- Email: [email protected]
Interferon-γ +874 Polymorphism in the First Intron of the Human Interferon-γ Gene and Kidney Allograft Outcome
Interferon-␥ ⴙ874 Polymorphism in the First Intron of the Human Interferon-␥ Gene and Kidney Allograft Outcome J.C.O. Crispim, I.J. Wastowski, D.M. Rassi, C.T. Mendes-Junior Silva, C. Bassi, E.C. Castelli, R.S. Costa, L.T. Saber, T.G.A. Silva, and E.A. Donadi
ABSTRACT Background. Despite advances in immunosuppressive therapy in the past decade, allograft rejection remains an important cause of kidney graft failure. Cytokines play a major role in the inflammatory and immune responses that mediate allograft outcomes. Several studies have shown that the production of cytokines varies among individuals. These variations are determined by genetic polymorphisms, most commonly within the regulatory region of cytokine genes. The aim of the present study was to assess the effect of allelic variation on acute rejection episodes (ARE) or chronic allograft nephropathy (CAN) after kidney transplantation. Methods. To determine a possible correlation between the interferon (INF)-␥ ⫹874 polymorphism and kidney allograft outcome, we isolated genomic DNA from 74 patients who underwent isolated kidney allografts and were classified into 2 groups—a rejection and a nonrejection group—for comparison with a control group of 163 healthy subjects. Results. We genotyped INF-␥ ⫹874 polymorphisms in all groups. The transplant group showed a significantly increased homozygous genotype T/T (P ⫽ .0118) compared with healthy controls. Similarly, considering only patients with CAN, the homozygous genotype T/T (P ⫽ .0067) was significantly increased compared with the healthy controls. The rejection group indicated a significant increased homozygous genotype T/T compared with the control group (P ⫽ .0061). Conclusion. Homozygous genotype T/T was associated with increased levels of INF-␥ and greater numbers among the rejection and CAN cohorts.
T
RANSPLANTATION IS THE current treatment of choice for end-stage renal disease. Unfortunately, acute and chronic allograft rejection remain an important
morbidity associated with this procedure. To reduce the risk of acute rejection episodes (ARE), potent immunosuppressive agents have been added to clinical protocols, conse-
From the Department of Biochemistry and Immunology (J.C.O.C.), School of Medicine of Ribeirão Preto, University of São Paulo, São Paulo, and the Department of Clinical and Toxicology Analysis (J.C.O.C., D.M.R.), Faculty of Pharmaceutical Sciences, (UFRN), Rio Grande do Norte; the Division of Clinical Immunology (I.J.W., E.C.C., E.A.D.), Department of Medicine, School of Medicine of Ribeirão Preto, University of São Paulo, São Paulo; the Department of Chemistry (C.T.M.-J.S.), Faculty of Philosophy, Science and Letters of Ribeirão Preto, University of São Paulo, São Paulo; the Department of Basics Sciences in Health (C.B.), School of Pharmaceutical Sciences, University Federal of Mato Grosso; the Department of Pathology (R.S.C.), School of Medicine of Ribeirão Preto, University of São Paulo, São Paulo; the Renal
Transplant Unity (L.T.S.), Clinical Nephrology, School of Medicine of Ribeirão Preto, University of São Paulo, (FMRP-USP), São Paulo; and the Department of Clinical Analysis (T.G.A.S.), School of Pharmaceutical Sciences, UNESP, São Paulo, Brazil. Supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Apoio ao Ensino, Pesquisa e Assistência do HCFMRP-USP. J.O.C. was support by a doctoral fellowship from CAPES/Brazil. Address reprint requests to Janaina Cristiana de Oliveira Crispim, PhD, Department of Biochemistry and Immunology, School of Medicine of Ribeirão Preto, University of São Paulo, Av. Bandeirantes, 3900, 14049-900, Ribeirão Preto, SP, Brazil. E-mail: [email protected]
© 2010 Published by Elsevier Inc. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/–see front matter doi:10.1016/j.transproceed.2010.10.014
Transplantation Proceedings, 42, 4505– 4508 (2010)
4505
4506
quently increasing the risk of drug-related side effects.1 The identification of risk factors that influence the incidence and severity of acute and chronic rejection is an important goal, because it may permit individualization of immunosuppressive therapy.1,2 There is ample evidence that a patient’s genetic background has a role to determine allograft outcome. Cytokine genotypes have previously been studied in patients undergoing solid organ transplantation; certain polymorphisms have been implicated in the development of graft failure versus survival.3,4 The alloimmune response plays a key role in transplant rejection and its T-lymphocyte dependency has been well-documented.1–3 Cytokines and certain cell membrane proteins collectively known as costimulatory molecules modulate immune responses by regulating lymphocyte activation, proliferation, differentiation, and survival.2,5–7 Interferon (IFN)-␥ is a signature cytokine of the T-helper (Th)1 subset. Enhanced production of IFN-␥ by mononuclear cells triggers inflammatory responses.8 Several studies have indicated that IFN-␥ levels have significant effects on susceptibility to various autoimmune diseases, infections, and allergies.9 It is now recognized that cytokine production shows wide variations among individuals. The IFN-␥ gene is located on chromosome 12q24.1. Among ⫹874 A/T single nucleotide polymorphisms (SNP) in the first intron of IFN-␥, the T allele of this SNP is associated with high INF-␥ production.10 Some investigators have observed an imbalance of Th1/Th2 cells during rejection, indicating that Th1 cells predominate among patients undergoing ARE11 or chronic allograft nephropathy (CAN), similar to results in a mouse model.12 These data suggest that cytokine gene polymorphisms are important determinants of disease risk or severity among conditions in which the immune system plays a pivotal role.13 In light of these findings, it seems plausible that genetic polymorphisms for various cytokines genes influence host responses to allografts. Cytokines are known to play a key role to mediate many events associated with rejection. These data suggest that genetic variations in INF-␥ may influence the disease course rather than be a disease-causing gene.10 –13 The ⫹874 A/T SNP has been associated with pediatric heart allograft outcomes.12 Among kidney transplantations, Gendzekhadze et al14 failed to observe an association between CTLA-4 ⫹49 or tumor necrosis factor (TNF)-␣ polymorphism with graft rejection. In the present study, we performed a crosssectional study correlating INF-␥ ⫹874 polymorphisms and clinical outcomes. Specifically, we sought an association between cytokine genotype profile and the risk of ARE or CAN.
