Original Clinical Science
Association Between a Gain-of-Function Variant of PTPN22 and Rejection in Liver Transplantation Raphael Dullin,1 Martina Koch,1 Martina Sterneck,2 Björn Nashan,1 and Hansjörg Thude1 Background. The protein tyrosine phosphatase nonreceptor 22 gene (PTPN22) encodes a strong T-cell regulator called lymphoid protein tyrosine phosphatase. Previously, PTPN22 was described as a susceptibility gene for autoimmunity because it contains single nucleotide polymorphisms (SNPs) associated with several autoimmune diseases. One SNP (rs2476601; 1858G>A) has emerged as a particularly potent risk factor for autoimmunity. We address the question whether PTPN22 polymorphisms are also associated with acute rejection after liver transplantation. Methods. We investigated the influence of six PTPN22 SNPs on the susceptibility to acute liver allograft rejection. Consequently, we carried out a retrospective study genotyping 345 German liver recipients at six SNP loci, which include rs2488457 (−1123G>C), rs33996649 (788C>T), rs2476601 (1858G>A), rs1310182 (−852A>G), rs1217388 (−2200G>A), rs3789604 (64434T>G). Our study enrolled 165 recipients who did not develop rejection, 123 who showed one rejection episode, and 57 patients who suffered from multiple acute rejections after transplantation. Results. The 1858A allele containing genotypes (GA+AA) and the 1858A allele had a significantly higher frequency in the group of patients with multiple rejection episodes (35.1% and 18.4%) compared to rejection-free patients (15.8% and 7.9%; P=0.022 and 0.023). In contrast, we could not detect any association between rejection and the other tested SNPs. Additionally, we identified one haplotype contributing to risk of multiple rejections, however, exhibiting no stronger impact than the 1858A allele alone. Conclusion. We conclude that the 1858G>A SNP may confer susceptibility to multiple acute liver transplant rejections in the German population.
(Transplantation 2015;99: 431–437)
L
iver transplantation is the lifesaving treatment of choice for end-stage liver disease. In spite of well-developed immunosuppressive therapy, transplant rejection still occurs. Acute allograft rejection episodes are alloimmune responses, which usually occur within the first 6 months after transplantation and are mostly based on T cell reactivity.1). The T cell reactivity is controlled mainly by the activity of kinases and phosphatases.2 One negative key regulator is coded by the protein tyrosine phosphatase nonreceptor type 22 gene (PTPN22). The PTPN22 is located on chromosome 1 (1p13) Received 27 November 2013. Revision requested 17 December 2013. Accepted 21 May 2014. 1
Department of Hepatobiliary and Transplant Surgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
2
Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
This work was supported by grants from the Deutsche Forschungsgemeinschaft (“Entzündung und Regeneration—Integriertes Graduiertenkolleg im SFB 841 Leberentzündung”). The authors declare no conflicts of interest. R.D. wrote the article, collected data, analyzed data, and performed the study. M.K., M.S., and B.N. designed the study and contributed patients. H.T. performed the study, analyzed the data, designed the study, and wrote the article. Correspondence: Hansjörg Thude, Universitätsklinikum Hamburg-Eppendorf, Zentrum Innere Medizin, Klinik für Hepatobiliäre Chirurgie und Transplantationschirurgie, Martinistraße 52, 20246 Hamburg, Germany. ([email protected]) Copyright © 2014 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 0041-1337/15/9902-431 DOI: 10.1097/TP.0000000000000313
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and encodes the multiphosphatase lymphoid-specific protein tyrosine phosphatase (LYP), which is predominantly found in the cytoplasm. This protein exhibits a molecular weight of 105 kDa and consists of 807 amino acids. In addition to the catalytic domain, LYP also features four C-terminal proline-rich motifs (P1-4), which operate as binding sites for cytoplasmic adaptor proteins. Lymphoidspecific protein tyrosine phosphatase dephosphorylates numerous signal adaptor proteins at their tyrosine residues, finally to decrease the level of T cell activation. Well-known substrates of LYP are immunoreceptor tyrosine-based activation motifs, such as the T cell receptor ζ and CD3ε, the Src family kinase LCK, the Syk family kinase ZAP-70, the protooncogene Vav, and VCP.3 The Src homology 3 domain of the C-terminal Src kinase (CSK) binds the P1 region of LYP.4 Actually, both LYP and CSK regulate the T-cell activation in a synergistic manner because CSK phosphorylates a C-terminal inhibitory tyrosine residue of Src family kinases, causing an additional reduction of activity.5,6 However, Vang et al. pointed out yet another role of CSK. They observed that the dissociation of the LYP-CSK complex is important to locate LYP near the cell membrane to dephosphorylate its membrane-associated substrates, especially LSK and T cell receptor ζ7 resulting in downregulation of T-cell activation. Single nucleotide polymorphisms (SNP) are contained in most genes. So far, over six hundreds have been identified (NCBI’s SNP database) in the PTPN22 gene. Six important SNPs of the PTPN22 gene are displayed in Figure 1. The first SNP, rs2488457 (-1123G>C), lies in a putative transcription factor binding site (TFBS) for the activator www.transplantjournal.com
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FIGURE 1. Schematic gene map of PTPN22 gene including the selected SNP loci (vertical arrows), UTR (empty rectangles), exons (filled rectangles), introns (horizontal lines) 34×4 mm (600×600 DPI). UTR, untranslated regions.
protein 4 (AP-4) within the promoter region8 and influences the expression level of LYP, concerning the results of Chen et al.9 Association studies have described a susceptibility to contract type 1 diabetes (T1D),10,11 rheumatoid arthritis (RA),12,13 autoreactive urticaria,14 and ulcerative colitis.9 The second SNP, rs33996649 (788C>T), is a loss-offunction missense point mutation (R263Q)15 located in exon 10 operating as part of the catalytic domain. A protective contribution to autoimmunity was shown in at least three independent studies.15-17 Rs2476601 (1858G>A) is also a missense point mutation. However, in contrast to 788C>T, it belongs to the category associated with a gain of function.18 An arginine is substituted by tryptophan in exon 14, which functions as part of the P1 linkage domain. Hence, this LYP mutant has a threefold decrease in affinity to CSK and consequently an increased
enzyme activity (19). In spite of the gain of function, the SNP 1858G>A is strongly associated with T1D,8,19-22 RA,12,23 systemic lupus erythematosus,23,24 vitiligo,25 Addison’s disease,26 Wegener’s granulomatosis,27 progressive systemic sclerosis,16 Grave’s disease,28 and alopecia areata.29 The next depicted SNPs, rs1310182 (-852A>G) and rs1217388 (-2200G>A), are point mutations in intronic regions carrying putative TFBS, described by Carlton et al.30 This group detected an association of both SNPs with RA and two other groups found an association to T1D.31,32 The last selected SNP, rs3789604 (64434T>G), is part of a TFBS controlling the level of LYP expression.30 Some studies have reported an association with Grave’s disease33 and psoriasis.34,35 Because selected mutations have functional consequences and confer an increased risk to several autoimmune diseases,
TABLE 1.
Characteristics of liver transplant recipients collectives No rejection n=165
Sex (m/f) 101 (61%)/64 (39%) Age (mean yr±SD) 53±11 Primary liver disease Alcoholic cirrhosis 50 (30%) Cirrhosis after HCV 31 (19%) Cirrhosis after HBV 16 (10%) Primary biliary cirrhosis 8 (5%) Primary sclerosing cholangitis 13 (8%) Wilson disease 3 (2%) α1-AT deficiency 2 (1%) Budd-Chiari syndrome 3 (2%) Cryptogenic cirrhosis 11 (7%) (Acute) liver failure 6 (4%) ADPKD 6 (4%) Extrahepatic ductopenia 1 (1%) Inborn errors of metabolism 2 (1%) Autoimmune hepatitis 4 (2%) Others 9 (5%) Additional HCC 42 (25%) Immunosuppressive treatment at the time of discharge Double regimen CNI+STER 59 (36%) Steroid-free (CNI+mTORI/MMF/AZA) 9 (5%) CNI-free (mTORI/MMF/AZA+STER) 3 (2%) Triple regimen CNI+mTORI/MMF/AZA+STER 88 (53%) Others 6 (4%)
Rejection (single)
Rejection (multiple)
n=123
P
n=57
P
77 (63%)/46 (37%) 50±13
NS 0.024
34 (60%)/23 (40%) 45±17
NS 0.001
34 (28%) 22 (18%) 7 (6%) 3 (2%) 15 (12%) 0 (0%) 2 (2%) 2 (2%) 7 (6%) 16 (13%) 2 (2%) 2 (2%) 2 (2%) 2 (2%) 7 (6%) 22 (18%)
NS NS NS NS NS NS NS NS NS 0.006 NS NS NS NS NS NS
11 (19%) 7 (12%) 8 (14%) 4 (7%) 5 (9%) 1 (2%) 1 (2%) 1 (2%) 4 (7%) 5 (9%) 0 (0%) 2 (4%) 3 (5%) 2 (4%) 3 (5%) 11 (19%)
NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS
53 (43%) 2 (2%) 3 (2%)
NS NS NS
21 (37%) 2 (4%) 3 (5%)
NS NS NS
63 (51%) 2 (2%)
NS NS
29 (51%) 2 (4%)
NS NS
Mean length of follow-up=8.5 years. Further genotype and allele frequency comparisons (P values, OR, and 95% CI) were adjusted for significant differences shown in this table. Percentages approximated. SD, Standard deviation; AT; alpha 1-antitrypsin; ADPKD, autosomal dominant polycystic kidney disease; HCC, hepatocellular carcinoma; CNI, calcineurin inhibitors; STER, corticosteroids; mTORI, mTOR inhibitors; MMF, mycophenolate mofetil; AZA, azathioprine; NS, not significant; HCV, hepatitis C virus; HBV, hepatitis B virus; OR, odds ratio; 95% CI, 95% confidence interval.
