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The PTPN22 C1858T (R620W) Functional Polymorphism in Kidney Transplantation
The PTPN22 C1858T (R620W) Functional Polymorphism in Kidney Transplantation I. Sfar, Y. Gorgi, H. Aouadi, M. Maklouf, T. Ben Romdhane, S. Jendoubi-Ayed, R. Bardi, E. Abderrahim, T. Ben Abdallah, and K. Ayed ABSTRACT To investigate the association between kidney transplant rejection and PTPN22 (protein tyrosine phosphatase non-receptor 22) polymorphism, genomic DNA of 175 renal transplant recipients and 100 healthy blood donors were genotyped by restriction fragment length polymorphism-polymerase chain reaction. The patients were classified in two groups: G1 included 33 HLA-identical recipients and G2 included 142 with one or more HLA mismatches. Forty-nine patients developed an acute rejection episode (ARE): 8 in G1 and 41 in G2. The allelic frequencies of PTPN22 R620W revealed a significant difference between patients and controls. In fact, the W-allele was significantly more frequent in graft recipients than in blood donors (0.05 vs 0.01, P ⬍ .05). Furthermore, the frequency of this allele was increased in G1 patients with an ARE (0.188) compared with those without an ARE (0.040), but the difference was not statistically significant. Thus, we concluded that the PTPN22 W-variant allele could be involved in the susceptibility to acute allograft rejection in Tunisian kidney transplant patients.
T
RANSPLANTATION IS THE CURRENT TREATMENT of choice for end-stage renal disease. Unfortunately, acute and chronic rejection remain major causes of morbidity. The alloimmune response plays a key role in acute allograft rejection episodes (AREs), which and Tlymphocyte dependent.1 Genetic differences between donor and recipient HLA haplotypes are of major importance for transplant rejection. Other gene polymorphisms, coding for proteins involved in immune response, also appear to exert an influence on the immunologic mechanisms that lead to allograft loss. The PTPN22 (protein tyrosine phosphatase non-receptor 22) gene, located on chromosome 1p13, encodes lymphoid protein tyrosine phosphatase (LYP), which is important in the negative control of T-lymphocyte activation.2 LYP, an intracellular tyrosine phosphatase (PTP) expressed in T lymphocytes, is physically bound through a proline-rich motif to the SH3 C-terminal domain of the Src kinase (CSK). This complex is an important suppressor of lymphocyte-specific tyrosine kinase (Lck) and Fyn, a member of the Src family of tyrosine kinases, that mediate T-cell receptor signaling.3 Recently, C1858T polymorphism in the PTPN22 gene has been shown to be associated with a range of autoimmune diseases including: type 1 diabetes mellitus, systemic lupus erythematosus (SLE), and rheumatoid arthritis.4 The PTPN22 1858 C ¡ T SNP changes the amino © 2009 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 41, 657– 659 (2009)
acid at position 620 from an arginine (R) to a tryptophan (W), disrupting the interaction between LYP and CSK, presenting formation of the complex and, therefore, the suppression of T-cell activation.5 In vitro experiments have shown that the W-allele of PTPN22 binds less efficiently to CSK than the R-allele suggesting that T cells expressing the W-allele may be hyperresponsive. Consequently, individuals carrying this allele may be prone to allograft rejection. PATIENTS AND METHODS Patients and Controls The study included DNA samples from 100 sex-matched healthy controls and 175 renal transplant recipients (107 men and 68 women) whose mean age was 30 ⫾ 10.2 years. The grafts came from living donors in 140 cases (136 related and four unrelated donors) and a deceased donor in 35 cases. Among the 175 renal recipients, 33 patients received HLA-identical kidney transplantations (G1) and 142, grafts with one or more mismatches (G2). Forty-nine patients (28%) developed ARE within the first 6 From the Laboratory of Immunology (I.S., Y.G., H.A., M.M., T.B.R., S.J.-A., R.B., K.A.) and the Departement of Nephrology (E.A., T.B.A.), Charles Nicolle Hospital, Tunis, Tunisia. Address reprint requests to Dr Sfar Imen, Immunology Lab, Charles Nicolle Hospital, Bd 9 Avril, 1006 Tunis, Tunisia. E-mail: [email protected] 0041-1345/09/$–see front matter doi:10.1016/j.transproceed.2009.01.029 657
658
SFAR, GORGI, AOUADI ET AL Table 1. Demographics of Recipients
Mean age ⫾ standard deviation Sex Male Female Primary disease Chronic glomerulonephritis Hypertensive nephropathy Diabetes mellitus
G1 (n ⫽ 33)
G2 (n ⫽ 142)
P
31 ⫾ 10.2
29.5 ⫾ 10.14
NS NS
25 (75%) 8 (25%)
82 (58%) 60 (42%)
15 (45.4%) 15 (45.4%) 2 (6%)
43 (30.8%) 29 (20.4%) 11 (7%)
NS
months posttransplantation: 8 in group 1 (24.2%) and 41 in group 2 (28.8%). The ARE diagnosis was established by clinical, histological (Banff criteria), and biochemical assessment. The study was approved by the local ethics committee and informed consent was obtained from all subjects.
