December 2009
Volume 50, Issue 12
Free
Glaucoma  |   December 2009
Primary Open-Angle Glaucoma: Association with Cholesterol 24S-Hydroxylase (CYP46A1) Gene Polymorphism and Plasma 24-Hydroxycholesterol Levels
Author Affiliations & Notes
  • Cynthia Fourgeux
    From the Eye and Nutrition Research Group, UMR1129 FLAVIC (Flaveur, vision et comportement du consommateur; Flavor, Vision, and Consumer Behavior), INRA (French National Institute for Agricultural Research), Dijon, France;
  • Lucy Martine
    From the Eye and Nutrition Research Group, UMR1129 FLAVIC (Flaveur, vision et comportement du consommateur; Flavor, Vision, and Consumer Behavior), INRA (French National Institute for Agricultural Research), Dijon, France;
  • Ingemar Björkhem
    the Department of Laboratory Medicine, Division of Clinical Chemistry, Karolinska University Hospital Huddinge, Stockholm, Sweden;
  • Ulf Diczfalusy
    the Department of Laboratory Medicine, Division of Clinical Chemistry, Karolinska University Hospital Huddinge, Stockholm, Sweden;
  • Corinne Joffre
    From the Eye and Nutrition Research Group, UMR1129 FLAVIC (Flaveur, vision et comportement du consommateur; Flavor, Vision, and Consumer Behavior), INRA (French National Institute for Agricultural Research), Dijon, France;
  • Niyazi Acar
    From the Eye and Nutrition Research Group, UMR1129 FLAVIC (Flaveur, vision et comportement du consommateur; Flavor, Vision, and Consumer Behavior), INRA (French National Institute for Agricultural Research), Dijon, France;
  • Catherine Creuzot-Garcher
    the Department of Ophthalmology, University Hospital, Dijon, France; and
    the Eye and Nutrition Research Group, UMR1129 FLAVIC, University de Burgundy, Dijon, France.
  • Alain Bron
    the Department of Ophthalmology, University Hospital, Dijon, France; and
    the Eye and Nutrition Research Group, UMR1129 FLAVIC, University de Burgundy, Dijon, France.
  • Lionel Bretillon
    From the Eye and Nutrition Research Group, UMR1129 FLAVIC (Flaveur, vision et comportement du consommateur; Flavor, Vision, and Consumer Behavior), INRA (French National Institute for Agricultural Research), Dijon, France;
  • Corresponding author: Lionel Bretillon, INRA, UMR1129 FLAVIC, Equipe Oeil et Nutrition, 17 rue Sully, BP86510, F-21065 Dijon, France; lionel.bretillon@dijon.inra.fr
Investigative Ophthalmology & Visual Science December 2009, Vol.50, 5712-5717. doi:10.1167/iovs.09-3655
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Cynthia Fourgeux, Lucy Martine, Ingemar Björkhem, Ulf Diczfalusy, Corinne Joffre, Niyazi Acar, Catherine Creuzot-Garcher, Alain Bron, Lionel Bretillon; Primary Open-Angle Glaucoma: Association with Cholesterol 24S-Hydroxylase (CYP46A1) Gene Polymorphism and Plasma 24-Hydroxycholesterol Levels. Invest. Ophthalmol. Vis. Sci. 2009;50(12):5712-5717. doi: 10.1167/iovs.09-3655.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose.: Genetics has made significant contributions to the study of glaucoma over the past few decades. Cholesterol-24S-hydroxylase (CYP46A1) is a cholesterol-metabolizing enzyme that is especially expressed in retinal ganglion cells. CYP46A1 and its metabolic product, 24S-hydroxycholesterol, have been linked to neurodegeneration. A single-nucleotide polymorphism (SNP) in the CYP46A1 gene, designated as rs754203 and associated with Alzheimer disease, was evaluated as a genetic risk factor for primary open-angle glaucoma (POAG), as well as plasma 24S-hydroxycholesterol levels.

Methods.: The frequency of the CYP46*C and CYP46*T alleles was analyzed in 150 patients with POAG and 118 control subjects. Plasma 24S-hydroxycholesterol levels were quantified. Sex, age, alleles, and genotype frequencies between patients with POAG and control subjects were compared by using the χ2 and Student's t-tests. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by logistic regression to assess the relative association between disease and age, sex, and genotypes.

Results.: The frequency of the TT genotype was significantly higher in patients with POAG than in control subjects (61.3% versus 48.3%, respectively, OR = 1.26; 95% CI = 1.006–1.574, P < 0.05). Plasma 24S-hydroxycholesterol levels did not differ between control subjects and patients with POAG. The ratio of estimated brain weight to liver volume as an estimate of the capacity of the human body to synthesize and metabolize 24S-hydroxycholesterol was found to correlate to plasma 24S-hydroxycholesterol in control subjects and patients with POAG.

Conclusions.: The rs754203 SNP in CYP46A1 was associated with a risk for POAG. This polymorphism was not associated with changes in plasma 24S-hydroxycholesterol, highlighting that despite its retinal origin, 24S-hydroxycholesterol cannot be used as a biomarker for POAG.