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a predisposing effect to rejection episodes after liver transplantation is also probable. The aim of the study is to clarify whether an association with acute rejection episodes after liver transplantation exists. Therefore, we examined the SNPs in 345 German liver transplant recipients to test our hypothesis. RESULTS The demographic and clinical data of the liver transplant recipients without any rejection (n=165), with at least one (n=123) and with more than one rejection episode (n=57) are summarized in Table 1. When patients without and patients with multiple rejection episodes were compared, the data differed in age (53±11 years vs. 45±17 years; P=0.001). Demographic and clinical determinants between patients without rejection and patients who exhibited one rejection episode displayed significant differences in the age (53±11 years vs. 50±13 years; P=0.024) and in the prevalence of acute liver failure as reason for transplantation (4% vs. 13%; P=0.006). Considering the number of retransplanted recipients in the cohorts, we obtained the following results. The cohort of
patients without rejection contains 14 (8.5%) retransplanted patients. Twelve (9.8%) retransplanted recipients were in the group of patients with one rejection episode (compared to the rejection free group: P=0.836), and in the group with multiple rejections, we found 12 (21.1%) retransplanted individuals (compared to the rejection-free group: P=0.016). The complete genotyping results for the three groups are shown in Table 2. Genotype data of each analyzed group did not deviate from the Hardy-Weinberg equilibrium at all six analyzed polymorphic sites (data not shown). Sequencing conferred concordance with polymerase chain reaction with allele-specific restriction analysis (PCR-ASRA) results. Frequency analysis revealed that the SNPs rs2488457, rs3996649, rs1310182, rs1217388, and rs3789604 were not associated with rejection risk at all. We could not observe any significant differences for patients with one and for patients with more than one rejection episode when they were each compared to rejection-free individuals. SNP rs2476601 (1858G>A) did not differ significantly in genotype (P=0.177) and allele frequency (P=0.151) between patients without and patients with a single rejection episode as well. In contrast, a significant difference between patients without and patients
TABLE 2.
Genotype and allele frequencies of the different collectives of liver transplant recipients
PTPN22 SNP
rs2488457 −1123G>C
rs3996649 788C>T
rs2476601 1858G>A
rs1310182 −852A>G
rs1217388 −2200G>A
rs3789604 64434T>G
GG GC CC G C CC CT TT C T GG GA AA G A AA AG GG A G GG GA AA G A TT TG GG T G
No rejection
Rejection (single)
n=165
n=123
10 (6.1%) 64 (38.8%) 91 (55.2%) 84 (25.5%) 246 (74.5%) 160 (97.0%) 5 (3.0%) 0 (0%) 325 (98.5%) 5 (1.5%) 139 (84.2%) 26 (15.8%) 0 (0.0%) 304 (92.1%) 26 (7.9%) 41 (24.8%) 79 (47.9%) 45 (27.3%) 161 (48.8%) 169 (51.2%) 10 (6.1%) 58 (35.2%) 97 (58.8%) 78 (23.6%) 252 (76.4%) 111 (67.3%) 53 (32.1%) 1 (0.6%) 275 (83.3%) 55 (16.7%)
6 (4.9%) 60 (48.8%) 57 (46.3%) 72 (29.3%) 174 (70.7%) 118 (95.9%) 5 (4.1%) 0 (0%) 241 (98.0%) 5 (2.0%) 95 (77.2%) 27 (22.0%) 1 (0.8%) 217 (88.2%) 29 (11.8%) 29 (23.6%) 67 (54.5%) 27 (22.0%) 125 (50.8%) 121 (49.2%) 6 (4.9%) 54 (43.9%) 63 (51.2%) 66 (26.8%) 180 (73.2%) 80 (65.0%) 40 (32.5%) 3 (2.4%) 200 (81.3%) 46 (18.7%)
Rejection (multiple) P c
NS
NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS
n=57
P
3 (5.3%) 26 (45.6%) 28 (49.1%) 32 (28.1%) 82 (71.9%) 56 (98.2%) 1 (1.8%) 0 (0%) 113 (99.1%) 1 (0.9%) 37 (64.9%) 19 (33.3%) 1 (1.8%) 93 (81.6%) 21 (18.4%) 9 (15.8%) 33 (57.9%) 15 (26.3%) 51 (44.7%) 63 (55.3%) 3 (5.3%) 26 (45.6%) 28 (49.1%) 32 (28.1%) 82 (71.9%) 43 (75.4%) 14 (24.6%) 0 (0%) 100 (87.7%) 14 (12,3%)
NS
ORa (95CIb)
NS NS NS NS NS 0.022
2.329 (1.132-4.792)
NS 0.023
2.135 (1.109-4.109)
NS NS NS NS NS NS NS NS NS
Test conditions: allele A (major allele) versus allele B (minor allele); AA+AB versus BB (dominant model); AA versus AB+BB (recessive model). Percentages approximated; P values, OR and 95CI were adjusted for clinical data effects via logistic regression. OR, odds ratio; 95% CI, 95% confidence interval; NS, not significant.
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with multiple rejection episodes could be detected. The results demonstrated that the A allele containing genotypes were overrepresented in recipients who rejected more than once (35.1%) compared to recipients without rejection (15.8%). This difference achieved statistical significance in a recessive inheritance (recessive model GA+AA vs. GG, odds ratio [OR], 2.329, 95% confidence interval [95% CI], 1.132-4.792; P=0.022). The power of this result was above 80%. Concerning the allele distribution, a similar predication was found. The A allele frequencies were 18.4% and 7.9% in patients with more than one rejection and patients without rejection, respectively (A vs. G; OR, 2.135; 95% CI, 1.109-4.109; P=0.023). The power reached also a value of over 80%. We found one homozygous AA carrier in each rejection group. Subsequently, a haplotype analysis was performed based on statistically significant differences in 1858G>A genotypes and alleles. Estimated haplotype frequencies and P values are summarized in Table 3. We obtained 19 haplotypes, five of which had an overall frequency above 5%. One haplotype carrying the 1858A risk allele was significantly overrepresented in patients with multiple rejections compared to patients without rejection (G-C-A-A-G-T, 13.7% vs. 7.0%; OR, 2.281; 95% CI, 1.074-4.754; P=0.023).
