Departments of 1Dermatology and 3Pathology, College of Medicine, University of Baghdad, Baghdad, Iraq; 2Department of Dermatology, Jena University Hospital, Jena, Germany; 4Department of Dermatology, Baghdad Teaching Hospital, Baghdad, Iraq *Corresponding Author e-mail: amarfaisal1976@yahoo.com الرتابط بني أعداد النسخ اجلينومية املرتفعة لربوتني ديفنسني بيتا DEFB4( 4( وبني داء هبجت عند املرضى العراقيني عمار حميد، �سامح جردات، ب�سام املو�سوي، خليفة ال�رسقي، مازن اإبراهيم، رافع احلياين، جونز نورقوار abstract: Objectives: Behçet’s disease (BD) is an immune-mediated small vessel systemic vasculitis. Human β-defensins are antimicrobial peptides associated with many inflammatory diseases and are encoded by the β-defensin family of multiple-copy genes. However, their role in BD necessitates further investigation. The aim of the present study was to investigate the possible association of BD in its various clinical forms with defensin β-4 (DEFB4) genomic copy numbers. Methods: This case-control study was conducted from January to September 2011 and included 50 control subjects and 27 unrelated Iraqi BD patients registered at Baghdad Teaching Hospital, Bagdad, Iraq. Copy numbers of the DEFB4 gene were determined using the comparative cycle threshold method by duplex real-time polymerase chain reaction technology at the Department of Dermatology of Jena University Hospital, Jena, Germany. Results: DEFB4 genomic copy numbers were significantly higher in the BD group compared to the control group (P = 0.010). However, no statistically significant association was found between copy numbers and clinical variables within the BD group. Conclusion: The DEFB4 copy number polymorphism may be associated with BD; however, it is not associated with different clinical manifestations of the disease. Keywords: Behçet Disease; beta-Defensins; Genetic Polymorphisms; Gene Copy Numbers; Iraq. امللخ�ص: الهدف: داء بهجت هو التهاب مناعي جهازي ي�سيب الأوعية الدموية ال�سغرية. وبيتا ديفن�سني هي بيبتيدات م�سادة للميكروبات ترتبط بكثري من الأمرا�ض اللتهابية، وتكون م�سفرة يف عائلة بيتا ديفين�سني املكونة من مورثات )جينات( متعددة الن�سخ. غري اأن الدور الذي قد تلعبه تلك املورثات يف داء بهجت ما زال يحتاج ملزيد من الدرا�سة. ويهدف هذا البحث لدرا�سة الرتابط املمكن بني داء بهجت ب�سوره الإكلينيكية املختلفة وبني اأعداد الن�سخة اجلينومية DEFB4. الطريقة: اأجريت هذه الدرا�سة ال�ستعادية من يناير اإىل �سبتمرب 2011م و�سملت 50 فردا �سحيحا )جمموعة �سابطة( و27 مري�سا عراقيا )لي�سوا باأقرباء( م�سابني بداء بهجت من امل�سجلني مب�ست�سفى بغداد التعليمي يف العراق. وحددت اأرقام مورثات DEFB4 بتقنية تفاعل الت�سل�سل البلومريزي بق�سم الأمرا�ض اجللدية يف م�ست�سفى اجلامعة بجينا يف اأملانيا. النتائج: وجد اأن اأرقام الن�سخة اجلينومية DEFB4 كانت اأعلى معنويا عند مر�سى داء بهجت عند املقارنة مع املجموعة ال�سابطة )0.010 = P(. غري اأنه مل تكن هنالك اأي فروقات معنوية بني ن�سخ اأرقام مورثات DEFB4 والأعرا�ض الإكلينيكية للم�سابني باملر�ض. اخلال�صة: هنالك ارتباط بني تعدد اأ�سكال اأرقام DEFB4 وبني داء بهجت، رغم عدم وجود اأي ارتباط لها مع الأعرا�ض الإكلينيكية. مفتاح الكلمات: داء بهجت؛ بيتا ديفن�سني؛ تعدد الأ�سكال الوراثية؛ اأعداد ن�سخ املورثات؛ العراق. Association of Higher Defensin β-4 Genomic Copy Numbers with Behçet’s Disease in Iraqi Patients *Ammar F. Hameed,1 Sameh Jaradat,2 Bassam M. Al-Musawi,3 Khalifa Sharquie,1 Mazin J. Ibrahim,3 Raafa K. Hayani,4 Johannes Norgauer2 clinical & basic research Advances in Knowledge - As part of its genetic pathogenesis, Behçet’s disease (BD) may be associated with the presence of a high number of human defensin β-4 (DEFB4) genomic copy numbers, as observed among this sample of unrelated Iraqi BD patients. - The DEFB4 genomic copy numbers in the healthy Iraqi control group were comparable to those observed in other populations. Application to Patient Care - BD has neither a specific diagnostic test nor a targeted course of therapy. As such, exploring the genetic basis of this autoimmune disease could improve understanding of its pathological mechanisms and promote the development of a more accurate diagnostic and therapeutic approach. Sultan Qaboos University Med J, November 2015, Vol. 15, Iss. 4, pp. e491–495, Epub. 23 Nov 15 Submitted 13 Feb 15 Revision Req. 4 Jun 15; Revision Recd. 11 Jun 15 Accepted 21 Jul 15 doi: 10.18295/squmj.2015.15.04.008 Behçet’s disease (bd) is a chronic auto-immune disease characterised by multiple inflammatory lesions of the orogenital mucosa, eyes and skin with additional involvement of other organs in the body.1 BD is prevalent in the Mediterranean region and