Methods Genomic DNA isolated from EDTA-anticoagulated peripheral blood samples of unrelated healthy blood donors and renal recipients was extracted by a salting-out process. Genotyping was performed using the restriction fragment length polymorphism-polymerase chain reaction (RPCR) method. To a 10-uL PCR volume containing 50 ng DNA, 10 pmol of each primer, 1 U of Taq polymerase (Promega), and 0.2 mmol/L of each desoxynucleoside triphosphate was added following primers: forward (5=-TGCCCATCCCACACTTTAT-3=) and reverse (5=-ACCTCCTGGGTTTGTACCTTA-3=). Thermal cycling was performed with an initial activation step at 95°C for 15 minutes, 35 cycles of denaturation at 94°C for 1 minute, annealing temperature 55°C for 1 minute, extension at 72°C for 1 minute, and a final extension at 72°C for 10 minutes. The PCR product was incubated with 1 U enzyme RsaI (Promega) in a 15-L volume at 37°C for 1.5 hours. The PCR generated a 326-bp fragment containing a restriction site for RsaI, which permited differentiation of the R620-allele (228 bp) and the 620W-allele (272 bp).
Statistical Analysis Allele frequencies were estimated by direct gene counting. Comparisons were performed between patients and controls using Fisher exact test with a P value ⬍ .05 considered to be significant.
RESULTS
No significant differences were observed between groups of patients for gender, age, and primary disease (Table 1). Table 2 summarizes the genotype and allele frequencies for
PTPN22 gene polymorphisms in donors and patients (G1 and G2 and in ARE allografts). PTPN22 W/R heterozygous genotype frequency showed a significant difference between renal recipients and healthy controls (P ⬍ .05). In addition, the W-allele frequency also showed a significant difference between the two groups (P ⬍ .05). However, in comparison with G2, patients of G1 displayed no significant difference in PTPN22 genotype or allele frequencies. Among G1 patients the W-allele frequency was more increased among AR recipients (0.188) than those patients without ARE (0.04); (Table 3). However, the difference was not statistically significant. DISCUSSION
A specific role of PTPN22 in T-cell regulation has been confirmed in knockout mice that show reduced thresholds for T-cell-receptor signaling.6 The PTPN risk-associated variant, W620, results in a gain of PTPN22 phosphatase activity in T cells, opening new avenues for exploring disease mechanisms.7 Despite the association of PTPN22 C1858T SNP with multiple autoimmune disorders, a role of this polymorphism in susceptibility to acute allograft rejection does not appear to be clear.8 In this study, we compared PTPN22 gene polymorphisms among a group of renal transplant recipients who experienced AREs and a cohort who had well-functioning renal grafts, as well as 100 healthy controls. PTPN22 W/R genotype and W-allele frequencies showed a significant difference between patients and controls. This observation might be explained by the fact that primary nephropathic disease is often due to an autoimmune disorder such SLE and diabetes mellitus, which have been characterized to involve PTPN22 in their pathophysiology.9 We also encountered an increased frequency of the W-allele among patients with acute rejection compared to nonrejectors in G1 recipients, but it was not significant. These results must be treated with caution because of the small number of ARE among HLA-identical kidney recipients. Further the increased incidence of episodes may be explained by the mismatching of HLA antigens in G2. However, despite these limitations, the present data might suggest a role of the W-allele to confer susceptibility to AREs. Further studies are needed to evaluate this association and determine the mechanisms by which this polymorphism affects the graft failure in kidney transplantation.
