|Molecular Vision 2005;
Received 22 July 2005 | Accepted 26 October 2005 | Published 23 November 2005
Association of a common coding polymorphism (N453S) of the cytochrome P450 1B1 (CYP1B1) gene with optic disc cupping and visual field alteration in French patients with primary open-angle glaucoma
1INSERM, Unit 580, Paris, France; 2Université Paris Descartes, Faculté de médecine René Descartes, Paris, France; 3AP HP, Hôpital Cochin, Ophthalmology Unit, Paris, France
Correspondence to: Henri-Jean Garchon, INSERM, Unit 580, Hôpital Necker, 161 Rue de Sèvres, 75743 Paris Cedex 15, France; Phone: 33 1 44 49 53 67; FAX: 33 1 44 49 41 00; email: firstname.lastname@example.org
Purpose: To investigate a role of common polymorphisms of the CYP1B1 gene in French patients with primary open-angle glaucoma (POAG).
Methods: Six common CYP1B1 variants, 5 coding and one in promoter, were compared in 224 unrelated French Caucasian POAG patients, excluding those with a CYP1B1 mutation, and in 47 population-matched controls with a normal ophthalmic examination. Allelic associations were assessed with the D' and r2 parameters. An effect of the representative variants on subphenotypes, including the age and the intraocular pressure at diagnosis, the cup to disk ratio, and the visual field alteration, was tested by multivariate analyses.
Results: Allele and haplotype frequencies were similar in patients and in controls. Five variants formed two groups with tightly correlated alleles while the sixth one, N453S, was independent. The age and the intraocular pressure at diagnosis were not influenced by any of the variants. In contrast, the 453*Serine allele was associated with decreased cupping of the optic disk (Odds ratio=0.32, 95% CI: 0.15-0.70; p=0.0036) and with a milder alteration of the visual field (p=0.025).
Conclusions: The common N453S coding variant of CYP1B1 is potentially a factor of severity in POAG patients.
Primary open-angle glaucoma (POAG) is the most common form of glaucoma and one of the leading causes of irreversible blindness worldwide . It is characterized by cupping of the optic nerve head, degeneration of ganglion cells, and progressive visual field damage . The condition is frequently associated with an increase in intraocular pressure (IOP) which, however, is neither necessary nor sufficient for the onset or the progression of the disease.
Genetic factors play an important but complex role in POAG predisposition. Seven loci were mapped by linkage analysis of large families [3,4]. Three of these loci, MYOCILIN (GLC1A/MYOC), OPTINEURIN (GLC1E/OPTN), and GLC1G/WDR36 were identified at the molecular level [4-6]. Typically, mutations of MYOC are associated with juvenile-onset glaucoma, and markedly elevated IOP [5,7], whereas mutations of OPTN are associated with adult onset and normal or moderately elevated IOP . WDR36 mutations were identified in both high and low pressure adult-onset POAG .
In addition to MYOC mutations that account for a small proportion of POAG cases in the French population (7%) , we recently reported that mutations in a fourth gene, CYP1B1, a member of the cytochrome P450 superfamily that is strongly inducible by dioxins , could be observed in 4.6% of French POAG patients, preferentially with an early onset of disease . Prior to our report, this gene was demonstrated to be the major gene, recessively mutated, in patients from different populations with primary congenital glaucoma, a rare but severe form of glaucoma [11-14]. The pathogenesis of CYP1B1 mutations is presently unknown.
Apart from mutations, the CYP1B1 gene harbors several common single-nucleotide polymorphisms (SNPs), five of which are coding [13,14]. Some of them have been associated with cancer predisposition in different populations [15-17]. Here, in light of these findings, we have investigated whether common SNPs in the CYP1B1 gene, including an SNP in the upstream region and the 5 coding SNPs, might influence susceptibility to POAG or modify the patient phenotype.
