Cytokine 67 (2014) 1–6

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Cytokine

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The role of IL-4 gene 70 bp VNTR and ACE gene I/D variants in FamilialMediterranean fever

http://dx.doi.org/10.1016/j.cyto.2014.01.0071043-4666/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Address: Ondokuz Mayis University, Faculty of Medicine,Department of Medical Biology, Section of Medical Genetics, Samsun 55130, Turkey.Tel.: +90 362 3121919/2277; fax: +90 362 4576041.

E-mail addresses: serbulent.yigit@gmail.com (S. Yigit), stural@omu.edu.tr(S. Tural), akintekcan@hotmail.com (A. Tekcan), turtasliyurt@hotmail.com(T. Tasliyurt), ainanir@gmail.com (A. Inanir), gorkemkismali@yahoo.com (G. Kismali).

1 Tel.: +90 362 3121919/2277; fax: +90 362 4576041.

Serbülent Yigit a, Sengul Tural b,⇑, Akın Tekcan b,1, Turker Tasliyurt c, Ahmet Inanir d, Süheyla Uzunkaya c,Gorkem Kismali e

a Gaziosmanpas�a University, Faculty of Medicine, Department of Medical Biology, Tokat, Turkeyb Ondokuz Mayis University, Faculty of Medicine, Department of Medical Biology and Genetics, Section of Medical Genetics, Samsun, Turkeyc Gaziosmanpas�a University, Faculty of Medicine, Department of Internal Medicine, Tokat, Turkeyd Gaziosmanpas�a University, Faculty of Medicine, Department of Physical Medicine and Rehabilitation, Tokat, Turkeye Ankara University, Veterinary Faculty, Department of Biochemistry, Ankara, Turkey

a r t i c l e i n f o

Article history:Received 19 December 2012Received in revised form 4 December 2013Accepted 20 January 2014Available online 22 February 2014

Keywords:ACE geneIL-4 geneFamilial Mediterranean feverPolymorphism

a b s t r a c t

Familial Mediterranean fever (FMF) is characterized by recurrent attacks of fever and inflammation in theperitoneum, synovium, or pleura, accompanied by pain. It is an autosomal recessive disease caused bymutations in the MEFV (MEditerranean FeVer) gene. Patients with similar genotypes exhibit phenotypicdiversity. As a result, the variations in different genes could be responsible for the clinical findings of thisdisease. In previous studies genes encoding Angiotensin-Converting Enzyme (ACE) and IL-4 (Interleukin-4) were found to be associated with rheumatologic and autoimmune diseases. In the present study wehypothesized whether ACE I/D or IL-4 70 bp variable tandem repeats (VNTR) genes are associated withFMF and its clinical findings in Turkish patients. Genomic DNA obtained from 670 persons (339 patientswith FMF and 331 healthy controls) was used in the study. Genotypes for an ACE gene I/D polymorphismand IL-4 gene 70 bp VNTR were determined by polymerase chain reaction with specific primers. To ourknowledge, this is the first study examining ACE gene I/D polymorphism and IL-4 gene 70 bp VNTR poly-morphism in FMF patients. As a result, there was a statistically significant difference between the groupswith respect to genotype distribution (p < 0.001). According to our results, ACE gene DD genotype wasassociated with an increased risk in FMF [p < 0.001; OR (95%): 7.715 (4.503–13.22)]. When we examinedACE genotype frequencies according to the clinical characteristics, we found a statistically significantassociation between DD + ID genotype and fever (p = 0.04). In addition IL-4 gene P1P1 genotype was asso-ciated with FMF (p < 0.001). We propose that D allele or DD genotype of ACE gene and P1 allele or P1P1

genotype of IL-4 gene may be important molecular markers for susceptibility of FMF.� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Familial Mediterranean fever (FMF, MIM No. 249100) is anautosomal recessive auto inflammatory disease, which character-ized by recurrent, self-limiting attacks of fever and serositis [1,2].The episodes of inflammation are mediated by an influx of neutro-phils into serous cavities and are accompanied by an elevation inthe levels of acute-phase inflammatory products and cytokines.

Around 30% of patients with FMF show subclinical inflammationbetween acute attacks [3]. The disease is caused by mutations inthe MEFV gene which encodes a protein called pyrin/marenostrin.This protein is likely to have a down-regulating influence on the re-sponse of neutrophils to inflammatory stimuli [4,5]. The diseaseoccurs most commonly in four populations of eastern Mediterra-nean descent, namely Jews, Armenians, Turks and Arabs [6,7]. Col-chicine has proven effective in preventing the attacks of FMF [8,9].