Table 2. Genotype and Allele Frequencies of PTPN22 Polymorphism in Controls and Patients Genotype Frequency (%)
Allele Frequency
Groups
n
R/R
W/R
W/W
R
W
P
Controls Patients G1 G2 AR (⫹) AR (⫺)
100 175 33 142 49 126
98 (98) 158 (90.3) 28 (84.8) 130 (91.5) 44 (89.8) 114 (90.5)
2 (2) 17 (9.7) 5 (15.2) 12 (8.5) 5 (10.2) 12 (9.5)
0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
0,990 0,950 0,924 0,957 0,948 0,952
0,010 0,050 0,076 0,043 0,052 0,048
P/C (P ⬍ .05)
AR, acute rejection.
G1/G2 (NS) AR⫹/AR⫺ (NS)
PTPN22 POLYMORPHISM
659
Table 3. Distribution of PTPN22 (C1858T) Genotypes in Renal Transplant Recipients With Acute Allograft Rejection G1 (n ⫽ 33) Genotypes (%)
R/R W/R W/W Alleles R W
G2 (n ⫽ 142)
n
AR⫹(n ⫽ 8)
AR⫺ (n ⫽ 25)
n
AR⫹ (n ⫽ 41)
AR⫺ (n ⫽ 101)
28 5 0
5 (17.8%) 3 (60%) 0
23 (82.2%) 2 (40%) 0
130 12 0
39 (30%) 2 (16%) 0
91 (70%) 10 (84%) 0
0.812 0.188
0.960 0.040
0.975 0.025
0.950 0.050
P
AR⫹/AR⫺ (NS)
AR, acute rejection.
REFERENCES 1. Dai Z, Lakkis FG: The role of cytokines, CTLA-4 and costimulation in transplant tolerance and rejection. Curr Opin Immunol 11:504, 1999 2. Cohen S, Dadi H, Shaoul E, et al: Cloning and characterization of a lymphoid-specific, inducible human protein tyrosine phosphatase LYP. Blood 93:2013, 1999 3. Cloutier JF, Veillette A: Cooperative inhibition of T-cell antigen receptor signalling by a complex between a kinase and a phosphatase. J Exp Med 189:111, 1999 4. Lee YH, Rho YH, Choi SJ, et al: The PTPN22 C1858T functional polymorphism and autoimmune diseases: a meta-analysis. Rheumatology 46:49, 2007
5. Begovich AB, Carlton VE, Honigberg LA, et al: A misesense single-nucleotide polymorphism in a gene encoding a protein tyrosine phosphatase (PTPN22) is associated with rheumatoid arthritis. Am J Hum Genet 75:330, 2004 6. Hasegawa K, Martin F, Huang G, et al: PEST domainenriched tyrosine phosphatase (PEP) regulation of effector/memory T cells. Science 303:685, 2004 7. Zhernakova A, Eerligh P, Wijmenga C: Differential association of the PTPN22 coding variant with autoimmune diseases in a dutch population. Genes Immun 6:459, 2005 8. Trivedi HL: Immunobiology of rejection and adaptation. Transplant Proc 39:647, 2007 9. Gumuscu SO, Buckner JH, Concannon P: A haplotype based analysis of the PTPN22 locus in type 1 diabetes. Diabetes 55:2883, 2006
T
RANSPLANTATION IS THE CURRENT TREATMENT of choice for end-stage renal disease. Unfortunately, acute and chronic rejection remain major causes of morbidity. The alloimmune response plays a key role in acute allograft rejection episodes (AREs), which and Tlymphocyte dependent.1 Genetic differences between donor and recipient HLA haplotypes are of major importance for transplant rejection. Other gene polymorphisms, coding for proteins involved in immune response, also appear to exert an influence on the immunologic mechanisms that lead to allograft loss. The PTPN22 (protein tyrosine phosphatase non-receptor 22) gene, located on chromosome 1p13, encodes lymphoid protein tyrosine phosphatase (LYP), which is important in the negative control of T-lymphocyte activation.2 LYP, an intracellular tyrosine phosphatase (PTP) expressed in T lymphocytes, is physically bound through a proline-rich motif to the SH3 C-terminal domain of the Src kinase (CSK). This complex is an important suppressor of lymphocyte-specific tyrosine kinase (Lck) and Fyn, a member of the Src family of tyrosine kinases, that mediate T-cell receptor signaling.3 Recently, C1858T polymorphism in the PTPN22 gene has been shown to be associated with a range of autoimmune diseases including: type 1 diabetes mellitus, systemic lupus erythematosus (SLE), and rheumatoid arthritis.4 The PTPN22 1858 C ¡ T SNP changes the amino © 2009 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 41, 657– 659 (2009)
