Association of donor TNFRSF6 (FAS) gene polymorphism with acute rejection in renal transplant patients: a case-control study
Sandrine Cappellesso1,2,6,,
Jean-François Valentin1,3,6,,
Bruno Giraudeau4,6,
Marie-Denise Boulanger5,6,
Azmi Al Najjar3,6,
Mathias Büchler1,3,6,
Jean-Michel Halimi1,3,4,6,
Hubert Nivet3,6,
Pierre Bardos1,2,6,
Yvon Lebranchu1,3,6 and
Hervé Watier1,2,6
1UPRES-EA 3249 Cellules Hématopoïétiques, Hémostase et Greffe, Université de Tours, Faculté de Médecine, Tours cedex, 2Laboratoire dImmunologie and 3Service de Néphrologie et Immunologie Clinique, CHRU de Tours, Tours cedex, 4Centre de Recherche Clinique, CHU et Faculté de Médecine de Tours, Tours cedex, 5EFS Centre-Atlantique, Tours cedex and 6Institut Fédératif de Recherche (IFR) 120, Faculté de Médecine de Tours, Tours cedex, France
Correspondence and offprint requests to: Prof. Y. Lebranchu, UPRES-EA 3249 Cellules Hématopoïétiques, Hémostase et Greffe, Faculté de Médecine, 2 bis boulevard Tonnellé, F-37032 Tours cedex, France. Email: lebranchu{at}med.univ-tours.fr
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Abstract
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Background. Genetic factors other than HLA have been reported to be associated with the outcome of organ transplantations. Because binding of FasL to its receptor Fas could play an important role in tubulitis and in the death of graft tubular epithelial cells during kidney allograft rejection, a gene polymorphism recently identified in position 671 in the promoter of the TNFRSF6 gene coding for Fas was investigated in donors.
Methods. A case-control study was performed within a cohort of non-hyperimmunized adult patients who had received cadaveric kidney transplants based on the occurrence or absence of acute cellular rejection in the first 6 months after renal transplantation. Each recipient from the acute rejection group (n = 35) was matched for age (± 5 years) and number of HLA-DR mismatches with two recipients within the non-acute rejection group (n = 70).
Results. The TNFRSF6-GG genotype was more frequent in donors in the group without rejection episodes. In contrast, patients who received a kidney from a TNFRSF6-A carrier were more likely to experience acute rejection episodes (relative risk nearly 2.1).
Conclusion. This study suggests that donor TNFRSF6 polymorphism directly or indirectly influences acute kidney rejection episodes.
Keywords: acute rejection; case-control study; kidney; polymorphism; TNFRSF6; transplantation
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Introduction
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Allograft rejection primarily results from human leukocyte antigen (HLA) polymorphism between donor and recipient and subsequent T cell recognition, either via direct recognition of allogeneic HLA on donor antigen-presenting cells (APC) or via indirect recognition of donor HLA-derived peptides presented by major histocompatibility complex class II antigens on recipient APCs. However, genetic factors other than HLA have been reported to be associated with graft rejection. Allograft rejection is indeed a complex multistage process involving not only T cell activation and proliferation but also multiple inflammatory and immune components such as cytokines and cytokine receptors, chemokines and chemokine receptors, and costimulatory and adhesion molecules that might themselves be encoded by polymorphic genes. Allelic polymorphisms in recipient genes coding for these molecules have been reported to be associated with variations in the outcome of kidney, heart, liver and lung transplantations [1,2]. These findings suggest that interindividual variations in the production of cytokines such as TNF-
and others members of the TNF super family have an important role in accelerating or limiting rejection processes. In view of the interactions between graft cells and graft-infiltrating leukocytes, it is likely that susceptibility to rejection is also influenced by allelic polymorphisms in genes coding for cytokines or cytokine receptors not only in the recipient but also in the donor.
