Influence of Nonsynonymous Polymorphisms of UGT1A8 and
UGT2B7 Metabolizing Enzymes on the Formation of Phenolic and
Acyl Glucuronides of Mycophenolic Acid
Olivier Bernard, Jelena Tojcic, Kim Journault, Louis Perusse, and Chantal Guillemette
Canada Research Chair in Pharmacogenomics, Oncology and Molecular Endocrinology Research Center, CHUL Research
Center, Faculty of Pharmacy (O.B., J.T., K.J., C.G.) and Division of Kinesiology, Department of Preventive Medicine (L.P.),
Laval University, Quebec, Canada
Received April 19, 2006; accepted June 20, 2006
Mycophenolic acid (MPA) is the active metabolite of mycopheno-
late mofetil (MMF), a standard immunosuppressive drug approved
for clinical use in the prevention of acute allograft rejection after
organ transplantation. This study examines the role of the genetic
variants of UDP-glucuronosyltransferase (UGT) 1A8 and 2B7 en-
zymes involved in the formation of the primary metabolite of MPA,
the inactive phenolic glucuronide (MPAG), and the reactive acyl
glucuronide (AcMPAG). The first exon of UGT1A8 was first rese-
quenced in the region encoding for the substrate binding domain in
254 Caucasians and 41 African Americans. Eight nonsynonymous
changes were observed and led to the following amino acid sub-
stitutions: S43L, H53N, S126G, A144V, A173G, A231T, T240A, and C277Y.
Thirteen haplotypes were inferred, comprising only two previously
described alleles, namely, UGT1A8*2 (A173G) and UGT1A8*3
(C277Y). Upon stable expression in human embryonic kidney 293
cells, the UGT1A8*3 (C277Y), *5 (G173A240), *7 (A231T), *8 (S43L), and
*9 (N53G) proteins were associated with the most profound de-
creases in the formation of MPAG and AcMPAG, indicating that
these amino acids are critical for substrate binding and enzyme
function. Altogether, the low-activity UGT1A8 enzymes are carried
by 2.8 to 4.8% of the population. The variant of the UGT2B7 protein
(UGT2B7*2 Y268), the main enzyme involved in the formation of
AcMPAG, demonstrated a catalytic efficiency comparable with
that of UGT2B7*1 (H268). In conclusion, although the common
UGT2B7*2 variant is predicted to have limited impact, several
UGT1A8 variants identified may potentially account for the large
interindividual variance in MMF pharmacokinetics and deserve
further clinical investigations.
Mycophenolate mofetil (MMF; Cellcept, Hoffmann-La Roche,
Nutley, NJ), an immunosuppressive drug, is approved for clinical use
in the prevention of acute allograft rejection after organ transplanta-
tion, as well as hematopoietic stem cell transplantation (Sollinger,
1995; Bullingham et al., 1998; Cohn et al., 1999). Mycophenolic acid
(MPA), its active metabolite, is a selective inhibitor of inosine mono-
phosphate dehydrogenase (IMPDH). The metabolism of MPA in-
volves mainly its conjugation by UDP-glucuronosyltransferase (UGT)
enzymes, yielding two glucuronide conjugates; namely, the major
derivative MPAG and the minor metabolite AcMPAG (Bullingham et
al., 1998; Shipkova et al., 2001b). MPAG has no inhibitory effects on
IMPDH and is the major urinary excretion product of MPA (Bulling-
ham et al., 1996; Schutz et al., 1999). In contrast, AcMPAG may be
biologically active by inhibiting IMPDH and leukocyte proliferation,
and by inducing cytokine release (Schutz et al., 1999; Wieland et al.,
2000; Shipkova et al., 2001b). A relationship between plasma levels
of MPA and clinical outcomes in transplant patients has been dem-
onstrated (Hale et al., 1998; van Gelder et al., 1999; Oellerich et al.,
2000; Weber et al., 2002). Besides, AcMPAG has been suggested to
be involved in some of the toxicities experienced by patients receiving
MMF, including neutropenia and gastrointestinal disorders (Wieland
et al., 2000; Maes et al., 2002). Therefore, factors affecting the extent
of MPA glucuronidation are likely to be clinically significant.
