Cathepsin A deficiency in galactosialidosis: studies of patients and carriers in 16 families.

Deficiency of lysosomal protective protein/cathepsin A in humans is the primary cause of galactosialidosis, a lysosomal storage disease characterized by combined deficiency of beta-galactosidase and neuraminidase. We have investigated 20 galactosialidosis patients and nine of their obligate heterozygous parents. A group of 12 patients with the early infantile type of the disease exhibited practically complete absence of cathepsin A activity, whereas eight patients with either the late infantile or the juvenile/adult type had 2-5% residual activity. Highest levels (5%) were present in two patients with milder clinical manifestations and later onset of the disease. In most fibroblast strains, beta-galactosidase activity was 10-15% of normal levels, whereas neuraminidase was reduced to less than 4%. Interestingly, a substantial residual activity (10%) of the latter enzyme was detected in the patient with the mildest phenotype and the highest cathepsin A activity. Heterozygous values for cathepsin A were reduced on average to half of normal levels. However, in two cell strains, the activity was far below control range, and in these cases, neuraminidase activity was severely depressed. Finally, we showed that cathepsin A had considerable activity in chorionic villi and amniocytes, but was deficient in amniocytes from a pregnancy with an affected fetus, indicating the relevance of cathepsin A assay for prenatal diagnosis of galactosialidosis.

Concanavalin A variants of alpha-fetoprotein in first trimester fetuses with trisomy 21 and with normal karyotypes.

Total alpha-fetoprotein (AFP) concentrations and proportions of AFP non-reactive with the lectin concanavalin A (Con A) were studied in extracellular fluid of 22 first-trimester fetuses. Total AFP concentrations were significantly lower in fetuses with Down's syndrome than in those with Mendelian-inherited diseases and normal karyotypes. The proportion of non-Con-A-reactive AFP was low in the fetal compartment (< or = 3.5%) irrespective of the fetal karyotype. So the AFP production in the fetal liver as well as in the yolk sac seems to be reduced in Down's syndrome. The fetus itself contributes only marginally to the non-Con-A-reactive AFP pool of amniotic fluid, which is therefore almost entirely of yolk sac origin. This pool is preserved well into the second trimester of pregnancy, despite cessation of yolk sac AFP production at the end of the first trimester, indicating a very slow rate of disappearance of proteins out of amniotic fluid.

A fluorimetric enzyme assay for the diagnosis of Sanfilippo disease C (MPS III C).

Both the alpha- and beta-anomers of 4-methylumbelliferyl-D-glucosaminide were synthesized and shown to be substrates for the lysosomal acetyl-CoA:glucosaminide N-acetyltransferase. Using the beta-anomer, fibroblasts and leukocytes from 11 different Sanfilippo C patients showed < 1% of mean normal N-acetyltransferase activity. Heterozygotes showed intermediate activities. The enzymatic liberation of the fluorochrome from 4-methylumbelliferyl-beta-D-glucosaminide requires the sequential action of the N-acetyltransferase and beta-hexosaminidase. Normal beta-hexosaminidase activity caused complete hydrolysis of the reaction intermediate 4-methylumbelliferyl-beta-D-N-acetylglucosaminide formed by the N-acetyltransferase. In cell extracts with a beta-hexosaminidase deficiency, however, a second incubation in the presence of excess beta-hexosaminidase is needed to avoid underestimation of the N-acetyltransferase activity.

Salla disease variant in a Dutch patient. Potential value of polymorphonuclear leucocytes for heterozygote detection.

A Dutch child with psychomotor retardation, impaired speech, ataxia, sialic acid storage and vacuolized skin fibroblasts and lymphocytes was diagnosed as having free sialic acid storage disease. Slight corneal opacities, pale optic disks at the fundus oculi and vertebral abnormalities, not earlier reported in Salla disease, were peculiar to this case. Free sialic acid was about tenfold increased in urine and cultured fibroblasts, without changes in the glycoconjugate-bound sialic acid pool. A subsequent pregnancy of the patient's mother was monitored by assay of sialic acid in chorionic villi and amniotic fluid. An unaffected foetus was predicted. Sialic acid was also assayed in peripheral blood total leucocytes, and in mononuclear and polymorphonuclear (PMN) leucocyte subpopulations. Each of these leucocyte fractions from the patient showed 10- to 30-fold increase in sialic acid content. The PMN subpopulation provided the most restricted range of control values and showed slightly increased values for the patient's parents. These results suggest that the assay of sialic acid in PMN might be useful for the identification of heterozygotes in sialic acid storage disease. Studies on a larger number of obligate heterozygotes are needed to confirm this observation.

