Novel splice-affecting variants in CYP27A1 gene in two Chilean patients with Cerebrotendinous Xanthomatosis

Cerebrotendinous Xanthomatosis (CTX), a rare lipid storage disorder, is caused by recessive loss-of-function mutations of the 27-sterol hydroxylase (CYP27A1), producing an alteration of the synthesis of bile acids, with an accumulation of cholestanol. Clinical characteristics include juvenile cataracts, diarrhea, tendon xanthomas, cognitive impairment and other neurological manifestations. Early diagnosis is critical, because treatment with chenodeoxycholic acid may prevent neurological damage. We studied the CYP27A1 gene in two Chilean CTX patients by sequencing its nine exons, exon-intron boundaries, and cDNA from peripheral blood mononuclear cells. Patient 1 is a compound heterozygote for the novel substitution c.256-1G > T that causes exon 2 skipping, leading to a premature stop codon in exon 3, and for the previously-known pathogenic mutation c.1183C > T (p.Arg395Cys). Patient 2 is homozygous for the novel mutation c.1185-1G > A that causes exon 7 skipping and the generation of a premature stop codon in exon 8, leading to the loss of the crucial adrenoxin binding domain of CYP27A1.     

Exon skipping in the sterol 27-hydroxylase gene leads to cerebrotendinous xanthomatosis

We report a new mutation in the sterol 27-hydroxylase (CYP 27) gene in a Dutch family with cerebrotendinous xanthomatosis: a G→A transition in the splice donor site in intron 4. This mutation leads to skipping of exon 4, resulting in a loss of 66 amino acids in the CYP 27 enzyme molecule. Received: 15 March 1997 / Accepted: 26 March 1997     

Premature termination codon at the sterol 27-hydroxylase gene causes cerebrotendinous xanthomatosis in a French family

Cerebrotendinous xanthomatosis (CTX) is an autosomal recessive lipid-storage disease caused by mutations in the sterol 27-hydroxylase gene (CYP27). So far several mutations causing CTX have been identified and characterized. A new mutation creating an insertion of cytosine at position 6 in the cDNA, which is expected to result in a frameshift and a premature termination codon at codon 179, has been identified in a French family. The mutation creates a new site for the restriction endonuclease HaeIII.     

Two new mutations in the sterol 27-hydroxylase gene in two families lead to cerebrotendinous xanthomatosis

This report concerns two new mutations in the sterol 27-hydroxylase gene in two patients with cerebrotendinous xanthomatosis (CTX). In a Surinam-Creole patient (patient A), a G deletion on position cDNA 546/547 in exon 3 led to a frameshift and the introduction of a premature termination codon. In a Dutch patient (patient B), a C→T transition at position 496 in exon 3 also led to a premature termination codon. Patient A was homozygous for the mutation, whereas patient B was compound heterozygous, a C→T transition also being found in exon 6 at position 1204. The two new mutations were confirmed by restriction analysis with the restriction enzymes FokI and MaeI, respectively. Received: 24 July 1996 / Revised: 9 August 1996     

Localization of sterol 27-hydroxylase immuno-reactivity in human atherosclerotic plaques

It has recently been shown that extrahepatic cells can eliminate intracellular cholesterol by enzymatic conversion into 27-hydroxy-cholesterol and 3β-hydroxy-5-cholestenoic acid. Using immunohistochemical methods, we studied the presence of the enzyme responsible for these conversions, sterol 27-hydroxylase, in human carotid atherosclerotic plaques. All plaques examined were found to contain sterol 27-hydroxylase immuno-reactive cells. While some endothelial cells stained for sterol 27-hydroxylase, the majority of the immunoreactive cells co-localized with macrophages. Accumulation of sterol 27-hydroxylase-positive cells were often observed in macrophage-rich core regions of complicated lesions. High concentrations of 27-hydroxycholesterol were found in plaques, while the concentration in non-atherosclerotic human vessels was lower by two orders of magnitude. The rabbit, which is particularly sensitive to dietary cholesterol and easily develops fatty streaks, had low plasma levels of 27-hydroxycholesterol, 3 ng/ml compared to 150 ng/ml in humans. The concentration of 27-hydroxycholesterol in the atherosclerotic rabbit vessels was also lower compared to human atherosclerotic plaques. The results are consistent with our hypothesis that sterol 27-hydroxylase may be utilized by human macrophages as a defence towards a high cholesterol load. This mechanism may be less important in some other species.     

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 7α-hydroxy-4-cholesten-3-one, a precursor both to bile acids and cholestanol. Injection of a cyp27a1^−/− mouse with ²H₇-labeled 7α-hydroxy-4-cholesten-3-one resulted in a significant incorporation of ²H₇-cholestanol in the brain. The results are consistent with a concentration-dependent flux of 7α-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.     

