Transcription profiling of liver from wild type vs glycerol kinase knockout mice to understand the molecular pathogenesis of GKD

Glycerol kinase deficiency (GKD) is an X-linked inborn error of metabolism with metabolic and neurologic crises. Liver shows the highest level of glycerol kinase (GK) activity in humans and mice. Absence of genotype-phenotype correlations in patients with GKD indicate the involvement of modifier genes, including other network partners. To understand the molecular pathogenesis of GKD, we performed microarray analysis on liver mRNA from neonatal glycerol kinase (Gyk) knockout (KO) and wild type (WT) mice. Unsupervised learning revealed the overall gene expression profile of the KO mice was different from that of WT. Real time PCR confirmed differences for selected genes. Functional gene enrichment analysis was used to find 56 increased and 37 decreased gene functional categories. Pathway Assist analysis identified changes in gene expression levels of genes involved in organic acid metabolism indicating that GK was part of the same metabolic network which correlates well with the patients with GKD having metabolic acidemia during their episodic crises. Network component analysis (NCA) showed that transcription factors SREBP-1c, ChREBP, HNF-4alpha, and PPAR-alpha, had increased activity in the Gyk KO mice compared with WT mice; while SREBP-2 was less active in the Gyk KO mice. These studies show that Gyk deletion causes alterations in gene expression of genes in several regulatory networks and is the first time NCA has been used to expand on microarray data from a mouse knockout model of a human disease. Male WT and KO mouse pups were sacrificed on day of life (dol) 3 and each liver was harvested. Total RNA from 4 KO and 4 WT livers was isolated individually. cDNA was synthesized from the poly(A)+ mRNA in the total RNA, Biotin-tagged and fragmented to an average strand length of 100 bases (range 35-200 bases). Ten µg of each cRNA was hybridized onto an Affymetrix mus 430 2.0 GeneChip to analyze differences in liver gene expression between KO and WT mice. Day of life three was chosen because the mice are phenotypically symptomatic with statistically different parameters for hypoglycemia, acidosis; low bicarbonate and decreased base excess. On day of life 2 they are not significantly different from wild type in all of these important clinical phenotypes.甘油激酶缺乏症（Glycerol kinase deficiency, GKD）是一种X连锁遗传性代谢病，伴随代谢性与神经性危象。在人类与小鼠体内，肝脏的甘油激酶（glycerol kinase, GK）活性水平最高。GKD患者中未观察到基因型与表型的相关性，提示修饰基因（包括其他网络互作伴侣）参与了疾病进程。为阐明GKD的分子发病机制，本研究对新生期甘油激酶（Gyk）敲除（knockout, KO）与野生型（wild type, WT）小鼠的肝脏mRNA开展了基因芯片分析。无监督学习分析显示，KO小鼠的整体基因表达谱与WT小鼠存在显著差异。实时PCR验证了部分筛选基因的表达差异。功能基因富集分析共鉴定出56个上调功能类别与37个下调功能类别。Pathway Assist分析发现，有机酸代谢相关基因的表达水平发生改变，表明GK参与同一代谢网络，这与GKD患者在发作性危象期间出现代谢性酸中毒的临床表型高度吻合。网络组分分析（Network component analysis, NCA）结果显示：与WT小鼠相比，Gyk KO小鼠体内的转录因子SREBP-1c、ChREBP、HNF-4α及PPAR-α活性升高，而SREBP-2的活性则有所降低。本研究证实，Gyk基因缺失会导致多个调控网络中的基因表达发生改变，且本研究首次将网络组分分析（NCA）应用于人类疾病小鼠敲除模型的芯片数据拓展分析。本研究选取生后第3天（day of life, dol 3）的雄性WT与KO小鼠幼崽进行安乐死，采集其肝脏组织。分别从4只KO小鼠与4只WT小鼠的肝脏中独立提取总RNA。以总RNA中的poly(A)+ mRNA反转录合成cDNA，经生物素标记与片段化处理后，平均链长为100碱基（范围35-200碱基）。取10 μg每份的cRNA与Affymetrix小鼠基因组430 2.0基因芯片（Affymetrix mus 430 2.0 GeneChip）进行杂交，以分析KO与WT小鼠间肝脏基因表达的差异。选择生后第3天作为实验节点，是因为该时期小鼠已表现出典型症状，其低血糖、酸中毒、碳酸氢盐水平降低及碱剩余减少等参数均存在统计学差异；而生后第2天时，小鼠在上述关键临床表型上与野生型小鼠无显著差异。