Both rejection cohorts were significantly younger than the group of rejection-free patients (P=0.024 for single, P=0.001 for multiple rejection episodes). This phenomenon could be explained by the terminus immunosenescence, which is an agerelated weakening of immune functions resulting in lower rates of acute allograft rejections.37 Indeed, it was shown that costimulatory signal pathways are reduced in elderly individuals.38 The analysis of rs2488457 (-1123G>C), rs3996649 (788C>T), rs1310182 (-852A>G), rs1217388 (-2200G>A), and rs3789604 (64434T>G) did not provide a significant correlation—neither with single nor with multiple rejection. Thus, our data suggest that these SNPs are of minor importance for liver transplant rejection. In contrast, previous studies have sporadically shown an association with various autoimmune diseases, though these results are heterogeneous.8-17,30-35 In autoreactivity, autoantibodies and autoreactive T lymphocytes could target several self-proteins, whereas in alloreactivity (rejection) T cells respond directly to foreign human leukocyte antigen molecules on the graft cells’ surface. Additionally, autoimmune reactions are regarded as pathologic, whereas alloimmune reactions use the mechanisms of physiologic pathogen defense.39 Because of these differences, the comparability with previously mentioned autoimmunity studies is limited. In spite of inconsistent results in literature, the point mutation rs2476601 (1858G>A) was consequently established as a risk factor for numerous autoimmune diseases,8,12,16,19-29 except for the Asian population.10,11,13,33 The disease predisposing allele A leads to a gain of function in a dominant manner.18 Interestingly, we also observed the influence of 1858G>A in the recessive model (GA+AA vs. GG) exclusively, where the A allele is assumed to be dominant. Because of the gain of function, a higher regulatory activity of LYP increases the risk for developing autoimmune diseases particularly mediated by hyperreactive T cells. Two theories attempt to solve this contradiction. Firstly, the survival of autoreactive T cells in the thymus is better if their response to autoantigens is weaker resulting from higher LYP activity. Next, regulatory T cells (Tregs) also express LYP and are inhibited in their function.40 Our results illuminate a significant correlation between carrying the A allele and the risk for multiple acute rejections after liver transplantation. Because we could detect only one AA genotype in each rejection group, the GA genotype distribution made the majority of the difference. The frequency of the genotypes GA plus AA within this group was more than twice as high as that of the comparison group (35.1% vs. 15.8%). Also, A-carriers had a risk for multiple rejections
Transplantation
DISCUSSION Lymphoid-specific protein tyrosine phosphatase encoded by PTPN22 gene acts as a strong downregulator of T-cell activity. The PTPN22 characterizes one of the most important autoimmunity risk genes, only human leukocyte antigen genes have a higher impact toward autoimmune diseases.36 This result is derived from numerous studies performed worldwide concerning autoimmune diseases.8-17,19-35 In context of transplantation, PTPN22 SNP data are limited, and the impact of SNPs in PTPN22 gene on liver allotransplant rejection is still unclear until now. Autoimmune diseases are mainly characterized by a disregulated hyperreactive immune system. The described association between PTPN22 SNPs and hyperreactive immune systems found in autoimmune diseases prompted us to address the question whether PTPN22 SNPs are potential genetic biomarkers for predicting the risk of rejecting a liver allotransplant. By genotyping 345 liver recipients divided in patients without biopsy, patients with one biopsy, and patients with more than one biopsy proven rejection episode, we examined the influence of these SNPs on the risk of rejecting a liver allotransplant. TABLE 3.
Results of haplotype analysis
PTPN22 haplotype
C-C-G-G-A-T C-C-G-A-A-G G-C-G-A-G-T G-C-A-A-G-T C-C-G-A-A-T
No rejection
Rejection (multiple)
Overall frequency
n=330
n=114
P
47.6% 14.1% 11.9% 8.5% 6.0%
154.5 (46.5%) 49.7 (15.0%) 40.1 (12.1%) 22.3 (7.0%) 23.1 (6.9%)
56.8 (49.9%) 13.1 (11.5%) 12.6 (11.1%) 15.6 (13.7%) 3.7 (3.2%)
NS NS NS 0.023 NS
Haplotype SNP order: rs2488457-rs33996649-rs2476601-rs1310182-rs1217388-rs3789604; haplotypes with overall frequency beneath 5% are not shown. OR, odds ratio; 95% CI, 95% confidence interval; NS, not significant.
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OR (95% CI)
2.281 (1.074–4.754)
Dullin et al
© 2015 Wolters Kluwer
increased by more than the double (OR=2.329). The comparison of genotype and allele distributions of recipients with single and recipients without rejection showed a tendency (22.8% vs. 15.8% for GA+AA genotypes, 11.8% vs. 7.9% for A alleles) lacking statistical significance. If a larger cohort of patients is tested, this effect will probably become significant. In clinical practice, single liver transplant rejection episodes can be treated very well with corticosteroid bolus therapy. If rejection does not recur, it will not provoke any complications such as side effects of elevated immunosuppressive drug dosage and allograft tissue damage. However, multiple rejections indicate an increased immune reactivity against the allograft, which can merely be overcome through adapting the immunosuppressive therapy, otherwise more rejection episodes after damage of the organ threaten the patient. In turn, this could lead to an elevated retransplantation rate among patients with multiple rejection episodes, according to our results (21.1% vs. 8.5% retransplanted recipients, P=0.016). Furthermore, our data show that 1858G>A is particularly associated with this elevated alloimmune reactivity, which could be resulted from Tregs inhibited by LYP with a gain of function. Functional tests have demonstrated that PTPN22−/− mice have more active Tregs, showing that LYP is also a key regulator in Tregs.41 Two groups from Poland42 and Tunisia43 investigated the influence of 1858G>A on acute kidney transplant rejection, detecting no significant association. The reason why their results differ from ours in spite of similar sample sizes could only be speculated. The frequency of the 1858A allele in Europe is decreasing from north to south.40 Truly, only about 4% of the alleles found by Sfar et al.43 were A. The limited presence of A allele could have contributed to the difficulty in detecting an association in risk allele carriers. It is more likely that the cellular and molecular mechanisms of rejection in liver and kidney are different. In fact, Tregs play a much more pivotal role in liver than in kidney transplant rejection, according to previously published results.44-46 To further define the role of 1858G>A regarding multiple rejections, we analyzed whether the coherence with the other selected SNPs in a haplotype intensifies the association. Out of 19 estimated haplotypes, one was associated with multiple rejections. Interestingly, the haplotype G-C-A-A-G-T contains
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the rejection-associated 1858A allele and all other alleles which were described to be disease-predisposing12,15,30,33 with exception of rs2488457 (-1123G>C, first position) which was actually tested having C as autoimmunity-predisposing allele.13 However, the P value (P=0.023) and the odds ratio (OR=2.281) are similar to those of the singularly tested 1858G>A allele distribution test (P=0.023, OR=2.135). Thus, a stronger disease-predisposing effect of haplotype GC-A-A-G-T could not be observed. In contrast, the other five tested PTPN22 SNPs seem not to have any additional influence. This stresses the exclusive impact of 1858G>A. In summary, our data suggest that PTPN22 1858G>A could be a suitable biomarker for repeated liver transplant rejection episodes. If the 1858A carriers’ susceptibility to rejection after liver transplantation could be validated in further studies, preoperative diagnostics via adequate automated commercial genotyping procedures could enable a release from standard protocols in immunosuppression, which goes along with a lower rate of drug-driven side effects. Additionally, patients could even benefit from postoperative genotyping since immunosuppression could subsequently be reduced or elevated. This patient-individualized immunosuppressive therapy could imply a substantial advance in transplant medicine. However, it is unlikely that along with the age, only one single point mutation is responsible for rejection. Hence, it is advisable to test further SNPs in PTPN22 or other genes encoding important proteins involved in alloreactivity in the future. Additionally, because of the shown association of PTPN22 1858G>A with multiple rejection risk, LYP might be a potential target protein for future immunosuppressive drugs.
MATERIALS AND METHODS Subjects
The study comprised 345 liver allograft recipients transplanted at the University Medical Center Hamburg-Eppendorf. The patients received transplants from January 1986 to April 2013 and were predominantly (>90%) Caucasian. The examined population had a mean age at transplantation of 51 years (standard deviation±13 years) and consisted of 212 male and 133 female patients (ratio, 1.6:1). The primary clinical data such as mean age, sex, immunosuppressive
TABLE 4.
SNP-specific PCR-ASRA conditions SNP
−1123G>C
Rs-code
rs2488457
Primer (forward, reverse)
5′-CGCCACCTTGCTGACAACAT-3′ 5′-AGCTCGCATAACCTCTCAATGG-3′ 788C>T rs33996649 5′-GATGGAGCAAGACTCAGACAC-3′ 5′-ATCTGCTCTTCTAACATGGGG-3′ 1858G>A rs2476601 5′-GTTGCGCAGGCTAGTCTTG-3′ 5′-CCGTTGAAGCAACATTATCAGT-3′ 852A>G rs1310182 5′-GGGGATACAGATGGTATTCTACT-3′ 5′-ACATGCTAGGTAATACACACAAG-3′ −2200G>A rs1217388 5′-CTCCCCAGTTTTCAGTTCCCG-3′ 5′-AGGGGAATTTACTGGCTTGTAAC-3' 64434T>G rs3789604 5′-CTGCCGTTTAACTCCCCGCG-3′ 5'-GGCTCTGAGTGGAGGAGTGG-3'
Annealing temperature, °C Amplicon in bp Restriction enzyme
59
204
Sac I
58
235
Msp I
60
361
Rsa I
58
203
Pml I
63
463
HpyCH4 III
60
409
ApeK I
SNP, single nucleotide polymorphism; PCR-ASRA, polymerase chain reaction with allele-specific restriction analysis.
