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.