Recently, UGT1A9 has been identified as the main enzyme in-
volved in the hepatic formation of MPAG (Bernard and Guillemette,
2004). This enzyme was predicted to be the key determinant of
MPAG formation in vivo because the metabolism of MPA takes place
mainly in the liver (Bowalgaha and Miners, 2001; Shipkova et al.,
2001a; Bernard and Guillemette, 2004). Functional genetic variants
within the UGT1A9 gene have been uncovered recently by our group
(Villeneuve et al., 2003; Girard et al., 2004). In human liver micro-
somes, the presence of the variants ?275A?T and ?2152T?C of the
UGT1A9 promoter region were associated with a 2.3-fold higher
hepatic expression of UGT1A9 and a 2.1-fold increased glucuronida-
This work was supported by the Canadian Institutes of Health Research
(MOP-42392) and Canada Research Chair Program (C.G.). O.B. is the recipient of
a studentship award from the Fonds de la Recherche en Sante ´ du Que ´bec. C.G.
is the chairholder of the Canada Research Chair in Pharmacogenomics.
Part of this work has been presented at the 13th North American Meeting of
the International Society for the Study of Xenobiotics, 2005 October 23–27th,
Article,publication date,and citation
information canbefound at
ABBREVIATIONS: MMF, mycophenolate mofetil; MPA, mycophenolic acid; IMPDH, inosine monosphospate dehydrogenase; UGT, UDP-
glucuronosyltransferase; MPAG, mycophenolic acid phenolic glucuronide; AcMPAG, mycophenolic acid acyl glucuronide; SNP, single-nucleotide
polymorphism; PCR, polymerase chain reaction; HEK, human embryonic kidney; CLint, intrinsic clearance.
DRUG METABOLISM AND DISPOSITION
Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics
DMD 34:1539–1545, 2006
Vol. 34, No. 9
Printed in U.S.A.
tion activity to generate MPAG (Girard et al., 2004). These in vitro
observations were confirmed recently in a clinical setting by the group
of Kuypers et al. (2005). In renal transplant recipients carrying these
polymorphisms, a reduced MPA exposure and an increased MPA
clearance were observed, demonstrating the clinical importance of
genetic variability in the UGT genes involved in the in vivo metab-
olism of MPA.
MPAG is also produced by UGT1A8, which is expressed in the
gastrointestinal tract and not in the liver (Cheng et al., 1998; Tukey
and Strassburg, 2000; Zheng et al., 2002; Bernard and Guillemette,
2004). UGT1A8 has demonstrated the highest catalytic efficiency for
MPAG formation in vitro (Bernard and Guillemette, 2004). Based on
these metabolic studies, UGT1A8 could also play a role in the
formation of AcMPAG, along with UGT2B7, which appears as the
predominant enzyme responsible for its formation (Picard et al.,
2005). Other extrahepatic UGTs, namely, UGT1A7 and UGT1A10,
demonstrated a lower reactivity toward MPA glucuronidation and are
predicted to play a minor role compared with UGT1A8, UGT1A9,
and UGT2B7 (Basu et al., 2004; Bernard and Guillemette, 2004;
Picard et al., 2005).
