The Effect Of Genetic Polymorphisms In Predicting The Prognosis Of Chinese Liver Transplant Recipients

[Introduction]Tacrolimus is one kind of the calcineurin inhibitors with high effect in preventing acute rejection after solid organ transplantation. However, it has a narrow therapeutic index and shows highly variable pharmacokinetics. Much recent research has focused on the possible causes of these interindividual differences in pharmacokinetics of calcineurin ingibitors.It has been clear that the biological activity of the permeability-glycoprotein plays an important role in this respect. P-glycoprotein (PGP1) is the product of the multidrug-resistance 1 gene (MDR1)(ABCB1). The protein serves as a transporter and is capable of pummping a wide variety of endogenous substances, as well as drugs (including tacrolimus), from the cytoplasm to the exterior of the cell. Physiologically, PGP1 is present in liver, kidney and small intestine. In the small intestine, PGP1 is expressed at the apical surface of mature enterocytes, where it prevents the absorption of xenobiotics from the intestinal lumen by active extruson from the cell interior.The interindividual differences in the pharmacokinetics of calcineurin inhibitors have been attributed to interindividual heterogeneity in enzymatic activity of PGP1. It has been reported that the C to T variation at nucleotide 3435 in exon 26 of MDR1 gene is associated with decreased PGP1 expression. This single nucleotidepolymorphism (SNP) may partly explain the large interindividual variations in thepharmacokinetic characteristics of tacrolimus and may control the extent of its uptakeafter ingestion.[Objective]To identify the relationship between the polymorphism of multidrug resistance 1 genein the liver transplant donors and recipients and interindividual variations inpost-transplant recipients' tacrolimus dose requirement and drug concentration-to-doseratio.[Methods]50 patients who underwent orthotopic liver transplantation from cadaveric donorsbetween 2004 July and 2005 March in our center and treated with triple-regimentherapy including tacrolimus, MMF and steroid were enrolled in this retrospectivestudy. At three time spots after FK506 administration, 1 week, 2 weeks and 1 month,the daily dose of tacrolimus was recorded and the blood trough concentrationmeasured. The donor and recipient genomic DNA was extracted fromEDTA-anticoagulated blood, liver or spleen specimen. MDR1 gene was amplified byPCR. The occurrence of MDR1 3435(C→T) polymorphism was investigated bypolymerase chain reaction followed by restriction sites polymorphism analysis orDNA sequencing in 50 liver transplant recipients and their corresponding donors.Doses (mg/kg body weight) and dose-adjusted trough levels (ng/ml per mg/kg bodyweight) were compared according to allelic status for MDR1.[Results]Although inconsistence of MDR1 genotypes was observed in 17 cases between thedonors and recipients, the differece of compositon of genotypes was not statisticallysignificant. The MDR1 genotype CC was observed in 23 subjects (23%), whereas 64(64%) were CT and 13 (13%) were TT. There was a large interindividual variation inrequired tacrolimus dose of recipients. The dose of FK506 ranged from 0.014 to 0.175mg/kg/d, from 0.017 to 0.182mg/kg/d and from 0.022 to 0.179mg/kg/d at the end of the 1^st week, 2^nd week and 1^st month after drug administration, respectively. Tacrolimus doses required to achieve target blood concentrations were higher in the patients with MDR1 CC genotype than in the CT or TT genotype patients, and the dose-adjusted trough levels were lower. No significant differences were found in tacrolimus doses or dose-adjusted trough levels according to the donor's MDR1genotype. [Conclusions]1. To obtain a target tacrolimus concentration after liver transplantation,tacrolimus dose requirement varies greatly between individuals.2. The large inter-individual variation of FK506 dose requirement is influenced by the absorption and excretion activity of MDR1, which is correlated with gene polymorphisms.3. Polymorphisms of the recipient MDR1 gene seem to contribute more to such variation than that of donor in liver transplantation. Genotyping the MDR1 3435 C/T polymorphism of recipient rather than donor would provide useful information to individualize tacrolimus dose.