Genetics, Natural History, and Pathophysiology of Behcet's Disease

白塞病的遗传学、自然史和病理生理学

• 批准号: 8350025
• 负责人: Daniel Kastner
• 金额: $ 92.08万
• 依托单位: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8350025
• 关键词: 2-oxoglutarate 3-dioxygenase proline; Accounting; Affect; Agonist; Alleles; American; Ankylosing spondylitis; Arthritis; Asia; Behcet Syndrome; Binding; Biological Assay; Blindness; CASP1 gene; CCL3 gene; CCL4 gene; CCR1 gene; Cell Line; Cells; Chemokine (C-C Motif) Ligand 4; Chemotaxis; Chinese People; Chromosomes, Human, Pair 3; Clinical; Code; Collaborations; Collection; Complementary DNA; Computer software; Confidence Intervals; Copy Number Polymorphism; Cutaneous; DNA Resequencing; Data; Data Set; Dendritic Cells; Disease; Disease susceptibility; Economic Inflation; Endoplasmic Reticulum; Enzymes; Equilibrium; Ethnic group; Europe; European; Excision; Exons; Familial Mediterranean Fever; Family; Far East; Frequencies; Functional disorder; Gene Targeting; Genes; Genetic; Genetic Polymorphism; Genetic Predisposition to Disease; Genital system; Genome; Genomics; Genotype; Goals; Graft Rejection; HLA-B Antigens; HLA-Bw51; Haplotypes; Human; IL12RB2 gene; IL1R1 gene; Immune; Immunologics; Individual; Inflammation; Inflammatory; Interleukin-10; Introns; Japan; Japanese Population; Korea; Lesion; Ligands; Linkage Disequilibrium; Lipopolysaccharides; Major Histocompatibility Complex; Manuscripts; Maps; Measures; Meleagris gallopavo; Messenger RNA; Meta-Analysis; Middle East; Minor; Modeling; Morbidity - disease rate; Multiple Sclerosis; Mus; Natural History; Natural Immunity; Nature; Neuraxis; Neurologic; Odds Ratio; Oral; Pathway interactions; Patients; Peptide Hydrolases; Peptides; Peripheral Blood Mononuclear Cell; Phenotype; Population; Predisposition; Preparation; Prevalence; Production; Proteins; Psoriasis; Publishing; Quality Control; RANTES; Recurrence; Reporting; Rheumatoid Arthritis; Rheumatology; Risk; Role; Running; STAT4 gene; Sampling; Siblings; Signal Transduction; Single Nucleotide Polymorphism; Small Inducible Cytokine A3; Stratification; T-Lymphocyte; TLR4 gene; TNFRSF1A gene; Technology; Testing; Therapeutic; Time; Transcript; Tumor Necrosis Factor-alpha; Ulcer; Universities; Untranslated Regions; Uveitis; Variant; Vasculitis; Venous; Work; anakinra; case control; chemokine; chemokine receptor; cohort; college; disorder risk; gastrointestinal; gene interaction; genetic variant; genome wide association study; genome-wide; human TNF protein; mRNA Expression; meetings; monocyte; mortality; neutrophil; next generation; novel; professor

Imputation of BD SNP data To identify additional BD susceptibility loci, we carried out whole-genome imputation using as a reference 96 of the Turkish healthy controls genotyped on Illumina HumanOmni1M-Quad SNP chips. SNPs were excluded for deviation from Hardy-Weinberg equilibrium (P less than 5 times 10 to the negative fourth), low call rate (less than 95%), and low MAF (less than 5%). Imputation was conducted using MACH v1.0.15 providing 814,474 SNPs for analysis in the 1,215 BD cases and 1,278 healthy controls. Sequenom iPLEX assays were used to validate the imputation results and to fine map the associated region. Two independent replication sets were genotyped for the most significant SNP. Using a P value cut-off of 1 times 10 to the