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Published Ahead of Print on September 20, 2013, as doi:10.3324/haematol.2013.089888.
Copyright 2013 Ferrata Storti Foundation.
Glutathione transferase-A2 S112T polymorphism predicts survival,
transplant-related mortality, busulfan and bilirubin blood levels
after allogeneic stem cell transplantation
by Francesca Bonifazi, Gianluca Storci, Giuseppe Bandini, Elena Marasco, Elisa Dan,
Elena Zani, Fiorenzo Albani, Sara Bertoni, Andrea Bontadini, Sabrina De Carolis,
Maria Rosaria Sapienza, Simonetta Rizzi, Maria Rosa Motta, Martina Ferioli, Paolo Garagnani,
Michele Cavo, Vilma Mantovani, and Massimiliano Bonafè
Haematologica 2013 [Epub ahead of print]
Citation: Bonifazi F, Storci G, Bandini G, Marasco E, Dan E, Zani E, Albani F, Bertoni S, Bontadini A,
De Carolis S, Sapienza MR, Rizzi S, Motta MR, Ferioli M, Garagnani P, Cavo M, Mantovani V,
and Bonafè M. Glutathione transferase-A2 S112T polymorphism predicts survival, transplant-related
mortality, busulfan and bilirubin blood levels after allogeneic stem cell transplantation.
Haematologica. 2013; 98:xxx
doi:10.3324/haematol.2013.089888
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Glutathione transferase-A2 S112T polymorphism predicts survival, transplant-related
mortality, busulfan and bilirubin blood levels after allogeneic stem cell transplantation
Francesca Bonifazi1, Gianluca Storci2,3, Giuseppe Bandini1, Elena Marasco2, Elisa Dan1, Elena
Zani2, Fiorenzo Albani4, Sara Bertoni2, Andrea Bontadini5, Sabrina De Carolis3, Maria Rosaria
Sapienza3, Simonetta Rizzi1, Maria Rosa Motta1, Martina Ferioli1, Paolo Garagnani2, Michele
Cavo1, Vilma Mantovani2, and Massimiliano Bonafè3
1Institute of Hematology “L. and A. Seràgnoli”, University of Bologna, St. Orsola-Malpighi
University Hospital; Bologna, Italy.
2Center for Applied Biomedical Research (CRBA), St. Orsola-Malpighi University Hospital;
Bologna, Italy.
3Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna.
4 IRCCS Istituto delle Scienze Neurologiche di Bologna and Department of Biomedical and NeuroMotor Sciences, University of Bologna, Italy.
5 Transfusion Medicine and Immunohematology, S. Orsola-Malpighi Hospital; Bologna, Italy.
Statement of equal contribution: FB and GS contributed equally to this manuscript.
Running heads: GSTA2 S112T polymorphism in allogeneic HSCT
Correspondence
Francesca Bonifazi, MD, Institute of Hematology “Seràgnoli”, University-Hospital S.
Orsola-Malpighi,, University of Bologna, Bologna, Italy. E-mail: [email protected]
Funding
This work has been supported by Progetto Università Regione 2008-2011, N. 1412: “HSCT
transplant in the elderly” (G. Bandini and M. Bonafè), University of Bologna RFO funds, Cornelia
Pallotti and Roberto Pallotti Foundation (M. Bonafè).
Acknowledgments
We thank Dr. Gabriele Grossi, Daniela Bastia and Dr. Chiara Fazio for technical assistance.
Abstract
Busulfan liver metabolism depends on glutathione, a crucial mediator of cellular and
systemic stress. Here we investigated 40 polymorphisms at 27 loci involved in hepatic glutathione
homeostasis, with the aim to test their impact on the clinical outcome of 185 busulfan-conditioned
allogeneic transplants. GSTA2 S112T serine allele homozygosity is an independent prognostic
factor for poorer survival (RR=2.388), for increased any time- and 100-day Transplant Related
Mortality (RR=4.912 and RR=5.185, respectively). The genotype also predicts a wider busulfan
area under the concentration-time curve (1214.36+570.06 vs 838.10+282.40 µMol*min) and higher
post-transplant bilirubin serum levels (3.280+0.422 vs 1.874+0.197 mg/dL). In vitro, busulfan
elicits pro-inflammatory activation (increased NF-KappaB activity and interleukin-8 expression) in
human hepatoma cells. At the same time, the drug down-regulates a variety of genes involved in
bilirubin liver clearance: constitutive androstane receptor, multidrug resistance-associated protein,
solute carrier organic anion transporters, and even GSTA2. Worthy of note, is the fact that GSTA2
also acts as an intra-hepatic bilirubin binding protein. These data underline the prognostic value of
GSTA2 genetic variability in busulfan-conditioned allotransplants and suggest a pathophysiological model in which busulfan-induced inflammation leads to the impairment of posttransplant bilirubin metabolism.
Keywords: stem cell transplantation, busulfan, GSTA2, bilirubin, NF-KappaB.
Introduction
Hematopoietic Stem Cell transplantation (HSCT) is a curative procedure for a variety of
hematological diseases1. Transplant related mortality (TRM) after HSCT depends mainly on graftversus-host disease (GVHD), infections and preparative regimen toxicity. The assessment of
polymorphisms at Major Hystocompatibility Complex (MHC) loci has long been proven to be
crucial for successful allogeneic HSCT2. Polymorphisms at non-MHC loci have also been
associated with the HSCT outcome3,4. Major attention has been paid to the relationship between
genetic variation at innate immunity loci and GVHD5. Up until now, studies on genetic
polymorphisms on HSCT have generated contrasting data, likely due to insufficient sample size,
selection biases, patient and treatment heterogeneity2.
HSCT is a systemic stress: the achievement of a favourable HSCT outcome is attained via
the successful functioning of multiple stress response systems, and is likely to be affected by
genetic variability over a large array of loci3.
Busulfan, a bi-functional alkylating agent, is an alternative to total body irradiation, and it is
one of the most widely used drugs in allogeneic HSCT conditioning regimen1. Busulfan is used in
association with cyclophosphamide1 and more recently with fludarabine6. Systemic exposure to the
drug is an important parameter for monitoring therapeutic efficacy7,8. Large inter-individual
variability in busulfan pharmacokinetics has been observed9: the therapeutic window of busulfan,
expressed as area under the concentration-time curve (AUC), ranges from 900 to 1500μMol*min.
AUC levels lower than 900μMol*min correlate with disease relapse and graft-failure, while values
higher than 1500μMol*min are associated with extra-hematological hepatic toxicity10,11. The
clearance of the drug occurs in the liver by direct conjugation with glutathione (GSH), as well as via
enzymatic GSH conjugation by glutathione-S-transferases12,13 (GST). Liver GST activity, which is
mainly due to GSTA1 and GSTA2 iso-enzymes, correlates with busulfan elimination14. Depletion
of liver GSH, as consequence of busulfan conjugation, impairs the metabolism of other drugs such
as cyclophosphamide15. Plasma GST activity and GSH level associate with the variability in
busulfan clearance14,16.
In this paper, 40 polymorphisms at 27 loci involved in hepatic glutathione balance17 were
studied in a total of 185 patients who underwent allogeneic HSCT from 2005 to 2009, after a
busulfan-based preparative regimen (busulfan cohort). The impact of such loci was tested on overall
survival, TRM, busulfan AUC and serum liver function tests. The same polymorphisms were also
tested on 146 consecutive patients not receiving busulfan in the preparative regimen, concomitantly
undergone transplantation at the same Institution (comparator cohort).
