Accumulation Of 7-dehydrocholesterol Research Articles

Sterol physical properties and relationships to endo-/exocytosis.

Multiple diseases arise from sterol homeostasis disruption and are characterized at the molecular level by depletion or accumulation of sterol species. One such disease is Smith-Lemli-Opitz syndrome (SLOS) that results from genetic mutations in a particular cholesterol synthesis enzyme, which leads to depletion of cholesterol and accumulation of 7-dehydrocholesterol (7DHC). Recent in vivo work demonstrated that depleting/enriching specific sterol species modulated clathrin-mediated endocytosis (CME) that might contribute to the cellular phenotypes observed in disease states. The CME results were explained by softening mechanisms involving sterol lateral redistribution, but it remains a question as to why this occurs. Here, we take the outlook that differences in Gaussian curvature modulus and local lipid composition destabilize narrow necks in fission, reducing the membrane's apparent rigidity. Using membrane fluctuation analysis, we quantify sterol physical properties that partially define the fusion/fission energetic pathway, for example: the compressibility modulus, bending modulus, leaflet neutral surface, leaflet spontaneous curvature, bilayer thickness preference, and thickness contribution to the Gaussian modulus. Using these quantities, we make predictions on where sterol species will redistribute on strongly curved membranes. Finally, we use all-atom simulations of strongly curved fusion pore and hemifusion diaphragm geometries to quantify the sterol redistribution and compare to hypotheses. Comparing cholesterol and 7DHC physical properties, for example, the key statistically significant quantity is the thickness contribution to the Gaussian modulus, and we predicted that 7DHC would be less excluded from the fusion pore than cholesterol. All-atom molecular dynamics simulations of the fusion pore confirmed this hypothesis. The result correlates to in vivo experiments that showed reduced CME with 7DHC compared to cholesterol (p < 0.10). Other tests are ongoing.

Ergosterol increases 7-dehydrocholesterol, a cholesterol precursor, and decreases cholesterol in human HepG2 cells.

Current treatment approaches for hyperlipidemia rely mainly on reducing the cholesterol level by inhibiting 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), which is involved in the presqualene pathway of cholesterol biosynthesis. Finding a compound that instead targets the postsqualene pathway could aid in the treatment of hyperlipidemia and synergistically reduce the cholesterol level when used in conjunction with HMGCR inhibitors. Ergosterol is a fungal sterol that is converted to brassicasterol by 7-dehydrocholesterol reductase (DHCR7). DHCR7 is also a cholesterol biosynthesis enzyme, and thus ergosterol may cause the accumulation of 7-dehydrocholesterol, a precursor of cholesterol and vitamin D3 , by a competitive effect. In this study, we examined the effect of ergosterol on the postsqualene pathway by quantifying cholesterol precursors and related sterols using gas chromatography-mass spectrometry and by conducting quantitative RT-PCR and western blot analysis for human HepG2 hepatoma cells. We found that ergosterol is converted into brassicasterol by the action of DHCR7 from HepG2 cells and that it induced the accumulation of cholesterol precursors (lathosterol, 7-dehydrocholesterol, and desmosterol) and decreased the cholesterol level by altering the mRNA and protein levels of cholesterol biosynthesis enzymes (increase of sterol 8,7-isomerase [EBP] and decrease of DHCR7 and 24-dehydrocholesterol reductase [DHCR24]). These results demonstrate that ergosterol inhibits the postsqualene pathway and may be useful for the prevention of hyperlipidemia.

Late endosomal/lysosomal accumulation of a neurotransmitter receptor in a cellular model of Smith-Lemli-Opitz syndrome.

Smith-Lemli-Opitz syndrome (SLOS) is a congenital and developmental malformation syndrome associated with defective cholesterol biosynthesis. It is characterized by accumulation of 7-dehydrocholesterol (the immediate biosynthetic precursor of cholesterol in the Kandutsch-Russell pathway) and an altered cholesterol to total sterol ratio. Because SLOS is associated with neurological malfunction, exploring the function and trafficking of neuronal receptors and their interaction with membrane lipids under these conditions assume significance. In this work, we generated a cellular model of SLOS in HEK-293 cells stably expressing the human serotonin1A receptor (an important neurotransmitter G-protein coupled receptor) using AY 9944, an inhibitor for the enzyme 3β-hydroxy-steroid-∆7 -reductase (7-DHCR). Using a quantitative flow cytometry based assay, we show that the plasma membrane population of serotonin1A receptors was considerably reduced under these conditions without any change in total cellular expression of the receptor. Interestingly, the receptors were trafficked to sterol-enriched LysoTracker positive compartments, which accumulated under these conditions. To the best of our knowledge, our results constitute one of the first reports demonstrating intracellular accumulation and misregulated traffic of a neurotransmitter GPCR in SLOS-like conditions. We believe these results assume relevance in our overall understanding of the molecular basis underlying the functional relevance of neurotransmitter receptors in SLOS.

