Polymorphism of CYP46A1 and PPARγ2 Genes in Risk Prediction of Primary Open Angle Glaucoma Among North Indian Population.

PURPOSE: Glaucoma is the leading cause of irreversible blindness and the second most common cause of all blindness after cataracts. This study investigates the association of polymorphism in the CYP46A1 and PPARγ2 genes and primary open angle glaucoma (POAG).
MATERIALS AND METHODS: This study evaluated 122 POAG cases (POAG group) and 112 cases of nonglaucomatous patients (control group). Polymorphisms of the CYP46A1 gene and PPARγ2 gene were evaluated with the polymerase chain reaction-restriction fragment length polymorphism method in both groups.
RESULTS: The mean ages were 49.88 ± 12.34 years and 53.74 ± 11.87 years for the POAG group and control group, respectively. The CYP46A1 gene CC, CT, TT genotype frequencies were 13.93%, 58.2%, 27.87% in the POAG group and 19.6%, 40.19%, 40.19% in the control group, respectively. The PPARγ2 gene CC, CG, GG genotype frequencies were 16.83%, 54.45%, 28.71% in cases and 3.92%, 28.43%, 67.64% in the control group, respectively. Statistically, significant differences in the frequencies of CYP46A1 CC, CT, TT and PPARγ2 CC, CG, GG (P < 0.05) genotype were found between groups (P < 0.05, all comparisons).
CONCLUSION: Findings of this study suggest that CYP46A1 gene and PPARγ2 gene polymorphisms can be a predictive marker for early identification of population at risk of POAG, although a larger sample size is required to determine the role of these polymorphisms in the pathogenesis and course of POAG.

INTRODUCTION

Glaucoma is the leading cause of irreversible blindness and the second most common cause of all blindness after cataracts. Globally, glaucoma affects at least 90 million people. There were 60.5 million people with primary glaucoma in 2010 which is forecasted to increase to 79.6 million by 20201 causing blindness in 11.2 million people.1 Primary open angle glaucoma (POAG) is the most common type of glaucoma and with no obvious ocular abnormality that indicates a cause. Although mutations in several genes, including myocilin, optineurin, and CYP1B1, have been reported to cause POAG, these genes account for <10% of cases worldwide.23 Genetic analysis has substantially contributed to the study of glaucoma over the past few decades. However, the link between genes and the development of the disease is not always clearly demonstrated.4 Other associations such as diabetes or systemic hypertension are still being debated.

Debate has also been sparked by a study that found cholesterol-lowering statins may decrease the risk of glaucoma development and progression.567 The CYP46A1 gene is almost located to neurons in the brain and retina. The gene encoding cholesterol 24-hydroxylase plays a key role in the hydroxylation of cholesterol and thereby mediates its removal from the brain.8 CYP46A1 is a member of the cytochrome P450 family, detected in rodent and bovine retinal ganglion cells, indicating a physiologic role in cholesterol metabolism of the mammalian retina.910

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors in the nuclear hormone receptor superfamily related to retinoid, steroid, and thyroid hormone receptors.11 Three subtypes of PPAR have been identified as PPARα, PPARβ, PPARγ. The PPAR subfamily is involved in many cellular processes including lipid and glucose homeostasis, cellular proliferation, differentiation, and control of inflammation. The PPAR0γ gene is located on chromosome 3p25 and encodes a nuclear transcription factor involved in the expression of hundreds of genes. PPARγ ligands contain antiangiogenic properties; it can inhibit the proangiogenic effects of vascular endothelial growth factor and endothelial cell migration.12 PPARγ agonist can also inhibit fibrotic changes by suppressing transforming growth factor beta signaling.13 Several corneal studies on PPARγ using the micropellet technique or gene transfer have been performed.1415 However, no ocular study used an ophthalmic solution of the PPAR0γ agonist. Hence, the present study was performed to investigate the association of CYP46A1 and PPARγ2 gene polymorphism with POAG cases and controls.

MATERIALS AND METHODS

Patient selection

Blood samples were collected from 122 patients with POAG (POAG group) and 112 healthy subjects without glaucoma (control group). Phlebotomy was performed at the Department of Ophthalmology of Era's Lucknow Medical College and Hospital, Lucknow, India. Data were collected on clinical variables including age, alcohol consumption, body mass index, height, weight, cigarette smoking, and family history. Each subject underwent a complete ophthalmological examination. Patients with POAG were defined by the presence of an open angle, pathological cupping of the optic disc, a glaucoma hemifield test (GHT) outside normal limits with reproducible visual field defects at the same location on two consecutive visits, and an intraocular pressure >21 mm Hg without topical glaucoma therapy. The cup-to-disc ratios were between 0.4 and 0.9. Patients with a history of eye surgery before the diagnosis of glaucoma, evidence of secondary glaucoma including exfoliation, pigment dispersion or uveitis, and other causes were excluded. The control group was comprised nonsmokers who were not diabetes and had no systemic illness. They had no family or personal history of glaucoma. In the control group, optic discs appeared normal on indirect ophthalmoscope with a cup-to-disc ratio of 0.3 or lower and GHT within normal limits. Ethical Committee clearance was obtained from the respective departments, prior to patient recruitment.

