Specific Substrate for the Assay of Lysosomal Acid Lipase.

BACKGROUND: Deficiency of lysosomal acid lipase (LAL) causes Wolman disease and cholesterol ester storage disease. With the recent introduction of enzyme replacement therapy to manage LAL deficiency comes the need for a reliable assay of LAL enzymatic activity that can be applied to dried blood spots (DBS). METHODS: We prepared and tested a library of analogs of palmitoyl 4-methylumbelifferyl esters to find a highly active and specific substrate for LAL in DBS. The LAL assay was optimized leading to both LC-MS/MS and fluorometric assay of LAL. We tested the new assay on DBS from healthy and LAL-deficient patients. RESULTS: The ester formed between palmitic acid and 4-propyl-8-methyl-7-hydroxycoumarin (P-PMHC) was found to be >98% selective for LAL in DBS based on the sensitivity of its activity to the LAL-specific inactivator Lalistat-2 and the fact that the activity was close to zero using DBS from patients previously shown to be LAL-deficient. Use of P-PMHC and heavy isotope-labeled internal standard with optimized assay conditions led to an approximately 2-fold increase in the specific activity of LAL compared with the previously reported LAL assay. Patients deficient in LAL were readily distinguished from normal persons with the new LAL assay using UPLC-MS/MS or fluorometric assay platforms. CONCLUSIONS: The new assay can measure LAL in DBS with a single measurement compared with the previous method involving 2 assays done in parallel.

Lysosomal lipase deficiency: molecular characterization of eleven patients with Wolman or cholesteryl ester storage disease.

Wolman Disease (WD) and cholesteryl ester storage disease (CESD) represent two distinct phenotypes of the same recessive disorder caused by the complete or partial deficiency of lysosomal acidic lipase (LAL), respectively. LAL, encoded by the LIPA gene, hydrolyzes cholesteryl esters derived from cell internalization of plasma lipoproteins. WD is a rapidly progressive and lethal disease characterized by intestinal malabsorption, hepatic and adrenal failure. CESD is characterized by hepatic fibrosis, hyperlipidemia and accelerated atherosclerosis. Aim of the study was the identification of LIPA mutations in three WD and eight CESD patients. The WD patients, all deceased before the first year of age, were homozygous for two novel mutations (c.299+1G>A and c.419G>A) or a mutation (c.796G>T) previously reported as compound heterozygosity in a CESD patient. The two mutations (c.419G>A and c.796G>T) resulting in truncated proteins (p.W140* and p.G266*) and the splicing mutation (c.229+1G>A) were associated with undetectable levels of LIPA mRNA in fibroblasts. All eight CESD patients carried the common mutation c.894G>A known to result not only in a major non-functional transcript with the skipping of exon 8 (p.S275_Q298del), but also in a minor normally spliced transcript producing 5-10% residual LAL activity. The c.894G>A mutation was found in homozygosity in four patients and, as compound heterozygosity, in association with a known (p.H295Y and p.G342R) or a novel (p.W140*) mutation in four other CESD patients. Segregation analysis performed in all patients harboring c.895G>A showed its occurrence on the same haplotype suggesting a common founder ancestor. The other WD and CESD mutations were associated with different haplotypes.

Wolman disease and cholesteryl ester storage disease diagnosed by histological and ultrastructural examination of intestinal and liver biopsy.

Deficient activity of lysosomal acid lipase (LAL) results in massive accumulation of cholesteryl esters and triglycerides in most tissues of the body. The deficiency state is expressed in two major phenotypes: Wolman disease (WD) and cholesteryl ester storage disease (CESD). WD occurs in infancy and is nearly always fatal before the age of 1 year, whereas CESD can be more benign and may not be detected until adulthood. Since there are no specific routine laboratory observations that suggest these metabolic diseases, diagnosis is based on the clinical picture combined with LAL deficiency in cultured skin fibroblasts or peripheral lymphocytes. Both disorders are rather rare, considering that about a hundred of cases have been described up to now. This study describes the histological and ultrastructural aspects disclosed by intestinal or liver biopsy in three cases of WD and in two cases of CESD. Furthermore, it emphasizes the role of morphological findings in pointing the diagnosis towards a metabolic storage disease.

Lysosomal acid lipase deficiency: correction of lipid storage by adenovirus-mediated gene transfer in mice.

