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Articles |
Departments of
1
Clinical Chemistry,
2
Nephrology and Rheumatology, and
3
Transplant Surgery, Georg-August-University, Robert-Koch-Strasse 40, 37075 Göttingen, Germany.
aAuthor for correspondence. Fax 49-551-39-12504; e-mail nahsen{at}gwdg.de.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: We investigated the occurrence of both polymorphisms in 124 stable Caucasian renal transplant recipients (>6 months after transplantation) on CsA as the primary immunosuppressant. Real-time, rapid-cycle PCR methods were developed and used for genotyping.
Results: The estimated allele frequencies for the MDR-1 C3435T allele (54%) and the CYP3A4-V allele (4.8%) were similar to those reported for Caucasian populations. No significant differences were found for the CsA doses needed to maintain similar CsA trough concentrations in patients with and without the CYP3A4-V allele or in patients with different MDR-1 C3435T genotypes. Furthermore, neither of the polymorphisms investigated was associated with renal function as assessed by creatinine plasma concentration or, in a retrospective analysis, the incidence of acute rejection.
Conclusions: These findings suggest that the MDR-1 C3435T mutation and the CYP3A4-V variant are not major determinants of CsA efficacy in renal transplant recipients.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cytochrome P-450 3A4 (CYP3A4) (2), the most abundant CYP enzyme in the human liver and intestine with wide intra- and interindividual differences in activity [for a review see Ref. (3)], makes a substantial contribution to the metabolism of CsA. This remarkable variation in drug metabolism may be caused by genetic polymorphisms, induction or inhibition of enzyme activity, physiological status, and concomitant diseases (4). CsA is also a substrate for intestinal P-glycoprotein (P-gp), the product of the multidrug resistance (MDR) gene (5). As with CYP3A4, significant interindividual variation has been observed in P-gp (6). It is therefore not surprising that a substantial proportion of the variability in the oral clearance of CsA could be attributed to variability in liver CYP3A4 activity and intestinal P-gp concentration (6).
Functional polymorphisms have been described for both of these
pharmacogenetically interesting genes. A polymorphism in the
CYP3A4 promoter region was identified and termed
"variant" allele CYP3A4-V (7). The mutation
is an A
G transition at position -290 and is situated in the
nifedipine-specific element of the gene. The presence of the
CYP3A4-V allele was associated with higher grade prostate
cancer (7), and it was proposed that people with the
CYP3A4-V genotype may have decreased CYP3A4 protein
activity. However, in an in vitro system, higher expression of a
reporter gene assay for the CYP3A4-V allele was observed as
compared with the -W allele (8). The induction of
P-gp by rifampin decreases the area under the curve of orally
administered digoxin, another substrate of P-gp (9).
Recently, Hoffmeyer et al. (10) found that a polymorphism in
exon 26 (C3435T) of the MDR-1 gene was correlated with
intestinal expression and in vivo activity of P-gp.
Because these two polymorphisms might be expected to have an impact on the metabolism and therapeutic efficacy of CsA, we undertook a study to determine their occurrence in 124 stable renal transplant recipients and to establish whether the presence of either polymorphism was correlated with the following: (a) an altered CsA dose requirement to achieve the target therapeutic range; (b) acute rejection as determined by biopsy; or (c) renal function as assessed by plasma creatinine concentrations.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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routine analyses
During a visit to the transplant outpatient clinic, blood samples
were drawn for the determination of CsA trough concentrations, as well
as for routine laboratory analyses. Plasma creatinine concentrations
were measured on a Hitachi 917 analyzer using reagents from Roche
Diagnostics. CsA concentrations were determined using the Emit assay
(Dade Behring) on a Cobas Mira analyzer (Roche) (11).
Dose-adjusted trough concentrations were calculated by dividing CsA
trough concentrations by the corresponding 12-h dose on a mg/kg basis.
genotype determination
Genotyping for the MDR-1 C3435T and CYP3A4-V
polymorphisms was performed using homogeneous hybridization probe
assays on the LightCyclerTM (Roche Biochemica).