Glaucoma is the second leading cause of blindness worldwide, affecting more than 50 million people. 1 Glaucoma is a progressive optic neuropathy characterized by optic nerve head changes and visual field loss. 2 Improving case identification and providing a better understanding of the causes of glaucoma has brought about great progress in defining risk factors such as high intraocular pressure, age, familial history, ethnicity, and myopia, which have been associated frequently with primary open-angle glaucoma (POAG). 3 Genetics has substantially contributed to the study of glaucoma over the past few decades. Myocilin has been identified and validated as the first gene linked to more than 10% of juvenile-onset POAG cases and 4% of adult-onset POAG cases. 4,5 Other genes such as optineurin, WDR36, and CYP1B1 have been found more frequently in certain clinical presentations of glaucoma. Nevertheless, the link between the genes and the development of the disease is not always clearly demonstrated. 6 Other associations such as diabetes or systemic hypertension are still being debated. The links between dyslipidemia and particularly cholesterol abnormalities have already been described. 7 Recent studies have reported the efficacy of statins and nonstatin cholesterol-lowering drugs in the treatment of POAG. 810 Although the mechanism underlying this association remains unclear, these data suggest that reducing cholesterol biosynthesis may be a target for POAG treatment. Increasing cholesterol efflux and/or enhancing cholesterol catabolism may also be critical. 
We 11 and others 12 recently reported that cholesterol 24S-hydroxylase (CYP46A1) is exclusively expressed in the neuronal part of the retina, most particularly in retinal ganglion cells. This finding consistently supports the possible role played by CYP46A1 in cholesterol homeostasis in ocular structures. Cholesterol-24S-hydroxylase was initially identified as a cholesterol-metabolizing enzyme in the brain. Therein, it catalyzes the formation of 24S-hydroxycholesterol, a polar metabolite of cholesterol that can cross the blood–brain barrier. 1317 In addition to their role in disease-free conditions, cholesterol-24S-hydroxylase (CYP46A1) and 24S-hydroxycholesterol have been linked to neurologic disorders of the central nervous system 1820 and potentially to neuronal death. 19 Single-nucleotide polymorphisms (SNPs) have been identified in the CYP46A1 gene. In some 2132 but not all 28,3339 studies, this type of polymorphism has been associated with increased risk for late-onset Alzheimer disease. Surprisingly, whether this intronic polymorphism affects the P450 mRNA and protein levels has not yet been studied. Therefore, today the mechanistic relation between the polymorphism and the apparent effect on the risk of Alzheimer disease remains unknown. 
The purpose of this study was to explore the association of rs754203 SNP in intron 2 of the CYP46A1 gene with the risk of POAG in a population from a single center in France and to evaluate plasma levels of the cholesterol metabolite formed by CYP46A1 activity, 24S-hydroxycholesterol. 
Methods
Subjects
The protocol adhered to the tenets of the Declaration of Helsinki. This cross-sectional study was approved by the Ethics Committee of Burgundy (France). Informed consent was obtained from all subjects before participation. 
Blood samples were collected from 150 patients with glaucoma and 118 control subjects after an overnight fast at the Department of Ophthalmology, Dijon University Hospital, Dijon, France. None of the subjects was related to any other. All patients included in the study were examined by a glaucoma specialist and underwent a thorough ocular examination. The patients had been observed in our clinic for several years. They did not have a history of neurologic disease or dyslipidemia. Those with angle-closure glaucomas and secondary glaucomas were excluded. POAG was defined by an open angle on gonioscopy, characteristic glaucomatous optic nerve head abnormalities, and corresponding reliable visual field defects recorded with SITA standard white-on-white perimetry (Humphrey Field Analyzer HFA-II; Carl Zeiss Meditec, Paris, France). Among the 150 patients, 18 had normal-tension glaucoma and four had exfoliative glaucoma. The glaucoma stages were defined according to the classification of Hoddap et al. 40 The control subjects were age matched and recruited among patients operated on for cataract or lid surgery and willing to participate in the study. They had an IOP below 21 mm Hg, no evidence of glaucoma on optic disc examination, and a normal frequency-doubling technique screening test result. The brain weight and liver volume were calculated using size and weight data and established formulas from the literature. 4143  
CYP46A1 Genotyping
Buffy coats were placed on DNA-preserving paper (FTA; Whatman, Kent, UK) and stored at room temperature in the dark according to the manufacturer's instructions. A 1.25-mm sample disc was taken for each PCR reaction. A 285-bp PCR product was amplified by using specific primers for rs754203 SNP (forward primer: 5′-TGAAAACGAGTTTCCCGTCC-3′; reverse primer: 5′-GTGTGACCAGGTAACAGTCA-3′) in a 12.5-μL volume reaction with 1.25 U of DNA polymerase, 1.5 mM of MgCl2, 1.25 μL of 10× NH4 buffer, 200 μM dNTPs (BioTaq; Bioline GmbH, Luckenwalde, Germany). 
After initial denaturation at 95°C for 8 minutes, the reaction mixture was subjected to 50 cycles of 1-minute denaturation at 95°C, 1-minute annealing at 53°C, and a 2-minute extension at 72°C, followed by a final extension at 72°C for 5 minutes. The PCR products were digested by MspI (Promega, Charbonnières, France). The fragment amplification and digestion results were revealed by 1.8% agarose gel electrophoresis and ethidium bromide staining. The CYP46A1*T allele corresponded to the uncut 285-bp fragment, whereas the CYP46A1*C allele was characterized by two fragments of 209 and 76 bp. 
Quantification of Plasma 24S-hydroxycholesterol Levels
Plasma 24S-hydroxycholesterol levels were assayed by isotope dilution mass spectrometry using racemic [23,23,24-2H3]-24-hydroxycholesterol, as previously described. 14,17,18,44 Briefly, deuterated 24-hydroxycholesterol (200 ng, [23,23,24-2H3]-24-hydroxycholesterol) was added to 0.5 mL of plasma. After alkaline hydrolysis with 1 N KOH in 90% ethanol for 2 hours, the solution was neutralized with 65 μL of phosphoric acid, and the sterols were extracted with 9 mL of chloroform in the presence of 3 mL of 0.9% sodium chloride. The organic phase was removed, and the solvent was evaporated to dryness. The sterols were dissolved in 1 mL of toluene. A 100 μL aliquot was removed for quantification of the cholesterol concentration. For this purpose, 30 μg of 5α-cholestane was added, the solvents were evaporated to dryness, and the sterols were derivatized to trimethylsilyl ethers by heating at 60°C after the addition of 200 μL of pyridine and 200 μL of BSTFA (Supelco, Bellafonte, PA). The solvents were evaporated under nitrogen gas, and the samples were resuspended in hexane and analyzed on a gas chromatography-electron ionization mass spectrometer (HP6890 series II; Agilent, Palo Alto, CA) plus a chromatograph combined with a mass selective detector (HP; Agilent) operated in selected ion monitoring mode. A 1-μL aliquot was injected by automated injection in a splitless mode at an injection temperature of 300°C on a fused silica capillary column (30 m × 0.25 mm ID; 0.25 μm film thickness; DB-5MS; J&W Scientific, Agilent Technologies, Massy, France). The initial oven temperature was kept at 50°C for 1 minute; then it was increased at a rate of 20°C · min−1 to 250°C and thereafter at 5°C · min−1 to a final temperature of 300°C. The temperature of the transfer line was kept at 300°C. Electron impact ionization was used at 70 eV ionization energy. Trimethylsilyl-cholesterol and 5α-cholestane were measured at m/z 368 and 372 atomic mass units (amu), respectively. Absolute amounts of cholesterol were determined by interpolation from a standard curve generated in each experiment. 
The remainder of the saponified sterols was purified on silica columns (Supelco) for quantification of 24S-hydroxycholesterol. Cholesterol was eluted with 8 mL of 0.5% isopropanol in hexane. Purified oxysterols were subsequently eluted with 5 mL of 30% isopropanol in hexane and derivatized to trimethylsilyl ether as just described. The samples were analyzed by gas chromatography-mass spectrometry as described. Both 24S-hydroxycholesterol and deuterated 24-hydroxycholesterol were measured at m/z 413 and 416 amu, respectively. Absolute amounts of 24S-hydroxycholesterol were determined by interpolation from a standard curve generated in each experiment. A typical mass spectrometry-gas chromatogram obtained from a plasma sample is displayed in Figure 1, as well as a typical standard curve in the inset in Figure 1
Figure 1.
 
