Characterization of a Novel Polymorphism in PPARG Regulatory Region Associated with Type 2 Diabetes and Diabetic Retinopathy in Italy

Peroxisome proliferator-activated receptor gamma polymorphisms have been widely associated with type 2 diabetes, although their role in the pathogenesis of vascular complications is not yet demonstrated. In this study, a cohort of 211 type 2 diabetes, 205 obese, and 254 control individuals was genotyped for Pro12Ala, C1431T, C-2821T polymorphisms, and for a newly identified polymorphism (A-2819G). The above-mentioned polymorphisms were analyzed by gene-specific PCR and direct sequencing of all samples. A significant difference was found for -2819G frequency when patients with type 2 diabetes—particularly diabetic women with the proliferative retinopathy—were compared with healthy control individuals. In conclusion, we identified a novel polymorphism, A-2819G, in PPARG gene, and we found it to be associated with type 2 diabetes and proliferative retinopathy in diabetic females. In the analyzed population, this variant represents a genetic risk factor for developing the diabetic retinopathy, whereas Pro12Ala and C1431T do not.


Introduction
Peroxisome proliferator-activated receptor gamma (PPARG) gene is a member of nuclear hormone receptor superfamily, and it regulates the transcription of several genes involved in glucose metabolism, adipocyte differentiation, lipid oxidation, angiogenesis, and inflammation [1]. PPARγ displays different protein isoforms generated by different promoters and alternative splicing [2,3]. The functional role of PPARγ is well documented, and its alterations have been widely associated with metabolic diseases, such as type 2 diabetes and obesity [4,5].
Indeed, the most analyzed PPARG polymorphism, Pro12Ala in the exon B [6], exerts an unclear effect on rec-eptor structure, and it seems to reduce the receptor binding affinity with the responsive elements, also inducing a decrease in transcriptional activity both with and without PPARγ agonists [7,8]. The common Pro12Ala polymorphism has been associated with increased risk of developing type 2 diabetes, even though a recent hypothesis suggests Pro12Ala could be involved in the aetiology of metabolic disorders not by itself, but because of linkage disequilibrium with other single nucleotide polymorphisms (SNPs) in the PPARG promoter [9,10].
Moreover, PPARG gene polymorphisms have also been associated with micro-and macrovascular complications of diabetes, although the few available studies do not unambiguously confirm the role of PPARG in the pathogenesis of 2 Journal of Biomedicine and Biotechnology these complications, especially diabetic retinopathy [11][12][13].
Indeed, recent studies have demonstrated that PPARγ ligands could both inhibit intraocular angiogenesis and upregulate VEGF gene expression in several cell types [14].
To date, most of the mutation analyses focused on the coding region of the PPARG gene [9], and only few studies have analyzed SNPs in the regulatory region of PPARG possibly associated with metabolic disorder susceptibility [15,16]. A novel functional variant in the PPARγ 2 promoter, C-2821T, that associates with predictors of type 2 diabetes and obesity, was recently reported in Pima Indians population [16].
In attempt to analyze this variant in Italian individuals affected by type 2 diabetes, we have performed an SNP analysis on 211 unrelated type 2 diabetic patients from Campania County, Southern Italy. Subsequently, we have extended this analysis to a cohort of 205 unrelated obese patients and 254 control individuals.
Furthermore, we have genotyped the above-mentioned cohort (diabetic, obese, and control individuals), for two extensively studied SNPs in high linkage disequilibrium, Pro12Ala and C1431T.

Patients and Clinical
Characteristics. Genomic DNA was extracted from blood samples by standard procedures [17]. SNP analysis was performed on genomic DNA from 211 unrelated Italian type 2 diabetic patients (102 of which suffered from proliferative retinopathy), 205 unrelated obese patients, and 254 control individuals. All the subjects were recruited from the Department of Geriatrics and Metabolic Diseases, Second University of Naples, Italy, and from the Department of Neuroscience, "Federico II" University of Naples, Italy. All procedures were approved by the Ethics Board of the participating institutes and adhered to the tenets of the Declaration of Helsinki. All samples were acquired after informed consent was obtained from the patients.
The diabetes and obese status were determined according to the criteria of World Health Organization [18]. Patients with type 2 diabetes were eligible for the study if they had background or proliferative diabetic retinopathy, similar age (±1 year) and similar duration of diabetes (±1 year). No other inclusion criteria had to be met. The clinical characteristics (age, sex, duration of T2DM, 2h-plasma glucose, HbA1c and BMI) of the 102 diabetic patients with proliferative retinopathy were not different from those of the 109 diabetic patients with background retinopathy (see Table 1). Retinopathy condition was evaluated by ocular fundus photography.   [16]. PCR reactions set-up with AmpliTaq Gold (Applied Biosystems, Calif, USA) were performed using an ABI 9700 automatic thermal cycler under the following conditions: 95 • C for 10 minutes; then 35 cycles of 95 • C for 1 minute, at annealing temperature (T a ) for 1 minute, of 72 • C for 2 minutes; and a final step of 72 • C for 10 minutes. PCR products were analyzed by denaturing high performance liquid chromatography (DHPLC), and those with a DHPLC pattern different from controls were sequenced using the ABI Prism Big Dye Terminator Cycle Sequencing V2.0 (Applied Biosystems).