acid at position 620 from an arginine (R) to a tryptophan (W), disrupting the interaction between LYP and CSK, presenting formation of the complex and, therefore, the suppression of T-cell activation.5 In vitro experiments have shown that the W-allele of PTPN22 binds less efficiently to CSK than the R-allele suggesting that T cells expressing the W-allele may be hyperresponsive. Consequently, individuals carrying this allele may be prone to allograft rejection. PATIENTS AND METHODS Patients and Controls The study included DNA samples from 100 sex-matched healthy controls and 175 renal transplant recipients (107 men and 68 women) whose mean age was 30 ⫾ 10.2 years. The grafts came from living donors in 140 cases (136 related and four unrelated donors) and a deceased donor in 35 cases. Among the 175 renal recipients, 33 patients received HLA-identical kidney transplantations (G1) and 142, grafts with one or more mismatches (G2). Forty-nine patients (28%) developed ARE within the first 6 From the Laboratory of Immunology (I.S., Y.G., H.A., M.M., T.B.R., S.J.-A., R.B., K.A.) and the Departement of Nephrology (E.A., T.B.A.), Charles Nicolle Hospital, Tunis, Tunisia. Address reprint requests to Dr Sfar Imen, Immunology Lab, Charles Nicolle Hospital, Bd 9 Avril, 1006 Tunis, Tunisia. E-mail: [email protected] 0041-1345/09/$–see front matter doi:10.1016/j.transproceed.2009.01.029 657
658
SFAR, GORGI, AOUADI ET AL Table 1. Demographics of Recipients
Mean age ⫾ standard deviation Sex Male Female Primary disease Chronic glomerulonephritis Hypertensive nephropathy Diabetes mellitus
G1 (n ⫽ 33)
G2 (n ⫽ 142)
P
31 ⫾ 10.2
29.5 ⫾ 10.14
NS NS
25 (75%) 8 (25%)
82 (58%) 60 (42%)
15 (45.4%) 15 (45.4%) 2 (6%)
43 (30.8%) 29 (20.4%) 11 (7%)
NS
months posttransplantation: 8 in group 1 (24.2%) and 41 in group 2 (28.8%). The ARE diagnosis was established by clinical, histological (Banff criteria), and biochemical assessment. The study was approved by the local ethics committee and informed consent was obtained from all subjects.
Methods Genomic DNA isolated from EDTA-anticoagulated peripheral blood samples of unrelated healthy blood donors and renal recipients was extracted by a salting-out process. Genotyping was performed using the restriction fragment length polymorphism-polymerase chain reaction (RPCR) method. To a 10-uL PCR volume containing 50 ng DNA, 10 pmol of each primer, 1 U of Taq polymerase (Promega), and 0.2 mmol/L of each desoxynucleoside triphosphate was added following primers: forward (5=-TGCCCATCCCACACTTTAT-3=) and reverse (5=-ACCTCCTGGGTTTGTACCTTA-3=). Thermal cycling was performed with an initial activation step at 95°C for 15 minutes, 35 cycles of denaturation at 94°C for 1 minute, annealing temperature 55°C for 1 minute, extension at 72°C for 1 minute, and a final extension at 72°C for 10 minutes. The PCR product was incubated with 1 U enzyme RsaI (Promega) in a 15-L volume at 37°C for 1.5 hours. The PCR generated a 326-bp fragment containing a restriction site for RsaI, which permited differentiation of the R620-allele (228 bp) and the 620W-allele (272 bp).
Statistical Analysis Allele frequencies were estimated by direct gene counting. Comparisons were performed between patients and controls using Fisher exact test with a P value ⬍ .05 considered to be significant.