To this day, two coding region polymorphisms have been reported
in the UGT1A8 gene, namely the variants A173G (UGT1A8*2) and
C277Y (UGT1A8*3) (Huang et al., 2002). In vitro metabolic studies
with heterologous expression of these variant allozymes revealed that
the polymorphism at codon 277 induces a drastic reduction in the
formation of MPAG, whereas no significant effect was observed for
the codon 173 variation (Bernard and Guillemette, 2004). The effect
of these variations on the formation of the AcMPAG remains to be
determined. As for the UGT2B7 gene, a frequent polymorphism
(UGT2B7*2; H268Y) has been reported in more than 50% of Cauca-
sian individuals (Jin et al., 1993; Lampe et al., 2000). The functional
impact of this polymorphism on the formation of AcMPAG has never
The aim of this study was to further investigate genetic variations
in the UGT1A8 gene by resequencing the first exon and assessing the
functional impact of newly found and known variants on the forma-
tion of both MPAG and AcMPAG. As a secondary aim, we explored
the role of known UGT2B7*1 (H268) and UGT2B7*2 (Y268) variant
enzymes in the formation of AcMPAG. Together, results of this study
identify new genetic factors resulting in structural changes in the
UGT1A8 protein that could potentially alter MPA metabolism in
extrahepatic tissues. In contrast, the UGT2B7*2 common variant
allozyme is predicted to have a modest influence on drug metabolism
Materials and Methods
Reagents and Chemicals. MPA was obtained from Sigma Diagnostics
Canada (Mississauga, ON, Canada). MPAG and AcMPAG were generous gifts
from Hoffmann-La Roche (Mississauga, ON, Canada). All other chemicals and
reagents were of the highest grade and commercially available.
Genomic DNA Samples. DNA samples from 254 healthy unrelated Cau-
casian subjects were obtained from the Quebec Family Study for UGT1A8
single-nucleotide polymorphism (SNP) genotyping (Simonen et al., 2002).
Additional random DNA samples from African-American subjects (n ? 41)
used in a previous study were sequenced (Butler et al., 2005). Subject identi-
fiers for these samples had been removed before their reception in our labo-
ratory. All subjects provided written consent for experimental purposes and the
present study was reviewed and approved by the Institutional Review Boards
(CHUL Research Center and Laval University).
Resequencing of the UGT1A8 Gene and Genotyping. The first exon of
UGT1A8 (?34/?935) was amplified using a previously described strategy
(Thibaudeau et al., 2006). In brief, three pairs of primers were designed to
amplify overlapping fragments covering the coding region of the first exon, a
small portion of the 5?-flanking region, and the intron-exon junction. PCR
conditions for the amplification primers were 3 min at 95°C for denaturation,
followed by 35 cycles at 95°C for 30 s, a 30-s annealing period, and 72°C for
30 s, with a final extension at 72°C for 7 min. PCR products were sequenced
using an ABI 3700 automated sequencer (Applied Biosystems, Foster City,
CA). Samples with ambiguous sequencing chromatograms and samples with
SNPs were subjected to a second independent amplification, followed by DNA
sequencing. Sequences were analyzed with the Staden preGap4 and Gap4
programs (Staden, Cambridge, UK). Allelic and genotype frequencies were
calculated for all alleles. Haplotypes and their respective frequencies were
inferred using the Phase 1.0.1 software (Stephens et al., 2001).
UGT-HEK293 Microsomal Preparations. The UGT1A8*1, UGT2B7*1,
and UGT2B7*2 constructions were kindly provided by Dr. Thomas Tephly
(Cheng et al., 1998). The UGT1A8*2 and UGT1A8*3 variants were prepared
as described previously (Bernard and Guillemette, 2004). All other UGT1A8
variants were generated by PCR site-directed mutagenesis using the primers
presented in Table 1 and inserted in the pcDNA3 vector. Before enzymatic
assays, Western blot analyses and catalytic activities on known substrates were
performed for each preparation. Stable HEK293 cells transfection with variant
pcDNA3-UGT expression plasmids, preparation of microsomes by differential
centrifugation, and determination of UGT protein levels by Western blot has
been described previously (Villeneuve et al., 2003). Protein expression levels
for UGT1A8 alleles were determined by Western blot using a polyclonal
anti-UGT1A antibody (in-house; RC-71) and were *1 (1.0), *2 (0.7), *3 (0.5),
*4 (0.4), *5 (0.5), *6 (0.5), *7 (0.2), *8 (1.4), *9 (1.5), H53N (1.1), A144V (0.4),
and T240A (0.4). Protein expression levels for UGT2B7 allozymes were
determined previously (Thibaudeau et al., 2006).