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Fragments in bp
G=186+18 C=204 C=91+144 T=235 G=141+176+44 A=141+220 A=203 G=113+90 G=179+147+137 A=179+284 T=113+296 G=113+56+240
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therapy at the time of discharge, and indications for liver transplantation are demonstrated in Table 1. The subjects were divided into two groups according to the presence or absence of acute rejection episodes. Additionally, the group consisting of patients with acute rejections was further subdivided into one subgroup of patients with single and another with multiple acute rejection episodes. Finally, we obtained 165 individuals without any rejection episode defining the comparison group, 123 patients with one and 57 with more than one acute rejection episode. In all included rejection cases, the diagnosis of acute rejection was based on the results of a liver biopsy demonstrating rejection and an increase in liver enzyme activities (aminotransferases and bilirubin) with no other identifiable cause and a response to high-dose corticosteroid treatment. We included patients who had one or more acute rejection episodes within one and a half years after transplantation. As we observed our subjects for a minimum of 1 year, we can affirm that rejection-free patients had no rejection within this period. The study design was approved by the Ethics Commission of the Medical Association of Hamburg, and all involved patients gave their written informed consent.
comparisons such as gender, immunosuppressive therapy, primary disease, genotype, and allele frequencies and the count of retransplanted recipients were calculated with Fisher’s exact test. Haplotype association analysis was carried out using HAPLOVIEW v4.1. P values below 0.05 were assumed to be statistically significant. The strength of association was estimated by OR and 95% confidence interval (95% CI). P value, OR, and 95% CI calculations were performed by R v3.0.0 (Foundation for Statistical Computing, Vienna, Austria). The power calculation was executed by G*Power 3.1 (Institute for Experimental Psychology, Heinrich Heine University, Düsseldorf, Germany) according to the programmer’s description.47 P values, OR, and 95% CI in genotype and allele frequency comparisons were adjusted for statistically significant differences of primary data items using the logistic regression feature of SPSS 20 software (SPSS Inc., Chicago, IL).
Transplantation
Genotyping
All 345 subjects were genotyped for the selected SNPs using PCR-ASRA. Genomic DNA was prepared from leukocytes in EDTA-anticoagulated venous blood samples using the innuPREP Blood DNA kit (Analytik Jena Biometra, Jena, Germany) according to the instruction manual. The PCR was performed with approximately 120 ng DNA in a final volume of 50 μL, additionally containing 40 pmol primers and 25 μL Mastermix (Roche, Mannheim, Germany) consisting of 1.13 U Taq polymerase in 20 mM Tris–HCl, 100 mM KCl, 3 mM MgCl2, dNTP mix (dATP, dCTP, dGTP, and dTTP each 0.4 mM). Primer sequences are listed in Table 4. The following amplification protocol was applied: initial activation at 95°C for 5 min followed by 34 cycles of denaturation at 95°C for 60 sec, annealing at the specific temperature displayed in Table 4 for 60 sec and extension at 72°C for 60 sec. All PCR programs implied a final extension step at 72°C for 3 min. After precipitation, the PCR product was incubated with the specific restriction endonuclease according to the manufacturer’s instructions. Specific restriction endonucleases are demonstrated in Table 4. The restriction enzymes SacI, MspI, RsaI, and PmlI were purchased from Thermo Scientific (Thermo Fisher Scientific Inc, Waltham, MA), and in case of HpyCH4III and ApeKI, we used products from NEB (New England Biolabs GmbH, Frankfurt am Main, Germany). Subsequently, 10 μL of the reaction solution was loaded on a 2% agarose gel containing ethidium bromide. Ten percent of these were randomly selected from all subjects and were regenotyped by sequencing to check the accuracy of PCR-ASRA results. Sequencing was performed by the company Seqlab (Göttingen, Germany) using the same primers as in PCR-ASRA. Statistical Analysis
The Hardy-Weinberg equilibrium was checked by HAPLOVIEW v4.1 (Broad Institute of MIT and Harvard, Boston, USA). Differences in age between case and control group were tested by Student’s t test. Further statistical
ACKNOWLEDGMENTS The authors thank Ms. Petra Tiede for her excellent technical assistance and Ms. Stefanie Ahrens as well as Mr. Sanjay Kumar for their suggestions on the manuscript. REFERENCES 1. Epstein F, Sayegh M, Turka L. The role of T-cell costimulatory activation pathways in transplant rejection. N Engl J Med 1998;338:1813. 2. Graves J, Krebs E. Protein phosphorylation and signal transduction. Pharmacol Ther 1999;82:111. 3. Wu J, Katrekar A, Honigberg LA, et al. Identification of substrates of human protein-tyrosine phosphatase PTPN22. J Biol Chem 2006;281:11002. 4. Vang T, Miletic AV, Bottini N, Mustelin T. Protein tyrosine phosphatase PTPN22 in human autoimmunity. Autoimmunity 2007;40:453. 5. Gjörloff-Wingren A, Saxena M, Williams S, Hammi D, Mustelin T. Characterization of TCR-induced receptor-proximal signaling events negatively regulated by the protein tyrosine phosphatase PEP. Eur J Immunol 1999;29:3845. 6. Cloutier JF, Veillette A. Cooperative inhibition of T-cell antigen receptor signaling by a complex between a kinase and a phosphatase. J Exp Med 1999;189:111. 7. Vang T, Liu W, Delacroix L, Wu S. LYP inhibits T-cell activation when dissociated from CSK. Nat Chem Biol 2012;8:437. 8. Fichna M, Zurawek M, Januszkiewicz-Lewandowska D, Fichna P, Nowak J. PTPN22, PDCD1 and CYP27B1 polymorphisms and susceptibility to type 1 diabetes in Polish patients. Int J Immunogenet 2010;37:367. 9. Chen Z, Zhang H, Xia B, Wang P. Association of PTPN22 gene (rs2488457) polymorphism with ulcerative colitis and high levels of PTPN22 mRNA in ulcerative colitis. Int J Colorectal Dis 2013;28:1351. 10. Kawasaki E, Awata T, Ikegami H, et al. Systematic search for single nucleotide polymorphisms in a lymphoid tyrosine phosphatase gene (PTPN22): association between a promoter polymorphism and type 1 diabetes in Asian populations. Am J Med Genet 2006;140:586. 11. Liu F, Liu J, Zheng T, et al. The -1123G>C variant of PTPN22 gene promoter is associated with latent autoimmune diabetes in adult Chinese Hans. Cell Biochem Biophys 2012;62:273. 12. Viken MK, Olsson M, Flåm ST, et al. The PTPN22 promoter polymorphism -1123G>C association cannot be distinguished from the 1858C>T association in a Norwegian rheumatoid arthritis material. Tissue Antigens 2007;70:190. 13. Huang JJ, Qiu YR, Li HX, Sun DH, Yang J, Yang CL. A PTPN22 promoter polymorphism -1123G>C is associated with RA pathogenesis in Chinese. Rheumatol Int 2012;32:767. 14. Brzoza Z, Grzeszczak W, Rogala B, Trautsolt W, Moczulski D. PTPN22 polymorphism presumably plays a role in the genetic background of chronic spontaneous autoreactive urticaria. Dermatology 2012;224:340. 15. Orrú V, Tsai SJ, Rueda B, et al. A loss-of-function variant of PTPN22 is associated with reduced risk of systemic lupus erythematosus. Hum Mol Genet 2009;18:569. 16. Diaz-Gallo LM, Gourh P, Broen J, et al. Analysis of the influence of PTPN22 gene polymorphisms in systemic sclerosis. Ann Rheum Dis 2011;70:454.
