Dense genotyping of immune-related loci implicates host responses to microbial exposure in Behçet's disease susceptibility.

We analyzed 1,900 Turkish Behçet's disease cases and 1,779 controls genotyped with the Immunochip. The most significantly associated SNP was rs1050502, a tag SNP for HLA-B*51. In the Turkish discovery set, we identified three new risk loci, IL1A-IL1B, IRF8, and CEBPB-PTPN1, with genome-wide significance (P < 5 × 10-8) by direct genotyping and ADO-EGR2 by imputation. We replicated the ADO-EGR2, IRF8, and CEBPB-PTPN1 loci by genotyping 969 Iranian cases and 826 controls. Imputed data in 608 Japanese cases and 737 controls further replicated ADO-EGR2 and IRF8, and meta-analysis additionally identified RIPK2 and LACC1. The disease-associated allele of rs4402765, the lead marker at IL1A-IL1B, was associated with both decreased IL-1α and increased IL-1ß production. ABO non-secretor genotypes for two ancestry-specific FUT2 SNPs showed strong disease association (P = 5.89 × 10-15). Our findings extend the list of susceptibility genes shared with Crohn's disease and leprosy and implicate mucosal factors and the innate immune response to microbial exposure in Behçet's disease susceptibility.

IL10 low-frequency variants in Behçet's disease patients.

AIM: To explain the missing heritability after the genome-wide association studies era, sequencing studies allow the identification of low-frequency variants with a stronger effect on disease risk. Common variants in the interleukin 10 gene (IL10) have been consistently associated with Behçet's disease (BD) and the goal of this study is to investigate the role of low-frequency IL10 variants in BD susceptibility. METHODS: To identify IL10 low-frequency variants, a discovery group of 50 Portuguese BD patients were Sanger-sequenced in a 7.7 kb genomic region encompassing the complete IL10 gene, 0.9 kb upstream and 2 kb downstream, and two conserved regions in the putative promoter. To assess if the novel variants are BD- and/or Portuguese-specific, they were assayed in an additional group of BD patients (26 Portuguese and 964 Iranian) and controls (104 Portuguese and 823 Iranian). RESULTS: Rare IL10 coding variants were not detected in BD patients, but we identified 28 known single nucleotide polymorphisms with minor allele frequencies ranging from 0.010 to 0.390, and five novel non-coding variants in five heterozygous cases. ss836185595, located in the IL10 3' untranslated region, was also detected in one Iranian control individual and therefore is not specific to BD. The remaining novel IL10 variants (ss836185596 and ss836185602 in intron 3, ss836185598 and ss836185604 in the putative promoter region) were not found in the replication dataset. CONCLUSION: This study highlights the importance of screening the whole gene and regulatory regions when searching for novel variants associated with complex diseases, and the need to develop bioinformatics tools to predict the functional impact of non-coding variants and statistical tests which incorporate these predictions.

Brief report: association of CCR1, KLRC4, IL12A-AS1, STAT4, and ERAP1 With Behçet's disease in Iranians.

OBJECTIVE: To independently replicate the top findings from 4 published genome-wide association studies (GWAS) of susceptibility genes in Behçet's disease (BD). METHODS: We tested 14 single-nucleotide polymorphisms (SNPs) in 13 genomic loci (excluding the major histocompatibility complex [MHC], IL10, and IL23R-IL12RB2, which have already been associated with BD in Iranians) for allelic and genotypic associations with BD in 973 patients and 828 controls from Iran and performed meta-analyses of the significantly associated markers. RESULTS: Six SNPs (in decreasing order of significance, rs7616215 located 38 kb downstream of CCR1, rs2617170 [p.Asn104Ser] in KLRC4, rs17810546 in IL12A-AS1, rs7574070 in STAT4, and rs10050860 [p.Asp575Asn] and rs13154629 in ERAP1) were nominally associated with BD in both allelic association tests (5.05 × 10(-9) ≤ Pallele ≤ 7.55 × 10(-3) ) and sex-adjusted genotypic association tests (6.01 × 10(-9) ≤ adjusted P value ≤ 1.30 × 10(-2) ). For all 6 SNPs tested by meta-analysis (Pmeta ), the association with BD was strengthened, because the direction and magnitude of association were similar across populations (e.g., for rs7574070, odds ratio [OR] for A allele 1.29 [95% confidence interval (95% CI) 1.21-1.37], Pmeta = 2.34 × 10(-16) ; for rs7616215, OR for C allele 0.70 [95% CI 0.65-0.76], Pmeta = 1.54 × 10(-19) ; for rs17810546, OR for A allele 0.60 [95% CI 0.52-0.70], Pmeta = 6.34 × 10(-11) ; for rs2617170, OR for T allele 0.76 [95% CI 0.70-0.81], Pmeta = 2.75 × 10(-14) ; for rs13154629, OR for TT genotype 2.76 [95% CI 2.01-3.80], Pmeta = 3.57 × 10(-10) ). CONCLUSION: This study reinforces the notion that CCR1, KLRC4, IL12A-AS1, STAT4, and ERAP1 are bona fide susceptibility genes for BD, in addition to the MHC, IL10, and IL23R-IL12RB2 loci. Future genetic and functional studies are now warranted to uncover the roles of these genes in the pathogenesis of BD.