[Introduction]With the development of more effective immunosuppressive strategies and prophylaxis of the primary disease recurrence to avoid or control graft failure, survival after liver transplantation (LT) has dramatically improved in the decade. Despite this success, graft failure occurs in up to 13% of the patients during the first year, rising to 35% in 5 years. Graft rejection and recurrence of the primary disease are the two major immunological complications. The outcome of any immune response is determined at least in part by the microenvironment in which the response occurs. A network of shortacting cytokines, which are produced by immunologically active cells and stromal tissue, in turn determines this environment. Cytokines have a central role in the immunologic events after transplantation and are intimately implicated in graft rejection and recurrence of primary disease.In view of the critical role of these cytokines and growth factors in regulating immune responses, subtle differences in cytokine expression may have a major effect on the outcome of the response. Cytokine production is under genetic control. Polymorphisms in cytokine gene regulatory regeins have shown to correlate with individual variations in cytokine production. For example, a G-to-A polymorphism at position -308 in the promoter region of the tumor necrosis factor alpha (TNF-α) gene increases transcription and in vitro production 6- to 7-fold. However, a similar G-to-Apolymorphism at position -1082 of the interleukin (IL)-10 promoter reduces IL-10 production. Alloimmune responses and variations in susceptibility to rejection may be influenced by individual variations in cytokine genes. A number of studies have investigated cytokine genes polymorphisms and their association with graft rejection in transplant patients. The results are controversial, and these studies mainly focused on Caucasian patients.Identifying genetic risk factors maybe can predict an outcome for the liver transplantation recipients, which may be used to develop a personalized immunosuppressive and prevent Hepatitis B Virus (HBV) recurrence regimens. [Objective]This study was to identify potential genetic markers that can be associated with Acute Rejection (AR) and recurrence of HBV and analyze how these polymorphisms affect the post-transplant outcome. For this purpose, we analyzed genetic polymorphism in three different cytokines, TNF-α, IL-10, and TGF-β1 genes. [Methods]A total of 186 Chinese patients with HBV (165 males and 21 females, aged 19-67 years, mean 46±9 years old) were included in this study, all of whom received LT at the Liver Transplant Centre of the First Affiliated Hospital of Zhejiang University College of Medicine between January 2004 and December 2005. Immunosuppression used Triple protocol including Cyclosporin or Tacrolimus. Lamivudine combined with Hepatits B Immunoglobin was used to prevent HBV recurrence. AR was diagnosed by histological findings when biopsy was performed for clinical parameters. HBV recurrence is characterized by the appearance of HBsAg in serum after liver transplantation.Genomic DNA was isolated using a standard proteinase K digestion of frozen explant liver tissue or peripheral blood from recipients. The TNF-α (-308/-238), IL-10(-1082/-819/-592), and TGF-β1 (-988/-800/-509/+869/+915) genetypings were performed using genomic DNA sequencing (ABI 3037). Single nucleotide polymorphisms were detected by Polyphred. To check the genomic DNA sequencing, the polymorphism at position IL-10-819 was detected by polymerase chain reaction-restriction fragment length polymorphinsm (PCR-RFLP). A chi square test (x2) was used to compare the genes frequency of occurrence between different groups. A P value less than 0.05 was considered statistically significant. [Results]In this study of the 186 patients, 41 patients suffered at least one acute rejection(AR) episode in the foliowed-up, with a risk of 22%; 29 patients occurred HBV recurrence, with a risk of 15.6%. Them, based on AR, we divided these patients into AR group(41) and non-AR group(145). And based on HBV recurrence, divided them into HBV recurrence group(29) and non-recurrence group(157).None of patients was positive for the TNF-α-308AA/-238AA and IL-10-1082GG genotypes in our study. The distribution of TNF-α-308GA/-238GA and IL-10-1082GA genotype frequency was 14.6%, 2.4% and 12.2% in the AR group respectively, while 10.3%, 3.4% and 11.7% in the non-AR group. The difference was not statistically significant (P>0.05). The distribution of TNF-α-308GA/-238GA and IL-10-1082GA genotype frequency was 20.7%, 6,9% and 20.7% in the HBV recurrence group respectively, while 9.6%, 2.6% and 10.2% in the non-recurrence group. The difference was not statistically significant yet(P>0.05). The IL-10 gene at position -819 and -592 was linkage disequilibrium (LD). In the AR and the non-AR groups, the distribution frequency of TT(AA)was 31.7% vs 44.8 %, TC(AC)was 51.2 %vs 49.7%, and CC(CC)was 17.1%vs 5.5%. The difference in distribution frequency of TT(AA)and TC+CC(AC+CC)group was not statistically significant (P>0.05). In the HBV recurrence and non-recurrence groups, the distributionfrequency of TT(AA)was 44.8% vs 41.4%, TC(AC)was 44.8% vs 51.0%, CC(CC)was 10.4% vs 7.6%, showing no statistically significant