Dietary cholesterol supplementation and inhibitory factor 1 serum levels in two dizygotic Smith-Lemli-Opitz syndrome twins: a case report

BackgroundSmith-Lemli-Opitz syndrome (SLOS) is a rare genetic neurodevelopmental disorder caused by the defect in the 7-dehydrocholesterol reductase. This defect leads to the deficiency of cholesterol biosynthesis with accumulation of 7-dehydrocholesterol. Inhibitory factor 1 (IF1) is a well-known mitochondrial protein. Recently, it has been discovered in the human serum where it is reported to be involved in the HDL-cholesterol intake. Here we report the IF1 presence in the serum of two paediatric SLOS dizygotic twins treated with dietary cholesterol supplementation.Case presentationThe patients showed a typical phenotype. They started dietary supplementation with cholesterol when 2 months old. The cholesterol intake was periodically titrated on the basis of weight increase and the twin 1 required a larger supplementation than the twin 2 during the follow-up. When 6.4-year-old, they underwent IF1 assay that was 7-fold increased in twin 2 compared to twin 1 (93.0 pg/ml vs 13.0 pg/ml, respectively).ConclusionsWe report, for the first time, the presence of circulating IF1 in the serum of SLOS patients, showing different levels among them. Our findings confirm that IF1 could be a novel research target in cholesterol-related disorders and also in SLOS, and could contribute to the general debate on IF1 as a new modulator of cholesterol levels.

Vulnerability of DHCR7+/− mutation carriers to aripiprazole and trazodone exposure

Smith-Lemli-Opitz syndrome is a recessive disorder caused by mutations in 7-dehydrocholesterol reductase (DHCR)7 with a heterozygous (HET) carrier frequency of 1-3%. A defective DHCR7 causes accumulation of 7-dehydrocholesterol (DHC), which is a highly oxidizable and toxic compound. Recent studies suggest that several antipsychotics, including the highly prescribed pharmaceuticals, aripiprazole (ARI) and trazodone (TRZ), increase 7-DHC levels in vitro and in humans. Our investigation was designed to compare the effects of ARI and TRZ on cholesterol (Chol) synthesis in fibroblasts from DHCR7+/- human carriers and controls (CTRs). Six matched pairs of fibroblasts were treated and their sterol profile analyzed by LC-MS. Significantly, upon treatment with ARI and TRZ, the total accumulation of 7-DHC was higher in DHCR7-HET cells than in CTR fibroblasts. The same set of experiments was repeated in the presence of 13C-lanosterol to determine residual Chol synthesis, revealing that ARI and TRZ strongly inhibit de novo Chol biosynthesis. The results suggest that DHCR7 carriers have increased vulnerability to both ARI and TRZ exposure compared with CTRs. Thus, the 1-3% of the population who are DHCR7 carriers may be more likely to sustain deleterious health consequences on exposure to compounds like ARI and TRZ that increase levels of 7-DHC, especially during brain development.

A placebo-controlled trial of simvastatin therapy in Smith-Lemli-Opitz syndrome

BackgroundSmith-Lemli-Opitz syndrome (SLOS) is a multiple malformation/cognitive impairment syndrome characterized by the accumulation of 7-dehydrocholesterol (7DHC), a precursor sterol of cholesterol. Simvastatin, an HMG-CoA reductase inhibitor that crosses the blood-brain-barrier, has been proposed for treatment of SLOS based on in vitro and in vivo studies suggesting that simvastatin increases expression of hypomorphic DHCR7 alleles.MethodsSafety and efficacy of simvastatin therapy in 23 mild to typical SLOS patients was evaluated in a randomized, double-blind, placebo-controlled trial. The cross-over trial consisted of two 12 month treatment phases separated by a 2 month wash-out period.ResultsNo safety issues were identified in this study. Plasma dehydrocholesterol levels decreased significantly 8.9 ± 8.4% on placebo to 6.1 ± 5.5% on simvastatin (p<0.005) and we observed a trend toward decreased cerebral spinal fluid dehydrocholesterol levels. A significant improvement (p=0.017, paired t-test) was observed in the Aberrant Behavior Checklist-C Irritability when subjects were on simvastatin.ConclusionsThis paper reports the first randomized, placebo-controlled trial designed to test the safety and efficacy of simvastatin therapy in SLOS. Simvastatin appears to be relatively safe in SLOS patients, improves the serum dehydrocholesterol/total sterol ratio, and significantly improves irritability symptoms in mild to classical SLOS patients.