Many of the genetic and non-genetic risk factors can be relatedto FMF. The correlation of genotype with its phenotype is verycomplex for this disease. Ethnic and environmental factors alsoplay a role in the clinical outcome. In FMF the phenotype is affectedby MEFV mutations, background modifier genes and environmentalfactors in an estimated ratio of about 6:1, 5:1 respectively [10]. Thesame genotypes show different phenotypic features; as well asenvironmental factors; different genetic factors might play a role

Table 1Demographic variables and baseline characteristics of patients and controls.

Individual characteristics Patients (n = 339)(%)/(mean ± SD)

Controls (n = 331)(%)/(mean ± SD)

SexFemale n (%) 188 (55.45%) 204 (61.63%)

2 S. Yigit et al. / Cytokine 67 (2014) 1–6

in the pathogenesis of this disease. We suggest that in these pa-tients, the mutations in other parts of the MEFV gene or other geneshave not been identified yet may be related to this disease. There-fore, we aimed to investigate the ACE and IL-4 genes in FMF be-cause these genes have been particularly related rheumatic andautoimmune diseases.

The local renin-angiotensin system (RAS) in the vessel wallplays a prominent role in the endothelial control of vascular tonusand contributes to inflammatory processes. ACE is the regulatorycomponent of the RAS. ACE (also known as peptidyl dipeptidaseA or kininase II) is encoded by the ACE gene (Gene Bank accessionnumber: NM 000789.2). The ACE gene is located on the long arm ofchromosome 17 and can be expressed in multiple tissues [11]. Thegene contains a polymorphism as a result of insertion (I) or dele-tion (D) of a 287 base pair ALU repeat sequence within intron16; resulting in 3 genotypes: DD and II homozygous and ID hetero-zygous. Plasma ACE levels vary with polymorphism; individualshomozygous for the D allele have the highest levels of enzyme,those homozygous for the insertion allele have the lowest and het-erozygous subjects have an intermediate level [12–16]. ACE con-verts angiotensin I to angiotensin II. The stimulation ofmonocytes with angiotensin II causes the activation of NF-jBand subsequent increase in the TNF-a production. Additionally, itis possible that activation of NF-jB occurs through the up-regula-tion of expressions of MEFV gene [17]. We think that it is essentialto investigate the associations between ACE gene polymorphismsand FMF in order to delineate the pathophysiology of the disease.

Cytokines are signaling molecules contributing to the inflam-matory response and protecting the body from pathogens andother environmental factors [19–21]. The cytokine network is acti-vated during attacks of FMF. Anti-inflammatory cytokines appearto play an important role in the evolution of FMF attacks (suchas IL-6) [22]. Interleukin-4 (IL-4) is a key cytokine that inducesthe activation and differentiation of B cells and involved in thedevelopment of the T helper-2 subset of lymphocytes. IL-4 hasanti-tumor effects, inhibiting induction of nitric oxide synthaseand release of superoxide by macrophages and has numerousanti-inflammatory effects [19–21]. IL-4 also plays a role in chemo-taxis of macrophages, B cells and T cells as well as, formation ofendothelial cell adhesion molecules and hematopoiesis.

Based on these findings we hypothesized that the genotypesof IL-4 and ACE in FMF patients may be a determining factorof pathogenesis. To our knowledge, ACE gene I/D and IL-4 gene70 bp VNTR polymorphism have not yet been investigated inFMF in any ethnic group. This is the first study investigatedthe interactions between ACE and IL-4 gene polymorphismsand FMF.

Male n (%) 151 (44.55%) 127 (38.37%)Mean age, years 31.64 ± 11.95 35.54 ± 11.85Initial symptom age 10.81 ± 8.34 –Diagnosis age 16.81 ± 11.06 –

Table 2The clinical findings of FMF patients.

