Disease chronicity in juvenile dermatomyositis (JDM): Epigenetic clues

描述(申请人提供)：幼年皮肌炎(JDM)是最常见的儿科炎症性肌病，发病于幼儿(平均发病年龄为6.7岁；女童：男童比例为2.3：1)，死亡率为1-2%，继发于营养不良钙化和继发性肌挛缩。这些儿童的生活质量有所下降，但慢性炎症的作用尚未得到评估。未经治疗的炎症持续时间是一个关键因素：在基因表达谱研究中，未经治疗的症状超过两个月与慢性皮疹和血管重塑基因上调有关。慢性皮疹还与甲皱毛细血管末端环丢失、药物吸收障碍有关，而且作为年轻人，患者似乎容易过早地出现心血管损害。对内皮细胞的损伤是由干扰素-1和肿瘤坏死因子-1介导的，这两种细胞因子在JDM血清和组织中均升高。肿瘤坏死因子-1水平的升高与肿瘤坏死因子-1-308启动子区域的多态性有关。我们还发现在JDM儿童中，OPN和TNF-1启动子区之间存在复杂的基因-基因-性别相互作用。这在JDM中定义了一个高血清干扰素-1亚群，这可能是导致JDM在女孩中发病率增加的原因之一。在治疗JDM时，公认的治疗指南是血清肌酶水平，它很快就会正常化，留下一个空白。为JDM提供有效治疗的一个主要障碍是缺乏炎症的生物标志物以及疾病严重程度的预后指标。这项研究的目的是确定表观遗传学机制-全球甲基化和miRNA表达的差异-在解剖慢性炎症和性别对JDM微血管病变的影响方面至关重要。我们假设：1)慢性JDM患者的生活质量下降与其说与虚弱有关，不如说与长期未经治疗的疾病相关的慢性皮肤受累有关，2)识别JDM肌肉和内皮前体细胞(EPC)中的表观遗传调节机制和特异性miRNAs将揭示新的致病机制，从而指导更有效的治疗方法的发展。为了实现这一目标，将对未经治疗的JDM儿童的特征良好的肌肉活检进行比较：1)病程短(1个月)与长(2个月)，以及2)年龄匹配的男孩与女孩，病程也匹配。在特定目标1，全球甲基化研究中，将JDM与对照组进行比较，将确定低甲基化区域，如WT1和Hox基因，并测试它们与miRNA表达强度水平的关系。具体目标2将确定miRNA表达水平与儿童的生活质量、疾病活动评分(DAS)以及他们的肿瘤坏死因子-1-308 AA/AG与GG状态的关系。例如，我们发现miR-34a在JDM GG中上调，病程短，调节转录激活物Bcl12的表达，这在凋亡途径中很重要。在具体目标3中，将从JDM和年龄性别匹配的对照组的血液中分离内皮前体细胞(EPC)，并对其表观遗传学特征进行表征。此外，它们的功能将在体外进行研究，并与儿童甲皱毛细血管末端排环结构丧失的程度进行比较。然后，将对分离的EPC进行功能增减实验，目的是恢复正常。预计从拟议工作中获得的科学知识将为我们今后的决策提供信息，并导致更合理和有效的干预措施。 公共卫生相关性：青少年皮肌炎(JDM)是一种儿童自身免疫性疾病，其中小血管受损；慢性炎症与持续性皮疹、活动范围丧失、钙化沉积甚至执行爬楼梯或举起物体等任务的能力有关，因此影响儿童的生活质量。在这项研究中，JDM儿童的生活质量将被确定并与他们的表观遗传状态相关--由基因和环境决定的表观遗传表型(个体的可观察特征)的遗传变化--通过测试来自未治疗的JDM儿童的诊断肌肉活检，与疾病持续时间(d 2个月)较短(d 2个月)的JDM儿童和男孩(与女孩(病程匹配)和年龄、性别匹配的健康对照组相比)。目标是确定特定的标记物，如microRNA，或可能是修复血管损伤和确定更有效的医疗干预的关键的“开启基因”。

DESCRIPTION (provided by applicant): Juvenile dermatomyositis (JDM) is the most common pediatric inflammatory myopathy and occurs in young children (mean age at onset of symptoms, 6.7 years; girl: boy ratio=2.3:1) with 1-2% mortality and increased morbidity secondary to dystrophic calcifications and secondary contractures. The children have a decreased quality of life, but the role of chronic inflammation has not been evaluated. The duration of time of the untreated inflammation is a critical factor: untreated symptoms of greater than two months are associated with a chronic rash and upregulation of vascular remodeling genes on gene expression profile studies. The chronic rash is also associated with loss of nailfold capillary end row loops, impaired absorption of medications, and as young adults, the patients appear prone to premature cardiovascular damage. Damage to endothelial cells is mediated by IFN- 1 and TNF-1, and both cytokines are increased in JDM sera and tissue. Increased TNF-1 levels are associated with a polymorphism in the promoter region of TNF-1-308. We also found a complex gene-gene-gender interaction between the OPN and TNF-1 promoter regions in JDM children. This defined a high serum IFN-1 subgroup within JDM, which may contribute to the increased incidence of JDM in girls. In treating JDM, the accepted guide to therapy is serum levels of muscle enzymes, which normalize rapidly, leaving a void. A major barrier to providing effective therapy in JDM is the lack of biomarkers of inflammation as well as prognostic indicators of disease severity. The purpose