Analytical Procedures for MPA, MPAG, and AcMPAG Detection.
Detection of MPA, MPAG, and AcMPAG was supported by a previously
published high-performance liquid chromatography coupled with mass spec-
trometry protocol used with slight modifications (Bernard and Guillemette,
2004). In brief, the analysis system consisted of a high-performance liquid
chromatography module (Alliance model 2690; Waters Corporation, Milford,
MA) and a triple-quadrupole mass spectrometer (API 3000). Acidified assays
were centrifuged for 6 min at 14,000g, and 250 ?l of supernatant was
collected. Ten-microliter samples, maintained at 4°C, were injected on a 100 ?
4.6 mm (4.0-?m diameter) Synergic RP-Hydro C-18 reversed-phase column
(Phenomenex, Torrance, CA). The mobile phase consisted of solution A
(MeOH ? 3 mM ammonium formate) and solution B (H2O ? 3 mM ammo-
nium formate) using the following gradient: 62% A (0–4.5 min), 95% A
(4.5–6.5 min), and 62% A (6.5–9.5 min). The flow rate was 0.9 ml/min. MS
detection of MPA was followed in the multiple reactions monitoring positive
ion mode with mass fragmentation of 321.1 ? 207.2 (MPA) and 514.3 ?
321.1 (MPAG and AcMPAG). Under these conditions, retention times for
MPAG, AcMPAG, and MPA were 1.66, 2.47, and 4.41 min, respectively. The
signals were found to be linear from 10 to 5000 ng/ml for MPAG, AcMPAG,
and MPA. The limit of quantification was 10 ng/ml using a signal-to-noise
ratio of 3. The within-day precision was ?5.0%, and the between-day preci-
sion was ?10%.
Site-directed mutagenesis primers sequences for UGT1A8 variants
Variant positions are in bold.
UGT1A8 VariantsPrimer Sequencea
F, forward; R, reverse.
aAccession number for the UGT1A8 reference sequence: AF297093.
BERNARD ET AL.
Enzymatic Assays and Kinetic Parameters Determination. The proce-
dure for enzymatic assays was described previously (Bernard and Guil-
lemette, 2004). In brief, incubations were performed for 1 h at 37°C with
50 ?g of UGT protein, 50 mM Tris-HCl, pH 6.8, 10 mM MgCl2, 2 mM
UDP-glucuronic acid, pepstatin, and phosphatidylcholine. Determination
of Vmaxand Kmwas performed for all UGT1A8 and UGT2B7 variants with
MPA ranging from 25 to 1250 ?M. Absolute velocity values were adjusted
according to protein expression levels relative to the corresponding UGT*1
allele determined by Western blot. Visual inspection of fitted functions (V
as a function of [S]) and Eadie-Hofstee plots (V as a function of V/[S]) was
used to select the best-fit enzyme kinetic model (Venkatakrishnan et al.,
2001). Kinetic parameters calculations were performed with the SigmaPlot
8.0 software assisted by the Enzyme Kinetics 1.1 software (SPSS, Chicago,
IL). Values were expressed as the mean of two to five experiments
performed in duplicate. Means comparisons were performed with the JMP
4.0.2 software (SAS Institute, Cary, NC) using Student’s t test with a
statistical significance threshold of p ? 0.05.
Identification of Novel Missense Mutations in the UGT1A8 First
Exon. The resequencing of the first exon of UGT1A8 led to the
discovery of four novel missense mutations in the Caucasian popula-
tion (n ? 254) at nucleotides 376 (A?G), 431 (C?T), 691 (G?A),
and 718 (A?G) relative to the start codon and at nucleotides 128
(C?T) and 157 (C?A) in the African-American population (n ? 41).
