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Association of chemokine gene variants with end stage renal disease in North Indian population
TRIM-00841; No of Pages 4 Transplant Immunology xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Transplant Immunology journal homepage: www.elsevier.com/locate/trim
Association of chemokine gene variants with end stage renal disease in North Indian population Vibha Singh, Praveen Kumar Jaiswal, Poonam Tiwari, Rakesh Kapoor, Rama Devi Mittal ⁎ Department of Urology and Renal Transplantation, Sanjay Gandhi Post Graduate Institute of Medical Science, Raebareli Road, Lucknow 226014, Uttar Pradesh, India
a r t i c l e
i n f o
Article history: Received 11 January 2013 Received in revised form 12 April 2013 Accepted 15 April 2013 Available online xxxx Keywords: Chemokine gene polymorphism ESRD PCR–RFLP ARMS-PCR Haplotype
a b s t r a c t Background & aim: The progression rate of chronic kidney disease (CKD) to its end-stage renal disease (ESRD), and the development and severity of various complications, are indirectly influenced by genetic and epigenetic factors. Chemokines are small inducible pro-inflammatory cytokines, which are implicated in many biological processes like migration of leukocytes, angiogenesis, tumor growth and metastasis. We tested association of four single nucleotide polymorphisms (SNPs) viz. CCL2I/D, CCL2A2518G, CXCL12G801A and CXCR2(+ 1208)C/T among individuals with ESRD (end stage renal disease) and normal healthy controls from North Indian population. Materials and method: CCL2I/D, CCL2A2518G, CXCL12G801A and CXCR2(+ 1208)C/T were genotyped in blood samples of hospital-based case–control study comprising of 200 ESRD cases and 200 healthy controls using Restriction Fragment Length Polymorphism (RFLP) and ARMS (Amplification Refractory Mutation Specific) PCR methodology. Results: A significant association was found in CXCL12G801A with ESRD risk. In case of CXCL12G801A polymorphism heterozygous (GA) genotype showed significant risk (p = 0.039; OR = 1.55) whereas A allele carrier (GA + AA) also exhibited risk with ESRD (p = 0.045, OR = 1.59). In CXCR2(+1208)C/T polymorphism, the heterozygous genotype (CT) showed significant risk for ESRD (p = 0.028; OR = 1.65) and combination of CT + TT demonstrated significant high risk for ESRD (p = 0.036; OR = 1.52). In case of CCL2I/D, the variant genotype (D/D) showed reduced risk for ESRD patients. Upon analyzing the gene–gene interaction between CXCR2 and CXCL12, the combination (CT–GA) showed 2.65 fold risk for ESRD (p = 0.018). Conclusion: Our results indicated that polymorphism in CXCL12G801A and CXCR2(+ 1208)C/T showed high risk for ESRD in North Indian population. However, CCL2I/D showed reduced risk and CCL2A2518G exhibited no association. Study with large sample size and diverse ethnicity is required to validate these observations. © 2013 Published by Elsevier B.V.
1. Introduction Renal failure refers to temporary or permanent damage to the kidneys that results in loss of normal kidney function. The prevalence of chronic kidney disease (CKD) and end-stage renal disease (ESRD) is growing worldwide and therefore, constitutes a serious public health problem, which causes substantial morbidity and mortality [1]. Chronic kidney disease progression has been linked to pro-inflammatory chemokine and markers of inflammation. These markers are also elevated in end-stage renal disease (ESRD). Persistent proteinuria, dyslipidemia, hypertension and smoking are considered established conventional risk factors along with a low estimated glomerular filtration rate (eGFR) and albuminuria which are known risk factors for end-stage renal disease (ESRD) [2,3]. However, it has been assumed that oxidative stress, inflammation and immune processes, may also be important contributors to ⁎ Corresponding author. Tel.: +91 522 2668004 8x4116; fax: +91 522 2668017. E-mail addresses: [email protected], [email protected] (R.D. Mittal).
the pathogenesis of cardiovascular disease as well as progression to ESRD [4]. The inflammatory response involved in renal damage produces a release of pro-inflammatory cytokines and chemokines, which cause an increased inflow of leukocytes, intensification of interstitial nephritis and progressive fibrosis. Alternatively, genetic susceptibility is also considered an important determining factor for the appearance and/or progression of ESRD and its complications [5,6]. Kidney cells produce MCP-1 (monocyte chemoattractant protein-1) in response to a variety of pro-inflammatory stimuli and predictably, its expression has been identified in kidney diseases which involve significant inflammation. A biallelic genetic variation (A/G) in the MCP-1 gene distal regulatory region at position − 2518 affects the level of MCP-1 expression in response to an inflammatory stimulus [7]. CXCL12 has been found to be involved in cell proliferation, cell migration, and cell invasion. The CXCL12 also known as stromal cell derived factor (SDF-1) has been revealed that a single nucleotide polymorphism (SNP), in this gene may affect the expression of SDF-1 chemokine [8].