PATIENTS AND METHODS Our research ethics committee approved the study protocol; written informed consent was obtained from all subjects, including permission to obtain 1 biopsy. The study included 74 patients who had undergone renal transplantation and 163 healthy controls.
CRISPIM, WASTOWSKI, RASSI ET AL
Patients INF-␥ ⫹874 genotyping was performed among primary kidney allograft patients who were stratified into 2 groups: a rejection group and a nonrejection group. Recipients were screened for specific human leukocyte antigen (HLA) antibodies before transplantation; no patient presented donor-specific HLA antibodies. All subjects and donors were typed for the HLA-A, HLA-B, and HLA-DR loci using the sequence-specific primer method (One Lambda, Canoga Park, Calif).
Controls Peripheral blood was obtained from a control group of 163 unrelated healthy bone marrow donors of both genders, from the same Brazilian population (São Paulo State), seeking to compare similar numbers of individuals in 2 populations (cases and controls). All donors (100%) were typed at a single center.
Specimens of Kidney Graft Biopsies Graft biopsies are not routinely performed in these renal transplanted patients, unless they presented clinical and/or laboratory evidence of acute or chronic graft dysfunction. In the absence of a biopsy indication, only noninvasive laboratory tests were obtained to monitor graft function. We studied 74 single biopsy specimens from patients who underwent isolated kidney grafts and gave informed consent. The biopsy sample was stained with hematoxylin and eosin, Masson-trichrome, periodic acid-Schiff and silver. Acute rejection and CAN stigmata were present in 28 specimens (37.80%), which were classified as the rejection group, whereas 46 (62.20%) showed no sign of rejection, but rather other histologic and pathologic features of fibrosis, acute tubular necrosis, Berger diséase, nephritis, interstitial inflammation, or glomerulonephritis. The histologic analyses of specimens were classified according to the Banff 1997 classification.15 All renal biopsies were classified by a single pathologist who was unaware of the results of the other analyses.
INF-␥ ⫹874 Polymorphism Evaluation DNA was extracted from whole blood or from lymphocytes preparations using the salt precipitation method.16 The INF-␥ ⫹874 polymorphism genotyping was identified according to conditions reported by Pravica et al,10 and the polymerase chain reaction products were analyzed upon nondenaturing 7% polyacrylamide gel electrophoresis followed by silver staining.
Immunosuppressive Therapy Immunosuppressive treatment consisted of cyclosporine or tacrolimus, methylprednisone, and mycophenolate mofetil. Cyclosporine was started at a dosage of 4 mg/kg per day (administered in 2 doses) within the first 24 hours posttransplantation; thereafter, the dose was adjusted to maintain target concentrations at 1 month of 200 – 400 ng/mL; 2–3 months, 200 –300 ng/mL; 4 – 6 months, 150 – 250 ng/mL; and 6 –12 months, 100 –200 ng/mL. Cyclosporine levels were measured by a fluorescence polarization immunoassay (Axsym, Abbott, Abbott Park, Ill). Tacrolimus was administered for the first 1–3 days after transplantation at 0.3 mg/kg per day divided into 2 doses with the aim of targeting whole-blood tacrolimus trough concentration of 10 –20 ng/mL. Dosages were adjusted by measuring blood levels. Mycophenolate mofetil at a starting dose between 2 and 3 g/d and a maintenance dose of 1–2 g/d (adminis-
INF-␥ ⫹874 POLYMORPHISM
4507
tered in divided doses). Additional immunosuppression was obtained with methylprednisone.
Statistical Analysis Allele and genotype frequencies were computed by the direct counting method. It adhered to the phenotypical proportions expected under Hardy–Weinberg equilibrium as tested by the complete enumeration method using GENEPOP 3.4 software.17 The frequency of each allele or genotype was compared between patients and controls by means of 2-sided Fisher’s exact tests, with the aid of the GraphPad Instat 3.05 software, which was also used to estimate the odds ratio (OR) and its 95% confidence interval (CI).
