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Abstract

Objective

Acute rejection resulting from alloimmune responses is a major risk factor affecting patient survival following liver transplantation. Since interleukin (IL)-6 can mediate acute rejection, the association between IL-6 gene single nucleotide polymorphisms (SNPs) and incidence of acute rejection in liver transplant recipients was investigated.

Methods

Patients who received liver transplant between January 2005 and December 2010 were typed for IL6-572C/G (rs1800796) polymorphisms using the snapshot technique. Association between genotype and acute rejection was analysed using the SNP Statistics website: http://bioinfo.iconcologia.net/snpstats/start.htm. Allelic and genotypic distributions for rs1800796 were compared among 335 patients with or without acute rejection within the first 6 months following liver transplant.

Results

Incidence of acute rejection was 11.94%. A heterozygous CG genotype for IL6-572C/G was associated with a lower acute rejection rate compared with homozygous CC or GG genotypes.

Conclusion

IL6-572 CG genotype may be related to protection from acute rejection following liver transplant in Han Chinese patients.

Keywords

Acute rejection interleukin-6 (IL-6)liver transplantation rs1800796 single nucleotide polymorphism (SNP)

Introduction

Liver transplantation is regarded to be an effective therapeutic option for patients with end-stage liver disease due to improvements in surgical techniques and postoperative management, and the introduction of new immunosuppressive agents and protocols.¹ Survival rates following liver transplantation are continually improving, with 1-year graft survival rates exceeding 80%. Graft failure occurs in up to 15% of patients during the first year, however, rising to 25% within 5 years following transplant.^2,3 Acute rejection may result in graft loss, increased risk of chronic graft dysfunction, and suboptimal long-term outcome.⁴ Despite improvements in immunosuppressive therapy, 20–40% of patients still have one or more acute rejection episodes.⁵

There is accumulating evidence to suggest that cytokines play a central role in the immunological events that occur following transplantation, and are intimately linked to graft rejection. For example, T-helper subtype 1 cytokines including interleukin (IL)-2, IL-15 and tumour necrosis factor (TNF)-α are associated with graft rejection, and T-helper subtype 2 cytokines such as IL-10 are associated with graft tolerance.^6–10 Polymorphisms with functional significance in the promoter and coding regions of cytokine genes have been suggested as possible factors in graft rejection. The polymorphisms of TNFA-308 G/A, IL10-1082 G/A, IL6-174 G/C and interferon-γ (IFNG) +874 T/A have all been reported to be associated with acute rejection.^11–13

The multifunctional cytokine IL-6 provokes a broad range of cellular and physiological responses, including the development of the nervous and haematopoietic systems, acute-phase responses, inflammatory and immune responses through regulation of gene activation, cell growth, proliferation, survival, and differentiation.¹⁴ Polymorphisms in the promoter region of the IL-6 gene may result in variations in transcription and expression between individuals. Genetic variants could, therefore, influence an individual’s susceptibility to a diverse range of diseases. Several studies have identified IL-6 to be associated with acute allograft rejection in patients¹³ and in experimental models.¹⁵ It has been reported that IL6-174 G/C polymorphisms have predictive values for acute rejection following liver transplantation,¹⁶ however, there are no published studies on the influence of IL-6 promoter single nucleotide polymorphisms (SNPs) on acute rejection following liver transplantation in China.

The present study investigated the association between IL-6 gene single nucleotide polymorphisms (SNPs) and incidence of acute rejection in liver transplant recipients.

Patients and methods

Study Population

Consecutive patients who received liver transplants at the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China, between January 2005 and December 2010, were enrolled. Patient characteristics and clinical data were retrospectively derived from medical records, or extracted from an electronic database that is prospectively maintained. All patients were Han Chinese. The inclusion criteria for the study were availability of adequate sample and patient survival >6 months after liver transplantation. All patients were followed up for ≥6 months.

This study was approved by the Ethical Review Committee of the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China; written informed consent was obtained from all patients.

