Ophthalmic Genetics, Early Online, 1–6, 2014 ! Informa Healthcare USA, Inc. ISSN: 1381-6810 print / 1744-5094 online DOI: 10.3109/13816810.2014.907921
RESEARCH REPORT
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Polymorphism of GST and FTO Genes in Risk Prediction of Cataract among a North Indian Population Anu Chandra1, Syed Tasleem Raza1, Shania Abbas1, Luxmi Singh2, Saliha Rizvi1, Faisal Ahmed1, Ale Eba1, and Farzana Mahdi1 1
Departments of Biochemistry and 2Opthalmology, Era’s Lucknow Medical College and Hospital, Lucknow, India
ABSTRACT Background: The present study was carried out to investigate the association of GST and FTO gene polymorphisms with cataract cases and controls. Materials and methods: The study included 131 cases and 126 controls. GST and FTO gene polymorphisms were evaluated by PCR-RFLP. Results: The frequency of the GSTM1-positive and GSTT1-positive in cataract cases were 62.13% and 86.40% while in the controls it was 46.39% and 95.87% with odds ratios of 1.9 (95% CI, 1.08–3.32; p value 0.025) and 0.27 (95% CI, 0.09–0.86; p value, 0.019) respectively. There was a statistically significant association between the GSTM1 null genotype and the risk of cataract development with an odds ratio of 0.43 (95% CI, 0.24–0.76; p value, 0.003). Significant differences were obtained in the frequencies of FTO AA and TT genotype (p = 0.023 and 0.023) between cases and controls. Conclusion: The present study suggested that GSTM1, GSTT1 and FTO gene polymorphisms are associated with increased risk for cataract in North Indian populations. Due to the limited sample size, the finding on GST and FTO gene polymorphisms need further investigation. Keywords: Cataract, FTO, genetic polymorphism, GST
INTRODUCTION
important roles in the development of senile cataract.9,10 Oxidative stress as a result of increased generation of reactive oxygen species and free radicals in the lens has been considered one of the main causes of senile cataract.11,12 The reducing compound glutathione is one of the essential antioxidants.13,14 Glutathione peroxidase scavenges the highly reactive lipid hydroperoxide in the aqueous phase of the cell membrane. During aging, the lens loses its antioxidant potencies with the decrease of glutathione or the decrease of expression of the antioxidant enzymes.15 Glutathione S-transferases (GSTs) are a group of dimeric detoxification enzymes that play an important role in
India contributes approximately 1.20 crore blind people, which is about one-fourth of the total blind population of the world and it shows that 14.9 out of every 1000 Indian are blind compared to about 3 per 1000 in most developed countries. Despite taking into account differing rates of cataract surgery, populationbased studies have reported higher prevalence rates of cataract in India.1–3 Epidemiologic studies have shown that cataract is associated with many environmental factors such as ultraviolet B light exposure, smoking, alcohol consumption, and use of steroids.4–8 Recently, genetic factors have been found to play
Received 10 January 2014; revised 10 February 2014; accepted 16 March 2014; published online 22 April 2014 Correspondence: Syed Tasleem Raza, PhD, Department of Biochemistry, Era’s Lucknow Medical College and Hospital, Lucknow, India 226025. Tel: +91 522 2408122, 2408123 Fax: +91 5222407824. E-mail: [email protected]
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detoxifying exogenous and endogenous toxic compounds under conditions of oxidative stress.16–19 Human GSTs are divided into different classes; including mu, theta and omega. Genetic polymorphisms in genes encoding Glutathione S-transferase M1 (a member of class mu; OMIM 138350), Glutathione S-transferase T1 (a member of class theta; OMIM 600436), and Glutathione S-transferase O2 (a member of class omega, OMIM: 612314) have been defined.20–22 The GST isozymes existing in human tissues and human lens tissue have been reported to express the mu, theta, and pi classes of this enzyme.23 Genes for Glutathione S-transferase M1 (GSTM1) and glutathione S transferase T1 (GSTT1) have been found to have functional polymorphisms and have been studied extensively in context to eye diseases.19,24–26 Also, studies have been conducted on the association between polymorphisms of several classes of glutathione S-transferases (such as GSTM1, GSTT1) with cataract risk in different populations.15,26,27 Recently, part of a genome-wide association study found that single nucleotide polymorphisms in the FTO gene was strongly associated with obesity.28,29 Several studies have reported the association between obesity and cataract.30–32 The fat mass and obesityassociated (FTO) gene, which is located on chromosome 16q12.2, has nine exons and emerged 450 million years ago.33 FTO is mainly expressed in the hypothalamus and encodes a 2-oxoglutarate-dependent nucleic acid demethylase. It may play important roles in the management of energy homeostasis,33,34 nucleic acid demethylation, and the regulation of body fat masses by lipolysis.35 A number of single nucleotide polymorphisms (SNPs) in tight linkage disequilibrium with rs9939609, and residing in the first intron of the FTO gene, have been associated with obesity in large populations of adults and children. These variants have also been shown to be associated with type 2 diabetes in an obesity-dependent manner.29,36,37 In the present study, we examined the association between GST and FTO gene polymorphisms in cataract cases among a North Indian population.
MATERIALS AND METHODS Selection of Patients A total of 131 blood samples of cataract cases and 126 healthy controls were collected from the Department of Ophthalmology of Era’s Lucknow Medical College & Hospital, Lucknow, India. Data collection was done for each patient on clinical variables including age, alcohol consumption, body mass index, height, weight, cigarette smoking and family history etc. All subjects with senile cataract (72 males, 59 females) 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. The age-matched control subjects were collected from unrelated volunteers in the same clinic. Informed consent was obtained from each subject before the study. Ethical committee’s clearances were obtained from the respective departments, earlier to the recruitment of subjects in this study.
