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

 描述(由申请人提供)：产前酒精暴露后酒精诱导的改变的类型和严重程度受到遗传的强烈影响。然而，由于母亲和胎儿都有独特的基因类型，遗传学的作用变得复杂起来。在评估治疗方法时，遗传学的作用可能是一个更大的考虑因素，因为任何治疗方法的新陈代谢在早期发育阶段都完全由母亲调节。这一提议将检验以下假设：1)母亲的基因型在决定酒精诱导的发育中脑细胞死亡的水平以及胆碱治疗在调节乙醇诱导的细胞死亡方面的有效性方面具有重要作用；2)胚胎的表观基因组在调节对酒精暴露影响的遗传差异方面具有重要作用。我们一直在检查由C57BL/6J(B6)和DBA/2J(D2)品系杂交产生的BXD小鼠小组，并在同等乙醇暴露后显示出不同的敏感性，从而促进了这些假设的测试。具体目标1将检验这一假设，即母体基因对发育中的神经管中乙醇诱导的细胞死亡水平有影响。将使用两种互补的方法--正反杂交和胚胎移植。胚胎将在胚胎第9天被暴露在乙醇中，并在最初的乙醇暴露后7小时收集。在发育中的端脑和脑干中，细胞死亡将通过TUNEL和激活的caspase-3标记的切片进行量化。如果在这两种方法中，细胞死亡水平根据母亲的基因型别而改变，这将表明母体基因型别是一个重要的介体。《特定目标2》将检验这一假说，即在DAM中存在有助于胆碱疗效的遗传差异。 减轻乙醇对细胞死亡的影响。B6和BXD菌株在酒精暴露后表现出高水平的细胞死亡，将用不同剂量的胆碱进行测试，以找到在改善酒精效果方面有效的最低剂量。如果不同菌株之间的剂量有显着差异，就会证明基因是关键。相反，如果胆碱对不同菌株的影响是相同的，这将表明基因型并不重要，胆碱应该对所有FASD儿童同样有益，一旦在人类群体中发现了相关剂量，它可能会在人群中的更大范围内同样有效。特定的Ai 3将检验以下假设，即酒精暴露引起的表观遗传变化因基因和母体环境的不同而不同，从而导致乙醇诱导的神经毒性易感性的遗传变异。端脑中的整体甲基化将通过减少代表性的亚硫酸氢盐测序和亚硫酸氢盐焦磷酸测序进行检查，并与基因表达的变化进行比较。这项建议的信息对于确定1)检查哪个基因组以确定儿童在特定类型的酒精诱导的神经畸形学影响方面风险最大，以及2)在实施胆碱作为治疗酒精诱导的脑损伤的药物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.