The impact of CTLA4 and PTPN22 genes polymorphisms on long-term renal allograft function and transplant outcomes

Abstract

Cytotoxic T-Lymphocyte Antigen 4 (CTLA4) downregulates the immune system. Lymphoid tyrosine phosphatase (Lyp)—PTPN22 protein—is suggested to be negative regulator of T-cell reaction. There are several polymorphisms in the CTLA4 and PTPN22 genes, which can influence the immune response and allograft function after kidney transplantation. The aim of this study was to examine the impact of CTLA4 and PTPN22 genes polymorphisms on the long-term renal transplant function and recipients’ outcomes during a 5-year follow-up observation. The study enrolled 268 Caucasian renal transplant recipients. Genotyping of the rs231775 (+49AG) CTLA4 gene polymorphism was performed using real-time PCR and rs2476601 (C1858T) PTPN22 gene polymorphism using PCR-RFLP method. The 5-year graft survival rate was 81.7%. Dialysis was necessary in 22 (8%) patients, 7 (2.6%) patients died and 20 (7.4%) switched to another transplantation center. We found no association between studied polymorphisms and graft loss/dialysis. Comparison of the distribution of the +49AG CTLA4 and C1858T PTPN22 genes polymorphisms genotypes among dead and living patients showed no statistically significant differences. Above results suggest that the rs231775 (+49AG) CTLA4 and rs2476601 (C1858T) PTPN22 genes polymorphisms are not associated with long-term allograft failure, graft loss and mortality after transplantation.

• CTLA4
• gene
• polymorphism
• PTPN22
• renal transplant

Introduction

The pathogenesis of rejection after transplantation is the consequence of immune response activation, which results in graft tissue and endothelial injury.1,2 Antigen presentation triggers the inflammation process.3 However, signal transmitted to the T cell can vary. It may be either activating, which has a beneficial effect on the proliferation and diversification of lymphocytes and cytokines secretion (mainly IL-2, INF-γ and TNF), as well as may possess an inhibitory character, restraining the immune response. The effect of this action is the reduction of cytokines and other mediators release and peripheral tolerance increase.4

Cytotoxic T-Lymphocyte Antigen 4 (CTLA4) has a suppressive role in the lymphocyte activation process.5 This molecule is a member of immunoglobulin superfamily, expressed on the surface of T helper cells, encoded by CTLA4 gene, located on the short arm of the second chromosome (2q33).5 Lymphoid tyrosine phosphatase (Lyp) known as protein PTPN22 is suggested to be negative regulation of T-cell reaction through dephosphorylation of Src family kinases—Lck and Fyn, regulatory kinase Csk and other signaling molecules.6 The mechanism of this regulation remains unclear. PTPN22 is an intracellular enzyme, encoded by PTPN22 gene located on the long arm of chromosome 1 (1p13.3–p.13.1).7 The rate of CTLA4 and PTPN22 synthesis has genetic background. There are several polymorphisms in the CTLA4 and PTPN22 genes, which can influence the expression of these proteins and may therefore affect the immune response and allograft function after kidney transplantation.8,9 Those polymorphisms were associated with autoimmune diseases.10,11 However, the area of transplantation still needs further studies. Important matter is that the distribution of CTLA4 and PTPN22 gene polymorphisms varies in different populations. The frequency of the rs231775 (+49AG) CTLA4 gene polymorphism in European and Oriental populations has been recorded, but the frequency among different ethnic groups is yet to be established.12 The rs2476601 (C1858T) PTPN22 gene polymorphism is observed less frequently among Asian and African population7, while in the European population it occurs with higher frequency (Table 1).6

Table 1. The frequency distribution of the rs231775 (+49AG) CTLA4 and rs2476601 (C1858T) PTPN22 genes polymorphisms genotypes among different ethnic groups27–30.