Expression of sterol 27-hydroxylase (CYP27A1) enhances cholesterol efflux

Cholesterol efflux from CHOP cells transfected with sterol 27-hydroxylase (CYP27A1) was compared with non-transfected and mock-transfected cells. Transfection caused expression of CYP27A1, formation of 27-hydroxycholesterol, and inhibition of cholesterol biosynthesis. Transfection enhanced cholesterol efflux to apolipoprotein A-I or human plasma by 2-3-fold but did not affect the efflux in the absence of acceptor. The analysis of released sterols revealed that 27-hydroxycholesterol represented only a small proportion of sterols, most of which was non-oxidized cholesterol. Time course and dose dependence studies showed that expression of CYP27A1 in CHOP cells mostly affected the efflux of the "fast" cholesterol pool, and relatively more cholesterol was released with low concentrations of an acceptor. Preincubation of non-transfected cells with exogenous 27-hydroxycholesterol (10(-9) and 10(-7) m) led to the stimulation of cholesterol efflux by 24-60%. Expression of CYP27A1 in CHOP cells did not affect ABCA1 expression and abundance of ABCA1 protein. Thus, introduction of CYP27A1 into cells stimulates cholesterol efflux and therefore may increase protection against atherosclerosis.     

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

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.     

Novel double deletions in the MECP2 gene in Tunisian Rett patient

Rett syndrome (RTT) is a severe neurodevelopmental disorder affecting almost exclusively girls. Rett patients present an apparently normal psychomotor development during the first 6-18 months of life. Thereafter, they show a short period of developmental stagnation followed by a rapid regression in language and motor development. RTT is currently considered as monogenic X-linked dominant disorder due to mutations in the MECP2 gene, encoding the methyl-CpG binding protein 2. The aim of this study was to perform a mutational analysis of the MECP2 gene in a classical Rett patient.The results showed the presence of a novel point mutation c.C1142T (p.P381L) and two deletions at the heterozygous state: a novel deletion c.1075delTTC (p.S359) and a known one c.1157del44 (p.L386Q fs X2) in the C-terminal region of MeCP2.     

Assay method for mitochondrial sterol 27-hydroxylase with 7alpha-hydroxy-4-cholesten-3-one as a substrate in the rat liver

Mitochondrial sterol 27-hydroxylase (EC 1.14.13.15) is an important enzyme, not only in the formation of bile acids from cholesterol intermediates in the liver but also in the removal of cholesterol by side chain hydroxylation in extrahepatic tissues. The enzyme has been assayed by complicated methods using radiolabeled substrates or deuterium-labeled tracers. These methods may be inaccurate for measuring enzyme activity, because the amount of electron-transferring proteins may be insufficient for maximal velocity. To solve this problem, after solubilization of the enzyme from rat liver mitochondria with n-octyl-beta-d-glucopyranoside (OGP), we measured the enzyme activity by incubating the solubilized enzyme with saturated amounts of electron-transferring proteins. In our assay system, using 7alpha-hydroxy-4-cholesten-3-one (HCO) as a substrate, we could easily measure the product, 7alpha,27-dihydroxy-4-cholesten-3-one, with HPLC monitoring absorbance at 240 nm. The product formation was proportionate to the time up to 5 min and the protein concentration up to 0.5 mg of protein/ml. The maximal velocity of the enzyme was 1.1 nmol/min/mg of protein, which was 4- to 16-fold higher than previously reported values. A simple and accurate assay method for sterol 27-hydroxylase in rat liver mitochondria is herein described.     

Human sterol 27-hydroxylase (CYP27) overexpressor transgenic mouse model. Evidence against 27-hydroxycholesterol as a critical regulator of cholesterol homeostasis

CYP27-overexpressed transgenic mice were generated with the use of a human full-length CYP27 coding region cloned into a ubiquitous expression vector. Positive transgenic mice were identified by tail DNA genotyping and high fecal 27-hydroxycholesterol content. The levels of 27-hydroxycholesterol were found to be 3-5 times higher in the circulation and the tissues of the overexpressed mice when compared with littermate controls. There were no gross morphological differences between the overexpressed mice and their controls. Total cholesterol and triglyceride levels were not affected by overexpression of CYP27. Serum lathosterol was also normal, suggesting a normal rate of cholesterol synthesis. Serum levels of 7alpha-hydroxycholesterol were unaffected, suggesting a normal rate of bile acid formation in the pathway involving cholesterol 7alpha-hydroxylase. Biliary bile acid composition was slightly affected by CYP27 overexpression in female but not in male mice. Fecal levels of neutral steroids were slightly but significantly increased in overexpressor female mice but not in male mice. Levels of 24-hydroxycholesterol in the circulation were significantly reduced in the overexpressed mice, probably as a consequence of a recently described catabolic pathway involving CYP27. Combined with the results of our previous work on mice with a disruption of the CYP27 gene, the present results suggest that the levels of 27-hydroxycholesterol are not of critical importance for cholesterol homeostasis in mice.     

