Mycophenolic acid pharmacogenomics in kidney transplantation

UGT1A1 and UGT1A7

Given the known minimal contribution of UGT1A1, UGT1A7 and UGT1A10 to MPA metabolism, there are few pharmacogenetic studies with these isoforms.

Satoh et al.^[23] investigated the influence of the UGT1A1*28 (rs3064744) variant in 30 Japanese adult patients. The authors expected to find an association between this UGT1A1 polymorphism and MPA toxicity, but they did not obtain any significant result.

Four other studies evaluated the impact of UGT1A7 variants in kidney transplant recipients. In three cohorts, UGT1A7 rs7586110 (-57T>G), UGT1A7*2 and UGT1A7*3 did not influence MPA pharmacokinetics or MMF-related gastrointestinal adverse events or leukopenia^[27,33,34]. However, UGT1A7 c.622CC (rs11692021) genotype was associated with an accumulation of MPAG (higher AUC_0-12) without detectable changes in MPA exposure (AUC_0-12) in Chinese patients^[35].

UGT1A8

The variant UGT1A8 rs1042597 (p.Ala173Gly, c.518C>G), also known as UGT1A8*2, is a missense variant that has a negligible role on protein activity in generating MPAG, but shows decreased production of AcMPAG^[31]. Consistent with this finding, no association of UGT1A8*2 variant with MPA or MPAG pharmacokinetics was found in kidney transplant recipients^[14,36-38]. Nevertheless, in agreement with reduced enzyme activity, UGT1A8*2 variant was associated with higher MPA AUC_0-12, in cyclosporine-treated patients from the FDCC Study^[39], and lower MPAG AUC_0-12 in Chinese on tacrolimus based treatment^[35].

UGT1A8 rs17863762 (p.Cys277Tyr, c.830G>A, UGT1A8*3) is another missense variant, that also induced a reduction in the formation of MPAG in vitro^[9,31], and did not impact MPA pharmacokinetics in pediatric or adult patients, probably because of its low frequency in the studied populations (UGT1A8 c.830A: 1% or 2%)^[39,40].

Regarding the clinical outcomes, Woillard et al.^[34] confirmed that MMF treatment combined with tacrolimus and sirolimus increased the risk of diarrhea when compared with cyclosporine. Moreover, only in cyclosporine-treated patients, did the authors demonstrate that UGT1A8*2 variant (c.518G allele) was associated with lower risk of MMF-related diarrhea. The supposed mechanism for this protection involved the decrease in intestinal exposure to AcMPAG^[34]. It is known that local (not systemic) exposure to AcMPAG contributes to the toxicity to the intestinal mucosa^[3]. However, in North American kidney transplant recipients up two weeks, UGT1A8 c.518GG (UGT1A8*2/*2) genotype carriers had higher severity of gastrointestinal disorders (abdominal pain, acid reflux, indigestion, diarrhea and constipation) compared to UGT1A8 c.518C allele carriers^[41]. This finding was not confirmed in final adjusted analysis (P = 0.069). Woillard et al.^[34] found that the results were not similar when they studied the relationship of UGT1A8*2 with diarrhea alone or together with abdominal pain, nausea/vomiting and anorexia, which constituted overly heterogeneous phenotypes.

The occurrence of infections, but not diarrhea or blood disorders, was associated with high dose (2 g/day) MMF treatment and the UGT1A8*3/*3 (c.830AA) genotype^[42]. The authors suggested that this finding was due to increased MPA levels and immunosuppression, in accordance with the lower enzyme activity associated with this variant.

As described in Table 1, UGT1A8 c.518C>G and c.830G>A variants were not associated with AR, diarrhea, leukopenia or anemia in other investigations with kidney recipients^{[27,29,39,43]}.

UGT1A9

UGT1A9 but not UGT1A8 is expressed in the liver at high levels and is considered the most important hepatic UGT enzyme involved in MPAG formation from MPA^[9,32].

The variants UGT1A9 rs6714486 (c.-275T>A) and rs17868320 (c.-2152C>T) lead to increased protein expression and activity in liver microsomes^[44]. Accordingly, these polymorphisms were associated with lower MPA exposure and enterohepatic recirculation (AUC_6-12 used as marker) in kidney patients on MPA and tacrolimus treatment^[39,45,46]. Kuypers et al.^[47] also reported this finding but only among patients treated with 2 g MMF, and van Schaik et al.^[39] reported that the association was dependent on tacrolimus treatment. On the contrary, in patients treated with cyclosporine or macrolides, no association with MPA or MPAG pharmacokinetics was found by others^{[14,40,48,49]}.

