B.R Achyut, Akanksha Srivastava, Sandeep Bhattacharya, Balraj Mittal. Sanjay Gandhi Postgraduate Institute of Medical Sciences Lucknow , India

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1 Clinica Chimica Acta 377 (2007) Genetic association of interleukin-1β ( 511C/T) and interleukin-1 receptor antagonist (86 bp repeat) polymorphisms with Type 2 diabetes mellitus in North Indians B.R Achyut, Akanksha Srivastava, Sandeep Bhattacharya, Balraj Mittal Sanjay Gandhi Postgraduate Institute of Medical Sciences Lucknow , India Received 10 July 2006; received in revised form 18 September 2006; accepted 18 September 2006 Available online 27 October 2006 Abstract Background: Type 2 diabetes mellitus (T2DM) is associated with a subclinical systemic inflammation and development of complications like nephropathy, retinopathy, neuropathy and hypertension. We studied the genetic association of bi-allelic polymorphism ( 511C/T) of interleukin (IL)-1β and 86 bp variable number tandem repeat (VNTR) polymorphism of natural receptor antagonist (IL-1RN) with T2DM and associated complications in North Indians. Methods: We genotyped 200 patients with T2DM and 223 healthy control subjects for IL-1β ( 511C/T) by PCR-RFLP. Genotyping of IL-1RN (VNTR) polymorphism was determined by gel electrophoresis after PCR amplification. Results: Interleukin-1β ( 511C/T) and IL-1RN (VNTR) polymorphisms were significantly associated with T2DM. IL-1β 511T, IL-1RN 2 and IL-1RN 3 alleles were associated with high risk of T2DM whereas; individuals with IL-1β 511C and IL-1RN 1 alleles were at low risk. Haplotype frequency analysis showed that T2 (IL-1β 511T/IL-1RN 2) haplotype was associated with the high risk ( p = 0.000; OR = 2.4, 95% CI ) and C1 (IL-1β 511C/IL-1RN 1) haplotype showed low risk ( p=0.000; OR=0.38, 95% CI ). Further, CT, TT genotypes of IL-1β ( 511C/T) and 1/2 genotype of IL-1RN (VNTR) were found to be associated with risk of complications particularly with nephropathy in T2DM. Conclusion: The IL-1β ( 511C/T) and IL-1RN (VNTR) polymorphisms are significantly associated with increased risk of T2DM as well as associated complications in North Indians Published by Elsevier B.V. Keywords: Type 2 diabetes; Proinflammatory cytokines; Genetic association; Gene polymorphism; Diabetic complications 1. Introduction Type 1 and type 2 diabetes mellitus (T2DM) are influenced by different genetic factors, but individuals with either of them are prone to developing complications including nephropathy, retinopathy, peripheral neuropathy, and hypertension [1,2]. T2DM is the major contributor to the epidemic rise in diabetes because it accounts for N90% of diabetes. T2DM is a polygenic Abbreviations: T2DM, Type 2 diabetes mellitus; RFLP, Restriction fragment length polymorphism; VNTR, Variable number of tandem repeat; OR, Odds ratio. Corresponding author. Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences Lucknow , India. Tel.: x2322; fax: / addresses: bml_pgi@yahoo.com, balraj@sgpgi.ac.in (B. Mittal). and multifactorial disease. Both environmental and genetic factors contribute to the inter-individual differences in disease susceptibility [3]. Besides factors like lifestyle, the prevalence of T2DM also varies by ethnicity [4,5]. Familial clustering of complications has also been reported in diabetes [6]. T2DM is associated with a subclinical systemic inflammation that might be attributable to a dysregulation of the immune system [7,8]. The cytokine proteins IL-1β and IL-1Ra are potent mediators of the inflammatory response and play an important role in tissue remodelling in chronic inflammation. Therefore, genes encoding these cytokines are important candidates which may be implicated in pathogenesis of T2DM and associated complications. Cytokine gene polymorphisms have recently attracted considerable interest since it has been discovered that distinct /$ - see front matter 2006 Published by Elsevier B.V. doi: /j.cca