0966-3274/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.trim.2013.04.004
Please cite this article as: Singh V, et al, Association of chemokine gene variants with end stage renal disease in North Indian population, Transpl Immunol (2013), http://dx.doi.org/10.1016/j.trim.2013.04.004
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V. Singh et al. / Transplant Immunology xxx (2013) xxx–xxx
CXCR-2 is a receptor of interleukin-8, which is involved in acute and chronic inflammatory processes. Polymorphisms in the CXCR2(+1208) C/T gene have been associated with chronic inflammatory conditions [9]. We selected these polymorphic markers as they have been reported to be functional and associated with altered immune responses. CCL2, also known as monocyte chemoattractant protein 1 (MCP-1) is the strongest known chemotactic factor for monocytes. Polymorphism in CCL2 gene at distal regulatory region at position −2518 affects the level of CCL2 expression in response to an inflammatory stimulus [7]. Exact mechanism of CCL2I/D gene polymorphism affecting the expression of gene is still unclear. CXCL12 gene is associated with cell proliferation, migration and invasion. Single nucleotide polymorphism (SNP) in this gene at CXCL12G801A may affect the expression of CXCL12 chemokine [8]. CXCR-2 gene is a receptor of interleukin 8 and is involved in acute and chronic inflammatory process. Polymorphisms at CXCR2(+1208)C/T gene have been associated with chronic inflammatory conditions [9]. Our preliminary data suggest that four polymorphisms showed an association with ESRD protection or development, which could help to predict the risk of developing ESRD. The aim of the present study was to investigate whether single nucleotide polymorphisms (SNPs) of CCL2I/D (rs3917887), CCL22518A/G (rs1024611), CXCL12G801A (rs1801157) and CXCR2(+1208)C/T (rs1801032), associated with different immune and inflammatory process could also be associated with development of ESRD in North Indian populations. 2. Materials & method 2.1. Patients and clinical data The demographic characteristics of the subjects are represented in Table 1. Healthy controls (n = 200), (mean age 33.8 ± 12.8, male = 192 and female = 8) for the study were recruited from Northern India with similar ethnicity having no history of hypertension, diabetes, renal failure, vascular diseases, stroke, and/or cardiomyopathy. They had no recognizable autoimmune disease at the time of assessment. A total number of 200 ESRD patients on hemodialysis (mean age 33.9 ± 11.3; male = 168 and female = 32) were recruited from Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India. Patients were selected having >4.0 mg/dl serum creatinine that had been dialyzing for at least 3 months with no evidence of active inflammation. Stringent diagnostic criteria were used for diagnosing ESRD. Data collected included basic demographic information, pattern, onset of disease, and use of medications. The Institutional Review Board and ethical committee approved the protocol, and informed consent was obtained from the patients and the controls participating in the study. 2.2. DNA extraction and genotyping Blood samples were collected from patients with ESRD and healthy controls in ethylenediamine tetraacetic acid (EDTA) anticoagulant coded vials. DNA was extracted from peripheral blood lymphocytes using ‘salting-out’ method [10]. Polymorphisms in chemokines CCL2I/D, Table 1 Demographic details of the ESRD patients and healthy controls. Variables
Controls (n = 200)
ESRD patients (n = 200)
p value
Age (mean ± SD) Gender (male/female)
33.8 ± 12.8 192/8
33.9 ± 11.3 168/32
0.103 0.348
Causes of ESRD Chronic glomerulonephritis (CGN) Chronic kidney disease (CKD) Diabetic nephropathy (DN) Diffuse global sclerosis (DGS)
– – – –
115 (57.5) 58 (29.0) 23 (11.5) 4 (2.0)
CCL2A2518G and CXCL12G801A and CXCR2(+1208)C/T genes were analyzed using PCR–RFLP (Polymerase Chain Reaction–Restriction Fragment Length Polymorphism) and ARMS-PCR (Amplification Refractory Mutation System-Polymerase Chain Reaction) method. Primer detail and PCR conditions of CCL2I/D, CCL2A2518G [11], CXCL12G801A [12] and CXCR2(+1208)C/T [13] were respectively. The genotyping for all the gene taken in present study was done on 15% Poly Acrylamide Gel Electrophoresis (PAGE) using molecular weight markers and further visualized after staining with ethidium bromide. Positive and negative controls were used in each genotyping assay, and 10% of the samples were randomly selected and run in duplicates with 100% concordance. The results were reproducible with no discrepancy in genotyping. 2.3. Statistical analysis The sample size was calculated using Quanto software, version 1.0 (available from: http://hydra.usc.edu/gxe) with input of following variables: case–control study design, significance level (alpha) > 0.05 (2 sided), model of inheritance was log additive, allele frequency was 0.28, and the genetic effect for odds ratio (OR) was 1.50 or greater. The present study achieved 80% of the statistical power. The goodness-of-fit chi square test was used to analyze any deviation from the Hardy– Weinberg Equilibrium in controls. A binary logistic regression model was used to estimate the risk as the OR at the 95% confidence interval. Haplotypes of each individual consisting of single nucleotide polymorphisms (SNPs) in chemokines were constructed, and the maximal likelihood haplotype frequencies were estimated using the expectation– maximization algorithm using the SNP analyzer ver. 1.2 program. Bonferroni's correction was applied in the case of multiple comparisons using the formula pc = p × n (pc represents corrected value where n is the number of comparisons performed). The statistical analysis was done using the Statistical Package for Social Sciences software, version 15.0 (SPSS, Chicago, IL), and p b 0.05 was considered statistically significant. 3. Results 3.1. Chemokine gene polymorphisms and ESRD The present study achieved 80% of the statistical power. The genotype and allele frequencies of chemokine gene polymorphisms in healthy individuals (controls) and ESRD patients are presented in (Table 2). The genotype frequencies of controls were in Hardy–Weinberg Equilibrium (HWE). We found significant association of CXCL12G801A and CXCR2(+1208)C/T polymorphism with ESRD risk whereas CCLI/D polymorphism showed reduced risk with ESRD. CCL2A2518G gene polymorphism showed no association with ESRD. In CXCR2(+1208)C/T polymorphism, the heterozygous genotype (CT) showed significant risk for ESRD (p = 0.020 OR = 1.65; 95% CI; 1.08–2.51). Combining heterozygous and variant genotype, (CT + TT) exhibited marginal risk for ESRD (p = 0.036, OR = 1.52; 95% CI; 1.02–2.61). In CXCL12G801A polymorphism the heterozygous genotype (GA) showed significant risk for ESRD (p = 0.039, OR = 1.55; 95% CI; 1.02–2.36). Combining heterozygous and variant genotype (GA + AA) demonstrated high risk for ESRD (p = 0.045, OR = 1.59; 95% CI; 1.04–3.68) However, at allelic level no significant association of any chemokine genes was observed with ESRD risk. 3.2. Association of CCL2I/D–CCL2A2518G haplotypes with ESRD risk Recent studies have demonstrated that haplotype analysis may be more affirmative in predicting the disease association compared with an analysis of a single polymorphism, as individual polymorphism is likely to confer modest effects to the risk of ESRD. We, therefore, examined the effects of CCL2 gene polymorphisms by constructing haplotype sets taking combination IA as a reference. However there was no significant association observed in case of any set of haplotype combinations with ESRD in North Indian population (Fig. 1). 3.3. Gene–gene interaction of chemokine gene polymorphism To analyze the combined effect of chemokine SNPs taken in the present study, we conducted gene–gene interaction. Five combinations were constructed for chemokines taken for the present study viz. CXCL12G801A–CCL2I/D, CXCL12G801A–CCL2A2518, CXCR2(+1208) C/T–CCL2I/D, CXCR2(+1208)C/T–CCL2A2518G & CXCR2(+1208)C/T–CXCL12G801A. Combination of CXCR2(+1208)C/T–CXCL12G801A only showed statistically significant results viz. CT–GA combination. After applying the Bonferroni correction for multiple variable CT–GA combination remains significant for ESRD risk (pc = 0.018). None of the other combinations
Please cite this article as: Singh V, et al, Association of chemokine gene variants with end stage renal disease in North Indian population, Transpl Immunol (2013), http://dx.doi.org/10.1016/j.trim.2013.04.004
V. Singh et al. / Transplant Immunology xxx (2013) xxx–xxx Table 2 Genotypic frequency of CCL2I/D, CCL22518A/G, CXCL12G801A and CXCR2(+1208)C/T in ESRD patients and control.