difference(P>0.05).There was no single nucleotide polymorphisms at position -988/-800/+915 of TGF-β1 gene. In the AR group and the non-AR group, the distribution frequency of TGF-β1 -509TT was 24.4% vs 27.6%, TC, 61.0% vs 53.8%, while CC, 14.6% v.s18.6%; and the distribution frequency of TGF-β1 +869CC was 24.4% vs 28.3%, CT, 53.7% vs 40.7%, while TT genotypes, 21.9% vs 31.0%. There was no statistically significant difference between the two groups (P>0.05). In the HBV recurrence group and non-recurrence group, the distribution frequency of TGF-β1 -509TT was 27.6% vs 26.8%, TC, 44.8% vs 57.3%, while CC, 27.6% vs 15.9%, and the distribution frequency of TGF-β1 +869CC was 31.0% vs 26.7%, CT, 31.0% vs 45.9%, while TT, 38.0% vs 27.4%. There was also no statistically significant difference (P>0.05). At position -509 and +869 of TGF-β1 gene was linkage disequilibrium too. [Conclusions]1. There was no statistically significant correlation between acute rejection and cytokine gene polymorphisms of TNF-α, IL-10, and TGF-β1 in liver transplant recipients.2. There was also no statistically significant correlation between HBV recurrence and cytokine gene polymorphisms of TNF-α, IL-10, and TGF-β1 in liver transplant recipients.3. TNF-α -308AA/-238AA and IL-10-1082GG genotypes, which are considered to be correlated with high cytokine production, are scarce in Chinese people.4. TGF-β1 gene at positions -988/-800/+915 were few single nucleotide polymorphisms in Chinese people.[Introduction]The use of hepatitis B immune globulin (HBIG) led to a significant reduction in recurrent Hepatitis B Virus (HBV) infection and improved survival in patients who received liver transplantations (LT) for HBV related diseases, but the recurrence remains. In the clinic practice, the patients with the same disease and same therapys, achieved different curative effect. We supposed that HBIG may have individual effect on prevention of HBV graft reinfection following liver transplantation. HBIG, one kind of IgG bind receptor, is intravenous or intramuscular injection. Studies showed human leukocyte FcγReceptor IIIa (CD16) present on position 158 (Phe or Val) in the membrane-proximal, IgG-binding domain, which is related to the process of antibody-dependent cell-mediated cytotoxicity (ADCC). The nonconservative T to G substitution at nucleotide 559 predicts (FCGR3A gene) accompanies a change of phenylalanine (F) to valine (V) at amino acid position 176. Compared with F/F homozygotes, FcγRIIIa expressed on NK cells and monocytes in V/V homozygotes bound more IgG1 and IgG3 despite identical levels of receptor expression. So the efficacy of HBIG must be influenced by the genetypings.[Objective]This study was to investigate whether the efficacy of HBIG is associated with FCGR3A gene polymorphisms in Chinese liver transplant patients.[Methods]A total of 77 patients received liver transplantation for hepatitis B related diseases between January 1st 2004 and November 30th 2005 and survived more than one year after surgery were included in this study. HBV recurrence is characterized by the appearance of HBsAg in serum after the operation. The FCGR3A genetyping was performed using genomic DNA sequencing (ABI 3037). Single nucleotide polymorphism at nucleotide 559 was detected by Polyphred. A chi square test (x²) was used to compare the genes frequency between the HBV recurrence group and non-recurrence group. The recurrence-free survival was compared between groups (be grouped according to the polymorphisms of FCGR3A) by Kaplan-Meier method. A P value less than 0.05 was considered statistically significant.[Results]There were 77 patients in the follow-up (70 men and 7 women). Their average age was 45.16±8.134 years old, and the average follow-up time was 623±169 days (360 to 996 days) . Of them, 14 patients complicated with HBV recurrence post-transplant. The FCGR3A at nucleotide 559 TT was observed in 35(45.4%) subjects, whereas 31(40.3%) were TG and 11(14.3%) were GG In 559G carrier group (42, 54.5%), the risk of HBV recurrence was 9.5%, and 1- and 2-year recurrence-free survival rates were 95.2% and 88.7%, respectively. In 559G noncarrier group (35, 45.5%), the risk of HBV recurrence was 28.6%, and 1- and 2-year recurrence-free survival rates were 74.3% and 69.3%, respectively. The risk of HBVrecurrence and the recurrence-free survival rate were both statistically significant different between 559G carrier and noncarrier groups (P<0.05).[Conclusions]1. A single nucleotide polymorphism at position 559 of FCGR3A gene was found in Chinese patients, and it is not scarce.2. The efficacy of HBIG in prophylaxis Hepatitis B Virus recurrence after liver transplantation is associated with FCGR3A gene at nucleotide 559 single nucleotide polymorphism (T/G).3. Detecting FCGR3A genotypes is useful to adjust the dosage of HBIG.