Patients and controls
The study included 224 unrelated French Caucasian POAG patients previously described [8,10] and was approved by the Ethics Committee of the Necker Medical School. Informed consent was obtained from all subjects according to the European Legislation. Clinical assessment included slit-lamp biomicroscopy, IOP measurement with a Goldman applanation tonometer, and automated perimetry with a Humphrey visual field analyzer, Octopus, or Moniteur Ophtalmologique. Diagnosis and clinical classifications were done prior to the genetic analysis. POAG was defined with the following criteria; normally open iridocorneal angle (grade III or IV gonioscopy), characteristic optic disc cupping, and an alteration of the visual field. The IOP level was not a criterion of selection. Because different perimetry devices were used, visual field alterations were grouped by consensus of two ophthalmologists into three robust categories of increasing severity to allow for comparison, including (1) mild alteration (including early defect, arcuate scotoma or nasal step), (2) advanced scotoma, and (3) no light perception. This ordinal variable was concordant with cupping of the optic disc to a highly significant level (Gamma coefficient=0.72, Kendall τ=0.44, p<10-10). Cup to disc ratios were estimated by glaucoma specialists and were available for 197 patients and visual field findings for 201 patients. Individuals with a cause of secondary glaucoma including glucocorticoid treatment, history of trauma or surgery, media opacity, pigment dispersion, and exfoliation syndrome were excluded. In addition, patients with a CYP1B1 mutation were excluded from the data set. The control group consisted of 47 unrelated Caucasian spouses with a normal ophthalmic examination from GLC1A/MYOC linked-glaucoma French families.
Alleles of the common coding SNPs shown in Table 1 were determined, blindly to the clinical data, as a result of the exhaustive characterization of the polymorphism of the CYP1B1 coding region in both the patients and the controls, as reported in our previous work . In addition, an SNP (T>C) in the upstream region, situated at 265 bp before the transcription start site [18,19], at position 2,805 (EMBL/GenBank U56438) was typed by PCR-RFLP. A 328 bp fragment was amplified with the following primers: 5'-GGT TGT ACC GAG CGT GGT TC-3' (forward) and 5'-TCT CAC AAC TGG AGT CGC AG-3' (reverse). The reaction was performed in a 25 μl mixture containing 100 ng of genomic DNA, 0.4 μmol/l of forward and reverse primers, 1.5 mmol/l MgCl2, 5% dimethylsulfoxide, 200 μmol/l of each dNTP, and 0.5 U of Taq DNA polymerase (Invitrogen, Life Technologies, Carlsbad, CA). Cycling conditions were for each cycle, 1 min at 94 °C, 1 min at 55 °C, and 1 min at 72 °C, for 35 cycles. The alternative 2805C allele creates a unique Taq I restriction site, yielding two fragments of 288 and 40 bp following cleavage of the PCR product, which were detected after migration on a 2% agarose gel. All SNPs were in Hardy-Weinberg equilibrium.
Allele frequencies in patients and in controls were compared with the COCAPHASE program of the UNPHASED suite . Haplotypes were deduced from unphased genotype data and compared between cases and controls with similar findings using COCAPHASE and the PHASE v2.0 program [21,22]. Measures of pairwise linkage disequilibrium (LD) between SNPs, including Lewontin's standardized disequilibrium coefficient (D') and the squared correlation coefficient (r2), were computed with the LDMAX software provided with the Graphical Overview of Linkage Disequilibrium (GOLD) package .
The effect of representative SNPs on subphenotypes was assessed through multivariate analyses. The age and IOP at the time of diagnosis were continuous variables following a normal distribution. Their dependency on representative SNPs was tested with a multi-way analysis of variance with the Statistica 6.0 software (Statsoft Inc., Tulsa, OK). The cup to disk ratio was a discrete variable taking values between 0.4 and 1 and with a skewed distribution. Its median was 0.8 and its mode was 0.9. It was therefore transformed into a binary variable, allowing for multivariate analysis. Patients were grouped into two categories, considering 0.8 as the threshold for severe cupping. Findings were very similar if 0.7 was used instead as the threshold. The dependency of this binary variable on representative SNPs was tested by logistic regression, using the LogXact version 6 software (Cytel, Cambridge, MA). Because the time period between diagnosis and examination influenced the cup to disk ratio (OR=1.05, p=0.025), effects of the SNPs were estimated after adjustement for this parameter. The p value produced by this analysis was multiplied by 3, using a Bonferroni correction for the number of subphenotypes tested (age, IOP at the time of diagnosis, and cup to disk ratio). Finally, since the visual field was tightly correlated with cup to disk ratios (see above), its dependency on the genetic polymorphism was assessed secondarily to cup to disk ratios, using the nonparametric Mann-Whitney test.
