EPOXIDE HYDROLASE AND PHENYTOIN INDUCED TERATOGENESIS

胎儿乙内酰脲综合征（FHS）是一种苯妥英钠相关的畸形 以颅面异常、肢体畸形和 通常是中枢神经系统受累。该综合征可能与 遗传多态性或突变引起的变异， 外源性微粒体环氧化物水解酶（mEH），一种负责 苯妥英（苯妥英）的芳烃氧化物代谢物的解毒。 在这份个人国家研究培训奖申请中， 申请人提出测试该假设， 下面讨论。 申请人将从符合以下条件的个人处获取血液样本： 胎儿乙内酰脲综合征诊断标准 影响（FHE），或有口腔裂，先天性心脏病，或 与妊娠早期病史相关的其他主要畸形 子宫苯妥英暴露，并有类似的暴露，但未受影响， 兄弟姐妹用作对照。 分离白细胞后，他将 使用申办方的标准酶指示剂测定和高性能 液相色谱法（HPLC）测定白细胞mEH活性， 受影响和未受影响的兄弟姐妹，并评估这些数据， 两组之间有显著性差异。 然后他将决定 mEH DNA序列多态性的性质和发生率， 受试者基因组DNA，使用聚合酶链扩增mEH外显子 反应，通过单链构象分析筛选外显子突变 多态性（SSCP）分析，使用等位基因- 特异性寡核苷酸（阿索）杂交和等位基因特异性 限制性酶消化以及候选片段的测序。作为 在预计支持期限允许的情况下，申请人还将 开始应用体外表达系统和转基因动物 mEH基因型变异性的表征及其随后的 表型 完成本建议书将方便申请人 她接受过分子生物学和医学遗传学方面的培训， 有助于了解苯妥英诱导的胚胎病以及 因为， 可能与其他相关因子有关的致畸作用。

Fetal hydantoin syndrome (FHS) is a phenytoin-related malformation syndrome characterized by craniofacial anomalies, limb dysmorphisms, and often central nervous system involvement. The syndrome may be associated with genetic polymorphisms or mutations causing variability in xenobiotic microsomal epoxide hydrolase (mEH), an enzyme responsible for detoxification of the arene oxide metabolite of phenytoin (Dilantin). In this Individual National Research Training Award application, the applicant proposes to test this hypothesis pursuing the aims and methods discussed below. The applicant will obtain blood samples from individuals who fulfill diagnostic criteria for fetal hydantoin syndrome (FHS), fetal hydantoin effect (FHE), or who have oral clefting, congenital heart disease, or other major malformations associated with history of first trimester in utero phenytoin exposure, and have similarly exposed, but unaffected, siblings to be used as controls. After isolating leukocytes, he will use the sponsor's standard enzyme-indicator assay and high performance liquid chromatography (HPLC) to determine leukocyte mEH activity in affected and unaffected siblings, and evaluate these data for a significant difference between two groups. He will then determine the nature and incidence of mEH DNA sequence polymorphisms by isolating subjects genomic DNA, amplifying the mEH exons using polymerase chain reaction, screening for exon mutations via single-strand conformational polymorphism (SSCP) analysis, distinguishing alleles using allele- specific oligonucleotide (ASO) hybridization and an allele-specific restriction enzyme digest, and sequencing of candidate fragments. As permitted by projected duration of support, the applicant will also begin application of in vitro expression systems and transgenic animals to characterization of mEH genotypic variability and its subsequent phenotypes. Completion of this proposal will facilitate the applicant s training in molecular biology and medical genetics, and will contribute to the understanding of phenytoin-induced embryopathy as well as, possibly, teratogenesis associated with other related agents.