type 2 diabetes ( t2d ) is the most common form of diabetes and an increasingly prevalent metabolic disease .
it is associated with microvascular and macrovascular complications and is considered one of the major causes of morbidity and mortality . according to the international diabetes federation ( idf ) , there were approximately 382 million people worldwide with diabetes in 2013 , and this number is expected to reach more than 592 million by 2035 . in recent decades , several studies have shown the role of chronic low - grade inflammation and activation of the immune system in the pathogenesis of t2d and its complications [ 37 ] .
however , the mechanisms by which chronic inflammation is involved in t2d are not completely clear .
it has been reported that synthesis and release of pro- and anti - inflammatory cytokines such as tumor necrosis factor- ( tnf- ) , interleukin- ( il- ) 10 , and il-6 and growth factors such as transforming growth factor-1 ( tgf-1 ) [ 813 ] may be involved in pathogenesis of t2d and its complications .
single nucleotide polymorphisms ( snps ) in the cytokine genes are usually located in their regulatory regions and affect the levels of their expression [ 14 , 15 ] .
various studies have associated cytokine gene expression alterations with obesity , changes in insulin sensitivity , and risk of t2d [ 6 , 7 , 16 ] .
some works have investigated the association between the tnf- -308g / a polymorphism ( rs1800629 ) and diabetic retinopathy ( drp ) , nephropathy ( dnp ) , and neuropathy ( dnr ) in different populations [ 1724 ] .
only a recent brazilian study   found an association between proliferative drp and these tnf- polymorphism .
dnp was associated with the polymorphisms il-10 -592c / a ( rs1800872 )   and il-10 -819t / c ( rs1800871 ) .
in addition , paine et al .   associated the polymorphism il-10 -1082g / a ( rs1800896 ) with drp and kolla et al .   observed an association with dnr
the human tgf-1 gene presents two hotspot focuses : codon 10t / c ( rs1800470 ) and codon 25c / g ( rs1800471 ) .
both polymorphisms were associated with drp by bernek et al . , and el - sherbini et al
( 2014 ) associated the tt genotype of tgf-1 codon 10t / c polymorphism with dnp risk .
il-6 gene polymorphism -174g / c ( rs1800795 ) was associated with dnp in study conducted by muammer et al .
the few studies that evaluated the association of the polymorphism ifn- + 874t / a ( rs2430561 ) with diabetes complications found association between this polymorphism and drp   and with dnr . in spite of the existence of several reports examining the association of polymorphisms in various cytokine genes , much controversy remains about their role in diabetes complications .
no studies to date have examined in a single population a large number of polymorphisms ( tnf-  -308g / a , il-10 -1082g / a , il-10 -819t / c ,
il-10 -592c / a , tgf-1 codon 10t / c , tgf-1 codon 25c / g , il-6  -174g / c , and ifn- + 874t / a ) and their association with the t2d complications and comorbidities . in this study
, we investigated if these polymorphisms are associated with drp , dnp , and dnr and with comorbidities ( hypertension , dyslipidemia , and obesity ) in a group of brazilian t2d patients .
this study was conducted with 102 brazilian individuals , including 19 men and 83 women with clinical and laboratory diagnosis of t2d , aged from 32 to 70 years ( 54.99  8.97 years ) , recruited from the clinic of endocrinology , santa casa hospital ( belo horizonte , minas gerais , brazil ) in the period from june 2012 to september 2013 .
drp was diagnosed by ophthalmoscopic examination through fundoscopic examination and slit lamp microscopic examination with present lens .
dnp was defined as albumin excretion rate ( aer ) > 30  mg/24  h and without coexisting renal diseases from causes other than diabetes ; and no dnp was defined at an aer < 30  mg/24  h , at least 2 out of 3 urine collections over a 3-month period .
dnr was defined according to diabetes control and complication trial ( dcct )   criteria .
the physical examination by neurologist and the presence of signs and symptoms ( dysesthesias , paresthesias , hypersensitivity to touch , or burning pain ) or absent deep tendon reflexes configured dnr presence .
two patients could not be classified according to the presence or absence of dnp due to lack of laboratory data .
the determination of hypertension ( systolic blood pressure  140  mmhg or diastolic blood pressure  80  mmhg or use of antihypertensive drugs ) and dyslipidemia ( ldl - cholesterol  100  mg / dl , hdl - cholesterol  50  mg / dl , and triglycerides  150  mg / dl or treatment of hyperlipidemia ) is also in accordance with the criteria adopted by ada .
bmi , t2d onset ) and laboratory data ( fasting and postprandial glucose , hba1c ) were obtained from medical records .
