diabetes research and clinical practice 103 (2014) 474–481

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Diabetes Research and Clinical Practice jou rnal hom ep ag e: w ww.e l s e v i er . c om/ loca te / d i ab r es

Association of RAGE gene polymorphism with vascular complications in Indian type 2 diabetes mellitus patients Ashok Kumar Tripathi a,*, Diwesh Chawla a, Savita Bansal a, Basu Dev Banerjee a, Sri Venkata Madhu b, Om Prakash Kalra b a

Biochemistry and Immunology Laboratory, Department of Biochemistry, University College of Medical Sciences (University of Delhi) and G.T.B. Hospital, Dilshad Garden, Delhi 110095, India b Department of Medicine, University College of Medical Sciences (University of Delhi) and G.T.B. Hospital, Dilshad Garden, Delhi 110095, India

article info

abstract

Article history:

Aims: The study was designed to evaluate the association of 374T/A and 429T/C poly-

Received 11 July 2013

morphism in the promoter region and Gly82Ser polymorphism in exon 3 region of RAGE gene

Received in revised form

with diabetic vascular complications in Indian population.

19 September 2013

Methods: We screened 603 subjects which includes 176 healthy controls, 140 type 2 diabetes

Accepted 18 December 2013

mellitus (T2DM) subjects without any vascular complications (DM), 152 T2DM subjects with

Available online 25 December 2013

microvascular complications (DM-micro) and 135 T2DM subjects with macrovascular com-

Keywords:

DNA isolated from the enrolled subjects were genotyped by PCR-RFLP. Logistic regression

Type 2 diabetes mellitus

analysis was used to evaluate the association of single nucleotide polymorphisms (SNPs).

plications (DM-macro) for 374T/A, 429T/C and Gly82Ser polymorphisms of RAGE gene.

RAGE gene Polymorphism

Results: The 429 T/C and Gly82Ser RAGE polymorphisms were found to be significantly

Vascular complications

associated with the development of macrovascular and microvascular complications, respectively, in T2DM subjects while 374A allele showed reduced risk towards the development of macrovascular complications. Further, 429T/C, 374T/A and Gly82Ser haplotype analysis revealed association of CTG haplotype with development of macrovascular complications while haplotype TAG was observed to be significantly protective towards development of macrovascular complications in T2DM subjects (OR = 0.617, p = 0.0202). Conclusions: Our data indicates significant association of RAGE SNPs and haplotypes with vascular complications in North Indian T2DM subjects. # 2013 Elsevier Ireland Ltd. All rights reserved.

1.

Introduction

Hyperglycemia associated with diabetes mellitus stimulate non-enzymatic glycation and oxidation of proteins and lipids leading to enhanced formation of advanced glycation end

products (AGEs) [1,2]. High levels of AGEs are reported in diabetes mellitus (DM) patients compared to healthy individuals. The formation and accumulation of AGEs constitute the characteristic feature in diabetes [3–5]. There is growing evidence that production and accumulation of AGEs is involved in the initiation and development of micro and

* Corresponding author. Tel.: +91 11 22582972 74x5210/+91 9811259019 (mob); fax: +91 11 22590495. E-mail addresses: [email protected] (A.K. Tripathi), [email protected] (D. Chawla), [email protected] (S. Bansal), [email protected] (B.D. Banerjee), [email protected] (S.V. Madhu), [email protected] (O.P. Kalra). 0168-8227/$ – see front matter # 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.diabres.2013.12.004

