(Arch Dis Child 1997;76:92-99)

No abstract

Bone marrow transplantation (BMT) was performed in 10 patients with Hunter disease (mucopolysaccharidosis type II, iduronate-2-sulphatase deficiency). The donor was an HLA-identical sibling in 2 cases, an HLA-nonidentical relative in 6 cases, a volunteer unrelated donor in 1 case, and details were not available in 1 case. Only three patients have survived for more than 7 years post BMT; however, this high mortality probably resulted from poor donor selection. In two, there has been a steady progression of physical disability and mental handicap. One patient has maintained normal intellectual development, with only mild physical disability. It is possible that BMT may be useful in selected patients with MPS II.

The lysosomal system is the main intracellular mechanism for the catabolism of naturally occurring endogenous and exogenous macromolecules and the subsequent recycling of their constituent monomeric components. It also plays an important part in processing essential metabolites. A genetic defect in a protein responsible for maintaining the lysosomal system results in the accumulation within lysosomes of partially degraded molecules, the initial step in the process leading to a lysosomal storage disease. The defective protein can be a luminal lysosomal enzyme or protein cofactor, a lysosomal membrane protein or a protein involved in the post-translational modification or transport of lysosomal proteins. Over 40 lysosomal storage diseases are known and they have a collective incidence of approximately 1 in 7000-8000 live births. Most of the genes for the lysosomal proteins have been cloned, permitting mutation analysis in individual cases. This information can be used for genotype/phenotype correlation, genetic counselling and the selection of patients for novel forms of therapy, such as substrate deprivation or dispersal, enzyme replacement, bone-marrow transplantation and gene transfer.

By presenting the liver with lower collagenase exposure than for the isolation of conventional single hepatocytes (singlets), a preparation is obtained containing -20% of hepatocytes which have not separated from their neighhour at the junctional complex and thus remain as pairs (couplets. doublets) or small multiples; subsequent centrifugal clutriation can enrich the proportion of couplets to -70%.The junctions between the adjacent cells in the couplet are repaired during short-term (4-5h) culture; a sulcd canalicular vacuole is formed from the canalicular components of the two cells. Materials destined for bile can be pumped into this vacuole and accumulatc; the couplets are therefore polarized cells and are acting as rn wino models for the upper pan of the hepatobiliary system. Though possessing no advantage over singlets (indeed they are more difficult to obtain and use) for many metabolic and metabolic control applications couplets have, however, made substantial contributions to our understanding of polarized and hcpatobiliary aspects of hepatocytc function. Singlet hepatocytes have lost this polarity and are thus inappropriate for such studies. In addition to conventional biochemical techniques, work with hepatocyte couplets has made extensive use of visible and fluorescence microscopy, to elucidate: 1) discrimination of hepatounalicular from systemic or ductular effects on bile formation. 2) transcellular movements and canalicular destinations of fluorescent cholephilcs.3) recruitment of transporter molecules to the canalicular membrane and its control. 4) role, control and maturation of tight junctions sealing the bile canaliculus. 5) control and performance of gap junctions between hepatocytes. 6) distribution and control of the cytoskcleton in polarized hepatocytcs. 7) relative contributions of zonal hepatocytc functions in bile formation. 8)m vztro effects and mechanisms of action of cholestatic and hepatotoxic agents.In recent years much has been learned about the functions and hiogenesis of peroxisomes. This increased knowledge has greatly been stimulated by studies on a genetic disease called Zellwegcr syndrome in which peroxisomer are completely lacking due to a defect in pcroxisomc biogcnesis. Most of thc functions of peroxisomes have to do with lipid metabolism and include: 1. Fatty acid beta-oxidation: peroxisomcs catalyse the baa-oxidative chainshortening of a distinct set of substrates including very-long-chain fatty acids, di-and trihydroxycholestanoic acid and 3. pristanic acid, which is the product of the alfa-oxidation of phytanic acid. 2. Etherphospholipid biosynthesis: synthesis of ether-linked phospholipids including plasmalogens is initiated in pcroxisomes since the first 2 enzymatic reactions catalysed by dihydroxyacetone phosphate acyltransferase (DHAPAT) and alkyl DHAP synthase are strictly peroxisomal. 3. Biosynthcsis of isoprenoids including cholesterol: evidence is increasing that pcroxisomcs play an indispensable role in isoprcnoid formation since enzymes such as medonate kinas...