MDR-1 C3435T primers and probes
A 252-bp fragment of the MDR-1 gene (GenBank Accession
No. AC005068) was generated by PCR amplification with the primers
(forward, 0.5 µM) 5'-TGT TTT CAG CTG CTT GAT GG-3' and (reverse, 0.5
µM) 5'-CAT GCT CCC AGG CTG TTT AT-3'. The reaction mixture also
included the wild-type probe (0.1 µM) 5'-AAG AGA TCG TGA
GGG CA-3'-fluorescein and the anchor probe (0.3 µM) 5'-Cy5.5-AAG GAG
GCC AAC ATA CAT GCC-3'-phosphate. The anchor and probe are derived from
the sequence of the sense strand. The underlined position in the probe
defines a CT polymorphism designated C3435T. The resulting C:A
mismatch is very unstable and destabilizes the probe. Heterozygous
samples are therefore well resolved (Fig. 1A
). The designation MDR-1 wild type is used
throughout this report to designate the allele that contains no 3435T
mutation. The presence of the expected mutation was confirmed by
sequencing the products (sequenase cycle sequencing reagent set;
Amersham) on an automated DNA sequencer (Licor 4200; Licor). The
resulting characterized samples were used as controls in the assay.
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CYP3A4-V allele primers and probes
A 427-bp fragment from the CYP3A4 gene promoter
(GenBank Accession No. D11131) was amplified by PCR with the primers
(forward, 0.5 µM) (12) 5'-AAC AGG CGT GGA AAC ACA AT-3'
and (reverse, 0.5 µM) 5'-CCA CTC ACT GAC CTC CTT TGA-3'. The
wild-type probe (0.1 µM) 5'-AGG GCA AGA GAG AGG
C-3'-fluorescein and the anchor probe (0.3 µM) 5'-LC-Red640-TTA ATA
GAT TTT ATG CCA ATG GCT CCA CTT GAG-3'-phosphate were included in the
reaction mixture. The probes were modified as indicated by the addition
of fluorescein, the LC-Red640 dye (Roche Biochemica), or
phosphorylation. The probe and anchor bind to the antisense strand. The
probe is specific for the sequence of the CYP3A4 gene and
has no hybridization site on the related CYP3A5 and
CYP3A7 genes. The CYP3A4-V allele is
characterized by an AG polymorphism at the underlined position. The
resulting A:C mismatch significantly destabilizes the probe and allows
for a good assignment of heterozygous samples (Fig. 1B
). The presence
of the expected mutation was also confirmed by the sequencing of PCR
products. Resulting characterized samples were used as controls in the
assay.
general PCR procedure
Genomic DNA was isolated from the EDTA-anticoagulated whole blood
not required for CsA measurements using a rapid method based on
alkaline lysis (13). DNA prepared by alternative methods is
equally suitable. We do not routinely quantify DNA before the assay
because the assay is very robust at different concentrations
(13). The reaction mixture for PCR amplification consisted
of 1 µL of genomic DNA solution, primers and probes at the
concentrations indicated above, 0.5 U of Taq DNA polymerase (Life
Technologies), 1 µL of 10x PCR buffer (Life Technologies), 0.2 mM
each dNTP (Roche Biochemica), 2.5 mM MgCl2, 500
mg/L bovine serum albumin (New England BioLabs), and 50 mL/L dimethyl
sulfoxide (Sigma). PCR-grade water was added to a final volume of 10
µL. The cycling program consisted of 30 s of initial
denaturation at 95 °C and 45 cycles of the following with the
maximum ramp rate: 95 °C for 0 s; 55 °C for 5 s; and
72 °C for 10 s. Melting curve acquisition was from 40 to
65 °C at 0.2 °C in channel two (LC-Red640) or channel three
(Cy5.5), respectively. For the MDR-1 C3435T polymorphism,
the melting point of the detection probe was 57.0 °C when hybridized
to the wild-type allele and 45.5 °C when hybridized to the mutant
allele. For the CYP3A4 allele, the melting point of the
detection probe was 56.5 °C when hybridized to the wild-type allele
and 47.5 °C when hybridized to the mutant allele (Fig. 1
).
statistical analysis
All calculations were performed with SPSS for Windows.