Combinations
Ref 0.703 0.035 0.762 Ref 0.203
Ref 1.08 0.45 0.94 Ref 0.83
(47.0) (44.0) (9.0) (53.0) (69.0) (31.0)
Ref 0.185 0.339 0.190 Ref 0.202
Ref 0.75 0.71 0.77 Ref 0.83
CXCR2(+1208)C/T–CXCL12G801A CC/GG 55 (27.5) CC/GA 48 (24.0) CC/AA 9 (4.5) CT/GG 39 (19.5) CT/GA 28 (14.0) CT/AA 6 (3.0) TT/GG 5 (2.5) TT/GA 9 (4.5) TT/AA 1 (0.5)
81 106 13 119 268 132
(40.5) (53.0) (6.5) (62.5) (67.0) (33.0)
Ref 0.039 0.866 0.045 Ref 0.194
Ref 1.55 0.96 1.59 Ref 1.22
91 95 14 109 277 123
(45.5) (47.5) (7.0) (54.5) (69.3) (30.7)
Ref 0.020 0.955 0.036 Ref 0.117
Ref 1.65 1.02 1.52 Ref 1.28
p-Value
CCL2I/D (rs3917887) I/I 84 (42.0) I/D 90 (45.0) D/D 26 (13.0) I/D + D/D 116 (58.0) I allele 258 (64.5) D allele 142 (35.5)
87 101 12 113 275 125
(43.5) (50.5) (6.0) (56.5) (68.8) (31.3)
CCL22518A/G (rs1024611) AA 81 (40.5) AG 97 (48.5) GG 22 (11.0) AG + GG 119 (59.5) A allele 259 (64.8) G allele 141 (35.3)
94 88 18 106 276 124
CXCL12G801A (rs1801157) GG 101 (50.5) GA 83 (41.5) AA 16 (8.0) GG + GA 99 (49.5) G allele 285 (71.3) A allele 115 (28.8) CXCR2(+1208)C/T (rs1801032) CC 112 (56.0) CT 73 (36.5) TT 15 (7.5) CT + TT 88 (44.0) C allele 297 (74.2) T allele 103 (25.8) a
Table 3 Gene–gene interaction of CXCR2(+1208)C/T–CXCL12G801A gene polymorphism in ESRD patients and healthy controls.
ORa (95% CI)
Cases n (%) (n = 200)
Controls n (%) (n = 200)
(0.72–1.64) (0.21–0.95) (0.63–1.39) (0.62–1.12)
3
Controls N (%)
Patients N (%)
OR (95%CI)
p value
37 (18.5) 48 (24.0) 6 (3.0) 38 (19.0) 50 (25.0) 7 (3.5) 6 (3.0) 8 (4.0) 0 (0.0)
Ref 1.49 0.99 1.45 2.65 1.73 1.78 1.32 NC
Ref 0.179 0.987 0.235 0.002a 0.355 0.367 0.599 NC
(0.84–2.69) (0.33–3.02) (0.78–2.67) (1.42–4.95) (0.54–5.57) (0.50–6.27) (0.47–3.73)
(0.49–1.15) (0.35–1.43) (0.52–1.14)
NC = Not calculated. a pc = 0.018, after Bonferroni correction for multiple variables.
(0.61–1.11)
gene. Whereas Ahluwalia et al. reported a risk in diabetic nephropathy comparatively [11]. While the other polymorphic site CCL2A2518G of CCL2 taken in the present study showed no significant association at genotypic or allelic level with ESRD. This was complimented by the observations of Steinmetz et al. in IgA nephropathy [14]. In case of CXCL12G801A, heterozygous GA genotype and the variant allele carrier i.e. GA + AA showed 1.6 fold increased risk for ESRD in our population. To the best of our knowledge this is probably the first study involving this polymorphism being reported for association with end stage renal disease. Our observations matched the study of Hirata et al. which showed that GA + AA genotype was significantly associated with prostate cancer and that of Warchoł et al. who found significant association of heterozygous G/A with systemic lupus erythematosus patients respectively [12,15]. Single nucleotide polymorphism at CXCR2(+1208)C/T which is located in the non-coding region of CXCR2 gene might provide valuable information for the pathogenesis and the susceptibility to chronic inflammatory disease. Some studies have reported that CXCR2 was over expressed in renal, prostate, pancreatic, colon, nasopharyngeal and gastric cancers [16]. The heterozygous genotype (CT) of CXCR2(+1208)C/T gene showed significant 1.65 fold risk along with the variant T allele carrier (CT + TT) showing 1.52 fold risk with ESRD risk in the present study. The reasons could be probably due to variation in ethnicity of the subjects worldwide comparatively. Another reason could be the presence of one risk allele in heterozygous condition (CT) and variant allele carrier genotype (CT + TT). This result is in line with the risk for acute pyelonephritis; a form of severe urinary tract infection showing risk at variant allele carrier level (CT + TT) and CXCR2(+1208)C/T gene [17]. To analyze the synergistic effect of chemokine SNPs and ESRD risk, we conducted gene–gene interaction. Five combinations were constructed for chemokines taken for present study viz. CXCL12G801A–CCL2I/D, CXCL12G801A–CCL2A2518, CXCR2(+1208)C/T–CCL2I/D, CXCR2(+1208) C/T–CCL2A2518G & CXCR2(+1208)C/T–CXCL12G801A. Combination of CXCR2(+1208)C/T–CXCL12G801A only showed statistically significant results viz. CT–GA combination. The ability of haplotypes to further substantiate the detection of association over the single locus analysis incited us to analyze haplotypes and their association with ESRD susceptibility. Thus we analyzed the haplotype combination of CCL2 gene with their two polymorphic sites and ESRD risk. However, no significant association was observed. The strength of our study was probably reporting for the first time CXCL12G801A polymorphism and its association with end stage renal disease. There were some limitations though like small sample size; that could have lead to a relatively lower statistical power, particularly in subgroups; nevertheless, it reflected some important findings in a pilot study. Therefore, analysis in combination of genetic variations using advanced technology such as microarrays or real time PCR may lead to a better understanding of genetic role in end stage renal disease.
