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Interleukin-2 gene polymorphism is associated with renal but not cardiac transplant outcome
Interleukin-2 Gene Polymorphism Is Associated With Renal but Not Cardiac Transplant Outcome A. Morgun, N. Shulzhenko, G.F. Rampim, J.O.P. Medina, P.G.P. Machado, R.V.Z. Diniz, D.R. Almeida, and M. Gerbase-DeLima ABSTRACT It was recently shown that IL-2 gene single nucleotide polymorphism (SNP) at position ⫺330 (G3 T) is related to in vitro cytokine production levels, with the T/T and T/G genotypes being associated with low production and the G/G genotype associated with high production. The objective of this study was to investigate a possible influence of this polymorphism on renal and cardiac allograft outcomes. IL-2 SNP G-T (⫺330) was determined by PCR-RFLP in 67 recipients of heart allografts and in 63 recipients of renal grafts from HLA-haplo-identical, related donors. A higher frequency of the T/T genotype was observed in renal transplant patients who experienced at least one acute rejection episode during the first 3 months after transplantation than in those without rejection during this period (80% vs 49%, respectively, P ⬍ .05). Accordingly, the same genotype tended to be more frequent in renal recipients with a 6-month serum creatinine level above 1.5 mg/dL (median value for the whole group of kidney recipients) than in patients with lower creatinine levels (79% vs 45%, P ⬍ .08). Regarding cardiac transplant recipients, no associations were observed concerning acute rejection or graft survival. The finding of the association of T/T but not T/G genotype with acute kidney rejection was unexpected considering that both genotypes were shown to be associated with equal (low) IL-2 in vitro production. Further studies are necessary not only to dissect the nature of IL-2 T/T genotype association with kidney rejection, but also to explain why this genotype does not apparently influence cardiac allograft outcome.
T
HERE IS INCREASING evidence that the level of production of a series of molecules involved in the immune response, particularly cytokines, can be modulated by polymorphisms of the corresponding genes.1 Since cytokines play a major role in the inflammatory and immune responses that mediate allograft outcome, the relationship between cytokine polymorphisms and occurrence of rejection following solid organ transplantation has been investigated in several studies.2,3 IL-2 is one of principal cytokines of the adaptive immune response that functions as a growth factor early in T-cell activation. In addition, it promotes T-cell apoptosis by activation-induced cell death.4 It was recently shown that IL-2 gene single nucleotide polymorphism (SNP) at position ⫺330 (G3 T) is related to in vitro cytokine production levels, with the T/T and T/G genotypes being associated with low production and the G/G genotype being associated with high production.5 The objective of the present study 0041-1345/03/$–see front matter doi:10.1016/S0041-1345(03)00366-X 1344
was to explore a possible influence of this polymorphism on renal and cardiac allograft outcomes. MATERIALS AND METHODS The study was conducted on 67 recipients of heart allografts and on 63 recipients of HLA-haploidentical renal grafts. All acute rejecFrom the Division of Immunogenetics, Department of Pediatrics (A.M., N.S., G.F.R., M.G.-D.); Division of Nephrology (J.O.P.M., P.G.P.M.); and Division of Cardiology (R.V.Z.D., D.R.A.), Department of Medicine, Universidade Federal de Sa˜o Paulo (UNIFESP-EPM), Sa˜o Paulo, Brazil. This work was supported by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientı´fico e Technolo´gico (CNPq). A.M. and N.S. contributed equally to this work. Address reprint requests to Maria Gerbase-DeLima, MD, Rua Napolea˜o de Barros, 1038, 04024-003 Sa˜o Paulo, SP, Brazil. E-mail: [email protected] © 2003 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 35, 1344-1345 (2003)
INTERLEUKIN-2 GENE POLYMORPHISM
1345
Table 1. Distribution of Genotype Frequencies of IL-2 (ⴚ330) Gene Polymorphism in Renal Transplant Recipients
Genotype
Putative Phenotypea
Acute Rejectionb (n ⫽ 20)
No Acute Rejection (n ⫽ 43)
Creatininec ⬎1.5 mg/dL (n ⫽ 14)
Creatininec ⱕ1.5 mg/dL (n ⫽ 22)
T/T T/G G/G
low low high
80%d 15% 5%
49% 39% 12%
79%e 21% 0%
45% 41% 14%
a
According to Hoffmann et al.5 At least one rejection confirmed by renal biopsy. c Six-month serum creatinine. d P ⬍ .05, recipients with rejection vs recipients without rejection. e P ⬍ .08, recipients with creatinine level ⬎ 1.5 mg/dL vs recipients with creatinine level ⱕ1.5 mg/dL. b
tions were diagnosed by histologic evaluation of endomyocardial or renal biopsies. DNA was extracted from whole blood by the dodecyl trimethylammonium bromide/cetyl trimethyl-ammonium bromide (DTAB/ CTAB) technique.6 IL-2 ⫺330 T3 G SNP genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP).7 Statistical analysis was performed by Fisher’s exact test.
