Discussion
Present data strongly suggest that the variant ABCG2
c.421C>A (rs2231142) allele increases
AUCτ,ss of MPA in stable renal transplant patients (by
around 40%, with a high probability that the effect is
>20%) in agreement with proportionally reduced
CLT/F,ss. The estimates are consistent based on raw data
(patients free of relevant interfering comorbidities and co-medication)
and in matched/adjusted analysis, where a number of further potential
confounders, “classical” and pharmacogenetic, were controlled for.
Considering the latter, we did not account for the SLCO1B3
c.334T>G (rs4149117) and UGT1A9*3(c.98T>C , rs72551330) SNPs. OATP1B3 mediates MPAG
uptake, and variant SLCO1B3 c.334T> G shows around
40% reduced activity in vitro [33]. We identified 4 studies
(two in European patients [31, 33], and one each in Chinese [34]
and Japanese [35] patients) reporting crude mean±SD dose-adjusted
MPA AUCτ,ss in TT/TG vs. GG patients on IR MMF
co-treated with CsA (3 cohorts) or macrolactam immunosuppressants (3
cohorts): pooled TT/TG vs. GG differences in the co-treatment subgroups
(consistently) and overall suggested a slight tendency of higher
exposure (by some 10-15%) in TT/TG subjects (see Figure S2). The most
compelling individual study findings were those [31] suggesting by
around 24% higher (crude) AUC in 56 TT/TG vs. 111 GG patients
co-treated with CsA, and around 18% higher AUC in 54 TT/TG vs. 107 GG
patients co-treated with macrolactams. The UGT1A9
c.98T>C SNP results in reduced enzyme activity in
vitro [36]. We identified 3 studies (European patients) [32, 33,
37] reporting crude AUCτ,ss in TC vs. TT patients on
IR MMF co-treated with CsA (2 cohorts) or with macrolactams (3 cohorts):
pooled TC vs. TT differences consistently suggested a mild tendency of
higher (by ∼10%) exposure in TC subjects (see Figure S3). The most
compelling individual study findings were those [32] reporting
around 50% higher AUC (time-averaged estimate of 6 measurements over 1
year) in 5 TC vs. 170 TT patients co-treated with CsA and in 5 TC vs.
158 TT patients co-treated with tacrolimus. The present sensitivity
analysis (Figure 3) demonstrates: even with a marked simultaneous
imbalance between ABCG2 c.421C>A variant and wt
patients regarding both TT/TG (SLCO1B3 ) and TC (UGT1A9 )
genotypes and assuming their maximum reported effects, bias-adjusted
estimate of the ABCG2 c.421C>A variant allele effect
would still be >1.25 (i.e., above the conventional upper
limit of equivalent exposure). However, it is not very likely that the
present estimate was biased by these two SNPs to such an extent: (i) all
the reported values were crude, unadjusted values; ii) there is no
biologically plausible reason to expect such a huge simultaneous
imbalance in prevalence of the two genotypes between ABCG2variant and wt subjects; (iii) UGT1A9 c.98T>C SNP is
rare, and a reasonably expected number of TC subjects in the present
sample is 2-3; (iv) population pharmacokinetic models in French [38]
and Chinese patients [39] found no association between these two
SNPs and MPA clearance. Also, it does not seem likely that other
enzyme/transporter SNPs could explain the present observations. ThreeUGT1A9 promoter SNPs [beyond -275T>A(rs6714486) and -2152C>T (rs17868320) that we
controlled for] are associated with increased UGT1A9 levels in the
liver: - 440C>T (rs2741045), -331T>C
(rs2741046) and -665C>T (rs10176426) [4,5,40]. However,
studies have failed to provide consistent signals about association of
any of these SNPs and exposure to MPA; moreover, rs6714486 and
rs17868320 are in complete LD with these SNPs and form two haplotypes
(UGT1A9*1l and *1n ) [40]. Therefore, by controlling
for rs6714486 and rs17868320, one controls also for several SNPs that
were not directly genotyped. No consistent signal of association with
MPA exposure has been found for several other UGT1A9 SNPs
(rs6731242, rs13418420, rs3832043, rs2741049, rs13418420, rs17868323)
[4,5,39,41]. Moreover, rs6714486 and rs17868320 are in LD with some
of them (haplotypes UGT1A91v and *1w ) [40]. Apart fromUGT2B7 802C>T (rs7439366), here “represented” by
rs7668258 (since in complete LD), studies have consistently failed to
yield a clear, reproducible signal of association of any otherUGT2B7 SNP and exposure to MPA. The same applies for a number of
evaluated UGT1A1, 1A7 and 1A8 SNPs [4,5,39,41]. In the
present analysis, we evaluated the effect of one of the ABCG2polymorphisms (rs2231142). Reduced transporter function has been
reported associated with three further SNPs (rs34783571, rs192169062 and
rs34264773), for three SNPs no effect on function is reported and for
the rest functional consequences are unknown [8]. The estimated
global cumulative minor allele prevalence of all “reduced function”
SNPs is 0.68%, and for combined “unknown” and “reduced” it is 1.3%
[8] – this implies that at most one of the present patients should
be reasonably expected to carry any of these SNPs, and it is highly
unlikely that this possibility affected the present estimates.
