Transcriptional profiling in childhood diseases

描述(申请人提供)：线粒体脑肌病是一组遗传异质性疾病，其分子诊断仍然非常困难。线粒体脑膜肌病的一种特殊形式称为Leigh综合征(LS)，是一种儿科神经退行性疾病，是多种能量代谢紊乱的表型表现，最常见的是细胞色素C氧化酶(COX)缺乏。由于与临床表型相关的基因座异质性，确定该病和其他线粒体疾病的分子基础仍然是一个巨大的挑战。目前，评估疑似线粒体疾病患者的最先进方法是进行肌肉活组织检查和呼吸链生化分析。然而，它并没有提供特定的基因诊断，这仍然是一项研究努力。研究人员希望探索利用培养的皮肤成纤维细胞的总RNA转录图谱来建立特定诊断的可行性。据推测，一些单基因线粒体疾病将有独特的转录图谱。要将其应用于LS和其他线粒体疾病，需要开发已知基因缺陷的数据集。来自研究人员实验室的初步数据显示，使用带有SURF1突变的COX缺陷成纤维细胞，聚类分析识别出以一致的方式上调或下调的基因亚集。研究人员建议使用额外的SURF1缺陷成纤维细胞来验证这些观察结果，方法是使用三个不同的软件包比较输出：Rosetta、GeneSpring和dChip。将进行三重比较，并与同样以三重分析的单个野生型对照进行比较。考察培养条件和传代次数对培养效果的影响。临床上可疑的样本将被分析，并根据已知的模式进行基因预测。在那些表现在培养细胞中的疾病中，这种方法提供了一种侵入性小得多的方法来确定特定的核基因缺陷。这些结果将有助于咨询，开发潜在的治疗方法，并评估长期结果。

DESCRIPTION (provided by applicant): Mitochondrial encephalomyopathies are a group of genetically heterogeneous disorders for which molecular diagnosis remains remarkably difficult. One particular form of mitochondrial encepaholmyopathy termed Leigh syndrome (LS) is a pediatric neurodegenerative condition and is a phenotypic manifestation of a variety of disorders of energy metabolism, most commonly cytochrome c oxidase (COX) deficiency. Because of locus heterogeneity associated with the clinical phenotype, identifying the molecular basis for this disease and other mitochondrial disorders remains an enormous challenge. Currently, the state-of-the-art in evaluating patients with suspected mitochondrial disorders is to perform a muscle biopsy and carry out biochemical assays of the respiratory chain. However, it does not provide a specific gene diagnosis, which remains a research endeavor. The investigators would like to explore the feasibility of establishing specific diagnoses using transcriptional profiling of total RNA from cultured skin fibroblasts. It is hypothesized that there will be unique transcriptional profiles for a number of single gene mitochondrial disorders. To apply this to LS and other mitochondrial diseases, data sets of known gene defects need to be developed. Preliminary data from the investigator's laboratory using COX deficient fibroblasts with mutations in SURF1 demonstrates that cluster analysis identifies subsets of genes that are either up or down regulated in a consistent fashion. The investigators propose to validate these observations using additional SURF1 deficient fibroblasts by comparing the output using three different software packages: Rosetta, GeneSpring, and dChip. Triplicate comparison will be made and compared to a single wild-type control also analyzed in triplicate. The effect of culture conditions and passage number will be examined. Clinically suspected samples will be analyzed and gene predictions made based upon known patterns. In those disorders manifested in cultured cells, this approach offers the potential for a much less invasive means of determining the specific nuclear gene defect. These results will help in counseling, the development of potential therapies, and assessing long-term outcomes.