Typical trace obtained by gas chromatography-mass spectrometry for quantification of 24S-hydroxycholesterol in a human plasma sample using an internal deuterated standard of 24-hydroxycholesterol. Inset: a standard curve.
Figure 1.
 
Typical trace obtained by gas chromatography-mass spectrometry for quantification of 24S-hydroxycholesterol in a human plasma sample using an internal deuterated standard of 24-hydroxycholesterol. Inset: a standard curve.
Statistical Analysis
The Student's t-test and the χ2 test were performed to compare sex, age, alleles, and genotype frequencies between POAG cases and controls. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by logistic regression analysis to assess the relative association between disease and age, sex, and genotypes. The percentage of population-attributable risk of carrying the TT genotype was calculated with the formula f(R − 1)/R, where f is the fraction of cases with the TT-genotype and R is the measure of OR. The SAS Software (SAS Institute, Cary, NC) was used for statistical analysis. P < 0.05 was considered to be statistically significant. 
Results
The clinical characteristics of control subjects and patients with POAG are presented in Table 1. Both groups were similar except for the female-to-male ratio in moderate POAG compared with control subjects and other POAG subgroups (Table 1). The genotypic OR and 95% CI for POAG were estimated assuming a dominant genetic model (major TT homozygote versus the others). The frequency of the TT-genotype and T-allele in the CYP46A1 intron 2 rs754203 polymorphism was significantly higher in the population of patients with POAG than in the control group: 61.3% versus 48.3%, respectively, for the TT genotype (P < 0.05). This frequency was even greater in patients with normal-tension glaucoma since 13 of the 18 carried the TT genotype. The rs754203 SNP in intron 2 of CYP46A1 was associated with a significant risk for POAG (OR = 1.26; 95% CI = 1.006–1.574, P < 0.05, Table 1). 
Table 1.
 
Clinical Characteristics, Plasma 24S-Hydroxycholesterol Levels and SNP in Intron 2 of the CYP46A1 Gene in the Study Subjects
Table 1.
 
Clinical Characteristics, Plasma 24S-Hydroxycholesterol Levels and SNP in Intron 2 of the CYP46A1 Gene in the Study Subjects
Control Subjects Patients with POAG
All Early* Moderate* Advanced*
n 118 150 31 49 70
Female-male ratio 1.3 1.1 1.1 1.6 0.9
Age, y (mean ± SEM) 70.9 ± 1.1 69.5 ± 1 66.1 ± 0.9 68.4 ± 0.9 71.5 ± 1
Plasma 24S-hydroxycholesterol
    ng · mL of plasma−1 (mean ± SEM) 97.4 ± 3.7 94 ± 2.6 102.8 ± 3.2 92.1 ± 2.5 91.5 ± 2.5
    ng · mg of cholesterol−1 (mean ± SEM) 123.1 ± 5.1 126 ± 5.6 120.6 ± 4.9 124.6 ± 5.7 129 ± 5.8
rs754203 SNP in CYP46A1 gene (percentage of total samples in each category)
    CC 2.5 3.3 3.2 4.1 2.9
    CT 49.2 35.3 29 42.9 32.9
    TT 48.3 61.3 67.7 53.1 64.3
Allele frequency
    CYP46A1*C 0.27 0.19 0.18 0.26 0.19
    CYP46A1*T 0.73 0.81 0.82 0.74 0.81
Patients with POAG had no significant differences in plasma 24S-hydroxycholesterol levels and the 24S-hydroxycholesterol-to-cholesterol ratio compared with control subjects (P > 0.05, Table 1). Similarly, the plasma 24S-hydroxycholesterol levels and the 24S-hydroxycholesterol-to-cholesterol ratio were not different between CC, CT, and TT carriers when considering patients with POAG and control subjects independently (P > 0.05, Table 2) or together (P > 0.05, data not shown). Plasma absolute and cholesterol-related 24S-hydroxycholesterol levels did not correlate to visual field parameters such as MD (mean defect) or PSD (pattern standard deviation) (data not shown). The subjects' ages, corresponding to the age when blood was sampled, were used as the cofactor. Figure 2 shows the plasma cholesterol-related levels of 24S-hydroxycholesterol in control subjects (Fig. 2A) and patients with POAG (Fig. 2B) from the age of 44 years onward. The ratio between brain weight and liver volume for the different ages, based on data from the literature, 4143 is also indicated in the figure. The levels of 24S-hydroxycholesterol closely followed the brain weight-to-liver volume ratio during the different decades of life. The only exception was observed in patients with POAG older than 80 years who had lower plasma 24S-hydroxycholesterol levels than expected from their brain weight-to-liver volume ratio. 
Table 2.
 