diabetes research and clinical practice 103 (2014) 474–481

macrovascular complications observed in diabetes mellitus [6–8]. AGEs bind to specific receptor known as receptor for advanced glycation end products (RAGE) which is expressed in many of the cell types such as endothelial cells, monocytes and lymphocytes, including b cells of pancreas [9–11]. RAGE is a 45kd transmembrane receptor of immunoglobin super family, has 332 amino acid components, consisting of 2 ‘C’ type domains preceded by 1 ‘V’ type immunoglobin like domain. RAGE has a single transmembrane domain followed by a highly charged 43 amino acid cytosolic tail [12,13]. AGE– RAGE interaction was demonstrated to augment cytokine induction, adhesion molecule expression, smooth and fibroblast proliferation and chemoattraction of inflammatory cells by upregulating multiple signalling pathways [14–16]. The up regulation and pathogenic effects of RAGE in vascular disease highlight the RAGE gene as a candidate for involvement in the pathogenesis of diabetes. The gene for RAGE is located on the chromosome 6p21.3 near the HLA locus, and at least 30 polymorphisms have been identified of which 9 are in promoter region, 11 in exon region and 10 in intron region. The two prominent polymorphisms in promoter region are 374T/A and 429T/C which affects RAGE expression and a 63 bp deletion spanning from 407 to 345, the use of later as genetic marker is limited due to its incidence being 5 years who regularly attended medical outpatient department and/or diabetic clinic of University College of Medical Sciences (UCMS) and GTB hospital, Delhi, were enrolled in this study. These patients were divided into three groups. The first group referred to as ‘DM’ comprised of 140 T2DM subjects without any vascular complications. The second group referred to as ‘DM-micro’ consisted of 152 T2DM subjects with microvascular complications (retinopathy and nephropathy). The third group referred to as ‘DM-macro’ consisted of 135 T2DM subjects with macrovascular complications. 176 healthy controls were also enrolled in the study by voluntary participation. The controls were departmental staff members, community participants or unrelated attendants of the patients. The study was approved by Institutional Ethics Committee-Human Research (IEC-HR) of UCMS. Written informed consent was obtained from each subject prior to entering the study. Diagnosis of diabetes was based on current criteria of the American Diabetes Association, 2010. Presence of microvascular complications was evaluated by assessment of retinopathy and/or nephropathy. Retinopathy was diagnosed on the basis of history and fundus examination for the presence of micro aneurysms, dot and blot hemorrhages or retinal changes such as edema and thickening. Nephropathy was diagnosed on the basis of history and clinical examination and presence of proteinuria (on the basis of routine urine analysis). If proteinuria was found negative then microalbuminuria was assessed by dipstick (Micral dipstick, Roche Diagnostic). Presence of macro-vascular complications was evaluated by history of cerebral vascular accidents (CVA)/ stroke, coronary artery disease (CAD) based on electrocardiographic changes suggestive of ST segment depression, Q2 wave changes and/or T wave inversion using appropriate Minnesota code or positive treadmill testing/stress echocardiography or positive coronary angiography wherever available and peripheral vascular disease (PVD) based on clinical findings of decreased peripheral arterial pulsation and/or ankle/brachial index of 130/80 mmHg or if antihypertensive drugs were being taken. Detailed clinical history was taken and relevant investigations including hemogram, blood glucose (fasting and 2 h post prandial), glycosylated hemoglobin, liver function test, blood urea, serum creatinine, chest X-ray, electrocardiography and urine examination was obtained for every subject. Fasting blood sample were withdrawn from ante cubital vein, collected into tubes containing EDTA and were stored at 20 8C. Isolation of DNA was performed within a week.

2.2.

RAGE genotyping

Three polymorphisms of the RAGE gene namely 374T/A, 429T/C and Gly82Ser were carried out using DNA extracted from 200 ml peripheral venous blood by using DNA isolation kit (Himedia, Mumbai, India).

