Human Immunology 76 (2015) 1–5

Contents lists available at ScienceDirect

www.ashi-hla.org

journal homepage: www.elsevier.com/locate/humimm

Association between interleukin 1 receptor antagonist gene 86-bp VNTR polymorphism and sepsis: A meta-analysis Fang Fang, Jian Pan, Yiping Li, Lixiao Xu, Guanghao Su, Gang Li, Jian Wang ⇑ Institute of Pediatric Research, Children’s Hospital of Soochow University, Suzhou 215003, China

a r t i c l e

i n f o

Article history: Received 10 January 2014 Accepted 3 December 2014 Available online 11 December 2014 Keywords: Interleukin 1 receptor antagonist Polymorphism Sepsis Mortality Meta-analysis

a b s t r a c t Objective: Many studies have focused on the relationship between interleukin 1 receptor antagonist (IL1RN) gene 86-bp VNTR polymorphism and sepsis, but the results remain inconsistent. Thus, a metaanalysis was carried out to derive a more precise estimation of the association between IL1RN 86-bp VNTR polymorphism and risk of sepsis and sepsis-related mortality. Methods: Relevant publications were searched in several widely used databases and six eligible studies were included in the meta-analysis. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to evaluate the strength of the association between IL1RN 86-bp VNTR polymorphism and risk of sepsis and sepsis-related mortality. Results: Significant associations between IL1RN 86-bp VNTR polymorphism and sepsis risk were observed in both overall meta-analysis for L2 versus 22 (OR = 0.75, 95% CI = 0.59–0.94) and severe sepsis subgroup for LL + L2 versus 22 (OR = 0.67, 95% CI = 0.47–0.93). L stands for long alleles containing three to six repeats; 2 stands for short allele containing two repeats. However, no significant sepsis mortality variation was detected for all genetic models. Conclusions: According to the results of our meta-analysis, the IL1RN 86-bp VNTR polymorphism probably associates with sepsis risk but not with sepsis-related mortality. Ó 2014 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.

1. Introduction The interleikin-1 system plays an important role in the protection against various insults such as microbial colonization, infection, and malignant transformation [1]. The interleukin 1 receptor antagonist (IL1RN) gene encodes a protein which belongs to the interleikin-1 family cytokines and functions as a competitive inhibitor in the regulation of interleukin-1-induced proinflammatory activity [1–3]. A variable number of tandem repeats (VNTR) of 86 bp polymorphism (rs2234663) has been reported to locate in intron 2 of the IL1RN gene, with five alleles identified. Those five alleles were short allele VNTR⁄2 (containing two repeats), and long alleles VNTR⁄L (containing three to six repeats) [4]. So far the IL1RN 86-bp VNTR polymorphism has drawn a lot of interest. It is reported that the proinflammatory immune response of individuals homozygous for the IL1RN VNTR⁄2 allele is more prolonged and more severe than those with other IL1RN VNTR genotypes [1], and the influence of the IL1RN VNTR⁄2 allele has been widely studied in a variety of diseases (inflammatory bowel disease,

systemic lupus erythematosus, Graves’ disease, nephropathy in diabetes mellitus, lichen sclerosis, ulcerative colitis, alopecia areata, and psoriasis) [5–12]. In this study, we investigated the association between IL1RN 86-bp VNTR polymorphism and the risk of sepsis and sepsisrelated mortality. Sepsis is a complex disease with dysregulated inflammatory response and high mortality rate. Current knowledge of the pathogenesis of sepsis is limited [13,14]. In the past decade, several studies have focused on the relationship between IL1RN 86bp VNTR polymorphism and sepsis, but the results of those individual studies provided limited information and could not draw a convincible conclusion [15–20]. Therefore, we performed a meta-analysis with a relatively large sample size of 6 eligible studies (1731 cases and 2199 controls in all) to provide a more reliable conclusion of the association between IL1RN 86-bp VNTR polymorphism and the risk of sepsis and sepsis-related mortality. 2. Materials and methods 2.1. Literature search, selection, and data collection

⇑ Corresponding author at: Institute of Pediatric Research, Children’s Hospital of Soochow University, Suzhou 215003, Jiangsu Province, China. E-mail address: [email protected] (J. Wang).

In this study, we searched papers published before Oct 18, 2013 according to the keywords ‘‘interleukin 1 receptor antagonist’’/

http://dx.doi.org/10.1016/j.humimm.2014.12.013 0198-8859/Ó 2014 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.

