[Laboratory diagnosis of a rare congenital neurodegenerative disease: cerebrotendinous xanthomatosis]. / Egy ritka, veleszületett neurodegeneratív betegség: a cerebrotendinosus xanthomatosis laboratóriumi diagnosztikája.

Cerebrotendinous xanthomatosis is a rare neurodegenerative disease characterized by the accumulation of cholesterol and cholestanol in the brain and the tendons caused by mutations of the gene encoding sterol 27-hydroxylase (CYP27A1), which is involved in bile acid synthesis. The diagnosis is often missed and delayed because of the variable clinical presentation of the disease. Blood testing for cerebrotendinous xanthomatosis is routinely performed using gas chromatography-mass spectrometry measurement of elevated cholestanol level, and the diagnosis is confirmed by molecular genetic analysis. Early recognition and initiation of chenodeoxycholic acid therapy with hydoxymethyl­glutaryl­Coenzyme-A reductase inhibitors is critical to prevent irreversible neurological damage and permanent disability. The authors summarize the current knowledge about the pathomechanism, laboratory diagnosis and therapeutic options of cerebrotendinous xanthomatosis.

A novel mutation in the sterol 27-hydroxylase gene of a woman with autosomal recessive cerebrotendinous xanthomatosis.

Mutations of the gene encoding the mitochondrial enzyme sterol 27-hydroxylase (CYP27A1 gene) cause defects in the cholesterol pathway to bile acids that lead to the storage of cholestanol and cholesterol in tendons, lenses and the central nervous system. This disorder is the cause of a clinical syndrome known as cerebrotendinous xanthomatosis (CTX). Since 1991 several mutations of the CYP27A1 gene have been reported. We diagnosed the clinical features of CTX in a caucasian woman. Serum levels of cholestanol and 7α-hydroxycholesterol were elevated and the concentration of 27-hydroxycholesterol was reduced. Bile alcohols in the urine and faeces were increased. The analysis of the CYP27A1 gene showed that the patient was a compound heterozygote carrying two mutations both located in exon 8. One mutation is a novel four nucleotide deletion (c.1330-1333delTTCC) that results in a frameshift and the occurrence of a premature stop codon leading to the formation of a truncated protein of 448 amino acids. The other mutation, previously reported, is a C - > T transition (c. c.1381C > T) that converts the glutamine codon at position 461 into a termination codon (p.Q461X). These truncated proteins are expected to have no biological function being devoid of the cysteine residue at position 476 of the normal enzyme that is crucial for heme binding and enzyme activity.

Identification of a novel missense mutation in the sterol 27-hydroxylase gene in two Japanese patients with cerebrotendinous xanthomatosis.

Cerebrotendinous xanthomatosis (CTX) is a rare autosomal recessive sterol storage disease caused by a mutated sterol 27-hydroxylase (CYP27A1) gene. We analyzed the CYP27A1 gene in two Japanese CTX patients. The CYP27A1 gene was amplified by PCR and screened by PCR-SSCP. The nucleotide sequence was analyzed to confirm mutations. Case 1 was a compound heterozygote for Arg104Gln in exon 2 and Arg441Gln in exon 8. To our knowledge, this is the first report in which the Arg104Gln mutation is identified in CTX patients. Probably case 2 would be a compound heterozygote for Arg441Trp in exon 8 and a mutation that was not identified.

On the mechanism of accumulation of cholestanol in the brain of mice with a disruption of sterol 27-hydroxylase.

The rare disease cerebrotendinous xanthomatosis (CTX) is due to a lack of sterol 27-hydroxylase (CYP27A1) and is characterized by cholestanol-containing xanthomas in brain and tendons. Mice with the same defect do not develop xanthomas. The driving force in the development of the xanthomas is likely to be conversion of a bile acid precursor into cholestanol. The mechanism behind the xanthomas in the brain has not been clarified. We demonstrate here that female cyp27a1(-/-) mice have an increase of cholestanol of about 2.5- fold in plasma, 6-fold in tendons, and 12-fold in brain. Treatment of cyp27a1(-/-) mice with 0.05% cholic acid normalized the cholestanol levels in tendons and plasma and reduced the content in the brain. The above changes occurred in parallel with changes in plasma levels of 7alpha-hydroxy-4-cholesten-3-one, a precursor both to bile acids and cholestanol. Injection of a cyp27a1(-/-) mouse with (2)H(7)-labeled 7alpha-hydroxy-4-cholesten-3-one resulted in a significant incorporation of (2)H(7)-cholestanol in the brain. The results are consistent with a concentration-dependent flux of 7alpha-hydroxy-4-cholesten-3-one across the blood-brain barrier in cyp27a1(-/-) mice and subsequent formation of cholestanol. It is suggested that the same mechanism is responsible for accumulation of cholestanol in the brain of patients with CTX.

