Womack Edward P, Reynolds Kristen K, Valdes Roland, Linder Mark W
Department of Pathology and Laboratory Medicine, University of Louisville, Louisville, Kentucky, USA.
Ther Drug Monit. 2007 Oct;29(5):607-11. doi: 10.1097/FTD.0b013e3181354b9b.
Cytochrome P450 2C9 (CYP4502C9) genotyping is useful in dosage adjustments for several critical drug therapies, including warfarin. Potential interference compromising these genotyping results could lead to inappropriate dose adjustments that may result in adverse drug reactions. During routine clinical CYP4502C9 genotyping using multiplex allele-specific primer extension, an ambiguous result was obtained for determination of the CYP2C9 430C>T substitution, which defines the CYP2C92 allele. In this one patient sample submitted for CYP2C9 genotyping, the ratio for the variant 430T allele signal to the total signal (C+T alleles) was 0.29. This is above the expected ratio to be classified as wild-type (<0.15) and below the minimum expected ratio (>0.3) when the genotype is heterozygous at the 430 position. The mean fluorescence intensity for the 430C allele was consistent with that observed in subjects who are heterozygous at this nucleotide position. However, the corresponding signal for the 430T allele indicated the absence of the CYP2C92 allele. This suggests the assay was not able to determine the correct nucleotide at position 430 for one of the two alleles in this patient. Subsequent sequencing to investigate the allele-specific primer extension failure revealed the presence of a rare C>T nucleotide substitution at position 429. We tested this subject's CYP2C9 genotype using AvaII restriction endonuclease digestion and found that this rare substitution causes false-positive identification of the CYP2C92 allele when using this method. We developed a DpnII endonuclease digestion assay to specifically detect the CYP2C9 429C>T substitution and tested 100 randomly selected samples obtained from unrelated individuals. The 429C>T polymorphism was not identified in this sample set, which indicates an allele frequency of less than 2.0% (95% confidence interval, 0.0-1.8%) in the general population. Despite the rarity of this polymorphism, it has important implications for the accuracy of assays using allele-specific primers and the Ava II restriction endonuclease, when it occurs, which are two common methods currently applied for detecting the presence of the CYP2C92 allele.
细胞色素P450 2C9（CYP4502C9）基因分型对于包括华法林在内的几种关键药物治疗的剂量调整很有用。可能影响这些基因分型结果的干扰因素可能导致不适当的剂量调整，进而可能引发药物不良反应。在使用多重等位基因特异性引物延伸进行常规临床CYP4502C9基因分型过程中，对于确定定义CYP2C92等位基因的CYP2C9 430C>T替换，获得了一个模糊的结果。在这个提交进行CYP2C9基因分型的患者样本中，变异的430T等位基因信号与总信号（C+T等位基因）的比率为0.29。这高于预期的野生型分类比率（<0.15），且低于430位点基因型为杂合子时的最低预期比率（>0.3）。430C等位基因的平均荧光强度与该核苷酸位置杂合子受试者中观察到的一致。然而，430T等位基因的相应信号表明不存在CYP2C92等位基因。这表明该检测方法无法确定该患者两个等位基因中一个在430位点的正确核苷酸。随后进行测序以调查等位基因特异性引物延伸失败的原因，结果显示在429位点存在罕见的C>T核苷酸替换。我们使用AvaII限制性内切酶消化检测该受试者的CYP2C9基因型，发现这种罕见的替换在使用该方法时会导致CYP2C92等位基因的假阳性鉴定。我们开发了一种DpnII内切酶消化检测方法来特异性检测CYP2C9 429C>T替换，并对从无关个体中随机选取的100个样本进行了检测。在这个样本集中未鉴定出429C>T多态性，这表明在一般人群中的等位基因频率低于2.0%（95%置信区间，0.0 - 1.8%）。尽管这种多态性很罕见，但当它出现时，对于使用等位基因特异性引物和Ava II限制性内切酶的检测准确性具有重要影响，而这两种方法是目前用于检测CYP2C92等位基因存在的两种常用方法。
