CORRESPONDENCE |
Re: Modification of Clinical Presentation of Prostate Tumors by a Novel Genetic Variant in CYP3A4
Yuichi Ando,
Tomonori Tateishi,
Yoshitaka Sekido,
Toshimichi Yamamoto,
Tetsuo Satoh,
Yoshinori Hasegawa,
Shinichi Kobayashi,
Yoshinao Katsumata,
Kaoru Shimokata,
Hidehiko Saito
Affiliations of authors: Y. Ando, Y. Hasegawa, H. Saito,
First Department of Internal Medicine, Nagoya University School of Medicine, Japan; T. Tateishi,
S. Kobayashi, Department of Pharmacology, St. Marianna University School of Medicine,
Kawasaki, Japan; Y. Sekido, K. Shimokata (Department of Preventive Clinical Medicine), T.
Yamamoto, Y. Katsumata (Department of Legal Medicine and Bioethics, Postgraduate School of
Medicine), Nagoya University; T. Satoh, The Biomedical Research Institute, HAB Discussion
Group, Chiba, Japan.
Correspondence to: Yuichi
Ando, M.D., First Department of Internal Medicine, Nagoya University School of Medicine, 65
Tsurumai, Showa-ku, Nagoya 466-8550, Japan (e-mail: yando{at}tsuru.med.nagoya-u.ac.jp).
In the recent study on a new variant of the cytochrome P450 3A4
(CYP3A4) gene by Rebbeck et al. (1), the authors did not
provide data that demonstrate an alteration of CYP3A4 function
as a consequence of the polymorphism. They suggested that the
variant allele might alter disposition of the androgenic substrates
of CYP3A4 as a result of decreased enzymatic activity. Worse clinical
presentation of prostate cancer (1) and a decreased risk
for treatment-related leukemia (2) were reported to be
associated with the variant allele, the former probably due to
increased bioavailability of testosterone and the latter probably due
to reduced production of leukemogenic metabolites of anticancer drugs.
Thus, we investigated the possible relationship between CYP3A4
genotypes and nifedipine oxidation activity, a prototype reaction of the
encoded enzyme, by use of a human liver microsome system in vitro.
Fifteen liver samples from Caucasian transplant donors were obtained from the National
Disease Research Interchange (Philadelphia, PA) through the Biomedical Research Institute,
Human and Animal Bridge Discussion Group (Chiba, Japan). The nifedipine oxidation activity and
the expression levels of CYP3A4 protein of the samples have been reported elsewhere (3). A variant sequence of the CYP3A4 gene was distinguished from
wild-type by a nested polymerase chain reaction-restriction fragment length polymorphism assay
by the use of genomic DNA prepared from the liver samples (Fig. 1,
A).
The institutional review board of St. Marianna University School of Medicine has approved the
study. The genotyping analysis revealed one homozygote and four heterozygotes for the variant.
The remaining 10 subjects were homozygous for the wild-type allele. No apparent relationships
between the genotypes and the activity or amount of CYP3A4 were found (Fig. 1
, B).


View larger version (88K):
[in this window]
[in a new window]
|
Fig. 1. A) Representative patterns of MboII
restriction fragment length polymorphisms (RFLPs) of the CYP3A4 gene.
The first-step polymerase chain reaction (PCR) amplification of a
592-base-pair (bp) fragment (nucleotide -570 to +22) was performed by
the use of previously described methods (1,2). The second
set of PCR amplifications was carried out by the use of the nested
primers designed to amplify a 168-bp segment. The mismatched
forward and the reverse primer was
5'-GGACAGCCATAGAGACAAGGGGA-3' (-290 to
-312;
underlining indicates artificial mismatched site) and
5'-CACTCACTGACCTCCTTTGAGTTCA-3' (-145 to -169),
respectively. The forward primer was designed to introduce a
MboII (Takara Shuzo Co., Ltd., Otsu, Japan) restriction site
in wild-type allele (GAAGA, -287 to -291), not
in the variant one carrying an A to G transition at
-289 from the initial site of the transcription (4). The
1000-fold diluted product of the first PCR was subjected to nested PCR
in a volume of 50 µL containing 0.2 mM of each
deoxynucleoside triphosphate, 50 mM KCl, 10 mM
Tris-HCl (pH 8.3), 1.5 mM MgCl2, 0.5 M of
each primer, and 1.3 U of Taq polymerase (Takara Shuzu Co.,
Ltd.). PCR conditions were 94 °C for 5 minutes followed by
25 cycles of 94 °C for 30 seconds, 60 °C for 40
seconds, and 72 °C for 40 seconds (PCR Thermal Cycler MP;
Takara Shuzo Co., Ltd.,). A 2-µL PCR product was digested with 6 U
of MboII for 1 hour at 37 °C. Restriction fragments
were analyzed by 4% agarose gel electrophoresis and ethidium bromide
staining. DNA from wild-type homozygotes (W/W) was digested
into 134- and 34-bp fragments; DNA from homozygotes for the variant
allele (V/V) gave an undigested 168-bp fragment and DNA from
the heterozygous (W/V) genotype gave all three fragments. To
corroborate genotyping results of the variant, another mismatched
forward primer 5'-GGACAGCCATAGAGACAAGGCCA-3'
(-290 to -312; underlining indicates artificial mismatched site)
was designed to amplify a 168-bp segment introducing a ScrF I
(New England Biolabs, Inc., Beverly, MA) site in variant allele (CCNGG,
-288 to -292), that is not in wild-type. The set of PCR
amplifications was identical with that for MboII RFLP
described above. Digestion of PCR products with
ScrF I gave 146- and 22-bp fragments from the variant allele
or an undigested 168-bp from wild-type. The heterozygous genotype gave
all three fragments. B) Nifedipine oxidation activity and
cytochrome P450 3A4 (CYP3A4) protein level by each genotype. Nifedipine
oxidation activity was measured according to the method of Guengerich
et al. (5). CYP3A4 protein levels were obtained by western
blot analysis. Circles indicate subjects given dexamethasone or
phenytoin in their last hospitalization. The median nifedipine
oxidation activity of the five subjects carrying the variant allele was
2080 pmol/minute/mg protein (interquartile range, 1430-2430
pmol/minute/mg protein; range, 1120-5190), which did not differ
statistically significantly from that of the wild-type subjects (1255
pmol/minute/mg protein; interquartile range, 675-1785; range,
100-3440; two-sided P = .11, Mann-Whitney U test).