On the genetics of the Silk Route: association analysis of HLA, IL10, and IL23R-IL12RB2 regions with Behçet's disease in an Iranian population.

Despite that the association of Behçet's disease (BD) with the HLA-B5 was first established in the 1970s, a number of recent genome-wide association studies have both confirmed and furthered this association--in various populations--to individual SNPs both inside and outside the HLA. The former include HLA-B, MICA, and HLA-A, while the latter encompass IL10 and IL23R-IL12RB2 regions. The present study examined whether some of these SNPs could be replicated in an Iranian population, where the prevalence of disease is amply documented. Eight SNPs were selected and tested in 552 patients and 417 controls. These were rs7539328, rs12119179, rs1495965, rs1518111, and rs1800871 in IL10 and IL23R-IL12RB2 regions and rs114854070, rs12525170, and rs76546355 (formerly rs116799036) in the HLA locus. The well-known BD-associated genes HLA-B and MICA were independently genotyped. Although we were not able to formally replicate the association with IL10 and IL23R-IL12RB2, we do report that BD in Iran is strongly associated with HLA-B*51, MICA-A6, and the three HLA-linked SNPs (odds ratio (OR) = 3.38, P = 6.21 × 10(-14); OR = 2.08, P = 1.58 × 10(-13); and OR = 1.67-4.05, P = 1.45 × 10(-04) to 4.79 × 10(-34), respectively). Our data further indicate that the robust HLA-B/MICA association may be explained by a single variant (rs76546355) between the two genes. Overall, these data contribute to a better appraisal of the intriguing linkage between BD and the ancient Silk Route, spanning from the Mediterranean shores to Japan.

The effect of Ramadan fasting on quiescent systemic lupus erythematosus (SLE) patients' disease activity, health quality of life and lipid profile: a pilot study.

SLE is a common autoimmune disease with considerable morbidity. Ramadan fasting is a religious custom Muslims regularly practice. We aimed to evaluate the effect of Ramadan fasting on SLE patients' disease activity, health quality of life and lipid profile. We conducted this case control study as a pilot study in 40 quiescent SLE patients, 21 cases who decided to fast and 19 controls who decided not to have Ramadan fasting between August and November 2009 in lupus unit of Rheumatology Research Center in Tehran University of Medical Sciences, Iran. They were assessed for SLE Disease Activity Index, lipid profile and quality of life with Short-Form 36 (SF-36) Health Survey, 1 day before Ramadan, the day after and 3 months after Ramadan fasting. After 24.1 ± 5.4 (mean ± SD) days of fasting, anti-ds DNA increased for 0.34 ± 0.41 mmol/dL in cases versus 0.07 ± 0.31 in controls (P = 0.026). Likewise C3 increased more dramatically in cases (16.8 ± 17.5 vs. 2.3 ± 13.2 mg/dL, P = 0.006). Three months after fasting, anti-ds DNA was still increased 0.28 ± 0.46 mmol/dL in cases while a 0.02 ± 0.43 mmol/dL drop in controls was detected (P = 0.04). On the contrary, C3 returned to baseline. These changes were not accompanied with significant changes in disease activity and health quality of life. Ramadan fasting had no effect on lipid profile except for delayed total cholesterol decrease in cases in comparison with controls (16.4 ± 29.4 decrease vs. 4.6 ± 23.9 mg/dL decrease, P = 0.018). Ramadan fasting probably has no detrimental effect on SLE patients' disease activity and their quality of life in the quiescent phase of disease.

Characterization of the major histocompatibility complex locus association with Behçet's disease in Iran.

