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”.