UGT1A9 rs2741046 (c.-331T>C), in complete linkage with rs2741045 (c.-440C>T)^[44], rs10176426 (-665C>T) and rs13418420 (-1818T>C) variants in the promoter region, also showed conflicting results. In an Italian cohort treated with MMF, cyclosporine and corticosteroid-free regimen, UGT1A9 c.-440 TT and -665CT genotypes were associated with higher MPA exposure (AUC_{0-2, 0-4, 0-12} or C/D), but not with MPAG AUC_0-12^[48]. On the contrary, UGT1A9 c.-440CT and -1818CC genotypes were associated with higher and lower MPAG AUC_0-12, respectively, in Chinese patients on tacrolimus treatment^[35]. Moreover, no association was found with MPA or MPAG pharmacokinetics in other studies^[14,38].

The variants UGT1A9 rs3832043 (-118delT), rs72551330 (c.98T>C) and rs2741049 (I399T>C or IVS1 + 399T>C) do not seem to influence MPA or MPAG pharmacokinetics in kidney transplant patients^{[14,33,35,38,40,47,49,50]}. Nevertheless, higher MPA AUC_0-12 was associated with the presence of UGT1A9 c.98TC genotype in one study with 338 patients^[39]. Although this result is in accordance with the low activity related to UGT1A9 c.98T>C variant, the c.98C allele frequency was 2%^[9,39]. Furthermore, the low frequency of the UGT1A9 c.-275T>A, c.-2152C>T, -665C>T and c.98T>C polymorphisms was an important limitation of the investigations included in Table 1.

Regarding the clinical outcomes, many investigations showed that the UGT1A9 c.-2152C>T, -665C>T, c.-275T>A, c.-440C>T, and c.98T>C variants were not associated with AR and MPA-related adverse events^{[13,22,27,29,34,47-49]}.

In line with the aforementioned subtherapeutic levels of immunosuppression, UGT1A9 c.-2152C>T and c.-275T>A variants were associated with increased risk for AR (OR: 13.3, 95%CI: 1.1-162.3, P = 0.042)^[39] in patients treated with MPA and tacrolimus. However, another investigation found that UGT1A9 c.-275A (or c.-2152T) allele carriers had more incidence of gastrointestinal adverse events, which included diarrhea, abdominal pain, reflux, heartburn and constipation, compared to non-carriers^[46]. A greater severity of diarrhea and heartburn was also associated with these variants. Moreover, the UGT1A9 rs6744284CC genotype was associated with higher severity of constipation (Gastrointestinal Symptom Rating Scale score) at week 1 after transplantation of a North American cohort^[41].

In pediatric kidney patients, UGT1A9 c.-331C allele was associated with leukopenia^[43], possibly because of increased systemic MPA exposure^[48], which was speculated but not evaluated by the authors.

Pazik et al.^[51,52] brought to light the important role of UGT1A9 in reducing exposure to dietary toxins and carcinogens. They showed that UGT1A9 c.98TC genotype was associated with decreased graft function (proteinuria and diminished eGFR), possibly due to reduced detoxifying potential of the UGT1A9 c.98T>C encoded enzyme.

UGT2B7

UGT2B7 is the key enzyme involved in AcMPAG formation. Djebli et al.^[53] investigated the in vivo and in vitro effect of UGT2B7 rs7438135 (c.-900G>A, otherwise termed c.-840G>A or c.−842G>A) and rs7439366 (802C>T) variants, which were in complete reverse linkage disequilibrium. The authors found that AcMPAG production was higher in the presence of UGT2B7 c.-900A allele compared to c.-900GG genotype in human liver microsomes. Moreover, the same authors showed that in patients treated with sirolimus (n = 40), but not with calcineurin inhibitors (CNIs), UGT2B7 c.-900AA (or 802CC) genotype was associated with higher AcMPAG AUC_0-9 at month 1 and 3 after transplantation^[53]. Likewise, no association with exposure to AcMPAG was found in other studies with adult patients treated with tacrolimus or cyclosporine^[48,54].