Prenatal diagnosis of Niemann-Pick disease type C.

Niemann-Pick disease type C (NPC) was demonstrated in two successive pregnancies by strongly reduced activity of sphingomyelinase in amniotic fluid cells. By contrast, chorionic villi from the first pregnancy had shown normal sphingomyelinase activity. The prenatal diagnosis of NPC in the two fetuses was confirmed, after termination of the pregnancies, by (phospho)lipid analyses of the fetal livers, by the assay of sphingomyelinase in the fetal fibroblasts and by the demonstration of a defective esterification of exogenous cholesterol and of cholesterol accumulation by filipin staining. Retrospective analysis of cultured amniocytes for cholesterol esterification and filipin staining confirmed the feasibility of these methods for prenatal diagnosis. In a recent pregnancy in the same mother the three available methods were applied to amniotic fluid cells and an unaffected child was correctly predicted. Lipid analysis of liver tissue from the patient with NPC and the two fetuses showed a 3-5 times increased level of cholesterol, a 2-3 times increased level of sphingomyelin and a remarkable increase of bis (monoacylglyceryl) phosphate.

Biosynthesis of human alpha-N-acetylgalactosaminidase: defective phosphorylation and maturation in infantile alpha-NAGA deficiency.

The biosynthesis of human alpha-N-acetylgalactosaminidase (alpha-NAGA) was studied in normal fibroblasts and in cells from patients with infantile alpha-NAGA deficiency. Normal alpha-NAGA is synthesized as a 52 kDa precursor which matures to a 49 kDa species through phosphorylation and carbohydrate triming. Fibroblasts from the patients synthesize normal amounts of a 52 kDa precursor, however phosphorylation does not occur and this precursor is subsequently degraded intracellularly.

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

4-Methylumbelliferyl-beta-D-galactopyranoside-6-sulphate was synthesized and used for the determination of galactose-6-sulphate sulphatase activity. Fibroblasts and leucocytes from 12 different Morquio A patients, showed 0.0-2.7% of mean normal galactose-6-sulphate sulphatase activity. Heterozygotes showed intermediate activities. The enzymatic liberation of the fluorochrome from 4-methylumbelliferyl-beta-D-galactopyranoside-6-sulphate requires the sequential action of galactose-6-sulphate sulphatase and beta-galactosidase. Normal beta-galactosidase activity caused nearly complete hydrolysis of non-fluorescing 4-methylumbelliferyl-galactoside, formed during incubation. In cell extracts with a beta-galactosidase deficiency however, a second incubation in the presence of excess beta-galactosidase is needed to avoid underestimation of galactose-6-sulphate sulphatase activity.

First trimester diagnosis of Wolman's disease.

Wolman's disease was diagnosed in the first trimester of pregnancy by the direct demonstration of acid lipase deficiency in chorionic villi. The diagnosis was confirmed by studies on cultured chorionic villus cells and fetal skin fibroblasts. Acid lipase activity was assayed with both 4-methylumbelliferyl-palmitate and radiolabelled cholesterol oleate as substrates. The higher specificity of the enzyme for the latter, natural, substrate makes it superior in prenatal diagnosis.

Late-onset globoid cell leucodystrophy (Krabbe's disease). Clinical and genetic delineation of two forms and their relation to the early-infantile form.

A girl is described with a late-onset form of globoid cell leucodystrophy (GLD, Krabbe's disease). Data of this patient and seventeen reported patients with late-onset GLD and cerebroside-beta-galactosidase deficiency were compared with those of patients with classical early-infantile GLD. Three phenotypes of GLD are proposed, an early-infantile form, and two late-onset forms. Biochemical studies demonstrated residual activities of cerebroside-beta-galactocerebrosidase in the late-onset forms. The KM values were identical in the three GLD phenotypes. Autosomal recessive inheritance is likely for each of the subtypes. Complementation studies by somatic cell hybridization suggest that the mutations in early-infantile and late-onset GLD are allelic.