	Genotypes frequency (%)
Polymorphism	rs231775 (+49AG) CTLA4
Ethnic group	AA	AG	GG
African Americans	33.5	54.5	12.0
Hispanic	37.5	52.5	10.0
Indians	50.0	40.5	9.5
Chinese	8.5	44.5	47.0
Central Europeans	36.5	50.0	13.5
Poles	34.57	48.7	16.73
Polymorphism	rs2476601 (C1858T) PTPN22
Ethnic group	CC	CT	TT
African Americans	97.0	3.0	–
Hispanic	91.78	7.54	0.68
White Americans	84.41	14.65	0.94
Scandinavians	80.3	18.9	0.8
Central Europeans	78	20	2.0
Poles	75.47	20.07	4.46

The aim of this study was to examine the impact of the rs231775 (+49AG) CTLA4 and rs2476601 (C1858T) PTPN22 gene polymorphisms on the long-term renal transplant function and recipients’ outcomes during a 5-year follow-up observation.

Material and methods

The study enrolled 268 Caucasian renal transplant recipients (165 males, 103 females, mean age 47.63 ± 12.96 years; transplantation performed between 2000 and 2006). The subjects were selected randomly from among first cadaveric renal transplant recipients. Recipients of more than one renal transplantation and with graft functioning for less than 6 months were excluded from the study. The longest period of observation lasted for 5 years in regard to 220 patients with functioning renal transplant. Patients were observed in the Clinical Department of Nephrology, Transplantology and Internal Medicine of the Pomeranian Medical University in Szczecin, Poland. The causes of renal failure were: chronic glomerulonephritis (58%), hypertension (9%), diabetes (9%), chronic pyelonephritis (5%), systemic lupus erythematosus (4%), autosomal dominant polycystic kidney disease (4%), vesico-ureteral reflux (3%) and Alport syndrome (1%). The cause of renal failure remained unknown in 7% of studied patients. The majority of patients (94%) needed hemodialysis before the transplantation, the rest of studied subjects were undergoing peritoneal dialysis (3%) or were qualified to preemptive kidney transplantation (3%). Concomitant diseases were: arterial hypertension, diabetes mellitus type 1 and 2, atherosclerosis, cardiovascular disease, autoimmune disorders, osteoarthritis and osteoporosis. The mean number of matching HLA-A and HLA-B alleles was 1.5 ± 0.8 (out of 4 possible) and the mean number of matching HLA-DR alleles was 1.2 ± 0.5 (out of 2 possible). The following parameters were recorded in each case: the recipient’s age and gender, delayed graft function, acute rejection13 and chronic allograft nephropathy,14 switching to another transplantation center, graft loss/return to dialysis, death of the patient. Blood samples were collected from all patients for genetic analysis at the start of the study and for creatinine concentrations evaluation 12, 24, 36, 48 and 60 months after kidney transplantation. Creatinine concentrations assessment was performed with colorimetric method. Deteriorating renal transplant function was verified with a biopsy, which was performed with PAJUNK DeltaCut Biopsy System (Geisingen, Germany). Biopsies were reviewed by a renal pathologist, and the Banff working classification criteria were used. All patients received standard immunosuppressive protocol with triple drug therapy including calcineurin inhibitor (cyclosporine A in 75%, or tacrolimus in 24% recipients), azathioprine (55%) or mycophenolate mofetil (37%) and steroids (91%). Informed consent was obtained from all patients. The local ethics committee of the Pomeranian Medical University in Szczecin, Poland, approved protocol of the study.