DNA Extraction Five millilitres of peripheral blood was collected from all the subjects in 0.5 M EDTA tubes. Genomic DNA was isolated from whole blood using the standard phenol-chloroform extraction method.38 The DNA concentration was determined by spectrophotometer and stored at 20 C.
Analysis of Polymorphisms GSTM1 and GSTT1 polymorphisms GSTM1 and GSTT1 genetic polymorphisms were evaluated using multiplex polymerase chain reaction (PCR) technique (MJ Mini Thermo Cycler- BioRad).39 Primers for GSTM1 were 50 -GAA CTC CCT GAA AAG CTAA AGC and 50 GTT GGG CTC AAA TAT ACG GTG G and for GSTT1 were 50 -TTC CTT ACT GGT CCT CAC ATC TC and 50 -TCA CCG GACAT GGC CAG CA. The b-globin locus was used as an internal control to avoid false-negative readings. Primers for b-globin were 50 -CAA CTT CAT CCA CGT TCA CC and 50 -GAA GAG CCA AGG ACA GGT AC. PCR reaction was carried out in a total volume of 25 ml containing 10 pmol of each primer, 2.5 mmol/L of MgCl2, 0.2 mmol/L of each deoxynucleotide triphosphate, 1 unit of Taq polymerase (Bioline Ltd., London, UK) and 100 ng of genomic DNA. Amplification was performed by initial denaturation at 94 C for 5 minutes, followed by 30 cycles at 94 C for 1 minute, 64 C for 1 minute and 72 C for 1 minute and a final extension of 72 C for 7 minutes. The amplified products were identified by electrophoresis in a 1.5% agarose gel and stained with 0.5 ug/ml ethidium bromide. The product lengths were 215 bp, 480 bp, and 268 bp for GSTM1, GSTT1 and b-globin, respectively (Figure 1). FTO Polymorphism The FTO SNP (rs9939609) was genotyped by PCR (MJ Mini Thermo Cycler- BioRad) and restriction fragment length polymorphism analysis.40 Primers for FTO were forward (50 -AACTGGCTCTTGAATG AAATAGGATTCAGA-30 ) and reverse (50 0 AGAGTAACAGAGACTATCCAAGTGCAGTAC-3 ). Ophthalmic Genetics
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GST and FTO Genes Polymorphism in Cataract PCR reaction was carried out in a total volume of 25 ml containing 10 pmol of each primer, 2.5 mmol/L of MgCl2, 0.2 mmol/L of each deoxynucleotide triphosphate, 1 unit of Taq polymerase (Bioline Ltd., London, UK) and 100 ng of genomic DNA. Amplification was performed by initial denaturation at 94 C for 5 min followed by 20 cycles of 94 C for 45 sec, 61 C for 45 sec (dropping 0.5 C per cycle), and 72 C for 45 sec. After this, the PCR mix was incubated for 15 cycles of 94 C for 45 sec, 51 C for 45 sec and 72 C for 45 sec, followed by a final incubation at 72 C for 10 min. The PCR products thus obtained were incubated at 37 C for 16 h with 2 U ScaI (New England Biolabs, Hitchin, UK). Upon running the final products on a 3% agarose gel, the T allele produced a 182-bp band and the A allele produced 154 and 28-bp bands (Figure 2).
Statistical Analysis All the figures are presented as means ± SD. The genotyping data were compared between cases and controls using the Chi-square test. Other variables were compared using Student’s t-test for normallydistributed variables. All statistical tests were performed using SPSS (Statistical Package for the Social Sciences) version 12 software.
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TABLE 1. Clinical and biochemical parameters of cataract cases and controls. Parameters
Cataract cases (n = 131)
Control (n = 126)
Gender (M/F) Age (yrs) Weight (kg) SBP (mm Hg) DBP (mm Hg) RBS (mg/dl) Hb (gm%) TLC (cell/cumm) Neutrophils (%) Lymphocytes (%) Eosinophils (%) Monocytes (%) Pl. count (Lakh/cumm)
72/59 52.02 ± 12.11 51.98 ± 10.43 126.04 ± 16.92 81.82 ± 8.83 129.31 ± 30.35 11.65 ± 1.19 7792 ± 2029 65.51 ± 9.35 29.14 ± 8.00 4.83 ± 3.94 2.47 ± 13.81 2.22 ± 0.77
69/57 53.74 ± 11.87 57.57 ± 11.39 122.30 ± 2019 82.15 ± 19.30 124 ± 61.47 14.45 ± 1.65 7571.70 ± 1690.06 65.35 ± 6.73 28.70 ± 6.73 4.58 ± 2.89 1.19 ± 3.20 1.99 ± 0.68
n, number of subjects SBP = Systolic blood pressure; DBP = Diastolic blood pressure; RBS = Random Blood Sugar; TLC = Total leukocyte count
24.74% respectively. The frequency of A and T allele in cataract cases was 30.10% and 69.90% as compared to 37.63% and 62.37% in the controls. Odds Ratio for AT was 0.5 (95%CI 0.27–0.91, p = 0.023, 2 = 5.17, p = 0.023, 2 = 5.17, power = 0.905). The genotype, allele’s frequencies of GSTM1, GSTT1 and FTO and statistical analysis among the cases and controls are also shown in Table 2.