negative fifth, we identified 114 non-HLA gene SNPs suggestive of association with BD. One imputed SNP, rs7616215 on chromosome 3, located about 38 kb from the 3 UTR of the chemokine receptor-1 gene (CCR1), (odds ratio = 0.71, P = 1.9 times 10 to the negative eighth) exceeded genome-wide significance. Fine mapping of the CCR1 region confirmed the imputation results for rs7616215 and identified 2 additional SNPs in strong linkage disequilibrium with rs7616215 that also exceeded genome-wide significance. The association of rs7616215 also replicated using additional Turkish and Japanese BD cases and controls (in a meta-analysis of 2,641 cases and 2,593 controls OR = 0.73, 95% CI = 0.660.79, P = 5.19 times 10 to the negative thirteenth). CCR1 belongs to the family of CC-motif chemokine receptors, is expressed on neutrophils, monocytes, and T lymphocytes and binds several chemokine ligands, including CCL5/RANTES, CCL3/MIP-1alpha, and CCL4/MIP-1beta. A role for CCR1 has been characterized in several inflammatory conditions, such as rheumatoid arthritis, multiple sclerosis, and transplantation rejection. ENCODE data indicate that rs7616215 resides in a putative regulatory genomic domain, and analysis of CCR1 transcripts from the HapMap European (CEU), Chinese (CHB) and Japanese (JPT) subjects showed that the protective minor allele (C) correlated with significantly increased CCR1 expression (p less than 0.03). This finding was also noted in cDNA from human monocytes. Individuals with the risk allele showed reduced chemotaxis of monocytes to MIP1alpha. In addition, we found associations at STAT4 (rs7574070, OR = 1.27, 95% CI = 1.17-1.37, P = 8.58 tiems 10 to the negative tenth) and KLRK1 (rs2733852, OR=0.79, 95% CI = 0.72-0.86, P = 2.81 times 10 negative eighth) loci. This work will be presented as a plenary podium presentation by Dr. Kirino at the annual meeting of the American College of Rheumatology in November, 2011, and we are currently preparing a manuscript describing our findings. Clinical Subset Analyses and Gene-Gene Interactions SNP genotype data from the previously reported Behets disease GWAS was re-analyzed in the subset of uveitis-positive patients. When we applied a recessive model, we found an association signal at the ERAP1 locus with close to genome-wide significance (1.7 times 10 to the negative seventh). Fine mapping of this region was performed with tag-SNPs selected from HapMap CEU data. We identified a haplotype containing two non-synonymous SNPs in ERAP1 that were both associated with increased risk for BD with uveitis (rs17482078 and rs10050860, OR = 4.18, 95% CI = 2.44-7.19, P = 2.38 times 10 to the negative eighth). These SNPs were recently shown to be protective against ankylosing spondylitis and psoriasis. This recessive model-dependent increased BD risk replicated in an independent collection of additional Turkish samples (including uveitis negative and positive patients) (OR = 4.07, P = 2.70 times 10 to the negative fourth). Meta-analysis including all the Turkish BD samples (cases n = 2028, controls n = 1876) showed significant association of the homozygous minor allele genotype with disease (OR = 3.07, P = 4.81 times 10 to the negative eighth). ERAP1 encodes the endoplasmic reticulum-associated amino peptidase 1, an enzyme involved in peptide trimming and loading onto HLA