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.
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