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For amplification of promoter region containing the 374T/ A and 429T/C polymorphism the following primers (Sigma) were used— Forward primer 50 AAA ACA TGA GAA ACC CCA GA 30 Reverse primer 50 CCC CGA TCC TAT TTA TTC CA 30 Polymerase chain reaction (PCR) was performed for amplification using the Eppendorf Mastercycler Gradient—5331 Thermocycler (Eppendorf AG, Hamburg, Germany). It was conducted in 25 ml reaction mixture containing 100 ng of genomic DNA and 10 pmol of each primer. Annealing temperature was 56 8C and final extension occurred at 72 8C for 10 min. The 222 bp PCR product was digested by using 5 units of restriction nucleases Tas1 (Tsp5091) (MBI Fermentas, Germany) for 374T/A polymorphism at 658C for 16 h yielding 101 + 121 bp fragments for T allele (wild type) and 222 bp fragment for A allele (mutant type). For 429T/C polymorphism, the PCR product (222 bp) was digested with Alu1 (MBI Fermentas, Germany) at 37 8C for 16 h yielding 222 bp fragment for T allele (wild type) and 176 + 46 bp fragments for C allele (mutant type). For Gly82Ser polymorphism, the exon 3 region of the RAGE gene was amplified by PCR using primers 50 GTA AGC GGG GCT CCT GTT GCA 30 and 50 GGC CAA GGC TGG GGT TGA AGG 30 . The 397 bp PCR product was digested by Alu I enzyme at 37 8C for 16 h yielding 248 + 149 bp fragments for wild type allele (Gly82) and 181 + 67 + 149 bp fragments for mutant allele (Ser82). The restricted product were separated by 2% agarose gel electrophoresis and visualized on UVP DIGI– DOC IT gel documentation system (UVP, Upland, CA, USA) after ethidium bromide staining. The genotypic analysis was repeated in at least 20% of the samples to rule out genotypic error and sequencing was carried out in some representative samples of each type of polymorphism using capillary based Genetic analysis system (GeXP, BECKMAN COULTER, USA).

2.3.

Statistical analysis

Statistical analysis was carried out using standard statistical methods (SPSS software version 16.0). Demographic data and routine biochemical parameters among the study groups were analyzed using one way analysis of variance (ANOVA) and p-values obtained from F-test was adjusted as per Bonferroni correction followed by post-hoc Tukey’s test. Data were expressed as mean  standard deviation. Concerning the evaluation of RAGE gene polymorphisms, the significance of differences from Hardy– Weinberg equilibrium (HWE) was tested using the x2 test. The frequency of all the three SNPs among study groups were found compatible with HWE ( p > 0.05). Power of the sample size for each SNP at 5% significance level and 80% power were calculated by genetic power calculator and sample size was kept accordingly. For comparison of prevalence of different genotypes in different subject groups the odds ratio and 95% confidence interval (CI) were calculated by using logistic regression analysis. Linkage disequilibrium (LD) and haplotype analysis of the three SNPs among different study groups was done by using SHEsis software [23]. p Value of less than 5% was considered significant.

3.

Results

We studied the three polymorphisms namely 429T/C (rs1800625) and 374T/A (rs1800624) in the promoter region and Gly82Ser (rs2070600) in exon 3 region of the RAGE gene in 603 study subjects. The clinical characteristics of the study groups are shown in Table 1. There was no significant difference in the age or sex distribution and duration of diabetes between the study groups. Fasting blood glucose, post prandial blood glucose and HbA1C were significantly higher in DM subjects compared to healthy individuals. Among the lipid profile, the serum level of cholesterol was significantly higher and level of HDL was lower in DM patients compared to healthy individuals while LDL and triglyceride level were significantly higher in DM patients having vascular complication. Serum level of urea and creatinine were also significantly higher in DM patients having vascular complications. Among the DM groups these parameters were more elevated in patients with microvascular and macrovascular complications compared to those without vascular complications. The genotype and allele frequency distribution of 374T/A and 429T/C polymorphism are shown in Table 2. In 374T/A polymorphism, wild type (TT) genotype was found to be significantly lower in DM groups as compared to healthy controls. In the evaluation of the association at the genotype level, the rare homozygous mutant group was collapsed with the heterozygous group, and logistic regression analysis was carried out. Mutant variant was found to be significantly associated with T2DM compared to healthy controls (OR = 2.312, p = 0.0004). The association persisted when we combined the three DM groups together and compared with healthy controls ( p = 0.0004). Also 374A allele was less prevalent in DM-macro group as compared to DM group. Logistic regression analysis revealed protective nature of 374A allele towards development of macrovascular complications (OR = 0.580, p = 0.028) in T2DM subjects (Table 3). In 429T/C polymorphism, the frequency of wild genotype (TT) was 65% lesser in T2DM patients with macrovascular complication and the mutant variant was found three times more in this group as compare to controls. RAGE 429C allele was associated with macrovascular complications in T2DM subjects in a highly significant manner indicating that mutant diabetic population has three fold more chances of developing macrovascular complications (OR = 2.898, p = 0.0001, CI = 1.717 to 4.892). In Gly82Ser polymorphism, the frequency of mutant variant was found three times higher in DM subjects with microvascular complications as compared to healthy controls (Table 2). The risk of having microvascular complications was significantly higher in T2DM patients with minor allele Ser82 as shown in Table 3 ( p = 0.004). Tests of association were repeated adjusting for confounding factors like age, sex and duration of diabetes in the logistic regression model, and no significant alterations was observed even after adjustment. The pairwise linkage disequilibrium (LD) values suggested that promoter region SNPs i.e. 429T/C and 374T/A are in strong LD among diabetic subjects (D0 = 0.791). However Gly82Ser SNP showed relatively low LD with promoter SNPs. We also evaluated association of 429T/C, 374T/A and