2 164 B.R. Achyut et al. / Clinica Chimica Acta 377 (2007) alleles of cytokine genes are associated with different immunoinflammatory diseases [9,10]. Therefore, genetic polymorphisms in these inflammatory cytokines may have a particular impact on the inter-individual differences and pathogenesis of T2DM. IL-1 cytokine genes are present in the form of cluster, consist of 3 linked genes; mapping to chromosome 2q13-24 [11] encodes glycoproteins IL-1α, IL-1β and IL-1 receptor antagonist (IL-1Ra). IL-1α and IL-1β are pro-inflammatory cytokines, whereas IL-1Ra is an anti-inflammatory cytokine that inhibits IL-1 by competing for receptor binding and appears to play a major role in determining the pro-inflammatory activities of IL-1α and IL-1β in vivo [12]. The balances between IL-1β and IL-1Ra in local tissues play an important role in many diseases. The IL-1β gene has bi-allelic polymorphism in the promoter region at position 511 representing C/T transition [13]. The variant allele at this locus (IL-1β 511T) has been found to be associated with various inflammatory diseases and patients carrying IL-1β 511T allele have increased concentrations of IL-1β [14]. Interleukin-1 receptor antagonist (IL-1RN) gene encodes for IL-1Ra protein. In intron 2 of this gene, 86 bp tandem repeat polymorphism (VNTR) leads to existence of 5 different alleles with allele 1 (4 repeats), allele 2 (2 repeats), allele 3 (5 repeats), allele 4 (3 repeats), and allele 5 (6 repeats) [15]. The 4-repeat (IL-1RN 1) and 2-repeat (IL-1RN 2) alleles are most common, while the other alleles occur at a combined frequency of b5%. A possible relationship of the IL-1RN (VNTR) polymorphism with lower C-reactive protein concentrations may exist in coronary disease patients [16]. Hypertension, poor glycemic control and albuminuria are known risk factors for diabetic nephropathy, but they do not explain all inter-individual variabilities in the rate of progression to kidney failure. In Korean patients with T2DM, IL-1β ( 511C/T), IL-1RN (VNTR) and TNF-α (-308G/A) polymorphisms have been found to be associated with diabetic nephropathy [17]. Increased frequency of IL-1RN 2 has been associated with more severe clinical outcome in several chronic inflammatory diseases including systemic lupus-erythematosus [18], rheumatoid arthritis, and ulcerative colitis [9]. Association studies of IL-1β and IL-1RN cytokine gene polymorphisms with T2DM are limited [17,35] and no study has been reported from India. In present study, we genotyped IL-1β ( 511C/T) and IL-1RN (VNTR) polymorphisms to find out the susceptibility of these polymorphisms with T2DM. The patients were stratified according to the presence of complications like nephropathy, neuropathy, retinopathy, and hypertension and T2DM without complication and association with polymorphisms was analyzed. 2. Subjects and methods 2.1. Subjects T2DM patients were enrolled from the outpatients attending the clinics of Sanjay Gandhi Postgraduate Institute of Medical Sciences, India from November 2003 to April Out of 325 patients screened 200 were included in this study. Most of the patients were from the State of Uttar Pradesh in North India. Subjects were categorized in diabetes group if they had fasting glucose concentrations 126 mg/dl or 2-h glucose concentrations 200 mg/dl after a 75-g oral glucose tolerance test [19]. All the patients were on oral hypoglycemic agent glimepride or metformin or both. Medical records of these patients were reviewed to ascertain diabetes-associated complications like neuropathy, nephropathy, hypertension and retinopathy. Neuropathy was defined as the presence of distal sensory loss in feet. Nephropathy was defined as the presence of gross albumin in urine N300 mg/d in 24-h collection of urine. Retinopathy