|Year : 2017 | Volume
| Issue : 1 | Page : 21-25
Association of angiotensin-converting enzyme, CYP46A1 genes polymorphism with senile cataract
Syed Tasleem Raza1, Shania Abbas1, Anu Chandra1, Luxmi Singh2, Saliha Rizvi1, Farzana Mahdi1
1 Department of Biochemistry, Era's Lucknow Medical College and Hospital, Lucknow, Uttar Pradesh, India
2 Department of Opthalmology, Era's Lucknow Medical College and Hospital, Lucknow, Uttar Pradesh, India
|Date of Web Publication||21-Feb-2017|
Syed Tasleem Raza
Department of Biochemistry, Era's Lucknow Medical College and Hospital, Lucknow - 226 025, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Senile cataract is the most common type of cataract characterized by gradual progressive thickening of the lens of the eye. Previously, many studies investigated the association between genetic polymorphism and senile cataract. Angiotensin-converting enzyme (ACE) I/D polymorphism is the potential risk factor for many eye-related diseases such as retinopathy and glaucoma. CYP46A1 enzyme converts cholesterol to 24S-hydroxycholesterol; human lens' membranes contain the highest cholesterol content. Defects in enzymes of cholesterol metabolism can be associated with cataracts. Hence, the present study was carried out to investigate the association of ACE and CYP46A1 genes polymorphism with senile cataract cases and controls.
Materials and Methods: ACE (rs 4646994) and CYP46A1 (rs 754203) genes polymorphism in cases and controls were evaluated by polymerase chain reaction and restriction fragment length polymorphism.
Results: This study included 103 senile cataract cases (55 were males and 48 were females) and 102 controls (53 were males and 49 were females). Mean age of cases in this study was 52.02 ± 12.11 years while in control group 53.74 ± 11.87 years. Frequencies of ACE ID, DD, and II genotypes in senile cataract cases were 64.07%, 4.85%, and 31.06% and controls were 61.76%, 26.47%, and 11.76%, respectively. The CYP46A1 gene CT, CC, and TT genotype frequencies were 48.54%, 8.73%, and 42.71% in senile cataract cases and 28.43%, 3.92%, and 67.64% in healthy controls, respectively. ACE DD and II genotypes (P < 0.001,P = 0.0008) and CYP46A1 CT and TT genotypes (P = 0.003,P = 0.0003) were significantly associated with senile cataract cases compared to the controls.
Conclusion: Findings of this study suggest that ACE and CYP46A1 genes polymorphism may be a predictive marker for early identification of population at risk of senile cataract. This potential role of ACE and CYP46A1 genes polymorphism as a marker of susceptibility to senile cataract needs further validation in studies involving larger number of patients from different regions.
Keywords: Angiotensin-converting enzyme, CYP46A1, genetic polymorphism, renin-angiotensin system, senile cataract
|How to cite this article:|
Raza ST, Abbas S, Chandra A, Singh L, Rizvi S, Mahdi F. Association of angiotensin-converting enzyme, CYP46A1 genes polymorphism with senile cataract. Oman J Ophthalmol 2017;10:21-5
|How to cite this URL:|
Raza ST, Abbas S, Chandra A, Singh L, Rizvi S, Mahdi F. Association of angiotensin-converting enzyme, CYP46A1 genes polymorphism with senile cataract. Oman J Ophthalmol [serial online] 2017 [cited 2017 Mar 29];10:21-5. Available from: http://www.ojoonline.org/text.asp?2017/10/1/21/200699
| Introduction|| |
A cataract is a clouding that develops in the crystalline lens of the eye or in its envelope; varying in degree from slight to complete opacity and obstructing the passage of light. According to the World Health Organization (WHO), cataract is the most common cause of blindness; accounting for 47.8% of the 161 million visually disabled people worldwide. Previously, many studies investigated the association between genetic polymorphism and senile cataract. Angiotensin-converting enzyme (ACE) is a key enzyme that converts inactive angiotensin I into a vasoactive and aldosterone-stimulating peptide angiotensin II. In some cases, the increase of ACE protein is responsible for the elevation of angiotensin II level. Previously, ACE Alu polymorphism has been associated with proliferative diabetic retinopathy. Most of the recognized renin-angiotensin system (RAS) components such as ACE, prorenin, renin, and angiotensinogen have already been detected in the human eye. In addition to circulatory RAS, there is a tissue-localized system, and local RAS may have a significant role in the formation of aqueous humor and also in its drainage. The ACE gene has been the main probable candidate gene predisposing the development of diabetic retinopathy. Findings of Movva et al. also demonstrated D allele carriers with type 2 diabetes to be more vulnerable to the development of diabetic nephropathy.