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Table 1 – Demographic data and routine biochemical parameter among diabetic subjects and control subjects. Parameter

Diabetic subjects

Control subjects

DM

DM-micro

DM-macro

Healthy subjects

Subjects

140

152

135

176

Age (years)

51.6  9

53.7  7.7

53.8  8.2

51.8  7

Sex distribution (male/female)

66/74

70/82

72/63

94/82

BMI (kg/m2)

25.1  2.6

24  2.3

25.3  3.0

24  2.1

Duration (years)

8.4  4.9

10  4.2

9.6  4.8

0

Fasting sugar (mmol/l)

8.6  2.9a

9.3  2.5a

9.1  2.5a

4.8  0.4

Post prandial sugar (mmol/l)

11.9  3.8a

13.3  3.0a,b

13.4  3.7a,b

5.9  0.7

HbA1C (%)

7.9  1.7a

8.6  1.6a,b

9.7  1.6a,b

5.3  0.5

Serum creatinine (mmol/l)

78.7  16.8

194.5  141.4a,b

128.2  122.0a,b

74.3  11.5

Urea (mmol/l)

4.2  1.4

9.9  6.7a,b

6.0  4.1a,b

4.1  1.0

Total serum cholesterol (mmol/l)

4.2  1.1

5.2  1.3a,b

4.8  1.2a,b

3.9  0.7

LDL cholesterol (mmol/l)

3.1  0.8

3.6  1.1a,b

3.1  0.9a

2.7  0.7

HDL cholesterol (mmol/l)

1.1  0.3a

1.0  0.3a

0.9  0.2a,b

1.3  0.2

Triglycerides (mmol/l)

1.7  0.7

2.1  0.6a,b

2.2  0.9a,b

1.4  0.4

Data are presented as mean  SD. Comparison between the groups was performed with one-way ANOVA and p-values obtained from F-test was adjusted as per Bonferroni correction ( p < 0.005) followed by post hoc Tukey’s analysis. DM, diabetes mellitus; DM-micro, diabetes mellitus with microvascular complications; DM-macro, diabetes mellitus with macrovascular complications; BMI, body mass index; HbA1C, glycated hemoglobin; LDL, low density lipoprotein; HDL, high density lipoprotein. a p < 0.005, diabetic patients vs. healthy subjects. b p < 0.005, diabetic patients with complications vs. DM.

Gly82Ser haplotypes with diabetic complications. The haplotype frequency >1% of different haplotypes were shown in Table 4. Significant association of different haplotypes of RAGE gene with the development of vascular complications among DM subjects has been observed (Table 5). Haplotype CTG was found to be associated with high risk of development of macrovascular complications using TTG haplotype as reference, while haplotype TAG was associated with reduced risk of

macrovascular complications (OR = 0.617, p = 0.0202).

