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We have investigated the utility of bone marrow-derived mesenchymal stem cells (MSCs) as targets for gene therapy of the autosomal recessive disorder mucopolysaccharidosis type IH (MPS-IH, Hurler syndrome). Cultures of MSCs were initially exposed to a green fluorescent protein-expressing retrovirus. Green fluorescent protein-positive cells maintained their proliferative and differentiation capacity. Next we used a vector encoding alpha-L-iduronidase (IDUA), the enzyme that is defective in MPS-IH. Following transduction, MPS-IH MSCs expressed high levels of IDUA and secreted supernormal levels of this enzyme into the extracellular medium. Exogenous IDUA expression led to a normalization of glycosaminoglycan storage in MPS-IH cells, as evidenced by a dramatic decrease in the amount of (35)SO(4) sequestered within the heparan sulfate and dermatan sulfate compartments of these cells. Finally, gene-modified MSCs were able to cross-correct the enzyme defect in untransduced MPS-IH fibroblasts via protein transfer.

Cholesteryl ester storage disease and Wolman disease are rare autosomal recessive lipoprotein-processing disorders caused by mutations in the gene encoding human lysosomal acid lipase. Thus far we have elucidated the genetic defects in 15 unrelated CESD patients. Seven were homozygotes for the prevalent hLAL exon 8 splice junction mutation which results in incomplete exon skipping, while eight probands were compound heterozygotes for E8SJM and a rare mutation on the second chromosome. In this report, we describe the molecular basis of CESD in three compound heterozygous subjects of Czech and Irish origin. RFLP and DNA sequence analysis revealed that they were heteroallelic for the common G(934)-->A substitution in exon 8 of the hLAL gene and a mutation which, if inherited on both alleles, would be expected to result in complete loss of enzyme activity and to cause Wolman disease. In patients A. M. and J. J., two nucleotide deletions in exons 7 and 10 were detected, involving a T at position 722, 723, or 724 and a G in a stretch of five guanosines at positions 1064;-1068 of the hLAL cDNA. Both mutations result in premature termination of protein translation at residues 219 and 336, respectively, and in the production of truncated, inactive enzymes. Subject D. H., in contrast, is a compound heterozygote for the Arg(44)-->Stop mutation previously described in a French CESD proband. Combined with data in the literature, our results demonstrate that compound heterozygosity for a mutation causing Wolman disease is common among cholesteryl ester storage disease patients.

Using a histochemical technique, we have demonstrated a consistent deficiency of alcohol (hexanol) dehydrogenase activity within the epidermis and jejunal mucosa of patients with Sjögren-Larsson syndrome. Biochemical assay of the fatty alcohol: NAD oxidoreductase activity in cultured fibroblasts and leukocytes from these patients showed deficient activities compared with controls. The histochemical and biochemical results are complementary, and the simpler histochemical method can be used reliably for initial screening of patients with ichthyosis in whom a diagnosis of Sjögren-Larsson syndrome is suspected.

Derivatives of galactocerebroside were prepared containing coloured (w-2,4,6-trinitrophenylaminolauric acid) or fluorescent (11-(9-anthroyloxy) undecanoic acid) fatty acid moieties.. These cerebrosides were used as substrates for galactocerebrosidase activity. By overcoming problems associated with the radioactively labelled substrates normally used, yet retaining good enzyme-substrate specificity, these derivatives provided useful and reliable alternative substrates for galactocerebrosidase activity. Enzyme activities in whole extracts of brain, liver, fibroblasts and cultured amniotic fluid cells were compared, using as substrates the novel cerebrosides as well as [3H]galactocerebroside. Good correlation of activities was obtained. In extracts derived from patients with Krabbe's disease marked deficiency of galactocerebrosidase activity was observed with each substrate, whereas extracts from heterozygous carriers exhibited a partial reduction in enzyme activity. The results show that these coloured and fluorescent galactocerebrosides may be used with confidence in the diagnosis and carrier detection of Krabbe's disease.

Fibroblast phosphodiesterase activity was studied using 4-methylumbelliferyl pyrophosphate diester as substrate. Release of the fluorogen, 4-methylumbelliferone, was found to be dependent on acid phosphatase activity, normally present in excess in crude cell extracts. Phosphodiesterase activity had an acid pH optimum, was deficient in Niemann-Pick disease fibroblasts, and, when assayed in the presence of exogenous acid phosphatase, had an identical electrofocusing profile to that of sphingomyelinase. These findings suggest that 4-methylumbelliferyl pyrophosphate diesterase and acid sphingomyelinase activities are dependent on the same enzyme.

A chromogenic substrate, 2-hexadecanoylamino-4-nitrophenyl-beta-D-galactopyranoside, has recently been described for the diagnosis of Krabbe's disease. Hydrolysis of this substrate by extracts of cultured cells and tissues was compared with the activities of lactocerebrosidase I and non-specific beta-galactosidase. Under appropriate conditions, hydrolysis of the chromogenic analogue was markedly reduced in extracts of cultured amniotic fluid cells and skin fibroblasts derived from cases of Krabbe's disease. Activity was also markedly deficient in extracts of Krabbe's brain, although only a partial reduction was measured in liver extracts. Generally activities were higher in tissues of fetal origion. Unfortunately, the new analogue proved less specific and less sensitive than the natural substrates used to diagnose Krabbe's disease. Consequently, the analogue does not provide a satisfactory alternative substrate for the prenatal diagnosis of Krabbe's disease.

Phosphodiesterase activity of cultured cells was determined with bis-(4-methylumbelliferyl) phosphate as substrate. In the presence of Triton X-100 an acid component was evident and results indicated that this enzyme was identical with sphingomyelinase. Acid phosphodiesterase activity was specifically inhibited by sphingomyelin. In fibroblasts from patients with Niemann-Pick diseases types A, B and C, acid phosphodiesterase activity was deficient whereas neutral activity was normal. Neutral activity could, however, be removed by acid precipitation or by binding to DEAE-cellulose. Hence a simple and sensitive fluorimetric method is described for the assay of sphingomyelinase activity in the diagnosis of Niemann-Pick disease.