class I. Recent reports have shown a strong protective interaction between HLA class I variants and ERAP1 variants in ankylosing spondylitis and psoriasis. We found that increased BD risk was associated with the ERAP1 minor allele homozygous genotype only in HLA-B51 positive individuals. This study establishes ERAP1 as a novel BD susceptibility locus and suggests that peptide production and presentation are important mechanisms in this and the other class I associated diseases. A manuscript reporting these findings is in preparation. Copy Number Variation Analysis SNP intensity data from our previous BD genome-wide association study was used for the analysis. We evaluated homozygously deleted regions in a genome-wide fashion, thus applying a recessive model for copy number variation association. The detected common CNV was validated with real-time PCR and break-point assay. Meta-analysis across various inflammatory diseases was performed. We identified a haplotype tagged by a 3.2 kb deletion polymorphism within the first intron of the LEPREL1 gene that is associated with protection against BD in multiple populations (combined cases = 2056, controls = 2120, OR = 0.63, P = 7.89 times 10 to the negative sixth). The LEPREL1 homozygous deletion was also found to be protective against various inflammatory diseases (cases = 9082, controls = 9243, OR = 0.85, P = 3.4 times 10 to the negative fourth). LEPREL1, which encodes prolyl 3-hydroxylase 2 (P3H2), was found to be expressed primarily in plasmacytoid/lymphocytoid dendritic cells (DCs) and lipopolysaccharide induced its expression in a murine DC line. Allelic expression analysis in cells heterozygous for the deletion showed that LEPREL1 mRNA is lower from the allele with the deletion. Individuals with the homozygous deletion produced lower levels of TNF-alpha in PBMC stimulated with innate-immune agonists. A manuscript reporting these findings is in preparation. Deep Resequencing of Targeted Genes Genes identified by genome-wide association studies (IL23R and IL10) and those involved in innate immunity (IL1B, IL1R1, IL1RN, NLRP3, MEFV, TNFRSF1A, PSTPIP1, CASP1, PYCARD, NOD2, and TLR4) were selected for resequencing in Japanese and Turkish BD collections. We first prepared pooled case DNAs and pooled control DNAs from these two populations (48 samples per pool, total of 384 cases and 384 controls), and PCR amplified the exons. We then deeply resequenced them using next-generation sequencing. The result was analyzed with CASAVA software and all of the low-frequency (less than 5%) non-synonymous coding variants were validated by Sequenom genotyping the individual samples and evaluated after genotyping the Japanese and Turkish BD collections. Applying the c-alpha test for statistical analysis, we found that rare and low-frequency non-synonymous coding variants in the IL23R and TLR4 genes associated with disease. We also found that the MEFV M694V variant (which, in the homozygous state, causes a severe form of familial Mediterranean fever) confers strong risk for BD in the Turkish population (OR = 2.70, P = 5.15 times 10 to the negative thirteenth). A manuscript describing these findings is in preparation. During the upcoming reporting period, our efforts will be directed towards: (1) completing four manuscripts reporting the findings described above; (2) following up on functional analyses of genetic variants identified by GWAS; (3) further delineating the role of HLA and interacting genes in BD; and (4) developing therapeutic strategies that target the immunologic pathways identified by these genetic studies.