Clinical characteristics Patients (n = 339)

Fever 276 (81.42%)Colchicine using 177 (52.21%)Colchicine response 173 (97.74%)Abdominal pain 312 (92.03%)Joint pain 242 (71.38%)Chest pain 86 (25.37%)Erythema 62 (18.28%)Amyloidosis 19 (5.60%)Appendicitis operation history 40 (11.79%)Positive family history 136 (40.11%)Mean age of diagnosis 16.81 ± 11.06Mean of first symptom age 10.81 ± 8.33

2. Materials and methods

2.1. Study population

This study included 339 FMF patients and 331 controls re-cruited from the department of physical medicine and rehabilita-tion and department of internal medicine from Gazi Osmanpas�aUniversity (Tokat, Turkey). Informed consent is in accordance withthe study protocol, approved by the ethics committee. The diagno-sis of FMF in these patients was made according to establishedclinical criteria [23]. All patients signed a written consent formafter being informed about the details of the study. The clinicalevaluation was done for FMF in all patients. The controls were se-lected by excluding the diagnosis of FMF. All individuals in the con-trol group were healthy. Data related to age, fever, colchicine usageand response, abdominal pain, joint pain, chest pain, erythema,amyloidosis, positive family history and appendicitis operation

history. Individual features of patients with FMF were summarizedin Tables 1 and 2.

2.2. Genotype determination

DNA was extracted from 2 ml venous blood according to kitprocedure (Sigma, USA) and stored at �20 �C. ACE genotypes weredetermined by polymerase chain reaction (PCR). Reactions wereperformed with 10 pmol of each primer: sense oligo: 50CTGGAGACCACT CCCATC CTT TCT 30 and antisense oligo: 50GAT GTGGCC ATC ACATTC GTC AGAT 30 in a final volume of 50 ll, containing3 mM MgCl2, 50 mM KCl, 10 mM Tris–HCl pH 8.4, 0.1 mg/ml gela-tin, 0.5 mM of each dNTP (Geneun, Germany), 2.5 U Taq DNA poly-merase (Fermentas, Germany). DNA was amplified for 30 cycleswith denaturation at 94 �C for 1 min, annealing at 60 �C for 1 min45 s, and extension at 72 �C for 1 min 30 s using a thermal cycler(Techne, USA). PCR products were analyzed on 2% agarose gelsafter staining with ethidium bromide. In the absence of the287 bp insertion in intron 16 of the ACE gene, this PCR method re-sulted in a 190 bp product (D allele) and in the presence of inser-tion, produced a 490 bp product (I allele). In heterozygoussamples, 2 bands (490 and 190 bp) were detected along with athird fragment of intermediate size. To detect 70 bp VNTR poly-morphism in third intron of IL-4 gene PCR assay was used as de-scribed by Mout et al. [17]. PCR was performed with a 25 llreaction mixture containing 50 ng DNA, 0.8 lM of each primer,200 lM of each dNTP, 2.5 mM MgCl2, 0.5 U Taq polymerase, 109KCl buffer (MBI, Fermentas). Amplification was carried out usingprimers F50AGGCTGAAAGGGGGAAAGC-30, R50-CTG TTCACCT-CAACTGCTCC-30 with initial denaturation at 95 �C for 5 min, 30 cy-cles of denaturation at 94 �C for 30 s, annealing at 58 �C for 45 s,extension at 72 �C for 1 min and final extension at 72 �C for10 min. The PCR products were visualized on 3% agarose gelstained with ethidium bromide. PCR product was of 183 bp for P1

allele and 253 bp for P2 allele. In order to validate the accuracyand reproducibility of this method, each PCR reaction includednegative and positive control. In addition, a second PCR was per-formed to confirm samples which results are not clear. Also, toconfirm the accuracy of the genotyping, repeated analysis was

S. Yigit et al. / Cytokine 67 (2014) 1–6 3

performed on randomly selected samples. No discrepancies werefound.

2.3. Statistical analysis

Analysis of the data was performed using the computer soft-ware SPSS 15.0 (SPSS, Chicago, IL, USA) and OpenEpi Info softwarepackage program. Continuous data were given as mean ± SD (stan-dard deviation) and (min–max). The frequencies of the alleles andgenotypes (Hardy–Weinberg) in patients and controls were com-pared with X2 analysis. Odds ratio (OR) and 95% confidence inter-vals (CIs) were calculated. P value smaller than 0.05 (two-tailed)was regarded as statistically significant. Power analysis was madeby using Minitab 15.0 package program.