Lysosomal acid lipase (LAL) is the essential enzyme for hydrolysis of triglycerides (TGs) and cholesteryl esters (CEs) in lysosomes. Its deficiency produces two human phenotypes: Wolman disease (WD) and cholesteryl ester storage disease (CESD). The LAL null (lal(-/-)) mouse mimicks aspects of human WD and CESD. The potential for gene therapy of LAL deficiency was tested with first-generation adenoviral vectors containing human LAL cDNA (Ad-hLAL) by intravenous injection into lal(-/-) mice. Compared with phosphate-buffered saline-injected controls, the mice receiving Ad-hLAL had increased hepatic LAL activity, decreased hepatomegaly, and normalization of histopathology. hLAL protein and mRNA were detected by immunohistochemical staining and in situ hybridization in hepatic parenchymal and sinusoid lining cells, splenic sinusoidal cells, lung macrophages, and adrenal cortical cells. Mice showed TG reductions in liver, spleen, and small intestine of 68, 54, and 50%, respectively, and cholesterol reductions of 55, 52, and 34%, respectively, at 20 days postinjection. These studies provide the basis for the use of gene therapy, in the form of gene transfer via intravenously administered adenovirus, to correct deficiency states, such as WD and CESD, and histopathology of a variety of tissues.

Enzyme therapy for lysosomal acid lipase deficiency in the mouse.

Lysosomal acid lipase (LAL) is the critical enzyme for the hydrolysis of the triglycerides (TG) and cholesteryl esters (CE) delivered to lysosomes. Its deficiency produces two human phenotypes, Wolman disease (WD) and cholesteryl ester storage disease (CESD). A targeted disruption of the LAL locus produced a null (lal( -/-)) mouse model that mimics human WD/CESD. The potential for enzyme therapy was tested using mannose terminated human LAL expressed in Pichia pastoris (phLAL), purified, and administered by tail vein injections to lal( -/-) mice. Mannose receptor (MR)-dependent uptake and lysosomal targeting of phLAL were evidenced ex vivo using competitive assays with MR-positive J774E cells, a murine monocyte/macrophage line, immunofluorescence and western blots. Following (bolus) IV injection, phLAL was detected in Kupffer cells, lung macrophages and intestinal macrophages in lal( -/-) mice. Two-month-old lal( -/-) mice received phLAL (1.5 U/dose) or saline injections once every 3 days for 30 days (10 doses). The treated lal( -/-) mice showed nearly complete resolution of hepatic yellow coloration; hepatic weight decreased by approximately 36% compared to PBS-treated lal( -/-) mice. Histologic analyses of numerous tissues from phLAL-treated mice showed reductions in macrophage lipid storage. TG and cholesterol levels decreased by approximately 50% in liver, 69% in spleen and 50% in small intestine. These studies provide feasibility for LAL enzyme therapy in human WD and CESD.

Lysosomal acid lipase mutations that determine phenotype in Wolman and cholesterol ester storage disease.

Mechanisms producing the divergent phenotypes, Wolman disease (WD) and cholesterol ester storage disease (CESD), associated with the genetic deficiency of human lysosomal acid lipase/cholesterol ester hydrolase (hLAL) function were investigated with the determination of HLAL activity levels, mRNA and protein expression, and defects in structural gene sequences in cells from three WD and five CESD patients. Measured with natural substrates, HLAL activities were all below 2% of normal, regardless of phenotype. Immunoblotting showed a lack of detectable hLAL protein in all mutant fibroblasts. Four CESD, but no WD genomes contained at least one allele with a specific exon 8 splice junction mutation, c.894 G>A, that encodes a shortened form of hLAL mRNA. Other CESD mutations were identical in type to the WD defects: nucleotide deletions (positions 397, 684, 980), insertions (594), or substitutions (193, 347) that result in premature terminations precluding any function. The only exception was a substitution at nucleotide 866 in the CESD case without an exon 8 splicing mutation; expression of the predicted S289C change in a transfection assay produced a low, but clearly measurable, level of acid esterase activity. Although it is not easily demonstrated in conventional assays, CESD is distinct from WD in that at least one mutant allele has the potential to produce enough residual enzymatic function to ameliorate the phenotype; in the majority of CESD cases this may come from a single, easily detected, splicing mutation in one allele.

Molecular defects underlying Wolman disease appear to be more heterogeneous than those resulting in cholesteryl ester storage disease.