(1.02–2.36) (0.40–2.10) (1.04–3.68) (0.90–1.65)
(1.08–2.51) (0.45–2.30) (1.02–2.61) (0.94–1.75)
Age–gender–smoking adjusted odds ratio.
for gene–gene interaction showed any significant associations with ESRD in the present study (Table 3).
4. Discussion Genetic susceptibility is considered an important determining factor for the appearance and/or progression of ESRD and its complications may help us to look further into the disease pathogenesis and its underlying causes, as well as to predict the predisposition to developing the disease in order to identify the at-risk population. In our study, we have found strong suggestion of associations between ESRD and SNPs of CCL2I/D (rs3917887), CXCL12G801A (rs1801157) and CXCR2(+1208)C/T (rs1801032), associated with different immune and inflammatory processes. All of these sites involve genes related to inflammation and immune response pathways. The variant (D/D) in CCL2I/D in the present study demonstrated reduced risk for ESRD as insertion/deletion sequence was located in Intron 1 region of CCL2 gene that affects the transcriptional activity of
Fig. 1. Haplotypic combination of CCL2 gene polymorphisms in ESRD patients and healthy controls.
Please cite this article as: Singh V, et al, Association of chemokine gene variants with end stage renal disease in North Indian population, Transpl Immunol (2013), http://dx.doi.org/10.1016/j.trim.2013.04.004
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V. Singh et al. / Transplant Immunology xxx (2013) xxx–xxx
5. Conclusion Our results suggested CXCL12G801A and CXCR2(+1208)C/T to be associated with ESRD risk whereas CCL2I/D gene polymorphism showed reduced risk and CCL2A2518G gene polymorphism was unaffected. Acknowledgment We are thankful to the urologists in our department for providing ESRD samples and relevant clinical information of patients. Vibha Singh is thankful to ICMR for providing Senior research fellowship (SRF) and Praveen Kumar Jaiswal is thankful to UGC for providing Junior research fellowship (JRF). References [1] Köttgen A. Genome-wide association studies in nephrology research. Am J Kidney Dis 2010;56:743–58. [2] Gansevoort RT, Matsushita K, Van der Velde M, Astor BC, Woodward M, Levey AS, et al. Lower estimated GFR and higher albuminuria are associated with adverse kidney outcomes. A collaborative meta-analysis of general and high-risk population cohorts. Kidney Int 2011;80:93–104. [3] Reich HN, Gladman DD, Urowitz MB, Bargman JM, Hladunewich MA, Lou W, et al. Persistent proteinuria and dyslipidemia increase the risk of progressive chronic kidney disease in lupus erythematosus. Kidney Int 2011;9:914–20. [4] Rao M, Wong C, Kanetsky P, Girndt M, Stenvinkel P, Reilly M, et al. Cytokine gene polymorphism and progression of renal and cardiovascular diseases. Kidney Int 2007;72:549–56. [5] Kottgen A, Pattaro C, Boger CA, Fuchsberger C, Olden M, Glazer NL, et al. New loci associated with kidney function and chronic kidney disease. Nat Genet 2010;42: 376–84.
[6] Chambers JC, Zhang W, Lord GM, van der Harst P, Lawlor DA, Sehmi JS, et al. Genetic loci influencing kidney function and chronic kidney disease. Nat Genet 2010;42:373–5. [7] Rovin BH, Lu L, Saxena R. A novel polymorphism in the MCP-1 gene regulatory region that influences MCP-1 expression. Biochem Biophys Res Commun 1999;259: 3448. [8] Apostolakis S, Baritaki S, Kochiadakis GE, Igoumenidis NE, Panutsopulos D, Spandidos DA. Effects of polymorphisms in chemokine ligands and receptors on susceptibility to coronary artery disease. Thromb Res 2007;119:63–71. [9] Viana AC, Kim YJ, Curtis KM, Renzi R, Orrico SR, Cirelli JA, et al. Association of haplotypes in the CXCR2 gene with periodontitis in a Brazilian population. DNA Cell Biol 2010;29:191–200. [10] Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988;16:1215. [11] Ahluwalia TS, Khullar M, Ahuja M, Kohli HS, Bhansali A, Mohan V, et al. Common variants of inflammatory cytokine genes are associated with risk of nephropathy in type 2 diabetes among Asian Indians. PLoS One 2009;4:51–68. [12] Hirata H, Hinoda Y, Kikuno N, Kawamoto K, Dahiya AV, Suehiro Y, et al. CXCL12 G801A polymorphism is a risk factor for sporadic prostate cancer susceptibility. Clin Cancer Res 2007;13:5056–62. [13] Snoussi K, Mahfoudh W, Bouaouina N, Fekih M, Khairi H, Helal AN, et al. Combined effects of IL-8 and CXCR2 gene polymorphisms on breast cancer susceptibility and aggressiveness. BMC Cancer 2010;10:283. [14] Steinmetz OM, Panzer U, Harendza S, Mertens PR, Ostendorf T, Floege J, et al. No association of the −2518 MCP-1 A/G promoter polymorphism with incidence and clinical course of IgA nephropathy. Nephrol Dial Transplant 2004;19:596–601. [15] Warchoł T, Lianeri M, Łacki JK, Jagodziński PP. SDF1–3′ G801A polymorphisms in Polish patients with systemic lupus erythematosus. Mol Biol Rep 2010;7:3121–5. [16] Mestas J, Burdick MD, Reckamp K, Pantuck A, Figlin RA, Streiter RM. The role of CXCR2/CXCR2 ligand biological axis in renal cell carcinoma. J Immunol 2005;175: 5351–7. [17] Javor J, Bucova M, Cervenova O, Kralinsky K, Sadova E, Suchankova M, et al. Genetic variations of interleukin-8, CXCR1 and CXCR2 genes and risk of acute pyelonephritis in children. Int J Immunogenet 2012;39:338–45.