Methods
Patients
185 consecutive patients (busulfan cohort, Table 1) were transplanted at the Institute of Hematology
“L. and A. Seràgnoli”, University of Bologna, between 2005 and 2009, using busulfan as a
conditioning drug. Myeloablative conditioning (0.8 mg/kg intravenous busulfan, 2h infusions, four
times a day, four consecutive days plus cyclophosphamide, 120mg/kg) was administered to 167
patients. Lower (6 patients) or standard busulfan doses plus fludarabine 160mg/m2 (12 patients)
were also used. Ideal body weight (IBW) was calculated as 0.91x (height in cm–152) +50 (for men)
or +45 (for women). IBW was used when lower than the actual weight and when body mass index
(BMI) was lower/equal to 27. When BMI was greater than 27, IBW was adjusted as IBW+ 0.25 x
(actual weight – IBW).
146 consecutive patients (comparator cohort, Table 1) transplanted at the same Institution were also
studied. In such cohort, the myeloablative conditioning was cyclophosphamide (120mg/kg) plus
unfractioned total body irradiation (8Gy).
GVHD prophylaxis was Cyclosporin-A and short-term methotrexate plus rabbit anti-thymocyte
globulin (3-5 mg/kg/die, Fresenius, Bad-Homburg, Germany), from day -6 to day -2. Patients with
acute leukemia in 1st complete remission or with chronic myeloid leukemia in 1st chronic phase
were classified as in early phase at transplant, the remaining patients were classified in advanced
phase. Written informed consent for the study was obtained from the patients. The study was
approved by the Ethics Committee of S. Orsola-Malpighi University Hospital, Bologna, Italy.
Busulfan pharmacokinetics
Plasma busulfan kinetics (64 patients) was assessed before administration, and at 15, 60, 120, 180,
240 minutes, after dose dose 9 (day 3)
19
. Plasma busulfan concentrations were determined by
HPLC (Perkin Elmer, USA)20. The area under the concentration-time curve (AUC) was calculated
by Kinetica software (Thermo Scientific, Waltham, MA, USA).
Genetic analysis
Forty polymorphisms at 27 loci were analyzed (Supplementary Table 1 and 2). 36 were genotyped
using MassARRAY high-throughput DNA analysis (Sequenom, Inc., San Diego, CA, US). Three
(CBS rs72958776-68bpIns, GST-T1 and GST-M1 null alleles) were genotyped by PCR; GPX1
SNP rs1050450 was assessed by tetra-ARMS PCR.
In vitro study
Human hepatoma HepG2 cells were grown in DMEM 10% FBS (Life Technologies, Carlsbad, CA,
USA). GSTA2-specific siRNA and GC-matched control siRNA were transfected by Lipofectamine
2000 (Life Technologies) to HepG2 cells (10x104/3cm2 well, 72h). Cells were exposed to busulafan
(2.5 to 400uM, 96h) and cell death was assessed by Trypan blue.
Real-time PCR
mRNA was extracted by Trizol (Life Technologies). Real-time PCR primers and probes for
GSTA2, interleukin-8 (IL8), constitutive androstane receptor (CAR), multidrug resistanceassociated protein ABCC2, solute carrier organic anion transporters SLCO1B1 and SLCO1B3 and
β-glucuronidase mRNAs were from Life Technologies. The amount of the target genes was
calculated by the 2-ddcT method.
Luciferase assay
NF-kappaB luciferase21 reporter activity was assessed in HepG2 (1 µg/well, 24h) by DualLuciferase® Reporter Assay System (Promega, Madison, WI).
Statistical analysis
Cox-proportional and general linear model (GLM) for repeated measures were employed.
p=0.00125 was considered significant in multiple comparisons. The analyses were performed by
PASW18
(SPSS-IBM,
(http://genecanvas.ecgene.net)22.
NY).
Haplotypes
were
estimated
by
Thesias
RESULTS
GSTA2 S112T locus impacts TRM and survival, but not GVHD and relapse, in patients
receiving busulfan.
Clinical characteristics of transplanted patients are reported in Table 1. Multivariate Cox
regression was performed using the five clinical variables included in the EBMT score (age, sex
mismatch, disease phase, type of donor, interval diagnosis-transplant) plus intensity of conditioning.
The analysis revealed that disease phase at transplant (advanced vs early) is a significant prognostic
factor for both TRM (advanced vs early, RR= 2.689, 95%CI: 1.078-6.703, p=0.034) and overall
survival (OS, advanced vs early, RR= 3.975, 95%CI: 2.262-6.986, p=1.61*10-6). All the genotypes
investigated (Supplementary Tables 1 and 2) were singularly tested in a Cox model implementing
clinical variables as covariates (Supplementary Table 3). The serine (ser) to threonine (thr)
aminoacid substitution at GSTA2 Codon 112 (S112T) was the only locus which remained
significant for TRM after adjustment for multiple comparisons (n=40, p=0.001), while the effect on
OS was only marginally significant (p=0.005, Supplementary Table 3). GSTA2 is a glutathione
transferase, which is expressed in the liver and metabolizes busulfan in cooperation with GSTA113.
GSTA2 S112T genotypes were then recoded as dichotomous variables, namely serine homozygotes
(ser/ser, n=38) and threonine carriers (thr+, n=142). We observed that GSTA2 S112T locus was
predictive of OS (ser/ser vs thr+, RR=2.388, 95%CI: 1.407-4.502, p=0.001), TRM (ser/ser vs thr+,
RR=4.912, 95%CI: 2.083-11.563, p=0.0002) and 100-day TRM (ser/ser vs thr+, RR=5.185,
95%CI:1.971-13.641, p=0.001, Table 2). The Kaplan and Meyer plot showed the poorer survival
and higher TRM of GSTA2 S112T ser/ser patients (Figure 1A, 1B and 1C). Furthermore, because
deaths mostly occurred in the first post-transplant weeks, we also calculated 100-day TRM (Figure
1C). In line with expectations, the analysis of 100-day TRM death causes revealed a significantly
higher number of deaths due to conditioning-related toxicity23 in GSTA2 S112T ser/ser patients (6
out of 9, 66.6%), compared to thr+ ones (2 out of 12, 16.6%, p=0.032, Fisher exact test , Figure
1D).
Interestingly, GSTA2 S112T locus did not significantly affect either TRM nor OS in the
comparator cohort (Supplementary Table 3), even when the analysis was performed separately on
the subset of patients receiving myeloablative conditioning regimen (ser/ser, n=9 vs thr+, n=52, OS:
RR=0.361 95%CI: 0.115-2.201, p=0.361; TRM: RR=0.558 95%CI: 0.070-4.412, p=0.580) or
reduced-intensity conditioning regimen (ser/ser, n=21 vs thr+, n=64; OS: RR=0.891 95%CI: 0.4932.237, p=0.891; TRM: RR=0.797 95%CI: 0.245-2.593, p=0.706).