Aim: The aim of this study was to determine whether globotriaosylceramide (Gb3) is a useful biomarker in Fabry disease. Methods: The levels of Gb3 were measured in plasma and urine by tandem mass spectrometry in untreated hemizygotes and heterozygotes with Fabry disease and in healthy controls, and the levels were monitored in patients on treatment with enzyme replacement therapy (ERT). Results: Hemizygotes with classic Fabry disease showed elevated levels of Gb3 in both plasma and urine and could readily be distinguished from normal controls. Male patients with the N215S mutation had normal levels in their plasma but 50% had marginally elevated levels in their urine. Thirty‐three percent of proven heterozygotes had elevated Gb3 concentrations in plasma but 97% of those without the N215S mutation (36/37) had an elevated level in urine. The four heterozygotes with the N215S mutation had normal Gb3 levels in urine. The level of Gb3 in plasma initially fell following the start of ERT in all patients who had an elevated level before treatment. However, in a few patients the level subsequently rose. Similar results were found for the levels of Gb3 in urine. Conclusion: Gb3 is not an ideal marker of Fabry disease or the response to treatment in all patients. Plasma and urine levels of Gb3 cannot be used as a marker of Fabry disease in patients with the N215S mutation and many heterozygotes do not have elevated Gb3 levels in plasma. The urine concentration is more informative in heterozygotes and can be used as a measure of the response to therapy. The fall in Gb3 levels in patients receiving ERT was not sustained in some patients, despite a clinical improvement. Additionally, Gb3 cannot be used to monitor the response to treatment in patients who initially have normal plasma and urine concentrations of this glycolipid.

Fabry disease is an X-linked disorder of glycosphingolipid metabolism resulting from a deficiency of the lysosomal enzyme alpha-galactosidase A. This deficiency leads to the progressive accumulation, in lysosomes of visceral tissues and in body fluids of hemizygotes, of the glycosphingolipids globotriaosylceramide (CTH, Gb(3) or GL-3) and galabiosylceramide (CDH) and to a lesser extent the blood group AB and B related glycolipids. Elevated levels of the glycosphingolipids are found in the urine of hemizygous males with the classic phenotype, but it is not known whether all symptomatic or asymptomatic heterozygotes have elevated levels. We have therefore measured CTH and CDH quantitatively in a multiplex assay using tandem mass spectrometry in urine from a large cohort (44) of genetically proven or obligate heterozygotes including four with the N215S mutation, from classic hemizygotes (28), from cardiac variant hemizygotes with the N215S mutation (6) and from normal controls. The levels of CTH and CDH were related to both creatinine and sphingomyelin. Urinary CTH was elevated in all 28 classic hemizygotes but only in 4/6 of the cardiac variants. The level was within or just above the normal reference range in the four individuals heterozygous for the N215S mutation but was elevated in 38/40 of the other heterozygotes. Similar results were obtained for CDH, except that only 34/40 heterozygotes had an elevated level. The level of CDH was not elevated in the four heterozygotes and 4/6 of the hemizygotes for the N215S mutation. Combining the levels of CTH and CDH did not improve the discrimination of heterozygotes from controls. The ratio of CDH to CTH was higher in heterozygotes than in hemizygotes. Measurement of urinary CTH gave the best discrimination of heterozygotes from controls.

Sanfilippo syndrome type B or mucopolysaccharidosis type IIIB (MPS IIIB) is one of a group of lysosomal storage disorders that are characterised by the inability to breakdown heparan sulphate. In MPS IIIB, there is a deficiency in the enzyme a-N-acetylglucosaminidase (NAGLU) and early clinical symptoms include aggressive behaviour and hyperactivity followed by progressive mental retardation. The disease is autosomal recessive and the gene for NAGLU, which is situated on chromosome 17q21, is approximately 8.5 kb in length and contains six exons. Primers were designed to amplify the entire coding region and intron/exon boundaries of the NAGLU gene in 10 fragments. The PCR products were analysed for sequence changes using SSCP analysis and fluorescent DNA sequencing technology. Sixteen different putative mutations were detected in DNA from 14 MPS IIIB patients, 12 of which have not been found previously. The mutations include four deletions (219-237de119, 334-358del25, 1335delC, 2099delA), two insertions (1447( -1448insT, 1932, two nonsense mutations (R297X, R626X), and eight missense mutations (F48C, Y140C, R234C, W268R, P521L, R565W, L591P, E705K). In this study, the Y140C, R297X, and R626X mutations were all found in more than one patient and together accounted for 25% of mutant alleles. (7Med Genet 1998;35:910-914)