Four novel CYP27A1 mutations in seven Italian patients with CTX.

BACKGROUND AND PURPOSE: Cerebrotendinous xanthomatosis (CTX) is a rare autosomal recessive disease, because of sterol 27-hydroxylase deficiency. Clinical manifestations of CTX are tendon xanthomas, juvenile cataracts, osteoporosis, diarrhoea and multiple progressive neurological dysfunctions. More than 300 patients with CTX have been reported to date worldwide and about fifty different mutations identified in CYP27A1 gene. This study describes the clinical and laboratory findings of seven new patients. METHODS: We report the molecular and clinical characterization of seven new Italian patients with CTX carrying four novel mutations. RESULTS: We identified four novel mutations located in different exons, in particular in the region of exons 2-5 of the CYP27A1 gene. Phenotypical expression did not differ from classical CTX presentation except for absence of tendon xanthomas in two patients.

An alternative pathway of reverse cholesterol transport: the oxysterol 27-hydroxycholesterol.

Reverse cholesterol transport, although not well understood, is an important mechanism in the pathophysiology of atherosclerosis. Macrophages can eliminate some cholesterol from atherosclerotic lesions by an oxidative mechanism involving sterol 27-hydroxylase. Patients with inherited "cerebrotendinous xanthomatosis" lack sterol 27-hydroxylase (CYP27A1) and develop severe premature atherosclerosis despite normal serum cholesterol concentrations. Thus, it has been speculated that sterol 27-hydroxylase is an anti-atherosclerotic enzyme. Here, we report the case of a 25-year-old patient who presented to our emergency room with an acute non-ST elevation myocardial infarction due to severe coronary heart disease. Lipid analysis revealed dramatically increased 27-hydroxycholesterol and low high-density lipoprotein (HDL)-cholesterol levels. Previous reports suggest that 27-hydroxylase is upregulated to protect peripheral cells from severe cholesterol accumulation, especially in cases of ineffective reverse cholesterol transport due to low HDL-cholesterol levels. Our findings indicate that oxysterols could play an important and so far underestimated role in reverse cholesterol transport.

Cerebrotendinous xanthomatosis (CTX): a treatable lipid storage disease.

Cerebrotendinous xanthomatosis (CTX) is a rare autosomal recessive lipid storage disease with multi-organ involvement. The clinical manifestations usually start at infancy and develop during the first and second decades of life; infantile-onset diarrhea may be the earliest clinical manifestation of CTX. Additional clinical manifestations are juvenile cataracts, tendon xanthomas, and multiple progressive neurological symptoms. Systemic manifestations that are often found include osteoporosis, heart involvement and premature arteriosclerosis. CTX is caused by mutations in the sterol 27 hydroxylase gene (CYP27) on chromosome 2q35-qter, which is responsible for conversion of cholesterol to cholic and chenodeoxycholic acid. Reduced synthesis of cholic and chenodeoxycholic acid results in failed feedback inhibition of cholesterol production, which in turn leads to increased serum cholestanol concentration and elevated urinary bile alcohols. Early treatment with chenodeoxycholic acid (CDCA) prevents the clinical symptoms and prevents deterioration. Although CTX is rare world wide, genetic islands of high frequency have been reported. In this review we would like to familiarize the reader with this fatal inborn error of metabolism that is possibly under-diagnosed and is preventable once recognized and treated.

Effectors of rapid homeostatic responses of endoplasmic reticulum cholesterol and 3-hydroxy-3-methylglutaryl-CoA reductase.

The cholesterol content of the endoplasmic reticulum (ER) and the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) imbedded therein respond homeostatically within minutes to changes in the level of plasma membrane cholesterol. We have now examined the roles of sterol regulatory element-binding protein (SREBP)-dependent gene expression, side chain oxysterol biosynthesis, and cholesterol precursors in the short term regulation of ER cholesterol levels and HMGR activity. We found that SREBP-dependent gene expression is not required for the response to changes in cell cholesterol of either the pool of ER cholesterol or the rate of cholesterol esterification. It was also found that the acute proteolytic inactivation of HMGR triggered by cholesterol loading required the conversion of cholesterol to 27-hydroxycholesterol. High levels of exogenous 24,25-dihydrolanosterol drove the inactivation of HMGR; lanosterol did not. However, purging endogenous 24,25-dihydrolanosterol, lanosterol, and other biosynthetic sterol intermediates by treating cells with NB-598 did not greatly affect either the setting of their ER cholesterol pool or the inactivation of their HMGR. In summary, neither SREBP-regulated genes nor 27-hydroxycholesterol is involved in setting the ER cholesterol pool. On the other hand, 27-hydroxycholesterol, rather than cholesterol itself or biosynthetic precursors of cholesterol, stimulates the rapid inactivation of HMGR in response to high levels of cholesterol.