Human lysosomal acid lipase/cholesteryl ester hydrolase (hLAL) is essential for the intralysosomal metabolism of cholesteryl esters and triglycerides taken up by receptor-mediated endocytosis of lipoprotein particles. The key role of the enzyme in intracellular lipid homeostasis is illustrated by two lysosomal storage diseases inherited as autosomal recessive traits. Wolman disease, associated with deficient hLAL activity, leads to massive intracellular substrate accumulation and is always fatal in early infancy. Cholesteryl ester storage disease (CESD), in contrast, is characterized by very low levels of enzymic activity sufficient to allow survival of the affected patients into adulthood. In order to elucidate the underlying molecular defects in Wolman disease, we have characterized the hLAL gene in two female Wolman patients of German and Turkish origin by SSCP and DNA sequence analysis. Our results demonstrate that the German proband was compound heterozygous for an 8-bp deletion in exon 3 and a 2-bp deletion in exon 4 of the hLAL gene. These frameshift mutations lead to protein truncation at amino acid positions 24 and 116 and to complete loss of hydrolytic activity. The Turkish proband, in contrast, was homozygous for a G(1064)-->T substitution in exon 10 of the hLAL gene which converts the completely conserved glycine (GGG) residue at position 321 of the mature enzyme to tryptophan (TGG). In vitro expression of the hLAL(Gly(321)-->Trp) cDNA construct revealed that the amino acid replacement results in a more than 99% reduction of neutral lipid hydrolysis. The mutations provide new insights into the molecular basis of Wolman disease which is apparently more heterogeneous at the genetic level than cholesteryl ester storage disease.-Lohse, P., S. Maas, P. Lohse, A. C. Sewell, O. P. van Diggelen, and D. Seidel. Molecular defects underlying Wolman disease appear to be more heterogeneous than those resulting in cholesteryl ester storage disease.

Targeted disruption of the mouse lysosomal acid lipase gene: long-term survival with massive cholesteryl ester and triglyceride storage.

Lysosomal acid lipase (LAL) is essential for the hydrolysis of the triglycerides and cholesteryl esters in lysosomes. Its deficiency produces two phenotypes, a severe infantile-onset variant, Wolman disease (WD), and a later onset variant, cholesteryl ester storage disease (CESD). A mouse model with a LAL null mutation was produced by targeting disruption of the mouse gene. Homozygote knockout mice (lal -/lal-) produce no LAL mRNA, protein or enzyme activity. The lal-/lal- mice are born in Mendelian ratios, are normal appearing at birth, and follow normal development into adulthood. However, massive accumulation of triglycerides and cholesteryl esters occurs in several organs. By 21 days, the liver develops a yellow-orange color and is approximately 1.5-2.0x larger than normal. The accumulated cholesteryl esters and triglycerides are approximately 30-fold greater than normal. The lal+/lal- mice have approximately 50% of normal LAL activity and do not show lipid accumulation. Male and female lal-/lal- mice are fertile and can be bred to produce progeny. This mouse model is a phenotypic model of human CESD, and a biochemical and histopathologic mimic of human WD. The lal-/lal- mice provide a model to determine the role of LAL in lipid metabolism and the pathogenesis of its deficiency states.

New lysosomal acid lipase gene mutants explain the phenotype of Wolman disease and cholesteryl ester storage disease.

Deficiency of lysosomal acid lipase (LAL) leads to either Wolman disease (WD) or the more benign cholesteryl ester storage disease (CESD). To identify the molecular basis of the different phenotypes we have characterised the LAL gene mutations in three new patients with LAL deficiency. A patient with WD was homozygote for a null allele Y303X. The other two patients, with CESD, presented either homozygosity for T267I or compound heterozygosity consisting of Q64R and an exon 8 donor splice site substitution (G-->A in position -1). The mutants T267I and Q64R and the previously reported L273S, G66V, and H274Y CESD substitutions, overexpressed in stable clones, were found to be fully glycosylated and show an enzymatic activity of 3-8% of that of normal LAL. On the other hand, the delta254-277 mutant protein derived from exon 8 skipping and the Y303X protein were totally inactive. By transient transfection of hybrid minigene constructs, the CESD G-->A (-1) substitution resulted in partial exon inclusion, thus allowing the production of a small amount of normal LAL mRNA and hence of a functional enzyme. In contrast, a G-->A substitution observed in WD at position + 1 of the same exon 8 donor site resulted in complete exon skipping and the sole production of an inactive delta254-277 protein. In conclusion, LAL genotypes determine the level of residual enzymatic activity, thus explaining the severity of the phenotype.

Altered mononuclear phagocyte differentiation associated with genetic defects of the lysosomal acid lipase.