RESULTS
Histopathologic analyses revealed 10 patients (13.5%) to exhibit ARE and 18 (24.3%) CAN. The remaining 46 patients (62.2%) showed no rejection; however, they displayed other histopathologic findings, including isolated fibrosis, acute tubular necrosis, interstitial inflammation, or recurrent kidney disease. We analyzed the INF-␥ ⫹874 polymorphism among kidney allograft recipients and healthy controls. There was no difference in the allele and genotype frequencies of INF-␥ ⫹874 polymorphism between healthy controls versus the rejection and nonrejection cohorts. We evaluated allele and genotype frequencies and Hardy–Weinberg equilibrium in Table 1 There was no departure from Hardy– Weinberg equilibrium. No differences (P ⬎ .05) were observed between kidney allograft recipients and healthy controls, indicating that the INF-␥ ⫹874 polymorphism was not correlated with kidney allograft outcomes. However, the analysis of the INF-␥ ⫹874 polymorphism in the rejection group indicated an increase in the homozygous genotype T/T (P ⫽ .0118) among the ARE cohort compared with healthy controls (P ⫽ .0118; OR, 7.095; 95% CI, 1.787–28.17; Table 2). Similarly, considering only patients with CAN compared with the healthy controls, there was an increase in the homozygous genotype T/T (P ⫽ .0067; OR, 5.321; 95% CI, 1.731–16.359; Table 2). Analysis of the rejection group showed a significant increase compared with the controls in the homozygous genotype T/T group (P ⫽ .0061; OR, 4.257; 95% CI, 1.588 –11.415; Table 2). DISCUSSION
It is well-recognized that ethnic background influences the outcome of or susceptibility to certain diseases.13 Cytokines
play an important role in the regulation of allograft outcomes. We investigated IFN-␥ polymorphism in the first intron among renal allograft recipients in a Brazilian population. Our aim was to investigate the relationship between cytokine gene polymorphism and ARE or CAN. We showed a significant association between the high INF-␥ genotype and susceptibility to ARE or CAN among the Brazilian population. Studies have shown that certain cytokine gene polymorphisms confers susceptibility to early acute rejection in adult heart, lung, and kidney transplant recipients. Turner et al18 reported that a combination of polymorphisms at ⫺308 in the TNF gene and at ⫺1082 in the interleukin (IL)-10 gene (high TNF/low IL-10) was associated with a greater level of acute graft rejection in adult heart transplant recipients.12,18 Similar findings were noted in pediatric heart transplant recipients.10 Among kidney allografts, Sankaran et al11 reported that high IL-10 and high TNF genotypes were each associated with a greater number of ARE. In pediatric heart transplant recipients, the INF-␥ ⫹874 polymorphism failed to show an association with the frequency of ARE.10 A previous study indicated that individuals homozygous for the allele 2; 12 CA repeat showed a high level of INF-␥ production.8,19 This allele 2 was later reported to be protective for arthritis in systemic lupus erythematosus.19 Because there was an absolute correlation between the presence of the ⫹874T allele and high-producing allele 2, our result confirms the previous observation regarding the protective role of INF-␥ on arthritis. It is interesting to note that this SNP coincides with the putative nuclear factorB– binding site, which may have a functional consequence for the INF-␥ gene.13 This observation strengthens the prior suggestion that genetic variations in INF-␥ expression influence the disease course rather than being a disease-causing gene. In addition, a positive association was observed between the amino acid polymorphism (Val14Met) within the INF-␥ receptor 1 gene and systemic lupus erythematosus.8 The genetic contribution to determine INF-␥ function may come from the receptor gene or a combination of cytokine and receptor genes. No significant differences were noted between the rejection and nonrejection groups regarding the T/T homozygous genotype. However, in the comparison with healthy controls, the rejection group was significantly increased in proportion among this group (P ⫽ .0061). Because the rejection group was composed of only 28 individuals, a larger sample would be required to prove
Table 1. Samples Sizes (n), INF-␥ ⴙ874 Polymorphism Genotype Frequencies, and Hardy–Weinberg Equilibrium Alleles
HWE
Genotypes
Posttransplantation Status
n
T
A
(P)
TT
AA
TA
Rejection group Acute rejection Chronic allograft nephropathy Nonrejection group Healthy controls
28 10 18 46 163
0.48 0.55 0.44 0.37 0.45
0.52 0.45 0.56 0.63 0.55
.2716 .2455 .6566 .0000 .1598
0.29 0.40 0.22 0.13 0.09
0.32 0.30 0.33 0.39 0.41
0.39 0.30 0.44 0.48 0.50
4508
CRISPIM, WASTOWSKI, RASSI ET AL
Table 2. Probability Values Obtained by Means of the 2-Sided Fisher’s Exact Test on the Comparisons of INF-␥ Allele/Genotype Frequencies Between Different Sample Groups Genotypes Posttransplantation Status
AR vs CAN AR vs NRG AR vs HC CAN vs NRG CAN vs HC RG vs NRG RG vs HC NRG vs HC
AA
AT
1.0000 0.7272 0.7418 0.7778 0.6179 0.6233 0.4104 0.8661
0.6888 0.4849 0.3299 1.0000 0.8046 0.6301 0.3118 0.8676
Allele TT
0.4004 0.0660 0.0118* 0.0805 0.0067† 0.1291 0.0061‡ 0.3961
A or T
0.5785 0.2075 0.0879 0.5459 0.2028 0.2281 0.0442§ 0.6195
Abbreviations: AR, acute rejection; CAN, chronic allograft nephropathy; RG, rejection group; NRG, nonrejection group; HC, healthy controls. *P ⬍ .05 (Fisher’s exact test; GraPhPad Instat). Odds ratio ⫽ 7.095 (95% CI, 1.787–28.175). † P ⬍ .05 (Fisher’s exact test; GraPhPad Instat). Odds ratio ⫽ 5.321 (95% CI, 1.731–16.359). ‡ P ⬍ .05 (Fisher’s exact test; GraPhPad Instat). Odds ratio ⫽ 4.257 (95% CI, 1.588 –11.415). § P ⬍ .05 (Fisher’s exact test; GraPhPad Instat). Odds ratio ⫽ 1.828 (95% CI, 1.031–3.240).
