中国人S－美芬妥英／奥美拉唑羟化代谢多态性的分子机制探讨

THE MOLECULAR BASIS OF GENETIC VARIATION OF S-MEPHENYTOIN/ OMEPRAZOLE OXIDATIVE POLYMORPHISM IN CHINESE SUBJECTS

为探讨中国人Ｓ－美芬妥英／奥美拉唑氧化代谢多态性的分子机制，本文用多聚酶链式反应（ＰＣＲ）方法扩增出ＣＹＰ２Ｃ１９ｉｎｔｒｏｎ４／ｅｘｏｎ５特异的ＤＮＡ片段，并用限制性内切酶ＳｍａＩ进行酶切分析。研究结果表明，１６位健康志愿者（９名Ｓ－ＭＰＥＭｓ，７名Ｓ－ＭＰＰＭｓ）的ＣＹＰ２Ｃ１９存在ＳｍａＩ酶切位点的多态性，且与Ｓ－ＭＰ羟化代谢表型有相关性。在９名ＥＭｓ中，有３名为ＣＹＰ２Ｃ１９ｗｔ／ｗｔ的纯合子，而在７名ＰＭｓ中，有４名为ＣＹＰ２Ｃ１９ｍ／ｍ的纯合子，余下９位均为杂合子（ＣＹＰ２Ｃ１９ｗｔ／ｍ）。因此中国人导致Ｓ－ＭＰ弱代谢表型主要由于ＣＹＰ２Ｃ１９基因外显子５的ＳｍａＩ酶切位点的突变。但这种突变只能解释Ｓ－ＭＰＰＭｓ的８０％等位基因突变（ＣＹＰ２Ｃ１９ｍ）或６０％的中国受试者弱代谢表型。对中国人ＣＹＰ２Ｃ１９基因ｉｎｔｒｏｎ４／ｅｘｏｎ５ＤＮＡ序列进行测定的结果表明，在ＥＭｓ的基因序列中有一个ＳｍａＩ酶位切点，而在ＰＭｓ中，由于Ｇ→Ａ突变，使ＳｍａＩ酶位切点消失。

It is well known now that the hydroxylative metabolism of S-MP is catalized by cytochrome P450 2C19(CYP 2C19). For genotyping analysis, DNA was isolated from the blood of 16 Chinese normal subjects who had been previously phenotyped(9 were S-MP EMs and 7 were S-MPPMs). Then the specific DNA fragment of CYP 2C19 was amplified by technique of PCR and digested with restrictive endoclease Sma I.000 Our results demonstrated that among the 16 subjects,4 were homozygous for the wild-type gene(CYP 2C19 wt/wt), 4 of them were homozygous for mutant gene(CYP 2C19 m/m)and 9 others were heterozygous(CYP 2C19 wt/m). Thus, the CYP 2C19m defect accounted for 11 of 14 alleles(80%)in Chinese poor metabolizers and could explain about 60% PMs phenotypes. This defect was not able to explain all PMs.It is therefore indicated the existence of another, yet unidentified mutation. DNA from two individuals, including 1 homozygous EMs and 1 hormozygous PMs was amplifled, subcloned into pGEM-T vector and sequenced.Our results indicated that only CYP 2C19 intron4/exon5 was amplified in the genotyping test. The principal defect in poor metabolizer is a single base pair(G→A)mutation in exon5 of CYP2C19, which created an aberrant splicing site and resulted in a non-functional enzyme protein, but the nucleotide sequence of intron 4 of the mutated gene is identical to that of the normal gene.