subjects older than 70 years , with cancer or autoimmune disease , with current or recent infectious process , with history of cardiovascular disease , and with treatment of anti - inflammatory drugs , were excluded .
the project was approved by the ethics committee of the federal university of minas gerais and santa casa hospital .
genomic dna was obtained from a sample of peripheral blood collected in edta . the biopur mini spin kit ( biometrix )
the polymorphisms were determined using the cytokine genotyping tray kit ( one lambda ) which employs polymerase chain reaction - sequence specific primers ( pcr - ssp ) , followed by electrophoresis in 2.5% agarose gel stained with gelgreen stain ( biotium ) .
we investigated the polymorphism tnf-  -308g / a , il-10 -1082g / a , il-10 -819t / c , il-10 -592c / a , tgf-1 codon 10t / c , tgf-1 codon
25c / g , il-6 -174g / c , and ifn- + 874t / a , which were selected due to evidence from previous studies that showed their association with the regulation in cytokines gene expression . for association analyses of polymorphisms
, patients were divided into groups according to the complication ( drp , dnp , and dnr ) and comorbidity presented ( hypertension , dyslipidemia , and bmi  categorized into bmi <
25  kg / m , bmi 2530  kg / m , and bmi  30 
deviations from hardy - weinberg equilibrium ( hwe ) were tested using an exact test ( available at http://genepop.curtin.edu.au/genepop_op1.html ) .
all of the statistical analyses were performed with statistical package of the social sciences ( spss ) version 13.0 .
data are presented as mean  standard deviation ( normal variables ) , median ( interquartile range ( iqr ) ) ( no normal variables ) , or percentage of total ( categorical variables ) .
differences in the allele and genotype frequencies between the groups ( drp , dnp , dnr , hypertension , dyslipidemia , and bmi ) were tested by pearson 's -test or fisher 's exact test with residuals test for three groups . for normal variables ,
student 's t - test was used to determine differences in means ; for no normal variables , kruskal - wallis and mann - whitney tests were used to determine differences in medians .
the differences in the haplotype frequencies between the groups ( drp , dnp , dnr , hypertension , dyslipidemia , and bmi ) were tested by -test .
as expected , individuals with drp were older than those without this complication ( p = 0.013 ) . a slight tendency toward higher fasting glucose was observed in patients with drp ( p = 0.074 ) ; however , postprandial glucose level was significantly increased in this group ( p = 0.013 ) when compared to individuals without drp .
this complication was significantly more frequent in patients with more than 10 years since t2d diagnosis when compared to patients diagnosed less than 5 or between 5 and 10 years earlier ( p < 0.0001 ) .
no significant difference was observed between the groups regarding gender , hba1c , and the frequency of hypertension and dyslipidemia ( p > 0.05 ) ( table 1 ) .
t2d patients with dnp presented significantly increased fasting and postprandial glucose levels and hba1c% . as seen in the drp group ,
patients with nephropathy were older and the bmi was lower than the group without this complication ( p < 0.0001 for both ) .
patients with t2d onset > 10 years were the most frequent group presenting dnp ( p = 0.001 ) .
no significant differences were observed for gender , hypertension , and dyslipidemia frequencies between dnp positive and negative patients , similarly to the results for the drp group reported above ( table 2 ) .
regarding dnr , we observed a significant difference concerning age and gender between the groups presenting this complication or not ( p = 0.012 and p = 0.003 , resp . ) .
the other variables were not significantly different between dnr positive and negative patients ( p > 0.05 ) , although a trend toward more dyslipidemic individuals was observed in the dnr group ( p = 0.079 ) ( table 3 ) .
next , we performed an analysis of genotype and allele frequencies involving a large number of polymorphisms for cytokine genes including tnf- , il-10 , tgf-1 , il-6 , and inf- for all complication groups .
we found that all polymorphisms were under hardy - weinberg equilibrium ( p > 0.05 for all ) .
we observed that drp is associated with the gg genotype and g allele in tgf-1 codon 25c / g polymorphism ( p = 0.004 , or 5.865 , and ci 95% 1.68520.414 and p = 0.018 , or 3.387 , and ci 95% 1.06911.064 ) .
the drp patients presented the following genotype and allele frequencies : gg 92.42% , gc 6.06% , cc 1.52% , g 95.45% , and c 4.55% , whereas patients without drp showed the following frequencies : gg 72.22% , gc 27.78% , cc 0% , g 86.11% , and c 13.89% .
