association of rage (p.gly82ser) and mnsod (p.val16ala) polymorphisms with diabetic retinopathy in...

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Title: Association of RAGE (p.Gly82Ser) and MnSOD(p.Val16Ala) Polymorphisms with Diabe<!–<queryid="Q1">Please provide a reduced form of the main title thatdoesn’t exceed 80characters.</query>–><!–<RunningTitle>Association ofRAGE (p.Gly82Ser) and MnSOD (p.Val16Ala)Polymorphisms with Diabe</RunningTitle>–>tic Retinopathyin T2DM patients from North India

Author: Vanita Vanita

PII: S0168-8227(14)00020-5DOI: http://dx.doi.org/doi:10.1016/j.diabres.2013.12.059Reference: DIAB 5978

To appear in: Diabetes Research and Clinical Practice

Received date: 26-4-2013Revised date: 30-8-2013Accepted date: 29-12-2013

Please cite this article as: V. Vanita, Association of RAGE (p.Gly82Ser)and MnSOD (p.Val16Ala) Polymorphisms with Diabetic Retinopathy in T2DMpatients from North India, Diabetes Research and Clinical Practice (2014),http://dx.doi.org/10.1016/j.diabres.2013.12.059

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http://dx.doi.org/doi:10.1016/j.diabres.2013.12.059

http://dx.doi.org/10.1016/j.diabres.2013.12.059

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Association of RAGE (p.Gly82Ser) and MnSOD (p.Val16Ala) Polymorphisms with Diabetic Retinopathy in T2DM patients from North India

Vanita VanitaDepartment of Human Genetics, Guru Nanak Dev University, Amritsar, Punjab, India

Running head short title: Association of RAGE and MnSOD polymorphisms with DR

Running head author’s title: Vanita V

Corresponding author:

Dr. Vanita VanitaProfessor and Head,Department of Human Genetics,Guru Nanak Dev University, Amritsar,Punjab, IndiaPhone: 0183-2258802-09, 2450601 Ext. 3279Fax: 0183-2258819-20 (Univ.); 2258863(O)E-mail: [email protected]

Grant supportThis work was in part supported by grant no. SR/FT/LS-025 sanctioned from DST, India

under SERC FAST Track Scheme for Young Scientists to VV and grant from DBT, India

BT/IN/German/13/VK/2010 and Bundesministerium für Bildung und Forschung BMBF,

IND 10/036 under the framework of Indo-German bilateral cooperation for research.

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Abstract

Aims: The present study aimed to examine the association of RAGE (p.Gly82Ser) and

MnSOD (p.Val16Ala) polymorphisms with diabetic retinopathy (DR) in north Indian

T2DM patients.

Methods: In this case-control association study, 758 T2DM patients were recruited. 446

with retinal neovascularization, microneurysms and hemorrhages were considered as cases

(DR) and 312 patients with T2DM and no clinical signs of retinopathy (DNR), were

recruited as controls. Genotypes for RAGE (p.Gly82Ser) and MnSOD (p.Val16Ala)

polymorphisms were generated by direct sequencing of amplified products.

Results: Genotype distribution of p.Gly82Ser (RAGE) and p.Val16Ala (MnSOD)

polymorphisms were significantly different between DR and DNR (p<0.05) whereas

distribution of allele frequency did not differ significantly (p>0.05). A significantly higher

frequency of homozygous Ser82 genotype in DR patients was detected compared with

DNR (2.4% vs 0.64%) for p.Gly82Ser (RAGE) polymorphism whereas there was a higher

frequency of homozygous Ala16 genotype for p.Val16Ala (MnSOD) polymorphism in DR

patients compared with DNR (22.6% vs 19.3%). Binary logistic analyses showed an

association of homozygous recessive genotype Ser82 with DR (OR: 2.63, 95% CI: 0.16-

15.88, p<0.033) for p.Gly82Ser (RAGE) polymorphism. However, we did not find a

significant association of p.Val16Ala polymorphism in MnSOD with retinopathy.

Conclusions: The findings indicate a statistically significant association of p.Gly82Ser

polymorphism in RAGE with DR in T2DM patients.