Multiparameter flow cytometry reveals a complex heterogeneity of mononuclear phagocyte differentiation within the peripheral blood compartment. In this study, the relation of abnormal cellular lipid metabolism to the phenotype of peripheral blood mononuclear phagocytes, which finally may be related to atherogenesis, was analyzed using recently characterized autosomal recessive defects of lysosomal acid lipase (LAL) expression as model system. The reduction of LAL activity in nine heterozygote, disease free carriers of mutations from two cholesteryl ester storage disease (CESD) pedigrees and the family of a patient with Wolman disease was associated with an increased fraction of monocytes which expressed CD56 (N-CAM) (4.1 +/- 2.7% of monocytes, compared to 2.2 +/- 0.5% in ten controls, P < 0.05), an antigen characteristic of immature myeloid cells, suggesting an increased turnover of monocytes. Furthermore, a trend was observed towards an enhanced blood pool of more mature mononuclear phagocytes which show decreased expression of the 55 kD lipopolysaccharide receptor (CD14) together with either expression of the Fc-gamma-receptor III (CD16) or a high expression of CD33. A similar phenotype of peripheral mononuclear phagocytes was observed in the two CESD patients analyzed. In conclusion, our data suggest that these monogenetic defects of lysosomal lipoprotein metabolism are associated with complex alterations of mononuclear phagocyte differentiation and extravasation.

Genetic and biochemical evidence that CESD and Wolman disease are distinguished by residual lysosomal acid lipase activity.

Cholesteryl ester storage disease (CESD) and Wolman disease are both autosomal recessive disorders associated with reduced activity and genetic defects of lysosomal acid lipase (LAL). We provide evidence that the strikingly more severe course of Wolman disease is caused by genetic defects of LAL that leave no residual enzyme activity. In a CESD patient, a G --> A mutation at position -1 of the exon 8 splice donor site results in skipping of exon 8 in 97% of the LAL hnRNA originating from this allele, while 3% are spliced correctly, resulting in full-length LAL enzyme. The mutant LAL mRNA codes for a protein lacking amino acids 254 to 277. On the other allele, a G --> T mutation leads to a premature stop codon at Gly245, resulting in inactive LAL enzyme. In addition, the previously identified Leu179 --> Pro mutation is present on this allele, and the LAL mRNA is rendered unstable by the premature stop codon. Analysis of two children with Wolman disease showed that both were homozygous for a G --> A mutation at position +1 of the same splice donor site as for the CESD patient, leading to skipping of exon 8. In contrast to the CESD patient, no correctly spliced mRNA was detectable. We have also expressed a wildtype LAL cDNA and the mutant LAL cDNA from one Wolman patient in Sf9 and H5 insect cells. We demonstrate that the LAL enzyme generated from the wildtype LAL cDNA was active in homogenates from Sf9 and H5 cells, while the enzyme with the internal deletion of 24 amino acids originating from the LAL cDNA of the Wolman patient was not. The combined data provide evidence that the only functionally relevant genetic difference between the Wolman patients and the CESD patient is that the splice defect in Wolman, which affects one of the invariable nucleotides of the splice consensus sequences (position +1), does not permit any correct splicing, whereas the defect observed in CESD (position -1) allows some correct splicing (3% of total LAL mRNA) and therefore the synthesis of functional enzyme.

Cholesteryl ester storage disease: hepatopathology and effects of therapy with lovastatin.

We describe three patients with cholesteryl ester storage disease. Diagnosis was confirmed by demonstrating a deficiency in lysosomal acid cholesteryl hydrolase activity in cultured skin fibroblasts from each of these patients. All had hepatomegaly, elevated serum aminotransferase activities and hyperlipoproteinemia. Histological examination of liver biopsy specimens before treatment revealed accumulation of fat within hepatocytes, bile duct epithelium and endothelial and Kupffer cells. Cholesterol crystals were recognized by their birefringence in frozen sections. A striking feature was the presence of markedly hypertrophied Kupffer cells and portal macrophages with foamy, tan-colored cytoplasm that stained readily with the periodic acid-Schiff reagent and aldehyde fuchsin. Periportal fibrosis was noted in all cases; incomplete cirrhosis was present in one case. Distinctive and hitherto undescribed lysosomal accumulations of triglyceride and cholesterol crystals were noted. The patients were treated with lovastatin, a cholesterol-lowering agent, for at least 12 mo. No significant changes were seen in serum lipoprotein concentrations or liver histopathology after therapy. Thus lovastatin did not have an obviously beneficial effect on abnormal lipid metabolism in these patients.