The effect of UGT2B7 polymorphisms on MPA exposure in kidney recipients also was investigated. UGT2B7 c.-900G>A and 802C>T variants had no effect on MPA pharmacokinetics in patients treated with or without corticosteroids, which may induce glucuronidation and result in low MPA exposure^{[36,39,49,53,55]}. Baldelli et al.^[48] found that the UGT2B7 802TT genotype was associated with higher MPA peak concentration without differences in AUC_0-12 MPA. Likewise, UGT2B7 -79G>A variant, which is linked to the 802C>T and shows a decline in the transcriptional activity of the reporter gene in Caco-2 and HepG2 cells, was not associated with MPA exposure^[39,56]. On the contrary, MMF apparent oral clearance (CL/F) was significantly higher in pediatric patients with UGT2B7 802T allele compared to UGT2B7 802CC genotype carriers, early after transplantation (60 days)^[40]. It is important to consider here that differences in metabolism between adults and children may modify the effect of the genetic variants^[27,57].

UGT2B7 rs62298861 (IVS1 + 985A>G), rs12233719 (c.211G > T) and rs7662029 (c.-327G > A) variants were also investigated in kidney transplantation. UGT2B7 IVS1 + 985AG and c.211GT genotypes were found to be associated with higher MPA exposure^[35,50], but the association with UGT2B7 c.211G > T was not confirmed by another study^[49]. Li et al.^[38] found an association between UGT2B7 rs7662029 (c.-327G > A) GG genotype and a higher MPA C/D (days 3-8) in 408 Chinese treated with tacrolimus or cyclosporine, but this result was not confirmed through multiple comparison tests correction.

In a retrospective study of Brazilian adult patients, UGT2B7 c.-900G allele was associated with prevention of AR (OR: 0.41, 95%CI: 0.19-0.92, P = 0.030)^[22]. Moreover, the UGT2B7 c.-900G allele was associated with increased risk of leukopenia (OR: 5.3, 95%CI: 1.1-25.0, P = 0.038)^[27], only on treatment with non-depleting antibodies, and anemia (GA vs. AA, OR: 1.7, 95%CI: 1.1-2.4, P = 0.010; GG vs. AA, OR: 1.9, 95%CI: 1.2-2.0, P = 0.003)^[29]. All these findings suggest that the UGT2B7 c.-900G allele is associated with higher exposure to MPA, known to favor leukopenia and anemia, and decreased incidence of AR^[3]. Woillard et al.^[29] also suggested that the relationship between the UGT2B7 c.-900 G>A variant and anemia could be because of the increased production of AcMPAG.

Another investigation failed to find an association of UGT2B7 c.-900G>A, 802C>T and -79G>A variants with AR, diarrhea, leukopenia, or other adverse events^{[13,34,39,43,49,54]}. Likewise, the variants rs12233719 (c.211G > T) and rs28365063 (c.372A>G) in UGT2B7 were not associated with neutrophil count or graft function in adult patients^[30,49].

ABCB1

ABCB1 is located on chromosome 7, with many variants described so far. Pharmacokinetic studies of MPA have addressed the main investigated polymorphisms of ABCB1: rs1128503 (c.1236C>T), rs2032582 (c.2677G > T/A), and rs1045642 (c.3435C>T), which are in linkage disequilibrium^[58].

In studies with kidney recipients, no association of these ABCB1 variants with exposure to MPA, and MPAG or AcMPAG were observed up to one year after transplantation^[23,24,59]. Bouamar et al.^[59] found that this result was independent of cyclosporine or tacrolimus treatment. It is known that co-administration of cyclosporine leads to lower MPA exposure, by inhibiting enterohepatic recycling of MPA, compared to co-administration of tacrolimus^[3]. The inhibition of ABCC2 and OATPs by cyclosporine possibly contributes to this drug-drug interaction^[18,60]. Moreover, cyclosporine is a substrate of ABCB1^[25].

Satoh et al.^[23] found that the ABCB1 c.3435T allele was associated with greater requirement of MMF dose reduction due to diarrhea in adult patients treated with tacrolimus. Nevertheless, the authors did not find differences in dose-adjusted MPA AUC in ABCB1 c.3435T allele carriers compared to c.3435CC genotype individuals. Thus, they speculated that the interaction of ABCB1 with other transporters and enzymes might be involved in MPA-induced gastrointestinal toxicity in their small sample size (n = 30)^[23]. ABCB1 c.3435TT genotype may decrease protein expression or activity, leading to increased drug absorption across the intestine and its higher systemic and intracellular concentrations^[61].