24S-hydroxycholesterol Levels as a Function of rs754203 SNP in Intron 2 of the CYP46A1 Gene in Control Subjects and Patients with POAG
Table 2.
 
24S-hydroxycholesterol Levels as a Function of rs754203 SNP in Intron 2 of the CYP46A1 Gene in Control Subjects and Patients with POAG
Plasma 24S-hydroxycholesterol Age (y)
ng · mL plasma−1 ng · mg cholesterol−1
Control subjects (n = 118) 97.4 ± 3.7 123.1 ± 5.1 70.9 ± 1.1
    TT (n = 57) 104 ± 6.6 123 ± 7.5 71.2 ± 1.6
    CC+CT (n = 61) 91 ± 3.5 123.2 ± 6.7 71 ± 1.4
All POAG patients 94.2 ± 2.6 124.8 ± 5.5 69.5 ± 0.9
    TT (n = 92) 98.3 ± 3.4 123.7 ± 6.9 68 ± 1.2
    CC+CT (n = 58) 87.9 ± 3.1 126.5 ± 9.7 68.2 ± 1.6
Classified in early glaucoma* 103.4 ± 1.3 122.8 ± 11.1 66.3 ± 0.4
    TT (n = 21) 106.8 ± 9.8 126.35 ± 14.6 65.2 ± 2.3
    CC+CT (n = 10) 94.9 ± 7.2 109.15 ± 15.6 67.8 ± 3.7
Moderate glaucoma* 92.1 ± 2.1 124.6 ± 10 68.3 ± 1.6
    TT (n = 26) 96.8 ± 6.9 127.7 ± 14.1 70.3 ± 2.1
    CC+CT (n = 23) 86.9 ± 5.1 121.2 ± 14.5 66 ± 2.3
Advanced glaucoma* 91.4 ± 4.6 128.9 ± 8.5 71.5 ± 1.2
    TT (n = 45) 94.4 ± 5 122.4 ± 9.9 72.2 ± 1.8
    CC+CT (n = 25) 86.5 ± 4.9 139.9 ± 15.7 70.2 ± 2.7
Figure 2.
 
Plasma cholesterol-related 24S-hydroxycholesterol levels in normal subjects (A) and patients with POAG (B) as a function of age. Results are expressed as the mean ± SEM. Estimated ratio between brain weight and liver volume is indicated.
Figure 2.
 