Statistical Analysis.
Statistical analysis was performed using standard chi-square, Fisher, and student's t-tests. For continuous variables, the general estimating equation procedure was used to adjust for covariates. The association of polymorphisms with type 2 diabetes, obesity, and retinopathy was analyzed by exact chi-square test, comparing SNPs frequencies.
Allele and genotype frequencies (Pearson χ 2 statistics), O.R., and P values, as well as dominant and recessive genetic models, were analyzed using FINETTI (http://ihg2.helmholtz-muenchen.de/cgi-bin/hw/hwa1.pl). Hardy-Weinberg equilibrium of tested groups and Armitage's trend test (ATT) were also calculated using FINETTI. ATT takes into account genotypes rather than alleles, avoiding a possible bias due to doubling of sample size [19]. It assumes additive (or codominant) disease model where all disease allele are independent and have the same contribution to the disease risk.
Continuous clinical data, such as duration of type 2 diabetes (years), HbA1c and BMI (body mass index-Kg/m 2 ), different between background and proliferative retinopathy populations, were compared by unpaired student's t-test (two-tailed test); a resulting P value less than 0.05 (C.I. 95%) was considered significant.

Results
To investigate whether SNPs in the PPARG gene could be a risk factor for type 2 diabetes and obesity, an extensive  We genotyped three SNPs, Pro12Ala, C1431T-in the coding region of PPARG gene-and C-2821T in the regulatory region [16], in 211 individuals with type 2 diabetes, 205 suffering from obesity, and 254 control individuals. Our analysis revealed that T-2821 allele was not found in 670 individuals, and consequently, C-2821 allele frequency was 100%.
Allelic frequencies of Pro12Ala and C1431T variants (listed in Table 2) are in agreement to the data previously reported by Doney et al., 2004 [20], although 1431T allele was shown to be less frequent in obese than in control individuals in the analyzed population.
Moreover, during the SNP analysis of the C-2821T SNP, we have characterized a novel SNP, A-2819G, located two base pair downstream, included within the same putative E2 box [16]. This novel SNP was genotyped on the entire cohort of 670 individuals using the same standard procedures. Genotypes and allelic frequencies of A-2819G are reported in Table 2.
The -2819G minor allele frequency, observed in 211 individuals with type 2 diabetes, was about 2-fold than the frequency of the risk allele in the control and obese groups (see Table 2). In logistic regression analyses, aimed to test the hypothesis of A-2819G as risk factor for type 2 diabetes under the assumption of a codominant, recessive and dominant model, the dominant model best fitted the data (P = 1.9 × 10 −4 , Table 3). According to this model, -2819G allele carriers (AG and GG genotype) showed an O.R. = 2.24  (95% C.I. 1.46-3.44) of developing type 2 diabetes compared to the noncarriers ( Table 3). The A-2819G allele is in Hardy-Weinberg disequilibrium (DHW) in the control group (P = .01 see Table 3). The newly identified risk allele -2819G was not found to be associated with obesity (Table 2) and it was estimated to be not in linkage disequilibrium with Pro12Ala and C1431T SNPs. The possible association of A-2819G SNP with some specific clinical features of both type 2 diabetes and obesity was also considered. Specifically, BMI in obese individuals, blood glucose, and HA1c levels and the presence of proliferative retinopathy in type 2 diabetic patients were evaluated. The analysis revealed that -2819G frequency in diabetic females with proliferative retinopathy was 2-fold increased with respect to background retinopathy female (P = .02; see Table 4 and Figure 1).
In logistic regression analyses, the dominant model best fitted the data (P = .03; see Table 5). According to this model, type 2 diabetes female with -2819G allele (AG and GG genotype) showed an O.R. = 2.42 (95% C.I. = 1.057-5.56) of developing proliferative retinopathy compared to the noncarrier diabetic female ( Table 5). The A-2819G allele is in Hardy-Weinberg equilibrium in both background and proliferative groups (Table 5). Moreover, diabetic females with proliferative retinopathy had more than 3-fold risk compared to control females (data not shown).
In conclusion, our results show that a novel nucleotide variant A-2819G, in the regulatory region of PPARG, is associated with type 2 diabetes and with proliferative retinopathy in females in an Italian cohort.