this association. Despite the smaller sample size of the ARE group (n ⫽ 10), the T/T homozygous genotype was significantly increased in proportion compared with healthy controls (P ⫽ .0118). Similarly, considering only the CAN group (n ⫽ 18), the homozygous genotype T/T (P ⫽ .0067) was significantly increased compared with healthy controls. This observation indicates that the T/T homozygous genotype may be a susceptibility factor at least for ARE and CAN. Another study by our group suggested that the T/T genotype may be associated with a better prognosis of systemic sclerosis, because this polymorphism seemed to favor INF-␥ gene transcription and consequently greater cytokine production.20 After cold ischemia and after approximately 20 –30 minutes of reperfusion, kidney tissue shows high glomerular and tubular mRNA expression of IL-2, IL-6, IL-10, transforming growth factor-1, platelet-derived growth factor-B, and IFN-␥.21 Our findings may result from the high INFG production associated with the T/T homozygous genotype of the INF-␥ gene.8 In fact, these findings are consistent with the view that INF-␥ is a proinflammatory Th1 type cytokine that upregulates cell-mediated responses, therefore favoring acute cellular rejection and CAN. Taken together, our data indicated that the INF-␥ ⫹874 polymorphism may be important for the outcome of kidney transplantations. REFERENCES 1. Davies JD, Martin G, Phillips J, et al: T cell regulation in adult transplantation tolerance. J Immunol 157:529, 1996
2. Nikolic B, Sykes M: Clonal deletion as a mechanism of transplantation tolerance. J Heart Lung Transplant 15:1171, 1996 3. Cripim JC, Wastowski IJ, Faggione LP, et al: Microchimerism evaluation in recipients of living-related or unrelated deceased allograft renal transplants. Transplant Proc 38:2828, 2006 4. Mendes-Júnior CT, Castelli EC, Simões RT, et al: HLA-G 14bp polymorphism at exon 8 in Amerindian populations from the Brazilian Amazon. Tissue Antigens 69:255, 2007 5. Di Giovine FS, Taskhsh E, Blakemore AIF, et al: Single base polymorphism at -511 in the human interleukin-1b gene (IL1b). Human Mol Gen 1:450, 1992 6. Kaminska D, Tyran B, Mazanowska O, et al: Cytokine gene expression in kidney allograft biopsies after donor brain death and ischemia-reperfusion injury using in situ reverse-transcription polymerase chain reaction analysis. Transplantation 84:1118, 2007 7. Perco P, Pleban C, Kainz A, et al: Gene expression and biomarkers in renal transplant ischemia reperfusion injury Transpl Int 20:2, 2007 8. Pravica V, Asderakis A, Perrey C, et al: In vitro production correlates with CA repeat polymorphism in the human interferon gene. Eur J Immunogen 26:1, 1999 9. Bidwell J, Keen L, Gallagher G, et al: Cytokine gene polymorphism in human disease: on-line databases, supplement 1. Genes Immun 2:61, 2001 10. Awad MR, Webber S, Boyle G, et al: The effect of cytokine gene polymorphisms on pediatric heart allograft outcome. J Heart Lung Transplant 20:625, 2001 11. Sankaran D, Asderakis A, Ashraf S, et al: Cytokine gene polymorphisms predict acute graft rejection following renal transplantation. Kidney Int 56:281, 1999 12. Hutchinson IV, Pravica V, Sinnott PJ: Genetic regulation of cytokines synthesis: consequences for acute and chronic organ allograft rejection. Graft 1:186, 1998 13. Kittles RA, Weiss KM: Race, ancestry, and genes: implications for defining disease risk. Annu Rev Genomics Hum Genet 4:33, 2003 14. Gendzekhadze K, Rivas-Vetencourt P, Montano RF: Risk of adverse post-transplant events after kidney allograft transplantation as predicted by CTLA-4 ⫹49 and TNF-alpha-308 single nucleotide polymorphisms: a preliminary study. Transpl Immunol 16:194, 2006 15. Racusen LC, Solezz K, Colvin RB, et al: The Bannf97 working classification of renal allograft pathology. Kidney Int 55:713, 1999 16. Higuchi R: Simple and rapid preparation of samples for PCR. In: Erlich HA, ed. PCR technology: Principles an applications for DNA amplification. New York: Stockton Press; 1989. p. 31 17. Raymond M, Rousset F: Genepop (Version-1.2): populationgenetics software for exact tests and ecumenicism. J Hered 86:248, 1995 18. Turner D, Grant SC, Yonan N, et al: Cytokine gene polymorphism and heart transplant rejection. Transplantation 64:776, 1997 19. Tangwattanachuleeporn M, Sodsai P, Avihingsanon Y, et al: Association of interferon-gamma gene polymorphism (⫹874A) with arthritis manifestation in SLE. Clin Rheumatol 26:1921, 2007 20. Wastowski IJ, Sampaio-Barros PD, Martelli-Palomino G, et al: Association of Interferon-gamma gene polymorphism (⫹874 T/A) with systemic sclerosis. Dis Markers 27:93, 2009 21. Kaminska D, Tyran B, Mazanowska O, et al: Gene expression of INF-gamma, IL- 10, IL-2, IL-6, PDGF-B, TGF-beta in kidney tissue after renal transplantation. Pol Merkur Lekarski 21:148, 2006