eastern Asia;2 Iraq is also known to have a relatively high prevalence of the disease.3 Although the precise pathogenesis Association of Higher Defensin β-4 Genomic Copy Numbers with Behçet’s Disease in Iraqi Patients e492 | SQU Medical Journal, November 2015, Volume 15, Issue 4 of BD remains unknown, genetic, immunological and environmental factors have been suggested to contribute to its development.2 A genetic basis for BD is supported by its high prevalence in certain geographic areas, the familial aggregation of cases and a strong association with human leukocyte antigen-B51.4,5 Moreover, single nucleotide polymor- phism studies have revealed associations with several genes, including interferon regulatory factor 1 and tumour necrosis factor-α (TNF-α).6,7 Human β-defensins (hBDs) are antimicrobial peptides expressed by various epithelial tissues. They are encoded by the multiple-copy β-defensin gene family, copies of which are clustered on the short arm of chromosome eight, including the human defensin β-2 gene synthesised by defensin β-4 (DEFB4).8 This β-defensin cluster has a genomic copy number of between two and 12 copies per genome, with a mode of four copies.9 Such variation in genomic copy numbers may play a role in innate immunity; research has shown that hBD-2 has the ability to function as a chemokine that attracts inflammatory cells, such as dendritic cells and T lymphocytes, to the inflamed tissues.10,11 Thus, it is possible that variations in the DEFB4 gene might influence an individual’s susceptibility to immune-mediated diseases. The antimicrobial and proinflammatory nature of these β-defensins has led many researchers to hypothesise that variations in gene dosage may affect the pathogenesis of many immunological diseases.12 Hence, the present study aimed to investigate genomic copy numbers of DEFB4 in Iraqi BD patients in comparison with normal healthy controls. Various clinical manifestations of BD were also studied in association with DEFB4 genomic copy numbers. Methods This case-control study was conducted between January and September 2011 and included all unrelated Iraqi patients registered at the BD Clinic in Baghdad Teaching Hospital, Baghdad, Iraq (n = 27), and randomly selected healthy control subjects (n = 50). All of the BD patients fulfilled the International Study Group criteria for a diagnosis of BD, which requires the presence of recurrent oral ulcers plus at least two of the following symptoms: recurrent genital ulcerations, typical defined eye or skin lesions or a positive skin pathergy test (a skin hypersensitivity reaction to a non-specific physical insult).13,14 Any patient who did not fulfil these criteria was excluded from the study. The control group included healthy individuals who donated their blood to the central blood bank after confirmation that they were free from any symptoms or signs suggestive of BD. Blood was collected from all of the subjects and genomic DNA was extracted from peripheral blood mononuclear cells by a standard phenol-chloroform method at the Department of Pathology in Baghdad Medical College, Baghdad. Samples were then stored at -20 °C and delivered to the Department of Dermato- logy at Jena University Hospital, Jena, Germany. DNA concentrations and quality were then determined with a NanoDrop 1000 spectrophotometer (Nanodrop Technologies Inc., Thermo Fisher Scientific Inc., Wilmington, Delaware, USA) using the following settings: optical density (OD) at 260/280 ~1.8 and OD at 260/230 = 2.0–2.2. Samples were anonymised and the genomic copy number analysis was performed by individuals who were blinded to the relevant clinical information. Diploid DEFB4 genomic copy numbers (GenBank: AF040153.1)15 were determined by a duplex real- time polymerase chain reaction (PCR) technique as described by Jaradat et al.16 Briefly, PCR was perfor- med in 96-Well Microplates (Applied Biosystems, Thermo Fisher Scientific Inc.) in a 7500 Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific Inc.). All assays were performed in quadruplicate. Each plate included templates for genomic DNA, negative controls and NA07048HM calibrator DNA (Coriell Institute for Medical Research, Camden, New Jersey, USA) at four copies per genome. Each well contained a 20 µL reaction mixture including 4 µL of genomic DNA (5 ng/µL), 10 µL of TaqMan® Genotyping Master Mix (Thermo Fisher Scientific Inc.) 1 µL of TaqMan® Copy Number Assay Mix (Applied Biosystems, Thermo Fisher Scientific Inc.), 1 µL of TaqMan® Copy Number