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Keywords: Diabetic retinopathy, Type 2 diabetes mellitus, RAGE, MnSOD,

Polymorphism, Case-control association study, DNA sequence analysis, Genotyping

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Introduction

Diabetic retinopathy (DR) is a major vascular complication affecting the retina and a

leading cause of blindness worldwide [1]. Longer duration of diabetes and higher levels of

glycated hemoglobin increase the risk for retinopathy [2-3]. Clinically, retinopathy is

characterized by the presence of microneursyms, hemorrhages, hard exudates, cotton wool

spots, pericyte loss and intraretinal microvascular abnormalities. Advanced glycation end

products (AGEs) and oxidative stress have been reported to play an important role in the

pathogenesis of DR [4]. AGEs are heterogeneous molecules formed by the reaction

between reducing sugar with free amino groups of proteins, lipids and nucleic acids

nonenzymatically (glycation). AGEs are also reported to play an important role

neurological, cardiovascular, and various other diseases [5]. AGEs mediate their effect by

binding to cellular receptor RAGE (receptor for advanced glycation end products), which

in truncated form, is a 35kDa transmembrane protein of the immunoglobulin superfamily

of cell surface molecules, and is expressed mainly in endothelium,

monocytes/macrophages, T-lymphocytes, neuronal cells and glomerular epithelial cells [6-

9]. Various other RAGE ligands are also known [10-14]. The AGEs-RAGE interaction

alters the intracellular signaling, gene expression, release of pro-inflammatory molecules

and free radicals, which are the major factors contributing to the pathology of DR [15-16].

To date, at least 30 polymorphisms in RAGE have been reported to be associated with

different vascular complications including DR. Of these, p.Gly82Ser (exon 3) is of interest

due to its localization in the N-linked glycation site (81 position) and hence influences

AGE-RAGE interaction [17].

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A large amount of reactive oxygen species (ROS) is produced in mitochondrial electron

transport chain as a result of oxidative stress, and is assumed to damage the mitochondrial

DNA and influence the respiratory chain by lipid peroxidation of nerve cells [18-19]. The

excessive production of ROS such as superoxide is prevented by over expression of

mitochondrial manganese superoxide dismutase (MnSOD) that catalyses dismutation of

superoxide radicals into hydrogen peroxide and hence defends the retinal endothelial cells

from oxidative damage [20]. The p.Val16Ala variant in MnSOD (exon 2) that under

oxidative stress leads to conformational change from a β sheet to α helical structure in

mitochondrial targeting sequence (MTS), affects mitochondrial processing efficiency [21].

There are only two studies indicating association of p.Val16Ala polymorphism in MnSOD

with DR in Caucasian and Finnish populations [22-23]. For p.Gly82Ser polymorphism in

RAGE there is only a single report indicating association of Ser82 allele with DR in Han

Chinese population [24]. However, two published reports in south Indian populations [25-

26] documented Ser82 as a protective allele for DR. Ng et al. [27] investigated p.Gly82Ser,

1704G/T and 2184A/G polymorphisms in RAGE in Malaysian T2DM patients and found

no association with the development of retinopathy. The present study aimed to investigate

the association of two candidate gene polymorphisms (p.Gly82Ser in RAGE and

p.Val16Ala in MnSOD) with DR in T2DM patients from north India.

Material and Methods

Subjects

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In this case control association study, 758 individuals with confirmed T2DM who visited

Dr. Daljit Singh Eye Hospital, Amritsar for ophthalmic examination were randomly

recruited. Ophthalmic examination that included visual acuity testing, Humphrey’s

perimetry, ocular coherence tomography and fundus examination followed by fundus

photography, was conducted on all individuals. Fundus examination revealed that 446

patients had hard exudates, vitreous hemorrhage, maculopathy or neovascularization and

thus were considered as DR (cases). 312 patients with T2DM without evidence of

retinopathy (DNR) were considered as controls. Information such as age, sex, weight,

height, body mass index (BMI), age of onset of diabetes mellitus, random blood glucose

levels, duration of diabetes mellitus, any other associated anomalies and blood pressure

were collected from on a pre-designed questionnaire. Fundus examination after pupil

dilation (tropicamide and phenylphrine 2.5%) with binocular ophthalmoscope and fundus

photographs with 50º-angle taking fovea as centre were taken by retina experts. Severity of

retinopathy was determined in each patient’s fundus photographs according to Early

Treatment Diabetic Retinopathy Study Research Group [28].