Mutational analysis of CYP27A1: assessment of 27-hydroxylation of cholesterol and 25-hydroxylation of vitamin D.

The CYP27A1 gene encodes a mitochondrial enzyme that modulates the acidic biosynthetic pathway for bile acids beginning with the 27-hydroxylation of cholesterol. CYP27A1 also 25-hydroxylates vitamin D(3). Gene mutations cause cerebrotendinous xanthomatosis (CTX), an autosomal recessive disorder, and may cause 25-hydroxyvitamin D deficiency and early-onset osteoporosis and fractures in affected patients. To examine the effects of mutations of CYP27A1 on vitamin D and cholesterol hydroxylating activity, recombinant CYP27A1 and mutant complementary DNAs produced by site-directed mutagenesis were stably expressed in either Escherichia coli or COS-1 cells. Activities of wild-type and mutant enzymes were determined with cholesterol, vitamin D(3), and 1alpha-hydroxyvitamin D(3) (1alphaOHD(3)) as substrates. Of the 15 mutants tested, 11 expressed protein and 4 expressed little or no protein. Functional heme activity, estimated by reduced CO difference spectra at 450 nm, was absent in 12 mutants. When expressed in E. coli, 3 mutants, K226R, D321G, and P408S, each known to cause clinically CTX, showed modest decreases in reduced CO spectra peak and either no change or decreases of less than 50% in hydroxylation of cholesterol, vitamin D(3), and 1alphaOHD(3) compared with wild type. When expressed transiently in COS-1 cells, each of these mutants showed 25-hydroxylation activity for 1alphaOHD(3) as well as wild type. Thus, 3 mutants, K226R, D321G, and P408S, known to occur clinically with nonfunctioning mutants, hydroxylated cholesterol, vitamin D(3), and 1alphaOHD(3). How they contribute to the pathogenesis of CTX despite being biologically active in vitro remains to be determined.

Rifampicin-induced CYP3A4 activation in CTX patients cannot replace chenodeoxycholic acid treatment.

Cerebrotendinous xanthomatosis (CTX) is a rare neurodegenerative disorder with cholestanol accumulation resulting from mutations in the sterol 27-hydroxylase gene (CYP27A). Conventional treatment includes chenodeoxycholic acid and HMG-CoA reductase inhibitors. Mice with disrupted Cyp27A (Cyp27 KO) do not show elevated cholestanol levels nor develop CTX manifestations. This phenomenon was proposed to be due to murine CYP3A overexpression leading to an alternative pathway for degradation of bile alcohols including cholestanol. Our objective was to examine the influence of CYP3A4 induction on cholestanol elimination in CTX patients. Rifampicin (600 mg/day x 7 days), known to induce the PXR, and thereby to increase CYP3A activity, was used. The degree of CYP3A4 induction was assessed by comparing midazolam pharmacokinetics before and after rifampicin treatment. Cholestanol levels and cholestanol/cholesterol ratios were assayed during the experimental period and compared to a 3 weeks period without treatment. The results show that despite 60% increase in CYP3A4 activity following rifampicin treatment, there is no significant change in cholestanol levels. We conclude that up-regulated expression of CYP3A affects cholestanol elimination in mice differently as compared to its effect in CTX patients. Therefore, CYP3A4 inducers cannot replace chenodeoxycholic acid for the treatment of CTX.

Mechanism of accumulation of cholesterol and cholestanol in tendons and the role of sterol 27-hydroxylase (CYP27A1).

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.

Two novel mutations in the sterol 27-hydroxylase gene causing cerebrotendinous xanthomatosis.