Please cite this article as: Singh V, et al, Association of chemokine gene variants with end stage renal disease in North Indian population, Transpl Immunol (2013), http://dx.doi.org/10.1016/j.trim.2013.04.004
Contents lists available at SciVerse ScienceDirect
Transplant Immunology journal homepage: www.elsevier.com/locate/trim
Association of chemokine gene variants with end stage renal disease in North Indian population Vibha Singh, Praveen Kumar Jaiswal, Poonam Tiwari, Rakesh Kapoor, Rama Devi Mittal ⁎ Department of Urology and Renal Transplantation, Sanjay Gandhi Post Graduate Institute of Medical Science, Raebareli Road, Lucknow 226014, Uttar Pradesh, India
a r t i c l e
i n f o
Article history: Received 11 January 2013 Received in revised form 12 April 2013 Accepted 15 April 2013 Available online xxxx Keywords: Chemokine gene polymorphism ESRD PCR–RFLP ARMS-PCR Haplotype
a b s t r a c t Background & aim: The progression rate of chronic kidney disease (CKD) to its end-stage renal disease (ESRD), and the development and severity of various complications, are indirectly influenced by genetic and epigenetic factors. Chemokines are small inducible pro-inflammatory cytokines, which are implicated in many biological processes like migration of leukocytes, angiogenesis, tumor growth and metastasis. We tested association of four single nucleotide polymorphisms (SNPs) viz. CCL2I/D, CCL2A2518G, CXCL12G801A and CXCR2(+ 1208)C/T among individuals with ESRD (end stage renal disease) and normal healthy controls from North Indian population. Materials and method: CCL2I/D, CCL2A2518G, CXCL12G801A and CXCR2(+ 1208)C/T were genotyped in blood samples of hospital-based case–control study comprising of 200 ESRD cases and 200 healthy controls using Restriction Fragment Length Polymorphism (RFLP) and ARMS (Amplification Refractory Mutation Specific) PCR methodology. Results: A significant association was found in CXCL12G801A with ESRD risk. In case of CXCL12G801A polymorphism heterozygous (GA) genotype showed significant risk (p = 0.039; OR = 1.55) whereas A allele carrier (GA + AA) also exhibited risk with ESRD (p = 0.045, OR = 1.59). In CXCR2(+1208)C/T polymorphism, the heterozygous genotype (CT) showed significant risk for ESRD (p = 0.028; OR = 1.65) and combination of CT + TT demonstrated significant high risk for ESRD (p = 0.036; OR = 1.52). In case of CCL2I/D, the variant genotype (D/D) showed reduced risk for ESRD patients. Upon analyzing the gene–gene interaction between CXCR2 and CXCL12, the combination (CT–GA) showed 2.65 fold risk for ESRD (p = 0.018). Conclusion: Our results indicated that polymorphism in CXCL12G801A and CXCR2(+ 1208)C/T showed high risk for ESRD in North Indian population. However, CCL2I/D showed reduced risk and CCL2A2518G exhibited no association. Study with large sample size and diverse ethnicity is required to validate these observations. © 2013 Published by Elsevier B.V.
1. Introduction Renal failure refers to temporary or permanent damage to the kidneys that results in loss of normal kidney function. The prevalence of chronic kidney disease (CKD) and end-stage renal disease (ESRD) is growing worldwide and therefore, constitutes a serious public health problem, which causes substantial morbidity and mortality [1]. Chronic kidney disease progression has been linked to pro-inflammatory chemokine and markers of inflammation. These markers are also elevated in end-stage renal disease (ESRD). Persistent proteinuria, dyslipidemia, hypertension and smoking are considered established conventional risk factors along with a low estimated glomerular filtration rate (eGFR) and albuminuria which are known risk factors for end-stage renal disease (ESRD) [2,3]. However, it has been assumed that oxidative stress, inflammation and immune processes, may also be important contributors to ⁎ Corresponding author. Tel.: +91 522 2668004 8x4116; fax: +91 522 2668017. E-mail addresses: [email protected], [email protected] (R.D. Mittal).
the pathogenesis of cardiovascular disease as well as progression to ESRD [4]. The inflammatory response involved in renal damage produces a release of pro-inflammatory cytokines and chemokines, which cause an increased inflow of leukocytes, intensification of interstitial nephritis and progressive fibrosis. Alternatively, genetic susceptibility is also considered an important determining factor for the appearance and/or progression of ESRD and its complications [5,6]. Kidney cells produce MCP-1 (monocyte chemoattractant protein-1) in response to a variety of pro-inflammatory stimuli and predictably, its expression has been identified in kidney diseases which involve significant inflammation. A biallelic genetic variation (A/G) in the MCP-1 gene distal regulatory region at position − 2518 affects the level of MCP-1 expression in response to an inflammatory stimulus [7]. CXCL12 has been found to be involved in cell proliferation, cell migration, and cell invasion. The CXCL12 also known as stromal cell derived factor (SDF-1) has been revealed that a single nucleotide polymorphism (SNP), in this gene may affect the expression of SDF-1 chemokine [8].