INTRODUCTION: The aim of this study was to characterize the association of human leukocyte antigen (HLA) B alleles and major histocompatibility complex (MHC) single nucleotide polymorphisms (SNPs) with Behçet's disease (BD) in an Iranian dataset. METHODS: The association of three SNPs in the MHC region previously identified as the most associated in high-density genotyping studies was tested in a case-control study on 973 BD patients and 825 controls from Iran, and the association of HLA-B alleles was tested in a subset of 681 patients and 414 controls. RESULTS: We found that HLA-B*51 (P = 4.11 × 10(-41), OR [95% CI] = 4.63[3.66-5.85]) and B*15 confer risk for BD (P = 2.83 × 10(-2), OR [95% CI] = 1.75[1.08-2.84]) in Iranian, and in B*51 negative individuals, only the B*15 allele is significantly associated with BD (P = 2.51 × 10(-3), OR [95% CI] = 2.40[1.37-4.20]). rs76546355, formerly known as rs116799036, located between HLA-B and MICA (MHC class I polypeptide-related sequence A), demonstrated the same level of association with BD as HLA-B*51 (P adj = 1.78 × 10(-46), OR [95% CI] = 5.46[4.21-7.09], and P adj = 8.34 × 10(-48), OR [95% CI] = 5.44[4.20-7.05], respectively) in the HLA-B allelotyped subset, while rs2848713 was less associated (P adj = 7.14 × 10(-35), OR [95% CI] = 3.73[2.97-4.69]) and rs9260997 was not associated (P adj = 1.00 × 10(-1)). Additionally, we found that B*51 genotype-phenotype correlations do not survive Bonferroni correction, while carriers of the rs76546355 risk allele predominate in BD cases with genital ulcers, positive pathergy test and positive BD family history (2.31 × 10(-4) ≤ P ≤ 1.59 × 10(-3)). CONCLUSIONS: We found that the HLA-B*51 allele and the rs76546355/rs116799036 MHC SNP are independent genetic risk factors for BD in Iranian, and that positivity for the rs76546355/rs116799036 risk allele, but not for B*51, does correlate with specific demographic characteristics or clinical manifestations in BD patients.

FUT2: filling the gap between genes and environment in Behçet's disease?

OBJECTIVES: To identify new susceptibility loci for Behçet's disease (BD), we performed a genome-wide association study (GWAS) using DNA pooling. METHODS: Two replicate pools of 292 Iranian BD cases and of 294 age- and sex-matched controls were allelotyped in quadruplicate on the Affymetrix Genome-Wide Human SNP Array 6.0. Of the 51 top markers, 47 were technically validated through individually genotyping. Replication of validated single nucleotide polymorphisms (SNPs) was performed in an independent Iranian dataset (684 cases and 532 controls). RESULTS: In addition to the well-established HLA-B locus, rs7528842 in a gene desert on chromosome 1p21.2, and rs632111 at the 3'UTR of FUT2 were associated in both the discovery and replication datasets (individually and in combination). However, only the FUT2 SNP was associated in a previous GWAS for BD in Turkish people. Fine-mapping of FUT2 in the full Iranian dataset showed additional associations in five coding SNPs (2.97E-06

Validation of the revised International Criteria for Behcet's Disease (ICBD) in Iran.

The first internationally agreed criteria for Behcet's disease were the International Study Group (ISG) criteria. It had very high specificity, but lacked good sensitivity, missing an important subset of patients. The International Criteria for Behcet's Disease (ICBD) were created in 2006 to overcome this lack of sensitivity. It was revised in 2010. The objective of this study was to evaluate the performance of the revised International Criteria for Behcet's Disease (rICBD) in Iran. In this study, the ISG and ICBD were evaluated and compared to the rICBD. All patients from the Behcet's Disease Registry (7,011) and controls (5,226), up to March 2013, entered the study. The diagnosis was clinical, by expert opinion. Sensitivity, specificity, and accuracy were calculated for ISG, ICBD, and rICBD. A 95% confidence interval (95%CI) was calculated for percentages. For ISG, the sensitivity was 77.5% (95%CI = 76.5-78.5). It was 98.3% for ICBD (95%CI = 98.0-98.6) and 96.8% for rICBD (95%CI = 96.4-97.2). Specificity was 99.2% (95%CI = 99.0-99.4) for ISG, 96.2% for ICBD (95%CI = 95.7-96.7), and 97.2% for rICBD (95%CI = 96.8-97.6). Accuracy was 86.7% (95%CI = 86.1-87.3) for ISG, 97.4% for ICBD (95%CI = 97.1-97.7), and 97.0% for rICBD (95%CI = 96.7-97.3). In Iranian patients, ICBD has 20.8% and rICBD 19.3% higher sensitivity than ISG. Although the specificity was lower than ISG by 3% for ICBD and 2% for rICBD, the accuracy was higher respectively by 10.7 and 10.3%. ICBD has by far better performance than ISG. The difference was even more prominent in Iranian patients than for the ICBD cohort of patients and controls.