FasL (TNFSF6, CD95L) and its death receptor Fas are involved in kidney damage [3]. Fas (CD95, TNFRSF6 and APO-1) is constitutively expressed on tubular cells and may be up-regulated in acute rejection or after injury such as cold ischaemia [4,5]. FasL is expressed by graft-infiltrating recipient T cells and it has been reported to be a specific graft rejection marker [6]. Therefore, binding FasL to its death receptor Fas could play an important role in the death of graft tubular epithelial cells and tubulitis, a major component of acute rejection. Two single nucleotide polymorphisms located in the promoter region of the TNFRSF6 (FAS) gene have been reported by Huang et al. [7]. One of these, a G to A nucleotide substitution at position 671, is located in a putative nuclear transcription element GAS binding site and could influence the expression of the TNFRSF6 gene [7]. We demonstrated in a previous study that there was no association between the recipient TNFRSF6 671 genotype and the occurrence of acute cellular rejection (AR) in renal transplant patients [8]. However, FAS genotype distribution in patients with rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis and primary Sjögren's syndrome [9,10] differs from normal controls. These findings suggest that the 671 TNFRSF6 gene polymorphism itself or polymorphisms in close linkage disequilibrium are of pathophysiological importance.
We have therefore tested the hypothesis of an association between donor TNFRSF6 671 genotype and the occurrence of acute cellular rejection episodes in non-hyperimmunized patients. A case-control study was performed by matching cadaveric kidney recipients on well-known predictable factors of allograft rejection, i.e. recipient age and number of HLA-DR mismatches. TNFRSF6 671 genotype was then determined in the donors corresponding to each recipient.
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Subjects and methods
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Study population and clinical data
222 patients underwent renal transplantation with a cadaveric kidney in the University Hospital of Tours between 1994 and 1998. Preoperative and postoperative clinical data for each transplanted patient were retrospectively collected. The DNA from each corresponding donor was available in the Blood Bank histocompatibility laboratory. 185 anonymous healthy blood donors from the Blood Bank were also studied to determine the frequency of TNFRSF6 alleles in the general population.
Selection of cases and controls
Of the 222 patients, a sub-cohort of 183 adults was first selected on the basis of a similar quadruple sequential immunosuppressive regimen. This sub-cohort was studied previously for an association between the recipient TNFRSF6 genotype and the occurrence of acute rejection [8]. Immunized patients defined as having >25% reactive anti-HLA antibodies were secondarily excluded from this cohort in order to avoid cases of vascular rejection. Of the resulting 162 recipients, the DNA for the corresponding donors was available in only 151 cases. All patients who had had at least one episode of AR within the first 6 months after transplantation were then identified and constituted a group of 35 patients (AR group). Only patients with AR within the first 6 months were selected because the rare AR episodes after 6 months are often due to non-immunological circumstances such as non-compliance to the immunosuppressive treatment. Because the incidence of acute rejection episodes is linked to the degree of HLA incompatibility, and particularly to the number of HLA-DR mismatches, each of the 35 patients from the AR group was matched for number of HLA-DR mismatches and for age (±5 years) with two patients from the non-acute rejection group (non-AR).
Immunosuppressive treatment
All patients received a quadruple sequential immunosuppressive regimen with induction therapy by antithymocyte globulin (ATG, Thymoglobulin®). ATG was monitored by CD3+ T cell counts, and was adapted to maintain CD3+ T cells below 20/mm3. Cyclosporin A (CsA; Neoral®) or tacrolimus (Prograf®) were introduced when serum creatinine levels were below 250 µM/l. Prednisolone (1 mg/kg/day) and anti-proliferative agents, mycophenolate mofetil (MMF; CellCept®) (2 g/day) or azathioprine (Imurel®) (12 mg/kg/day) were started on the day of transplantation. Prednisolone was progressively tapered and then withdrawn between months 6 and 9. Daily doses of CsA or tacrolimus were adjusted according to morning trough blood levels.
Clinical data
Acute cellular rejection was established in all cases by core biopsy according to the Banff criteria and treated with high dose pulse of corticosteroids for 5 days. Delayed graft function was defined as the need for at least one dialysis session during the first 7 postoperative days. Creatinine clearance was calculated according to the Gault Cockcroft formula.
DNA extraction
DNA extraction was performed from cadaveric donor spleens using the simple salting out procedure as described previously [11]. Genomic DNA from the general population was prepared from blood cells using a standard column-extraction technique (QIAamp Blood Midi Kit, Qiagen, Courtaboeuf, France).