Allele frequencies at the six common SNPs of the CYP1B1 gene did not differ significantly between the POAG patients and the controls (Table 1). Other alleles that had been detected in our exhaustive analysis of CYP1B1 sequence were too rare (frequency less than 2%) to contribute to a statistical finding . Therefore they were not included in the present study.
Haplotypes were reconstructed from the genotypic data. Four major haplotypes, among the 18 estimated by COCAPHASE, showed a frequency >5% (Table 2). They accounted for 91% of estimated haplotypes in patients and for 97% in controls. Their frequencies were similar in both groups. All other haplotypes had frequencies less than 2%. Because of the small size of the control group, we cannot rule out that we did not detect a small difference in allele or haplotype frequencies between patients and controls. Therefore larger samples will have to be tested to observe such differences.
There were fewer haplotypes than expected (26=64), indicating a strong linkage disequilibrium across the CYP1B1 locus. As shown in Figure 1, the D' coefficient was equal or close to 1 for all pairs of SNPs, both in patients and in controls (Figure 1A), strongly suggesting that there has been no or little historical recombination in the region over time. The r2 parameter that measures the correlation between alleles showed that 5 of the 6 SNPs formed two groups of tightly correlated SNPs (Figure 1B). The first group included the SNP of the upstream region, 2805C>T, and the two coding SNPs in exon II, R48G and A119S. The second group included another two SNPs in exon III, L432V and D449D. The remaining SNP, N453S, also in exon III, was weakly correlated to the other five SNPs and was therefore independent. This pattern of association was very similar in patients and in controls.
One SNP from each group of associated SNPs, including 2805T>C and L432V, and the independent SNP, N453S, were selected and their collective effect on important clinical parameters was assessed by means of multivariate analyses. There was no significant effect of the three representative SNPs on the IOP and the age at diagnosis, tested by a three factor ANOVA. In contrast, severe cupping of the optic disk was significantly altered by the SNPs, as assessed by logistic regression (p=0.0016; corrected p=0.005, for 3 phenotypes tested). Parameter estimation indicated that this effect was associated with the N453S SNP (p=0.0036) and not with the other two SNPs (Table 3). The 453*Serine allele was observed in 76 of the 197 patients with measured cup to disk ratios. It was associated with a decreased cupping (60 cases with cup to disk ratio less than or equal to 0.8 and 16 cases with cup to disk ratio>0.8; odds ratio=0.32, 95% confidence interval: 0.15-0.70). There were too few Ser/Ser homozygous patients (n=8) to efficiently discriminate between a dominant and a partially dominant effect of the Serine allele.
As seen in Table 4, the 453*Serine allele was also associated with a milder alteration of the visual field (Mann-Whitney test, one sided p=0.025), which was somehow expected given the concordance between alteration of the visual field and cupping of the optic disc.