Genotyping

Genomic DNA was extracted (precipitation with trimethylammonium bromide salts) from leukocytes contained in 450 μL whole-blood samples with ethylenediaminetetraacetic acid (EDTA) as an anticoagulant using a nonorganic and non-enzymatic extraction method.15 DNA was then precipitated in 99.5% ethanol and dissolved in distilled water. We standardized the DNA with a Nanodrop ND-1000 spectrophotometer (Wilmington, DE) and equalized DNA concentrations to 20 ng/μL. This material was used as a matrix for the amplification in the real-time polymerase chain reaction (RT-PCR) for the CTLA4 gene polymorphism and restriction fragment length polymorphism (PCR-RFLP) for the PTPN22 gene polymorphism. We analyzed a single-nucleotide polymorphism (SNP): rs231775 (+49AG) of the CTLA4 gene with specific pair of primers – 5′-GCTCTACTTCCTGAAGACCT-3′ and 5′-AGTCTCACTCACCTTTGCAG-3′ and SNP: rs2476601 (C1858T) of the PTPN22 gene with specific pair of primers – 5′-ACCGCGCCCAGCCCTACTTTTG-3′ and 5′-AGCCACCATGCCCATCCCACACT-3′ in Master cycler Gradient (Eppendorf). The substrates for the first reaction were 40 ng of the genomic DNA, PCR buffer (10 mM Tris-HCl, 50 mM KCl, 0.08% Nonidet P40) (MBI Fermentas), dNTP (200 µM) (MBI Fermentas), MgCl₂ (1.5 mM) (MBI Fermentas), 4 pmol of sense primer and antisense primer (TIB MOLBIOL, Poznań), 0.5 U polymerase Taq (MBI Fermentas). The substrates for the second reaction were 40 ng of the genomic DNA, PCR buffer (QIAGEN), Q-Solution (QIAGEN), dNTP [200 µM] (MBI Fermentas), 4 pmol of sense primer and antisense primer (TIB MOLBIOL, Poznań), 1 U of polymerase Hot Star Taq (QIAGEN). The reactions were performed at 94 °C for 5 min, then 37 cycles of denaturation (94 °C for 20 s), annealing (25–60 °C for 40 s) and elongation (72 °C for 40 s). PCR products were separated through agarose gel electrophoresis with ethidium bromide as dye. The final step was the documentation of the gel by taking a photo with use of camera (DS-34 Direct Screen Camera, Minnetonka, MN) on Polaroid, under UV light (Transilluminator 4000, Stratagene, Heidelberg, Germany). Photos were scanned and saved in the form of graphic files with the extension jpeg.

Statistical analysis

The associations of studied genotypes with patients graft loss, return to dialysis and death were analyzed with univariate and multivariate Cox proportional hazards model. The serum concentrations of creatinine were compared between genotype groups with Kruskal–Wallis test followed by Mann–Whitney test. p-Value <0.05 was considered as statistically significant.

Results

After the period of 60 months, graft survival rate was 81.7%. Dialysis after transplantation was necessary in 22 (8%) patients, 7 (2.6%) patients died and 20 (7.4%) switched to another transplantation centre.

The genotype distribution of rs231775 (+49AG) CTLA4 gene polymorphism: 92 (34.3%) AA, 131 (48.9%) AG, 45 (16.8%) GG was consistent with Hardy–Weinberg equilibrium (p = 1.0). However, the distribution of rs2476601 (C1858T) PTPN22 genotypes, 202 (75.4%) CC, 54 (20.1%) CT, 12 (4.5%) TT, significantly deviated from Hardy–Weinberg equilibrium (p = 0.005) due to excess of TT homozygotes (12 observed vs. 6 expected).

Creatinine concentrations 12, 24, 36, 48 and 60 months after transplantation did not differ significantly between rs231775 (+49AG) CTLA4 gene polymorphism genotypes (p = 0.71, p = 0.65, p = 0.32, p = 0.71, p = 0.63, respectively) (Table 2). Similarly, there were no statistically significant differences between the rs2476601 (C1858T) PTPN22 gene polymorphism genotypes 12, 24, 36, 48 and 60 months after transplantation in regard to creatinine concentrations (p = 0.69, p = 0.77, p = 0.86, p = 0.93, p = 0.95, respectively) (Table 3).

Table 2. Creatinine serum concentrations after transplantation in patients stratified according to rs231775 CTLA4 gene polymorphism genotype.