Immunosuppressant Regimen and Rejection Diagnosis

The routine immunosuppressant regimen comprised 1–2 mg/day tacrolimus, together with either 1 mg/day rapamycin or 500 mg/day mycophenolate mofetil, and 120 mg prednisone q12h; all drugs were administered orally.

Acute rejection diagnosis was based on conventional clinical parameters (fever and elevation of bilirubin and/or transaminase levels in the absence of vascular or biliary problems) and biopsy findings, according to Banff criteria.¹⁷ Acute rejection episodes were managed with high doses (500 mg) of methylprednisolone, administered orally for 3 days.

Patients who suffered acute rejection within 6 months following transplantation were classified into the acute rejection (AR) group. All other patients were classified into the nonacute rejection (NAR) group.

DNA Extraction

Venous whole blood was collected using commercial collection tubes containing ethylenediaminetetra-acetic acid (REF367841; BD Biosciences, San Jose, CA, USA) and stored at −80°C before isolation of genomic DNA.

Genomic DNA was isolated from 500 µl of blood using a Maxwell® 16 Blood DNA Purification kit (Promega, Madison, WI, USA), according to the manufacturer’s instructions. DNA was stored in elution buffer at −80°C until analysis of cytokine genotypes by polymerase chain reaction (PCR).

Polymorphism Genotyping

Details of IL6 promoter region SNPs for the Chinese population of Beijing were searched for in the public genotyping database http://www.hapmap.org. The search criteria included a minor allele frequency ≥20% and r² ≥0.8. The current study focused on IL6-572 C/G (rs1800796), and SNPs at loci IL6-572 C/G were detected by DNA sequencing.

The fragment was first amplified by PCR. Allele-specific forward primers and generic reverse primers were designed using the IL6 gene sequence published in the nucleotide database at http://www.ncbi.nlm.nih.gov. The primer pairs for this multiplex PCR were the following: forward, 5′-TGGGCTGAAGCAGGTGAAGA-3′; reverse, 5′-GCTGAAGTCATGCACGAAGTTTTACA-3′. The PCR reaction system included 1 × HotStarTaq® buffer (QIAGEN GmbH, Hilden, Germany), 3.0 mM Mg²⁺, 0.3 mM dNTP, 0.1 µM of each primer, 1 U HotstarTaq® polymerase (QIAGEN GmbH), and 5–10 ng genomic DNA, in a total volume of 20 µl. The cycling programme was carried out using an Applied Biosystems® 2720 Thermal Cycler (Applied Biosystems, Foster City, CA, USA) as follows: denaturation at 95°C for 15 min followed by 12 cycles of 94°C for 20 s, 65°C (reduced by 0.5°C per cycle) for 40 s and 72°C for 100 s, then 23 cycles of 94°C for 20 s, 57°C for 30 s and 72°C for 90 s, with a final extension at 72°C for 2 min. Ultrapure water was used as a negative control. The PCR products were separated on 1–2% agarose gels and visualized using ethidium bromide staining and ultraviolet light.

A mixture containing 1 U of shrimp alkaline phosphatase (Promega) and 6 U of exonuclease I was used to purify 10 µl of PCR product. The mixture was incubated at 37°C for 60 min followed by inactivation at 75°C for 15 min. Then, 2 µl of purified PCR product was mixed with 5 µl SNaPshot® Multiplex kit (Applied Biosystems), 2 µl ultrapure water, and 1 µl sequencing primer for further reaction. The sequencing primer was: 5′-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTTCTGGCTCTCCCTGTGAG-3′. The cycling programme was as follows: denaturation at 96°C for 1 min followed by 28 cycles of 96°C for 10 s, 50°C for 5 s, 60°C for 30 s, then a final extension at 72°C for 2 min. Next, 1 U of shrimp alkaline phosphatase was mixed with 10 µl of PCR product, incubated at 37°C for 60 min and inactivated at 75°C for 15 min for purification. Next, 0.5 µl of the purified PCR product was mixed with 0.5 µl Genescan™ –120 LIZ® size standard and 9 µl HiDi™ (Applied Biosystems). Denaturing of extension products was performed at 95°C for 5 min and the mixture was sequenced using an ABI 3130xl Genetic Analyser (Applied Biosystems). Sequence data were analysed using Gene Mapper® software, version 4.0 (Applied Biosystems).

