Wilson Disease: Effect of High-Choline Feeding on Fetal and Offspring Mouse Liver

描述(申请人提供)：肝豆状核变性(WD)是一种遗传性、常染色体隐性遗传性铜(铜)蓄积性疾病，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的临床表现具有显著的异质性。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。这项拟议研究的假设是，妊娠期饮食中甲基基团的供应改变了胎儿DNA甲基化机制，该机制调节与WD肝损伤相关的基因的表达，并持续到后代的后续生命。我们将在WD的TX-j小鼠模型中验证这一假设，我们之前的特征是显示出全局DNA低甲基化和与肝脏蛋氨酸代谢相关的基因表达的变化。我们的特定目标是测试1)特定目标1：孕期母体甲基化状态是否会影响胎儿肝脏的转录组和甲基组状态；2)特定目标2：孕期母体甲基化状态会影响蛋氨酸代谢参数以及4月龄仔鼠肝脏整体DNA甲基化和基因表达的持续性变化。这项研究将在交配前和妊娠期间饲喂补充胆碱或对照饲料的TX-j和野生型母鸡的胎儿肝脏(特定目标1)，以及TX-j和野生型母鸡后代的肝脏进行。拟议的实验将使用表观遗传学技术，用于全球DNA甲基化、RNA测序分析和甲基C-seq分析，以确定全基因组CpG位点的基因特异性甲基化状态。预期的发现是，胎儿蛋氨酸代谢异常和改变的全球和基因特异性DNA甲基化将在后代中持续存在，并通过母亲饮食提供胆碱来防止。这些发现将提供重要的证据，证明WD不仅具有铜蓄积的特征，而且具有全局DNA低甲基化的特征，这可以通过在母体饮食中提供甲基来纠正。这项研究的积极结果将为可能使用药物来改变癌症的基因甲基化状态提供理论基础。 预防和/或治疗WD的临床表现。本建议是基于目前K08进展的一个新的起点，并将成为随后的R01应用的基础，通过探索在传统的抗铜治疗中添加饮食甲基供体(如胆碱)来研究动物模型和患有WD的人类的甲基化状态，以改变疑难和反应较差病例的病程，并作为传统抗铜制剂的佐剂。

DESCRIPTION (provided by applicant): Wilson disease (WD) is an inherited, autosomal recessive, copper (Cu) accumulation disorder, which is caused by the dysfunction of a Cu-transporting P-type ATPase that is crucial in the hepatocellular utilization and biliary excretion f Cu. WD is characterized by a remarkable heterogeneity in its clinical hepatic presentation. The failure to identify specific genotype-to-phenotype correlations could be due in part to the presence of epigenetic factors affecting phenotype expression. The HYPOTHESIS of the proposed research is that the gestational supply of dietary methyl groups modifies fetal DNA methylation mechanisms that regulate the expression of genes involved in liver damage in WD and persist through subsequent life of the offspring. We will test this hypothesis in the tx-j mouse model of WD, which we have previously characterized as showing global DNA hypomethylation and changes in the expression of genes related to hepatic methionine metabolism. Our SPECIFIC AIMS will be to test if 1) Specific Aim 1: maternal methyl status during pregnancy will affect transcriptome and methylome status in fetal liver; 2) Specific Aim 2: maternal methyl status during pregnancy will affect parameters of methionine metabolism and persisting changes in on hepatic global DNA methylation and gene expression in 4 month old offspring. The study will be conducted on livers of fetuses from tx-j and wild-type dams fed a choline-supplemented or control diets before mating and during gestation (Specific Aim 1), and on livers from offspring of tx-j and wild-type dams. The proposed experiments will use epigenetic techniques of dot-blot for global DNA methylation, RNA-sequencing analysis, and MethylC-seq analysis to determine the gene specific methylation status of CpG sites genome-wide. The anticipated findings are that aberrant fetal methionine metabolism and altered global and gene-specific DNA methylation will persist in offspring and be prevented by maternal dietary provision of choline. These findings will provide important evidence that WD is a condition characterized not only by Cu accumulation but also by global DNA hypomethylation which can be corrected by provision of methyl groups in the maternal diet. Positive results from this study will provide a rationale for the potential use of agents that modify gene methylation status in the prevention and/or treatment of clinical manifestations of WD. The present proposal is a novel departure based on current K08 progress and will form the basis for a subsequent R01 application to study methylation status over the lifespan of animal models and humans with WD by exploring the addition of dietary methyl donors such as choline to the traditional anti-Cu treatment in order to modify the disease course in difficult and less responsive cases and as adjuvant to traditional anti-Cu agents.