EPOXIDE HYDROLASE AND PHENYTOIN INDUCED TERATOGENESIS

胎儿海因综合征(FHS)是一种苯妥英相关畸形 以颅面畸形、肢体畸形和 通常是中枢神经系统受累。这一证候可能与 基因多态或突变导致基因变异 异源生物微粒体环氧化物水解酶(MEH)是一种负责 苯妥英(苯妥英)的芳烃氧化代谢物的解毒。 在这个个人国家研究培训奖的申请中， 申请者建议对这一假设进行检验，以追求目标和方法 下面将进行讨论。 申请人将从符合以下条件的个人那里获取血液样本 胎儿海因综合征(FHS)的诊断标准 影响(Fhe)，或有口腔裂伤、先天性心脏病或 其他与妊娠早期病史有关的重大畸形 子宫内苯妥英钠暴露，也有类似的暴露，但没有受到影响， 要用作控件的同级。在分离白细胞后，他将 使用赞助商的标准酶指示剂分析和高性能 高效液相色谱法测定人外周血白细胞甲氧基异亮氨酸活性 受影响和未受影响的兄弟姐妹，并评估这些数据以 两组比较差异有统计学意义。然后他将决定 分离的mEH DNA序列多态的性质和发生率 受试者基因组DNA，用聚合酶链法扩增meh外显子 反应，通过单链构象筛选外显子突变 多态(SSCP)分析，使用等位基因- 特异性寡核苷酸(ASO)杂交和等位基因特异性 限制性内切酶和候选片段的测序。AS 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 关于meh基因变异性及其后续的特征 表型。完成这项建议将为申请人提供便利 S接受了分子生物学和医学遗传学的培训，并将 也有助于理解苯妥英钠所致胚胎病变 作为， 可能是与其他相关因素有关的致畸作用。

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.