Regulation of sterol 27-hydroxylase in human monocyte-derived macrophages: up-regulation by transforming growth factor beta1

Regulatory mechanisms for human CYP27A1 enzyme have not yet been fully investigated. Our approach was to add different hormones and cytokines to cultured human monocyte-derived macrophages, and assess the effects on the CYP27A1 by measuring the production of 27-hydroxylated cholesterol in the media. Of the different hormones and cytokines tested, only transforming growth factor beta1 (TGF-beta1) had a clear effect on CYP27A1. Further experiments showed a significant increase in 27-hydroxylated cholesterol products (27-hydroxycholesterol and 3beta-hydroxy-5-cholestenoic acid). A concomitant increase in CYP27A1 mRNA levels was also seen and this positive effect was confirmed using a human CYP27A1 luciferase reporter gene expressed in HepG2 cells. Experiments with progressive deletion/luciferase reporter gene constructs indicated that a TGF-beta1 responsive sequence might be localized in a region about 400 bp upstream of the CYP27A1 translation start. The possibility is discussed that induction of CYP27A1 by TGF-beta1 may be responsible for some of the anti-atherogenic properties of this cytokine.     

Identification of a Novel Mutation in the COL2A1 Gene in a Chinese Family with Spondyloepiphyseal Dysplasia Congenita

Spondyloepiphyseal dysplasia congenita (SEDC) is an autosomal dominant chondrodysplasia characterized by disproportionate short-trunk dwarfism, skeletal and vertebral deformities. Exome sequencing and Sanger sequencing were performed in a Chinese Han family with typical SEDC, and a novel mutation, c.620G>A (p.Gly207Glu), in the collagen type II alpha-1 gene (COL2A1) was identified. The mutation may impair protein stability, and lead to dysfunction of type II collagen. Family-based study suggested that the mutation is a de novo mutation. Our study extends the mutation spectrum of SEDC and confirms genotype-phenotype relationship between mutations at glycine in the triple helix of the alpha-1(II) chains of the COL2A1 and clinical findings of SEDC, which may be helpful in the genetic counseling of patients with SEDC.     

Modulation of cholesterol 7alpha-hydroxylase and sterol 27-hydroxylase activities by steroids and physiological conditions in hamster.

Our purpose was to examine the in vitro modulation of liver mitochondrial sterol 27-hydroxylase (S27OHase) and microsomal cholesterol 7alpha-hydroxylase (CH7alphaOHase) activities by certain drugs, sterols, oxysterols and bile acids, and to compare the influence of sex, age, diet and cholestyramine on these activities, in the hamster. In vitro, 7beta-hydroxycholesterol and 5alpha-cholestan-3beta-ol (cholestanol) were strong inhibitors (at 2 microM) of both enzyme activities, while 5beta-cholestan-3alpha-ol (epicoprostanol, 2 microM) and cyclosporin A (20 microM) inhibited S27OHase, but not CH7alphaOHase. These data suggest that a hydroxyl group at the 7alpha position is not required to inhibit CH7alphaOHase and that the presence of an aliphatic CH2-CH-(CH3)2 chain appears to be structurally important for S27OHase activity. Both enzyme activities remained unchanged by hyodeoxycholic acid (40 or 80 microM) while epicoprostanol inhibited only S27OHase and chenodeoxycholic acid only CH7alphaOHase. Adult (9-week old) male or female hamsters displayed similar S27OHase activity but the CH7alphaOHase activity was lower in females than in males, suggesting that the neutral bile acid pathway has a less important role in females. In male hamsters, S27OHase activity did not change with age, while CH7alphaOHase activity significantly increased (one-year vs 9-week old). A semi-purified sucrose-rich (lithogenic) diet significantly lowered both enzyme activities compared to the commercial diet. Cholestyramine induced a stimulation of both enzymes, slightly more vigorously however for the key enzyme involved in the neutral pathway. Taken together, these data indicate that the two enzymes are separately regulated and that certain drugs or steroid compounds can be useful for specifically inhibiting or stimulating the neutral or acidic bile acid pathway.     

