Henry L. Nadler

Radiometric assays of N-acetylglucosaminylphosphotransferase and α-N-acetylglucosaminyl phosphodiesterase with substrates labeled in the glucosamine moiety

The assay of fibroblast and leukocyte-N-acetylglucosaminylphosphotransferase with alpha-methylmannoside acceptor and commercially available UDP-[3H or 14C]N-acetylglucosamine donor was modified to yield low background and consequently high sensitivity and reliability comparable to those obtained with the synthetically made [beta-32P]UDP-N-acetylglucosamine donor. This was achieved by an additional elution step that removed free [3H or 14C]N-acetylglucosamine which appeared to be the breakdown product responsible for the high background. In addition, the [3H or 14C]N-acetylglucosamine-1-phospho-6-alpha-methylmannoside product of the transfer reaction was then isolated and, following desalting, could serve as a substrate for the assay of alpha-N-acetylglucosaminyl phosphodiesterase. Cell preparations of patients with I-cell disease and pseudo-Hurler polydystrophy demonstrated severe to moderate deficiency of transferase activity and normal phosphodiesterase activity toward the respective substrates labeled with 3H or 14C in the glucosamine moiety.

First trimester prenatal evaluation for I-cell disease by N-acetyl-glucosamine 1-phosphotransferase assay

First trimester prenatal diagnosis was offered to a couple at risk for having a child with I‐cell disease (mucolipidosis II). The prenatal evaluation was based for the first time on examination of N‐acetylglucosamine 1‐phosphotransferase activity, deficiency of which is the primary biochemical defect in both I‐cell disease and pseudo‐Hurler polydystrophy (mucolipidosis III). Heterozygote levels of this enzyme activity were determined in chorionic villi obtained at 9 weeks of gestation, as well as in cultured trophoblasts derived from this specimen, and led to the diagnosis of an unaffected fetus. This procedure has advantages over that based on detection of abnormal intracellular‐extracellular distribution of lysosomal enzyme activities, which is expressed only in homozygotes and fully expressed only in cell culture specimens.

Lysosomal enzyme activities in fresh and frozen chorionic villi and in cultured trophoblasts

Fifteen lysosomal enzyme activities were compared in 14 presumed normal chorionic villus specimens that were each divided, processed and analyzed as fresh tissue, tissue frozen for 1 week, and cultures established from minced whole villi. Most of the activities determined in the chorionic villus tissue were not affected significantly by freezing. However, activities for most enzymes were significantly different from those determined in the cultured cells. Our experience with first trimester prenatal evaluations for several lysosomal disorders showed that the limited amount of tissue obtained is not always sufficient for thorough analysis and thus, cultured trophoblasts derived from the tissue specimen should also be examined. The results of this study stress the importance of using appropriate tissue-type and cell-type controls to establish the normal range in the respective analyses.

Thermal activation of hexosaminidase A in a genetic compound with Tay-Sachs disease

Increase in total hexosaminidase activity has been observed during heat treatment of serum and leukocyte specimens from a 1-year-old boy with cherry-red spot and severe and progressive mental and motor deterioration. The activity increased 40% in the first 40–70 min of incubation at 50°C and pH 4.3, but declined thereafter and was only slightly above the initial activity in the final 2–3 h of incubation. Heat treatment of specimens from family members revealed very reduced rates of inactivation of hexosaminidase in the probands father and some paternal relatives, whereas those of the mother and some maternal relatives were indistinguishable from those of Tay-Sachs carriers. Mixing experiments with enzyme preparations from the proband, normal controls and patients with Tay-Sachs disease resulted in additive values and did not support the possibility of inhibitor- or activator-related defect. Fractionation of heat-treated samples by ion exchange chromatography and electrophoresis, as well as examination of the separated fractions for their thermostability, have shown that hexosaminidase A is the activated component and hexosaminidases B, I1 and I2 are not affected. These findings suggest that the patient is a genetic compound and the apparent thermal activation is probably due to formation of hexosaminidase A from altered α-subunits produced by the paternal mutant α-allele and β-subunits produced by the normal β-alleles.