We also observed a weak association between TRM and other polymorphisms located at the
GSTA1-GSTA2 genomic region, namely GSTA1 SNPs (rs1051775, rs3957356, rs4715332,
Supplementary Table 3). As previously reported24,25, owing to the significant linkage disequilibrium
among polymorphisms at GSTA2 and GSTA1 loci (Supplementary Table 4), the two loci haplotype
frequency distribution of GSTA2 S112T and GSTA1 loci was estimated: the ser allele was highly
associated with the so-called GSTA1*B rs3957356-rs4715332 functional haplotype25,26
(Supplementary Table 5). Nevertheless, survival analysis of GSTA2 S112T-GSTA1*A/B
haplotypes reinforced the conclusion that GSTA1*B haplotype does not significantly modify the
impact of the GSTA2 S112T locus on TRM and survival in our case set (Table 3 and
Supplementary Table 6). Moreover, GSTA2 S112T locus does not affect relapse rate, which was
instead associated with the phase at transplant (Supplementary Table 7). Furthermore, incidence of
aGVHD III-IV grades and hepatic acute GVHD III-IV stages were similar between GSTA2 S112T
ser/ser and thr+ patients (18.9% vs 19.1%, p=0.591, 5.4% vs 5.6%, p=0.472, respectively).
Impact of the GSTA2 S112T locus on busulfan-AUC and the post transplant serum bilirubin
level.
GSTA2 S112T polymorphism is not located in the enzyme catalytic site but it has been supposed to
affect protein stability27. Busulfan is almost exclusively metabolized in the liver, where GSTA2 is
expressed15,28. Anova analysis implementing age, sex, BMI and source of HSC as covariates,
revealed higher systemic exposure to busulfan (day-3 AUC) in GSTA2 S112T ser/ser patients
compared to thr+ (1214.36+570.06 vs 838.10+282.40 μMol*min, F=9.185, p=0.001, Figure 2A).
Notably, BMI was not affected by S112T GSTA2 polymorphism, given that ser/ser patients show
the same BMI distribution as thr+ patients, in each gender group (males: 27.70+2.62 vs 26.5+3.85,
females: 21.13+1.51 vs 24,21+4.55, p=0.85, GLM analysis). Moreover, busulfan AUC was not
different between patients with BMI lower than/equal to 27 vs those with BMI greater than 27
(mean values: 871.00+343.09 vs 903.96+355.21 μMol*min, p=0.29, adjusted for gender).
Because glutathione depletion causes liver damage18, we then evaluated the association
between GSTA2 S112T SNP and liver function tests: total bilirubin, alanine transaminase, aspartate
transaminase, cholinesterase, alkaline phosphatase and gamma-glutamyl transferase were examined
weekly from the day of conditioning, up to the 35th post-transplant day. Higher un-fractioned
plasma bilirubin levels for the first five weeks after transplant were found in GSTA2 S122T ser/ser
patients compared to thr+ ones (GLM-estimated marginal means: 3.280+0.422 vs 1.874+0.197
mg/dL, F=8.728, p=0.004, Figure 2B). Interestingly, no association was found between GSTA2
S112T locus and all the other liver functional tests (Supplementary Table 8).
Exposure of human hepatoma cells to busulfan induces a pro-inflammatory response
and reduces the expression of genes involved in bilirubin clearance.
The data above suggest that, in patients receiving busulfan, the difference in post-transplant
plasma bilirubin level between ser/ser vs thr+ patients is not correlated with the extent of liver
necrosis. In this regard, negligible cell death was observed in human hepatoma cells HepG2
exposed to busulfan at concentrations comparable to those occurring in vivo, even when such cells
were knocked down by a GSTA2-specific short interfering RNA oligonucleotide (siGSTA2, Figure
3A). In HepG2 cells, busulfan administration elicited the up-regulation of NF-kappaB, the master
controller of the inflammatory response29 (Figure 3B). Moreover, siGSTA2 transfected HepG2 cells
disclosed the up-regulation of NF-kappaB activity and of the pro-inflammatory cytokine
interleukin-8, compared to controls (Figure 3C).
At cellular and systemic levels, inflammation reduces the expression of a variety of genes
involved in liver bilirubin metabolism29,30, namely constitutive androstane receptor31 (CAR),
multidrug resistance associated protein MRP2/ABCC232 and solute organic carriers SLCO1B1 and
SCLO1B332 . We also included GSTA2 in this gene set, since it is the intra-hepatic bilirubin
ligandin, a function which is independent of its enzymatic activity33. We found that exposure of
HepG2 cells to non-cytotoxic concentrations of busulfan elicited a dramatic decrease in GSTA2,
CAR, ABCC2, SLCO1B1 and SCLO1B3 expression (Figure 3D). Notably, SLCO1B1 and
SCLO1B3 became almost undetectable upon busulfan exposure. Moreover, in siGSTA2-transfected
HepG2, we observed a further decrease in CAR and ABCC2 expression compared to controls
(Figure 3E). These data led us to argue that the pro-inflammatory activation of busulfan-exposed
hepatocytes causes the down-regulation of GSTA2 and of bilirubin-metabolizing enzymes (Figure
3F). This metabolic reshaping is expected to impair bilirubin clearance in the post-transplant phase
and to be dependent on the individual genetic landscape.
Discussion
In this paper we report that the GSTA2 S112T polymorphism affects survival of HSCT
patients receiving a busulfan-based conditioning regimen, serine allele homozygotes being more
prone to higher transplant-related and overall mortality compared to threonine allele carriers. The
frequency of GSTA2 S112T serine homozygotes here reported (21%) is close to that found in the
Italian population34 (19.8%) and in Caucasians24 (18.5%), suggesting that the locus does not
represent a risk factor for developing hematopoietic malignancies. Interestingly, the association of
GSTA2 S112T with TRM and survival is not significant in the cohort of HSCT patients not
receiving busulfan (comparator cohort). Therefore, the data suggest that the polymorphism changes
the individual response to the drug. In support of this hypothesis, we report that GSTA2 S112T
serine homozygotes show higher busulfan plasma levels compared to threonine carriers, indicating
that such a group of patients display a reduced busulfan clearance capability. In fact, busulfan
metabolism mainly involves its conjugation with GSH in the liver, via alpha class cytosolic
GST12,13. Busulfan administration depletes the content of hepatocyte GSH, the cofactor of GST
enzymes18,35. Plasma GSH correlates with busulfan clearance capacity16 and hepatic GST activity
negatively correlates with plasma busulfan and positively associates with its clearance14. In line
with these data, the association between high busulfan plasma levels and post-transplant mortality
has already been described7,8,10. Consistent with the above reasoning, we found that the proportion
of deaths due to drug toxicity in GSTA2 S112T serine homozygotes is higher than in other patients.
Hence, the GSTA2 S112T locus may be regarded as a reliable predictor for individual susceptibility
to the adverse effects of busulfan. It might also be argued that GSTA2 S112T locus can be used to
tailor busulfan dosages. However,our series is based on the standard association of busulfan and
cyclophosphamide, the GSTA2 S112T locus should be assessed in patients receiving other
busulfan-based drugs combinations.
Our data do not confirm the previously reported association between busulfan AUC and GVHD9.
Insufficient power or heterogeneity of patients’ GVHD risk (i.e. type of donor, source of HSC,
HLA distance) may explain these findings.
The GSTA2 S112T aminoacid change is not located at the enzyme catalytic site36.
Moreover, serine and threonine residues are both polar non-charged aminoacids and their reciprocal
substitution is not expected to yield any predictable change in the protein structure36. Nevertheless,
in the liver, the GSTA2 S112T locus has been found to impact the GSTA2 protein level and
themostability24,27. These data suggest that the GSTA2 S112T locus affects the individual capability
to metabolize busulfan, independently of the alteration of GSTA2 enzyme activity.
