W.F. Carey

FIRST‐TRIMESTER DIAGNOSIS OF SMITH–LEMLI–OPITZ SYNDROME

We report here the prenatal diagnosis of Smith–Lemli–Opitz (SLO) syndrome in the first trimester by direct measurement of 7‐dehydrocholesterol (7‐DHC) in a chorionic villus (CV) biopsy. The proband was diagnosed clinically at birth and the diagnosis was confirmed biochemically by demonstrating elevated 7‐DHC in plasma. The family pursued prenatal diagnosis in their fourth, fifth, and sixth pregnancies. The fourth pregnancy spontaneously miscarried at 9 weeks gestation. Analysis in both direct and cultured curetting tissue (identified as similar to CV tissue) showed an abnormal tissue neutral sterol pattern with an elevated 7‐DHC concentration. The fifth pregnancy also miscarried spontaneously at 9 weeks but no tissue of unequivocal fetal origin could be identified to allow biochemical investigation. In the sixth pregnancy, ultrasound examination at the time of CV sampling showed a thickened nuchal fold. Direct analysis of the CV sample revealed elevated levels of 7‐DHC consistent with the diagnosis of SLO. The pregnancy was terminated and both fetal tissue and cultured fetal cells showed marked increases in 7‐DHC, confirming the prenatal diagnosis.

Prenatal diagnosis of mucolipidosis II - Electron microscopy and biochemical evaluation

Prenatal diagnosis of mucolipidosis type II (I‐cell disease) can be performed quickly and reliably by electron microscopy of chorionic villus tissue. This study reports the results of studies in three prenatal assessments (two families) where the pregnancy was at one in four risk of the disorder. In all three cases, electron microscopy showed marked vacuolation in chorionic villus cells, consistent with the fetus being affected by the disorder. Further studies in cultured chorionic villus cells showed a marked deficiency of a number of lysosomal enzymes. All pregnancies were terminated. Follow‐up studies in fetal tissue (where available) confirmed the prenatal diagnosis as correct. Copyright

Diagnosis of Tay-Sachs disease using [3H]N-acetylneuraminic acid labelled GM2 ganglioside as substrate

GM2 ganglioside labelled with tritium in the N-acetylneuraminic acid moiety was prepared and used to measure beta-hexosaminidase A activity in cultured humans skin fibroblasts extracts. The latter convert this substrate to the correspondingly labelled GM3 ganglioside which can easily be separated from the substrate by thin-layer chromatography. No cleavage of the N-acetylneuraminic acid group was observed under our conditions. Two methods are described for the determination of GM2-beta-hexosaminidase A activity in fibroblasts. The application of these methods to the diagnosis of Tay-Sachs disease is discussed.

A micro-radiochemical assay for α-1,4-glucosidase and its use in the assessment of type II glycogenosis (pompe's disease)

An assay for alpha-1,4-glucosidase (acid maltase) activity which is deficient in Pompes disease is described. The assay can be used to measure the enzyme in cultured skin fibroblasts, cultured amniotic cells and peripheral blood leucocytes. [U-14 C]Maltose is used as the substrate in a total assay volume of 8 microliter. The product, [U-14C]glucose, is separated from the substrate by cellulose thin-layer chromatography. The procedure permits replicate assays from 400 microliter whole blood and from amniotic cells in primary culture. Discrimination of the heterozygous Pompe state appears to be facilitated.

Prenatal diagnosis of lysosomal storage diseases: A review of 23 cases

During the previous 2 yr, 23 pregnancies were monitored for the presence of 9 different lysosomal storage diseases. There were 3 possible cases of GM1 gangliosidosis, 4 of GM2 gangliosidosis Type I and one of Type II, 4 of glucosylceramidosis, 3 of sulphatide lipidosis, one of galactosylceramidosis, 2 of glycogen storage disease Type II, 2 of mucolipidosis Type II and 3 of nephropathic cystinosis. Twelve foetuses were predicted to be homozygous affected and these pregnancies were terminated. Analysis of tissues (cultured fibroblasts, brain, liver, spleen) from 11 of these foetuses confirmed the prenatal diagnosis: one foetus was lost to biochemical follow-up. Of the remaining 11 pregnancies, one terminated spontaneously (4 wk after amniocentesis). Five children have been born and considered to be unaffected by the disease for which they were at risk both by clinical and biochemical evaluation: 5 further children have not yet been tested. In 22 cases cultured amniotic cells were used for the diagnosis and sufficient cells were grown to perform the analysis before the 20th wk of gestation. In one case of mucolipidosis Type II, cells failed to proliferate in culture and cell free amniotic fluid was used for assay. It is concluded that the techniques currently employed for the above tests are diagnostic. Even so, a multidirectional approach to each diagnosis, bringing to bear all available technology in our laboratory, is still used to ensure that diagnostic errors are not made.