3. Results

Demographic variables and baseline characteristics of patientsand controls were given in Table 1. The mean age ± standard devi-ation (SD) was 31.64 ± 11.95 in patients and 35.54 ± 11.85 in con-trol group respectively. There were 188 (55.45%) females and 151(44.55%) males in patient group and in the control group therewere 204 (61.63%) and 127 (38.37%) respectively. Table 2 presentsthe clinical findings of FMF patients. Table 3 shows the distributionof ACE gene I/D genotypes in patients and control groups. Statisti-cally significant difference was found between the groups with re-spect to ACE genotype distribution (p < 0.001). Table 4 shows thedistribution of IL-4 gene 70 bp VNTR genotypes in patients andcontrol groups. There was statistically significant difference be-tween the groups with respect to IL-4 genotype distribution(p < 0.001). A significant difference was found in frequencies ofACE I/D alleles between patients and controls, with FMF patientshaving a higher representation of D and lower representation of Ialleles compared to controls (p < 0.001). The D allele frequencywas 69% and I allele was 31% in the patients; it was 49% and 51%respectively in the control group (Table 3). According to our re-sults, ACE gene DD genotype was associated with an increased riskin FMF [p < 0.001; OR (95%): 7.715 (4.503–13.22)]. We examinedACE genotype frequencies according to the clinical characteristics.

Table 3The distribution of ACE gene I/D genotypes in patient and control groups.

Genotype Patients (n = 339) (%) Controls (n = 331) (%)

DD 152 (44.83) 91 (27.49)II 21 (6.19) 97 (29.30)ID 166 (48.96) 143 (42.18)DD + ID:II 318:21 234:97DD:ID + II 152:187 91:240Allele frequencyD 470 (69) 325 (49)I 208 (31) 337 (51)

The results that are statistically significant are typed in bold.

Table 4The distribution of IL-4 gene 70 bp VNTR polymorphism and allele frequencies between F

Genotype Patients (n = 339) (%) Controls (n = 331) (

P1P1 16 (4.7) 4 (1.2)P1P2 77 (22.7) 21 (6.3)P2P2 246 (72.6) 306 (92.4)P1P1 + P1P2: P2P2 93:246 25:306I P1P2 + P2P2: P1P1 223:16 327:4Allele frequencyP1 109 (16) 29 (4)P2 569 (84) 633 (96)

The results that are statistically significant are typed in bold.

Statistically significant correlation was found between DD + IDgenotype and fever (p = 0.04) (Table 5). Also, it is remarkable thatthere is an association between fever and ACE genotype, whilethere is not correlation between ACE, IL-4 gene variations andabdominal pain, colchicine response that is frequently seen inFMF patients in this study (Table 2 and 5). Additionally, we exam-ined IL-4 genotype frequencies according to the clinical character-istics and any statistically correlation was not found (p > 0.05). Themean IL-4 serum levels were 22.65 ± 20.35 in patients and22.50 ± 20.06 in control group respectively. A significant differencewas not found in IL-4 serum levels between patients and controls(p > 0.05). The ACE and IL-4 gene polymorphisms are required toevaluate together with MEFV polymorphisms. We found that itwas not significant association in the distribution of combinedgenotypes of MEFV, IL-4 and ACE gene polymorphisms in FMF pa-tients (p > 0.05) (Table 6). Also, the distribution of MEFV mutationsin FMF patients and controls were given Table 7. It is seen that therate of MEFV gene M694 V mutation is high in FMF patients.

IL-4 gene P1 allele frequency was 16% and P2 allele was 84% inthe patients; it was 4% and 96% respectively in the control group(Table 3). According to our results, ACE gene DD genotype wasassociated with FMF [p < 0.001; OR (95%):7.715 (4.503–13.22)].IL-4 gene P1 allele is also associated with FMF in Turkishpopulation.

4. Discussion

The estimated prevalence of FMF in Turkey is 1/1000 [24]. FMFhas a wide ranging clinical spectrum. Although FMF has been ac-cepted as a single gene disorder, recently it has been discussedwhether it is a syndrome rather than a disease. Environmental fac-tors and many other genetic factors that influence the course andthe emergence of the disease are considered [7]. FMF is a diseasethat can be seen in combination of several clinical symptoms. Envi-ronmental factors might play a role of this disease, and unidenti-fied gene mutations in different genes may be associated withFMF disease [25–28]. In addition, mutations in different genescould be responsible for the clinical findings of this disease. Theseveral genes have been investigated in FMF patients. Many of

v2 p Value OR (95%CI)

63.75 <0.001 7.715 (4.503–13.22)

61.65 <0.001 6.277 (3.804–10.36)19.25 <0.001 0.466 (0.337–0.644)

12.54 <0.001 2.343 (1.874–2.929)

MF patients and controls.