4.

among

T2DM

subjects

Discussion

Considering the importance of AGE–RAGE interaction in the pathology of complications in T2DM, various polymorphisms

Table 2 – Allelic and genotypic frequency of S374T/A, S429T/C and Gly82Ser polymorphism among the different subject groups. Genotype 374T/A polymorphism TT TA AA T-allele A-allele 429T/C polymorphism TT TC CC T allele C allele Gly82Ser polymorphism GG (82G/82G) AG (82S/82G) AA (82S/82S) G allele A allele

Controls subjects (N = 176)

DM (N = 140)

DM-micro (N = 152)

DM-macro (N = 135)

127 (72%) 46 (26%) 3 (2%) 85% 15%

74 (53%) 56 (40%) 10 (7%) 73% 27%

78 (51%) 68 (45%) 6 (4%) 73.5% 26.5%

89 (66%) 40 (30%) 6 (4%) 81% 19%

148 (84.1%) 27 (15.3%) 1 (0.6%) 91.75% 8.25%

109 (78%) 30 (21%) 1 (1%) 88.5% 11.5%

120 (79%) 31 (20.3%) 1 (0.7%) 89.15% 10.85%

74 (55%) 56 (41%) 5 (4%) 75.5% 24.5%

159 (90%) 17 (10%) 0 95% 5%

126 (90%) 13 (9.3%) 1 (0.7%) 94.65% 5.35%

117 (77%) 30 (20%) 5 (3%) 87% 13%

122 (90.3%) 12 (9%) 1 (0.7%) 94.8% 5.2%

Values are presented as no. (%). Genotype distributions were in Hardy–Weinberg equilibrium. DM: type 2 diabetes mellitus without complications; DM-micro: type 2 diabetes mellitus with microvascular complications; DM-macro: type 2 diabetes mellitus with macrovascular complications.

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Table 3 – Association of S374T/A, S429T/C and Gly82Ser polymorphisms in RAGE gene with vascular complications in DM. 374T/A polymorphism a

OR

95% CI

DM vs. controlref.

2.312

1.448–3.690

After adjustmentb DM-micro vs. DMref. After adjustmentb DM-macro vs. DMref. After adjustmentb DM + DM-micro + DM-macro vs. controlref. After adjustmentb

2.304 1.064 1.026 0.580 0.597 2.000 1.975

1.440–3.688 0.672–1.684 0.642–1.637 0.356–0.943 0.365–1.007 1.366–2.928 1.347–2.895

p Value

429T/C polymorphism a

OR

95% CI

0.0004

1.503

0.852–2.652

0.001 0.792 0.916 0.028 0.040 0.0004 0.0006

1.537 0.938 0.975 2.898 2.915 2.163 2.196

0.868–2.724 0.537–1.638 0.554–1.714 1.717–4.892 1.717–4.949 1.373–3.409 1.391–3.466

Gly82Ser polymorphism ORa

95% CI

0.159

1.039

0.493–2.189

0.919

0.141 0.821 0.929 0.0001 0.0001 0.0009 0.001

0.997 2.692 2.778 0.959 0.956 1.589 1.566

0.471–2.111 1.379–5.256 1.411–5.469 0.433–2.124 0.430–2.130 0.900–2.804 0.885–2.769

0.995 0.004 0.003 0.918 0.913 0.110 0.123

p Value

p Value

OR: Odds ratio; CI: Confidence interval. ref. = Reference baseline. To find out the association at genotype level, the rare homozygous mutant group was collapsed with the heterozygous group for all three polymorphisms. b Test of association was repeated in logistic regression model adjusting for age, duration and gender, and no significant alterations was observed even after adjustment. a