RNase P Reference Assay mix (Applied Biosystems, Thermo Fisher Scientific Inc.) and 4 µL of nuclease-free water (Ambion® GmbH, Thermo Fisher Scientific Inc.). The reaction conditions were as follows: 95 °C for 10 minutes for initial denaturation and enzyme activation followed by 40 cycles of 95 °C for 15 seconds and 60 °C for one minute. Cycle threshold values were calculated using the ABI PRISM® 7700 Sequence Detection System, Version 1.3 (Applied Biosystems, Thermo Fisher Scientific Inc.). Relative quantitation was performed using Copy CallerTM Software (Applied Biosystems) to estimate the genomic copy number in each sample according to the comparative cycle threshold method (∆∆Ct). Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS), Version 16.0, (IBM Corp., Chicago, Illinois, USA). Ammar F. Hameed, Sameh Jaradat, Bassam M. Al-Musawi, Khalifa Sharquie, Mazin J. Ibrahim, Raafa K. Hayani and Johannes Norgauer Clinical and Basic Research | e493 Data were presented as the mean estimated copy numbers of DEFB4. Each single genomic copy number estimate was rounded to the nearest integer number. Comparisons between the study groups were performed using an unpaired Student’s t-test to assess the differences in the means ± standard deviation of DEFB4 genomic copy number values. Pearson’s Chi- squared test was used to analyse categorical data. All P values were two-sided and statistical significance was set at P ≤0.05. This study was approved by the Ethical Committee of the Iraqi Board for Medical Specializations (approval #3) according to the principles of the Declaration of Helsinki. All subjects gave informed consent for inclusion in this study. Results A total of 27 BD patients were recruited along with 50 healthy controls. No significant difference in age (P = 0.33) or gender (P = 0.57) was found between the groups [Table 1]. The DEFB4 integer genomic copy number range was 3–7 copies per diploid genome in BD cases compared with 2–6 copies in the control group. The mean was 4.44 ± 1.15 copies versus 3.80 ± 0.92 copies in the BD and control groups, respectively. The DEFB4 genomic copy number was significantly higher in BD cases than in controls (P = 0.01) [Figure 1]. No association was found between the clinical presentations of BD and DEFB4 genomic copy numbers [Table 2]. Discussion High DEFB4 genomic copy number values have been reported in patients with psoriasis and chronic obstructive pulmonary disease.17,18 Previous studies have also demonstrated increased DEFB4 messenger ribonucleic acid expression of these copy number variations on the cellular level and in the mucosal tissues of the colon, upper airways and psoriatic skin.9,18–20 In a large Chinese sample, significantly higher DEFB4 gene numbers were observed among patients with systemic lupus erythematosus and anti-neutrophil cytoplasmic antibody-associated small vasculitis with a mean genomic copy number of 3.98 and 4.05, respectively.21 Jansen et al. presented evidence that both genomic copy number and pro-inflammatory cytokines affect the biological levels of hBD-2.20 BD is a good example of a disease rooted in immunological disturbance mediated by cytokines derived from T-helper lymphocytes, such as TNF-α, which acts as a mediator in the initiation and propaga- tion of BD.22 In this context, TNF-α is a common inducer of DEFB4 expression.18 Possible mechanisms underlying the uncontrolled inflammatory response seen in BD to infection or other environmental triggers are either a high basal level of hBD-2 occurring in Table 2: Defensin β-4 genomic copy numbers by clinical variables among patients with Behçet’s disease in Baghdad, Iraq (N = 27) Clinical variable Present Absent P value* n Mean GCN ± SD n Mean GCN ± SD Genital ulcer 17 4.39 ± 9.94 10 4.18 ± 1.19 0.21 Ocular lesion 7 4.15 ± 0.20 20 4.36 ± 1.17 0.64 Skin lesion 18 4.21 ± 1.03 9 4.46 ± 0.09 0.47 Arthritis 23 4.53 ± 1.09 4 4.46 ± 1.02 0.72 Positive skin pathergy test 20 4.47 ± 1.11 7 3.79 ± 0.10 1.22 GCN = gene copy number; SD = standard deviation. *Determined using the unpaired Student’s t-test. Figure 1: Frequency of defensin β-4 (DEFB4) genomic copy numbers among Iraqi patients with Behçet's disease (n = 27) in comparison to healthy controls (n = 50). The mean DEFB4 genomic copy number was significantly higher in the BD group (P = 0.01). Table 1: Demographic variables of patients with Behçet’s disease in comparison to healthy controls in Baghdad, Iraq (N = 77) Variable Control group (n = 50) BD group (n = 27) P value Mean