%) v2 p Value OR (95%CI)

45.63 p < 0.001

45.62 p < 0.001 4.627 (2.885–7.421)12.34 p = 0.0002 5.865 (1.935–17.78)

87.24 p = 0.00001 13.47 (6.764–26.84)

Table 5ACE genotype frequencies according to the clinical characteristics.

Clinical characteristics ACE gene p Value

DD + ID genotype II Genotype

Fever Yes 257 (75.8) 198 (5.6) 0.04No 53 (15.6) 10 (2.9)

Colchicine using Yes 166 (48.9) 11 (3.2) 0.834No 152 (44.8) 10 (2.9)

Colchicine response Yes 162 (47.7) 11 (3.2) 0.717No 5 (1.4) 0 (0)Not using 151 (44.5) 10 (2.9)

Abdominal pain Yes 193 (56.9) 19 (5.6) 0.851No 25 (7.3) 2 (0.5)

Joint pain Yes 228 (67.2) 14 (4.1) 0.806No 90 (26.5) 7 (2.0)

Chest pain Yes 82 (24.1) 4 (1.1) 0.692No 236 (69.6) 17 (5.0)

Erythema Yes 57 (16.8) 5 (1.4) 0.603No 281 (82.8) 16 (0.8)

Amyloidosis Yes 17 (5.0) 2 (0.5) 0.664No 301 (88.7) 19 (5.6)

Positive family history Yes 194 (57.22) 10 (2.9) 0.325No 124 (36.57) 11 (3.24)

Table 6The distribution of combined genotypes of MEFV, IL-4 and ACE gene polymorphisms in FMF patients.

MEFV genotype IL-4 genotype ACE genotype

DD ID II Total

Without mutation P2P2 37 46 7 90P1P2 13 13 2 28P1P1 4 3 1 8Total 54 62 10 126

Homozygote or compound heterozygote P2P2 60 59 7 126P1P2 19 20 1 40P1P1 6 1 0 7Total 85 80 8 173

Heterozygote P2P2 9 19 2 30P1P2 4 4 1 9P1P1 0 1 0 1Total 13 24 3 40

Pearson Chi-Square (p): 0.937.

4 S. Yigit et al. / Cytokine 67 (2014) 1–6

the genetic factors are linked to modifier genes (e.g., SAA – Seroam-yloid A gene; MICA – MHC class I chain-related A gene) and canplay a role in FMF phenotypes [9,25,29–31]. Kobak et al. reportedthat TNF-a/238 and TNF-a/308 promoter polymorphisms were notgenetic risk factors for FMF [32]. Gunesacar et al. suggested thatVEGF gene 936 C/T polymorphism is not associated with FMF sus-ceptibility and its clinical manifestations [33]. Erken et al. showedthat C5aR gene 450 CT genotype tended to associate with the pres-ence of Henoch-Schonlein purpura in FMF [34]. In this study, thedistribution of ACE gene I/D and IL-4 gene 70 bp VNTR polymorphicgenotypes were analyzed in FMF patients in a Turkish populationto assess its possible role in the pathogenesis of FMF.

The present study indicates that the allele and genotype distri-bution of ACE gene polymorphism are significantly different be-tween patients and controls (Table 3). Most of the studies haveshown that no relationship exists between FMF and other genesexcept MEVF. ACE gene found to be associated with rheumatologicand autoimmune diseases, such as rheumatoid arthritis (RA) [18],spondylarthropathies [35], Behçet Disease (BD) [36] and FMF asseen in this study. It is known that ACE converts angiotensin I toangiotensin II and the stimulation of monocytes with angiotensin

II causes the activation of NF-jB. Additionally, it was expressedthat the activation of NF-jB associated with angiotensin II, causesthe up-regulation of expressions of MEFV gene [17]. According tothis information, albeit indirectly, it is seen that the expression ofMEFV gene is affected by the ACE gene I/D polymorphism associ-ated with the formation of angiotensin II and this seems to supportour results. The results of Lapteva et al. are important in terms ofinvestigation of associations between ACE gene polymorphismand FMF. The investigation of the ACE gene polymorphism mayprovide significant benefits in determination of genetic susceptibil-ity to FMF. Also, the ACE gene polymorphisms should be evaluatedtogether MEFV polymorphisms (Table 6). In our study, we no asso-ciation was found between combined genotypes of MEFV, IL-4 andACE gene polymorphisms in FMF patients. The Çalgüneri et al.study involved 50 patients with FMF and suggested no associationexists between the ACE gene polymorphism and development ofFMF. The study stated that more FMF patients should be included[37]. There is no concordance between our results and Çalgüneriet al. results.