Furthermore, the difference in the CYP3A4 protein levels was not
statistically significant (two-sided P = .18, Mann-Whitney
U test), i.e., the median was 30.8 pmol/mg protein
(interquartile range, 18.9-45.3 pmol/mg protein; range, 5.5-110.9) in
the subjects homozygous for the wild-type allele and 64.6 pmol/mg
(interquartile range, 35.9-75.5 pmol/mg protein; range, 15.4-165.5)
in those who were heterozygous or homozygous for the variant allele.
|
|
Moreover, we examined the distribution of the new polymorphism in the Japanese population.
DNA samples were prepared from 128 unrelated, healthy volunteers (median age, 22 years; range,
21-40 years) who had given written informed consent for their blood to be used in this study; the
study had obtained approval from the Ethics Committee of Nagoya University School of
Medicine. Although the frequency of the variant allele is 9.4% in Caucasian individuals (1), the population study revealed that all of the Japanese subjects were
homozygous for wild-type.
In our in vitro analysis, we did not find any relationship between the CYP3A4
genotype and the level of nifedipine oxidation activity, which seems inconsistent with the findings
presented by Rebbeck et al. (1). The reason for the disagreement is
unclear. This may be due to the small sample size in our study. Alternatively, the variant allele
may be in linkage disequilibrium with another mutation or gene that more strongly influences
prostate carcinogenesis and leukemogenic drug effects. However, CYP3A4 activity is known to
exhibit wide interindividual variability due to physiologic factors (e.g., age, food), pathologic
conditions (e.g., hepatic diseases), environmental factors (e.g., smoking), and concomitant drug
intake (e.g., steroids, anticonvulsants, antifungal agents), aside from any genetic factors (6). We consider the difference in enzymatic activity between the CYP3A4
genotypes, if any, as too small to explain such a wide variation in the observed activity.
It cannot be ruled out that the genetic variant in a 5' regulatory element might be
related to a variability in the inducibility of CYP3A4 by such agents as rifampin or
anticonvulsants. It is also possible that the induction of CYP3A4 in subjects with the variant allele
might be weak compared with those with wild-type, given exposure to the same amount of
inducer.
This analysis demonstrates a great difference in the distribution of the variant allele between
Caucasian and Japanese populations. Recently, increased attention has been focused on racial
variability in drug disposition and sensitivity. The recognized ethnic difference in the distribution
of pharmacogenetic polymorphisms should be important in determining drug dosages in different
populations. Because the variant allele examined here does not seem to be strongly related to
CYP3A4 activity, the ethnic variability in the distribution of the polymorphism would be irrelevant
in determining, in different populations, the dosage of drugs that are primarily metabolized by
CYP3A4.
REFERENCES
1
Rebbeck TR, Jaffe JM, Walker AH, Wein AJ, Malkowicz SB.