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17. Diaz-Gallo L, Espino-Paisán L, Fransen K, et al. Differential association of two PTPN22 coding variants with Crohn’s disease and ulcerative colitis. Inflamm Bowel Dis 2011;17:2287. 18. Vang T, Congia M, Macis MD, et al. Autoimmune-associated lymphoid tyrosine phosphatase is a gain-of-function variant. Nat Genet 2005;37:1317. 19. Zheng W, She JX. Genetic association between a lymphoid tyrosine phosphatase (PTPN22) and type 1 diabetes. Diabetes. 2005;54:906. 20. Bottini N, Musumeci L, Alonso A, et al. A functional variant of lymphoid tyrosine phosphatase is associated with type I diabetes. Nat Genet 2004;36:337. 21. Saccucci P, Del Duca E, Rapini N, et al. Association between PTPN22 C1858T and type 1 diabetes: a replication in continental Italy. Tissue Antigens 2008;71:234. 22. Korolija M, Renar IP, Hadzija M, et al. Association of PTPN22 C1858Tand CTLA-4 A49G polymorphisms with type 1 diabetes in Croatians. Diabetes Res Clin Pract 2009;86:e54. 23. Orozco G, Sánchez E, González-Gay MA, et al. Association of a functional single-nucleotide polymorphism of PTPN22, encoding lymphoid protein phosphatase, with rheumatoid arthritis and systemic lupus erythematosus. Arthritis Rheum 2005;52:219. 24. Kyogoku C, Langefeld CD, Ortmann WA, et al. Genetic association of the R620W polymorphism of protein tyrosine phosphatase PTPN22 with human SLE. Am J Hum Genet 2004;75:504. 25. Cantón I, Akhtar S, Gavalas NG, et al. A single-nucleotide polymorphism in the gene encoding lymphoid protein tyrosine phosphatase (PTPN22) confers susceptibility to generalised vitiligo. Genes Immun 2005;6:584. 26. Skinningsrud B, Husebye ES, Gervin K, et al. Mutation screening of PTPN22: association of the 1858T-allele with Addison’s disease. Eur J Hum Genet 2008;16:977. 27. Jagiello P, Aries P, Arning L, et al. The PTPN22 620W allele is a risk factor for Wegener’s granulomatosis. Arthritis Rheum 2005;52:4039. 28. Velaga MR, Wilson V, Jennings CE, et al. The codon 620 tryptophan allele of the lymphoid tyrosine phosphatase (LYP) gene is a major determinant of Graves’ disease. J Clin Endocrinol Metab 2004;89:5862. 29. Kemp EH, McDonagh AJG, Wengraf DA, et al. The non-synonymous C1858T substitution in the PTPN22 gene is associated with susceptibility to the severe forms of alopecia areata. Hum Immunol 2006;67:535. 30. Carlton VEH, Hu X, Chokkalingam AP, et al. PTPN22 genetic variation: evidence for multiple variants associated with rheumatoid arthritis. Am J Hum Genet 2005;77:567.
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31. Taniyama M, Maruyama T, Tozaki T, Nakano Y, Ban Y. Association of PTPN22 haplotypes with type 1 diabetes in the Japanese population. Hum Immunol 2010;71:795. 32. Onengut-Gumuscu S, Buckner JH, Concannon P. A haplotype-based analysis of the PTPN22 locus in type 1 diabetes. Diabetes 2006;55:2883. 33. Ichimura M, Kaku H, Fukutani T, Koga H, Mukai T, Miyake I. Associations of protein tyrosine phosphatase with susceptibility to Graves’’ disease in a Japanese population. Thyroid. 2008;18:625. 34. Smith RL, Warren RB, Eyre S, et al. Polymorphisms in the PTPN22 region are associated with psoriasis of early onset. Br J Dermatol. 2008;158:962. 35. Li Y, Liao W, Chang M, et al. Further genetic evidence for three psoriasis-risk genes: ADAM33, CDKAL1, and PTPN22. J Invest Dermatol 2009;129:629. 36. Anis SK, Abdel Ghany EA, Mostafa NO, Ali AA. The role of PTPN22 gene polymorphism in childhood immune thrombocytopenic purpura. Blood Coagul Fibrinolysis 2011;22:521. 37. Heinbokel T, Elkhal A, Liu G, Edtinger K, Tullius SG. Immunosenescence and organ transplantation. Transplant Rev 2013;27:65. 38. Effros RB. Costimulatory mechanisms in the elderly. Vaccine. 2000;18:1661. 39. Felix NJ, Allen PM. Specificity of T-cell alloreactivity. Nat Rev Immunol 2007;7:952. 40. Vang T, Miletic AV, Arimura Y, Tautz L, Rickert RC, Mustelin T. Protein tyrosine phosphatases in autoimmunity. Annu Rev Immunol 2008;26:29. 41. Brownlie R, Miosge L. Lack of the phosphatase PTPN22 increases adhesion of murine regulatory T cells to improve their immunosuppressive function. Sci Signal 2012;5:87. 42. Domański L, Bobrek-Lesiakowska K, Kłoda K, et al. Lack of association of the rs2476601 PTPN22 gene polymorphism with transplanted kidney function. Ann Transplant 2011;16:63. 43. Sfar I, Gorgi Y, Aouadi H, et al. The PTPN22 C1858T (R620W) functional polymorphism in kidney transplantation. Transplant Proc 2009;41:657. 44. Heidt S, Wood K. Biomarkers of operational tolerance in solid organ transplantation. Expert Opin Med Diagn 2012;6:281. 45. Sánchez-Fueyo A, Strom TB. Immunologic basis of graft rejection and tolerance following transplantation of liver or other solid organs. Gastroenterology 2011;140:51. 46. Londoño MC, Danger R, Giral M, Soulillou JP, Sánchez-Fueyo A, Brouard S. A need for biomarkers of operational tolerance in liver and kidney transplantation. Am J Transplant 2012;12:1370. 47. Faul F, Erdfelder E, Lang A-G, Buchner A. G*Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav Res Methods 2007;39:175.
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Association Between a Gain-of-Function Variant of PTPN22 and Rejection in Liver Transplantation Raphael Dullin,1 Martina Koch,1 Martina Sterneck,2 Björn Nashan,1 and Hansjörg Thude1 Background. The protein tyrosine phosphatase nonreceptor 22 gene (PTPN22) encodes a strong T-cell regulator called lymphoid protein tyrosine phosphatase. Previously, PTPN22 was described as a susceptibility gene for autoimmunity because it contains single nucleotide polymorphisms (SNPs) associated with several autoimmune diseases. One SNP (rs2476601; 1858G>A) has emerged as a particularly potent risk factor for autoimmunity. We address the question whether PTPN22 polymorphisms are also associated with acute rejection after liver transplantation. Methods. We investigated the influence of six PTPN22 SNPs on the susceptibility to acute liver allograft rejection. Consequently, we carried out a retrospective study genotyping 345 German liver recipients at six SNP loci, which include rs2488457 (−1123G>C), rs33996649 (788C>T), rs2476601 (1858G>A), rs1310182 (−852A>G), rs1217388 (−2200G>A), rs3789604 (64434T>G). Our study enrolled 165 recipients who did not develop rejection, 123 who showed one rejection episode, and 57 patients who suffered from multiple acute rejections after transplantation. Results. The 1858A allele containing genotypes (GA+AA) and the 1858A allele had a significantly higher frequency in the group of patients with multiple rejection episodes (35.1% and 18.4%) compared to rejection-free patients (15.8% and 7.9%; P=0.022 and 0.023). In contrast, we could not detect any association between rejection and the other tested SNPs. Additionally, we identified one haplotype contributing to risk of multiple rejections, however, exhibiting no stronger impact than the 1858A allele alone. Conclusion. We conclude that the 1858G>A SNP may confer susceptibility to multiple acute liver transplant rejections in the German population.
(Transplantation 2015;99: 431–437)
L
iver transplantation is the lifesaving treatment of choice for end-stage liver disease. In spite of well-developed immunosuppressive therapy, transplant rejection still occurs. Acute allograft rejection episodes are alloimmune responses, which usually occur within the first 6 months after transplantation and are mostly based on T cell reactivity.1). The T cell reactivity is controlled mainly by the activity of kinases and phosphatases.2 One negative key regulator is coded by the protein tyrosine phosphatase nonreceptor type 22 gene (PTPN22). The PTPN22 is located on chromosome 1 (1p13) Received 27 November 2013. Revision requested 17 December 2013. Accepted 21 May 2014. 1
Department of Hepatobiliary and Transplant Surgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
2
Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
This work was supported by grants from the Deutsche Forschungsgemeinschaft (“Entzündung und Regeneration—Integriertes Graduiertenkolleg im SFB 841 Leberentzündung”). The authors declare no conflicts of interest. R.D. wrote the article, collected data, analyzed data, and performed the study. M.K., M.S., and B.N. designed the study and contributed patients. H.T. performed the study, analyzed the data, designed the study, and wrote the article. Correspondence: Hansjörg Thude, Universitätsklinikum Hamburg-Eppendorf, Zentrum Innere Medizin, Klinik für Hepatobiliäre Chirurgie und Transplantationschirurgie, Martinistraße 52, 20246 Hamburg, Germany. ([email protected]) Copyright © 2014 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 0041-1337/15/9902-431 DOI: 10.1097/TP.0000000000000313
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and encodes the multiphosphatase lymphoid-specific protein tyrosine phosphatase (LYP), which is predominantly found in the cytoplasm. This protein exhibits a molecular weight of 105 kDa and consists of 807 amino acids. In addition to the catalytic domain, LYP also features four C-terminal proline-rich motifs (P1-4), which operate as binding sites for cytoplasmic adaptor proteins. Lymphoidspecific protein tyrosine phosphatase dephosphorylates numerous signal adaptor proteins at their tyrosine residues, finally to decrease the level of T cell activation. Well-known substrates of LYP are immunoreceptor tyrosine-based activation motifs, such as the T cell receptor ζ and CD3ε, the Src family kinase LCK, the Syk family kinase ZAP-70, the protooncogene Vav, and VCP.3 The Src homology 3 domain of the C-terminal Src kinase (CSK) binds the P1 region of LYP.4 Actually, both LYP and CSK regulate the T-cell activation in a synergistic manner because CSK phosphorylates a C-terminal inhibitory tyrosine residue of Src family kinases, causing an additional reduction of activity.5,6 However, Vang et al. pointed out yet another role of CSK. They observed that the dissociation of the LYP-CSK complex is important to locate LYP near the cell membrane to dephosphorylate its membrane-associated substrates, especially LSK and T cell receptor ζ7 resulting in downregulation of T-cell activation. Single nucleotide polymorphisms (SNP) are contained in most genes. So far, over six hundreds have been identified (NCBI’s SNP database) in the PTPN22 gene. Six important SNPs of the PTPN22 gene are displayed in Figure 1. The first SNP, rs2488457 (-1123G>C), lies in a putative transcription factor binding site (TFBS) for the activator www.transplantjournal.com
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FIGURE 1. Schematic gene map of PTPN22 gene including the selected SNP loci (vertical arrows), UTR (empty rectangles), exons (filled rectangles), introns (horizontal lines) 34×4 mm (600×600 DPI). UTR, untranslated regions.