Identification of SNPs in the genes that are associated with glaucoma or with glaucoma severity should contribute to better understand the disease mechanisms. In addition, one potentially important application of such information is an improved management of the patients, and hopefully rationalized and individualized treatments. Accordingly, SNPs in the MYOC, OPTN, APOE, and OPA1 genes were associated with POAG predisposition or with subphenotypes, including visual-field damage, IOP control, or age at onset of the disease [24-29]. Our present study reveals an association of a common coding polymorphism of CYP1B1 as a potential factor of severity in POAG patients for the first time. Remarkably, the N453S variant is functional and is therefore a good candidate to explain our findings. A recent study demonstrated that the change of the asparagine into a serine at position 453 resulted into a three fold shorter half-life and a two fold lower cellular level of the CYP1B1 protein, as a consequence of a higher rate of proteasomal degradation . The Serine allele was recently associated with a decreased risk of endometrial cancer . In our study, it was also associated with less severe cupping of the optic disc and milder alteration of the visual field. This protective association, however, is intriguing in light of the effect of deleterious CYP1B1 mutations on predisposition to primary congenital glaucoma and to early-onset POAG [10,14,31]. Most of these mutations also cause a lower level or even an absence of functional protein and therefore an SNP causing a decrease in protein level would be expected to be associated with more severe symptoms. We propose that this discrepancy could be explained by the fact that, contrary to gene-inactivating mutations, the N453S variant is unlikely to affect the regulation of the CYP1B1 gene. The CYP1B1 gene is highly inducible by dioxins , and probably also by other still unknown factors, endogenous or environmental. Inability to upregulate CYP1B1 appropriately in response to such factors might be a critical mechanism in glaucoma pathogenesis. In contrast, a low steady-state level of CYP1B1 could exert a long-term protective effect on ocular tissues.
Although the N453S variant is independent of the other SNPs investigated in this study, we cannot rule out that another, yet to be identified, SNP associated with N453S is actually responsible for our findings. Since we have characterized the coding region of CYP1B1 exhaustively, this putative SNP would have to be noncoding. Importantly, the high values, close or equal to unity, taken by the D' measure of pairwise linkage disequilibrium strongly suggests that the DNA segment lying between the 2805T>C promoter variant and N453S behaves as a single haplotypic block. In this regard, it is striking that the 2805T>C promoter SNP is tightly correlated with the R48G and A119S variants. These variants are associated with altered enzymatic activity of CYP1B1  and with susceptibility to several cancers [15,16]. Thus, the 2805T>C SNP, or other SNPs recently identified in the upstream region of CYP1B1 , might also contribute to these reported risks. Recent data from the international HapMap project shed more light on this point and show that the CYP1B1 gene belongs to a large block of linkage disequilibrium of 99 Kb extending between SNPs rs336035 and rs727631, and covered with a density greater than 1 SNP per 2 Kb, in Caucasian populations. The N453S (rs1800440) and L432V (rs1056836) variants, but not the other four variants of our present study, have been characterized in HapMap (release 16c.1/PhaseI June 05). Using the r2 parameter of correlation between alleles, the N453S variant is not associated with any other SNP of the current dataset. This indicates that the other SNPs currently typed in the HapMap project are unlikely to account for the association reported herein.
In conclusion, this finding is a first observation of an association of CYP1B1 with POAG subphenotypes in the French population. It needs to be replicated in larger groups and in other populations to clarify the significance of the N453S variant in POAG pathogenesis and to evaluate its usefulness in the clinical management of the patients.
This work was supported by INSERM (Institut National de la Santé et de la Recherche Médicale), by the Assistance Publique des Hôpitaux de Paris (grant DRRC-AOM96110), by the Fondation pour la Recherche Médicale, and by Insite Vision Inc. (Alameda, CA). RM was awarded a fellowship of the Comite Mixte Inter-Universitaire Franco Marocain (AI 237/SVS/2000). We are grateful to Evelyne Colomb for expert technical assistance, to Dr. Merouane Berkani, Dr. Françoise Valtot, Dr. Jean Claude Dascotte, and Pr. Alain Béchetoille for providing access to clinical records of their patients.
1. Quigley HA. Number of people with glaucoma worldwide. Br J Ophthalmol 1996; 80:389-93.
2. Quigley HA. Open-angle glaucoma. N Engl J Med 1993; 328:1097-106.
3. WuDunn D. Genetic basis of glaucoma. Curr Opin Ophthalmol 2002; 13:55-60.
4. Monemi S, Spaeth G, DaSilva A, Popinchalk S, Ilitchev E, Liebmann J, Ritch R, Heon E, Crick RP, Child A, Sarfarazi M. Identification of a novel adult-onset primary open-angle glaucoma (POAG) gene on 5q22.1. Hum Mol Genet 2005; 14:725-33.
