Objectives: This study sought to investigate the associations between polymorphisms in the chemokine receptor 5 gene and acute rejection in liver transplant.

Materials and Methods: A total of 266 patients who underwent a liver transplant between January 2006 and March 2009 were enrolled in this study. Genomic DNA was extracted from whole blood, and chemokine receptor 5Δ32 was detected by polymerase chain reaction. Eight nucleotide polymorphism loci in the chemokine receptor 5 gene were detected by Applied Biosystems SNaP-Shot and TaqMan technologies.

Results: Chemokine receptor 5Δ32 mutation was not detected in all the individuals from China. There was no significant association between the single nucleotide polymorphism in chemokine receptor 5 gene and acute rejection.

Conclusions: Single nucleotide polymorphisms in a single gene of the chemokine receptor 5 might not play a role in acute rejection after liver transplant.

Key words : Chemokine receptor 5, Single nucleotide polymorphism, Acute rejection, Liver transplant, Chemokine receptor 5Δ32 polymorphism

Introduction

Liver transplant has become an effective method of curing end-stage liver disease; however, allograft rejection remains a major risk factor for long-term allograft dysfunction.¹ Recruitment of allo-leukocytes to the graft is a key feature in developing acute and chronic rejection in solid organ transplant. Chemokine receptors are important for regulating migration and activation of allo-leukocytes by interacting with its respective chemokine.² Some genetic variants of chemokines and chemokine receptors have been demonstrated to be associated with functional and biological effects including susceptibility to numerous diseases. Polymorphisms in chemokine receptors could be factors that affect allograft rejection.³

Chemokine receptor 5 (CCR5) is a functional GPCR (the 7-transmembrane G-protein coupled receptor) that is antagonized by several C-C motif chemokines. It is expressed mainly in granulocytes, macrophages, immature dendritic cells, CD8+ lymphocytes, and Th1 lymphocytes. It influences migration and activation of cells that express the receptor by interacting with its ligands.⁴ Chemokine receptor 5 has been demonstrated to be associated with susceptibility to allograft rejection. In a murine transplant model, deficiency or blockage of CCR5 can attenuate rejection, and prolong allograft survival. The benefit might be associated with a diminished infiltration of allo-reactive leukocytes and a shift in the immune response from Th1 to Th2. The null functional genetic variant CCR5Δ32 and the decreased functional genotype CCR5-59029A>G (rs1799987) have been reported as protective genetic factors against transplant rejection.⁵ In this study, we genotyped the polymorphisms in CCR5 gene, and investigated the associations between polymorphisms of CCR5 and allograft rejection risk in liver transplant recipients from China.

Materials and Methods

Study population
This study was conducted at our center. A total of 266 adult liver transplant recipients were enrolled. Liver transplants were performed between January 2006 and March 2009 at the Department of Hepatobiliary Pancreatic Surgery of the First Affiliated Hospital, Zhejiang University School of Medicine. Piggy-back liver transplants were performed in the deceased donor. In the living donor, right-lobe liver volume was preoperatively estimated by computed tomography scans. The graft-to-recipient weight ratio of 0.8% or graft volume/standard liver volume of 35% was the lowest limit for a transplant. Details of donor hepatectomy and the recipients’ surgical technique, including middle hepatic vein reconstruction, agreed with our previous report.⁶ All individuals were of Chinese ethnicity. This study was approved by the Ethical Review Committee of the First Affiliated Hospital, School of Medicine; written, informed consent was obtained from all patients, and the study protocol adhered to the Declaration of Helsinki.

Maintenance immunosuppressive therapy consisted of triple drugs of tacrolimus, mycophenolate, and prednisone. Patients were highly suspicious of acute rejection when they presented the symptoms of fever, change in color and amount of bile, hyperbilirubinemia, or hyper­transaminasemia in the absence of vascular and biliary complications. All acute rejection cases were confirmed by percutaneous liver biopsy. Banff criteria7 were applied to classify acute rejection, and rejection active index was introduced to score the severity of acute rejection. In this system, the rejection active index comprised 3 components, each scored from 0 to 3: venous endothelial inflammation, bile duct damage, and portal inflammation. Each was graded semiquantitatively on a scale ranging from 0 (absent) to 3 (severe). Individual scores were added to produce an overall rejection score ranging from 0 to 9. A score ≥ 3 was considered acute rejection. Once acute rejection was confirmed with grades, patients were treated with an intravenous steroid (3 × 500 mg methylprednisolone); patients with steroid-resistant rejections were treated with OKT3 (muromonab-CD3, 5 mg for 5 to 7 days). Patients with ongoing rejection were confirmed by repeat biopsy.

DNA extraction and genotyping of chemokine receptor 5
The genomic DNA was extracted from 0.2 mL EDTA-anticoagulated whole blood by QIAamp DNA Blood Mini Kits (Qiagen, Hilden, Germany) according to the manufacturer’s protocol.

