SummaryPrenatal diagnosis was requested by a family at risk for metachromatic leukodystrophy (MLD). An examination of the family leukocyte arylsulfatase A profile revealed that the mother had pseudo arylsulfatase A deficiency. Cultured amniotic fluid cells were deficient in arylsdfatase A, so two possibilities were indicated. the fetus was affected with MLD or had the pseudodeficiency phenotype. The only known biochemical test to differentiate the two enzyme deficient phenotypes is cerebroside sulfate loading of growing fibroblasts. The pseudodeficient cells hydrolyze the incorporated sulfatide as efficiently as control cells, whereas MLD cells show no hydrolysis. Application of this test to the at risk cultured amniotic fluid cells resulted in appreciable uptake of the sulfolipid, but no hydrolysis. Control amniotic fluid cell cultures hydrolyzed 82 to 95% of the incorporated sulfatide. Therefore, an affected fetus was indicated. Fibroblasts derived from the aborted fetus sbowed a deficiency of arylsulfatase A and a similar inability to hydrolyze cerebroside sulfate i n the loading test. The loading technique allowed the prenatal diagnosis of MLD when the arylsulfatase A analysis was equivocal. Speculation I n metachromatic leukodystrophy families with pseudo arylsulfatase A deficiency, the usual enzyme assays on cultured amniotic fluid cell extracts fail to differentiate between the fetus with the affected phenotype and the fetus with the pseudodeficiency phenotype. The cerebroside sulfate loading test in growing cultured amniotic fluid cells allowed this discrimination. I t is important to examine the family enzyme profile for the peudodeficiency phenotype as a prerequisite in the prenatal diagnosis of metachromatic leukodystrophy to avoid the erroneous identification of a pseudodeficient fetus as a metachromatic leukodystrophy fetus.In metachromatic leukodystrophy (MLD), the profound deficiency of arylsulfatase A (ajlsuifaie ~ulfohydrola&, EC 3.1.6.1) leads to the accumulation of cerebroside sulfate in neural tissue ~ resulting in progressive neurological degeneration (5). Based on the age of onset of symptoms, three classical types of MLD are recognized: late infantile, juvenile, and adult. Each type appears to be an independent autosomal recessive disorder, so allelism is implied. Although no treatment is available, the disorder can be prevented in at risk families because MLD is amenable to prenatal diagnosis. In affected pregnancies, cultured amniotic fluid cells are deficient in arylsulfatase A (10,14,17,(21)(22)(23)(24).Dubois el al. (3, 4). Lott el al. (15). and Fluharty el al. (8) described four MLD families in which one of the parents and some of the unaffected children had very low arylsulfatase A activities which overlapped the range of probands. These individuals showed no neurologic dysfunction and were healthy, so it would be appropriate to iypifi the apparent enzyme deficikncy as a ~seudodeficiencv. The attenuated enzyme activity was observed in' leukocyte anda fibroblast extracts khether thk ...

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N-[7-Nitrobenz-2-oxa-1,3-diazol-4-yl]psychosine sulfate (NBD-PS), a fluorescent analog of cerebroside sulfate (CS), was synthesized and tested as an alternative to the radiolabeled forms of CS used for assaying arylsulfatase A (ASA) in its physiological role as a cerebroside sulfate sulfohydrolase. NBD-PS simulates the natural substrate for ASA. Protocols have been developed for its use in differentiating low enzyme activities in diagnostic samples. Hydrolysis of NBD-PS is specific for ASA and optimal assay parameters were identical to those determined for CS. Differentiations between each of the major phenotypes for ASA activity were possible in the set of samples tested. One particular advantage was the ability to discriminate between individuals exhibiting arylsulfatase A pseudodeficiency and the truly deficient individuals with metachromatic leukodystrophy. Differential diagnosis was possible with fibroblast extracts by an assay that is more sensitive than procedures employing radioisotopes. Reaction products may be analyzed quantitatively by HPLC, or semiquantitatively with TLC. NBD-PS provides a simpler, safer, and more cost-effective means of performing natural substrate enzyme assays for ASA. Phenotyping with the fluorescence assay is an effective alternative to the laborious radioactive CS preparations and tissue culture loading studies that have previously been necessary.

Prenatal diagnosis was performed on a pregnancy at risk for metachromatic leukodystrophy (MLD) in a family with the pseudo arylsulphatase A deficiency trait. Extracts of cultured amniotic fluid cells were deficient in arylsulphatase A indicating that the fetus was either affected with MLD or had the benign pseudodeficiency trait. In the cerebroside sulphate loading test, the at risk cells hydrolysed sulphatide like control cultured amniotic fluid cells implying that the fetus had pseudodeficiency. The pregnancy was carried to term and a male child was delivered. Placenta, urine and fibroblasts had very low activities of arysulphatase A. However, no sulphatide could be detected in urine and growing fibroblasts responded normally in the cerebroside sulphate loading test, suggesting pseudodeficiency. At 29 months, the infant is healthy and shows no stigmata of MLD. The prediction based on the results of the cerebroside sulphate loading test on cultured amniotic fluid cells appeared to be borne out.