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Impact of a Polymorphism in the IL-12p40 Gene on the Outcome of Kidney Transplantation
Impact of a Polymorphism in the IL-12p40 Gene on the Outcome of Kidney Transplantation T.W. Hoffmann, J.-M. Halimi, M. Büchler, F. Velge-Roussel, A. Al-Najjar, J.-F. Marliere, Y. Lebranchu, and C. Baron ABSTRACT A number of factors interfere with the outcome of renal transplantation. Revealing genetic factors that impact on graft outcome may have consequences for clinical practice. Interleukin-12 (IL-12), by stimulating interferon gamma (IFN␥) production, plays a crucial role in immune responses against both graft and viral agents. An A-to-C single nucleotide polymorphism (SNP) within the 3=-untranslated region (3=UTR) of the IL-12p40 gene has been reported to be both functionally and clinically relevant. Since the impact of this SNP on kidney graft outcome has never been reported, we investigated the impact of the 3=UTR polymorphism on clinical events after transplantation among 253 kidney recipients transplanted between 1995 and 2003. The polymorphism was genotyped using the restriction fragment length polymorphism method. Our results showed that the 3=UTR polymorphism affected neither graft survival (P ⫽ .768) nor the occurrence of delayed graft function (DGF; P ⫽ .498). C allele carriers in our study displayed more acute rejections in the first year than patients with the A/A genotype, but it did not reach statistical significance (P ⫽ .108). In contrast, the C allele appeared to be a significant risk factor for cytomegalovirus infection (odds ratio ⫽ 1.77; P ⫽ .027). In conclusion, IL12B 3=UTR polymorphism did not affect graft survival, DGF, or acute rejection episodes, but had an impact on the occurrence of cytomegalovirus infection.
A
FTER TRANSPLANTATION, a number of donorderived proteins are recognized as foreign antigens by the recipient immune system, resulting in acute rejection whereby grafted cells are rapidly and specifically killed by recipient immunity.1 Immunosuppressive drugs are used to prevent this reaction, but such treatments are generally associated with increased risks for infection, neoplasia, and toxic effects. Some events such as acute rejection, ischemiareperfusion injuries, cytomegalovirus (CMV) infection, and chronic nephropathy alter graft survival. The immune response established in the graft is complex, representing an important factor in these transplant injuries. The T-cellmediated immune response is regulated through an intricate network of molecular signals, such as interleukin-12 (IL-12), which stimulates interferon gamma (IFN␥) production in T and natural killer (NK) cells, acting as a major actor in the balance between Th1-type and Th2-type immune responses.2 IL-12 (IL-12p70) is composed of 2 subunits (p35 and p40) encoded by IL12A and IL12B genes located in 2 different chromosomes. A single nucleotide polymorphism (SNP) has recently been described in the 3=-untranslated region (3=UTR) 0041-1345/09/$–see front matter doi:10.1016/j.transproceed.2008.12.002 654
of IL12B (rs3212227). This A-to-C substitution has been shown to reduce the stability of the p40 mRNA in transfected cell lines,3 and to decrease the production of the IL-12p40 protein in stimulated peripheral blood mononuclear cells (PBMC).4 In addition, this SNP appears to be clinically relevant, since it has been found to be associated with multiple sclerosis5 and psoriasis vulgaris.6 However, the impact of this SNP on clinical events after organ transplantation has never been reported. Our study among a population of kidney graft recipients sought to examine whether this SNP in the IL-12p40
From the Université François Rabelais de Tours, EA4245 Cellules Dendritiques, Immunomodulation et Greffes (T.W.H., J.-M.H., M.B., F.V.-R., Y.L., C.B.), Tours, France and CHRU de Tours, Service de Néphrologie et Immunologie Clinique (J.-M.H., M.B., A.A.-N., J.-F.M., Y.L., C.B.), Tours, France. This work was supported by a grant from the Société Francophone de Transplantation. Address reprint requests to Dr Christophe Baron, Service de Néphrologie et Immunologie Clinique, CHRU de Tours, 37044 Tours Cedex 9, France. E-mail: [email protected] © 2009 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 41, 654 – 656 (2009)
IMPACT OF IL12B 3=UTR POLYMORPHISM
655
gene had an impact on clinical events after kidney transplantation.
