Study outline
We included consecutive adult and adolescent (age ≥16 years) de
novo renal transplant recipients submitted to routine therapeutic drug
monitoring (TDM) of immunosuppressants after completion of the initial
week of treatment. All participants provided signed informed consent for
genotyping of pharmacogenes. Clinical and bioanalytical procedures were
described in detail previously [12, 13]. Briefly, patients on
standard immunosuppressant protocols including MPA (IR MMF or EC-MPS),
CNI (CsA [microemulsion] or tacrolimus) and glucocorticoids were
closely monitored over 5-7 post-transplant days; on the subsequent day
(steady-states of MPA, CsA/tacrolimus achieved), after overnight fast,
at 08:00 hours blood samples were taken for quantification of MPA and
CsA/tacrolimus, treatments were administered and 6 blood samples were
taken over the 12-hour dosing interval (at 0.5, 1, 2, 3, 8 and 12 hours
post-dose) for quantification of MPA. They were included in the present
analysis if: 1) clinical status was considered stable during the
observed period based on (i) lack of surgical complications and signs of
graft dysfunction or rejection; (ii) no severe comorbidity
(cardiovascular, hepatic, metabolic, infectious, gastrointestinal);
(iii) low immunological risk, (iv) stably improving renal function
(serum creatinine ≤300 µmol/L and by at least 1/3 lower than on the
1st postoperative day, with stable diuresis at around
60 mL/hour); (v) serum albumin >31 g/L; 2) were not treated
with drugs that affect exposure to MPA (proton pump inhibitors,
antacids, phosphate binders, oral iron, magnesium or calcium, rifampicin
or any antibiotics) during the prestudy and study days. Patients were
genotyped for the ABCG2 c.421C>A (rs2231142) and
further SNPs suggested (although not unambiguously) to be associated
with MPA pharmacokinetics: UGT1A9 -275T>A(rs6714486) and -2152C>T (rs17868320); UGT2B7
-161C>T (rs7668258) [in complete LD with UGT2B7
802C>T (rs7439366)] [14]; ABCB1
2677G>T/A (rs2032582), 3435C>T(rs1045642) and 1236C>T (rs1128503); SLCO1B1
c.521T>C (rs4149065) [in complete LD with
c.388A>G (rs2306283)] [4]; CYP3A4*22(rs35599367) and CYP3A5*3 (rs776746); ABCC2
-24C>T (rs717620) and 1249G>A(rs2273697) (both recipients and donors). To estimate the effect of theABCG2 c.421C>A SNP on exposure to MPA at
steady-state, patients were classified as c.421C>Avariant carriers (“treated”) and wild-type (wt) subjects
(“controls”), and we used matching to achieve conditional
exchangeability. We followed the principles introduced by Pearl [15]
with operational development [16, 17] and implementation in packagedaggity [18] in R [19] [see Electronic supplementary
material (ESM) – Supplemental Methods A, for details].
Study was approved by the Ethics Committee of the University Hospital
Center Zagreb (approval No. 8.1-17/242-2 02/21, January 30, 2018). All
procedures performed in the study were in accordance with the 1964
Declaration of Helsinki and its later amendments. All patients included
in the present analysis underwent standard routine therapeutic drug
monitoring in their post-transplant period. Those meeting inclusion
criteria were included only if they signed an informed consent for
genotyping of pharmacogenes for research purposes.