DNA extraction

Five milliliters of peripheral blood was collected from all the subjects and stored in 0.5 M ethylenediaminetetraacetic acid tubes. Genomic DNA was isolated from whole blood using the standard phenol-chloroform extraction method. The DNA concentration was determined with a spectrophotometer and stored at −20°C.

Analysis of polymorphisms

CYP46A1 Polymorphism

Genotyping was performed using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) with the following primer: 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 min, the reaction mixture was subjected to 50 cycles of 1 min denaturation at 95°C, 1 min annealing at 53°C, and a 2 min extension at 72°C, followed by a final extension at 72°C for 5 min. The PCR products were digested by MspI (Promega, Charbonnie’res, 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 [Figure 1].

Figure 1

Agarose gel picture showing polymerase chain reaction product for CYP46A1 gene polymorphism. The CYP46A1*T allele corresponds to uncut 285-bp fragment, whereas the CYP46A1*C allele characterized by two fragments of 209-bp and 76-bp. Lane 1, 5, 6 shows C/T genotype, lane 2, 7, 8 shows C/C genotype, lane 3 shows T/T genotype, and lane 4 shows 100-bp ladder

PPARγ2 Polymorphism

Genotyping was performed using PCR-RFLP with the following primer: The forward primer 5’- CAA GCC CAG TCC TTT CTG TG-3’, the reverse primer 5’- AGT GAA GGA ATC GCT TTC CG-3.’ All reactions were performed in a total volume of 50 μl containing 10 mmol/L Tris-HCl (PH 8.8), 50 mmol/L KCl, 1.5 mmol/L MgCl2, 0.2 mmol of each dNTP, 50 pmol of each of the two primers, unit of Taq DNA polymerase, 200 μg of genomic DNA were added last to PCR. The amplification program was started as initial precycling denaturation by holding at 94°C for 3 min, denaturation for 40 cycles at 94°C for 30 s followed by annealing at 53°C for 30 s, extension at 72°C for 1 min, and a final extension period at 72°C for 9 min. RFLP was detected after digestion overnight with 2 U of the restriction enzyme Hpa II which cuts the mutant allele at a site introduced by the reverse primer. Samples were applied to 3% agarose gel and subjected to electrophoresis for about half an hour and visualized on an ultraviolet transilluminator [Figure 2].

Figure 2

Agarose gel picture showing polymerase chain reaction-restriction fragment length polymorphism LP product of PPARγ 2 gene, lane1 shows undigested product lane 2, 3, 7, 8 shows CG (+/−) genotype lane 4, 6 shows CC (+/+), and lane 6 shows 100-bp ladder

Statistical analysis

All the figures are presented as means ± standard deviation. The genotyping data were compared between cases and controls with the Chi-square test. Variables with normal distribution were compared with Student's t-test. All statistical tests were performed using Statistical Package for the Social Sciences version 12 software (IBM Corp., Armonk, New York, USA). A P < 0.05 was considered statistically significant.