Statistical Analyses

Statistical analyses were performed using the SPSS® software package, version 16.0 (SPSS Inc., Chicago, IL, USA) for windows®. Between-group differences in clinical characteristics and the frequencies of alleles in patients with or without acute rejection were compared using Pearson’s χ²-test, with Yates’ correction for continuity (or Fisher’s exact test where appropriate). Genotypes were analysed using the website for SNP Statistics: http://bioinfo.iconcologia.net/snpstats/start.htm. Multivariate logistic regression analysis was used to analyse the association of genotypes in four inheritance models (codominant, dominant, recessive, overdominant) in the AR and NAR groups. All tests were two-tailed and P values ≤0.05 were considered to be statistically significant.

Results

A total of 335 patients were included in this study: 290 males and 45 females, with a mean ± SD age at transplantation of 46.6 ± 9.9 years. Patients were classified into two groups: 295 (88.06%) into the NAR group and 40 (11.94%) into the AR group. The mean ± SD time of acute rejection onset was 46.2 ± 42.1 days. The primary indications for liver transplantation included hepatocellular carcinoma, alcoholic cirrhosis and primary biliary hepatitis. No significant between-group differences were found regarding sex, blood type, model for end-stage liver disease (MELD) score¹⁸ and primary disease relating to the need for liver transplantation (Table 1).

Table 1.

Baseline characteristics and primary pre-liver transplant disease for 335 patients assigned to an acute rejection (AR) group or nonacute rejection (NAR) group following liver transplantatio.

Characteristic	Patient group		Statistical significance^a
Characteristic	AR n = 40	NAR n = 295	Statistical significance^a
Age range, years	31–71	13–66	NA
Survival time, days	775.9 ± 726.0	1093.8 ± 638.8	NA
Sex			NS
Male	36 (90.0)	254 (86.1)
Female	4 (10.0)	41 (13.9)
Blood type			NS
A	9 (22.5)	89 (30.2)
B	13 (32.5)	80 (27.1)
AB	3 (7.5)	26 (8.8)
O	15 (37.5)	100 (33.9)
MELD score¹⁸			NS
<10	10 (25.0)	48 (16.3)
10–19	12 (30.0)	115 (39.0)
20–29	10 (25.0)	94 (31.9)
≥30	8 (20.0)	38 (12.9)
Pre-liver transplant disease			NS
Hepatocellular carcinoma	16 (40.0)	109 (36.9)
Fulminant hepatitis	11 (27.5)	46 (15.6)
Decompensated liver cirrhosis	13 (32.5)	134 (45.4)
Other	0 (0.0)	6 (2.0)

Data expressed as mean or n (%) of patients.

Pearson’s χ²-test with Yates’ correction for continuity (or Fisher’s exact test).

MELD, model for end-stage liver disease; NA, not applicable; NS, no statistically significant between-group differences (P > 0.05).

Liver transplant patients were genotyped for IL6-572 C/G (rs1800796), which met the search criteria of minor allele frequency ≥20% and r² ≥0.8. There was no significant difference in rs1800796 allele frequency between the AR and NAR groups (Table 2).

Table 2.

Distribution of genotypes and allele frequency for IL-6-572 C/G (rs1800796) in 335 patients assigned to an acute rejection (AR) group or nonacute rejection (NAR) group following liver transplantatio.

