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 . 

ppar displays different protein isoforms generated by different promoters and 
 alternative splicing [ 2 , 3 ] .
the functional role of ppar 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 , 
 exerts an unclear effect on receptor 
 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 ppar 
 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 these complications , especially diabetic retinopathy [ 1113 ] . 

indeed , recent studies have demonstrated that ppar ligands could both inhibit intraocular 
 angiogenesis and upregulate vegf gene expression in several cell types . to date , most of the mutation analyses focused on the coding region of 
 the pparg gene , 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 ppar2 promoter , c-2821 t , that associates with predictors of type 2 diabetes and 
 obesity , was recently reported in pima indians population . 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 c1431 t .
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 .
the diabetes and obese status were determined 
 according to the criteria of world health organization .
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 ) .
the clinical characteristics ( age , sex , duration of t2 dm , 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 ) .
pro12ala snp analysis was performed with specific 
 primers pairs ( pro12alaf 5-ttt taa cgg att gat ctt ttg c-3 and pro12alar 
 5-acc ctt aca taa atg ccc cc-3 ) amplifying a fragment of 214 bp .
c1431 t snp 
 in the exon 6 of pparg gene was 
 analyzed by pcr amplification of a 358 bp fragment using specific primers pairs 
 ( c1431tf 5-ctg aac ccc ctg ttg tgt tt-3 and c1431tr 5-ggt gtc aga ttt tcc 
 ctc aga-3 ) .
the screening of c-2821 t and a-2819 g snps was carried out on a 453 bp genomic region of ppar2-specific promoter , pcr amplified with specific 
 oligonucleotides ( c-2821tf 5-tac acc aat ggg ttg tca-3 ; c-2821tr 5-gta gcc 
 aaa gac agg ttc tg-3 ) by muller et al .
pcr reactions set - up with amplitaq gold ( applied biosystems , calif , usa )   were 
 performed using an abi 9700 automatic thermal cycler under the following 
 conditions : 95c for 10 minutes ; then 35 cycles of 95c for 1 minute , at 
 annealing temperature ( ta ) for 1 minute , of 72c for 2 
 minutes ; and a final step of 72c 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 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  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 .
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 ) , 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 .
to investigate whether snps in the pparg gene could be a risk factor for type 2 diabetes and 
 obesity , an extensive polymorphism analysis in a population of 670 individuals 
 from campania county , in southern italy , was performed .
the diabetes and obese status were determined 
 according to the criteria of world health organization .
we genotyped three snps , pro12ala , c1431t  in the 
 coding region of pparg gene  and
c-2821 t in the regulatory region , 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 c1431 t variants ( listed 
 in table 2 ) are in agreement to the data previously reported by doney et al . , 2004 , although 1431 t 
 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-2821 t snp , we 
 have characterized a novel snp , a-2819 g , located two base pair downstream , 
 included within the same putative e2 box .
this novel 
 snp was genotyped on the entire cohort of 670 individuals using the same 
 standard procedures .
the -2819 g 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-2819 g 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 , table 3 ) . according to this model
, -2819 g allele carriers ( ag 
 and gg genotype ) showed an o.r .  
1.463.44 ) of 
 developing type 2 diabetes compared to the noncarriers ( table 3 ) .
the a-2819 g 
 allele is in hardy - weinberg disequilibrium ( dhw ) in the control group ( p = .01 
 see table   3 ) .
the newly identified risk allele -2819 g was not found 
 to be associated with obesity ( table 2 ) and it 
 was estimated to be not in linkage disequilibrium with pro12ala and c1431 t snps .
the possible association of 
 a-2819 g 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 -2819 g 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 -2819 g allele ( ag and gg genotype ) 
 showed an o.r .  
= 1.0575.56 ) of developing proliferative retinopathy compared to the noncarrier diabetic female   ( table 5 ) .
the a-2819 g 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-2819 g , in the 
 regulatory region of pparg , is 
 associated with type 2 diabetes and with proliferative retinopathy in females 
 in an italian cohort .
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 . 
 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 c1431 t 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 .
the 
 same variation has been associated with high bmi values in obese female from 
 finland in coassociation with ala12 allele . in the present study , we demonstrated that pro12ala 
 and c1431 t 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 
 -2821 t allele , described in the pima 
 indians population from arizona , was not present in the analyzed population .
in contrast , a novel nucleotide variation , a-2819 g , 
 two base pair downstream the c-2821 t snp described by muller et al . , was 
 identified .
we demonstrated that the a-2819 g nucleotide variant is associated 
 with type 2 diabetes , but not with obesity or bmi variations ( tables 2 and 3 ) .
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-2819 g 
 snp , as well as for all the examined snps ,
we can not 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 .
g 
 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 . 
 1.094.83 ;   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-2819 g snp and the c-2821 t   are both located in a binding 
 site for a transcriptional repressor ef1 .
the polymorphisms described by muller et al . , 2003 , 
 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 .
this finding is supported by the notion that 
 supraphysiological activation of ppar 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 . 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 ppar intracellular pathway
. 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 ppar 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 -2821 t 
 allele , via its effect on ppar expression , may exert functional consequences on 
 different ppar2-activated 
 pathways and that its association with the well - known pro12ala may contribute 
 to ppar2-related phenotypes .
full and partial tzd agonists can induce distinct receptor 
 combinations , leading to selective gene expression .
ligand - receptor complexes , 
 assuming different three - dimensional conformations , show unique and 
 differential interactions with cofactors and other transcription factors .
particularly , ppar2 is highly 
 and heterogeneously expressed in the human eye , where its ligand - dependent activation evokes potent and robust 
 inhibition of corneal angiogenesis
. 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 c1431 t polymorphism , also involved in the susceptibility to 
 premature myocardial infarction .
g 
 nucleotide variation  possibly altering 
 ppar2 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 ppar could be a novel pharmacological 
 target of angiostatic agents in the treatment of diabetic retinopathy
 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 , c1431 t , c-2821 t polymorphisms , and for a newly identified polymorphism ( a-2819 g ) . 
 the above - mentioned polymorphisms were analyzed by gene - specific pcr and direct sequencing of all samples . 
 a significant difference was found for -2819 g 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-2819 g , 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 c1431 t do not . 