Tacrolimus is an immunosuppressive drug used in liver and kidney transplant recipients to prevent the rejection of transplanted organs (1). Tacrolimus has a narrow therapeutic range, and the drug pharmacokinetics show wide interindividual variability (2, 3). The oral bioavailability and clearance of tacrolimus are dependent on cytochrome P450 3A (CYP3A) and P-glycoprotein (P-gp), which are encoded by the CYP3A and ATP-binding cassette sub-family B member 1 (ABCB1) genes, respectively.
In selecting the optimal dosage of a drug for an individual patient, dose adjustments may be made by monitoring drug concentration in the blood. This type of dose adjustment, however, requires considerable time, because a steady-state level must be reached first. For tacrolimus, therefore, it is difficult to determine the optimal dosage in the first few days after organ transplantation. This may lead to acute rejection of an organ if the tacrolimus concentration is too low, or drug toxicity if the tacrolimus concentration is too high. By understanding the impact of CYP3A and ABCB1 gene variants on drug metabolism, genotyping of individual patients or liver donors can allow the design of individualized tacrolimus dosage regimens, thus optimizing treatment of transplant recipients. We have therefore analyzed the frequencies of common functional single nucleotide polymorphisms (SNPs) of the CYP3A4, CYP3A5, CYP3AP1, and ABCB1 genes in Korean transplantation recipients and donors, and compared the blood concentrations of tacrolimus in patients with different genotypes to determine the impact of these polymorphisms on the metabolism of the drug.
This study presents the results of a search for both ABCB1 and CYP3A variants in a large panel of Korean individuals and show the relationship between the functional SNPs and tacrolimus concentration.
MATERIALS AND METHODS
Patients and Laboratory Analysis
Study participants included 506 recipients and 62 donors of solid organ transplants (259 liver transplants, 185 renal transplants, and 62 pairs of liver transplant recipients and donors) treated between 2001 and 2003 at Asan Medical Center (Seoul, Korea), for whom records of tacrolimus therapeutic drug monitoring were available. All study subjects were ethnically Korean. Blood sampling was performed when drug blood concentrations were in the steady state. The steady state was considered to be attained when constant or similar tacrolimus blood concentrations were measured serially for at least 7 days when tacrolimus was taken at a constant dosage. Blood concentrations of tacrolimus were measured using the Abbott IMx system and the tacrolimus II reagent (Abbott Diagnostics Laboratories, Abbott-Park, IL). Measured concentrations were converted to concentration-to-adjusted dose ratio (ng/mL per mg/kg/day), with the latter value used as the parameter for calculation using blood tacrolimus concentrations (4, 5). Demographic characteristics, laboratory test results, drug administration history, and pathologic data were obtained from the electronic medical records of Asan Medical Center. When liver transplant recipients showed any sign of rejection, this was explored by liver biopsy. The study protocol was approved by the Institutional Review Board of the Asan Medical Center.
Preparation of Genomic DNA
Genomic DNA was extracted from lymphocytes using the PUREGENE DNA Purification Kit (Gentra Systems, Inc., Minneapolis, MN), according to the manufacturer's protocol.
CYP3A4 c.830_831insA (CYP3A4*6), CYP3A4 c.878T>C (CYP3A4*18) (rs28371759), CYP3A5 c.306-237A>G(CYP3A5*3) (rs776746), CYP3A5P1 c.-44G>A (CYP3A5P1*3), ABCB1 c.2677G>A/T (rs2032582), and ABCB1 c.3435C>T (rs1045642) sequences were retrieved from the dbSNP database (http://www.ncbi.nlm.nih.gov/SNP).
The detection of SNPs was based on the analysis of primer extension products generated from previously amplified genomic DNA using a chip-based MALDI-TOF mass spectrometry platform (Sequenom Inc., San Diego, CA), with procedures performed according to the manufacturer's standard protocols.
Polymerase Chain Reaction
Polymerase chain reaction (PCR) primers were designed using the Primer3 program (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi) and are listed in Table 1. Each PCR reaction was performed in a volume of 5 μL containing 1× PCR buffer (TAKARA Biomedicals, Otsu, Japan), 2.5 mM MgCl2, 0.2 mM of each dNTP, 0.1 U HotStar Taq Polymerase (Qiagen GmbH, Hilden, Germany), 8 pM of each primer, and 4.0 ng of genomic DNA. The amplification protocol consisted of an initial denaturation at 95°C for 15 min, followed by 45 cycles of denaturation at 95°C for 20 sec, annealing at 56°C for 30 sec, and extension at 72°C for 1 min, with a final extension at 72°C for 3 min.
Homogeneous MassEXTEND (hME)
After PCR, all unincorporated dNTPs were removed by incubation with 0.3 U of shrimp alkaline phosphatase for 20 min at 37°C, followed by incubation for 5 min at 85°C to inactivate the enzyme.
The extension primers were designed manually and are also listed in Table 1. The choice of forward or reverse DNA strand was dependent on several factors, including characteristics of the polymorphism, guanosine cytosine content, and possible primer dimerization and hairpin structures.
