DNA POLYMORPHIC FORMS OF HUMAN SERUM CHOLINESTERASE

人类血清胆碱酯酶具有药物遗传学意义，因为 这种酶的变异形式的缺陷会引起夸大的反应 肌肉松弛药琥珀酰胆碱。我们的实验室已经研究了 这种酶在过去的25年里。我们已经测定了完整的氨基 酶的酸性序列，从人脑cDNA中克隆了该基因 文库，并对该蛋白的编码区进行了测序。在过去的几年里 两年来，我们发现结构性基础最常见 质的变异，非典型的(抗地布卡因)变异，是一个点 核苷酸碱基209位的突变(GAT-&GT；GGT)；这会改变位于 密码子70来自Glu-gt；Gly。同样的突变在每一个 具有非典型表型的个体。我们使用了聚合酶链条 扩增选定的白细胞DNA片段的反应技术，包括 突变区域，然后直接测序。在接下来的三年里 几年后，我们将确定负责的结构基础(DNA改变) 对于其他一些定性的变体，例如氟化物- 抗性变异体和几个数量变异体，如K-变异体 (减少1/3)；J-变量(减少2/3)，以及H-变量 (减少了90%)以及一些“无声”变体 由第351位核苷酸的移码突变(GGT-&GT；GGAG)形成。 这些分析将为分类提供DNA结构基础 胆碱酯酶基因分型，并提高我们识别这些基因的能力 对琥珀胆碱敏感的个人，如果给药的话。 简单的非放射性、等位基因特异的生物素化探针正在被 开发用于在聚合酶链式反应后轻松、快速地鉴定变异的基因类型 从白细胞DNA中扩增各自的DNA片段。 这些研究将确定一些氨基酸具有关键的 在酶的催化活性中的作用，特别是一些 影响酶的半衰期或稳定性。

Human serum cholinesterase is of pharmacogenetic interest since deficiencies of variant forms of this enzyme cause an exaggerated response to the muscle-relaxant drug, succinylcholine. Our laboratory has studied this enzyme for the past 25 years. We have determined the complete amino acid sequence of the enzyme, cloned the gene from a human brain cDNA library, and sequenced the coding region of this protein. During the last two years, we found that the structural basis for the most common qualitative variant, the atypical (dibucaine-resistant) variant, is a point mutation at nucleotide base 209 (GAT->GGT); this changes the amino acid at codon 70 from Glu->Gly. This same mutation has been found in every individual with the atypical phenotype. We used the polymerase chain reaction technique to amplify a selected segment of WBC DNA which included the mutation region, followed by direct sequencing. During the next three years we will identify the structural basis (DNA alterations) responsible for a number of the other qualitative variants, such as the fluoride- resistant variant, and several quantitative variants, such as the K-variant (reduction of 1/3); the J-variant (reduction of 2/3), and the H-variant (reduction of 90%) as well as some of the "silent" variant which accounted for by a frame-shift mutation at nucleotide 351 (GGT->GGAG). These analyses will provide a DNA structural basis for classifying cholinesterase genotypes, and improve our ability to identify those individuals who would be sensitive to succinylcholine, if given the drug. Simple non-radioactive, allele-specific biotinylated probes are being developed to easily and quickly identify the variant genotypes, after PCR amplification of the respective DNA segments from white blood cell DNA. These studies will identify a number of amino acids that have a critical role in the catalytic activity of the enzyme, and some that particularly influence the half-life or stability of the enzyme.