RESULTS
Table 1 shows graft survival and the occurrence of DGF, acute rejection episodes, and CMV infections according to the presence or absence of the 3=UTR C allele. Mean graft survival was estimated to be 99 ⫾ 4 months in C carriers and 102 ⫾ 4 months in the A/A patients, indicating that the 3=UTR polymorphism did not affect graft survival (P ⫽ .768). Similar results were obtained concerning DGF (18% and 21%, respectively; P ⫽ .498). C allele carriers had more acute rejections in the first year than patients with the A/A genotype (35% and 26%, respectively), but it did not reach statistical significance (P ⫽ .108). In contrast, CMV infection was significantly more frequent among C carriers than the other group (50% and 36%, respectively; odds ratio [OR] ⫽ 1.77 [95% confidence interval 1.07–2.92]; P ⫽ .027).
PATIENTS AND METHODS Genotyping was performed on DNA samples from 253 Caucasian patients who had received a deceased-donor kidney transplantation between 1995 and 2003. Blood samples for DNA analysis from these patients were taken after obtaining signed informed consent. The patients were divided into 2 groups according to the presence versus absence of the A-to-C substitution. Most patients received induction with either antithymocyte globulin or anti-IL2R and immunosuppressive treatment based on a calcineurin inhibitor, azathioprine/mycophenolate mofetil, and steroids. CMV seronegative patients who had received an organ from a seropositive donor received CMV prophylaxis with either acyclovir, valacyclovir or gancyclovir. The 2 groups were not different concerning major baseline characteristics such as recipient age, HLA matching, graft rank, anti-HLA immunization, induction and immunosuppressive treatment, CMV prophylaxis, and CMV serological status before the graft. Clinical data for each patient were extracted from the renal transplant database. We studied delayed graft function (DGF), acute rejection episodes in the first year, CMV infection, and graft survival. DGF was defined by dialysis in the first postoperative week; rejection episodes, by clinical diagnosis, namely, an elevation in serum creatinine levels by ⬎20% compared with baseline, with a confirmation by biopsy according to Banff classification. CMV infection was defined as positive pp65 antigenemia (ⱖ2 positive circulating leukocytes). Graft survival was calculated using KaplanMeier life-table analysis. The SNP was genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Primers were designed to amplify a 421-bp region containing the 3=UTR polymorphism, with 5=-GGCATGAAATCCCTGAAACC-3= as forward primer and 5=-TACATCCTGGCAGACAAACG-3= as reverse primer. The obtained fragment was digested with 5 units of TaqI (Invitrogen, Cergy-Pontoise, France) for 1h40 at 65°C; the digestion product was loaded on a 2% agarose gel to obtain the genotype as deduced from the digestion profile. We used a 607-bp region of the GAPDH gene containing a TaqI restriction site as an internal control to assess the efficiency of the TaqI digestion. Chi-square test was used to compare the occurrence of DGF, acute rejection episodes, and CMV infections between IL12B 3=UTR genotypes (presence or absence of the C allele), whereas log-rank test was used to compare graft survivals. P ⬍ .05 was considered significant.
DISCUSSION
We analyzed the relationship between the IL12B 3=UTR SNP and graft outcome. No significant association was revealed between this polymorphism and DGF, acute rejection in the first year, or graft survival, indicating that this polymorphism may not play a major role in these parameters. Concerning acute rejection, the P value was near significance (.108). The impact of the SNP on acute rejection might demand a larger cohort to be revealed. Our findings revealed that the C allele of this SNP was associated with an increased risk for CMV infection after renal transplantation. Acute rejection was not a risk factor for CMV infection in our study (OR ⫽ 1.27; P ⫽ .387). In order to carry out a multivariate analysis, we plan to study a larger patient cohort. The results of the present study addressed the question of the pathogenic link between the IL-12p40 gene polymorphism and CMV infection. This SNP has been shown to be functionally relevant since the C allele is associated with lower mRNA stability3 and less production of the p40 polypeptide.4 We suggest that this lower p40 expression in C carriers might explain the pathogenic link between the IL12B genotype and CMV replication. Indeed, direct activating roles of the p40/p40 homodimer have recently been reported, especially as an IFN␥ stimulator in CD8⫹ T cells and T-cell response initiator.7 As both CD8⫹ T cells and IFN␥ have been shown to be major mediators of host
Table 1. Impact of IL12B 3=UTR Genotype on Graft Outcome in 253 Kidney Graft Recipients 3=UTR Genotype Clinical Events
A/A (n ⫽ 141)
A/C and C/C (n ⫽ 112 [100 ⫹ 12])
Graft survival (months)* Delayed graft function‡ Acute rejection in the first year‡ Positive CMV pp65 antigenemia‡
102 ⫾ 4 30 (21%) 36 (26%) 51 (36%)
99 ⫾ 4 20 (18%) 39 (35%) 56 (50%)
Odds Ratio (95% Confidence Interval) †
NA 0.80 (0.43–1.50) 1.56 (0.91–2.67) 1.77 (1.07–2.92)
P
.768 .498 .108 .027
*Graft survivals were expressed as mean values ⫾ standard deviations by Kaplan-Meier life-table analysis and compared between 3=UTR genotypes by log-rank test. † Not applicable. ‡ The occurrence of delayed graft function, acute rejection episodes, and CMV infections was compared between 3=UTR genotypes by chi-square test.