Analyses of RAGE (p.Gly82Ser) and MnSOD (p.Val16Ala) polymorphisms

The study was approved by our Institutional review board in accordance with the

declaration of Helsinki and written informed consent was obtained from each individual.

10 ml of peripheral venous blood was collected and genomic DNA extracted using a

standard protocol. For the amplification of exon 3 of RAGE that harbors p.Gly82Ser

substitution, following forward ‘5-CACTGTTTAGGCCCTGCTTC-3’ and reverse primers

‘5-GGAATTCTTACGGTAGACACGG-3’ [29] were used. For PCR, 10μl reaction

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mixture consisting 1xTaq buffer A with 1.5mM MgCl2, 0.125mM of each dNTP, 1.0 pmole

of each forward and reverse primer, 10ng of genomic DNA and 0.075 units of Taq DNA

polymerase (Bangalore Genei) was used. After an initial denaturation at 95 °C for 5 min,

each PCR consisted of 2 cycles at 95 ºC for 1 min, 66 °C for 1 min, 72 ºC for 1 min

followed by 30 cycles at 95 °C for 1 min, 64 ºC for 1 min, 72 ºC for 1 min and final

extension at 72 °C for 10 min. For the amplification of exon 2 of MnSOD that harbors

p.Val16Ala substitution following forward ‘5-CAGCCCAGCCTGCGTAGACGG-3’ and

reverse ‘5-CTTGGCCAACGCCTCCTGGTACTT-3’ primers were used [30]. Each PCR

cycle consisted of initial denaturation at 95 °C for 5 min followed by 25 cycles of 95 °C for

1 min, 63 °C for 1 min, 72 °C for 1 min followed by final extension at 72 °C for 10 min.

Amplified products were purified and sequenced bi-directionally following conditions as

described elsewhere [31]. The sequencing reaction products were purified by the

isopropanol precipitation method (Applied Biosystems (ABI) protocol), resuspended in 10

µl of loading buffer (5:1 ratio of deionized formamide and 25 mM EDTA with blue dextran

(50 mg/ml)), denatured at 95 oC for 5 min, and electrophoresed on 4% denaturing

polyacrylamide gels on DNA sequencer (ABI-Prism 377). Sequencing results were

assembled and analyzed using the SeqMan II program of the Lasergene package

(DNASTAR Inc., Madison, WI).

Statistical Analyses

All the statistical analyses were performed using SPSS for windows version 16 (SPSS Inc).

Hardy-Weinberg equilibrium was tested using the Cochran-Armitage trend test (2x3

contingency table) based on a linear regression model. Genotype and allelic frequency

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between DR and DNR were compared using chi-square test. Student’s t-test and one way

ANOVA were performed to compare different clinical characteristics as expressed mean ±

SD. All statistical tests were considered significant with p<0.05 (two-tailed). Binary

logistic regression analyses were used to estimate Ors for the association of p.Gly82Ser

polymorphism in RAGE and p.Val16Ala in MnSOD with DR compared with DNR after

adjusting for potential confounders. The present analysis with a total sample size of 758

including 446 cases (DR) and 312 controls (DNR) had a statistical power of more than

80% to detect an association with an OR of 1.5 at p=0.05. The corrections for multiple

testing were done by the Bonferroni method with adjusted z-scores where needed.

Results

Comparisons of mean values for clinical characteristics between DR (cases) and DNR

(controls) groups are presented in Table 1. The two groups (DR and DNR) were well-

matched for age (55.9 ± 8.9 yrs for DR; 55.8 ± 12.2 yrs for DNR) but mean age of onset of

diabetes and BMI were significantly higher in DNR group (p<0.001) whereas duration of

diabetes and systolic blood pressure were significantly higher (p<0.001) in DR group. The

random blood glucose levels between DR and DNR also were statistically significantly

different (p<0.001). The frequency of DR between males and females did not differ

significantly (p>0.05). Comparisons of clinical characteristics between DR and DNR

groups by gender (data not shown) showed that age of onset and duration of diabetes were

similar whereas there was a gender difference for systolic blood pressure (higher in males)

and BMI (higher in females) (p<0.05). Clinical characteristics of individuals stratified by

genotypes between DR and DNR groups were compared by one way ANOVA for both

RAGE (p.Gly82Ser) and MnSOD (p.Val16Ala) polymorphisms (Table 2, 3). Since the

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prevalence of Ser82 genotype (RAGE) was too low for sufficient statistical power, the

analyses focused on the comparison between wild type (Gly82) and Ser82 allele carriers