Expression and functional characterization of a C-7 cholesterol desaturase from Tetrahymena thermophila in an insect cell line

Tetrahymena thermophila transforms exogenous cholesterol into pro-vitamin D3 (7-dehydrocholesterol) with remarkable efficiency in a one-step reaction carried out by a C-7 cholesterol desaturase. The enzyme DES7 is encoded by the gene TTHERM_00310640, identified with RNAi and gene knock-out experiments, but has not yet been heterologously expressed actively in any organism. A model derived from its amino acid sequence classified DES7p as a Rieske-type oxygenase with transmembrane localization. The protein has catalytic activity, sequence and topological similarity to DAF-36/Neverland proteins involved in the synthesis of steroid hormones in insects and nematodes. Due to their structural and functional similarity, we analyzed the expression of a codon optimized DES7 gene from Tetrahymena in the insect Sf9 cell line, identified and measured the steroid metabolites formed, and extended the actual knowledge on its localization. We found that the accumulation of 7-dehydrocholesterol could be increased 16–40-fold in Spodoptera frugiperda, depending on physiological conditions, by overexpression of T. thermophila DES7. The protein was detected in the microsomal fraction, in accordance with previous reports. Although the electron transfer chain for Des7p/DAF-36/Neverland Rieske-type oxygenases is presently unknown, we identified possible donors in the ciliate and insect genomes by bioinformatic analysis. In spite of the large evolutionary distance between S. frugiperda and T. thermophila, the results indicate that there is significant functional conservation of the electron donors, since the ciliate’s sterol desaturase can function in the context of the insect electron transport system. The results achieved demonstrate that DES7 is the first gene from a ciliate, coding for a microsomal enzyme, expressed in active form in an insect cell line.

Adrenal function in Smith–Lemli–Opitz syndrome

Smith-Lemli-Opitz syndrome (SLOS) is a multiple malformation syndrome due to mutations of the 7-dehydrocholesterol reductase gene (DHCR7), which leads to a deficiency of cholesterol synthesis and an accumulation of 7-dehydrocholesterol. The SLOS clinical spectrum ranges from multiple major malformations to a mild phenotype with minor anomalies and intellectual disability. Several children with SLOS and adrenal insufficiency have been described. We performed ovine corticotropin (oCRH) testing in 35 SLOS patients and 16 age- and gender-matched controls. We reviewed prior adrenocorticotropin (ACTH) stimulation tests of our SLOS patients (19 of 35 available) and reviewed results of ACTH stimulation tests from 10 additional SLOS patients. Results from oCRH testing showed that patients with SLOS had significantly higher ACTH baseline values than healthy controls (24.8 ± 15.3 pg/ml vs. 17.8 ± 7.5 pg/ml, P = 0.034). However, no statistically significant differences were noted for peak ACTH values (74.4 ± 35.0 pg/ml vs. 64.0 ± 24.9 pg/ml, P = 0.303) and for baseline (14.2 ± 7.8 mcg/dl vs. 14.2 ± 6.3 mcg/dl, P = 0.992) and peak cortisol values (28.2 ± 7.9 mcg/dl vs. 24.8 ± 8.1 mcg/dl, P = 0.156). The area-under-the-curve (AUC) was not significantly different in SLOS patients compared to controls for both ACTH (250.1 ± 118.7 pg/ml vs. 195.3 ± 96.6 pg/ml, P = 0.121) as well as cortisol secretion (83.1 ± 26.1 mcg/dl vs. 77.8 ± 25.9 mcg/dl, P = 0.499). ACTH stimulation test results were normal in 28 of 29 tests. The individual with the abnormal test results had subsequent normal oCRH tests. The slightly increased baseline ACTH level seen during oCRH testing may be due to compensated adrenocortical insufficiency. However, we were able to show that our patients with SLOS had an adequate glucocorticoid response, and thus, in mild to moderate cases of SLOS stress steroid coverage may not be warranted.