5. Stone EM, Fingert JH, Alward WL, Nguyen TD, Polansky JR, Sunden SL, Nishimura D, Clark AF, Nystuen A, Nichols BE, Mackey DA, Ritch R, Kalenak JW, Craven ER, Sheffield VC. Identification of a gene that causes primary open angle glaucoma. Science 1997; 275:668-70.
6. Rezaie T, Child A, Hitchings R, Brice G, Miller L, Coca-Prados M, Heon E, Krupin T, Ritch R, Kreutzer D, Crick RP, Sarfarazi M. Adult-onset primary open-angle glaucoma caused by mutations in optineurin. Science 2002; 295:1077-9.
7. Adam MF, Belmouden A, Binisti P, Brezin AP, Valtot F, Bechetoille A, Dascotte JC, Copin B, Gomez L, Chaventre A, Bach JF, Garchon HJ. Recurrent mutations in a single exon encoding the evolutionarily conserved olfactomedin-homology domain of TIGR in familial open-angle glaucoma. Hum Mol Genet 1997; 6:2091-7.
8. Melki R, Belmouden A, Brezin A, Garchon HJ. Myocilin analysis by DHPLC in French POAG patients: increased prevalence of Q368X mutation. Hum Mutat 2003; 22:179.
9. Sutter TR, Tang YM, Hayes CL, Wo YY, Jabs EW, Li X, Yin H, Cody CW, Greenlee WF. Complete cDNA sequence of a human dioxin-inducible mRNA identifies a new gene subfamily of cytochrome P450 that maps to chromosome 2. J Biol Chem 1994; 269:13092-9.
10. Melki R, Colomb E, Lefort N, Brezin AP, Garchon HJ. CYP1B1 mutations in French patients with early-onset primary open-angle glaucoma. J Med Genet 2004; 41:647-51.
11. Sarfarazi M, Akarsu AN, Hossain A, Turacli ME, Aktan SG, Barsoum-Homsy M, Chevrette L, Sayli BS. Assignment of a locus (GLC3A) for primary congenital glaucoma (Buphthalmos) to 2p21 and evidence for genetic heterogeneity. Genomics 1995; 30:171-7.
12. Stoilov I, Akarsu AN, Sarfarazi M. Identification of three different truncating mutations in cytochrome P4501B1 (CYP1B1) as the principal cause of primary congenital glaucoma (Buphthalmos) in families linked to the GLC3A locus on chromosome 2p21. Hum Mol Genet 1997; 6:641-7.
13. Stoilov I, Akarsu AN, Alozie I, Child A, Barsoum-Homsy M, Turacli ME, Or M, Lewis RA, Ozdemir N, Brice G, Aktan SG, Chevrette L, Coca-Prados M, Sarfarazi M. Sequence analysis and homology modeling suggest that primary congenital glaucoma on 2p21 results from mutations disrupting either the hinge region or the conserved core structures of cytochrome P4501B1. Am J Hum Genet 1998; 62:573-84.
14. Bejjani BA, Lewis RA, Tomey KF, Anderson KL, Dueker DK, Jabak M, Astle WF, Otterud B, Leppert M, Lupski JR. Mutations in CYP1B1, the gene for cytochrome P4501B1, are the predominant cause of primary congenital glaucoma in Saudi Arabia. Am J Hum Genet 1998; 62:325-33.
15. Watanabe J, Shimada T, Gillam EM, Ikuta T, Suemasu K, Higashi Y, Gotoh O, Kawajiri K. Association of CYP1B1 genetic polymorphism with incidence to breast and lung cancer. Pharmacogenetics 2000; 10:25-33.