On the other hand, in a study with 237 Caucasian patients on MMF and cyclosporine treatment, the ABCB1 c.2677T allele and c.3435T/c.2677T/c.1236T was associated haplotype with increased risk of AR (OR: 3.2, 95%CI: 1.5-6.7, P = 0.003 and OR: 2.1, 95%CI: 1.3-3.4, P = 0.002; respectively), but MPA pharmacokinetic data were unavailable^[25]. Since low MPA AUC_0-12 was associated with increased incidence of biopsy-proven acute rejection^[3], this result from Grinyó et al.^[25] collaborates the complex mechanisms of clinical outcomes in immunosuppressive therapy.

Other investigations found no association of ABCB1 c.1236C>T, c.2677G > T/A, and c.3435C>T variants with AR, DGF, diarrhea, leukopenia or other MPA-related adverse events in pediatric or adult kidney recipients^{[13,24,26,27,59]}.

ABCC2

The extensively studied variant of ABCC2 (on chromosome 10), the rs717620 (c.-24C>T), is located in the 5ʹ-UTR. The effect of this polymorphism in several in vivo studies is contradictory, suggesting that it is highly tissue specific and dependent on regulatory factors, such as the epigenetics (miRNA expression)^[58]. Naesens et al.^[62] investigated the ABCC2 c.-24C>T variant, in linkage disequilibrium with rs3740066 (c.3972C>T), in adult kidney transplant recipients treated with MMF and tacrolimus for one year. At day 7, only non-carriers of the ABCC2 c.-24C>T variant and patients with mild liver dysfunction had significantly lower MPA exposure than those without liver disease. This difference was not observed in ABCC2 c.-24T allele carriers. The reasons were not clarified. In the same study, from day 42 post-transplantation, the ABCC2 c.-24T allele was associated with higher MPA C/D and AUC^[62]. The authors suggested that the variant was associated with increased protein expression and/or activity and enhanced enterohepatic recirculation. In line with this, the ABCC2 c.-24TT genotype was also associated with higher MPA C/D (days 3-8) in 408 Chinese patients on cyclosporine or tacrolimus treatment, but this result was not confirmed after Bonferroni correction in the multiple comparisons analysis^[38].

van Schaik et al.^[39] found that MPA AUC_0-12 was 17%-23% higher for ABCC2 c.-24C>T carriers, but this difference reached statistical significance only at 6 weeks post-transplantation. Conversely, the ABCC2 c.-24T allele was associated with a lower MPA AUC at month 1, in a study with 55 kidney transplant recipients on tacrolimus and sirolimus therapy^[63].

Božina et al.^[64] did not find any association between the ABCC2 c.-24T variant and MPA pharmacokinetics in kidney recipient or donors, but the A allele of the ABCC2 rs2273697 (Val417Ile, c.1249G>A) in kidney donors was associated with a reduced peak (29%) and early (AUC_0-2, 33%) exposure to MPA, suggesting an increased renal elimination. Moreover, the authors showed that the combination of cyclosporine and ABCC2 c.1249A allele reduced MPA AUC_0-2 by 49%, an additive effect of this immunosuppressant.

Lack of association of ABCC2 c.-24C>T, 1249G>A, c.3972C>T, and rs8187694 (3563T>A) variants with MPA or MPAG pharmacokinetics was reported by other studies^{[14,24,37,39,40,45,48,49,65,66]}.

Regarding clinical outcomes, Naesens et al.^[62] found that ABCC2 c.-24T allele carriers had more frequent episodes of diarrhea that non-carriers in the first year after transplantation. The authors considered the limitation of the sample size but stated that an increased enterohepatic recirculation of MPAG associated with the ABCC2 c.-24C>T variant could lead to diarrhea. It is important to mention that the local exposure and not the systemic concentration of MPA is relevant for gastrointestinal adverse events. Moreover, AcMPAG, produced in gastrointestinal cells and also substrate of ABCC2, contributes to cell toxicity^[3,15].

Other studies failed to confirm associations between ABCC2 polymorphisms and clinical outcomes (AR, DGF, graft function) or MPA-related adverse events in pediatric or adult kidney recipients^{[13,22,24,25,27,29,30,34,39,49,65]}.

ABCG2

Brazilian, Chinese, and North American cohorts have been studied regarding two ABCG2 variants in pediatric and adult kidney transplant patients [Table 2]. The ABCG2 c.421C > A (rs2231142) variant leads to the amino acid exchange Gln141Lys, whereas ABCG2 -20 + 11790G>A (rs4491984) is an intronic polymorphism.