RESULTS
A higher frequency of the T/T genotype was observed in renal transplant patients who experienced at least one acute rejection episode during the first 3 months after transplantation than in those without rejection during this period (80% vs 49%, respectively, P ⬍ .05) (Table 1). Accordingly, the same genotype tended to be more frequent in renal recipients with a 6-month serum creatinine level above 1.5 mg/dL (median value for the whole group of kidney recipients) than in patients with lower creatinine levels (79% vs 45%, P ⬍ .08) (Table 1). However, we did not detect any difference in genotype distribution when analyzing creatinine levels at 1- and 3-month (data not shown). Regarding cardiac transplant recipients, no associations were observed regarding acute rejection or graft survival (Table 2). DISCUSSION AND CONCLUSIONS
In agreement with our data on ⫺330 IL-2 gene polymorphism in heart transplants, lack of association with acute cardiac rejection was also recently reported by Holweg and colleagues.8 However, this group demonstrated an association between acute cardiac rejection and a dinucleotide repeat polymorphism located at the 3⬘-flanking region of the IL-2 gene.9 In addition, some cytokine gene polymorphisms, such as TNF-␣, IL-10, and TGF-1 polymorphisms, were shown to influence cardiac transplant outcome.2,3,10
The finding of T/T but not T/G genotype association with acute kidney rejection was unexpected considering that both genotypes were shown to be associated with equal (low) in vitro IL-2 production.5 One possible explanation is that in vivo, under conditions of allostimulation and immunosuppression, the influence of the T/T and T/G genotypes on IL-2 production could be different from that observed in vitro. On the other hand, the association could also be related to another polymorphism, in linkage disequilibrium with the studied IL-2 ⫺330T. Further studies are necessary not only to clarify the nature of the IL-2 T/T genotype association with kidney rejection but also to explain why this genotype apparently does not influence cardiac allograft outcome. Nevertheless, the genotyping of (⫺330) IL-2 polymorphism in renal transplant recipients seems to help identify recipients at a higher risk of acute rejection.
REFERENCES 1. Bidwell J, Keen L, Gallagher G, et al: Genes Immun 2:61, 2001 2. Marshall SE, Welsh KI: Genes Immun 2:297, 2001 3. Akalin E, Murphy B: Curr Opin Immunol 13:572, 2001 4. Li XC, Wells AD, Strom TB, et al: Curr Opin Immunol 12:522, 2000 5. Hoffmann SC, Stanley EM, Cox ED, et al: Transplantation 72:1444, 2001 6. Gustincich S, Manfiolett G, Del Sal G, et al: Bio Techniques 11:298, 1991 7. Cox ED, Hoffmann SC, DiMercurio BS, et al: Transplantation 72:720, 2001 8. Holweg C, Peeters A, Balk A, et al: American J Transplantation 2(Suppl):215, 2002 9. Holweg C, Peeters A, Balk A, et al: Transplantation 73:1354, 2002 10. Morgun A, Shulzhenko N, Rampim GF, et al: American J Transplantation 2(Suppl):313, 2002
Table 2. Distribution of Genotype Frequencies of IL-2 (ⴚ330) Gene Polymorphism in Cardiac Transplant Recipients Survival (years) Genotype
Putative Phenotypea
Acute Rejectionb (n ⫽ 25)
No Acute Rejection (n ⫽ 42)
⬍2 (n ⫽ 12)
⬎2 (n ⫽ 48)
⬍5 (n ⫽ 15)
⬎5 (n ⫽ 35)
T/T T/G G/G
low low high
48% 48% 4%
62% 31% 7%
34% 58% 8%
58% 36% 6%
40% 53% 7%
57% 34% 9%
a
According to Hoffmann et al.5 At least one biopsy graded 3A or more (International Society of Heart and Lung Transplantation criteria) during the first 6 months after transplantation.
