==== Front Ann Saudi MedAnn Saudi MedAnnals of Saudi Medicine0256-49470975-4466King Faisal Specialist Hospital and Research Centre 2418893610.5144/0256-4947.2013.437asm-5-437Original ArticleInsertion/deletion polymorphism of the ACE gene increased risk of Behcet disease: evidence from a meta-analysis Mandal Raju Kumar aYaday Suraj Singh bPanda Aditya K. cKhattri Sanjay b a College of Medicine, Riyadh, Saudi Arabia b Pharmacology, King George Medical University, Uttar Pradesh, India c Infectious Disease Biology Group, Institute of Life Science, Bhubneswar, IndiaCorrespondence: Dr. Sanjay Khattri, Pharmacology, King George Medical University, Chowk, Lucknow, Uttar Pradesh 226004 India, T:+918948577770, F:+91 522 2257539, drsanjaykhattri@gmail.comSep-Oct 2013 33 5 437 442 Copyright © 2013, Annals of Saudi Medicine2013This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.BACKGROUND AND OBJECTIVES Endothelial dysfunction has a role in the development of the Behcet disease (BD). Local renin–angiotensin system (RAS) plays a crucial role in the endothelial control, and angiotensin-converting enzyme (ACE) is the monitoring component of the RAS. We investigated the relationship between the ACE Ins/Del (I/D) variants and the risk of BD. DESIGN AND SETTINGS A meta-analysis was conducted from all published studies on the associations between the ACE I/D polymorphism and BD. METHODS We systemically searched all published studies from PubMed and EMBASE, and data were quantitatively synthesized. Pooled odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated for allele, homozygous, heterozygous, and combined genetic models. RESULTS Out of 5 eligible studies, 676 healthy controls and 534 BD cases were included in the present meta-analysis. D allele carrier was significantly associated with increased BD risk (D vs I: P=.002; OR=1.321, 95% CI=1.111–1.570). Homozygous mutant DD genotype also revealed 1.5-fold increased risk (DD vs II; P=.004; OR=1.573, 95% CI=1.156–2.141). In addition, the dominant genetic model demonstrated an increased risk of developing BD (DD vs II+ID: P=.001; OR=1.610, 95% CI=1.242–2.087) CONCLUSION The current study suggests that ACE gene polymorphism (Ins/Del) contributes an increased susceptibility to BD. However, larger studies with stratified case control population and biological characterization are needed to validate this finding. ==== Body Behcet disease (BD) is a chronic inflammatory disorder,1 characterized by a wide range of clinical manifestations, including recurrent oral and genital ulcers, skin lesions, and uveitis.2 BD also affects all types and sizes of blood vessels, various joints, the central nervous system, lungs, and gastrointestinal system. Despite of these clinical manifestations, the etiologies of BD remain elusive, and the host genetic factors and environmental features have been attributed to the development of BD.3 Angiotensin-converting enzyme (ACE), also known as peptidyl dipeptidase A or kininase II, encoded by the ACE gene (GenBank NM_000789.2), is located on the long arm of chromosome 17.4 The local renin–angiotensin system (RAS) in the vessel walls plays a crucial role in the endothelial control of vascular tonus and contributes to the inflammatory process via stimulation of cytokine production.5 ACE plays a key role in RAS as well as in kallikrein–kininogen systems by hydrolyzing inactive angiotensin I to active angiotensin II and inactivate the bradykinin (naturally occurring inflammatory peptides).6 Angiotensin II also acts as a potent pro-inflammatory modulator.7 A common 287 base pair Ins/Del (I/D) polymorphism (ALU repeat sequence) has been reported in intron 16 of ACE gene and known to be associated with serum levels of circulating ACE. Subjects having the extra fragment (Ins allele) is associated with lower circulating ACE and tissue activity, and the absence of this fragment (Del allele) is associated with a comparatively higher ACE activity. However, heterozygous (Ins/Del) subjects display an intermediate level of ACE activity.8 Given the functional significance of this genetic variant, it is expected that ACE I/D polymorphism is possibly associated with predisposition to BD. Thus several case–control studies have been conducted to investigate the association between ACE I/D polymorphism and BD in different populations.9–13 But the existing studies have yielded inconsistent or conflicting results. These controversies may be partly ascribed to small sample sizes, various types of genotyping quality, false-positive results, and publication biases. Meta-analysis is a powerful tool for analyzing cumulative data from studies where individual sample sizes are small and have lower statistical power.14 Hence, the