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Articles |
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Department of Clinical and Toxicological Analysis, Faculty of Pharmaceutical Sciences of the Sao Paulo University, Av. Lineu Prestes 580, B17, CEP 05508-900, Sao Paulo, SP, Brazil
a author for correspondence: fax 55-11-3813-2197, e-mail scavalli{at}usp.br
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Introduction |
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 Top Introduction References |
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G) transition at the 5' promoter region
(5'PR) of the human CYP3A4 gene (position
-290) that is located in a sequence known as the
nifedipine-specific element. The variant 5'PR allele was associated
with higher clinical stage and grade in men with prostate tumors,
possibly because of increased testosterone bioavailability
(5). On the other hand, the variant allele was observed to
be a protective factor for treatment-related leukemia, whereas the
wild-type allele for this polymorphism of CYP3A4 was
associated with increased metabolism of epipodophyllotoxin, a
chemotherapy agent, leading to leukemias with MLL gene
translocations (6). These studies suggest that the variant
allele of 5'PR is associated with decreased CYP3A4 expression or
decreased activity of the enzyme (5)(6). In
contrast, pharmacokinetic studies did not confirm these findings
(7)(8).
A high degree of variability in the frequency of the 5'PR polymorphism
has been observed among racial groups. The frequency of the variant
5'PR allele is higher in black subjects (
60%) than in Caucasians
(4%). Interestingly, this allele has not been found in Japanese and
Chinese subjects (8)(9). The effect caused by
the 5'PR polymorphism in the metabolism of drugs depends on the 5'PR
genotype. Therefore, the identification of this polymorphism is
fundamental for evaluation of the potential risk or protection for
cancer diseases or of therapeutic failure. The variant 5'PR allele
usually is screened by conformation-sensitive gel
electrophoresis of the product amplified by PCR
(5), which has to be confirmed by DNA sequencing
(7)(8). Therefore, methods that directly
identify this variant would greatly improve the screening of the 5'PR
polymorphic site of the CYP3A4 gene in individuals with
different therapeutic responses. In this study, we developed a method
for direct detection of the variant 5'PR allele of the
CYP3A4 gene by site-directed mutation PCR followed by
MboII restriction analysis.
Blood samples were obtained after informed consent from 90 healthy
white individuals, and genomic DNA was extracted from leukocytes by a
salting-out method (10). The forward
(5'-GGAATGAGGACAGCCATAGAGACAAGGGGA-3') and reverse
(5'-CCTTTCAGCTCTGTGTTGCTCTTTGCTG-3') primers were created based on the
published sequence [GenBank Accession No. D11131
(http://www.ncbi.nlm.nih.gov/GenBank)] (11)
for amplification of the flanking 5' promoter region of the human
CYP3A4 gene (nucleotides -318 to +67) by PCR. The sense
primer was designed to generate an MboII endonuclease
recognition site when the first base incorporated by extension is an
adenine (A; wild type). In contrast, incorporation of a guanine (G;
variant) does not create the recognition site for MboII
(Fig. 1A
). Each 50-µL PCR reaction contained 50 ng of genomic DNA,
1 U of DNA polymerase (Biotools), 10 pmol of each primer, 200
µM each dNTP (Amersham-Pharmacia Biotech do Brasil), 2.0 mM
MgCl2, and PCR buffer [50 mM KCl, 2 mM
MgCl2, 20 mM
(NH4)2SO4,
75 mM Tris-HCl, pH 9.0; Biotools]. After initial denaturation at
98 °C for 5 min, amplification was performed using 30 cycles of
95 °C for 1 min, 60 °C for 1.5 min, and 72 °C for 2 min,
followed by 72 °C for 10 min for final extension. The 385-bp
amplification product contained both polymorphic (position 41) and
constitutive (position 210) cleavage sites for MboII. The
constitutive site can be useful as an internal control for the
enzymatic activity.
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Amplification products were submitted to MboII cleavage (5U;
Amersham Pharmacia Biotech do Brasil) in a total reaction volume of 15
µL. The fragments were analyzed by 3% agarose gel electrophoresis.