b
T
HERE IS INCREASING evidence that the level of production of a series of molecules involved in the immune response, particularly cytokines, can be modulated by polymorphisms of the corresponding genes.1 Since cytokines play a major role in the inflammatory and immune responses that mediate allograft outcome, the relationship between cytokine polymorphisms and occurrence of rejection following solid organ transplantation has been investigated in several studies.2,3 IL-2 is one of principal cytokines of the adaptive immune response that functions as a growth factor early in T-cell activation. In addition, it promotes T-cell apoptosis by activation-induced cell death.4 It was recently shown that IL-2 gene single nucleotide polymorphism (SNP) at position ⫺330 (G3 T) is related to in vitro cytokine production levels, with the T/T and T/G genotypes being associated with low production and the G/G genotype being associated with high production.5 The objective of the present study 0041-1345/03/$–see front matter doi:10.1016/S0041-1345(03)00366-X 1344
was to explore a possible influence of this polymorphism on renal and cardiac allograft outcomes. MATERIALS AND METHODS The study was conducted on 67 recipients of heart allografts and on 63 recipients of HLA-haploidentical renal grafts. All acute rejecFrom the Division of Immunogenetics, Department of Pediatrics (A.M., N.S., G.F.R., M.G.-D.); Division of Nephrology (J.O.P.M., P.G.P.M.); and Division of Cardiology (R.V.Z.D., D.R.A.), Department of Medicine, Universidade Federal de Sa˜o Paulo (UNIFESP-EPM), Sa˜o Paulo, Brazil. This work was supported by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientı´fico e Technolo´gico (CNPq). A.M. and N.S. contributed equally to this work. Address reprint requests to Maria Gerbase-DeLima, MD, Rua Napolea˜o de Barros, 1038, 04024-003 Sa˜o Paulo, SP, Brazil. E-mail: [email protected] © 2003 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 35, 1344-1345 (2003)
INTERLEUKIN-2 GENE POLYMORPHISM
1345
Table 1. Distribution of Genotype Frequencies of IL-2 (ⴚ330) Gene Polymorphism in Renal Transplant Recipients
Genotype
Putative Phenotypea
Acute Rejectionb (n ⫽ 20)
No Acute Rejection (n ⫽ 43)
Creatininec ⬎1.5 mg/dL (n ⫽ 14)
Creatininec ⱕ1.5 mg/dL (n ⫽ 22)
T/T T/G G/G
low low high
80%d 15% 5%
49% 39% 12%
79%e 21% 0%
45% 41% 14%
a
According to Hoffmann et al.5 At least one rejection confirmed by renal biopsy. c Six-month serum creatinine. d P ⬍ .05, recipients with rejection vs recipients without rejection. e P ⬍ .08, recipients with creatinine level ⬎ 1.5 mg/dL vs recipients with creatinine level ⱕ1.5 mg/dL. b
tions were diagnosed by histologic evaluation of endomyocardial or renal biopsies. DNA was extracted from whole blood by the dodecyl trimethylammonium bromide/cetyl trimethyl-ammonium bromide (DTAB/ CTAB) technique.6 IL-2 ⫺330 T3 G SNP genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP).7 Statistical analysis was performed by Fisher’s exact test.
