Genetic Analysis of Complex Inflammatory Disorders

复杂炎症性疾病的遗传分析

• 批准号: 7319595
• 负责人: Daniel L Kastner
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 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/7319595
• 关键词: Genetic; Analysis; Complex; Inflammatory; Disorders

The Complex Disease Genetics Unit (CDGU) of the Genetics and Genomics Branch was established to identify genes conferring susceptibility to genetically complex rheumatic and inflammatory diseases. The strategy often entails nonparametric linkage analysis of large numbers of sibling pairs concordant for the disease in question and/or association studies on collections of independent cases and controls using a dense map of markers drawn from a given chromosomal region or the whole genome. The CDGU has focused on rheumatoid arthritis (RA), which affects as much as 1% of the population worldwide. This work builds on our long-standing participation in the North American Rheumatoid Arthritis Consortium (NARAC), a large collaborative group that has collected samples from over 1000 sibling pairs concordant for RA, as well as from cohorts of singleton RA cases and ethnically-matched controls. Genome-wide linkage data from previous reporting periods have confirmed a major genetic effect in the HLA region, and also showed evidence of linkage (p less than 0.005) for chromosomes 1p13, 1q43, 6q21, 10q21, 12q12, 17p13, and 18q21. Because the 18q21 region has been replicated in independent French and Canadian cohorts, we have concentrated association studies for linkage disequilibrium (LD) on this segment of the genome. NARAC previously conducted a dense single-nucleotide polymorphism (SNP) analysis of the chromosome 18q21 candidate region using a bead-based optical technology (Illumina), examining 460 cases and 460 controls. The average marker density for the SNPs successfully typed was 4.3 kb over the 10 Mb region. Two LD clusters were identified, each with several SNPs with evidence for linkage to RA (p value less than or equal to 0.001). Results of the Last Year Genome-wide analyses: During the past year we completed a second genome-wide analysis of the NARAC sibling pairs using >5700 SNPs (Illumina) (the previous genome-wide linkage analysis used 379 microsatellite markers). This more informative analysis identified two new regions on 11p12 and 2q33 with strong evidence for linkage to a disease susceptibility locus (LOD > 3.0). Furthermore, new regions with suggestive evidence for linkage were found on chromosomes 4q25, 5p12, and 10q21, and additional support was obtained for previously identified regions on 18q21, 20p13, and 1q41-42. Analysis of the chromosome 18q21 candidate region: During the past year, we further examined the two 18q21 regions we identified last year and also a third region, in which we found a 4-SNP haplotype with evidence for association with an RA susceptibility locus. Addition of 656 independent cases and 604 controls to the analysis of previously identified regions strengthened the statistical significance to p = 0.0005 and p = 0.0009, respectively, retaining nominal significance when corrected for possible stratification among European subpopulations. The most strongly associated SNPs in region 1 are located between a transcription factor gene, ONECUT2, and the gene encoding ferrochelatase, FECH. The minor allele frequency of the most strongly-associated SNP in this region has a higher frequency in RA cases than in controls (0.35 in RA cases vs. 0.29 in controls). The most strongly-associated SNPs in region 2 are distal to