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Cerebrotendinous xanthomatosis (CTX) is an autosomal recessive lipid-storage disorder caused by a deficiency of the mitochondrial sterol 27-hydroxylase. Genetic analysis utilizing SSCP and direct DNA sequencing identified a new mutation. One base-pair of cytosine was deleted at codon 326 on exon 2 of CYP27 in all CTX patients while their father was heterozygotic. This novel point deletion predicts a frameshift in mRNA (Pro(102) -->Leu) and results in the appearance of a premature termination codon (TGA) to substitute for Val(106) (GTG). To characterize the pathological mechanism of CTX patients, the protein profiles of serum and leukocytes extracted from these subjects were presented by means of proteomic technologies including 2-DE and MALDI-TOF analysis. According to the results, the amount of vinculin, ABP-280, talin and vimentin in leukocytes of CTX patients had changed significantly, reflecting the changes in membrane dynamics concerning cholestanol accumulation. The expression of target proteins in CTX patients and control was further verified by western blotting which indicated the same tendency as 2-DE data. This is the first paper to integrate both genomic and proteomic concepts for analyzing the possible mechanism of CTX and provides more information for related study in the future.

Cerebrotendinous xanthomatosis (CTX) is a rare autosomal recessive lipid-storage disorder characterised by xanthomas, neurological dysfunctions and premature atherosclerosis. A case of a well differentiated adenocarcinoma of the gallbladder occurring in a 57-year-old Japanese man with CTX, confirmed clinically, biochemically and at autopsy is reported together with analyses of the sterol 27-hydroxylase (CYP27) and p53 genes. A missense mutation of the p53 (G for C) was detected in the gallbladder adenocarcinoma. Direct sequence analysis also showed a silent mutational substitution of unknown significance, C for A, in CYP27 at codon 89. In the past, CTX patients have only demonstrated this infrequently, indicating no direct relationship between CYP27 dysfunction and tumour development. Thus, the present case of gallbladder cancer appears to be a chance occurrence.

Objective: Tendon xanthomas are deposits of lipids and connective tissue commonly found in hypercholesterolemic patients. Macrophages are likely to be responsible for the lipid accumulation. Normolipidemic patients with the rare disease cerebrotendinous xanthomatosis, lacking the enzyme sterol 27-hydroxylase (CYP27A1), develop prominent xanthomas in tendons and brain containing both cholestanol and cholesterol, with a cholestanol:cholesterol ratio higher than that in the circulation. Because of its ability to convert cholesterol into polar metabolites that leave the cells faster, CYP27A1 has been suggested to be an antiatherogenic enzyme. The hypothesis was tested that tendons contain CYP27A1 that may be of importance for the normal efflux of both steroids., Methods and Results: Western blotting and combined gas chromatography-mass spectrometry showed that human tendons contain significant amounts of CYP27A1 and its product, 27-hydroxycholesterol. Immunohistochemistry showed that CYP27A1 is present in macrophages and tenocytes. The tendons also contained cholestanol, with a cholestanol:cholesterol ratio slightly higher than that in the circulation. Recombinant human CYP27A1, and cultured human macrophages containing this enzyme, had similar activity toward cholesterol and cholestanol. After loading of macrophages with labeled cholesterol and cholestanol, there was an efflux of these steroids in both unmetabolized and 27-oxygenated form, resulting in a significant cellular accumulation of cholestanol compared with cholesterol., Conclusion: The results are consistent with the possibility that CYP27A1 is of importance for the efflux of both cholesterol and cholestanol from tendons.

Cerebrotendinous xanthomatosis (CTX) is a lipid storage disease caused by a deficiency of the mitochondrial enzyme 27-sterol hydroxylase (CYP 27), due to mutations in its gene. In this study we report on mutations in 58 patients with CTX out of 32 unrelated families. Eight of these were novel mutations, two of which were found together with two already known pathogenic mutations. Twelve mutations found in this patient group have been described in the literature. In the patients from 31 families, mutations were found in both alleles. In the literature, 28 mutations in 67 patients with CTX out of 44 families have been described. Pooling our patient group and the patients from the literature together, 37 different mutations in 125 patients out of 74 families were obtained. Identical mutations have been found in families from different ethnic backgrounds. In 41% of all the patients, CYP 27 gene mutations are found in the region of exons 6-8. This region encodes for adrenodoxin and haem binding sites of the protein. Of these 125 patients, a genotype-phenotype analysis was done for 79 homozygous patients harbouring 23 different mutations, out of 45 families. The patients with compound heterozygous mutations were left out of the genotype-phenotype analysis. The genotype-phenotype analysis did not reveal any correlation.

We describe seven Dutch patients from six families with a slowly progressive, mainly spinal cord syndrome that remained for many years the sole expression of cerebrotendinous xanthomatosis (CTX). MRI demonstrated white matter abnormalities in the lateral and dorsal columns of the spinal cord. Post-mortem examination of one of the patients showed extensive myelin loss in these columns. An array of genotypes was found in these patients. We conclude that 'spinal xanthomatosis' is a clinical and radiological separate entity of CTX that should be included in the differential diagnosis of 'chronic myelopathy'.

Cultured macrophages and endothelial cells have been reported to secrete 27-oxygenated metabolites of cholesterol. This mechanism was compared with the classical high density lipoprotein (HDL)-dependent reverse cholesterol transport. Under standard conditions, macrophage preparations had considerably higher capacity to secrete 27-hydroxycholesterol and 3beta-hydroxy-5-cholestenoic acid than had endothelial cells and fibroblasts. Western blotting showed that lung macrophages contained the most sterol 27-hydroxylase protein of the cells tested. The relative amounts of 3beta-hydroxy-5-cholestenoic acid produced by the macrophages were also highest. Macrophages derived from monocytes of patients with sterol 27-hydroxylase deficiency did not secrete 27-oxygenated products, demonstrating that sterol 27-hydroxylase is the critical enzyme for the conversion of cholesterol into the 27-oxygenated steroids. That sterol 27-hydroxylase is responsible not only for 27-hydroxylation of cholesterol but also for the further oxidation of this steroid into 3beta-hydroxy-5-cholestenoic acid was shown with use of tritium-labeled 27-hydroxycholesterol and an inhibitor of sterol 27-hydroxylase. Secretion of 27-oxygenated products by the cultured macrophages as well as the ratio between the alcohol and the acid appeared to be dependent upon total 27-hydroxylase activity, the availability of substrate cholesterol, and the presence of an acceptor for 27-hydroxycholesterol in the medium. With albumin as extracellular acceptor, the major secreted product was 3beta-hydroxy-5-cholestenoic acid. Under such conditions, secretion of labeled 27-oxygenated products was higher than that of labeled cholesterol from lung alveolar macrophages preloaded with [4-14C]cholesterol. With HDL as acceptor, 27-hydroxycholesterol was the major secreted product, and the total secretion of labeled 27-oxygenated products was only about 10% of that of labeled cholesterol. Thus, 27-hydroxycholesterol and cholesterol may compete for HDL-mediated efflux from the cells. The results support the contention that the sterol 27-hydroxylase-mediated elimination of cholesterol is more important in macrophages than in endothelial cells. This mechanism may be an alternative and/or a complement to the classical HDL-mediated reverse cholesterol transport in macrophages, in particular when the concentration of HDL is low.