	GG	AG	AA	K–W test	GG + AG vs. AA	GG vs. AA
Creatinine (mg/dL)	Mean ± SD	Mean ± SD	Mean ± SD	p	p*	p*
12 months	1.75 ± 0.53	1.77 ± 0.64	1.71 ± 0.55	0.71	0.53	0.42
24 months	1.75 ± 0.47	1.75 ± 0.59	1.68 ± 0.55	0.65	0.41	0.32
36 months	1.72 ± 0.45	1.76 ± 0.62	1.66 ± 0.62	0.32	0.14	0.17
48 months	1.74 ± 0.41	1.77 ± 0.66	1.76 ± 0.68	0.71	0.63	0.43
60 months	1.71 ± 0.49	1.76 ± 0.71	1.66 ± 0.62	0.63	0.35	0.44

p-Value calculated with the Kruskal–Wallis (K–W) and Mann–Whitney* tests; SD, standard deviation.

Table 3. Creatinine serum concentrations after transplantation in patients stratified according to rs2476601 PTPN22 gene polymorphism genotype.

	TT	CT	CC	K–W test	TT + CT vs. CC	TT vs. CC
Creatinine (mg/dL)	Mean ± SD	Mean ± SD	Mean ± SD	p	p*	p*
12 months	1.61 ± 0.53	1.75 ± 0.48	1.76 ± 0.62	0.69	0.85	0.50
24 months	1.69 ± 0.63	1.77 ± 0.55	1.72 ± 0.55	0.77	0.52	0.99
36 months	1.74 ± 0.62	1.67 ± 0.49	1.73 ± 0.62	0.86	0.96	0.69
48 months	1.72 ± 0.60	1.75 ± 0.55	1.76 ± 0.66	0.93	0.71	0.83
60 months	1.64 ± 0.69	1.75 ± 0.66	1.71 ± 0.64	0.95	0.81	0.92

p-Value calculated with the Kruskal–Wallis (K–W) and Mann–Whitney* tests; SD, standard deviation.

Univariate Cox regression analysis showed no statistically significant associations between the risk of graft loss/dialysis and rs231775 (+49AG) CTLA4 genotype (HR = 0.896, 95% CI = 0.376–2.137, p = 0.81 for GG+AG vs. AA) or rs2476601 (C1858T) PTPN22 genotype (HR = 0.659, 95% CI = 0.223–1.946, p = 0.45 for TT+CT vs. CC). Multivariate Cox regression model adjusted for recipients’ age and gender also revealed no association between studied polymorphisms and graft loss/dialysis (Table 4). Similarly, univariate Cox regression analysis showed no statistically significant associations between the risk of patient’s death and rs231775 (+49AG) CTLA4 genotype (HR = 0.694, 95% CI = 0.155–3.101, p = 0.63 for GG+AG vs. AA) or rs2476601 (C1858T) PTPN22 genotype (HR = 0.497, 95% CI = 0.060–4.132, p = 0.52 for TT+CT vs. CC). There was no statistical significance of studied gene polymorphisms as risk factors of death after transplantation in multivariate Cox regression analysis adjusted for recipients’ age and gender (Table 5).

Table 4. Multivariate Cox regression model for the risk of graft loss/dialysis after transplantation.

Risk factor	HR	−95% CI	+95% CI	p
Male gender	0.589	0.250	1.392	0.23
Age (years)	0.962	0.929	0.996	0.03
rs231775 CTLA4 (GG+AG vs. AA)	0.909	0.378	2.187	0.83
rs2476601 PTPN22 (TT+CT vs. CC)	0.806	0.270	2.406	0.70

HR, hazard ratio.

Table 5. Multivariate Cox regression model for the risk of death after transplantation.

Risk factor	HR	−95% CI	+95% CI	p
Male gender	1.479	0.277	7.890	0.65
Age (years)	1.030	0.968	1.096	0.34
rs231775 CTLA4 (GG+AG vs. AA)	0.678	0.149	3.081	0.61
rs2476601 PTPN22 (TT+CT vs. CC)	0.441	0.052	3.704	0.45

HR, hazard ratio.