0966-3274/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.trim.2013.04.004
Please cite this article as: Singh V, et al, Association of chemokine gene variants with end stage renal disease in North Indian population, Transpl Immunol (2013), http://dx.doi.org/10.1016/j.trim.2013.04.004
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V. Singh et al. / Transplant Immunology xxx (2013) xxx–xxx
CXCR-2 is a receptor of interleukin-8, which is involved in acute and chronic inflammatory processes. Polymorphisms in the CXCR2(+1208) C/T gene have been associated with chronic inflammatory conditions [9]. We selected these polymorphic markers as they have been reported to be functional and associated with altered immune responses. CCL2, also known as monocyte chemoattractant protein 1 (MCP-1) is the strongest known chemotactic factor for monocytes. Polymorphism in CCL2 gene at distal regulatory region at position −2518 affects the level of CCL2 expression in response to an inflammatory stimulus [7]. Exact mechanism of CCL2I/D gene polymorphism affecting the expression of gene is still unclear. CXCL12 gene is associated with cell proliferation, migration and invasion. Single nucleotide polymorphism (SNP) in this gene at CXCL12G801A may affect the expression of CXCL12 chemokine [8]. CXCR-2 gene is a receptor of interleukin 8 and is involved in acute and chronic inflammatory process. Polymorphisms at CXCR2(+1208)C/T gene have been associated with chronic inflammatory conditions [9]. Our preliminary data suggest that four polymorphisms showed an association with ESRD protection or development, which could help to predict the risk of developing ESRD. The aim of the present study was to investigate whether single nucleotide polymorphisms (SNPs) of CCL2I/D (rs3917887), CCL22518A/G (rs1024611), CXCL12G801A (rs1801157) and CXCR2(+1208)C/T (rs1801032), associated with different immune and inflammatory process could also be associated with development of ESRD in North Indian populations. 2. Materials & method 2.1. Patients and clinical data The demographic characteristics of the subjects are represented in Table 1. Healthy controls (n = 200), (mean age 33.8 ± 12.8, male = 192 and female = 8) for the study were recruited from Northern India with similar ethnicity having no history of hypertension, diabetes, renal failure, vascular diseases, stroke, and/or cardiomyopathy. They had no recognizable autoimmune disease at the time of assessment. A total number of 200 ESRD patients on hemodialysis (mean age 33.9 ± 11.3; male = 168 and female = 32) were recruited from Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India. Patients were selected having >4.0 mg/dl serum creatinine that had been dialyzing for at least 3 months with no evidence of active inflammation. Stringent diagnostic criteria were used for diagnosing ESRD. Data collected included basic demographic information, pattern, onset of disease, and use of medications. The Institutional Review Board and ethical committee approved the protocol, and informed consent was obtained from the patients and the controls participating in the study. 2.2. DNA extraction and genotyping Blood samples were collected from patients with ESRD and healthy controls in ethylenediamine tetraacetic acid (EDTA) anticoagulant coded vials. DNA was extracted from peripheral blood lymphocytes using ‘salting-out’ method [10]. Polymorphisms in chemokines CCL2I/D, Table 1 Demographic details of the ESRD patients and healthy controls. Variables
Controls (n = 200)
ESRD patients (n = 200)
p value
Age (mean ± SD) Gender (male/female)
33.8 ± 12.8 192/8
33.9 ± 11.3 168/32
0.103 0.348
Causes of ESRD Chronic glomerulonephritis (CGN) Chronic kidney disease (CKD) Diabetic nephropathy (DN) Diffuse global sclerosis (DGS)
– – – –
115 (57.5) 58 (29.0) 23 (11.5) 4 (2.0)
CCL2A2518G and CXCL12G801A and CXCR2(+1208)C/T genes were analyzed using PCR–RFLP (Polymerase Chain Reaction–Restriction Fragment Length Polymorphism) and ARMS-PCR (Amplification Refractory Mutation System-Polymerase Chain Reaction) method. Primer detail and PCR conditions of CCL2I/D, CCL2A2518G [11], CXCL12G801A [12] and CXCR2(+1208)C/T [13] were respectively. The genotyping for all the gene taken in present study was done on 15% Poly Acrylamide Gel Electrophoresis (PAGE) using molecular weight markers and further visualized after staining with ethidium bromide. Positive and negative controls were used in each genotyping assay, and 10% of the samples were randomly selected and run in duplicates with 100% concordance. The results were reproducible with no discrepancy in genotyping. 2.3. Statistical analysis The sample size was calculated using Quanto software, version 1.0 (available from: http://hydra.usc.edu/gxe) with input of following variables: case–control study design, significance level (alpha) > 0.05 (2 sided), model of inheritance was log additive, allele frequency was 0.28, and the genetic effect for odds ratio (OR) was 1.50 or greater. The present study achieved 80% of the statistical power. The goodness-of-fit chi square test was used to analyze any deviation from the Hardy– Weinberg Equilibrium in controls. A binary logistic regression model was used to estimate the risk as the OR at the 95% confidence interval. Haplotypes of each individual consisting of single nucleotide polymorphisms (SNPs) in chemokines were constructed, and the maximal likelihood haplotype frequencies were estimated using the expectation– maximization algorithm using the SNP analyzer ver. 1.2 program. Bonferroni's correction was applied in the case of multiple comparisons using the formula pc = p × n (pc represents corrected value where n is the number of comparisons performed). The statistical analysis was done using the Statistical Package for Social Sciences software, version 15.0 (SPSS, Chicago, IL), and p b 0.05 was considered statistically significant. 