Quantitative Proteomics Analysis of Inborn Errors of Cholesterol Synthesis: Identification of Altered Metabolic Pathways in DHCR7 and SC5D Deficiency

Smith-Lemli-Opitz syndrome (SLOS) and lathosterolosis are malformation syndromes with cognitive deficits caused by mutations of 7-dehydrocholesterol reductase (DHCR7) and lathosterol 5-desaturase (SC5D), respectively. DHCR7 encodes the last enzyme in the Kandutsch-Russel cholesterol biosynthetic pathway, and impaired DHCR7 activity leads to a deficiency of cholesterol and an accumulation of 7-dehydrocholesterol. SC5D catalyzes the synthesis of 7-dehydrocholesterol from lathosterol. Impaired SC5D activity leads to a similar deficiency of cholesterol but an accumulation of lathosterol. Although the genetic and biochemical causes underlying both syndromes are known, the pathophysiological processes leading to the developmental defects remain unclear. To study the pathophysiological mechanisms underlying SLOS and lathosterolosis neurological symptoms, we performed quantitative proteomics analysis of SLOS and lathosterolosis mouse brain tissue and identified multiple biological pathways affected in Dhcr7(Delta3-5/Delta3-5) and Sc5d(-/-) E18.5 embryos. These include alterations in mevalonate metabolism, apoptosis, glycolysis, oxidative stress, protein biosynthesis, intracellular trafficking, and cytoskeleton. Comparison of proteome alterations in both Dhcr7(Delta3-5/Delta3-5) and Sc5d(-/-) brain tissues helps elucidate whether perturbed protein expression was due to decreased cholesterol or a toxic effect of sterol precursors. Validation of the proteomics results confirmed increased expression of isoprenoid and cholesterol synthetic enzymes. This alteration of isoprenoid synthesis may underlie the altered posttranslational modification of Rab7, a small GTPase that is functionally dependent on prenylation with geranylgeranyl, that we identified and validated in this study. These data suggested that although cholesterol synthesis is impaired in both Dhcr7(Delta3-5/Delta3-5) and Sc5d(-/-) embryonic brain tissues the synthesis of nonsterol isoprenoids may be increased and thus contribute to SLOS and lathosterolosis pathology. This proteomics study has provided insight into the pathophysiological mechanisms of SLOS and lathosterolosis, and understanding these pathophysiological changes will help guide clinical therapy for SLOS and lathosterolosis.

232. Gene Therapy for Deficient Cholesterol Synthesis in a Mouse Model of SLOS

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive disorder, characterized by dysmorphology and mental retardation. It is a relatively high frequency inherited disease (1:20,000-1:40,000). The cause is a relative inactivity of 7-dehydrocholesterol-|[Delta]|7 -reductase (DHCR7), the terminal enzyme in cholesterol (C) synthesis. Affected individuals have low levels of C in blood and tissues and an accumulation of 7-dehydrocholesterol (7-DHC) and its isomer 8-dehydrocholesterol (8-DHC). Current treatment for SLOS, administration of dietary C, shows limited improvement in clinical symptoms. Enzyme replacement therapy is not feasible as the reductase is membrane bound; however, gene therapy for SLOS could be effective. Our goal is to develop gene therapy by using mouse models that mimic human SLOS and delivering the DHCR7 gene using adeno-associated virus (AAV) vector. Mouse models for SLOS were developed at NIH and are now established at CHORI. The first model is a Dhcr7 null mutation with coding exons 3-5 deleted. Homozygous pups of this model, Dhcr7 |[Delta]|3-5/|[Delta]|3-5, die on the day of birth. The second model has a missense mutation (T93M) and is associated with a mild phenotype. We used compound heterozygotes, Dhcr7 |[Delta]|3-5/T93M for our studies. These animals demonstrate the biochemical abnormality, but survive into adulthood. Concentrations of C, 7-DHC and 8-DHC in liver, brain and serum were quantified by gas chromatography-mass spectrometry (GC/MS). A reduction in the DHC/C ratio compared to untreated controls was an indicator for a positive response to gene therapy. An AAV-DHCR7 plasmid using the CMV promoter/enhancer, a woodchuck hepatitis virus post-translational regulatory element and the cDNA coding for human DHCR7 was constructed. It was used in a triple transfection protocol followed by gradient centrifugation and heparin column chromatography to make AAV-DHCR7. An initial preparation of the viral vector had a titer of 2|[times]|10 11 particles/ml as determined by quantitative real time PCR. It was tested in HEK293 cells for expression. Transduced cells had increased levels of DHCR7 mRNA, detected by RT-PCR and they showed the presence of AAV DNA by PCR. For in vivo evaluation, Dhcr7 |[Delta]|3-5/T93M adult mice were administered AAV-DHCR7 intravenously. Three weeks later mice were sacrificed and tissues recovered. PCR analysis showed that vector DNA was present in the livers of treated mice but not in controls. RT-PCR analysis of liver mRNA showed increased transcript in the treated animals. Treated animals had improved DHC/C ratios compared to their controls. Data at injection time and three weeks later were used to determine the relative change in the DHC/C ratio. The results indicate that the vector can deliver DNA to target cells. They imply that the DHCR7 sequence is transcribed and translated to yield an active enzyme that at least partially restores cholesterol metabolism. We conclude that gene therapy may provide a promising treatment for SLOS. It remains to be seen if it can ameliorate the neurological and developmental problems associated with SLOS.