protein 4 (AP-4) within the promoter region8 and influences the expression level of LYP, concerning the results of Chen et al.9 Association studies have described a susceptibility to contract type 1 diabetes (T1D),10,11 rheumatoid arthritis (RA),12,13 autoreactive urticaria,14 and ulcerative colitis.9 The second SNP, rs33996649 (788C>T), is a loss-offunction missense point mutation (R263Q)15 located in exon 10 operating as part of the catalytic domain. A protective contribution to autoimmunity was shown in at least three independent studies.15-17 Rs2476601 (1858G>A) is also a missense point mutation. However, in contrast to 788C>T, it belongs to the category associated with a gain of function.18 An arginine is substituted by tryptophan in exon 14, which functions as part of the P1 linkage domain. Hence, this LYP mutant has a threefold decrease in affinity to CSK and consequently an increased
enzyme activity (19). In spite of the gain of function, the SNP 1858G>A is strongly associated with T1D,8,19-22 RA,12,23 systemic lupus erythematosus,23,24 vitiligo,25 Addison’s disease,26 Wegener’s granulomatosis,27 progressive systemic sclerosis,16 Grave’s disease,28 and alopecia areata.29 The next depicted SNPs, rs1310182 (-852A>G) and rs1217388 (-2200G>A), are point mutations in intronic regions carrying putative TFBS, described by Carlton et al.30 This group detected an association of both SNPs with RA and two other groups found an association to T1D.31,32 The last selected SNP, rs3789604 (64434T>G), is part of a TFBS controlling the level of LYP expression.30 Some studies have reported an association with Grave’s disease33 and psoriasis.34,35 Because selected mutations have functional consequences and confer an increased risk to several autoimmune diseases,
TABLE 1.
Characteristics of liver transplant recipients collectives No rejection n=165
Sex (m/f) 101 (61%)/64 (39%) Age (mean yr±SD) 53±11 Primary liver disease Alcoholic cirrhosis 50 (30%) Cirrhosis after HCV 31 (19%) Cirrhosis after HBV 16 (10%) Primary biliary cirrhosis 8 (5%) Primary sclerosing cholangitis 13 (8%) Wilson disease 3 (2%) α1-AT deficiency 2 (1%) Budd-Chiari syndrome 3 (2%) Cryptogenic cirrhosis 11 (7%) (Acute) liver failure 6 (4%) ADPKD 6 (4%) Extrahepatic ductopenia 1 (1%) Inborn errors of metabolism 2 (1%) Autoimmune hepatitis 4 (2%) Others 9 (5%) Additional HCC 42 (25%) Immunosuppressive treatment at the time of discharge Double regimen CNI+STER 59 (36%) Steroid-free (CNI+mTORI/MMF/AZA) 9 (5%) CNI-free (mTORI/MMF/AZA+STER) 3 (2%) Triple regimen CNI+mTORI/MMF/AZA+STER 88 (53%) Others 6 (4%)
Rejection (single)
Rejection (multiple)
n=123
P
n=57
P
77 (63%)/46 (37%) 50±13
NS 0.024
34 (60%)/23 (40%) 45±17
NS 0.001
34 (28%) 22 (18%) 7 (6%) 3 (2%) 15 (12%) 0 (0%) 2 (2%) 2 (2%) 7 (6%) 16 (13%) 2 (2%) 2 (2%) 2 (2%) 2 (2%) 7 (6%) 22 (18%)
NS NS NS NS NS NS NS NS NS 0.006 NS NS NS NS NS NS
11 (19%) 7 (12%) 8 (14%) 4 (7%) 5 (9%) 1 (2%) 1 (2%) 1 (2%) 4 (7%) 5 (9%) 0 (0%) 2 (4%) 3 (5%) 2 (4%) 3 (5%) 11 (19%)
NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS
53 (43%) 2 (2%) 3 (2%)
NS NS NS
21 (37%) 2 (4%) 3 (5%)
NS NS NS
63 (51%) 2 (2%)
NS NS
29 (51%) 2 (4%)
NS NS
Mean length of follow-up=8.5 years. Further genotype and allele frequency comparisons (P values, OR, and 95% CI) were adjusted for significant differences shown in this table. Percentages approximated. SD, Standard deviation; AT; alpha 1-antitrypsin; ADPKD, autosomal dominant polycystic kidney disease; HCC, hepatocellular carcinoma; CNI, calcineurin inhibitors; STER, corticosteroids; mTORI, mTOR inhibitors; MMF, mycophenolate mofetil; AZA, azathioprine; NS, not significant; HCV, hepatitis C virus; HBV, hepatitis B virus; OR, odds ratio; 95% CI, 95% confidence interval.
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a predisposing effect to rejection episodes after liver transplantation is also probable. The aim of the study is to clarify whether an association with acute rejection episodes after liver transplantation exists. Therefore, we examined the SNPs in 345 German liver transplant recipients to test our hypothesis. RESULTS The demographic and clinical data of the liver transplant recipients without any rejection (n=165), with at least one (n=123) and with more than one rejection episode (n=57) are summarized in Table 1. When patients without and patients with multiple rejection episodes were compared, the data differed in age (53±11 years vs. 45±17 years; P=0.001). Demographic and clinical determinants between patients without rejection and patients who exhibited one rejection episode displayed significant differences in the age (53±11 years vs. 50±13 years; P=0.024) and in the prevalence of acute liver failure as reason for transplantation (4% vs. 13%; P=0.006). Considering the number of retransplanted recipients in the cohorts, we obtained the following results. The cohort of
patients without rejection contains 14 (8.5%) retransplanted patients. Twelve (9.8%) retransplanted recipients were in the group of patients with one rejection episode (compared to the rejection free group: P=0.836), and in the group with multiple rejections, we found 12 (21.1%) retransplanted individuals (compared to the rejection-free group: P=0.016). The complete genotyping results for the three groups are shown in Table 2. Genotype data of each analyzed group did not deviate from the Hardy-Weinberg equilibrium at all six analyzed polymorphic sites (data not shown). Sequencing conferred concordance with polymerase chain reaction with allele-specific restriction analysis (PCR-ASRA) results. Frequency analysis revealed that the SNPs rs2488457, rs3996649, rs1310182, rs1217388, and rs3789604 were not associated with rejection risk at all. We could not observe any significant differences for patients with one and for patients with more than one rejection episode when they were each compared to rejection-free individuals. SNP rs2476601 (1858G>A) did not differ significantly in genotype (P=0.177) and allele frequency (P=0.151) between patients without and patients with a single rejection episode as well. In contrast, a significant difference between patients without and patients
TABLE 2.