quantitative synthesis may provide clearer evidence on the association of such genetic polymorphisms with BD. We therefore performed a meta-analysis of the published studies to clarify this inconsistency and to establish a comprehensive picture of the relationship between ACE I/D polymorphisms and BD. METHODS Published reports search strategy and data extraction We carried out a PubMed (Medline) and EMBASE searches and covered all research papers published with a combination of the following key words: “ACE gene or ACE polymorphisms and Behcet disease” (last updated on Februrary 2013). We evaluated potentially relevant genetic association studies by examining their titles and abstracts, and all published studies matching the eligible criteria were retrieved. Inclusion and exclusion criteria To minimize heterogeneity and facilitate the interpretation of our results, studies included in the current meta-analysis had to meet all the following criteria: (a) evaluation of the ACE I/D and BD risk, (b) use of a case–control design, (c) recruitment of confirmed BD patients and BD free controls, (d) have available genotype frequency in case and control, and (e) have publication in English language. Additionally, when the case–control study was included by more than 1 article using the same case series, we selected the study that included the largest number of individuals. The major reasons for excluding studies were as follows: (a) overlapping data, (b) case-only studies, (c) family-based studies, and (d) review articles. Data extraction and quality assessment For each publication, the methodological quality assessment and data extraction were independently abstracted in duplicate by 2 independent investigators using a standard protocol and data-collection form according to the inclusion criteria listed above to ensure the accuracy of the data. In the case of disagreement on any item of the data, the problem was fully discussed to reach a consensus. Characteristics abstracted from the studies included the name of the first author, the year of publication, the country of origin, the sources of cases and controls, the number of cases and controls, the types of studies, genotype frequencies, and minor allele frequencies in the controls with Hardy–Weinberg (HWE) P value. Statistical analysis We calculated the combined ORs and corresponding 95% CIs to evaluate the association between the ACE I/D polymorphism and BD risk. Heterogeneity in meta-analysis refers to the variation in study outcomes between different studies. Heterogeneity assumption was checked by the chi-square–based Q-test,15 and a P value >.10 indicates a lack of heterogeneity among the studies. Besides this, the pooled OR was calculated by the fixed effects model;16 otherwise, the random-effects model was used.17 In addition, I2 statistics was used to quantify inter-study variability.18 HWE in the control group was assessed via chi-square test and a P value <.05 was considered significant. Publication bias was assessed by the visual inspection of funnel plots in which the standard error of log (OR) of each study was plotted against its log (OR). An asymmetric plot suggests a possible publication bias. Funnel plot asymmetry was also assessed by Egger linear regression test, a linear regression approach to measure funnel plot asymmetry on the natural logarithm scale of the OR. The significance of the intercept was determined by the t-test (P<.05 was considered representative of statistically significant publication bias).19 All statistical analysis for meta-analysis was performed by comprehensive meta-analysis (CMA) V2 software (Biostat, USA). CMA V2 has several advantages over other software available for computing meta-analyses (http://www.meta-analysis.com/pages/comparisons.html). RESULTS Characteristics of published studies A total 13 articles were achieved by published report searches from PubMed (Medline) and EMBASE. All retrieved articles were examined by reading the titles and abstracts, and the full texts for the potentially relevant publications were further checked for their suitability for this meta-analysis (Figure 1). Besides the database search, the reference lists of the retrieved articles were screened for other potential articles. Studies either using ACE polymorphism to predict survival or considering ACE variants as indicators for response to therapy were excluded. Studies investigating the levels of ACE mRNA or protein expression or review article were also excluded. We included only case–control or cohort design studies having frequency of all 3 genotypes. After careful screening and following inclusion and exclusion criteria, 5 eligible original published studies were included in this study (Table 