Oxysterols in the circulation of patients with the Smith-Lemli-Opitz syndrome: abnormal levels of 24S- and 27-hydroxycholesterol

Infants with the cholesterol synthesis defect Smith-Lemli-Opitz syndrome (SLO) have reduced activity of the enzyme 7-dehydrocholesterol-7-reductase and accumulate 7-dehydrocholesterol, with the highest concentration in the brain. As a result of the generally reduced content of cholesterol, plasma levels of oxysterols would be expected to be reduced. 24S-hydroxycholesterol is almost exclusively formed in the brain, whereas 27-hydroxycholesterol is mainly formed from extrahepatic and extracerebral cholesterol. In accordance with the expectations, sterol-correlated plasma levels of 24S-hydroxycholesterol were reduced in infants with SLO (by about 50%). In contrast, the sterol-correlated levels of 27-hydroxycholesterol in the circulation were markedly increased. No side-chain oxidized metabolites of 7-dehydrocholesterol were detected in the circulation. Recombinant human CYP27 had markedly lower 27-hydroxylase activity toward 7-dehydrocholesterol than towards cholesterol. HEK293 cells expressing 24S-hydroxylase active toward cholesterol had no significant activity towards 7-dehydrocholesterol. The plasma levels of 3β,7α-dihydroxy-5-cholestenoic in the patients acid were reduced, suggesting a generally reduced metabolism of 27-oxygenated steroids. It is concluded that the accumulation of 7-dehydrocholesterol in the brains of patients with SLO reduces formation of 24S-hydroxycholesterol. The condition is associated with markedly increased circulating levels of 27-hydroxycholesterol, most probably due to reduced metabolism of this oxysterol. We discuss the possibility that the circulating levels of 24S-hydroxycholesterol may be used as a marker for the severity of the disease. —Björkhem, I., L. Starck, U. Andersson, D. Lütjohann, S. von Bahr, I. Pikuleva, A. Babiker, and U. Diczfaulsy. Oxysterols in the circulation of patients with the Smith-Lemli-Opitz syndrome: abnormal levels of 24S- and 27-hydroxycholesterol.

Bile acid synthesis in the Smith-Lemli-Opitz syndrome: effects of dehydrocholesterols on cholesterol 7α-hydroxylase and 27-hydroxylase activities in rat liver