Chemokine receptor 5Δ32 genotyping was performed using polymerase chain reaction (PCR) amplification of genomic DNA. Primers for CCR5 were forward, cttcattacacctgcagc-3’, and reverse, 5’-cctctcatttcgacaccgaagc-3’. The PCR mixture was Ampli Taq Gold 360 Master Mix (Invitrogen Ltd., Shanghai, PR China), and the reaction volume was 25 µL. Forty PCR cycles were run on the thermal cycler (Eppendorf AG, Hamburg, Germany) using the following temperature profile: Denaturation, 95°C for 4 minutes; amplification, 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds (35 cycles), and terminal longation, 72°C for 10 minutes. Polymerase chain reaction products were separated by electrophoresis in a 3% agarose gel (169 base pairs for the wild-type allele, and 137 base pairs for the 32 base pair deletion allele).

Eight single nucleotide polymorphism (SNP) loci of the CCR5 gene (rs2734648, rs1799863, rs1799987, rs1799988, rs1800023, rs1800024, rs1800452, and rs746492) were detected using Applied Biosystems SNaP-Shot (Applied Biosystems, Foster City, CA, USA) and TaqMan (Applied Biosystems) technologies. Polymerase chain reaction amplification was performed in a 10-µL reaction system, which contained 1 × GC buffer I (Takara Biotechnology [Dalian], Shiga, Japan), 3.0 mM Mg2⁺, 0.3 mM dNTP, 1 U Takara Taq polymerase (Takara), 1 µL genomic DNA; and 1 µM, each, forward and reverse primers. The thermal cycle was set at 95°C for 2 minutes; 11 cycles × (94°C for 20 s, 65°C-0.5°C/cycle for 40 s, and 72°C for 1.5 min); 24 cycles × (94°C for 20 s, 59°C for 30 s, and 72°C for 1.5 min); 72°C for 2 minutes; and 4°C forever.

Multiplex PCR products were purified to remove the remaining primers and dNTPs through the following procedures. The 10-µL purification reaction volume consisted of 9 µL of PCR product, 1 U of SAP (Promega Corporation, Madison, WI, USA), and 1 U of ExoI (Epicentre Biotechnologies, Madison, WI, USA). Incubation of the reaction for 60 minutes at 37°C, then inactivation of the enzymes for 15 minutes at 75°C. A single base extension reaction was performed using the SNaPShot Multiplex Kit, with 5 µL SNaPShot ready reaction mix of 1 µL of extension primer for each polymorphism, and 2 µL of purified PCR products, 2 µL ddH₂O in 10 µL total volume. Extension was done using 28 cycles of denaturation at 96°C for 10 seconds, annealing at 50°C for 10 seconds, and extension at 60°C for 30 seconds. After the reaction, postextension treatment helped remove the ddNTPs from the extension products. The 0.5-µL purified products were mixed with 9 µL of Hi-Di formamide and 0.5 µL of GeneScan 120 LIZ internal size standard (Applied Biosystems). We denatured the mixture at 95°C for 5 minutes and then put it on the ice for 5 minutes. The allele and genotype frequency were determined by analyzing raw data from ABI3130XL with Gene Mapper 4.0 (Applied Biosystems). Polymerase chain reaction and extension primers are listed in Table 1. To assess the reliability of genotyping, a random selection of samples > 10% was genotyped for a second time with 100% concordance.

Statistical Analyses
Statistical analyses were performed with SPSS software for Windows (Statistical Product and Service Solutions, version 15.0, SSPS Inc, Chicago, IL, USA). Qualitative data are demonstrated as means ± standard deviation; continuous variables were analyzed with a t test; and categorical variables were analyzed with chi-square test or Fisher exact test. Haploview version 4.2 was used to assess disequilibrium of the linage.⁸ All tests were 2-tailed. Values for P < .05 were considered statistically significant.

Results

Demographic and clinical characteristics
Demographic and clinical characteristics are shown in Table 2. A total 266 patients (218 male and 48 female) was enrolled in this study, the mean age was 47.0 ± 11.5 years. The overall incidence of patients with acute rejection was 12.8% (34/266). Mean follow-up after liver transplant was 24.2 ± 13.8 months. Analyses of the recipients’ characteristics and factors that might affect acute rejection including sex, age, preoperative Model for End-Stage Liver Disease score, donor type, and hepatitis B infection history showed no differences between those with acute rejection and those without acute rejection. Of the 34 rejection episodes, 2 were severe grade, 14 were moderate grade, and 18 were mild grade. After antirejection therapy, 32 were reversed, 2 patients died with severe liver congestion and acute rejection, 2 patients died of recurrent hepatocellular carcinoma during follow-up. The 1- and 2-year survival rates for the rejection and nonrejection transplant recipients were 91.2% versus 92.2%, 88.2% versus 88.4%. No statistical significance existed between patients with acute rejection and those without it.

No associations were detected between poly­morphisms in chemokine receptor 5 with acute rejection
Among the 8 SNP loci, all the SNP loci were in Hardy-Weinberg equilibrium. Minor allelic frequency of rs1799863 and rs1800452 were less than 1% (Table 3). rs1799988 and rs1800023 were in completely linkage disequilibrium with rs1799987 and rs2734648. The genotype of CCR5Δ32 was not detected in all the individuals from China. For these reasons, the SNP loci rs1799863, rs1800452, rs1799988, rs1800023, and CCR5Δ32 were excluded from the next analysis.