Analysis of TNFRSF6 promoter region polymorphism
The BstNI restriction fragment length polymorphism was studied by polymerase chain reaction (PCR) amplification as described previously [7]. Briefly, PCR reactions were carried out in a final volume of 50 µl. Reaction mixtures consisted of Taq buffer (10 mM TrisHCl pH 9.0, 50 mM KCl, 0.1% Triton X-100), 1.5 mM MgCl2, 200 µM dNTP, 1 µM of each reverse (5'-GGCTGTCCATGTTGTGGCTGC-3') and forward primers (5'-CTACCTAAGAGCTATCTACCG TTC-3') specific for Fas, and 0.5 U of Taq DNA polymerase (Promega, Charbonnières, France). DNA was amplified by 35 cycles of denaturation at 94°C, annealing at 58°C and extension at 72°C, each for 30 s. Following PCR, 10 µl of reaction products were digested by 10 U of BstNI (New England Biolabs, Hertfordshire, UK), resulting in one fragment of 233 bp for the TNFRSF6-A allele and two fragments of 188 and 45 bp for the TNFRSF6-G allele identified on 8% non-denaturing polyacrylamide gel electrophoresis. For both alleles, digestion also produced a 99 bp fragment that was used as an inner digestion control.
Statistical analysis
Demographic and clinical data are expressed as means ± SD or percentage, and comparisons between groups were performed by means of univariate conditional logistic regressions. The association between donor TNFRSF6 gene polymorphism and acute rejection was tested in the framework of a conditional logistic regression [12] and the result was expressed as a point estimate of the odds ratio with its associated 95% confidence interval. As for the association between TNFRSF6 gene allele and acute rejection, this association was tested by means of a re-sampling scheme (bootstrap) [13], taking into account the correlation between the two alleles of a subject. Analyses were performed using the SAS software (SAS institute Inc, Cary, NC).
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Results
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Baseline characteristics
Baseline characteristics of the final AR and non-AR groups were compared (Table 1). No differences were observed in terms of age and sex in the donors (Table 1). Moreover, no statistically significant differences were found between the two groups of recipients in terms of sex distribution, percentage of first kidney transplantation and cause of end-stage renal disease (Table 1). Concerning the recipients clinical data displayed in Table 2, there were no significant differences between the AR group and the non-AR group for total number of HLA-mismatches, ischaemia times, serum creatinine levels and creatinine clearance at 7 days post-transplantation, or CMV disease (Table 2). The A and B loci mismatch for the rejection group was 2.75 ± 0.8 (mean ± SD), and for the control group 2.81 ± 0.9. The difference was not statistically different (P = ns). Delayed graft function was more frequent in the non-AR group (21.4 vs 14.28%) but the difference was not statistically significant (Table 2). There was also no significant difference between the two groups in terms of immunosuppressive treatment except for the distribution of azathioprine and MMF (Table 2). More patients received MMF in the non-AR group [P = 0.029; OR (CI 95%) = 3.66 (1.14; 11.71)].
Allele frequencies
Of the 105 donors studied in both groups, 33 proved to be AA (31.4%), 47 AG (44.8%) and 25 GG (23.8%). TNFRSF6 genotype distribution in the three genotype groups was very close to that found in 185 blood donors (genotype distribution: AA = 33.5%, AG = 47.0%, GG = 19.5%). Allele frequencies in both cases were in HardyWeinberg equilibrium (data not shown).
Acute rejection episode and TNFRSF6 polymorphism
When the 671 TNFRSF6 allele frequencies in the donors of AR and non-AR groups were compared (Table 3), the frequency of the G allele appeared higher in the donors of the non-AR group than in the donors of the AR group, reaching a frequency similar to that of the A allele in the latter group (Table 3). However, the difference did not reach statistical significance. When the frequency of acute rejection according to the 671 TNFRSF6 genotype distribution in donors was studied (Tables 4 and 5), a lesser risk of acute rejection was evidenced in donors with GG genotype with an odds ratio of 3.27 [95% CI (1.04; 10.32)]. The difference did not appear statistically significant when we compared the three groups together (Table 4) but was significant when GG genotype was compared with AA and AG donors together, referred to as A carriers (16 vs 38.8% in the GG and A carrier groups respectively; Table 5). The GG genotype was three times more frequent in the donors of the non-AR group than in the donors of the AR group, and represented nearly one-third of donors of this group (Table 5). In contrast, the occurrence of AR was similar in the G carrier group (i.e. GG and AG) and AA donors (data not shown). As the distribution of patients receiving MMF was statistically more frequent in the non-AR group, odds ratio were adjusted for this treatment. The result revealed that the risk of AR was still nearly 2.1 times less frequent in the GG donors without reaching significance because of the weakness of the sample size [P = 0.227; OR (CI 95%) = 2.11 (0.63; 7.07)].