2

F. Fang et al. / Human Immunology 76 (2015) 1–5

‘‘il1rn’’/‘‘il-1rn’’/‘‘il1ra’’/‘‘il-1ra’’, ‘‘sepsis’’/‘‘septic’’, and ‘‘polymorphism’’ in three widely used databases (PubMed, Web of Science, and EBSCO) independently. The papers obtained were further selected for the meta-analysis and our selection criteria were: (i) full text English-written study, (ii) study providing complete case and control data about the relationship between IL1RN 86-bp VNTR polymorphism and sepsis, (iii) studies sharing the same sample of cases and controls were compared and the most complete study from them was included in our meta-analysis, (iv) studies with control group genotypes in Hardy–Weinberg equilibrium (HWE). HWE was tested by v2 test, and when v2 test reported a P value of more than 0.05, the control group genotypes were consistent with HWE. In this study, two investigators independently collected data from each eligible paper. The data were composed of first author, published year, ethnicity, source of controls, sepsis type, and numbers of cases and controls. Through checking between the two investigators, a final data collection was determined. 2.2. Meta-analysis methods According to the data collected from each eligible paper, we performed meta-analysis to evaluate the relationship between IL1RN 86-bp VNTR polymorphism and the risk of sepsis and sepsis-related mortality. In the meta-analysis, pooled odds ratios (ORs) and 95% confidence intervals (CIs) for dominant, recessive, and codominant genetic models were all calculated by fixed effects model or random effects model. The model chosen was based on the heterogeneity test. For the heterogeneity test, we performed the v2-based Q-test in this study [21]. When Q-test reported a P value of more than 0.10, fixed effects model was used to calculate the pooled ORs [22], otherwise random effects model was used [23]. Publication bias was also tested using the Begg’s funnel plot and the Egger’s test [24]. If the funnel plot was asymmetric and the

Fig. 1. Flow chart of study selection.

Egger’s test reported a P value of less than 0.05, the publication bias probably exists. In this study, we used the software Stata version 12.0 (Stata Corporation, College Station, TX, USA) to carry out the metaanalysis. 3. Results 3.1. Studies and data included in this meta-analysis Through searching and selection, a final list of 6 eligible studies [15–20] was collected for meta-analysis (see Fig. 1). All 6 studies collected were case-control studies with various ethnicities (1 study of Asians, and 5 studies of Caucasians), and source of controls (4 studies of population-based controls, and 2 studies of hospital and population mixed controls). Sepsis was defined as sepsis (1 study), severe sepsis (2 studies), and mixed (3 studies). 2 studies focused on sepsis risk, and 4 studies evaluated both sepsis risk and sepsis-related mortality risk. The control groups of the 6 eligible studies were all in Hardy–Weinberg equilibrium (P > 0.05). The information of these 6 studies and the numbers of cases and controls with different genotypes reported in each study were all presented in Table 1. In total, the 6 eligible studies provided 1731 cases and 2199 controls about the relationship between IL1RN 86-bp VNTR polymorphism and sepsis. 3.2. Overall and subgroup meta-analysis results To evaluate the association between IL1RN 86-bp VNTR polymorphism and sepsis risk, we performed both the overall metaanalysis and the subgroup meta-analysis based on ethnicity and sepsis type according to the 6 eligible studies. The results of the overall meta-analysis provided some evidence of the association between IL1RN 86-bp VNTR polymorphism and sepsis risk (OR = 0.75, 95% CI = 0.59–0.94 for L2 versus 22, see Table 2 and Fig. 2). The subgroup meta-analysis based on sepsis type further showed that IL1RN 86-bp VNTR polymorphism was significantly associated with severe sepsis risk (OR = 0.67, 95% CI = 0.47–0.93 for LL + L2 versus 22, see Table 2 and Fig. 3), while no such significant association was detected in either non-severe sepsis or septic shock subgroup (see Table 2). In the stratified meta-analysis based on ethnicity, no obvious association existed in either Asians or Caucasians (see Table 2). As for sepsis-related mortality risk, 4 studies were involved in the overall meta-analysis. The results did not suggest any association between IL1RN 86-bp VNTR polymorphism and sepsis-related mortality risk for all the genetic models (see Table 2). Further stratified analyses based on ethnicity and sepsis type were not performed because of the insufficient study number. In summary, according to the results of our meta-analysis, the IL1RN 86-bp VNTR polymorphism probably associates with sepsis risk especially severe sepsis risk, with the VNTR⁄2 allele acting as a risk factor. However, the IL1RN 86-bp VNTR polymorphism is not significantly associated with sepsis-related mortality.