We characterized the linkage disequilibrium block between the 4 CCR5 SNP loci using the pairwise D’ values.⁹ The D’ values of every 2 SNP loci ranged from 0.94 to 0.99, indicating a strong linkage disequilibrium between each paired SNP loci. Single nucleotide polymorphism distributions of the CCR5 gene in transplant recipients are listed in Table 4. No associations were found between SNP and rejection in liver transplant.

In addition, analyses of haplotypes were performed to investigate the combined effect of the 4 SNP loci (rs2734648, rs1788887, rs1800024, and rs746492) on allograft rejection. The most common haplotypes were TGCT, GGCT, and TACG (> 5%); no association was found between the haplotype and rejection risk from a conditional logistic regression test.

Discussion

Almost all patients need lifelong immuno­suppressive therapy after a liver transplant. Despite advances in immunosuppressant drugs and technology, the incidence of rejection remains a risk factor for long-term allograft dysfunction. Clarifying the mechanism of rejection is crucial. Recently, the interaction between chemokines and chemokine receptors in allograft rejection has been intensively studied.

Chemokine receptor 5 was one of the most-studied chemokine receptors recognized as the coreceptor of human immunodeficiency virus (HIV).⁴ Recently, several transplant models, such as renal,¹⁰ cardiac,¹¹ and islet¹² were performed in mice. Results suggest that the absence and blockage of CCR5 could prolong allograft survival time and attenuate the allograft rejection. Chemokine receptor 5 also has been suggested as the target of modulating the immune response of the host. Chemokine receptor 5Δ32, a 32-base pair deletion in the CCR5 gene, which leads to an inactive receptor and up-regulation of ligands for CCR5,13 has been demonstrated as a protective genetic factor against rejection in solid organ transplant.¹⁴

The influence of CCR5Δ32 in allograft rejection after liver transplant remains controversial. Moench and associates¹⁵ have demonstrated that the CCR5Δ32 allele in adult liver transplant is associated with a high risk for ischemic-type biliary lesions. Heidenhain and associates indicated that patients with the CCR5Δ32 allele had a benefit effect of reducing rejection episodes, suggesting CCR5 as a target of therapy and characterization of rejection potential.¹⁶ However, a study on pediatric liver transplants¹⁷ revealed that the CCR5Δ32 allele were not associated with allograft rejection. These controversial results might be caused by a difference in design and epidemiologic factors. In our study, the CCR5Δ32 mutation was not detected in all individuals, and we could not assess the association with rejection in liver transplant.

The SNP in rs1799987A>G correlated with a decreased expression level and was reportedly associated with pathogenesis and progression of HIV infection.^{18, 19} Renal transplant recipients with the A allele were reported to have a 2-fold reduction in the incidence of acute rejection and higher number of rejection episodes.²⁰ Another study in a Turkish cohort had similar results.²¹ However, neither of these studies explained the relations between reduction of rejection risk and increased expression level in the renal recipients carrying the A allele. The result from a Korean cohort suggests that renal transplant recipients with the A allele have a higher risk of early acute rejection, the genotype of donor in rs1799987 did not correlate with rejection risk.²² Whether the SNP in the CCR5 gene can be associated with rejection in liver transplant remains unknown.

In the present study, we genotyped 8 SNP loci in the CCR5 gene; among these, 5 SNP loci were in the upstream in the open reading frame—the haplotype-based researches have demonstrated as being associated with the progression of HIV infection. However, in our study, rs1799988 and rs1800023 were nearly in complete linkage disequilibrium with rs1799987 and rs2734648. The 2 loci in the extron correlated with missense function had no polymorphisms (minor allele frequency < 1). Thereafter, these 4 SNPs were excluded, and we assessed the associations of 4 SNP loci rs1799987, rs2734648, rs1800024, and rs746492 with acute rejection in liver transplant recipients. Neither the analyses of a single SNP locus nor the haplotypes of the 4 combined SNP loci were associated with acute rejection.

Chemokine receptor 5 might play a role in selective recruitment of T cells to infiltrate the graft, leading to mutant alleles correlating with a higher expression level and increased rejection risk. Unlike previous reports on kidney transplant, we failed to find an association between rejection risk and SNP in the CCR5 gene. This negative result might be caused by differences in the study design and demographics. Apart from the liver allograft serving as an immunologically privileged organ, several genetic conditions might contribute to differences in the results: The results of this study might be confounded by sample selection, genetic admixture, and regional differences in gene frequencies and gene-environment interactions. It also suggests that progression of acute rejection in a liver transplant might be affected by multiple, interactive genes. Polymorphisms in a single gene of CCR5 might not play a role in rejection, but genetic effects would be displayed when other genes are taken into consideration. The main limitation of this study was that the mRNA and functional chemokine receptor expression level in peripheral blood and/or donor tissue was not included. We need more functional data to support future research.

Although the role of CCR5 in transplant rejection has been demonstrated, several genetic variants have been reportedly associated with risk of rejection in renal transplant, the results of our study suggest that SNPs in a single gene of CCR5 might not play a role in acute rejection in Chinese patients after a liver transplant.

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