CYP46A1 is a member of the cytochrome P-450 family. It converts cholesterol to 24S-hydroxycholesterol. 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. Cholesterol-24S-hydroxylase was initially identified as a cholesterol-metabolizing enzyme in the brain. Accumulating evidence suggests that cholesterol is a risk factor for Alzheimer's disease;,,CYP46A1 has also been detected in rodent and bovine retinal ganglion cells, indicating a physiologic role in the cholesterol metabolism of the mammalian retina., Human lens' membranes contain the highest cholesterol content of any known biological membrane. Defects in enzymes of cholesterol metabolism and use of drugs which inhibit lens cholesterol biosynthesis can be associated with cataracts. The aim of the study was to investigate the association between ACE and CYP46A1 genes polymorphism and senile cataract cases.
| Materials and Methods|| |
The study enrolled 103 senile cataract cases from the Outpatient Department of Cataract Clinic Department of Ophthalmology at Era's Lucknow Medical College and Hospital, Lucknow. Clinical and biochemical parameters were also done for each patient. Data collection was done for each patient on clinical variables including age, alcohol consumption, body mass index, height, weight, cigarette smoking, and family history. All patients with senile cataract had visual disturbance and their corrected visual acuities were under 6/24. We excluded patients with secondary cataract due to diabetes, trauma, steroid administration, and other causes. One hundred and two healthy volunteers without age-related cataract or other age-related ocular diseases were enrolled as the control group in the same clinic. Informed consent was obtained from each patient before the study.
Protocol and procedures employed were reviewed and approved by the Institutional Ethical/Review Committee.
Five milliliters of peripheral blood was collected from all the patients in 0.5 M EDTA tubes. Genomic DNA was isolated from whole blood using the standard phenol-chloroform extraction method. The DNA concentration was determined by spectrophotometer and stored at −20°C.
Analysis of polymorphisms
Angiotensin-converting enzyme polymorphism
Polymerase chain reaction (PCR) was employed for genotyping of the ACE I/D polymorphism. Reactions were performed with 10 pmol of each primer: forward primer 5'-CTGGAGACCACTCCCATCCTTTCT-3', reverse primer 5'-GATGTGGCCATCTTCGTCAGAT -3', in a final volume of 20 μl containing 3 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl (pH 8.4), 0.5 mM of each dNTPs, and 2U Taq polymerase. PCR amplification was carried out under the conditions: Initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 45 s, annealing at 60°C for 1.15 min, extension at 72°C for 2.30 min, and final extension at 72°C for 5 min. PCR products were separated on 2.0% ethidium bromide-stained agarose gel and visualized by UVP BioImaging gel doc system. The product was 490 bp for allele I and 190 bp for allele D [Figure 1].
|Figure 1: Agarose gel picture showing polymerase chain reaction product for angiotensin-.converting enzyme polymorphism. Lane 1, 2 and 8 is I/I genotype (490 bp), lane 6, 7 is I/D genotype, lane 3 and 5 is D/D genotype (190 bp), and lane 4 is 100 bp ladder|
Click here to view
Genotyping was performed using PCR-restriction fragment length polymorphism 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 10X 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, 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 [Figure 2].
|Figure 2: Agarose gel picture showing PCR-RFLP product for CYP46A1 gene polymorphism. The CYP46A1*T allele corresponds to uncut 285 bp fragment, whereas the CYP46A1*C allele characterized by two fragment of 209 bp and 76 bp. Lane 3 shows C/T genotype, lane 6 shows C/C genotype, lane 1, 2, 5, and 7 shows T/T genotype, and lane 4 shows 100 bp ladder|
Click here to view
All the figures are presented as means ± standard deviation. The genotyping data were compared between cases and controls using Chi-square test. Other variables were compared using Student's t-test for normally distributed variables. Hardy–Weinberg (HW) equilibrium has been calculated using HW Diagnostics-Version 1 beta (Fox Chase Cancer Center, Philadelphia, PA, USA). All statistical tests were performed using Statistical Package for the Social Sciences version 12 software International Business Machines Corp, New Orchard Road, Armonk, New York.
| Results|| |
Our study included 103 senile cataract cases (55 were males and 48 were females) and 102 controls (53 were males and 49 were females). Mean age of the cases in this study was 52.02 ± 12.11 years, while in control group, it was 53.74 ± 11.87 years. Clinical and biochemical parameters of cases and controls are shown in [Table 1].
|Table 1: Clinical and biochemical parameters of cataract cases and controls|
Click here to view
Hardy–Weinberg equilibrium test
ACE and CYP46A1 genotype distributions in all cases and controls were in line with HW equilibrium (all P > 0.05, data not shown).
