Enzyme replacement therapy in 12 patients with MPS I-H/S with homozygous p.Leu490Pro mutation.

We describe a cohort of 14 Hurler-Scheie patients homozygous for the p.Leu490Pro missense mutation in the alpha-L-iduronidase gene. Now based in the UK, they are all of Pakistani/Kashmiri descent; 64% were female; 11/14 (79%) had a sibling or cousin with MPS I and the parents are consanguineous in all cases. The median age at diagnosis was 1.8 years (range from antenatal diagnosis to 16.5 years). Twelve were on ERT with recombinant human alpha-L-iduronidase (IDUA; Laronidase, Genzyme) for a median duration of 22.5 months (range 2-71 months) and median age at commencement of ERT was 8.6 years (range 0.4-23.1 years). There was clear improvement in the size of liver and spleen as well as reduction in urine glycosaminoglycans (GAGs). The mean (range) urine GAG levels in mg/mmol creatinine were 63.4 (28.9-105.6), 28.3 (10.9-41.4), 22.8 (12.1-43.1), 15.7 (9.2-24.8) and 16.3 (10.1-21.0) at commencement, 3 months post ERT, 6 months post ERT, 12 months post ERT and 24 months post ERT, respectively. Effects on growth were not clear as there does not seem to be an obvious trend of increase or decrease in height after commencement of ERT and this seems to be the case regardless of the age at which ERT was started.

Biochemical monitoring after haemopoietic stem cell transplant for Hurler syndrome (MPSIH): implications for functional outcome after transplant in metabolic disease.

Hurler Syndrome is corrected by allogeneic BMT by the action of donor enzyme on recipient tissue. In this paper, we describe monitoring of 39 patients transplanted in two centres to determine donor chimerism, enzyme level and residual substrate - expressed as dermatan sulphate to chondroitin sulphate ratio. We show that in fully engrafted recipients, the enzyme level, expressed as mumol/g total protein/h, post-transplant is 24.2 from an unrelated donor and 10.2 from a heterozygote family donor (P<0.0001). There is a tight relationship between mean post-transplant enzyme level and residual substrate - Spearman's rank correlation coefficient (Rho) was -0.76 and -0.80 at 12 and 24 months, respectively (P<0.0001). We propose that these differences affect patient outcome. As unrelated donor transplant outcomes improve and especially given the higher levels of donor cell engraftment following cord transplants, our data might influence donor selection where only heterozygote-matched family members are available.

Heparan sulfate levels in mucopolysaccharidoses and mucolipidoses.

Glycosaminoglycans are accumulated in both mucopolysaccharidoses (MPS) and mucolipidoses (ML). MPS I, II, III and VII and ML II and ML III patients cannot properly degrade heparan sulphate (HS). In spite of the importance of HS storage in the metabolic pathway in these diseases, blood and urine HS levels have not been determined systematically using a simple and economical method. Using a new ELISA method using anti-HS antibodies, HS concentrations in blood and urine were determined in MPS and ML II and ML III patients. HS concentrations were determined in 156 plasma samples from MPS I (n = 23), MPS II (n = 26), MPS III (n = 24), MPS IV (n = 62), MPS VI (n = 5), MPS VII (n = 5), ML II (n = 8) and ML III (n = 3), and 205 urine samples from MPS I (n = 33), MPS II (n = 33), MPS III (n = 30), MPS IV (n = 82), MPS VI (n = 7), MPS VII (n = 9), ML II (n = 8) and ML III (n = 3). The ELISA method used monoclonal antibodies against HS. MPS I, II, III and VII and ML II and III patients had significant elevation in plasma HS, compared to the age-matched controls (p < 0.0001). Eighty-three out of 89 (93.3%) of individual values in the above MPS types and ML were above the mean +2SD of the controls. In urine samples, 75% of individual values in patients with those types were above the mean +2SD of the controls. In contrast to the previous understanding of the HS metabolic pathway, plasma HS levels in all five MPS VI and 15% of MPS IV patients were elevated above the mean +2SD of the controls. These findings suggest that HS concentration determined by ELISA, especially in plasma, could be a helpful marker for detection of the most severe MPS I, II, III, VI and VII and ML II, distinguishing them from normal populations.

Keratan sulphate levels in mucopolysaccharidoses and mucolipidoses.