RESULTS
No significant differences were observed between groups of patients for gender, age, and primary disease (Table 1). Table 2 summarizes the genotype and allele frequencies for
PTPN22 gene polymorphisms in donors and patients (G1 and G2 and in ARE allografts). PTPN22 W/R heterozygous genotype frequency showed a significant difference between renal recipients and healthy controls (P ⬍ .05). In addition, the W-allele frequency also showed a significant difference between the two groups (P ⬍ .05). However, in comparison with G2, patients of G1 displayed no significant difference in PTPN22 genotype or allele frequencies. Among G1 patients the W-allele frequency was more increased among AR recipients (0.188) than those patients without ARE (0.04); (Table 3). However, the difference was not statistically significant. DISCUSSION
A specific role of PTPN22 in T-cell regulation has been confirmed in knockout mice that show reduced thresholds for T-cell-receptor signaling.6 The PTPN risk-associated variant, W620, results in a gain of PTPN22 phosphatase activity in T cells, opening new avenues for exploring disease mechanisms.7 Despite the association of PTPN22 C1858T SNP with multiple autoimmune disorders, a role of this polymorphism in susceptibility to acute allograft rejection does not appear to be clear.8 In this study, we compared PTPN22 gene polymorphisms among a group of renal transplant recipients who experienced AREs and a cohort who had well-functioning renal grafts, as well as 100 healthy controls. PTPN22 W/R genotype and W-allele frequencies showed a significant difference between patients and controls. This observation might be explained by the fact that primary nephropathic disease is often due to an autoimmune disorder such SLE and diabetes mellitus, which have been characterized to involve PTPN22 in their pathophysiology.9 We also encountered an increased frequency of the W-allele among patients with acute rejection compared to nonrejectors in G1 recipients, but it was not significant. These results must be treated with caution because of the small number of ARE among HLA-identical kidney recipients. Further the increased incidence of episodes may be explained by the mismatching of HLA antigens in G2. However, despite these limitations, the present data might suggest a role of the W-allele to confer susceptibility to AREs. Further studies are needed to evaluate this association and determine the mechanisms by which this polymorphism affects the graft failure in kidney transplantation.
Table 2. Genotype and Allele Frequencies of PTPN22 Polymorphism in Controls and Patients Genotype Frequency (%)
Allele Frequency
Groups
n
R/R
W/R
W/W
R
W
P
Controls Patients G1 G2 AR (⫹) AR (⫺)
100 175 33 142 49 126
98 (98) 158 (90.3) 28 (84.8) 130 (91.5) 44 (89.8) 114 (90.5)
2 (2) 17 (9.7) 5 (15.2) 12 (8.5) 5 (10.2) 12 (9.5)
0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
0,990 0,950 0,924 0,957 0,948 0,952
0,010 0,050 0,076 0,043 0,052 0,048
P/C (P ⬍ .05)
AR, acute rejection.
G1/G2 (NS) AR⫹/AR⫺ (NS)
PTPN22 POLYMORPHISM
659
Table 3. Distribution of PTPN22 (C1858T) Genotypes in Renal Transplant Recipients With Acute Allograft Rejection G1 (n ⫽ 33) Genotypes (%)
R/R W/R W/W Alleles R W
G2 (n ⫽ 142)
n
AR⫹(n ⫽ 8)
AR⫺ (n ⫽ 25)
n
AR⫹ (n ⫽ 41)
AR⫺ (n ⫽ 101)
28 5 0
5 (17.8%) 3 (60%) 0
23 (82.2%) 2 (40%) 0
130 12 0
39 (30%) 2 (16%) 0
91 (70%) 10 (84%) 0
0.812 0.188
0.960 0.040
0.975 0.025
0.950 0.050
P
AR⫹/AR⫺ (NS)
AR, acute rejection.
REFERENCES 1. Dai Z, Lakkis FG: The role of cytokines, CTLA-4 and costimulation in transplant tolerance and rejection. Curr Opin Immunol 11:504, 1999 2. Cohen S, Dadi H, Shaoul E, et al: Cloning and characterization of a lymphoid-specific, inducible human protein tyrosine phosphatase LYP. Blood 93:2013, 1999 3. Cloutier JF, Veillette A: Cooperative inhibition of T-cell antigen receptor signalling by a complex between a kinase and a phosphatase. J Exp Med 189:111, 1999 4. Lee YH, Rho YH, Choi SJ, et al: The PTPN22 C1858T functional polymorphism and autoimmune diseases: a meta-analysis. Rheumatology 46:49, 2007
5. Begovich AB, Carlton VE, Honigberg LA, et al: A misesense single-nucleotide polymorphism in a gene encoding a protein tyrosine phosphatase (PTPN22) is associated with rheumatoid arthritis. Am J Hum Genet 75:330, 2004 6. Hasegawa K, Martin F, Huang G, et al: PEST domainenriched tyrosine phosphatase (PEP) regulation of effector/memory T cells. Science 303:685, 2004 7. Zhernakova A, Eerligh P, Wijmenga C: Differential association of the PTPN22 coding variant with autoimmune diseases in a dutch population. Genes Immun 6:459, 2005 8. Trivedi HL: Immunobiology of rejection and adaptation. Transplant Proc 39:647, 2007 9. Gumuscu SO, Buckner JH, Concannon P: A haplotype based analysis of the PTPN22 locus in type 1 diabetes. Diabetes 55:2883, 2006