The major aims of this project are to: 1) Further elucidate the specific defects in inherited disorders of cholesterol and bile acid synthesis, i.e. the Smith-Lemli-Opitz (SLO) syndrome, sitosterolemia, and cerebrotendinous xanthomatosis (CTX). In the SLO syndrome, the abnormally low concentrations of tissue cholesterol and accumulation of 7-dehydrocholesterol (7DHC, the penultimate cholesterol precursor) have suggested a block in the conversion of 7DHC to cholesterol due to a defect in 7-dehydrocholesterol 7-reductase (7DHCR). The purification of 7DHCR and sequencing of tryptic peptides will allow the isolation of 7DHCR cDNA clones and correction of SLO fibroblasts by DNA transfection. The SLO gene will also be isolated by expression cloning, and causative mutations of the 7DHCR gene will be identified by reverse transcription polymerase chain reaction (PCR) of poly A+RNA from SLO tissues. In sitosterolemia, the suggestion of an inherited defect in 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) can be further examined by the analysis and cloning of PCR products from sitosterolemic tissues and tranfection of sitosterolemic fibroblasts with clones of the HMGR gene to test the role of this gene in the inherited defect; 2) Develop a test for diagnosis of heterozygotes (carriers) of the defective SLO gene. Experiments will be carried out in fibroblasts grown under conditions where the differences in cellular sterol composition (concentrations of cholesterol, 7DHC and other metabolites) and 7DHCR activities are maximized and discriminatory between normal, heterozygous, and homozygous cells; 3) Use a rat animal model with inhibited 7DHCR activity (fed inhibitors BM 15.766 or AY 9944) that reproduces the biochemical defect of the SLO syndrome to examine the relationship between brain and plasma sterol composition. 7DHCR activity, and cerebral function (sensory reactivity and motor learning), and to test different treatment strategies; 4) Study the mechanisms of regulation of key enzymes involved in cholesterol and bile acid metabolic disorders, i.e. 7DHCR, lathosterol 5-dehydrogenase, cholesterol 7 alpha hydroxylase, sterol 27-hydroxylase, and 5 beta cholestane, 3 alpha, 7 beta, 12 alpha, 25-tetrol 24S-hydroxylase (a defective enzyme in CTX). The proposed research extends our work in the past 18 years of grant support in areas relating to the formation of cholesterol and bile acids and its regulation in inherited disorders.
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