Expression data from quadriceps muscle of young DMD patients and age matched controls

Albeit increased serum CK level and abnormal muscle histology are always present, boys with DMD are phenotipically indistinguishable from the normal ones at birth and, in their first years of life, acquire early motor milestones at normal times. A clear defect in muscle function becomes generally apparent by the end of the second year. As the disease is typically diagnosed between the ages of 3 and 7, the first two years are often considered and referred to as clinically presymptomatic. As a defined gene expression signature was shown to characterize these symptomatic patients we sought to investigate whether and to which extent alterations may be also present in muscle from presymptomatic DMD infants. To this aim, we used the Affymetrix technology to compare the individual expression profiles of 19 DMD patients with age at biopsy scattered along the first two years of the disease with those of 14 age matched controls. This approach allowed us to describe with high resolution the altered transcriptional state that characterizes this early, presymptomatic phase of the disease and highlight some molecular pathways as potential critical targets in the pathophysiology of the disease. Keywords: two populations study -Subjects Relevant data about the participants are posted under supplementary material (Table ST1). All DMD patients (n=22) had diagnosis of Duchenne Muscular Dystrophy based on the absence of dystrophin immunoreactivity on quadriceps muscle sections. None of the participants at the time of biopsy was or had been under corticosteroid treatment. Control biopsies (n=14) were selected from diagnostic specimens which were not given a diagnosis of neuromuscular disease nor showed any non-specific myopathic signs. A MIAME compliant description of this study is posted under supplementary Material (MIAME compliance). -RNA extraction. Total RNA was extracted from frozen quadriceps muscle biopsies by TriZol. (TriZol reagent, Invitrogen). RNA was further purified using the RNAeasy mini kit following the RNA cleanup protocol as indicated by the manufacturer (QIAGEN). RNA purity and integrity was assessed by spectophotometric analysis and agarose gel electrophoresis. -Affymetrix Genechips. In this work we made use of one chip type: the affymetrix HG-U133A genechipCRNA synthesis was performed using 5ug of total RNA as template, as described in the Affymetrix Gene Expression Manual. Genechips were washed and stained in an Affymetrix fluidic station 430. To avoid overcorrelation, samples were processed 8 at the time and arranged to have both DMD and control samples in each experimental session.尽管血清肌酸激酶（Creatine Kinase, CK）水平升高与肌肉组织学异常是杜氏肌营养不良症（Duchenne Muscular Dystrophy, DMD）的典型表现，但患儿在出生时与正常男婴表型无差异，且在生命最初数年的运动发育里程碑达成时间与正常儿童一致。通常在2岁末时，肌肉功能的明显缺陷才会显现。由于该病通常在3至7岁时确诊，因此患病最初两年常被视为临床前症状期。 鉴于已有研究证实症状期患者存在特征性基因表达特征，本研究旨在探究临床前症状期的杜氏肌营养不良症婴儿的肌肉组织中是否存在此类表达改变，以及改变的程度如何。为此，我们采用Affymetrix技术，对比了19例活检时处于患病最初两年的杜氏肌营养不良症患者与14例年龄匹配对照者的个体基因表达谱。该方法使我们得以高分辨率地刻画该病早期临床前症状期的特征性转录状态改变，并明确了若干分子通路作为该病病理生理学的潜在关键靶点。 关键词：双队列研究——研究对象 受试者的相关数据已上传至补充材料（表ST1）。所有22例杜氏肌营养不良症患者均通过股四头肌肌肉切片中肌营养不良蛋白免疫反应性缺失确诊。所有受试者在活检时均未接受或既往未接受糖皮质激素治疗。14例对照活检样本选自未被诊断为神经肌肉疾病、且无任何非特异性肌病表现的诊断性标本。本研究符合MIAME（微阵列实验最少信息规范）的描述已上传至补充材料（MIAME合规性说明）。 ——RNA提取 采用TriZol试剂（Invitrogen公司出品）从冰冻股四头肌活检标本中提取总RNA。随后按照制造商QIAGEN提供的RNA纯化方案，使用RNAeasy迷你试剂盒完成RNA的进一步纯化。通过分光光度分析与琼脂糖凝胶电泳评估RNA的纯度与完整性。 ——Affymetrix基因芯片 本研究使用一款芯片类型：Affymetrix HG-U133A基因芯片。按照Affymetrix基因表达实验手册的方法，以5μg总RNA为模板进行cRNA合成。基因芯片在Affymetrix流体工作站430中完成洗涤与染色。为避免样本过度相关，实验以8个样本为一批进行处理，且每批实验中均同时包含杜氏肌营养不良症样本与对照样本。