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Technical Briefs |
Departments of
1
Clinical Chemistry and
2
Pediatrics, University Hospital Rotterdam, PO Box 2040, 3000 CA Rotterdam, The Netherlands
aauthor for correspondence: fax 31-10-4367894, e-mail vanschaik{at}ckcl.azr.nl
Enzymes of the cytochrome P450 system are involved in the
metabolism of a broad range of foreign compounds, such as drugs,
environmental pollutants, and carcinogens (1). The most
abundant enzyme in the human liver is cytochrome P450 3A4 (CYP3A4)
(2). This enzyme is involved in the metabolism of >50% of
all drugs used in humans (3)(4), and the
interindividual differences in the pharmacokinetics of these drugs are
thought to be related to variations in CYP3A4 activity
(4)(5)(6). These variations may be caused by age and
disease-related differences, by drugs inducing or repressing
transcription/translation, or by genetic polymorphisms. Although the
CYP3A4 gene was initially thought not to be polymorphic,
recent reports have described three genetic variants of this gene:
CYP3A41B, CYP3A42, and CYP3A43
(7)(8). The allelic frequency for the
CYP3A41B allele, which contains an A(-290)G
substitution in the promoter region of CYP3A4, ranges from
0.0% in Chinese and Japanese Americans to >54% in African Americans
(8)(9). American and European Caucasians were
reported to have an allelic frequency of 
4–5% (8)(9)(10)(11).
The CYP3A42 allele, which encodes a Ser222Pro change, has
an allelic frequency of 2.7% in the white (Finnish) population
(8). Because variant alleles that are found in >1% of the
population are defined as genetic polymorphisms (12), both
the CYP3A41B and the CYP3A42 allele are
considered to be genetic polymorphisms of CYP3A4. In
addition, a variant allele found in the DNA of a single Chinese subject
contained a T1437C substitution (8). Because this allele,
encoding a Met445Thr change, was not found in any other of the 91
subjects investigated in that study, it was referred to as a rare
allele.
In this study, we developed a PCR-restriction fragment length
polymorphism (RFLP) procedure for the detection of the
CYP3A43 allele. We used this assay to determine the allelic
frequency of CYP3A43. EDTA-whole blood was obtained from
499 healthy Dutch Caucasian volunteers after informed consent. The
study was approved by the Medical Ethical Committee of the University
Hospital Rotterdam. We isolated genomic DNA from 300 µL of blood,
using the GenomicPrep Blood DNA Isolation reagent set (Amersham
Pharmacia Biotech). DNA yields were estimated by measuring the
absorbance at 260 nm (A260). A total
of 50 ng of genomic DNA was used in a PCR volume of 50 µL. The PCR
mixture contained 1x buffer [10 mM Tris-HCl (pH 8.3), 1.5 mM
MgCl2, 50 mM KCl, and 0.01 g/L gelatin
(Perkin-Elmer)], 0.2 mM each dNTP (Roche), 1.25 U of AmpliTaq Gold
(Perkin-Elmer), and 40 pmol of each of forward primer (5'-TGG ACC CAG
AAA CTG CAT ATG C-3'; nucleotides 23255–23276; GenBank
sequence AF209389) and reverse primer (5'-GAT CAC AGA TGG GCC TAA
TTG-3'; nucleotides 23483–23503; GenBank sequence AF209389). The
nucleotides underlined are mismatches with the CYP3A4
sequence, creating a NsiI restriction site in the wild-type
CYP3A4 PCR product. When the CYP3A43 allele is
amplified, this NsiI site is disrupted. PCR conditions were
as follows: 7 min at 94 °C; 35 cycles of 1 min at 94 °C, 1 min at
55 °C, and 1 min at 72 °C; and finally 7 min at 72 °C. The
249-bp PCR products of five DNA samples were sequenced, confirming that
indeed only the CYP3A4 gene was amplified.
The PCR product (15 µL) was digested with 10 U of NsiI
(Roche) for 2 h at 37 °C, and analyzed on a 3% MP
agarose/Tris-borate-EDTA gel with ethidium bromide staining. The
fragments produced were 226 and 23 bp for the wild-type sequence (Fig. 1
, lane wt/wt) and 249, 226, and 23 bp for heterozygous sequences
(Fig. 1
, lane wt/3); the 23-bp fragments are not visible on the gel.
Samples that produced a heterozygous signal were reanalyzed using the
PCR-RFLP assay. Subsequently, heterozygosity for CYP3A43
was confirmed by sequencing of the PCR product with the reverse primer
on an automated ABI 310 capillary sequencer (Perkin-Elmer) using the
Big Dye Terminator Cycle Sequencing reagent set (Perkin-Elmer). To
check reproducibility, the CYP3A43 assay was performed 10
times for a wild-type and 10 times for a heterozygous sample; the
genotype was identified correctly each time.
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The CYP3A43 PCR-RFLP assay was applied to 499 genomic DNA samples obtained from Dutch Caucasians. In 488 cases, digestion of the 249-bp PCR product produced the 226- and 23-bp fragments, as expected for wild-type samples, whereas in 11 cases (2.2%), the heterozygous signal was produced. No homozygotes were detected. The allelic frequency of CYP3A43 in these Caucasians was therefore 1.1%. These allelic and genotypic frequencies are in Hardy-Weinberg equilibrium (P = 0.80). In the heterozygous samples, direct sequencing showed a mixed T/C peak corresponding to position 1437, indicating that the nucleotide change was indeed T1437C in all cases.