Table 1 Studies and data included in this meta-analysis.

a

First author

Published year

Ethinicity

Source of controls

Sepsis type

Sample size (case/control)

Fang Arnalich Ma Balding García-Segarra Solé-Violán

1999 2002 2002 2003 2007 2010

Caucasian Caucasian Asian Caucasian Caucasian Caucasian

Population Population and hospital Population Population Population Population and hospital

Severe sepsis Severe sepsis Sepsis Mixed Mixed Mixed

93/261 78/186 60/60 183/389 165/80 1152/1223

P value for Hardy–Weinberg equilibrium test in each control group.

Cases

Controls

LL/L2/22

LL/L2/22

37/43/13 32/33/13 26/27/7 88/70/25 84/76/5 617/428/107

152/92/17 88/81/17 36/21/3 198/160/31 30/41/9 631/495/97

PHWEa

0.544 0.790 0.978 0.867 0.367 0.995

F. Fang et al. / Human Immunology 76 (2015) 1–5

3

0.006 0.71 (0.27,1.89)b

The results of Begg’s funnel plot (see Supplementary Figs. 1 and 2) and Egger’s test showed no publication bias for both sepsis risk (LL versus 22: P = 0.707, L2 versus 22: P = 0.876, LL + L2 versus 22: P = 0.840, LL versus L2 + 22: P = 0.308) and sepsis-related mortality risk (LL versus 22: P = 0.170, L2 versus 22: P = 0.273, LL + L2 versus 22: P = 0.218, LL versus L2 + 22: P = 0.402). 4. Discussion

c

b

P value for heterogeneity test. If P > 0.1, ORs were calculated using fixed effects model, otherwise the random effects model was used. ORs calculated using random effects model. Results which are statistically significant. If 95% CI does not cross 1.0, the result is statistically significant. a

4 Risk of mortality Overall analysis

148/1320

0.34 (0.05,2.30)b

0.000

0.48 (0.11,2.22)b

0.004

0.44 (0.09,2.10)b

0.001

0.410 0.053 0.165 1.08 (0.92,1.27) 0.84 (0.59,1.19)b 1.24 (0.93,1.66) 3 5 2 Sepsis type Non-severe sepsis Severe sepsis Septic shock

986/1692 433/2139 252/1303

0.89 (0.67,1.19) 0.59 (0.33,1.07)b 1.71 (0.31,9.34)b

0.138 0.062 0.022

0.80 (0.59,1.07) 0.71 (0.49,1.02) 1.20 (0.30,4.89)b

0.281 0.308 0.056

0.85 (0.64,1.12) 0.67 (0.47,0.93)c 1.45 (0.31,6.68)b

0.186 0.143 0.032

– 0.004 0.51 (0.25,1.05) 0.91 (0.66,1.27)b 1 5 Ethnicity Asian Caucasian

60/60 1671/2139

0.31 (0.07,1.31) 0.75 (0.40,1.39)b

– 0.001

0.55 (0.13,2.39) 0.75 (0.49,1.16)b

– 0.075

0.40 (0.10,1.62) 0.75 (0.45,1.25)b

– 0.009

0.002 0.86 (0.62,1.18)b 6 Risk of sepsis Overall analysis

1731/2199

0.68 (0.38,1.22)b

0.002

0.75 (0.59,0.94)c

0.123

0.71 (0.44,1.14)b

0.014

Pa LL versus L2 + 22 LL + L2 versus 22 L2 versus 22 LL versus 22 Sample size (case/control) No. of studies Meta-analysis groups

Table 2 Detailed results of the meta-analysis.

OR (95% CI)

Pa

OR (95% CI)

Pa

OR (95% CI)

Pa

OR (95% CI)

