Scleroderma Subtyping from Fresh and Archived Biopsies using NextGen Sequencing

描述（由申请人提供）：系统性硬化症（SSc）是一种自身免疫性疾病，表现为异质性和复杂的表型。该疾病的主要表现是皮肤纤维化、血管功能障碍和免疫系统激活。没有经过验证的诊断标志物。没有治愈性的治疗方法。三分之一的患者在诊断后10年内死亡。一些药物（开发用于其他适应症）目前正在进行临床试验，但是，结果指标（如改良的Rodnan皮肤评分）不足，患者人群的异质性阻碍了试验的有效性，因为只有一部分患者可能对任何给定的药物有反应。Celdara Medical正在通过将基因特征与临床表型相关联来分割这种异质性或“亚型”SSc，以提供患者预后的定量测量，临床试验期间的疗效评估，以及对疾病机制的深入了解，这将导致为每种SSc亚型设计药物。由于SSc是一种罕见疾病，皮肤活检的需求很高，用于DNA微阵列分析的新鲜活检样本的纵向积累很慢，这严重阻碍了测试的临床验证。对来自单个临床位点的存档福尔马林固定石蜡包埋（FFPE）组织块的回顾性分析是解决该问题的一种方法，然而，这些样品中RNA降解的程度（维斯维斯新鲜冷冻（FF）样品）使它们不适合于DNA微阵列分析。因此，我们建议开发一个强大的SSc子集的方法，使用测量基因表达的短读超高通量（UHTP）从FFPE和FF皮肤活检测序预测SSc亚型。在第一阶段，我们将实现以下目标：目标1。比较UHTS与NanoString技术在测量FFPE和FF样本基因表达谱方面的实用性。目标2.使用UHTS或Nanostring提高临床纵向数据的质量和样本量，并分析与内在子集相关的临床协变量。成功完成本文所述的工作将：1。允许临床验证，2.简化临床实施，3.降低患者分型的成本，以及4.通过纳入存档的样本，极大地快速扩展了基因表达样本数据库，可能导致对这种可怕而复杂的疾病的新的基于基因组学的见解。 公共卫生相关性：硬皮病是一种知之甚少的疾病，影响了30万美国人，并在诊断后10年内杀死了1/3的患者。对患者样本的基因分析揭示了这种疾病的亚型，每种亚型都有不同的症状，每种亚型都提出了不同的潜在治疗方法。该项目的目标是简化目前复杂的样品制备方案，同时提高遗传分析的鲁棒性和降低成本。

DESCRIPTION (provided by applicant): Systemic sclerosis (SSc) is an autoimmune disease that presents with a heterogeneous and complex phenotype. Major manifestations of the disease are skin fibrosis, vascular dysfunction, and immune system activation. There are no validated diagnostic markers. There are no curative treatments. One in three patients dies within 10 years of diagnosis. Some drugs (developed for other indications) are currently in clinical trials however, outcome measures such as the modified Rodnan skin score are inadequate, and heterogeneity in patient populations hinders trial effectiveness since only a subset of patients is likely to respond to any given drug. Celdara Medical is segmenting this heterogeneity or "subtyping" SSc by associating gene signatures with clinical phenotypes to provide a quantitative measure for patient prognosis, assessment of efficacy during clinical trials, and insight into disease mechanisms which will lead to drugs designed for each SSc subtype. Because SSc is a rare disease, skin biopsies are in high demand and longitudinal accrual of fresh biopsy samples for DNA microarray analysis is slow, which severely hampers clinical validation of the test. A retrospective analysis of archived formalin fixed paraffin embedded (FFPE) tissue blocks from a single clinical site is one solution to this problem, however, the degree of RNA degradation in these samples (vis- `-vis fresh frozen (FF) samples) makes them unsuitable for DNA microarray analysis. We therefore propose to develop a robust SSc subsetting method using measurement of gene expression by short read ultra high-throughput (UHTP) sequencing from FFPE and FF skin biopsies to predict SSc subtype. In Phase I we will accomplish the following Aims: Aim 1. Compare the utility of UHTS to NanoString technology for the measurement of gene expression profiles from FFPE and FF samples. Aim 2. Increase the quality and sample size of clinical longitudinal data using either UHTS or Nanostring and analyze the clinical covariates associated with the intrinsic subsets. Successful completion of the work described herein will: 1. allow for clinical validation, 2. simplify clinical implementation, 3. reduce the cost of patient subtyping, and 4. dramatically and quickly expand the gene expression sample database through inclusion of archived samples, potentially leading to novel genomics- based insights into this terrible and complex disease. PUBLIC HEALTH RELEVANCE: Scleroderma is a poorly-understood disease which affects 300,000 Americans, and kills 1/3 of those afflicted within 10 years of diagnosis. Genetic analyses of patient samples have revealed subtypes of the disease, each with different prognoses, and each suggesting different potential therapeutics. The goal of this project is to simplify the currently complex sample preparation protocol while improving the robustness and decreasing the cost of the genetic analyses.