Variant CYP3A4 alleles in the population may contribute to
interindividual variability in CYP3A4 activity, and detecting genetic
polymorphisms may help to predict an individual’s ability to respond
to certain drugs. The CYP3A43 allele, which has a T1473C
change that produces a Met445Thr substitution in exon 12, was found in
only 1 Chinese subject from Shanghai and could not be detected in 91
other individuals (8). Because of this, CYP3A43
was described as being a rare allele, which may lead researchers to
assign a low priority to performing functional studies on this allele.
Our data indicate that the CYP3A43 allele is not limited to
a single individual, but has an allelic frequency of 1.1% in
Caucasians. This implies that the variant CYP3A43 allele is
not a rare allele, but instead represents a genetic polymorphism that
can be found in a substantial part of the population. The
identification of the CYP3A43 variant allele as a genetic
polymorphism, in addition to the CYP3A41B and 2
polymorphisms, has implications for the number of variant CYP3A4alleles to be expected in the population. The CYP3A41B
allele potentially alters the transcription efficiency and thus the
overall enzymatic activity of CYP3A4. Although initial reports
suggested decreased activity in vivo
(7)(13)(14), increased activity in
vitro (15)(16) and no effect
(10)(14)(17) have also been
reported. For the variant allele CYP3A42, a decreased
enzymatic activity was observed for nifedipine, but not for
testosterone (8). For CYP3A43, the location of
the amino acid that is changed in the CYP3A4 protein is near the
cysteine that is involved in the active site of the enzyme
(8). This might induce structural differences, leading to
alteration in enzymatic activity. However, expression studies need to
be performed to confirm this. Taking into account the allelic
frequencies of the genetic polymorphisms in CYP3A4 (10%
heterozygous for CYP3A41B, 5.4% heterozygous for
CYP3A42, and 2.2% heterozygous for CYP3A43),
this implies that 15% of the (Caucasian) population may carry a
genetic polymorphism in this allele. Because genetic polymorphisms may
exhibit strong differences in occurrence among different ethnic groups,
other populations need to be investigated to determine the allelic
frequency of CYP3A43.
In conclusion, we have described and validated a PCR-RFLP assay for the CYP3A43 allele. The frequency of this variant allele in the Caucasian population (1.1%) indicates that it might be important in predicting CYP3A4 activity based on genotype. Future research should be directed toward elucidating the effect of this polymorphism on CYP3A4 enzymatic activity and toward establishing whether this is solely a genetic, or also a functional, polymorphism.
Acknowledgments
We thank the Bloodbank ZWN Rotterdam for their cooperation in collecting blood samples. We also thank Dr. Y. Fang for technical assistance.
References
The following articles in journals at HighWire Press have cited this article:
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  P. Gladding, M. Webster, I. Zeng, H. Farrell, J. Stewart, P. Ruygrok, J. Ormiston, S. El-Jack, G. Armstrong, P. Kay, et al. The Pharmacogenetics and Pharmacodynamics of Clopidogrel Response: An Analysis From the PRINC (Plavix Response in Coronary Intervention) Trial J. Am. Coll. Cardiol. Intv., December 1, 2008; 1(6): 620 - 627. [Abstract] [Full Text] [PDF]
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 S. De Fazio, L. Gallelli, A. De Siena, G. De Sarro, and M. G. Scordo Role of CYP3A5 in Abnormal Clearance of Methadone Ann. Pharmacother., June 1, 2008; 42(6): 893 - 897. [Abstract] [Full Text] [PDF]
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 A. Henningsson, S. Marsh, W. J. Loos, M. O. Karlsson, A. Garsa, K. Mross, S. Mielke, L. Vigano, A. Locatelli, J. Verweij, et al. Association of CYP2C8, CYP3A4, CYP3A5, and ABCB1 Polymorphisms with the Pharmacokinetics of Paclitaxel Clin. Cancer Res., November 15, 2005; 11(22): 8097 - 8104. [Abstract] [Full Text] [PDF]
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E. R. Lepper, S. D. Baker, M. Permenter, N. Ries, R. H.N. van Schaik, P. W. Schenk, D. K. Price, D. Ahn, N. F. Smith, G. Cusatis, et al. Effect of Common CYP3A4 and CYP3A5 Variants on the Pharmacokinetics of the Cytochrome P450 3A Phenotyping Probe Midazolam in Cancer Patients Clin. Cancer Res., October 15, 2005; 11(20): 7398 - 7404. [Abstract] [Full Text] [PDF]
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 O. Q. P. Yin, X. J. Shi, B. Tomlinson, and M. S. S. Chow EFFECT OF CYP2D6*10 ALLELE ON THE PHARMACOKINETICS OF LORATADINE IN CHINESE SUBJECTS Drug Metab. Dispos., September 1, 2005; 33(9): 1283 - 1287. [Abstract] [Full Text] [PDF]
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 R. H. J. Mathijssen, F. A. de Jong, R. H. N. van Schaik, E. R. Lepper, L. E. Friberg, T. Rietveld, P. de Bruijn, W. J. Graveland, W. D. Figg, J. Verweij, et al. Prediction of Irinotecan Pharmacokinetics by Use of Cytochrome P450 3A4 Phenotyping Probes J Natl Cancer Inst, November 3, 2004; 96(21): 1585 - 1592. [Abstract] [Full Text] [PDF]
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 K. Kim, J. A. Johnson, and H. Derendorf Differences in Drug Pharmacokinetics Between East Asians and Caucasians and the Role of Genetic Polymorphisms J. Clin. Pharmacol., October 1, 2004; 44(10): 1083 - 1105. [Abstract] [Full Text] [PDF]
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