(Gly82Ser+Ser82) for RAGE and between wild type (Val16) and Ala16 allele carriers

(Val16Ala+Ala16) for MnSOD. No significant differences were observed among DR and

DNR groups for different genotypes in RAGE (Table 2). However, for different genotypes

in MnSOD, significant mean differences were observed for age of onset (p<0.038) and

duration of diabetes (p<0.05) in the DR group (Table 3). However, no difference was

observed between wild type and risk allele carriers in DNR group. The alleles, genotypes

frequency and adjusted OR distribution between DR and DNR for RAGE and MnSOD

polymorphisms (p.Gly82Ser and p.Val16Ala), respectively, are presented in Table 4. There

was also evidence of an association of homozygous recessive genotype Ser82 (RAGE) with

the presence of diabetic retinopathy (adjusted OR: 2.63, 95% CI: 0.16-15.88, p<0.033).

However, Gly82 dominant genotype showed a significant protective association (adjusted

OR: 0.11, 95% CI: 0.02-0.54, p<0.0007) (Table 4). In addition, homozygosity for Ser82

genotypes tended to be more common in cases (DR group) than in the control (DNR) group

(2.4 vs 0.64%), suggestive of an association of the recessive model. The present study also

found an indication of dominant mode of action for a protective role of the Gly82

genotype. The genotype distribution of RAGE polymorphism (p.Gly82Ser) between DR

and DNR was statistically significant (χ2=13.57; p=0.001), however, the distribution of

allelic frequency between DR and DNR was not (χ2=1.42; p=0.233).

For MnSOD p.Val16Ala polymorphism the homozygosity for Ala16 genotypes tended to

be more common in cases (DR) than in controls (DNR) (22.6 vs 19.3%) whereas, in the

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dominant genetic model, Val16 genotype showed a significant protective association with

DR (adjusted OR: 0.05, 95% CI: 0.01-0.38, p<0.0004). The genotype distribution of

MnSOD polymorphism (p.Val16Ala) between DR and DNR was significant (χ2=14.33;

p=0.0008), however, the distribution of allele frequency between DR and DNR was not

(χ2=1.54; p=0.214). Hence, the RAGE polymorphism (p.Gly82Ser) showed positive and

significant associations with DR, whereas, MnSOD polymorphism (p.Val16Ala) showed no

statistically significant association with DR, although the genotype frequency of the risk

allele (Ala16) of MnSOD was observed to be higher in the DR group compared with the

DNR group (22.6% vs 19.3%) (Table 4).

Hardy-Weinberg equilibrium of these two SNPs (p.Gly82Ser (rs2070600) in RAGE and

p.Val16Ala (rs4880) in MnSOD) in cases (DR) and controls (DNR) was examined using

Cochran-Armitage trend test (2x3 contingency table) based on a linear regression model.

Co-dominant model has been used for weight age analysis as under this model each

genotype gives a diverse and non-additive risk.. A significant deviation from HWE was

observed (p=0.0011). The deviation from HWE for combined samples among both the

analyzed SNPs may point to hospital based sampling bias. However, the distribution of the

allele frequencies for both SNPs did not deviate significantly from HWE (p=0.233 for

rs2070600, p=0.214 for rs4880) indicating that there were no serious concerns about the

genetic structure of the samples. Furthermore, no significant deviation from HWE was

observed in the control groups for both SNPs. Therefore, only genotype distributions of the

patients groups showed deviations from HWE which may provide additional support for an

association of these two SNPs loci with DR.

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Discussion

In the present study, the association of RAGE (p.Gly82Ser) and MnSOD (p.Val16Ala)

polymorphisms has been evaluated with DR in T2DM patients from north India. An

association between age of onset and duration of diabetes with retinopathy was observed in

the present study and is in accordance with previous reports [32, 33]. Also, an earlier onset

and longer duration of diabetes as observed in our analysis compared with DNR is in

accord with the findings reported by Blum et al. [34] on DR patients from Germany,

Slovenian DR patients [22] and also is in agreement with the UKPDS 22 and UKPDS 50

studies [35-36]. In the present study, BMI, blood glucose level and systolic blood pressure

were also observed to be significantly associated with DR. These findings are in

accordance with previous reports which also showed an association between severity of DR

and BMI, blood glucose level and systolic blood pressure [37-38].

