Functional Genomics of Psoriasis

描述（由申请人提供）：最近的银屑病全基因组关联研究（GWAS）已确定了 41 个银屑病易感位点，我们目前正在将多种遗传和基因组资产应用于银屑病的分子遗传解剖，这为识别 PsC 和 PsA 的因果变异提供了一个独特的强大平台。寻找疾病相关遗传变异的功能基础是分析牛皮癣和所有复杂遗传性疾病疾病关联的一个主要目标，因为它提供了基因型和表型之间的分子联系。然而，迄今为止，在银屑病以及其他复杂的遗传性疾病中，令人信服地证明疾病相关的变异特异性功能并不常见。在许多情况下，这是由于功能变体的确定不完整和/或不准确造成的。我们将利用我们独特的数据集提供的统计专业知识将有助于更好地定义功能研究的分子靶标。此外，我们的实验室在角质形成细胞生物学和免疫学方面拥有强大的专业知识，为已识别变异的功能探索提供补充平台。最后，值得注意的是，个体变体的功能分析并不是本研究的唯一目标。为了通过开发新疗法使患者受益并实现个性化医疗的承诺，严格识别它们所描绘的分子途径至关重要。 基于这些考虑，我们提出这样的假设：利用当前或即将获得的数据，并采用整合与银屑病相关的生物统计学和生物学专业知识的综合方法，可以识别个体遗​​传变异的功能及其描绘的途径。为了检验这一假设，我们提出以下具体目标： 1. 使用新型统计工具准确识别银屑病的候选因果变异。这将通过 (a) 应用多轮来完成 对 GWAS-IChip 荟萃分析、靶向重测序、PsA GWAS 和外显子组阵列数据集进行条件分析和优先连锁不平衡 (PLD) 分析； (b) PsA 和 PsC 之间 MHC 差异的扩展分析； (c) 对扩展样本中已识别的变异进行进一步分析。 2. 系统地预测目标 1 中确定的疾病相关变异的潜在影响。这将通过 (a) 预测功能来实现 新发现的编码变体的影响； (b) 利用 ENCODE 数据库对非编码变体进行系统评估； (c) 利用我们的 RNASeq 数据评估候选顺式作用变体的效果； (d) 对可能在功能方面连接多个变体的途径进行系统评估。 3. 使用已识别的变体进行功能测试 基于角质形成细胞（KC）的平台。这将通过测量信号转导反应作为 TRAF3IP2 中 D10N 变体基因型的函数来实现，使用 (a) 从携带不同 TRAF3IP2 基因型的个体收获的正常人角质形成细胞 (NHK) 和 (b) 将“野生型”与变体等位基因转染到永生化 KC 中，无论是否沉默内源 TRAF3IP2。我们还将 (c) 将此方法扩展到从目标 1 和 2 中识别出的预测破坏性变异，这些变异可能在 KC 中很重要。 4. 使用基于血液的活细胞平台对已识别的变体进行功能测试。这将实现 (a) 从 TYK2 中 P1104A 和 I684S 变体已知基因型的个体的血液中分离单核细胞和 T 细胞，然后测量作为基因型函数的信号转导反应。我们还将 (b) 将这些研究扩展到预测目标 1 和 2 中确定的其他基因座上可能对免疫细胞重要的破坏性变异。

DESCRIPTION (provided by applicant): Recent genome-wide association studies (GWAS) of psoriasis have identified 41 psoriasis susceptibility loci, and we are currently applying multiple genetic and genomic assets to the molecular genetic dissection of psoriasis, which provide a uniquely powerful platform for identification of causal variants in PsC and PsA. Finding the functional basis of disease-associated genetic variation is a major goal in the analysis of disease associations in psoriasis and all complex genetic disorders, because it provides a molecular link between genotype and phenotype. To date, however, convincing demonstration of disease associated variant-specific function is uncommon in psoriasis as well as in other complex genetic disorders. In many instances, this has been due to incomplete and/or inaccurate determination of the functional variants. The statistical expertise that we will bring t bear on our unique datasets will allow much better definition of molecular targets for functional studies. Moreover, our laboratories have strong expertise in keratinocyte biology and immunology, providing complementary platforms for the functional exploration of identified variants. Finally, it is important to note that functional analysis of individual variants is not te only goal of this research. In order to benefit patients via the development of new therapies and fulfill the promise of personalized medicine, it is of key importance to rigorously identify the molecular pathways that they delineate. Based on these considerations, we advance the hypothesis that the functions of individual genetic variants and the pathways they delineate can be identified, using currently or soon-to-be available data, given an integrated approach that integrates biostatistical and biological expertise relevant to psoriasis. To test this hypothesis, e propose the following specific aims: 1. To accurately identify causal variant candidates in psoriasis using novel statistical tools. This will be accomplished by (a) applying multiple rounds of conditional analysis and preferential linkage disequilibrium (PLD) analysis to GWAS-IChip meta-analysis, targeted resequencing, PsA GWAS, and exome array datasets; (b) expanded analysis of MHC differences between PsA and PsC; and (c) further analysis of identified variants in an expanded sample. 2. To systematically predict the potential effects of the disease-associated variants identified in Aim 1. This will be accomplished by (a) prediction of functional effects of newly-identified coding variants; (b) systematic assessment of non-coding variants utilizing the ENCODE database; (c) utilization of our RNASeq data for assessment of the effects of candidate cis-acting variants; and (d) systematic assessment of pathways likely to connect multiple variants in functional terms. 3. Functional testing of identified variants using a keratinocyte (KC)-based platform. This will be accomplished by measurement of signal transduction responses as a function of genotype for the D10N variant in TRAF3IP2 using (a) normal human keratinocytes (NHK) harvested from individuals bearing different TRAF3IP2 genotypes and (b) transfection of "wild type" vs variant alleles into immortalized KC with or without silencing of endogenous TRAF3IP2. We will also (c) extend this approach to predicted damaging variants identified from Aims 1 and 2 as likely to be important in KC. 4. Functional testing of identified variants using a blood-based, living-cell platform. This will be accomplished by (a) separation of monocytes and T-cells from the blood of individuals of known genotypes for the P1104A and I684S variants in TYK2, followed by measurement of signal transduction responses as a function of genotype. We will also (b) extend these studies to predicted damaging variants at other loci identified from Aims 1 and 2 as likely to be important in immunocytes.