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

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.