Plasma cholesterol-related 24S-hydroxycholesterol levels in normal subjects (A) and patients with POAG (B) as a function of age. Results are expressed as the mean ± SEM. Estimated ratio between brain weight and liver volume is indicated.
Discussion
Glaucoma encompasses a group of diseases that appear to be genetically heterogeneous. Different glaucoma genes have been identified, but these genes account for only a small proportion of glaucomas. 45 Myocilin mutations have been linked to POAG and associated with 10% of juvenile-onset and 4% of adult-onset cases of POAG. 4,5 Nevertheless, contradictory data have been obtained on other genes such as apolipoprotein E. 4656 Most glaucoma cases appear to be multifactorial and are probably affected by multiple interacting loci. Several genetic susceptibility factors have been suggested to contribute to glaucoma. These factors fit into two broad groups, those affecting intraocular pressure and those that are important in modulating retinal ganglion cell viability. 45 Recent data suggest that cholesterol and cholesterol metabolism may be involved in the pathogenesis of glaucoma. 8,9,57 Cholesterol-24S-hydroxylase (CYP46A1) is a cholesterol-metabolizing enzyme involved in the removal of cholesterol from neuronal structures. 13,14,17 Among various SNPs in the CYP46A1 gene, 2139,58 the TT genotype in intron 2 of CYP46A1, designated as rs754203 SNP, has been found to be a risk factor for Alzheimer disease. 25,26,30,32 Several observations have suggested a possible relationship between Alzheimer disease and glaucoma, 47,5963 with possible pathogenic similarities. Among the common features of these two diseases are overproduction of Alzheimer-Aβ peptide, which has been reported in the aqueous humor of patients with glaucoma, 64 and deposition of which has been associated with retinal degeneration. 65 Brown et al. 66 reported that 24S-hydroxycholesterol inhibits Aβ and amyloid precursor protein secretion in cortical neurons via LXR activation. Increased 24S-hydroxycholesterol production would therefore be expected to decrease neuron degeneration. 
Most 24S-hydroxycholesterol in human circulation originates from the brain 14,17 and is metabolized in the liver. 14,67 Hence, the ratio of brain weight-to-liver volume has been found to be a powerful indicator of the age-related variations of plasma 24S-hydroxycholesterol levels in humans. 18 In our population of patients with POAG and control subjects, we also found a correlation between plasma 24S-hydroxycholesterol and subject age (Fig. 2), suggesting that even though the neural retina also expresses CYP46A1 and is enriched in 24S-hydroxycholesterol, 11 it accounts for only a minor part of the plasma 24S-hydroxycholesterol levels. Furthermore, our data showed that 24S-hydroxycholesterol was not a relevant glaucoma biomarker. 
The biological importance of 24S-hydroxycholesterol in the eye has not been clearly elucidated until now. We 11 and others 12 have found that the neural retina exclusively expresses CYP46A1 and is enriched in 24S-hydroxycholesterol. 11 Of note, Ikeda et al. 68 reported the existence of an enzyme in the nonpigmented ciliary epithelium capable of converting 24S-hydroxycholesterol into 7α,24S-dihydroxycholesterol. 24S-hydroxycholesterol may also bind oxysterol-binding protein, which has already been reported to be expressed in the retina, 69 and thereby participate in cellular signaling. As in the brain, 13,14,17 production of 24S-hydroxycholesterol may be considered a metabolic pathway for neurons to eliminate cholesterol. Indeed, by triggering the addition of a hydroxyl group, CYP46A1 renders the cholesterol molecule more hydrophilic and facilitates its flux through biological membranes. Based on data from animal models and human observations, other mechanisms involving lipoproteins are also crucial for cholesterol metabolism in the retina. 7074  
In view of the present controversy with respect to the possible link between the intronic polymorphism in the CYP46A1 gene and Alzheimer disease, 2131,3339 it is important that the results of the present study be confirmed in other populations of patients with glaucoma. Indeed, glaucoma can be considered an optic nerve neuropathy characterized by neurodegenerative processes. 2 But among the various subtypes of glaucoma, normal-tension glaucoma may share the closest features with Alzheimer disease. Therefore, further investigations of CYP46 polymorphisms in those patients are warranted to verify the higher frequency of the TT genotype. Since we did not find a correlation between the levels of 24S-hydroxycholesterol in the circulation and the polymorphism, we do not have a mechanistic explanation for the apparent link between glaucoma and the polymorphism. If the present results can be confirmed, however, the polymorphism can be added as an additional risk factor for the disease. 
Footnotes
 Supported by a grant from the University Hospital in Dijon, France (Programme Hospitalier de Recherche Clinique, Direction de la Recherche Clinique).
Footnotes
 Disclosure: C. Fourgeux, None, L. Martine, None; I. Björkhem, None; U. Diczfalusy, None; C. Joffre, None; N. Acar, None; C. Creuzot-Garcher, None; A. Bron, None; L. Bretillon, None
Footnotes
 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
The authors thank Linda Northrup (PhD, ELS; English Solutions, Voiron, France) for skillful assistance in English language editing. 
References
Quigley HA Broman AT . The number of people with glaucoma worldwide in 2010 and 2020. Br J Ophthalmol. 2006;90:262–267. [CrossRef] [PubMed]
Weinreb RN Khaw PT . Primary open-angle glaucoma. Lancet. 2004;363:1711–1720. [CrossRef] [PubMed]
Quigley HA . Glaucoma: macrocosm to microcosm: The Friedenwald Lecture. Invest Ophthalmol Vis Sci. 2005;46:2663–2670. [CrossRef]
Kwon YH Fingert JH Kuehn MH Alward WL . Primary open-angle glaucoma. New Engl J Med. 2009;360:1113–1124. [CrossRef] [PubMed]
Resch ZT Fautsch MP . Glaucoma-associated myocilin: a better understanding but much more to learn. Exp Eye Res. 2009;88:704–712. [CrossRef] [PubMed]
Wiggs JL . Genetic etiologies of glaucoma. Arch Ophthalmol. 2007;125:30–37. [CrossRef] [PubMed]
Stewart WC Sine C Sutherland S Stewart JA . Total cholesterol and high-density lipoprotein levels as risk factors for increased intraocular pressure. Am J Ophthalmol. 1996;122:575–577. [CrossRef] [PubMed]
McGwin GJr McNeal S Owsley C Girkin C Epstein D Lee PP . Statins and other cholesterol-lowering medications and the presence of glaucoma. Arch Ophthalmol. 2004;122:822–826. [CrossRef] [PubMed]