RESULTS

There were (65 males and 57 females) in POAG group. The control group was comprised 59 males and 53 females. The mean age was 49.88 ± 12.34 years for the POAG group and 53.74 ± 11.87 years for the control group. Clinical and biochemical parameters of cases and controls are presented in Table 1. The mean red blood cell lysate glutathione levels were statistically, significantly lower in the POAG group compared to the control group (P < 0.05). The CYP46A1 gene CC, CT, TT genotype frequencies were 13.93%, 58.2%, 27.87% in POAG group and 19.6%, 40.19%, 40.19% in healthy controls, respectively. Odds ratio (OR) for CC was 0.66 (95% confidence interval [CI] 0.33-1.32, P = 0.242, χ2 = 1.37, power = 0.880), for CT 2.07 (95% CI 1.23-3.49, P = 0.006, χ2 = 7.58, power = 0.95), and for TT 0.58 (95% CI 0.33-0.99, P = 0.047, χ2 = 3.96, power = 0.894). The frequencies of C and T allele in the POAG group were 43.03% and 56.97% as compared to 39.70% and 60.29% in the control group. OR for C was 1.15 (95% CI 0.79-1.66, P = 0.470, χ2 = 0.52, power = 0.844), and for T 0.87 (95% CI 0.60-1.26, P = 0.470, χ2 = 0.52, power = 0.844). The PPARγ2 gene CC, CG, GG genotype frequencies were 16.83%, 54.45%, 28.71% for the POAG group and 3.92%, 28.43%, 67.64% in the control group, respectively. OR for CC was 4.96 (95% CI 1.61-15.31, P = 0.003, χ2 = 9.12, power = 0.964), for CG 3.01 (95% CI 1.68-5.39, P = 0.0002, χ2 = 14.17, power = 0.978), and for GG 0.19 (95% CI 0.11-0.35, P < 0.0001, χ2 = 30.81, power = 0.995). The frequencies of C and G allele in the POAG group were 44.05% and 55.94% as compared to 18.13% and 81.86% in the control group. OR for C was 3.55 (95% CI 2.62-5.58, P < 0.0001, χ2 = 31.87, power = 0.996) and for G 0.28 (95% CI 0.18-0.44, P < 0.0001, χ2 = 31.87, power = 0.996). The genotype, allele's frequencies of CYP46A1, PPARγ2, and statistical analysis among the cases and controls are also shown in Table 2. Genotype distribution for all investigated single nucleotide polymorphisms was in Hardy-Weinberg equilibrium in both groups.

Table 1

Clinical and biochemical profile of POAG cases and controls

Table 2

Genotypes and alleles frequency of CYP46A1 and PPARγ2 genes in POAG cases and controls

DISCUSSION

Glaucoma is defined as progressive optic neuropathies with apoptotic retinal ganglion cell death leading to cupping of the optic nerve with typical visual field defects. If it is untreated, the natural course leads to blindness, or at least significant visual disability.16 Although much effort has been made, the exact pathogenesis remains elusive.2

CYP46A1 polymorphism

Recently, controversy has arisen over the association of polymorphisms in the CYP46 locus with increased risk of developing Alzheimer's disease.1718 Several observations have suggested a possible relationship between Alzheimer disease and glaucoma192021222324 with possible pathogenic similarities. In the current study, we have found a significant association of CYP46A1 CT, TT genotype between POAG cases and control (P < 0.0001). We have observed that frequency of CYP46A1 CC genotype was 13.93% in our population which is significantly lower in comparison with 29.9% for a Hong Kong cohort, 30.4% for a Shantou cohort (Southern Chinese) 30.4%, and 37.5% for a Northern Chinese (Beijing cohort).25 The frequency of the computed tomography genotype was 58.2% which is significantly higher 35.3% in the French population and 48.8% in Caucasians.2627 We have observed that the frequency of the TT genotype was 27.87% in our cases which are lower than 61.3% for the French population and 41.2% for Caucasians.2829 In the present study, we have found a possible association of CYP46A1 gene with POAG which was also observed in the French population while no such association was observed in the Hong Kong cohort, Shantou cohort (Southern Chinese), Northern Chinese (Beijing cohort), and Caucasians [Table 3].252627

Table 3

CYP46A1 gene polymorphism in different ethnic groups

PPARγ2 polymorphism

PPARG is a key transcription factor involved in adipocyte differentiation, lipid, and glucose homeostasis.28 It is an important therapeutic target for type 2 diabetes and metabolic syndrome.28 The association between the substitution of G (alanine) for C (proline) at codon 12 of PPARγ2 and risk for type 2 diabetes mellitus has been widely studies since Yen et al. first reported this polymorphism.29 We have observed that PPARγ2 CC, CG, GG genotype frequencies were 16.83%, 54.45%, 28.71% in the POAG group and 3.92%, 28.43%, 67.64% in the control group, respectively. A statistically significant association was found in the genotype frequency of CYP46A1 gene between POAG group and the control group (P < 0.05). We have observed that the genotype frequencies of PPARγ2 CC and CG genotype were 16.83% and 54.45% in the POAG group which is significantly higher than 3.92% for CC and 28.43% for CG in the control group. The frequency of the GG genotype was 55.94% in the POAG group which is lower in comparison to 67.64% for the control group. We also observed the possible association between PPARγ2 C and G allele and POAG (P < 0.0001). In our population, the frequency of PPARγ2 C allele was higher in the POAG group at 44.05% as compared to 18.13% for the control group. However, the G (55.94%) allele was lower in the POAG group compare to the control group (81.86%).

The findings of this study suggest that CYP46A1 and PPARγ2 gene polymorphisms can be a predictive marker for early identification of a population at risk of POAG, although a larger sample is required to elucidate the role of these polymorphisms in the pathogenesis and course of glaucoma.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.