Genetic polymorphism analysis
Angiotensin-converting enzyme polymorphism analysis
ACE gene ID, DD, II genotype frequencies were 64.07%, 4.85%, 31.06% in cases and 61.76%, 26.47%, 11.76% in controls, respectively. Odds ratio (OR) for ID genotype was 1.104 (P = 0.732), for DD genotype OR = 0.142 (P < 0.001), for II genotype OR was 3.38 (P = 0.0008). The frequency of I and D allele in cases was 63.11% and 36.89% as compared to 42.65% and 57.35% in the controls. OR for I and D was 2.3.
Significant differences were observed in the genotypes and alleles frequencies of ACE gene on comparing senile cataract cases with healthy control group [Table 2] for details].
|Table 2: Genotype and allele frequencies of angiotensin-converting enzyme and CYP46A1 genes in cataract cases and healthy controls|
Click here to view
CYP46A1 polymorphism analysis
The CYP46A1 gene CT, CC, TT genotypes frequencies were 48.54%, 8.73%, 42.71% in cases and 28.43%, 3.92%, 67.64% in healthy controls, respectively. OR for CT genotype was 2.375 (P = 0.912), for CC genotype was 2.346 (P = 0.157), and for TT genotype was 0.357 (P = 0.0003). The frequencies of C and T alleles were 33.01% and 66.99% in cases as compared to 18.14% and 81.86% in the controls. OR for C was 2.224 and for T allele was 0.449 (P < 0.001).
Significant association was found between CYP46A1 genes polymorphism with senile cataract where CT, TT genotypes and C, T allele were found to increase the risk of senile cataract whereas CC genotype and C allele conferred a low risk for the same [Table 2] for details].
| Discussion|| |
Age-related cataracts are responsible for 48% of world blindness, which represents about 18 million people, according to the WHO. Previously, many studies investigated the association between genetic polymorphisms and cataract. According to our data, we have found a significant difference in weight (P < 0.001), intraocular pressure (P < 0.001), diastolic blood pressure (P = 0.016), serum bilirubin (P < 0.001), hemoglobin (P < 0.001), total leukocytes count (P < 0.001), eosinophils (P < 0.001), platelet count (P = 0.009) between cases and control.
Angiotensin-converting enzyme polymorphism
Numerous studies have previously investigated ACE I/D polymorphism as a potential risk factor for many eye-related diseases such as retinopathy and glaucoma., In our study, on ACE gene polymorphism, we observed no significant relationship between ACE ID genotype and cataract (P = 0.732). We found that the frequency of occurrence of II genotypes was higher in our senile cataract cases (31.06%) as compared to controls (11.76%), whereas the frequency of DD genotype was lower in cases (4.85%) as compared to controls (26.47%), respectively. On studying the allele frequencies between cases and controls, we found that the frequency of carrying I allele was significantly higher in cases (63.11%, P < 0.001) as compared to controls (42.65%) suggesting I allele to be a positive risk factor for senile cataract. On the other hand, frequency of D allele was significantly lower in cases (36.89%, P < 0.001) as compared to controls (57.35%) suggesting D allele to be a negative risk factor for senile cataract. The above results are shown in [Table 2].
CYP46A1 gene was initially identified as a cholesterol-metabolizing enzyme in the brain. The TT genotype in intron 2 of CYP46A1, designated as the rs754203 SNP, has been identified as a risk factor for Alzheimer disease,,, and more recently, for POAG.CYP46A1 and its metabolic product 24S-hydroxycholesterol are specific for the neural retina, and especially for retinal ganglion cells., We have observed significant association between CYP46A1 gene polymorphism and cataract. According to our data, the frequency of CT genotype and C allele was significantly higher in cases than control (P = 0.003, P < 0.001). We have found that the frequency of TT genotype and T allele was significantly lower in cases as compared to the control (P < 0.001). In our study, we found that the frequency of CT genotype in senile cataract cases was 48.54% which was similar to the frequency of CT genotype in French cataract cases 49.2%. The frequency of CC genotype in senile cataract cases in our study was 8.73% which was slightly higher than French cataract cases (2.5%). The frequencies of TT genotype in our population (42.71%) were found to be lower as compared to the Australian population with cataract (47.8%). Our results show that CC genotype was not significant between cases and control (P = 0.157).
| Conclusion|| |
Findings of this study suggest that ACE and CYP46A1 genes polymorphism can be a predictive marker for early identification of population at risk of cataract. This potential role of ACE and CYP46A1 genes polymorphism as a marker of susceptibility to senile cataract needs further validation in studies involving larger number of patients from different regions.
