected patients. Metachromatic leukodystrophy phenotypes were distinguished by quantification of sulfatide and other secondarily altered lipids in urine and skin fibroblasts; this enabled further differentiation of the late-infantile form of the disorder from the juvenile and adult forms. Prediction of the rate of disease progression for metachromatic leukodystrophy requires a combination of information on genotype, residual arylsulfatase A protein and activity and the measurement of sulfatide and other lipids in urine and cultured skin fibroblasts.

Mucopolysaccharidosis type I (Hurler syndrome): linkage disequilibrium indicates the presence of a major allele.

SummaryTwo polymorphisms exist in the α-l-iduronidase (IDUA) gene, the gene that is defective in mucopolysaccharidosis type I (MPS I), viz. aKpnI polymorphism and a variable number of tandem repeats (VNTR) polymorphism with three common alleles. The analysis of allele and haplotype frequencies for these two polymorphisms in the normal population and in MPS I patients revealed the presence of linkage disequilibrium. The frequency of the 2,2 (VNTR,KpnI) allele in MPS I patients was 57% compared with only 37% in the normal population. The implications for the presence of a major MPS I allele and the ability to predict patient phenotype are discussed.

Gene diagnosis and carrier detection in hunter syndrome by the iduronate-2-sulphatase cDNA probe

Hunter disease (McKusick 309900) is an X-chromosomal mucopolysaccharidosis due to deficiency of the lysosomal enzyme iduronate-2-sulphatase (IDS; EC 3.1.6.13). Diagnosis is based on both the typical clinical features of patients and the lack/reduction of IDS activity. Female carriers show no symptoms of the disease. In the past, several different assays were elaborated for measuring enzyme activity in carriers but none of them proved to be suitable for detecting heterozygotes reliably (Zlotogora and Bach 1984)

A specific fluorogenic assay for N-acetylgalactosamine-4-sulphatase activity using immunoadsorption

SummaryA method combining immune capture and enzyme detection by fluorochemistry has been developed for the diagnostic assay of N-acetylgalactosamine-4-sulphatase (4-sulphatase). The procedure uses a monoclonal antibody 4-S 4.1 to immunoadsorb 4-sulphatase specifically from complex protein samples containing other sulphatases, and 4-methylumbelliferyl sulphate to detect captured 4-sulphatase. The assay provides an accurate and simple method for the diagnosis of Maroteaux-Lamy syndrome (Mucopolysaccharidosis type VI).