Abstract
Human cytochrome P450 3A4 is a major P450 enzyme in the liver and gastrointestinal tract. It plays important roles in the metabolism of a wide variety of drugs, some endogenous steroids, and harmful environmental contaminants. CYP3A4 exhibits a remarkable interindividual activity variation as high as 20-fold. To investigate whether the interindividual variation in CYP3A4 levels can be partly explained by genetic polymorphism, we analyzed DNA samples from 102 Chinese subjects by polymerase chain reaction (PCR)-single-strand conformation polymorphism analysis for novel point mutation in the CYP3A4 coding sequence and promoter region. Using PCR and directed sequencing method to establish the complete intron sequence ofCYP3A4 from leukocytes, the complete genomic sequence from exon 1 through 13 of CYP3A4 was determined and published in the GenBank database (accession no. AF209389). CYP3A4-specific primers were designed accordingly. After PCR-single-strand conformation polymorphism and restriction fragment length polymorphism screening, we found three novel mutations; two are point mutations and one is insertion. The first variant allele (CYP3A4*4), an Ile118Val change, was found in 3 of 102 Chinese subjects. The next allele (CYP3A4*5), which causes a Pro218Arg amino acid change, was found in 2 of 102 subjects. We found an insertion in A17776, designated asCYP3A4*6, which causes frame shift and an early stop codon in exon 9, in one heterozygous subject. We also investigated the CYP3A4 activity in these mutant subjects by measuring the morning spot urinary 6β-hydroxycortisol to free cortisol ratio with the enzyme-linked immunosorbent assay method. When compared with healthy Chinese population data, the 6β-hydroxycortisol to free cortisol ratio data suggested that these alleles (CYP3A4*4,CYP3A4*5, and CYP3A4*6) may decrease the CYP3A4 activity. Incidences of these mutations in Chinese subjects are rare. The prevalence of these point mutations in other ethnic groups and its effect on the metabolic activity of CYP3A4 remain to be further evaluated.
Footnotes
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Send reprint requests to: Jin-ding Huang, Ph.D., Dept. of Pharmacology, National Cheng Kung University, Medical College, 1 University Rd., Tainan 70101, Taiwan. E-mail:jinding{at}mail.ncku.edu.tw
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The work is supported by Grants NHRI-GT-EX89S831L and DOH88-HR-831 of the National Health Research Institute and Department of Health of Republic of China (Taipei).
- Abbreviations used are::
- P450
- cytochrome P450
- 6βOHF
- 6β-hydroxycortisol
- 6βOHF/F
- the ratio of 6βOHF to free cortisol
- dNTP
- deoxynucleotide triphosphates
- PCR
- polymerase chain reaction
- SSCP
- single-strand conformation polymorphism
- bp
- base pair
- RFLP
- restriction fragment length polymorphism
- Taq
- Thermus aquaticus
- Received August 17, 2000.
- Accepted October 31, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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