GSTA2 and GSTA1 share 95% aminoacid identity but GSTA2 is endowed with lower
busulfan-GSH conjugation activity13,28. The GSTA2 S112T locus is closely associated with the
functional (rs395376)-52bp GSTA1 polymorphism, which modifies the gene transcription rate34.
Consequently, the tight linkage disequilibrium among GSTA1 and GSTA2 polymorphisms24,25,37
might explain the functional association here reported. The GSTA1 promoter hosts functional *A
and *B haplotypes25,26,37: the GSTA1*B haplotype has been associated with four-fold reduction of
GSTA1 enzyme activity and higher busulfan plasma concentration8,26,37-39. Though the GSTA2
S112T serine allele is in linkages with the GSTA1 *B haplotype24-26, it seems to be the sole cause
for the results here obtained. Even the GSTA2 thr-*B haplotype, which is expected to combine the
low activity of both isoenzymes24,25, seems not to impact patients’ survival. Intriguingly, the
association between the GSTA1*B haplotype and reduced busulfan clearance was observed with
oral but not intravenous administration40,41. On the contrary, other authors failed to find any
association between GSTA1 polymorphisms and busulfan metabolism42,43. Nevertheless, our data
support the role of GSTA1-GSTA2 genomic region in the inter-individual variability in busulfan
metabolism.
Here we show that 35 days post-transplant serum bilirubin levels are increased in GSTA2
S112T serine homozygotes patients receiving a preparative regimen containing busulfan. Since
GSTA2 S112T serine homozygotes do not display significantly different alterations in other liver
function tests, we speculate that such patients might undergo specific alterations of bilirubin
clearance. In this regard, busulfan is not hepatotoxic itself, at least within the therapeutic
concentrations range44. According to this notion, we did not find cell death in human hepatoma cells
exposed to therapeutic concentrations of busulfan, even upon siRNA-mediated GSTA2 knockdown.
We observed that busulfan triggers the pro-inflammatory activation of human hepatoma cells. At
cellular and systemic levels29,30, inflammation has been linked to the down-regulation of a variety of
genes involved in bilirubin metabolism. In particular, we show here that busulfan downregulates: i.
CAR, the nuclear transcription factor for several liver bilirubin clearance genes32; ii.
MRP2/ABCC2, a member of the ATP-binding cassette family of membrane transporters localized
at the apical border of hepatocytes, which is involved in the transport of conjugated bilirubin and
glutathione trafficking to the bile29; iii. SLCO1B1 and SLCO1B3, trans-membrane transporters at
hepatocytes’ baso-lateral border, which control the uptake of conjugated and/or unconjugated serum
bilirubin32. Intriguingly, busulfan also downregulates GSTA2 which is part of the bilirubin
metabolism, owing to its ability to act as intrahepatocytic ligandin, which prevents the backflow of
hepatic bilirubin into blood circulation31. In addition, we found that the exposure of GSTA2
knockdown cells elicits higher inflammatory activation and lower expression of CAR and ABCC2,
compared to controls. Since it has been previously reported that post-transplant interleukin-8 serum
level is highly correlated with serum bilirubin45 it can be hypothesized that the pro-inflammatory
status, which develops in busulfan prepared patients, might be linked to the reduced clearance of
un-conjugated bilirubin, as well as to the increase of serum bilirubin. On the basis of this reasoning,
these findings lead us to speculate that post transplant hyper-bilirubinemia in GSTA2 S112T serine
homozygotes may be the consequence of their peculiar liability to the development of busulfandependent pro-inflammatory liver injury (Figure 3F).
In conclusion, our data suggest that the GSTA2 S112T polymorphism is predictive of
transplant outcome in patients receiving busulfan in the preparative regimen, at least in association
with cyclophosphamide.
The study is retrospective and mono-center: this analysis warrants
validation by a prospective clinical study to gauge the role of genotyping at GSTA S112T locus in
the adjustment of busulfan dosages.
Authorship and disclosure
FB, GS, GB and MB designed the study; FB, GB, MC and MF recruited patients; GS performed the
in vitro studies; EZ and FA assessed busulfan dosage and kinetics; SB, SDC performed cell
cultures, Real Time PCR, gene knockdown; EM, PG and VM made genotype design and analysis;
AB, SR, ED, MRM MRS were in charge of cell banking, DNA banking and extraction; FB and MB
carried out statistical analysis; FB, GS, GB and MB wrote the manuscript; all the authors reviewed
and approved the manuscript. The authors report no potential conflicts of interest.
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busulphan on the pharmacokinetics of cyclophosphamide and its 4-hydroxy metabolite: time
interval influence on therapeutic efficacy and therapy-related toxicity. Bone Marrow Transplant.
2000;25(9):915-24.
16. Almog S, Kurnik D, Shimoni A, Loebstein R, Hassoun E, Gopher A, et al. Linearity and
stability of intravenous busulfan pharmacokinetics and the role of glutathione in busulfan
elimination. Biol Blood Marrow Transplant. 2011;17(1):117-23.
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19. Vaughan WP, Carey D, Perry S, Westfall AO, Salzman DE. A limited sampling strategy for
pharmacokinetic directed therapy with intravenous busulfan. Biol Blood Marrow Transplant.
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20. Rifai N, Sakamoto M, Lafi M, Guinan E. Measurement of plasma busulfan concentration by
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21. Storci G, Sansone P, Mari S, D'Uva G, Tavolari S, Guarnieri T, et al. TNFalpha up-regulates
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the human glutathione S-transferase A1 promoter on hepatic GSTA1 and GSTA2 expression.
Pharmacogenetics. 2001;11(8):663-9.
27. Zhang W, Modén O, Mannervik B. Differences among allelic variants of human glutathione
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34. Landi S, Gemignani F, Neri M, Barale R, Bonassi S, Bottari F, et al. Polymorphisms of
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36. Tetlow N, Board PG. Functional polymorphism of human glutathione transferase A2.
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Tables
Table 1. Clinical characteristics of patients
Busulfan cohort
Comparator cohort
(n=185)
(n=146)
median
range
Age (years)
41
(18-59)
43
(16-61)
Interval diagnosis-transplant (months)
18
(2-128)
20
(2-240)
Follow up (months)
31
(1-238.7)
38.9
(1-239.2)
§
Years of transplant
median
§
range
na
(2005-2009)
na
N
(%)
N
(%)
Gender (males/females)
110/75
(59.4/60.6)
85/59
(58.2/61.8)
Type of donor (Sibling/Unrelated)
97/88
(52.4/47.6)
65/81
(44.5/55.5)
Sex mismatch (male recipient/female donor)
39
(21.1)
32
(21.9)
Early phase at transplant*
85
(45.9)
47
(32.2)
Myeloablative conditioning regimen
179
(96.8)
61
(41.8)
Anti-thymocyte globulin
126
(68.1)
99
(67.8)
Acute Myeloid Leukemia
90
(48.7)
29
(19.9)
Acute Lymphoblastic leukemia
35
(18.9)
23
(15.8)
Myelodisplastic syndromes
8
(4.3)
4
(2.7)
Multiple Myeloma
9
(4.9)
28
(19.2)
Lymphomas
10
(5.4)
50
(34.2)
Chronic Myeloid Leukemia
27
(14.6)
10
(6.8)
Other
6
(3.2)
2
(1.4)
Bone Marrow
78
(42.2)
68
(46.6)
Peripheral Blood
101
(54.6)
76
(52.1)
Cord Blood
6
(3.2)
2
(1.3)
(2005-2009)
Diagnosis
Source of HSC
Footnote: §not applicable; *early phase at transplant: acute leukemia in 1st complete remission or
myeloid leukemia in1st chronic phase.