Between-group comparisons were done with the Mann–Whitney
U-test for the mutant CYP3A4 allele.
Between-group frequencies of alleles were compared by Fisher’s exact
test or Pearson’s 
2, as appropriate.
Between-group comparisons for the MDR-1 genotypes were
performed by one-way ANOVA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2=3.01; P = 0.08). As can be
seen from Table 1
, the current total CsA doses and weight-adjusted doses were
similar, irrespective of the MDR-1 genotype. Patients who
were homozygous for the MDR-1 wild-type genotype (3435 CC)
were maintained at CsA trough concentrations similar to those for
patients who were homozygous for the MDR-1 3435 TT mutation.
Consequently, there was no significant difference in the dose-adjusted
trough concentrations. Neither the incidence of acute rejection
(P = 0.602) nor the plasma creatinine concentrations
(P = 0.238) differed between the groups with different
MDR-1 3435 alleles. Acute rejection was confirmed by biopsy.
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The variant CYP3A4-V allele was found in 12 of 124
transplant recipients. All 24 subjects were heterozygous for the
mutation. There was no gender-related difference for the prevalence of
the mutation (P = 0.232) and the population was in
Hardy–Weinberg equilibrium (2=0.32;
P = 0.57). Patients who were heterozygous for the
CYP3A4-V allele were maintained at CsA doses and trough CsA
concentrations similar to those for patients who were homozygous for
the wild-type allele (Table 2
). The incidence of acute rejection was similar
(P = 0.533) between the two groups and the plasma
creatinine concentrations did not differ (P = 0.842).
To investigate a possible interaction between the CYP3A4-V
allele and the MDR-1 3435 genotype, we compared eight
patients carrying the heterozygous CYP3A4-V allele, as well
as a heterozygous (n = 5) or a homozygous (n = 3)
MDR-1 3435 genotype. These cases were not different from
those having both wild-type alleles (n = 27) with respect to the
CsA daily dose (P = 0.862), the CsA dose per kg
(P = 0.269), the dose-adjusted trough concentration
(P = 0.499), or acute rejections (P =
0.236).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The recently reported functional polymorphism of the MDR-1 gene (C3435T) correlated with MDR-1 in vivo expression and activity (10). The mutant allele, although caused by a wobble base, was associated with significantly decreased intestinal P-gp expression, higher peak digoxin concentrations, and, in a small group of individuals, a higher digoxin area under the concentration–time curve after rifampin induction of P-gp. This mutation appears to be fairly common with an estimated allele frequency of 48% in a Caucasian population (10). In our collective of 124 Caucasian renal transplant recipients, the estimated allele frequency of the MDR-1 C3435T allele of 54% was similar to that reported by Hoffmeyer et al. (10). However, no association was observed between the occurrence of this mutation and the dose-adjusted CsA trough concentration. Thus, the CsA dose required to maintain CsA whole-blood trough concentrations within the therapeutic range (100–150 µg/L) at our institution for renal transplant recipients was independent of the MDR-1 genotype. Because trough CsA concentrations are a product of absorption, distribution, and elimination, it may be possible that the MDR-1 C3435T mutation is associated with higher peak CsA concentrations during the early postabsorptive phase leading to increased exposure to CsA. A retrospective analysis revealed no correlation between the MDR-1 genotype and the incidence of acute rejection in this collective. The rejection incidence in the group of patients who were homozygous for the MDR-1 C3435T genotype was almost identical to that found in patients who were homozygous for the wild-type allele (41% vs 42%, respectively). Furthermore, plasma creatinine concentrations did not differ between patients who were homozygous for the MDR-1 C3435T genotype and those who were homozygous for the wild-type genotype. Because the nephrotoxicity of CsA is associated with increased exposure to the drug, these findings suggest that the MDR-1 C3435T mutation may not be an important determinant for CsA concentrations in renal transplant recipients receiving the microemulsion formulation Neoral. Because we investigated only stable renal transplant patients who received transplants at least 6 months before this study, the possibility of a selection bias must be considered. However, the allele frequencies in this transplant collective for both MDR-1 and CYP3A4 were in Hardy–Weinberg equilibrium and were also in agreement with published allele frequencies for healthy controls (10). This argues against a major selection bias.