The Smith-Lemli-Opitz syndrome (SLOS) is a congenital birth defect syndrome caused by a deficiency of 3β-hydroxysterol Δ7-reductase, the final enzyme in the cholesterol biosynthetic pathway. The patients have reduced plasma and tissue cholesterol concentrations with the accumulation of 7-dehydrocholesterol and 8-dehydrocholesterol. Bile acid synthesis is reduced and unnatural cholenoic and cholestenoic acids have been identified in some SLOS patients. To explore the mechanism of the abnormal bile acid production, the activities of key enzymes in classic and alternative bile acid biosynthetic pathways (microsomal cholesterol 7α-hydroxylase and mitochondrial sterol 27-hydroxylase) were measured in liver biopsy specimens from two mildly affected SLOS patients. The effects of 7- and 8-dehydrocholesterols on these two enzyme activities were studied by using liver from SLOS model rats that were treated with the Δ7-reductase inhibitor (BM15.766) for 4 months and were comparable with more severe SLOS phenotype in plasma and hepatic sterol compositions. In the SLOS patients, cholesterol 7α-hydroxylase and sterol 27-hydroxylase were not defective. In BM15.766-treated rats, both enzyme activities were lower than those in control rats and they were competitively inhibited by 7- and 8-dehydrocholesterols. Rat microsomal cholesterol 7α-hydroxylase did not transform 7-dehydrocholesterol or 8-dehydrocholesterol into 7α-hydroxylated sterols. In contrast, rat mitochondrial sterol 27-hydroxylase catalyzed 27-hydroxylation of 7- and 8-dehydrocholesterols, which were partially converted to 3β-hydroxycholestadienoic acids. Addition of microsomes to the mitochondrial 27-hydroxylase assay mixture reduced 27-hydroxydehydrocholesterol concentrations, which suggested that 27-hydroxydehydrocholesterols were further metabolized by microsomal enzymes.▪These results suggest that reduced normal bile acid production is characteristic of severe SLOS phenotype and is caused not only by depletion of hepatic cholesterol but also by competitive inhibition of cholesterol 7α-hydroxylase and sterol 27-hydroxylase activities by accumulated 7- and 8-dehydrocholesterols. Unnatural bile acids are synthesized mainly by the alternative pathway via mitochondrial sterol 27-hydroxylase in SLOS.— Honda, A., G. Salen, S. Shefer, A. K. Batta, M. Honda, G. Xu, G. S. Tint, Y. Matsuzaki, J. Shoda, and N. Tanaka. Bile acid synthesis in the Smith-Lemli-Opitz syndrome: effects of dehydrocholesterols on cholesterol 7α-hydroxylase and 27-hydroxylase activities in rat liver. J. Lipid Res. 1999. 40: 1520–1528.

Rapid identification of smith-lemlip-opitz syndrome homozygotes and heterozygotes (carriers) by measurement of deficient 7-dehydrocholesterol-Δ 7-reductase activity in fibroblasts

To extend the enzyme deficiency in Smith-Lemli-Opitz syndrome (SLOS) to extrahepatic tissues, 7-dehydrocholesterol-Δ 7-reductase activity was measured in fibroblasts from 10 controls, five SLOS homozygotes, and five obligate heterozygotes. In cells grown almost to confluence in cholesterol-containing medium (4 mg/dL), the conversion of [1,2- 3H]7-dehydrocholesterol to cholesterol (7-dehydrocholesterol-Δ 7-reductase activity) was 3.8 times higher in control than in homozygote cells and 2.2 times higher than in heterozygote cells. After 24 hours' exposure of the fibroblasts to cholesterol-deficient medium supplemented with lovastatin, 7-dehydrocholesterol-Δ 7-reductase activity increased twofold in controls, but did not change significantly in either heterozygous or homozygous cells. In contrast, the activities of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and lathosterol 5-dehydrogenase, two key enzymes that precede 7-dehydrocholesterol-Δ 7-reductase in the cholesterol biosynthetic pathway, and low-density lipoprotein (LDL) receptor—mediated binding were equal in control, homozygote, and heterozygote fibroblasts. Further, HMG-CoA reductase activity and LDL receptor—mediated binding increased after exposure to the cells to cholesterol-deficient medium. Fibroblast cholesterol concentrations were approximately equal, although homozygote cells contained 30 times more 7-dehydrocholesterol. Thus, markedly reduced 7-dehydrocholesterol-Δ 7-reductase activity that cannot be upregulated after exposure of the cells to cholesterol-deficient medium is diagnostic for the biochemical defect in SLOS. Significantly reduced enzyme activity between the levels in controls and homozygotes without accumulation of 7-dehydrocholesterol in fibroblasts identified heterozygotes.