The total volume of each reaction was 9 μL and included hME enzyme (Thermosequenase; GE Healthcare, Bucks, UK), the appropriate termination mix, and 5 μM of the appropriate extension primer. The primer extension protocol consisted of an initial denaturation at 94°C for 2 min, followed by 55 cycles of denaturation at 94°C for 5 sec, annealing at 52°C for 5 sec, and extension at 72°C for 5 sec. After desalting of the reaction product with SpectroCLEAN (Sequenom), the samples were dispensed on 384 well SpectroCHIPs (Sequenom) using SpectroJET (Sequenom), and the SpectroCHIPs were analyzed in the fully automated mode using the MALDI-TOF MassARRAY system (Bruker-Sequenom, San Diego, CA). Assays with bad peaks were reanalyzed manually.
Haplotype frequency, linkage disequilibrium (LD), and Hardy-Weinberg analysis were performed with PHASE (UW TechTransfer Digital Ventures, Seattle, WA) and Arlequin version 3.11 software (http://CMPG.unibe.ch/software/arlequin3). Drug concentrations in groups of patients with different genotypes were compared with Student's t test or analysis of variance using SPSS software for Windows version 11.5 (SPSS Inc., Chicago, IL). P values less than 0.05 were considered statistically significant.
Of the 506 liver transplant recipients (377 men, 129 women), 321 had liver transplants and 185 had kidney transplants. In liver transplant recipients, mean (±SD) patient age was 53.8±12.89 years (range, 0.8–98.0 years) and body weight was 61.2±11.7 kg. In these patients, the tacrolimus concentration-to-adjusted dose ratio was 181.50±243.3 ng/mL per mg/kg/day, and the adjusted drug dose at the time of blood drug concentration measurement was 0.06±0.06 mg/kg per day (range, 0.01–0.53 mg/kg per day).
In renal transplant recipients, patient age was 55.4±11.89 years (range, 31–98.0 years) and body weight was 62.8±10.08 kg. In these patients, the tacrolimus concentration-to-adjusted dose ratio was 116.7±85.75 ng/mL per mg/kg/day and the adjusted drug dose at the time of blood drug concentration measurement was 0.07±0.05 mg/kg per day (range, 0.01–0.53 mg/kg per day).
Genotype Frequencies of CYP3A and ABCB1
The distributions of CYP3A and ABCB1 genotypes are shown in Tables 2 to 4. All genotype frequencies were in Hardy-Weinberg equilibrium (P>0.18). Frequencies of variant alleles among these transplant recipients were CYP3A5*3 76.8%, CYP3A5P1*3 75.9%, ABCB1 c.2677A/T 1.9%, ABCB1 c.3435T 36.9%, CYP3A4*18 1.9%, and CYP3A4*6 0.1%.
Linkage Disequilibrium and Haplotype Analyses
Pairwise analysis of the LD of the four variant alleles with a frequency greater than 10% showed that CYP3A5 c.306-237A/G was strongly linked to CYP3A5P1 c.-44A/G (r2=0.816) and ABCB1 c.3435C/T was strongly linked to ABCB1 c.2677G/T (r2=0.312). The other combinations showed no LD (r2<0.05).
Using the genotypes of the six SNP sites, haplotype analysis showed that five haplotypes were represented with a frequency greater than 5%, covering 88.5% of all study subjects (Table 5).
Tacrolimus Concentration-to-Adjusted Dose Ratio Versus SNP Genotypes and Haplotypes
The tacrolimus blood concentration-to-adjusted dose ratios in groups with different genotypes are shown in Tables 2 to 4. Separate analyses by liver and kidney transplant recipients and donors showed the same results.
Among the CYP3A genotypes, the presence or absence of the CYP3A5*3 and CYP3A5P1*3 alleles was associated with differential levels of tacrolimus concentration-to-adjusted dose ratios in both liver transplant donors and kidney transplant recipients. For the CYP3A5 c.306-237 genotype, the tacrolimus concentration-to-adjusted dose ratio was significantly higher in transplant recipients with CYP3A5 *1/*3 than in those with *1/*1, and significantly higher in recipients with *3/*3 than in patients with *1/*3 (P<0.001 for each comparison). Similarly, for the CYP3A5P1 pseudogene, recipients with *3/*3 had significantly greater tacrolimus concentrations than did those with *1/*3 or *1/*1 (P<0.001).
There were no differences in tacrolimus concentration-to-adjusted dose ratios in transplant recipients with the CYP3A4*18 or ABCB1 c.2677G>T/A alleles compared with recipients with wild-type alleles. One patient with the CYP3A4*6 allele had a high tacrolimus concentration-to-adjusted dose ratio, but statistical analysis using single-patient data was not possible.
The average tacrolimus concentration-to-adjusted dose ratio did not differ among the five high-frequency haplotypes and the corresponding nonhaplotypes.