These nonsynonymous changes led to the following amino acid sub-
stitutions: S43L, H53N, S126G, A144V, A231T, and T240A (Fig. 1). The
allelic frequencies of these variants were 0.2 to 1.4% (Table 2). The
two previously reported single-nucleotide polymorphisms (SNPs) of
UGT1A8 at codons 173 and 277 were confirmed with genotypic
frequencies of 0.238 and 0.012, respectively. In addition to these, four
synonymous variations were found at nucleotides 90 (G?A; V30V),
441 (T?G; L147L), 765 (A?G; T255T), and 804 (T?C; N268N), and
one intronic missense mutation was found at nucleotide 883 relative to
the start codon, which is 27 base pairs downstream of the end of exon
1 (IVS1?27). All SNP frequencies were found to follow the Hardy-
Weinberg equilibrium (data not shown).
Eleven haplotypes were inferred in the Caucasian population
(n ? 508 chromosomes). In contrast, five haplotypes, of which two
were not encountered in the Caucasians, were observed in the
African Americans (n ? 82 chromosomes) (Table 3). These hap-
lotypes generated ten different diplotypes (Table 4). The
UGT1A8*1a, UGT1A8*1b (T255T), and UGT1A8*2a (A173G) al-
leles were found to be the most frequent at 59, 13, and 22%,
respectively. The other haplotypes with nonsynonymous variations
were found at frequencies of 0.2 to 1.4%. The subjects with the
A144V variant also presented the A173G variant, resulting in the
UGT1A8*4 allele. The T240A variant was found only once and in
the presence of the A173G variant, representing the UGT1A8*5
allele. The S126G and A231T variants were found alone generating
the UGT1A8*6 and UGT1A8*7 alleles, respectively. The
UGT1A8*1, *2, and *4 alleles were also found in the African-
American population at frequencies of 92.7, 3.7, and 1.2%, respec-
tively. The S43L polymorphism (UGT1A8*8) was found only in
one individual, whereas the H53N polymorphism was found in
combination with the A173G variant, generating the UGT1A8*9
Kinetic Analyses of UGT1A8 Variant Allozymes on MPAG and
AcMPAG Formation. To assess the impact of nonsynonymous
UGT1A8 polymorphisms on the glucuronidation of MPA, kinetic
analyses were performed on all variants for the determination of Km,
Vmax, and CLintvalues. Kinetic estimates are presented in Table 5. A
novel finding of this study is the observation that UGT1A8 has the
capability to generate both MPAG and AcMPAG. The UGT1A8*3,
*7, *8, and *9 enzymes were associated with the most profound
effects on the level of MPAG formation, with 3.3 to 82.5-fold reduced
CLintvalues. This decrease was mostly explained by an altered
velocity of the enzyme, except for the codon 277 variation (*3), which
affects both the affinity and the velocity of the protein, consistent with
our previous observations (Bernard and Guillemette, 2004). In con-
trast, the UGT1A8*4 protein appears as a high-activity enzyme for the
formation of MPAG with a 1.8-fold higher CLintvalue, explained by
both a significantly better affinity and an increased velocity caused by
the V144G173mutations compared with the reference *1 protein. In the
FIG. 1. UGT1A8 genetic variants. A, sche-
matic representation of polymorphisms lo-
cated in UGT1A8. Amino acid position is
relative to UGT1A8 according to the refer-
ence sequence (AF297093). B, amino acid
alignment of UGT1A proteins at UGT1A8
polymorphic sites. ref, reference sequence;
var, variant sequence.
MPA GLUCURONIDATION BY UGT1A8 AND UGT2B7 VARIANTS
case of the UGT1A8*5 protein, despite an enhanced affinity for the
formation of MPAG, the velocity of the enzyme was drastically
reduced, leading to a clearance value similar to the *1 protein. In turn,
the UGT1A8*2 and *6 proteins demonstrated modest modifications
of their kinetic parameters compared with the reference *1 protein.