BD SNP数据的插补 为了识别其他BD易感基因座，我们使用作为在Illumina honsomni1m-Quad SNP芯片上的土耳其健康对照组的参考96进行了全基因组插补。 SNP因偏离Hardy-Weinberg平衡而排除在外（P小于5倍至负四倍），低呼叫率（小于95％）和低MAF（小于5％）。使用MACH V1.0.15进行插补，在1,215个BD病例和1,278个健康对照组中提供814,474个SNP进行分析。 序列IPLEX测定法用于验证插奖结果并详细绘制相关区域。 针对最重要的SNP对两个独立的复制集进行了基因分型。 使用P值截止为1倍至负第五，我们确定了114个非HLA基因SNP，建议与BD关联。 3染色体上的一个估算的SNP，rs7616215，位于趋化因子受体-1基因的3 UTR（CCR1）的38 kb（占0.71，p = 0.71，p = 1.9乘以1.9倍至负第八）超过基因组显着性。 CCR1区域的精细映射证实了RS7616215的插奖结果，并在强链路上鉴定了2个其他SNP与rs7616215的强烈连接不平衡，这也超过了全基因组的意义。 RS7616215的关联也使用其他土耳其和日本的BD病例和对照组（在2,641例荟萃分析和2,593例对照组或= 0.73，95％CI = 0.660.79，p = 5.19倍至负3次）。 CCR1属于CC-MOTIF趋化因子受体的家族，在中性粒细胞，单核细胞和T淋巴细胞上表达，并结合几种趋化因子配体，包括CCL5/Rantes，CCL3/MIP-1Alpha和CCL4/MIP-1Beta。 CCR1的作用已经在几种炎症状态（例如类风湿关节炎，多发性硬化症和移植排斥反应）中表征。编码数据表明，RS7616215驻留在推定的调节基因组域中，并且分析来自HAPMAP欧洲（CEU），中国（CHB）和日语（JPT）受试者的CCR1转录本表明，保护性次要等位基因（C）与CCR1表达显着增加了CCR1的表达（P小于0.03）。人类单核细胞中的cDNA也发现了这一发现。具有风险等位基因的个体显示单核细胞对MIP1Alpha的趋化性降低。此外，我们在STAT4（rs7574070，OR = 1.27，95％CI = 1.17-1.37，p = 8.58 TIEMS 10至负数）和KLRK1（rs2733852，rs2733852，OR = 0.79，95％ci = 0.72 ci = 0.72-0.86，p = 2.86，p = 81 tierm = 81 tierme = 81 tierme = 81 tierme = 81 tierme。 基里诺博士将在2011年11月的美国风湿病学院年会上作为基里诺博士作为全体讲台演讲的介绍，我们目前正在准备一份描述我们发现的手稿。 临床子集分析和基因基因相互作用 先前报道的Behets疾病GWAS的SNP基因型数据在葡萄膜炎阳性患者的子集中被重新分析。当我们应用隐性模型时，我们在ERAP1基因座上发现了一个接近全基因组显着性的关联信号（1.7乘以1.7倍至第七元）。使用从HAPMAP CEU数据中选择的TAG-SNP对该区域进行细微映射。我们确定了一种单倍型，其中包含ERAP1中的两个非同义SNP，这些SNP均与葡萄膜炎的BD风险增加有关（RS17482078和RS10050860，OR = 4.18，95％CI = 2.44-7.19，p = 2.38次至负Eighth）。这些SNP最近被证明是针对强直性脊柱炎和牛皮癣的保护作用。这种隐性模型依赖性增加的BD风险在独立的土耳其样本（包括葡萄膜炎和阳性患者）的独立集中复制（OR = 4.07，p = 2.70倍至2.70倍至负四）。包括所有土耳其BD样品（病例n = 2028，对照n = 1876）的荟萃分析显示，纯合小等位基因基因型与疾病的显着关联（OR = 3.07，p = 4.81，p = 4.81倍10倍至第八次负）。 ERAP1编码内质网相关的氨基肽酶1，一种参与肽修剪的酶，并在HLA类I上加载。最近的报道显示，HLA I类变体与紧密的脊柱炎和psoryasis中的HLA I类变体和ERAP1变体之间存在强烈的保护性相互作用。我们发现，仅在HLA-B51阳性个体中，BD风险增加与ERAP1小等位基因基因型有关。 这项研究将ERAP1确定为一种新型的BD敏感性基因座，并表明肽的产生和表现是该疾病和其他I类疾病的重要机制。 报告这些发现的手稿正在准备。 拷贝数变化分析 我们以前的BD基因组关联研究中的SNP强度数据用于分析。我们以全基因组方式评估了纯合区域，从而应用了隐性模型，以实现拷贝数变化关联。通过实时PCR和断点测定法对检测到的常见CNV进行了验证。进行了各种炎症性疾病的荟萃分析。 我们确定了在LEPREL1基因的第一个内含子中标记为3.2 Kb缺失多态性的单倍型，该内含子与多个种群中的BD保护相关（组合病例= 2056，对照= 2120，OR = 0.63，或= 0.63，p = 7.89乘以p = 7.89次至负第六）。还发现Leprel1纯合缺失具有针对各种炎症性疾病的保护作用（病例= 9082，对照= 9243，OR = 0.85，p = 3.4倍10至阴性第四倍）。 LEPREL1编码了三羟化酶2（P3H2）的Leprel1，主要在浆细胞类/淋巴细胞生长的树突状细胞（DCS）和脂多糖中表达，在鼠DC系中诱导了其表达。缺失的细胞杂合子中的等位基因表达分析表明，Leprel1 mRNA与缺失的等位基因低。具有纯合缺失的个体在PBMC中产生了较低水平的TNF-Alpha，其先天免疫激动剂刺激。 报告这些发现的手稿正在准备。 对靶基因的深度重新纠正 Genes identified by genome-wide association studies (IL23R and IL10) and those involved in innate immunity (IL1B, IL1R1, IL1RN, NLRP3, MEFV, TNFRSF1A, PSTPIP1, CASP1, PYCARD, NOD2, and TLR4) were selected for resequencing in Japanese and Turkish BD collections.我们首先从这两个人群（每个池48个样本，总计384例和384个对照组）准备了合并的病例DNA和汇总对照DNA，并且PCR扩增了外显子。然后，我们使用下一代测序将它们重新置于它们。通过Casava软件对结果进行了分析，并通过序列化基因分型分型单个样本验证了所有低频（少于5％）的非同义编码变体，并在基因分型后进行了日本和土耳其BD集合后进行了评估。应用C-Alpha检验进行统计分析，我们发现与疾病相关的IL23R和TLR4基因中的罕见和低频非义义编码变体。我们还发现，MEFV M694V变体（在纯合状态下会引起严重的家族性地中海发烧形式）在土耳其人群中赋予BD强风险（OR = 2.70，p = 5.15倍10倍10至第十三个负数）。描述这些发现的手稿正在准备。 在即将到来的报告期间，我们的努力将致力于：（1）完成四个手稿，报告上述发现； （2）跟进GWAS确定的遗传变异的功能分析； （3）进一步描述了BD中HLA和相互作用基因的作用； （4）制定针对这些遗传研究确定的免疫途径的治疗策略。