Cerebrotendinous xanthomatosis (CTX) is a rare recessive autosomal disease caused by mutations of the sterol 27-hydroxylase gene. Clinically, CTX is characterized by tendon xanthomas, cataracts and progressive neurological deficits. Because of the disruption of the 27-hydroxylase activity, CTX patients have elevated plasma levels of cholestanol, a by-product of abnormal bile acid synthesis. The present authors describe a female patient with CTX. The proband in this study presented with elevated cholestanol levels, markedly reduced mitochondrial 27-hydroxylase activity and altered bile acid composition. The 27-hydroxylase gene was analysed for mutations by polymerase chain reaction amplification of the exons and the splice-junction regions of the gene. The proband was found to be a compound heterozygote for two different mutations which have not been previously described: (1) a G --> A transition at nucleotide 455 that is responsible for converting a glycine to a glutamic acid residue at amino acid position 112 (G112E); and (2) a five-nucleotide deletion in exon 5 (from nucleotide 965 to 969) that is responsible for a shift in the reading frame and the insertion of a premature codon at position 296, and consequently, the synthesis of a truncated protein lacking the heme-binding and andrenodoxin-binding domains. Long-term (18-year) treatment of the proband with chenodeoxycholic acid (750 mg day-1) has been effective in preventing any progression of the disease.

Structure-function analysis of CYP27B1 and CYP27A1. Studies on mutants from patients with vitamin D-dependent rickets type I (VDDR-I) and cerebrotendinous xanthomatosis (CTX).

We have determined eight types of missense mutants of CYP27B1 from Japanese vitamin D-dependent rickets type I (VDDR-I) patients [Kitanaka, S., Takeyama, K., Murayama, A., Sato, T., Okumura, K., Nogami, M., Hasegawa, Y., Niimi, H., Yanagisawa, J., Tanaka, T. & Kato, S. (1998) New England J. Med., 338, 653-661 and Kitanaka, S., Murayama, A., Sakaki, T., Inouye, K., Seino, Y., Fukumoto, S., Shima, M., Yukizane, S., Takayanagi, M., Niimi, H., Takeyama, K. & Kato, S. (1999) J. Clin. Endocrine Metab., 84, 4111-4117]. None of the CYP27B1 mutants showed 1alpha-hydroxylase activity towards 25-hydroxyvitamin D3. Thus, it was assumed that the mutated amino-acid residues play important roles in the 1alpha-hydroxylase activity, such as substrate binding, activation of molecular oxygen, interaction with adrenodoxin, and folding of the cytochrome P450 structure. To examine our hypothesis, we generated various mutants of CYP27B1 and studied their enzymatic properties. In addition, the corresponding mutations were introduced to CYP27A1, which belongs to the same family as CYP27B1. As CYP27A1 showed much higher expression level than CYP27B1 in Escherichia coli, further analysis including heme-binding and substrate-binding was performed with CYP27A1 in place of CYP27B1. Western blot analysis, spectral analysis including reduced CO-difference spectra and substrate-induced difference spectra, and enzymatic analysis of the mutant CYP27A1 gave information on the structure-function relationships of both CYP27A1 and CYP27B1. Although the sequence alignment suggested that Arg107, Gly125, and Pro497 of CYP27B1 might be involved in substrate binding, the experimental data strongly suggested that mutations of these amino-acid residues destroyed the tertiary structure of the substrate-heme pocket. It was also suggested that Arg389 and Arg453 of CYP27B1 were involved in heme-propionate binding, and Asp164 stabilized the four-helix bundle consisting of D, E, I and J helices, possibly by forming a salt bridge. Thr321 was found to be responsible for the activation of molecular oxygen.

Sterol 27-hydroxylase acts on 7-ketocholesterol in human atherosclerotic lesions and macrophages in culture.

27-Hydroxycholesterol (27OH) is the major oxysterol in human atherosclerotic lesions, followed by 7-ketocholesterol (7K). Whereas 7K probably originates nonenzymically, 27OH arises by the action of sterol 27-hydroxylase, a cytochrome P450 enzyme expressed at particularly high levels in the macrophage and proposed to represent an important pathway by which macrophages eliminate excess cholesterol. We hypothesized and here show that 27-hydroxylated 7-ketocholesterol (270H-7K) is present in human lesions, probably generated by the action of sterol 27-hydroxylase on 7K. Moreover, [(3)H]27OH-7K was produced by human monocyte-derived macrophages (HMDMs) supplied with [(3)H]7K but not in HMDMs from a patient with cerebrotendinous xanthomatosis (CTX) shown to have a splice-junction mutation of sterol 27-hydroxylase. Whereas [(3)H]27OH-7K was predominantly secreted into the medium, [(3)H]-27OH formed from [(3)H]-cholesterol was mostly cell-associated. The majority of supplied [(3)H]7K was metabolized beyond 27OH-7K to aqueous-soluble products (apparently bile acids derived from the sterol 27-hydroxylase pathway). Metabolism to aqueous-soluble products was ablated by a sterol 27-hydroxylase inhibitor and absent in CTX cells. Sterol 27-hydroxylase therefore appears to represent an important pathway by which macrophages eliminate not only cholesterol but also oxysterols such as 7K. The fact that 7K (and cholesterol) still accumulates in lesions and foam cells indicates that this pathway may be perturbed in atherosclerosis and affords a new opportunity for the development of therapeutic strategies to regress atherosclerotic lesions.