RESULTS DISCUSSION Our study included 131 cataract cases (72 were males and 59 were females) and 126 controls (69 were males and 57 were females). Mean age of the cases in this study was 53.74 ± 11.87 years, while in the control group it was 52.02 ± 12.11 years. Clinical and biochemical parameters of cases and controls are shown in Table 1. The frequency of GSTM1 and GSTT1 null genotypes was 33.01% and 13.60% in cataract cases while it was 53.61% and 4.12% in the controls, respectively. The frequency of GSTM1-positive and GSTT1-positive was 62.13% and 86.40% in cataract cases as compared to 46.39% and 95.87% in the controls. The odds ratio for GSTM1 null genotypes was 0.43 (95%CI 0.24–0.76, p = 0.003, 2 = 8.65, power = 0.867), and for GSTT1 null genotypes 2.23 (95%CI 0.66–7.48, p = 0.186, 2 = 1.75, power = 0.864). The odds ratio for GSTM1-positive genotypes was 1.9 (95%CI 1.08–3.32, p = 0.025, 2 = 4.99, power = 0.848), for GSTT1-positive genotypes 0.27 (95%CI 0.09–0.86, p = 0.019, 2 = 5.47, power = 0.793) and the frequency of GSTM1, GSTT1 wild genotype was 53.39% in cataract cases and 42.27% in control. The odds ratio for GSTM1, GSTT1 wild genotype was 1.56 (95%CI 0.86–2.74, p = 0.115, 2 = 2.48, power = 0.774). The frequencies of the FTO genotypes AA, AT, TT in cataract cases were 0%, 60.19%, 39.81% while in the control group these frequencies were 0%, 75.26% and !
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Cataract is one of the leading causes of visual impairment and blindness in the world today.41 Genetic factors are considered the most important factors in the development of senile cataract. Previously, many studies have investigated the association between genetic polymorphisms and cataract.
GST Gene Polymorphism Many studies have attempted to show an association between GST polymorphic variants and disease susceptibility. The GSTM1 and GSTT1 polymorphisms were reported to be associated with many diseases, including glaucoma,42,43 acute leukemia,44 senile macular degeneration45 and cataract in the different populations tested.46 There are several reasons to account for the association between the GSTM1positive genotype and cortical age-related cataract. GST enzymes are very useful for detoxification and defense against oxidative stress, but they are also involved in reactions that result in toxic products. The toxic products may lead to structural damage of the proteins in the lens that then cause lens opacification.47,48 Also, the GST activity is significantly decreased in the cataractous lens compared with
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TABLE 2. Genotype and allele frequency of GSTT1, GSTM1 and FTO (rs9939609) genes in cataract cases and controls. Controls (126)
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GST Genotype GSTT1-positive, GSTM1-positive GSTT1-null GSTM1-positive GSTT1-Positive GSTM1-null FTO Genotype AA AT TT Allele A T
Cases (131)
n
Frequency (%)
n
Frequency (%)
OR
95% CI
2
p Values
power
53 5 58 121 68
42.27 4.12 46.39 95.87 53.61
70 18 81 113 43
53.39 13.60 62.13 86.40 33.01
1.56 2.23 1.9 0.27 0.43
0.86–2.74 0.66–7.48 1.08–3.32 0.09–0.86 0.24–0.76
2.48 1.75 4.99 5.47 8.65
0.115 0.186 0.025 0.019 0.003
0.774 0.864 0.848 0.793 0.867
N/A 95 31
N/A 75.26 24.74
N/A 79 52
N/A 60.19 39.81
N/A 0.5 2.01
N/A 0.27–0.91 1.10–3.69
N/A 5.17 5.17
N/A 0.023 0.023
N/A 0.905 0.905
94 158
37.63 62.37
79 183
30.10 69.90
0.71 1.4
0.47–1.08 0.92–2.12
2.54 2.54
0.111 0.111
0.83 0.83
that in the normal lens, the GSTM1-positive genotype may have lost its ability to detoxify the harmful substances under oxidative stress. In our study, we found that the frequencies of GSTM1 null, GSTT1 null genotypes in cases and controls were 33.01%, 13.60% and 53.61%, 4.12%, respectively, while the frequency of GSTM1-positive and GSTT1-positive were 62.13% and 86.40% in the cases as compared to 46.39% and 95.87% in controls. These results agree with previous findings reporting on cataract in the Chinese population, Iranian population, and Estonian population.47,49,50 The result showed that the GSTT1 null genotype was not associated with cataract (p = 0.186). Similarly, no association was found between the GSTT1 null genotype and cataract in Iranian,15 Estonian,47 and Turkish48 populations. We have observed that the genotype frequency of GSTT1positive was 86.4% which is similar to the Iranian and Estonian populations (73.65%, 85.5%), while it is higher in comparison with the Chinese population (47.6%).49,50 The frequency of GSTM1 null genotype was (13.6%) in our population which is lower in comparison with the Chinese population (52.4%).49 Thus, in our study, GSTM1-positive (p = 0.025), GSTM1-null (p = 0.003) and GSTT1-positive (p = 0.019) genotypes, were found to be associated with cataract.
FTO Gene Polymorphism Obesity is one of the most prevalent disorders in the world. It constitutes an important risk for several diseases such as type 2 diabetes, hypertension, stroke, osteoarthritis, and sleep apnea syndrome.51 Some eye diseases like cataract,52,53 glaucoma,30,54 diabetic retinopathy,55 and age-related macular degeneration56 were reported to have potential relation to obesity. In our study, we found that the frequencies of AT and TT
FIGURE 1. PCR products analyzed on 2% agarose gel. The presence or absence (null) of GSTM1 and GSTT1 was detected by the presence or absence of a band at 480 bp (corresponding to GSTT1) and a band at 210 bp (corresponding to GSTM1). b-Globin is considered an internal control (260 bp). 100 bp ladder Lane 5; GSTM1 and GSTT1 wild type Lane 2, 3, 6; GSTM1 null Lane 4; b-Globin bands Lane 1, 7, 8.