in addition , dnp is associated with lower frequency of gg genotype in il-10 -1082g / a polymorphism ( p = 0.049 , or 2.357 , and ci 95% 1.6129.076 ) .
the frequencies observed for patients with dnp , were gg 8.75% , ga 50.00% , and aa 41.25% , and patients without dnp , gg 25.00% , ga 25.00% , and aa 50.00% . no other polymorphism was associated with drp , dnp , or dnr ( see table 1 in supplementary material available online at http://dx.doi.org/10.1155/2015/605965 ) . since il-10 -1082g / a polymorphism showed association with dnp , an analysis of haplotype frequencies of il-10 polymorphisms ( -1082g / a , -819t / c , and -512c / a ) was performed in the patients grouped according to the t2d complications .
however , no haplotype was associated with drp , dnp , or dnr ( table 4 ) .
interestingly , individuals who presented the aa genotype in ifn- + 874t / a polymorphism presented lower fasting glucose levels when compared to the other genotypes : tt [ median ( iqr ) = 146.0 ( 106.0 )  mg / dl ] , ta [ 134.0 ( 65.0 )  mg / dl ] , and aa [ 114.0 ( 53.5 )  mg / dl ] ; [ tt  ta ( p = 0.783 ) ; tt  aa ( p = 0.045 ) ; ta  aa ( p = 0.015 ) ] . the patients also were evaluated according to the presence of comorbidities such as hypertension , dyslipidemia , and bmi categories concerning the presence of the same polymorphisms .
the analysis of the genotype frequencies revealed the association of hypertension with the cc genotype and c allele for il-10 -592c / a polymorphism ( p = 0.013 , or 9.400 , and ci 95% 1.50958.568 and p = 0.009 , or 3.830 , and ci 95% 1.20612.546 , resp . ) .
the frequencies observed were patients with hypertension ( cc 50.00% , ca 39.36% , aa 10.54% , c 69.68% , and a 30.32% ) and patients without hypertension ( cc 25.00% , ca 25.00% , aa 50.00% , c 37.50% , and a 62.50% ) . a higher frequency of t allele for the tgf-1 codon 10t / c ( p = 0.047 , or 2.810 , and ci 95% 1.8909.148 ) and c allele for the il-10 819t / c ( p = 0.033 , or 0.338 , and ci 95% 1.1071.154 ) was also observed in patients with hypertension .
moreover , a significant association was found between the polymorphism tgf-1 codon 10t / c and bmi groups ( p = 0.026 , or 1.963 and ci 95% 1.0073.826 and or 1.546 and ci 95% 1.8582.786 ) .
the cc genotype was more frequent in the group with bmi < 25  kg / m , while the tc genotype was more frequent in the group with bmi  30 
the frequencies observed were bmi < 25  kg / m ( tt 26.32% , tc 42.10% , and cc 31.58% ) , bmi 2530  kg / m
( tt 48.00% , tc 40.00% , and cc 12.00% ) , and bmi  30  kg / m ( tt 31.04% , tc 62.07% , and cc 6.89% ) .
we also found a trend for association of the polymorphism il-10 -592c / a and the bmi groups ( p = 0.058 ) . in this case , the aa genotype was less frequent in the bmi  30  kg / m group .
no other polymorphism was associated with hypertension , dyslipidemia , or bmi categories ( table 2 in supplementary material ) . since il-10 -592c / a polymorphism showed an association and a tendency for association with hypertension and bmi groups , respectively , an analysis of haplotype frequencies for il-10 polymorphisms ( -1082g / a
, -819t / c , and -512c / a ) was performed in the patients grouped according to t2d comorbidities . for this analysis
, bmi was categorized into two groups : bmi < 30  kg / m and bmi  30  kg / m .
however , no haplotype was associated with hypertension , dyslipidemia , and bmi categories ( table 4 ) .
this study investigated the association of cytokine gene polymorphisms in t2d patients including t2d complications drp , dnp , and dnr , as well as comorbidities commonly observed in these patients : hypertension , dyslipidemia , and obesity . when the patients were grouped according to t2d complications
, the clinical and laboratory characteristics revealed , as expected , that drp , dnp , and dnr are more common in older individuals with longer diagnostic t2d , because of their association with disease progression .
significant difference in the levels of postprandial glucose was observed in the drp and dnp cases when compared to the t2d group without complications .
these results suggest a relationship between the hyperglycemic status and the development of microvascular complications of t2d . although higher levels of fasting glucose , postprandial glucose , and hba1c were observed in patients with dnr , these levels were not significantly different from those found in t2d individuals without this complication .
the small sample of individuals presenting dnr may explain the lack of significance concerning these variables in this group .