Genotype and allele frequencies of the different collectives of liver transplant recipients
PTPN22 SNP
rs2488457 −1123G>C
rs3996649 788C>T
rs2476601 1858G>A
rs1310182 −852A>G
rs1217388 −2200G>A
rs3789604 64434T>G
GG GC CC G C CC CT TT C T GG GA AA G A AA AG GG A G GG GA AA G A TT TG GG T G
No rejection
Rejection (single)
n=165
n=123
10 (6.1%) 64 (38.8%) 91 (55.2%) 84 (25.5%) 246 (74.5%) 160 (97.0%) 5 (3.0%) 0 (0%) 325 (98.5%) 5 (1.5%) 139 (84.2%) 26 (15.8%) 0 (0.0%) 304 (92.1%) 26 (7.9%) 41 (24.8%) 79 (47.9%) 45 (27.3%) 161 (48.8%) 169 (51.2%) 10 (6.1%) 58 (35.2%) 97 (58.8%) 78 (23.6%) 252 (76.4%) 111 (67.3%) 53 (32.1%) 1 (0.6%) 275 (83.3%) 55 (16.7%)
6 (4.9%) 60 (48.8%) 57 (46.3%) 72 (29.3%) 174 (70.7%) 118 (95.9%) 5 (4.1%) 0 (0%) 241 (98.0%) 5 (2.0%) 95 (77.2%) 27 (22.0%) 1 (0.8%) 217 (88.2%) 29 (11.8%) 29 (23.6%) 67 (54.5%) 27 (22.0%) 125 (50.8%) 121 (49.2%) 6 (4.9%) 54 (43.9%) 63 (51.2%) 66 (26.8%) 180 (73.2%) 80 (65.0%) 40 (32.5%) 3 (2.4%) 200 (81.3%) 46 (18.7%)
Rejection (multiple) P c
NS
NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS
n=57
P
3 (5.3%) 26 (45.6%) 28 (49.1%) 32 (28.1%) 82 (71.9%) 56 (98.2%) 1 (1.8%) 0 (0%) 113 (99.1%) 1 (0.9%) 37 (64.9%) 19 (33.3%) 1 (1.8%) 93 (81.6%) 21 (18.4%) 9 (15.8%) 33 (57.9%) 15 (26.3%) 51 (44.7%) 63 (55.3%) 3 (5.3%) 26 (45.6%) 28 (49.1%) 32 (28.1%) 82 (71.9%) 43 (75.4%) 14 (24.6%) 0 (0%) 100 (87.7%) 14 (12,3%)
NS
ORa (95CIb)
NS NS NS NS NS 0.022
2.329 (1.132-4.792)
NS 0.023
2.135 (1.109-4.109)
NS NS NS NS NS NS NS NS NS
Test conditions: allele A (major allele) versus allele B (minor allele); AA+AB versus BB (dominant model); AA versus AB+BB (recessive model). Percentages approximated; P values, OR and 95CI were adjusted for clinical data effects via logistic regression. OR, odds ratio; 95% CI, 95% confidence interval; NS, not significant.
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with multiple rejection episodes could be detected. The results demonstrated that the A allele containing genotypes were overrepresented in recipients who rejected more than once (35.1%) compared to recipients without rejection (15.8%). This difference achieved statistical significance in a recessive inheritance (recessive model GA+AA vs. GG, odds ratio [OR], 2.329, 95% confidence interval [95% CI], 1.132-4.792; P=0.022). The power of this result was above 80%. Concerning the allele distribution, a similar predication was found. The A allele frequencies were 18.4% and 7.9% in patients with more than one rejection and patients without rejection, respectively (A vs. G; OR, 2.135; 95% CI, 1.109-4.109; P=0.023). The power reached also a value of over 80%. We found one homozygous AA carrier in each rejection group. Subsequently, a haplotype analysis was performed based on statistically significant differences in 1858G>A genotypes and alleles. Estimated haplotype frequencies and P values are summarized in Table 3. We obtained 19 haplotypes, five of which had an overall frequency above 5%. One haplotype carrying the 1858A risk allele was significantly overrepresented in patients with multiple rejections compared to patients without rejection (G-C-A-A-G-T, 13.7% vs. 7.0%; OR, 2.281; 95% CI, 1.074-4.754; P=0.023).
Both rejection cohorts were significantly younger than the group of rejection-free patients (P=0.024 for single, P=0.001 for multiple rejection episodes). This phenomenon could be explained by the terminus immunosenescence, which is an agerelated weakening of immune functions resulting in lower rates of acute allograft rejections.37 Indeed, it was shown that costimulatory signal pathways are reduced in elderly individuals.38 The analysis of rs2488457 (-1123G>C), rs3996649 (788C>T), rs1310182 (-852A>G), rs1217388 (-2200G>A), and rs3789604 (64434T>G) did not provide a significant correlation—neither with single nor with multiple rejection. Thus, our data suggest that these SNPs are of minor importance for liver transplant rejection. In contrast, previous studies have sporadically shown an association with various autoimmune diseases, though these results are heterogeneous.8-17,30-35 In autoreactivity, autoantibodies and autoreactive T lymphocytes could target several self-proteins, whereas in alloreactivity (rejection) T cells respond directly to foreign human leukocyte antigen molecules on the graft cells’ surface. Additionally, autoimmune reactions are regarded as pathologic, whereas alloimmune reactions use the mechanisms of physiologic pathogen defense.39 Because of these differences, the comparability with previously mentioned autoimmunity studies is limited. In spite of inconsistent results in literature, the point mutation rs2476601 (1858G>A) was consequently established as a risk factor for numerous autoimmune diseases,8,12,16,19-29 except for the Asian population.10,11,13,33 The disease predisposing allele A leads to a gain of function in a dominant manner.18 Interestingly, we also observed the influence of 1858G>A in the recessive model (GA+AA vs. GG) exclusively, where the A allele is assumed to be dominant. Because of the gain of function, a higher regulatory activity of LYP increases the risk for developing autoimmune diseases particularly mediated by hyperreactive T cells. Two theories attempt to solve this contradiction. Firstly, the survival of autoreactive T cells in the thymus is better if their response to autoantigens is weaker resulting from higher LYP activity. Next, regulatory T cells (Tregs) also express LYP and are inhibited in their function.40 Our results illuminate a significant correlation between carrying the A allele and the risk for multiple acute rejections after liver transplantation. Because we could detect only one AA genotype in each rejection group, the GA genotype distribution made the majority of the difference. The frequency of the genotypes GA plus AA within this group was more than twice as high as that of the comparison group (35.1% vs. 15.8%). Also, A-carriers had a risk for multiple rejections
Transplantation
DISCUSSION Lymphoid-specific protein tyrosine phosphatase encoded by PTPN22 gene acts as a strong downregulator of T-cell activity. The PTPN22 characterizes one of the most important autoimmunity risk genes, only human leukocyte antigen genes have a higher impact toward autoimmune diseases.36 This result is derived from numerous studies performed worldwide concerning autoimmune diseases.8-17,19-35 In context of transplantation, PTPN22 SNP data are limited, and the impact of SNPs in PTPN22 gene on liver allotransplant rejection is still unclear until now. Autoimmune diseases are mainly characterized by a disregulated hyperreactive immune system. The described association between PTPN22 SNPs and hyperreactive immune systems found in autoimmune diseases prompted us to address the question whether PTPN22 SNPs are potential genetic biomarkers for predicting the risk of rejecting a liver allotransplant. By genotyping 345 liver recipients divided in patients without biopsy, patients with one biopsy, and patients with more than one biopsy proven rejection episode, we examined the influence of these SNPs on the risk of rejecting a liver allotransplant. TABLE 3.
Results of haplotype analysis
PTPN22 haplotype
C-C-G-G-A-T C-C-G-A-A-G G-C-G-A-G-T G-C-A-A-G-T C-C-G-A-A-T
No rejection
Rejection (multiple)
Overall frequency
n=330
n=114
P
47.6% 14.1% 11.9% 8.5% 6.0%
154.5 (46.5%) 49.7 (15.0%) 40.1 (12.1%) 22.3 (7.0%) 23.1 (6.9%)
56.8 (49.9%) 13.1 (11.5%) 12.6 (11.1%) 15.6 (13.7%) 3.7 (3.2%)
NS NS NS 0.023 NS
Haplotype SNP order: rs2488457-rs33996649-rs2476601-rs1310182-rs1217388-rs3789604; haplotypes with overall frequency beneath 5% are not shown. OR, odds ratio; 95% CI, 95% confidence interval; NS, not significant.
