Mutations in the bile acid biosynthetic enzyme sterol 27-hydroxylase underlie cerebrotendinous xanthomatosis

Abstract

The sterol storage disorder cerebrotendinous xanthomatosis (CTX) is characterized by abnormal deposition of cholesterol and cholestanol in multiple tissues. Deposition in the central nervous system leads to neurological dysfunction marked by dementia, spinal cord paresis, and cerebellar ataxia. Deposition in other tissues causes tendon xanthomas, premature atherosclerosis, and cataracts. In two unrelated patients with CTX, we have identified different point mutations in the gene (CYP27) encoding sterol 27-hydroxylase, a key enzyme in the bile acid biosynthesis pathway. Transfection of mutant cDNAs into cultured cells results in the synthesis of immunoreactive sterol 27-hydroxylase protein with greatly diminished enzyme activity. We have localized the CYP27 gene to the q33-qter interval of human chromosome 2, and to mouse chromosome 1, in agreement with the autosomal recessive inheritance pattern of CTX. These findings underscore the essential role played by sterols in the central nervous system and suggest that mutations in other sterol metabolizing enzymes may contribute to diseases with neurological manifestations.

Fig. 1. Southern blot hybridization of genomic DNA isolated from subjects CTX1 and CTX2

Genomic DNA (10 μg) from normal and CTX fibroblasts was digested with the indicated restriction enzyme and subjected to Southern blot analysis as described by Lehrman et al. (17). Radioactive single-stranded DNA probes (45) corresponding to approximately nucleotides 500 to 710, 711 to 914, and 1400 to 1594 of the human sterol 27-hydroxylase cDNA (10) were used in the hybridization reactions. The positions to which HindIII fragments of bacteriophage λ-DNA migrated in the agarose gel and their sizes in kilobases (kb) are indicated on the left side of the figure. Filters were exposed to Kodak XRP x-ray film for 120 h in the presence of an intensifying screen.

Fig. 2. Blot hybridization analysis of normal and CTX sterol 27-hydroxylase mRNAs

Different amounts of total cellular RNA isolated from normal and CTX fibroblasts were analyzed by blot hybridization after electrophoresis through 1.5% (w/v) agarose gels. Hybridization probes labeled with ³²P were derived from nucleotides 510 to 710, 711 to 914, and 1400 to 1594 of the sterol 27-hydroxylase cDNA. After washing, the filters were exposed to Kodak XAR-5 x-ray film for 96 h in the presence of an intensifying screen. The positions to which RNAs of known size (kilobases, kb) migrated to in the gel are shown on the left of the figure.

Fig. 3. CTX mutations in sterol 27-hydroxylase

The 532-amino-acid coding region of the sterol 27-hydroxylase mRNA specifies a 33-residue mitochondrial signal sequence (hatched region) and a 499-residue mature protein (white block). Noncoding sequences (thick black lines) are present at the 5′ and 3′ ends of the mRNA. The locations of the amino acid changes detected in subjects CTX1 and CTX2 after amplification and DNA sequencing are indicated above the line. Two protein domains of this mitochondrial cytochrome P-450 that may be affected by these changes are indicated below the schematic.

Fig. 4. Expression of normal and CTX sterol 27-hydroxylase cDNAs

COS cells were transfected with the indicated cDNA in the pCMV2 vector. Forty-eight hours after transfection, the medium was made 2.5 μM in substrate (5β-[7β-³H]cholestane-3α,7α,12α-triol) as outlined under “Experimental Procedures” and left on the cells for the indicated time periods. The conversion of substrate into products (5β-[7β-³H]cholestane-3α,7α,12α,27-tetrol plus 3α,7α,12α-trihydroxy-5β-[7β-³H]cholestanoic acid) was determined by thin layer chromatography analysis of media sterols. The results shown are representative of two separate transfection experiments.

Fig. 5. Immunoblotting of proteins synthesized from transfected sterol 27-hydroxylase cDNAs

COS cells were transfected with the indicated cDNA. Forty-eight hours after transfection, the cells were lysed with detergent-containing buffers and fractionated by centrifugation into a nuclear, mitochondrial, and microsomal pellet (46). Twenty micrograms of the mitochondrial pellet was electrophoresed and subjected to immunoblot analysis as described under “Experimental Procedures.” In lane 7, 20 μg of mitochondrial pellet from cells transfected with the normal cDNA was electrophoresed and subjected to immunoblot analysis as described above, except that the antisera was preincubated with 10 μg/ml peptide prior to inclusion in the blotting reactions.

Fig. 6. Regional mapping of sterol 27-hydroxylase gene (CYP27) on human chromosome 2

The regions present in four analyzed hybrid cell lines are indicated by vertical bars next to an idiogram of chromosome 2. Southern blot analysis of hybrid cell line DNA using a sterol 27-hydroxylase cDNA probe revealed the presence (+) or absence (−) of the human CYP27 fragments as indicated. The bracket demarks region q33-qter that contains the CYP27 locus.