With regard to AcMPAG, its formation was undetectable for the *3
and *7 proteins and severely reduced for the *5 protein (20-fold
reduction) because of an altered velocity. Velocities were also signif-
icantly reduced for the *2, *4, *6, and *8 enzymes by 2- to 4-fold
compared with the UGT1A8*1 protein. The affinity of variant pro-
teins *2, *4, *5, *6, *8, and *9 was not significantly altered compared
with UGT1A8*1. In the individuals tested, 2.8% of Caucasians and
4.8% of African Americans carry at least one of the low-activity
alleles (*3, *7, *8, and *9).
To gain insight into the nucleic acid positions responsible for
critical changes in the kinetic parameters of the UGT1A8 protein, few
mutations were tested alone. Results indicate that the replacement of
a threonine by an alanine at codon 240 abolishes the ability of the
UGT1A8 protein to form the acyl glucuronide and drastically com-
promised the formation of the phenolic glucuronide. In turn, the H53N
change acts only on the velocity of the protein, whereas the A144V
mutation alters specifically the formation of the MPAG (Kmand Vmax)
with no significant detectable effect on the kinetic parameters for the
formation of the acyl.
UGT1A8 haplotypes in Caucasian and African-American subjects
Haplotypes (H) and their respective frequencies were inferred with the Phase 1.00.1. Newly identified SNPs and nucleotide changes are in bold.
IVS1?27 Caucasians (n ? 508)African Americans (n ? 82)
n, number of chromosomes analyzed.
aHaplotypes *2b, *5, and *9 could not be confirmed in more than one individual and therefore are considered as hypothetical alleles.
UGT1A8 diplotypes in Caucasian and African-American subjects
Caucasians (n ? 254)African Americans (n ? 41)
Frequency of UGT1A8 variants in the Caucasian and African-American populations
Newly identified SNPs are in bold.
Nucleic Acid Changes Amino Acid Changes
HomozygousHeterozygous Homozygous Variant
Caucasians (n ? 254 subjects)
African Americans (n ? 41 subjects)
aIVS1?27: intronic variation 27 base pairs downstream of exon 1.
BERNARD ET AL.
Kinetic Analyses of the UGT2B7*1 and *2 Allele AcMPAG
Formation. The UGT2B7 enzyme was found to generate high levels
of AcMPAG with no detectable formation of MPAG. Both the affinity
and the capacity of the UGT2B7*1 protein were higher for the
formation of the acyl glucuronide compared with the UGT1A8*1
protein, with a 31-fold higher Clintvalue. No significant changes in
the kinetic parameters were associated with the UGT2B7*2 protein,
with Kmand Vmaxvalues similar to those observed for the UGT2B7*1
In a recent study, we revealed the existence of common variations
in the upstream region of the UGT1A9 gene (?275T?A and
?2152C?T) associated with higher protein expression and higher
glucuronidation activity in human liver samples (Girard et al., 2004).
These genetic variants were further shown to influence the pharma-
cokinetics of MMF in transplant recipients (Kuypers et al., 2005).
Based on in vitro data, two additional UGTs, UGT1A8 and UGT2B7,
are proposed to play a critical role in the metabolism of MPA (Basu
et al., 2004; Bernard and Guillemette, 2004; Picard et al., 2005). In
this study, polymorphisms conferring a low-activity phenotype were
identified in UGT1A8, the extrahepatic MPA-metabolizing enzyme
that demonstrates the highest catalytic efficiency for MPAG forma-
tion. It is thus predicted that specific UGT1A8 variants identified here
may have an impact on the pharmacokinetics of MMF and potentially
on the metabolism of other UGT1A8 substrates.
Six novel nonsynonymous variations in the coding region of
UGT1A8 (S43L, H53N S126G, A144V, A231T, and T240A) were iden-
tified, and the two previously described variants *2 (A173G) and *3
(C277Y) were also observed. The A173G and C277Y variants were
initially reported with frequencies of 14.5 and 2.2%, respectively
(Huang et al., 2002). Although the C277Y variant is reported in this
study with a similar frequency, a 2-fold higher frequency was ob-
served for the A173G and could be attributed to several differences in
the population studied (254 healthy subjects of French-Canadian
origin versus 69 individuals with lung cancer patients, their family
members, and other volunteers).
