Polymorphism of FABP2 and PPARG2 genes in risk prediction of cataract among North Indian population

Background Cataract is the leading cause of bilateral blindness in India. It has been reported that cataract is responsible for 50–80% of the bilaterally blind in the country. Cataract formation is a natural part of the ageing process. At present, adequate data are not available regarding the FABP2 and PPARG2 gene polymorphisms and their susceptibility with cataract cases in the North Indian population. Thus, the present study was carried out to investigate the association of FABP2 and PPARG2 gene polymorphisms with cataract cases and controls. Materials and methods This study includes 130 cataract cases and 118 controls. FABP2 and PPARG2 gene polymorphisms in cases and controls were evaluated by PCR-RFLP. Results Frequencies of Ala54Ala, Ala54Thr and Thr54Thr genotypes in FABP2 gene in cataract cases and controls were 50.76%, 39.23%, 10% and 25.42%, 61.86%, 12.71% respectively. The PPARG2 gene CC, CG, GG genotype frequencies were 11.53%, 87.69% and 0.76% in cases and 21.18%, 39.83% and 38.98% in healthy controls respectively. Significant differences were observed in the frequencies of FABP2 Ala54Ala, Ala54Thr genotype (p < 0.05) and PPARG2 CC, CG, GG genotype (p < 0.05) between cases and controls. Conclusion The findings of this study suggest that FABP2 and PPARG2 gene polymorphisms can be an informative marker for early identification of population at risk of cataract. The potential role of FABP2 and PPARG2 gene polymorphisms as a marker of susceptibility to cataract needs further studies in a larger number of patients.


Introduction
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. Age related cataracts are responsible for 48% of world blindness, which represents about 18 million people, according to the World Health Organization (WHO) (Dua et al., 2009). It is a multifactorial disease which is associated with many environmental (McCarty and Taylor, 2002) and genetic variations (Hejtmancik and Kantorow, 2004;McCarty and Taylor, 2001;Shiels and Hejtmancik, 2007). 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 (Hiratsuka and Li, 2001;Klein et al., 2001;McCarty and Taylor, 2002;Tan et al., 2008;Wang et al., 2008). Recently, genetic factors have been found to play important roles in the development of senile cataract (Hejtmancik and Kantorow, 2004;McCarty and Taylor, 2001;Shiels and Hejtmancik, 2007).
The intestinal fatty acid-binding protein-2 (FABP2) gene codes a protein expressed in enterocytes and is responsible for the absorption of long-chain fatty acids (Weiss et al., 2002a(Weiss et al., , 2002b. The gene for FABP2 is located in the long arm of chromosome 4. The G to A polymorphism (rs1799883) of codon 54 results in the substitution of threonine (Thr) for alanine (Ala) (Baier et al., 1995). A single nucleotide polymorphism (SNP) in the FABP2 gene at codon 54 causes an amino acid change (Ala to Thr). Carriers of the Thr54 allele in FABP2 gene have a twofold greater affinity for the absorption of long-chain fatty acids than those with the Ala54 allele (Agren et al., 2001), which supports the role of the FABP2 Ala54Thr polymorphism in the etiology of obesity and metabolic disorders. Thr54 allele is significantly associated with higher total cholesterol, stroke incidence (Carlsson et al., 2000), elevation of fasting and postprandial triglyceride (Georgopoulos et al., 2000), insulin resistance (Baier et al., 1995;Weiss et al., 2002aWeiss et al., , 2002b, and higher non-esterified fatty acid concentrations (Pratley et al., 2000).
Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors in the nuclear hormone receptor superfamily related to retinoid, steroid and thyroid hormone receptors (Abdelrahman et al., 2005). The PPAR family is represented by three members: PPARα, PPARβ/δ, and PPARγ (Tyagi et al., 2011). The PPARγ gene contains 9 exons which spans more than 100 kb, and because of alternative mRNA splicing it results in the production of 2 protein isoforms: PPARγ1 and PPARγ2 (Fajas et al., 1997). The anti-inflammatory effects of PPARγ have been observed in various organs, although previous investigations mainly focused on internal organs, such as the kidneys (Taguchi et al., 2012), heart (Yu et al., 2012), and lungs (Reddy et al., 2012). In addition, a PPARγ agonist inhibited fibrotic changes by suppressing transforming growth factor beta (TGF-β) signaling (Hatanaka et al., 2012). In the present study, we examined the association between FABP2 and PPARG2 gene polymorphisms in cataract cases among North Indian population.

Patient's selection
A total of 130 blood samples of cataract cases and 118 healthy controls were collected from the Department of Ophthalmology of Era's Lucknow Medical College & Hospital, Lucknow. Data collection was done for each patient on clinical variables including age, alcohol consumption, body mass index, height, weight, cigarette smoking, 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 (institutional ethics committee no. ELMC/E-1/2010/3942) clearances were obtained from the respective departments earlier before the recruitment of subjects in this study.

DNA extraction
Five milliliters 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 (Sambrook et al., 1989). The DNA concentration was determined by spectrophotometer and stored at −20°C.