RESULTS
A higher frequency of the T/T genotype was observed in renal transplant patients who experienced at least one acute rejection episode during the first 3 months after transplantation than in those without rejection during this period (80% vs 49%, respectively, P ⬍ .05) (Table 1). Accordingly, the same genotype tended to be more frequent in renal recipients with a 6-month serum creatinine level above 1.5 mg/dL (median value for the whole group of kidney recipients) than in patients with lower creatinine levels (79% vs 45%, P ⬍ .08) (Table 1). However, we did not detect any difference in genotype distribution when analyzing creatinine levels at 1- and 3-month (data not shown). Regarding cardiac transplant recipients, no associations were observed regarding acute rejection or graft survival (Table 2). DISCUSSION AND CONCLUSIONS
In agreement with our data on ⫺330 IL-2 gene polymorphism in heart transplants, lack of association with acute cardiac rejection was also recently reported by Holweg and colleagues.8 However, this group demonstrated an association between acute cardiac rejection and a dinucleotide repeat polymorphism located at the 3⬘-flanking region of the IL-2 gene.9 In addition, some cytokine gene polymorphisms, such as TNF-␣, IL-10, and TGF-1 polymorphisms, were shown to influence cardiac transplant outcome.2,3,10
The finding of T/T but not T/G genotype association with acute kidney rejection was unexpected considering that both genotypes were shown to be associated with equal (low) in vitro IL-2 production.5 One possible explanation is that in vivo, under conditions of allostimulation and immunosuppression, the influence of the T/T and T/G genotypes on IL-2 production could be different from that observed in vitro. On the other hand, the association could also be related to another polymorphism, in linkage disequilibrium with the studied IL-2 ⫺330T. Further studies are necessary not only to clarify the nature of the IL-2 T/T genotype association with kidney rejection but also to explain why this genotype apparently does not influence cardiac allograft outcome. Nevertheless, the genotyping of (⫺330) IL-2 polymorphism in renal transplant recipients seems to help identify recipients at a higher risk of acute rejection.
REFERENCES 1. Bidwell J, Keen L, Gallagher G, et al: Genes Immun 2:61, 2001 2. Marshall SE, Welsh KI: Genes Immun 2:297, 2001 3. Akalin E, Murphy B: Curr Opin Immunol 13:572, 2001 4. Li XC, Wells AD, Strom TB, et al: Curr Opin Immunol 12:522, 2000 5. Hoffmann SC, Stanley EM, Cox ED, et al: Transplantation 72:1444, 2001 6. Gustincich S, Manfiolett G, Del Sal G, et al: Bio Techniques 11:298, 1991 7. Cox ED, Hoffmann SC, DiMercurio BS, et al: Transplantation 72:720, 2001 8. Holweg C, Peeters A, Balk A, et al: American J Transplantation 2(Suppl):215, 2002 9. Holweg C, Peeters A, Balk A, et al: Transplantation 73:1354, 2002 10. Morgun A, Shulzhenko N, Rampim GF, et al: American J Transplantation 2(Suppl):313, 2002
Table 2. Distribution of Genotype Frequencies of IL-2 (ⴚ330) Gene Polymorphism in Cardiac Transplant Recipients Survival (years) Genotype
Putative Phenotypea
Acute Rejectionb (n ⫽ 25)
No Acute Rejection (n ⫽ 42)
⬍2 (n ⫽ 12)
⬎2 (n ⫽ 48)
⬍5 (n ⫽ 15)
⬎5 (n ⫽ 35)
T/T T/G G/G
low low high
48% 48% 4%
62% 31% 7%
34% 58% 8%
58% 36% 6%
40% 53% 7%
57% 34% 9%
a
According to Hoffmann et al.5 At least one biopsy graded 3A or more (International Society of Heart and Lung Transplantation criteria) during the first 6 months after transplantation.
b