ABSTRACT Background. Despite advances in immunosuppressive therapy in the past decade, allograft rejection remains an important cause of kidney graft failure. Cytokines play a major role in the inflammatory and immune responses that mediate allograft outcomes. Several studies have shown that the production of cytokines varies among individuals. These variations are determined by genetic polymorphisms, most commonly within the regulatory region of cytokine genes. The aim of the present study was to assess the effect of allelic variation on acute rejection episodes (ARE) or chronic allograft nephropathy (CAN) after kidney transplantation. Methods. To determine a possible correlation between the interferon (INF)-␥ ⫹874 polymorphism and kidney allograft outcome, we isolated genomic DNA from 74 patients who underwent isolated kidney allografts and were classified into 2 groups—a rejection and a nonrejection group—for comparison with a control group of 163 healthy subjects. Results. We genotyped INF-␥ ⫹874 polymorphisms in all groups. The transplant group showed a significantly increased homozygous genotype T/T (P ⫽ .0118) compared with healthy controls. Similarly, considering only patients with CAN, the homozygous genotype T/T (P ⫽ .0067) was significantly increased compared with the healthy controls. The rejection group indicated a significant increased homozygous genotype T/T compared with the control group (P ⫽ .0061). Conclusion. Homozygous genotype T/T was associated with increased levels of INF-␥ and greater numbers among the rejection and CAN cohorts.
T
RANSPLANTATION IS THE current treatment of choice for end-stage renal disease. Unfortunately, acute and chronic allograft rejection remain an important
morbidity associated with this procedure. To reduce the risk of acute rejection episodes (ARE), potent immunosuppressive agents have been added to clinical protocols, conse-
From the Department of Biochemistry and Immunology (J.C.O.C.), School of Medicine of Ribeirão Preto, University of São Paulo, São Paulo, and the Department of Clinical and Toxicology Analysis (J.C.O.C., D.M.R.), Faculty of Pharmaceutical Sciences, (UFRN), Rio Grande do Norte; the Division of Clinical Immunology (I.J.W., E.C.C., E.A.D.), Department of Medicine, School of Medicine of Ribeirão Preto, University of São Paulo, São Paulo; the Department of Chemistry (C.T.M.-J.S.), Faculty of Philosophy, Science and Letters of Ribeirão Preto, University of São Paulo, São Paulo; the Department of Basics Sciences in Health (C.B.), School of Pharmaceutical Sciences, University Federal of Mato Grosso; the Department of Pathology (R.S.C.), School of Medicine of Ribeirão Preto, University of São Paulo, São Paulo; the Renal
Transplant Unity (L.T.S.), Clinical Nephrology, School of Medicine of Ribeirão Preto, University of São Paulo, (FMRP-USP), São Paulo; and the Department of Clinical Analysis (T.G.A.S.), School of Pharmaceutical Sciences, UNESP, São Paulo, Brazil. Supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Apoio ao Ensino, Pesquisa e Assistência do HCFMRP-USP. J.O.C. was support by a doctoral fellowship from CAPES/Brazil. Address reprint requests to Janaina Cristiana de Oliveira Crispim, PhD, Department of Biochemistry and Immunology, School of Medicine of Ribeirão Preto, University of São Paulo, Av. Bandeirantes, 3900, 14049-900, Ribeirão Preto, SP, Brazil. E-mail: [email protected]
© 2010 Published by Elsevier Inc. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/–see front matter doi:10.1016/j.transproceed.2010.10.014
Transplantation Proceedings, 42, 4505– 4508 (2010)
4505
4506
quently increasing the risk of drug-related side effects.1 The identification of risk factors that influence the incidence and severity of acute and chronic rejection is an important goal, because it may permit individualization of immunosuppressive therapy.1,2 There is ample evidence that a patient’s genetic background has a role to determine allograft outcome. Cytokine genotypes have previously been studied in patients undergoing solid organ transplantation; certain polymorphisms have been implicated in the development of graft failure versus survival.3,4 The alloimmune response plays a key role in transplant rejection and its T-lymphocyte dependency has been well-documented.1–3 Cytokines and certain cell membrane proteins collectively known as costimulatory molecules modulate immune responses by regulating lymphocyte activation, proliferation, differentiation, and survival.2,5–7 Interferon (IFN)-␥ is a signature cytokine of the T-helper (Th)1 subset. Enhanced production of IFN-␥ by mononuclear cells triggers inflammatory responses.8 Several studies have indicated that IFN-␥ levels have significant effects on susceptibility to various autoimmune diseases, infections, and allergies.9 It is now recognized that cytokine production shows wide variations among individuals. The IFN-␥ gene is located on chromosome 12q24.1. Among ⫹874 A/T single nucleotide polymorphisms (SNP) in the first intron of IFN-␥, the T allele of this SNP is associated with high INF-␥ production.10 Some investigators have observed an imbalance of Th1/Th2 cells during rejection, indicating that Th1 cells predominate among patients undergoing ARE11 or chronic allograft nephropathy (CAN), similar to results in a mouse model.12 These data suggest that cytokine gene polymorphisms are important determinants of disease risk or severity among conditions in which the immune system plays a pivotal role.13 In light of these findings, it seems plausible that genetic polymorphisms for various cytokines genes influence host responses to allografts. Cytokines are known to play a key role to mediate many events associated with rejection. These data suggest that genetic variations in INF-␥ may influence the disease course rather than be a disease-causing gene.10 –13 The ⫹874 A/T SNP has been associated with pediatric heart allograft outcomes.12 Among kidney transplantations, Gendzekhadze et al14 failed to observe an association between CTLA-4 ⫹49 or tumor necrosis factor (TNF)-␣ polymorphism with graft rejection. In the present study, we performed a crosssectional study correlating INF-␥ ⫹874 polymorphisms and clinical outcomes. Specifically, we sought an association between cytokine genotype profile and the risk of ARE or CAN.