The mucopolysaccharidoses (MPS) is characterized by accumulation of glycosaminoglycans (GAGs), and mucolipidosis (ML) by accumulation of GAGs and sphingolipids. Each type of MPS accumulates specific GAGs. The lysosomal enzymes N-acetylgalactosamine-6-sulphate sulphatase and beta-galactosidase involve the stepwise degradation of keratan sulphate (KS). Deficiency of these enzymes results in elevation of KS levels in the body fluids and in tissues, leading to MPS IV disease. In this study, we evaluated blood and urine KS levels in types of MPS and ML other than MPS IV. Eighty-five plasma samples came from MPS I (n = 18), MPS II (n = 28), MPS III (n = 20), MPS VI (n = 3), MPS VII (n = 5) and ML (n = 11) patients while 127 urine samples came from MPS I (n = 34), MPS II (n = 34), MPS III (n = 32), MPS VI (n = 7), MPS VII (n = 9) and ML (n = 11) patients. KS levels were determined using the ELISA method. Plasma KS levels varied with age in both control and patient populations. In all age groups, the mean values of plasma KS in MPS and ML patients were significantly higher than those in the age-matched controls. Plasma KS values in four newborn patients were above the mean + 2SD of the age-matched controls (mean, 41 ng/ml). Overall, 85.9% of individual values in non-type IV MPS and ML patients were above the mean + 2SD of the age-matched controls. For urine KS levels, 24.4% of individual values in patients were above the mean + 2SD of the age-matched controls. In conclusion, KS in blood is elevated in each type of non-type IV MPS examined, in contrast to the conventional understanding. This finding suggests that measurement of KS level provides a new diagnostic biomarker in a wide variety of mucopolysaccharidoses and mucolipidoses in addition to MPS IV.

Application of low dose rate pulsed fluoroscopy in cardiac pacing and electrophysiology: patient dose and image quality implications.

The performance of a low dose rate pulsed fluoroscopy option and its successful application to cardiac pacing and electrophysiology is reported. Low dose rate 6.25 frames per second pulsed fluoroscopy was made available in two catheter laboratories at a specialist cardiac centre in February 2003, and was adopted as the standard imaging technique for cardiac pacing procedures. The image quality was found to be considerably poorer than conventional modern units, being very similar to that which would have been accepted as adequate performance 20 years ago, but at less than one-tenth of the dose rate. No problems with the clinical acceptance of this imaging mode for cardiac pacing and electrophysiology have been reported. The already low median patient dose-area product for pacing at this cardiac centre was further reduced by 50% with the introduction of this fluoroscopy option.

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.

Mutation analysis in 57 unrelated patients with MPS II (Hunter's disease).

Genomic DNA from 57 unrelated MPS II (Hunter's disease) patients was analysed for mutations of the iduronate sulphatase (IDS) gene. The aim of the study was threefold: to identify the primary genetic lesion in patients, to investigate the correlation between genotype and phenotype, and most importantly, to provide reliable carrier testing for female members once the family mutation was identified. In 42 patients, point mutations were identified involving single base substitutions, deletions, or insertions. These included four new nonsense mutations (R8X, C84X, E245X, Y466X), six new missense mutations (D45N, N115Y, P228L, P266R, E434K, I485K, W502C), three new insertions (c70C71ins, c652C654ins, c709G710ins), six new deletions (c500delC, c705delC, c1023delA, c1049delA, c1141delC, c1576delG), and five new mutations involving splice sites (IVS1-2 a-->g, IVS2-10 t-->g, IVS5 + 2 t-->g L236L, IVS7 + 2 t-->c). One patient had a new seven base deletion in exon 9 (c1482-1488del). Four patients were shown to have complete deletions of the IDS gene and two deletions involved one or more exons. Previously described mutations present in these patients were Q80X, P86L, R172X, G374G, S333L, R443X, and R468Q. In eight patients, no mutation was detected throughout the entire coding region. Most mutations that result in MPS II appear to be unique. Absence of the probands' mutations in eight of nine maternal grandmothers suggests many mutations have arisen recently. Prediction of the clinical phenotype from the identified genotype was difficult in some families, and further studies using reverse transcription polymerase chain reaction are needed to confirm the predicted effects on the IDS mRNA suggested by genomic analysis.

First-trimester diagnosis of Hunter syndrome: very low iduronate sulphatase activity in chorionic villi from a heterozygous female fetus.

Iduronate sulphatase activity was determined in uncultured chorionic villi from four fetuses at risk for Hunter syndrome. All fetuses were shown to be female by chromosome analysis. Biopsy material from three fetuses showed iduronate sulphatase activity within normal limits whilst the fourth fetus showed activity reduced to 7 per cent of our control mean. The importance of fetal sexing in prenatal diagnosis of this condition is emphasized as female carrier fetuses may show iduronate sulphatase activity reduced to levels observed in affected males.

The clinical phenotype of two patients with a complete deletion of the iduronate-2-sulphatase gene (mucopolysaccharidosis II--Hunter syndrome).

