Department of Neurology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA.
Hum Mol Genet. 2010 Aug 15;19(16):3233-53. doi: 10.1093/hmg/ddq232. Epub 2010 Jun 8.
Advances in genomics and proteomics permit rapid identification of disease-relevant genes and proteins. Challenges include biological differences between animal models and human diseases, high discordance between DNA and protein expression data and a lack of experimental models to study human complex diseases. To overcome some of these limitations, we developed an integrative approach using animal models, postmortem human material and a combination of high-throughput microarray methods to identify novel molecular markers of amyotrophic lateral sclerosis (ALS). We used laser capture microdissection coupled with microarrays to identify early transcriptome changes occurring in spinal cord motor neurons or surrounding glial cells. Two models of familial motor neuron disease, SOD1(G93A) and TAU(P301L), transgenic mice were used at the presymptomatic stage. Identified gene expression changes were predominantly model-specific. However, several genes were regulated in both models. The relevance of identified genes as clinical biomarkers was tested in the peripheral blood transcriptome of presymptomatic SOD1(G93A) animals using custom-designed ALS microarray. To confirm the relevance of identified genes in human sporadic ALS (SALS), selected corresponding protein products were examined by high-throughput immunoassays using tissue microarrays constructed from human postmortem spinal cord tissues. Genes that were identified by these experiments and located within a linkage region associated with familial ALS/frontotemporal dementia were sequenced in several families. This large-scale gene and protein expression study pointing to distinct molecular mechanisms of TAU- and SOD1-induced motor neuron degeneration identified several new SALS-relevant proteins (CNGA3, CRB1, OTUB2, MMP14, SLK, DDX58, RSPO2) and putative blood biomarkers, including Nefh, Prph and Mgll.
基因组学和蛋白质组学的进展使得快速鉴定与疾病相关的基因和蛋白质成为可能。挑战包括动物模型和人类疾病之间的生物学差异、DNA 和蛋白质表达数据之间的高度不一致以及缺乏研究人类复杂疾病的实验模型。为了克服其中的一些限制，我们开发了一种综合方法，该方法使用动物模型、尸检人类材料和高通量微阵列方法的组合，以鉴定肌萎缩侧索硬化症 (ALS) 的新型分子标志物。我们使用激光捕获显微切割与微阵列相结合的方法来鉴定脊髓运动神经元或周围神经胶质细胞中发生的早期转录组变化。使用两种家族性运动神经元疾病模型，SOD1(G93A)和 TAU(P301L)转基因小鼠在无症状前阶段进行研究。鉴定的基因表达变化主要是模型特异性的。然而，有几个基因在两种模型中都受到调节。使用定制的 ALS 微阵列，在无症状 SOD1(G93A)动物的外周血转录组中测试了鉴定基因作为临床生物标志物的相关性。为了确认鉴定基因在人类散发性 ALS (SALS) 中的相关性，使用组织微阵列构建物，通过高通量免疫测定法检查了选定的对应蛋白产物，这些组织微阵列来自人类尸检脊髓组织。通过这些实验鉴定的位于与家族性 ALS/额颞叶痴呆相关的连锁区域内的基因在几个家族中进行了测序。这项大规模的基因和蛋白质表达研究表明，TAU 和 SOD1 诱导的运动神经元变性具有不同的分子机制，确定了几个新的与 SALS 相关的蛋白（CNGA3、CRB1、OTUB2、MMP14、SLK、DDX58、RSPO2）和潜在的血液生物标志物，包括 Nefh、Prph 和 Mgll。
