Maternal genotype, choline intervention,& epigenetics in Fetal Alcohol Syndrome

 描述（由申请人提供）：产前乙醇暴露后乙醇诱导的改变的类型和严重程度受到遗传学的强烈影响。然而，遗传学的作用是复杂的事实，母亲和胎儿都有独特的基因型。遗传学的作用可能是评估治疗的一个更大的考虑因素，因为任何治疗药物的代谢都完全由母亲在早期发育中调节。该提议将测试以下假设：1）母亲的基因型在确定发育中的脑中乙醇诱导的细胞死亡的水平以及胆碱处理在调节乙醇诱导的细胞死亡中的功效方面具有重要作用，以及2）胚胎的表观基因组在调节对乙醇暴露影响的易感性的遗传差异方面具有重要作用。我们一直在研究通过杂交C57 BL/6 J（B6）和DBA/2 J（D2）品系产生的BXD小鼠组，并在同等乙醇暴露后显示出不同的敏感性，从而有助于检验这些假设。具体目标1将检验母体基因型影响发育中神经管中乙醇诱导细胞死亡水平的假设。将使用两种互补的方法，即正反交和胚胎移植。胚胎将在胚胎第9天暴露于乙醇，并在初始乙醇暴露后7小时收集。将从发育中的端脑和脑干中的TUNEL和活化的半胱天冬酶-3标记切片定量细胞死亡。如果在这两种方法中，细胞死亡的水平根据母亲的基因型而改变，这将表明母亲的基因型是一个重要的媒介。具体目标2将测试的假设，有遗传差异，在大坝，有助于胆碱的疗效 减轻乙醇对细胞死亡的影响。将用不同剂量的胆碱测试暴露于乙醇后显示高水平细胞死亡的B6和BXD菌株，以找到有效改善乙醇作用的最低剂量。如果剂量在不同菌株间存在显著差异，则表明基因型至关重要。相比之下，如果胆碱的作用在不同菌株之间是等效的，这将表明基因型是不重要的，胆碱应该在所有FASD儿童中同样有益，并且一旦在人群中发现相关剂量，它可能在广泛的人群中同样有效。特定Ai 3将检验以下假设：乙醇暴露诱导的表观遗传变化基于基因型以及母体环境而不同，从而导致对乙醇诱导的神经毒性易感性的遗传变异性。将使用还原代表性亚硫酸氢盐测序和亚硫酸氢盐焦磷酸测序检查端脑中的整体甲基化，并与基因表达的变化进行比较。来自该提案的信息对于确定1）检查哪个基因组以识别特定类型乙醇诱导的神经畸形效应的风险最大的儿童以及2）在实施胆碱作为乙醇诱导的脑损伤的治疗时是否需要考虑基因型将是重要的。

 DESCRIPTION (provided by applicant): The type and severity of ethanol-induced alterations following prenatal ethanol exposure is strongly impacted by genetics. However, the role of genetics is complicated by the fact that both the mother and fetus have unique genotypes. The role of genetics could be an even bigger consideration in the evaluation of treatments, since the metabolism of any therapeutic is completely regulated by the mother in early development. This proposal will test the following hypotheses: 1) the genotype of the mother has a significant role in determining the level of ethanol-induced cell death in the developing brain as well as in the efficacy of choline treatment in modulating ethanol-induced cell death and 2) the epigenome of the embryo has an important role in modulating genetic differences in susceptibility to the effects of ethanol exposure. We have been examining the BXD panel of mice, generated by crossing C57BL/6J (B6) and DBA/2J (D2) strains, and show differential sensitivity following equivalent ethanol exposure thereby facilitating the testing of these hypotheses. Specific Aim 1 will test the hypothesis that the maternal genotype influences the level of ethanol-induced cell death in the developing neural tube. Two complementary approaches, reciprocal crosses and embryo transplants, will be used. Embryos will be exposed to ethanol on embryonic day 9 and collected 7 hours after the initial ethanol exposure. Cell death will be quantified from TUNEL and activated caspase-3 labeled sections in the developing telencephalon and brain stem. If, in both approaches, the level of cell death is altered depending upon the genotype of the mother, it will show that the maternal genotype is an important mediator. Specific Aim 2 will test the hypothesis that there are genetic differences in the dam that contribute to the efficacy of choline in mitigating ethanol's effects on cell death. B6 and BXD strains that show high levels of cell death following ethanol exposure will be tested with different doses of choline to find the lowest dose that is efficacious in ameliorating ethanol's effects. If the dose differs significantly acros the strains it will demonstrate that genotype is critical. In contrast, if choline's effects are equivalent across strains, it will suggest that genotype is unimportant and that choline should be equally beneficial in all FASD children and that once a relevant dose is found within the human population, it will likely be equally effective across a wide sector of the population. Specific Ai 3 will test the hypotheses that epigenetic changes induced by ethanol exposure differ based on genotype, as well as maternal environment, thus contributing to genetic variability in susceptibility to ethanol- induced neurotoxicity. Global methylation will be examined in the telencephalon using reduced representation bisulfite sequencing and bisulfite pyrosequencing and be compared to changes in gene expression. Information from this proposal will be important for determining 1) which genome to examine to identify children most at risk for specific types of ethanol-induced neuroteratological effects and 2) whether genotype needs to be considered in implementing choline as a therapeutic for ethanol-induced brain damage.