FABP2 polymorphism
PCR was employed for genotyping of the FABP2 gene polymorphism. Reactions were performed using 10 pmol of each primer: forward primers 5′-ACAGGTGTTAATATAGTGAAAAG-3′ and reverse primer 5′-TACCCTGAGTTCAGTTCCGTC-3′. The final volume of PCR (MJ Mini Thermo Cycler-BioRad) reaction mixture was 20 μl containing 200 ng of genomic DNA, 0.3 U of Taq DNA polymerase (Bioline Ltd., London, UK), 10 mmol/l Tris-HCl pH 8.3, 50 mmol/l of KCl, 1.5 mmol/l of MgCl 2 , 100 mmol/l of dNTPs and 10 pmol of each primer. PCR amplification was carried out under the conditions: 35 cycles for 1 min at 94°C, 1 min at 5°C and 1 min at 72°C. The PCR products thus obtained were analyzed on 2% agarose gel stained with ethidium bromide to certify the proper amplification. The amplified PCR products of 180 bp were digested with the addition of 2 U HhaI (New England Biolabs), 10 mmol/l Tris-HCl pH 7.9, 50 mmol/l NaCl, 10 mmol/l MgCl 2 and 1 mmol/l dithiothreitol. After incubation at 37°C for 2 h with restriction enzyme HhaI, digested samples were separated by polyacrylamide gel electrophoresis on 10% ethidium bromide stained polyacrylamide gel and visualized by UVP BIOLMAGING gel doc system. PCR products having an intact HhaI site were cleaved into 99 and 81 bp fragments; the Ala54Thr substitution abolished the restriction site (Fig. 2).

PPARG2 polymorphism
Genotyping was performed using PCR-RFLP (MJ Mini Thermo Cycler-BioRad). with the following primers: forward primer 5′-CAA GCC CAG TCC TTT CTG TG-3′ and reverse primer 5′-AGT GAA GGA ATC GCT TTC CG-3′ (Ek et al., 1999). 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 MgCl 2 , 0.2 mmol of each dNTPs, 50 pmol of each of the two primers, 0.3 U of Taq DNA polymerase (Bioline Ltd., London, UK) and 200 ng of genomic DNA. PCR amplification was carried out under the conditions: 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. The PCR products were incubated over night with 2 U of the restriction enzyme Hpa II (New England Biolabs, Hitchin, UK). This cuts the mutant allele at a site introduced by the reverse primer. The digested samples were applied to 3% agarose gel, subjected to electrophoresis for about half an hour and visualized on a UV transilluminator (Fig. 1). PPARG2 homozygous for CC genotype corresponded to the 106-bp fragment, whereas the heterozygous CG was characterized by two fragments of 106 and 130 bp (Fig. 2).

Statistical analysis
All the figures are presented as means ± SD. 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. All statistical tests were performed using SPSS (Statistical Package for the Social Sciences) version 12 software.

Discussion
Cataracts are a clinically and genetically heterogeneous group of eye disorders that causes visual impairment. At least 34 loci and mutations in 22 genes have been reported to be linked with different forms of cataracts. 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.

FABP2 gene polymorphism
The FABP2 gene has been proposed as a candidate gene for diabetes and insulin resistance because the protein it encodes is involved in fatty acid absorption and metabolism (Weiss et al., 2002a(Weiss et al., , 2002b. Among Pima Indians, who are known to have the highest prevalence of type 2 diabetes mellitus (T2DM), the Thr encoding allele (Thr54) is associated with insulin resistance and enhanced fat oxidation rates (Baier et al., 1996;Knowler et al., 1990). The intestinal fatty acid-binding protein (FABP2) gene located at chromosome 4q28-31 is a candidate gene possibly implicated in insulin resistance and the pathogenesis of type 2 diabetes mellitus. Several studies have reported associations between this polymorphism and insulin resistance, dyslipidemia, stroke, metabolic syndromes and hypertriglyceridemia (Carlsson et al., 2000;Vimaleswaran et al., 2006;Weiss et al., 2002aWeiss et al., , 2002b. The frequency of FABP2 Ala54Ala genotype was 50.76% in cases which is significantly higher in comparison with control 25.42% (p b 0.0001). The frequency of Ala54Thr genotype in our study was 39.23% in cases which is significantly lower in comparison with controls where it was 61.86% (p = 0.0004).

PPARG2 gene polymorphism
The association between the substitution of G (alanine) for C (proline) at codon 12 of PPARG2 gene and the risk for type 2 diabetes mellitus has been widely studied since Yen first reported this polymorphism (Yen et al., 1997). Cataracts are more common in people with diabetes than in the general population under the age of 40 years and they are morphologically similar to senile cataracts. The exact correlation between cataracts and T2DM is without a definite conclusion. Cataracts are a significant complication of T2DM (Liu et al., 2009;McDonough et al., 2009;Unoki et al., 2008). Most have found that the carrier of the G (Ala 12) allele had a lower risk for T2DM and insulin resistance (Deeb et al., 1998). In our study we have reported that the frequency CG genotype (Pro12Ala) was 87.69% in cases which is significantly higher in comparison with the controls 39.83% (p b 0.0001). The frequency of PPARG2 CC and GG genotypes in our study group was 11.53%, 0.76% in cases which is considerably lower in comparison to the controls where it was 21.18% and 38.98% (p = 0.039, p b 0.0001), respectively. According to our data, the frequency of C allele in cases was 55.38% which is significantly higher than the controls 41.10% (p = 0.001).

Conclusion
The findings of this study suggest that FABP2 and PPARG2 gene polymorphisms can be an informative marker for the early identification of population at risk of cataract. The potential role of FABP2 and PPARG2 gene polymorphisms as a marker of susceptibility to cataract needs further studies in a larger number of patients.