A Novel Cysteine-Sparing NOTCH3 Mutation in a Chinese Family with CADASIL

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an adult onset cerebral small vessel disorder caused by the mutations of the neurogenic locus notch homolog protein 3 (NOTCH3) gene. The extracellular part of NOTCH3 is composed of 34 epidermal growth factor-like (EGF-like) repeat domains. Each EGF-like domain is rich of cysteine and glycine to produce three loops that are essential for high-affinity binding to its ligand. Nearly all reported CADASIL-associated mutations result in gain or loss of a cysteine residue within the EGF-like domains. Only a few cysteine-sparing NOTCH3 mutations have been documented in the patients with CADASIL to date. Here, we reported a Chinese CADASIL family with a cysteine-sparing NOTCH3 mutation. In this family, affected patients had dizziness, memory loss, gait instability, or hemiplegia. Brain magnetic resonance imaging (MRI) showed diffuse leukoencephalopathy with confluent signal abnormalities in the periventricular white matter, basal ganglia, and centrum semiovale bilaterally. By screening the entire coding region of NOTCH3, a novel missense mutation p.G149V (c.446G>T) was found. This mutation was not detected in 400 normal controls. Considering the critical position of glycine within the C-loop of EGF-like domain and its high conservation through evolution, p.G149V mutation could be a potential pathogenic cause for CADASIL.     

Regulation of sterol 27-hydroxylase and an alternative pathway of bile acid biosynthesis in primary cultures of rat hepatocytes

In man, hepatic mitochondrial sterol 27-hydroxylase and microsomal cholesterol 7-hydroxylase initiate distinct pathways of bile acid biosynthesis from cholesterol, the “acidic” and “neutral” pathways, respectively. A similar acidic pathway in the rat has been hypothesized, but its quantitative importance and ability to be regulated at the level of sterol 27-hydroxylase are uncertain. In this study, we explored the molecular regulation of sterol 27-hydroxylase and the acidic pathway of bile acid biosynthesis in primary cultures of adult rat hepatocytes. mRNA and protein turnover rates were approximately 10-fold slower for sterol 27-hydroxylase than for cholesterol 7-hydroxylase. Sterol 27-hydroxylase mRNA was not spontaneously expressed in culture. The sole requirement for preserving sterol 27-hydroxylase mRNA at the level of freshly isolated hepatocytes (0 h) after 72 h was the addition of dexamethasone (0.1 μM; > 7-fold induction). Sterol 27-hydroxylase mRNA, mass and specific activity were not affected by thyroxine (1.0 μM), dibutyryl-cAMP (50 μM), nor squalestatin 1 (150 nM-1.0 μM), an inhibitor of cholesterol biosynthesis. Taurocholate (50 μM), however, repressed sterol 27-hydroxylase mRNA levels by 55%. Sterol 27-hydroxylase specific activity in isolated mitochondria was increased > 10-fold by the addition of 2-hydroxypropyl-β-cyclodextrin. Under culture conditions designed to maximally repress cholesterol 7-hydroxylase and bile acid synthesis from the neutral pathway but maintain sterol 27-hydroxylase mRNA and activity near 0 h levels, bile acid synthesis from [¹⁴C]cholesterol remained relatively high and consisted of β-muricholate, the product of chenodeoxycholate in the rat. We conclude that rat liver harbors a quantitatively important alternative pathway of bile acid biosynthesis and that its initiating enzyme, sterol 27-hydroxylase, may be slowly regulated by glucocorticoids and bile acids.     

Marked induction of sterol 27-hydroxylase activity and mRNA levels during differentiation of human cultured monocytes into macrophages

Sterol 27-hydroxylase has been suggested to be involved in an alternative pathway for the elimination of cholesterol from macrophages and early atherosclerotic lesions. We have previously shown that human lung macrophages as well as monocyte-derived macrophages have a relatively high activity of sterol 27-hydroxylase (CYP27). This enzyme converts intracellular cholesterol into 27-hydroxycholesterol and cholestenoic acid that flux from cultured cells into the medium. It is shown here that human monocytes have very low CYP27 activity and CYP27 mRNA levels. During differentiation into macrophages, both CYP27 activity and CYP27 mRNA levels increase markedly after 4 days of culture in serum-free medium. Addition of macrophage-colony stimulating factor had no significant effect on the induction and addition of fetal calf serum had an inhibitory effect. Cholesterol synthesis was found to be a critical factor for the production of 27-oxygenated products by the macrophages cultured in serum-free medium. The increased capacity of the differentiated cells to eliminate intracellular cholesterol is of interest and supports the contention that CYP27 is an antiatherogenic factor.