Sata F, Sapone A, Elizondo G, Stocker P, Miller V P, Zheng W, Raunio H, Crespi C L, Gonzalez F J
Laboratory of Metabolism, National Cancer Institute, Bethesda, MD 20892, USA.
Clin Pharmacol Ther. 2000 Jan;67(1):48-56. doi: 10.1067/mcp.2000.104391.
To determine the existence of mutant and variant CgammaP3A4 alleles in three racial groups and to assess functions of the variant alleles by complementary deoxyribonucleic acid (cDNA) expression.
A bacterial artificial chromosome that contains the complete CgammaP3A4 gene was isolated and the exons and surrounding introns were directly sequenced to develop primers to polymerase chain reaction (PCR) amplify and sequence the gene from lymphocyte DNA. DNA samples from Chinese, black, and white subjects were screened. Mutating the affected amino acid in the wild-type cDNA and expressing the variant enzyme with use of the baculovirus system was used to functionally evaluate the variant allele having a missense mutation.
To investigate the existence of mutant and variant CgammaP3A4 alleles in humans, all 13 exons and the 5'-flanking region of the human CgammaP3A4 gene in three racial groups were sequenced and four alleles were identified. An A-->G point mutation in the 5'-flanking region of the human CgammaP3A4 gene, designated CgammaP3A41B, was found in the three different racial groups. The frequency of this allele in a white population was 4.2%, whereas it was 66.7% in black subjects. The CgammaP3A41B allele was not found in Chinese subjects. A second variant allele, designated CgammaP3A42, having a Ser222Pro change, was found at a frequency of 2.7% in the white population and was absent in the black subjects and Chinese subjects analyzed. Baculovirus-directed cDNA expression revealed that the CYP3A42 P450 had a lower intrinsic clearance for the CYP3A4 substrate nifedipine compared with the wild-type enzyme but was not significantly different from the wild-type enzyme for testosterone 6beta-hydroxylation. Another rare allele, designated CgammaP3A4*3, was found in a single Chinese subject who had a Met445Thr change in the conserved heme-binding region of the P450.
These are the first examples of potential function polymorphisms resulting from missense mutations in the CgammaP3A4 gene. The CgammaP3A4*2 allele was found to encode a P450 with substrate-dependent altered kinetics compared with the wild-type P450.
确定三个种族群体中CgammaP3A4等位基因突变体和变体的存在，并通过互补脱氧核糖核酸（cDNA）表达评估变体等位基因的功能。
分离出包含完整CgammaP3A4基因的细菌人工染色体，直接对其外显子和周围内含子进行测序，以设计引物用于聚合酶链反应（PCR）扩增并对淋巴细胞DNA中的该基因进行测序。对来自中国、黑人和白人受试者的DNA样本进行筛查。通过在野生型cDNA中突变受影响的氨基酸，并利用杆状病毒系统表达变体酶，对具有错义突变的变体等位基因进行功能评估。
为研究人类中CgammaP3A4等位基因突变体和变体的存在情况，对三个种族群体中人类CgammaP3A4基因的全部13个外显子和5'侧翼区域进行了测序，共鉴定出四个等位基因。在三个不同种族群体中均发现了人类CgammaP3A4基因5'侧翼区域的一个A→G点突变，命名为CgammaP3A41B。该等位基因在白人人群中的频率为4.2%，而在黑人受试者中为66.7%。在中国受试者中未发现该等位基因。发现了第二个变体等位基因，命名为CgammaP3A42，其发生了Ser222Pro变化，在白人人群中的频率为2.7%，在分析的黑人受试者和中国受试者中均未出现。杆状病毒介导的cDNA表达显示，与野生型酶相比，CYP3A42 P450对CYP3A4底物硝苯地平的内在清除率较低，但对睾酮6β-羟化与野生型酶无显著差异。在一名中国受试者中发现了另一个罕见等位基因，命名为CgammaP3A43，其P450保守血红素结合区域发生了Met445Thr变化。
这些是CgammaP3A4基因错义义突变导致潜在功能多态性的首个实例。与野生型P450相比，发现CgammaP3A4*2等位基因编码的P450具有底物依赖性的动力学改变。
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