The perspectives of Iranian physicians and patients towards patient decision aids: a qualitative study.

BACKGROUND: Patient preference is one of the main components of clinical decision making, therefore leading to the development of patient decision aids. The goal of this study was to describe physicians' and patients' viewpoints on the barriers and limitations of using patient decision aids in Iran, their proposed solutions, and, the benefits of using these tools. METHODS: This qualitative study was conducted in 2011 in Iran by holding in-depth interviews with 14 physicians and 8 arthritis patient. Interviewees were selected through purposeful and maximum variation sampling. As an example, a patient decision aid on the treatment of knee arthritis was developed upon literature reviews and gathering expert opinion, and was presented at the time of interview. Thematic analysis was conducted to analyze the data by using the OpenCode software. RESULTS: The results were summarized into three categories and ten codes. The extracted categories were the perceived benefits of using the tools, as well as the patient-related and physician-related barriers in using decision aids. The following barriers in using patient decision aids were identified in this study: lack of patients and physicians' trainings in shared decision making, lack of specialist per capita, low treatment tariffs and lack of an exact evaluation system for patient participation in decision making. CONCLUSIONS: No doubt these barriers demand the health authorities' special attention. Hence, despite patients and physicians' inclination toward using patient decision aids, these problems have hindered the practical usage of these tools in Iran--as a developing country.

Gene expression profiling and association studies implicate the neuregulin signaling pathway in Behçet's disease susceptibility.

Behçet's disease (BD) is a complex disease with genetic and environmental risk factors implicated in its etiology; however, its pathophysiology is poorly understood. To decipher BD's genetic underpinnings, we combined gene expression profiling with pathway analysis and association studies. We compared the gene expression profiles in peripheral blood mononuclear cells (PBMCs) of 15 patients and 14 matched controls using Affymetrix microarrays and found that the neuregulin signaling pathway was over-represented among the differentially expressed genes. The Epiregulin (EREG), Amphiregulin (AREG), and Neuregulin-1 (NRG1) genes of this pathway stand out as they are also among the top differentially expressed genes. Twelve haplotype tagging SNPs at the EREG-AREG locus and 15 SNPs in NRG1 found associated in at least one published BD genome-wide association study were tested for association with BD in a dataset of 976 Iranian patients and 839 controls. We found a novel association with BD for the rs6845297 SNP located downstream of EREG, and replicated three associations at NRG1 (rs4489285, rs383632, and rs1462891). Multifactor dimensionality reduction analysis indicated the existence of epistatic interactions between EREG and NRG1 variants. EREG-AREG and NRG1, which are members of the epidermal growth factor (EGF) family, seem to modulate BD susceptibility through main effects and gene-gene interactions. These association findings support a role for the EGF/ErbB signaling pathway in BD pathogenesis that warrants further investigation and highlight the importance of combining genetic and genomic approaches to dissect the genetic architecture of complex diseases.

Association study of IL10 and IL23R-IL12RB2 in Iranian patients with Behçet's disease.

OBJECTIVE: Independent replication of the findings from genome-wide association studies (GWAS) remains the gold standard for results validation. Our aim was to test the association of Behçet's disease (BD) with the interleukin-10 gene (IL10) and the IL-23 receptor-IL-12 receptor ß2 (IL23R-IL12RB2) locus, each of which has been previously identified as a risk factor for BD in 2 different GWAS. METHODS: Six haplotype-tagging single-nucleotide polymorphisms (SNPs) in IL10 and 42 in IL23R-IL12RB2 were genotyped in 973 Iranian patients with BD and 637 non-BD controls. Population stratification was assessed using a panel of 86 ancestry-informative markers. RESULTS: Subtle evidence of population stratification was found in our data set. In IL10, rs1518111 was nominally associated with BD before and after adjustment for population stratification (odds ratio [OR] for T allele 1.20, 95% confidence interval [95% CI] 1.02-1.40, unadjusted P [P(unadj) ] = 2.53 × 10(-2) ; adjusted P [P(adj) ] = 1.43 × 10(-2) ), and rs1554286 demonstrated a trend toward association (P(unadj) = 6.14 × 10(-2) ; P(adj) = 3.21 × 10(-2) ). Six SNPs in IL23R-IL12RB2 were found to be associated with BD after Bonferroni correction for multiple testing, the most significant of which were rs17375018 (OR for G allele 1.51, 95% CI 1.27-1.78, P(unadj) = 1.93 × 10(-6) ), rs7517847 (OR for T allele 1.48, 95% CI 1.26-1.74, P(unadj) = 1.23 × 10(-6) ), and rs924080 (OR for T allele 1.29, 95% CI 1.20-1.39, P = 1.78 × 10(-5) ). SNPs rs10489629, rs1343151, and rs1495965 were also significantly associated with BD in all tests performed. Results of meta-analyses of our data combined with data from other populations further confirmed the role of rs1518111, rs17375018, rs7517847, and rs924080 in the risk of BD, but no epistatic interactions between IL10 and IL23R-IL12RB2 were detected. Results of imputation analysis highlighted the importance of IL23R regulatory regions in the susceptibility to BD. CONCLUSION: These findings independently confirm, extend, and refine the association of BD with IL10 and IL23R-IL12RB2. These associations warrant further validation and investigation in patients with BD, as they may have implications for the development of novel therapies (e.g., immunosuppressive therapy targeted at IL-23p19).