656
defenses against CMV,8,9 these experimental findings suggest that the level of p40 expression may play an important role during CMV infection. Finally, since the strategy to manage the risk for CMV infection in seropositive recipients remains a matter of debate, the description of a genetic risk factor might improve this management. Our results suggest that 3=UTR C allele carriers might greatly benefit from CMV prophylaxis. REFERENCES 1. Benichou G: Direct and indirect antigen recognition: the pathways to allograft immune rejection. Front Biosci 4:D476, 1999 2. Trinchieri G: Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat Rev Immunol 3:133, 2003 3. Morahan G, Huang D, Ymer SI, et al: Linkage disequilibrium of a type 1 diabetes susceptibility locus with a regulatory IL12B allele. Nat Genet 27:218, 2001
HOFFMANN, HALIMI, BU¨CHLER ET AL 4. Stanilova S, Miteva L: Taq-I polymorphism in 3=UTR of the IL-12B and association with IL-12p40 production from human PBMC. Genes Immun 6:364, 2005 5. van Veen T, Crusius JB, Schrijver HM, et al: Interleukin12p40 genotype plays a role in the susceptibility to multiple sclerosis. Ann Neurol 50:275, 2001 6. Cargill M, Schrodi SJ, Chang M, et al: A large-scale genetic association study confirms IL12B and leads to the identification of IL23R as psoriasis-risk genes. Am J Hum Genet 80:273, 2007 7. Cooper AM, Khader SA: IL-12p40: an inherently agonistic cytokine. Trends Immunol 28:33, 2007 8. Presti RM, Pollock JL, Dal Canto AJ, et al: Interferon gamma regulates acute and latent murine cytomegalovirus infection and chronic disease of the great vessels. J Exp Med 188:577, 1998 9. Reddehase MJ, Mutter W, Munch K, et al: CD8-positive T lymphocytes specific for murine cytomegalovirus immediate-early antigens mediate protective immunity. J Virol 61:3102, 1987
A
FTER TRANSPLANTATION, a number of donorderived proteins are recognized as foreign antigens by the recipient immune system, resulting in acute rejection whereby grafted cells are rapidly and specifically killed by recipient immunity.1 Immunosuppressive drugs are used to prevent this reaction, but such treatments are generally associated with increased risks for infection, neoplasia, and toxic effects. Some events such as acute rejection, ischemiareperfusion injuries, cytomegalovirus (CMV) infection, and chronic nephropathy alter graft survival. The immune response established in the graft is complex, representing an important factor in these transplant injuries. The T-cellmediated immune response is regulated through an intricate network of molecular signals, such as interleukin-12 (IL-12), which stimulates interferon gamma (IFN␥) production in T and natural killer (NK) cells, acting as a major actor in the balance between Th1-type and Th2-type immune responses.2 IL-12 (IL-12p70) is composed of 2 subunits (p35 and p40) encoded by IL12A and IL12B genes located in 2 different chromosomes. A single nucleotide polymorphism (SNP) has recently been described in the 3=-untranslated region (3=UTR) 0041-1345/09/$–see front matter doi:10.1016/j.transproceed.2008.12.002 654
of IL12B (rs3212227). This A-to-C substitution has been shown to reduce the stability of the p40 mRNA in transfected cell lines,3 and to decrease the production of the IL-12p40 protein in stimulated peripheral blood mononuclear cells (PBMC).4 In addition, this SNP appears to be clinically relevant, since it has been found to be associated with multiple sclerosis5 and psoriasis vulgaris.6 However, the impact of this SNP on clinical events after organ transplantation has never been reported. Our study among a population of kidney graft recipients sought to examine whether this SNP in the IL-12p40
From the Université François Rabelais de Tours, EA4245 Cellules Dendritiques, Immunomodulation et Greffes (T.W.H., J.-M.H., M.B., F.V.-R., Y.L., C.B.), Tours, France and CHRU de Tours, Service de Néphrologie et Immunologie Clinique (J.-M.H., M.B., A.A.-N., J.-F.M., Y.L., C.B.), Tours, France. This work was supported by a grant from the Société Francophone de Transplantation. Address reprint requests to Dr Christophe Baron, Service de Néphrologie et Immunologie Clinique, CHRU de Tours, 37044 Tours Cedex 9, France. E-mail: [email protected] © 2009 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 41, 654 – 656 (2009)
IMPACT OF IL12B 3=UTR POLYMORPHISM
655
gene had an impact on clinical events after kidney transplantation.