The present study indicates that the percentage of IL-4 genepolymorphism allele and the distribution of genotypes are

Table 7The distribution of MEFV polymorphisms in FMF patients and relatives.

Polymorphism status Patients Relatives Total

Normal 126 168 294M694V/M694V 59 11 70M694V/– 30 55 85M694V/M680I 29 10 39M694V/V726A 9 5 14M694V/E148Q 2 4 6M694V/A744S 1 1 2M694V/R761H 2 1 3M680I/M680I 22 5 27M680I/– 26 29 55M680I/V726A 12 4 16M680I/R761H 1 0 1M680I/E148Q 2 3 5V726A/– 4 8 12V726A/P479L 1 1 2V726A/E148Q 1 1 2E148Q/– 4 11 15E148Q/P369S 3 2 5A744S/– 3 2 5K695R/– 1 3 4F479L/– 1 1 2P369S/– 0 4 4R761H/– 0 1 1M694I/E148Q 0 1 1

S. Yigit et al. / Cytokine 67 (2014) 1–6 5

significantly different between patients and controls (Table 4).Association studies of VNTR polymorphism of the IL-4 gene involveautoimmune diseases in various populations, immunologic dis-eases of RA, immune thrombocytopenic purpura, end-stage renaldisease (ESRD), systemic lupus erythematosus (SLE), type-2 diabe-tes, vitiligo, periodontitis and multiple sclerosis [38–43]. A recentstudy showed that IL4 gene promoter polymorphisms might affectsusceptibility to BD, while IL4-590 and IL4-1098 polymorphismsmight be valuable markers to predict the risk for the developmentof BD and increase risk of developing the disease in a Turkishpopulation [44]. Nevertheless there has been no report about therelationship of IL-4 gene intron 3 VNTR polymorphism and FMF.The present study was performed a possible association betweenthe functional IL-4 VNTR polymorphism with FMF susceptibilityand disease progression. It was reported that IL-4 production wassignificantly increased in IL-4 VNTR P1P1 genotype compared toP2P2 and P1P2 genotypes in different populations [45,46]. It wassuggested that the pro-inflammatory activators (such as the Th1cytokines; IFN-g, TNF, and LPS) up-regulated monocyte MEFVmessage levels; anti-inflammatory cytokines (such as the Th2cytokines; IL-4 and IL-10 and TGF-b) down-regulated monocyticMEFV expression [47]. MEFV gene expression is decreased in FMFpatients [48]. The increased IL-4 levels and IL-4 gene P1P1 genotypeexacerbate the pathogenesis of FMF. Also, it is expressed in somestudies that IL-4 cytokines are not effective in FMF inflammation[49]. Difference in IL-4 serum levels between patients and controlsin this study (p > 0.05) was insignificant, while the IL-4 genepolymorphism genotypes differ significantly. It is specified thatthe autoimmune diseases are accompanied by a dysregulation oflymphokine secretion at the level of circulating leukocytes or cellssituated outside of the local inflammatory event [50]. Therefore,the diagnostic measurements of local inflammation may help inthe management of FMF symptoms such as amyloidosis. The localcytokine secretion may be playing an important role in the devel-opment of FMF. Considering this information, IL-4 polymorphismsshould be investigated along with IL-4 levels in FMF patients. Therehas been no report about the relationship of IL-4 gene intron 3VNTR, ACE gene I/D polymorphisms and FMF.

In the present study, we found a significant association betweenthe ACE gene I/D polymorphism and FMF in Turkish population. We

found that DD + ID genotype was associated with fever. In additionIL-4 gene P1P1 genotype was associated with FMF (p < 0.001). Wepropose that D allele or DD genotype of ACE gene and P1 allele orP1P1 genotype of IL-4 gene may be an important molecular markerto predict FMF susceptibility and FMF risk in Turkish population.This is the first study of ACE and IL-4 gene polymorphisms investi-gated in FMF and more investigations are required.

Acknowledgement

We would like to thank Abhisek Gangrade for editing manu-script in terms of English grammar.

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