Mesenchymal stem cell therapy for knee osteoarthritis. Preliminary report of four patients.

BACKGROUND: Osteoarthritis (OA) is a cartilage degenerative process, involving the immune system, producing local inflammatory reactions, with production of pro-inflammatory cytokines and metalloproteinases. No treatment is still available to improve or reverse the process. Stem cell therapy opened new horizons for treatment of many incurable diseases. Mesenchymal stem cells (MSCs) due to their multi-lineage potential, immunosuppressive activities, limited immunogenicity and relative ease of growth in culture, have attracted attentions for clinical use. AIM: The aim of this study was to examine whether MSC transplantation could reverse the OA process in the knee joint. The project was approved by the Tehran University of Medical Sciences Research Committee and Ethical Committee. PATIENTS AND METHODS: Four patients with knee osteoarthritis were selected for the study. They were aged 55, 57, 65 and 54 years, and had moderate to severe knee OA. After their signed written consent, 30 mL of bone marrow were taken and cultured for MSC growth. After having enough MSCs in culture (4-5 weeks) and taking in consideration all safety measures, cells were injected in one knee of each patient. RESULTS: The walking time for the pain to appear improved for three patients and remained unchanged for one. The number of stairs they could climb and the pain on visual analog scale improved for all of them. On physical examination, the improvement was mainly for crepitus. It was minor for the improvement of the range of motion. CONCLUSION: Results were encouraging, but not excellent. Improvement of the technique may improve the results.

Behcet's disease in Iran: analysis of 6500 cases.

OBJECTIVE: To identify the clinical picture of Behcet's disease in a large cohort of patients (6500) in Iran, over a period of 35 years, and compare them with other large series from around the world. METHODS: Patients with Behcet's disease from all over Iran were seen in the Behcet's Disease Research Unit by a multidisciplinary team (rheumatologists, dermatologists, and ophthalmologists). Diagnosis was based on 'expert opinion'. Data were collected on a standardized data sheet (105 items), and stored in an electronic database. Data were updated at each follow-up. RESULTS: Male to female ratio was 1.22 : .00. The mean age at onset was 26 years ± 11.3. The frequency of symptoms were: oral aphthosis 97.3%, genital aphthosis 64.6%, skin manifestations 64.9% (pseudofolliculitis 54.5%, erythema nodosum 22.5%, other lesions 7%), pathergy phenomenon 52.5%, ophthalmologic manifes-tations 56.8% (anterior uveitis 41.2%, posterior uveitis 44.9%, retinal vasculitis 32.1%), joint manifestations 37.4% (arthralgia 17.2%, monoarticular arthritis 7.6%, oligoarthritis 16.8%, ankylosing spondylitis 2%), neurological manifestations 3.8% (central manifestations 3.5%, mononeuritis multiplex 0.3%), gastrointestinal manifestations 7.4%, vascular involvement 8.3% (phlebitis 5.7%, superficial phlebitis 2.2%, large vein thrombosis 1.1%, arterial thrombosis 0.154%, aneurysm 0.5%), epididymitis 4.7%, cardiac involvement 0.6%, and pulmonary involvement 0.9%. Sedimentation rate was normal in 46.5% of patients. Abnormal urine sediment was detected in 12.2%. HLA-B5 was present in 53.3% and HLA-B51 in 47.9% of patients. CONCLUSION: Behcet's disease is mainly seen in young people. The most frequent symptoms are mucocutaneous, ocular and joint manifestations. Comparison with large series did not show major differences.