patients with drp and dnp showed lower bmi when compared to individuals without these complications .
these findings seem contradictory , because obesity is considered a main risk factor for t2d and its complications .
this inconsistency may be explained by the fact that individuals with higher bmi were also those with shorter t2d onset and therefore presented fewer complications ( data not shown ) .
curiously , a higher frequency of women was observed in the dnr group , although the reason underlying this observation is not clear .
studies involving a much larger sample may be necessary to confirm this finding and explore the possible causes for this observation .
drp is the most frequent cause of newly detected cases of blindness in adults . in the present study ,
drp was associated with the gg genotype and g allele of tgf-1 codon 25c / g polymorphism .
this polymorphism is located in the region of the gene encoding the signal peptide and causes a change in the amino acid sequence ( g / arg  c / pro ) .
tgf-1 modulates ocular cell migration and proliferation by inducing fibroblast growth factor - like and platelet - derived growth factor , which accelerate the process of retinal neovascularization .
tgf-1 is also involved in extracellular matrix deposition ( an essential step in new vessel formation ) and stimulates angiogenesis in patients with ischemia and proliferative drp .
another case - control study with t2d patients with diabetic proliferative retinopathy found an association of the polymorphisms tgf-1 codon 25c / g ( g allele ) and tgf-1 codon 10t / c ( t allele ) with proliferative diabetic retinopathy . on the contrary ,
a recent meta - analysis found an association between tgf-1 codon 10t / c polymorphism and drp .
although the present study did not find significant association with other polymorphisms , the studies published by paine et al .
[ 18 , 31 ] showed the association of the polymorphisms il-10 -1082g / a and ifn- + 874t / a with proliferative diabetic retinopathy .
this discrepancy may be due to the genetic background of the populations studied since paine et al .
dnp is a common cause of end - stage renal disease and the major cause of morbidity and premature mortality in patients with t2d .
structurally , dnp is characterized by renal hypertrophy , mesangial matrix expansion , glomerulosclerosis , and tubulointerstitial fibrosis .
recently , it has become evident that chronic inflammatory mechanisms contribute to the development and progression of dnp , such as infiltration of renal compartments by lymphocytes and monocytes ( or macrophages ) as well as local production of cytokines and chemokines in the kidney [ 41 , 42 ] .
studies have observed that acute phase markers of inflammation ( c reactive protein , fibrinogen , and il-6 ) were correlated to proteinuria in t2d patients [ 43 , 44 ] and showed that increased tnf- levels were linked with dnp progression . in our study , dnp was associated with lower frequency of gg genotype polymorphism il-10 -1082g / a , which is related with higher expression of this cytokine .
in fact , il-10 is an anti - inflammatory cytokine and downregulates proinflammatory production of tnf- , il-6 , and mcp-1 .
thereby , lower production of il-10 may be associated with a high production of proinflammatory cytokines and an exacerbated inflammatory response with subsequent renal injury in t2d patients .
ezzidi et al . , in a case - control study with t2d patients from tunisia , found a higher frequency of t allele ( il-10 -819t / c ) in the group with dnp .
.   found higher frequency of the genotypes aa and cc for the il-10 -592c / a polymorphism in individuals with the dnp in a taiwanese population .
in contrast to the current results , no association was found between the il-10 -1082g / a polymorphism and dnp in patients from turkey .
the polymorphism tgf-1 codon 10t / c was associated with dnp risk in a meta - analysis conducted by zhou et al . .
thus , it is possible that the different polymorphisms may reflect the genetic background of the population studied .
ifn- is a pivotal proinflammatory cytokine implicated in the induction of immune mediated inflammatory response .
studies suggest that ifn- participates in the pathogenesis of diabetes mellitus by upregulating the expression of mhc i / mhc ii antigens and adhesion molecules on pancreatic  cells [ 4951 ] .
herein we report an association of the aa genotype polymorphism ifn- + 874t / a with lower glucose levels than those presented by patients carrying other genotypes .
it is known that the allele + 874 t is associated with high ifn- levels , whereas the allele + 874a is associated with low production of this cytokine .
thus , decreased expression of ifn- may contribute to the downregulation inflammatory response in t2d patients and consequently to allowing better glycemic control .
it is known that obesity and visceral fat contribute to the development of hypertension , insulin resistance , and diabetes mellitus . typically , hypertension is a clinical condition commonly present in the patient at diagnosis of t2d , and the elevation of blood pressure often occurs before the onset of microalbuminuria . in our study
we found an association between the cc genotype and c allele for il-10 -592c / a polymorphism and hypertension ; furthermore we found a trend for association of the same polymorphism and bmi groups , since the -592aa genotype was less frequent in the obese t2d patients .