Modification of clinical presentation of prostate tumors by a novel genetic variant in CYP3A4. J Natl Cancer Inst 1998;90:1225-9.[Abstract/Free Full Text]
2
Felix CA, Walker AH, Lange BJ, Williams TM, Winick NJ,
Cheung NV, et al. Association of CYP3A4 genotype with treatment-related leukemia. Proc
Natl Acad Sci U S A 1998;95:13176-81.[Abstract/Free Full Text]
3
Tateishi T, Watanabe M, Moriya H, Yamaguchi S, Sato T,
Kobayashi S. No ethnic difference between Caucasian and Japanese hepatic samples in the
expression frequency of CYP3A5 and CYP3A7 proteins. Biochem Pharmacol 1999;57:935-9.[Medline]
4
Hashimoto H, Toide K, Kitamura R, Fujita M, Tagawa S, Itoh S,
et al. Gene structure of CYP3A4, an adult-specific form of cytochrome P450 in human livers, and
its transcriptional control. Eur J Biochem 1993;218:585-95.[Abstract]
5
Guengerich FP, Martin MV, Beaune PH, Kremers P, Wolff T,
Waxman DJ. Characterization of rat and human liver microsomal cytochrome P-450 forms
involved in nifedipine oxidation, a prototype for genetic polymorphism in oxidative drug
metabolism. J Biol Chem 1986;261:505-160.[Abstract/Free Full Text]
6
Soons PA, Schellens JH, Breimer DD. Variability in
pharmacokinetics and metabolism of nifedipine and other dihydropyridine calcium entry blockers.
In: Kalow W, editor. Pharmacogenetics of drug metabolism. New York: Pergamon Press, Inc.;
1992. p. 769-89.
This article has been cited by other articles in HighWire Press-hosted journals:
-
Zeigler-Johnson, C., Friebel, T., Walker, A. H., Wang, Y., Spangler, E., Panossian, S., Patacsil, M., Aplenc, R., Wein, A. J., Malkowicz, S. B., Rebbeck, T. R.
(2004). CYP3A4, CYP3A5, and CYP3A43 Genotypes and Haplotypes in the Etiology and Severity of Prostate Cancer. Cancer Res
64: 8461-8467
[Abstract]
[Full Text]
-
Keshava, C., McCanlies, E. C., Weston, A.
(2004). CYP3A4 Polymorphisms--Potential Risk Factors for Breast and Prostate Cancer: A HuGE Review. Am. J. Epidemiol.
160: 825-841
[Abstract]
[Full Text]
-
Plummer, S. J., Conti, D. V., Paris, P. L., Curran, A. P., Casey, G., Witte, J. S.
(2003). CYP3A4 and CYP3A5 Genotypes, Haplotypes, and Risk of Prostate Cancer. Cancer Epidemiol Biomarkers Prev
12: 928-932
[Abstract]
[Full Text]
-
Guengerich, F. P.
(2003). Cytochromes P450, Drugs, and Diseases. Mol Interv
3: 194-204
[Abstract]
[Full Text]
-
Hotta, K., Sekine, I., Tamura, T., Sawada, M., Watanabe, H., Kusaba, H., Akiyama, Y., Inoue, A., Shimoyama, T., Nokihara, H., Ueda, Y., Yamamoto, N., Kunitoh, H., Ohe, Y., Kodama, T., Saijo, N.
(2001). A Phase I/II Study of Cisplatin and Vinorelbine Chemotherapy in Patients with Advanced Non-small Cell Lung Cancer. Jpn. J. Clin. Oncol.
31: 596-600
[Abstract]
[Full Text]
-
van Schaik, R. H.N., de Wildt, S. N., Brosens, R., van Fessem, M., van den Anker, J. N., Lindemans, J.
(2001). The CYP3A4*3 Allele: Is It Really Rare?. Clin Chem
47: 1104-1106
[Full Text]
-
von Ahsen, N., Richter, M., Grupp, C., Ringe, B., Oellerich, M., Armstrong, V. W.
(2001). No Influence of the MDR-1 C3435T Polymorphism or a CYP3A4 Promoter Polymorphism (CYP3A4-V Allele) on Dose-adjusted Cyclosporin A Trough Concentrations or Rejection Incidence in Stable Renal Transplant Recipients. Clin Chem
47: 1048-1052
[Abstract]
[Full Text]
-
Naoe, T., Takeyama, K., Yokozawa, T., Kiyoi, H., Seto, M., Uike, N., Ino, T., Utsunomiya, A., Maruta, A., Jin-nai, I., Kamada, N., Kubota, Y., Nakamura, H., Shimazaki, C., Horiike, S., Kodera, Y., Saito, H., Ueda, R., Wiemels, J., Ohno, R.
(2000). Analysis of Genetic Polymorphism in NQO1, GST-M1, GST-T1, and CYP3A4 in 469 Japanese Patients with Therapy-related Leukemia/Myelodysplastic Syndrome and de novo Acute Myeloid Leukemia. Clin Cancer Res
6: 4091-4095
[Abstract]
[Full Text]
-
van Schaik, R. H.N., de Wildt, S. N., van Iperen, N. M., Uitterlinden, A. G., van den Anker, J. N., Lindemans, J.
(2000). CYP3A4-V Polymorphism Detection by PCR-Restriction Fragment Length Polymorphism Analysis and Its Allelic Frequency among 199 Dutch Caucasians. Clin Chem
46: 1834-1836
[Full Text]
-
Rebbeck, T. R.
(2000). More About: Modification of Clinical Presentation of Prostate Tumors by a Novel Genetic Variant in CYP3A4. J Natl Cancer Inst
92: 76-76
[Full Text]
 |
|