AGEs occur under physiological conditions however, their level is reported to increase

during hyperglycemia, oxidative stress and inflammation leading to its binding to cellular

receptors which further induces signal transduction and cellular dysfunctions [15, 39].

AGEs have been documented as key substances in diabetic vascular remodeling [40] and

increased levels of AGEs are assumed for the development and progression of retinopathy

due to increased permeability of retinal endothelial cells leading to vascular leakage,

induction of growth factors such as VEGF, leading to neovascularization and angiogenesis

[41]. The AGE-RAGE interaction dictates various signaling cascades (MAPKs, Rho

GTPases, NF-κB, Ras pathway, Rac/Cdc42 and JAK/STAT, activation of protein kinase C)

that alter gene expression [42-47]. Expression of RAGE is reported to be blocked by

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recombinant soluble RAGE and a decrease in the acceleration of atherosclerosis has been

documented when sRAGE is engineered in animal models [48].

Hudson et al., [17] reported a comparatively higher frequency of RAGE p.Gly82Ser

polymorphism in Asian (90%GG, 10%GS, 0%SS) and Caucasian (87%GG, 12%GS,

1%SS) non-diabetic control subjects and reported associations of two polymorphisms -

374T/A and -429T/C in RAGE with DR in a British population [49]. Lindholm et al. [50-

51] also documented a higher frequency of -374T/A and TA genotypes in Caucasian DR

patients and Ramprasad et al. [52] reported a modest association of -374T/A in RAGE in

south Indian subjects with non-prliferative retinopathy. However, three different studies

have documented no association of -429T/C and -374T/A polymorphisms with DR in

Chienes, Caucasians and Malaysian TADM patients with retinopathy [53-55]. Liu and

Xiang [56] showed that p.Gly82Ser is not associated with diabetic microangiopathy in

Chinese population with T2DM. However, Zhang et al.,[24] showed an association of the

Ser82 allele with DR in Han Chinese population, suggesting the possibilities of either

different molecular pathogenesis of DR in the two populations or differences in the

population history, thus leading to different haplotype structure of the associated region.

Yoshioka et al. [57] and Ng et al. [27] demonstrated that p.Gly82Ser was not associated

with DR in patients with T2DM in Japanese and Malaysian population.

However, in the present study the genotype frequency of homozygous Ser82 was observed

to be higher among DR patients (2.4% vs 0.64%) compared with controls (DNR) with an

odds ratio=2.63 (p<0.033) suggesting that Ser82 allele is significantly associated with DR

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in our north Indian T2DM patients. Interestingly, Kumaramanickavel et al., [25] and

Balasubbu et al., [26] reported Gly82Ser to be associated with the decreased risk of DR and

that Ser82 was a protective allele for DR in their south Indian population. This difference

might be attributed to genetic, cultural and marriage structure which are completely

different in these two populations. Balasubbu et al., [26] have also hypothesized that

associations seen in other populations might not be present in southern Indians due to

different population histories which may alter the haplotype block structure and possibly

due to genetic and environmental factors in different ethnic and geographic groups.

p.Gly82Ser substitution in RAGE has also been reported to be associated with skin

complications, gastric cancer, rheumatoid arthritis and nephropathy, patients with type 1

and type 2 diabetes mellitus [29, 58-60]. Since RAGE is localized in the HLA region in the

MHC Class III on chromosomes 6p21.3, the possibility that any association with RAGE

(p.Gly82Ser) due to linkage disequilibrium cannot be excluded.

MnSOD is a homotetramer of two identical subunits, each consisting of N-terminal helical

loop and C-terminal α/β domain [61]. MnSOD is a major antioxidant enzyme that catalyzes

the dismutation of superoxide to hydrogen peroxide in mitochondria. Kowluru et al., [20]

documented that over-expression of MnSOD prevents an increase in glucose induced

oxidative stress, apoptosis of the retinal endothelial cells suggesting a protective role of

MnSOD in the pathogenesis of DR. Various other biochemical markers of oxidative stress

such as malondialdehyde, thiobarbituric acid reacting substances, conjugated diene,

advanced oxidation protein products, protein carbonyl, 8-hydroxydeoxyguanosin,

nitrotyrosine, F(2) isoprostanes and pro-apoptosis molecules are also known to be

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associated with DR [62]. Therefore, these findings prompted us to initiate the present study

to elucidate the role of p.Val16Ala variant in MnSOD in a north Indian population.