Song J Deng PF Stinnett SS Epstein DL Rao PV . Effects of cholesterol-lowering statins on the aqueous humor outflow pathway. Invest Ophthalmol Vis Sci. 2005;46:2424–2432. [CrossRef] [PubMed]
De Castro DK Punjabi OS Bostrom AG . Effect of statin drugs and aspirin on progression in open-angle glaucoma suspects using confocal scanning laser ophthalmoscopy. Clin Exp Ophthalmol. 2007;35:506–513. [CrossRef]
Bretillon L Diczfalusy U Bjorkhem I . Cholesterol-24S-hydroxylase (CYP46A1) is specifically expressed in neurons of the neural retina. Curr Eye Res. 2007;32:361–366. [CrossRef] [PubMed]
Ramirez DM Andersson S Russell DW . Neuronal expression and subcellular localization of cholesterol 24-hydroxylase in the mouse brain. J Comp Neurol. 2008;507:1676–1693. [CrossRef] [PubMed]
Björkhem I Lütjohann D Breuer O Sakinis A Wennmalm A . Importance of a novel oxidative mechanism for elimination of brain cholesterol. Turnover of cholesterol and 24(S)-hydroxycholesterol in rat brain as measured with 18O2 techniques in vivo and in vitro. J Biol Chem. 1997;272:30178–30184. [CrossRef] [PubMed]
Björkhem I Lütjohann D Diczfalusy U Stahle L Ahlborg G Wahren J . Cholesterol homeostasis in human brain: turnover of 24S-hydroxycholesterol and evidence for a cerebral origin of most of this oxysterol in the circulation. J Lipid Res. 1998;39:1594–1600. [PubMed]
Lund EG Guileyardo JM Russell DW . cDNA cloning of cholesterol 24-hydroxylase, a mediator of cholesterol homeostasis in the brain. Proc Natl Acad Sci USA. 1999;96:7238–7243. [CrossRef] [PubMed]
Lund EG Xie C Kotti T Turley SD Dietschy JM Russell DW . Knockout of the cholesterol 24-hydroxylase gene in mice reveals a brain-specific mechanism of cholesterol turnover. J Biol Chem. 2003;278:22980–22988. [CrossRef] [PubMed]
Lutjohann D Breuer O Ahlborg G . Cholesterol homeostasis in human brain: evidence for an age-dependent flux of 24S-hydroxycholesterol from the brain into the circulation. Proc Natl Acad Sci USA. 1996;93:9799–9804. [CrossRef] [PubMed]
Bretillon L Lütjohann D Stahle L . Plasma levels of 24S-hydroxycholesterol reflect the balance between cerebral production and hepatic metabolism and are inversely related to body surface. J Lipid Res. 2000;41:840–845. [PubMed]
Kolsch H Lutjohann D Tulke A Bjorkhem I Rao ML . The neurotoxic effect of 24-hydroxycholesterol on SH-SY5Y human neuroblastoma cells. Brain Res. 1999;818:171–175. [CrossRef] [PubMed]
Lutjohann D Papassotiropoulos A Bjorkhem I . Plasma 24S-hydroxycholesterol (cerebrosterol) is increased in Alzheimer and vascular demented patients. J Lipid Res. 2000;41:195–198. [PubMed]
Borroni B Archetti S Agosti C . Intronic CYP46 polymorphism along with ApoE genotype in sporadic Alzheimer Disease: from risk factors to disease modulators. Neurobiol Aging. 2004;25:747–751. [CrossRef] [PubMed]
Combarros O Infante J Llorca J Berciano J . Genetic association of CYP46 and risk for Alzheimer's disease. Dement Geriatr Cogn Disord. 2004;18:257–260. [CrossRef] [PubMed]
Johansson A Katzov H Zetterberg H . Variants of CYP46A1 may interact with age and APOE to influence CSF Abeta42 levels in Alzheimer's disease. Hum Genet. 2004;114:581–587. [CrossRef] [PubMed]
Kolsch H Lutjohann D Ludwig M . Polymorphism in the cholesterol 24S-hydroxylase gene is associated with Alzheimer's disease. Mol Psychiatry. 2002;7:899–902. [CrossRef] [PubMed]
Papassotiropoulos A Streffer JR Tsolaki M . Increased brain beta-amyloid load, phosphorylated tau, and risk of Alzheimer disease associated with an intronic CYP46 polymorphism. Arch Neurol. 2003;60:29–35. [CrossRef] [PubMed]
Wang B Zhang C Zheng W . Association between a T/C polymorphism in intron 2 of cholesterol 24S-hydroxylase gene and Alzheimer's disease in Chinese. Neurosci Lett. 2004;369:104–107. [CrossRef] [PubMed]
Helisalmi S Vepsalainen S Koivisto AM . Association of CYP46 intron 2 polymorphism in Finnish Alzheimer's disease samples and a global scale summary. J Neurol Neurosurg Psychiatry. 2006;77:421–422. [CrossRef] [PubMed]
Ma SL Tang NL Lam LC Chiu HF . Polymorphisms of the cholesterol 24-hydroxylase (CYP46A1) gene and the risk of Alzheimer's disease in a Chinese population. Int Psychogeriatr. 2006;18:37–45. [CrossRef] [PubMed]
Fernandez Del Pozo V Alvarez Alvarez M Fernandez Martinez M . Polymorphism in the cholesterol 24S-hydroxylase gene (CYP46A1) associated with the APOEpsilon3 allele increases the risk of Alzheimer's disease and of mild cognitive impairment progressing to Alzheimer's disease. Dement Geriatr Cogn Disord. 2006;21:81–87. [CrossRef] [PubMed]
Li Y Chu LW Chen YQ . Intron 2 (T/C) CYP46 Polymorphism Is associated with Alzheimer's disease in Chinese patients. Dement Geriatr Cogn Disord. 2006;22:399–404. [CrossRef] [PubMed]
Papassotiropoulos A Wollmer MA Tsolaki M . A cluster of cholesterol-related genes confers susceptibility for Alzheimer's disease. J Clin Psychiatry. 2005;66:940–947. [CrossRef] [PubMed]
Fu BY Ma SL Tang NL . Cholesterol 24-hydroxylase (CYP46A1) polymorphisms are associated with faster cognitive deterioration in Chinese older persons: a two-year follow up study. Int J Geriatr Psychiatry. Published online February 11, 2009.
Vega GL Weiner M Kolsch H . The effects of gender and CYP46 and apoE polymorphism on 24S-hydroxycholesterol levels in Alzheimer's patients treated with statins. Curr Alzheimer Res. 2004;1:71–77. [CrossRef] [PubMed]
Tedde A Rotondi M Cellini E . Lack of association between the CYP46 gene polymorphism and Italian late-onset sporadic Alzheimer's disease. Neurobiol Aging. 2006;27:773 e771–e773. [CrossRef] [PubMed]
Ingelsson M Jesneck J Irizarry MC Hyman BT Rebeck GW . Lack of association of the cholesterol 24-hydroxylase (CYP46) intron 2 polymorphism with Alzheimer's disease. Neurosci Lett. 2004;367:228–231. [CrossRef] [PubMed]
Kabbara A Payet N Cottel D Frigard B Amouyel P Lambert JC . Exclusion of CYP46 and APOM as candidate genes for Alzheimer's disease in a French population. Neurosci Lett. 2004;363:139–143. [CrossRef] [PubMed]
Chalmers KA Culpan D Kehoe PG Wilcock GK Hughes A Love S . APOE promoter, ACE1 and CYP46 polymorphisms and beta-amyloid in Alzheimer's disease. Neuroreport. 2004;15:95–98. [CrossRef] [PubMed]
Desai P DeKosky ST Kamboh MI . Genetic variation in the cholesterol 24-hydroxylase (CYP46) gene and the risk of Alzheimer's disease. Neurosci Lett. 2002;328:9–12. [CrossRef] [PubMed]
Juhasz A Rimanoczy A Boda K . CYP46 T/C polymorphism is not associated with Alzheimer's dementia in a population from Hungary. Neurochem Res. 2005;30:943–948. [CrossRef] [PubMed]