Debranching enzyme in fibroblasts, amniotic fluid cells and chorionic villi: pre- and postnatal diagnosis of glycogenosis type III.

Glycogenosis type III is characterized by a deficiency of debranching enzyme in most tissues, and it can be detected by the inability to liberate glucose from limit dextrin. However, using this assay, the deficiency is not expressed in cultured fibroblasts from patients with glycogenosis type III. We have demonstrated that the failure to detect debranching enzyme deficiency in fibroblasts is entirely due to interference of acid alpha-glucosidase, which can also hydrolyse limit dextrin. A method is described to remove specifically acid alpha-glucosidase allowing clear discrimination between fibroblasts from patients and controls, whereas heterozygotes showed intermediate values. The results with amniotic fluid cells and chorionic villi suggest the feasibility of first- and second-trimester prenatal diagnosis of glycogenosis III.

Amniotic fluid disaccharidases in the prenatal detection of cystic fibrosis.

Intestinal disaccharidases in amniotic fluid were studied in 41 pregnancies with a recurrence risk for cystic fibrosis (CF). In 11 out of 13 pregnancies with CF fetuses the maltase and sucrase activities were either below the control range (8 cases) or below the 10th percentile of control values (3 cases). Trehalase and lactase were slightly less informative indicators of CF. Of the other 28 pregnancies 3 had low amniotic fluid activities of several intestinal enzymes and were terminated, 12 resulted in the birth of a healthy child and 13 are continuing. The findings in fetal CF suggest an impairment of the defaecation of intestinal contents into the amniotic fluid. Reduced or low amniotic fluid disaccharidase activities were also found in other fetal disorders with demonstrated or presumed intestinal anomalies: e.g. anal atresia (2 cases), anencephaly (3 our of the 7 cases), trisomy 13 (5 cases), trisomy 18 (3 of the 5 cases) and trisomy 21 (19 of the 22 cases). Reduced amniotic fluid disaccharidase activities, although not specific for CF, are highly informative in pregnancies at high risk for CF. Using the 10th percentile of the normal range for amniotic fluid disaccharidase activities as an action line, the sensitivity of CF detection is estimated at 80 to 90 per cent, which could in high risk pregnancies reduce the risk of having another affected child from 1 in 4 to 1 in 20.

Two genetically different MU-NANA neuraminidases in human leucocytes.

Human leucocytes contain two different MU-NANA neuraminidases, which can be distinguished by Concanavalin A binding. The Con A binding form is predominant in lymphocytes (more than 80%) and the non-binding form predominates in granulocytes. The pH optima of both these neuraminidases as well as their subcellular localization as determined by Percoll gradient centrifugation suggest that they are both lysosomal. Immunological studies indicate that the Con A binding form is present in a complex with beta-galactosidase whereas the non-binding form is not. Leucocytes from patients with sialidosis or galactosialidosis are deficient in the Con A binding neuraminidase, whereas the non-binding form is normal. In sialolipidosis both forms are normal. These results demonstrate that leucocytes contain at least two genetically different MU-NANA neuraminidases. Thus, the use of leucocytes should be avoided for the diagnosis of sialidosis and galactosialidosis, and isolated lymphocytes should be used to obtain reliable results.

Comparison of growth characteristics of experimental tumours and derived cell cultures.

Cells from seven different rat tumours and a mouse sarcoma have been transplanted in syngeneic animals and were cultured in vitro. Tumours produced by inoculation of cultured cells in animals have been compared with the primary tumours. For the transplanted tumours, volume doubling times, T d, have been compared with doubling times, T d(cult), of cell numbers in cultures. Volume doubling times of the transplanted tumours generally decrease with increasing volume. At volumes of about 0-5 cm3, T d values range from 2-2 days to 10 days, while T d(cult) values ranged from 11 to 24 hr. A systematic correlation between T d and T d(cult) could not be established. During sequential transplantation of the tumours for many generations, as well as during continuous propagation of derived cell cultures, significant changes occurred which resulted in a decrease in the expression of differentiation characteristics in tumours.