The study was supported by an intramural grant from the Era's Lucknow Medical College and Hospital, Lucknow, Uttar Pradesh, India.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Resnikoff S, Pascolini D, Etya'ale D, Kocur I, Pararajasegaram R, Pokharel GP, et al.
Global data on visual impairment in the year 2002. Bull World Health Organ 2004;82:844-51.
Shiels A, Hejtmancik JF. Genetic origins of cataract. Arch Ophthalmol 2007;125:165-73.
Oktem F, Sirin A, Bilge I, Emre S, Agaçhan B, Ispir T. ACE I/D gene polymorphism in primary FSGS and steroid-sensitive nephrotic syndrome. Pediatr Nephrol 2004;19:384-9.
Pucci L, Lucchesi D, Stellini D, Bandinelli S, Giannarelli R, Penno G, et al
. Alu DNA polymorphism in ACE gene is protective for age-related macular degeneration. Biochem Biophys Res Commun 2002;295:668-72.
Vaajanen A, Vapaatalo H. Local ocular renin-angiotensin system – A target for glaucoma therapy? Basic Clin Pharmacol Toxicol 2011;109:217-24.
Movva S, Alluri RV, Komandur S, Vattam K, Eppa K, Mukkavali KK, et al.
Relationship of angiotensin-converting enzyme gene polymorphism with nephropathy associated with type 2 diabetes mellitus in Asian Indians. J Diabetes Complications 2007;21:237-41.
Garcia AN, Muniz MT, Souza e Silva HR, da Silva HA, Athayde-Junior L. Cyp46 polymorphisms in Alzheimer's disease: A review. J Mol Neurosci 2009;39:342-5.
Wang B, Zhang C, Zheng W, Lu Z, Zheng C, Yang Z, et al.
Association between a T/C polymorphism in intron 2 of cholesterol 24S-hydroxylase gene and Alzheimer's disease in Chinese. Neurosci Lett 2004;369:104-7.
Li Y, Chu LW, Chen YQ, Cheung BM, Leung RY, Yik PY, et al.
Intron 2 (T/C) CYP46 polymorphism is associated with Alzheimer's disease in Chinese patients. Dement Geriatr Cogn Disord 2006;22:399-404.
Fu BY, Ma SL, Tang NL, Tam CW, Lui VW, Chiu HF, et al.
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 2009;24:921-6.
Bretillon L, Diczfalusy U, Bjorkhem I, Ståhle L, Ahlborg G, Wahren J, et al
. Cholesterol-24S-hydroxylase (CYP46A1) is specifically expressed in neurons of the neural retina. Curr Eye Res 2007;32:361-6.
Ramirez DM, Andersson S, Russell DW. Neuronal expression and subcellular localization of cholesterol 24-hydroxylase in the mouse brain. J Comp Neurol 2008;507:1676-93.
Cenedella RJ. Cholesterol and cataracts. Surv Ophthalmol 1996;40:320-37.
Sambrook J, Frisch EF, Maniatis T, editors. Molecular Cloning: A Labortory Manual. 2nd
ed., Vol. 9. New York: Cold Spring Harbor Laboratory Press; 1989. p. 9.14-9.19.
Dua HS, Said DG, Otri AM. Are we doing too many cataract operations? Cataract surgery: A global perspective. Br J Ophthalmol 2009;93:1-2.
Hashizume K, Mashima Y, Fumayama T, Ohtake Y, Kimura I, Yoshida K, et al.
Genetic polymorphisms in the angiotensin II receptor gene and their association with open-angle glaucoma in a Japanese population. Invest Ophthalmol Vis Sci 2005;46:1993-2001.
Ozkur M, Erbagci I, Gungor K, Nacak M, Aynacioglu S, Bekir NA. Angiotensin-converting enzyme insertion-deletion polymorphism in primary open-angle glaucoma. Ophthalmologica 2004;218:415-8.
Fourgeux C, Martine L, Björkhem I, Diczfalusy U, Joffre C, Acar N, et al.
Primary open-angle glaucoma: Association with cholesterol 24S-hydroxylase (CYP46A1) gene polymorphism and plasma 24-hydroxycholesterol levels. Invest Ophthalmol Vis Sci 2009;50:5712-7.
Mossböck G, Weger M, Faschinger C, Schmut O, Renner W, Wedrich A, et al.
Role of cholesterol 24S-hydroxylase gene polymorphism (rs754203) in primary open angle glaucoma. Mol Vis 2011;17:616-20.
[Figure 1], [Figure 2]
[Table 1], [Table 2]