Table 2. Multivariate Cox analysis of overall survival (OS), transplant related mortality (TRM) and 100-day TRM.
OS
[95% CI]
TRM
Variables
RR
p
Age
1.004 [0.980-1.029] 0.746
Interval diagnosis-transplant
RR
[95% CI]
100-day TRM
p
RR
[95% CI]
p
1.040 [0.996-1.085]
0.074
1.030 [0.982-1.080]
0.226
0.995 [0.981-1.010] 0.525
1.004 [0.983-1.026]
0.716
1.003 [0.980-1.026]
0.801
Sex donor/recipient (female/male vs others)
1.579 [0.834-2.989] 0.161
1.443 [0.475-4.384]
0.518
0.432 [0.096-1.938]
0.273
Type of donor (unrelated vs sibling)
1.467 [0.871-2.470] 0.150
1.173 [0.500-2.571]
0.714
1.010 [0.388-2.631]
0.983
Conditioning regimen (myeloablative vs reduced intensity) 0.216 [0.029-1.597] 0.133
0.522 [0.068-4.020]
0.533
1.665 [0.210-13.172] 0.629
Phase at transplant (advanced vs early)
4.083 [2.295-7.265] 0.0001 3.547 [1.331-9.452]
0.011
6.269 [1.734-22.659] 0.005
GSTA2 S112T (ser/ser vs thr+)
2.388 [1.407-4.052] 0.001
4.912 [2.083-11.583] 0.0002 5.185 [1.971-13.641] 0.001
Footnote: The effect of GSTA2 S112T locus remained significant in multivariate analysis even when patients who received reduced intensity
conditioning regimen (n=6) were removed: OS, RR=2.183 95%CI: 1.283-3.716, p=0.004; TRM, RR=3.821 95%CI: 1.645-9.018, p=0.002
Table 3 Multivariate Cox analysis of TRM and OS according to GSTA2 S112T- GSTA1*A/B
haplotypes.
Haplotype
TRM
OS
RR
[95% CI]
p
RR
[95% CI]
p
Thr- GSTA1*A
1
/
/
1
/
/
Thr- GSTA1*B
1.744
[0.533-5.695]
0.356
1.5428
[0.733-3.243]
0.252
Ser- GSTA1*A
3.081
[1.281-7.431]
0.011
2.3663
[1.348-4.145]
0.002
Ser- GSTA1*B
1.889
[1.046-3.388]
0.034
1.6771
[1.116-2.427]
0.005
25
Figure legends
Figure 1. GSTA2 S112T locus affects OS and TRM in patients prepared with busulfan. A,
Kaplan-Meyer analysis of OS, B, TRM at any time and C, 100-day TRM according to GSTA2
S112T genotypes (ser/ser, n=38; thr+, n=142); p-values refer to log-rank test; D: pie-chart
representation of 100-day TRM causes of death in ser/ser (n=9) and thr+ (n=12) patients. In
particular, in serine homozygotes the organ toxicity was: liver (2 patients), liver and renal (1
patient), gut (2 patients), heart (1 patient). In threonine carriers the organ involvement was liver (1
patient), hearth (1 patient).
Figure 2. GSTA2 S112T locus impacts serum bilirubin and plasma busulfan levels.
A, busulfan AUC in patients receiving busulfan according to GSTA2 S112T genotypes (ser/ser,
n=8, thr+, n=56); p values refer to multivariate GLM adjusted for age, sex, BMI and source of HSC.
B, bilirubin serum levels according to GSTA2 S112T genotypes (ser/ser, n=20, thr+, n=99) from
pre-transplant to 35 days post-transplant; p value is referred to GLM Anova for repeated measures,
adjusted for age, sex mismatch, interval from diagnosis to transplant, intensity of conditioning and
disease phase at transplant.
Figure 3. Exposure to busulfan and GSTA2 knockdown induce a pro-inflammatory response
and reduce the expression of CAR, ABCC2, SLCO1B1 and SLCO1B3 in human hepatoma
cells (HepG2).
A, cell death analysis of HepG2 cells transfected with control (Ctr) or GSTA2 specific siRNA
(siGSTA2) exposed to increasing busulfan concentrations; B, NF-kappaB luciferase assay in
HepG2 cells exposed to increasing busulfan concentrations; C, NF-kappaB luciferase assay and
Interleukin-8 (IL-8) real time PCR analysis in Ctr/siGSTA2 transfected HepG2 cells exposed to
increasing busulfan concentrations; D, real-time PCR analysis of GSTA2, CAR, ABCC2 SLCO1B1
26
and SLCO1B3 mRNA level in Ctr/siGSTA2 transfected HepG2 cells exposed to increasing
busulfan concentrations; E, real-time PCR analysis of GSTA2, CAR and ABCC2 mRNA level in
HepG2 cells exposed to increasing busulfan concentrations; F, descriptive picture of the proposed
mechanism: busulfan exposure triggers hepatic inflammatory activation which associates with the
down-regulation of genes involved in bilirubin metabolism, namely GSTA2, CAR, ABCC2,
SLCO1B1, SCLO1B3. This phenomenon is amplified by decreased levels of GSTA2 and,
speculatively, in serine GSTA2S112T allele carriers.
27
Supplementary Table 1. Genotype frequency distributions in the Busulfan and in the Comparator cohort.