Two patients with a complete deletion of the iduronate-2-sulphatase (IDS) gene are described. In both patients, the resulting phenotype was that of very severe Hunter syndrome (mucopolysaccharidosis II). In addition, both had features not commonly seen in this disorder, e.g. early onset of seizures in one patient and ptosis in the other. It is speculated that loss of adjacent loci may contribute to the unusual findings and that the severe features present in both patients may represent contiguous gene syndromes. Further analysis of IDS cDNA from other patients with Hunter's syndrome may eventually enable phenotype to be predicted more accurately.

beta-mannosidase deficiency in a female infant with epileptic encephalopathy.

We report a female infant with an isolated deficiency of beta-mannosidase activity. At nine months of age dysmorphism was absent except for brachecephaly. There was moderate developmental delay and a startle response to sound. At 12 months there was a sudden onset of tonic-clonic seizures which were unresponsive to drug therapy, requiring paralysis and mechanical ventilation for control. The child died suddenly aged 15 months. beta-mannosidase activity was markedly reduced in white cells and cultured skin fibroblasts whilst other lysosomal enzymes were normal. The disaccharide ManGlcNAc was excreted in urine but urinary mucopolysaccharides were normal.

Alpha- and beta-mannosidoses.

Clinical, pathological and biochemical findings in the mannosidoses are described. Family studies showed granulocyte-rich white cell fractions to be the tissue of choice for carrier detection in beta-mannosidosis. Metabolic labelling studies using [3H] mannose demonstrated accumulation of Man beta 1-4GlcNAc in cultured skin fibroblasts from a patient with this condition. Alternative methods of egress from lysosomes were suggested for this compound by its secretion into culture medium and apparent reduction of storage with time in cultures. beta-mannosidase deficient goats are not thought to be a true animal model of the human condition, as although they showed a similar enzyme deficiency, the clinical presentation is much more severe and the major storage material (Man beta 1-4GlcNAc beta 1-4GlcNAc) is different.

Human beta-mannosidase deficiency: biochemical findings in plasma, fibroblasts, white cells and urine.

Marked deficiencies of beta-mannosidase activity were demonstrated in plasma, leukocytes, fibroblasts and urine of a patient with beta-mannosidosis, similar deficiencies were observed in the proband's sibling. All other lysosomal enzymes measured, including sulphamidase, exhibited normal activity. Both parents showed reduced plasma and leukocyte beta-mannosidase activity. Urinary glycosaminoglycan excretion was normal but TLC of urinary oligosaccharides revealed an abnormal band with the mobility of a disaccharide. This finding was confirmed by Bio-Gel P2 column chromatography. Further purification of this compound revealed two disaccharides, both of which yielded mannose and glucosamine following acid hydrolysis and mannose and N-acetylglucosamine following enzymic digestion. These two compounds are thought to be structural isomers of the disaccharide Man beta-GlcNAc.

Detection of Chlamydia psittaci from various animal species by reverse passive haemagglutination.

A reverse passive haemagglutination test (RPH) has previously been developed to detect the genus-specific antigen of Chlamydia. Clinical samples were obtained from various sites of different animal species. The RPH test detected chlamydial antigen from clinical cases of conjunctivitis in cats, abortion in sheep and psittacosis in birds. Although not as sensitive as cell culture isolation, this test has the advantages of rapidity and of detecting antigen from dead chlamydiae.

Properties of monoclonal antibodies to the genus-specific antigen of Chlamydia and their use for antigen detection by reverse passive haemagglutination.

The chlamydial genus-specific antigen was extracted with phenol/chloroform/petroleum ether (PCP) from preparations of Chlamydia trachomatis and C. psittaci, and quantities measured using an assay for lipopolysaccharide (LPS). The LPS from C. trachomatis contained 2.2% (w/w) of ketodeoxyoctanoic acid. Five IgG monoclonal antibodies reacted in an ELISA with LPS from both species, the antigen being periodate-sensitive and heat-resistant, confirming that all antibodies were against the genus-specific antigen. All the antibodies bound to the PCP extract of C. trachomatis on an immunoblot, at a position corresponding to the periodate-Schiff-stained bands of both C. trachomatis extract and Salmonella Re-LPS. When linked to trypsin-treated sheep erthrocytes and used in reverse passive haemagglutination tests, all antibodies gave indicator cells capable of detecting chlamydial LPS or crude preparations of chlamydiae grown in McCoy cells, the sensitivity varying with the antibody used. The antibodies varied in IgG subclass (either IgG2a or IgG3), and in ability to precipitate in immunodiffusion tests. Two antibodies cross-reacted with one strain of Acinetobacter in ELISA and with Salmonella Re-LPS in both ELISA and immunodiffusion tests. The other three did not react in ELISA with Acinetobacter strains or Salmonella Re-LPS, and none of the five reacted with LPS of E. coli or Pseudomonas morsprunorum.

A study by high-resolution two-dimensional polyacrylamide gel electrophoresis of relationships between Neisseria gonorrhoeae and other bacteria.