RESULTS
Table 1 shows graft survival and the occurrence of DGF, acute rejection episodes, and CMV infections according to the presence or absence of the 3=UTR C allele. Mean graft survival was estimated to be 99 ⫾ 4 months in C carriers and 102 ⫾ 4 months in the A/A patients, indicating that the 3=UTR polymorphism did not affect graft survival (P ⫽ .768). Similar results were obtained concerning DGF (18% and 21%, respectively; P ⫽ .498). C allele carriers had more acute rejections in the first year than patients with the A/A genotype (35% and 26%, respectively), but it did not reach statistical significance (P ⫽ .108). In contrast, CMV infection was significantly more frequent among C carriers than the other group (50% and 36%, respectively; odds ratio [OR] ⫽ 1.77 [95% confidence interval 1.07–2.92]; P ⫽ .027).
PATIENTS AND METHODS Genotyping was performed on DNA samples from 253 Caucasian patients who had received a deceased-donor kidney transplantation between 1995 and 2003. Blood samples for DNA analysis from these patients were taken after obtaining signed informed consent. The patients were divided into 2 groups according to the presence versus absence of the A-to-C substitution. Most patients received induction with either antithymocyte globulin or anti-IL2R and immunosuppressive treatment based on a calcineurin inhibitor, azathioprine/mycophenolate mofetil, and steroids. CMV seronegative patients who had received an organ from a seropositive donor received CMV prophylaxis with either acyclovir, valacyclovir or gancyclovir. The 2 groups were not different concerning major baseline characteristics such as recipient age, HLA matching, graft rank, anti-HLA immunization, induction and immunosuppressive treatment, CMV prophylaxis, and CMV serological status before the graft. Clinical data for each patient were extracted from the renal transplant database. We studied delayed graft function (DGF), acute rejection episodes in the first year, CMV infection, and graft survival. DGF was defined by dialysis in the first postoperative week; rejection episodes, by clinical diagnosis, namely, an elevation in serum creatinine levels by ⬎20% compared with baseline, with a confirmation by biopsy according to Banff classification. CMV infection was defined as positive pp65 antigenemia (ⱖ2 positive circulating leukocytes). Graft survival was calculated using KaplanMeier life-table analysis. The SNP was genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Primers were designed to amplify a 421-bp region containing the 3=UTR polymorphism, with 5=-GGCATGAAATCCCTGAAACC-3= as forward primer and 5=-TACATCCTGGCAGACAAACG-3= as reverse primer. The obtained fragment was digested with 5 units of TaqI (Invitrogen, Cergy-Pontoise, France) for 1h40 at 65°C; the digestion product was loaded on a 2% agarose gel to obtain the genotype as deduced from the digestion profile. We used a 607-bp region of the GAPDH gene containing a TaqI restriction site as an internal control to assess the efficiency of the TaqI digestion. Chi-square test was used to compare the occurrence of DGF, acute rejection episodes, and CMV infections between IL12B 3=UTR genotypes (presence or absence of the C allele), whereas log-rank test was used to compare graft survivals. P ⬍ .05 was considered significant.