3.3. Publication bias test results

In this study, the results of our overall meta-analysis and stratified analysis based on sepsis type suggest that the IL1RN 86-bp VNTR polymorphism probably associates with sepsis risk especially severe sepsis risk, with the VNTR⁄2 allele acting as a risk factor. This conclusion is supported by the reported potential biological function of IL1RN 86-bp VNTR polymorphism. In 1993, Tarlow et al. identified the IL1RN 86-bp VNTR polymorphism and observed significant VNTR⁄2 allele frequency changes in several inflammatory diseases [4]. In 2002, Arnalich et al. detected significantly lower levels of IL1RN protein produced from peripheral blood mononuclear cells of IL1RN VNTR⁄2 homozygous severe sepsis patients [16]. Since the IL1RN protein plays an important role in the regulation of interleukin-1-induced proinflammatory activity [1–3], it is probably biologically reasonable to speculate that the IL1RN 86-bp VNTR polymorphism may influence sepsis risk through affecting the protein production of IL1RN. In addition, the IL1RN 86-bp VNTR polymorphism is in linkage disequilibrium with some other polymorphisms [25], hence it may not function independently in sepsis development. Instead, combined effects of this polymorphism with other biological and environmental factors probably exist. In the subgroup meta-analysis based on ethnicity, however, no significant association between IL1RN 86-bp VNTR polymorphism and sepsis risk was detected in Asians or Caucasians. In the Asian subgroup, only one study with 60 cases and 60 controls was involved, hence convincible result could not be achieved. In the Caucasian subgroup, there were five studies with 1671 cases and 2139 controls in all. However, due to the heterogeneity in data, pooled ORs and 95% CIs for dominant, recessive, and codominant genetic models were all calculated by random effects model and no significant results were obtained. For mortality risk, the results of our meta-analysis did not suggest any association between IL1RN 86-bp VNTR polymorphism and sepsis-related mortality risk either. In addition, all the results of our meta-analysis should be considered prudently due to the existence of several limitations. One limitation is the insufficient sample size used in our meta-analysis especially in the subgroup analysis based on ethnicity and sepsis type, which may lead to false-positive and false-negative results. For example, the result reported in article García-Segarra et al. [19] differ quite a lot from the rest studies especially in the subgroup analysis based on sepsis type. However, due to the inadequate sample size in this meta-analysis, it is not certain whether such a different result of article García-Segarra et al. would be supported by future studies or it is only an abnormal result. Therefore our meta-analysis result that the IL1RN 86-bp VNTR polymorphism probably associates with severe sepsis risk might be obtained by chance, and its biological implication should be taken cautiously. A second limitation is the lack of case-control data adjustment according to detailed individual information such as age, sex and lifestyle in our meta-analysis. The third limitation is that the exact molecular basis of the association between the IL1RN 86-bp VNTR polymorphism and sepsis is still not clear enough at present and needs further investigation. Hence, in order to achieve a more convincible conclusion, further analysis using larger sample size and adjusted individual data is required, and further experimental research on molecular mechanism should also be performed.

4

F. Fang et al. / Human Immunology 76 (2015) 1–5

Fig. 2. Forest plot for L2 versus 22 of the overall meta-analysis using fixed-effects model. The squares and horizontal lines correspond to the study-specific OR and 95% CI. The area of the squares reflects the weight (inverse of the variance). The diamond represents the summary OR and 95% CI.

Fig. 3. Forest plot for LL + L2 versus 22 of the severe sepsis subgroup meta-analysis using fixed-effects model. The squares and horizontal lines correspond to the study-specific OR and 95% CI. The area of the squares reflects the weight (inverse of the variance). The diamond represents the summary OR and 95% CI.

In conclusion, supported by a meta-analysis with a total of 6 eligible studies (1731 cases and 2199 controls in all), our study indicates that the IL1RN 86-bp VNTR polymorphism probably associates with sepsis risk especially severe sepsis risk, with the VNTR⁄2 allele acting as a risk factor. Although there are some limitations, our meta-analysis can still provide valuable information for studying the relationship between the IL1RN 86-bp VNTR polymorphism and sepsis. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.humimm.2014. 12.013.

References [1] Witkin SS, Gerber S, Ledger WJ. Influence of interleukin-1 receptor antagonist gene polymorphism on disease. Clin Infect Dis 2002;34:204–9. [2] Arend WP, Malyak M, Guthridge CJ, Gabay C. Interleukin-1 receptor antagonist: role in biology. Annu Rev Immunol 1998;16:27–55. [3] Dinarello CA. Biologic basis for interleukin-1 in disease. Blood 1996;87:2095–147. [4] Tarlow JK, Blakemore AI, Lennard A, Solari R, Hughes HN, Steinkasserer A, et al. Polymorphism in human IL-1 receptor antagonist gene intron 2 is caused by variable numbers of an 86-base pair tandem repeat. Hum Genet 1993;91:403–4. [5] Bioque G, Crusius JB, Koutroubakis I, Bouma G, Kostense PJ, Meuwissen SG, et al. Allelic polymorphism in IL-1 and IL-1 receptor antagonist (IL-1Ra) genes in inflammatory bowel disease. Clin Exp Immunol 1995;102:379–83. [6] Blakemore AIF, Tarlow JK, Cork MJ, Gordon C, Emery P, Duff GW. Interleukin-1 receptor antagonist gene polymorphism as a disease severity factor in systemic lupus erythematosus. Arthritis Rheum 1994;37:1380–5.