the TCF4 gene in a partially characterized transcript expressed in lung, cerebellum, and primary cells cultured from cartilage. One of these SNPs would result in the substitution of glutamine for arginine at residue 95 (R95Q) of the predicted protein (p = 0.002). In a third, newly identified 18q region, we found a relatively rare 4-SNP haplotype with evidence for association with RA (the haplotype was found at 1.6% frequency in 460 controls and 4.4% frequency in 459 independent cases p=0.0006). We genotyped additional samples for a total of 1386 controls and 667 independent cases and found the haplotype frequency was 2.1% in controls and 3.9% in cases (Chi square analysis p = 0.001, permutation analysis, p = 0.008). The genomic region containing this haplotype was located distal to region 1 and the 4 SNPs were within FECH, encoding ferrochelatase, a mitochondrial enzyme responsible for the final step in the heme synthesis pathway, and which is mutated in erythropoietic protoporphyria. Further evaluation of these three regions entails the genotyping of an additional 1000 cases and controls that is currently underway. Association of variants within the peptidyl arginine deiminase, type IV (PADI4) gene: Earlier studies from another group indicated that a specific variant and haplotype of the peptidyl arginine deiminase, type IV (PADI4) gene are associated with RA among the Japanese. During the previous reporting period we found that a different PADI4 haplotype associated with RA among Caucasians. In order to thoroughly evaluate the variants within the PADI4 gene region we genotyped the HapMap CEU samples for 69 polymorphic SNPs located within this 75 kb gene region. We then selected 22 tag SNPs that non-redundantly represented all 69 SNPs and genotyped the NARAC cases and controls for these 22 SNPs. We found two of the tag SNPs to be more strongly associated with RA than padi4-94, the most strongly associated SNP reported in the Japanese population. We genotyped all the SNPs in strong LD with the two disease associated SNPs and identified a total of 3 SNPs, all located in the first intron of PADI4, with minor allele frequencies of 5-6 percent in controls and 10-11 percent in cases (p equals 0.00006 to 0.00009). When we limited the analysis to the 453 rheumatoid factor positive cases, the p value was 0.000005, suggesting that the association was even stronger in this phenotypically more homogeneous group. Current studies are focused on determining whether these non-coding SNPs are associated with a difference in transcriptional efficiency. Analysis of the type I diabetes-associated variant in the interferon-induced helicase gene: A nonsynonymous variant, A946T, in the interferon-induced helicase gene (IFIH1) was recently reported to be associated with type I diabetes. We genotyped this variant in the NARAC cohort and controls and found results similar to those observed in type I diabetes patients, that the minor allele (T) was reduced in cases compared with healthy controls. In the type I diabetes cases the minor allele frequency was 0.35 in cases and 0.39 in controls. We found the minor allele frequency was 0.37 in RA cases and 0.41 in controls (p = 0.02 in 653 independent cases and 1344 controls, p = 0.005 in an analysis including all affecteds that discounts sibs for their relatedness, i.e., the effective number of cases = 824). Replication studies are underway. Other candidate genes evaluated: Additional candidate genes evaluated included FCRL3 (Fc receptor-like 3), which is associated with RA in the Japanese, NFKB1 (encoding the p50 NF kappa B subunit), and FCGR3A, which is associated with RA in the UK. In the NARAC cohort none of the polymorphisms we evaluated were associated with RA. Conclusions and Significance The data of the last year support the notion that multiple non-HLA genes confer modest levels of risk for RA, and continue to support the likelihood of an RA susceptibility locus on chromosome 18q. During the next year, we plan to continue studies of this region. We will also continue studies of genes not on chromosome 18, including PADI4 and IFIH1. As a part of the NARAC collaboration, we will also continue to provide in-depth analysis of regions within the newly identified linkage peaks and new regions identified by more global analyses, such as whole-genome association studies. Finally, we plan replication studies for those genomic regions that currently give equivocal results.

建立了遗传学和基因组分支的复杂疾病遗传学单位（CDGU），以鉴定赋予对遗传复杂的风湿性和炎症性疾病的易感性的基因。该策略通常需要使用从给定的染色体区域或整个基因组中绘制的密集的标记物或整个基因组的标记图，对所讨论的疾病和/或关联研究的大量兄弟姐妹对的非参数链接分析和/或关联研究。 CDGU专注于类风湿关节炎（RA），该关节炎影响了全球多达1％的人口。这项工作以我们长期参与北美类风湿关节炎联盟（NARAC）为基础，这是一个大型协作小组，该小组从1000多个同胞配对中收集了RA的样本，以及来自Singleton RA病例和种族匹配的控制的同类。来自先前报告期的全基因组联系数据已经证实了HLA区域的主要遗传作用，并且还显示了染色体1P13、1Q43、6Q21、10Q21、10Q21、12Q12、12Q12、17P13、17p13和18Q21的连接的证据（P小于0.005）。由于18q21区域已在独立的法国和加拿大队列中复制，因此我们对基因组的这一部分进行了集中的联系不平衡（LD）研究。 NARAC先前使用基于珠子的光学技术（Illumina）进行了460例和460个对照组，对染色体18q21候选区域进行了密集的单核苷酸多态性（SNP）分析。成功键入的SNP的平均标记密度在10 MB区域为4.3 kb。鉴定了两个LD簇，每个簇都有几个SNP，并有证据表明与RA连接（P值小于或等于0.001）。 去年的结果 全基因组分析：在过去的一年中，我们使用> 5700 SNP（Illumina）（以前的全基因组链接分析使用了379个微映射标记）对NARAC同胞对进行了第二个全基因组分析。这项更有信息的分析确定了11p12和2q33上的两个新区域，并有很强的证据表明与疾病易感性基因座有联系（LOD> 3.0）。此外，在染色体4q25、5p12和10q21上发现了具有暗示性证据的新区域，并在18Q21、20P13和1Q41-42的先前鉴定的区域获得了额外的支持。 对18Q21候选染色体候选区域的分析：在过去的一年中，我们进一步研究了我们去年确定的两个18q21地区，也研究了第三个区域，在该区域中，我们发现了4-SNP单倍型，并有证据表明与RA易感性基因座相关的证据。在对先前识别区域的分析中增加了656例独立病例和604个对照，加强了p = 0.0005和p = 0.0009的统计显着性，当校正欧洲亚群中可能分层时，保持了名义意义。区域1中最密切相关的SNP位于转录因子基因，OneCut2和编码铁螯合酶Fech的基因之间。在该区域中最强相关的SNP的次要等位基因频率在RA病例中的频率高于对照组（在RA病例中为0.35，对照中的0.29）。区域2中最强烈的SNP在肺，小脑和因软骨培养的原代细胞中表达的部分转录本中是TCF4基因的远端。这些SNP之一将导致在预测蛋白的残基95（R95Q）处取代谷氨酰胺代替精氨酸（P = 0.002）。在三分之一的新确定的18q区域中，我们发现了一个相对罕见的4-SNP单倍型，并具有与RA相关的证据（在460个对照中发现单倍型为1.6％的频率，而在459个独立病例P = 0.0006的频率为4.4％）。我们对总共1386个对照组和667个独立病例进行了基因分型样品，发现对照组的单倍型频率为2.1％，病例分别为3.9％（Chi Square Analysis P = 0.001，置换分析，P = 0.008）。包含该单倍型的基因组区域位于区域1的远端，4个SNP位于Fech内，编码铁胆管酶，这是一种导致血红素合成途径的最后一步的线粒体酶，并且在红血病中突变在异性含量的蛋白质中。对这三个区域的进一步评估需要进行目前正在进行的另外1000例病例和对照的基因分型。 肽基精氨酸脱氨酸酶（IV型（PADI4）基因的变异缔合：来自另一组的早期研究表明，肽基精氨酸脱氨酸酶的特定变异和单倍型，IV型（PADI4）基因与日本人之间的RA相关。在上一个报告期间，我们发现高加索人中与RA相关的PADI4单倍型。为了彻底评估PADI4基因区域内的变体，我们为位于该75 Kb基因区域内的69个多态性SNP进行了基因分型的HAPMAP CEU样品。然后，我们选择了22个标签SNP，这些标签SNP非偿还了所有69个SNP，并为这22个SNP的NARAC病例和对照组进行了基因分型。我们发现，两个TAG SNP与RA相比，与PADI4-94相比，这是日本人口中报告的最密切相关的SNP。我们以两种疾病与SNP相关的强LD中的所有SNP进行了基因分型，并确定了总共3个SNP，全部位于PADI4的第一个内含子中，对照组的次要等位基因频率为5-6％，情况下为10-11％（P等于0.00006至0.00009）。当我们将分析限制为453个类风湿因子阳性病例时，P值为0.000005，这表明该缔合在这种表型上更均匀的组中更强。当前的研究重点是确定这些非编码SNP是否与转录效率的差异有关。 在干扰素诱导的解旋酶基因中与I型糖尿病相关的变体的分析：非同义变体A946T在干扰素诱导的解旋酶基因（IFIH1）中最近据报道与I型糖尿病相关。我们在NARAC队列和对照组中对这种变体进行了基因分型，发现与I型糖尿病患者中观察到的结果相似，与健康对照相比，在病例中降低了未成年等位基因（T）。在I型糖尿病病例中，次要等位基因频率为0.35，对照组为0.39。我们发现，在RA病例中，次要等位基因频率为0.37，对照组为0.41（653例独立病例和1344个对照中的p = 0.02，分析中的p = 0.005，包括所有对其相关性打折SIB的受影响的人，即有效的病例数= 824）。复制研究正在进行中。 评估的其他候选基因：评估的其他候选基因包括与日语中的RA相关的FCRL3（FC受体样3），NFKB1（编码P50 NF Kappa B亚基）和FCGR3A，与RA有关。在NARAC队列中，我们评估的多态性均与RA有关。 结论和意义 去年的数据支持以下观点：多个非HLA基因赋予RA的适度风险水平，并继续支持RA易感性基因座的可能性18Q。在明年，我们计划继续对该地区进行研究。我们还将继续研究未在包括PADI4和IFIH在内的不包括染色体的基因。作为NARAC合作的一部分，我们还将继续对新确定的链接峰和通过更多全球分析（例如全基因组协会研究）确定的新区域内的区域进行深入分析。最后，我们计划为当前带来模棱两可结果的基因组区域的复制研究。