Among the variants found, UGT1A8*3 (C277Y), *5 (G173A240), *7
(A231T), *8 (S43L), and *9 (N531G) were associated with the most
profound decreases in the formation of MPA glucuronides in vitro.
The formation of MPAG in the gastrointestinal tract is predicted to be
reduced in the presence of those alleles, occurring in 2.8% of Cauca-
sians and 4.8% of African Americans. As for AcMPAG, although
UGT2B7 is the most active UGT compared with UGT1A8 (CLint?
12 versus 0.39 ?l/min/mg), it is predicted that UGT1A8 variants
would have a limited impact on the formation of the acyl glucuronide
in vivo. Before this study, UGT1A8*3 had been identified as a
low-activity protein on various substrates, including a dramatic re-
duction in MPAG formation, but its impact on AcMPAG formation
was not assessed (Huang et al., 2002; Bernard and Guillemette, 2004).
Our study further shows that a similar effect can be observed for
AcMPAG. Such an impact could be explained by the fact that this
amino acid variation involves the substitution of a highly conserved
cysteine for a tyrosine. Likewise, the T240A variant (not encountered
alone in the population studied) seems to be responsible for the
reduced capacity observed for the UGT1A8*5 protein (G173A240) to
generate MPA glucuronides, as the activity of the *5 (G173A240) and
T240A variants are similar. This also suggests a negligible role of the
A173G variation (*2 allele) on UGT1A8 protein activity, as seen
previously (Huang et al., 2002; Bernard and Guillemette, 2004). The
dramatic activity reduction associated with the UGT1A8*7 (A231T)
protein is somehow surprising because the alanine-threonine substi-
tution represents a fairly conservative change. Nevertheless, the ve-
locity of the UGT1A8*7 (A231T) protein was reduced by 281-fold for
MPAG compared with UGT1A8*1, indicating a critical role of this
amino acid for enzyme function. As for the UGT1A8*8 (S43L) and *9
(N53G173) proteins, they were both associated with similar decreases
in velocity, with 2.8- and 3.3-fold reduced Vmaxvalues, respectively.
The H53N variation involves the substitution of a highly conserved
histidine for an asparagine, which could explain the reduced activity
observed with the variant protein.
One of the UGT1A8 variant allozymes, UGT1A8*4 (V144G173),
demonstrated an effect specific to the glucuronide product formed.
The combination of variations at codons 144 and 173 led to an
enhanced activity of the protein specifically for the formation of
MPAG whereas the formation of AcMPAG was lowered. According
Kinetic estimates for MPAG and AcMPAG formation by UGT1A8 and UGT2B7 variant allozymes
Kinetic parameter determination was performed by incubating MPA (25 to 1250 ?M) with HEK293 cells stably expressing UGT1A8 and UGT2B7 variants. Velocity values were adjusted
according to protein expression levels relative to the *1 allele determined by Western blot. All values are expressed as the mean ? S.E.M. of two to five experiments performed in duplicate.
112,826 ? 5434
87,992 ? 4263
3296 ? 359 **
30,470 ? 1102**
9273 ? 400**
48,880 ? 8735*
402 ? 1**
40,451 ? 416*
33,773 ? 3705*
44,809 ? 1387*
66,479 ? 9498*
7261 ? 1386**
532 ? 39
242 ? 5**
120 ? 13*
26 ? 5*
128 ? 20*
164 ? 12*
166 ? 8*
204 ? 23*
403 ? 101
411 ? 65
296 ? 31
89 ? 11*
105 ? 13*
75 ? 10*
333 ? 120
288 ? 136
531 ? 18
683 ? 262
441 ? 74
130 ? 25*
75 ? 18*
703 ? 144
566 ? 9
468 ? 40
278 ? 36
483 ? 95
1125 ? 226
672 ? 125
773 ? 188
553 ? 43
183 ? 99
146 ? 61
1993 ? 193
2610 ? 805
Significant differences from the *1 allele; * p ? 0.05, ** p ? 0.001. N.D., not detectable.
bNot encountered alone in the population studied.