Tacrolimus Concentration-to-Adjusted Dose Ratio Versus Rejection of Transplanted Liver
In our study, liver biopsy was performed on 67 of 259 liver transplant patients and 20.1% showed findings of acute or chronic rejection (Table 6). The rejection incidence did not significantly correlate with the mean tacrolimus concentration-to-adjusted dose ratio or genotype variance. This may be because many factors, including genotype, are involved in the rejection of transplanted livers.
We have shown here that allelic frequencies of genotypes related to drug metabolism in Korean individuals were similar to those reported for other Asian ethnic groups (6–13). The frequency of the most common allele in this study, CYP3A5*3, was 76.8%, much higher than that of the wild-type allele (*1). The allele frequency of CYP3A5*3 in Asian individuals has been reported to be 60% to 76% (6–8), similar to our findings. In particular, a study of a Japanese population (8) reported a CYP3A5*3 allele frequency almost the same as the frequency found in Korean individuals in the present work.
The frequency distribution of the 3435C>T SNP of the ABCB1 gene is lower than that of the C/T or C/C genotypes in our study. This suggests ethnic variability in this SNP. Both C and T alleles are found at approximately similar frequencies in whites (14), and the T/T genotype is common in Malaysian patients (14). This ethnic variability in SNP, and any effects on drug pharmacokinetics or pharmacodynamics, should be explored further in Asian populations.
Recently, it has been reported that CYP3A5 gene polymorphisms of donors rather than recipients affect individual tacrolimus dose requirements in liver transplantation recipients (15, 16). This study performed genotype analysis on 62 pairs of liver transplant recipients and donors. The data show that CYP3A5 and CYP3AP1 gene polymorphisms of donors rather than recipients influence individual tacrolimus dose requirements in liver transplantation.
CYP3A5 encodes a truncated CYP3A5 protein that is expressed in the kidney and intestine and which shows polymorphic expression in the liver. Individuals with CYP3A5*3 allele may have a lower rate of tacrolimus metabolism and thus higher steady-state tacrolimus concentrations. Indeed, previous studies have shown that subjects with the CYP3A5*3 allele have higher blood tacrolimus concentrations than those with the CYP3A5*1 allele (5–7, 17). We also found that the tacrolimus concentration-to-adjusted dose ratio was much higher in subjects with the CYP3A5*3 allele than in those with CYP3A5*1 alleles and we observed a dosage effect of this allele.
CYP3A5P1 is a pseudogene located in the CYP3A7-CYP3A5 intergenic region; although the gene is involved in hepatic CYP3A activity, the genetic mechanism has not yet been determined (9, 12, 18, 19). We found that the tacrolimus concentration-to-adjusted dose ratio was much lower in subjects with the −44G allele (*1) compared with those with the A allele (*3).
CYP3A4*6 is a rare variant, in which the insertion of an adenine residue in exon 9 causes a frameshift and generates an early TGA stop codon, thus possibly lowering blood tacrolimus concentration by impeding CYP3A4 enzyme activity (20). Only one of our patients had the CYP3A4*6 allele, and this patient had a tacrolimus concentration-to-adjusted dose ratio 4.3-fold higher than the mean ratio seen in wild-type patients.
CYP3A4*18 is a nonsynonymous SNP that is present only in Asian individuals at an allele frequency of 2% and causes a change from leucine to proline at codon 293. Moreover, a recombinant CYP3A4*18 allele was shown to enhance the in vitro metabolism of testosterone and chlorpyrifos (11). Although we found that the frequency of this allele was 1.9%, as shown previously, the presence of this allele had no effect on tacrolimus concentration-to-adjusted dose ratio relative to wild-type individuals.
Significant interinstitutional variations exist in recommended doses and blood target levels of tacrolimus in solid organ transplant recipients. In our institution, the recommended oral doses for liver and kidney recipients are 0.1 to 0.2 and 0.15 to 0.3 mg/kg per day, respectively, and the target blood drug concentrations are 5 to 20 and 8 to 20 ng/mL, respectively (3). Overall, the maintenance dose of tacrolimus in our patients was 0.01 to 0.58 mg/kg per day and blood drug concentration at the steady state was 2.2 to 105.1 ng/mL, with the wide variability in both values possibly arising, at least in part, from genotype effects. The allele frequencies of significant genes have been found to range from 38.4% to 76.8%, so it may be necessary to determine genotype-specific target ranges for tacrolimus.
We assessed transplant recipients with two ABCB1 SNPs, c.2677G>A/T and c.3435C>T, both of which are associated with drug metabolism. Although the absorption of tacrolimus is regulated by P-gp, the roles of the SNPs of ABCB1, which encodes P-gp, are not yet clear (5, 18, 21–24).
In summary, we found that the CYP3A5*3 and pseudogene CYP3A5P1*3 alleles are common in the Korean population and are correlated with tacrolimus blood concentrations in solid organ transplant patients. In renal transplant patients, tacrolimus concentration was associated with patient CYP3A5 genotype, but in liver transplant patients, it was associated with the CYP3A5 genotype of the donor only. Therefore, the CYP3A5 genotype of the functioning liver shows the most important association with tacrolimus concentrations.
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