Discussion

In this study, we examined the impact of CTLA4 and PTPN22 gene polymorphisms on the long-term renal transplant function and recipients outcomes during a 5-year follow-up observation. The studied polymorphisms were not associated with creatinine concentrations, dialysis frequency or mortality after transplantation.

Antigen presentation is crucial for immune response after kidney transplantation. Interaction of the T cell with antigen-presenting cell (APC) requires signal transmission. Reaction of co-stimulant molecules, CD28, CTLA4 (CD152), PD-1, CD40L, ICOS, CD134, with complementary ligands on APC, B7.1 (CD80), B7.2 (CD86), PDL1, PDL2, OX40L, B7RP1, CD40 (CD154), is a part of the transmission. This signal can modulate the inflammatory process.3 Knockout CTLA4 gene mice died, because of massive lymphoproliferation and multiorgan tissue destruction.16 Clonal expansion by CTLA4 was observed in vivo.17 In experimental study, monoclonal antibodies (MAbs) against CTLA were used and it was stated that the inhibition of interaction between CTLA4 and B.7 results with increased activation of previously stimulated (through anti-CD3 and anti-CD28 MAbs) T cells.18 The distribution of three CTLA4 gene polymorphisms, which can change the expression of CTLA-4, were examined. There were differences in the genotypes and alleles distribution among rejectors and nonrejectors of allogenic kidney transplant recipients.19 The impact of CTLA4 gene polymorphisms on the kidney transplantation outcome was also evaluated, but there was no relationship between the allele variants and acute rejection or graft function.20 There are studies indicating that good long-term graft function promoted by treatment with CTLA4-Ig can be the result of reduced number of donor reactive cells and reduction in clone expansion.21 However, in our study the rs231775 (+49AG) CTLA4 gene polymorphism was not associated with graft function or graft survival.

Suppressive role of PTPN22 is based on a complex mechanism. Negative regulatory kinase Csk together with Lyp reduces activity of the Src family kinases and Zeta-chain-associated protein kinase 70 (ZAP-70).22 Moreover, Lyp dephosphorylates Src family tyrosine kinases (Lck and Fyn) directly and affects the ZAP-70 along with immunoreceptor tyrosine-based activation motif (ITAM). This pathway stops the T-cell receptor signaling and inhibits the immunological response.23 There is another, still not well-known mechanism of PTPN22 interaction with proteins involved in cell signaling—Growth factor receptor-bound protein 2 (Grb2) and Casitas B-lineage Lymphoma (Cbl). The result of these proteins activation is ubiquitination cell transformation and proliferation.24,25 A retrospective meta-analysis covering 39 studies evaluating the association between the functional rs2476601 (C1858T) polymorphism of the PTPN22 gene and autoimmune-based diseases was conducted. Allele T was significantly associated with rheumatoid arthritis, systemic lupus, diabetes mellitus type 1 and Graves’ disease. There was no association of studied polymorphism with psoriasis, multiple sclerosis or Addison’s disease.26 A study on Tunisian patients after kidney transplantation investigated the relationship between rs2476601 (C1858T) PTPN22 gene polymorphism and occurrence of acute rejection. The frequency of T allele was increased in HLA-identical individuals with acute rejection compared to those without acute rejection episode, but this difference was not statistically significant.24 In our earlier study, we also found no correlation between rs2476601 (C1858T) PTPN22 gene polymorphism and acute rejection, delayed graft function or chronic allograft nephropathy.9 In the current study, we did not analyze the cause of adverse graft outcome, but the total number of patients requiring dialysis after transplantation was too low to allow stratification into subgroups with different rejection types and other causes of graft failure. Further studies should also analyze potential associations between CTLA4 and PTPN22 genes polymorphism and the number or function of T cells circulating in recipients. Above results suggest that the rs231775 (+49AG) CTLA4 and rs2476601 (C1858T) PTPN22 genes polymorphisms are not associated with long-term allograft failure, graft loss and mortality after transplantation.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.