Hamzeiy Hossein, Vahdati-Mashhadian Nasser, Edwards Helen J, Goldfarb Peter S
Molecular Toxicology Group, School of Biomedical and Life Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK.
Mutat Res. 2002 Mar 20;500(1-2):103-10. doi: 10.1016/s0027-5107(01)00305-0.
Human CYP3A4 is the major cytochrome P450 isoenzyme in adult human liver and is known to metabolise many xenobiotic and endogenous compounds. There is substantial inter-individual variation in the hepatic levels of CYP3A4. Although, polymorphic mutations have been reported in the 5' regulatory region of the CYP3A4 gene, those that have been investigated so far do not appear to have any effect on gene expression. To determine whether other mutations exist in this region of the gene, we have performed a new population screen on a panel of 101 human DNA samples. A 1140 bp section of the 5' proximal regulatory region of the CYP3A4 gene, containing numerous regulatory motifs, was amplified from genomic DNA as three overlapping segments. The 300 bp distal enhancer region at -7.9kb containing additional regulatory motifs was also amplified. Mutation analysis of the resulting PCR products was carried out using non-radioactive single strand conformation polymorphism (SSCP) and confirmatory sequencing of both DNA strands in those samples showing extra SSCP bands. In addition to detection of the previously reported CYP3A41B allele in nine subjects, three novel alleles were found: CYP3A41E (having a T-->A transversion at -369 in one subject), CYP3A41F (having a C-->G tranversion at -747 in 17 subjects) and CYP3A415B containing a nine-nucleotide insertion between -845 and -844 linked to an A-->G transition at -392 and a G-->A transition in exon 6 (position 485 in the cDNA) in one subject. All the novel alleles were heterozygous. No mutations were found in the upstream distal enhancer region. Our results clearly indicate that this rapid and simple SSCP approach can reveal mutant alleles in drug metabolising enzyme genes. Detection and determination of the frequency of novel alleles in CYP3A4 will assist investigation of the relationship between genotype, xenobiotic metabolism and toxicity in the CYP3A family of isoenzymes.
人CYP3A4是成人肝脏中主要的细胞色素P450同工酶，已知其可代谢多种外源性和内源性化合物。CYP3A4的肝脏水平存在显著的个体间差异。尽管在CYP3A4基因的5'调控区已报道有多态性突变，但迄今为止所研究的那些突变似乎对基因表达没有任何影响。为了确定该基因的这一区域是否存在其他突变，我们对一组101份人类DNA样本进行了新的群体筛查。从基因组DNA中扩增出CYP3A4基因5'近端调控区的一段1140 bp片段，该片段包含许多调控基序，作为三个重叠片段进行扩增。还扩增了位于-7.9 kb处的300 bp远端增强子区域，其包含额外的调控基序。使用非放射性单链构象多态性（SSCP）对所得PCR产物进行突变分析，并对那些显示额外SSCP条带的样本中的两条DNA链进行验证性测序。除了在9名受试者中检测到先前报道的CYP3A41B等位基因外，还发现了三个新的等位基因：CYP3A41E（一名受试者在-369处发生T→A颠换）、CYP3A41F（17名受试者在-747处发生C→G颠换）以及CYP3A415B，其在一名受试者中，于-845和-844之间有一个九核苷酸插入，与-392处的A→G转换以及外显子6（cDNA中的第485位）中的G→A转换相关联。所有新的等位基因均为杂合子。在上游远端增强子区域未发现突变。我们的结果清楚地表明，这种快速且简单的SSCP方法能够揭示药物代谢酶基因中的突变等位基因。检测和确定CYP3A4中新等位基因的频率将有助于研究CYP3A同工酶家族中基因型、外源性物质代谢与毒性之间的关系。