Antenatal diagnosis of cystinosis

Cystinosis is a recessively inherited metabolic disorder characterized biochemically by a high intracellular content of free cystine; this results in cystine crystal deposition in many body organs. The primary genetic defect has not been identified. This disorder is diagnosable in cultured human skin fibroblasts and cultured amniotic fluid cells by radiochemically measuring the increased intracellular cystine levels, thus allowing antenatal detection of the disorder. Mrs P., who had had a previous child with cystinosis, presented at 16 wk gestation for antenatal diagnosis. Amniocentesis was performed, the amniotic fluid cells cultured for 6 wk, then 35 S-cystine was added to the culture medium of the test and control cells for 24xa0h. Mrs P.s cultured amniotic fluid cells demonstrated elevated intracellular 35 S-cystine levels compared with the controls. Subsequently, the pregnancy was terminated. The diagnosis of cystinosis was confirmed by histopathological examination of fetal tissues and by elevated 35 S-cystine accumulation in cultured fibroblasts derived from the umbilical cord and pericardium of the abortus.

Radioisotope-labelled substrates in enzyme assays with special reference to the preparation of radiolabelled glycosphingolipids and their use in the diagnosis of inborn errors of complex lipid metabolism

Radioactive substrates are being used increasingly in assays for enzymes that are present at very low levels of activity, particularly if artificial chromogenic or fluorogenic substrates are unavailable, or have varying sensitivity towards different isoenzymes. Frequently, these radioactive substrates are commercially unavailable, and must be prepared. Over the past few years fluorogenic substrates (based on 4-methylumbelliferyl derivates) have become available for the assay of the lysosomal enzymes associated with glycosphingolipid catabolism. However, it has recently been shown that such assays may be diagnostically misleading and more consideration is now placed on the use of suitably radiolabelled naturally occurring lipids (corresponding to those accumulated in lysosomal disease states) as substrates in aqueous in vitro assay system. Six of these lipids are of immediate importance—sphingomyelin (accumulated in Niemann-Pick disease), galactosylceramide (Krabbes), glucosyl-ceramide (Gauchers), galactosyl-sulphate-ceramide (metachromatic leukodystrophy), GM 1 ganglioside (generalized gangliosidosis) and GM 2 ganglioside (Tay Sachs). There are 2 principal methods of labelling these lipids to render them suitable for use as substrates. The first involves incorporation of tritium into the water soluble hydrophilic part of the molecule (e.g. terminal carbohydrate or phosphoryl-choline residue). The water soluble label released by enzyme action may then be partitioned from the hydrophobic substrate by relatively simple means although more complex systems must be used when GM 1 or GM 2 gangliosides are used as substrates. Alternatively, the hydrophobic part of the molecule may be labelled by catalytic tritiation. In our hands, more than 80% of the tritium incorporated goes into the fatty acids of the ceramides, with the remaining 20% to the long chain base. After enzymatic cleavage, the labelled, hydrophobic ceramide may be rapidly separated from the unchanged hydrophobic substrate by chromatography on micro silica gel plates. As before, however, the use of GM 1 and GM 2 gangliosides require more complicated procedures. Problems which have been overcome in using these tritiated lipids include their limited ‘solubility in aqueous assay systems and their non-specific adsorption of silica gel, which necessitates special precautions prior to scintillation counting.

A micro-radiochemical assay forα-1,4-glucosidase

An assay system for α-l,4-glucosidase (acid maltase) which is deficient in glycogen storage disease Type 2 (Pompes disease) has been developed. The assay, which represents an improvement in sensitivity of at least 100-fold over the more conventional colorimetric methods is used to measure the enzyme activity in peripheral blood leucocytes, cultured amniotic cells and skin fibroblasts, has a total volume of 8xa0μl and uses (U- 14 C) maltose as the substrate, The product (U- 14 C) glucose is separated from the substrate by cellulose thin layer chromatography. The procedure allows the assay of acid maltase in cell extracts equivalent to about 2000–5000 cultured cells or 0.5-1.0 × 10 5 leucocytes isolated from 400xa0μl of whole blood. The assay has been used to measure the enzyme activity in fibroblasts and leucocytes derived from normal subjects, an affected patient and to assess the genotype of two foetuses suspected of being affected by Pompes disease.

A transient variant of serum lactate dehydrogenase isoenzymes

An unusual variant of the serum lactate dehydrogenase (LDH) isoenzyme pattern is described in an apparently healthy young woman. The abnormal pattern consisted of a single zone of LDH activity, having the same mobility as, but more diffuse than, normal LDH-4. The molecular weight of the abnormal LDH complex is approximately 280 000, but the nature of the additional component remains unknown, as the isoenzyme pattern spontaneously reverted to normal six weeks after it was first noticed.

The enzymatic diagnosis of some inborn errors of lipid metabolism

Work is currently in progress in this laboratory to establish and extend existing methods for the perinatal diagnosis of inborn errors of metabolism by the measurement of a wide range of enzyme activities, with particular emphasis on those associated with sphingolipid storage disorders. The current enzyme estimations are performed on extracts of leukocytes, cultured skin fibroblasts and amniotic fluid cells. To date, data have been accumulated from almost 200 leukocyte extracts. Less information is available for skin fibroblasts, and the wide range of activity for some enzymes would appear to make diagnosis difficult. However, data from both of these cell types has been used in the confirmation of cases of GM 2 gangliosidosis (Type II), glucosyl ceramidosis, sulphatide lipidosis and of 4 cases of GM 1 gangliosidosis. The data on amniotic fluid cells at present have not been used in the prenatal detection of disease and one attempt at culturing such cells from a GM 2 gangliosidosis (Type I) heterozygote was unsuccessful.