Epigenetic regulation of Wilson Disease

 描述（由申请方提供）：Wilson病（WD）是一种常染色体隐性遗传的肝铜蓄积性疾病，由铜转运P型ATP酶功能障碍引起，该酶在肝细胞利用和胆汁排泄铜中至关重要。WD的特点是其临床肝脏严重程度和神经精神表现的显著异质性。未能确定特定的基因型与表型的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表型和其潜在的反应，抗铜治疗可能是由母亲的饮食甲基对后代肝脏蛋氨酸代谢和氧化磷酸化机制的影响。我们的目标是：1）确定母亲甲基状态对肝脏基因组甲基化的影响，氧化磷酸化途径基因的转录水平和后代的线粒体功能及其对铜螯合的反应; 2）表征人类肝脏DNA甲基化和血浆酰基肉碱作为不同表型WD患者线粒体功能的标志物。我们将在TX-J小鼠模型中测试第一个目标 WD，我们以前的特点是显示全球DNA低甲基化和肝甲硫氨酸代谢，基因组DNA甲基化和肝损伤相关基因的表达变化。我们将为杂合子母鼠（Atp 7 b +/-）提供对照或胆碱补充饮食，并研究这些饮食对肝脏铜蓄积的24周野生型和纯合子（Atp 7 b- /-）tx-j后代小鼠肝脏基因组DNA甲基化和基因表达及其对铜螯合剂的反应的影响。此外，我们将与美国和欧洲的医疗机构合作，在未染色的肝活检切片和WD患者组织块的DNA中进行全球DNA甲基化研究。我们将特别关注线粒体功能基因表达途径，我们已经表明，从胎儿到成年的生活在tx-j小鼠肝脏受到影响。所提出的实验将需要使用表观遗传学技术，包括通过MethylC-seq和焦磷酸测序在肝脏中进行的甲基化组分析，以确定小鼠中全基因组CpG位点的甲基化状态，使用人肝脏活检载玻片的免疫染色用于全局DNA甲基化模式，甲基化组分析用于全局DNA甲基化定量，以及基因特异性甲基化分析，和测量来自WD受试者的血清/血浆样品中的酰基肉毒碱作为线粒体功能的指标。预期的发现是，异常的甲硫氨酸代谢和改变的基因特异性DNA甲基化将通过在小鼠中提供胆碱的母体饮食来防止，并且具有不同表型的WD患者将具有不同的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.