Busulfan color
(n=185)
Comparator cohort
(n=146)
Polymorphism
Nucleotide
change
MAF
Region
Protein
Genotypes N (%)
Genotypes N (%)
HW (p)
ABCB1
rs1045642
T>A
0.43
coding
I1145I
TT 62 (34)
TA 87 (47)
AA 36 (19)
TT 38 (26)
TA 76 (52)
AA 32 (22)
>0.05
ABCC1
rs45511401
G>T
0.04
coding
G671V
GG 175 (95)
GT 10 (5)
TT 0 (0)
GG 131 (91)
GT 13 (9)
TT 0 (0)
>0.05
ABCC4
rs2274407
C>A
0.02
coding
N304K
CC 154 (84)
AC 28 (15)
AA 1(1)
AA 124 (86)
AC 21 (14)
AA 0 (0)
>0.05
ABCC4
rs11568658
G>T
0
coding
G187W
GG 167 (90)
GT 18 (10)
TT 0 (0)
GG 135 (94)
GT 8 (5)
TT 1 (1)
>0.05
ABCC4
rs9561778
G>T
0.18
intronic
---
GG 118 (64)
GT 63 (34)
TT 3 (2)
GG 106 (73)
GT 38 (26)
TT 2 (1)
>0.05
CBS
rs5742905
T>C
0.00
coding
I278T
TT 172 (93)
CT 13 (7)
CC 0 (0)
TT 134 (92)
CT 11 (8)
CC 0 (0)
>0.05
CBS
rs234706
C>T
0.33
coding
Y233Y
CC 87 (47)
CT 84 (45)
TT 14 (8)
CC 74 (51)
CT 54 (37)
TT 18 (12)
>0.05
CBS
rs72958776
ins68bp
0.03
intronic/coding
---
wt 156 (85)
wt/ins 27 (15)
-
wt 132 (92)
wt/ins 11 (8)
-
--
CTH
rs482843
G>A
0.41
upstream gene
---
GG 66 (36)
AG 96 (52)
AA 23 (12)
GG 49 (34)
AG 62 (43)
AA 34 (23)
>0.05
CYP2B6
rs8192719
C>T
0.28
intronic
---
CC 92 (50)
TC 77 (42)
TT 16 (8)
CC 79 (54)
TC 59 (40)
TT 8 (6)
>0.05
CYP2C9
rs1057910
A>C
0.06
coding
I359L
AA 177 (96)
CA 8 (4)
CC 0 (0)
AA 137 (94)
CA 9 (6)
CC 0 (0)
>0.05
CYP2C19
rs4244285
G>A
0.16
coding
P227P
GG 135 (73)
AG 46 (25)
AA 4 (2)
GG 97 (67)
AG 45 (31)
AA 3 (2)
>0.05
GCLM
rs2301022
G>A
0.40
intronic
---
GG 78 (42)
AG 87 (47)
AA 20 (11)
GG 55 (38)
AG 73 (50)
AA 18 (12)
>0.05
GCLC
rs17883901
C>T
0.09
upstream gene
---
CC 156 (85)
CT 27 (15)
TT 0 (0)
CC 118 (81)
CT 27 (18)
TT 1 (1)
>0.05
GGT1
rs17004876
A>G
0.11
coding
N419D
AA 166 (90)
AG 19 (10)
GG 0 (0)
AA 133 (91)
AG 13 (9)
GG 0 (0)
>0.05
GPX1
rs1050450
C>T
0.12
coding
P200L
CC 82 (44.5)
CT 82 (44.5)
TT 20 (11)
CC 62 (43)
CT 59 (41)
TT 22 (16)
>0.05
GPX2
rs17881652
C>T
0.01
coding
P126L
CC 180 (97)
CT 5 (3)
TT 0 (0)
CC 143 (98)
CT 3 (2)
TT 0 (0)
>0.05
GPX3
rs3763013
A>G
0.33
upstream gene
---
AA 80 (43)
AG 77 (42)
GG 27 (15)
AA 61 (42)
AG 70 (48)
GG 15 (10)
>0.05
GSTA1
rs4715332
T>G
0.41
upstream gene
---
TT 53 (29)
GT 97 (53)
GG 34 (18)
TT 47 (32)
GT 75 (51)
GG 24 (17)
>0.05
GSTA1
rs3957356
G>A
0.37
upstream gene
---
GG 54 (30)
GA 96 (52)
AA 33 (18)
GG 48 (33)
GA 73 (50)
AA 24 (17)
>0.05
GSTA1
rs1051775
A>G
0.31
coding
K125K
AA 60 (33)
GA 89 (49)
GG 34 (18)
AA 47 (32)
GA 76 (53)
GG 22 (15)
>0.05
GSTA2
rs2180314
C>G
0.43
coding
S112T
CC 57 (32)
CG 85 (47)
GG 38 (21)
CC 45 (33)
CG 63 (46)
GG 30 (23)
>0.05
Gene/Locus
GSTM1
null allele
gene
deletion
0.47
coding
---
wt/wt or
wt/null 77(44)
null 99 (56)
---
wt/wt or
wt/null 67
(49)
null 71 (51)
---
---
GSTP1
rs1138272
C>T
0.097
coding
A114V
CC 160 (86)
TC 24 (13)
TT 1 (1)
CC 129 (89)
TC 16 (11)
TT 0 (0)
>0.05
GSTP1
rs1695
A>G
0.41
coding
I105V
AA 101 (54)
AG 66 (36)
GG 18 (10)
AA 74 (51)
AG 56 (39)
GG 14 (10)
>0.05
null 40 (23)
---
wt/wt or
wt/null 112
(81)
null 26 (19)
---
---
GSTT1
null allele
gene
deletion
0.2
coding
---
wt/wt or
wt/null 136
(77)
MGST1
rs7970208
G>A
0.49
upstream gene
---
AA 63 (34)
AG 89 (48)
GG 33 (18)
AA 40 (27)
AG 76 (52)
GG 30 (21)
>0.05
MGST1
rs2239676
C>G
0.08
intronic
---
CC 166 (89)
CG 18 (10)
GG 1 (1)
CC 128 (88)
CG 16 (11)
GG 2 (1)
>0.05
MGST1
rs11875
G>A
0.09
3' UTR
---
GG 156 (84)
AG 27 (15)
AA 2 (1)
GG 129 (88)
AG 16 (11)
AA 1 (1)
>0.05
MTHFR
rs1801131
A>C
0.36
coding
E429A
AA 95 (51)
CA 77 (42)
CC 13 (7)
AA 73 (50)
CA 57 (39)
CC 15 (10)
>0.05
MTR
rs1805087
A>G
0.16
coding
D919G
AA 129 (70)
GA 51 (28)
GG 4 (2)
AA 103 (71)
GA 41 (28)
GG 2 (1)
>0.05
MTRR
rs1532268
C>T
0.31
coding
S175L
CC 75 (40)
CT 83 (45)
TT 27 (15)
CC 70 (48)
CT 62 (42)
TT 14 (10)
>0.05
MTRR
rs1801394
A>G
0.45
coding
I22M
AA 63 (34)
GA 91 (49)
GG 31 (17)
AA 48 (33)
GA 60 (41)
GG 37 (26)
>0.05
NAT1
rs4986782
G>A
0.01
coding
R187Q
GG 180 (98)
GA 4 (2)
AA 0 (0)
GG 138 (95)
GA 7 (5)
AA 0 (0)
>0.05
NAT2
rs1801280
T>C
0.44
coding
I114T
TT 67 (36)
CT 88 (48)
CC 30 (16)
TT 47 (32)
CT 71 (49)
CC 28 (19)
>0.05
NAT2
rs1799930
G>A
0.30
coding
R197Q
GG 94 (51)
AG 71 (38)
AA 20 (11)
GG 73 (50)
AG 59 (40)
AA 14 (10)
0.013
PON1
rs854560
T>A
0.38
coding
L55M
AA 76 (41)
TA 77 (42)
TT 32 (17)
AA 60 (41)
TA 59 (41)
TT 26 (18)
0.021
TCNI
rs34324219
G>T
0.125
coding
D301Y
GG 141 (77)
GT 38 (21)
TT 4 (2)
GG 113 (77)
GT 30 (21)
TT 3 (2)
>0.05
TCN2
rs1801198
C>G
0.45
coding
P259R
CC 67 (36)
GC 88 (48)
GG 30 (16)
CC 58 (40)
GC 62 (42)
GG 26 (18)
>0.05
TCN2
rs4820889
G>A
0.042
coding
R399Q
GG 174 (94)
AG 11 (6)
AA 0 (0)
GG 136 (93)
AG 10 (7)
AA 0 (0)
>0.05
Footnote: MAF: minor allele frequency in Caucasian European (www.ncbi.nlm.nih.gov/snp ); HW: Hardy-Weinberg; ins: insertion; wt: wild-type.
Supplementary Table 2. List of primers for SequenomTM analysis.
