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

对来自日本的200个个体样本进行变性高效液相色谱分析和新snp序列分析。第一个SNP被鉴定为位于第5外显子的2556C>T，导致Thr261Ile的氨基酸改变。单倍型分析表明，100C>T、1039C>T、1661G>C和4180G>C存在于CYP2D6基因的同一等位基因中。在这200个个体中，有1个是2556C >t SNP的杂合，表明该等位基因频率在日本人群中为0.002。第二个SNP被鉴定为3835A>C，位于第8外显子，导致Lys404G1n的氨基酸改变。单倍型分析表明，在CYP2D6基因的同一等位基因中存在1661G >c、2850C >t和4180G >c。在这200个个体中，有一个人是3835A>C SNP的杂合，这表明等位基因频率在日本人群中为0.002。这些新的snp位于CYP2D6基因的外显子上，导致氨基酸替换。CYP2D6中的Thr261和Lys404分别位于g -螺旋和K"-螺旋上。这些氨基酸残基没有定位在底物识别位点，但在哺乳动物的CYP2D亚家族中是保守的。我们成功地在COS-7细胞中表达了在日本人群中检测到的12种CYP2D6变异蛋白。我们证明了它们对右美沙芬的催化活性。由11个CYP2D6蛋白变体催化的底物代谢活性降低到CYP2D6.1活性的0% - 64%。另一方面，CYP2D6.53表现出2倍高的活性。其中两个等位变异（CYP2D6.27和CYP2D6.54）改变了CYP2D6蛋白的表达水平。这些结果应该有助于我们了解与CYP2D6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.