Fig. 1B
shows the electrophoretic profile for individuals
carrying different genotypes of the MboII polymorphism at
5'PR of the CYP3A4 gene. DNA from homozygous wild-type
subjects (genotype M+M+) produced two fragments of 175 and 169 bp,
whereas DNA from individuals homozygous for the variant type (genotype
M-M-) produced 210- and 175-bp fragments, and the heterozygous
genotype (genotype M+M-) was identified by the presence of 210-, 175-,
and 169-bp fragments. As demonstrated, the presence of the 210-, 175-,
and 169-bp fragments allows discrimination among the MboII
genotypes. For this reason, visualization of the 41-bp fragment is not
necessary for identification of 5'PR genotypes.
One sample of each different genotype was sequenced in the ABI Prism 377 DNA Sequencer (Applied Biosystem) to confirm the expected sequences of the M+M+, M+M-, and M-M- genotypes.
2 analysis was used for comparison of allelic
frequencies and genotype distribution between the studied individuals
and data in the literature, with the level of significance set at 5%.
Analysis of the MboII polymorphism showed that the frequency
of the variant allele (M-; 13.3%) in the studied group was higher
than the frequency found in white individuals from the United States
(3.6%; P = 0.0021) (8). Therefore, future
studies are necessary to confirm these data.
The aim of this study was to establish a rapid and reliable method for detection of the CYP3A4 5'PR polymorphism in a manner suitable for studies of the prevalence of the polymorphism in a population. Single nucleotide polymorphisms of various genes, such as apolipoprotein E, apolipoprotein B, and LDL receptor (12)(13)(14), can be directly identified by genotyping using restriction endonucleases. The polymorphism of the 5'PR of the CYP3A4 gene does not undergo cleavage by currently available endonucleases. Therefore, using appropriate primers, we introduced a cleavage site for MboII into the PCR product by changing a single base. This strategy allows direct detection of the variant 5'PR allele, which can be easily identified by agarose gel electrophoresis. Moreover, the presence of the constitutive cleavage site for MboII improves the accuracy of the test, controlling the efficiency of the restriction reaction.
MboII restriction analysis of the 5'PR polymorphism of the CYP3A4 gene, as described here, is less complex than conformation-sensitive gel electrophoresis and DNA sequencing (5)(7)(8). Therefore, it can easily be introduced into a large number of laboratories. The proposed method is rapid, does not require confirmatory tests, and can be useful for screening the CYP3A4 polymorphism in pharmacogenetic research.
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Acknowledgments |
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References |
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TopIntroductionReferences |
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The following articles in journals at HighWire Press have cited this article:
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  E. Goekkurt, S.-E. Al-Batran, U. Mogck, C. Pauligk, J. T. Hartmann, M. Kramer, E. Jaeger, G. Ehninger, and J. Stoehlmacher Pharmacogenetic analyses of hematotoxicity in advanced gastric cancer patients receiving biweekly fluorouracil, leucovorin, oxaliplatin and docetaxel (FLOT): a translational study of the Arbeitsgemeinschaft Internistische Onkologie (AIO) Ann. Onc., March 1, 2009; 20(3): 481 - 485. [Abstract] [Full Text] [PDF]
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 J. M. Vagace, G. Gervasini, F. Morais, J. Benitez, N. Alonso, D. de Argila, I. Arranz, and R. Bajo Retinal Toxic Reactions Following Photopheresis Arch Dermatol, May 1, 2007; 143(5): 622 - 625. [Abstract] [Full Text] [PDF]
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3']
that is not present when the variant allele is amplified.
MboII also has a constitutive cleavage site. After
MboII digestion, fragments of 210 (variant) or 169 (wild
type) bp are produced. (B), after electrophoresis, the
3% agarose gel was stained with ethidium bromide to visualize PCR
amplification and MboII restriction products.
Lane 1, M-M- genotype (210 and 175 bp); lane
2, M+M- genotype (210, 175, and 169 bp); lane
3, M+M+ genotype (175 and 169 bp); lane M,
100-bp DNA ladder.