PATIENTS AND METHODS Our research ethics committee approved the study protocol; written informed consent was obtained from all subjects, including permission to obtain 1 biopsy. The study included 74 patients who had undergone renal transplantation and 163 healthy controls.
CRISPIM, WASTOWSKI, RASSI ET AL
Patients INF-␥ ⫹874 genotyping was performed among primary kidney allograft patients who were stratified into 2 groups: a rejection group and a nonrejection group. Recipients were screened for specific human leukocyte antigen (HLA) antibodies before transplantation; no patient presented donor-specific HLA antibodies. All subjects and donors were typed for the HLA-A, HLA-B, and HLA-DR loci using the sequence-specific primer method (One Lambda, Canoga Park, Calif).
Controls Peripheral blood was obtained from a control group of 163 unrelated healthy bone marrow donors of both genders, from the same Brazilian population (São Paulo State), seeking to compare similar numbers of individuals in 2 populations (cases and controls). All donors (100%) were typed at a single center.
Specimens of Kidney Graft Biopsies Graft biopsies are not routinely performed in these renal transplanted patients, unless they presented clinical and/or laboratory evidence of acute or chronic graft dysfunction. In the absence of a biopsy indication, only noninvasive laboratory tests were obtained to monitor graft function. We studied 74 single biopsy specimens from patients who underwent isolated kidney grafts and gave informed consent. The biopsy sample was stained with hematoxylin and eosin, Masson-trichrome, periodic acid-Schiff and silver. Acute rejection and CAN stigmata were present in 28 specimens (37.80%), which were classified as the rejection group, whereas 46 (62.20%) showed no sign of rejection, but rather other histologic and pathologic features of fibrosis, acute tubular necrosis, Berger diséase, nephritis, interstitial inflammation, or glomerulonephritis. The histologic analyses of specimens were classified according to the Banff 1997 classification.15 All renal biopsies were classified by a single pathologist who was unaware of the results of the other analyses.
INF-␥ ⫹874 Polymorphism Evaluation DNA was extracted from whole blood or from lymphocytes preparations using the salt precipitation method.16 The INF-␥ ⫹874 polymorphism genotyping was identified according to conditions reported by Pravica et al,10 and the polymerase chain reaction products were analyzed upon nondenaturing 7% polyacrylamide gel electrophoresis followed by silver staining.
Immunosuppressive Therapy Immunosuppressive treatment consisted of cyclosporine or tacrolimus, methylprednisone, and mycophenolate mofetil. Cyclosporine was started at a dosage of 4 mg/kg per day (administered in 2 doses) within the first 24 hours posttransplantation; thereafter, the dose was adjusted to maintain target concentrations at 1 month of 200 – 400 ng/mL; 2–3 months, 200 –300 ng/mL; 4 – 6 months, 150 – 250 ng/mL; and 6 –12 months, 100 –200 ng/mL. Cyclosporine levels were measured by a fluorescence polarization immunoassay (Axsym, Abbott, Abbott Park, Ill). Tacrolimus was administered for the first 1–3 days after transplantation at 0.3 mg/kg per day divided into 2 doses with the aim of targeting whole-blood tacrolimus trough concentration of 10 –20 ng/mL. Dosages were adjusted by measuring blood levels. Mycophenolate mofetil at a starting dose between 2 and 3 g/d and a maintenance dose of 1–2 g/d (adminis-
INF-␥ ⫹874 POLYMORPHISM
4507
tered in divided doses). Additional immunosuppression was obtained with methylprednisone.
Statistical Analysis Allele and genotype frequencies were computed by the direct counting method. It adhered to the phenotypical proportions expected under Hardy–Weinberg equilibrium as tested by the complete enumeration method using GENEPOP 3.4 software.17 The frequency of each allele or genotype was compared between patients and controls by means of 2-sided Fisher’s exact tests, with the aid of the GraphPad Instat 3.05 software, which was also used to estimate the odds ratio (OR) and its 95% confidence interval (CI).