genotype in cataract cases and controls were 79%, 52% and 95%, 31% respectively. The frequencies of A and T alleles in cases and controls were 79%, 183% and 94%, 158% respectively. Our results show a positive association between the AT and TT genotype with cataract (p = 0.023), however adequate data of FTO polymorphism and cataract was not available. These findings are consistent with data from some existing studies.52,53,57
CONCLUSION In this case-control study, positive associations were found between GSTM1-null, GSTM1-positive and GSTT1-positive genotypes with cataract. FTO gene polymorphism was also significantly associated in Ophthalmic Genetics
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GST and FTO Genes Polymorphism in Cataract
FIGURE 2. Polyacrylamide gel picture showing digested PCR products for FTO gene polymorphism. Lane 2, 3, 6, 7 shows AT genotype (182 and 154), lane 4 shows TT genotype (182 bp), lane 5 shows 100 bp ladder and lane 1 shows undigested PCR product of FTO (182 bp).
cataract cases. The present study thus suggests that GST and FTO gene polymorphism is associated with cataract and may be a genetic risk factor for the prediction of this disease. Due to the limited sample size, the findings need further investigation to test the validity of this study.
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. We are grateful for the support of an intramural grant from the Era’s Lucknow Medical College and Hospital, Lucknow, Uttar Pradesh, India.
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28. Hinney A. Genome wide association (GWA) study for early onset extreme obesity supports the role of fat mass and obesity associated gene (FTO) variants. PloS ONE 2007;2:e1361. 29. Scuteri A, Sanna S, Chen WM, et al. Genome-wide association scan shows genetic variants in the FTO gene are associated with obesity-related traits. Plos Genet 2007; 3:1200–1210. 30. Cheung N, Wong TY. Obesity and eye diseases. Surv Ophthalmol 2007;52:180–195. 31. Weintraub JM, Willett WC, Rosner B, et al. A prospective study of the relationship between body mass index and cataract extraction among US women and men. Int J Obes Relat Metab Disord 2002;26:1588–1595. 32. Schaumberg DA, Glynn RJ, Christen WG, et al. Relations of body fat distribution and height with cataract in men. Am J Clin Nutr 2000;72:1495–1502. 33. Fredriksson R, Hagglund M, Olszewski PK, et al. The obesity gene, FTO, is of ancient origin, up-regulated during food deprivation and expressed in neurons of feedingrelated nuclei of the brain. Endocrinology 2008;149: 2062–2071. 34. Stratigopoulos G, Padilla SL, Leduc CA, et al. Regulation of Fto/Ftm gene expression in mice and humans. Am J Physiol – Regulatory Integr Compar Physiol 2008;294: R1185–R1196. 35. Wahlen K, Sjolin E, Hoffstedt J. The common rs9939609 gene variant of the fat mass- and obesity-associated gene FTO is related to fat cell lipolysis. J Lipid Res 2008;49: 607–661. 36. Frayling TM, Timpson NJ, Weedon MN, et al. A common variant in the FTO gene is associated with body mass index and predisposes to childhood and adult obesity. Science 2007;316:889–894. 37. Dina C, Meyre D, Gallina S, et al. Variation in FTO contributes to childhood obesity and severe adult obesity. Nat Genet 2007;39:724–726. 38. Sambrook J, Frisch EF, Maniatis T. Molecular cloning; a laboratory manual. 2nd edition. New York: Cold Sping Harbor Laboratory Press; 1989:9.14–9.19. 39. Arand M, Muhlbauer R, Hengstler J, et al. Multiplex polymerase chain reaction protocol for the simultaneous analysis of the glutathione S-transferase GSTM1 and GSTT1 polymorphisms. Anal Biochem 1996; 236:184–186. 40. Hao K, Niu T, Sangokoya C, et al. SNP kit: an efficient approach to systematic evaluation of candidate single nucleotide polymorphisms in public databases. Biotechniques 2002;33:822–828. 41. Brian G, Taylor H. Cataract blindness – challenges for the 21st century. Bull World Health Organ 2001;79:249–256. 42. Izzotti A, Sacca SC, Cartiglia C, De Flora S. Oxidative deoxyribonucleic acid damage in the eyes of glaucoma patients. Am J Med 2003;114:638–646.
