D.M. Broadhead

Clinical findings in 12 patients with MPS IV A (Morquio's disease). Further evidence for heterogeneity. Part I: Clinical and biochemical findings.

Clinical heterogeneity in MPS IV A (Mucopolysaccharidosis IV A, Morquio Disease Type A) has become more clearly identified in recent years. The clinical findings in 12 cases of MPS IV A are described. Clinical presentation was variable, and some cases were only mildly affected. All showed deficiency of N‐acetylgalactosamine‐6‐sulphate sulphatase in fibroblasts, but the patient with the mildest clinical presentation showed a high residual enzyme activity. The urinary glycosaminoglycans (GAGs) were examined on all patients by a two‐dimensional electrophoresis technique which proved to be highly reliable and efficient. In particular, no false negative results were obtained, a problem often encountered with routine screening methods. These cases support the division of MPS IV A into three subgroups: the severe “classical” type, an intermediate type and a mild type, all caused by N‐acetylgalactosamine‐6‐sulphate sulphatase deficiency. Residual enzyme activity may be an important prognostic indicator.,

Recognition of abnormal lysosomal enzyme patterns in childhood leukaemia by isoelectric focusing, with special reference to some properties of abnormally expressed components.

Abstract Isoelectric focusing profiles of six lysosomal enzymes, β-hexosaminidase, α-fucosidase, α-mannosidase, β-glucuronidase, acid phosphatase and α-galactosidase, were studied in lymphoid cells derived from childhood leukaemic patients and control subjects. Altered β-hexosaminidase patterns were observed in nine out of eleven patients with common ALL, in one patient with null-cell ALL and two with acute undifferentiated leukaemia. These changes were not confined to β-hexosaminidase, for other enzymes including α-fucosidase, α-mannosidase, β-glucuronidase and acid phosphatase were commonly involved. Altered enzyme patterns returned to normal with remission. With the exception of acid phosphatase abnormally expressed enzymes appeared to represent more anodic forms of the parent enzymes. For β-hexosaminidase the leukaemic enzymes unlike the normal forms were found to be neuraminidase-sensitive whereas other properties of the enzymes were found to be similar to their native forms. It is suggested that studies on lysosomal enzymes may be valuable in the differentiation of sub-types of leukaemia.

Diagnosis of Pompe's disease in cultured skin fibroblasts and primary amniotic fluid cells using 4-methylumbelliferyl-α-d-glucopyranoside as substrate

The possible interference of neutral alpha-D-glucosidase in the diagnosis of Pompes disease using 4-methylumbelliferyl-alpha-D-glucopyranoside as substrate for the assay of acid alpha-D-glucosidase was investigated. The pH profile of alpha-D-glucosidase in control skin fibroblasts and amniotic fluid cells showed two peaks of activity. The shape of the pH profile depended upon whether or not the extract was added to the buffer before the substrate. If extract was added to the buffer before the substrate, a greater separation was obtained between the two peaks of activity. The neutral alpha-D-glucosidase activity could be totally removed by preliminary precipitation at pH 5.0. Following acid region whilst Pompes cells had no activity enabling a clear distinction to be made between carriers and the disease state.

Lysosomal enzymes of cultured amniotic fluid cells

Abstract 1. 1. The levels of some lysosomal enzymes in a minimum of 38 amniotic fluid cell strains cultured to the third passage were as follows (mean ± standard deviation): N- acetyl -β- d - glucosaminidase 52.1 ± 16.70 , acid phosphatase 7.73 ± 2.82, β-galactosidase 4.59 ± 1.97, β-glucuronidase 1.30 ± 0.52, β-glucosidase 0.072 ± 0.030, α-galactosidase 0.42 ± 0.15, α-arabinosidase 0.53 ± 0.25, α-glucosidase 0.38 ± 0.18 and α-mannosidase 0.65 ± 0.30 nmole 4-methylumbelliferone/min/mg protein. 2. 2. No correlation was observed between the levels of these enzymes and gestational age. 3. 3. The levels of these enzymes in uncultured cells tended to be lower than in the corresponding cultured cells. Occasionally, very low levels of certain of the enzymes were found in uncultured cells, although normal levels were found in the corresponding cultured cells.