IL-6 -572C/G characteristic	Patient group		OR (95% CI)	Statistical significance	Model
IL-6 -572C/G characteristic	AR n = 40	NAR n = 295	OR (95% CI)	Statistical significance	Model
Allele			—	NS^a	—
C	63 (78.75)	444 (75.25)
G	17 (21.25)	146 (24.75)
Genotype				NS^c	Codominant
CC	27 (67.5)	164 (55.6)	1.00
CG	9 (22.5)	116 (39.3)	0.47 (0.21, 1.04)
GG	4 (10)	15 (5.1)	1.62 (0.50, 5.25)
CC vs	27 (67.5)	164 (55.6)	1.00	NS^b	Dominant
CG + GG	13 (32.5)	131 (44.4)	0.60 (0.30, 1.21)
CC + CG vs	36 (90.0)	280 (94.9)	1.00	NS^b	Recessive
GG	4 (10.0)	15 (5.1)	2.07(0.65, 6.59)
CC + GG vs	31 (77.5)	179 (60.7)	1.00	P = 0.033^b	Overdominant
CG	9 (22.5)	116 (39.3)	0.45 (0.21, 0.98)

Data presented as within-group n (%) incidence.

Pearson’s χ²-test with Yates’ correction for continuity.

Genotypes analysed using multivariate logistic regression analysis, using the website for SNP Statistics: http://bioinfo.iconcologia.net/snpstats/start.htm.

OR, odds ratio; CI, confidence intervals; NS, no statistically significant between-group differences (P > 0.05); vs, versus.

Analysis of genotype distribution, to assess whether there was an association between genotype and acute rejection, is shown in Table 2

Multivariate logistic regression analysis of the association of genotypes in four inheritance models showed that, in the overdominant model, the heterozygous CG genotype was associated with a significantly decreased acute rejection rate when compared with the homozygous CC and GG genotypes (P = 0.033) (Table 2).

Discussion

Studies have focused on the identification of individual immune response characteristics following liver transplantation, in an attempt to predict acute rejection and adapt immunosuppressive therapy accordingly.^11,19 The current study investigated IL6-572 C/G in liver transplant recipients, and examined whether polymorphisms in the IL6 promoter region had a predictive value for acute rejection in these patients. There were no statistically significant between-group differences in the distribution of IL6-572 C/G alleles, but the IL6-572 CG genotype was strongly associated with a significantly lower rate of acute rejection.

Interleukin-6 is a mediator of the acute-phase response and differentiation or activation of macrophages (and B and T cells), and has both pro- and anti-inflammatory properties.²⁰ Furthermore, IL-6 can activate the signal transducer and activator of transcription 3 and tyrosine-protein phosphatase nonreceptor type 11/growth factor-receptor-bound protein 2-associated binder/mitogen-activated protein kinase signalling pathways, via the glycoprotein 130 signal transducer.²¹ IL-6 has been associated with various diseases. For example, increased levels of IL-6 are observed in diseases such as rheumatoid arthritis,²² chronic inflammatory proliferative disease, Graves’ disease, and systemic lupus erythematosus.¹⁴ High levels of plasma IL-6 have been associated with acute rejection,²³ and high levels of IL-6 have been consistently associated with increased graft rejection following liver transplantation.^24,25 Because several lines of evidence support the involvement of IL-6 in various diseases, genetic polymorphisms related to these diseases have been extensively studied. The human IL6 gene is located on chromosome 7p21. Four polymorphisms, −597 G/A, −572 C/G, −373 A(n)T(n) and −174 G/C, have been identified in the promoter region of the human IL-6 gene. A polymorphism in the 5′ flanking region of the IL-6 gene at position −174 G/C will affect IL6 gene transcription.²⁶ The polymorphism at position −174 (rs1800795) in the IL6 promoter region results in a G to C transition, leading to a subsequent decrease in IL6 expression and plasma levels.²⁶ Following coronary artery bypass surgery, patients with the −174 C/C allele show higher plasma IL-6 levels than patients with the −174 G/G allele.²⁷ Both −174 G/C and −597 G/A in the IL6 gene have been related to a higher risk of developing type 2 diabetes mellitus.²⁸ Studies on the impact of recipient −174 G/C polymorphism on acute rejection following renal transplantation have provided conflicting results. One study found a lower frequency of G/G or G/C genotype in recipients who developed acute rejection,²⁹ while another found a higher frequency of these genotypes,^30,31 although the results of these studies were not statistically significant. High levels of the G/G and G/C IL-6 promoter genotypes have been shown to be significantly associated with decreased risk of acute rejection following liver transplant.¹⁶

