Mutation of senataxin alters disease-specific transcriptional networks in patients with ataxia with oculomotor apraxia type 2 [04_Cerebellum_and_brain]

Senataxin, encoded by the SETX gene, contributes to multiple aspects of gene expression, including transcription and RNA processing. Mutations in SETX cause the recessive disorder ataxia with oculomotor apraxia type 2 (AOA2) and a dominant juvenile form of amyotrophic lateral sclerosis (ALS4). To assess the functional role of senataxin in disease, we examined differential gene expression in AOA2 patient fibroblasts, identifying a core set of genes showing altered expression by microarray and RNA-sequencing. To determine whether AOA2 and ALS4 mutations differentially affect gene expression, we overexpressed disease-specific SETX mutations in senataxin-haploinsufficient fibroblasts and observed changes in distinct sets of genes. This implicates mutation-specific alterations of senataxin function in disease pathogenesis and provides a novel example of allelic neurogenetic disorders with differing gene expression profiles. Weighted gene co-expression network analysis (WGCNA) demonstrated these senataxin-associated genes to be involved in both mutation-specific and shared functional gene networks. To assess this in vivo, we performed gene expression analysis on peripheral blood from members of 12 different AOA2 families and identified an AOA2-specific transcriptional signature. WGCNA identified two gene modules highly enriched for this transcriptional signature in the peripheral blood of all AOA2 patients studied. These modules were disease-specific and preserved in patient fibroblasts and in the cerebellum of Setx knockout mice demonstrating conservation across species and cell types, including neurons. These results identify novel genes and cellular pathways related to senataxin function in normal and disease states, and implicate alterations in gene expression as underlying the phenotypic differences between AOA2 and ALS4. Total RNA samples obtained from 1) an AOA2 patient and carrier fibroblast cell lines, 2) 2 biological replicates of haploinsufficient SETX fibroblast cell lines transfected with one of 4 different wild-type and mutant SETX constructs, 3) peripheral blood from 33 patients and carriers across 12 families, and 4) 2 tissues from 2 Setx knockout and 2 control mice were analyzed using expression microarray. This submission represents the microarray component of study.由SETX基因编码的Senataxin蛋白参与基因表达的诸多进程，涵盖转录与RNA加工环节。SETX基因的突变可引发两种神经遗传病：隐性遗传的2型眼运动共济失调（ataxia with oculomotor apraxia type 2, AOA2），以及显性遗传的青少年型肌萎缩侧索硬化症（amyotrophic lateral sclerosis 4, ALS4）。 为探究Senataxin在疾病中的功能作用，本研究对AOA2患者的成纤维细胞进行差异基因表达分析，通过基因芯片（microarray）与RNA测序（RNA-sequencing）筛选得到一组核心差异表达基因。 为明确AOA2与ALS4致病突变是否对基因表达存在差异化影响，本研究在Senataxin单倍体不足（senataxin-haploinsufficient）的成纤维细胞中过表达疾病特异性SETX突变体，观察到不同基因集的表达发生改变。该结果提示Senataxin功能的突变特异性改变参与疾病发病机制，同时为存在差异化基因表达谱的等位基因神经遗传病提供了全新研究范例。 加权基因共表达网络分析（Weighted gene co-expression network analysis, WGCNA）结果显示，这些与Senataxin相关的基因既参与突变特异性功能基因网络，也参与共享功能基因网络。 为在体内验证上述发现，本研究对12个AOA2家系成员的外周血进行基因表达分析，筛选得到AOA2特异性转录特征谱。 通过WGCNA分析，本研究在所有受试AOA2患者的外周血中鉴定出两个显著富集该转录特征谱的基因模块。 这两个基因模块具有疾病特异性，且在患者成纤维细胞以及Setx基因敲除小鼠的小脑组织中均保持稳定，表明其在不同物种与细胞类型（包括神经元）中具有保守性。 本研究结果鉴定出与正常及疾病状态下Senataxin功能相关的全新基因与细胞通路，并提示基因表达改变是AOA2与ALS4表型差异的潜在分子基础。 本研究采用基因芯片对以下四类总RNA样本进行表达分析：1）AOA2患者及其携带者的成纤维细胞系；2）转染4种野生型与突变型SETX构建体之一的SETX单倍体不足成纤维细胞系的2个生物学重复样本；3）来自12个家系的33名患者及携带者的外周血样本；4）2只Setx基因敲除小鼠与2只野生型对照小鼠的2份组织样本。 本提交数据为该研究的基因芯片分析部分。