Similarly, the three ABCB1 (linked) SNPs controlled for are by
far the most prevalent (among Caucasians) coding ABCB1 variants.
Cumulative prevalence of other six coding ABCB1 SNPs in
Caucasians is around 10% [42], suggesting that at most 6-7 patients
in the current sample might have harbored any of those SNPs. In order to
be accountable to any relevant part of the present observations, all
such (hypothetical) SNPs should have had marked and synergistic effects
– not a likely scenario: as recently reviewed [43], most of them
have no practical relevance in drug pharmacokinetics. The same is
applicable to the ABCC2 SNPs (beyond those controlled for in the
present study) and a wide range of investigated ABCC1 andABCC3 SNPs [43]. Specifically, in respect to MPA, apart fromABCC2 1249G>A (rs2273697) and-24C>T (rs71762) controlled for in the present
analysis, studies have consistently failed to identify a relevant signal
of association between MPA exposure and a range of investigatedABCC2 SNPs (rs3740066, rs8187710, rs1885301, rs7910642,
rs113646094, rs8187694, rs17222723, rs3740066, rs2804402) andABCC3 SNPs (rs4793665, rs2277624) [4,5,39,41]. Finally,
(apart from the SLCO1B1 c.521T>C , in LD withc.388A>G , controlled in the present study, and
already discussed SLCO1B3 c334T>G ), no consistent
signal of association between a range of SLCO1B1 and 1B3SNPs and MPA exposure has been detected across numerous individual
studies [4,5,39,41]. To attribute the observed effect to these
unmeasured but unlikely confounders, one needs to assume their
simultaneous synergistic effects. The present sensitivity analysis
suggests: even if it existed, and even if really marked (GMR=1.60), such
a (hypothetical) cumulative confounding effect would not completely
explain away the observed effect since GMR for the variant ABCG2
c421C>A allele vs. wild type would still be 1.20. Overall,
it is justified to state that present data reasonably validly document
an effect of the ABCG2 c.421C>A variant allele on
steady-state exposure to MPA in renal transplant patients. Discrepancy
between the present results and earlier studies not detecting
associations between exposure to MPA and ABCG2
c.421C>A SNP might, at least in part, be due to
methodological differences. A study in Chinese patients co-treated with
CsA reported slightly higher crude dose-adjusted AUCτ,ssin17 variant carriers than in 20 wt controls (30.9±13.0 vs. 27.7±10.7 mg
× h/L) [44]. Our patients were co-treated with CsA or tacrolimus
(and matched for CNI and CNI troughs). Neither CsA nor tacrolimus are
ABCG2 substrates, but both are ABCG2 inhibitors, and their inhibitory
effect might differ, particularly under c.421 SNP (with reduced
transporter numbers) [45-47]. Two larger studies (Chinese [48]
and Brazilian [49] patients) reporting no association between thec.421 SNP and MPA measured only trough concentrations while
present data refer to AUCτ,ss (note: in the present
analysis, dose-adjusted MPA troughs tended to be higher in variant
carriers, but variability was high), while a Chinese population
pharmacokinetic model included only patients co-treated with tacrolimus
[39]. Clearly, it is difficult to directly compare results from
observational studies differing in methodology and design, sampling
populations and sample sizes, outcomes and control of confounding –
each should be evaluated on its own merit. We believe that the present
analysis reasonably supports a conclusion that the observed difference
in AUCτ,ss between the ABCG2
c.421C>A variant and wt subjects is attributable to the
fact of variant allele carriage.
Present study is limited by a modestly sized single-center sample, the
fact that MPAG was not measured (as not a part of routine TDM), and,
relatedly, by no insight into possible mechanisms of the observed
effect. A study in Japanese patients [11] reported higher
steady-state MPAG concentrations in 44 c.421C>Avariant carriers than in 36 wt controls (median 1540 vs. 1195 mg × h/L;
P=0.029; corresponds to ROM=1.29), and suggested involvement of ABCG2 in
MPAG-MPA recirculation. Current observations (Figure 1, Table 5) of
closely similar Cmax (at around 2 hours post-dose), but
clearly larger AUCτ,ss in variant carriers vs. wt
controls indirectly support such a possibility: the difference in AUC is
primarily due to differences that occurred between 3 and 12 hours
post-dose, which is in agreement with hypothetical differences in MPAG
recirculation.