in the RAGE gene have been identified and screened for their association with vascular complications [24,25]. Of the many polymorphisms that have been screened, we studied 429T/C and 374T/A polymorphism in the promoter region and Gly82Ser polymorphism in the exon 3 region of the RAGE gene to look for their association with DM and accompanied complications in North Indian population. Upon stratifying the T2DM subjects into three groups namely DM, DM-micro and DM-macro group based on absence or presence of vascular complications, we observed that 374T/A polymorphism was significantly associated with T2DM as well as with reduced risk towards the development of macrovascular complication in T2DM subjects; whereas 429T/C and Gly82Ser polymorphism were found to be significantly associated with progression of macrovascular and microvascular complications, respectively, in T2DM subjects. Worthy to note that this is the first comprehensive study where association of RAGE polymorphism with vascular complications in North Indian T2DM population has been reported. Compared to healthy controls, 374T/A polymorphism significantly associated with T2DM subjects ( p = 0.0004). The association persisted when we combined the three DM groups together and compared with healthy controls ( p = 0.0004). Among black Americans association of mutant allele has been shown with insulin resistance thereby indirectly showing association with occurrence of T2DM [26]. However, majority of reports available did not show any significant association of 374T/A polymorphism with diabetes in various ethnic populations [27–29]. A recent study has claimed 374T/A as

monomorphic in Indian population [30]. This is surprising as we observed 15% of mutant variant in the control group; also 13–20% of mutant variant in healthy Indian population have been reported in two early studies [31,32]. Another interesting finding that emerged from our study is that 374A allele has significantly lesser occurrence of macrovascular complications, indicating the protective nature of this polymorphism among North Indian DM subjects. Similar observations that presence of 374A allele reduce the risk of coronary artery disease have been reported in African–Brazilian and Caucasian–Brazilian population [33,34]. A meta analysis report have reaffirmed the protective nature of 374A allele towards development of macrovascular complications in DM patients [35]. Analysis of genotype distribution of 429T/C polymorphism revealed significant association of mutant variant with progression of macrovascular complications in T2DM subjects (OR = 2.898, p = 0.0001). The mutant DM population has three fold more chances of developing macrovascular complications in comparison to DM patients with wild genotype. In two early reports association of 429T/C SNP with macrovascular complication in T2DM patients have been carried out in Chinese and Slovene population, and no association have been reported [36,37]. In contrast, Hudson et al. found an increase risk of diabetic retinopathy among patients with the TC or CC genotypes [18]. Not many studies are available on 429T/C promoter polymorphism in Indian population. However, Prasad et al. have reported association of 429T/C SNP with diabetic chronic renal insufficiency [30] unlike our

Table 4 – Haplotype frequency among study groups. Haplotype TTG TAG CTG TTA

Healthy control (%)

DM (%)

DM-micro (%)

DM-macro (%)

74.8 12.3 5.9 4.5

56.8 26.4 10.7 5.3

56.0 22.3 9.6 9.1

54.0 17.9 22.2 3.7

Haplotype with frequency >1% shown.

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Table 5 – Association of haplotypes of S429T/C, S374T/A and Gly82Ser polymorphisms in RAGE gene with vascular complications in DM. Haplotype

DM-macro vs. DM Odds ratio

TTG CTG TAG TTA

Reference 2.427 0.617 0.685

DM-micro vs. DM

95% CI

p Value

– 1.507–3.907 0.409–0.929 0.301–1.556

Reference 0.0002 0.0202 0.3632

Odds ratio – 0.889 0.804 1.576

95% CI 0.519–1.524 0.550–1.175 0.812–3.061

p Value 0.6696 0.2592 0.1759

CI: Confidence Interval. TTG as reference haplotype.