43. Jansson M, Rada A, Tomic L, et al. Analysis of the glutathione S-transferase M1 gene using pyrosequencing and multiplex PCR – no evidence of association to glaucoma. Exp Eye Res 2003;77:239–243. 44. Glass DC, Gray CN, Jolley DJ, et al. Leukemia risk associated with low-level benzene exposure. Epidemiology 2003;14:569–577. 45. Oz O, Aras Ates N, Tamer L, et al. Glutathione S-transferase M1, T1, and P1 gene polymorphism in exudative age-related macular degeneration: a preliminary report. Eur J Ophthalmol 2006;16:105–110. 46. Hao Y, He S, Gu Z, et al. Relationship between GSTM1 genotype and susceptibility to senile cataract. Zhonghua Yan Ke Za Zhi 1999;35:104–106. 47. Juronen E, Tasa G, Veromann S, et al. Polymorphic glutathione S-transferases as genetic risk factors for senile cortical cataract in Estonians. Invest Ophthalmol Vis Sci 2000;41:2262–2267. 48. Gu¨ven M, Unal M, Sarici A, et al. Glutathione-S-transferase M1 and T1 genetic polymorphisms and the risk of cataract development: a study in the Turkish population. Curr Eye Res 2007;32:447–454. 49. Jiang Z, Liang K, Zhang Q, Tao L. Glutathione S-transferases polymorphisms confer susceptibility to senile cortical cataract in the Han Chinese population. Mol Vis 2012;18: 1247–1252. 50. Saadat I, Ahmadi Z, Farvardin-Jahromi M, Saadat M. Association between cataract and genetic polymorphisms of GSTM1, GSTT1, and GSTO2 with respect of work place. Mol Vis 2012;18:1996–2000. 51. Haslam DW, James WP. Obesity. Lancet 2005;366: 1197–1209. 52. Leske MC, Wu SY, Hennis A. Diabetes, hypertension, and central obesity as cataract risk factors in a black population. The Barbados Eye Study. Ophthalmology 1999;106: 35–41. 53. Kuang TM, Tsai SY, Hsu WM, et al. Body mass index and age-related cataract: the Shihpai Eye Study. Arch Ophthalmol 2005;123:1109–1114. 54. Leske MC, Connell AM, Wu SY, et al. Risk factors for openangle glaucoma. The Barbados Eye Study. Arch Ophthalmol 1995;113:918–924. 55. van Leiden HA, Dekker JM, Moll AC, et al. Blood pressure, lipids, and obesity are associated with retinopathy: the Hoorn study. Diabet Care 2002;25:1320–1325. 56. Clemons TE, Milton RC, Klein R, et al.; Age-Related Eye Disease Study Research Group. Risk factors for the incidence of advanced age-related macular degeneration in the Age-Related Eye Disease Study (AREDS). AREDS report no. 19. Ophthalmology 2005;112:533–539. 57. Hiller R, Podgor MJ, Sperduto RD, et al. A longitudinal study of body mass index and lens opacities. The Framingham Studies. Ophthalmology 1998;105:1244–1250.
Ophthalmic Genetics
RESEARCH REPORT
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Polymorphism of GST and FTO Genes in Risk Prediction of Cataract among a North Indian Population Anu Chandra1, Syed Tasleem Raza1, Shania Abbas1, Luxmi Singh2, Saliha Rizvi1, Faisal Ahmed1, Ale Eba1, and Farzana Mahdi1 1
Departments of Biochemistry and 2Opthalmology, Era’s Lucknow Medical College and Hospital, Lucknow, India
ABSTRACT Background: The present study was carried out to investigate the association of GST and FTO gene polymorphisms with cataract cases and controls. Materials and methods: The study included 131 cases and 126 controls. GST and FTO gene polymorphisms were evaluated by PCR-RFLP. Results: The frequency of the GSTM1-positive and GSTT1-positive in cataract cases were 62.13% and 86.40% while in the controls it was 46.39% and 95.87% with odds ratios of 1.9 (95% CI, 1.08–3.32; p value 0.025) and 0.27 (95% CI, 0.09–0.86; p value, 0.019) respectively. There was a statistically significant association between the GSTM1 null genotype and the risk of cataract development with an odds ratio of 0.43 (95% CI, 0.24–0.76; p value, 0.003). Significant differences were obtained in the frequencies of FTO AA and TT genotype (p = 0.023 and 0.023) between cases and controls. Conclusion: The present study suggested that GSTM1, GSTT1 and FTO gene polymorphisms are associated with increased risk for cataract in North Indian populations. Due to the limited sample size, the finding on GST and FTO gene polymorphisms need further investigation. Keywords: Cataract, FTO, genetic polymorphism, GST
INTRODUCTION
important roles in the development of senile cataract.9,10 Oxidative stress as a result of increased generation of reactive oxygen species and free radicals in the lens has been considered one of the main causes of senile cataract.11,12 The reducing compound glutathione is one of the essential antioxidants.13,14 Glutathione peroxidase scavenges the highly reactive lipid hydroperoxide in the aqueous phase of the cell membrane. During aging, the lens loses its antioxidant potencies with the decrease of glutathione or the decrease of expression of the antioxidant enzymes.15 Glutathione S-transferases (GSTs) are a group of dimeric detoxification enzymes that play an important role in
India contributes approximately 1.20 crore blind people, which is about one-fourth of the total blind population of the world and it shows that 14.9 out of every 1000 Indian are blind compared to about 3 per 1000 in most developed countries. Despite taking into account differing rates of cataract surgery, populationbased studies have reported higher prevalence rates of cataract in India.1–3 Epidemiologic studies have shown that cataract is associated with many environmental factors such as ultraviolet B light exposure, smoking, alcohol consumption, and use of steroids.4–8 Recently, genetic factors have been found to play
Received 10 January 2014; revised 10 February 2014; accepted 16 March 2014; published online 22 April 2014 Correspondence: Syed Tasleem Raza, PhD, Department of Biochemistry, Era’s Lucknow Medical College and Hospital, Lucknow, India 226025. Tel: +91 522 2408122, 2408123 Fax: +91 5222407824. E-mail: [email protected]
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detoxifying exogenous and endogenous toxic compounds under conditions of oxidative stress.16–19 Human GSTs are divided into different classes; including mu, theta and omega. Genetic polymorphisms in genes encoding Glutathione S-transferase M1 (a member of class mu; OMIM 138350), Glutathione S-transferase T1 (a member of class theta; OMIM 600436), and Glutathione S-transferase O2 (a member of class omega, OMIM: 612314) have been defined.20–22 The GST isozymes existing in human tissues and human lens tissue have been reported to express the mu, theta, and pi classes of this enzyme.23 Genes for Glutathione S-transferase M1 (GSTM1) and glutathione S transferase T1 (GSTT1) have been found to have functional polymorphisms and have been studied extensively in context to eye diseases.19,24–26 Also, studies have been conducted on the association between polymorphisms of several classes of glutathione S-transferases (such as GSTM1, GSTT1) with cataract risk in different populations.15,26,27 Recently, part of a genome-wide association study found that single nucleotide polymorphisms in the FTO gene was strongly associated with obesity.28,29 Several studies have reported the association between obesity and cataract.30–32 The fat mass and obesityassociated (FTO) gene, which is located on chromosome 16q12.2, has nine exons and emerged 450 million years ago.33 FTO is mainly expressed in the hypothalamus and encodes a 2-oxoglutarate-dependent nucleic acid demethylase. It may play important roles in the management of energy homeostasis,33,34 nucleic acid demethylation, and the regulation of body fat masses by lipolysis.35 A number of single nucleotide polymorphisms (SNPs) in tight linkage disequilibrium with rs9939609, and residing in the first intron of the FTO gene, have been associated with obesity in large populations of adults and children. These variants have also been shown to be associated with type 2 diabetes in an obesity-dependent manner.29,36,37 In the present study, we examined the association between GST and FTO gene polymorphisms in cataract cases among a North Indian population.