was defined as the presence of retinal vascular microaneurysm, blot hemorrhages, or cotton wool spots. Hypertension was defined as the presence of blood pressure of 140/90 mm Hg at 3 different readings on different days. Two-hundred twenty-three age-matched controls were healthy staff members of institute with normal oral glucose tolerance test. All the subjects with similar ethnicity were randomly selected from same source population. Those patients and controls having the history of coronary artery disease, albuminuria between 30 and 300 mg/d, neoplasia, or other metabolic disorder (including obesity), were excluded. Study was approved from the ethical committee of the institute. After an informed consent, 5-ml blood sample was taken in EDTA for analysis of DNA. The genomic DNA was extracted from peripheral blood leucocytes pellet using the standard salting out method [20] Genotyping of IL-1β ( 511C/T) polymorphism PCR amplification was conducted in a total volume of 50 μl with 25 pmol of each primer; as reported by Rad et al. [21], (F) 5 -TGG CAT TGA TCT GGT TCA TC-3 and (R) 5 -GTT TAG GAA TCT TCC CAC TT-3 ; genomic DNA ( ng); 10 Taq polymerase buffer (5 μl) and (1.5) units of Taq DNA polymerase (Bangalore Genei, India). PCR conditions were as follows: initial denaturation of 95 C for 5 min, 35 cycles of 95 C for 30 s, 55 C for 30 s and 72 C for 30 s; followed by final extension of 72 C for 5 min. PCR products were digested by restriction endonuclease Aval (MBI Fermentas) at 37 C for overnight and then analyzed by 10% polyacrlamide gel electrophoresis. Alleles were coded as follows: 304 bp for T/T; 190 and 114 bp for C/C; and 114 bp for C/T. Twenty percent of samples were regenotyped by other laboratory personnel. Approximately 3% heterozygote samples showed discrepancy were excluded from analysis. Then randomly selected 5% samples were sequenced and no inconsistency was observed Genotyping of IL-1-RN (VNTR) polymorphism Polymerase chain reaction (PCR) analysis was carried out in a total volume of 50 μl, containing genomic DNA ( ng); 10 pmol of each primer; 10 Taq polymerase buffer (5 μl) and (1.5) units of Taq DNA polymerase (Bangalore Genei, India). The primers for the IL-1-RN VNTR (86 bp) in intron 2 were (F)5 -CTC AGC AAC ACT CCT AT-3 and (R)5 -TCC TGG TCT GCA GGT AA-3 according to Tarlow et al. [15]. PCR amplification was performed under the following conditions: denaturing step at 95 C for 5 min, 35 cycles of 95 C for 30 s, 58 C for 30 s and 72 C for 30 s and one cycle of extension at 72 C for 10 min. The PCR products were separated by electrophoresis on 2% agarose gel and stained with ethidium bromide. Gel documentation was done by Alphaimager 1220, Alpha Innotech Corporation, San Leandro, CA Statistical analysis Data was analyzed using the statistical software SPSS ver (SPSS, Chicago, IL). Direct gene counting method was used to determine the frequency of genotypes and alleles. The χ 2 test or Fisher's exact test was used to determine differences in allele and genotype frequencies of IL-1β ( 511C/T) polymorphism. Observed and expected genotype frequencies were compared by χ 2 test to check deviation from Hardy Weinberg Equilibrium and p-value was considered significant at b0.05 concentration. The haplotype frequencies were calculated from the observed genotypes by using the Arlequin software vers [22]. The allele and genotype frequencies of IL-1RN (VNTR) and haplotype distributions between IL-1β ( 511C/T) and IL1-RN (VNTR) were evaluated using the Monte Carlo Style CLUMP analysis program for the presence of multivariant alleles (when the standard χ 2 test on the relevant contingency table was invalid because of small expected values) [23]. Interaction between IL-1β ( 511C/T) and IL-1RN