1). The distribution of genotypes in the controls did not deviate from HWE (Table 2). Publication bias Begg funnel plot and Egger test were performed to assess the publication bias in the studies included for meta-analysis. The shape of funnel plots (figures not shown) and Egger test did not show any evidence of publication bias (Table 3). Test of heterogeneity Heterogeneity among studies was assessed by Q test and I2 statistics. Results are shown in Table 3. Heterogeneity was not observed in all the models, thus the fixed effects model was used for calculating OR and 95% CI. Meta-analysis results We pooled all the 5 studies that comprised 676 controls and 534 BD cases and used fixed effects model (based on heterogeneity test) to assess the overall association between the ACE I/D polymorphism and the risk of BD. The variant D allele was significantly associated with the risk of developing BD in terms of frequency with when compared with wild allele (D vs I: P=.002; OR=1.321, 95% CI=1.111–1.570). Similarly homozygous mutant genotype DD significantly altered the risk for the occurrence of BD as compared with the wild-type homozygous II genotype (DD vs II; P=.004; OR=1.573, 95% CI=1.156–2.141). In addition, the analysis of the dominant genetic model indicated 1.6-fold increased risk of developing BD (DD vs II+ID: P=.001; OR=1.610, 95%CI=1.242–2.087) (Figure 2). However, Heterozygous genotype ID (ID vs II: P=.322; OR=1.047, 95% CI=0.772–1.420) and recessive model (TT+CT vs CC: P=.194; OR=0.853, 95%CI=0.623–1.169) did not demonstrate an increased risk of developing BD compared with the II genotype (Figure 3). DISCUSSION It is well known that BD is a multifactorial disease in which multiple genetic factors in combination with environmental factors and infectious agents are probably of importance in determining susceptibility. As a result, the number of candidate genes was investigated to assess the probable association between modulations of BD risk across different populations. The prevalence and incidence of the condition and its constituent manifestations show marked variability among different populations.20 Unbiased epidemiological investigations with large the sample sizes of gene polymorphisms can provide insights into the relationship between candidate genes and diseases. In the present study, we performed a meta-analysis to examine the relationship between the ACE I/D variant in the intron 16 of the ACE gene and the risk of BD. The purpose of this study was to summarize the collected data of 5 studies and explore whether an association exists between the ACE I/D and the occurrence of BD risk. ACE is a membrane-bound enzyme localized in endothelial cells and it is also present in the smooth muscle cells and the adventitial layers of the blood vessels.21 Endothelial cell injury and/or pathologic activation are characteristic features of BD, and this enzyme plays an integral role in the regulatory system responsible for endothelial control.22,23 Increased ACE gene expression and its activity in the vessel wall led to an increased conversion of angiotensin I to angiotensin II. Angiotensin II is a growth factor that plays an active role in vascular inflammation.24,25 Additionally, early response to inflammation is associated with an upregulation of angiotensinogen levels. A high activity of ACE has been observed in inflammatory sites of monocytes/macrophages.26,27 Studies have indicated that ACE inhibition improves endothelial function. 28 The levels of tissue and circulating ACE activities are regulated under tight genetic control.6 Genetic variation in ACE gene plays a major role in determining ACE levels in human T lymphocytes.29 Hence, ACE I/D polymorphism could be a genetic factor for inter-individual differences in susceptibility to BD. In the present study, we combined published results from 5 case–control studies and found an overall increased BD risk for carriers of 1 and 2 variants allele compared with the wild allele (I) and homozygous (II) genotype. When we stratified by dominant and recessive genetic model, the dominant model (DD vs II+ID) had an increased risk of BD (1.6-fold). It has been speculated that ACE I/D polymorphism accounts for approximately one-half of the variance in ACE plasma levels.30 In earlier studies, Lee et al also reported the overall risk of ACE I/D polymorphism with BD risk.31 Chang et al was the first to investigate the association between the incidence of BD and ACE I/D polymorphism. 