16. Chang BL, Zheng SL, Isaacs SD, Turner A, Hawkins GA, Wiley KE, Bleecker ER, Walsh PC, Meyers DA, Isaacs WB, Xu J. Polymorphisms in the CYP1B1 gene are associated with increased risk of prostate cancer. Br J Cancer 2003; 89:1524-9.
17. Sasaki M, Tanaka Y, Kaneuchi M, Sakuragi N, Dahiya R. CYP1B1 gene polymorphisms have higher risk for endometrial cancer, and positive correlations with estrogen receptor alpha and estrogen receptor beta expressions. Cancer Res 2003; 63:3913-8.
18. Wo YY, Stewart J, Greenlee WF. Functional analysis of the promoter for the human CYP1B1 gene. J Biol Chem 1997; 272:26702-7.
19. Han W, Pentecost BT, Spivack SD. Functional evaluation of novel single nucleotide polymorphisms and haplotypes in the promoter regions of CYP1B1 and CYP1A1 genes. Mol Carcinog 2003; 37:158-69.
20. Dudbridge F. Pedigree disequilibrium tests for multilocus haplotypes. Genet Epidemiol 2003; 25:115-21.
21. Stephens M, Smith NJ, Donnelly P. A new statistical method for haplotype reconstruction from population data. Am J Hum Genet 2001; 68:978-89.
22. Stephens M, Donnelly P. A comparison of bayesian methods for haplotype reconstruction from population genotype data. Am J Hum Genet 2003; 73:1162-9.
23. Abecasis GR, Cookson WO. GOLD--graphical overview of linkage disequilibrium. Bioinformatics 2000; 16:182-3.
24. Colomb E, Nguyen TD, Bechetoille A, Dascotte JC, Valtot F, Brezin AP, Berkani M, Copin B, Gomez L, Polansky JR, Garchon HJ. Association of a single nucleotide polymorphism in the TIGR/MYOCILIN gene promoter with the severity of primary open-angle glaucoma. Clin Genet 2001; 60:220-5.
25. 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; 70:1575-81.
26. Melki R, Belmouden A, Akhayat O, Brezin A, Garchon HJ. The M98K variant of the OPTINEURIN (OPTN) gene modifies initial intraocular pressure in patients with primary open angle glaucoma. J Med Genet 2003; 40:842-4.
27. Polansky JR, Juster RP, Spaeth GL. Association of the myocilin mt.1 promoter variant with the worsening of glaucomatous disease over time. Clin Genet 2003; 64:18-27.
28. Powell BL, Toomes C, Scott S, Yeung A, Marchbank NJ, Spry PG, Lumb R, Inglehearn CF, Churchill AJ. Polymorphisms in OPA1 are associated with normal tension glaucoma. Mol Vis 2003; 9:460-4 <http://www.molvis.org/molvis/v9/a58/>.
29. Fan BJ, Wang DY, Fan DS, Tam PO, Lam DS, Tham CC, Lam CY, Lau TC, Pang CP. SNPs and interaction analyses of myocilin, optineurin, and apolipoprotein E in primary open angle glaucoma patients. Mol Vis 2005; 11:625-31 <http://www.molvis.org/molvis/v11/a74/>.
30. Bandiera S, Weidlich S, Harth V, Broede P, Ko Y, Friedberg T. Proteasomal degradation of human CYP1B1: effect of the Asn453Ser polymorphism on the post-translational regulation of CYP1B1 expression. Mol Pharmacol 2005; 67:435-43.
31. Vincent AL, Billingsley G, Buys Y, Levin AV, Priston M, Trope G, Williams-Lyn D, Heon E. Digenic inheritance of early-onset glaucoma: CYP1B1, a potential modifier gene. Am J Hum Genet 2002; 70:448-60.
32. Aklillu E, Oscarson M, Hidestrand M, Leidvik B, Otter C, Ingelman-Sundberg M. Functional analysis of six different polymorphic CYP1B1 enzyme variants found in an Ethiopian population. Mol Pharmacol 2002; 61:586-94.