Although there is no published research relating IL6-597 G/A (rs1800797) polymorphisms and acute rejection, some studies report an association with osteoarthritis.^32,33 Most studies have focused on the IL6-174 G/C polymorphism, which has been identified as having an association with acute rejection following liver transplantation.¹⁶ Investigations into the IL6-572 C/G polymorphism have demonstrated IL-6 levels to be significantly lower in subjects carrying the −572 G allele compared with those carrying the −572 C allele, and the C→G variation was shown to reduce the transcriptional activity of the IL6 promoter significantly.^27,34 Other research suggests that individuals carrying the GG genotype have a significantly higher risk of chronic periodontitis,³⁵ and the CC genotype may increase the risk for Alzheimer's disease.³⁶ The present study investigated the association between SNPs of the IL6 promoter region and the incidence of acute rejection in liver transplant recipients. Only the −572 C/G (rs1800796) polymorphism met the criteria of minor allele frequency ≥20% and r² ≥0.8 for the Chinese population of Beijing. Thus, further research focused on IL6-572 C/G.

Studies on transcriptional regulation of the IL6 gene have identified at least four transcription factor sites located within the IL-6 promoter region, including nuclear factor-κB, adenosine 3′5′ cyclic monophosphate response element binding protein, CCAAT-enhancer-binding protein, and activator protein 1.³⁷ Under normal conditions, some transcription factors play a key role, whereas others play a cofactor role. Although research has suggested that subjects carrying the −572 G allele could possess significantly reduced IL6 promoter transcriptional activity, the role of each binding site and its cognate transcription factor(s) in the regulation of IL-6 expression is complex and dependent on cell type and stimulus.³⁷ The heterozygous CG genotype in IL6-572 C/G in the present study appeared to be a protection factor for acute rejection after liver transplantation. With regard to acute rejection of the liver, the CG genotype may reduce the function of the key transcription factor and this co-operative function may reduce the transcriptional activity of the IL-6 promoter, subsequently reducing IL-6 levels. When this variation occurs in both alleles, the genotype for CC or GG, the key transcription factor may lose its function and the cotranscription factor may compensate, resulting in both unchanged IL-6 promoter transcriptional activity and unchanged IL-6 levels. Thus, IL6-572 C/G may play a role in modifying the risk of acute rejection.

In conclusion, this is the first report to evaluate the relationship between polymorphisms of IL6-572 C/G and acute rejection following liver transplant. The true biological function of IL-6 in acute rejection is still unclear, so further research should focus on the function of the IL6-572 C/G SNP. There is a possibility that genotyping could be used as a predictor of acute rejection. This may have significant implications for future immunosuppressive therapy to prevent acute rejection, following liver transplant procedures, in the Han Chinese population.

Footnotes

Acknowledgements

We thank all patients who participated in this study, and Zhang Lin and Fu Jilei for collecting the clinical data.

Declaration of Conflicting Interest

The Authors declare that there are no conflicts of interest.

Funding

This work was supported by the National Basic Research Program for Innovative Research Group of China (No.81121002), National Basic Research Program of China (973 Program) (No.2009CB522407), National Hi-tech Project 863 (No.2011AA020103) and the Nonprofit Technology and Application Research Program of Zhejiang Province, grant number 2011C37013.

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Recipient IL-6-572C/G genotype is associated with reduced incidence of acute rejection following liver transplantation

Abstract

Objective

Methods

Results

Conclusion

Keywords

Introduction

Patients and methods

Study Population

Immunosuppressant Regimen and Rejection Diagnosis

DNA Extraction

Polymorphism Genotyping

Statistical Analyses

Results

Discussion

Footnotes

Acknowledgements

Declaration of Conflicting Interest

Funding

References