age in years ± SD 34.20 ± 8.88 36.39 ± 10.00 0.33* Gender, n Female Male 20 30 9 18 0.57† BD = Behçet’s disease; SD = standard deviation. *Calculated using the unpaired Student’s t-test. †Calculated using the Pearson’s Chi-squared test. Association of Higher Defensin β-4 Genomic Copy Numbers with Behçet’s Disease in Iraqi Patients e494 | SQU Medical Journal, November 2015, Volume 15, Issue 4 genetically susceptible individuals with high DEFB4 genomic copy numbers or high levels of hBD-2 induced through the stimulation of TNF-α. It is notable that the oral epithelium, the main site of involvement in BD, has enriched hBD-2 expression.23 The BD patients in the present study showed significantly higher DEFB4 copy numbers than the healthy controls. Such an association between DEFB4 copy numbers and BD has not been previously reported. However, no significant relationship was observed between genomic copy number variations of DEFB4 and the various clinical manifestations of BD. The results obtained from the current study were not consistent with previous research from South Korea, which found no significant correlation between DEFB4 copy numbers and BD.24 These contradictory results could be due to several reasons. It is well known that the effect of variation in β-defensin genomic copy numbers is regarded as complex, rather than simple, copy number variation; this is difficult to evaluate in small- or moderate-sized case-control studies. As a result, very few good candidate genes have been accurately studied in genomic association trials.25 The results of Aldhous et al. support this observation; in spite of a large sample size, they failed to replicate both the previously reported high and low genomic copy numbers of DEFB4 among patients with Crohn’s disease.19,26,27 In addition, discrepancies in assay reliability could play a role in explaining differences in the obtained results as well as the difference between the paralog ratio test used in the Korean study and the quantitative PCR assay used in the present study.24 However, Fernandez-Jimenez et al. demonstrated that both techniques can produce comparable DEFB4 results with the use of optimum DNA normalisation and high-quality genomic DNA.28 Nevertheless, it is known that DEFB4 copy numbers vary between ethnicities;29 this might account for the differences observed between the studied Iraqi and Korean populations.24 Genetic heterogeneity was observed in the β-defensin gene cluster among 67 populations; this may have been due to selection.30 Such genetic heterogeneity may differ along geographical or racial lines in different populations. One of the limitations of this study was the small sample size. It is important to note that the current study simply presents the significantly higher counts of mean DEFB4 genomic copies in BD, drawing attention to a possible pathological association. Further research should focus on the influence of these results on hBD-2 levels both in the peripheral tissues (especially the orogenital mucosa) and serum of BD individuals. Nevertheless, the present study is the first to analyse DEFB4 in a cohort of healthy Iraqi men and women. The median genomic copy number found among this group was similar to the modal diplotype genomic copy numbers observed in seven different populations.12 A large-scale multicentre study is recommended to validate the results of the present study and to investigate the functional consequences of these copy number variations in terms of hBD-2 protein expression. Conclusion There was an association between BD and DEFB4 genomic copy numbers among the studied Iraqi subjects, with BD patients having significantly higher DEFB4 genomic copy numbers than the control group. This suggests that high DEFB4 genomic copy numbers may be associated with susceptibility to BD. However, no association was found between DEFB4 genomic copy numbers and the different clinical manifestations of BD. Among the healthy Iraqi control group, the median DEFB4 genomic copy number was comparable to numbers observed in other populations. c o n f l i c t o f i n t e r e s t The authors declare no conflicts of interest. References 1. Sibley C, Yazici Y, Tascilar K, Khan N, Bata Y, Yazici H, et al. Behçet syndrome manifestations and activity in the United States versus Turkey: A cross-sectional cohort comparison. J Rheumatol 2014; 41:1379–84. doi: 10.3899/jrheum.131227. 2. Hatemi G, Seyahi E, Fresko I, Talarico R, Hamuryudan V. 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