4-Methylumbelliferyl-alpha-iduronate 2-sulphate was synthesized and shown to be a specific substrate for the lysosomal iduronate-2-sulphate sulphatase (IDS). Fibroblasts (n = 17), leukocytes (n = 3) and plasmas (n = 9) from different MPS II patients showed < 5% of mean normal IDS activity. The enzymatic liberation of the fluorochrome from 4-methylumbelliferyl-alpha-iduronate 2-sulphate requires the sequential action of IDS and alpha-iduronidase. A normal level of alpha-iduronidase activity was insufficient to complete the hydrolysis of the reaction intermediate 4-methylumbelliferyl-alpha-iduronide formed by IDS. A second incubation step in the presence of excess purified alpha-iduronidase is needed to avoid underestimation of the IDS activity.

A fluorimetric enzyme assay for the diagnosis of MPS II (hunter disease)

Background: Pompe disease (acid maltase deficiency, glycogen storage disease type II; OMIM 232300) is an autosomal recessive lysosomal storage disorder characterized by acid α-glucosidase deficiency due to mutations in the GAA gene. Progressive skeletal muscle weakness affects motor and respiratory functions and is typical for all forms of Pompe disease. Cardiac hypertrophy is an additional fatal symptom in the classic infantile subtype. c.-32-13T→G is the most common mutation in adults. Objective: To delineate the disease variation among patients with this mutation and to define the c.-32-13T→G haplotypes in search for genotype–phenotype correlations. Methods: We studied 98 compound heterozygotes with a fully deleterious mutation (11 novel mutations are described) and the common c.-32-13T→G mutation. Results: All patients were Caucasian. None had the classic infantile form of Pompe disease. The clinical course varied far more than anticipated (age at diagnosis GAA gene. In 76% of the cases, c.-32-13T→G was encountered in the second most common GAA core haplotype (DHRGEVVT). In only one case was c.-32-13T→G encountered in the major GAA core haplotype (DRHGEIVT). Conclusion: Patients with the same c.-32-13T→G haplotype (c.q. GAA genotype) may manifest first symptoms at different ages, indicating that secondary factors may substantially influence the clinical course of patients with this mutation.

Broad spectrum of Pompe disease in patients with the same c.-32-13T→G haplotype

Review of clinical presentation and diagnosis of mucopolysaccharidosis IVA

Lysosomal enzymes sialidase (alpha-neuraminidase), beta-galactosidase, and N-acetylaminogalacto-6-sulfate sulfatase are involved in the catabolism of glycolipids, glycoproteins, and oligosaccharides. Their functional activity in the cell depends on their association in a multienzyme complex with lysosomal carboxypeptidase, cathepsin A. We review the data suggesting that the integrity of the complex plays a crucial role at different stages of biogenesis of lysosomal enzymes, including intracellular sorting and proteolytic processing of their precursors. The complex plays a protective role for all components, extending their half-life in the lysosome from several hours to several days; and for sialidase, the association with cathepsin A is also necessary for the expression of enzymatic activity. The disintegration of the complex due to genetic mutations in its components results in their functional deficiency and causes severe metabolic disorders: sialidosis (mutations in sialidase), GM1-gangliosidosis and Morquio disease type B (mutations in beta-galactosidase), galactosialidosis (mutations in cathepsin A), and Morquio disease type A (mutations in N-acetylaminogalacto-6-sulfate sulfatase). The genetic, biochemical, and direct structural studies described here clarify the molecular pathogenic mechanisms of these disorders and suggest new diagnostic tools.

Lysosomal multienzyme complex: biochemistry, genetics, and molecular pathophysiology.

A Rapid Fluorogenic Palmitoyl-Protein Thioesterase Assay: Pre- and Postnatal Diagnosis of INCL

A fluorimetric enzyme assay for the diagnosis of Morquio disease type A (MPS IV A)

textA new fluorogenic substrate, 4 methylumbelliferyl B-D-6-sulphogalactoside, was used for the
assay of galactose-6-sulphate sulphatase activity in chorionic villi, cultured villus cells, and
amniocytes. The fluorometric assay is much more convenient than the conventional assay
using radiolabelled, sulphated oligosaccharides. Both types of substrate were used in the
prenatal diagnosis of three pregnancies at risk for Morquio type A disease using amniocytes.
These enzyme tests, as well as electrophoresis of glycosaminoglycans in the amniotic fluid,
indicated affected fetuses in two pregnancies and a non-affected fetus in one.

/pdf/prenatal-diagnosis-of-morquio-disease-type-a-using-a-simple-574oep85k8.pdf

H. Zhao

Papers

A fluorimetric enzyme assay for the diagnosis of Morquio disease type A (MPS IV A)

Prenatal diagnosis of morquio disease type a using a simple fluorometric enzyme assay

Enzymatic diagnosis of Morquio A syndrome with a new fluorimetric substrate.