Cholesterol ester storage disease in an adult presenting with sea-blue histiocytosis

An adult patient is described with hepatomegaly and sea‐blue histiocytes in the bone marrow. A diagnosis of cholesterol ester storage disease was established following enzyme and lipid analyses on liver biopsy and cultured skin fibroblasts. Acid esterase activity was deficient (approx. 5% of controls) in liver and fibroblasts using [14C]‐triolein or 4‐methylumbelliferyl palmitate as substrates. Cholesterol ester levels were raised about 70‐fold in liver, whereas triglyceride levels were only marginally raised. Marked accumulation of cholesterol esters was also demonstrated in cultured fibroblasts. Clinically, the patient responded favourably to phenobarbitone treatment. However, this was not reflected in liver acid esterase or lipid levels.

Lysosomal enzyme levels in human amniotic fluid cells in tissue culture. I. α-glucosidase and β-glucosidase

Abstract A number of factors which may correlate with the levels of α-glucosidase and β-glucosidase in cultured amniotic fluid cells have been investigated. Fluctuations in enzyme activity occurred as passage numbers increased. Whereas α-glucosidase showed a consistently lower activity in the earlier passages compared to the later ones, the results for β-glucosidase were equivocal. Both enzymes showed an increase in activity correlated with the time taken by the cells to reach confluency in the third passage. When replicate cultures were assayed daily after subculture, neither enzyme showed any change correlated with time. When cultures were grown in parallel in Hams F10 and Eagles M.E.M. tissue culture media, the activity of both enzymes was unaffected. Cell strains cultured from serial samples of amniotic fluid from the same woman had differing enzyme levels unrelated to gestational age.

The diagnosis of gaucher's disease in liver using 4-methylumbelliferyl-β-d-glucopyranoside

1. Cases of Gauchers disease could not be distinguished from controls by the assay of beta-glucosidase activity in water homogenates of liver using 4-methylumbelliferyl-beta-D glucopyranoside. 2. Two peaks of beta-glucosidase activity were separated by Sephadex G-150 gel filtration in control and Gaucher livers. In the presence of the elution buffer pH profiles of peak I showed a deficiency at pH 3.5-4.5 in Gauchers disease. Gaucher and control peak II had similar pH profiles with little or no activity at pH 3.0-4.0. 3. A clear distinction between homogenates of Gaucher and control liver was obtained by assay at pH 4.0 in the presence of elution buffer, or of sodium chloride, a component of the elution buffer.

Effect of serum concentration, type of culture medium and pH on the lysosomal enzyme activity of cultured human amniotic fluid cells.

Abstract 1. 1. Possible changes in lysosomal enzyme activity of cultured amniotic fluid cells with serum concentration, type of medium and the pH of the medium were studied. 2. 2. Apart from a small, but significant, decrease in the activity of β-galactosidase, none of the enzymes changed with increasing serum concentration. 3. 3. No significant changes in enzyme activity were found between cells cultured in Hams F10 and Eagles MEM medium. 4. 4. Lysosomal enzyme levels were unaffected by culturing cells for up to 9 days at pH 7.0, 7.4 and 7.9.

Lysosomal enzyme levels in human amniotic fluid cells in tissue culture: III, β-glucuronidase, N-acetyl-β-D-glucosaminidase, α-mannosidase and acid phosphatase

Fluctuations in the levels of β‐glucuronidase, N‐acetyl‐βp‐D‐glucosaminidase, rr‐mannosi dase and acid phosphatase in amniotic fluid cells with time in culture were studied. The four enzymes fluctuated markedly with passage; no consistent trends were apparent. The activity of a‐mannosidase in amniotic fluid cell strains at the third passage was correlated with the time taken to reach confluency. There was no consistent pattern of enzyme activity associated with the time after subculture. Enzyme levels in cell strains derived from serial samples of amniotic fluid from several women showed large differences in activity unrelated to gestational age. The significance of the findings are discussed in relation to antenatal diagnosis of inborn errors of lysosomal metabolism.

Elucidation of an unbalanced chromosome translocation by gene dosage studies.

A chromosome abnormality, 46, XY, lp +, was detected in cultured amniotic fluid cells. The chromosomes of both parents were normal and it was impossible to recognise the extra chromosomal material using current banding techniques. The activity of acid a‐glucosidase was found to be consistently higher than controls whereas activity of several other lysosomal enzymes, galactokinase and thymidine kinase was not. The results suggest that the extra material is that part of the long arm of chromosome 17 bearing the gene for acid a‐glucosidase but not the genes for galactokinase and thymidine kinase. This would narrow the assignment of the acid α‐glucosidase locus to 17q2 2–17qter.