3 B.R. Achyut et al. / Clinica Chimica Acta 377 (2007) Table 1 Demographic profile of T2DM patients and controls Demographic profile Patients Controls No. of Subjects Age (y) a 49.67± ±12.14 Male 122 (61%) 147 (65.92%) Female 78 (39%) 76 (34.08%) Hight (cm) a ± ±6.65 Weight (Kg) a 62.75± ±9.26 BMI (Kg/M 2 ) a 24.37± ±2.68 Duration of Diabetes (y) a 9.3±3.4 a Values in mean±sd. (VNTR) alleles was calculated using logistic regression. Age, height and BMI variables were expressed as mean±sd. The sample size was calculated using the QUANTO ver. 1 program ( hydra.use.edu/gxe). Calculated sample size was adequate to study both polymorphisms. Log additive inheritance model was used for T2DM as a polygenic disease. Desired power of our study was set at 80% with at pb0.05 in a 2-sided test. 3. Results A total of 200 patients (122 male/78 female: mean age 49.67±13.97 y) and 223 healthy controls (147 male and 76 female: mean age ± y) were evaluated for the IL-1β ( 511C/T) and IL-1RN (VNTR) polymorphism study. Duration of diabetes was 9.3±3.4 y (Table 1). Out of 200, 33 (16.5%), 11 (5.5%), 21 (10.5%) and 30 (15.0%) patients showed history of Table 2 Complication status of T2DM patients Complication status n (%) BP# (mm Hg) systolic/diastolic HbA1c# (%) Fasting blood glucose# (mg %) HT 15 (7.5) 135±8.72/85± DN 15 (7.5) 137±14.31/ DR 1 (0.5) 145/ DN+NEU 3 (1.5) / DN+DR 3 (1.5) / DR+HT 8 (4.0) / DN+DR+ 5 (2.5) / NEU DN+HT+ NEU 3 ( / DN+DR+ 4 (2.0) / HT DN$ 33 (16.50) NEU$ 11 (5.50) DR$ 21 (10.50) HT$ 30 (15.0) T2DM@ 68 (34.0) Others 37 (18.5) n=number of patients with particular complication. HT: Hypertension, DN: Diabetic nephropathy, DR: Diabetic retinopathy, NEU: Neuropathy. T2DM patients with history of more than one complication. $ Total count of patients with particular type of T2DM patients without any complication. T2DM patients whom complication status was unavailable. # Values expressed in Mean±SD are taken at one point of time during treatment and will not indicate a life long trend of the concentrations in the given patients. Table 3 Genotype and allele frequencies of IL-1β ( 511C/T) polymorphism in T2DM patients and controls Genotypes/alleles Patients n a (%) Controls n a (%) P-value OR (95% CI) CC 9 (4.5) 29 (13.0) ( ) CT 90 (45.0) 117 (52.5) ( ) TT 101 (50.5) 77 (34.5) ( ) C 108 (27.0) b 175 (39.3) b ( ) T 292 (73.0) b 271 (60.8) b ( ) a Total T2DM patients 200 and controls 223. b Total chromosome number. nephropathy, neuropathy, retinopathy and hypertension complications respectively. Sixty-eight (34.0%) patients had T2DM (without complications). We did not have data for 37 patients regarding complication, therefore, excluded from analysis. However, T2DM patients with one or more than one type of complications were observed. The mean blood pressure, HbA1c and glucose plasma concentrations for T2DM patients are listed in Table 2. Represented values were taken at 1 point of time during the management of diabetes with appropriate drugs. In controls, all the frequencies of tested genotypes, alleles and haplotypes were in Hardy Weinberg equilibrium. Association of the polymorphism was first done in the pooled patient and control populations and further analysis were performed by Table 4 Genotype and allele frequencies of IL-1RN (VNTR) polymorphism in T2DM patients and controls Genotype/allele Patient n a (%) Control n a (%) P-value OR (95% CI) 1/1 60 (30.00) 138 (61.88) ( ) 1/2 107 (53.50) 64 (28.70) ( ) 1/3 5 (2.50) 1 (0.45) ( ) 1/4 0 (0) 5 (2.24) /2 20 (10.00) 14 (6.28) ( ) 2/3 2 (1.00) 0 (0) /4 3 (1.50) 0 (0) /3 3 (1.50) 0 (0) /4 0 (0) 1 (0.45) (58) b 346 (77.58) b ( ) (38) b 92 (20.63) b ( ) 3 13 (3.25) b 1 (0.23) b ( ) 4 3 (0.75) b 7 (1.56) b ( ) a Total T2DM patients 200 and controls 223, b total chromosome number. c CLUMP (T1) χ 2 =54.72, p= (for genotypes), CLUMP (T1) χ 2 =46.76, p= (for alleles). c χ 2 calculated by CLUMP statistical package, when multiple allele variants are present. T1=2 by 6 contingency table.