Hoddap E Parrish R Anderson D . Clinical Decisions in Glaucoma. Saint Louis, MO: Mosby Year Book; 1993: 53.
Dekaban AS . Changes in brain weights during the span of human life: relation of brain weights to body heights and body weights. Ann Neurol. 1978;4:345–356. [CrossRef] [PubMed]
Urata K Kawasaki S Matsunami H . Calculation of child and adult standard liver volume for liver transplantation. Hepatology. 1995;21:1317–1321. [CrossRef] [PubMed]
Wynne HA Cope LH Mutch E Rawlins MD Woodhouse KW James OF . The effect of age upon liver volume and apparent liver blood flow in healthy man. Hepatology. 1989;9:297–301. [CrossRef] [PubMed]
Bretillon L Sidén A Wahlund LO . Plasma levels of 24S-hydroxycholesterol in patients with neurological diseases. Neurosci Lett. 2000;293:87–90. [CrossRef] [PubMed]
Libby RT Gould DB Anderson MG John SW . Complex genetics of glaucoma susceptibility. Annu Rev Genomics Hum Genet. 2005;6:15–44. [CrossRef] [PubMed]
Lam CY Fan BJ Wang DY . Association of apolipoprotein E polymorphisms with normal tension glaucoma in a Chinese population. J Glaucoma. 2006;15:218–222. [CrossRef] [PubMed]
Tamura H Kawakami H Kanamoto T . High frequency of open-angle glaucoma in Japanese patients with Alzheimer's disease. J Neurol Sci. 2006;246:79–83. [CrossRef] [PubMed]
Fan BJ Wang DY Fan DS . SNPs and interaction analyses of myocilin, optineurin, and apolipoprotein E in primary open angle glaucoma patients. Mol Vis. 2005;11:625–631. [PubMed]
Mabuchi F Tang S Ando D . The apolipoprotein E gene polymorphism is associated with open angle glaucoma in the Japanese population. Mol Vis. 2005;11:609–612. [PubMed]
Ressiniotis T Griffiths PG Birch M Keers SM Chinnery PF . Apolipoprotein E promoter polymorphisms do not have a major influence on the risk of developing primary open angle glaucoma. Mol Vis. 2004;10:805–807. [PubMed]
Junemann A Bleich S Reulbach U . Prospective case control study on genetic association of apolipoprotein epsilon2 with intraocular pressure. Br J Ophthalmol. 2004;88:581–582. [CrossRef] [PubMed]
Lake S Liverani E Desai M . Normal tension glaucoma is not associated with the common apolipoprotein E gene polymorphisms. Br J Ophthalmol. 2004;88:491–493. [CrossRef] [PubMed]
Ressiniotis T Griffiths PG Birch M Keers S Chinnery PF . The role of apolipoprotein E gene polymorphisms in primary open-angle glaucoma. Arch Ophthalmol. 2004;122:258–261. [CrossRef] [PubMed]
Tomarev SI Wistow G Raymond V Dubois S Malyukova I . Gene expression profile of the human trabecular meshwork: NEIBank sequence tag analysis. Invest Ophthalmol Vis Sci. 2003;44:2588–2596. [CrossRef] [PubMed]
Vickers JC Craig JE Stankovich J . The apolipoprotein epsilon4 gene is associated with elevated risk of normal tension glaucoma. Mol Vis. 2002;8:389–393. [PubMed]
Copin B Brezin AP Valtot F Dascotte JC Bechetoille A Garchon HJ . Apolipoprotein E-promoter single-nucleotide polymorphisms affect the phenotype of primary open-angle glaucoma and demonstrate interaction with the myocilin gene. Am J Hum Genet. 2002;70:1575–1581. [CrossRef] [PubMed]
Yucel I Akar Y Yucel G Ciftcioglu MA Keles N Aslan M . Effect of hypercholesterolemia on inducible nitric oxide synthase expression in a rat model of elevated intraocular pressure. Vision Res. 2005;45:1107–1114. [CrossRef] [PubMed]
Wang F Jia J . Polymorphisms of cholesterol metabolism genes CYP46 and ABCA1 and the risk of sporadic Alzheimer's disease in Chinese. Brain Res. 2007;1147:34–38. [CrossRef] [PubMed]
Bayer AU Ferrari F . Severe progression of glaucomatous optic neuropathy in patients with Alzheimer's disease. Eye. 2002;16:209–212. [CrossRef] [PubMed]
Bayer AU Ferrari F Erb C . High occurrence rate of glaucoma among patients with Alzheimer's disease. Eur Neurol. 2002;47:165–168. [CrossRef] [PubMed]
Karseras AG . Progressive glaucoma in patients with Alzheimer's disease. Eye. 2004;18:229. [CrossRef] [PubMed]
Nesher R Trick GL . The pattern electroretinogram in retinal and optic nerve disease: a quantitative comparison of the pattern of visual dysfunction. Doc Ophthalmol. 1991;77:225–235. [CrossRef] [PubMed]
Parisi V . Correlation between morphological and functional retinal impairment in patients affected by ocular hypertension, glaucoma, demyelinating optic neuritis and Alzheimer's disease. Semin Ophthalmol. 2003;18:50–57. [PubMed]
Janciauskiene S Krakau T . Alzheimer's peptide: a possible link between glaucoma, exfoliation syndrome and Alzheimer's disease. Acta Ophthalmol Scand. 2001;79:328–329. [CrossRef] [PubMed]
Ning A Cui J To E Ashe KH Matsubara J . Amyloid-beta deposits lead to retinal degeneration in a mouse model of Alzheimer disease. Invest Ophthalmol Vis Sci. 2008;49:5136–5143. [CrossRef] [PubMed]
Brown J3rd Theisler C Silberman S . Differential expression of cholesterol hydroxylases in Alzheimer's disease. J Biol Chem. 2004;279:34674–34681. [CrossRef] [PubMed]
Bjorkhem I Andersson U Ellis E . From brain to bile: evidence that conjugation and omega-hydroxylation are important for elimination of 24S-hydroxycholesterol (cerebrosterol) in humans. J Biol Chem. 2001;276:37004–37010. [CrossRef] [PubMed]
Ikeda H Ueda M Ikeda M Kobayashi H Honda Y . Oxysterol 7alpha-hydroxylase (CYP39A1) in the ciliary nonpigmented epithelium of bovine eye. Lab Invest. 2003;83:349–355. [CrossRef] [PubMed]
Moreira EF Jaworski C Li A Rodriguez IR . Molecular and biochemical characterization of a novel oxysterol-binding protein (OSBP2) highly expressed in retina. J Biol Chem. 2001;276:18570–18578. [CrossRef] [PubMed]
Gordiyenko N Campos M Lee JW Fariss RN Sztein J Rodriguez IR . RPE cells internalize low-density lipoprotein (LDL) and oxidized LDL (oxLDL) in large quantities in vitro and in vivo. Invest Ophthalmol Vis Sci. 2004;45:2822–2829. [CrossRef] [PubMed]
Li CM Chung BH Presley JB . Lipoprotein-like particles and cholesteryl esters in human Bruch's membrane: initial characterization. Invest Ophthalmol Vis Sci. 2005;46:2576–2586. [CrossRef] [PubMed]
Li CM Presley JB Zhang X . Retina expresses microsomal triglyceride transfer protein: implications for age-related maculopathy. J Lipid Res. 2005;46:628–640. [CrossRef] [PubMed]
Tserentsoodol N Sztein J Campos M . Uptake of cholesterol by the retina occurs primarily via a low density lipoprotein receptor-mediated process. Mol Vision. 2006;12:1306–1318.
Bretillon L Acar N Seeliger MW . ApoB100, LDLR−/− mice exhibit reduced electroretinographic response and cholesteryl esters deposits in the retina. Invest Ophthalmol Vis Sci. 2008;49:1307–1314. [CrossRef] [PubMed]
Figure 1.
 
