Expression Profiling of Ehlers-Danlos Fibroblasts

描述（由申请人提供）： Ehlers-Danlos综合征（EDS）是一种全身性结缔组织疾病 由纤维胶原代谢缺陷引起。 EDS的诊断是 由于几种描述形式的临床特征重叠而复杂化 和基因座异质性。 我们假设真皮成纤维细胞来源于 形式EDS患者将显示基因表达的改变， 这是每个患者中导致EDS的突变基因的特征。 这些 表达模式可以通过微阵列技术鉴定， 用于指导EDS中的突变分析和识别新的候选基因。 在目标1中，将定义成纤维细胞系的基因表达变化 来源于COL 5A 1和COL 5A 2基因突变的患者， 典型EDS; C 0 L3 A1突变导致血管型EDS的患者; 而生腱蛋白X突变导致最近描述的 经典EDS的隐性变体。细胞系将通过以下方式进行表征： 少数基因的错误表达与 EDS基因之一，或通过使用聚类的全局表达模式， 算法 在目标2中，表达谱分析指导突变的能力被证明是有效的。 分析将进行测试。将对细胞进行表达谱分析 以前没有研究过的线路。 表现出表达变化的细胞系 已知EDS基因的特征将通过以下方法进行突变分析： 直接测序合适的基因。 表达谱分析也可能导致 在EDS中识别新的候选基因。预计部分 基因在所有或大多数细胞系中会一直被错误调节 携带已知的突变 虽然这些基因对于区分 在已知的突变中，它们本身就是良好的EDS候选基因。 在Aim中 3，这些基因将被测试其破坏胶原蛋白沉积的能力 在体外，其是EDS表型的细胞标志物。 为了做到这一点， 将产生腺病毒载体，其表达0.5至1.5kb的反义核酸， 来源于候选基因的序列，作为候选基因的3'非翻译区。 LacZ 这些结构使我们能够监测野生型成纤维细胞的感染 并产生对基因表达的有效抑制。胶原蛋白沉积将是 通过免疫组织化学和代谢标记分析。

DESCRIPTION (provided by applicant): The Ehlers-Danlos syndrome (EDS) is a generalized connective tissue disorder caused by defects in fibrillar collagen metabolism. Diagnosis in EDS is complicated by overlap in the clinical features of the several described forms and by locus heterogeneity. We hypothesize that dermal fibroblasts derived form EDS patients will display alterations in gene expression that are characteristic of the mutant gene responsible for EDS in each patient. These expression patterns can be identified by microarray technology and will be used to guide mutational analysis in EDS and to identify new candidate genes. In Aim 1, changes in gene expression will be defined for fibroblast cell lines derived from patients with mutations in the COL5A1 and COL5A2 genes causing classical EDS; patients with mutations in C0L3A1 causing vascular type EDS; and patients with mutations in tenascin-X causing a recently described recessive variant of classical EDS. Cell lines will be characterized either by a small number of genes whose mis-expression is specifically associated with one of the EDS genes, or by global patterns of expression using a clustering algorithm. In Aim 2, the ability of expression profiling to direct mutational analysis will be tested. Expression profiling will be carried out on cell lines not previously studied. Cell lines that demonstrate expression changes characteristic of known EDS genes will be subjected to mutational analysis by direct sequencing of the appropriate gene. Expression profiling may also lead to the identification of new candidate genes in EDS. It is expected that some genes will be consistently mis-regulated in all or most of the cell lines carrying known mutations. While these genes are not useful for distinguishing between known mutations, they are themselves good EDS candidate genes. In Aim 3, these genes will be tested for their ability to disrupt collagen deposition in vitro, which is a cellular marker for the EDS phenotype. To do this, adenoviral vectors will be created that express 0.5 to 1.5 kb of antisense sequence derived from the candidate gene as the 3' untranslated region of LacZ. These constructs allow us to monitor infection of wild-type fibroblasts and produce potent inhibition of gene expression. Collagen deposition will be analyzed by immunohistochemistry and metabolic labeling.