F. Fang et al. / Human Immunology 76 (2015) 1–5 [7] Blakemore AIF, Watson PF, Weetman AP, Duff GW. Association of Graves’ disease with an allele of the interleukin-1 receptor antagonist gene. J Clin Endocrinol Metab 1995;80:111–5. [8] Blakemore AIF, Cox A, Gonzalez AM, Maskill JK, Hughes ME, Wilson RM, et al. Interleukin-1 receptor antagonist allele (IL1RN⁄2) associated with nephropathy in diabetes mellitus. Hum Genet 1996;97:369–74. [9] Clay FE, Cork MJ, Tarlow JK, Blakemore AIF, Harrington CI, Lewis F, et al. Interleukin 1 receptor antagonist gene polymorphism association with lichen sclerosis. Hum Genet 1994;94:407–10. [10] Mansfield JC, Holden H, Tarlow JK, Di Giovine FS, McDowell TL, Wilson AG, et al. Novel genetic association between ulcerative colitis and the antiinflammatory cytokine interleukin-1 receptor antagonist. Gastroenterology 1994;106:637–42. [11] Tarlow JK, Clay FE, Cork MJ, Blakemore AIF, McDonagh AJG, Messenger AG, et al. Severity of alopecia areata is associated with a polymorphism in the interleukin-1 receptor antagonist gene. J Invest Dermatol 1994;103:387–90. [12] Tarlow JK, Cork MJ, Clay FE, Schmitt-Egenolf M, Crane AM, Stierle C, et al. Association between interleukin-1 receptor antagonist (IL-1ra) gene polymorphism and early and late-onset psoriasis. Br J Dermatol 1997;136:147–8. [13] Stearns-Kurosawa DJ, Osuchowski MF, Valentine C, Kurosawa S, Remick DG. The pathogenesis of sepsis. Annu Rev Pathol 2011;6:19–48. [14] Faix JD. Biomarkers of sepsis. Crit Rev Clin Lab Sci 2013;50:23–36. [15] Fang XM, Schröder S, Hoeft A, Stüber F. Comparison of two polymorphisms of the interleukin-1 gene family: interleukin-1 receptor antagonist polymorphism contributes to susceptibility to severe sepsis. Crit Care Med 1999;27:1330–4.

5

[16] Arnalich F, López-Maderuelo D, Codoceo R, Lopez J, Solis-Garrido LM, Capiscol C, et al. Interleukin-1 receptor antagonist gene polymorphism and mortality in patients with severe sepsis. Clin Exp Immunol 2002;127:331–6. [17] Ma P, Chen D, Pan J, Du B. Genomic polymorphism within interleukin-1 family cytokines influences the outcome of septic patients. Crit Care Med 2002;30:1046–50. [18] Balding J, Healy CM, Livingstone WJ, White B, Mynett-Johnson L, Cafferkey M, et al. Genomic polymorphic profiles in an Irish population with meningococcaemia: is it possible to predict severity and outcome of disease? Genes Immun 2003;4:533–40. [19] García-Segarra G, Espinosa G, Tassies D, Oriola J, Aibar J, Bové A, et al. Increased mortality in septic shock with the 4G/4G genotype of plasminogen activator inhibitor 1 in patients of white descent. Intensive Care Med 2007;33:1354–62. [20] Solé-Violán J, de Castro FR, García-Laorden MI, Blanquer J, Aspa J, Borderías L, et al. Genetic variability in the severity and outcome of community-acquired pneumonia. Respir Med 2010;104:440–7. [21] Lau J, Ioannidis JP, Schmid CH. Quantitative synthesis in systematic reviews. Ann Intern Med 1997;127:820–6. [22] Mantel N, Haenszel W. Statistical aspects of the analysis of data from retrospective studies of disease. J Natl Cancer Inst 1959;22:719–48. [23] DerSimonian R, Laird N. Meta-analysis in clinical trials. Control Clin Trials 1986;7:177–88. [24] Egger M, Davey Smith G, Schneider M, Minder C. Bias in meta-analysis detected by a simple, graphical test. BMJ 1997;315:629–34. [25] Cox A, Camp NJ, Nicklin MJ, di Giovine FS, Duff GW. An analysis of linkage disequilibrium in the interleukin-1 gene cluster, using a novel grouping method for multiallelic markers. Am J Hum Genet 1998;62:1180–8.