The investigations pointed out that these variants did not influence MPA or MPAG exposure or clinical outcomes during treatment with different immunosuppressive therapies^{[24,27,30,37,38]}.

SLCO1B1

A large number of SLCO1B1 variants have been described. The two SLCO1B1 common variants rs2306283 (p.Asn130Asp, c.388A>G) and rs4149056 (p.Val174Ala, c.521T>C) form together four distinct haplotypes: SLCO1B1*1A (c.388A-c.521T, reference haplotype), *1B (c.388G-c.521T), *5 (c.388A-c.521C) and *15 (c.388G-c.521C)^[69].

Kidney transplantation with macrolide or cyclosporine-based treatment has shown no association between the SLCO1B1 c.388A>G, c.521T>C, and rs11045819 (463C > A) variants or haplotypes with MPA, MPAG or AcMPAG pharmacokinetics^{[14,24,49,59,65]}.

Likewise, SLCO1B1 variants have not been associated with AR, DGF, diarrhea, leukopenia or neutrophil count in the studies summarized in Table 2^{[22,24,27,49,59,65]}. Nevertheless, SLCO1B1 c.521C allele (SLCO1B1*5) was associated with reduced MPA-related adverse events, including leukopenia, anemia, thrombocytopenia, diarrhea, nausea, vomiting or infection in French patients on cyclosporine-free treatment^[13]. Possibly because cyclosporine inhibits OATP1B1, the effect of the SLCO1B1 c.521C allele was not detectable in the therapy with this immunosuppressant^[13,60]. Indeed, cyclosporine-based treatment and SLCO1B1 c.521C allele were independent factors related to risk reduction of adverse events (OR: 0.22, 95%CI: 0.08-0.56, P = 0.002 and OR: 0.38, 95%CI: 0.21-0.69, P = 0.001; respectively)^[13]. It was also demonstrated in vitro the reduced uptake of MPAG and AcMPAG related to SLCO1B1 c.521C variant. The authors hypothesized that carriers of the SLCO1B1 c.521C allele had impaired hepatic uptake of MPAG and AcMPAG, which leads to less enterohepatic recycling and, consequently, reduced exposure to MPA and less adverse events^[13]. However, this study did not evaluate MPA exposure, and other investigations did not find an association between the SLCO1B1 c.521T>C variant and MPA pharmacokinetics, as previously mentioned.

SLCO1B3

Four variants in SLCO1B3 were investigated in pediatric and adult recipients of kidney transplantation [Table 2]. Three are missense variants, SLCO1B3 rs4149117 (Ser112Ala, c.334T>G), rs7311358 (Met233Ile, c.699G>A) and rs60140950 (Gly256Ala, c.767G > C).

SLCO1B3 c.334T>G and c.699G>A are in linkage disequilibrium, and the SLCO1B3 c.334G-c.699A haplotype was associated with reduced MPAG uptake in HEK293 cells in vitro^[14]. In a study with 70 patients on tacrolimus and sirolimus treatment, but not on cyclosporine, the SLCO1B3 c.334GG genotype was associated with lower MPA peak concentration and exposure (AUC_0-12) and higher MPAG/MPA ratio^[14]. On the basis of in vitro and in vivo results, the authors suggested that reduced OATP1B3 activity would decrease hepatic uptake of MPAG, reducing reabsorption of MPA through enterohepatic cycling in SLCO1B3 c.334GG genotype carriers.

Other investigations did not find an association between SLCO1B3 c.334T>G (or c.699G>A) variant and exposure to MPA, MPAG, or AcMPAG during treatment with either cyclosporine, in line with the previous findings^[14], or tacrolimus^[37,38,59]. On the contrary, the SLCO1B3 c.334GG (699AA) genotype was associated with higher MPA AUC_6-12, considered a marker of MPA recirculation, in Japanese patients with tacrolimus-based therapy at day 28 after transplantation^[65].

SLCO1B3 c.334T>G (or 699G>A) and rs1104585 variants were not associated with AR, leukopenia, diarrhea or other adverse events^{[13,27,29,59,65]}.

SLCO2B1

In Brazilian or Japanese kidney recipients, early or long after transplantation, SLCO2B1 c.-71T > C (rs2851069) and SLCO2B1*3 (rs2306168) variants did not influence MPA pharmacokinetics or clinical outcomes of tacrolimus based-therapy^[24,65].