MATERIALS AND METHODS Selection of Patients A total of 131 blood samples of cataract cases and 126 healthy controls were collected from the Department of Ophthalmology of Era’s Lucknow Medical College & Hospital, Lucknow, India. Data collection was done for each patient on clinical variables including age, alcohol consumption, body mass index, height, weight, cigarette smoking and family history etc. All subjects with senile cataract (72 males, 59 females) 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. The age-matched control subjects were collected from unrelated volunteers in the same clinic. Informed consent was obtained from each subject before the study. Ethical committee’s clearances were obtained from the respective departments, earlier to the recruitment of subjects in this study.
DNA Extraction Five millilitres of peripheral blood was collected from all the subjects in 0.5 M EDTA tubes. Genomic DNA was isolated from whole blood using the standard phenol-chloroform extraction method.38 The DNA concentration was determined by spectrophotometer and stored at 20 C.
Analysis of Polymorphisms GSTM1 and GSTT1 polymorphisms GSTM1 and GSTT1 genetic polymorphisms were evaluated using multiplex polymerase chain reaction (PCR) technique (MJ Mini Thermo Cycler- BioRad).39 Primers for GSTM1 were 50 -GAA CTC CCT GAA AAG CTAA AGC and 50 GTT GGG CTC AAA TAT ACG GTG G and for GSTT1 were 50 -TTC CTT ACT GGT CCT CAC ATC TC and 50 -TCA CCG GACAT GGC CAG CA. The b-globin locus was used as an internal control to avoid false-negative readings. Primers for b-globin were 50 -CAA CTT CAT CCA CGT TCA CC and 50 -GAA GAG CCA AGG ACA GGT AC. PCR reaction was carried out in a total volume of 25 ml containing 10 pmol of each primer, 2.5 mmol/L of MgCl2, 0.2 mmol/L of each deoxynucleotide triphosphate, 1 unit of Taq polymerase (Bioline Ltd., London, UK) and 100 ng of genomic DNA. Amplification was performed by initial denaturation at 94 C for 5 minutes, followed by 30 cycles at 94 C for 1 minute, 64 C for 1 minute and 72 C for 1 minute and a final extension of 72 C for 7 minutes. The amplified products were identified by electrophoresis in a 1.5% agarose gel and stained with 0.5 ug/ml ethidium bromide. The product lengths were 215 bp, 480 bp, and 268 bp for GSTM1, GSTT1 and b-globin, respectively (Figure 1). FTO Polymorphism The FTO SNP (rs9939609) was genotyped by PCR (MJ Mini Thermo Cycler- BioRad) and restriction fragment length polymorphism analysis.40 Primers for FTO were forward (50 -AACTGGCTCTTGAATG AAATAGGATTCAGA-30 ) and reverse (50 0 AGAGTAACAGAGACTATCCAAGTGCAGTAC-3 ). Ophthalmic Genetics
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GST and FTO Genes Polymorphism in Cataract PCR reaction was carried out in a total volume of 25 ml containing 10 pmol of each primer, 2.5 mmol/L of MgCl2, 0.2 mmol/L of each deoxynucleotide triphosphate, 1 unit of Taq polymerase (Bioline Ltd., London, UK) and 100 ng of genomic DNA. Amplification was performed by initial denaturation at 94 C for 5 min followed by 20 cycles of 94 C for 45 sec, 61 C for 45 sec (dropping 0.5 C per cycle), and 72 C for 45 sec. After this, the PCR mix was incubated for 15 cycles of 94 C for 45 sec, 51 C for 45 sec and 72 C for 45 sec, followed by a final incubation at 72 C for 10 min. The PCR products thus obtained were incubated at 37 C for 16 h with 2 U ScaI (New England Biolabs, Hitchin, UK). Upon running the final products on a 3% agarose gel, the T allele produced a 182-bp band and the A allele produced 154 and 28-bp bands (Figure 2).
Statistical Analysis All the figures are presented as means ± SD. The genotyping data were compared between cases and controls using the Chi-square test. Other variables were compared using Student’s t-test for normallydistributed variables. All statistical tests were performed using SPSS (Statistical Package for the Social Sciences) version 12 software.