MPA GLUCURONIDATION BY UGT1A8 AND UGT2B7 VARIANTS
to the kinetic properties of the V144alone and the G173alone
(UGT1A8*2), the effect on AcMPAG formation would be a conse-
quence of the amino acid substitution at codon 173, whereas the
increased capacity of MPAG formation would mostly be caused by
the change at codon 144.
UGT2B7 has been identified as the main enzyme involved in the
formation of AcMPAG (Picard et al., 2005) and is expressed in the
liver and the intestine (Jin et al., 1993; Radominska-Pandya et al.,
1998). The common UGT2B7*2 (Y268) allele, carried by 27% of
Asians and up to 54% of Caucasians (Jin et al., 1993; Guillemette et
al., 2000; Lampe et al., 2000), demonstrated a catalytic efficiency
comparable with that of UGT2B7*1 in most studies for various
substrates, including opioids, androgens, 3?-azido-3?-deoxythimidine,
and morphine (Coffman et al., 1998; Barbier et al., 2000; Bhasker et
al., 2000; Innocenti et al., 2001). Thus, it is predicted that the in vivo
formation of AcMPAG in the liver and the gastrointestinal tract is
probably not significantly modulated by UGT2B7*2. Other variants
of UGT2B7, namely, SNPs of the 5?-regulatory region, could affect
the levels of expression of the gene and would deserve further con-
sideration with regards to AcMPAG formation. One example is the
?79G?A polymorphism in the UGT2B7 gene, in complete linkage
disequilibrium with UGT2B7*2, that results in a reduction of 2.5- to
7-fold lower promoter activity and found in approximately 5% of the
population (Duguay et al., 2004).
The pharmacokinetics of MPA and its metabolites have been shown
to be highly variable in various transplant subpopulations (Bulling-
ham et al., 1998; Ensom et al., 2002; Jacobson et al., 2005; Srinivas
et al., 2005). One of the factors likely involved is the genetic diversity
of UGT genes, such as the UGT1A9 ?275/?2152 variants recently
associated with significantly lower MPA exposure in renal transplant
patients (Kuypers et al., 2005). As proposed by the authors, the altered
pharmacokinetics related to the presence of these common polymor-
phisms is believed to be at least partially caused by a reduction of
enterohepatic recirculation, a process accounting for up to 40% of the
plasma MPA area under the concentration-time curve (Seifeldin,
1995; van Gelder et al., 2001). Given the important role of this process
in the pharmacokinetics of MMF, the intestinal conjugation of MPA
certainly deserves further attention (Bullingham et al., 1998).
UGT1A8 is one of few UGT enzymes to be specifically expressed in
the gastrointestinal tract (Cheng et al., 1998; Tukey and Strassburg,
2000; Zheng et al., 2002), along with UGT1A9 and 2B7 (Tukey and
Strassburg, 2000; Turgeon et al., 2001). This isoform could signifi-
cantly contribute to the intestinal formation of the inactive glucuro-
nide MPAG, but also the reactive and potentially toxic metabolite
AcMPAG, yet to a much lesser degree than UGT2B7 (31-fold lower
CLintvalue). In conclusion, although the common variant of UGT2B7
at codon 268 is predicted to have limited impact in vivo, several
UGT1A8 variants were identified and could contribute to the interin-
dividual variability in MMF pharmacokinetics and deserve further
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Address correspondence to: Chantal Guillemette, Canada Research Chair in
Pharmacogenomics, Pharmacogenomics Laboratory, CHUL Research Center,
T3-48, 2705 Boul. Laurier, QC, G1V 4G2, Canada. E-mail: chantal.guillemette@
MPA GLUCURONIDATION BY UGT1A8 AND UGT2B7 VARIANTS