Abstract
The activity of ergosterol Δ7-reductase (3β-hydroxysteroid Δ7-reductase) was measured in cultured skin fibroblasts from 7 controls, 10 Smith–Lemli–Opitz syndrome (SLOS) patients, and 10 parents (obligate carriers). The fibroblasts were exposed to delipidated medium supplemented with lovastatin for 24h and the enzyme activity was determined by incubating cell-free homogenate with ergosterol (ergosta-5,7,22-trien-3β-ol) and measuring the mass of brassicasterol (ergosta-5,22-dien-3β-ol) formed by gas chromatography–mass spectrometry with selected-ion monitoring. In carriers, the activity was significantly lower than in controls (22±2 vs 65±10 pmol/min per mg protein, p<0.0005), and no overlap was observed. The mean activity in carriers' fibroblasts was more than 100 times higher than in patients' cells (0.2 pmol/min per mg protein). The use of ergosterol avoids the many problems caused by the instability and lack of availability of radiolabelled 7-dehydrocholesterol. The present method makes it possible to discriminate SLOS carriers from both controls and patients using a commercially available substrate and common analytical equipment.
Similar content being viewed by others
REFERENCES
Bilheimer DW, Grundy SM, Brown MS, Goldstein JL (1983) Mevinolin and colestipol stimulate receptor-mediated clearance of low density lipoprotein from plasma in familial hypercholesterolemia heterozygotes. Proc Natl Acad Sci USA 80: 4124–4128.
Brown MS, Dana SE, Goldstein JL (1973) Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in human fibroblasts by lipoproteins. Proc Natl Acad Sci USA 70: 2162–2166.
Clejan S, Bittman R, Rottem S (1981) Effects of sterol structure and exogenous lipids on the transbilayer distribution of sterols in membrane of mycoplasma capricolum. Biochemistry 20: 2200–2204.
Curry CJR, Carey JC, Holland JS, et al (1987) Smith-Lemli-Opitz syndrome-type II: multiple congenital anomalies with male pseudohermaphroditism and frequent early lethality. Am J Med Genet 26: 45–57.
Honda A, Tint GS, Salen G, Batta AK, Chen TS, Shefer S (1995) Defective conversion of 7-dehydrocholesterol to cholesterol in cultured skin fibroblasts from Smith-Lemli-Opitz syndrome homozygotes. J L ipid Res 36: 1595–1601.
Honda A, Shefer S, Salen G, et al (1996) Regulation of the last 2 enzymatic reactions in cholesterol biosynthesis in rats: effects of BM 15.766, cholesterol, cholic acid, lovastatin, and their combinations. Hepatology 24: 435–439.
Honda A, Batta AK, Salen G, Tint GS, Chen TS, Shefer S (1997) Screening for abnormal cholesterol biosynthesis in the Smith-Lemli-Opitz syndrome: rapid determination of plasma 7-dehydrocholesterol by ultraviolet spectrometry. Am J Med Genet 68: 288–293.
Honda M, Tint GS, Honda A, et al (1996) Measurement of 3β-hydroxysteroid Δ7-reductase activity in cultured skin fibroblasts utilizing ergosterol as a substrate: a new method for the diagnosis of the Smith-Lemli-Opitz syndrome. J L ipid Res 37: 2433–2438.
Irons M, Elias ER, Salen G, Tint GS, Batta AK (1993) Defective cholesterol biosynthesis in Smith-Lemli-Opitz syndrome. L ancet 341: 1414.
Kelley RI (1995) Diagnosis of Smith-Lemli-Opitz syndrome by gas chromatography/mass spectrometry of 7-dehydrocholesterol in plasma, amniotic fluid and cultured skin fibroblasts. Clin Chim Acta 236: 45–58.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265–275.
Shefer S, Salen G, Batta AK, et al (1995) Markedly inhibited 7-dehydrocholesterol Δ7-reductase activity in liver microsomes from Smith-Lemli-Opitz homozygotes. J Clin Invest 96: 1779–1785.
Shefer S, Salen G, Honda A, et al (1997) Rapid identification of Smith-Lemli-Opitz syndrome homozygotes and heterozygotes (carriers) by measurement of deficient 7-dehydrocholesterol Δ7-reductase activity in fibroblasts. Metabolism 46: 844–850.
Smith DW, Lemli L, Opitz JM (1964) A newly recognized syndrome of multiple congenital anomalies. J Pediatr 64: 210–217.
Tint GS, Irons M, Elias ER, et al (1994) Defective cholesterol biosynthesis associated with the Smith-Lemli-Opitz syndrome. N Engl J Med 330: 107–113.
Tint GS, Salen G, Batta AK, et al (1995) Correlation of severity and outcome with plasma sterol levels in variants of the Smith-Lemli-Opitz syndrome. J Pediatr 127: 82–87.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Honda, M., Tint, G.S., Shefer, S. et al. Accurate detection of Smith–Lemli–Opitz syndrome carriers by measurement of the rate of reduction of the ergosterol C-7 double bond in cultured skin fibroblasts. J Inherit Metab Dis 21, 761–768 (1998). https://doi.org/10.1023/A:1005401317306
Issue Date:
DOI: https://doi.org/10.1023/A:1005401317306