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Discussion
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The findings of this case-control study suggest an association between donor TNFRSF6 671 polymorphism and the occurrence of acute renal allograft rejection during the initial 6-month post-transplantation period. Indeed, the frequency of the TNFRSF6 671*GG genotype was significantly higher in the donors of patients who did not experience acute rejection. Because of the study design, this result was independent of recipient age and HLA-DR mismatches which are known risk factors for rejection. In addition, the two groups were comparable in terms of the total number of HLA mismatches, cold ischaemia, delayed graft function and occurrence of CMV infection, which are reported to be risk factors for AR [14,15]. However, the distribution of MMF vs azathioprine was different between the two groups. When this factor was taken into account in this small sample, the adjusted relative risk of acute rejection remained nearly 2.1 in GG vs A carriers but lost statistical significance because of the small number of patients treated with MMF. Nevertheless, genotype does not depend on treatment, and this increased relative risk indicates that patients receiving a TNFRSF6 671*A carrier kidney might more likely experience AR episodes. However, multicentric studies including higher numbers of patients will be required to confirm this interesting genetic association.
The A to G substitution at position 671 is located in the enhancer region of the TNFRSF6 gene in a putative GAS binding site consensus sequence [16]. GAS sequences are binding elements for STATs (signal transducer and activator of transcription), a family of cytokine-responsive transcription factors. In contrast to what was initially described [7], only the G allele carries to the Stat 5 binding motif [17]. Whether the A to G transition is of importance for binding of Stat 5 or other Stats to GAS, and thus influences the regulation and expression of the TNFRSF6 gene in response to various cytokines, is not known. Interestingly, IFN-
-induced Fas and FasL up-regulation in the human adenocarcinoma cell line requires the expression and activation of Stat-1 protein [18]. Moreover, a recent study reported that exposure of cardiac myocytes to ischaemia-reperfusion results in the induction of Fas and FasL expression via a Stat-1-dependent pathway [19]. Recently, it was demonstrated that the A allele in the Fas promoter had a higher ability to bind STAT 1 than the G allele, without significant evidence that it could influence Fas expression [20]. Additional studies are needed to substantiate the functional consequences of the TNFRSF6 671 polymorphism, and to support the hypothesis that the association observed with AR is attributable to TNFRSF6 671 polymorphism and not to other polymorphism(s) in linkage disequilibrium.
This study has identified a new genetic factor possibly associated with AR that belongs to the cytokine/cytokine receptor system and could be added to other known genetic risk factors such as TNF-
and IL-10 [1]. Associations have been found with the recipient genotype and these factors [21], whereas acute rejection in our study was associated with donor alleles. These findings are consistent with the common view that most cytokines are expressed by recipient infiltrating leukocytes (genetic factors in the recipient) whereas the cytokine receptors are located in the graft tissue (genetic factors in the donor). It would therefore be interesting to analyse recipient FasL (TNFSF6) polymorphism [10] and/or donor TNFRI polymorphism [22] to further understand the relationships between pro-apoptotic mechanisms and AR. We can hypothesize that a comprehensive analysis of these polymorphisms in both, the donor and the recipient could predict the risk of AR and influence therapeutic strategies.
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Acknowledgments
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S. Cappellesso was supported by a grant from the Fondation Mérieux and from Ouest Transplant. This study was supported by MENRT and the Fondation Langlois.
Conflict of interest statement. None declared.
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Notes
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S. Cappellesso and J. F. Valentin contributed equally to the work presented in this report. 
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Received for publication: 10. 2.03
Accepted in revised form: 17. 9.03