Sterol concentrations in cultured Smith-Lemli-Opitz syndrome skin fibroblasts: Diagnosis of a biochemically atypical case of the syndrome

The Smith-Lemli-Opitz syndrome is a common birth defect syndrome caused by a deficiency of 7-dehydrocholesterol delta 7-reductase, an essential enzyme in the biosynthesis of cholesterol. The syndrome can usually be diagnosed easily from the plasma markers of markedly elevated 7-dehydrocholesterol and reduced cholesterol concentrations. However, atypical cases with normal plasma levels of cholesterol with only moderately elevated 7-dehydrocholesterol have been reported. To establish a sensitive method for the biochemical diagnosis of the atypical cases of the syndrome, we measured sterol concentrations of cultured skin fibroblasts. 7-Dehydrocholesterol concentrations in patients' fibroblasts grown in the presence of 10% fetal bovine serum were significantly higher than those in controls and parents (P < 0.0005), but they were not elevated proportionately as much as in plasma. To re-produce the accumulation of 7-dehydrocholesterol, the cells were exposed to delipidated medium to induce sterol biosynthesis. After 4 weeks, 7-dehydrocholesterol concentrations in patients' fibroblasts increased from 2.8 +/- 0.3% to 34 +/- 3% of total sterols (cholesterol + 7-dehydrocholesterol + 8-dehydrocholesterol). The increase was also observed in fibroblasts from an atypical patient who has a normal plasma cholesterol level and a 7-dehydrocholesterol concentration of only 0.15 mg/dl. In contrast, cells from parents and controls accumulated very little 7-dehydrocholesterol (< 1% of total sterols). These results demonstrate that cultured fibroblasts exhibit abnormally high accumulation of 7-dehydrocholesterol after cells are exposed to delipidated medium not only in typical patients, but also in an atypical case. The present method is a sensitive procedure for the biochemical diagnosis of this syndrome.

Clinical and biochemical screening for Smith-Lemli-Opitz syndrome. Italian SLOS Collaborative Group.

Smith-Lemli-Opitz syndrome (SLOS) is a multiple congenital anomalies/mental retardation disorder possibly due to a defect of delta 7-sterol reductase, leading to low plasma cholesterol levels and to the accumulation of 7-dehydrocholesterol (7-DHC) and other cholesterol precursors. This study aimed to identify clinical features that could potentially be specific indicators for the clinical diagnosis of SLOS, and to test the reliability of ultraviolet spectrophotometry (UVS) as a biochemical screening procedure for the syndrome. Twenty patients with clinical suspicion of SLOS, referred to 11 Italian paediatric and clinical genetic centres, were collected during 1994. In 10 patients the diagnosis was confirmed biochemically by gas chromatography/mass spectrometry (GC/MS) analysis of serum sterols, whereas in the other 10 patients the serum sterol profiles were normal. A comparison between confirmed SLOS patients and biochemically negative subjects did not show clinical signs specific for the syndrome. UVS measurement of 7-DHC correlated well with GC/MS profiles, showing 100% sensitivity and specificity. Four out of five patients had serum bile acid concentrations below the normal range of controls.

Changes in serum sterols of rats treated with 7-dehydrocholesterol-delta 7-reductase inhibitors: comparison to levels in humans with Smith-Lemli-Opitz syndrome.

The impaired conversion of 7-dehydrocholesterol to cholesterol, as a result of a permanent inhibition of the activity of 7-dehydrocholesterol-delta 7-reductase, has been reported in the Smith-Lemli-Opitz (SLO) syndrome (1, 2). For the purpose of experimental teratology, an animal disease model consisting of the offspring of pregnant rats treated with AY 9944 or BM 15766, inhibitors of 7-dehydrocholesterol-delta 7-reductase, was established. The present study compares the profiles of sterols in rat serum, obtained after transient treatment with inhibitors, with profiles of sterols obtained from patients with the permanent enzyme defect. AY 9944 (single dose of 50, 75, or 100 mg/kg) or BM 15766 (60, 75, or 90 mg/kg per day for 11 days) induces hypocholesterolemia and accumulation of 7-dehydrocholesterol and aberrant sterols in rat serum. The aberrant sterols in the treated rats are similar to those detected in human SLO patients by gas chromatography coupled to mass spectrometry (1, 3, 4) and were identified as 7- and 8-dehydrocholesterol, two trienols (I and II), and 19-nor-5,7,9(10)-cholestatrien-3 beta-ol. The time- and dose-dependences of the biochemical alterations are compared to the teratogenic abnormalities induced by inhibitors. The dietary cholesterol supplementation that suppresses embryo malformations induced by AY 9944 prevents severe hypocholesterolemia and decreases the aberrant sterol levels. As a function of time after intoxication, the 8-dehydrocholesterol to 7-dehydrocholesterol ratio increases, suggested that 8-dehydrocholesterol is derived from the gradual conversion of the accumulated 7-dehydrocholesterol. The ratio of 8-dehydrocholesterol to 7-dehydrocholesterol is higher in human SLO than in the animal disease model. This may be explained by a permanent block in 7-dehydrocholesterol-delta 7-reductase in SLO compared to a transient inhibition of this enzyme in the animal model.