of this study is to identify epigenetic mechanisms - differences in global methylation and miRNA expression - critical in dissecting the impact of chronic inflammation and gender on JDM microvasculopathy. We hypothesize: 1) Decreased quality of life in chronic JDM is related less to weakness than to the chronic skin involvement, associated with long untreated disease, and, 2) identification of epigenetic regulatory mechanisms and specific miRNAs in JDM muscle and in endothelial precursor cells (EPCs) will disclose novel pathogenetic mechanisms that will guide the development of more effective therapy. To accomplish this goal, well-characterized muscle biopsies from untreated children with JDM will be compared: 1) short duration (< 2 month) vs. long (e 2 months) duration, and 2) age-matched boys compared with girls, also matched for disease duration. In Specific Aim 1, global methylation studies, comparing JDM with controls will identify areas of hypomethylation, such as the WT1 and HOX genes, and test their relation to intensity levels of miRNA expression. Specific Aim 2 will determine the association of levels of miRNA expression with the child's quality of life, disease activity scores (DAS), and their TNF-1-308 AA/AG vs GG status. For example, we found that miR-34a, upregulated in JDM GG with short disease duration modulates the expression of the transcriptional activator Bcl12, important in the apoptotic pathway. In Specific Aim 3, endothelial precursor cells (EPCs) will be isolated from the blood of JDM and age-gender matched controls and their epigenetic features characterized. In addition, their function will be studied in vitro and compared with the extent of the child's structural loss of nailfold capillary end row loops. The isolated EPCs will then be tested in gain/loss of function experiments, with the goal of restoration of normality. It is anticipated that the scientific knowledge gained from the proposed work will inform our future decision making and lead to more rational and effective interventions. PUBLIC HEALTH RELEVANCE: Juvenile dermatomyositis (JDM) is an autoimmune disease of children, in which there is damage to the small blood vessels; chronic inflammation is associated with persistent rash, loss of range of motion, deposits of calcifications and even the ability to perform tasks like climbing stairs or lifting objects and, therefore, affects the child's quality of life. In this study, the quality of life of the children with JDM will be determined and correlated with their epigenetic status - inherited changes in phenotype (individual's observable traits) determined by genes and the environment - by testing diagnostic muscle biopsies from untreated children with JDM with long (>2 months) compared with short disease duration (d 2 months) and boys compared with girls (matched for disease duration) and age-, gender-matched healthy controls. The goal is to identify specific markers, like microRNA, or "turned on genes" that may be key to repair the vascular damage and to identify more effective medical interventions.