Gaedigk A, Spielberg S P, Grant D M
Division of Clinical Pharmacology and Toxicology, Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
Pharmacogenetics. 1994 Jun;4(3):142-53. doi: 10.1097/00008571-199406000-00005.
Therapy with the aromatic anticonvulsants phenytoin, phenobarbital and carbamazepine has been associated with the occurrence of rare idiosyncratic hypersensitivity reactions. These drugs are thought to be activated to potentially reactive arene oxide (epoxide) metabolites by cytochrome P450-dependent monooxygenation, while liver microsomal epoxide hydrolase (mEH) plays a detoxifying role by converting such reactive intermediates to non-toxic dihydrodiols. Evidence from in vitro lymphocyte toxicity tests and enzyme inhibitor studies has suggested that an inherited defect in mEH function may be responsible for the enhanced drug toxicity observed in affected individuals. To test this hypothesis we designed methods to directly compare mEH gene structure in patients presenting with anticonvulsant adverse reactions and in control subjects in which no in vivo or in vitro toxicity to anticonvulsants could be demonstrated. Southern analysis of peripheral lymphocyte DNA using a full-length mEH cDNA as hybridization probe revealed no gross differences in mEH gene structure in selected patients when compared with DNA samples from unaffected control subjects. The human mEH gene was then cloned and characterized from a control individual. Nine exons were identified within a 22 kb region and sequences of selected regions, including all exons, were determined. Single strand conformation polymorphism (SSCP) analysis was performed on all exonic regions in genomic DNA from each of 26 subjects, including six unrelated patients with previous toxicity to anticonvulsant therapy and seven siblings (three of whom had displayed toxicity). Several distinct SSCP patterns could be observed among the subjects tested, each corresponding to a specific point mutation within one of the amplified fragments of the mEH gene. However, none of the SSCP patterns reflecting point mutations was correlated with the occurrence of anticonvulsant toxicity. From these observations we conclude that a genetic defect altering the structure and function of the mEH protein is unlikely to be responsible for predisposing patients to anticonvulsant adverse reactions.
使用芳香族抗惊厥药苯妥英、苯巴比妥和卡马西平进行治疗与罕见的特异质性超敏反应的发生有关。这些药物被认为通过细胞色素P450依赖性单加氧作用被激活为潜在的反应性芳烃氧化物（环氧化物）代谢物，而肝微粒体环氧化物水解酶（mEH）通过将此类反应性中间体转化为无毒的二氢二醇发挥解毒作用。体外淋巴细胞毒性试验和酶抑制剂研究的证据表明，mEH功能的遗传缺陷可能是受影响个体中观察到的药物毒性增强的原因。为了验证这一假设，我们设计了方法，直接比较出现抗惊厥药物不良反应的患者和未表现出体内或体外抗惊厥药物毒性的对照受试者的mEH基因结构。使用全长mEH cDNA作为杂交探针对外周淋巴细胞DNA进行Southern分析，结果显示，与未受影响的对照受试者的DNA样本相比，所选患者的mEH基因结构没有明显差异。然后从一名对照个体中克隆并鉴定了人类mEH基因。在一个22 kb的区域内鉴定出9个外显子，并确定了包括所有外显子在内的选定区域的序列。对26名受试者的基因组DNA中的所有外显子区域进行了单链构象多态性（SSCP）分析，其中包括6名曾对抗惊厥治疗有过毒性反应且无亲缘关系的患者和7名兄弟姐妹（其中3人曾表现出毒性反应）。在所测试的受试者中可以观察到几种不同的SSCP模式，每种模式对应于mEH基因扩增片段之一内的一个特定点突变。然而，没有一种反映点突变的SSCP模式与抗惊厥药物毒性的发生相关。从这些观察结果中我们得出结论，改变mEH蛋白结构和功能的遗传缺陷不太可能是使患者易发生抗惊厥药物不良反应的原因。