Discussion
Previous studies have shown that nucleotide variations, both point mutations and SNPs, in the PPARG coding region, are associated with an increased susceptibility of developing type 2 diabetes and obesity [9]. Particularly, the well-known Pro12Ala substitution has been widely associated with both type 2 diabetes and obesity, or predictors of these diseases, in different populations [10,12]. The Pro12Ala polymorphism has been recently predicted to confer protection, through unknown mechanisms, from type 2 diabetes and nephropathy [12,15,21,22].
Conversely, it has also been shown that Pro12Ala does not associate with diabetic retinopathy [11,13]. The Pro12Ala SNP has been described in high linkage disequilibrium with C1431T polymorphism, located in exon 6 of PPARG gene. Several studies have shown T minor allele to be associated, in obese individuals, with low plasmatic leptin levels [14]. The same variation has been associated with high BMI values in obese female from Finland in coassociation with Ala12 allele [23].
In the present study, we demonstrated that Pro12Ala and C1431T variants are not associated with type 2 diabetes, obesity, BMI variation, and also microangiopatic complication of diabetes, in a cohort of 670 individuals from Campania County, in the Southern Italy. We also found that -2821T allele, described in the Pima Indians population from Arizona, was not present in the analyzed population.
In contrast, a novel nucleotide variation, A-2819G, two base pair downstream the C-2821T SNP described by Muller et al. [16], was identified. We demonstrated that the A-2819G nucleotide variant is associated with type 2 diabetes, but not with obesity or BMI variations (Tables 2 and 3). The A-2819G allele is in DHW in the control group. DHW may be due to several factors such as genotyping errors, population stratification, selection, inbreeding, the presence of a causative allele or simply by the chance. We can therefore exclude with high confidence that DHW in the control group could be due to genotyping errors. For A-2819G SNP, as well as for all the examined SNPs, all the subjects were analyzed by DHPLC and then sequenced. We cannot exclude that DHW is due to other confounding factors; however, at least for the autosome genes, the presence of DHW in controls is compatible with the segregation of a causative variant according to a dominant model [24].
The association tests for the newly identified A-2819G SNP, revealed statistical significant association with a common and detrimental microvascular complication of type 2 diabetes, namely, the diabetic proliferative retinopathy, in the diabetic females of the examined population (O.R. = 2.43; C.I. 1.09-4.83; P = 0.02). This microvascular complication represents a leading cause of blindness, characterized by an increased vascular permeability, haemostatic abnormalities, increased tissue ischemia, and also neoangiogenesis.
The newly identified A-2819G SNP and the C-2821T [16] are both located in a binding site for a transcriptional repressor δEF1. The polymorphisms described by Muller et al., 2003 [16], leading to higher levels of PPARG transcription, has been associated with an increased risk of development of type 2 diabetes and obesity in Pima Indians [16]. This finding is supported by the notion that supraphysiological activation of PPARγ by thiazolidinedione (TZD) agonists increases triglyceride (TG) content of white adipose tissue, thereby decreasing TG levels in the liver and muscles, resulting in increased insulin sensitivity and type 2 diabetes [25].
In this study, we demonstrated that a novel polymorphism, in the regulatory region of PPARG, associates to proliferative retinopathy in diabetic females, suggesting a possible involvement of the hormonal female component, such as the estrogen receptor signaling that interferes with the physiological PPARγ intracellular pathway [26]. We can reject sex differences due to genotype bias, since males and females were analyzed together, in blind fashion, and the gender was assigned after the analysis.
We postulate that PPARγ may be involved in the development and progression of diabetic retinopathy via a plethora of mechanisms, although its pathogenic effect on the development of the microvascular complications in type 2 diabetic patients is not yet known.
Indeed, Muller et al. (2003) concluded that -2821T allele, via its effect on PPARγ expression, may exert functional consequences on different PPARγ 2 -activated pathways and that its association with the well-known Pro12Ala may contribute to PPARγ 2 -related phenotypes [16].
PPARγ is a multivalent receptor able to bind different ligands. Full and partial TZD agonists can induce distinct receptor combinations, leading to selective gene expression. Ligand-receptor complexes, assuming different threedimensional conformations, show unique and differential interactions with cofactors and other transcription factors [27].
Particularly, PPARγ 2 is highly and heterogeneously expressed in the human eye, where its ligand-dependent activation evokes potent and robust inhibition of corneal angiogenesis [27]. Moreover, altered lipid oxidation in the retina supporting cells (retinal pigment epithelium, RPE) and in the eye vessels, and an increased glucose uptake from RPE cells, has pathological effects on eye vessels [28,29]. However, it was already shown that lipid peroxidation is influenced by C1431T polymorphism, also involved in the susceptibility to premature myocardial infarction [30].
We hypothesize that the newly identified A-2819G nucleotide variation-possibly altering PPARγ 2 transcript abundance-may affect the complex and highly regulated gene expression network in the eye, conferring in turn the susceptibility to diabetic retinopathy.
Further studies enrolling a greater number of subjects, both patients and controls, would be useful to better elucidate the involvement of this novel SNP in the susceptibility of developing diabetic retinopathy. However, these findings strengthen the hypothesis that PPARγ could be a novel pharmacological target of angiostatic agents in the treatment of diabetic retinopathy.