Mannosylation of glycoproteins and dolichol derivatives in fibroblasts from patients with cystic fibrosis

Increased incorporation of mannose into endogenous glycoprotein fractions has been found in whole cell lysates and crude membrane preparations of cultured skin fibroblasts from patients with cystic fibrosis (1.3-2.3-times normal) when GDP[14C]mannose served as the mannosyl donor. In contrast, the incorporation of mannose from GDPmannose into lipid fractions containing dolichol phosphate and dolichol pyrophosphate oligosaccharides as well as the incorporation of mannose from dolichol phospho[3H]mannose into both glycoproteins and dolichol derivatives were not significantly different among cell preparations from patients with cystic fibrosis and normal controls. Mannosyltransferase activity toward exogenous glycoproteins as well as the activities of soluble and membranous alpha-mannosidase and beta-mannosidase appeared to be normal and could not account for the observed differences. The altered incorporation of mannose into endogenous glycoprotein may reflect changes in glycosylation processes other than mannosylation.

Km defect in neuraminidase of dysmorphic type sialidosis with and without β-galactosidase deficiency

Kinetic studies of 4-methylumbelliferyl neuraminidase activity were carried out in cultured skin fibroblasts from patients with various disorders of neuraminidase deficiency. Cell extracts from two patients with dysmorphic type sialidosis of infantile onset, with isolated deficiency of neuraminidase activity, and three patients with dysmorphic type sialidosis of juvenile onset, with combined deficiency of neuraminidase and beta-galactosidase activities, demonstrated 7-12 times higher apparent Km values than those of normal controls (1.0-1.5 mmol/l as compared with 0.12-0.15 mmol/l). The apparent Ki values for N-acetylneuraminic acid and colominic acid were also increased in the dysmorphic type (7-15 and 7-11 times the normal values, respectively). In contrast, in the normomorphic type, normal apparent Km and Ki values were found for 4-methylumbelliferyl neuraminidase activity in fibroblasts from one patient with isolated neuraminidase deficiency and two patients with combined deficiency of neuraminidase and beta-galactosidase. The altered kinetics in the dysmorphic cases indicates a primary defect in neuraminidase with a secondary deficiency of beta-galactosidase in patients with combined deficiency. It is not clear if the primary defect in the normomorphic cases involves a defect in neuraminidase other than a Km defect or if neuraminidase or both neuraminidase and beta-galactosidase deficiencies are secondary to another defect as yet undetermined.

Biochemical and Biologic Problems and Pitfalls in the Prenatal Diagnosis of Inborn Errors of Metabolism

Rapid expansion of our knowledge of the biochemical basis of inherited metabolic disorders during recent years and the development of new diagnostic methods have led to rapid advances in the intrauterine diagnosis of the inborn errors of metabolism. Amniocentesis continues to be the primary tool. Cell-free amniotic fluid, noncultivated amniotic fluid cells, and cultivated amniotic fluid cells have all been utilized in the prenatal diagnosis of inborn errors of metabolism, and, given the appropriate circumstances, each may provide important information regarding the status of the fetus. The accuracy of prenatal biochemical diagnosis has generally been very high, but errors have occurred. It is of paramount importance that extreme caution be exercised in the interpretation of prenatal studies. In many instances, a decision as to whether or not a pregnancy will be continued is based on the result of a single laboratory study. The responsibility entrusted to those involved in prenatal diagnostic studies is therefore great. It is essential that everyone involved in such endeavors be thoroughly familiar with the origin and characteristics of normal amniotic fluid and amniotic fluid cells. The normal variability in any biochemical or cytological characteristic must be well defined before the results obtained from a particular analysis can be assumed to represent fetal abnormality.