Characterization of beta-galactosidase in leukocytes and fibroblasts of GM1 gangliosidosis heterozygotes compared to normal subjects.

OBJECTIVES: Characterization of beta-galactosidase in leukocytes and fibroblasts of heterozygotes for GM1 type I. DESIGN AND METHODS: Leukocyte and fibroblast beta-galactosidase activity was determined fluorimetrically using 4-methylumbelliferyl-beta-D-galactoside as an artificial substrate. Optimum pH, Km, Vmax and thermostability of the enzyme at 42 degrees C were determined. RESULTS: The leukocyte and fibroblast enzyme of heterozygotes have an optimum pH of 4.0 and 4.2, respectively. In normal subjects, the optimum pH was 4.2 in both cells, according to previous studies. The Km of the enzyme of heterozygotes was determined to be 0.65 mM in leukocytes and 0.59 mM in fibroblasts. The Vmax was determined in 167.21 nmol/h/mg of protein in heterozygotes leukocytes and 541.2 nmol/h/mg of protein in heterozygotes fibroblasts compared to 291.7 and 1768.1 nmol/h/mg of protein in controls leukocytes and fibroblasts, respectively. When leukocyte and fibroblast heterozygote beta-galactosidase was preincubated at 42 degrees C, after 80 min the residual activity was determined to be 25 to 30% of the initial activity. These results are similar to the control group. CONCLUSIONS: We have found significant differences between the two groups in some investigated parameters. Both fibroblasts and leukocytes showed a virtually similar level of reliability as source of enzyme for the detection of heterozygotes.

Biochemical studies on leukocyte and fibroblast human beta-galactosidase.

OBJECTIVES: Some biochemical characteristics of the human leukocyte and fibroblast beta-galactosidase were studied. DESIGN AND METHODS: Leukocyte and fibroblast enzyme activity was determined fluorometricaly using 4-methylumbelliferyl-beta-D-galactoside as artificial substrate. Optimum pH, Km, Vmax and thermostability of the enzyme at 42 degrees C were determined. RESULTS: The leukocyte and fibroblast enzyme has an optimum pH at 4.2, which is in agreement with the lysosomal origin of the enzyme. The Km of the enzyme was 0.62 in leukocytes and 0.67 in fibroblasts, and Vmax was 289.9 nmol/h/mg of protein and 1779.2 nmol/h/mg of protein in the two tissues, respectively. When fibroblast or leukocyte beta-galactosidase was pre-incubated at 42 degrees C, it did not retain its activity because the residual activity after 80 minutes of pre-incubation at this temperature was lower than 30% of the initial activity both in leukocytes and fibroblasts. CONCLUSIONS: This was the first study of Km, Vmax and thermostability of beta-galactosidase performed on leukocytes and provided data for a better characterization of the enzyme beta-galactosidase, allowing the improvement of the analytical conditions.

Six novel beta-galactosidase gene mutations in Brazilian patients with GM1-gangliosidosis.

GM1-gangliosidosis is a lysosomal storage disease caused by a deficiency of acid beta-galactosidase. Three clinical forms are recognized-infantile, juvenile, and adult-based on age of onset and severity of the symptoms. We have performed molecular analysis of a large cohort of GM1 patients (19 Brazilian and one Uruguayan), using nonradioactive single-strand conformation polymorphism (SSCP) and restriction enzyme analysis of genomic DNA. Six novel mutations (R121S, V240M, D491N, 638-641insT, 895-896insC, 1622-1627insG) and two previously described point mutations (R59H, R208C) were identified. Together they accounted for 90% of the disease alleles of the patients. Two mutations, 1622-1627insG and R59H, were present in 18 of 20 patients. In addition, four polymorphisms (L10P, L12L, R521C, S532G) were identified. All cases reported are infantile GM1 gangliosidosis. This report constitutes the most comprehensive molecular study to date of this disorder in infantile patients. Since GM1-gangliosidosis is the most common lysosomal storage disorder in Southern Brazil, molecular diagnosis will be important for genetic counseling, carrier detection and prenatal diagnosis in index families.

Early onset bilateral calcifications and epilepsy.

Bilateral occipital calcifications associated with epilepsy and sometimes with celiac disease have been described previously. A boy with bilateral frontal and occipital diffuse calcifications accompanied by failure to thrive, nephrogenic diabetes insipidus, developmental delay and seizures, but without celiac disease is presented. Follow-up at 3 years of age disclosed neurodevelopmental delay, height and weight less than expected for age, and seizures controlled with carbamazepine. The uncommon association of these features and the early onset of symptoms is discussed. Although bilateral occipital calcifications share some clinical features with bilateral fronto-occipital calcifications, it is arguable whether the two are on a spectrum of a single disease or represent separate entities.