RESULTS
Histopathologic analyses revealed 10 patients (13.5%) to exhibit ARE and 18 (24.3%) CAN. The remaining 46 patients (62.2%) showed no rejection; however, they displayed other histopathologic findings, including isolated fibrosis, acute tubular necrosis, interstitial inflammation, or recurrent kidney disease. We analyzed the INF-␥ ⫹874 polymorphism among kidney allograft recipients and healthy controls. There was no difference in the allele and genotype frequencies of INF-␥ ⫹874 polymorphism between healthy controls versus the rejection and nonrejection cohorts. We evaluated allele and genotype frequencies and Hardy–Weinberg equilibrium in Table 1 There was no departure from Hardy– Weinberg equilibrium. No differences (P ⬎ .05) were observed between kidney allograft recipients and healthy controls, indicating that the INF-␥ ⫹874 polymorphism was not correlated with kidney allograft outcomes. However, the analysis of the INF-␥ ⫹874 polymorphism in the rejection group indicated an increase in the homozygous genotype T/T (P ⫽ .0118) among the ARE cohort compared with healthy controls (P ⫽ .0118; OR, 7.095; 95% CI, 1.787–28.17; Table 2). Similarly, considering only patients with CAN compared with the healthy controls, there was an increase in the homozygous genotype T/T (P ⫽ .0067; OR, 5.321; 95% CI, 1.731–16.359; Table 2). Analysis of the rejection group showed a significant increase compared with the controls in the homozygous genotype T/T group (P ⫽ .0061; OR, 4.257; 95% CI, 1.588 –11.415; Table 2). DISCUSSION
It is well-recognized that ethnic background influences the outcome of or susceptibility to certain diseases.13 Cytokines
play an important role in the regulation of allograft outcomes. We investigated IFN-␥ polymorphism in the first intron among renal allograft recipients in a Brazilian population. Our aim was to investigate the relationship between cytokine gene polymorphism and ARE or CAN. We showed a significant association between the high INF-␥ genotype and susceptibility to ARE or CAN among the Brazilian population. Studies have shown that certain cytokine gene polymorphisms confers susceptibility to early acute rejection in adult heart, lung, and kidney transplant recipients. Turner et al18 reported that a combination of polymorphisms at ⫺308 in the TNF gene and at ⫺1082 in the interleukin (IL)-10 gene (high TNF/low IL-10) was associated with a greater level of acute graft rejection in adult heart transplant recipients.12,18 Similar findings were noted in pediatric heart transplant recipients.10 Among kidney allografts, Sankaran et al11 reported that high IL-10 and high TNF genotypes were each associated with a greater number of ARE. In pediatric heart transplant recipients, the INF-␥ ⫹874 polymorphism failed to show an association with the frequency of ARE.10 A previous study indicated that individuals homozygous for the allele 2; 12 CA repeat showed a high level of INF-␥ production.8,19 This allele 2 was later reported to be protective for arthritis in systemic lupus erythematosus.19 Because there was an absolute correlation between the presence of the ⫹874T allele and high-producing allele 2, our result confirms the previous observation regarding the protective role of INF-␥ on arthritis. It is interesting to note that this SNP coincides with the putative nuclear factorB– binding site, which may have a functional consequence for the INF-␥ gene.13 This observation strengthens the prior suggestion that genetic variations in INF-␥ expression influence the disease course rather than being a disease-causing gene. In addition, a positive association was observed between the amino acid polymorphism (Val14Met) within the INF-␥ receptor 1 gene and systemic lupus erythematosus.8 The genetic contribution to determine INF-␥ function may come from the receptor gene or a combination of cytokine and receptor genes. No significant differences were noted between the rejection and nonrejection groups regarding the T/T homozygous genotype. However, in the comparison with healthy controls, the rejection group was significantly increased in proportion among this group (P ⫽ .0061). Because the rejection group was composed of only 28 individuals, a larger sample would be required to prove
Table 1. Samples Sizes (n), INF-␥ ⴙ874 Polymorphism Genotype Frequencies, and Hardy–Weinberg Equilibrium Alleles
HWE
Genotypes
Posttransplantation Status
n
T
A
(P)
TT
AA
TA
Rejection group Acute rejection Chronic allograft nephropathy Nonrejection group Healthy controls
28 10 18 46 163
0.48 0.55 0.44 0.37 0.45
0.52 0.45 0.56 0.63 0.55
.2716 .2455 .6566 .0000 .1598
0.29 0.40 0.22 0.13 0.09
0.32 0.30 0.33 0.39 0.41
0.39 0.30 0.44 0.48 0.50
4508
CRISPIM, WASTOWSKI, RASSI ET AL
Table 2. Probability Values Obtained by Means of the 2-Sided Fisher’s Exact Test on the Comparisons of INF-␥ Allele/Genotype Frequencies Between Different Sample Groups Genotypes Posttransplantation Status
AR vs CAN AR vs NRG AR vs HC CAN vs NRG CAN vs HC RG vs NRG RG vs HC NRG vs HC
AA
AT
1.0000 0.7272 0.7418 0.7778 0.6179 0.6233 0.4104 0.8661
0.6888 0.4849 0.3299 1.0000 0.8046 0.6301 0.3118 0.8676
Allele TT
0.4004 0.0660 0.0118* 0.0805 0.0067† 0.1291 0.0061‡ 0.3961
A or T
0.5785 0.2075 0.0879 0.5459 0.2028 0.2281 0.0442§ 0.6195
Abbreviations: AR, acute rejection; CAN, chronic allograft nephropathy; RG, rejection group; NRG, nonrejection group; HC, healthy controls. *P ⬍ .05 (Fisher’s exact test; GraPhPad Instat). Odds ratio ⫽ 7.095 (95% CI, 1.787–28.175). † P ⬍ .05 (Fisher’s exact test; GraPhPad Instat). Odds ratio ⫽ 5.321 (95% CI, 1.731–16.359). ‡ P ⬍ .05 (Fisher’s exact test; GraPhPad Instat). Odds ratio ⫽ 4.257 (95% CI, 1.588 –11.415). § P ⬍ .05 (Fisher’s exact test; GraPhPad Instat). Odds ratio ⫽ 1.828 (95% CI, 1.031–3.240).