High-resolution two-dimensional polyacrylamide gel electrophoresis was used to analyse the soluble proteins from seven strains of Neisseria gonorrhoeae, six strains of Neisseria meningitidis and one or two strains of twelve other species. Approximately 200 individual polypeptides could be visualized as Coomassie Blue stained spots on an electrophoretogram of N. gonorrhoeae and similar numbers were found for the other bacteria. Each species of bacterium had a distinctly different pattern of spots which could be recognized. Quantitative comparisons of 48 selected spots derived from one strain of N. gonorrhoeae with those of five other strains of gonococcus, three strains of N. meningitidis and one of Branhamella catarrhalis, showed relationships in agreement with their current taxonomic classification but with a higher level of discrimination than that of previously used methods. It was also possible to distinguish the individual gonococcal strains. It is suggested that the method could be useful for bacterial classification and identification.

Surface antigens of gonococci: correlation with virulence and serum resistance.

Encapsulated and non-encapsulated variants of one strain of gonococcus were compared for their capacity to produce infection in chambers implanted subcutaneously in mice, for their reactions with specific antibody and for their precipitation with wheat germ agglutinin. Only the encapsulated variant could infect implanted chambers. Specific rabbit antiserum raised against the non-encapsulated variant killed both variants. Saline extracts and lipopolysaccharide preparations of the encapsulated variant differed from those of the non-encapsulated one in their reactions with wheat germ agglutinin and antibody in diffusion and electrophoresis tests. Preparations from infective encapsulated gonococci reacted with wheat germ agglutinin while those from the non-encapsulated variant did not. Immunodiffusion tests showed that lipopolysaccharides from both variants share a common antigenic determinant, but saline extracts and lipopolysaccharides from encapsulated gonococci possess an additional determinant. The significance of these findings is discussed.

Comparison of reverse passive antiglobulin haemagglutination with double antibody radioimmunoassay for estimation of total human serum IgE.

Serum IgE levels can be measured by reverse passive antiglobulin haemagglutination (RPAH) using trypsin-treated human red cells coupled to anti-human IgE by chronic chloride. The results are read after only 90 min incubation. RPAH and double antibody radioimmunoassay have been used to measure IgE levels in 100 sera, with levels ranging from 5 to 43,000 international units (i.u.)/ml. Correlation between the two assays was high over the whole range, provided that affinity-purified anti-IgE was used in the RPAH test. When two non-affinity-purified anti-IgE reagents were used in the RPAH, correlation was poor for sera with levels below 1000 i.u./ml. It is concluded that RPAH tests for IgE are of comparable sensitivity and specificity to radioimmunoassay procedures, and provide a useful simple, yet more rapid alternative.

Comparison of red-cell linked anti-IgE and 125I-labelled anti-IgE in a solid-phase system for the measurement of IgE specific for castor bean allergen.

The report described the development of a mixed reverse (solid-phase) passive antiglobulin haemadsorption (MRsPAH) test for specific IgE antibody to castor bean allergen. The allergen is immobilised by formalin fixation in the wells of polyvinyl chloride microtitre plates. After allowing allergen-specific antibodies in the test serum to bind to the allergen, plates are washed thoroughly, and red cells coupled by chromic chloride to sheep IgG anti-human IgE are used to detect specifically bound IgE. This system was compared with a solid-phase radioimmunoassay in which 125I-labelled anti-IgE was substituted for the antiglobulin-linked red cells of the MRsPAH test; the earlier stages of both tests being the same. 12 sera, 10 from patients with allergic asthma to castor bean allergen and two from non-allergic controls, were tested for castor-bean-specific serum IgE by both methods and the results showed high correlation. The MRsPAH tst for allergen-specific serum IgE provides a useful alternative to the RAST system, being free of the disadvantages inherent in the use of radio-labelled materials.

Measurement of human serum IgE and IgA by reverse passive antiglobulin haemagglutination.

Serum IgE levels can be measured by reverse passive antiglobulin haemagglutination (RPAH) of trypsin-treated human or sheep red cells coupled to sheep IgG anti-human IgE by chromic chloride. The results show a high correlation with those obtained by the radioactive single radial immunodiffusion method. Interfering anti-sheep IgG factors can be easily removed by absorption with small amounts of whole sheep or bovine serum cross-linked with glutaraldehyde. Standardisation with the British standard for IgE shows that the detection limit of the RPAH method is 0.5 IU/ml (1.2 ng/ml). The system is therefore comparable in sensitivity to the paper radio-immunosorbent test, and has the advantages of being simple, rapid and cheap. The RPAH method can be used to measure any class of immunoglobulin. For IgA the detection limit is found to be 10(-4) IU/ml (1.4 ng/ml).