3. Results 3.1. Chemokine gene polymorphisms and ESRD The present study achieved 80% of the statistical power. The genotype and allele frequencies of chemokine gene polymorphisms in healthy individuals (controls) and ESRD patients are presented in (Table 2). The genotype frequencies of controls were in Hardy–Weinberg Equilibrium (HWE). We found significant association of CXCL12G801A and CXCR2(+1208)C/T polymorphism with ESRD risk whereas CCLI/D polymorphism showed reduced risk with ESRD. CCL2A2518G gene polymorphism showed no association with ESRD. In CXCR2(+1208)C/T polymorphism, the heterozygous genotype (CT) showed significant risk for ESRD (p = 0.020 OR = 1.65; 95% CI; 1.08–2.51). Combining heterozygous and variant genotype, (CT + TT) exhibited marginal risk for ESRD (p = 0.036, OR = 1.52; 95% CI; 1.02–2.61). In CXCL12G801A polymorphism the heterozygous genotype (GA) showed significant risk for ESRD (p = 0.039, OR = 1.55; 95% CI; 1.02–2.36). Combining heterozygous and variant genotype (GA + AA) demonstrated high risk for ESRD (p = 0.045, OR = 1.59; 95% CI; 1.04–3.68) However, at allelic level no significant association of any chemokine genes was observed with ESRD risk. 3.2. Association of CCL2I/D–CCL2A2518G haplotypes with ESRD risk Recent studies have demonstrated that haplotype analysis may be more affirmative in predicting the disease association compared with an analysis of a single polymorphism, as individual polymorphism is likely to confer modest effects to the risk of ESRD. We, therefore, examined the effects of CCL2 gene polymorphisms by constructing haplotype sets taking combination IA as a reference. However there was no significant association observed in case of any set of haplotype combinations with ESRD in North Indian population (Fig. 1). 3.3. Gene–gene interaction of chemokine gene polymorphism To analyze the combined effect of chemokine SNPs taken in the present study, we conducted gene–gene interaction. Five combinations were constructed for chemokines taken for the present study viz. CXCL12G801A–CCL2I/D, CXCL12G801A–CCL2A2518, CXCR2(+1208) C/T–CCL2I/D, CXCR2(+1208)C/T–CCL2A2518G & CXCR2(+1208)C/T–CXCL12G801A. Combination of CXCR2(+1208)C/T–CXCL12G801A only showed statistically significant results viz. CT–GA combination. After applying the Bonferroni correction for multiple variable CT–GA combination remains significant for ESRD risk (pc = 0.018). None of the other combinations
Please cite this article as: Singh V, et al, Association of chemokine gene variants with end stage renal disease in North Indian population, Transpl Immunol (2013), http://dx.doi.org/10.1016/j.trim.2013.04.004
V. Singh et al. / Transplant Immunology xxx (2013) xxx–xxx Table 2 Genotypic frequency of CCL2I/D, CCL22518A/G, CXCL12G801A and CXCR2(+1208)C/T in ESRD patients and control.
Combinations
Ref 0.703 0.035 0.762 Ref 0.203
Ref 1.08 0.45 0.94 Ref 0.83
(47.0) (44.0) (9.0) (53.0) (69.0) (31.0)
Ref 0.185 0.339 0.190 Ref 0.202
Ref 0.75 0.71 0.77 Ref 0.83
CXCR2(+1208)C/T–CXCL12G801A CC/GG 55 (27.5) CC/GA 48 (24.0) CC/AA 9 (4.5) CT/GG 39 (19.5) CT/GA 28 (14.0) CT/AA 6 (3.0) TT/GG 5 (2.5) TT/GA 9 (4.5) TT/AA 1 (0.5)
81 106 13 119 268 132
(40.5) (53.0) (6.5) (62.5) (67.0) (33.0)
Ref 0.039 0.866 0.045 Ref 0.194
Ref 1.55 0.96 1.59 Ref 1.22
91 95 14 109 277 123
(45.5) (47.5) (7.0) (54.5) (69.3) (30.7)
Ref 0.020 0.955 0.036 Ref 0.117
Ref 1.65 1.02 1.52 Ref 1.28
p-Value
CCL2I/D (rs3917887) I/I 84 (42.0) I/D 90 (45.0) D/D 26 (13.0) I/D + D/D 116 (58.0) I allele 258 (64.5) D allele 142 (35.5)
87 101 12 113 275 125
(43.5) (50.5) (6.0) (56.5) (68.8) (31.3)
CCL22518A/G (rs1024611) AA 81 (40.5) AG 97 (48.5) GG 22 (11.0) AG + GG 119 (59.5) A allele 259 (64.8) G allele 141 (35.3)
94 88 18 106 276 124
CXCL12G801A (rs1801157) GG 101 (50.5) GA 83 (41.5) AA 16 (8.0) GG + GA 99 (49.5) G allele 285 (71.3) A allele 115 (28.8) CXCR2(+1208)C/T (rs1801032) CC 112 (56.0) CT 73 (36.5) TT 15 (7.5) CT + TT 88 (44.0) C allele 297 (74.2) T allele 103 (25.8) a
Table 3 Gene–gene interaction of CXCR2(+1208)C/T–CXCL12G801A gene polymorphism in ESRD patients and healthy controls.
ORa (95% CI)
Cases n (%) (n = 200)
Controls n (%) (n = 200)
(0.72–1.64) (0.21–0.95) (0.63–1.39) (0.62–1.12)
3
Controls N (%)
Patients N (%)
OR (95%CI)
p value
37 (18.5) 48 (24.0) 6 (3.0) 38 (19.0) 50 (25.0) 7 (3.5) 6 (3.0) 8 (4.0) 0 (0.0)
Ref 1.49 0.99 1.45 2.65 1.73 1.78 1.32 NC
Ref 0.179 0.987 0.235 0.002a 0.355 0.367 0.599 NC
(0.84–2.69) (0.33–3.02) (0.78–2.67) (1.42–4.95) (0.54–5.57) (0.50–6.27) (0.47–3.73)
(0.49–1.15) (0.35–1.43) (0.52–1.14)
NC = Not calculated. a pc = 0.018, after Bonferroni correction for multiple variables.
