Supplemental Methods A – Methods to achieve conditional
exchangeability
Currently, ABCG2 is not considered in the context of MPA (or MPAG)
pharmacokinetics [1] and there is no explicit evidence that either
MPA or MPAG are ABCG2 substrates – but there is also no explicit
evidence that they are not [2]. We aimed to estimate effect of theABCG2 c.421C>A (rs2231142) SNP (results in reduced
transporter numbers and function), i.e., of carrying a variant allele,
on steady-state exposure to MPA. As reviewed, studies (so far) have
failed to detect association between this SNP and exposure to MPA (as
AUCτ or as trough concentrations) [3]. However, in
one study in Japanese renal transplant recipients, variant carriers
(n=44) had higher (adjusted for MMF dose; expressed per 1000 mg) MPAG
AUC0-12 than 36 wt controls (median 1540 vs. 1195 mg ×
h/L; P=0.029) [4]. Authors suggested that ABCG2 might be included in
biliary excretion of MPAG [4]. By analogy with reports of
association between OATP1B1 and OATP1B3 polymorphisms (MPAG is a
substrate, MPA is not) [1] and systemic bioavailability of MPA
(reviewed in [3]), the ABCG2 c.421 SNP might (as well)
reflect on the systemic exposure to MPA. Current “failures” in this
respect might be due to methodological study characteristics, and/or
could indicate that even if it existed, the effect was mild-moderate,
i.e., not robust enough as to be spotted under certain methodological
circumstances.
Figures S1A and S1B schematically represent the setting in which the
effect of variant c.421C>A allele was to be assessed
and measures undertaken to control for confounding. We followed the
concepts developed and presented by Pearl [5] and further elaborated
by VanderWeele [6, 7], and implemented in R package dagitty[8]. Major elements are depicted in Figure S1A (some are based on
explicit in vitro and/or in vivo evidence, some are
implied based on circumstantial evidence and are partly hypothetical):
1. For simplicity, in the main text ABCG2 c.421C>ASNP is designated as a binary treatment (variant allele=1,
treated; wt homozygous=0, control). Its immediate consequence is reduced
number (and activity) of ABCG2 (increased degradation). Hence, the
actual treatment (or exposure) is ABCG2 activity (may be
dichotomized as “reduced” [variant allele]=treatment; and
“preserved” [wt homozygous]= control). However, in vivoABCG2 activity cannot be measured, hence the true exposure remains
unobserved, and we use ABCG2 c.421 genotype as an instrumental
variable – it has no other effect on the outcome or on any other
variable (in vitro , both CsA and tacrolimus are potent ABCG2
inhibitors [9], but neither is an ABCG2 substrate [10, 11])
beyond that conveyed through reduced transporter function; 2.
Steady-state MPA pharmacokinetic indicators (MPA PK) are continuous
outcomes; 3. The connection between the exposure (ABCG2 activity,c.421 SNP) and the outcome might be a direct one (assumes that
MPA is an ABCG2 substrate), and/or an indirect one (as suggested in the
Japanese study [4]) with MPAG as a hypothetical mediator (unmeasured
in the present study). The setting is such that it can estimate the
total effect of exposure (instrument) on the outcome.