Bhasker C R, Miners J O, Coulter S, Birkett D J
Department of Clinical Pharmacology, Flinders University of South Australia, Australia.
Pharmacogenetics. 1997 Feb;7(1):51-8. doi: 10.1097/00008571-199702000-00007.
Single nucleotide substitutions are known to result in a different amino acid at one of four sites in cytochrome P4502C9 (CYP2C9) namely: residue 144: Arg/Cys; residue 358: Tyr/Cys; residue 359: Ile/Leu and residue 417: Gly/Asp. Polymerase chain reaction (PCR)-based amplification of the nucleotide fragments encompassing the four residues (144, 358-359 and 417) in 18 samples of human genomic DNA from a liver bank and one sample of DNA extracted from the blood of a known poor metabolizer of tolbutamide has been carried out. The products of PCR amplification were analysed by either allele-specific restriction endonucleases or probed with radioactively labelled allele-specific oligonucleotides in dot blot hybridizations. Fourteen individuals were homozygous for Arg144 and four were heterozygous Arg/Cys144. All individuals analysed were homozygous for Tyr358 (n = 17) and for Gly417 (n = 18). With the exception of one heterozygote the other 17 subjects were homozygous for Ile359. The genotype of the known poor metabolizer of tolbutamide was homozygous for Arg144, Leu359 and Gly417. The relative levels of expression of the Cys and Arg144 alleles was studied in the heterozygotes. A relative 5- to 10-fold greater expression of the Cys- over the Arg144 allele was noted in two heterozygotes. There was no apparent correlation of genotype to the hydroxylation of the known CYP2C9 substrates phenytoin, tolbutamide, torasemide and diclofenac. Apparent K(m) values for the cDNA-expressed Arg144/Ile359, Cys144/ Ile359 and Arg144/Leu359 variants towards tolbutamide were 91 microM, 62 microM and 229 microM, respectively. It is likely that functional changes occurring as a result of the Ile359Leu transition are responsible for the tolbutamide poor metabolizer phenotype.
已知单核苷酸替换会导致细胞色素P4502C9（CYP2C9）四个位点之一出现不同的氨基酸，即：第144位残基：精氨酸/半胱氨酸；第358位残基：酪氨酸/半胱氨酸；第359位残基：异亮氨酸/亮氨酸；第417位残基：甘氨酸/天冬氨酸。已对来自肝脏库的18份人类基因组DNA样本以及从已知甲苯磺丁脲代谢不良者血液中提取的一份DNA样本中包含这四个残基（144、358 - 359和417）的核苷酸片段进行了基于聚合酶链反应（PCR）的扩增。PCR扩增产物通过等位基因特异性限制性内切酶进行分析，或在斑点印迹杂交中用放射性标记的等位基因特异性寡核苷酸进行探针检测。14名个体为第144位精氨酸纯合子，4名个体为第144位精氨酸/半胱氨酸杂合子。所有分析个体均为第358位酪氨酸纯合子（n = 17）和第417位甘氨酸纯合子（n = 18）。除一名杂合子外，其他17名受试者为第359位异亮氨酸纯合子。已知甲苯磺丁脲代谢不良者的基因型为第144位精氨酸、第359位亮氨酸和第417位甘氨酸纯合子。在杂合子中研究了第144位半胱氨酸和精氨酸等位基因的相对表达水平。两名杂合子中，第144位半胱氨酸等位基因的表达比精氨酸等位基因相对高5至10倍。基因型与已知的CYP2C9底物苯妥英、甲苯磺丁脲、托拉塞米和双氯芬酸的羟化作用之间没有明显相关性。cDNA表达的第144位精氨酸/第359位异亮氨酸、第144位半胱氨酸/第359位异亮氨酸和第144位精氨酸/第359位亮氨酸变体对甲苯磺丁脲的表观K(m)值分别为91 microM、62 microM和229 microM。很可能是第359位异亮氨酸向亮氨酸的转变导致的功能变化造成了甲苯磺丁脲代谢不良者的表型。