4 166 B.R. Achyut et al. / Clinica Chimica Acta 377 (2007) Table 5 Haplotype frequency (%) distributions of IL-1β ( 511C/T) and IL-1RN (VNTR) polymorphisms in T2DM patients and controls Haplotype Patient n a (%) Control n a (%) P-value OR (95% CI) C 1 30 (15.32) 72 (32.33) ( ) T 1 85 (42.68) 102 (45.70) ( ) C 2 20 (10.00) 15 (6.68) ( ) T 2 56 (28.00) 31 (14.17) ( ) C 3 3 (1.32) 1 (0.22) ( ) T 3 4 (1.93) C 4 1 (0.36) T 4 1 (0.39) 2 (0.90) ( ) n a Total chromosome number (patients 400 and controls 446). CLUMP (T1) χ 2 =61.67, p= (for haplotypes). stratifying the data according to associated complications in T2DM subjects. IL-1β ( 511C/T) polymorphism was significantly associated with T2DM. The frequencies of TT genotype and T allele were significantly higher in patients than controls (50.5% vs. 34.5% and 73.0% vs %, Table 3) and associated with high risk ( p =0.001; OR= 2.0, 95% CI and p =0.000; OR=1.8, 95% CI respectively). On the other hand, the CC genotype and C allele had low risk ( p =0.002; OR=0.32, 95% CI and p=0.000; OR=0.57, 95% CI ). When data was stratified according to gender, we did not get any gender specific association. After dividing study population into different age groups, the risk due to TT genotype was similar in different age groups (data not shown). Out of 5, only 4 alleles (allele 1, 2, 3, and 4) of IL-1RN (VNTR) were observed in the present study. As shown in Table 4, the genotype and allele frequencies of IL1RN (VNTR) were significantly different between cases and controls [CLUMP (T1) χ 2 =54.72, p=0.000 and χ 2 =46.76, p=0.000 respectively)]. Frequencies of IL-1RN 1/2 genotype and IL-1RN 2 allele were higher in patients (53.5% and 38%) than the controls (28.7% and 20.63%), ( p=0.000 and p=0.000) with increased risk of the disease (OR=2.9, 95% CI and OR=2.4, 95% CI ). On the other hand, the frequencies of IL-1RN 1/1 genotype and IL-1RN 1 allele were higher in control group (61.88% and 77.58%) than the patient group (30% and 58%) thus conferred low risk ( p=0.000; OR=0.3, 95% CI and p=0.000; OR = 0.4, 95% CI for genotype and allele respectively). Allele 3 ofil-1rn VNTR was also found to be associated with T2DM ( p = 0.001; OR = 14.98, 95% CI ). IL1RN polymorphism analysis separately in male and female subjects did not differ from combined analysis (Data not shown). Similarly, we did not get much effect of age on diabetes due to IL-1RN (VNTR) polymorphism (Data not shown). Haplotype frequency estimation of IL-1β ( 511C/T) and IL-1RN (VNTR) polymorphisms are shown in Table 5. Haplotype frequencies were significantly different between patients and controls [CLUMP (T1) χ 2 =61.67, p=0.0000)]. C1 (IL-1β 511C/IL-1RN 1) haplotype was more commonly observed in controls than in patients (32.3% vs. 15.3%) and significantly conferred protection ( p = 0.000; OR = 0.38, 95% CI ). T2 (IL-1β 511T/ IL-1RN 2) haplotype frequency was higher in patients than in controls (28.0% vs. 14.1%) and significantly associated with the high risk ( p=0.000; OR=2.4, 95% CI ). T3 (IL-1β 511T/ IL-1RN 3) and C4 (IL-1β 511C/IL-1RN 4) haplotypes were found only in patients (1.9% and 0.36%). The interaction between IL-1β 511C/T and IL-1RN (VNTR) allele carriers was studied in order to evaluate whether an epistatic effect between 2 polymorphisms was present. The result showed IL-1β 511T/IL-1RN 2 alleles conferred high risk of developing T2DM (data not shown). Patient population was also subdivided according to the associated complications viz. neuropathy, nephropathy, retinopathy, hypertension and T2DM patients without complication (Table 6). In IL-1β polymorphism, CT and TT genotypes were significantly ( p=0.000 and p=0.009) associated with risk of nephropathy in patient group (OR=1.53, 95% CI and OR= 1.2, 95% CI ). Patients having retinopathy ( p = 0.006; OR= 1.3, 95% CI and p =0.019; OR = 1.2, 95% CI respectively) and hypertension (p =0.001; OR=1.4, 95% CI and p=0.003; OR=1.3, 95% CI respectively) complications also showed association with CT and TT genotypes. However, patients without complication did not show any association with IL-1β genotypes. In case of IL-1RN (VNTR) polymorphism, those genotypes having frequency b 10% were excluded in further analysis. Table 7 shows that IL-1RN 1/1 and 1/2 genotypes were Table 6 IL-1β ( 511C/T) genotypes and risk of associated complications in T2DM patients Genotype Nephropathy (n=33) Neuropathy (n=11) Retinopathy (n=21) Hypertension (n=30) No complication (n=68) CC (6) OR (95% CI) 2.0 ( ) 1.2 ( ) 1.2 ( ) 0.5 ( ) p-value p=0.134 p=0.453 p=0.453 p=0721 CT (58) OR (95% CI) 1.53 ( ) 1.14 ( ) 1.3 ( ) 1.4 ( ) 0.7 ( ) p value p=0.000 p=0.041 p=0.006 p=0.001 p=0.280 TT (65) OR (95% CI) 1.2 ( ) 1.0 ( ) 1.2 ( ) 1.3 ( ) 1.5 ( ) p-value p=0.009 p=0.191 p=0.019 p=0.003 p=0.164 T2 DM patients without complications.