12 Thereafter, more and more studies were conducted to further assess the association of this polymorphism with BD; however, the results are inconsistent. Our meta-analysis result suggested significant association of ACE I/D polymorphism with the risk of developing BD. However, the etiology of BD is not fully understood and the single genetic variant is usually insufficient to predict the risk of this disease. One important property of this gene polymorphism is that their incidence can vary substantially between different racial or ethnic populations.32 Meta-analysis is a highly cost-effective method that combines the findings of independent similar studies and derives a definitive conclusion.33 In this study we investigated the well-known I/D polymorphism of ACE gene and BD risk and found that this polymorphism is associated with an increased risk of BD. Our study has some advantages; first, it provides an update for this polymorphism and BD risk. Second, our results indicate that this polymorphism is associated with an increased risk of BD. Third, the methodological issues for meta-analysis, such as heterogeneity and publication bias, are well investigated. Some limitations of our meta-analysis should be acknowledged when interpreting the results. First, since our assessment included published studies in English that were indexed by the selected electronic databases for data analysis, it is possible that some relevant studies in other databases or some unpublished studies were not included, and this may have biased our conclusions. Second, we did not study the possible association with BD severity due to unavailability of data in the included studies. In conclusion, we found that I/D polymorphism of ACE gene could be a risk factor for BD. However, the sample size was small in our study, future well-designed studies including larger sample sizes with environmental factors are necessary to validate the role of this association in different populations. Such studies might eventually lead to a better and more comprehensive understanding of the association between the ACE gene polymorphism and BD risk. Figure 1 Flow diagram identifying potential studies for meta-analysis. Figure 2 Forest plot of overall BD risk associated with ACE Ins/Del polymorphism. The squares and horizontal lines correspond to the study-specific OR and 95% CI. The area of the squares reflects the weight of the respective study. Figure 3 Forest plot of overall BD risk associated with ACE Ins/Del polymorphism. The squares and horizontal lines correspond to the study-specific OR and 95% CI. The area of the squares reflects the weight of the respective study. Table 1 Main characteristics of all five studies included in the meta-analysis. First authors Year Country Study design Genotyping method Controls Cases Source of genotyping Yigit et al 2013 Turkey HB PCR 300 266 Blood Dursun et al 2009 Turkey HB PCR 90 73 Blood Ozturk et al 2004 Turkey HB PCR 30 90 Blood Chang et al 2004 Korea HB PCR 106 70 Blood Turgut et al 2005 Turkey HB PCR 150 35 Blood HB-Hospital-based Table 2 Distribution of gene polymorphism of studies included in the meta-analysis. Authors and year Control Case HWE Genotype Minor allele Genotype Minor allele II ID DD MAF II ID DD MAF P value Yigit et al 20138 128 95 77 0.41 112 50 104 0.48 <.001 Dursun et al 20099 23 35 32 0.55 12 29 32 0.63 .26 Ozturk et al 200410 5 16 9 0.56 12 56 22 0.55 .63 Chang et al 200411 42 44 20 0.39 25 28 17 0.44 .17 Turgut et al 200512 19 44 87 0.72 2 7 26 0.84 .008 MAF-Minor allele frequency, HWE-Hardy Weinberg equilibrium Table 3 Statistics to test publication bias and heterogeneity in meta-analysis. Comparisons Egger’s regression analysis Heterogeneity analysis Model used for meta-analysis Intercept 95% Confidence Interval P value Q value Pheterogeneity I2 (%) D vs I 0.19 −3.44 to 3.83 .87 2.88 0.57 <0.0001 Fixed DD vs II 0.21 −1.72 to 2.14 .75 1.27 0.86 <0.0001 Fixed ID vs II 2.23 −0.30 to 4.78 .06 6.28 0.17 36.30 Fixed DD+ID vs II 1.26 −0.09 to 2.63 .05 2.52 0.64 <0.0001 Fixed DD vs II+ID −1.59 −5.10 to 1.91 .24 3.96 0.41 <0.0001 Fixed ==== Refs REFERENCES 1 McGonagle D McDermott MF A proposed classification of the immunological diseases PLoS Med 2006 3 e297 16942393 2 Mendoza-Pinto C García-Carrasco M Jiménez-Hernández M Jiménez Hernández C Riebeling-Navarro C Nava Zavala A Vera Recabarren M Espinosa G Jara Quezada J Cervera R Etiopathogenesis of Behcet’s disease Autoimmun Rev 2010 9 241 2455 19879978 3 Sakane T Takeno M Suzuki N Inaba G Behçet’s disease N Engl J Med 1999 341 1284 1291 10528040 4 Mattei MG Hubert C Alhenc-Gelas F Roeckel N Corvol P Soubrier F Angiotensin I converting enzyme gene is on chromosome 17 Cytogenet Cell Genet 1989 51 1041 5 Henrion D Benessiano J Lévy BI In vitro modulation of a resistance artery diameter by the tissue renin-angiotensin system of a large donor artery Circ Res 1997 80 189 195 9012741 6 Holla L Vašků A Znojil V Sisková L Vácha J Association of 3 gene