Deborah A. Mitchell

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.

Prenatal Diagnosis of I-cell Disease in the First and Second Trimesters

First trimester prenatal diagnosis of I-cell disease (1 case) was based on demonstration of profound deficiency of N-acetylglucosamine 1-phosphotransferase in chorionic villi and in cultured trophoblasts derived from the chorionic villus specimen. Deficiency of this enzyme in cultured amniotic fluid cells obtained via amniocentesis was the basis for prenatal diagnosis of I-cell disease in the second trimester (2 cases). In both procedures, the diagnosis was corroborated by the finding of intracellular deficiency and extracellular elevation of multiple lysosomal enzymes in the fetal cell cultures (trophoblasts and amniotic fluid cells), as well as a significant increase in several lysosomal enzyme activities in the maternal serum.

Discrimination Between Metachromatic Leukodystrophy and Pseudo-Deficiency of Arylsulfatase A by Restriction Digest of Amplified Gene Fragments

Mutations causing metachromatic leukodystrophy and pseudo-deficiency were detected in the arylsulfatase A gene by methods based on different wild-type and mutant restriction sites. After polymerase chain reaction amplification of fragments of the arylsulfatase A gene and digestion by the appropriate endonuclease, the mixtures were separated by polyacrylamide gel electrophoresis and visualized by ethidium bromide staining. The common splice mutation in intron 2 (459 + 1G→A) causing, in homozygosity, late-infantile metachromatic leukodystrophy and the common missense mutation in exon 8 (P426L) causing, in homozygosity, adult or juvenile metachromatic leukodystrophy were found to abolish Bst NI and Aci I sites, respectively. The polyadenylation pseudo-deficiency mutation (1619A→G) was found to create a Mae III restriction site. The N-glycosylation pseudo-deficiency mutation (N350S) does not produce or destroy any known restriction site, and in this case, introduction of a single nucleotide mismatch in one of the primers enabled the authors to create a Bfa I site in the mutant allele.

Characterization of the mutant N-acetylglucosaminylphosphotransferase in I-cell disease and pseudo-Hurler polydystrophy: complementation analysis and kinetic studies.

Complementation was examined among various types of I-cell disease and pseudo-Hurler polydystrophy by monitoring N-acetylglucosaminylphosphotransferase activity in multinucleated cells produced by fusing pair combinations of cultured skin fibroblasts. Patients with the classical forms of these disorders (5 I-cell disease and 3 pseudo-Hurler polydystrophy cell lines) comprised one complementation group and 5 cell lines from patients with variant forms of pseudo-Hurler polydystrophy comprised a distinct complementation group. In the first group, total or partial deficiency of the transferase activity was demonstrated with both natural (lysosomal enzymes) and artificial (alpha-methylmannoside) acceptor substrates with low Vmax but apparently normal Km values for the donor (UDP-GlcNAc) and acceptor (alpha-methylmannoside) substrates. The activity toward artificial substrate could be inhibited by adding exogenous lysosomal enzyme preparations to the reaction mixture. In the second group, the cells demonstrated deficiency of the transferase activity toward lysosomal enzyme acceptors but had normal activity toward alpha-methylmannoside acceptor and this activity could not be inhibited by the addition of exogenous lysosomal enzyme preparations. These findings suggest that N-acetylglucosaminylphosphotransferase is composed of at least two distinct subunits, a catalytic subunit which is absent or defective in the first complementation group, and a recognition subunit which is altered or deficient in the second group.

Stimulation of GM3 ganglioside sialidase activity by an activator protein in patients with mucolipidosis IV and controls.

An activator protein that stimulates the enzymic hydrolysis of sialic acid from gangliosides by ganglioside sialidase was fractionated from human liver. This fraction was distinct from those stimulating the hydrolysis of galactose from GM1 ganglioside by beta-galactosidase and the hydrolysis of N-acetylgalactosamine from GM2 ganglioside by hexosaminidase A. This fraction was highly specific for the hydrolysis of sialic acid from GM3 ganglioside, and was equally effective in fibroblasts from patients with mucolipidosis IV and in fibroblasts from controls.

First Trimester Maternal Serum Lysosomal Enzymes: Implications for Carrier Testing and Prenatal Diagnosis

Carrier detection for lysosomal storage diseases is sometimes possible by evaluating maternal serum levels of specific enzymes. However, lysosomal enzymes (LE) can be modified by maternal hormonal changes in pregnancy or embryonic contributions. Maternal serum was obtained prospectively in the follicular phase and at 2-5 and 7-11 weeks after conception from 13 infertility patients with precisely known ovulation dates. Eleven enzyme activities were determined fluorimetrically using 4-methylumbelliferyl substrates. Using repeated measures ANOVA, alpha-N-acetyl-glucosaminidase (p less than 0.05), hexosaminidase A (p less than 0.005) and hexosaminidase A and B (p less than 0.005) increased during the first trimester, and 8 enzymes did not change significantly. Our data show differing patterns of LE in the first trimester. These may be explained by: (1) variability of maternal reaction to hormonal changes of pregnancy, or (2) variable embryonic contributions suggesting differential ontogeny and placental transfer of these enzymes. The increase in levels of the 3 specific LE in maternal serum may interfere with the accuracy of carrier testing in early pregnancy, but pregnancy should not interfere with the other 8.

Detection of kinetically abnormal argininosuccinate synthase in neonatal citrullinemia by conversion of citrulline to arginine in intact fibroblasts

The apparent Km values of argininosuccinate synthase toward citrulline and aspartate were significantly increased in cultured skin fibroblasts from one patient with neonatal citrullinemia, whereas, those determined in cells from three other patients were within the normal range. The abnormal apparent Km of the mutant enzyme toward aspartate was determined by a ureagenesis assay system (0.14 mmol/l as compared with 0.010-0.018 mmol/l for the normal enzyme), and the abnormal values toward citrulline were measured by both arginine synthesis (6.1 mmol/l as compared with 0.21-0.26 mmol/l for the normal enzyme) and ureagenesis (0.63 as compared with 0.043-0.067) assay systems.