DISCUSSION
We analyzed the relationship between the IL12B 3=UTR SNP and graft outcome. No significant association was revealed between this polymorphism and DGF, acute rejection in the first year, or graft survival, indicating that this polymorphism may not play a major role in these parameters. Concerning acute rejection, the P value was near significance (.108). The impact of the SNP on acute rejection might demand a larger cohort to be revealed. Our findings revealed that the C allele of this SNP was associated with an increased risk for CMV infection after renal transplantation. Acute rejection was not a risk factor for CMV infection in our study (OR ⫽ 1.27; P ⫽ .387). In order to carry out a multivariate analysis, we plan to study a larger patient cohort. The results of the present study addressed the question of the pathogenic link between the IL-12p40 gene polymorphism and CMV infection. This SNP has been shown to be functionally relevant since the C allele is associated with lower mRNA stability3 and less production of the p40 polypeptide.4 We suggest that this lower p40 expression in C carriers might explain the pathogenic link between the IL12B genotype and CMV replication. Indeed, direct activating roles of the p40/p40 homodimer have recently been reported, especially as an IFN␥ stimulator in CD8⫹ T cells and T-cell response initiator.7 As both CD8⫹ T cells and IFN␥ have been shown to be major mediators of host
Table 1. Impact of IL12B 3=UTR Genotype on Graft Outcome in 253 Kidney Graft Recipients 3=UTR Genotype Clinical Events
A/A (n ⫽ 141)
A/C and C/C (n ⫽ 112 [100 ⫹ 12])
Graft survival (months)* Delayed graft function‡ Acute rejection in the first year‡ Positive CMV pp65 antigenemia‡
102 ⫾ 4 30 (21%) 36 (26%) 51 (36%)
99 ⫾ 4 20 (18%) 39 (35%) 56 (50%)
Odds Ratio (95% Confidence Interval) †
NA 0.80 (0.43–1.50) 1.56 (0.91–2.67) 1.77 (1.07–2.92)
P
.768 .498 .108 .027
*Graft survivals were expressed as mean values ⫾ standard deviations by Kaplan-Meier life-table analysis and compared between 3=UTR genotypes by log-rank test. † Not applicable. ‡ The occurrence of delayed graft function, acute rejection episodes, and CMV infections was compared between 3=UTR genotypes by chi-square test.
656
defenses against CMV,8,9 these experimental findings suggest that the level of p40 expression may play an important role during CMV infection. Finally, since the strategy to manage the risk for CMV infection in seropositive recipients remains a matter of debate, the description of a genetic risk factor might improve this management. Our results suggest that 3=UTR C allele carriers might greatly benefit from CMV prophylaxis. REFERENCES 1. Benichou G: Direct and indirect antigen recognition: the pathways to allograft immune rejection. Front Biosci 4:D476, 1999 2. Trinchieri G: Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat Rev Immunol 3:133, 2003 3. Morahan G, Huang D, Ymer SI, et al: Linkage disequilibrium of a type 1 diabetes susceptibility locus with a regulatory IL12B allele. Nat Genet 27:218, 2001
HOFFMANN, HALIMI, BU¨CHLER ET AL 4. Stanilova S, Miteva L: Taq-I polymorphism in 3=UTR of the IL-12B and association with IL-12p40 production from human PBMC. Genes Immun 6:364, 2005 5. van Veen T, Crusius JB, Schrijver HM, et al: Interleukin12p40 genotype plays a role in the susceptibility to multiple sclerosis. Ann Neurol 50:275, 2001 6. Cargill M, Schrodi SJ, Chang M, et al: A large-scale genetic association study confirms IL12B and leads to the identification of IL23R as psoriasis-risk genes. Am J Hum Genet 80:273, 2007 7. Cooper AM, Khader SA: IL-12p40: an inherently agonistic cytokine. Trends Immunol 28:33, 2007 8. Presti RM, Pollock JL, Dal Canto AJ, et al: Interferon gamma regulates acute and latent murine cytomegalovirus infection and chronic disease of the great vessels. J Exp Med 188:577, 1998 9. Reddehase MJ, Mutter W, Munch K, et al: CD8-positive T lymphocytes specific for murine cytomegalovirus immediate-early antigens mediate protective immunity. J Virol 61:3102, 1987