Sudden death due to cerebrotendinous xanthomatosis confirmed by mutation analysis.

A case of sudden death of a 52-year-old mentally retarded Caucasian male is described where the rectal temperature was 43.4 degrees C 3 h postmortem. The autopsy revealed cerebrotendinous xanthomatosis (CTX), a rare hereditary metabolic disorder, as the primary disease. The diagnosis was confirmed by postmortem identification of two mutations (compound heterozygosity for R237X and IVS6+1G-->A) in the sterol 27-hydroxylase (CYP27) gene. Both mutations have already been described in patients with CTX and can be considered the most likely cause of the disease. The pathomechanism of the excessive hyperthermia could not be completely elucidated.

A novel Arg362Ser mutation in the sterol 27-hydroxylase gene (CYP27): its effects on pre-mRNA splicing and enzyme activity.

A novel C to A mutation in the sterol 27-hydroxylase gene (CYP27) was identified by sequencing amplified CYP27 gene products from a patient with cerebrotendinous xanthomatosis (CTX). The mutation changed the adrenodoxin cofactor binding residue 362Arg to 362Ser (CGT 362Arg to AGT 362Ser), and was responsible for deficiency in the sterol 27-hydroxylase activity, as confirmed by expression of mutant cDNA into COS-1 cells. Quantitative analysis showed that the expression of CYP27 gene mRNA in the patient represented 52.5% of the normal level. As the mutation occurred at the penultimate nucleotide of exon 6 (-2 position of exon 6-intron 6 splice site) of the gene, we hypothesized that the mutation may partially affect the normal splicing efficiency in exon 6 and cause alternative splicing elsewhere, which resulted in decreased transcript in the patient. Transfection of constructed minigenes, with or without the mutation, into COS-1 cells confirmed that the mutant minigene was responsible for a mRNA species alternatively spliced at an activated cryptic 5' splice site 88 bp upstream from the 3' end of exon 6. Our data suggest that the C to A mutation at the penultimate nucleotide of exon 6 of the CYP27 gene not only causes the deficiency in the sterol 27-hydroxylase activity, but also partially leads to alternative pre-mRNA splicing of the gene. To our knowledge, this is the first report regarding effects on pre-mRNA splicing of a mutation at the -2 position of a 5' splice site.

Markedly reduced bile acid synthesis but maintained levels of cholesterol and vitamin D metabolites in mice with disrupted sterol 27-hydroxylase gene.

Sterol 27-hydroxylase is important for the degradation of the steroid side chain in conversion of cholesterol into bile acids and has been ascribed a regulatory role in cholesterol homeostasis. Its deficiency causes the autosomal recessive disease cerebrotendinous xanthomatosis (CTX), characterized by progressive dementia, xanthomatosis, and accelerated atherosclerosis. Mice with a disrupted cyp27 (cyp27(-/-)) had normal plasma levels of cholesterol, retinol, tocopherol, and 1,25-dihydroxyvitamin D. Excretion of fecal bile acids was decreased (<20% of normal), and formation of bile acids from tritium-labeled 7alpha-hydroxycholesterol was less than 15% of normal. Compensatory up-regulation of hepatic cholesterol 7alpha-hydroxylase and hydroxymethylglutaryl-CoA reductase (9- and 2-3-fold increases in mRNA levels, respectively) was found. No CTX-related pathological abnormalities were observed. In CTX, there is an increased formation of 25-hydroxylated bile alcohols and cholestanol. In bile and feces of the cyp27(-/-) mice only traces of bile alcohols were found, and there was no cholestanol accumulation. It is evident that sterol 27-hydroxylase is more important for bile acid synthesis in mice than in humans. The results do not support the contention that 27-hydroxylated steroids are critical for maintenance of cholesterol homeostasis or levels of vitamin D metabolites in the circulation.