Epigenetic regulation of Wilson Disease

 描述(申请人提供)：肝豆状核变性(WD)是一种常染色体隐性遗传性肝脏铜蓄积疾病，由铜转运P型ATPase功能障碍引起，该酶在肝细胞利用和胆汁铜排泄中起关键作用。WD的特点是其临床、肝脏严重程度和神经精神症状具有显著的异质性。未能确定特定的基因型-表型相关性可能部分是由于存在影响表型表达的表观遗传因素。我们的假设是，WD的表型及其对抗铜治疗的潜在反应可能受到母体饮食甲基基团对子代肝脏蛋氨酸代谢的影响和氧化磷酸化机制的调节。我们的目标将是1)在TX-j WD小鼠模型中确定母体甲基状态对肝脏基因组甲基化、氧化磷酸化途径基因转录水平和子代线粒体功能的影响以及它们对铜螯合的反应；2)表征人肝DNA甲基化和血浆酰肉碱作为不同表型WD患者线粒体功能标志的特征。我们将在TX-J小鼠模型中测试第一个目标 我们以前的特征是显示出全球DNA低甲基化和与肝脏蛋氨酸代谢、基因组DNA甲基化和肝损伤相关的基因表达的变化。我们将为杂合子鼠(ATP7B+/-)提供对照或添加胆碱的日粮，并研究这些日粮对肝脏铜蓄积的24周龄野生型和纯合子(ATP7B-/-)TX-j子代小鼠肝脏基因组DNA甲基化和基因表达的影响，以及它们对铜络合剂的反应。此外，我们将与美国和欧洲的医疗机构合作，对未染色的肝活检切片和WD患者组织块中的DNA进行全球DNA甲基化研究。我们将特别关注线粒体功能基因表达途径，我们已经显示了在TX-j小鼠从胚胎到成年生命的肝脏中受到影响。拟议的实验将需要使用表观遗传学技术，包括利用甲基C-seq的甲基组分析和肝脏中的焦磷酸测序来确定小鼠全基因组中CpG位点的甲基化状态，使用人肝活检切片的免疫染色来确定全球DNA甲基化模式，使用甲基组分析来进行全球DNA甲基化量化，以及基因特异性甲基化分析，以及测量WD受试者血清/血浆样本中的酰卡尼汀作为线粒体功能的指标。预期的发现是，母体饮食供应胆碱可以防止小鼠蛋氨酸代谢异常和基因特异性DNA甲基化改变，不同表型的WD患者将有不同的DNA甲基化和线粒体损伤模式。

 DESCRIPTION (provided by applicant): Wilson disease (WD) is an autosomal recessive disorder of hepatic copper accumulation, which is caused by the dysfunction of a copper-transporting P-type ATPase that is crucial in the hepatocellular utilization and biliary excretion f copper. WD is characterized by a remarkable heterogeneity in its clinical hepatic severity and neuropsychiatric presentations. The failure to identify specific genotype-to-phenotype correlations could be due in part to the presence of epigenetic factors affecting phenotype expression. Our hypothesis is that WD phenotype and its potential response to anti copper treatment may be regulated by maternal dietary methyl group effects on offspring hepatic methionine metabolism and oxidative phosphorylation mechanisms. Our aims will be 1) To determine the effects of maternal methyl status on hepatic genomic methylation, transcript levels of oxidative phosphorylation pathway genes and mitochondrial function of offspring and their response to copper chelation in the tx-j mouse model of WD; 2) To characterize human liver DNA methylation and plasma acyl carnitines as markers of mitochondrial function in WD patients with different phenotypic presentation. We will test the first aim in the tx-j mouse model of WD, which we have previously characterized as showing global DNA hypo-methylation and changes in the expression of genes related to hepatic methionine metabolism, genomic DNA methylation, and liver injury. We will provide control or choline supplemented diets to heterozygous dams (Atp7b +/-) and study the effects of these diets on hepatic genomic DNA methylation and gene expressions and their response to copper chelating agents in 24 week wild type and homozygous (Atp7b - /-) tx-j offspring mice with hepatic copper accumulation. In addition, we will conduct global DNA methylation studies in unstained liver biopsy slides and DNA from tissue blocks from WD patients in collaboration with medical institutions in both the United States and Europe. We will focus particularly on mitochondrial function gene expression pathways which we have shown are affected in tx-j mice liver from fetal to adult life. The proposed experiments will entail the use of epigenetic techniques including methylome analysis by MethylC- seq and pyro-sequencing in liver to determine the methylation status of CpG sites genome-wide in mice, the use of immune staining of human liver biopsy slides for global DNA methylation patterns, methylome analysis for global DNA methylation quantification, and gene specific methylation analysis, and measurements of acylcarnitines in serum/plasma samples from WD subjects as indices of mitochondrial function. The anticipated findings are that aberrant methionine metabolism and altered gene-specific DNA methylation will be prevented by maternal dietary provision of choline in mice and that WD patient with different phenotypes will have different DNA methylation and mitochondrial impairment patterns.