Gene/Locus
Polymorphism
First Primer
Second Primer
Extension Primer
Multiplex
ABCB1
rs1045642
ACGTTGGATGACTGCAGCATTGCTGAGAAC
ACGTTGGATGTATGTTGGCCTCCTTTGCTG
CTCCTTTGCTGCCCTCAC
1
ABCC1
rs45511401
ACGTTGGATGCGTTTCAGCATCACCTTCTC
ACGTTGGATGTGAGAGCAGGGACGACTTTC
ccaACGGCCACCAAAGCA
2
ABCC4
rs2274407
ACGTTGGATGTATCTGGTTGACATCACTGC
ACGTTGGATGGGCAGGAACTTCTCAGAATC
ctaAGAATCTTGGAAATCTCCTT
1
ABCC4
rs11568658
ACGTTGGATGTTTCAGGCACTTCGTCTTAG
ACGTTGGATGCAGCAGATTGACTATCTGGC
GGCCTGTGGTTGTCTTCC
2
ABCC4
rs9561778
ACGTTGGATGAGTAGGAAGCATAGAGAACG
ACGTTGGATGAGCCATAACTGTACTTGGTC
CATTCTCCTTCCCTTCC
3
CBS
rs234706
ACGTTGGATGACGGGCTCTGCTCTCTTTC
ACGTTGGATGGGCAGGAACTTCTCAGAATC
gagaATCAGCGGTGGTGTC
1
CBS
rs5742905
ACGTTGGATGTCTGCGAGGATGGACCCTTC
ACGTTGGATGTTTTGCTGGCCTTGAGCCCT
CTGAAGCCGCGCCCTCTGCAGATCA
3
CTH
rs482843
ACGTTGGATGGCAATTGAAAAGCTCTTGAC
ACGTTGGATGAACATGGAGAATTGCTCCCC
CCCCTAAAATGTTTTCTCTCTGATAT
4
CYP2B6
rs8192719
ACGTTGGATGTAAGCTGGACCCACAATTTC
ACGTTGGATGGTATCTCTCGTTGTTTTTCTC
ccCTCGTTGTTTTTCTCAAGTTG
4
CYP2C19
rs4244285
ACGTTGGATGCACTTTCCATAAAAGCAAGG
ACGTTGGATGGCAATAATTTTCCCACTATC
CCCACTATCATTGATTATTTCCC
4
CYP2C9
rs1057910
ACGTTGGATGGGCAGGCTGGTGGGGAGAA
ACGTTGGATGACATGCCCTACACAGATGCT
ttTGGTGCACGAGGTCCAGAGATAC
5
GCLC
rs17883901
ACGTTGGATGAGGCGTGTGCAAGGGTGAT
ACGTTGGATGTTTGCGTAAAGCGAGGCCGA
gTCTCGCGAGCTGCTCCCCTCAACTG
3
GCLM
rs2301022
ACGTTGGATGGAAGCACCCTAAATAAAACAC
ACGTTGGATGGAGTCACACACCACAGTTTG
gggcAAACATTGTTCAAAGGACTA
5
GGT1
rs17004876
ACGTTGGATGATGCTGGGAGAGCTGAAGTC
ACGTTGGATGTCTACTTTGGCTCCAAGGTC
ggGCGGGATCCTGTTCAAT
1
GPX2
rs17881652
ACGTTGGATGTCTTCGCCTACCTGAAGGAC
ACGTTGGATGAATGATGAGCTTGGGATCGG
GGGAAAATGGGTCATCATAA
3
GPX3
rs3763013
ACGTTGGATGGTGGACAACTGAGATCAGAG
ACGTTGGATGCCAGAGACCTGAGATGCTAC
acattCTACCCCTGGATTGCTAC
5
GSTA1
rs1051775
ACGTTGGATGCCACCTGAGGAAAAAGATGC
ACGTTGGATGTTCAAAGGCAGGGAAGTAGC
AGTAGCGATTTTTTATTTTCTC
6
GSTA1
rs4715332
ACGTTGGATGGAGTGACGCAAAGAGGATAG
ACGTTGGATGACATTTAGGTGGGTATCCTG
ttTATCCTGTATTTTATCTGACAAATC
5
GSTA1
rs3957356
ACGTTGGATGGCTTTTCCCTAACTTGACTC
ACGTTGGATGATAAGCTCTTTGTTCCTCTC
CTTTGTTCCTCTCAATAGTTC
7
GSTA2
rs2180314
ACGTTGGATGGAAGGTATAGCAGATTTGGG
ACGTTGGATGGCAAGCTTGGCATCTTGTTC
gaATCTTGTTCCTCAGGTTGA
8
GSTP1
rs1138272
ACGTTGGATGTGATACATGGTGGTGTCTGG
ACGTTGGATGTCAAAAGGCTTCAGTTGCCC
ttccCATAGTCATCCTTGCCC
8
GSTP1
rs1695
ACGTTGGATGTGGTGCAGATGCTCACATAG
ACGTTGGATGTGGTGGACATGGTGAATGAC
ACCTCCGCTGCAAATAC
2
MGST1
rs2239676
ACGTTGGATGCCAAACCCCTCCTCTAAATC
ACGTTGGATGAGAAGGGCTCAAAGGGAATC
tttgtAATCAGCAGGCGATGGTTACT
5
MGST1
rs11875
ACGTTGGATGGTACAGAATTCTTAAAAAGCC
ACGTTGGATGGCTTACAGGTTGCTGAAAAG
CCTGTAAAGAAAATCATACAACTCA
5
MGST1
rs7970208
ACGTTGGATGATGGATCTATCTTTCATGGG
ACGTTGGATGGAGATATAGCACTAGGAGAG
gagACAGAAATCCTTAAACATTTCTC
4
MTHFR
rs1801131
ACGTTGGATGTCTCCCGAGAGGTAAAGAAC
ACGTTGGATGAGGAGCTGCTGAAGATGTGG
ggaGAGCTGACCAGTGAAG
6
MTR
rs1805087
ACGTTGGATGCTTTGAGGAAATCATGGAAG
ACGTTGGATGTACCACTTACCTTGAGAGAC
CCTTGAGAGACTCATAATGG
8
MTRR
rs1532268
ACGTTGGATGTGTAGCAGCTCTGACTTCAC
ACGTTGGATGACAAGAGGAGATAAGTGGCG
CATCACCTGCATCCT
1
MTRR
rs1801394
ACGTTGGATGCTATATGCTACACAGCAGGG
ACGTTGGATGGCAGAAAATCCATGTACCAC
CACAGCTTGCTCACA
1
NAT1
rs4986782
ACGTTGGATGCTGATCTCCTAGAAGACAGC
ACGTTGGATGAATCTTCAATTGTTCGAGGC
gTAAGAGTAAAGGAGTAGATTTTT
8
NAT2
rs1801280
ACGTTGGATGTCTGGGAGGAGCTTCCAGAC
ACGTTGGATGCATGGTTCACCTTCTCCTGC
TCCTGCAGGTGACCA
8
NAT2
rs1799930
ACGTTGGATGTCATAGACTCAAAATCTTC
ACGTTGGATGCCTGCCAAAGAAGAAACACC
tcatcTACTTATTTACGCTTGAACCTC
3
PON1
rs854560
ACGTTGGATGGAGCTAATGAAAGCCAGTCC
ACGTTGGATGTTTCTGGCAGAAACTGGCTC
cagcAACTGGCTCTGAAGAC
6
TCN2
rs1801198
ACGTTGGATGCCTCACTCTATCACCAGTTC
ACGTTGGATGGCCTTGAGACATGCTGTTCC
CCCAGTTCTGCCCCA
8
TCN2
rs4820889
ACGTTGGATGCATGACTCACCTTGCAACAG
ACGTTGGATGTACTTAACCTCCGTGATGGG
ccacGTTCTGGCAGCTTCTCC
1
TCNI
rs34324219
ACGTTGGATGAGGTCTTACCTGCCCTGATG
ACGTTGGATGTATACCTGAAGCAGAGACGC
GACGCAAGAAGAGTCTTTGTTAATAT
1
Supplementary Table 3. Multivariate Cox analysis of TRM and OS in busulfan and comparator cohorts.