(0.61–1.11)
gene. Whereas Ahluwalia et al. reported a risk in diabetic nephropathy comparatively [11]. While the other polymorphic site CCL2A2518G of CCL2 taken in the present study showed no significant association at genotypic or allelic level with ESRD. This was complimented by the observations of Steinmetz et al. in IgA nephropathy [14]. In case of CXCL12G801A, heterozygous GA genotype and the variant allele carrier i.e. GA + AA showed 1.6 fold increased risk for ESRD in our population. To the best of our knowledge this is probably the first study involving this polymorphism being reported for association with end stage renal disease. Our observations matched the study of Hirata et al. which showed that GA + AA genotype was significantly associated with prostate cancer and that of Warchoł et al. who found significant association of heterozygous G/A with systemic lupus erythematosus patients respectively [12,15]. Single nucleotide polymorphism at CXCR2(+1208)C/T which is located in the non-coding region of CXCR2 gene might provide valuable information for the pathogenesis and the susceptibility to chronic inflammatory disease. Some studies have reported that CXCR2 was over expressed in renal, prostate, pancreatic, colon, nasopharyngeal and gastric cancers [16]. The heterozygous genotype (CT) of CXCR2(+1208)C/T gene showed significant 1.65 fold risk along with the variant T allele carrier (CT + TT) showing 1.52 fold risk with ESRD risk in the present study. The reasons could be probably due to variation in ethnicity of the subjects worldwide comparatively. Another reason could be the presence of one risk allele in heterozygous condition (CT) and variant allele carrier genotype (CT + TT). This result is in line with the risk for acute pyelonephritis; a form of severe urinary tract infection showing risk at variant allele carrier level (CT + TT) and CXCR2(+1208)C/T gene [17]. To analyze the synergistic effect of chemokine SNPs and ESRD risk, we conducted gene–gene interaction. Five combinations were constructed for chemokines taken for present study viz. CXCL12G801A–CCL2I/D, CXCL12G801A–CCL2A2518, CXCR2(+1208)C/T–CCL2I/D, CXCR2(+1208) C/T–CCL2A2518G & CXCR2(+1208)C/T–CXCL12G801A. Combination of CXCR2(+1208)C/T–CXCL12G801A only showed statistically significant results viz. CT–GA combination. The ability of haplotypes to further substantiate the detection of association over the single locus analysis incited us to analyze haplotypes and their association with ESRD susceptibility. Thus we analyzed the haplotype combination of CCL2 gene with their two polymorphic sites and ESRD risk. However, no significant association was observed. The strength of our study was probably reporting for the first time CXCL12G801A polymorphism and its association with end stage renal disease. There were some limitations though like small sample size; that could have lead to a relatively lower statistical power, particularly in subgroups; nevertheless, it reflected some important findings in a pilot study. Therefore, analysis in combination of genetic variations using advanced technology such as microarrays or real time PCR may lead to a better understanding of genetic role in end stage renal disease.
(1.02–2.36) (0.40–2.10) (1.04–3.68) (0.90–1.65)
(1.08–2.51) (0.45–2.30) (1.02–2.61) (0.94–1.75)
Age–gender–smoking adjusted odds ratio.
for gene–gene interaction showed any significant associations with ESRD in the present study (Table 3).
4. Discussion Genetic susceptibility is considered an important determining factor for the appearance and/or progression of ESRD and its complications may help us to look further into the disease pathogenesis and its underlying causes, as well as to predict the predisposition to developing the disease in order to identify the at-risk population. In our study, we have found strong suggestion of associations between ESRD and SNPs of CCL2I/D (rs3917887), CXCL12G801A (rs1801157) and CXCR2(+1208)C/T (rs1801032), associated with different immune and inflammatory processes. All of these sites involve genes related to inflammation and immune response pathways. The variant (D/D) in CCL2I/D in the present study demonstrated reduced risk for ESRD as insertion/deletion sequence was located in Intron 1 region of CCL2 gene that affects the transcriptional activity of
Fig. 1. Haplotypic combination of CCL2 gene polymorphisms in ESRD patients and healthy controls.
Please cite this article as: Singh V, et al, Association of chemokine gene variants with end stage renal disease in North Indian population, Transpl Immunol (2013), http://dx.doi.org/10.1016/j.trim.2013.04.004
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V. Singh et al. / Transplant Immunology xxx (2013) xxx–xxx
5. Conclusion Our results suggested CXCL12G801A and CXCR2(+1208)C/T to be associated with ESRD risk whereas CCL2I/D gene polymorphism showed reduced risk and CCL2A2518G gene polymorphism was unaffected. Acknowledgment We are thankful to the urologists in our department for providing ESRD samples and relevant clinical information of patients. Vibha Singh is thankful to ICMR for providing Senior research fellowship (SRF) and Praveen Kumar Jaiswal is thankful to UGC for providing Junior research fellowship (JRF). References [1] Köttgen A. Genome-wide association studies in nephrology research. Am J Kidney Dis 2010;56:743–58. [2] Gansevoort RT, Matsushita K, Van der Velde M, Astor BC, Woodward M, Levey AS, et al. Lower estimated GFR and higher albuminuria are associated with adverse kidney outcomes. A collaborative meta-analysis of general and high-risk population cohorts. Kidney Int 2011;80:93–104. [3] Reich HN, Gladman DD, Urowitz MB, Bargman JM, Hladunewich MA, Lou W, et al. Persistent proteinuria and dyslipidemia increase the risk of progressive chronic kidney disease in lupus erythematosus. Kidney Int 2011;9:914–20. [4] Rao M, Wong C, Kanetsky P, Girndt M, Stenvinkel P, Reilly M, et al. Cytokine gene polymorphism and progression of renal and cardiovascular diseases. Kidney Int 2007;72:549–56. [5] Kottgen A, Pattaro C, Boger CA, Fuchsberger C, Olden M, Glazer NL, et al. New loci associated with kidney function and chronic kidney disease. Nat Genet 2010;42: 376–84.
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Please cite this article as: Singh V, et al, Association of chemokine gene variants with end stage renal disease in North Indian population, Transpl Immunol (2013), http://dx.doi.org/10.1016/j.trim.2013.04.004