However we did not observe a statistically significant association of p.Val16Ala

polymorphism in MnSOD with DR compared with controls. Lee and Choi [63] in a Korean

population, reported p.Val16Ala polymorphism in MnSOD to be associated with macular

edema but not with the development or progression of DR. The results in the present and

previous studies [22-23, 63] suggest an inter-population variance in the frequency of the

p.Val16Ala allele of MnSOD according to ethnicity and geographic location. Also the

association of p.Val16Ala variant in MnSOD has previously been reported with diseases

related to oxidative stress and abnormal free radical defense mechanisms [64-70].

We also performed a sub-analysis to test the significance of a possible association under

different genetic models of inheritance of p.Gly82Ser and p.Val16Ala variants of RAGE

and MnSOD respectively. There is a possibility that any association seen in different ethnic

groups could be due to a different haplotype block and diverse genetic as well as

environmental factors, which may further enhance the risk of disease. Further genetic

association studies are liable to statistical errors and population related genotype which

may vary in different populations. However, present results are sufficiently encouraging

for more extensive population based studies.

In conclusion, p.Gly82Ser polymorphism in RAGE seem to have a major effect on the

susceptibility to DR in north Indian T2DM patients. However, the p.Val16Ala variant in

MnSOD has no significant effect on susceptibility to DR. However, in the view of

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significant effect of this variant (p.Val16Ala) in MnSOD on disease risk in other

populations, it is possible that our study was underpowered to detect an effect, if it exists.

Further studies with larger sample size of patients and controls are required to explore this

further.

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Acknowledgments

Special thanks are to the patients for their cooperation and ophthalmologist at the Dr. Daljit

Singh Eye Hospital, Amritsar, for providing ophthalmic and clinical details of these

patients. Thanks are also to Dr. Badaruddoza, Assistant Professor, Department of Human

Genetics, for assisting in statistical analysis of the data and Ms. Nisha Gupta, Project

Fellow (DST Project) for assisting in genotyping. This work was in part supported by grant

no. SR/FT/LS-025 sanctioned from DST, India under SERC FAST Track Scheme for

Young Scientists to VV and grant from DBT, India BT/IN/German/13/VK/2010 and

Bundesministerium für Bildung und Forschung BMBF, IND 10/036 under the framework

of Indo-German bilateral cooperation for research.

Conflict of Interest: None

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Table 1. Comparison of descriptive statistics for different variables through t-test with 95% confidence level between diabetic retinopathy (cases) and diabetic without retinopathy (control) groups.

Diabetic retinopathy(n=446)

Diabetic without retinopathy(n=312)

t P value 95% confidence level

Characteristics

Mean SD Mean SD

Age (yrs) 55.92 8.90 55.84 12.2 0.104 0.917 -1.42 to 1.59

Age of onset (yrs) 44.34 11.05 47.24 11.80 3.45 <0.001 -4.54 to -1.25Random blood glucose level (gm/dl)

200 89.9 140 39.5 11.06 <0.001 49.35 to 70.64

Duration of diabetes (yrs)

12.2 10.0 8.70 6.46 3.50 <0.001 2.24 to 4.76

Systolic blood pressure(mm Hg)

134.6 15.2 129.2 9.7 5.50 <0.001 3.84 to 7.32

Diastolic blood pressure (mm Hg)

82.0 9.70 81.0 8.40 1.47 0.141 -0.33 to 2.33

BMI (kg/m2) 22.63 7.70 25.03 7.9 4.17 <0.001 -3.53 to 1.27

(Male/Female) 303/143 - 174/138 - - >0.05 -

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Table 2. Comparison of mean values for clinical characteristics stratified by genotypes between diabetic retinopathy (cases) and diabetic without retinopathy (control) groups for RAGE (p.Gly82Ser) polymorphism.