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TABLE 1. Clinical and biochemical parameters of cataract cases and controls. Parameters
Cataract cases (n = 131)
Control (n = 126)
Gender (M/F) Age (yrs) Weight (kg) SBP (mm Hg) DBP (mm Hg) RBS (mg/dl) Hb (gm%) TLC (cell/cumm) Neutrophils (%) Lymphocytes (%) Eosinophils (%) Monocytes (%) Pl. count (Lakh/cumm)
72/59 52.02 ± 12.11 51.98 ± 10.43 126.04 ± 16.92 81.82 ± 8.83 129.31 ± 30.35 11.65 ± 1.19 7792 ± 2029 65.51 ± 9.35 29.14 ± 8.00 4.83 ± 3.94 2.47 ± 13.81 2.22 ± 0.77
69/57 53.74 ± 11.87 57.57 ± 11.39 122.30 ± 2019 82.15 ± 19.30 124 ± 61.47 14.45 ± 1.65 7571.70 ± 1690.06 65.35 ± 6.73 28.70 ± 6.73 4.58 ± 2.89 1.19 ± 3.20 1.99 ± 0.68
n, number of subjects SBP = Systolic blood pressure; DBP = Diastolic blood pressure; RBS = Random Blood Sugar; TLC = Total leukocyte count
24.74% respectively. The frequency of A and T allele in cataract cases was 30.10% and 69.90% as compared to 37.63% and 62.37% in the controls. Odds Ratio for AT was 0.5 (95%CI 0.27–0.91, p = 0.023, 2 = 5.17, p = 0.023, 2 = 5.17, power = 0.905). The genotype, allele’s frequencies of GSTM1, GSTT1 and FTO and statistical analysis among the cases and controls are also shown in Table 2.
RESULTS DISCUSSION Our study included 131 cataract cases (72 were males and 59 were females) and 126 controls (69 were males and 57 were females). Mean age of the cases in this study was 53.74 ± 11.87 years, while in the control group it was 52.02 ± 12.11 years. Clinical and biochemical parameters of cases and controls are shown in Table 1. The frequency of GSTM1 and GSTT1 null genotypes was 33.01% and 13.60% in cataract cases while it was 53.61% and 4.12% in the controls, respectively. The frequency of GSTM1-positive and GSTT1-positive was 62.13% and 86.40% in cataract cases as compared to 46.39% and 95.87% in the controls. The odds ratio for GSTM1 null genotypes was 0.43 (95%CI 0.24–0.76, p = 0.003, 2 = 8.65, power = 0.867), and for GSTT1 null genotypes 2.23 (95%CI 0.66–7.48, p = 0.186, 2 = 1.75, power = 0.864). The odds ratio for GSTM1-positive genotypes was 1.9 (95%CI 1.08–3.32, p = 0.025, 2 = 4.99, power = 0.848), for GSTT1-positive genotypes 0.27 (95%CI 0.09–0.86, p = 0.019, 2 = 5.47, power = 0.793) and the frequency of GSTM1, GSTT1 wild genotype was 53.39% in cataract cases and 42.27% in control. The odds ratio for GSTM1, GSTT1 wild genotype was 1.56 (95%CI 0.86–2.74, p = 0.115, 2 = 2.48, power = 0.774). The frequencies of the FTO genotypes AA, AT, TT in cataract cases were 0%, 60.19%, 39.81% while in the control group these frequencies were 0%, 75.26% and !
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Cataract is one of the leading causes of visual impairment and blindness in the world today.41 Genetic factors are considered the most important factors in the development of senile cataract. Previously, many studies have investigated the association between genetic polymorphisms and cataract.
GST Gene Polymorphism Many studies have attempted to show an association between GST polymorphic variants and disease susceptibility. The GSTM1 and GSTT1 polymorphisms were reported to be associated with many diseases, including glaucoma,42,43 acute leukemia,44 senile macular degeneration45 and cataract in the different populations tested.46 There are several reasons to account for the association between the GSTM1positive genotype and cortical age-related cataract. GST enzymes are very useful for detoxification and defense against oxidative stress, but they are also involved in reactions that result in toxic products. The toxic products may lead to structural damage of the proteins in the lens that then cause lens opacification.47,48 Also, the GST activity is significantly decreased in the cataractous lens compared with
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TABLE 2. Genotype and allele frequency of GSTT1, GSTM1 and FTO (rs9939609) genes in cataract cases and controls. Controls (126)
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GST Genotype GSTT1-positive, GSTM1-positive GSTT1-null GSTM1-positive GSTT1-Positive GSTM1-null FTO Genotype AA AT TT Allele A T
Cases (131)
n
Frequency (%)
n
Frequency (%)
OR
95% CI
2
p Values
power
53 5 58 121 68
42.27 4.12 46.39 95.87 53.61
70 18 81 113 43
53.39 13.60 62.13 86.40 33.01
1.56 2.23 1.9 0.27 0.43
0.86–2.74 0.66–7.48 1.08–3.32 0.09–0.86 0.24–0.76
2.48 1.75 4.99 5.47 8.65
0.115 0.186 0.025 0.019 0.003
0.774 0.864 0.848 0.793 0.867
N/A 95 31
N/A 75.26 24.74
N/A 79 52
N/A 60.19 39.81
N/A 0.5 2.01
N/A 0.27–0.91 1.10–3.69
N/A 5.17 5.17
N/A 0.023 0.023
N/A 0.905 0.905
94 158
37.63 62.37
79 183
30.10 69.90
0.71 1.4
0.47–1.08 0.92–2.12
2.54 2.54
0.111 0.111
0.83 0.83
that in the normal lens, the GSTM1-positive genotype may have lost its ability to detoxify the harmful substances under oxidative stress. In our study, we found that the frequencies of GSTM1 null, GSTT1 null genotypes in cases and controls were 33.01%, 13.60% and 53.61%, 4.12%, respectively, while the frequency of GSTM1-positive and GSTT1-positive were 62.13% and 86.40% in the cases as compared to 46.39% and 95.87% in controls. These results agree with previous findings reporting on cataract in the Chinese population, Iranian population, and Estonian population.47,49,50 The result showed that the GSTT1 null genotype was not associated with cataract (p = 0.186). Similarly, no association was found between the GSTT1 null genotype and cataract in Iranian,15 Estonian,47 and Turkish48 populations. We have observed that the genotype frequency of GSTT1positive was 86.4% which is similar to the Iranian and Estonian populations (73.65%, 85.5%), while it is higher in comparison with the Chinese population (47.6%).49,50 The frequency of GSTM1 null genotype was (13.6%) in our population which is lower in comparison with the Chinese population (52.4%).49 Thus, in our study, GSTM1-positive (p = 0.025), GSTM1-null (p = 0.003) and GSTT1-positive (p = 0.019) genotypes, were found to be associated with cataract.
