Genotyping of cytochrome P450 3A5
Genomic DNA was extracted from whole blood samples using a QIAamp DNA
Mini kit (Qiagen Inc., Germantown, MD, USA), and stored at - 20°C. The
genotypes of the CYP3A5 gene (6986 A>G in
intron 3) and the ABCB1 gene (1236C>T in exon
12 and 3435C>T in exon 26) were determined using the
TaqMan allelic discrimination assay. Primers and probes were designed
with Primer Express Software Version 3.0 (Applied Biosystems, Foster
City, CA, USA). The polymerase chain reaction (PCR) mixture included 50
ng of genomic DNA in a 1 μL volume, along with the following reagents:
FAM and TET probes (5 pmol/μL, respectively), primers (20 pmol/μL for
sense and antisense primers, respectively), and 2X TaqMan Universal PCR
Master Mix (Applied Biosystems). PCR cycling reactions were conducted in
an ABI 9700 PCR system (Applied Biosystems); the reactions consisted of
initial 10-min denaturation at 95°C, followed by 40 cycles of 15-sec
denaturation at 95°C and 1-min annealing and extension at 60°C. The
results were analyzed with the ABI 7900HT Sequence Detection System
(Applied Biosystems). To control the genotyping quality, 10% of the
samples were randomly selected for retyping with a double-blind check.
Genetic polymorphisms were analyzed on the day of transplantation. The
study patients were blinded to the genetic polymorphisms they carried.
Considering the low frequency of CYP3A5*1 allele, its carriers(CYP3A5*1/*1 or CYP3A5*1/*3) were selected first and classified
as the “expressors”. The patients who carried CYP3A5*3/*3genotype, responsible for the lack of CYP3A5 expression, were
selected second, and were classified as the “non-expressors”.