finding where we observed highly significant association with macrovascular complications in T2DM subjects. Many authors including Prasad et al. however, did not include DM-macro in their study group. It is interesting to note that the two promoter polymorphisms i.e. 374T/A and 429T/C that we have studied showed dissimilar association with respect to diabetic complications. While 374T/A polymorphism found to be protective in nature towards development of macrovascular complication, 429T/ C showed significant association towards the development of macrovascular complication in diabetic patients. The opposite phenotypic characteristics of these two promoter region polymorphisms may be attributed to their dissimilarity in association to diabetic complications and also due to strong LD as shown in our results. 374T/A polymorphism have been shown to cause repression of RAGE gene expression [20]. The lesser the RAGE gene expression, lesser will be its binding with ligand and consequent lesser signal transduction for procoagulant or pro-thrombotic gene and thereby imparting protection towards vasculature. On the other hand 429T/C promoter polymorphism have been shown to increase RAGE gene transcript [18]. The enhanced availability of RAGE augment AGE binding and induce downstream signalling resulting in release of proinflammatory cytokines and adhesion protein that favours thrombosis and eventually capillary leakage and occlusion. Recently we have reported association of RAGE polymorphism with blood level of paraoxonase and AGEs level [38]. We also observed significant association between Ser82 allele and microvascular complications in T2DM patients (OR = 2.692, p = 0.004). Similar to our finding, the mutant allele (Ser82) has been shown to be associated with development of diabetic nephropathy and diabetic retinopathy in Chinese and Caucasian population, respectively [39,40]. A meta analysis report have also reaffirmed the significant association of Gly82Ser polymorphism towards development of diabetic retinopathy among Asian populations [41]. Although, several studies found no association between Gly82Ser polymorphism and diabetic complications in various other populations such as Malaysian, Brazilian and Japanese [42–44]. Very few studies are available on Indian population, however we have come across two reports where Ser82 mutant allele have been shown as a low risk allele for diabetic retinopathy in South Indian population [31,45]. Neither any report is available to corroborate the low risk allele for diabetic retinopathy, nor the consequence of G82S mutation on the AGE binding suggests the presence of low risk allele as mentioned below.

Gly82Ser polymorphism is a naturally occurring polymorphism where glycine at position 82 in N-terminal variable domain is involved in ligand binding to RAGE. This polymorphism occurs with relatively high incidence compared with other RAGE polymorphism [19]. G82S mutant RAGE displays enhanced ligand binding and downstream signalling leading to microvascular complications. How RAGE signalling induces microvascular complications in T2DM subjects has not been fully elucidated. Various reports have suggested that NF-kB mediated enhanced expression of pro-inflammatory and pro-fibrotic cytokine TGF-b is responsible for retinal and renal damage, characteristics of microvascular complications [46,47]. Haplotype based analysis are generally believed to be more powerful tool to detect genes with modest effects. We therefore carried out haplotype analysis of three SNPs of RAGE gene and observed that out of possible eight haplotypes combination only four haplotypes have >1% haplotype frequency. The study have revealed that haplotype CTG was associated with high risk of development of macrovascular complications among diabetic subjects, while haplotype TAG was associated with reduced risk of macrovascular complications in T2DM patients confirming the results on association of individual SNPs with vascular complications in T2DM patients. In an early report Zee et al. have reported the association of haplotype TAG with reduced risk of ischemic stroke (OR = 0.63, CI = 0.40 to 0.99, p = 0.05) [48]. Although Peng et al. found no haplotype association with severity of CAD among Chinese population [36]. In conclusion, considering that the 40% of world diabetic population is present in India and in absence of significant report on association of RAGE SNPs with diabetic complications, the result of this report showing significant association of RAGE SNPs with vascular complications in North Indian T2DM patients is of great importance towards identification of candidate gene. The pathogenetic association reported in our study must be interpreted with precaution in view of moderate sample size. Extrapolation of any conclusions from the present study may be insufficient and further large, multiethnic, linkage and haplotype studies should be performed to confirm the role of RAGE gene polymorphism in disease susceptibility and pathogenesis.

Conflict of interest statement The authors declare that there is no conflict of interest.

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Acknowledgement This work is supported through a project funded by Indian Council of Medical Research (ICMR Project No. 5/3/8/60/2008RHN), Government of India, New Delhi.

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Association of RAGE gene polymorphism with vascular complications in Indian type 2 diabetes mellitus patients.

The study was designed to evaluate the association of -374T/A and -429T/C polymorphism in the promoter region and Gly82Ser polymorphism in exon 3 regi...
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