FTO Gene Polymorphism Obesity is one of the most prevalent disorders in the world. It constitutes an important risk for several diseases such as type 2 diabetes, hypertension, stroke, osteoarthritis, and sleep apnea syndrome.51 Some eye diseases like cataract,52,53 glaucoma,30,54 diabetic retinopathy,55 and age-related macular degeneration56 were reported to have potential relation to obesity. In our study, we found that the frequencies of AT and TT
FIGURE 1. PCR products analyzed on 2% agarose gel. The presence or absence (null) of GSTM1 and GSTT1 was detected by the presence or absence of a band at 480 bp (corresponding to GSTT1) and a band at 210 bp (corresponding to GSTM1). b-Globin is considered an internal control (260 bp). 100 bp ladder Lane 5; GSTM1 and GSTT1 wild type Lane 2, 3, 6; GSTM1 null Lane 4; b-Globin bands Lane 1, 7, 8.
genotype in cataract cases and controls were 79%, 52% and 95%, 31% respectively. The frequencies of A and T alleles in cases and controls were 79%, 183% and 94%, 158% respectively. Our results show a positive association between the AT and TT genotype with cataract (p = 0.023), however adequate data of FTO polymorphism and cataract was not available. These findings are consistent with data from some existing studies.52,53,57
CONCLUSION In this case-control study, positive associations were found between GSTM1-null, GSTM1-positive and GSTT1-positive genotypes with cataract. FTO gene polymorphism was also significantly associated in Ophthalmic Genetics
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GST and FTO Genes Polymorphism in Cataract
FIGURE 2. Polyacrylamide gel picture showing digested PCR products for FTO gene polymorphism. Lane 2, 3, 6, 7 shows AT genotype (182 and 154), lane 4 shows TT genotype (182 bp), lane 5 shows 100 bp ladder and lane 1 shows undigested PCR product of FTO (182 bp).
cataract cases. The present study thus suggests that GST and FTO gene polymorphism is associated with cataract and may be a genetic risk factor for the prediction of this disease. Due to the limited sample size, the findings need further investigation to test the validity of this study.
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. We are grateful for the support of an intramural grant from the Era’s Lucknow Medical College and Hospital, Lucknow, Uttar Pradesh, India.
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43. Jansson M, Rada A, Tomic L, et al. Analysis of the glutathione S-transferase M1 gene using pyrosequencing and multiplex PCR – no evidence of association to glaucoma. Exp Eye Res 2003;77:239–243. 44. Glass DC, Gray CN, Jolley DJ, et al. Leukemia risk associated with low-level benzene exposure. Epidemiology 2003;14:569–577. 45. Oz O, Aras Ates N, Tamer L, et al. Glutathione S-transferase M1, T1, and P1 gene polymorphism in exudative age-related macular degeneration: a preliminary report. Eur J Ophthalmol 2006;16:105–110. 46. Hao Y, He S, Gu Z, et al. Relationship between GSTM1 genotype and susceptibility to senile cataract. Zhonghua Yan Ke Za Zhi 1999;35:104–106. 47. Juronen E, Tasa G, Veromann S, et al. Polymorphic glutathione S-transferases as genetic risk factors for senile cortical cataract in Estonians. Invest Ophthalmol Vis Sci 2000;41:2262–2267. 48. Gu¨ven M, Unal M, Sarici A, et al. Glutathione-S-transferase M1 and T1 genetic polymorphisms and the risk of cataract development: a study in the Turkish population. Curr Eye Res 2007;32:447–454. 49. Jiang Z, Liang K, Zhang Q, Tao L. Glutathione S-transferases polymorphisms confer susceptibility to senile cortical cataract in the Han Chinese population. Mol Vis 2012;18: 1247–1252. 50. Saadat I, Ahmadi Z, Farvardin-Jahromi M, Saadat M. Association between cataract and genetic polymorphisms of GSTM1, GSTT1, and GSTO2 with respect of work place. Mol Vis 2012;18:1996–2000. 51. Haslam DW, James WP. Obesity. Lancet 2005;366: 1197–1209. 52. Leske MC, Wu SY, Hennis A. Diabetes, hypertension, and central obesity as cataract risk factors in a black population. The Barbados Eye Study. Ophthalmology 1999;106: 35–41. 53. Kuang TM, Tsai SY, Hsu WM, et al. Body mass index and age-related cataract: the Shihpai Eye Study. Arch Ophthalmol 2005;123:1109–1114. 54. Leske MC, Connell AM, Wu SY, et al. Risk factors for openangle glaucoma. The Barbados Eye Study. Arch Ophthalmol 1995;113:918–924. 55. van Leiden HA, Dekker JM, Moll AC, et al. Blood pressure, lipids, and obesity are associated with retinopathy: the Hoorn study. Diabet Care 2002;25:1320–1325. 56. Clemons TE, Milton RC, Klein R, et al.; Age-Related Eye Disease Study Research Group. Risk factors for the incidence of advanced age-related macular degeneration in the Age-Related Eye Disease Study (AREDS). AREDS report no. 19. Ophthalmology 2005;112:533–539. 57. Hiller R, Podgor MJ, Sperduto RD, et al. A longitudinal study of body mass index and lens opacities. The Framingham Studies. Ophthalmology 1998;105:1244–1250.
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