It can be concluded that any influence of the MDR-1 polymorphism on CsA exposure is not sufficient to have a major effect on the clinical outcome of patients whose CsA dosage is monitored according to trough CsA concentrations. The possibility must be considered that the microemulsion formulation could potentially prevent the interaction of CsA with P-gp. Choc et al. (15) investigated the influence of a CYP3A/P-gp inhibitor on the bioavailability of CsA and concluded that for Neoral and Sandimmun, there was no discriminating formulation-dependent effect on the CYP3A and P-gp systems.
The CYP3A4-V allele has an estimated frequency of 3.6–9.6% in the Caucasian population (7)(17)(18). The observed frequency of 4.8% in our collective of renal transplant recipients is in accordance with these previous findings. On the basis of clinical studies (7)(12), it was postulated that the CYP3A4-V allele may be associated with decreased CYP3A4 expression or decreased enzymatic activity. However, in vitro studies do not support this hypothesis (8)(16)(17)(18). Our results are in agreement with these latter studies and show a lack of functional significance of the CYP3A4-V allele for the CsA dose required to achieve therapeutic concentrations of CsA, as well as for the incidence of acute rejection in renal transplant recipients on CsA immunosuppression. While this report was in preparation, the investigation of Rivory et al. (19) was published, reporting an incidence for the CYP3A4-V allele of 3% among 91 renal transplant recipients. The polymorphism did not influence the CsA pseudoclearance, which is in agreement with our findings. Data on rejection incidence or renal function were not reported in this study. From a comparison of patients carrying the heterozygous CYP3A4-V allele and also a heterozygous or homozygous MDR-1 3435 genotype with those having both wild-type alleles, we found no evidence for possible interactions between these genotypes.
In conclusion, our results support the notion that the investigated polymorphisms are not major determinants of interindividual CsA variability. We found no significant differences in the CsA doses needed for the maintenance of similar CsA trough concentrations in stable renal transplant recipients with and without the CYP3A4-V allele or in patients with different MDR-1 C3435T genotypes. Furthermore, after stratification according to genotype, we found no significant differences with respect to renal function as assessed by creatinine plasma concentration or with respect to the incidence of acute rejection. If there is a functional relevance of the CYP3A4-V allele, then this would only lead to minor changes in CYP3A4 expression. The manifestation of diseases eventually caused by this polymorphism might only be expected in the long term. In the case of the MDR-1 C3435T polymorphism, other factors may be necessary for the genotype to gain clinically functional relevance, such as induction of enzyme activity, physiological status, or concomitant disease. The genotyping of these polymorphisms is unlikely to be useful for the optimization of CsA drug therapy in the clinical setting.
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Acknowledgments |
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Footnotes |
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1 Nonstandard abbreviations: CsA, cyclosporin A; CYP3A4, cytochrome P-450 3A4; P-gp, P-glycoprotein; and MDR, multidrug resistance. 
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1-antitrypsin genotyping by multiplex real-time fluorescence PCR with the LightCycler. Clin Chem 2000;46:156-161.The following articles in journals at HighWire Press have cited this article:
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