Detection of defective 3 beta-hydroxysterol delta 7-reductase activity in cultured human fibroblasts: a method for the diagnosis of Smith-Lemli-Opitz syndrome.

Patients with the autosomal recessive disorder Smith-Lemli-Optiz syndrome (SLO) have recently been shown to have markedly increased tissue levels of certain cholesterol biosynthesis intermediates, most notably 7-dehydrocholesterol. The findings strongly suggest a block in the step that catalyses reduction of 7-dehydrocholesterol to cholesterol. The accumulation of 7-dehydrocholesterol can generally easily be detected in serum by gas chromatography-mass spectrometry. However, it could not be totally ruled out that SLO patients with less severe enzyme defects could escape detection by this method. A more direct way of diagnosing a defect in 7-dehydrocholesterol reduction would be to assay the conversion of 7-dehydrocholesterol to cholesterol in cultured fibroblasts from patients with suspected SLO. In the present work, an assay for the conversion of [3H]lathosterol to [3H]cholesterol in cultured human fibroblasts is described. Lathosterol is the immediate precursor of 7-dehydrocholesterol in the cholesterol biosynthetic pathway and was chosen for the assay instead of 7-dehydrocholesterol owing to the difficulty in preparation and handling of the latter compound. Fibroblasts from control subjects converted [3H]lathosterol to [3H]cholesterol efficiently, whereas in fibroblasts from SLO patients the conversion did not go beyond 7-dehydrocholesterol. It is concluded that the present method is useful for the diagnosis of SLO.

Experimental myotonia and hypocholesterolemic agents

Three hypocholesterolemic agents which inhibit cholesterol biosynthesis at different stages were administered to rats, and changes in plasma and muscle sterols, erythrocyte membrane (Na + + K +)-ATPase activity and electromyography were measured. Myotonia was induced in rats treated with triparanol (MER-29) and 20,25-diazacholesterol. These drugs block Δ 24-reductase and cause the accumulation of desmosterol in plasma and muscle. The (Na + + K +)-ATPase activity of the erythrocyte membranes isolated from rats treated with 20,25-diazacholesterol or triparanol was increased. Rats treated with AY9944, a drug which inhibits Δ 7-reductase and causes the accumulation of 7-dehydrocholesterol but not desmosterol, did not become myotonic. No significant changes in the erythrocyte membrane ATPase activities of these rats were observed. These results support the hypothesis that desmosterol accumulation in muscle is the cause of myotonia induced by 20,25-diazacholesterol and triparanol.

Changes in brain hydrolytic enzyme activities in rats treated with cholesterol biosynthesis inhibitor, AY9944

Intraperitoneal administration of AY9944 causes accumulation of 7-dehydrocholesterol, appearance of abnormal neuronal cytoplasmic lamellar inclusions containing acid phosphatase activities, and degeneration of oligodendroglial cells. In the present study, we attempted to correlate appearance and disappearance of abnormal inclusions, oligodendroglial degeneration, activities of lysosomal enzymes, and accumulation of 7-dehydrocholesterol. One group of 5-day-old rats received daily injection of AY9944, 30 mg/kg, for 30 days. Other groups received the same daily dosage but only for 10 consecutive days, starting from 5, 15, 25, and 35 days, respectively. Activities of 13 brain hydrolytic enzymes were determined. In the first group, activities of most enzymes increased over the controls, reaching the peak between the 15th and 25th day, corresponding to the maximum appearance of the neuronal inclusions and degenerating oligodendroglia. Despite the continued administration of AY9944 and the high tissue concentration of 7-dehydrocholesterol, activities of most enzymes declined after 25 days to relatively steady levels somewhat higher than controls. In the group which received 10-day injections, enzyme activities reached the peak at the end of the injections and then rapidly returned to normal within 10 days thereafter, corresponding to the appearance and disappearance of the abnormal inclusions. However, 7-dehydrocholesterol continued to increased for 10 days after the drug administration was discontinued. AY9944 had no direct effect on any of the enzymes in vitro. There appears to be generalized lysosomal activation concomitant with formation of abnormal neuronal inclusions in the experimental conditions.