this association. Despite the smaller sample size of the ARE group (n ⫽ 10), the T/T homozygous genotype was significantly increased in proportion compared with healthy controls (P ⫽ .0118). Similarly, considering only the CAN group (n ⫽ 18), the homozygous genotype T/T (P ⫽ .0067) was significantly increased compared with healthy controls. This observation indicates that the T/T homozygous genotype may be a susceptibility factor at least for ARE and CAN. Another study by our group suggested that the T/T genotype may be associated with a better prognosis of systemic sclerosis, because this polymorphism seemed to favor INF-␥ gene transcription and consequently greater cytokine production.20 After cold ischemia and after approximately 20 –30 minutes of reperfusion, kidney tissue shows high glomerular and tubular mRNA expression of IL-2, IL-6, IL-10, transforming growth factor-1, platelet-derived growth factor-B, and IFN-␥.21 Our findings may result from the high INFG production associated with the T/T homozygous genotype of the INF-␥ gene.8 In fact, these findings are consistent with the view that INF-␥ is a proinflammatory Th1 type cytokine that upregulates cell-mediated responses, therefore favoring acute cellular rejection and CAN. Taken together, our data indicated that the INF-␥ ⫹874 polymorphism may be important for the outcome of kidney transplantations. REFERENCES 1. Davies JD, Martin G, Phillips J, et al: T cell regulation in adult transplantation tolerance. J Immunol 157:529, 1996
2. Nikolic B, Sykes M: Clonal deletion as a mechanism of transplantation tolerance. J Heart Lung Transplant 15:1171, 1996 3. Cripim JC, Wastowski IJ, Faggione LP, et al: Microchimerism evaluation in recipients of living-related or unrelated deceased allograft renal transplants. Transplant Proc 38:2828, 2006 4. Mendes-Júnior CT, Castelli EC, Simões RT, et al: HLA-G 14bp polymorphism at exon 8 in Amerindian populations from the Brazilian Amazon. Tissue Antigens 69:255, 2007 5. Di Giovine FS, Taskhsh E, Blakemore AIF, et al: Single base polymorphism at -511 in the human interleukin-1b gene (IL1b). Human Mol Gen 1:450, 1992 6. Kaminska D, Tyran B, Mazanowska O, et al: Cytokine gene expression in kidney allograft biopsies after donor brain death and ischemia-reperfusion injury using in situ reverse-transcription polymerase chain reaction analysis. Transplantation 84:1118, 2007 7. Perco P, Pleban C, Kainz A, et al: Gene expression and biomarkers in renal transplant ischemia reperfusion injury Transpl Int 20:2, 2007 8. Pravica V, Asderakis A, Perrey C, et al: In vitro production correlates with CA repeat polymorphism in the human interferon gene. Eur J Immunogen 26:1, 1999 9. Bidwell J, Keen L, Gallagher G, et al: Cytokine gene polymorphism in human disease: on-line databases, supplement 1. Genes Immun 2:61, 2001 10. Awad MR, Webber S, Boyle G, et al: The effect of cytokine gene polymorphisms on pediatric heart allograft outcome. J Heart Lung Transplant 20:625, 2001 11. Sankaran D, Asderakis A, Ashraf S, et al: Cytokine gene polymorphisms predict acute graft rejection following renal transplantation. Kidney Int 56:281, 1999 12. Hutchinson IV, Pravica V, Sinnott PJ: Genetic regulation of cytokines synthesis: consequences for acute and chronic organ allograft rejection. Graft 1:186, 1998 13. Kittles RA, Weiss KM: Race, ancestry, and genes: implications for defining disease risk. Annu Rev Genomics Hum Genet 4:33, 2003 14. Gendzekhadze K, Rivas-Vetencourt P, Montano RF: Risk of adverse post-transplant events after kidney allograft transplantation as predicted by CTLA-4 ⫹49 and TNF-alpha-308 single nucleotide polymorphisms: a preliminary study. Transpl Immunol 16:194, 2006 15. Racusen LC, Solezz K, Colvin RB, et al: The Bannf97 working classification of renal allograft pathology. Kidney Int 55:713, 1999 16. Higuchi R: Simple and rapid preparation of samples for PCR. In: Erlich HA, ed. PCR technology: Principles an applications for DNA amplification. New York: Stockton Press; 1989. p. 31 17. Raymond M, Rousset F: Genepop (Version-1.2): populationgenetics software for exact tests and ecumenicism. J Hered 86:248, 1995 18. Turner D, Grant SC, Yonan N, et al: Cytokine gene polymorphism and heart transplant rejection. Transplantation 64:776, 1997 19. Tangwattanachuleeporn M, Sodsai P, Avihingsanon Y, et al: Association of interferon-gamma gene polymorphism (⫹874A) with arthritis manifestation in SLE. Clin Rheumatol 26:1921, 2007 20. Wastowski IJ, Sampaio-Barros PD, Martelli-Palomino G, et al: Association of Interferon-gamma gene polymorphism (⫹874 T/A) with systemic sclerosis. Dis Markers 27:93, 2009 21. Kaminska D, Tyran B, Mazanowska O, et al: Gene expression of INF-gamma, IL- 10, IL-2, IL-6, PDGF-B, TGF-beta in kidney tissue after renal transplantation. Pol Merkur Lekarski 21:148, 2006