in addition , we observed an association of the c allele for the il-10 -819t / c polymorphism and patients with hypertension . for both il-10 polymorphisms ,
reported that increased il-10 levels were associated with improved systemic endothelial vasoreactivity in patients with elevated serum crp levels , a condition commonly observed in t2d patients .
furthermore , zeyda et al .   demonstrated that human adipose tissue macrophages ( atms ) produce high levels of il-10 .
consequently , increased levels of il-10 could be associated with an exacerbated inflammatory profile in which the balance between pro- and anti - inflammatory cytokines contributes to chronic low - grade inflammation observed in obesity and t2d , as well as being involved in increased blood pressure .
the cc genotype was more frequent in the group with bmi < 25  kg / m , while the tc genotype was more frequent in the group with bmi  30  kg / m .
this polymorphism consists of a t  c transition at nucleotide 29 in the region encoding the signal peptide sequence , which results in a leu  pro substitution at amino acid 10 .
studies have shown that the c allele increases the production of the tgf-1 protein [ 55 , 56 ] .
the tgf-1 is a multifunctional cytokine and shows anti - inflammatory action such as suppressing generation of free radicals , as well as vasculoprotective properties .
tgf-1 can inhibit the adhesion and transmigration of neutrophils and t cells to the endothelium , inhibit production of adhesion molecules by the endothelial cells , and inhibit macrophage foam cell formation [ 5861 ] .
the higher frequency of the cc genotype found in patients with a bmi < 25 
kg / m suggests that the increased expression of tgf-1 and its potential anti - inflammatory effect can facilitate the control of adiposity , since obese individuals have higher frequency tc genotype . a higher frequency of t allele for the tgf-1 codon
although some studies have demonstrated that tgf-1 is associated with increased risk of essential hypertension through the stimulation of endothelin-1 expression in the vascular endothelium , release of renin from the juxtaglomerular cells in the kidney , and regulation of angiotensin ii expression [ 6264 ] , no study has evaluated the impact of this polymorphism on hypertension in patients with diabetes .
thus , our results suggest that lower expression of tgf-1 could predispose to diabetic hypertension due to lack of anti - inflammatory and protective tgf-1 effects in vascular endothelium .
in conclusion , this is the first study to evaluate the association of a large panel of polymorphisms of cytokine genes with complications and comorbidities in t2d patients .
however , the small sample of this study is considered a limitation and further studies including clinical classifications concerning nonproliferative / proliferative drp and autonomic / chronic sensorimotor dnr may improve our current understanding about the link between the cytokine polymorphisms , their expression levels , and the development or progression of these complications .
although the results are not conclusive regarding the association of polymorphisms of cytokine genes with microvascular complications in t2d and comorbidities , taken together , our results may be relevant for future molecular studies aiming to predict possible t2d complications .
 
 aims . 
 the polymorphisms of pro- and anti - inflammatory cytokines may be involved in type 2 diabetes ( t2d ) pathogenesis and its complications . 
 methods . 
 we investigated in 102 t2d patients the association of the cytokine polymorphisms in the tnf- , il-10 , il-6 , tgf-1 , and ifn- genes with the t2d microvascular complications and comorbidities ( hypertension , dyslipidemia , and obesity ) . 
 cytokine genotypes were determined by pcr using cytokine genotyping tray kit . 
 results . 
 diabetic retinopathy was associated with gg genotype and g allele in tgf-1 codon 25c / g polymorphism ( p = 0.004 and p = 0.018 ) and the nephropathy was associated the lower frequency of gg genotype in il-10 -1082g / a polymorphism ( p = 0.049 ) . 
 hypertension was associated with the cc genotype and c allele for il-10 -592c / a polymorphism ( p = 0.013 and p = 0.009 ) and higher frequencies of t ( p = 0.047 ) and c ( p = 0.033 ) alleles of the tgf-1 codon 10t / c and il-10 -819t / c polymorphisms , respectively . 
 the tgf-1 codon 10t / c polymorphism was associated with the bmi groups ( p = 0.026 ) : the cc genotype was more frequent in the group with bmi < 25  kg / m2 , while the tc genotype was more frequent in the group with bmi = 30  kg / m2 . conclusions . 
 our findings suggest that tgf-1 and il-10 polymorphisms are involved in complications and comorbidities in t2d patients . 
1. Introduction
2. Material and Methods
3. Results
4. Discussion
5. Conclusion