polymorphisms with atopic diseases J Allergy Clin Immunol 1999 103 702 708 10200023 7 Kranzhofer R Schmidt J Pfeiffer CA Hagl S Libby P Kubler W Angiotensin induces inflammatory activation of human vascular smooth muscle cells Arterioscler Thromb Vasc Biol 1999 19 1623 1629 10397679 8 Kritchevsky SB Nicklas BJ Visser M Simonsick EM Newman AB Harris TB Lange EM Penninx BW Goodpaster BH Satterfield S Colbert LH Rubin SM Pahor M Angiotensin-converting enzyme insertion/deletion genotype, exercise, and physical decline JAMA 2005 294 691 698 16091571 9 Yigit S Tural S Rüstemoglu A Inanir A Gul U Kalkan G Akkanet S Karakuş N Ateş O DD geno-type of ACE gene I/D polymorphism is associated with Behcet disease in a Turkish population Mol Biol Rep 2013 40 365 358 23065219 10 Dursun A Durakbasi-Dursun HG Dursun R Baris S Akduman L Angiotensin-converting enzyme gene and endothelial nitric oxide synthase gene polymorphisms in Behçet’s disease with or without ocular involvement Inflamm Res 2009 58 401 405 19255721 11 Ozturk MA Calgüneri M Kiraz S Ertenli I Onat AM Ureten K Ozbalkan Z Haznedaroğlu IC Angiotensin-converting enzyme gene polymorphism in Behçet’s disease Clin Rheumatol 2004 23 142 146 15045629 12 Chang HK Kim JU Lee SS Yoo DH Lack of association between angiotensin converting enzyme gene polymorphism and Korean Behçet’s disease Ann Rheum Dis 2004 63 106 107 14672905 13 Turgut S Turgut G Atalay EO Atalay A Angiotensin-converting enzyme I/D polymorphism in Behçet’s disease Med Princ Pract 2005 14 213 216 15961928 14 Cohn LD Becker BJ How Meta-Analysis Increases Statistical Power Psychological Methods 2003 8 243 253 14596489 15 Wu R Li B A multiplicative-epistatic model for analyzing interspecific differences in outcrossing species Biometrics 1999 55 355 365 11318188 16 Mantel N Haenszel W Statistical aspects of the analysis of data from retrospective studies of disease J Natl Cancer Inst 1959 22 719 748 13655060 17 DerSimonian R Laird N Meta-analysis in clinical trials Control Clin Trials 1986 7 177 188 3802833 18 Higgins JP Thompson SG Deeks JJ Altman DG Measuring inconsistency in meta-analyses BMJ 2003 327 557 560 12958120 19 Egger M Davey Smith G Schneider M Minder C Bias in meta-analysis detected by a simple, graphical test BMJ 1997 315 629 634 9310563 20 Barnes CG Yazici H Behcet’s syndrome Rheumatology 1999 38 1171 1174 10587540 21 Erdos EG Angiotensin I converting enzyme and the changes in our concepts through the years Hypertension 1990 16 363 370 2170273 22 Ozoran K Duzgun H Gurler A Tutkak H Tokgo ZG Plasma von Willebrand factor, tissue plasminogen activator, plasminogen activator inhibitor, and antithrombin II levels in Behçet’s disease Scan J Rheumatol 1995 24 376 382 23 Kaufman KM Kelly J Gray-McGuire C Asundi N Yu H Reid J Baird T Hutchings D Bruner G Scofield RH Moser K Harley JB Linkage analysis of angiotensin-converting enzyme (ACE) insertion/deletion polymorphism and systemic lupus erythematosus Mol Cell Endocrinol 2001 177 81 85 11377823 24 Muller DN Bohlender J Hilgers KF Dragun D Costerousse O Ménard J Luft FC Vascular angiotensin-converting enzyme expression regulates local angiotensin II Hypertension 1997 29 98 104 9039087 25 Dzau VJ Theodore Cooper Lecture: Tissue angiotensin and pathobiology of vascular disease: a unifying hypothesis Hypertension 2001 37 1047 1052 11304501 26 Brasier AR Li J Mechanisms for inducible control of angiotensinogen gene transcription Hypertension 1996 27 465 475 8613188 27 Diet F Pratt RE Berry GJ Momose N Gibbons GH Dzau VJ Increased accumulation of tissue ACE in human atherosclerotic coronary artery disease Circulation 1996 94 2756 2767 8941100 28 Nakamura M Funakoshi T Yoshida H Arakawa N Suzuki T Hiramori K Endothelium-dependent vasodilation is augmented by angiotensin converting enzyme inhibitors in healthy volunteers J Cardiovasc Pharmacol 1992 20 949 954 1282598 29 Costerousse O Allegrini J Lopez M Alhenc-Gelas F Angiotensin I-converting enzyme in human circulating mononuclear cells: genetic polymorphism of expression in T-lymphocytes Biochem J 1993 290 33 40 8382480 30 Rigat B Hubert C Alhenc-Gelas F Cambien F Corvol P Soubrier F An insertion/deletion polymorphism in the angiotensin I-converting enzyme gene accounting for half the variance of serum enzyme levels J Clin Invest 1990 86 1343 1346 1976655 31 Lee YH Choi SJ Ji JD Song GG Associations between the angiotensin-converting enzyme insertion/deletion polymorphism and susceptibility to vasculitis: a meta-analysis J Renin Angiotensin Aldosterone Syst 2012 13 196 201 22277255 32 Jorde B Wooding SP Genetic variation, classification and race Nat Genet 2004 36 28 33 33 Egger M Smith GD Phillips AN Meta-analysis: principles and procedures BMJ 1997 315 1533 1537 9432252