Characteristics Diabetic retinopathy(n=446)

Diabetic without retinopathy (n=312)

Gly82(n=410)

Gly82Ser+Ser82(n=36)

Gly82(n=272)

Gly82Ser+Ser82(n=40)

Mean SD Mean SD P value


Age (yrs) 56.0 8.5 57.35 8.8 0.362 55.7 12.5 56.37 10.07 0.746

Age of onset (yrs) 44.2 11.0 44.11 11.05 0.962 47.06 12.2 48.42 8.69 0.497


11.48 8.9 12.4 9.20 0.553 8.70 6.47 8.84 6.47 0.898

Systolic blood pressure (mm Hg)

134.5 15.4 135.0 14.11 0.707 127.0 33.4 128.0 12.4 0.852


81.75 8.9 84.5 6.57 0.071 81.82 10.2 80.5 6.77 0.433

BMI (kg/m2) 24.30 4.26 25.5 4.48 0.107 25.1 7.88 23.9 3.26 0.343

(Male/Female) 269/141 - 19/17 - 0.68 154/118

- 20/20 - 0.84

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Table 3. Comparison of mean values for clinical characteristics stratified by genotypes between diabetic retinopathy (cases) and diabetic without retinopathy (control) groups for MnSOD (p.Val16Ala) polymorphism.Characteristics Diabetic retinopathy

(n=446)Diabetic without retinopathy

(n=312)Val16

(n=127)Val16Ala+Ala16

(n=319)Val16(n=58)

Val16Ala+Ala16(n=254)



Age (yrs) 55.69 9.2 56.22 8.39 0.52 56.26 9.71 56.44 10.09 0.90

Age of onset (yrs)

43.02 13.7 45.4 10.4 <0.038 47.24 10.37 46.89 10.8 0.82


12.2 11.8 10.36 7.9 <0.05 8.86 6.95 9.24 6.59 0.69

Systolic blood pressure (mm Hg)

132.6 24.0 133.5 15.2 0.63 129.2 28.2 128.0 31.7 0.91


80.5 10.8 81.2 7.22 0.41 81.7 8.40 81.92 9.12 0.78

BMI (kg/m2) 24.5 4.57 24.11 3.83 0.327 24.98 6.34 24.87 7.00 0.86

(Male/Female) 70/57 - 180/139

- 0.89 20/38 - 120/134 - 0.83

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Table. 4 Distribution of frequency of genotypes and alleles for RAGE (p.Gly82Ser) and MnSOD (p.Val16Ala) polymorphisms.Data are number of subjects with each genotype and allele (frequency in percentage). OR-Odds Ratio, CI-Confidence Interval.ORs for different modes of inheritance were calculated.Study RAGE (p.Gly82Ser) polymorphism

(rs2070600)Genotype (%) Allele (%) P value Dominant model

(Gly82Ser/Ser82 vs Gly82)

Co-dominant model

(Ser82 vs Gly82Ser)=

(Gly82Ser vs Gly82)

Recessive model (Ser82 vs Gly82/

Gly82Ser)

Gly82 Gly82 Ser

Ser82 Gly82 Ser82 Genotype Allele *OR (95% CI)

p-value

*OR (95%

CI)

p-value *OR (95% CI)

P-value

DR (n=446)

410(91.9)

25(5.6)

11(2.4)

845(94.73)

47(5.27)

DNR(n=312)

272(87.17)

38(12.1)

2(0.64)

582(93.27)

426.73)

<0.001 0.279 0.110(0.023-0.542

<0.007 0.239(0.036-1.578)

0.137 2.629(0.161-15.88)

<0.033

MnSOD (p.Val16Ala) polymorphisms(rs4880)

Genotype (%) Allele (%) P value Dominant model (Val16Ala/Ala16 vs Val16)

Co-dominant model

(Ala16 vsVal16Ala)=

(Val16Ala vs Val16)

Recessive model(Ala16 vs Val16/

Val16Ala)

Val16 Val16 Ala

Ala16 Val16 Ala16 Genotype Allele *OR (95% CI)

p-value

*OR (95%

CI)

p-value *OR (95% CI)

P-value

DR (n=446)

127(28.47)

218(48.9)

101(22.6)

472(53.0)

420(47.0)

DNR(n=312)

58(18.5)

194(62.2)

60(19.3)

310(49.67)

314(50.3)

<0.009 0.235 0.051(0.007-0.382)

<0.004 0.889(0.308-2.561)

0.827 0.595(0.191-1.852)

0.370

* ORs were adjusted for age of onset, duration of diabetes, blood glucose level, hypertension and BMI

association of rage (p.gly82ser) and mnsod (p.val16ala) polymorphisms with diabetic retinopathy in...

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