5 B.R. Achyut et al. / Clinica Chimica Acta 377 (2007) Table 7 IL-1RN (VNTR) genotypes and risk of associated complications in T2DM patients #Genotype Nephropathy (n=33) Neuropathy (n=11) Retinopathy (n=21) Hypertension (n=30) No complication (n=68) 1/1 (42) OR (95% CI) 1.34 ( ) 1.05 ( ) 1.14 ( ) 1.2 ( ) 1.3 ( ) p-value p=0.016 p=0.346 p=0.126 p=0.065 p= /2 (66) OR (95% CI) 1.35 ( ) 1.12 ( ) 1.3 ( ) 1.4 ( ) 1.0 ( ) p-value p=0.001 p=0.035 p=0.003 p=0.000 p= /2 (14) OR (95% CI) 1.4 ( ) 1.1 ( ) 1.2 ( ) 1.3 ( ) 1.1 ( ) p-value p=0.143 p=0.502 p=0.329 p=0.219 p=0.921 T2 DM patients without complication. # Genotypes of IL-1RN (VNTR) having frequency N10%. significantly associated with the risk of nephropathy ( p = 0.016; OR=1.34, 95% CI and p=0.001; OR=1.35, 95% CI ). IL-1RN 1/2 genotype also showed high risk for diabetes associated hypertension complication ( p =0.000; OR = 1.4, 95% CI ). The risk for neuropathy and retinopathy was also marginally higher ( p=0.035; OR=1.12, 95% CI and p=0.003; OR=1.3, 95% CI ). However, patients without complication did not show any association with IL-1RN genotypes. 4. Discussion Inflammation has been reported to be increased in T2DM which contributes to the pathology of the disease [7]. Earlier studies have reported that hyperglycemia associated with T2DM, acutely increases circulating IL-6 and TNF-α [24] and IL-1 proteins. So far, most of the studies in T2DM are confined to genetic polymorphisms in TNF-α and IL-6, but no significant or only marginal association was observed [25,26]. The polymorphisms of IL-1RN and IL-1β genes could alter the ratios of IL-1β to IL-1Ra, thereby modulating the proinflammatory activity. IL-1RN 2 and IL-1β ( 511) T alleles could enhance the production of IL-1β which is known to cause inflammatory response [27 29]. Many studies have documented the association of IL-1β 511T and IL-1RN 2 alleles with inflammation. Mansfield et al. reported the association IL-1RN 2 allele with ulcerative colitis [9]. Cork et al. reported the association IL-1RN 2 allele with inflammatory skin diseases [10]. Furuta et al. found that IL-1β 511T allele conferred high risk of hypochlorohydria and gastritis [14]. IL-1RN polymorphism ( 2 allele) as a disease severity factor in systemic lupus erythematosus was reported [18]. Coronary heart disease and alcoholism were also found to be associated with IL-1β 511T and IL-1RN 2 alleles [16,30]. One study in Danish multiplex families showed the association of IL-1β 511T allele with T1DM [13]. Since there are limited studies on the influence of IL1-β and IL-1Ra cytokines in T2DM, we have carried out a detailed investigation. The analysis of our pooled data showed that IL-1β 511T allele, IL-1RN 1/2 genotype, IL-1RN 2 allele and T2 (IL-1β 511T/ IL-1RN 2) haplotype were significantly associated with the increased risk of T2DM. However, increased frequency of IL1RN 3 allele in patients, suggest that this allele along with IL-1RN 2 allele may be implicated in pathogenesis. The reason for higher risk due to IL1RN 3 allele is still unknown because its mechanism of action is not very clear. Association of IL1RN 3 allele has also been reported with other conditions like recurrent spontaneous abortion [31] and high IL-6 blood concentration in the critically ill patients from Japan [32]. It seems that these alleles act in tandem to bring about susceptibility of T2DM. Further, several studies on ethnic differences in diabetic complication have reported higher prevalence and incidence rates among ethnic minorities. For example nephropathy and end stage renal disease of diabetic patients have been reported to be more common among Asian [4,5]. Earlier reports suggest that increased concentrations of cytokines in patients with diabetes could lead to inappropriate metabolic effects causing various types of complications [33] which again signify that patients with diabetes are genetically susceptible to develop complications [3]. In various studies, IL-1RN and IL-1β alleles have been found to be associated with higher risk of neuropathy, retinopathy and hypertension which is otherwise more often in diabetic patients than controls. In order to minimize the error in association studies related to complications, we have used caseonly analysis rather than normal healthy volunteers as controls. The genes within the IL-1 gene cluster are potential candidates in the pathogenesis of diabetic nephropathy. In rat models of nephritis, both IL-1β and IL-1Ra are produced in the glomerulus, and treatment with recombinant IL-1Ra suppressed inflammation [34]. Several polymorphisms in cytokine genes such as IL-1β, IL-1RN, and TNF-α have been implicated in several kidney diseases [14,15]. It appears that IL-1β and IL-1RN polymorphism carriers have higher risk of diabetic nephropathy in certain populations. A study from Korea showed 511C/T in IL-1β, variable numbers of tandem repeat in IL-1RN and 308 G/A polymorphism in TNF-α gene were significantly associated with an increased risk of end-stage kidney failure [17]. Similar to the Korean study, we also analyzed our results using diabetic patients without nephropathy as controls. Interleukin-1β polymorphism (T allele) has also been found to be associated with diabetic nephropathy in the Northern Ireland population [35]. The association of IL-1 gene with kidney failure in diabetes has been attributed to pro-

6 168 B.R. Achyut et al. / Clinica Chimica Acta 377 (2007) inflammatory properties of IL-1. Stimulating effects of IL-1 probably influences proliferation of mesangial cells and synthesis of extracellular matrix which ultimately affect the progression of diabetic nephropathy. Interestingly, we got the association of IL-1RN 1/1 genotype with increase risk of nephropathy. Some studies also support this contradictory result where IL-1RN 1 allele was found to be associated with the other conditions like alcoholism, lung cancer and chronic rejection of transplant [27,36,37]. It is possible that the differences in findings reflect differences in ethnic background and marked ethnic variation is observed with diabetic nephropathy being more common in certain ethnic groups like India. Parental history of hypertension, diabetes or cardiovascular disease appears to predispose to nephropathy in patients with diabetes. In our study, IL-1β ( 511) CT, TT and IL-1RN 1/2 genotypes were also found to be significantly associated with neuropathy, retinopathy and hypertension with very marginal risk. However, limited number of patients in this subgroup compromises the power of such analyses. Further, other studies with more number of subjects are needed to confirm our findings. In conclusion, our results suggest that IL-1β ( 511C/T) and IL-1RN (VNTR) polymorphisms were significantly associated with increased risk of T2DM as well as associated complications in North Indians. The IL-1 gene cluster could become a good candidate for pharmacogenetic studies in T2DM. Acknowledgement The fellowship provided by CSIR (New Delhi) to Mr. B.R. Achyut is greatly acknowledged. References [1] Ritz E, Hasslacher C, Tschope W. Diabetic nephropathy are there differences between type I and type II? Miner Electrolyte Metab 1990;16: [2] Hendriksen PH, Oey PL, Wieneke GH, Bravenboer B, Banga JD. 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