Analysis of genetic polymorphisms in human drug-metabolizing enzyme genes and functions of enzymatic proteins

在来自日本受试者的200个个体样本中进行了变性HPLC测定和新型SNP的序列分析。第一个SNP被鉴定为外显子5中的2556 C>T，导致Thr 261 Ile的氨基酸改变。单倍型分析表明，100C>T、1039 C>T、1661 G>C和4180 G>C存在于CYP 2D 6基因的同一等位基因中。在这200个个体中，有一个是2556 C>T SNP的杂合子，表明在日本人群中等位基因频率为0.002。第二个SNP被鉴定为外显子8中的3835 A>C，导致Lys 404 G1 n的氨基酸改变。单倍型分析表明，CYP 2D 6基因的1661 G>C、2850 C>T和4180 G>C存在于同一等位基因中。在这200个个体中，有一个是3835 A>C SNP的杂合型，表明在日本人群中等位基因频率为0.002。这些新的SNP位于CYP 2D 6基因的外显子中，并导致氨基酸取代。CYP 2D 6的Thr 261和Lys 404分别位于G-螺旋和K”-螺旋。这些氨基酸残基未定位于底物识别位点，但在哺乳动物的CYP 2D亚家族中是保守的。我们在COS-7细胞中成功地表达了12种在日本人群中检测到的CYP 2D 6变异蛋白。我们证明了它们对二甲双胍的催化活性。11种CYP 2D 6蛋白变体催化的底物代谢活性降低至CYP2D6.1活性的0%-64%。另一方面，CYP2D6.53表现出2倍的活性。两个等位基因变体（CYP2D6.27和CYP2D6.54）改变了CYP 2D 6蛋白的表达水平。这些结果将有助于我们了解与CYP 2D 6功能相关的广泛的个体差异。

The denaturing HPLC assay and the sequence analysis for novel SNPs were performed in 200 individual samples from Japanese subjects. The first SNP was identified as 2556C>T in exon 5 resulting in an amino acid change of Thr261Ile. Haplotype analysis indicated that 100C>T, 1039C>T, 1661G>C, and 4180G>C existed in the same allele of the CYP2D6 gene. Of the 200 individuals, one was heterozygous for the 2556C>T SNP, suggesting that the allele frequency was 0.002 in the Japanese population. The second SNP was identified as 3835A>C in exon 8 resulting in an amino acid change of Lys404G1n. Haplotype analysis indicated that 1661G>C, 2850C>T, and 4180G>C existed in the same allele of the CYP2D6 gene. Of the 200 individuals, one was heterozygous for the 3835A>C SNP, suggesting that the allele frequency was 0.002 in the Japanese population. These novel SNPs were located in the exons of the CYP2D6 gene and result in amino acid substitutions. The Thr261 and Lys404 in CYP2D6 are located in G-helix and K"-helix, respectively. These amino acid residues are not mapped in substrate recognition sited, but are conserved in the CYP2D subfamily in mammals. We succeeded in expressing 12 variant CYP2D6 proteins, which have been detected in the Japanese population, in COS-7 cells. We demonstrated their catalytic activities for dextromethorphan. The metabolic activities with the substrates catalyzed by 11 variants of the CYP2D6 protein were reduced to between 0% and 64% of the activity observed with CYP2D6.1. On the other hand, CYP2D6.53 exhibited a 2-fold higher activity. Two of the allelic variants (CYP2D6.27 and CYP2D6.54) had altered expression levels of the CYP2D6 protein. These results should assist us in gaining an understanding of the extensive individual variations associated with CYP2D6 function.