DNA POLYMORPHIC FORMS OF HUMAN SERUM CHOLINESTERASE

人血清胆碱酯酶具有药物遗传学意义， 这种酶的变异形式的缺乏会引起过度的反应， 肌肉松弛药琥珀胆碱 我们的实验室研究了 这种酶已经有25年了 我们已经确定了完整的氨基 酶的氨基酸序列，从人脑cDNA中克隆出该基因 文库，并对该蛋白的编码区进行测序。 在过去 两年来，我们发现，最常见的结构基础 定性变异，非典型（二布卡因耐药）变异，是一个点 在核苷酸碱基209（GAT->GGT）突变;这改变了氨基酸在 密码子70从Glu → Gly。 这种突变在所有的 具有非典型表型的个体。 我们用聚合酶链 扩增WBC DNA选定片段的反应技术，包括 突变区，然后直接测序。 今后三 我们将在几年内确定负责的结构基础（DNA改变） 对于其他一些定性变量，例如氟化物， 抗性变体和几种定量变体，如K-变体 （减少1/3）; J-变体（减少2/3）和H-变体 （减少90%）以及一些“沉默”变体， 通过核苷酸351处的移码突变（GGT->GGAG）。 这些分析将为分类提供DNA结构基础 胆碱酯酶基因型，并提高我们识别这些基因型的能力。 对琥珀酰胆碱敏感的人，如果给予药物。 简单的非放射性，等位基因特异性生物素化探针正在被 在PCR后，开发了简单快速地鉴定变异基因型的方法， 从白色血细胞DNA扩增相应的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，以及一些特别是 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.