GENE
LOCUS
busulfan color
Comparator color
(n=185)
(n=146)
TRM
OS
TRM
OS
p
p
p
p
ABCB1
rs1045642
ns
ns
ns
ns
ABCC1
rs45511401
Ns
ns
ns
ns
ABCC 4
rs2274407
ns
ns
ns
ns
ABCC 4
rs11568658
ns
ns
ns
0.018
ABCC 4
rs9561778
ns
ns
ns
0.013
CBS
rs5742905
ns
ns
ns
ns
CBS
rs234706
ns
ns
ns
ns
CBS
ins 68bp
ns
ns
ns
ns
CTH
rs482843
ns
ns
ns
ns
CYP2B6
rs8192719
ns
ns
ns
ns
CYP2C9
rs1057910
ns
ns
ns
ns
CYP2C19
rs4244285
0.05
ns
ns
ns
GCLM
rs2301022
ns
ns
ns
ns
GCLC
rs17883901
ns
ns
ns
ns
GGT 1
rs17004876
ns
ns
0.014
ns
GPX 1
rs1050450
ns
ns
ns
ns
GPX2
rs17881652
ns
ns
ns
0.014
GPX3
rs3763013
ns
ns
ns
ns
GSTA1
rs1051775
0.04
ns
ns
ns
GSTA1
rs4715332
0.05
ns
ns
ns
GSTA1
rs3957356
0.05
ns
ns
ns
GSTA 2
rs2180314
0.001
0.005
ns
ns
GSTM 1
rs1065410
ns
0.04
ns
ns
GSTM 1
rs366631
ns
ns
ns
ns
GSTP 1
rs1138272
ns
ns
ns
ns
GSTP 1
rs1695
ns
ns
ns
0.014
GSTT 1
rs2266637
ns
0.02
ns
ns
mGST1
rs7970208
0.01
0.05
ns
ns
mGST1
rs2239676
ns
ns
ns
ns
MGST1
rs11875
ns
ns
ns
ns
MTHFR
rs1801131
ns
ns
ns
ns
MTR
rs1805087
ns
ns
ns
ns
MTRR
rs1532268
ns
ns
ns
ns
MTRR
rs1801394
ns
ns
ns
ns
NAT1
rs4986782
ns
ns
ns
ns
NAT2
rs1801280
ns
ns
ns
ns
NAT2
rs1799930
ns
ns
ns
ns
PON1
rs854560
ns
ns
ns
ns
TCN I
rs34324219
ns
0.04
ns
ns
TCN2
rs1801198
ns
ns
ns
ns
TCN2
rs4820889
ns
ns
ns
ns
Footnote: all p-values are referred to multivariate Cox analysis: for each polymorphism as covariate age, sex mismatch, disease phase, type of donor, interval
to transplant and intensity of conditioning were included with covariates. Adjustment for multiple comparisons was made, leading to significant p-value
threshold = 0.00125.
Supplementary Table 4. GSTA2 S112T-GSTA1 two loci haplotypes frequency estimates and linkage disequilibrium coefficients (D’).
GSTA2-GSTA1
S112T-rs1051775
S112T-rs3957356
S112T-rs4715332
Haplotype
Estimated frequency
Thr-A
0.478
Thr-G
0.072
Ser-A
0.092
Ser-G
0.358
Thr-G
0.485
Thr-A
0.067
Ser-G
0.093
Ser-A
0.355
Thr-T
0.475
Thr-G
0.076
Ser-T
0.077
Ser-G
0.372
Linkage
Disequilibrium
D’
p
0.70
<0.0001
0.72
<0.0001
0.69
<0.0001
Supplementary Table 5. GSTA2 S112T-GSTA1*A/B haplotype frequency estimation.
Haplotype
Estimated frequency
Thr-GSTA1*A
0.485
Thr-GSTA1*B
0.062
Ser-GSTA1*A
0.057
Ser-GSTA1*B
0.354
Others
0.042
Footnote: GSTA1*A haplotype is set up by the G allele at rs3957356 locus and the T allele at rs4715332 locus; GSTA1*B haplotype is set up by the A allele
at rs3957356 locus and the G allele at rs4715332 locus, as previously reported26.
Supplementary Table 6 Multivariate Cox analysis of TRM and OS according to two loci GSTA2-GSTA1 haplotypes.
TRM
OS
GSTA2-GSTA1
Haplotype
RR
[95% CI]
p
RR
[95% CI]
p
S112T-rs1051775
Thr-A
1
/
/
1
/
/
Thr-G
2.271
[0.635-8.116]
0.206
1.529
[0.724-3.232]
0.265
Ser-A
3.076
[1.305-7.248]
0.010
2.320
[1.312-4.104]
0.003
Ser-G
2.187
[1.163-4.113]
0.015
1.630
[1.129-2.354]
0.009
Thr-G
1
/
/
1
/
/
Thr-A
1.936
[0.563-6.654]
0.297
1.454
[0.693-3.053]
0.321
Ser-G
3.083
[1.387-6.851]
0.006
2.502
[1.268-3.787]
0.006
Ser-A
1.884
[1.016-3.492]
0.044
1.569
[1.063-2.221]
0.022
Thr-T
1
/
/
1
/
/
Thr-G
2.271
[0.635-8.117]
0.206
1.525
[0.722-3.220]
0.268
Ser-T
3.076
[1.305-7.248]
0.010
2.433
[1.403 -4.217]
0.001
Ser-G
2.187
[1.163-4.113]
0.015
1.529
[1.058 -2.211]
0.023
S112T-rs3957356
S112T-rs4715332
Supplementary Table 7. Multivariate Cox analysis of Relapse in Busulfan cohort.
Variables
RR
[95% CI]
p
Age
0.986
0.959-1.013
0.312
Interval diagnosis- transplant
0.989
0.972-1.007
0.233
Sex donor/recipient (female/male vs others)
0.939
0.472-1.867
0.857
Type of donor (unrelated vs sibling)
0.708
0.381-1.317
0.276
Conditioning regimen (myeloablative vs reduced-intensity)
3.315
0.436-25.219
0.247
Phase at transplant (advanced vs early*)
3.3976
2.111-7.491
0.0001
GSTA2 S112T (ser/ser vs thr+)
1.332
0.672-2.641
0.411
Supplementary Table 8. Multivariate analysis of GSTA2 S112T locus on liver function tests.
Liver function tests
Anova
for repeated measures
F-statistics
p
Aspartate transaminase (AST)
0.002
0.957
Alanine transaminase (ALT)
0.095
0.759
Alkaline phosphatase (ALP)
1.050
0.309
Gamma-glutamyl transferase (GGT)
0.118
0.732
Cholinesterase (AChE)
0.285
0.598
Footnote: ANOVA for repeated measures, adjusted for age, phase at transplant, intensity of conditioning, interval between diagnosis and transplant and sexmismatch. Time points for plasma liver function tests were: baseline (day of admittance at BMT Unit), day 0, +7, +14, +21, +28 and +35 after transplant.