Typical trace obtained by gas chromatography-mass spectrometry for quantification of 24S-hydroxycholesterol in a human plasma sample using an internal deuterated standard of 24-hydroxycholesterol. Inset: a standard curve.
Figure 1.
 
Typical trace obtained by gas chromatography-mass spectrometry for quantification of 24S-hydroxycholesterol in a human plasma sample using an internal deuterated standard of 24-hydroxycholesterol. Inset: a standard curve.
Figure 2.
 
Plasma cholesterol-related 24S-hydroxycholesterol levels in normal subjects (A) and patients with POAG (B) as a function of age. Results are expressed as the mean ± SEM. Estimated ratio between brain weight and liver volume is indicated.
Figure 2.
 
Plasma cholesterol-related 24S-hydroxycholesterol levels in normal subjects (A) and patients with POAG (B) as a function of age. Results are expressed as the mean ± SEM. Estimated ratio between brain weight and liver volume is indicated.
Table 1.
 
Clinical Characteristics, Plasma 24S-Hydroxycholesterol Levels and SNP in Intron 2 of the CYP46A1 Gene in the Study Subjects
Table 1.
 
Clinical Characteristics, Plasma 24S-Hydroxycholesterol Levels and SNP in Intron 2 of the CYP46A1 Gene in the Study Subjects
Control Subjects Patients with POAG
All Early* Moderate* Advanced*
n 118 150 31 49 70
Female-male ratio 1.3 1.1 1.1 1.6 0.9
Age, y (mean ± SEM) 70.9 ± 1.1 69.5 ± 1 66.1 ± 0.9 68.4 ± 0.9 71.5 ± 1
Plasma 24S-hydroxycholesterol
    ng · mL of plasma−1 (mean ± SEM) 97.4 ± 3.7 94 ± 2.6 102.8 ± 3.2 92.1 ± 2.5 91.5 ± 2.5
    ng · mg of cholesterol−1 (mean ± SEM) 123.1 ± 5.1 126 ± 5.6 120.6 ± 4.9 124.6 ± 5.7 129 ± 5.8
rs754203 SNP in CYP46A1 gene (percentage of total samples in each category)
    CC 2.5 3.3 3.2 4.1 2.9
    CT 49.2 35.3 29 42.9 32.9
    TT 48.3 61.3 67.7 53.1 64.3
Allele frequency
    CYP46A1*C 0.27 0.19 0.18 0.26 0.19
    CYP46A1*T 0.73 0.81 0.82 0.74 0.81
Table 2.
 
24S-hydroxycholesterol Levels as a Function of rs754203 SNP in Intron 2 of the CYP46A1 Gene in Control Subjects and Patients with POAG
Table 2.
 
24S-hydroxycholesterol Levels as a Function of rs754203 SNP in Intron 2 of the CYP46A1 Gene in Control Subjects and Patients with POAG
Plasma 24S-hydroxycholesterol Age (y)
ng · mL plasma−1 ng · mg cholesterol−1
Control subjects (n = 118) 97.4 ± 3.7 123.1 ± 5.1 70.9 ± 1.1
    TT (n = 57) 104 ± 6.6 123 ± 7.5 71.2 ± 1.6
    CC+CT (n = 61) 91 ± 3.5 123.2 ± 6.7 71 ± 1.4
All POAG patients 94.2 ± 2.6 124.8 ± 5.5 69.5 ± 0.9
    TT (n = 92) 98.3 ± 3.4 123.7 ± 6.9 68 ± 1.2
    CC+CT (n = 58) 87.9 ± 3.1 126.5 ± 9.7 68.2 ± 1.6
Classified in early glaucoma* 103.4 ± 1.3 122.8 ± 11.1 66.3 ± 0.4
    TT (n = 21) 106.8 ± 9.8 126.35 ± 14.6 65.2 ± 2.3
    CC+CT (n = 10) 94.9 ± 7.2 109.15 ± 15.6 67.8 ± 3.7
Moderate glaucoma* 92.1 ± 2.1 124.6 ± 10 68.3 ± 1.6
    TT (n = 26) 96.8 ± 6.9 127.7 ± 14.1 70.3 ± 2.1
    CC+CT (n = 23) 86.9 ± 5.1 121.2 ± 14.5 66 ± 2.3
Advanced glaucoma* 91.4 ± 4.6 128.9 ± 8.5 71.5 ± 1.2
    TT (n = 45) 94.4 ± 5 122.4 ± 9.9 72.2 ± 1.8
    CC+CT (n = 25) 86.5 ± 4.9 139.9 ± 15.7 70.2 ± 2.7
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×