Functional Genomics of Psoriasis

描述(申请人提供)：最近对牛皮癣的全基因组关联研究(GWAS)已经确定了41个牛皮癣易感基因座，我们目前正在将多种遗传和基因组资产应用于牛皮癣的分子遗传学解剖，这为鉴定PSC和PSA的因果变异提供了一个独特而强大的平台。寻找疾病相关基因变异的功能基础是分析银屑病和所有复杂遗传疾病疾病关联的主要目标，因为它提供了基因和表型之间的分子联系。然而，到目前为止，令人信服的疾病相关变异特异性功能的证明在牛皮癣以及其他复杂的遗传疾病中并不常见。在许多情况下，这是由于对功能变体的确定不完整和/或不准确。我们将为我们独特的数据集带来的统计专业知识将使我们能够更好地定义用于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，为识别的变体的功能探索提供了补充平台。最后，重要的是要注意到，对单个变体的功能分析并不是本研究的唯一目标。为了通过开发新的治疗方法使患者受益，并实现个性化药物的承诺，严格识别他们所描绘的分子通路是至关重要的。基于这些考虑，我们提出了一个假设，即利用目前或即将获得的数据，结合与牛皮癣相关的生物统计学和生物学专业知识，可以识别单个基因变异的功能和它们描绘的途径。为了验证这一假设，我们提出了以下具体目标：1.使用新的统计工具准确识别银屑病的因果变异候选。这将通过以下方式实现：(A)应用多轮 将条件分析和优先连锁不平衡(PLD)分析用于GWAS-iChip荟萃分析、靶向重测序、PSA GWAS和外显子组阵列数据集；(B)对PSA和PSC之间的MHC差异进行扩展分析；以及(C)在扩展样本中进一步分析已识别的变异。2.系统地预测目标1中确定的与疾病相关的变异的潜在影响。这将通过(A)功能预测来实现 这些研究包括：(A)对新发现的编码变体的影响进行评估；(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影响；(D)对可能在功能上连接多种变体的途径进行系统评估。3.使用 基于角质形成细胞(KC)的平台。这将通过使用(A)从携带不同TRAF3IP2基因的个体获得的正常人角质形成细胞(NHK)和(B)在内源性TRAF3IP2沉默或不沉默的情况下将“野生型”VS变异等位基因导入永生化KC来测量TRAF3IP2中D10N变体的信号转导反应作为基因型的函数。我们还将(C)将这一方法扩展到从目标1和目标2中确定的可能在KC中重要的预测破坏性变体。4.使用基于血液的活细胞平台对已确定的变异体进行功能测试。这将会实现的 通过(A)从已知基因型个体的血液中分离出TYK2中P1104A和I684S变异的单核细胞和T细胞，然后测量信号转导反应作为基因型的函数。我们还将(B)将这些研究扩展到从AIMS 1和AIMS 2中确定的其他可能对免疫细胞重要的基因座上预测的破坏性变异。

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.