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OR (95% CI)
2.281 (1.074–4.754)
Dullin et al
© 2015 Wolters Kluwer
increased by more than the double (OR=2.329). The comparison of genotype and allele distributions of recipients with single and recipients without rejection showed a tendency (22.8% vs. 15.8% for GA+AA genotypes, 11.8% vs. 7.9% for A alleles) lacking statistical significance. If a larger cohort of patients is tested, this effect will probably become significant. In clinical practice, single liver transplant rejection episodes can be treated very well with corticosteroid bolus therapy. If rejection does not recur, it will not provoke any complications such as side effects of elevated immunosuppressive drug dosage and allograft tissue damage. However, multiple rejections indicate an increased immune reactivity against the allograft, which can merely be overcome through adapting the immunosuppressive therapy, otherwise more rejection episodes after damage of the organ threaten the patient. In turn, this could lead to an elevated retransplantation rate among patients with multiple rejection episodes, according to our results (21.1% vs. 8.5% retransplanted recipients, P=0.016). Furthermore, our data show that 1858G>A is particularly associated with this elevated alloimmune reactivity, which could be resulted from Tregs inhibited by LYP with a gain of function. Functional tests have demonstrated that PTPN22−/− mice have more active Tregs, showing that LYP is also a key regulator in Tregs.41 Two groups from Poland42 and Tunisia43 investigated the influence of 1858G>A on acute kidney transplant rejection, detecting no significant association. The reason why their results differ from ours in spite of similar sample sizes could only be speculated. The frequency of the 1858A allele in Europe is decreasing from north to south.40 Truly, only about 4% of the alleles found by Sfar et al.43 were A. The limited presence of A allele could have contributed to the difficulty in detecting an association in risk allele carriers. It is more likely that the cellular and molecular mechanisms of rejection in liver and kidney are different. In fact, Tregs play a much more pivotal role in liver than in kidney transplant rejection, according to previously published results.44-46 To further define the role of 1858G>A regarding multiple rejections, we analyzed whether the coherence with the other selected SNPs in a haplotype intensifies the association. Out of 19 estimated haplotypes, one was associated with multiple rejections. Interestingly, the haplotype G-C-A-A-G-T contains
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the rejection-associated 1858A allele and all other alleles which were described to be disease-predisposing12,15,30,33 with exception of rs2488457 (-1123G>C, first position) which was actually tested having C as autoimmunity-predisposing allele.13 However, the P value (P=0.023) and the odds ratio (OR=2.281) are similar to those of the singularly tested 1858G>A allele distribution test (P=0.023, OR=2.135). Thus, a stronger disease-predisposing effect of haplotype GC-A-A-G-T could not be observed. In contrast, the other five tested PTPN22 SNPs seem not to have any additional influence. This stresses the exclusive impact of 1858G>A. In summary, our data suggest that PTPN22 1858G>A could be a suitable biomarker for repeated liver transplant rejection episodes. If the 1858A carriers’ susceptibility to rejection after liver transplantation could be validated in further studies, preoperative diagnostics via adequate automated commercial genotyping procedures could enable a release from standard protocols in immunosuppression, which goes along with a lower rate of drug-driven side effects. Additionally, patients could even benefit from postoperative genotyping since immunosuppression could subsequently be reduced or elevated. This patient-individualized immunosuppressive therapy could imply a substantial advance in transplant medicine. However, it is unlikely that along with the age, only one single point mutation is responsible for rejection. Hence, it is advisable to test further SNPs in PTPN22 or other genes encoding important proteins involved in alloreactivity in the future. Additionally, because of the shown association of PTPN22 1858G>A with multiple rejection risk, LYP might be a potential target protein for future immunosuppressive drugs.
MATERIALS AND METHODS Subjects
The study comprised 345 liver allograft recipients transplanted at the University Medical Center Hamburg-Eppendorf. The patients received transplants from January 1986 to April 2013 and were predominantly (>90%) Caucasian. The examined population had a mean age at transplantation of 51 years (standard deviation±13 years) and consisted of 212 male and 133 female patients (ratio, 1.6:1). The primary clinical data such as mean age, sex, immunosuppressive
TABLE 4.
SNP-specific PCR-ASRA conditions SNP
−1123G>C
Rs-code
rs2488457
Primer (forward, reverse)
5′-CGCCACCTTGCTGACAACAT-3′ 5′-AGCTCGCATAACCTCTCAATGG-3′ 788C>T rs33996649 5′-GATGGAGCAAGACTCAGACAC-3′ 5′-ATCTGCTCTTCTAACATGGGG-3′ 1858G>A rs2476601 5′-GTTGCGCAGGCTAGTCTTG-3′ 5′-CCGTTGAAGCAACATTATCAGT-3′ 852A>G rs1310182 5′-GGGGATACAGATGGTATTCTACT-3′ 5′-ACATGCTAGGTAATACACACAAG-3′ −2200G>A rs1217388 5′-CTCCCCAGTTTTCAGTTCCCG-3′ 5′-AGGGGAATTTACTGGCTTGTAAC-3' 64434T>G rs3789604 5′-CTGCCGTTTAACTCCCCGCG-3′ 5'-GGCTCTGAGTGGAGGAGTGG-3'
Annealing temperature, °C Amplicon in bp Restriction enzyme
59
204
Sac I
58
235
Msp I
60
361
Rsa I
58
203
Pml I
63
463
HpyCH4 III
60
409
ApeK I
SNP, single nucleotide polymorphism; PCR-ASRA, polymerase chain reaction with allele-specific restriction analysis.
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Fragments in bp
G=186+18 C=204 C=91+144 T=235 G=141+176+44 A=141+220 A=203 G=113+90 G=179+147+137 A=179+284 T=113+296 G=113+56+240
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February 2015
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Volume 99
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therapy at the time of discharge, and indications for liver transplantation are demonstrated in Table 1. The subjects were divided into two groups according to the presence or absence of acute rejection episodes. Additionally, the group consisting of patients with acute rejections was further subdivided into one subgroup of patients with single and another with multiple acute rejection episodes. Finally, we obtained 165 individuals without any rejection episode defining the comparison group, 123 patients with one and 57 with more than one acute rejection episode. In all included rejection cases, the diagnosis of acute rejection was based on the results of a liver biopsy demonstrating rejection and an increase in liver enzyme activities (aminotransferases and bilirubin) with no other identifiable cause and a response to high-dose corticosteroid treatment. We included patients who had one or more acute rejection episodes within one and a half years after transplantation. As we observed our subjects for a minimum of 1 year, we can affirm that rejection-free patients had no rejection within this period. The study design was approved by the Ethics Commission of the Medical Association of Hamburg, and all involved patients gave their written informed consent.
comparisons such as gender, immunosuppressive therapy, primary disease, genotype, and allele frequencies and the count of retransplanted recipients were calculated with Fisher’s exact test. Haplotype association analysis was carried out using HAPLOVIEW v4.1. P values below 0.05 were assumed to be statistically significant. The strength of association was estimated by OR and 95% confidence interval (95% CI). P value, OR, and 95% CI calculations were performed by R v3.0.0 (Foundation for Statistical Computing, Vienna, Austria). The power calculation was executed by G*Power 3.1 (Institute for Experimental Psychology, Heinrich Heine University, Düsseldorf, Germany) according to the programmer’s description.47 P values, OR, and 95% CI in genotype and allele frequency comparisons were adjusted for statistically significant differences of primary data items using the logistic regression feature of SPSS 20 software (SPSS Inc., Chicago, IL).
Transplantation
Genotyping
All 345 subjects were genotyped for the selected SNPs using PCR-ASRA. Genomic DNA was prepared from leukocytes in EDTA-anticoagulated venous blood samples using the innuPREP Blood DNA kit (Analytik Jena Biometra, Jena, Germany) according to the instruction manual. The PCR was performed with approximately 120 ng DNA in a final volume of 50 μL, additionally containing 40 pmol primers and 25 μL Mastermix (Roche, Mannheim, Germany) consisting of 1.13 U Taq polymerase in 20 mM Tris–HCl, 100 mM KCl, 3 mM MgCl2, dNTP mix (dATP, dCTP, dGTP, and dTTP each 0.4 mM). Primer sequences are listed in Table 4. The following amplification protocol was applied: initial activation at 95°C for 5 min followed by 34 cycles of denaturation at 95°C for 60 sec, annealing at the specific temperature displayed in Table 4 for 60 sec and extension at 72°C for 60 sec. All PCR programs implied a final extension step at 72°C for 3 min. After precipitation, the PCR product was incubated with the specific restriction endonuclease according to the manufacturer’s instructions. Specific restriction endonucleases are demonstrated in Table 4. The restriction enzymes SacI, MspI, RsaI, and PmlI were purchased from Thermo Scientific (Thermo Fisher Scientific Inc, Waltham, MA), and in case of HpyCH4III and ApeKI, we used products from NEB (New England Biolabs GmbH, Frankfurt am Main, Germany). Subsequently, 10 μL of the reaction solution was loaded on a 2% agarose gel containing ethidium bromide. Ten percent of these were randomly selected from all subjects and were regenotyped by sequencing to check the accuracy of PCR-ASRA results. Sequencing was performed by the company Seqlab (Göttingen, Germany) using the same primers as in PCR-ASRA. Statistical Analysis
The Hardy-Weinberg equilibrium was checked by HAPLOVIEW v4.1 (Broad Institute of MIT and Harvard, Boston, USA). Differences in age between case and control group were tested by Student’s t test. Further statistical
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